Faculty Profiles - KAWACHI Hiroshi

Papers - KAWACHI Hiroshi

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14-3-3 Proteins stabilize actin and vimentin filaments to maintain processes in renal glomerular podocyte. Reviewed International journal

Hidenori Yasuda, Yoshiyasu Fukusumi, Ying Zhang, Hiroshi Kawachi

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 37 ( 10 ) e23168 2023.10

Language：English Publishing type：Research paper (scientific journal)

14-3-3 proteins are a ubiquitously expressed family of adaptor proteins. Despite exhibiting high sequence homology, several 14-3-3 isoforms have isoform-specific binding partners and roles. We reported that 14-3-3β interacts with FKBP12 and synaptopodin to maintain the structure of actin fibers in podocytes. However, the precise localization and differential role of 14-3-3 isoforms in kidneys are unclear. Herein, we showed that 14-3-3β in glomeruli was restricted in podocytes, and 14-3-3σ in glomeruli was expressed in podocytes and mesangial cells. Although 14-3-3β was dominantly co-localized with FKBP12 in the foot processes, a part of 14-3-3β was co-localized with Par3 at the slit diaphragm. 14-3-3β interacted with Par3, and FKBP12 bound to 14-3-3β competitively with Par3. Deletion of 14-3-3β enhanced the interaction of Par3 with Par6 in podocytes. Gene silencing for 14-3-3β altered the structure of actin fibers and process formation. 14-3-3β and synaptopodin expression was decreased in podocyte injury models. In contrast, 14-3-3σ in podocytes was expressed in the primary processes. 14-3-3σ interacted with vimentin but not with the actin-associated proteins FKBP12 and synaptopodin. Gene silencing for 14-3-3σ altered the structure of vimentin fibers and process formation. 14-3-3σ and vimentin expression was increased in the early phase of podocyte injury models but was decreased in the late stage. Together, the localization of 14-3-3β at actin cytoskeleton plays a role in maintaining the foot processes and the Par complex in podocytes. In contrast, 14-3-3σ at vimentin cytoskeleton is essential for maintaining primary processes.

DOI： 10.1096/fj.202300865R

Th17 Cells Participate in Thy1.1 Glomerulonephritis Which Is Ameliorated by Tacrolimus. Reviewed International journal

Syuhei Watanabe, Ying Zhang, Yoshiyasu Fukusumi, Hidenori Yasuda, Akira Takada, Junichiro J Kazama, Hiroshi Kawachi

American journal of nephrology 53 ( 5 ) 388 - 396 2022

Language：English Publishing type：Research paper (scientific journal)

INTRODUCTION: Thy1.1 glomerulonephritis (Thy1.1 GN) in rats is widely used as an experimental model of mesangial proliferative glomerulonephritis (GN). We previously reported that T-helper (Th) cells were accumulated in glomeruli from the early phase of this model and that not Th2 cells but Th1 cells play an important role in the development of glomerular alterations. Although Th17 is reported to be involved in the pathogenesis of several autoimmune diseases, the role of Th17 cells in the pathogenesis of mesangial alterations in Thy1.1 GN remains unclear. METHODS: The kinetics of the infiltration of subsets of Th cells and the expression of IL-17 in Thy1.1 GN were analyzed. Next, the localization and the cell types of IL-17 receptor (IL-17R)-positive cells and IL-6-positive cells were analyzed. Then, the effect of tacrolimus on the expressions of Th17-related cytokines in Thy1.1 GN was analyzed. RESULTS: Not only Th1 cells but also Th17 cells were recruited into glomeruli from the early phase of the disease. mRNA expression of IL-17 in glomeruli was elevated. The increased positive expression of IL-17R was detected in the mesangial area, and some of IL-17R-positive cells were co-stained with IL-6. Tacrolimus treatment ameliorated mesangial alterations by suppressing the expressions of Th17-related cytokines such as IL-17 and IL-6. CONCLUSION: Th17 cells participate in the development of Thy1.1 GN, a mimic of mesangial proliferative GN, and Th17 cells and their related cytokines are pertinent therapeutic targets.

DOI： 10.1159/000524111

Tacrolimus ameliorates podocyte injury by restoring FK506 binding protein 12 (FKBP12) at actin cytoskeleton. Reviewed International journal

Hidenori Yasuda, Yoshiyasu Fukusumi, Veniamin Ivanov, Ying Zhang, Hiroshi Kawachi

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 35 ( 11 ) e21983 2021.11

Language：English Publishing type：Research paper (scientific journal)

FKBP12 was identified as a binding protein of tacrolimus (Tac). Tac binds to FKBP12 and exhibits immunosuppressive effects in T cells. Although it is reported that Tac treatment directly ameliorates the dysfunction of the podocyte in nephrotic syndrome, the precise pharmacological mechanism of Tac is not well understood yet. It is also known that FKBP12 functions independently of Tac. However, the localization and the physiological function of FKBP12 are not well elucidated. In this study, we observed that FKBP12 is highly expressed in glomeruli, and the FKBP12 in glomeruli is restricted in podocytes. FKBP12 in cultured podocytes was expressed along the actin cytoskeleton and associated with filamentous actin (F-actin). FKBP12 interacted with the actin-associated proteins 14-3-3 and synaptopodin. RNA silencing for FKBP12 reduced 14-3-3 expression, F-actin staining, and process formation in cultured podocytes. FKBP12 expression was decreased in the nephrotic model caused by adriamycin (ADR) and the cultured podocyte treated with ADR. The process formation was deteriorated in the podocytes treated with ADR. Tac treatment ameliorated these decreases. Tac treatment to the normal cells increased the expression of FKBP12 at F-actin in processes and enhanced process formation. Tac enhanced the interaction of FKBP12 with synaptopodin. These observations suggested that FKBP12 at actin cytoskeleton participates in the maintenance of processes, and Tac treatment ameliorates podocyte injury by restoring FKBP12 at actin cytoskeleton.

DOI： 10.1096/fj.202101052R

Podocyte-specific Crb2 knockout mice develop focal segmental glomerulosclerosis. International journal

Akiko Tanoue, Kan Katayama, Yugo Ito, Kensuke Joh, Masaaki Toda, Taro Yasuma, Corina N D'Alessandro-Gabazza, Hiroshi Kawachi, Kunimasa Yan, Masaaki Ito, Esteban C Gabazza, Karl Tryggvason, Kaoru Dohi

Scientific reports 11 ( 1 ) 20556 - 20556 2021.10

Language：English Publishing type：Research paper (scientific journal)

Crb2 is a cell polarity-related type I transmembrane protein expressed in the apical membrane of podocytes. Knockdown of crb2 causes glomerular permeability defects in zebrafish, and its complete knockout causes embryonic lethality in mice. There are also reports of Crb2 mutations in patients with steroid-resistant nephrotic syndrome, although the precise mechanism is unclear. The present study demonstrated that podocyte-specific Crb2 knockout mice develop massive albuminuria and microhematuria 2-month after birth and focal segmental glomerulosclerosis and tubulointerstitial fibrosis with hemosiderin-laden macrophages at 6-month of age. Transmission and scanning electron microscopic studies demonstrated injury and foot process effacement of podocytes in 6-month aged podocyte-specific Crb2 knockout mice. The number of glomerular Wt1-positive cells and the expressions of Nphs2, Podxl, and Nphs1 were reduced in podocyte-specific Crb2 knockout mice compared to negative control mice. Human podocytes lacking CRB2 had significantly decreased F-actin positive area and were more susceptible to apoptosis than their wild-type counterparts. Overall, this study's results suggest that the specific deprivation of Crb2 in podocytes induces altered actin cytoskeleton reorganization associated with dysfunction and accelerated apoptosis of podocytes that ultimately cause focal segmental glomerulosclerosis.

DOI： 10.1038/s41598-021-00159-z

Synbindin Downregulation Participates in Slit Diaphragm Dysfunction Reviewed International journal

Veniamin Ivanov, Yoshiyasu Fukusumi, Ying Zhang, Hidenori Yasuda, Meiko Kitazawa, Hiroshi Kawachi

American Journal of Nephrology 52 ( 8 ) 1 - 10 2021.8

Language：English Publishing type：Research paper (scientific journal) Publisher：S. Karger AG

<b><i>Introduction:</i></b> Synbindin, originally identified as a neuronal cytoplasmic molecule, was found in glomeruli. The cDNA subtractive hybridization technique showed the mRNA expression of synbindin in glomeruli was downregulated in puromycin aminonucleoside (PAN) nephropathy, a mimic of minimal-change nephrotic syndrome. <b><i>Methods:</i></b> The expression of synbindin in podocytes was analyzed in normal rats and 2 types of rat nephrotic models, anti-nephrin antibody-induced nephropathy, a pure slit diaphragm injury model, and PAN nephropathy, by immunohistochemical analysis and RT-PCR techniques. To elucidate the function of synbindin, a gene silencing study with human cultured podocytes was performed. <b><i>Results:</i></b> Synbindin was mainly expressed at the slit diaphragm area of glomerular epithelial cells (podocytes). In both nephrotic models, decreased mRNA expression and the altered staining of synbindin were already detected at the early phase when proteinuria and the altered staining of nephrin, a key molecule of slit diaphragm, were not detected yet. Synbindin staining was clearly reduced when severe proteinuria was observed. When the cultured podocytes were treated with siRNA for synbindin, the cell changed to a round shape, and filamentous actin structure was clearly altered. The expression of ephrin-B1, a transmembrane protein at slit diaphragm, was clearly lowered, and synaptic vesicle-associated protein 2B (SV2B) was upregulated in the synbindin knockdown cells. <b><i>Conclusion:</i></b> Synbindin participates in maintaining foot processes and slit diaphragm as a downstream molecule of SV2B-mediated vesicle transport. Synbindin downregulation participates in slit diaphragm dysfunction. Synbindin can be an early marker to detect podocyte injury.

DOI： 10.1159/000517975

Nephrin-Ephrin-B1-Na+/H+ Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury. Reviewed International journal

Yoshiyasu Fukusumi, Hidenori Yasuda, Ying Zhang, Hiroshi Kawachi

The American journal of pathology 191 ( 7 ) 1209 - 1226 2021.7

Language：English Publishing type：Research paper (scientific journal)

Ephrin-B1 is one of the critical components of the slit diaphragm of kidney glomerular podocyte. However, the precise function of ephrin-B1 is unclear. To clarify the function of ephrin-B1, ephrin-B1-associated molecules were studied. RNA-sequencing analysis suggested that Na+/H+ exchanger regulatory factor 2 (NHERF2), a scaffolding protein, is associated with ephrin-B1. NHERF2 was expressed at the apical area and the slit diaphragm, and interacted with the nephrin-ephrin-B1 complex at the slit diaphragm. The nephrin-ephrin-B1-NHERF2 complex interacted with ezrin bound to F-actin. NHERF2 bound ephrin-B1 via its first postsynaptic density protein-95/disks large/zonula occludens-1 domain, and podocalyxin via its second postsynaptic density protein-95/disks large/zonula occludens-1 domain. Both in vitro analyses with human embryonic kidney 293 cells and in vivo study with rat nephrotic model showed that stimulaiton of the slit diaphragm, phosphorylation of nephrin and ephrin-B1, and dephosphorylation of NHERF2 and ezrin, disrupted the linkages of ephrin-B1-NHERF2 and NHERF2-ezrin. It is conceivable that the linkage of nephrin-ephrin-B1-NHERF2-ezrin-actin is a novel critical axis in the podocytes. Ephrin-B1 phosphorylation also disrupted the linkage of an apical transmembrane protein, podocalyxin, with NHERF2-ezrin-actin. The phosphorylation of ephrin-B1 and the consequent dephosphorylation of NHERF2 are critical initiation events leading to podocyte injury.

DOI： 10.1016/j.ajpath.2021.04.004

Xanthine oxidoreductase inhibitor topiroxostat ameliorates podocyte injury by inhibiting the reduction of nephrin and podoplanin. Reviewed International journal

Ying Zhang, Yoshiyasu Fukusumi, Mutsumi Kayaba, Takashi Nakamura, Ryusuke Sakamoto, Naoki Ashizawa, Hiroshi Kawachi

Nefrologia 2021.3

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: Topiroxostat, an inhibitor of xanthine oxidoreductase (XOR) was shown to reduce urinary albumin excretion of hyperuricemic patients with chronic kidney disease. However, its pharmacological mechanism is not well understood. In this study, we examined the effects of topiroxostat on glomerular podocytes. Podocyte is characterized by foot process and a unique cell-cell junction slit diaphragm functioning as a final barrier to prevent proteinuria. METHODS: The effects of topiroxostat on the expressions of podocyte functional molecules were analysed in db/db mice, a diabetic nephropathy model, anti-nephrin antibody-induced rat podocyte injury model and cultured podocytes treated with adriamycin. RESULTS: Topiroxostat treatment ameliorated albuminuria in db/db mice. The expression of desmin, a podocyte injury marker was increased, and nephrin and podocin, key molecules of slit diaphragm, and podoplanin, an essential molecule in maintaining foot process were downregulated in db/db mice. Topiroxostat treatment prevented the alterations in the expressions of these molecules in db/db mice. XOR activity in kidney was increased in rats with anti-nephrin antibody-induced podocyte injury. Topiroxostat treatment reduced XOR activity and restored the decreased expression of nephrin, podocin and podoplanin in the podocyte injury. Furthermore, topiroxostat enhanced the expression of podoplanin in injured human cultured podocytes. CONCLUSIONS: Podocyte injury was evident in db/db mice. Topiroxostat ameliorated albuminuria in diabetic nephropathy model by preventing podocyte injury. Increase of XOR activity in kidney contributes to development of podocyte injury caused by stimulation to slit diaphragm. Topiroxostat has an effect to stabilize slit diaphragm and foot processes by inhibiting the reduction of nephrin, podocin and podoplanin.

DOI： 10.1016/j.nefro.2020.10.011

New insight into podocyte slit diaphragm, a therapeutic target of proteinuria. Reviewed

Hiroshi Kawachi, Yoshiyasu Fukusumi

Clinical and experimental nephrology 24 ( 3 ) 193 - 204 2020.3

Language：English Publishing type：Research paper (scientific journal)

Dysfunction of slit diaphragm, a cell-cell junction of glomerular podocytes, is involved in the development of proteinuria in several glomerular diseases. Slit diaphragm should be a target of a novel therapy for proteinuria. Nephrin, NEPH1, P-cadherin, FAT, and ephrin-B1 were reported to be extracellular components forming a molecular sieve of the slit diaphragm. Several cytoplasmic proteins such as ZO-1, podocin, CD2AP, MAGI proteins and Par-complex molecules were identified as scaffold proteins linking the slit diaphragm to the cytoskeleton. In this article, new insights into these molecules and the pathogenic roles of the dysfunction of these molecules were introduced. The slit diaphragm functions not only as a barrier but also as a signaling platform transfer the signal to the inside of the cell. For maintaining the slit diaphragm function properly, the phosphorylation level of nephrin is strictly regulated. The recent studies on the signaling pathway from nephrin, NEPH1, and ephrin-B1 were reviewed. Although the mechanism regulating the function of the slit diaphragm had remained unclear, recent studies revealed TRPC6 and angiotensin II-regulating mechanisms play a critical role in regulating the barrier function of the slit diaphragm. In this review, recent investigations on the regulation of the slit diaphragm function were reviewed, and a strategy for the establishment of a novel therapy for proteinuria was proposed.

DOI： 10.1007/s10157-020-01854-3

Partitioning-Defective-6-Ephrin-B1 Interaction Is Regulated by Nephrin-Mediated Signal and Is Crucial in Maintaining Slit Diaphragm of Podocyte. Reviewed International journal

Sayuri Takamura, Yoshiyasu Fukusumi, Ying Zhang, Ichiei Narita, Hiroshi Kawachi

The American journal of pathology 190 ( 2 ) 333 - 346 2020.2

Language：English Publishing type：Research paper (scientific journal)

Ephrin-B1 plays a critical role at slit diaphragm. Partitioning-defective (Par)-6 is down-regulated in podocyte of ephrin-B1 knockout mouse, suggesting that Par-6 is associated with ephrin-B1. Par polarity complex, consisting of Par-6, Par-3, and atypical protein kinase C, is essential for tight junction formation. In this study, the expression of Par-6 was analyzed in the normal and nephrotic syndrome model rats, and the molecular association of Par-6, Par-3, ephrin-B1, and nephrin was assessed with the human embryonic kidney 293 cell expression system. Par-6 was concentrated at slit diaphragm. Par 6 interacted with ephrin-B1 but not with nephrin, and Par-3 interacted with nephrin but not with ephrin-B1. The complexes of Par-6-ephrin-B1 and Par-3-nephrin were linked via extracellular sites of ephrin-B1 and nephrin. The Par-6-ephrin-B1 complex was delinked from the Par-3-nephrin complex, and Par-6 and ephrin-B1 were clearly down-regulated already at early phase of nephrotic model. The alteration of Par-6/ephrin-B1 advanced that of Par-3/nephrin. Stimulation to nephrin phosphorylated not only nephrin but also ephrin-B1, and consequently inhibited the interaction between ephrin-B1 and Par-6. Par-6 appeared at presumptive podocyte of early developmental stage and moved to basal area at capillary loop stage to participate in slit diaphragm formation at the final stage. Par-6-ephrin-B1 interaction is crucial for formation and maintenance of slit diaphragm of podocyte.

DOI： 10.1016/j.ajpath.2019.10.015

Synaptic Vesicle Protein 2B Negatively Regulates the Amyloidogenic Processing of AβPP as a Novel Interaction Partner of BACE1. Reviewed International journal

Masakazu Miyamoto, Akira Kuzuya, Yasuha Noda, Sakiho Ueda, Megumi Asada-Utsugi, Shinji Ito, Yoshiyasu Fukusumi, Hiroshi Kawachi, Ryosuke Takahashi, Ayae Kinoshita

Journal of Alzheimer's disease : JAD 75 ( 1 ) 173 - 185 2020

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: Given that amyloid-β (Aβ) peptide is produced and released at synapses, synaptic Aβ is one of the promising therapeutic targets to prevent synaptic dysfunction in Alzheimer's disease (AD). Although Aβ production begins with the cleavage of the amyloid-β protein precursor (AβPP) by β-site AβPP cleaving enzyme 1 (BACE1), the mechanism on how BACE1 is involved in AβPP processing at synapses remains unclear. OBJECTIVE: This study aimed to identify novel BACE1 interacting proteins regulating Aβ production at the synapse. METHODS: BACE1 interacting proteins were pulled down using a mass spectrometry-based proteomics of wild-type (WT) rat brain synaptoneurosome lysates utilizing anti-BACE1 antibody. Then, a novel BACE1 interactor was identified and characterized using experimental systems that utilized transfected cells and knockout (KO) mice. RESULTS: Synaptic vesicle protein 2B (SV2B) was identified as a novel presynaptic interaction partner of BACE1. In HEK293 cells, co-overexpression of SV2B with BACE1 significantly reduced the sAβPPβ and Aβ levels released in the media; thus, SV2B overexpression negatively affected the AβPP cleavage by BACE1. Compared with those of WT mice, the hippocampal lysates of SV2B knockout mice had significantly elevated Aβ levels, whereas the β-secretase activity and the AβPP and BACE1 protein levels remained unchanged. Finally, a fractionation assay revealed that BACE1 was mislocalized in SV2B KO mice; hence, SV2B may be involved in BACE1 trafficking downregulating the amyloidogenic pathway of AβPP. CONCLUSION: SV2B has a novel role of negatively regulating the amyloidogenic processing of AβPP at the presynapses.

DOI： 10.3233/JAD-200071

Phosphate binding by sucroferric oxyhydroxide ameliorates renal injury in the remnant kidney model. Reviewed International journal

Yoshikazu Nemoto, Takanori Kumagai, Kenichi Ishizawa, Yutaka Miura, Takeshi Shiraishi, Chikayuki Morimoto, Kazuhiro Sakai, Hiroki Omizo, Osamu Yamazaki, Yoshifuru Tamura, Yoshihide Fujigaki, Hiroshi Kawachi, Makoto Kuro-O, Shunya Uchida, Shigeru Shibata

Scientific reports 9 ( 1 ) 1732 - 1732 2019.2

Language：English Publishing type：Research paper (scientific journal)

Recent clinical studies indicate that the disturbed phosphate metabolism in chronic kidney disease (CKD) may facilitate kidney injury; nonetheless, the causal role of phosphate in CKD progression remains to be elucidated. Here, we show that intestinal phosphate binding by sucroferric oxyhydroxide (SF) ameliorates renal injury in the rat remnant kidney model. Sprague-Dawley rats received 5/6 nephrectomy (RK) and had a normal chow or the same diet containing SF (RK + SF). RK rats showed increased plasma FGF23 and phosphate levels, which were suppressed by SF administration. Of note, albuminuria in RK rats was significantly ameliorated by SF at both 4 and 8 weeks. SF also attenuated glomerulosclerosis and tubulointerstitial injury. Moreover, several different approaches confirmed the protective effects on podocytes, explaining the attenuation of glomerulosclerosis and albuminuria observed in this study. As a possible mechanism, we found that SF attenuated renal inflammation and fibrosis in RK rats. Interestingly, von Kossa staining of the kidney revealed calcium phosphate deposition in neither RK nor RK + SF rats; however, plasma levels of calciprotein particles were significantly reduced by SF. These data indicate that latent positive phosphate balance accelerates CKD progression from early stages, even when overt ectopic calcification is absent.

DOI： 10.1038/s41598-018-38389-3

Nephrin-binding Ephrin-B1 at the slit diaphragm controls podocyte function through the JNK pathway

Yoshiyasu Fukusumi, Ying Zhang, Ryohei Yamagishi, Kanako Oda, Toru Watanabe, Katsuyuki Matsui, Hiroshi Kawachi

Journal of the American Society of Nephrology 29 ( 5 ) 1462 - 1474 2018.5

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society of Nephrology

Background B-type ephrins are membrane-bound proteins that maintain tissue function in several organs. We previously reported that ephrin-B1 is localized at the slit diaphragm of glomerular podocytes. However, the function of ephrin-B1 at this location is unclear. Methods We analyzed the phenotype of podocyte-specific ephrin-B1 knockout mice and assessed the molecular association of ephrin-B1 and nephrin, a key molecule of the slit diaphragm, in HEK293 cells and rats with anti-nephrin antibody-induced nephropathy. Results Compared with controls, ephrin-B1 conditional knockoutmice displayed altered podocytemorphology, disarrangement of the slit diaphragm molecules, and proteinuria. Ephrin-B1 expressed in HEK293 cells immunoprecipitated with nephrin, which required the basal regions of the extracellular domains of both proteins. Treatment of cells with an anti-nephrin antibody promoted the phosphorylation of nephrin and ephrin-B1. However, phosphorylation of ephrin-B1 did not occur in cells expressing amutant nephrin lacking the ephrin-B1 binding site or in cells treated with an Src kinase inhibitor. The phosphorylation of ephrin-B1 enhanced the phosphorylation of nephrin and promoted the phosphorylation of c-Jun N-terminal kinase (JNK), which was required for ephrin-B1- promoted cell motility in wound-healing assays. Notably, phosphorylated JNK was detected in the glomeruli of control mice but not ephrin-B1 conditional knockout mice. In rats, the phosphorylation of ephrin-B1, JNK, and nephrin occurred in the early phase (24 hours) of anti-nephrin antibody-induced nephropathy. Conclusions Through interactions with nephrin, ephrin-B1 maintains the structure and barrier function of the slit diaphragm. Moreover, phosphorylation of ephrin-B1 and, consequently, JNK are involved in the development of podocyte injury.

DOI： 10.1681/ASN.2017090993

PACSIN2 accelerates nephrin trafficking and is up-regulated in diabetic kidney disease

Vincent Dumont, Tuomas A. Tolvanen, Sara Kuusela, Hong Wang, Tuula A. Nyman, Sonja Lindfors, Jukka Tienari, Harry Nisen, Shiro Suetsugu, Markus Plomann, Hiroshi Kawachi, Sanna Lehtonen

FASEB JOURNAL 31 ( 9 ) 3978 - 3990 2017.9

Language：English Publishing type：Research paper (scientific journal) Publisher：FEDERATION AMER SOC EXP BIOL

Nephrin is a core component of podocyte (glomerular epithelial cell) slit diaphragm and is required for kidney ultrafiltration. Down-regulation or mislocalization of nephrin has been observed in diabetic kidney disease (DKD), characterized by albuminuria. Here, we investigate the role of protein kinase C and casein kinase 2 substrate in neurons 2 (PACSIN2), a regulator of endocytosis and recycling, in the trafficking of nephrin and development of DKD. We observe that PACSIN2 is up-regulated and nephrin mislocalized in podocytes of obese Zucker diabetic fatty (ZDF) rats that have altered renal function. In cultured podocytes, PACSIN2 and nephrin colocalize and interact. We show that nephrin is endocytosed in PACSIN2-positive membrane regions and that PACSIN2 overexpression increases both nephrin endocytosis and recycling. We identify rabenosyn-5, which is involved in early endosome maturation and endosomal sorting, as a novel interaction partner of PACSIN2. Interestingly, rabenosyn-5 expression is increased in podocytes in obese ZDF rats, and, in vitro, its overexpression enhances the association of PACSIN2 and nephrin. We also show that palmitate, which is elevated in diabetes, enhances this association. Collectively, PACSIN2 is up-regulated and nephrin is abnormally localized in podocytes of diabetic ZDF rats. In vitro, PACSIN2 enhances nephrin turnover apparently via a mechanism involving rabenosyn-5. The data suggest that elevated PACSIN2 expression accelerates nephrin trafficking and associates with albuminuria.

DOI： 10.1096/fj.201601265R

Restricted nutrition-induced low birth weight, low number of nephrons and glomerular mesangium injury in Japanese quail Reviewed

H. Nishimura, E. Yaoita, M. Nameta, K. Yamaguchi, M. Sato, C. Ihoriya, L. Zhao, H. Kawachi, T. Sasaki, Y. Ikezumi, Y. Ouchi, N. Kashihara, T. Yamamoto

Journal of Developmental Origins of Health and Disease 8 ( 3 ) 287 - 300 2017.6

Language：English Publishing type：Research paper (scientific journal) Publisher：Cambridge University Press

Insufficient nutrition during the perinatal period causes structural alterations in humans and experimental animals, leading to increased vulnerability to diseases in later life. Japanese quail, Coturnix japonica, in which partial (8-10%) egg white was withdrawn (EwW) from eggs before incubation had lower birth weights than controls (CTs). EwW birds also had reduced hatching rates, smaller glomeruli and lower embryo weight. In EwW embryos, the surface condensate area containing mesenchymal cells was larger, suggesting that delayed but active nephrogenesis takes place. In mature EwW quail, the number of glomeruli in the cortical region (mm2) was significantly lower (CT 34.7±1.4, EwW 21.0±1.2)
capillary loops showed focal ballooning, and mesangial areas were distinctly expanded. Immunoreactive cell junction proteins, N-cadherin and podocin, and slit diaphragms were clearly seen. With aging, the mesangial area and glomerular size continued to increase and were significantly larger in EwW quail, suggesting compensatory hypertrophy. Furthermore, apoptosis measured by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling analysis was higher in EwWs than in CTs on embryonic day 15 and postnatal day 4 (D4). Similarly, plasma glucocorticoid (corticosterone) was higher (P&lt
0.01) on D4 in EwW quail. These results suggest that although nephrogenic activity is high in low-nutrition quail during the perinatal period, delayed development and increased apoptosis may result in a lower number of mature nephrons. Damaged or incompletely mature mesangium may trigger glomerular injury, leading in later life to nephrosclerosis. The present study shows that birds serve as a model for 'fetal programming,' which appears to have evolved phylogenetically early.

DOI： 10.1017/S2040174416000787

Possible role for glomerular-derived angiotensinogen in nephrotic syndrome

Mihoko Yamazaki, Yoshiyasu Fukusumi, Mutsumi Kayaba, Yukina Kitazawa, Sayuri Takamura, Ichiei Narita, Hiroshi Kawachi

JOURNAL OF THE RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM 17 ( 4 ) 2016.10

Language：English Publishing type：Research paper (scientific journal) Publisher：SAGE PUBLICATIONS LTD

Background and objective: Renin-angiotensin system (RAS) inhibitors reduce glomerular injury and proteinuria, indicating that angiotensin II (Ang II) is involved in glomerular diseases. Although the local RAS is reported to play an essential role in maintaining local tissue functions, the role of the local RAS in regulating glomerular function is not well evaluated. In this study, we analyzed the glomerular expression of RAS components in nephrotic models and the effect of Ang II receptor blockers (ARB) on the expression of angiotensinogen (AGT).
Methods: The levels of glomerular expression of RAS components were analyzed in two nephrotic models: anti-nephrin antibody-induced nephropathy and PAN nephropathy, a mimic of human minimal change nephrotic syndrome. The effect of the ARB irbesartan on the expression of AGT in the nephrotic model was analyzed.
Results: Glomerular expression of AGT and the receptors for Ang II was clearly increased in the nephrotic models, while the expression levels of renin, ACE and ACE2 were decreased. ARB treatment suppressed the increase of glomerular expression of AGT in the nephrotic model.
Conclusion: It is conceivable that the promoted local RAS action participated in the glomerular dysfunction, and that ARB treatment ameliorated slit diaphragm injury by inhibiting the positive feedback loop of the activated local Ang II action.

DOI： 10.1177/1470320316681223

Leptin deficiency down-regulates IL-23 production in glomerular podocytes resulting in an attenuated immune response in nephrotoxic serum nephritis

Kei Goto, Yoshikatsu Kaneko, Yuya Sato, Tadashi Otsuka, Suguru Yamamoto, Shin Goto, Keiko Yamamoto, Tadashi Yamamoto, Hiroshi Kawachi, Michael P. Madaio, Ichiei Narita

INTERNATIONAL IMMUNOLOGY 28 ( 4 ) 197 - 208 2016.4

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Leptin and IL-23 production in NTS nephritis.Leptin, one of the typical adipokines, is reported to promote T(h)17 cell responses and to enhance production of proinflammatory cytokines. To clarify the role of leptin in the regulation of the IL-23/IL-17 axis and the development of kidney disease, we used a murine model of nephrotoxic serum (NTS) nephritis (NTN). Sheep NTS was administered in wild-type C57BL/6J mice and food-restricted, leptin-deficient C57BL/6J-ob/ob (FR-ob/ob) mice after preimmunization with sheep IgG. The profile of mRNA expression relevant to T helper lymphocytes in the kidneys was analyzed by quantitative real-time PCR (qRT-PCR). Cultured murine glomerular podocytes and peritoneal exudate macrophages (PEMs) were used to investigate the direct effect of leptin on IL-23 or MCP-1 production by qRT-PCR. Kidney injury and macrophage infiltration were significantly attenuated in FR-ob/ob mice 7 days after NTS injection. The T(h)17-dependent secondary immune response against deposited NTS in the glomeruli was totally impaired in FR-ob/ob mice because of deteriorated IL-17 and proinflammatory cytokine production including IL-23 and MCP-1 in the kidney. IL-23 was produced in glomerular podocytes in NTN mice and cultured murine glomerular podocytes produced IL-23 under leptin stimulation. MCP-1 production in PEMs was also promoted by leptin. Induction of MCP-1 expression was observed in PEMs regardless of Ob-Rb, and the leptin signal was transduced without STAT3 phosphorylation in PEMs. Leptin deficiency impairs the secondary immune response against NTS and down-regulates IL-23 production and T(h)17 responses in the NTN kidney, which is accompanied by decreased MCP-1 production and macrophage infiltration in the NTN kidney.

DOI： 10.1093/intimm/dxv067

Role of calcineurin (CN) in kidney glomerular podocyte: CN inhibitor ameliorated proteinuria by inhibiting the redistribution of CN at the slit diaphragm

Ayako Wakamatsu, Yoshiyasu Fukusumi, Eriko Hasegawa, Masayuki Tomita, Toru Watanabe, Ichiei Narita, Hiroshi Kawachi

Physiological Reports 4 ( 6 ) 1 - 13 2016.3

Language：English Publishing type：Research paper (scientific journal) Publisher：Wiley-Blackwell Publishing Ltd

Although calcineurin (CN) is distributed in many cell types and functions in regulating cell functions, the precise roles of CN remained in each type of the cells are not well understood yet. A CN inhibitor (CNI) has been used for steroid-resistant nephrotic syndrome. A CNI is assumed to ameliorate proteinuria by preventing the overproduction of T-cell cytokines. However, recent reports suggest that CNI has a direct effect on podocyte. It is accepted that a slit diaphragm (SD), a unique cell-cell junction of podocytes, is a critical barrier preventing a leak of plasma protein into urine. Therefore, we hypothesized that CNI has an effect on the SD. In this study, we analyzed the expression of CN in physiological and in the nephrotic model caused by the antibody against nephrin, a critical component of the SD. We observed that CN is expressed at the SD in normal rat and human kidney sections and has an interaction with nephrin. The staining of CN at the SD was reduced in the nephrotic model, while CN activity in glomeruli was increased. We also observed that the treatment with tacrolimus, a CNI, in this nephrotic model suppressed the redistribution of CN, nephrin, and other SD components and ameliorated proteinuria. These observations suggested that the redistribution and the activation of CN may participate in the development of the SD injury.

DOI： 10.14814/phy2.12679

Avian Podocytes, Which Lack Nephrin, Use Adherens Junction Proteins at Intercellular Junctions

Eishin Yaoita, Hiroko Nishimura, Masaaki Nameta, Yutaka Yoshida, Hiroki Takimoto, Hidehiko Fujinaka, Hiroshi Kawachi, Sameh Magdeldin, Ying Zhang, Bo Xu, Tomizo Oyama, Fujio Nakamura, Tadashi Yamamoto

JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 64 ( 1 ) 67 - 76 2016.1

Language：English Publishing type：Research paper (scientific journal) Publisher：SAGE PUBLICATIONS LTD

Nephrin, a major intercellular junction (ICJ) molecule of mammalian podocytes in the renal glomerulus, is absent in the avian genome. We hypothesized that birds use ICJ molecules other than nephrin in their podocytes. Therefore, in the present study, we examined the possible involvement of adherens junction (AJ) proteins in the ICJs of avian podocytes. We found the AJ proteins N-cadherin and - and -catenins in podocytes of quail and chickens but not in those of rats, pigs or humans. The AJ proteins were prominent in avian glomerulus-rich fractions in immunoblot analyses, and in immunofluorescence microscopy analyses, they were localized along glomerular capillary walls appearing in at least two staining patterns: weakly diffuse and distinctly granular. Immunoelectron microscopy demonstrated that the significant accumulation of immunogold particles for the AJ proteins were especially evident in avian slit diaphragms and AJs. Furthermore, N-cadherin was found to be expressed in all nephron cells in the early developmental stage but became confined to podocytes during maturation. These results indicate that avian slit diaphragms clearly express AJ proteins as compared with that in the mammalwhere AJ proteins are suppressed to an extremely low leveland that avian podocytes are interconnected by AJs per se in addition to slit diaphragms.

DOI： 10.1369/0022155415611708

Alteration in the podoplanin-ezrin-cytoskeleton linkage is an important initiation event of the podocyte injury in puromycin aminonucleoside nephropathy, a mimic of minimal change nephrotic syndrome

Koichi Suzuki, Yoshiyasu Fukusumi, Mihoko Yamazaki, Hiroshi Kaneko, Kazushi Tsuruga, Hiroshi Tanaka, Etsuro Ito, Katsuyuki Matsui, Hiroshi Kawachi

CELL AND TISSUE RESEARCH 362 ( 1 ) 201 - 213 2015.10

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

Podoplanin was identified as a protein associated with the transformation of arborized foot processes of glomerular epithelial cells (podocytes) to flat feet. However, the function of podoplanin in the podocyte is not yet fully clarified. In this study, we analyzed the molecular nature of podoplanin, and its expression in rat nephrotic models and patients with minimal change nephrotic syndrome (MCNS). We demonstrated here that podoplanin has two forms: one contains abundant sialic acid and the other a lesser amount of sialic acid. Podoplanin bound ezrin to interact with the cytoskeleton. The silencing of podoplanin in cultured podocytes caused a change in the cell shape and the distribution of ezrin and actin. The expression of podoplanin was clearly reduced before the onset of proteinuria in puromycin aminonucleoside (PAN) nephropathy, a mimic of MCNS, and the decrease in the expression of podoplanin became more evident at the proteinuric stage. Podoplanin was detected in normal urine samples, and the amount of urinary podoplanin markedly increased on day 1 of PAN nephropathy. Urinary ezrin was also detected. The amount of the phosphorylated ezrin was reduced, while the amount of the podoplanin-interacting ezrin increased. The podoplanin expression was reduced in a patient with active-phase MCNS. It is conceivable that the alteration of the podoplanin-ezrin-cytoskeleton linkage is an important event of the podocyte injury in MCNS.

DOI： 10.1007/s00441-015-2178-8

SV2B is essential for the integrity of the glomerular filtration barrier

Yoshiyasu Fukusumi, Ayako Wakamatsu, Natsumi Takashima, Eriko Hasegawa, Naoko Miyauchi, Masayuki Tomita, Hiroshi Kawachi

Laboratory Investigation 95 ( 5 ) 534 - 545 2015.5

Language：English Publishing type：Research paper (scientific journal) Publisher：Nature Publishing Group

The glomerular visceral epithelial cell (podocyte) is characterized as a specialized structure of the interdigitating foot processes, covering the outer side of the glomerular basement membrane (GBM). The neighboring foot processes are connected by a slit diaphragm, which is a key structure regulating the barrier function of the glomerular capillary wall to prevent proteinuria. We have previously reported that synaptic vesicle protein 2 B (SV2B) is expressed in the podocyte and that the expression is clearly decreased in nephrotic models. However, the precise function of SV2B in the podocyte is unclear. To investigate the role of SV2B in maintaining the podocyte function and to better understand the function of the neuron-like vesicle expressing SV2B in the podocyte, we analyzed them with SV2B knockout (KO) mice. An increase in the amount of proteinuria, effacement of the foot process of the podocyte, and alterations of the GBM were detected in SV2B KO mice. It was also found that the expression of CD2AP, nephrin, and NEPH1, the functional molecules of the slit diaphragm, and laminin, a critical component of the GBM, is clearly altered in SV2B KO mice. Synaptotagmin and neurexin, which have a role in the synaptic vesicle docking in neurons, are downregulated in the kidney cortex of SV2B KO mice. We have previously reported that neurexin interacts with CD2AP, and the present study shows that SV2B interacts with CD2AP. These findings suggest that the SV2B-neurexin complex is involved in the formation and maintenance of the slit diaphragm. In addition, SV2B is densely expressed close to the cell surface in the presumptive podocyte in the early stage of glomerulogenesis. These results suggest that SV2B has an essential role in the formation and maintenance of the glomerular capillary wall.

DOI： 10.1038/labinvest.2015.39

Systematic implantation of dedifferentiated fat cells ameliorated monoclonal antibody 1-22-3-induced glomerulonephritis by immunosuppression with increases in TNF-stimulated gene 6

Takashi Maruyama, Noboru Fukuda, Taro Matsumoto, Koichiro Kano, Morito Endo, Minako Kazama, Tomohiko Kazama, Jin Ikeda, Hiroyuki Matsuda, Takahiro Ueno, Masanori Abe, Kazuyoshi Okada, Masayoshi Soma, Koichi Matsumoto, Hiroshi Kawachi

STEM CELL RESEARCH & THERAPY 6 2015.4

Language：English Publishing type：Research paper (scientific journal) Publisher：BIOMED CENTRAL LTD

Introduction: Implantation of mesenchymal stem cells (MSCs) has recently been reported to repair tissue injuries through anti-inflammatory and immunosuppressive effects. We established dedifferentiated fat (DFAT) cells that show identical characteristics to MSCs.
Methods: We examined the effects of 10(6) of DFAT cells infused through renal artery or tail vein on monoclonal antibody (mAb) 1-22-3-induced glomerulonephritis (as an immunological type of renal injury) and adriamycin-induced nephropathy (as a non-immunological type of renal injury) in rats. The mAb 1-22-3-injected rats were also implanted with 10(6) of DFAT cells transfected with TSG-6 siRNA through tail vein.
Results: Although DFAT cells transfused into blood circulation through the tail vein were trapped mainly in lungs without reaching the kidneys, implantation of DFAT cells reduced proteinuria and improved glomerulosclerosis and interstitial fibrosis. Implantation of DFAT cells through the tail vein significantly decreased expression of kidney injury molecule-1, collagen IV and fibronectin mRNAs, whereas nephrin mRNA expression was increased. Implantation of DFAT cells did not improve adriamycin-induced nephropathy, but significantly decreased the glomerular influx of macrophages, common leukocytes and pan T cells. However, the glomerular influx of helper T cells, was increased. Implantation of DFAT cells decreased expression of interleukin (IL)-6 and IL-12 beta mRNAs and increased expression of TNF-stimulated gene (TSG)-6 mRNA in renal cortex from mAb 1-22-3-injected rats. The basal level of TSG-6 protein was significantly higher in DFAT cells than in fibroblasts. Expression of TSG-6 mRNA in MCs cocultured with DFAT cells was significantly higher than in mesangial cells or DFAT cells alone. Systematic implantation of DFAT cells with TSG-6 siRNA through tail vein did not improve proteinuria, renal dysfunction and renal degeneration in the mAb 1-22-3-injected rats.
Conclusion: Systematic implantation of DFAT cells effectively ameliorated mAb 1-22-3-induced glomerulonephritis through immunosuppressive effects accompanied by the suppression of macrophage infiltration and expression of IL-6, IL-10 and IL-12 beta, and increased production of serum and renal TSG-6 that improved the mAb 1-22-3-induced renal degeneration by the immunosuppressive effects of TSG-6. Thus DFAT cells will be suitable cell source for the treatment of immunological progressive renal diseases.

DOI： 10.1186/s13287-015-0069-2

ループス腎炎におけるポドサイト関連分子とカルシニューリンの変化

若松彩子, 和田庸子, 野澤由貴子, 佐藤弘恵, 中枝武司, 今井直史, 中野正明, 河内裕, 成田一衛

日本腎臓学会誌 57 ( 3 ) 484 - 484 2015.4

Angiotensin II type 1 receptor blockade ameliorates proteinuria in puromycin aminonucleoside nephropathy by inhibiting the reduction of NEPH1 and nephrin Reviewed

Aya Takahashi, Yoshiyasu Fukusumi, Mihoko Yamazaki, Mutsumi Kayaba, Yukina Kitazawa, Masayuki Tomita, Hiroshi Kawachi

JOURNAL OF NEPHROLOGY 27 ( 6 ) 627 - 634 2014.12

Language：English Publishing type：Research paper (scientific journal) Publisher：WICHTIG EDITORE

The precise pathogenic mechanism and role of angiotensin II (Ang II) action in the development of proteinuria in minimal change nephrotic syndrome (MCNS) is uncertain.
The glomerular expressions of the slit diaphragm (SD) molecules nephrin, podocin and NEPH1 in rat puromycin aminonucleoside (PAN) nephropathy, a mimic of MCNS, were analyzed. The effects of Ang II receptor blockade (ARB) (irbesartan 15 mg/kg body weight/day) on proteinuria and on the expression of the SD molecules were analyzed.
mRNA expressions of nephrin, podocin and NEPH1 were decreased to an undetectable level at 1 h. The staining of these SD molecules shifted to a discontinuous pattern, and their intensity was reduced. NEPH1 staining was reduced to an undetectable level on day 10. ARB treatment ameliorated the peak value of proteinuria (237.6 +/- A 97.0 vs. 359.0 +/- A 63.3 mg/day, p < 0.05), and prevented the decrease in the mRNA expression of the SD molecules (nephrin 66.96 %, podocin 60.40 %, NEPH1 77.87 % of normal level). The immunofluorescence staining of NEPH1 was restored by ARB. ARB treatment enhanced the expression of NEPH1 of normal rats.
Dysfunction of the SD molecules including NEPH1 is a crucial initiation event of PAN nephropathy. ARB treatment ameliorates proteinuria in PAN nephropathy by inhibiting the reduction of NEPH1 and nephrin. Ang II action regulates the expression of NEPH1 and nephrin in not only the pathological but also physiological state.

DOI： 10.1007/s40620-014-0147-z

Therapeutic target for nephrotic syndrome: Identification of novel slit diaphragm associated molecules. International journal

Fukusumi Yoshiyasu, Miyauchi Naoko, Hashimoto Taeko, Saito Akira, Kawachi Hiroshi

World J Nephrol 3 ( 3 ) 77 - 84 2014.8

Language：English Publishing type：Research paper (scientific journal)

The slit diaphragm bridging the neighboring foot processes functions as a final barrier of glomerular capillary wall for preventing the leak of plasma proteins into primary urine. It is now accepted that the dysfunction of the sit diaphragm contributes to the development of proteinuria in several glomerular diseases. Nephrin, a gene product of NPHS1, a gene for a congenital nephrotic syndrome of Finnish type, constitutes an extracellular domain of the slit diaphragm. Podocin was identified as a gene product of NPHS2, a gene for a familial steroid-resistant nephrotic syndrome of French. Podocin binds the cytoplasmic domain of nephrin. After then, CD2 associated protein, NEPH1 and transient receptor potential-6 were also found as crucial molecules of the slit diaphragm. In order to explore other novel molecules contributing to the development of proteinuria, we performed a subtraction hybridization assay with a normal rat glomerular RNA and a glomerular RNA of rats with a puromycin aminonucleoside nephropathy, a mimic of a human minimal change type nephrotic syndrome. Then we have found that synaptic vesicle protein 2B, ephrin-B1 and neurexin were already downregulated at the early stage of puromycin aminonucleoside nephropathy, and that these molecules were localized close to nephrin. It is conceivable that these molecules are the slit diaphragm associated molecules, which participate in the regulation of the barrier function. These molecules could be targets to establish a novel therapy for nephrotic syndrome.

DOI： 10.5527/wjn.v3.i3.77

Therapeutic effect of anti-C-X-C motif chemokine 10 (CXCL10) antibody on C protein-induced myositis mouse Reviewed

Jinhyun Kim, Ji Y. Choi, Sung-Hye Park, Seung H. Yang, Ji A. Park, Kichul Shin, Eun Y. Lee, Hiroshi Kawachi, Hitoshi Kohsaka, Yeong W. Song

Arthritis Research and Therapy 16 ( 3 ) 2014.6

Language：English Publishing type：Research paper (scientific journal) Publisher：BioMed Central Ltd.

Introduction: C-X-C motif chemokine 10 (CXCL10) is a chemokine that plays a critical role in the infiltration of T cells in autoimmune diseases and is reported to be expressed in muscle tissue of polymyositis. To determine the therapeutic efficacy of CXCL10 blockade, we investigated the role of CXCL10 and the effect of anti-CXCL10 antibody treatment in C protein-induced myositis (CIM), an animal model of polymyositis.Methods: CIM was induced with human skeletal muscle C protein fragment in female C57BL/6 mice. Immunohistochemistry of CXCL10 and C-X-C motif chemokine receptor 3 (CXCR3) and measurement of serum CXCL10 were performed. Cell surface markers and interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) in CIM lymph node cells was investigated by flow cytometry. Mice with CIM were treated with anti-CXCL10 antibody or control antibody (anti-RVG1) and the inflammation in muscle tissue was assessed.Results: Immunohistochemistry showed increased expression of CXCL10 and CXCR3 in the inflammatory lesions of muscle in CIM. Especially, CD8+ T cells invading myofiber expressed CXCR3. Serum level of CXCL10 was increased in CIM compared to the level in normal mice (normal mouse, 14.3 ± 5.3 pg/ml vs. CIM, 368.5 ± 135.6 pg/ml, P &lt
0.001). CXCR3 positivity in CD8+ T cells was increased compared to that of CD4+ T cells in the lymph node cells of CIM (CXCR3+ among CD8+ T cell, 65.9 ± 2.1% vs. CXCR3+ among CD4+ T cell, 23.5 ± 4.7%, P &lt
0.001). Moreover, IFN-γ+ cells were increased among CXCR3+CD8+ T cells compared to CXCR3-CD8+ T cells (CXCR3+CD8+ T cell, 28.0 ± 4.2% vs. CXCR3-CD8+ T cell, 9.5 ± 1.5%, P = 0.016). Migration of lymph node cells was increased in response to CXCL10 (chemotactic index was 1.91 ± 0.45). CIM mice treated with anti-CXCL10 antibody showed a lower inflammation score in muscles than those with anti-RVG1 (median, anti-CXCL10 treatment group, 0.625 vs. anti-RVG1 treatment group, 1.25, P = 0.007).Conclusions: CXCL10/CXCR3 expression was increased in the inflammation of CIM model and its blockade suppressed inflammation in muscle. © 2014 Kim et al.
licensee BioMed Central Ltd.

DOI： 10.1186/ar4583

Twisted Gastrulation, a BMP Antagonist, Exacerbates Podocyte Injury

Sachiko Yamada, Jin Nakamura, Misako Asada, Masayuki Takase, Taiji Matsusaka, Taku Iguchi, Ryo Yamada, Mari Tanaka, Atsuko Y. Higashi, Tomohiko Okuda, Nariaki Asada, Atsushi Fukatsu, Hiroshi Kawachi, Daniel Graf, Eri Muso, Toru Kita, Takeshi Kimura, Ira Pastan, Aris N. Economides, Motoko Yanagita

PLOS ONE 9 ( 2 ) e89135 - e89135 2014.2

Language：English Publishing type：Research paper (scientific journal) Publisher：PUBLIC LIBRARY SCIENCE

Podocyte injury is the first step in the progression of glomerulosclerosis. Previous studies have demonstrated the beneficial effect of bone morphogenetic protein 7 (Bmp7) in podocyte injury and the existence of native Bmp signaling in podocytes. Local activity of Bmp7 is controlled by cell-type specific Bmp antagonists, which inhibit the binding of Bmp7 to its receptors. Here we show that the product of Twisted gastrulation (Twsg1), a Bmp antagonist, is the central negative regulator of Bmp function in podocytes and that Twsg1 null mice are resistant to podocyte injury. Twsg1 was the most abundant Bmp antagonist in murine cultured podocytes. The administration of Bmp induced podocyte differentiation through Smad signaling, whereas the simultaneous administration of Twsg1 antagonized the effect. The administration of Bmp also inhibited podocyte proliferation, whereas simultaneous administration of Twsg1 antagonized the effect. Twsg1 was expressed in the glomerular parietal cells (PECs) and distal nephron of the healthy kidney, and additionally in damaged glomerular cells in a murine model of podocyte injury. Twsg1 null mice exhibited milder hypoalbuminemia and hyperlipidemia, and milder histological changes while maintaining the expression of podocyte markers during podocyte injury model. Taken together, our results show that Twsg1 plays a critical role in the modulation of protective action of Bmp7 on podocytes, and that inhibition of Twsg1 is a promising means of development of novel treatment for podocyte injury.

DOI： 10.1371/journal.pone.0089135

Tyrosine kinases inhibition by Imatinib slows progression in chronic anti-thy1 glomerulosclerosis of the rat Reviewed

Yingrui Wang-Rosenke, Dymtro Khadzhynov, Tanja Loof, Alice Mika, Hiroshi Kawachi, Hans-H Neumayer, Harm Peters

BMC NEPHROLOGY 14 2013.10

Language：English Publishing type：Research paper (scientific journal) Publisher：BIOMED CENTRAL LTD

Background: Chronic progressive mesangioproliferative nephropathy represents a major cause of end-stage renal disease worldwide. Until now, effective approaches to stop or even slow its progression are limited. We tested the effects of an inhibitor of PDGF receptor, abl and c-kit tyrosine kinases, Imatinib, in a chronic progressive model of mesangioproliferative glomerulosclerosis.
Methods: Anti-thy1 glomerulosclerosis was induced by injection of anti-thy1 antibody into uninephrectomized Wistar rats. One week after disease induction, according to the degree of proteinuria, animals were stratified and assigned to chronic glomerulosclerosis (cGS) and cGS plus Imatinib (10 mg/kg body weight/day). In week 20, renoprotective actions of Imatinib were analyzed by a set of functional, histological and molecular biological parameters.
Results: Untreated cGS rats showed elevation of systolic blood pressure and marked progression in proteinuria, renal fibrosis, cell infiltration, cell proliferation and function lost. Administration of Imatinib went along significantly with lower systolic blood pressure (-10 mmHg) and proteinuria (-33%). Imatinib administration was paralled by significant reductions in tubulointerstitial accumulation of matrix proteins (-44%), collagen I deposition (-86%), expression of TGF-beta1 (-30%), production of fibronectin (-23%), myofibroblast differentiation (-87%), macrophage infiltration (-36%) and cell proliferation (-45%), respectively. In comparison with untreated cGS animals, Imatinib therapy lowered also blood creatinine (-41%) and blood urea concentrations (-36%) and improved creatinine clearance (+25%). Glomerular fibrotic changes were lowered moderately by Imatinib.
Conclusions: Therapy with Imatinib limits the progressive course of chronic anti-thy1 glomerulosclerosis towards tubulointerstitial fibrosis and renal insufficiency. This was paralleled by direct and indirect sign of TGF-beta 1 and PDGF inhibition. The findings suggest that the pharmacological principal of inhibition of tyrosine kinases with drugs such as Imatinib might serve as approach for limiting progression of human mesangioproliferative glomerulosclerosis.

DOI： 10.1186/1471-2369-14-223

Curcumin alleviates oxidative stress, inflammation, and renal fibrosis in remnant kidney through the Nrf2-keap1 pathway Reviewed

Vivian Soetikno, Flori R. Sari, Arun P. Lakshmanan, Somasundaram Arumugam, Meilei Harima, Kenji Suzuki, Hiroshi Kawachi, Kenichi Watanabe

MOLECULAR NUTRITION & FOOD RESEARCH 57 ( 9 ) 1649 - 1659 2013.9

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

Scope: We hypothesized that curcumin, by increasing the expression of nuclear factor-erythroid-2-related factor 2 (Nrf2), could reduce oxidative stress, inflammation, and renal fibrosis in remnant kidney.
Methods and resultsSprague-Dawley rats were subjected to 5/6 nephrectomy and randomly assigned to untreated (Nx), curcumin-treated (75 mg/kg/day, orally), and telmisartan-treated groups (10 mg/kg/day, orally; as positive control). Sham-operated rats also served as controls. Five/sixth nephrectomy caused renal dysfunction, as evidenced by elevated proteinuria, blood urea nitrogen, and plasma creatinine, and decreased creatinine clearance that were ameliorated by curcumin or telmisartan treatment. The Nx rats demonstrated reduced Nrf2 protein expression, whereas the Kelch-like ECH-associated protein 1 was upregulated and heme oxygenase-1 level was significantly diminished. Consequently, Nx animals had significantly higher kidney malondialdehyde concentration and lower glutathione peroxidase activity, which was associated with the upregulation of nicotinamide adenine dinucleotide phosphatase oxidase subunit (p67(phox) and p22(phox)), NF-kappaB p65, TNF-, TGF-1, cyclooxygenase-2, and fibronectin accumulation in remnant kidney. Interestingly, all of these changes were ameliorated by curcumin or telmisartan.
ConclusionThese findings demonstrate that, by modulating Nrf2-Keap1 pathway, the curcumin effectively attenuates oxidative stress, inflammation, and renal fibrosis, which suggest that curcumin hold promising potential for safe treatment of chronic kidney disease.

DOI： 10.1002/mnfr.201200540

Planar cell polarity pathway regulates nephrin endocytosis in developing podocytes Reviewed

Sima Babayeva, Brittany Rocque, Lamine Aoudjit, Yulia Zilber, Jane Li, Cindy Baldwin, Hiroshi Kawachi, Tomoko Takano, Elena Torban

Journal of Biological Chemistry 288 ( 33 ) 24035 - 24048 2013.8

Language：English Publishing type：Research paper (scientific journal)

Background: The PCP pathway controls many cell processes during development. Results: The PCP pathway induces nephrin endocytosis when cultured podocytes are treated with Wnt5a. Loss of PCP protein Vangl2 decreases nephrin endocytosis. Conclusion: During glomerular development, endocytosis of nephrin is regulated by the PCP pathway. Significance: Implicating the PCP pathway in nephrin endocytosis is important for understanding the complexity of PCP signaling during mammalian development. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M113.452904

Curcumin decreases renal triglyceride accumulation through AMPK-SREBP signaling pathway in streptozotocin-induced type 1 diabetic rats

Vivian Soetikno, Flori R. Sari, Vijayakumar Sukumaran, Arun Prasath Lakshmanan, Meilei Harima, Kenji Suzuki, Hiroshi Kawachi, Kenichi Watanabe

Journal of Nutritional Biochemistry 24 ( 5 ) 796 - 802 2013.5

Language：English Publishing type：Research paper (scientific journal)

Diabetic kidney disease has been associated with the presence of lipid deposits. We assumed that curcumin, a polyphenol, would attenuate the tissue dyslipidemic condition through activation of 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation and suppression of sterol regulatory element-binding protein (SREBP)-1c in the kidney and would prevent renal progression in experimental type 1 diabetic rats. Diabetes was induced with streptozotocin (STZ) (55 mg/kg) by intraperitoneal injection in male Sprague-Dawley rats. Three weeks after STZ injection, rats were divided into three groups, namely, control, diabetic and diabetic treated with curcumin (100 mg/kg/day) by gavage for 8 weeks. We found that curcumin decreased plasma triglyceride and the amount of renal triglyceride significantly. Furthermore, treatment of diabetic rats with curcumin increased the phosphorylation of AMPK and prevented the increased renal expression of SREBP-1c and, as a result, decreased the expression of acetyl CoA carboxylase and fatty acid synthase as well as adipose differentiation-related protein, a marker of cytoplasmic droplets. We also demonstrate that curcumin significantly suppressed the increased expression of transforming growth factor β, vascular endothelial growth factor and extracellular matrix proteins such as type IV collagen and fibronectin. In addition, curcumin treatment increased nephrin expression to near-normal levels in diabetic rats. These results demonstrated that curcumin protects against the development of diabetic nephropathy through the AMPK-SREBP pathway and the reduction of renal triglyceride accumulation which could be a possible mechanism by which curcumin preserves renal function in diabetes. © 2013 Elsevier Inc.

DOI： 10.1016/j.jnutbio.2012.04.013

The hyperglycemia stimulated myocardial endoplasmic reticulum (ER) stress contributes to diabetic cardiomyopathy in the transgenic-non-obese type 2 diabetic rats: A differential role of unfolded protein response (UPR) signaling proteins Reviewed

Arun Prasath Lakshmanan, Meilei Harima, Kenji Suzuki, Vivian Soetikno, Masaki Nagata, Takashi Nakamura, Toshihiro Takahashi, Hirohito Sone, Hiroshi Kawachi, Kenichi Watanabe

INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY 45 ( 2 ) 438 - 447 2013.2

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

It has been well demonstrated that excessive blood glucose level could be detrimental to the myocardial function through the variety of mechanisms, of which endoplasmic reticulum stress (ERS) could play an unprecedented role through the activation of unfolded protein response (UPR). Recently, reports are coming out with the evidences that UPR signaling proteins are regulated differentially depend on the experimental conditions and cell types. In addition, ERS has been proposed to be closely associated with the regulation of lipogenesis. Therefore, in this study we tried to find out the expressions of myocardial UPR signaling proteins as well as proteins involved in lipid and glucose metabolism in non-obese type 2 diabetic mellitus (DM) condition using Spontaneous Diabetic Toni (SDT) rat. We have found the significant up-regulation of oxidative, nitrosative and ERS marker proteins in the myocardium of the SDT rats, in comparison to its normal (Sprague-Dawley - SD) rats. In addition, the sub-arm-of UPR signaling proteins, such as p-PERK, p-eIF2 alpha, ATF6, CHOP/GADD153, TRAF2, apoptotic signaling proteins, such as BAD, cytochrome C, cleaved caspase-7 and -12, were significantly up-regulated in the SDT rats, in comparison to the SD rats. Interestingly, there were no significant changes in the phosphorylation of IRE-1 alpha, and XBP-1 protein expression. In addition, the proteins involved in lipid and glucose metabolisms, such as PPAR alpha, PPAR gamma, CPT1, PGC-1 alpha except GLUT4, and the proteins involved in insulin signaling, such as p-Akt and p-PI3K were shown significant attenuation in its expressions in the SDT rats, when compared with the SD rats. Taken together, it is suggested that the activation of PERK and ATF6 pathway are the major determinant rather than the IRE-1 alpha-XBP1 pathway for the ERS-mediated metabolic dysfunction, which might eventually leads to diabetic cardiomyopathy in non-obese type 2 DM. (c) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biocel.2012.09.017

Development of lymphatic vasculature and morphological characterization in rat kidney Reviewed

Maki Tanabe, Akira Shimizu, Yukinari Masuda, Mitue Kataoka, Arimi Ishikawa, Kyoko Wakamatsu, Akiko Mii, Emiko Fujita, Seiichiro Higo, Tomohiro Kaneko, Hiroshi Kawachi, Yuh Fukuda

Clinical and Experimental Nephrology 16 ( 6 ) 833 - 842 2012.12

Language：English Publishing type：Research paper (scientific journal)

Background: The mechanisms and morphological characteristics of lymphatic vascular development in embryonic kidneys remain uncertain. Methods: We examined the distribution and characteristics of lymphatic vessels in developing rat kidneys using immunostaining for podoplanin, prox-1, Ki-67, type IV collagen (basement membrane: BM), and α-smooth muscle actin (αSMA: pericytes or mural cells). We also examined the expression of VEGF-C. Results: At embryonic day 17 (E17), podoplanin-positive lymphatic vessels were observed mainly in the kidney hilus. At E20, lymphatic vessels extended further into the developing kidneys along the interlobar vasculature. In 1-day-old pups (P1) to P20, lymphatic vessels appeared around the arcuate arteries and veins of the kidneys, with some reaching the developing cortex via interlobular vessels. In 8-week-old adult rats, lymphatic vessels were extensively distributed around the blood vasculature from the renal hilus to cortex. Only lymphatic capillaries lacking continuous BM and αSMA-positive cells were present within adult kidneys, with none observed in renal medulla. VEGF-C was upregulated in the developing kidneys and expressed mainly in tubules. Importantly, the developing lymphatic vessels were characterized by endothelial cells immunopositive for podoplanin, prox-1, and Ki-67, with no surrounding BM or αSMA-positive cells. Conclusion: During nephrogenesis, lymphatic vessels extend from the renal hilus into the renal cortex along the renal blood vasculature. Podoplanin, prox-1, Ki-67, type IV collagen, and αSMA immunostaining can detect lymphatic vessels during lymphangiogenesis. © 2012 Japanese Society of Nephrology.

DOI： 10.1007/s10157-012-0637-z

Local delivery of a direct renin inhibitor into the kidney ameliorates progression of experimental glomerulonephritis

Ayako Sato, Hoglan Piao, Yukiko Nozawa, Tetsuo Morioka, Hiroshi Kawachi, Takashi Oite

Clinical and Experimental Nephrology 16 ( 4 ) 539 - 548 2012.8

Language：English Publishing type：Research paper (scientific journal)

Background: Increasing evidence indicates that locally blocking renin-angiotensin system activity exerts a beneficial effect on glomerulonephritis (GN) progression leading to irreversible glomerulosclerosis. This is the first study on the pharmacological effect of the renal delivery of aliskiren, a direct renin inhibitor, in a progressive model of anti-Thy-1 GN. Methods: Local blockade of renin activity was accomplished by subrenal capsular implantation of a collagen sponge with aliskiren. The pharmacological effect was evaluated by semiquantitative and quantitative analysis of immunohistological findings and by analysis of glomerular microcirculation using an intravital microscope system. Results: Quantitative mesangial matrix analysis showed that local treatment with aliskiren significantly suppressed mesangial matrix expansion and ameliorated the glomerular sclerotic index in the progressive model of ATS GN. Immunofluorescent studies revealed that renin expression at the juxtaglomerular region was enhanced in the ATS + aliskiren group, and pathological expressions of α-smooth muscle cell actin and type I collagen in ATS GN were remarkably decreased by local treatment with aliskiren. Furthermore, local delivery of aliskiren significantly improved glomerular blood flow levels. Conclusion: This study revealed that renally delivered aliskiren has a renoprotective effect on potentially progressive glomerulosclerosis. © 2012 Japanese Society of Nephrology.

DOI： 10.1007/s10157-012-0601-y

Irsogladine maleate ameliorates infl ammation and fi brosis in mice with chronic colitis induced by dextran sulfate sodium

Hana Yamaguchi, Kenji Suzuki, Masaki Nagata, Tomoyuki Kawase, Vijayakumar Sukumaran, Rajarajan A. Thandavarayan, Yusuke Kawauchi, Junji Yokoyama, Masayuki Tomita, Hiroshi Kawachi, Kenichi Watanabe, Hiroyuki Yoneyama, Hitoshi Asakura, Ritsuo Takagi

Medical Molecular Morphology 45 ( 3 ) 140 - 151 2012.6

Language：English Publishing type：Research paper (scientific journal)

Intestinal fi brosis is a common and severe complication of infl ammatory bowel disease (IBD), especially Crohn's disease (CD). To investigate the therapeutic approach to intestinal fi brosis, we have developed a mouse model of intestinal fi brosis by administering dextran sulfate sodium (DSS) and examining the effects of irsogladine maleate (IM) [2,4-diamino-6-(2,5- dichlorophenyl)-s-triazine maleate], which has been widely used as an antiulcer drug for gastric mucosa in Japan, on DDS-induced chronic colitis. In this experimental colitis lesion, several pathognomonic changes were found: increased deposition of collagen, increased number of profi brogenic mesenchymal cells such as fi broblasts (vimentin+, 〈-SMA) and myofi broblasts (vimentin+, 〈-SMA+) in both mucosa and submucosa of the colon with infi ltrating infl ammatory cells, and increased mRNA expressions of collagen type I, transforming growth factor (TGF)-®, matrix metalloproteinase (MMP)-2, and tissue inhibitor of matrix metalloproteinase (TIMP)-1. When IM was administered intrarectally to this colitis, all these pathological changes were signifi cantly decreased or suppressed, suggesting a potential adjunctive therapy for intestinal fi brosis. IM could consequently reduce fi brosis in DSS colitis by direct or indirect effect on profi brogenic factors or fi broblasts. Therefore, the precise effect of IM on intestinal fi brosis should be investigated further. © 2012 The Japanese Society for Clinical Molecular Morphology.

DOI： 10.1007/s00795-011-0550-7

Early treatment with everolimus exerts nephroprotective effect in rats with adriamycin-induced nephrotic syndrome Reviewed

Rawi Ramadan, Diana Faour, Hoda Awad, Eleanor Khateeb, Ravit Cohen, Ali Yahia, Rafael Torgovicky, Raanan Cohen, David Lazari, Hiroshi Kawachi, Zaid Abassi

Nephrology Dialysis Transplantation 27 ( 6 ) 2231 - 2241 2012.6

Language：English Publishing type：Research paper (scientific journal)

Background. Nephrotic syndrome (NS) is a clinical state characterized by massive proteinuria and excessive fluid retention. The effects of early versus late treatment with low or high doses of oral everolimus, a mammalian target of rapamycin inhibitor, on proteinuria in NS have not been previously described.Methods.The effects of early treatment (2 days prior to NS induction) versus late treatment (beginning 2 weeks following the establishment of NS) with a low (20 mg/L) or high (100 mg/L) dose of everolimus for 5-7 weeks on proteinuria and nephrin/podocin abundance were assessed in male adult SD rats with adriamycin-induced NS.Results.Adriamycin caused a significant increase in daily and cumulative proteinuria throughout the experimental period. Early, and to a lesser extent late treatment, with a low dose of everolimus, significantly decreased both daily and cumulative proteinuria and improved renal function. The anti-proteinuric effects of low-dose everolimus were associated with restoration of the disruptive glomerular nephrin/podocin abundance. In contrast, administration of a high dose of everolimus resulted in a decrease in proteinuria in NS rats, subsequently to deterioration of renal function.Conclusions.Early, and to a lesser extent late treatment, with a low but not a high dose of everolimus is effective in reducing proteinuria in nephrotic rats. The mechanism may be via nephrin/podocin protection. © 2012 The Author.

DOI： 10.1093/ndt/gfr581

Early treatment with olmesartan prevents juxtamedullary glomerular podocyte injury and the onset of microalbuminuria in type 2 diabetic rats. International journal

Tadashi Sofue, Hideyasu Kiyomoto, Hiroyuki Kobori, Maki Urushihara, Yoko Nishijima, Kumiko Kaifu, Taiga Hara, Sachiko Matsumoto, Atsuhiko Ichimura, Hiroyuki Ohsaki, Hirofumi Hitomi, Hiroshi Kawachi, Melvin R Hayden, Adam Whaley-Connell, James R Sowers, Sadayoshi Ito, Masakazu Kohno, Akira Nishiyama

American journal of hypertension 25 ( 5 ) 604 - 11 2012.5

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: Studies were performed to determine if early treatment with an angiotensin II (Ang II) receptor blocker (ARB), olmesartan, prevents the onset of microalbuminuria by attenuating glomerular podocyte injury in Otsuka Long-Evans Tokushima Fatty (OLETF) rats with type 2 diabetes mellitus. METHODS: OLETF rats were treated with either a vehicle, olmesartan (10 mg/kg/day) or a combination of nonspecific vasodilators (hydralazine 15 mg/kg/day, hydrochlorothiazide 6 mg/kg/day, and reserpine 0.3 mg/kg/day; HHR) from the age of 7-25 weeks. RESULTS: OLETF rats were hypertensive and had microalbuminuria from 9 weeks of age. At 15 weeks, OLETF rats had higher Ang II levels in the kidney, larger glomerular desmin-staining areas (an index of podocyte injury), and lower gene expression of nephrin in juxtamedullary glomeruli, than nondiabetic Long-Evans Tokushima Otsuka (LETO) rats. At 25 weeks, OLETF rats showed overt albuminuria, and higher levels of Ang II in the kidney and larger glomerular desmin-staining areas in superficial and juxtamedullary glomeruli compared to LETO rats. Reductions in mRNA levels of nephrin were also observed in superficial and juxtamedullary glomeruli. Although olmesartan did not affect glucose metabolism, it decreased blood pressure and prevented the renal changes in OLETF rats. HHR treatment also reduced blood pressure, but did not affect the renal parameters. CONCLUSIONS: This study demonstrated that podocyte injury occurs in juxtamedullary glomeruli prior to superficial glomeruli in type 2 diabetic rats with microalbuminuria. Early treatment with an ARB may prevent the onset of albuminuria through its protective effects on juxtamedullary glomerular podocytes.

DOI： 10.1038/ajh.2012.1

Successful treatment of icodextrin-single peritoneal dialysis for refractory nephrotic syndrome induced by idiopathic membranous nephropathy. Reviewed

Takada D, Mii A, Higo S, Obara Y, Kurabayashi Y, Kurosawa N, Miura S, Kawachi H, Shimizu A

CEN case reports 1 ( 1 ) 16 - 23 2012.5

Proteinuria in AMPD2-deficient mice Reviewed

Toyama Keiko, Morisaki Hiroko, Cheng Jidong, Kawachi Hiroshi, Shimizu Fujio, Ikawa Masahito, Okabe Masaru, Morisaki Takayuk

Genes To Cells 17 ( 1 ) 28 - 38 2012

Publishing type：Research paper (scientific journal)

The AMPD2 gene, a member of the AMPD gene family encoding AMP deaminase, is widely expressed in nonmuscle tissues including kidney, although its functions have not been fully elucidated. In this study, we studied the function of the AMPD2 gene by establishing AMPD2-deficient model animal. We established AMPD2 knockout mice by using gene transfer and homologous recombination in murine ES cells and studied phenotypes and functions in the kidneys of these animals. AMPD activity was decreased from 22.9mIU/mg protein to 2.5mIU/mg protein in the kidneys of AMPD knockout mice. In addition to changes in nucleotide metabolism in the kidneys, proteinuria was found in 3-week-old AMPD2 knockout mice, followed by a further increment up to a peak level at 6weeks old (up to 0.6g/dL). The major protein component in the urine of AMPD2 knockout mice was found to be albumin, indicating that AMPD2 may have a key role in glomerular filtration. Indeed, an ultrastructure study of glomerulus specimens from these mice showed effacement of the podocyte foot processes, resembling minimal-change nephropathy in humans. Based on our results, we concluded that AMPD2 deficiency induces imbalanced nucleotide metabolism and proteinuria, probably due to podocyte dysfunction. © 2012 The Authors. Journal compilation © 2012 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

DOI： 10.1111/j.1365-2443.2011.01568.x

Angiotensin II type I receptor blockade suppresses glomerular renin-angiotensin system activation, oxidative stress, and progressive glomerular injury in rat anti-glomerular basement membrane glomerulonephritis Reviewed

Yukiko Kinoshita, Shuji Kondo, Maki Urushihara, Kenichi Suga, Sato Matsuura, Masanori Takamatsu, Maki Shimizu, Akira Nishiyama, Hiroshi Kawachi, Shoji Kagami

TRANSLATIONAL RESEARCH 158 ( 4 ) 235 - 248 2011.10

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

Excessive renin-angiotensin system (RAS) activation within the kidney induces not only renal oxidative stress but also renal scarring and dysfunction. This study examined the effects of an angiotensin II (Ang II) type I receptor (AT1R) blocker (ARB) on the progression of renal injury in rat anti-glomerular basement membrane glomerulonephritis (GN), with a particular focus on the participation of glomerular RAS activation in glomerular structural alterations, inflammation, and oxidative stress. Nephritic rats were divided into 2 groups and treated with vehicle or ARB until day 28. Treatment with ARB improved proteinuria significantly in nephritic rats. Vehicle-treated nephritic rats developed crescentic GN accompanied by marked macrophage infiltration and the enhanced expression of glomerular alpha-smooth muscle actin (alpha-SMA), angiotensinogen (AGT), Ang II, AT1R, and NADPH oxidase (Nox2) on days 7 and 28 of GN. ARB improved pathologic alterations such as crescent formation and glomerulosclerosis, and it had a significant inhibitory effect on the levels of these parameters on day 28 of GN. Enhanced superoxide production in nephritic glomeruli was decreased also by ARB. Moreover, Ang II and transforming growth factor beta (TGF-beta) in the supernatant of cultured glomeruli was increased significantly in vehicle-treated nephritic rats whereas ARB inhibited the production of these compounds significantly on day 28. These results indicate that increased glomerular RAS activity and the resulting Ang II play important roles in progressive glomerular injury-the induction of oxidative stress and TGF-beta expression, and they suggest that AT1R blockade attenuates proteinuria and progressive glomerular remodeling via the suppression of glomerular RAS activation in GN. (Translational Research 2011;158:235-248)

DOI： 10.1016/j.trsl.2011.05.003

Curcumin ameliorates macrophage infiltration by inhibiting NF-kappa B activation and proinflammatory cytokines in streptozotocin induced-diabetic nephropathy

Vivian Soetikno, Flori R. Sari, Punniyakoti T. Veeraveedu, Rajarajan A. Thandavarayan, Meilei Harima, Vijayakumar Sukumaran, Arun Prasath Lakshmanan, Kenji Suzuki, Hiroshi Kawachi, Kenichi Watanabe

NUTRITION & METABOLISM 8 ( 1 ) 35 - 35 2011.6

Language：English Publishing type：Research paper (scientific journal) Publisher：BIOMED CENTRAL LTD

Background: Chronic inflammation plays an important role in the progression of diabetic nephropathy (DN) and that the infiltration of macrophages in glomerulus has been implicated in the development of glomerular injury. We hypothesized that the plant polyphenolic compound curcumin, which is known to exert potent anti-inflammatory effect, would ameliorate macrophage infiltration in streptozotocin (STZ)-induced diabetic rats.
Methods: Diabetes was induced with STZ (55 mg/kg) by intraperitoneal injection in rats. Three weeks after STZ injection, rats were divided into three groups, namely, control, diabetic, and diabetic treated with curcumin at 100 mg/kg/day, p.o., for 8 weeks. The rats were sacrificed 11 weeks after induction of diabetes. The excised kidney was used to assess macrophage infiltration and expression of various inflammatory markers.
Results: At 11 weeks after STZ injection, diabetic rats exhibited renal dysfunction, as evidenced by reduced creatinine clearance, increased blood glucose, blood urea nitrogen and proteinuria, along with marked reduction in the body weight. All of these abnormalities were significantly reversed by curcumin. Hyperglycemia induced the degradation of I kappa Ba and NF-kappa B activation and as a result increased infiltration of macrophages (52%) as well as increased proinflammatory cytokines: TNF-alpha and IL-1 beta. Curcumin treatment significantly reduced macrophage infiltration in the kidneys of diabetic rats, suppressed the expression of above proinflammatory cytokines and degradation of I kappa Ba. In addition, curcumin treatment also markedly decreased ICAM-1, MCP-1 and TGF-beta 1 protein expression. Moreover, at nuclear level curcumin inhibited the NF-kappa B activity.
Conclusion: Our results suggested that curcumin treatment protect against the development of DN in rats by reducing macrophage infiltration through the inhibition of NF-kappa B activation in STZ-induced diabetic rats.

DOI： 10.1186/1743-7075-8-35

Analysis of intestinal fibrosis in chronic colitis in mice induced by dextran sulfate sodium Reviewed

Kenji Suzuki, Xiaomei Sun, Masaki Nagata, Tomoyuki Kawase, Hana Yamaguchi, Vijayakumar Sukumaran, Yusuke Kawauchi, Hiroshi Kawachi, Takayoshi Nishino, Kenichi Watanabe, Hiroyuki Yoneyama, Hitoshi Asakura

Pathology International 61 ( 4 ) 228 - 238 2011.4

Language：English Publishing type：Research paper (scientific journal)

Fibrogenic mesenchymal cells including fibroblasts and myofibroblasts play a key role in intestinal fibrosis, however, their precise role is largely unknown. To investigate their role in intestinal fibrosis, we analyzed the lesions of chronic colitis in C57BL/6 (B6) mice induced by dextran sulfate sodium (DSS). B6 mice exposed to single cycle administration of DSS for 5days developed acute colitis that progressed to severe chronic inflammation with dense infiltrates of mononuclear cells, irregular epithelial structure, thickening of colonic wall, and persistent deposits of collagen. Increased mRNA expressions of proinflammatory cytokines are correlated with extensive cellular infiltration, and the mRNA expressions of collagen 1, transforming growth factor (TGF)-β, and matrix metalloproteinases were also enhanced in the colon. In the colon of chronic DSS colitis, fibroblasts (vimentin +, α-smooth muscle actin (α-SMA) -) were increased in both mucosal and submucosal layers, while myofibroblasts (vimentin +, α-SMA +) were increased in mucosal but not in submucosal layers. Primary mouse subcutaneous fibroblast cultures experiments revealed that exogenously added TGF-β 1 substantially augmented the expressions of both vimentin and α-SMA proteins with increased production of collagen. In conclusion, profibrogenic mesenchymal cells play an important role in the development of intestinal fibrosis in this chronic DSS-induced colitis model. © 2011 The Authors. Pathology International © 2011 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.

DOI： 10.1111/j.1440-1827.2011.02647.x

A notable case report of May-Hegglin anomaly with immune complex-related nephropathy: a genetic and histological analysis

Y. Ohtsuka, T. Kanaji, M. Nish, N. Sakai, T. Sato, S. Aoki, K. Wakayama, S. Nakazato, S. Hisano, Y. Sado, H. Kawachi, K. Izuhara, Y. Hamasaki

CLINICAL NEPHROLOGY 75 ( 3 ) 255 - 262 2011.3

Language：English Publishing type：Research paper (scientific journal) Publisher：DUSTRI-VERLAG DR KARL FEISTLE

May-Hegglin anomaly (MHA) is a rare autosomal dominant disease characterized by macrothrombocytopenia and leukocyte inclusions with microfilaments in the ribosomes. Mutations in the MYH9 gene, encoding non-muscle myosin heavy chain IIA (NMMHC-IIA) have been identified in patients with MHA and other MYH9-related diseases. Two young males (an older and younger brother) presented with macrothrombocytopenia and leukocyte inclusion bodies. Electron microscopy (EM) revealed parallel filaments in leukocyte inclusion bodies characteristic of MHA. Immunofluorescence microscopy (IF) showed NMMHC-IIA antibodies in 1 - 2 leukocyte inclusion bodies. These findings were consistent with MHA and they were identified to express the MYH9 mutation, D1424H. The older brother underwent a renal biopsy because of persistent proteinuria. Histology revealed mesangial proliferative glomerulonephritis with granular deposits of IgG and CIq. EM showed that the dense deposits were located in subendothelial cells, mesangial cells and Bowman's capsule. Immunocytochemistry revealed that NMMHC-IIA antibodies were localized in podocyte and endothelial cells in the glomerulus. Moreover, the expression of nephrin and podocin, slit diagram protein, was normal. An inflammatory mechanism may occur separately from MYH9-related disease. This report presents a case of MHA with immune complex-related nephropathy.

DOI： 10.5414/CNP75255

CD28 superagonist-induced regulatory T cell expansion ameliorates mesangioproliferative glomerulonephritis in rats. Reviewed

Kenro Miyasato, Yoshitsugu Takabatake, Junya Kaimori, Tomonori Kimura, Harumi Kitamura, Hiroshi Kawachi, Xiao-Kang Li, Thomas Hünig, Shiro Takahara, Hiromi Rakugi, Yoshitaka Isaka

Clinical and experimental nephrology 15 ( 1 ) 50 - 7 2011.2

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

BACKGROUND: Naturally occurring regulatory T cells (Treg) are essential for the prevention of autoimmunity and overshooting immune responses to pathogens; however, the involvement of Treg in mesangioproliferative glomerulonephritis, a major cause of chronic kidney disease, remains unclear. Superagonistic CD28-specific monoclonal antibodies (CD28SA) are highly effective activators of Treg in rats. METHOD: To confirm our hypothesis that CD28SA reduces the severity of experimental glomerulonephritis, anti-Thy1 nephritis model rats were treated with CD28SA or saline. RESULTS: CD28SA significantly suppressed the increase in proteinuria and serum creatinine levels. CD28SA-treated nephritic rats exhibited an increase in the infiltration of Treg in the glomeruli accompanied by infiltration of CD163-positive macrophages ("alternatively activated" macrophages). In addition, CD28SA significantly induced interleukin-10 mRNA expression in glomeruli, thereby ameliorating mesangial cell proliferation and extracellular matrix expansion. CONCLUSION: We established a new therapeutic approach to suppressing progressive glomerulonephritis. The therapeutic value of this approach warrants further attention and preclinical studies.

DOI： 10.1007/s10157-010-0370-4

Other Link： http://orcid.org/0000-0001-6508-4338

Neurexin-1, a presynaptic adhesion molecule, localizes at the slit diaphragm of the glomerular podocytes in kidneys Reviewed

Akira Saito, Naoko Miyauchi, Taeko Hashimoto, Tamaki Karasawa, Gi Dong Han, Mutsumi Kayaba, Tomoyuki Sumi, Masayuki Tomita, Yohei Ikezumi, Kenji Suzuki, Yasushi Koitabashi, Fujio Shimizu, Hiroshi Kawachi

AMERICAN JOURNAL OF PHYSIOLOGY-REGULATORY INTEGRATIVE AND COMPARATIVE PHYSIOLOGY 300 ( 2 ) R340 - R348 2011.2

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER PHYSIOLOGICAL SOC

Saito A, Miyauchi N, Hashimoto T, Karasawa T, Han GD, Kayaba M, Sumi T, Tomita M, Ikezumi Y, Suzuki K, Koitabashi Y, Shimizu F, Kawachi H. Neurexin-1, a presynaptic adhesion molecule, localizes at the slit diaphragm of the glomerular podocytes in kidneys. Am J Physiol Regul Integr Comp Physiol 300: R340-R348, 2011. First published November 3, 2010; doi: 10.1152/ajpregu.00640.2009.-The slit diaphragm connecting the adjacent foot processes of glomerular epithelial cells (podocytes) is the final barrier of the glomerular capillary wall and serves to prevent proteinuria. Podocytes are understood to be terminally differentiated cells and share some common features with neurons. Neurexin is a presynaptic adhesion molecule that plays a role in synaptic differentiation. Although neurexin has been understood to be specifically expressed in neuronal tissues, we found that neurexin was expressed in several organs. Several forms of splice variants of neurexin-1 alpha were detected in the cerebrum, but only one form of neurexin-1 alpha was detected in glomeruli. Immunohistochemical study showed that neurexin restrictedly expressed in the podocytes in kidneys. Dual-labeling analyses showed that neurexin was colocalized with CD2AP, an intracellular component of the slit diaphragm. Immunoprecipitation assay using glomerular lysate showed that neurexin interacted with CD2AP and CASK. These observations indicated that neurexin localized at the slit diaphragm area. The staining intensity of neurexin in podocytes was clearly lowered, and their staining pattern shifted to a more discontinuous patchy pattern in the disease models showing severe proteinuria. The expression and localization of neurexin in these models altered more clearly and rapidly than that of other slit diaphragm components. We propose that neurexin is available as an early diagnostic marker to detect podocyte injury. Neurexin coincided with nephrin, a key molecule of the slit diaphragm detected in a presumptive podocyte of the developing glomeruli and in the glomeruli for which the slit diaphragm is repairing injury. These observations suggest that neurexin is involved in the formation of the slit diaphragm and the maintenance of its function.

DOI： 10.1152/ajpregu.00640.2009

Identification of alternatively activated macrophages in new-onset paediatric and adult immunoglobulin A nephropathy: potential role in mesangial matrix expansion Reviewed

Yohei Ikezumi, Toshiaki Suzuki, Tamaki Karasawa, Hiroya Hasegawa, Takeshi Yamada, Naofumi Imai, Ichiei Narita, Hiroshi Kawachi, Kevan R. Polkinghorne, David J. Nikolic-Paterson, Makoto Uchiyama

HISTOPATHOLOGY 58 ( 2 ) 198 - 210 2011.1

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL PUBLISHING, INC

Aims:
New onset of the clinical symptoms of immunoglobulin A (IgA) nephropathy (IgAN) manifests with proliferative glomerular lesions in children, whereas adults exhibit mesangial matrix expansion and interstitial fibrosis. Alternatively, activated (M2) macrophages have been implicated in promoting tissue fibrosis in some settings. Therefore, the aim of this study was to investigate whether M2 macrophages are present in new-onset IgAN and if they are related to pathological differences between paediatric and adult disease.
Methods and results:
Biopsy specimens from paediatric (< 10 years, n = 14; > 12 years, n = 15) and adult (n = 27) IgAN showed a significant infiltrate of CD68+ macrophages. M2 macrophages, identified by CD163 or CD204 expression, were detected in glomeruli and the interstitium, being more prominent in adults versus young children. CD163+ and CD204+ macrophages were present in areas of fibrosis containing myofibroblasts, and double staining showed that CD163+ cells produced the profibrotic molecule, connective tissue growth factor. In young children, total CD68+ macrophages, but not M2 macrophages, correlated with glomerular hypercellularity. In contrast, in adults and older children, mesangial matrix expansion correlated with M2 macrophages but not with the total CD68+ macrophage infiltrate.
Conclusions:
Alternatively activated M2 macrophages are present in new-onset paediatric and adult IgAN, and this population may promote the development of fibrotic lesions.

DOI： 10.1111/j.1365-2559.2011.03742.x

Defective glycosylation of α-dystroglycan contributes to podocyte flattening Reviewed

Kenichiro Kojima, Hitonari Nosaka, Yuki Kishimoto, Yuri Nishiyama, Seiichi Fukuda, Masaru Shimada, Kenzo Kodaka, Fumiaki Saito, Kiichiro Matsumura, Teruo Shimizu, Tatsushi Toda, Satoshi Takeda, Hiroshi Kawachi, Shunya Uchida

Kidney International 79 ( 3 ) 311 - 316 2011

Language：English Publishing type：Research paper (scientific journal) Publisher：Nature Publishing Group

In addition to skeletal muscle and the nervous system, α-dystroglycan is found in the podocyte basal membrane, stabilizing these cells on the glomerular basement membrane. Fukutin, named after the gene responsible for Fukuyama-type congenital muscular dystrophy, is a putative glycosyltransferase required for the post-translational modification of α-dystroglycan. Chimeric mice targeted for both alleles of fukutin develop severe muscular dystrophy
however, these mice do not have proteinuria. Despite the lack of a functional renal defect, we evaluated glomerular structure and found minor abnormalities in the chimeric mice by light microscopy. Electron microscopy revealed flattening of podocyte foot processes, the number of which was significantly lower in the chimeric compared to wild-type mice. A monoclonal antibody against the laminin-binding carbohydrate residues of α-dystroglycan did not detect α-dystroglycan glycosylation in the glomeruli by immunoblotting or immunohistochemistry. In contrast, expression of the core α-dystroglycan protein was preserved. There was no statistical difference in dystroglycan mRNA expression or in the amount of nephrin and α3-integrin protein in the chimeric compared to the wild-type mice as judged by immunohistochemistry and real-time RT-PCR. Thus, our results indicate that appropriate glycosylation of α-dystroglycan has an important role in the maintenance of podocyte architecture. © 2011 International Society of Nephrology.

DOI： 10.1038/ki.2010.403

Ameliorating effects of L-carnitine on diabetic podocyte injury.

Fan Jiang Ping, Kim Dongyeop, Kawachi Hiroshi, Ha Tae-Sun, Han Gi Dong

J Med Food 13 ( 6 ) 1324 - 1330 2010.12

Language：English Publishing type：Research paper (scientific journal)

High glucose levels can change podocyte gene expression and subsequently induce podocyte damage through altered glucose metabolism. l-Carnitine is known to play a beneficial role in diabetes; however, there are no studies on the effects of l-carnitine on podocyte alteration under high glucose conditions. This study investigated whether l-carnitine can attenuate diabetic podocyte injury through the prevention of loss of slit diaphragm proteins. The l-carnitine treatment group showed increased glucose uptakes compared to the control group, suggesting that glucose utilization in the podocytes was increased by l-carnitine. l-Carnitine treatment also prevented decreased mRNA expressions of nephrin and podocin in the high glucose-stimulated podocytes. However, mRNA expressions of CD2AP and alpha-actinin-4 were not significantly changed by the high glucose conditions. When these data are taken together, l-carnitine can increase glucose uptake in podocytes under high glucose conditions, and its mechanism may be at least partly related to the up-regulation of nephrin and podocin. Our results help clarify the beneficial effects of l-carnitine in diabetic nephropathy.

DOI： 10.1089/jmf.2010.1079

Allogenic fetal membrane-derived mesenchymal stem cells contribute to renal repair in experimental glomerulonephritis. Reviewed

Tsuda H, Yamahara K, Ishikane S, Otani K, Nakamura A, Sawai K, Ichimaru N, Sada M, Taguchi A, Hosoda H, Tsuji M, Kawachi H, Horio M, Isaka Y, Kangawa K, Takahara S, Ikeda T

American journal of physiology. Renal physiology 299 ( 5 ) F1004 - 13 2010.11

p21-activated kinases regulate actin remodeling in glomerular podocytes

Jianxin Zhu, Ortal Attias, Lamine Aoudjit, Ruihua Jiang, Hiroshi Kawachi, Tomoko Takano

American Journal of Physiology - Renal Physiology 298 ( 4 ) F951 - F961 2010.4

Language：English Publishing type：Research paper (scientific journal)

The tyrosine phosphorylation of nephrin is reported to regulate podocyte morphology via the Nck adaptor proteins. The Pak family of kinases are regulators of the actin cytoskeleton and are recruited to the plasma membrane via Nck. Here, we investigated the role of Pak in podocyte morphology. Pak1/2 were expressed in cultured podocytes. In mouse podocytes, Pak2 was predominantly phosphorylated, concentrated at the tips of the cellular processes, and its expression and/or phosphorylation were further increased when differentiated. Overexpression of rat nephrin in podocytes increased Pak1/2 phosphorylation, which was abolished when the Nck binding sites were mutated. Furthermore, dominant-negative Nck constructs blocked the Pak1 phosphorylation induced by antibody-mediated cross linking of nephrin. Transient transfection of constitutively kinase-active Pak1 into differentiated mouse podocytes decreased stress fibers, increased cortical F-actin, and extended the cellular processes, whereas kinase-dead mutant, kinase inhibitory construct, and Pak2 knockdown by shRNA had the opposite effect. In a rat model of puromycin aminonucleoside nephrosis, Pak1/2 phosphorylation was decreased in glomeruli, concomitantly with a decrease of nephrin tyrosine phosphorylation. These results suggest that Pak contributes to remodeling of the actin cytoskeleton in podocytes. Disturbed nephrin-Nck-Pak interaction may contribute to abnormal morphology of podocytes and proteinuria. Copyrigh © 2010 the American Physiological Society.

DOI： 10.1152/ajprenal.00536.2009

Effects of mineralocorticoid and angiotensin II receptor blockers on proteinuria and glomerular podocyte protein expression in a model of minimal change nephrotic syndrome Reviewed

Akihiro Fukuda, Shouichi Fujimoto, Shuji Iwatsubo, Hiroshi Kawachi, Kazuo Kitamura

NEPHROLOGY 15 ( 3 ) 321 - 326 2010.4

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL PUBLISHING, INC

Aim:
Several proteins constituting the slit diaphragm are considered important for maintaining capillary wall permselectivity. Early intervention with blockers of angiotensin II receptors (AR) and mineralocorticoid receptors (MR) is effective against proteinuria in models of chronic hypertensive and protein-induced renal damage. However, the effects of AR and/or MR blockers in a model of acute nephrotic syndrome remain unknown. The effects of AR and MR blockers were examined in puromycin aminonucleoside (PAN)-treated rats.
Methods:
Six week old male Sprague-Dawley (SD) rats were injected with PAN or vehicle and assigned to groups as follows: vehicle (group C); PAN (group P); PAN followed 3 days later by administration of the MR blocker, eplerenone (group MR), and by the AR blocker, losartan (group AR). Blood pressure and urinary protein excretion were measured and all rats were killed for immunohistochemical investigation on day 14 after PAN administration.
Results:
Blood pressure did not change throughout the study period. Proteinuria was decreased in groups MR and AR compared with group P (on day 14 after PAN administration, respectively; group P vs AR, P < 0.01; group P vs MR, P < 0.05). Nephrin, podocin and podocalyxin staining was preserved in the glomeruli of groups MR and AR compared with group P.
Conclusion:
The MR and AR blockers decreased proteinuria in the acute model of nephrotic syndrome with preserved expression of glomerular podocyte protein independently of blood pressure.

DOI： 10.1111/j.1440-1797.2009.01256.x

Loss of the BMP antagonist USAG-1 ameliorates disease in a mouse model of the progressive hereditary kidney disease Alport syndrome. Reviewed International journal

Mari Tanaka, Misako Asada, Atsuko Y Higashi, Jin Nakamura, Akiko Oguchi, Mayumi Tomita, Sachiko Yamada, Nariaki Asada, Masayuki Takase, Tomohiko Okuda, Hiroshi Kawachi, Aris N Economides, Elizabeth Robertson, Satoru Takahashi, Takeshi Sakurai, Roel Goldschmeding, Eri Muso, Atsushi Fukatsu, Toru Kita, Motoko Yanagita

The Journal of clinical investigation 120 ( 3 ) 768 - 77 2010.3

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC CLINICAL INVESTIGATION INC

The glomerular basement membrane (GBM) is a key component of the filtering unit in the kidney. Mutations involving any of the collagen IV genes (COL4A3, COL4A4, and COL4A5) affect GBM assembly and cause Alport syndrome, a progressive hereditary kidney disease with no definitive therapy. Previously, we have demonstrated that the bone morphogenetic protein (BMP) antagonist uterine sensitization-associated gene-1 (USAG-1) negatively regulates the renoprotective action of BMP-7 in a mouse model of tubular injury during acute renal failure. Here, we investigated the role of USAG-1 in renal function in Col4a3-/- mice, which model Alport syndrome. Ablation of Usag1 in Col4a3-/- mice led to substantial attenuation of disease progression, normalization of GBM ultrastructure, preservation of renal function, and extension of life span. Immunohistochemical analysis revealed that USAG-1 and BMP-7 colocalized in the macula densa in the distal tubules, lying in direct contact with glomerular mesangial cells. Furthermore, in cultured mesangial cells, BMP-7 attenuated and USAG-1 enhanced the expression of MMP-12, a protease that may contribute to GBM degradation. These data suggest that the pathogenetic role of USAG-1 in Col4a3-/- mice might involve crosstalk between kidney tubules and the glomerulus and that inhibition of USAG-1 may be a promising therapeutic approach for the treatment of Alport syndrome.

DOI： 10.1172/JCI39569

Rectal administration of tranilast ameliorated acute colitis in mice through increased expression of heme oxygenase-1 Reviewed

Xiaomei Sun, Kenji Suzuki, Masaki Nagata, Yusuke Kawauchi, Masahiko Yano, Shogo Ohkoshi, Yasunobu Matsuda, Hiroshi Kawachi, Kenichi Watanabe, Hitoshi Asakura, Yutaka Aoyagi

PATHOLOGY INTERNATIONAL 60 ( 2 ) 93 - 101 2010.2

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY

Mast cells play a key role in the pathophysiology of inflammatory bowel disease (IBD). Tranilast, a mast cell stabilizer, has been empirically used for IBD in Japan, but its precise role in the treatment of IBD is largely unknown. To investigate the role of tranilast for the treatment of IBD, tranilast was administered intrarectally to mice with dextran sulfate sodium (DSS)-induced colitis. Tranilast ameliorated DSS colitis clinically and pathologically, as demonstrated by decreased number and degranulation of mast cells in the colon. mRNA expression was increased for tumor necrosis factor-a, interferon-g and interleukin (IL)-6, and decreased for IL-10 in the colon of DSS colitis mice. In contrast, tranilast markedly decreased expression of mRNAs for the pro-inflammatory cytokines, and increased that of the anti-inflammatory cytokines. Moreover, tranilast increased heme oxygenase (HO)-1 expression on colonic epithelial cells as well as on colon-infiltrating cells of DSS colitis. In conclusion, tranilast ameliorated DSS colitis by regulating mast cell degranulation, decreasing inflammatory cytokines and increasing anti-inflammatory cytokines. Tranilast might exert these effects partly through enhanced HO-1 expression in the colon, suggesting a potential adjunctive therapy for IBD.

DOI： 10.1111/j.1440-1827.2009.02490.x

ERK5 activation enhances mesangial cell viability and collagen matrix accumulation in rat progressive glomerulonephritis Reviewed

Maki Urushihara, Masanori Takamatsu, Maki Shimizu, Shuji Kondo, Yukiko Kinoshita, Kenichi Suga, Akiko Kitamura, Sato Matsuura, Masanori Yoshizumi, Toshiaki Tamaki, Hiroshi Kawachi, Shoji Kagami

AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY 298 ( 1 ) F167 - F176 2010.1

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER PHYSIOLOGICAL SOC

Urushihara M, Takamatsu M, Shimizu M, Kondo S, Kinoshita Y, Suga K, Kitamura A, Matsuura S, Yoshizumi M, Tamaki T, Kawachi H, Kagami S. ERK5 activation enhances mesangial cell viability and collagen matrix accumulation in rat progressive glomerulonephritis. Am J Physiol Renal Physiol 298: F167-F176, 2010. First published October 21, 2009; doi:10.1152/ajprenal.00124.2009.-The mitogen-activated protein kinase (MAPK) cascade plays an important role in the regulation of various cellular functions in glomerulonephritis (GN). Here, we investigated whether extracellular signal-regulated kinase 5 (ERK5), a member of the MAPK family, is involved in the pathogenesis of chronic mesangioproliferative GN, using a rat model induced by uninephrectomy and anti-Thy-1 antibody injection. Immunostaining of kidneys obtained at different time points revealed that phospho-ERK5 was weakly expressed in control glomeruli but dramatically increased in a typical mesangial pattern after 28 and 56 days of GN. A semiquantitative assessment indicated that glomerular phospho-ERK5 expression closely paralleled the accumulation of extracellular matrix (ECM), collagen type I, as well as glomerular expression of reactive oxygen species (ROS) and ANG II. On the other hand, phospho-ERK1/2 expression increased on day 7 during the phase of enhanced mesangial cell (MC) proliferation and decreased thereafter. H(2)O(2) and ANG II each induced ERK5 phosphorylation by cultured rat MCs. Costimulation with both H(2)O(2) and ANG II synergistically increased ERK5 phosphorylation in MCs. Cultured MCs transfected with ERK5-specific small interference RNA showed a significant decrease in H(2)O(2) or ANG II-induced cell viability and soluble collagen secretion compared with control cells. Treatment of GN rats with an ANG II type 1 receptor blocker resulted in significant decreases in phospho-ERK5 expression and collagen accumulation accompanied by remarkable histological improvement. Taken together, these results suggest that MC ERK5 phosphorylation by ANG II or H(2)O(2) enhances cell viability and ECM accumulation in an experimental model of chronic GN.

DOI： 10.1152/ajprenal.00124.2009

Rac1 Contributes to Actin Organization in Glomerular Podocytes

Ortal Attias, Ruihua Jiang, Lamine Aoudjit, Hiroshi Kawachi, Tomoko Takano

NEPHRON EXPERIMENTAL NEPHROLOGY 114 ( 3 ) E93 - E106 2010

Language：English Publishing type：Research paper (scientific journal) Publisher：KARGER

Background/Aims: The function of glomerular podocytes is closely associated with the actin cytoskeleton. In this study, we studied the role of the small Rho-GTPase, Rac1, in actin organization in podocytes. Methods: Conditionally immortalized mouse podocytes (MP) stably expressing nephrin or control plasmid were used. Results: In MP, Rac1 activity increased significantly at 1 week of differentiation. MP stably expressing nephrin showed Rac1 activity significantly higher and more sustained than vector-expressing control cells. Antibody-mediated cross-linking of nephrin also activated Rac1. Differentiated MP showed more distinct lamellipodia/cellular processes, as compared with undifferentiated cells, which was further augmented by nephrin expression. Transient transfection of constitutively active Rac1 markedly increased the number of lamellipodia/cellular processes in undifferentiated MP, while the Rac1 inhibitor caused actin cytoskeleton derangement in differentiating MP. In the rat model of puromycin aminonucleoside nephrosis, RhoA activity was increased at Day 7 (at the peak of proteinuria), while Rac1 activity increased significantly only at Day 14, when the recovery process had started. Conclusion: Rac1 is activated in differentiating MP and nephrin potentiates Rac1 activation. Rac1 likely contributes to lamellipodia formation in differentiating MP and may contribute to process formation in podocytes recovering from injuries. Copyright (C) 2009 S. Karger AG, Basel

DOI： 10.1159/000262317

Peroxisome Proliferator-Activated Receptor-gamma Agonist Rosiglitazone Prevents Albuminuria but Not Glomerulosclerosis in Experimental Diabetes Reviewed

Giorgia Setti, Anthea Hayward, Cecile Dessapt, Francesca Barone, Robin Buckingham, Kathryn White, Rudolf Bilous, Kawachi Hiroshi, Gabriella Gruden, GianCarlo Viberti, Luigi Gnudi

AMERICAN JOURNAL OF NEPHROLOGY 32 ( 5 ) 393 - 402 2010

Language：English Publishing type：Research paper (scientific journal) Publisher：KARGER

Backgrounds/Aims: Renal inflammation and nephrin downregulation contribute to albuminuria in diabetes. We studied, in streptozotocin-induced diabetic rats, the effect of rosiglitazone (RSG), a peroxisome proliferator-activated receptor-gamma agonist, on renal macrophage infiltration, MCP1, and nephrin expression in relation to albuminuria. Methods: We investigated control and diabetic rats treated or untreated with RSG. Animals were sacrificed at 1, 3, and 9 months. Renal MCP1 and nephrin expression were studied by immunoblotting, renal macrophage infiltration by immunohistochemistry, and albuminuria by ELISA. Electron microscopy was used to assess glomerular ultrastructural morphology. In vitro experiments were conducted in isolated cultured rat glomeruli. Results: Glycaemic control was similar in diabetic rats treated and untreated with RSG, and blood pressure was comparable in all groups. RSG prevented diabetes-induced albuminuria at 9 months, and renal macrophage infiltration and MCP1 upregulation at 3 and 9 months. Diabetes-mediated nephrin downregulation was abolished by RSG. Diabetes-induced glomerulosclerosis, glomerular basement membrane thickening, and foot process fusion were not affected by RSG. In isolated glomeruli, MCP1 directly induced nephrin downregulation and this was prevented by RSG. RSG had no effect on nephrin expression. Conclusion: RSG prevents albuminuria and nephrin downregulation in experimental diabetes independently of glycaemic and blood pressure control. This effect likely occurs via correction of diabetes-induced inflammatory processes. Copyright (C) 2010 S. Karger AG, Basel

DOI： 10.1159/000320129

Contrasting Effects of Steroids and Mizoribine on Macrophage Activation and Glomerular Lesions in Rat Thy-1 Mesangial Proliferative Glomerulonephritis Reviewed

Yohei Ikezumi, Toshiaki Suzuki, Tamaki Karasawa, Hiroya Hasegawa, Hiroshi Kawachi, David J. Nikolic-Paterson, Makoto Uchiyama

AMERICAN JOURNAL OF NEPHROLOGY 31 ( 3 ) 273 - 282 2010

Language：English Publishing type：Research paper (scientific journal) Publisher：KARGER

Background: Macrophages with a pro-inflammatory (M1) phenotype mediate renal injury in proliferative forms of glomerulonephritis, while alternatively activated (M2) macrophages are thought to be anti-inflammatory and promote repair. Glucocorticoids, the mainstay therapy for proliferative glomerulonephritis, can induce alternative macrophage activation in vitro, but it is unknown whether this occurs in vivo and if this is required for glucocorticoid responsiveness. In addition, clinical studies have suggested that the ability of mizoribine (MZR) to suppress steroid-resistant proliferative glomerulonephritis may operate via inhibiting pro-inflammatory macrophage activation. Methods: This study examined prednisolone (PSL) and/or MZR treatment of rat Thy-1 disease - a model in which macrophages promote mesangial proliferative glomerulonephritis. Results: PSL treatment of Thy-1 nephritis induced an M2-like macrophage phenotype, but failed to modify mesangial hypercellularity and actually exacerbated global glomerulosclerosis. In contrast, MZR treatment reduced hypercellularity and glomerulosclerosis and suppressing both M1 and M2 markers of macrophage activation, with a selective reduction in CD169+ macrophages. Combined PSL/MZR treatment suppressed glomerular lesions and prevented steroid induction of an M2-like macrophage phenotype. In vitro, MZR prevented steroid induction of an M2 macrophage phenotype. Conclusions: Glucocorticoid induced alternative macrophage activation failed to ameliorate rat mesangial proliferative glomerulonephritis, whereas MZR suppression of this disease model was attributed, in part, to inhibition of M1-like proinflammatory macrophage activation. Copyright (C) 2010 S. Karger AG, Basel

DOI： 10.1159/000279163

Tolvaptan, a selective oral vasopressin V2 receptor antagonist, ameliorates podocyte injury in puromycin aminonucleoside nephrotic rats Reviewed

Tadashi Okada, Toshifumi Sakaguchi, Ikuji Hatamura, Fumie Saji, Shigeo Negi, Haruhisa Otani, Yasuteru Muragaki, Hiroshi Kawachi, Takashi Shigematsu

CLINICAL AND EXPERIMENTAL NEPHROLOGY 13 ( 5 ) 438 - 446 2009.10

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

Proteinuria caused by glomerular disease is characterized by podocyte injury. Vasopressin V2 receptor antagonists are effective in reducing albuminuria, although their actions on glomerular podocytes have not been explored. The objective of this study was to evaluate the effects of tolvaptan, a selective oral V2 receptor antagonist, on podocytes in a puromycin aminonucleoside (PAN)-induced nephrosis rat model.
Rats were allocated to a control, PAN nephrosis, or tolvaptan-treated PAN nephrosis group (n = 9 per group). Urinary protein excretion and serum levels of total protein, albumin, creatinine, and total cholesterol were measured on day 10. The influence of tolvaptan on podocytes was examined in renal tissues by immunofluorescence and electron microscopy.
PAN induced massive proteinuria and serum creatinine elevation on day 10, both of which were significantly ameliorated by tolvaptan. Immunofluorescence studies of the podocyte-associated proteins nephrin and podocin revealed granular staining patterns in PAN nephrosis rats. In tolvaptan-treated rats, nephrin and podocin expressions retained their normal linear pattern. Electron microscopy showed foot process effacement was ameliorated in tolvaptan-treated rats.
Tolvaptan is protective against podocyte damage and proteinuria in PAN nephrosis. This study indicates that tolvaptan exerts a renoprotective effect by affecting podocyte morphology and probably function in PAN nephrosis. Tolvaptan is a promising pharmacological tool in the treatment of renal edema.

DOI： 10.1007/s10157-009-0196-0

Active vitamin D and its analogue, 22-oxacalcitriol, ameliorate puromycin aminonucleoside-induced nephrosis in rats. International journal

Isao Matsui, Takayuki Hamano, Kodo Tomida, Kazunori Inoue, Yoshitsugu Takabatake, Yasuyuki Nagasawa, Noritaka Kawada, Takahito Ito, Hiroshi Kawachi, Hiromi Rakugi, Enyu Imai, Yoshitaka Isaka

Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 24 ( 8 ) 2354 - 61 2009.8

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: Recent studies have demonstrated that podocyte injury, which results in proteinuria, leads to tubulointerstitial fibrosis. Although some studies have revealed that vitamin D administration protects renal structure and function in mesangial cell proliferative and/or excessive matrix productive models, the effects of vitamin D on podocyte injury have remained uncertain. METHODS: In this study, we examined whether administration of active vitamin D (calcitriol) or its analogue, 22-oxacalcitriol (maxacalcitol), is preventative in podocyte injury using the puromycin aminonucleoside nephrosis model with neither mesangial proliferation nor matrix accumulation. RESULTS: Before the onset of proteinuria, renal 1alpha-hydroxylase and 24-hydroxylase were markedly down-regulated and up-regulated, respectively, leading to impaired vitamin D activation. Thereafter, serum 25-hydroxyvitamin D decreased along with the increased excretion of vitamin D-binding protein in urine. After confirming that podocytes express vitamin D receptor and all retinoid X receptors (RXRs) except RXR-alpha, we found that daily administration of calcitriol or its analogue 22-oxacalcitriol ameliorated the nephrotic state by protecting podocytes, as shown by the reduced staining of desmin (podocyte injury marker) and the upregulation of nephrin and podocin. These data suggest that the impairment of the vitamin D system plays a role in increasing proteinuria in podocyte injury. CONCLUSIONS: We demonstrated the breakdown of the vitamin D activation system in podocyte injury, and established a preventative role for vitamin D in podocyte injury.

DOI： 10.1093/ndt/gfp117

Slit diaphragm dysfunction in proteinuric states: identification of novel therapeutic targets for nephrotic syndrome Reviewed

Hiroshi Kawachi, Koichi Suzuki, Naoko Miyauchi, Taeko Hashimoto, Yasuhiro Otaki, Fujio Shimizu

CLINICAL AND EXPERIMENTAL NEPHROLOGY 13 ( 4 ) 275 - 280 2009.8

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

Several recent studies have demonstrated that the slit diaphragm of the glomerular epithelial cell (podocyte) is the structure likely to be the principal barrier in the glomerular capillary wall. Nephrin identified as a gene product mutated in congenital nephrotic syndrome located at the outer leaflet of plasma membranes of the slit diaphragm. The anti-nephrin antibody is capable of inducing massive proteinuria, which indicates that nephrin is a key functional molecule in the slit diaphragm. Expression of nephrin was reduced in glomeruli of minimal change nephrotic syndrome. Some recent studies demonstrated that podocin, CD2-associated protein and NEPH1 are also functional molecules in the slit diaphragm, and their expressions are altered in membranous nephropathy and also in focal glomerulosclerosis. These observations suggested that the alteration of the molecular arrangement in the slit diaphragm is involved in the development of proteinuria in several kinds of glomerular diseases. Recent studies of our group have demonstrated that type 1 receptor-mediated angiotensin II action reduced the expression of the slit diaphragm-associated molecules and that type 1 receptor blockade ameliorated proteinuria by preventing the function of angiotensin II on the slit diaphragm. By the subtraction hybridization techniques using glomerular cDNA of normal and proteinuric rats, we detected that synaptic vesicle protein 2B and ephrin B1 are involved in the maintenance of the barrier function of the slit diaphragm.

DOI： 10.1007/s10157-009-0162-x

Therapeutic targets in the podocyte: findings in anti-slit diaphragm antibody-induced nephropathy

Hiroshi Kawachi, Gi Dong Han, Naoko Miyauchi, Taeko Hashimoto, Koichi Suzuki, Fujio Shimizu

JOURNAL OF NEPHROLOGY 22 ( 4 ) 450 - 456 2009.7

Language：English Publishing type：Research paper (scientific journal) Publisher：WICHTIG EDITORE

Recent studies have demonstrated that the slit diaphragm of the glomerular epithelial cell (podocyte) is the structure likely to be the barrier in the glomerular capillary wall. Murine monoclonal antibody against nephrin, a molecule constituting the extracellular site of the slit diaphragm, caused severe proteinuria if injected into rats, in a complement- or inflammatory cell-independent manner. In this proteinuric state, not only nephrin but also other slit diaphragm-associated molecules are down-regulated. These observations suggest that the antibody alters the molecular composition of the slit diaphragm and, thereby, affects the glomerular permeability barrier. Recently, it was found that IP-10, SV2B, ephrin B1 and the receptors of angiotensin II were expressed in the podocyte, and that their expressions were clearly altered in anti-nephrin antibody-induced nephropathy. It is conceivable that these molecules are involved in the development of proteinuria in this model. IP-10 is assumed to play a role in maintaining the slit diaphragm function by regulating the cell cycle balance of the podocyte. SV2B and ephrin B1 play pivotal roles in the proper localization of the slit diaphragm component. In vivo and in vitro studies demonstrated that angiotensin II type 2 receptor-mediated action enhanced the expression of nephrin. We propose that these molecules could be novel therapeutic targets for proteinuria.

Receptor expression modulates the specificity of transforming growth factor-beta signaling pathways. International journal

Masaru Murakami, Hiroyuki Kawachi, Kenji Ogawa, Yoshii Nishino, Masayuki Funaba

Genes to cells : devoted to molecular & cellular mechanisms 14 ( 4 ) 469 - 82 2009.4

Language：English Publishing type：Research paper (scientific journal)

In current models of transforming growth factor-beta (TGF-beta) family signaling, type II receptors activate specific activin receptor-like kinase (ALK) type I receptors. These serine/threonine kinases activate ligand-dependent receptor regulated (R)-Smad by phosphorylating carboxy-terminal serines. We found that the receptor expression levels affected the phosphorylation and activation of the two R-Smad subclasses, activin/TGF-beta-specific (AR-Smad) and bone morphogenetic protein (BMP)-specific (BR-Smad). Co-expressing constitutively active type I and type II receptors in COS7 cells resulted in the phosphorylation of both R-Smad subclasses in a ligand-independent manner. This was verified using in vitro kinase assays. In untransfected B16 melanoma cells, TGF-beta1 and BMP-2 induced phosphorylation of both R-Smad subclasses, and TGF-beta1 up-regulated the inhibitor of differentiation (Id) gene, which is usually regulated by BMP. By contrast, BMP-2 up-regulated plasminogen activator inhibitor-1 (PAI-1), which is an AR-Smad-regulated gene. Except for ALK4 and ALK6, levels of type I and type II receptor mRNAs were higher in B16 cells than in HeLa and HepG2 cells, in which TGF-beta1 and BMP-2 induced phosphorylation of only the expected R-Smad. These results help to explain the diverse effects of this ligand family.

DOI： 10.1111/j.1365-2443.2009.01283.x

Angiotensin II receptor blockade inhibits acute glomerular injuries with the alteration of receptor expression Reviewed

Akiko Mii, Akira Shimizu, Yukinari Masuda, Masamichi Ishizaki, Hiroshi Kawachi, Yasuhiko Iino, Yasuo Katayama, Yuh Fukuda

LABORATORY INVESTIGATION 89 ( 2 ) 164 - 177 2009.2

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

Angiotensin II receptor blockade (ARB) suppresses the progression of chronic kidney disease. However, the re-noprotective effect of ARB in the active phase of glomerulonephritis (GN) has not been evaluated in detail. We examined the alteration of angiotensin II receptors' expression and the action of ARB on acute glomerular injuries in GN. Thy-1 GN was induced in rats that were divided into three groups (n = 7, in each group); high dose ( 3 mg/kg/day) or low dose (0.3 mg/kg/day) olmesartan (Thy-1 GN+HD- or LD-ARB group), and vehicle (Thy-1 GN group). Renal function and histopathology were assessed by week 2. In the Thy-1 GN group, diffuse mesangiolysis and focal aneurysmal ballooning developed by day 3. Marked mesangial proliferation and activation progressed with glomerular epithelial injury. We confirmed that both angiotensin II type 1 receptor (AT1R) and type 2 receptor (AT2R) were expressed on glomerular endothelial, mesangial, epithelial cells, and macrophages, and increased 7 days after disease induction. However, ARB treatment caused a decrease in AT1R and a further increase in AT2R expression in glomeruli. ARB prevented capillary destruction and preserved eNOS expression after diffuse mesangiolysis. Mesangial proliferation and activation was suppressed markedly with low levels of PDGF-B expression. Glomerular desmin expression, which is a marker for injured glomerular epithelial cells, was diminished significantly with retained expression of nephrin and podoplanin. Glomerular macrophage infiltration was also inhibited. Proteinuria was suppressed significantly. Furthermore, these effects of ARB showed dose dependency. These results provide insights that ARB affects individual glomerular cells and macrophages through angiotensin II receptors, with the alteration of both AT1R and AT2R expressions, and leads to inhibition of the acute destructive and proliferative glomerular lesions in GN. Laboratory Investigation ( 2009) 89, 164-177; doi:10.1038/labinvest.2008.128; published online 12 January 2009

DOI： 10.1038/labinvest.2008.128

[Basic research for kidney disease].

Kawachi Hiroshi

Nihon Jinzo Gakkai Shi 51 ( 1 ) 7 - 12 2009

Dissociation of NEPH1 from nephrin is involved in development of a rat model of focal segmental glomerulosclerosis Reviewed

Yasuhiro Otaki, Naoko Miyauchi, Mutsumi Higa, Akira Takada, Takeshi Kuroda, Fumitake Gejyo, Fujio Shimizu, Hiroshi Kawachi

American Journal of Physiology - Renal Physiology 295 ( 5 ) F1376 - F1387 2008.11

Language：English Publishing type：Research paper (scientific journal) Publisher：5

Focal segmental glomerulosclerosis (FSGS) is a disease showing severe proteinuria, and the disease progresses to end-stage kidney failure in many cases. However, the pathogenic mechanism of FSGS is not well understood. The slit diaphragm (SD), which bridges the neighboring foot processes of glomerular epithelial cells, is understood to function as a barrier of the glomerular capillary wall. To investigate the role of SD dysfunction in the development of FSGS, we analyzed the expression of SD-associated molecules in rat adriamycin-induced nephropathy, a mimic of FSGS. The staining of the SD molecules nephrin, podocin, and NEPH1 had already shifted to a discontinuous dotlike pattern at the initiation phase of the disease, when neither proteinuria nor any morphological alterations were detected yet. The alteration of NEPH1 expression was the most evident among the molecules examined, and NEPH1 was dissociated from nephrin at the initiation phase. On day 28, when severe proteinuria was detected and sclerotic changes were already observed, alteration of the expressions of nephrin, podocin, and NEPH1 worsened, but no alteration in the expression of other SD-associated molecules or other podocyte molecules was detected. It is postulated that the dissociation of NEPH1 from nephrin initiates proteinuria and that the SD alteration restricted in these molecules plays a critical role in the development of sclerotic changes in FSGS. Copyright © 2008 the American Physiological Society.

DOI： 10.1152/ajprenal.00075.2008

Activated macrophages down-regulate podocyte nephrin and podocin expression via stress-activated protein kinases Reviewed

Yohei Ikezumi, Toshiaki Suzuki, Tamaki Karasaw, Hiroshi Kawachi, David J. Nikolic-Paterson, Makoto Uchiyam

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 376 ( 4 ) 706 - 711 2008.11

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

The development of proteinuria and glomerulosclerosis in kidney disease is associated with podocyte damage, including down-regulation of nephrin and podocin. Macrophages are known to induce renal injury, but the mechanisms involved are not fully understood. This study examined macrophage-mediated podocyte damage. Conditioned media (CM) from activated macrophages caused a 50-60% reduction in nephrin and podocin nnRNA and protein expression in cultured mouse podocytes and rat glomeruli. This was abolished by a neutralizing anti-TNF alpha antibody. The addition of recombinant TNF alpha to podocytes OF glomeruli caused a comparable reduction in podocyte nephrin and podocin expression to that of macrophage CM. Inhibition of c-Jun amino terminal kinase (JNK) or p38 kinase abolished the TNF alpha-induced reduction in nephrin and podocin expression. This study demonstrates that activated macrophages can induce podocyte injury via a TNF alpha-JNK/p38-dependent mechanism. This may explain, in part, the protective effects of JNK and p38 blockade in experimental kidney disease. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2008.09.049

Selective loss of podoplanin protein expression accompanies proteinuria and precedes alterations in podocyte morphology in a spontaneous proteinuric rat model Reviewed

Klaas Koop, Michael Eikmans, Markus Wehland, Hans Baelde, Daphne Ijpelaar, Reinhold Kreutz, Hiroshi Kawachi, Dontscho Kerjaschki, Emile de Heer, Jan Anthonie Bruijn

AMERICAN JOURNAL OF PATHOLOGY 173 ( 2 ) 315 - 326 2008.8

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC INVESTIGATIVE PATHOLOGY, INC

To evaluate changes during the development of proteinuria, podocyte morphology and protein expression were evaluated in spontaneously proteinuric, Dahl salt-sensitive (Dahl SS) rats. Dahl SS rats on a low-salt diet were compared with spontaneously hypertensive rats (SHR) at age 2, 4, 6, 8, and 10 weeks. Blood pressure, urinary protein excretion, urinary albumin excretion, and podocyte morphology were evaluated. In addition, the expression of 11 podocyte-related proteins was determined by analyzing protein and mRNA levels. in Dahl SS rats, proteinuria became evident around week 5, increasing thereafter. SHR rats remained non-proteinuric. Dahl SS rats showed widespread foot process effacement at 10 weeks. At:58 weeks, expression and distribution of the podocyte proteins was similar between the two strains, except for the protein podoplanin. At 4 weeks, podoplanin began decreasing in the glomeruli of Dahl SS rats in a focal and segmental fashion. Podoplanin loss increased progressively and correlated with albuminuria (r = 0.8, P < 0.001). Double labeling experiments revealed increased expression of the podocyte stress marker desmin in glomerular areas where podoplanin was lost. Dahl SS rats did not show podoplanin gene mutations or decreased mRNA expression. Thus, podocyte morphology and the expression and distribution of most podocyte-specific proteins were normal in young Dahl SS rats, despite marked proteinuria. Our study suggests that decreased expression of podoplanin plays a role in the decrease of glomerular permselectivity.

DOI： 10.2353/ajpath.2008.080063

Rosuvastatin is additive to high-dose candesartan in slowing progression of experimental mesangioproliferative glomerulosclerosis Reviewed

Stephanie Krämer, Susanne Kron, Yingrui Wang-Rosenke, Tanja Loof, Dmytro Khadzhynov, Stanislao Morgera, Hiroshi Kawachi, Fujio Shimizu, Sebastian Martini, Hans-H. Neumayer, Harm Peters

American Journal of Physiology - Renal Physiology 294 ( 4 ) F801 - F811 2008.4

Language：English Publishing type：Research paper (scientific journal)

Rosuvastatin is additive to high-dose candesartan in slowing progression of experimental mesangioproliferative glomerulosclerosis (GS). Progressive mesangioproliferative glomerulonephritis, mostly IgA nephropathy, is a major cause of end-stage kidney disease worldwide. In a chronic-progressive model of mesangioproliferative GS, we tested the renoprotective efficacy of rosuvastatin alone and in combination with a high-dose of the AT1 blocker candesartan. Treatment was started 1 wk after disease induction (anti-thy1 antibody injection into uninephrectomized rats) and continued until week 20. Tubulointerstitial expression of the key fibrosis mediator transforming growth factor (TGF)-β served as the main marker of disease progression. Compared with the untreated GS rats (475 ± 52 pg/ml), tubulointerstitial TGF-β1 protein expression was significantly reduced by both single therapies (rosuvastatin -47%, candesartan -51%, P &lt
0.01). Tubulointerstitial matrix accumulation (matrix score in GS: 64 ± 7%) was relatively reduced by -45 and -52%, respectively (P &lt
0.01). The combination of rosuvastatin and candesartan had significantly greater effects on tubulointerstitial TGF-β1 expression (-82% vs. GS) and matrix accumulation (-83% vs. GS) (P &lt
0.001 vs. GS, P &lt
0.05 vs. single therapy) than either drug alone. Similar additive beneficial effects were observed for renal fibronectin and tissue inhibitor of metalloproteinase-1 expression, cell proliferation, macrophage infiltration, proteinuria, and kidney function. In conclusion, rosuvastatin limits the progressive course of anti-thy1-induced GS toward chronic tubulointerstitial fibrosis and renal insufficiency to a degree comparable to the one achieved by a high dose of the AT1 antagonist candesartan. Combined treatment yields significantly greater actions on renal TGF-β overexpression and matrix accumulation, cell proliferation, and macrophage infiltration. The results suggest that rosuvastatin and an AT 1 blocker independently interfere with separate key pathways involved in the progression of chronic mesangioproliferative GS. Copyright © 2008 the American Physiological Society.

DOI： 10.1152/ajprenal.00148.2007

Use of mizoribine as a rescue drug for steroid-resistant pediatric IgA nephropathy Reviewed

Yohei Ikezumi, Toshiaki Suzuki, Tamaki Karasawa, Hiroshi Kawachi, David J. Nikolic-Paterson, Makoto Uchiyama

PEDIATRIC NEPHROLOGY 23 ( 4 ) 645 - 650 2008.4

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

Recent clinical trials have shown a beneficial effect of mizoribine (Miz), an immunosuppressive drug, in the treatment of new-onset pediatric IgA nephropathy (IgAN). In this study, we evaluated the efficacy of Miz treatment in three children with established steroid-resistant IgAN. The patients had IgAN featuring persistent proteinuria and diffuse mesangial proliferation and had failed to respond to 2 years of treatment with prednisolone. Based upon the second biopsy results, patients were given methylprednisolone (mPSL) pulse therapy that induced a transient reduction in proteinuria, which was reversed when the mPSL dose was tapered. Miz therapy was then instigated in place of pulse mPSL. All three patients showed a substantial reduction in proteinuria and resolution of hematuria within 5 months. A follow-up biopsy in two of the patients showed a substantial reduction in the severity of glomerular lesions and a decrease in the number of activated macrophages. In conclusion, Miz therapy was found to be a safe and effective therapy in three cases of steroid-resistant pediatric IgAN. The ability of Miz to reduce the number of activated macrophages may be an important mechanism by which this drug ameliorated renal disease in these patients.

DOI： 10.1007/s00467-007-0664-2

Eplerenone potentiates the antiproteinuric effects of enalapril in experimental nephrotic syndrome Reviewed

Farid Nakhoul, Eliyahu Khankin, Afif Yaccob, Hiroshi Kawachi, Tony Karram, Huda Awaad, Nakhoul Nakhoul, Aaron Hoffman, Zaid Abassi

American Journal of Physiology - Renal Physiology 294 ( 3 ) F628 - F637 2008.3

Language：English Publishing type：Research paper (scientific journal)

Nephrotic syndrome (NS) is a clinical state characterized by massive proteinuria and edema. It is believed that nephrin and podocin are involved in the development of proteinuria. The proteinuria and effects of eplerenone alone or combined with enalapril on nephrin/podocin abundance in rats with NS have not yet been studied. Therefore, the present study was designed to examine the early (beginning 2 days before NS induction) and late (beginning 2 wk after NS induction) effects of eplerenone and enalapril, alone or combined, on proteinuria and nephrin/podocin abundance in rats with adriamycin-induced NS. Adriamycin caused a significant increase in daily protein excretion (U prV
from 26.96 ± 3.43 to 958.57 ± 56.7 mg/day, P &lt
0.001) and cumulative proteinuria [from 900.33 ± 135.5 to 22,490.62 ± 931.26 mg (P &lt
0.001)] during 6 wk. Early treatment with enalapril significantly decreased UprV from 958.6 ± 56.7 to 600.31 ± 65.13 mg/day (P &lt
0.001) and cumulative proteinuria to 12,842.37 ± 1,798.17 mg/6 wk (P &lt
0.001). Similarly, early treatment with eplerenone produced a profound antiproteinuric effect: UprV decreased from 958.57 ± 56.7 to 593.38 ± 21.83 mg/day, P &lt
0.001, and cumulative proteinuria to 16,601.84 ± 1,334.31 mg/6 wk
P &lt
0.001. An additive effect was obtained when enalapril and eplerenone were combined: U prV decreased from 958.57 ± 56.69 to 424.17 ± 38.54 mg/day, P &lt
0.001, and cumulative protein excretion declined to 10,252.88 ± 1,011.3 mg/6 wk, P &lt
0.001. These antiproteinuric effects were associated with substantial preservation of glomerular nephrin and podocin. In contrast, late treatment with either enalapril or eplerenone alone or combined mildly decreased UprV and cumulative proteinuria. Thus pretreatment with eplerenone or enalapril is effective in reducing daily and cumulative protein excretion and preservation of nephrin/podocin. More profound antiproteinuric effects were obtained when enalapril and eplerenone were combined. Copyright © 2008 the American Physiological Society.

DOI： 10.1152/ajprenal.00524.2007

Low-dose mTOR inhibition by rapamycin attenuates progression in anti-thy1-induced chronic glomerulosclerosis of the rat Reviewed

Stephanie Krämer, Yingrui Wang-Rosenke, Valeska Scholl, Eva Binder, Tanja Loof, Dmytro Khadzhynov, Hiroshi Kawachi, Fujio Shimizu, Fritz Diekmann, Klemens Budde, Hans-H. Neumayer, Harm Peters

American Journal of Physiology - Renal Physiology 294 ( 2 ) F440 - F449 2008.2

Language：English Publishing type：Research paper (scientific journal)

Treatment options in human mesangioproliferative glomerulonephritis/ sclerosis, mostly IgA nephropathy, are limited. Progressive mesangioproliferative nephropathy represents a major cause of end-stage kidney disease. The present study explores the efficacy of low-dose mTOR inhibition by rapamycin in a chronic-progressive model of mesangioproliferative glomerulosclerosis (cGS). cGS was induced by high-dose anti-thy1 antibody injection into uninephrectomized rats. Rapamycin administration (2.5 mg·kg-1·body wt-1) was started 10 days after antibody injection and continued until week 20. cGS was characterized by advancing proteinuria, increased blood pressure, marked tubulointerstitial and glomerular fibrosis, cell proliferation and round cell infiltration, and impaired renal function. Kruskal-Wallis and Mann-Whitney U-tests were used for statistical analysis. The course of chronic anti-thy1-induced glomerulosclerosis was significantly attenuated by low-dose rapamycin treatment. In week 20, this was demonstrated by improvements in proteinuria (-38%), systolic blood pressure (-16 mmHg), tubulointerstitial and glomerular histological matrix accumulation (-61 and -24%), transforming growth factor-β1 overexpression (-41 and -47%), collagen I deposition (-53 and -65%), cell proliferation (-90 and -76%), and leukocyte number (macrophages -52 and -53%
lymphocytes -58 and 51%), respectively. Rapamycin improved renal function as well (blood creatinine -0.68 mg/dl, urea -66.7 mg/day, and creatinine clearance +0.13 ml·min -1·100 g body wt-1). In conclusion, low-dose mTOR inhibition by rapamycin limits the progressive course of anti-thy1-induced renal disease toward chronic glomerulosclerosis, tubulointerstitial fibrosis, and renal insufficiency. Renoprotection by rapamycin involved significant beneficial effects on multiple key pathways in the progression of chronic renal disease, i.e., proteinuria, extracellular matrix accumulation, renal cell proliferation, and inflammatory cell infiltration. Copyright © 2008 the American Physiological Society.

DOI： 10.1152/ajprenal.00379.2007

IFN-inducible protein 10 (CXCL10) regulates tubular cell proliferation in renal ischemia-reperfusion injury

Kengo Furuichi, Takashi Wada, Shinji Kitajikma, Tadasi Toyama, Toshiya Okumura, Akinori Hara, Hiroshi Kawachi, Fujio Shimizu, Takeshi Sugaya, Naofumi Mukaida, Shosaku Narumi, Kouji Matsushima, Shuichi Kaneko

NEPHRON EXPERIMENTAL NEPHROLOGY 109 ( 1 ) C29 - C38 2008

Language：English Publishing type：Research paper (scientific journal) Publisher：KARGER

Although renal tubular cell proliferation after acute tubular necrosis is an important and essential response in the recovery of renal dysfunction in acute renal failure, the precise factors and mechanisms of tubular cell regeneration remain unclear. Here, we describe our studies using a neutralizing antibody (Ab) against interferon-inducible protein of 10 kDa (IP-10; CXCL10) that indicate a role for CXCL10 in tubular cell proliferation after renal ischemia-reperfusion injury. Tissue necrosis and interstitial infiltrating numbers were comparable between anti-CXCL10 Ab-treated and control mice treated with IgG at the 24 and 48 h time points after reperfusion. In contrast, the numbers of Ki67-positive proliferating tubular cells were significantly increased in anti-CXCL10 Ab-treated mice 48 h after reperfusion. In accordance with the in vivo findings, in vitro studies using murine tubular epithelial cells indicated an antiproliferative effect of CXCL10 upon the intensity of cell proliferation and the number of Ki67-positive cells. These data suggest that CXCL10 plays a role in the regulation of tubular cell proliferation following renal ischemia-reperfusion injury. Copyright (C) 2008 S. Karger AG, Basel.

DOI： 10.1159/000135675

Suppressive activity of pemirolast potassium, an antiallergic drug, on glomerulonephritis: Studies in glomerulonephritis model rats and in patients with chronic glomerulonephritis concurrently affected by allergic rhinitis Reviewed

Tomohito Gohda, Chisei Ra, Chieko Hamada, Toshinao Tsuge, Hiroshi Kawachi, Yasuhiko Tomino

Arzneimittel-Forschung/Drug Research 58 ( 1 ) 18 - 23 2008

Language：English Publishing type：Research paper (scientific journal)

Background: It is still difficult to manage chronic glomerulonephritis with corticosteroids because of safety concerns, especially for patients with mild symptoms and infants. Therefore, an alternative approach is greatly required. Pemirolast potassium (CAS 100299-08-9) is an antiallergic drug with high safety. Methods: Two glomerulonephritis rat models were prepared to examine the pharmacological actions of pemirolast potassium: one reversible model prepared with the anti-Thy-1 antibody, and another irreversible model by unilateral nephrectomy and with the anti-Thy-1 antibody. Pemirolast potassium was administered to 10 Japanese chronic glomerulonephritis patients concurrently affected by allergic rhinitis in order to examine its efficacy for mild proteinuria. Results: Pemirolast potassium 1 and 10 mg/kg markedly inhibited proteinuria in the reversible model. In the irreversible model, pemirolast potassium 3 mg/kg showed a significant decrease in the incidence of glomerulosclerosis. In chronic glomerulonephritis patients, pemirolast potassium, 10 mg twice daily, for 6 months, significantly reduced the severity of proteinuria. Conclusion: Our research suggested the efficacy of pemirolast potassium in glomerulonephritis. A well-controlled study is considered necessary to validate pemirolast potassium as a therapeutic drug for glomerulonephritis. © ECV Editio Cantor Verlag.

Decreased tyrosine phosphorylation of nephrin in rat and human nephrosis

K. Uchida, K. Suzuki, M. Iwamoto, H. Kawachi, M. Ohno, S. Horita, K. Nitta

Kidney International 73 ( 8 ) 926 - 932 2008

Language：English Publishing type：Research paper (scientific journal) Publisher：Nature Publishing Group

Phosphorylation of tyrosine residue (Y1204) of rat nephrin by Fyn kinase allows Nck adaptor protein binding to nephrin motifs, which include the phosphorylated tyrosine. This phosphorylation-dependent switch induces actin polymerization in a cell culture system. Here, we generated an antibody recognizing phosphorylated nephrin at the Nck binding sites pY1204 and pY1228 to determine the phosphorylation status of nephrin using a rat model of puromycin aminonucleoside-induced nephrosis. Changes in globular actin (G-actin) and filamentous actin (F-actin) contents in isolated glomeruli were measured by western blot. Before experimental nephrosis, both Y1204 and Y1228 were phosphorylated, and most of the actin was filamentous. Before the onset of overt proteinuria, however, phosphorylation of both Y1204 and Y1228 rapidly decreased and became almost undetectable. During this period, the amount of F-actin in glomeruli began to decrease, whereas G-actin increased. Phosphorylation of nephrin at Y1228 in glomeruli of patients with minimal change nephrosis was significantly decreased compared with that in normal glomeruli. Our study suggests that tyrosine phosphorylation of nephrin by regulating F-actin formation may be important for the maintenance of normal podocyte morphology and function. © 2008 International Society of Nephrology.

DOI： 10.1038/ki.2008.19

The mTOR inhibitor everolimus attenuates the time course of chronic anti-Thy1 nephritis in the rat

Sandra Wittmann, Christoph Daniel, Andrea Braun, Regina Vogelbacher, Fuijo Shimizu, Hiroshi Kawachi, Christian Hugo

NEPHRON EXPERIMENTAL NEPHROLOGY 108 ( 2 ) 45 - 56 2008

Language：English Publishing type：Research paper (scientific journal) Publisher：KARGER

Background: The antiproliferative immunosuppressant everolimus adversely affects the acute reversible anti-Thy1 nephritis model. We hypothesized that everolimus treatment started after the acute proliferative phase could even be beneficial in the chronic anti-Thy1 nephritis model in the rat. Methods: Chronic anti-Thy1 nephritis was induced by injection of the monoclonal antibody 1-22-3 in 20 male Sprague-Dawley rats 7 days after uninephrectomy. Two weeks after disease induction, rats were randomly treated with either everolimus or vehicle for 14 weeks. Changes in progression of renal disease were investigated by immunohistochemistry and real-time PCR 16 weeks after disease induction. Results: During chronic anti-Thy1 nephritis, the formation of focal segmental glomerulosclerosis lesions, the degree of interstitial fibrosis as well as the increase in proteinuria over 14 weeks was ameliorated by everolimus treatment. Increased glomerular hypertrophy observed in the vehicle-treated rats was completely prevented in the everolimus-treated nephritic rats. Increased glomerular fibronectin mRNA and protein as well as the renal influx of monocytes/macrophages was significantly reduced in the evero-limus group. Everolimus reduced the pro-angiogenic factor vascular endothelial growth factor (VEGF) and VEGF mRNA in glomeruli, while the transforming growth factor-beta signaling pathway was not affected. Conclusion: 'Late' start of everolimus treatment demonstrates beneficial effects on the time course of chronic anti-Thy1 nephritis. Copyright (C) 2008 S. Karger AG, Basel.

DOI： 10.1159/000116112

Nephrin mediates actin reorganization via phosphoinositide 3-kinase in podocytes

J. Zhu, N. Sun, L. Aoudjit, H. Li, H. Kawachi, S. Lemay, T. Takano

Kidney International 73 ( 5 ) 556 - 566 2008

Language：English Publishing type：Research paper (scientific journal) Publisher：Nature Publishing Group

Nephrin is a slit diaphragm protein critical for structural and functional integrity of visceral glomerular epithelial cells (podocytes) and is known to be tyrosine phosphorylated by Src family kinases. We studied the role of phosphoinositide 3-kinase (PI3K), activated via the phosphorylation of nephrin, in actin cytoskeletal reorganization of cultured rat podocytes. Phosphorylation of rat nephrin by the Fyn kinase markedly increased its interaction with a regulatory subunit of PI3K. Stable transfection of rat nephrin in the podocytes with podocin led to nephrin tyrosine phosphorylation, PI3K-dependent phosphorylation of Akt, increased Rac1 activity, and an altered actin cytoskeleton with decreased stress fibers and increased lamellipodia. These changes were reversed with an inhibitor of PI3K and not seen when the nephrin-mutant Y1152F replaced wild-type nephrin. Rac1 and Akt1 contributed to lamellipodia formation and decreased stress fibers, respectively. Finally, in the rat model of puromycin aminonucleoside nephrosis, nephrin tyrosine phosphorylation, nephrin-PI3K association, and glomerular Akt phosphorylation were all decreased. Our results suggest that PI3K is involved in nephrin-mediated actin reorganization in podocytes. Disturbed nephrin-PI3K interactions may contribute to abnormal podocyte morphology and proteinuria. © 2008 International Society of Nephrology.

DOI： 10.1038/sj.ki.5002691

IFN-inducible protein 10 (CXCL10) regulates tubular cell proliferation in renal ischemia-reperfusion injury. Reviewed

Furuichi K, Wada T, Kitajikma S, Toyama T, Okumura T, Hara A, Kawachi H, Shimizu F, Sugaya T, Mukaida N, Narumi S, Matsushima K, Kaneko S

Nephron. Experimental nephrology 109 ( 1 ) c29 - 38 2008

スリット膜関連分子の異常による蛋白尿. Invited

河内裕, 宮内直子

腎と透析 62 370 - 373 2007.12

Function of the podocyte slit diaphragm Invited

KAWACHI Hiroshi

Jpn J Nephrol 49 ( 2 ) 77 - 81 2007.12

糸球体濾過とその調節機構：構造と機能. Invited

河内裕

細胞 39 190 - 194 2007.12

蛋白尿発症機序についての最近の知見. Invited

宮内直子, 河内裕

新潟医学会雑誌 112 219 - 225 2007.12

蛋白尿の成因最近の進歩. Invited

河内裕

綜合臨床 56 155 - 156 2007.12

Ephrin-B1 localizes at the slit diaphragm of the glomerular podocyte Reviewed

T. Hashimoto, T. Karasawa, A. Saito, N. Miyauchi, G. D. Han, K. Hayasaka, F. Shimizu, H. Kawachi

KIDNEY INTERNATIONAL 72 ( 8 ) 954 - 964 2007.10

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

Ephs and ephrins are a family of membrane-bound proteins that function as receptor-ligand pairs. Members of the Eph-ephrin-B family have recently been reported to regulate the paracellular permeability of epithelial cells. In this study, we analyzed the expression and the function of ephrin-B1 in glomeruli. Using immunofluorescence ( IF), we found that ephrin-B1 was expressed along the glomerular capillary loop. Immunoelectron microscopy revealed that ephrin-B1 expression was restricted at the slit diaphragm. Dual labeled IF showed ephrin-B1 colocalized with the slit diaphragm proteins nephrin and CD2-associated protein. Ephrin-B1 colocalized with nephrin at the late capillary loop stage of kidney development. Additionally, injection of rats with a nephritogenic anti-nephrin antibody ( ANA) reduced ephrin-B1 expression. When podocytes were cultured in vitro, they extruded processes that co-stained for ephrin-B1 and for CD2-associated protein. When these podocytes were treated in culture with small interfering RNA for ephrin-B1, CD2-associated protein was reduced in the processes, with a remaining faint perinuclear staining. We suggest that ephrin-B1 has a role in maintaining barrier function at the slit diaphragm.

DOI： 10.1038/sj.ki.5002454

Prevention of hypertension with or without renin-angiotensin system inhibition precludes nephrin loss in the early stage of experimental diabetes mellitus Reviewed

Roberto Bleuel Amazonas, Rodrigo De Almeida Sanita, Hiroshi Kawachi, José B. Lopes De Faria

Nephron - Physiology 107 ( 2 ) p57 - p64 2007.10

Language：English Publishing type：Research paper (scientific journal)

Background and Aims: Several lines of clinical evidence support the concept that the reduction of blood pressure may be useful in the prevention of diabetic kidney disease. In young diabetic spontaneously hypertensive rats (SHR), prevention of hypertension reduces several early renal abnormalities including albuminuria. However, the contribution of nephrin loss to albuminuria in this early stage of experimental diabetes is unknown. Therefore, we investigated whether elevation of albuminuria in young diabetic SHR is associated with nephrin loss, and if prevention of hypertension, with or without inhibition of the renin-angiotensin system, precludes these abnormalities. Methods: Diabetes was induced by streptozotocin injection in 4-week-old still normotensive SHR and their genetically normotensive control, Wistar-Kyoto rats. Diabetic SHR were randomized for no treatment, or treatment with captopril, losartan, or triple therapy (hydrochlorothiazide, reserpine and hydralazine) for 20 days. Results: The increase in systolic blood pressure was equally prevented by all treatments. Albuminuria was higher in diabetic SHR and similarly reduced (p &lt
0.05) by captopril, losartan, and triple therapy. Glomerular expression of nephrin was significantly reduced in diabetic SHR in comparison with non-diabetic controls. The antihypertensive treatment prevented the reduction in glomerular expression of nephrin. Conclusions: These results demonstrate that the loss of nephrin is associated with albuminuria in a model of genetic hypertension and diabetes, and that the prevention of development of hypertension restores nephrin and prevents albuminuria. This finding suggests a crucial role of blood pressure in diabetes as determinant of nephrin expression and albuminuria. Copyright © 2007 S. Karger AG.

DOI： 10.1159/000108642

Podocyte protection by darbepoetin: preservation of the cytoskeleton and nephrin expression Reviewed

N. Eto, T. Wada, R. Inagi, H. Takano, A. Shimizu, H. Kato, H. Kurihara, H. Kawachi, S. J. Shankland, T. Fujita, M. Nangaku

KIDNEY INTERNATIONAL 72 ( 4 ) 455 - 463 2007.8

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

Podocyte injury is a significant contributor to proteinuria and glomerulosclerosis. Recent studies have shown a renoprotective effect of erythropoietin (EPO) during ischemic kidney disease. In this study, we examine mechanisms by which a long acting recombinant EPO analog, darbepoetin, may confer renoprotection in the puromycin aminonucleoside-induced model of nephrotic syndrome. Darbepoetin decreased the proteinuria of rats treated with puromycin. This protective effect was correlated with the immunohistochemical disappearance of the podocyte injury markers desmin and the immune costimulator molecule B7.1 with the reappearance of nephrin expression in the slit diaphragm. Podocyte foot process retraction and effacement along with actin filament rearrangement, determined by electron microscopy, were all reversed by darbepoetin treatment. The protective effects were confirmed in puromycin-induced nephrotic rats that had been hemodiluted to normal hematocrit levels. Furthermore, puromycin treatment of rat podocytes in culture caused actin cytoskeletal reorganization along with deranged nephrin distribution. All these effects in vitro were reversed by darbepoetin. Our study demonstrates that darbepoetin treatment ameliorates podocyte injury and decreases proteinuria by a direct effect on podocytes. This may be accomplished by maintenance of the actin cytoskeleton and nephrin expression.

DOI： 10.1038/sj.ki.5002311

Tumor suppressor carcinoembryonic antigen-related cell adhesion molecule 1 potentates the anchorage-independent growth of human hepatoma HepG2 cells

Mariko Hokari, Yasunobu Matsuda, Toshifumi Wakai, Yoshio Shirai, Munehiro Sato, Atsunori Tsuchiya, Masaaki Takamura, Satoshi Yamagiwa, Kenji Suzuki, Shogo Ohkoshi, Takafumi Ichida, Hiroshi Kawachi, Yutaka Aoyagi

LIFE SCIENCES 81 ( 4 ) 336 - 345 2007.7

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), an adhesion molecule of the immunoglobulin superfamily, has been characterized as a putative tumor suppressor because it is frequently down-regulated in aggressive types of cancer cells. Recently, however, several studies have shown that CEACAM 1 actively contributes to malignant progression or migration in some types of tumor cells, suggesting that the role of CEACAM 1 might be diverse among different types of cancer cells. To investigate the functional consequences of CEACAM 1 expression in hepatocellular carcinoma, we analyzed the status of CEACAM 1 in hepatoma cell lines HLF, PLC/PRF/5, HepG2 and KYN-2. We found that CEACAM1 was only expressed in HepG2 cells, which show a unique property for enhanced anchorage-independent growth. When HepG2 cells were treated with small interfering RNA targeted against CEACAM1, the growth rate in monolayer culture was increased. In contrast, when HepG2 cells were cultured in suspension, inhibition of CEACAM1 expression significantly decreased the growth rate, and the speed of cell-cell attachment was repressed. Hyaluronidase treatment attenuated the growth rate of HepG2 cells in suspension culture, indicating that cell-cell attachment is a requisite for anchorage-independent growth. Our data may reveal the dual role of CEACAM1 on hepatocarcinogenesis, by showing that CEACAM1 acts as a tumor suppressor in HepG2 cells in anchorage-dependent growth conditions, while in anchorage-independent growth conditions, it augments cell proliferation by potentiating the cell-cell attachment. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.lfs.2007.06.002

Blockade of interferon-gamma-inducible protein-10 attenuates chronic experimental colitis by blocking cellular trafficking and protecting intestinal epithelial cells

Kenji Suzuki, Yusuke Kawauchi, Suresh S. Palaniyandi, Punniyakoti T. Veeraveedu, Masato Fujii, Satoshi Yamagiwa, Hiroyuki Yoneyama, Gi Dong Han, Hiroshi Kawachi, Yoshiaki Okada, Yoichi Ajioka, Kenichi Watanabe, Masamichi Hosono, Hitoshi Asakura, Yutaka Aoyagi, Shosaku Narumi

PATHOLOGY INTERNATIONAL 57 ( 7 ) 413 - 420 2007.7

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL PUBLISHING

The role of chemokines, especially CXCL10/interferon-gamma-inducible protein 10 kDa (IP-10), a chemokine to attract CXCR3(+) T-helper 1-type CD4(+) T cells, is largely unknown in the pathophysiology of inflammatory bowel disease; ulcerative colitis and Crohn's disease. The authors have earlier shown that IP-10 neutralization protected mice from acute colitis by protecting crypt epithelial cells of the colon. To investigate the therapeutic effect of neutralization of IP-10 on chronic colitis, an anti-IP-10 antibody was injected into mice with newly established murine AIDS (MAIDS) colitis. Anti-IP-10 antibody treatment reduced the number of colon infiltrating cells when compared to those mice given a control antibody. The treatment made the length of the crypt of the colon greater than control antibody. The number of Ki67(+) proliferating epithelial cells was increased by the anti-IP-10 antibody treatment. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)(+) apoptotic cells were observed in the epithelial cells of the luminal tops of crypts in control MAIDS colitis, whereas TUNEL+ apoptotic epithelial cells were rarely observed with anti-IP-10 antibody treatment. In conclusion, blockade of IP-10 attenuated MAIDS colitis through blocking cellular trafficking and protecting intestinal epithelial cells, suggesting that IP-10 plays a key role in the development of inflammatory bowel disease as well as in chronic experimental colitis.

DOI： 10.1111/j.1440-1827.2007.02117.x

Angiotensin II type 1 and type 2 receptors play opposite roles in regulating the barrier function of kidney glomerular capillary wall Reviewed

Koichi Suzuki, Gi Dong Han, Naoko Miyauchi, Taeko Hashimoto, Takeshi Nakatsue, Yumiko Fujioka, Hiroko Koike, Fujio Shimizu, Hiroshi Kawachi

AMERICAN JOURNAL OF PATHOLOGY 170 ( 6 ) 1841 - 1853 2007.6

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC INVESTIGATIVE PATHOLOGY, INC

Although angiotensin H (Ang II) type 1 receptor antagonist ameliorates proteinuria, its pharmacological mechanism and the differential roles of Ang H type 1 receptor (AT1R) and type 2 receptor (AT2R) are not well understood. We analyzed the effect of Ang H type 1 receptor antagonist on proteinuria caused by antibody against nephrin, a functional molecule of glomerular slit diaphragm and dysfunction of which is involved in the development of proteinuria in several glomerular diseases. We show here that AT1R antagonist ameliorated proteinuria by preventing a reduction in the functional molecules of the slit diaphragm. We also analyzed the role of AT1R- or AT2R-mediated actions on the expression of the slit diaphragm molecules in an in vivo study of normal rat and in an in vitro study of cultured podocytes. AT1R-mediated action hampered the mRNA expression of the slit diaphragm molecules, whereas AT2R-mediated action enhanced it. These findings indicate that Ang H receptor subtypes play opposite roles in regulating the barrier function of glomerular capillary wall and that the enhancement of AT2R stimulation may serve as a potential therapeutic strategy for proteinuria.

DOI： 10.2353/ajpath.2007.060484

Podocyte as the target for aldosterone - Roles of oxidative stress and Sgk1 Reviewed

Shigeru Shibata, Miki Nagase, Shigetaka Yoshida, Hiroshi Kawachi, Toshiro Fujita

HYPERTENSION 49 ( 2 ) 355 - 364 2007.2

Language：English Publishing type：Research paper (scientific journal) Publisher：LIPPINCOTT WILLIAMS & WILKINS

Accumulating evidence suggests that mineralocorticoid receptor blockade effectively reduces proteinuria in hypertensive patients. However, the mechanism of the antiproteinuric effect remains elusive. In this study, we investigated the effects of aldosterone on podocyte, a key player of the glomerular filtration barrier. Uninephrectomized rats were continuously infused with aldosterone and fed a high-salt diet. Aldosterone induced proteinuria progressively, associated with blood pressure elevation. Notably, gene expressions of podocyte-associated molecules nephrin and podocin were markedly decreased in aldosterone-infused rats at 2 weeks, with a gradual decrease thereafter. Immunohistochemical studies and electron microscopy confirmed the podocyte damage. Podocyte injury was accompanied by renal reduced nicotinamide-adenine dinucleotide phosphate oxidase activation, increased oxidative stress, and enhanced expression of aldosterone effector kinase Sgk1. Treatment with eplerenone, a selective aldosterone receptor blocker, almost completely prevented podocyte damage and proteinuria, with normalization of elevated reduced nicotinamide-adenine dinucleotide phosphate oxidase activity. In addition, proteinuria, podocyte damage, and Sgk1 upregulation were significantly alleviated by tempol, a membrane-permeable superoxide dismutase, suggesting the pathogenic role of oxidative stress. Although hydralazine treatment almost normalized blood pressure, it failed to improve proteinuria and podocyte damage. In cultured podocytes with consistent expression of mineralocorticoid receptor, aldosterone stimulated membrane translocation of reduced nicotinamide-adenine dinucleotide phosphate oxidase cytosolic components and oxidative stress generation in podocytes. Furthermore, aldosterone enhanced the expression of Sgk1, which was inhibited by mineralocorticoid receptor antagonist and tempol. In conclusion, podocytes are injured at the early stage in aldosterone-infused rats, resulting in the occurrence of proteinuria. Aldosterone can directly modulate podocyte function, possibly through the induction of oxidative stress and Sgk1.

DOI： 10.1161/01.HYP.0000255636.11931.a2

SM22 alpha: the novel phenotype marker of injured glomerular epithelial cells in anti-glomerular basement membrane nephritis Reviewed

Asa Ogawa, Minoru Sakatsume, Xingzhi Wang, Yunichi Sakamaki, Yutaka Tsubata, Bassam Alchi, Takeshi Kuroda, Hiroshi Kawachi, Ichiei Narita, Fujio Shimizu, Fumitake Gejyo

NEPHRON EXPERIMENTAL NEPHROLOGY 106 ( 3 ) 77 - 87 2007

Language：English Publishing type：Research paper (scientific journal) Publisher：KARGER

Background/Aims: Our previous comprehensive analysis of the genes expressed in kidneys with anti-glomerular basement membrane ( GBM) nephritis using DNA microarrays showed that SM22 alpha was one of the highly expressed genes. SM22 alpha is a 22-kDa cytoskeletal protein that is exclusively expressed in smooth muscle cells. We investigated the localization of SM22 alpha at mRNA and protein levels, and its pathological significance in anti-GBM nephritis kidneys. Methods: Northern blot analysis, in situ hybridization, immunohistochemistry and double immunofluorescence studies were performed. The specific antibody ( Ab) against SM22 alpha was obtained by immunization of rabbits with recombinant rat SM22 alpha protein. Results: SM22 alpha mRNA expression was up-regulated in kidneys and inducibly expressed in the parietal and visceral glomerular epithelial cells in anti-GBM nephritis kidneys. Immunohistochemistry with anti-SM22 alpha Ab showed that SM22 alpha protein was localized in the same series of cells. Double immunofluorescence with anti-SM22 alpha and anti- glomerular cell markers demonstrated that SM22 alpha might be expressed in epithelial cells of injured glomeruli. In visceral epithelial cells, SM22 alpha might be expressed in cells in which podocyte specific markers, podocalyxin and nephrin were lost. Conclusion: The injured glomerular epithelial cells in anti-GBM nephritis might undergo structural and functional alterations, including the expression of a smooth muscle marker, SM22 alpha. Copyright (C) 2007 S. Karger AG, Basel.

DOI： 10.1159/000103020

Blockade of IP-10/CXCR3 promotes progressive renal fibrosis Reviewed

Izaya Nakaya, Takashi Wada, Kengo Furuichi, Norihiko Sakai, Kiyoki Kitagawa, Hitoshi Yokoyama, Yuko Ishida, Toshikazu Kondo, Takeshi Sugaya, Hiroshi Kawachi, Fujio Shimizu, Shosaku Narumi, Makoto Haino, Craig Gerard, Kouji Matsushima, Shuichi Kaneko

NEPHRON EXPERIMENTAL NEPHROLOGY 107 ( 1 ) E12 - E21 2007

Language：English Publishing type：Research paper (scientific journal) Publisher：KARGER

Background/Aim: Fibrosis is a hallmark of progressive organ disease. The 10-kDa interferon-inducible protein IP-10/CXCL10 is a potent chemoattractant for activated T lymphocytes, natural killer cells, and monocytes. However, the involvement of IP-10 in the pathogenesis of renal diseases via its receptor, CXCR3, remains unclear. To contribute to the clarification of this issue was the aim of this study. Methods: The impacts of IP-10 on renal fibrosis were investigated in a unilateral ureteral obstruction model in CXCR3-deficient mice and mice treated with anti-IP-10-neutralizing monoclonal antibody. Anti-IP-10 monoclonal antibody (5 mg/kg/day) was injected intravenously once a day until sacrifice on days 1, 4, or 7 after treatment. The effects of IP-10 were confirmed in cultured tubular epithelial cells. Results: IP-10 and CXCR3 were upregulated in progressive renal fibrosis. Blockade of IP-10/CXCR3 promotes renal fibrosis, as evidenced by increases in interstitial fibrosis and hydroxyproline contents, concomitant decrease in hepatocyte growth factor expression, and converse increase in transforming growth factor-beta 1 in diseased kidneys. IP-10 blockade affected neither macrophage nor T cell infiltration in diseased kidneys. Conclusion: These results suggest that blockade of IP-10 via CXCR3 contributes to renal fibrosis, possibly by upregulation of transforming growth factor-beta 1, concomitant with downregulation of hepatocyte growth factor. Copyright (c) 2007 S. Karger AG, Basel.

DOI： 10.1159/000106505

Histological differences in new-onset IgA nephropathy between children and adults Reviewed

Yohei Ikezumi, Toshiaki Suzuki, Naofumi Imai, Mitsuhiro Ueno, Ichiei Narita, Hiroshi Kawachi, Fujio Shimizu, David J. Nikolic-Paterson, Makoto Uchiyama

NEPHROLOGY DIALYSIS TRANSPLANTATION 21 ( 12 ) 3466 - 3474 2006.12

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Background. It is suggested that IgA nephropathy (IgAN) manifests differently in children vs adults on the basis of biopsy findings. However, this has been difficult to establish owing to the uncertainty of the timing of disease onset in adult IgAN. We addressed this question by comparing both histology and leucocyte accumulation in biopsies of recently diagnosed childhood and adult IgAN.
Methods. Biopsies taken within 2 years from the onset of renal abnormalities in 33 childhood (10 +/- 3 years of age) and 38 adult (35 +/- 6 years) cases of IgAN were examined for histological changes (cellularity in mesangial, endocapillary and extracapillary areas, matrix expansion, adhesions/crescents and interstitial damage), glomerular deposition of immunoglobulin and complement, and the presence of macrophages, activated macrophages and T cells by immunohistochemistry.
Results. Glomerular hypercellularity owing to increased cells in mesangial area was prominent in paediatric IgAN and significantly greater than in adult IgAN. In contrast, glomerular matrix expansion, crescent formation and interstitial damage were more severe in adults compared to paediatric IgAN. Indeed, glomerular hypercellularity correlated with proteinuria in paediatric but not in adult IgAN, whereas glomerular matrix correlated with proteinuria and renal function in adult but not in paediatric IgAN. The degree of C3c deposition was significantly greater in paediatric IgAN, while deposition of fibrinogen was greater in adult IgAN. Glomerular and interstitial CD68+ macrophages and a subset of sialoadhesin (Sn)+ activated macrophages were identified in both paediatric and adult IgAN, being significantly greater in number in adult IgAN. Glomerular leucocyte infiltration correlated with proteinuria while interstitial leucocyte infiltration correlated with interstitial damage in both groups. However, only the subset of Sn+ macrophages gave a significant correlation with renal function, glomerular hypercellularity and glomerular matrix.
Conclusions. This study has demonstrated significant differences in the early glomerular lesions of IgAN in children vs adults. Furthermore, Sn+ activated macrophages are implicated in the pathogenesis of IgAN in both patient groups. The prognostic significance of these findings warrants further study.

DOI： 10.1093/ndt/gfl455

Expression of allograft inflammatory factor-1 in kidneys: A novel molecular component of podocyte Reviewed

Y. Tsubata, M. Sakatsume, A. Ogawa, B. Alchi, Y. Kaneko, T. Kuroda, H. Kawachi, I. Narita, T. Yamamoto, F. Gejyo

KIDNEY INTERNATIONAL 70 ( 11 ) 1948 - 1954 2006.12

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

Our comprehensive gene expression profiles of the kidneys in an anti-glomerular basement membrane (GBM) nephritis model using DNA arrays revealed that allograft inflammatory factor-1 (AIF-1) was one of the highly expressed genes. Here, we explored the pathological significance of AIF-1 expression in the kidneys. The expression pattern of AIF-1 mRNA and protein in the kidneys of normal and diseased rats, such as anti-GBM nephritis and puromycin aminonucleoside nephrosis, was investigated by in situ hybridization, immunohistochemistry, and immunoelectron microscopy. Furthermore, the expression of AIF-1 in human kidneys and urinary sediments was examined. AIF-1 was expressed at both mRNA and protein levels in podocytes of normal and diseased rats, and in infiltrating cells in anti-GBM nephritis kidneys. The expression of AIF-1 in podocytes was constitutive; positive in podocytes of both normal and diseased rats. In humans, AIF-1 was expressed in podocytes and infiltrating inflammatory cells, similarly. Moreover, it was detected in urinary podocytes from patients with immunoglobulin A nephropathy. These data document for the first time that AIF-1, a constitutively expressed protein in rat and human podocytes, is a novel molecular component of podocytes, and that the upregulation of AIF-1 in an anti-GBM nephritis model may mainly be a consequence of its expression in infiltrating cells.

DOI： 10.1038/sj.ki.5001941

蛋白尿発症の分子メカニズム. Invited

河内裕, 鈴木浩一

医学のあゆみ 216 680 - 684 2006.12

Nucleotidase improves glomerular autoregulation in Thy-1 nephritic rats Reviewed

Takenaka Tsuneo, Okada Hirokazu, Kanno Yoshihiko, Inoue Tsutomu, Ryuzaki Munekazu, Nakamoto Hidetomo, Kawachi Hiroshi, Shimizu Fujio, Suzuki Hiromichi

JOURNAL OF HYPERTENSION 24 188 2006.12

Rat nephrin modulates cell morphology via the adaptor protein Nck Reviewed

Hongping Li, Jianxin Zhu, Lamine Aoudjit, Mathieu Latreille, Hiroshi Kawachi, Louise Larose, Tomoko Takano

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 349 ( 1 ) 310 - 316 2006.10

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Nephrin is a transmembrane molecule essential for morphology and function of kidney podocytes. We and others reported previously that the cytoplasmic domain of human and mouse nephrin interacts with the adaptor protein, Nck, in a tyrosine phosphorylation-dependent manner. In the current study, we characterized the interaction of rat nephrin with Nck and further addressed its impact on cell morphology. Rat nephrin expressed in Cos-1 cells co-immunoprecipitated with Nck in a manner dependent on the phosphorylation of Y 1204 and Y 1228. Nephrin from normal rat glomeruli was also tyrosine phosphorylated and associated with Nck. Overexpression of rat nephrin in HEK293T cells induced morphological changes resembling process formation, which became more distinct when the extracellular domain of nephrin was cross-linked by antibodies. The morphological changes were attenuated by expression of dominant negative constructs of Nck. In the rat model of podocyte injury and proteinuria, nephrin tyrosine phosphorylation and nephrin-Nck interaction were both reduced significantly. Taken together, we propose that Nck couples nephrin to the actin cytoskeleton in glomerular podocytes and contributes to the maintenance of normal morphology and function of podocytes. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2006.08.053

Synaptic vesicle protein 2B is expressed in podocyte, and its expression is altered in proteinuric glomeruli Reviewed

Naoko Miyauchi, Akira Saito, Tamaki Karasawa, Yutaka Harita, Koichi Suzuki, Hiroko Koike, Gi Dong Han, Fujio Shimizu, Hiroshi Kawachi

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 17 ( 10 ) 2748 - 2759 2006.10

Language：English Publishing type：Research paper (scientific journal) Publisher：AMERICAN SOCIETY NEPHROLOGY

Synaptic vesicle protein 2B (SV2B) was identified by the subtraction hybridization technique as a molecule of which mRNA expression was decreased in puromycin aminonucleoside (PAN) nephropathy by glonterular cDNA subtraction assay. The expression of SV2B was detected in glomerular lysate with Western blot analysis. Dual-labeling immunofluorescence studies with glomerular cell markers demonstrated that SV2B is expressed in glomerular visceral epithelial cells (podocytes). The expression of SV2B is detected also in cultured podocyte and in human kidney section as podocytic pattern. The decrease of SV2B mRNA was already detected before the onset of proteinuria in PAN nephropathy. The mRNA expression of SV2B clearly is altered not only in PAN nephropathy but also in another proteinuric state that is caused by an antibody against nephrin, a functional molecule of the slit diaphragm. The decreased intensity in SV2B staining was already detected before the peak of proteinuria in both models with immunofluorescence study. A reduced amount of SV2B was detected in both models also with Western blot analysis. CD2AP, another functional molecule of the slit diaphragm, was observed in cytoplasm, including the processes area of the cultured podocyte, and when the podocyte was treated with small interfering RNA for SV2B, CD2AP staining at the process area was not detected. These results suggest that SV2B is a functional molecule of podocyte, and SV2B may play a role in the expression and proper localization of CD2AP.

DOI： 10.1681/ASN.2005121293

Contribution of nonproteolytically activated prorenin in glomeruli to hypertensive renal damage Reviewed

Atsuhiro Ichihara, Yuki Kaneshiro, Tomoko Takemitsu, Mariyo Sakoda, Tsutomu Nakagawa, Akira Nishiyama, Hiroshi Kawachi, Fujio Shimizu, Tadashi Inagami

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 17 ( 9 ) 2495 - 2503 2006.9

Language：English Publishing type：Research paper (scientific journal) Publisher：LIPPINCOTT WILLIAMS & WILKINS

Prorenin is activated without proteolysis by binding of prorenin receptor to the pentameric "handle region" (HR) of prorenin prosegment. It was hypothesized that such activation occurs in the kidneys of hypertensive rats and causes tissue renin-angiotensin system (RAS) activation and end-organ damage. Because the HR's binding to its binding protein made the adjacent tetrameric "gate region" (GR) accessible to its specific antibody, immunohistochemistry of the GR was performed to test the hypothesis. Methods also were devised specifically to inhibit the nonproteolytic activation by the decapeptide corresponding to the HR as a decoy. Immunohistochemistry of the GR demonstrated that the majority of nonproteolytically activated prorenin is present in podocytes of the kidneys from stroke-prone spontaneously hypertensive rats, in which activation of renal tissue RAS, proteinuria, and glomerulosclerosis occurred. Continuous subcutaneous administration of the HR decoy peptide completely inhibited both nonproteolytic activation of tissue prorenin and activation of tissue RAS without affecting circulating RAS or arterial pressure and significantly attenuated the development and progression of proteinuria and glomerulosclerosis. These studies clearly demonstrated that nonproteolytic activation of prorenin in glomeruli is critically involved in renal tissue RAS activation, leading to renal damage in hypertensive animals.

DOI： 10.1681/ASN.2005121278

Role of IP-10/CXCL10 in the progression of pancreatitis-like injury in mice after murine retroviral infection Reviewed

Yusuke Kawauchi, Kenji Suzuki, Shiro Watanabe, Satoshi Yamagiwa, Hiroyuki Yoneyama, Gi Dong Han, Suresh S. Palaniyandi, Punniyakoti T. Veeraveedu, Kenichi Watanabe, Hiroshi Kawachi, Yoshiaki Okada, Fujio Shimizu, Hitoshi Asakura, Yutaka Aoyagi, Shosaku Narumi

AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY 291 ( 2 ) G345 - G354 2006.8

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER PHYSIOLOGICAL SOC

Exocrinopathy and pancreatitis-like injury were developed in C57BL/6 (B6) mice infected with LP-BM5 murine leukemia virus, which is known to induce murine acquired immunodeficiency syndrome ( MAIDS). The role of chemokines, especially CXCL10/interferon (IFN)-gamma-inducible protein 10 (IP-10), a chemokine to attract CXCR3(+) T helper 1-type CD4(+) T cells, has not been investigated thoroughly in the pathogenesis of pancreatitis. B6 mice were inoculated intraperitoneally with LP-BM5 and then injected every week with either an antibody against IP-10 or a control antibody. Eight weeks after infection, we analyzed the effect of IP-10 neutralization. Anti-IP-10 antibody treatment did not change the generalized lymphadenopathy and hepatosplenomegaly of mice with MAIDS. The treatment significantly reduced the number of IP-10- and CXCR3-positive cells in the mesenteric lymph nodes (mLNs) but not the phenotypes and gross numbers of cells. In contrast, IP-10 neutralization reduced the number of mononuclear cells infiltrating into the pancreas. Anti-IP-10 antibody treatment did not change the numbers of IFN-gamma(+) and IL10(+) cells in the mLN but significantly reduced their numbers, especially IFN-gamma(+) and IL-10(+) CD4(+) T cells and IFN-gamma(+) Mac-1(+) cells, in the pancreas. IP-10 neutralization ameliorated the pancreatic lesions of mice with MAIDS probably by blocking the cellular infiltration of CD4(+) T cells and IFN-gamma(+) Mac-1(+) cells into the pancreas at least at 8 wk after infection, suggesting that IP-10 and these cells might play a key role in the development of chronic autoimmune pancreatitis.

DOI： 10.1152/ajpgi.00002.2006

Role of podocyte slit diaphragm as a filtration barrier Invited Reviewed

Hiroshi Kawachi, Naoko Miyauchi, Koichi Suzuki, Gi Dong Han, Michiaki Orikasa, Fujio Shimizu

NEPHROLOGY 11 ( 4 ) 274 - 281 2006.8

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL PUBLISHING

Although the role of glomerular basement membrane has been emphasised as the barrier for retaining plasma proteins in the past three decades, some recent studies have demonstrated that the slit diaphragm of the glomerular epithelial cell (podocyte) is the structure likely to be the barrier in the glomerular capillary wall. Nephrin and podocin were identified as gene products mutated in Finnish-type congenital nephrotic syndrome and autosomal recessive steroid-resistant nephrotic syndrome, respectively. Nephrin s located at the outer leaflet of plasma membranes of the slit diaphragm. Podocin is reported to have an interaction with nephrin. The anti-nephrin antibody is capable of inducing massive proteinuria, which indicates that nephrin is a key functional molecule in the slit diaphragm. The expression of nephrin and podocin was reduced in glomeruli of minimal change nephrotic syndrome, which suggested that the altered expression of these molecules contributes to the development of proteinuria also in acquired diseases. Some recent studies demonstrated that CD2-associated protein (CD2AP) is also a functional molecule in the slit diaphragm, and its expression is altered in membranous nephropathy. These observations suggested that alteration of the molecular arrangement in the slit diaphragm is involved in the development of proteinuria in several kinds of glomerular diseases.

DOI： 10.1111/j.1440-1797.2006.00583.x

Transcription factor Ets-1 is essential for mesangial matrix remodeling Reviewed

M. Mizui, Y. Isaka, Y. Takabatake, Y. Sato, H. Kawachi, F. Shimizu, S. Takahara, T. Ito, E. Imai

KIDNEY INTERNATIONAL 70 ( 2 ) 298 - 305 2006.7

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

Most advanced glomerular diseases are characterized by abnormal extracellular matrix (ECM) accumulation in the glomeruli, and matrix metalloproteinases ( MMPs) play a pivotal role in ECM remodeling in various glomerular diseases. The proto-oncogene, ets-1, is a transcription factor regulating the expression of various matrix proteinases, including MMP-1, MMP-3, and MMP-9. The goal of the present study was to characterize the role of Ets-1 in the progression of glomerular diseases. Overexpression of Ets-1 in cultured mesangial cells prevented transforming growth factor (TGF)-beta-induced inhibition of DNA-binding activity and TGF-beta-induced type I collagen production. In addition, exogenous Ets-1 abolished TGF-beta-induced collagen gel contraction. The in vivo transfection of the ets-1 gene into nephritic kidney resulted in the increases in glomerular MMP-1, MMP-3, and MMP-9 mRNA, decreases in mesangial ECM deposition, and attenuation of fibronectin extradomain A (EDA) and type I collagen expression. In contrast, knockdown of Ets-1 in glomeruli resulted in severe ECM deposition in diseased glomeruli. In conclusion, Ets-1 promotes degradation of ECM proteins and is critical for integral glomerular reorganization.

DOI： 10.1038/sj.ki.5001541

Blockade of VEGF accelerates proteinuria, via decrease in nephrin expression in rat crescentic glomerulonephritis Reviewed

A. Hara, T. Wada, K. Furuichi, N. Sakai, H. Kawachi, F. Shimizu, M. Shibuya, K. Matsushima, H. Yokoyama, K. Egashira, S. Kaneko

Kidney International 69 ( 11 ) 1986 - 1995 2006.6

Language：English Publishing type：Research paper (scientific journal)

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that maintains the glomerular and peritubular capillary (PTC) network in the kidney. The soluble form of the VEGF receptor-1 (soluble fms-like tyrosine kinase 1 (sFlt-1)) is known to regulate VEGF activity by binding VEGF in the circulation. We hypothesized that VEGF may be beneficial for maintaining glomerular filtration barrier and vascular network in rats with progressive glomerulonephritis (GN). For blockade of VEGF activity in vivo, rats were transfected twice with plasmid DNA encoding the murine sFlt-1 gene into femoral muscle 3 days before and 2 weeks after the induction of antiglomerular basement membrane antibody-induced GN. Inhibition of VEGF with sFlt-1 resulted in massive urinary protein excretion, concomitantly with downregulated expression of nephrin in nephritic rats. Further, blockade of VEGF induced mild proteinuria in normal rats. Administration of sFlt-1 affected neither the infiltration of macrophages nor crescentic formation. In contrast, treatment of sFlt-1 accelerated the progression of glomerulosclerosis and interstitial fibrosis accompanied with renal dysfunction and PTC loss at day 56. VEGF may play a role in maintaining the podocyte function as well as renal vasculature, thereby protecting glomeruli and interstitium from progressive renal insults. © 2006 International Society of Nephrology.

DOI： 10.1038/sj.ki.5000439

Attenuation of mouse acute colitis by naked hepatocyte growth factor gene transfer into the liver Reviewed

T Hanawa, K Suzuki, Y Kawauchi, M Takamura, H Yoneyama, GD Han, H Kawachi, F Shimizu, H Asakura, J Miyazaki, H Maruyama, Y Aoyagi

JOURNAL OF GENE MEDICINE 8 ( 5 ) 623 - 635 2006.5

Language：English Publishing type：Research paper (scientific journal) Publisher：JOHN WILEY & SONS LTD

Background Hepatocyte growth factor (HGF) has multiple biological effects on a wide variety of cells. It modulates intestinal epithelial proliferation and migration, and critically regulates intestinal wound healing. Aims To investigate the therapeutic effect of HGF gene transfer, we introduced the HGF gene into the liver of mice with acute colitis. Methods The rat HGF expression plasmid vector, pCAGGS-HGF, was injected via the tail vein into C57BL/6 mice, followed by dosing with dextran sulfate sodium in distilled water. Firstly, the HGF gene was injected once on day 0. Secondly, the HGF gene was injected on day 0 and again on day 2. Results Injection of the HGF gene ameliorated colitis with inhibition of both loss of body weight and shortening of colon length. It protected the colon from epithelial erosions and cellular infiltration. Expression of mRNAs for IFN-gamma, IL18, and TNF-alpha was reduced in the colon. In contrast, expression of mRNA for IL-10 was increased. The numbers of BrdU-positive intestinal epithelial cells were increased, and the numbers of TUNEL-positive apoptotic cells were decreased. Furthermore, a second injection prolonged the elevation of serum HGF levels, and ameliorated the symptoms better than a single injection. The empty pCAGGS plasmid did not ameliorate acute colitis. Conclusions HGF gene transfer attenuated acute colitis by facilitating intestinal wound repair as well as inhibiting inflammation, suggesting a new strategy for treatment of IBD. Copyright (c) 2006 John Wiley & Sons, Ltd.

DOI： 10.1002/jgm.880

Antagonism of PDGF-D by human antibody CR002 prevents renal scarring in experimental glomerulonephritis Reviewed

Tammo Ostendorf, Song Rong, Peter Boor, Stefanie Wiedemann, Uta Kunter, Ulrike Haubold, Claudia R. C. van Roeyen, Frank Eitner, Hiroshi Kawachi, Gary Starling, Enrique Alvarez, Glennda Smithson, Juergen Floege

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 17 ( 4 ) 1054 - 1062 2006.4

Language：English Publishing type：Research paper (scientific journal) Publisher：AMERICAN SOCIETY NEPHROLOGY

Glomerular mesangial cell proliferation and/or matrix accumulation characterizes many progressive renal diseases. PDGF-D was identified recently as a novel mediator of mesangial cell proliferation in vitro and in vivo. This study investigated the long-term consequences of PDGF-D inhibition in vivo. Rats with progressive mesangioproliferative glomerulonephritis (uninephrectomy plus anti-Thy-1.1 antibody) received the PDGF-D-neutralizing, fully human mAb CR002 on days 3, 10, and 17 after disease induction. Glomerular mesangioproliferative changes on day 10 were significantly reduced by anti-PDGF-D treatment as compared with control antibody. Eight weeks after disease induction, anti-PDGF-D therapy significantly ameliorated focal segmental glomerulosclerosis, podocyte damage (de novo desmin expression), tubulointerstitial damage, and fibrosis as well as the accumulation of renal interstitial matrix including type III collagen and fibronectin. Treatment with anti-PDGF-D also reduced the cortical infiltration of monocytes/macrophages on day 56, possibly related to lower renal cortical complement activation (C5b-9 deposition) and/or reduced epithelial-to-mesenchymal transition (preserved cortical expression of E-cadherin and reduced expression of vimentin and a-smooth muscle actin). In conclusion, these data provide evidence for a causal role of PDGF-D in the pathogenesis of renal scarring and point to a new therapeutic approach to progressive mesangioproliferative renal disease.

DOI： 10.1681/ASN.2005070683

Exogenous 5′-nucleotidase improves glomerular autoregulation in Thy-1 nephritic rats Reviewed

Tsuneo Takenaka, Hirokazu Okada, Yoshihiko Kanno, Tsutomu Inoue, Munekazu Ryuzaki, Hidetomo Nakamoto, Hiroshi Kawachi, Fujio Shimizu, Hiromichi Suzuki

American Journal of Physiology - Renal Physiology 290 ( 4 ) F844 - F853 2006.4

Language：English Publishing type：Research paper (scientific journal)

Experiments were performed to characterize renal hemodynamics in Thy-1 nephritic rats. A monoclonal antibody against Thy-1 was intravenously injected to induce mesangiolysis in rats, and 2 days later renal hemodynamic responses to variations in blood pressure were determined. In the first series of experiments, autoregulation of renal plasma flow (RPF) or glomerular filtration rate (GFR) was impaired in nephritic rats. In response to a reduction in blood pressure (98 ± 2 to 80 ± 1 mmHg), both RPF (4.17 ± 0.63 to 3.20 ± 0.45 ml·min-1·g kidney wt-1, P &lt
0.05, n = 6) and GFR (0.88 ± 0.05 to 0.75 ± 0.06 ml·min-1·g kidney wt-1, P &lt
0.05) were decreased in nephritic rats. Intravenous administration of furosemide and 30% albumin, both of which inhibit tubuloglomerular feedback, diminished renal autoregulation in control but not nephritic rats. In the second studies, the infusion of 5′-nucleotidase, an enzyme expressed on mesangial cells, into a renal artery ameliorated the magnitude of autoregulatory decrements in GFR in nephritic rats (-16 ± 5 to -6 ± 2%, P &lt
0.05, n = 6), but this enzyme failed to alter renal autoregulation in control rats. In the third studies, the effects of indomethacin were examined in nephritic rats. Inhibition of prostaglandin synthesis reduced RPF (4.07 ± 0.30 to 1.54 ± 0.22 ml·min-1·g kidney wt-1, P &lt
0.05, n = 5) and GFR (1.03 ± 0.18 to 0.69 ± 0.13 ml·min-1·g kidney wt-1, P &lt
0.05) in nephritic rats. However, cyclooxygenase inhibition failed to restore renal autoregulation in nephritic rats. Our results indicate that renal autoregulation is impaired in Thy-1 nephritis. Furthermore, the present data provide evidence that prostanoids contribute to maintain renal circulation in nephritic rats. Finally, our findings suggest that mesangial cells and/or 5′-nucleotidase plays an important role in mediating renal autoregulation. Copyright © 2006 the American Physiological Society.

DOI： 10.1152/ajprenal.00112.2005

Microarray analysis of a reversible model and an irreversible model of anti-Thy-1 nephritis Reviewed

M Tsuji, T Monkawa, J Yoshino, M Asai, S Fukuda, H Kawachi, F Shimizu, M Hayashi, T Saruta

KIDNEY INTERNATIONAL 69 ( 6 ) 996 - 1004 2006.3

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

A single intravenous injection of anti-Thy-1 monoclonal antibody (mAb) 1-22-3 is known to cause reversible mesangial proliferative glomerulonephritis. However, mAb 1-22-3 injection followed by unilateral nephrectomy leads to progressive glomerulosclerosis and tubulointerstitial change with an irreversible course. To identify genes that play an important role in the irreversible progression of renal injury, we used microarray technology to identify differences in gene expression between these models. Rats were intravenously injected with mAb 1-22-3 1 week after unilateral nephrectomy (irreversible model) or a sham operation (reversible model), and rats were killed on days 4, 7, 14, 42, and 56 after the injection. complementary DNA probes prepared from kidney messenger RNAs were hybridized with oligonucleotide microarrays containing 4854 rat genes. The microarray identified 189 differentially expressed genes, having at least a two-fold difference in expression level between the two models, and they were classified into five clusters. One of the clusters consisted of genes whose expression was markedly upregulated in the irreversible model. This cluster included the genes encoding osteopontin, kidney injury molecule-1, and thymosin beta 10. Increased expression of thymosin beta 10 was localized mainly in macrophages in the fibrotic interstitium, and upregulation of thymosin beta 10 expression was also observed in a unilateral ureteral obstruction model. The microarray analysis yielded information on the molecular mechanisms responsible for the difference in disease progression between the reversible and irreversible model of anti-Thy-1 nephritis. Thymosin beta 10 may play an important role in the progression of kidney disease.

DOI： 10.1038/sj.ki.5000191

Up-regulation of integrin-linked kinase activity in rat mesangioproliferative glomerulonephritis Reviewed

S Kagami, M Shimizu, S Kondo, A Kitamura, M Urushihara, M Takamatsu, S Yamaji, Y Ishigatsubo, H Kawachi, F Shimizu

LIFE SCIENCES 78 ( 16 ) 1794 - 1800 2006.3

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

This study investigated whether integrin-linked kinase (ILK) is involved in the pathogenesis of chronic glomerulonephritis (GN) by analyzing the expression and activity of glomerular ILK in a chronic rat model of mesangioproliferative GN. Double immunostaining of kidneys obtained at different time points with glomerular cell-specific markers revealed that ILK was primarily expressed by glomerular epithelial cells, and weakly by mesangial cells (MCs) and endothelial cells in control rats, but dramatically increased in a typical mesangial pattern at days 21 and 28 of GN. Semiquantitative assessment indicated that the level of glomerular ILK expression closely parallels the level of accumulation of glomerular extracellular matrix (ECM) as well as fibronectin (FN). Immunoprecipitation and kinase activity assays using isolated nephritic glomeruli indicated a striking increase of ILK activity on days 21 and 28 of GN. Further, cultured rat MCs overexpressing kinase-deficient ILK diminished FN assembly and collagen matrix remodeling as compared with control transfectants. The results showed that glomerular ILK expression and activity are markedly increased in an experimental model of chronic GN. Increased activity of ILK in MCs may contribute to the development of chronic mesangial alterations leading to glomerular sear-ring. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.lfs.2005.08.037

Addition of the antioxidant probucol to angiotensin II type I receptor antagonist arrests progressive mesangioproliferative glomerulonephritis in the rat Reviewed

Shuji Kondo, Maki Shimizu, Maki Urushihara, Koichiro Tsuchiya, Masanori Yoshizumi, Toshiaki Tamaki, Akira Nishiyama, Hiroshi Kawachi, Fujio Shimizu, Mark T. Quinn, David J. Lambeth, Shoji Kagami

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 17 ( 3 ) 783 - 794 2006.3

Language：English Publishing type：Research paper (scientific journal) Publisher：AMERICAN SOCIETY NEPHROLOGY

Angiotensin II (Ang II) and reactive oxidative species (ROS) that are produced by NADPH oxidase have been implicated in the progression of glomerulonephritis (GN). This study examined the effect of simultaneously interrupting Ang 11 and ROS with an Ang II receptor blocker (ARB), candesartan, and a free radical scavenger, probucol, in a model of progressive mesangioproliferative GN induced by the injection of anti-Thy-1 antibody into uninephrectomized rats. Nephritic rats were divided into four groups and given daily oral doses of the following: Vehicle, 1% probucol diet, 70 mg/L candesartan in drinking water, and probucol plus candesartan. These treatments lasted until day 56. Vehicle-treated nephritic rats developed progressively elevated proteinuria and glomerulosclerosis. Candesartan kept proteinuria significantly lower than vehicle or probucol. The addition of probucol to candesartan normalized urinary protein excretion. Increases in BP in nephritic rats were lowered by these treatments, except with probucol. It is interesting that both glomerular cell number and glomerulosclerosis were significantly decreased by candesartan and normalized by the addition of probucol. Immunohistochemical studies for TGF-beta 1, collagen type I, and fibronectin revealed that the combined treatment abolished glomerular fibrotic findings compared with candesartan. In addition, glomerular expression of NADPH oxidase components and superoxide production suggested that the combined treatment completely eliminated NADPH oxidase-associated ROS production. In conclusion, our study provides the first evidence that the antioxidant probucol, when added to an Ang II receptor blockade, fully arrests proteinuria and disease progression in GN. Furthermore, the data suggest that NADPH oxidase-associated ROS production may play a pivotal role in the progression of GN. The combination of probucol and candesartan may represent a novel route of therapy for patients with progressive GN.

DOI： 10.1681/ASN.2005050519

IFN-inducible protein-10 plays a pivotal role in maintaining slit-diaphragm function by regulating podocyte cell-cycle balance Reviewed

Gi Dong Han, Koichi Suzuki, Hiroko Koike, Kenji Suzuki, Hiroyuki Yoneyama, Shosaku Narumi, Fujio Shimizu, Hiroshi Kawachi

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 17 ( 2 ) 442 - 453 2006.2

Language：English Publishing type：Research paper (scientific journal) Publisher：AMERICAN SOCIETY NEPHROLOGY

IFN-inducible protein-10 (IP-10/CXCL10) is a potent chemoattractant for activated T lymphocytes and was reported recently to have several additional biologic activities. In this study, the pathophysiologic role of IP-10 in the glomerular visceral epithelial cell (podocyte) was investigated. In cultured podocytes subjected to recombinant IP-10 treatment, the expression of slit-diaphragm (SD) components nephrin and podocin clearly was heightened. Rats that had puromycin aminonucleoside nephropathy and anti-nephrin antibody-induced nephropathy and were subjected to anti-IP-10 function-blocking antibody (anti-IP-10 mAb) treatment displayed a decrease in the protein level of SD components, as well as exacerbated proteinuria. For exploration of the mechanisms of this process, the interaction between IP-10 and the cell-cycle regulatory proteins was investigated. Cultured podocytes subjected to recombinant IP-10 treatment displayed an increase in the protein level of P-27Kip1, whereas the levels of cyclins E and A decreased. The expression of IP-10 and SD components was heightened by the treatment of siRNA of cyclin A, whereas these expressions were lowered by the treatment of siRNA of p(27Kip1). Proteinuric rats subjected to anti-IP-10 mAb treatment displayed a heightened expression of cyclin A from the early phase of the disease, which indicates that the anti-IP-10 mAb treatment exacerbates podocyte injury by disturbing the cell-cycle balance. These results raise the possibility that IP-10 could become a novel therapeutic target in nephrotic syndrome and several diseases with altered cell-cycle balance.

DOI： 10.1681/ASN.2004090755

Altered expression of junctional adhesion molecule 4 in injured podocytes Reviewed

Yutaka Harita, Naoko Miyauchi, Tamaki Karasawa, Koichi Suzuki, Dong Han Gi, Hiroko Koike, Takashi Igarashi, Fujio Shimizu, Hiroshi Kawachi

American Journal of Physiology - Renal Physiology 290 ( 2 ) F335 - F344 2006.2

Language：English Publishing type：Research paper (scientific journal)

Recent investigations have revealed the importance of glomerular podocytes with its diaphragm as the major filtration barrier. Junctional adhesion molecule 4 (JAM4) has been identified as a protein that interacts with membrane-associated guanyl kinase inverted (MAGI)-1 and is reported to be expressed on podocytes. To elucidate the role of JAM4 on podocytes, we examined the expression of JAM4 and MAGI-1 in normal and two different proteinuric rat models: puromycin aminonucleoside (PAN) nephropathy and anti-nephrin antibody-induced (ANA) nephropathy, one model with and one without effacement of podocyte foot processes. JAM4 was detected by immunomicroscopy at the apical membrane of normal podocytes. JAM4 immunostaining was focally increased in the podocytes in PAN nephropathy but not in ANA nephropathy. In proteinuric podocytes, the expression of JAM4 was distinct from that of MAGI-1 or other slit diaphragm molecules such as nephrin and ZO-1. Close colocalization of JAM4 and ezrin was maintained in PAN nephropathy. By immunoelectron microscopy, the signals for JAM4 were detected at the free apical membrane of the podocytes with effaced foot processes. Studies with selective detergent extract revealed that the subcellular localization of JAM4 was altered in PAN nephropathy. Thus the altered expression of JAM4 appears to be associated with morphological changes in podocytes and can be a useful marker of injured podocytes. JAM4 may have a different role at the apical membrane besides the role as a junctional molecule and is likely associated with the unique structure of this epithelium. Copyright © 2006 the American Physiological Society.

DOI： 10.1152/ajprenal.00253.2005

Pathophysiologic implications of reduced podocyte number in a rat model of progressive glomerular injury Reviewed

D Macconi, M Bonomelli, A Benigni, T Plati, F Sangalli, L Longaretti, S Conti, H Kawachi, P Hill, G Remuzzi, A Remuzzi

AMERICAN JOURNAL OF PATHOLOGY 168 ( 1 ) 42 - 54 2006.1

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC INVESTIGATIVE PATHOLOGY, INC

Changes in podocyte number or density have been suggested to play an important role in renal disease progression. Here, we investigated the temporal relationship between glomerular podocyte number and development of proteinuria and glomerulosclerosis in the male Munich Wistar Fromter (MWF) rat. We also assessed whether changes in podocyte number affect podocyte function and focused specifically on the slit diaphragm-associated protein nephrin. Age-matched Wistar rats were used as controls. Estimation of podocyte number per glomerulus was determined by digital morphometry of WT1-positive cells. MWF rats developed moderate hypertension, massive proteinuria, and glomerulosclerosis with age. Glomerular hypertrophy was already observed at 10 weeks of age and progressively increased thereafter. By contrast, mean podocyte number per glomerulus was lower than normal in young animals and further decreased with time. As a consequence, the capillary tuft volume per podocyte was more than threefold increased in older rats. Electron microscopy showed important changes in podocyte structure of MWF rats, with expansion of podocyte bodies surrounding glomerular filtration membrane. Glomerular nephrin expression was markedly altered in MWF rats and inversely correlated with both podocyte loss and proteinuria. Our findings suggest that reduction in podocyte number is an important determinant of podocyte dysfunction and progressive impairment of the glomerular permselectivity that lead to the development of massive proteinuria and ultimately to renal scarring.

DOI： 10.2353/ajpath.2006.050398

Enhancing cGMP in experimental progressive renal fibrosis: Soluble guanylate cyclase stimulation vs. phosphodiesterase inhibition Reviewed

Yingrui Wang, Stephanie Krämer, Tanja Loof, Sebastian Martini, Susanne Kron, Hiroshi Kawachi, Fuijo Shimizu, Hans-H. Neumayer, Harm Peters

American Journal of Physiology - Renal Physiology 290 ( 1 ) F167 - F176 2006.1

Language：English Publishing type：Research paper (scientific journal)

cGMP serves as the main second messenger of nitric oxide (NO). Antifibrotic effects of enhancing renal cGMP levels have recently been documented in experimental acute anti-Thy-1 glomerulonephritis. The present study compares the effects of the cGMP production-increasing soluble guanylate cyclase (sGC) stimulator BAY 41-2272 with those of the cGMP degradation-limiting phosphodiesterase inhibitor pentoxifylline (PTX) in a progressive model of renal fibrosis. At 1 wk after induction of anti-Thy-1-induced chronic glomerulosclerosis (cGS), rats were randomly assigned to groups as follows: cGS, cGS + BAY 41-2272 (10 mg·kg body wt-1·day -1), or cGS + PTX (50 mg·kg body wt -1·day-1). BAY 41-2272 and PTX reduced systolic blood pressure significantly. At 16 wk, tubulointerstitial expressions of sGC mRNA and NO-induced cGMP synthesis were increased in untreated cGS animals, whereas their glomerular activity was depressed compared with normal controls. Tubulointerstitial and glomerular cGMP production in response to NO were significantly enhanced in animals treated with BAY 41-2272, but not in those treated with PTX. BAY 41-2272 administration resulted in marked reductions of glomerular and tubulointerstitial histological matrix accumulation, expression of TGF-β1 and fibronectin, macrophage infiltration, and cell proliferation as well as improved renal function. In contrast, only moderate and nonsignificant renoprotective changes were observed in the cGS + PTX group. In conclusion, increasing renal cGMP production through BAY 41-2272 significantly improved renal NO-cGMP signaling and limited progression in anti-Thy-1-induced chronic renal fibrosis, whereas inhibition of cGMP degradation by PTX was only moderately effective. The findings indicate that pharmacological enhancement of renal cGMP levels by sGC stimulation represents a novel and effective antifibrotic approach in progressive kidney disorders. Copyright © 2006 the American Physiological Society.

DOI： 10.1152/ajprenal.00197.2005

The sialoadhesin (CD169) expressing a macrophage subset in human proliferative glomerulonephritis Reviewed

Y Ikezumi, T Suzuki, S Hayafuji, S Okubo, DJ Nikolic-Paterson, H Kawachi, F Shimizu, M Uchiyama

NEPHROLOGY DIALYSIS TRANSPLANTATION 20 ( 12 ) 2704 - 2713 2005.12

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Background. Sialoadhesin ( Sn; CD169) is a lectin-like receptor whose expression is restricted to subsets of tissue and inflammatory macrophages. We have previously identified accumulation of Sn+ macrophages as an important marker of disease progression versus remission in rat mesangial proliferative nephritis. The current study examined the significance of Sn+ macrophages in human proliferative glomerulonephritis.
Methods. Frozen kidney sections from normal adult human kidney ( n = 4) and pediatric nephropathy ( n 40) were stained for total macrophages ( CD68+ cells), Sn+ macrophages, CD3+ T-cells and collagen type I by immunofluorescence. Leukocyte infiltration and the severity of glomerular lesions and interstitial damage were scored. A second protocol biopsy was performed in 27 cases and clinical and biopsy-based data obtained.
Results. Sn+ macrophages were absent from glomeruli in normal adult human kidney and in thin basement membrane disease ( n = 4), but were detected in 4 of 9 cases of purpura nephritis; 7 of 17 IgA nephropathy; 5 of 5 membranoproliferative glomerulonephritis, and 5 of 5 lupus nephritis. Sn+ macrophages were localized in areas of focal glomerular and interstitial damage. Two-colour immunostaining confirmed that Sn+ cells are a subset of total CD68+ macrophages. The number of glomerular Sn+ macrophages correlated with the degree of proteinuria and glomerular lesions ( r = 0.44, P = 0.0045 and r = 0.82, P < 0.0001; respectively), while interstitial Sn+ macrophages correlated with the degree of proteinuria and interstitial damage ( r = 0.59, P < 0.0001 and r = 0.75, P < 0.0001; respectively). Combined immunostaining revealed that interstitial Sn+ macrophages and CD3+ T-cells co-localized in areas of tubulointerstitial damage with increased type I collagen deposition. There was significant correlation between the number of interstitial Sn+ macrophages and CD3+ T-cells ( r = 0.74, P < 0.0001). Most patients responded to a 2 year period of glucocorticoid therapy with a reduction in proteinuria and glomerular lesions and this correlated with the reduction in the number of glomerular Sn+ macrophages.
Conclusion. This study has identified Sn+ cells as a macrophage subset whose accumulation in the kidney correlates with proteinuria and histologic damage. These results, together with recent findings from animal studies, suggest that Sn+ macrophages may play an important role in progressive renal disease.

DOI： 10.1093/ndt/gfi105

注目される腎関連蛋白5 catenin. Invited

河内裕, 張田豊

腎と透析 58 394 - 396 2005.12

注目される腎関連蛋白6 ZO-1. Invited

河内裕, 唐澤環

腎と透析 58 398 - 399 2005.12

注目される腎関連蛋白2 nephrin. Invited

河内裕, 鈴木浩一

腎と透析 58 118 - 119 2005.12

注目される腎関連蛋白3 αactinin-4 . Invited

河内裕, 宮内直子, 韓基東

腎と透析 58 250 - 254 2005.12

注目される腎関連蛋白13 B7-1 protein(CD80). Invited

河内裕, 韓基東

腎と透析 59 166 - 167 2005.12

腎疾患の展望 2.蛋白尿発症の分子生物学. Invited

河内裕

BIO Clinica 20 11 958 - 964 2005.12

MAGI-1 is a component of the glomerular slit diaphragm that is tightly associated with nephrin Reviewed

S Hirabayashi, H Mori, A Kansaku, H Kurihara, T Sakai, F Shimizu, H Kawachi, Y Hata

LABORATORY INVESTIGATION 85 ( 12 ) 1528 - 1543 2005.12

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

MAGUK with inverted domain structure-1 (MAGI-1) is a membrane-associated protein with one guanylate kinase, six PSD-95/Dlg-A/ZO-1 (PDZ), and two WW domains and is localized at tight junctions in epithelial cells. MAGI-1 interacts with various proteins and is proposed to function as a scaffold protein. In the previous study, we discovered a MAGI-1-interacting cell adhesion molecule junctional adhesion molecule 4 (JAM4). Both proteins are highly expressed in glomerular podocytes in the kidney and partially colocalized. In this study, we have further searched for a binding partner of MAGI-1 in the kidney through yeast two-hybrid screening and obtained nephrin. Nephrin is a cell adhesion molecule specifically localized at the slit diaphragm between neighboring foot processes of podocytes. Biochemical studies reveal that nephrin directly binds to the middle PDZ domains of MAGI-1 through its carboxyl terminus but does not bind to ZO-1. MAGI-1 forms a tripartite complex with nephrin and JAM4 in vitro. Immunoelectron microscopy shows that the localization of MAGI-1 is restricted to the slit diaphragm, whereas JAM4 is also distributed on apical membranes of podocytes. In puromycin aminonucleoside-induced nephrotic podocytes, MAGI-1 is localized with nephrin at the displaced slit diaphragm. These data indicate that MAGI-1 is a component of the slit diaphragm and tightly interacts with nephrin and JAM4 in vivo. MAGI-1 may play a role in determining the boundary between the apical and the bosolateral domain at the level of slit diaphragm.

DOI： 10.1038/labinvest.3700347

Monoclonal antibody 1-22-3-induced glomerulonephritis in uninephrectomized rats as a model of progressive renal failure Reviewed

Masayuki Tomita, Hajime Sogabe, Shoko Nakazato, Shunji Nakatsuji, Takahisa Noto, Kaori Hamada, Hiroshi Kawachi, Fujio Shimizu, Masahiko Matsuo, Seitaro Mutoh

Nephrology Dialysis Transplantation 20 ( 11 ) 2358 - 2367 2005.11

Language：English Publishing type：Research paper (scientific journal)

Background. At present, there are few available animal models of progressive renal failure originating from mesangial proliferative glomerulonephritis (GN). In the current study, we examined the usefulness of anti-Thy-1 monoclonal antibody (mAb) 1-22-3-induced GN in uninephrectomized rats as a model of progressive renal failure by analysing the similarities to human disease. Methods. GN was induced by intravenous injection of mAb 1-22-3 into uninephrectomized male Wistar rats. The natural course of the disease was analysed in this model for 47 weeks. The effect of treatment with the angiotensin-converting enzyme inhibitor, captopril, on renal functional outcome was also examined in this model for 23 weeks, beginning from 1 week after antibody injection. Results. Injection of mAb 1-22-3 induced a persistent proteinuria during the entire study period. Animals showed a progressive decline in renal function and 63% died by week 47. Severe glomerular and tubulointerstitial lesions were consistently observed. Treatment with captopril significantly inhibited increases in proteinuria and blood pressure, and attenuated renal injury. Captopril also retarded the progression of renal failure, and decreased mortality. Finally, the level of proteinuria was significantly correlated with the rate of decline in renal function, and the reduction in proteinuria by captopril was accompanied by a slower progression of renal failure. Conclusions. The mAb 1-22-3-induced GN in a uninephrectomized rat model simulates the clinical manifestations of human disease, indicating that this model may be useful for studying progressive renal failure and for investigating new therapeutic strategies against renal failure. © The Author [2005]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

DOI： 10.1093/ndt/gfi062

Continuous, noninvasive monitoring of local microscopic inflammation using a genetically engineered cell-based biosensor Reviewed

YM Meng, A Kasai, N Hiramatsu, K Hayakawa, K Yamauchi, M Takeda, H Kawachi, F Shimizu, J Yao, M Kitamura

LABORATORY INVESTIGATION 85 ( 11 ) 1429 - 1439 2005.11

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

Using an inflammation-responsive regulatory element as a molecular sensor, we established a cell-based biosensor for continuous, noninvasive monitoring of local microscopic inflammation in vivo. Glomerular mesangial cells were stably transfected with a marker gene encoding secreted alkaline phosphatase (SEAP) under the control of the kappa B enhancer elements. The established cells secreted SEAP in vitro in response to proinflammatory cytokines as well as to soluble factors produced by inflamed glomeruli. To examine feasibility of using the established cells for in vivo monitoring of local microscopic inflammation, the sensor cells were transferred selectively into rat glomeruli via the renal circulation. After induction of acute glomerulonephritis, the serum level of SEAP was increased transiently in cell-transferred nephritic rats. The kinetics of serum SEAP was closely correlated with the natural course of the inflammation, and the increase in SEAP was attenuated by suppression of inflammation using an immunosuppressive drug, cyclophosphamide. Neither cell-transferred normal rats nor nephritic rats without cell transfer exhibited increase in the serum level of SEAP. When the sensor cells were transferred extrarenally, elevation of serum SEAP was not observed in nephritic rats, confirming that the locally settled sensor cells responded only to local inflammation. These results suggested that, without invasive procedures like tissue biopsies, continuous monitoring of microscopic inflammation is feasible in vivo via locally created, cell-based biosensors.

DOI： 10.1038/labinvest.3700341

Role of p38 mitogen-activated protein kinase activation in podocyte injury and proteinuria in experimental nephrotic syndrome Reviewed

M Koshikawa, M Mukoyama, K Mori, T Suganami, K Sawai, T Yoshioka, T Nagae, H Yokoi, H Kawachi, F Shimizu, A Sugawara, K Nakao

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 16 ( 9 ) 2690 - 2701 2005.9

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC NEPHROLOGY

Podocytes play an important role in maintaining normal glomerular function and structure, and podocyte injury leads to proteinuria and glomerulosclerosis. The family of mitogen-activated protein kinases (MAPK; extracellular signal-regulated kinase [ERK], c-Jun N-terminal kinase, and p38) may be implicated in the progression of various glomerulopathies, but the role of MAPK in podocyte injury remains elusive. This study examined phosphorylation of p38 MAPK in clinical glomerulopathies with podocyte injury, as well as in rat puromycin aminonucleoside (PAN) nephropathy and mouse adriamycin (ADR) nephropathy. The effect of treatment with FR167653, an inhibitor of p38 MAPK, was also investigated in rodent models. In human podocyte injury diseases, the increased phosphorylation of p38 MAPK was observed at podocytes. In PAN and ADR nephropathy, the phosphorylation of p38 MAPK and ERK was marked but transient, preceding overt proteinuria. Pretreatment with FR167653 (day -2 to day 14, subcutaneously) to PAN or ADR nephropathy completely inhibited p38 MAPK activation and attenuated ERK phosphorylation, with complete suppression of proteinuria. Electron microscopy and immunohistochemistry for nephrin and connexin43 revealed that podocyte injury was markedly ameliorated by FR167653. Furthermore, early treatment with FR167653 effectively prevented glomerulosclerosis and renal dysfunction in the chronic phase of ADR nephropathy. In cultured podocytes, PAN or oxidative stress induced the phosphorylation of p38 MAPK along with actin reorganization, and FR167653 inhibited such changes. These findings indicate that the activation of MAPK is necessary for podocyte injury, suggesting that p38 MAPK and, possibly, ERK should become a potential target for therapeutic intervention in proteinuric glomerulopathies.

DOI： 10.1681/ASN.2004121084

Infusion of angiotensin II reduces loss of glomerular capillary area in the early phase of anti-Thy-1.1 nephritis possibly via regulating angiogenesis-associated factors Reviewed

Yuki Takazawa, Yohei Maeshima, Hiroyuki Kitayama, Yoshihiko Yamamoto, Hiroshi Kawachi, Fujio Shimizu, Hideki Matsui, Hitoshi Sugiyama, Yasushi Yamasaki, Hirofumi Makino

Kidney International 68 ( 2 ) 704 - 722 2005.8

Language：English Publishing type：Research paper (scientific journal)

Background. Although angiotensin II (Ang II) is involved in the progression of renal diseases, infusion of Ang II was reported to surprisingly ameliorate the early phase of anti-Thy-1.1 nephritis (Wenzel et al, Kidney Int 61:1020, 2002). Considering the known proangiogenic effect of Ang II and that angiogenic glomerular capillary repair is required for the recovery of damaged glomeruli in rat anti-Thy-1.1 nephritis, we hypothesized that Ang II infusion starting prior to the initiation of nephritis may induce the expression of angiogenic growth factors such as vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1), resulting in the increased glomerular capillary area in the early phase. Methods. Ang II was infused (170 ng/min) in rats, and 5 days later, nephritis was induced by the administration of monoclonal 1-22-3 antibodies. Ang II type 1 or type 2 receptor antagonist (AT1R or AT2R, respectively) (losartan or PD123319, respectively) was coadministered. Results. Ang II infusion affected on neither the deposition of Ig nor mesangiolysis in the initial phase, and resulted in the aggravation of creatinine clearance at day 14 and 35 after initiating anti-Thy-1.1 nephritis. Histologic alterations were ameliorated accompanied by reduced loss in rat endothelial cell antigen (RECA)-1(+) endothelial area in Ang II-infused nephritic rats on day 6 and 14 as compared to control nephritic group, and nephritic alterations were mostly resolved on day 35 in both groups. At the early stage (day 6), glomerular expression of VEGF and receptors flk-1 and flt-1 as well as Ang-1, and receptor Tie2 were increased, and glomerular monocyte infiltration and the expression of angiopoietin-2 (Ang-2), a natural antagonist of Ang-1, were reduced. Both Ang II receptors were involved in the regulation of angiogenic factors and receptors. Conclusion. These results demonstrate that infusion of exogenous Ang II starting prior to the induction of nephritis activates VEGF and Ang-1 signaling regulated via both Ang II receptors, potentially leading to the accelerated recovery of injured glomerular endothelial cells in the early phase of anti-Thy-1.1 nephritis. Increased expression of VEGF and Ang-1 on podocytes further suggests the crucial association of endothelial cells and podocytes in maintaining proper glomerular capillary structures. © 2005 by the International Society of Nephrology.

DOI： 10.1111/j.1523-1755.2005.00449.x

Mycophenolate mofetil slows progression in anti-thy1-induced chronic renal fibrosis but is not additive to a high dose of enalapril Reviewed

Stephanie Krämer, Tanja Loof, Sebastian Martini, Matthias Rückert, Yingrui Wang, Torsten Böhler, Fuijo Shimizu, Hiroshi Kawachi, Hans-H. Neumayer, Harm Peters

American Journal of Physiology - Renal Physiology 289 ( 2 ) F359 - F368 2005.8

Language：English Publishing type：Research paper (scientific journal)

Tubulointerstitial inflammation and fibrosis are hallmarks of chronic progressive renal diseases. To characterize the functional interaction between cell infiltration and matrix expansion, this study compared the immunosuppressant mycophenolate mofetil (MMF), intended as primarily anti-inflammatory intervention, the angiotensin-converting enzyme inhibitor enalapril, intended as primarily an anti-fibrotic drug, and a combination of both as anticipated anti-inflammatory/anti-fibrotic intervention. The model used was anti-thy1-induced chronic-progressive glomerulosclerosis (cGS) in the rat, where a brief anti-thy1-induced glomerular injury progresses spontaneously toward tubulointerstitial fibrosis and renal insufficiency. cGS was induced by injection of anti-thy1 antibody into uninephrectomized Wistar rats. One week after disease induction, animals were randomly assigned to the following groups: cGS, cGS plus MMF (20 mg·kg body wt-1·day -1), cGS plus high-dose enalapril (12 mg·kg body wt -1·day-1), and cGS plus both. At week 16 after disease induction, MMF or enalapril alone reduced signs of chronic renal disease significantly and similarly compared with the untreated cGS group. Variables measured included proteinuria, blood pressure, tubulointerstitial and glomerular matrix accumulation, expression of transforming growth factor- β1, fibronectin, and plasminogen activator inhibitor-1, infiltration of lymphocytes and macrophages, plasma creatinine and urea levels, and glomerular filtration rate. Combined MMF and enalapril treatment was not superior to single therapy. In conclusion, MMF slows the progression of chronic renal fibrosis and renal insufficiency as effectively as high-dose enalapril in the anti-thy1-induced chronic-progressive glomerulosclerosis model. The dual anti-inflammatory/anti-fibrotic intervention does not yield additive renoprotective effects, indicating that MMF and enalapril interfere with similar or very closely related pathways involved in progression of renal disease. Copyright © 2005 the American Physiological Society.

DOI： 10.1152/ajprenal.00442.2004

Real-time monitoring of mesangial cell-macrophage cross-talk using SEAP in vitro and ex vivo Reviewed

Yiman Meng, Ayumi Kasai, Nobuhiko Hiramatsu, Kunihiro Hayakawa, Masayuki Takeda, Fujio Shimizu, Hiroshi Kawachi, Jian Yao, Masanori Kitamura

Kidney International 68 ( 2 ) 886 - 893 2005.8

Language：English Publishing type：Research paper (scientific journal)

Background. Macrophage-mesangial cell interaction plays a crucial role in the pathogenesis of glomerulonephritis. We established a novel system for continuous, real-time monitoring of cross-talk between macrophages and mesangial cells in vitro and ex vivo. Methods. Rat mesangial cells were genetically engineered to produce secreted alkaline phosphatase (SEAP) under the control of the nuclear factor-κB (NF-κB) enhancer elements. The established sensor cells were exposed to macrophages or macrophage-derived factors, and the level of SEAP production was evaluated. Results. In vitro, the established cells expressed and secreted SEAP when exposed to activated macrophages or to cytokines produced by macrophages. The kinetics of SEAP activity in culture media was closely correlated with the expression level of SEAP mRNA. The sensor cells also secreted SEAP in response to media conditioned by macrophage-accumulating, inflamed rat glomeruli. When the sensor cells were transferred adoptively into rat glomeruli subjected to acute anti-Thy 1 glomerulonephritis, the isolated glomeruli containing sensor cells secreted SEAP rapidly and progressively. Conclusion. These data suggested that the established system provides simple and useful tools for monitoring of cross-talk between macrophages and mesangial cells in vitro and ex vivo. This approach would be useful for investigation of molecular mechanisms involved in mesangial cell-macrophage interaction and also for screening of therapeutic agents that efficiently interfere with the link between infiltrating leukocytes and resident glomerular cells. © 2005 by the International Society of Nephrology.

DOI： 10.1111/j.1523-1755.2005.00471.x

Stimulation of soluble guanylate cyclase slows progression in anti-thy1-induced chronic glomerulosclerosis Reviewed

YR Wang, S Kramer, T Loof, S Martini, S Kron, H Kawachi, F Shimizu, HH Neumayer, H Peters

KIDNEY INTERNATIONAL 68 ( 1 ) 47 - 61 2005.7

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL PUBLISHING INC

Background. A critical role of soluble guanylate cyclase and nitric oxide-dependent cyclic 3',5'-guanosine monophosphate (cGMP) production for glomerular matrix expansion has recently been documented in a rat model of acute anti-thy1 glomerulonephritis. The present study analyzes the renal activity of the nitric oxide-cGMP signaling cascade in and the effect of the specific soluble guanylate cyclase stimulator Bay 41-2272 on a progressive model of anti-thy1-induced chronic glomerulosclerosis.
Methods. Anti-thy1 glomerulosclerosis was induced by injection of anti-thy1 antibody into uninephrectomized rats. One week after disease induction, animals were randomly assigned to chronic glomerulosclerosis, chronic glomerulosclerosis plus Bay 41-2272 (10 mg/kg body weight/day) or chronic glomerulosclerosis plus hydralazine (15 mg/kg body weight/day). In week 16, analysis included effects on systolic blood pressure, proteinuria, kidney function, glomerular and tubulointerstitial matrix protein accumulation, expression of transforming growth factor-beta 1 (TGF-beta 1), fibronectin and plasminogen activator inhibitor type 1 (PAI-1), macrophage infiltration, cell proliferation, basal and nitric oxide-stimulated cGMP production as well as tubulointerstitial mRNA expression of alpha 1 and beta 1 soluble guanylate cyclase.
Results. The moderately elevated systolic blood pressure seen in the chronic glomerulosclerosis group was comparably decreased by both treatments. Compared to normal controls, soluble guanylate cyclase mRNA expression and nitric oxide-stimulated cGMP production were up-regulated in the tubulointerstitium of the untreated chronic glomerulosclerosis animals, while its activity was decreased in glomeruli. Bay 41-2272 treatment enhanced glomerular and tubulointerstitial nitric oxide-cGMP signaling significantly. This went along with markedly reduced glomerular and tubulointerstitial macrophage infiltration, number of proliferating cells, matrix expression and accumulation, as well as improved kidney function. In contrast, hydralazine therapy did not significantly affect renal nitric oxide-cGMP signaling, macrophage number, cell proliferation, matrix protein expression and accumulation.
Conclusion. Glomerular and tubulointerstitial soluble guanylate cyclase activity are discordantly altered in anti-thy1-induced chronic glomerulosclerosis. Stimulation of soluble guanylate cyclase signaling by Bay 41-2272 limits the progressive course of this model toward tubulointerstitial fibrosis and impaired renal function at least in part in a blood pressure-independent manner. The results suggest that soluble guanylate cyclase activation counteracts fibrosis and progression in chronic renal disease.

DOI： 10.1111/j.1523-1755.2005.00380.x

Exploring RNA inteference as a therapeutic strategy for renal disease Reviewed

Y. Takabatake, Y. Isaka, M. Mizui, H. Kawachi, F. Shimizu, T. Ito, M. Hori, E. Imai

Gene Therapy 12 ( 12 ) 965 - 973 2005.6

Language：English Publishing type：Research paper (scientific journal)

The short synthetic interfering RNA duplexes (siRNAs) can selectively suppress gene expression in somatic mammalian cells without nonselective toxic effects of double-stranded RNA (dsRNA). However, a selective in vivo delivery of siRNA transfer has not been reported in kidney. Here, we investigated whether injection of synthetic siRNAs via renal artery followed by electroporation could be effective and therapeutic in silencing specific gene in glomerulus. We investigated the effect of siRNA in rat cultured mesangial cells (MCs) and showed that siRNA sequence-specific suppression of transgene expression was over a 1000-fold more potent than that by antisense oligodeoxynucleotide (ASODN). Transfection of siRNA targeting luciferase into rat kidneys significantly inhibited expression of a cotransfected luciferase expression vector in vivo. The delivery of siRNA targeting enhanced green fluorescent protein (EGFP) in the transgenic 'green' rat reduced endogenous EGFP expression, mainly in glomerular MCs. Furthermore, RNAi targeting against TGF-β1 significantly suppressed TGF-β1 mRNA and protein expression, thereby ameliorated the progression of matrix expansion in experimental glomerulonephritis. In addition, vector-based RNAi also inhibited TGF-β1 expression in vitro and in vivo. In conclusion, siRNA-directed TGF-β1 silencing may be of therapeutic value in the prevention and treatment of fibrotic diseases. © 2005 Nature Publishing Group. All rights reserved.

DOI： 10.1038/sj.gt.3302480

Expression and regulation of adrenomedullin in renal glomerular podocytes Reviewed

M Hino, M Nagase, S Kaname, S Shibata, T Nagase, S Oba, M Funaki, N Kobayashi, H Kawachi, P Mundel, T Fujita

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 330 ( 1 ) 178 - 185 2005.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Adrenomedullin (AM) is postulated to exert organ-protective effects. It is expressed in the renal glomeruli, but its roles in the glomerular podocytes have been poorly elucidated. In the present study, we investigated the expression and regulation of AM in recently established conditionally immortalized mouse podocyte cell line in vitro and podocyte injury model in vivo. The cultured differentiated podocytes expressed AM mRNA and secreted measurable amount of AM. AM secretion from the podocytes was increased by H2O2, hypoxia, puromycin aminonucleoside (PAN), albumin overload, and TNF-alpha. Real-time RT-PCR analysis revealed that AM mRNA expression in the podocytes was enhanced by PAN and TNF-alpha, both of which were suppressed by mitochondrial antioxidants. Furthermore, AM expression was upregulated in the glomerular podocytes of PAN nephrosis rats. These results indicated that AM expression in the podocytes was upregulated by stimuli or condition relevant to podocyte injury, suggesting its potential role in podocyte pathophysiology. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2005.02.142

Novel molecules in regulating the barrier function of podocyte Reviewed

Hiroshi Kawachi

Japanese Journal of Nephrology 47 ( 3 ) 231 - 232 2005

Language：English Publishing type：Research paper (scientific journal)

Several recent studies indicated that slit diaphragm (SD) of glomerular epithelial cell (podocyte) plays a critical role in maintaining the permeability barrier of glomerular capillary wall. It is also reported that common proteinuric diseases such as minimal change type nephrotic syndrome (MCNS) and membranous nephropathy resulted from the dysfunction of SD. Although our group has reported that nephrin and podocin are critical components of SD, the precise molecular composition constituting the barrier of podocyte is not fully elucidated. To search for novel molecules involved in the development of proteinuria, we performed the subtraction hybridization techniques using glomerular cDNA materials from normal rats and the proteinuric rats with podocyte specific injuries. We identified 71 genes down-regulated in puromycin aminonucleoside nephropathy, an experimental model of MCNS. We confirmed that 11 genes of them were specifically expressed in podocyte in glomeruli and their expressions were already decreased before the onset of proteinuria. Decreased expressions of them are also observed in another proteinuric model induced by anti-nephrin antibody. The observations suggested that these molecules are involved in the development of proteinuria. In this symposium, we will introduce the nature of these molecules. © 2005, Japanese Society of Nephrology. All rights reserved.

DOI： 10.14842/jpnjnephrol1959.47.231

Multi-glycoside of Tripterygium wilfordii hook f. ameliorates proteinuria and acute mesangial injury induced by anti-Thy1.1 monoclonal antibody Reviewed

YG Wan, LB Gu, K Suzuki, T Karasawa, Y Fujioka, GD Han, H Koike, H Kawachi, F Shimizu

NEPHRON EXPERIMENTAL NEPHROLOGY 99 ( 4 ) E121 - E129 2005

Language：English Publishing type：Research paper (scientific journal) Publisher：KARGER

Background/Aims: Multi-glycoside from Tripterygium wilfordii Hook f. (GTW) is used for various immune and inflammatory diseases including renal diseases represented by mesangial proliferative glomerulonephritis (MsPGN) in China. However, there have been no fundamental studies on the operating mechanism of GTW on MsPGN. The aim of this study is to examine as the first step the effects of GTW on acute injurious process such as mesangial injury and proteinuria in an acute and reversible Thy. 1.1 glomerulonephritis (Thy1.1GN) model and then to clarify the action mechanism of GTW at molecular level by examining its effects on various injurious factors in this model. Methods: Thy1.1 GN was induced in rats by a single intravenous injection with 500 mu g of anti-Thy1.1 mAb 1-22-3. Daily oral administration of GTW and vehicle as a control was started from 3 days before injection of mAb to the day of sacrifice in each experiment. Fourteen rats were randomly divided into 2 groups, GTW-treated and vehicle-treated groups, and sacrificed on day 14 in experiment 1 or on day 7 in experiment 2 after induction of Thy1.1 GN. Proteinuria was determined on days 1, 3, 5, 7, 10 and 14 in experiment 1 or on 1, 3, 5 and 7 in experiment 2. From blood and kidneys taken at sacrifice, blood biochemical parameters, mesangial morphological changes, glomerular macrophage infiltration, and glomerular mRNA expression of cytokines were examined. Results: In experiment 1, proteinuria and mesangial matrix expansion were significantly attenuated by GTW treatment. In experiment 2, GTW treatment significantly ameliorated proteinuria, mesangial lesions and macrophage accumulation in glomerulus. In addition, it significantly reduced the glomerular expression of mRNA for PDGF, MCP-1 and IL-2. Conclusion: GTW ameliorated not only proteinuria but also mesangial alterations in Thy1.1 GN most likely by reducing expression of injurious cytokines, indicating that GTW has suppressive effects on acute inflammatory changes in glomeruli. Copyright (C) 2005 S. Karger AG, Basel.

DOI： 10.1159/000083980

Nephrin and podocin dissociate at the onset of proteinuria in experimental membranous nephropathy Reviewed

Takeshi Nakatsue, Hiroko Koike, Gi Dong Han, Koichi Suzuki, Naoko Miyauchi, Huaiping Yuan, David J. Salant, Fumitake Gejyo, Fujio Shimizu, Hiroshi Kawachi

Kidney International 67 ( 6 ) 2239 - 2253 2005

Language：English Publishing type：Research paper (scientific journal) Publisher：Blackwell Publishing Inc.

Background. The slit diaphragm plays a critical role in maintaining the barrier function of the glomerular capillary wall. The pathogenic mechanism of proteinuria in membranous nephropathy remains uncertain. This study was undertaken to analyze the pathogenic role of slit diaphragm in proteinuria in experimental membranous nephropathy. Methods. The expression and the localization of slit diaphragm-associated molecules (nephrin, podocin, and CD2AP) and other podocyte-associated molecules (podocalyxin and α3 integrin) in passive and active Heymann nephritis were analyzed by immunofluorescence and Western blot analysis. The interaction of slit diaphragm-associated molecules was investigated by the dual-labeling immunofluorescence method. The mRNA expression of these molecules was also analyzed. Results. Shifts in nephrin and podocin staining patterns, from linear to granular, were detected in the early stages of passive Heymann nephritis. These shifts were not parallel, and the dissociation of these molecules was detected by the dual-labeling immunofluorescence method in passive and active Heymann nephritis. Western blot analyses with sequentially solubilized materials indicated that the nephrin-rich fraction changed from being partly detergent-resistant to being predominantly detergent-soluble. This change did not occur with podocin. Nephrin excreted into urine was already detected in the early stages of passive Heymann nephritis. Decreased mRNA expression of nephrin and podocin was observed before the onset of proteinuria. By contrast, no extensive change in the expression of α3 integrin was observed in this study. Conclusion. Nephrin is dissociated from podocin and excreted into urine in the early stages of Heymann nephritis. The reduced expression of nephrin and podocin, along with their dissociation, may contribute to the development of proteinuria in Heymann nephritis. © 2005 by the International Society of Nephrology.

DOI： 10.1111/j.1523-1755.2005.00328.x

Intrarenal injection of bone marrow-derived angiogenic cells reduces endothelial injury and mesangial cell activation in experimental glomerulonephritis Reviewed

Hideki Uchimura, Takeshi Marumo, Osamu Takase, Hiroshi Kawachi, Fujio Shimizu, Matsuhiko Hayashi, Takao Saruta, Keiichi Hishikawa, Toshiro Fujita

Journal of the American Society of Nephrology 16 ( 4 ) 997 - 1004 2005

Language：English Publishing type：Research paper (scientific journal)

Loss of glomerular endothelial cells has been suggested to contribute to the progression of glomerular injury. Although therapeutic angiogenesis induced by administration of bone marrow-derived endothelial progenitor cells has been observed in disease models of endothelial injury, the effects on renal disease have not been clarified. Whether administration of culture-modified bone marrow mononuclear cells would mitigate the glomerular endothelial injury in anti-Thy1.1 nephritis was investigated. After cultivation under conditions that promote endothelial progenitor cell growth, bone marrow mononuclear cells were labeled with CM-DiI, a fluorescence marker, and injected into the left renal artery of Lewis rats with anti-Thy1.1 glomerulonephritis. The decrease in glomerular endothelial cells was significantly attenuated in the left kidney, as compared with the right, in nephritic rats that received the cell infusion. Glomerular injury score, the area positive for mesangial α-smooth muscle actin, and infiltration of macrophages were significantly decreased in the left kidney. CM-DiI-positive cells were distributed in glomeruli of the left kidney but not in those of the right kidney. Among CM-DiI-labeled cells incorporated into glomeruli, 16.5 ± 1.2% of cells were stained with an endothelial marker, rat endothelial cell antigen-1. Culture-modified mononuclear cells secreted 281.2 ± 85.0 pg of vascular endothelial growth factor per 105 cells per day. In conclusion, intra-arterial administration of culture-modified bone marrow mononuclear cells reduced endothelial injury and mesangial activation in anti-Thy1.1 glomerulonephritis. Incorporation into the glomerular endothelial lining and production of angiogenic factor(s) are likely to contribute to the protective effects of culture-modified mononuclear cells against glomerular injury. Copyright © 2005 by the American Society of Nephrology.

DOI： 10.1681/ASN.2004050367

Suppressive effects of GTW treatment on mesangial lesions in experimental irreversible glomerulosclerosis Reviewed

Yi-Gang Wan, Wei Sun, Fujio Shmizu, Liu-Bao Gu, Koichi Suziki, Tamaki Karasawa, Hiroshi Kawachi

Zhongguo Zhongyao Zazhi 30 ( 5 ) 361 - 365 2005

Language：Chinese Publishing type：Research paper (scientific journal)

Objective: To examine suppressive effects of multi-glycoside of Tripterygium wilfordii Hook. f. (GTW)on mesangial injury induced by two-injecti on of anti-Thy1. 1 monoclonal antibody (mAb) 1-22-3 in vitro. Method: We established the irreversible model of glomerulosclerosis with anti-Thy1. 1 mAb 1-22-3. After 42 days of oral treatment with GTW (50 mg·kg-1 BW) and vehicle (distilled water), to observe effects of GTW on proteinuria, renal function, mesangial morphological change, and mRNA expressions of collagen type I and TGF-β by light microscope (LM), immunofluorescence (IF), and Reverse Transcription Polymerase Chain Reaction (RT-PCR). Result: GTW ameliorated proteinuria (from day24 to day 42) and mesangial proliferation [total cell number, GTW group 65.67 ± 3.43 vs. control group 87.02 ± 2.41, P &lt
0.05
matrix expansion, GTW group 1.20±0.06 vs. control group 2.77±0.23, P&lt
0.05
α-smooth muscle actin(α-SMA) expression, GTW group 1.75±0.33 vs. control group 2.62±0.15, P&lt
0.05
collagen type I expression, GTW group 1.68±0.31 vs. control group 2.06±0.24, P&lt
0.05], moreover, significantly reduced the glomerular expression of mRNA for collagen type I(53.5% to the control group, P &lt
0.05)and TGF-β(14.7% to the control group, P &lt
0.05)on day 42day. Conclusion: GTW can not only decrease proteinuria, but also ameliorate mesangial alterations probably by the reduction of cytokines. GTW may be a promising agent for the prevention of progressive and irreversible glomerulosclerosis.

Spironolactone in combination with cilazapril ameliorates proteinuria and renal interstitial fibrosis in rats with anti-Thy-1 irreversible nephritis Reviewed

Masaki Asai, Toshiaki Monkawa, Takeshi Marumo, Seiichi Fukuda, Mihoko Tsuji, Jun Yoshino, Hiroshi Kawachi, Fujio Shimizu, Matsuhiko Hayashi, Takao Saruta

Hypertension Research 27 ( 12 ) 971 - 978 2004.12

Language：English Publishing type：Research paper (scientific journal)

Blockade of the renin-angiotensin system has been established as a treatment for heart failure with hypertension and left ventricular hypertrophy, and for progressive kidney diseases. The present study was conducted to examine whether spironolactone, a mineralocorticold receptor antagonist, alone or in combination with cilazapril, an angiotensin converting enzyme (ACE) inhibitor, ameliorates proteinuria and renal lesions in an immune-initiated progressive nephritis model. Wistar rats were uninephrectomized 7 days before injection of anti-Thy-1 monoclonal antibody 1-22-3 to induce progressive glomerulonephritis. The nephritic rats were untreated or treated with spironolactone (400 mg/kg body weight/day), cilazapril (11 mg/kg body weight/day), or both for 10 weeks. Proteinuria was increased in the untreated rats 1 week after nephritis induction and was maintained throughout the experiment. Compared with the untreated animals (212.9± 49.2mg/day), proteinuria was significantly reduced in the spironolactone-treated group (62.0±4.0 mg/day, p=0.0046) and the cilazapril-treated group (71.8±26.0 mg/day, p=0.0048) on day 70 after antibody injection. Further reduction of proteinuria (42.4±4.5 mg/day, p=0.0019 vs. the untreated group) and less renal cortex interstitial fibrotic change (fibrosis score: 142.0±18.4 vs. 80.3±18.5 in the untreated group, p=0.0123) were detected in the spironolactone plus cilazapril-treated group. Blood pressure did not differ among the three treatment groups. In conclusion, spironolactone ameliorates proteinuria to the same degree as cilazapril, and concomitant use of spironolactone and an ACE inhibitor further suppresses renal disease progression. These data suggest that concomitant treatment with spironolactone and an ACE inhibitor has beneficial effects on immune-initiated progressive kidney disease.

DOI： 10.1291/hypres.27.971

ネフローゼ症候群の成因 Invited

河内裕

内科 94 5 - 12 2004.12

蛋白尿の分子機序 Invited

河内裕

日本内科学会誌 93 ( 5 ) 954 - 959 2004.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：日本内科学会

腎糸球体係蹄壁は,内皮細胞,糸球体基底摸,そしてその外側を覆う糸球体上皮細胞足突起の3層からなり,足突起間にはスリット膜とよばれるフィルター様の構造物がある.従来,糸球体係蹄壁の蛋白透過を防ぐメインバリアーは,基底膜であるとする考え方が一般的であったが,近年,上皮細胞スリット膜の重要性を示唆する多くの報告がなされてきている. nephrin, podocinは,スリット膜の機能分子で多くの病態で,蛋白尿発症に関わっていると考えられてきており,今後の治療のターゲットとして有力視されている.

DOI： 10.2169/naika.93.954

Other Link： http://hdl.handle.net/10191/17974

腎炎・ネフローゼ「糸球体から蛋白尿が生じる機序についてお教え下さい」 Invited

河内裕

小児内科 36 241 - 243 2004.12

Src-family kinase Fyn phosphorylates the cytoplasmic domain of nephrin and modulates its interaction with podocin Reviewed

H Li, S Lemay, L Aoudjit, H Kawachi, T Takano

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 15 ( 12 ) 3006 - 3015 2004.12

Language：English Publishing type：Research paper (scientific journal) Publisher：LIPPINCOTT WILLIAMS & WILKINS

Visceral glomerular epithelial cells (GEC) are critical for normal permselectivity of the kidney. Nephrin is a molecule that is expressed specifically in GEC in a structure called the slit diaphragm and is required for normal morphology and permselectivity of GEC. However, the mechanisms of action of nephrin are not understood precisely. The intracellular domain of nephrin has six conserved tyrosine residues. It was hypothesized that these tyrosine residues are phosphorylated by Src-family kinases and that this phosphorylation modulates the function of nephrin. A transient transfection system was used to study the role of tyrosine phosphorylation of the cytoplasmic domain of nephrin in its function. When nephrin was cotransfected with Src-family kinases Fyn or Src in Cos-1 cells, nephrin was strongly tyrosine phosphorylated by Fyn and less so by Src. The results with tyrosine-to-phenylalanine mutations suggested that multiple tyrosine residues contribute to phosphorylation mediated by Src-family kinases. The intracellular domain of nephrin is known to interact with another slit diaphragm protein, podocin. When nephrin and podocin were transfected with Fyn, the interaction between nephrin and podocin was augmented significantly. Podocin was not tyrosine phosphorylated by Fyn; thus, the increased interaction is likely to be secondary to tyrosine phosphorylation of nephrin. Fyn also significantly augmented the activation of the AP-1 promoter induced by nephrin and podocin. In summary, Fyn phosphorylates the cytoplasmic domain of nephrin on tyrosine, leading to enhanced association with podocin and downstream signaling of nephrin.

DOI： 10.1097/01.ASN.0000146689.88078.80

スリット膜関連の新しい機能分子. Invited

河内裕

日本臨床 62 1823 - 1828 2004.12

Prevention and reversal of renal injury by leptin in a new mouse model of diabetic nephropathy Reviewed

T Suganami, M Mukoyama, K Mori, H Yokoi, M Koshikawa, K Sawai, S Hidaka, K Ebihara, T Tanaka, A Sugawara, H Kawachi, C Vinson, Y Ogawa, K Nakao

FASEB JOURNAL 18 ( 13 ) 127 - + 2004.10

Language：English Publishing type：Research paper (scientific journal) Publisher：FEDERATION AMER SOC EXP BIOL

Diabetic nephropathy is the leading cause of end-stage renal disease, for which effective therapy to prevent the progression at advanced stages remains to be established. There is also a long debate whether diabetic glomerular injury is reversible or not. Lipoatrophic diabetes, a syndrome caused by paucity of adipose tissue, is characterized by severe insulin resistance, dyslipidemia, and fatty liver. Here, we show that a genetic model of lipoatrophic diabetes (A-ZIP/F-1 mice) manifests a typical renal injury observed in human diabetic nephropathy that is associated with glomerular hypertrophy, diffuse and pronounced mesangial widening, accumulation of extracellular matrix proteins, podocyte damage, and overt proteinuria. By crossing A-ZIP/F-1 mice with transgenic mice overexpressing an adipocyte-derived hormone leptin, we also reveal that leptin completely prevents the development of hyperglycemia and nephropathy in A-ZIP/F-1 mice. Furthermore, continuous leptin administration to A-ZIP/F-1 mice by minipump beginning at 40 weeks of age significantly alleviates the glomerular injury and proteinuria. These findings demonstrate the therapeutic usefulness of leptin at least for a certain type of diabetic nephropathy. The model presented here will serve as a novel tool to analyze the molecular mechanism underlying not only the progression but also the regression of diabetic nephropathy.

DOI： 10.1096/fj.04-2183fje

DNAzyme for TGF-β suppressed extracellular matrix accumulation in experimental glomerulonephritis Reviewed

Yoshitaka Isaka, Hiroyuki Nakamura, Masayuki Mizui, Yoshitsugu Takabatake, Masaru Horio, Hiroshi Kawachi, Fujio Shimizu, Enyu Imai, Masatsugu Hori

Kidney International 66 ( 2 ) 586 - 590 2004.8

Language：English Publishing type：Research paper (scientific journal)

Background. We developed an electroporation-mediated gene transfer method targeting glomerular mesangial cells. Injecting DNA solution via renal artery followed by electric pulses using tweezers-type electrodes could result in efficient transfection in mesangial cells. Therefore, this gene transfer system opened a feasible strategy to manipulate the function of several cytokines and growth factors in mesangial cells. Recently, a new generation of catalytic nucleic acid composed of DNA, named DNA enzyme (DNAzyme), has been developed. Method. We generated a DNAzyme (TGFDE) targeting transforming growth factor-β1 (TGF-β1), and examined the therapeutic effect of TGFDE in vitro and in vivo. Results. In cultured rat mesangial cells, treatment with TGFDE blocked TGF-β1 mRNA expression, and thereby suppressed type I collagen mRNA expression. Next, we introduced TGFDE or scrambled DNAzyme (TGFSCR) into anti-Thy-1 model of nephritic rats by electroporation 3 days after disease induction. Northern blot analysis and immunohistochemical staining demonstrated that glomerular message and protein ex-pression of TGF-β1, α-smooth muscle actin (α-SMA), and type I collagen were suppressed in TGFDE-transfected nephritic rats compared with untreated nephritic rats and TGFSCR-transfected rats on day 7. Consequently, we observed significant reduction in glomerular matrix score in TGFDE-transfected nephritic rats. Conclusion. Inhibition of TGF-β1 expression by electroporation-mediated DNAzyme transfer might be useful for the therapy of glomerulonephritis.

DOI： 10.1111/j.1523-1755.2004.00777.x

Identification of hydroxyapatite deposits in the smooth muscle cells and ganglion cells of autopsied small intestines Reviewed

Hirotaka Tanabe, Takumi Akashi, Hiroshi Kawachi, Noboru Andou, Yoshinobu Eishi, Touichiro Takizawa, Morio Koike, Shizuko Ichinose

Journal of Medical and Dental Sciences 51 ( 2 ) 129 - 138 2004.6

Language：English Publishing type：Research paper (scientific journal)

We have studied the deposition of calcium salts in the autopsied intestines which have not been described previously as far as we were able to determine. In order to clarify the incidence, predisposing conditions, mineral compositions of the deposited materials and clinical significance of this phenomenon, we examined 76 cases of consecutive autopsied small intestines by von Kossa's staining. Deposited calcium salts were further examined by electron microscopically, energy-dispersive X-ray spectroscope and electron diffraction analysis. Deposition of calcium salts was observed in the small intestines of 13 cases. Among them, 10 cases were accompanied by hypercalcemia. Deposition of calcium salts was mainly observed in smooth muscle cells of the proper muscle layers and ganglion cells of the Auerbach's myenteric plexus. Intestinal calcinosis was frequently accompanied by deposition of calcium salts in the proper muscle layers of esophagus and large intestine, renal tubules and cardiac myocardial cells. Electron microscopically, the calcium salts were identified as needle-shaped crystals and located on the surface of the extracellular- collagen bundles, in the cytoplasm, mitochondria and nucleus of the smooth muscles cells. Energy-dispersive X-ray spectroscope and electron diffraction analysis suggested the deposited calcium salts as hydroxyapatite. Two patients among the six cases with moderate to severe calcium deposition showed clinical manifestation of paralytic ileus. In conclusion, intestinal calcinosis was frequently observed mostly associated with hypercalcemia. Calcium salts of hydroxyapatite were deposited to the smooth muscle cells and the Auerbach's myenteric plexus of the muscular layer. Deposition of calcium salts might occasionally causes the paralytic ileus but clinical significance of this lesion requires further examination.

Local Actions of Endogenous Angiotensin II in Injured Glomeruli Reviewed

Ji Ma, Taiji Matsusaka, Haichun Yang, Hiroshi Kawachi, Fujio Shimizu, Yoshitaka Isaka, Enyu Imai, Valentina Kon, Iekuni Ichikawa

Journal of the American Society of Nephrology 15 ( 5 ) 1268 - 1276 2004.5

Language：English Publishing type：Research paper (scientific journal)

A previous study showed that exogenous angiotensin II (AngII) induces proliferation of glomerular cells through systemic actions of AngII. In the present study, the authors examined the mode of actions of endogenous AngII in injured kidneys that were made deficient in AT1 by using in vivo transfection of antisense oligodeoxynucleotide (AS-ODN). Thy-1 nephritis was induced in rats by injection of mAb 1-22-3. Four days later, glomerular transfection was performed by unilateral whole-kidney electroporation after AT1 AS-ODN delivery through the left renal artery (n = 7). The expression of renal AT1 was assessed by autoradiography. The effect of the AS-ODN transfection was assessed 3 d later and compared with transfection with control ODN (n = 6), systemically administered pharmacologic AT1 antagonist losartan (n = 5) as well as untreated Thy-1 animals (n = 5). Fluorescence-labeled AS-ODN was found transfected in almost all glomeruli and localized primarily to the mesangium. Compared with the contralateral untransfected kidney in both normal and Thy-1 rats. AS-ODN suppressed cortical AT1 expression by some 70%. The AS-ODN transfected kidneys of Thy-1 rats had significantly lower glomerular mesangial cell proliferation (7.38 ± 0.68 cells/glomerulus) and extracellular matrix accumulation (0.262 ± 0.009) than kidneys transfected with control ODN (10.94 ± 0.51 cells/glomerulus and 0.342 ± 0.031), contralateral untransfected kidneys (9.56 ± 1.01 cells/glomerulus and 0.371 ± 0.011), or kidneys that were exposed to Thy-1 alone (10.45 ± 1.06 cells/glomerulus and 0.359 ± 0.013). There were no significant differences in systolic BP among groups. In glomeruli, immunohistochemistry detected no difference in AT2 receptor expression, number of ED1-positive macrophages or number of apoptotic cells among groups. Thus, in renal injury induced by Thy-1 nephritis, selective suppression of mesangial AT1 expression by ASODN significantly reduced mesangial cell proliferation and matrix. These data provide in vivo evidence that injured glomeruli are sensitive to local tissue actions of AngII, which promote proliferation and matrix accumulation within the glomerulus.

DOI： 10.1097/01.ASN.0000125675.70674.0F

Novel expression of sodium/myo-inositol co-transporter in podocytes in puromycin aminonucleoside nephrosis Reviewed

Y Watanabe, T Kobayashi, E Yaoita, H Kawachi, A Yamauchi, T Inoue, F Shimizu, Y Yoshida, AGA El-Shemi, H Okada, H Suzuki, T Yamamoto

NEPHROLOGY DIALYSIS TRANSPLANTATION 19 ( 4 ) 817 - 822 2004.4

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Background. How podocytes respond to injury is poorly understood, although podocyte injury in the glomerulus has been proposed as the crucial mechanism in the pathogenesis of proteinuria and focal segmental glomerulosclerosis. An increase in sodium/myo-inositol co-transporter (SMIT) transcripts, an osmoprotective gene, has been demonstrated in a variety of brain injury models. In the present study, we investigated SMIT expression in podocytes in experimental nephrosis.
Methods. Two types of nephrosis were induced in rats: puromycin aminonucleoside (PAN) nephrosis and monoclonal antibody (mAb) 5-1-6 nephropathy. Podocyte injury was morphologically distinct in the former type of nephrosis and limited to a minimum in the latter. SMIT expression in isolated glomeruli was estimated by ribonuclease protection assay. Localization of SMIT-expressing cells in glomeruli was examined by in situ hybridization.
Results. WIT transcripts in glomeruli increased conspicuously in the nephrotic stage of PAN nephrosis, whereas the transcripts in cortices and medullae did not show significant changes. In situ hybridization revealed that podocytes were predominant cells expressing SMIT in the glomerulus. Significant increase of SMIT mRNA in the glomeruli was detected before the onset of massive proteinuria. In contrast, up-regulation of SMIT expression was not observed in mAb 5-1-6 nephropathy, whose urinary protein levels were comparable with those in the nephrotic stage of PAN nephrosis.
Conclusions. These findings suggest that SMIT expression in podocytes is not provoked by an effect of massive proteinuria but by extensive cellular injury.

DOI： 10.1093/ndt/gfh026

Decreased collagen-degrading activity could be a marker of prolonged mesangial matrix expansion Reviewed

Masayuki Tomita, Hiroko Koike, Gi Dong Han, Fujio Shimizu, Hiroshi Kawachi

Clinical and Experimental Nephrology 8 ( 1 ) 17 - 26 2004.3

Language：English Publishing type：Research paper (scientific journal)

Background. Mesangial matrix expansion is caused by the overproduction and/or the impaired proteolytic degradation of the extracellular matrix. However, the relative contribution of these changes to the development of prolonged mesangial matrix expansion is still poorly understood. We aimed to elucidate the relative role of the matrix metalloproteinase (MMP)/tissue inhibitors of metalloproteinases (TIMPs) system in the development of prolonged mesangial matrix expansion. Methods. We prepared two rat models, showing reversible or prolonged mesangial matrix expansion, induced by a single injection or two consecutive injections of anti-Thy-1.1 monoclonal antibody 1-22-3, respectively. We analyzed the glomerular expression of type I and type IV collagens
MMP-2, -9, and -13
membrane type 1-MMP (MT1-MMP)
TIMP-1
and urinary type I collagen-degrading activity in both models. Results. There were no differences in glomerular mRNA levels of type I and type IV collagens between the reversible and the prolonged models. MMP-9 mRNA expression and protein level was lower in the prolonged model than in the reversible one, whereas there were no differences in mMRNA levels of MMP-2, -13, MT1-MMP, or TIMP-1 between the two models. Urinary type I collagen-degrading activity in the prolonged model was lower than that in the reversible one. Furthermore, there was a significant correlation between the mesangial matrix expansion and urinary type I collagen-degrading activity. Conclusions. Impaired expression of MMP-9 may contribute to the development of prolonged mesangial matrix expansion. Analysis of urinary type I collagen-degrading activity may provide additional diagnostic information in mesangial proliferative glomerulonephritis.

DOI： 10.1007/s10157-003-0258-7

Retinoic acid receptor alpha and retinoid X receptor specific agonists reduce renal injury in established chronic glomerulonephritis of the rat Reviewed

M Schaier, S Liebler, K Schade, F Shimizu, H Kawachi, HJ Grone, R Chandraratna, E Ritz, J Wagner

JOURNAL OF MOLECULAR MEDICINE-JMM 82 ( 2 ) 116 - 125 2004.2

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER-VERLAG

Retinoids, derivatives of vitamin A, inhibit mesangial cell proliferation, glomerular inflammation, and extracellular matrix deposition in acute anti-Thy1.1 glomerulonephritis (Thy-GN) of the rat. We examined a model, chronic mesangioproliferative Thy-GN (MoAb 1-22-3), which is more akin to human disease. Treatment started on day 23 when Thy-GN had already been established. Nonnephritic control and Thy-GN rats were treated orally for 67 days with vehicle or with two doses of either the retinoic acid receptor alpha-specific agonist AGN 195183 (RARalpha agonist) or the retinoid X receptor specific agonist AGN 194204 (RXR agonist). Doses of either the RARalpha or the RXR agonist significantly reduced albuminuria and normalized blood pressure during the course of treatment. The glomerulosclerosis index, glomerular cell and interstitial cell counts, and area of the interstitial space were significantly lower in nephritic rats treated with the RARalpha agonist or RXR agonist than with vehicle. The RARalpha and RXR agonist significantly reduced the infiltration of the glomerulus by macrophages. The increase in glomerular TGFbeta1 and prepro-ET1 gene expression in vehicle-treated nephritic rats was significantly attenuated by RARalpha or RXR agonists. Glomerular expression of RXRalpha and RARalpha receptor mRNA was significantly greater in vehicle-treated nephritic rats than in nonnephritic controls. Treatment with RARalpha or RXR agonists tended to normalize retinoid-receptor gene expression. Our data indicate that both RARalpha agonists and RXR agonists reduce renal damage in rats with established chronic glomerulonephritis. Receptor-specific retinoids may provide a novel therapeutic approach for the treatment of chronic glomerulonephritis.

DOI： 10.1007/s00109-003-0510-3

Superior renoprotective effects of combination therapy with ACE and AGE inhibition in the diabetic spontaneously hypertensive rat Reviewed

BJ Davis, JM Forbes, MC Thomas, G Jerums, WC Burns, H Kawachi, TJ Allen, ME Cooper

DIABETOLOGIA 47 ( 1 ) 89 - 97 2004.1

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER

Aims/hypothesis. Diabetic renal disease has been postulated to progress as a result of an interaction between metabolic and haemodynamic pathways. Our aim was to assess the functional, structural, molecular and cellular aspects of renal disease in an experimental model of diabetes with associated hypertension.
Method. Streptozotocin-induced diabetic spontaneously hypertensive rats were randomised to no treatment, the ACE inhibitor, perindopril (2 mg/l), the AGE formation inhibitor, aminoguanidine (1 g/l) and a combination of both agents and were followed for 32 weeks.
Results. Diabetes was associated with a considerable increase in albumin excretion rate. Both aminoguanidine and perindopril retarded the increase in albuminuria, which was completely abrogated by combination therapy. Glomerulosclerosis and tubulointerstitial damage was reduced by both monotherapies with further renoprotection afforded by combination therapy in both cases. Combination therapy was also associated with a superior restoration in diabetes-induced nephrin protein depletion compared to either monotherapy. TGFbeta1 expression as assessed by in situ hybridisation was increased in the diabetic rats and reduced by perindopril and aminoguanidine.
Conclusion/interpretation. These findings indicate that in the context of diabetes-related renal injury, blocking both the renin-angiotensin and advanced glycation pathways offers superior renoprotection and could be considered as a therapeutic strategy in the prevention and retardation of progressive-diabetic renal injury.

DOI： 10.1007/s00125-003-1256-8

The role of lymphocytes in the experimental progressive glomerulonephritis Reviewed

Yohei Ikezumi, Katsue Kanno, Tamaki Karasawa, G. I. Dong Han, Yumi Ito, Hiroko Koike, Shinichi Toyabe, Makoto Uchiyama, Fujio Shimizu, Hiroshi Kawachi

Kidney International 66 ( 3 ) 1036 - 1048 2004

Language：English Publishing type：Research paper (scientific journal) Publisher：Blackwell Publishing Inc.

Background. Glomerular accumulation of leukocytes, including lymphocytes, is a common feature in most types of glomerulonephritis. However, the role of lymphocytes in progressive glomerulonephritis has not been elucidated. We examined the role of lymphocytes in the development of progressive mesangial proliferative glomerulonephritis induced by two injections of monoclonal antibody 1-22-3 in rats. Methods. To elucidate the role of lymphocytes, circulating lymphocytes were depleted using specific monoclonal antibodies to rat lymphocytes prior to the induction of progressive glomerulonephritis. The effects of lymphocyte depletion on proteinuria and glomerular alterations were assessed 7 and 56 days after the induction of progressive glomerulonephritis. Results. Significant glomerular accumulation of CD4+ T cells, CD8+ T cells, and ED3+-activated macrophage were observed after the induction of glomerulonephritis. Depletion studies showed that continuous treatment with anti-CD5, anti-CD4, or anti-CD8 treatment reduced proteinuria and ameliorated the glomerular lesions on day 56. Depletion of CD4+ T cells also reduced glomerular accumulation of CD8+ T cells and ED3+-activated macrophages, and reduced glomerular expression of mRNA for interferon-gamma (INF-γ) (63.0% in anti-CD5 and 62.3% reduction in anti-CD4). Transit lymphocyte depletion limited in early stage of progressive glomerulonephritis demonstrated that CD4+ T-cell depletion, but not anti-CD8 treatment prevented glomerular injuries 56 days after the induction of progressive glomerulonephritis. Conclusion. CD4+ T cells played a central role in the development of progressive glomerulonephritis, controlling recruitment and activation of CD8+ cytotoxic cells and/or macrophages.

DOI： 10.1111/j.1523-1755.2004.00852.x

Moderate alcohol intake has no impact on acute and chronic progressive anti-Thy-1 glomerulonephritis Reviewed

Peters H, Martini S, Woydt R, Ruckert M, Shimizu F, Kawachi H, Liefeldt L, Kramer S, Neumayer HH

Am J Physio Renal Physiol 284 F1105 - F1114 2003.12

続々と登場する糸球体上皮細胞スリット膜関連分子-蛋白尿発現の分子機構解明に向けて Invited

河内裕

医学のあゆみ 206 287 - 288 2003.12

糸球体上皮細胞の機能因子最近明らかになった蛋白尿発症責任分子について Invited

河内裕

診断と治療 91 517 - 522 2003.12

IFN-inducible protein-10 has a differential role in podocyte during Thy 1.1 glomerulonephritis Reviewed

GD Han, H Koike, T Nakatsue, K Suzuki, H Yoneyama, S Narumi, N Kobayashi, P Mundel, F Shimizu, H Kawachi

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 14 ( 12 ) 3111 - 3126 2003.12

Language：English Publishing type：Research paper (scientific journal) Publisher：LIPPINCOTT WILLIAMS & WILKINS

IFN-inducible protein-10 (IP-10/CXCL10) is a potent chemoattractant for activated T lymphocytes and was recently reported to have several additional biologic activities. In this study, the expression and the function in normal glomeruli and in Thy1.1 glomerulonephritis (GN) were investigated. The expression of IP-10 was detected in normal rat glomeruli mainly in the podocyte. The expression of IP-10 was also detected on the cultured podocyte. The IP-10 expression was elevated at the early phase of Thyl.1 GN. The double staining immunofluorescence study clearly demonstrated that the elevated expression of IP-10 was mostly detected in the podocyte and very partly in mesangial area. A receptor for IP-10, CXCR3, showed similar expression patterns to that of IP-10. Expressions of neither of IP-10 nor of CXCR3 were detected on the inflammatory cells. For elucidating the role of IP-10, the blocking study was carried out with monoclonal anti-IP-10 antibody. The monoclonal anti-IP-10 antibody treatment decreased the expression of IP-10 and podocyte associated proteins such as nephrin and podocin that are reported to be essential for maintaining the podocyte function (IP-10, 53.0% to control; nephrin, 43.5%; podocin, 60.4%). The findings indicated that the anti-IP-10 treatment disturbed the podocyte function. The anti-IP-10 treatment given to the rats with Thy1.1 nephritis exacerbated proteinuria, mesangiolysis, and matrix expansion. Collectively, the findings indicated that IP-10 plays a role in maintaining the podocyte function. Also, the findings suggested that anti-IP-10 treatment exacerbated the glomerular alterations in Thyl.1 GN by disturbing the podocyte function.

DOI： 10.1097/01.ASN.0000097371.64671.65

スリット膜関連分子群の性状. Invited

河内裕

腎と透析 55 702 - 706 2003.12

蛋白尿発症の分子機構 Invited

河内裕

腎と透析 54 189 - 193 2003.12

蛋白尿の発症機序. Invited

河内裕

小児内科 35 815 - 819 2003.12

Urinary sediment podocalyxin in children with glomerular diseases Reviewed

Kanno K, Kawachi H, Uchida Y, Hara M, Shimizu F, Uchiyama M

Nephron Clin Pract 95 ( 3 ) c91 - c99 2003.12

蛋白尿発現の分子機構：ネフリンのネフローゼ症候群における臨床的意義 Invited

清水不二雄, 河内裕

日本内科学会誌 92 ( 7 ) 159 - 165 2003.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：日本内科学会

ラット腎糸球体上皮細胞(GEC)足突起間スリット膜に対する単クローン抗体投与によりラットに高度の蛋白尿が惹起されることやスリット膜構成分子ネフリンの遺伝子レベルにおける異常が重症の先天性ネフローゼ症候群をもたらすことなどから,スリット膜が,最終的に高分子透過阻止に関与することが明らかにされた.ネフリンが発端となり,ポドシン, CD2APなどGEC,とりわけスリット膜関達分子の遺伝子レベルにおける異常が蛋白尿ないし家族性の糸球体硬化症をもたらすことが相次いで報告されている.新しく登場する分子も含めたこれら諸分子の相互関係の解明をとうして, GECの構造・機能を制御する情報伝達系の詳細が明らかにされ,その結果,蛋白尿の合理的な抑制策の確立とそれによる慢性腎不全状態への進展阻止が可能となるものと期待され,その中心的な存在であるネフリンは新しい治療法開発への重要な鍵をにぎる対象として注目され続けていくと思われる.

DOI： 10.2169/naika.92.1337

Other Link： http://hdl.handle.net/10191/17972

Kinetic analysis of the development of pancreatic lesions in mice infected with a murine retrovirus Reviewed

S Watanabe, K Suzuki, Y Kawauchi, S Yamagiwa, H Yoneyama, H Kawachi, Y Okada, F Shimizu, H Asakura, Y Aoyagi

CLINICAL IMMUNOLOGY 109 ( 2 ) 212 - 223 2003.11

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE

Sjogren's syndrome (SjS)-like sialoadenitis and exocrine pancreatitis were induced in mice infected with LP-BM5 murine leukemia virus, which induces a severe immunodeficiency termed murine AIDS (MAIDS). All mice with MAIDS showed advancing cellular infiltration around the pancreatic ducts as well as systemic exocrinopathy. The primary target tissue of the pancreas was acinar cells, and the pancreatic islets were well preserved until a late phase of the disease. Immunofluorescence and flow cytometry demonstrated that CD4(+) T cells, Mac-1(+) cells, and B220(+) cells were major inflammatory components, and IFN-gamma and IL-10 were mainly detected on CD4(+) T and Mac-1(+) cells in the pancreas. Both Th1 and Th2 cells were found. TUNEL+ apoptotic cells were mostly detected among pancreas-infiltrating cells. Fas ligand and TNF-alpha were also detected among pancreas-infiltrating cells, whereas Fas was rarely expressed in the pancreatic acinar cells. Thus, MAIDS mice could be valuable for analyzing the pathogenesis of autoimmume-related pancreatitis associated with SjS. (C) 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/S1521-6616(03)00197-9

Monoclonal antibody against rat podocyte-derived macrophagic cells reacts with crescent-forming cells in an experimental model Reviewed

Michiaki Orikasa, Toshihiko Iwanaga, Hiroshi Kawachi, Akihisa Oyanagi, Ping Li, Qian Su, Hideo Kobayashi, Hitoshi Nakayama, Hiroshi Kikuchi, Fujio Shimizu

Nephrology 8 ( 5 ) 217 - 223 2003.10

Language：English Publishing type：Research paper (scientific journal)

The origin of crescent-forming cells in crescentic glomerulonephritis has not been clarified in spite of the application of monoclonal antibodies (mAbs) against glomerular epithelial cells or monocytes/macrophages. This study was undertaken to characterize the cellular composition of crescents using a new marker, mAb OS-3, produced against macrophagic cells derived from podocytes in normal rat glomerular culture. Monoclonal antibody OS-3 was confirmed to be reactive with some normal epithelial cells of Bowman's capsule. Female Wistar Kyoto rats were injected with rabbit anti-glomerular basement membrane (GBM) serum and killed at 2 h, 1, 3, 7, 14 days and 2 months, respectively. The mAb OS-3-positive cells were segmentally observed in glomeruli at 3 days, increased at 14 days, but decreased at 2 months. These cells lacked reactivity with antipodocalyxin in double immunofluorescence (IF) staining. In immunoelectron microscopy of a glomerulus on day 3 and 7, however, reaction products were observed within cells located on the outer surface of the GBM, which were considered to be podocyte in terms of its localization. In conclusion, we have shown a possibility that damaged podocytes partly constitute crescent-forming cells with phenotypic changes, visualized by positive staining with mAb OS-3. We propose a novel concept of crescent formation, suggesting that crescents may be partly composed of phenotypically changed cells, which could not be detected by typical markers for glomerular epithelial cells or monocytes/macrophages.

DOI： 10.1046/j.1440-1797.2003.00167.x

Genetic polymorphism of NPHS1 modifies the clinical manifestations of Ig a nephropathy Reviewed

Narita, I, S Goto, N Saito, J Song, D Kondo, K Omori, H Kawachi, F Shimizu, M Sakatsume, M Ueno, F Gejyo

LABORATORY INVESTIGATION 83 ( 8 ) 1193 - 1200 2003.8

Language：English Publishing type：Research paper (scientific journal) Publisher：LIPPINCOTT WILLIAMS & WILKINS

Nephrin, the molecule responsible for congenital nephrotic syndrome of Finnish type, is crucial in maintaining the glomerular filtration barrier. Recently, its complete gene structure and common gene polymorphisms in its exons have been reported, although the functional and clinical significance of these polymorphisms has not yet been elucidated. We investigated a possible association of the NPHS1 polymorphisms with the development of Ig A nephropathy (IgAN), as well as the clinical and histologic manifestations in IgAN. A total of 464 Japanese subjects, including 267 patients with histologically proven IgAN and 197 healthy controls with normal urinalysis, were genotyped for the NPHS1 G349A, G2289A, and T3315C polymorphisms. The frequencies of the genotypes, alleles, and estimated haplotypes of NPHS1 polymorphisms were no different between patients with IgAN and the controls. Within the IgAN group, patients carrying at least one G allele of G349A tended to present with more proteinuria, lower renal function, and more severe histopathologic injury than those with the AA genotype, although the time from the first urinary abnormality to the renal biopsy was no different between both groups. The logistic regression analysis indicated that even after adjusting for the effect of proteinuria and hypertension the GG genotype of NPHS1 G349A was an independent risk factor for the deteriorated renal function at the time of diagnosis. This study suggests that the NPHS1 G349A polymorphism may be associated with heavy proteinuria and a decline in renal function in patients with IgAN.

DOI： 10.1097/01.LAB.0000080600.49276.31

Hydrodynamics-based delivery of the viral interleukin-10 gene suppresses experimental crescentic glomerulonephritis in Wistar-Kyoto rats Reviewed

N Higuchi, H Maruyama, T Kuroda, S Kameda, N Iino, H Kawachi, Y Nishikawa, H Hanawa, H Tahara, J Miyazaki, F Gejyo

GENE THERAPY 10 ( 16 ) 1297 - 1310 2003.8

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

Gene therapy is expected to revolutionize the treatment of kidney diseases. Viral interleukin (vlL)-10 has a variety of immunomodulatory properties. We examined the applicability of vIL-10 gene transfer to the treatment of rats with crescentic glomerulonephritis, a T helper 1 (Th 1) predominant disease. To produce the disease, Wistar-Kyoto, rats were injected with a rabbit polyclonal anti-rat glomerular basement membrane antibody. After 3 h, a large volume of plasmid DNA expressing viL-10 (pCAGGS-vlL-10) solution was rapidly injected into the tail vein. pCAGGS solution was similarly injected into control rats (pCAGGS rats). We confirmed the presence of vector-derived vlL-10 mainly in the liver and observed high serum vIL-10 levels in pCAGGSvIL-10-injected rats. Compared with the pCAGGS rats, the pCAGGS-vlL-10 rats showed significant therapeutic effects: reduced frequency of crescent formation, decrease in the number of total cells, macrophages, and CD4(+) T cells in the glomeruli, decrease in urine protein, and attenuation of kidney dysfunction. Using quantitative real-time polymerase chain reaction, we also observed that this model was Th1-predominant in the glomeruli and that the ratio of the transcripts of CD4, interferon-gamma, tumor necrosis factor-a, and monocyte chemotactic protein-1 to the transcripts of glucose-6-phosphate dehydrogenase in the glomeruli were all significantly lower in the pCAGGS-vlL-10 rats than in the pCAGGS rats. These results demonstrate that pCAGGS-vlL-10 gene transfer by hydrodynamics-based transfection suppresses crescentic glomerulonephritis.

DOI： 10.1038/sj.gt.3301988

Expression of podocyte-associated molecules in acquired human kidney diseases Reviewed

K Koop, M Eikmans, HJ Baelde, H Kawachi, E De Heer, LC Paul, JA Bruijn

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 14 ( 8 ) 2063 - 2071 2003.8

Language：English Publishing type：Research paper (scientific journal) Publisher：LIPPINCOTT WILLIAMS & WILKINS

Proteinuria is a poorly understood feature of many acquired renal diseases. Recent studies concerning congenital nephrotic syndromes and findings in genetically modified mice have demonstrated that podocyte molecules make a pivotal contribution to the maintenance of the selective filtration barrier of the normal glomerulus. However, it is unclear what role podocyte molecules play in proteinuria of acquired renal diseases. This study investigated the mRNA and protein expression of several podocyte-associated molecules in acquired renal diseases. Forty-eight patients with various renal diseases were studied. including minimal change nephropathy, focal segmental glomerulosclerosis, IgA nephropathy, lupus nephritis, and diabetic nephropathy, together with 13 kidneys with normal glomerular function. Protein levels of nephrin, podocin, CD2-associated protein, and podocalyxin were investigated using quantitative immunohistochemical assays. Real-time PCR was used to determine the mRNA levels of nephrin, podocin, and podoplanin in microdissected glorneruli. The obtained molecular data were related to electron microscopic ultrastructural changes, in particular foot process width, and to clinical parameters. In most acquired renal diseases, except in IgA nephropathy, a marked reduction was observed at the protein levels of nephrin, podocin, and podocalyxin, whereas an increase of the glomerular mRNA levels of nephrin, podocin, and podoplanin was found, compared with controls. The mean width of the podocyte foot processes was inversely correlated with the protein levels of nephrin (r = -0.443, P < 0.05), whereas it was positively correlated with podoplanin mRNA levels (r = 0.468, P < 0.05) and proteinuria (r = 0.585, P = 0.001). In the diseases studied, the decrease of slit diaphragm proteins was related to the effacement of foot processes and coincided with a rise of the levels of the corresponding mRNA transcripts. This suggests that the alterations in the expression of podocyte-associated molecules represent a compensatory reaction of the podocyte that results from damage associated with proteinuria.

DOI： 10.1097/01.ASN.0000078803.53165.C9

Retinoids regulate the repairing process of the podocytes in puromycin aminonucleoside-induced nephrotic rats Reviewed

A Suzuki, T Ito, E Imai, M Yamato, H Iwatani, H Kawachi, M Hori

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 14 ( 4 ) 981 - 991 2003.4

Language：English Publishing type：Research paper (scientific journal) Publisher：LIPPINCOTT WILLIAMS & WILKINS

The foot processes forming the slit diaphragm are disrupted in diseases associated with proteinuria. Although they are often repairable, regulators for the repairing process remain unknown. By extrapolating from the fact that vitamin A is essential for the nephrogenesis, this study examined whether or not injured podocytes in the middle of the repairing process require retinaldehyde dehydrogenase type 2 (RALDH2), one of the key enzymes to produce all-trans-retinoic acid (ATRA). RALDH2 was dramatically upregulated in podocytes of puromycin aminonucleoside-induced nephrosis (PAN nephrosis) rats. On day 5 of PAN nephrosis, RALDH2 showed the remarkable induction, whereas glomerular expression levels of nephrin and midkine, one of the ATRA target genes, were downregulated. Daily administration of ATRA ameliorated proteinuria, which was accompanied by the improvement in the effacement of the foot processes and by the induction of nephrin and midkine. In contrast, recovery from PAN nephrosis was delayed in rats fed with a vitamin A-deficient diet. Consistently, the promoter region of human nephrin gene (NPHS1) contained three putative retinoic acid response elements (RARE) and showed the enhancer activity in response to ATRA in a dose-dependent manner. This transcriptional activation was regulated through the receptors for retinoids because BMS-189453, an antagonist to the retinoid receptors, counteracted it in a dose-dependent manner. In conclusion, active metabolites of vitamin A, especially ATRA produced by RALDH2 play relevant roles during the repairing process of injured podocytes. The results obtained from PAN nephrosis rats might be applicable to human renal diseases.

DOI： 10.1097/01.ASN.0000057857.66268.8F

Disparate effects of angiotensin II antagonists and calcium channel blockers on albuminuria in experimental diabetes and hypertension: Potential role of nephrin Reviewed

Belinda J. Davis, Zemin Cao, Marc De Gasparo, Hiroshi Kawachi, Mark E. Cooper, Terri J. Allen

Journal of Hypertension 21 ( 1 ) 209 - 216 2003.1

Language：English Publishing type：Research paper (scientific journal)

Objective and design: To explore the effects of various antihypertensive regimes which achieve similar blood pressure control using a range of agents including the angiotensin II type 1 receptor antagonist, valsartan, as monotherapy or in combination with two subclasses of calcium channel blockers (CCBs) (the dihydropyridine, amlodipine and the phenylalkylamine, verapamil) on the progression of renal disease and the expression of the podocyte slit pore protein, nephrin in an accelerated model of diabetic nephropathy. Results: Valsartan treatment reduced systolic blood pressure as assessed by radiotelemetry (135 ± 3 versus diabetic 153 ± 6 mmHg) as well as retarding the increase in albumin excretion rate by approximately 50%. Combination therapy of valsartan with either amlodipine or verapamil was equally effective in reducing blood pressure to valsartan monotherapy (valsartan + amlodipine 129 ± 4 valsartan + verapami1133 ± 6 mmHg
) but was not as effective at reducing albuminuria. A reduction in glomerulosclerosis was observed with valsartan monotherapy with less reduction in injury with the valsartan + amlodipine combination, despite a similar reduction in blood pressure. The decrease in nephrin, in diabetic rats was attenuated by valsartan monotherapy, but not by other treatments. Conclusions: The results of this study demonstrate that despite a similar reduction in blood pressure, the addition of the CCB amlodipine to the All antagonist failed to provide similar renoprotection to that observed with an equihypotensive regimen of valsartan as monotherapy. Furthermore, the depletion in glomerular nephrin expression in diabetic animals was only abrogated by valsartan treatment, the therapy which was most effective at retarding the development of albuminuria in this model. © 2003 Lippincott Williams &amp
Wilkins.

DOI： 10.1097/00004872-200301000-00031

Cloning of rat homologue of podocin: Expression in proteinuric states and in developing glomeruli Reviewed

H Kawachi, H Koike, H Kurihara, T Sakai, F Shimizu

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 14 ( 1 ) 46 - 56 2003.1

Language：English Publishing type：Research paper (scientific journal) Publisher：LIPPINCOTT WILLIAMS & WILKINS

Podocin is identified as a product of the gene mutated in a patient with autosomal recessive steroid-resistant nephrotic syndrome. Although podocin is reported to be located at the slit diaphragm area, the precise role of podocin for maintaining the barrier function of the slit diaphragm has not been clearly elucidated. A rat homologue of podocin was cloned, and the expression of podocin was investigated and then compared with the nephrin and the ZO-1 expressions in rat experimental proteinuric models and in developing glomeruli. Amino acid sequences of rat and human podocin are highly homologous (84.3% identity). The domain structure of podocin is also highly conserved between rat and human. The mRNA expression for podocin was detected in glomeruli and the nerve tissues. The localization of podocin has close proximity to that of nephrin in normal adult rat glomeruli. Podocin staining was restricted to the basal side of the podocyte of the early developing stage, whereas nephrin staining was detected on the basolateral surface of podocyte. The redistribution of podocin was observed in the anti-nephrin antibody (ANA)-induced nephropathy and puromycin aminonucleoside (PAN) nephropathy. The redistribution of podocin paralleled with nephrin in ANA nephropathy but not in PAN nephropathy. Podocin is observed at the site of tight junction newly formed in proteinuric state in PAN nephropathy. It is postulated that podocin is one of the critical components of a slit diaphragm for maintaining the barrier function of the glomerular capillary wall. kawachi@med.niigata-u.ac.jl.

DOI： 10.1097/01.ASN.0000037401.02391.76

抗体による蛋白尿. Invited

河内裕

腎と透析 53 569 - 573 2002.12

糸球体バリアの構造と機能蛋白尿のメカニズム. Invited

清水不二雄, 河内裕

Medical Practice(文光堂) 19 448 - 449 2002.12

遺伝子異常と蛋白尿. Invited

河内裕

小児科 43 617 - 623 2002.12

Therapeutic effect of sulphated hyaluronic acid, a potential selectin-blocking agent, on experimental progressive mesangial proliferative glomerulonephritis Reviewed

Mitsuhiro Matsuda, Kenichi Shikata, Fujio Shimizu, Yasuo Suzuki, Masayuki Miyasaka, Hiroshi Kawachi, Hiroto Kawashima, Jun Wada, Hikaru Sugimoto, Yasushi Shikata, Daisuke Ogawa, Shinichiro J. Tojo, Kazuo Akima, Hirofumi Makino

Journal of Pathology 198 ( 3 ) 407 - 414 2002.11

Language：English Publishing type：Research paper (scientific journal)

The initial event in the process of leukocyte infiltration is characterized by leukocyte rolling on the surface of the endothelium, which is mediated by selectins. P- and L-selectin bind to the sulphated sugar chains of their natural ligands, including sulphated glycolipids such as sulphatide. Recently, it has been demonstrated that sulphated glycolipids and sulphated oligosaccharides interfere with selectin binding pathways. This study synthesized sulphated hyaluronic acid (SHA), which is a potential selectin-blocking agent, and examined its therapeutic effect on the experimental progressive mesangial proliferative glomerulonephritis induced by anti-Thy-1 monoclonal antibody (1-22-3 MAb) after unilateral nephrectomy. The selectin-inhibitory effect of SHA in vitro was confirmed. SHA inhibited the binding of P- and L-selectin to sulphatide, which is a glycolipid ligand for P- and L-selectin, at a concentration of 1.5 μg/ml and 100 μg/ml. Immunohistochemical examination showed that P-selectin was up-regulated in the glomeruli in the 1-22-3 MAb nephritis model, while the ligands for L-selectin were not detected in the glomerular tufts. A single administration of SHA ameliorated proteinuria and glomerular leukocyte infiltration in 24 h after the injection of anti-Thy-1 MAb. Anti-P-selectin MAb, but not anti-L-selectin MAb, inhibited proteinuria and glomerular leukocyte infiltration. To examine further the therapeutic effect of SHA on chronic glomerulonephritis, SHA was administered daily from day 3 to day 14 in this model. Proteinuria and glomerular leukocyte infiltration were significantly diminished in SHA-treated rats on day 14. These results suggest that SHA ameliorated rat progressive mesangial proliferative glomerulonephritis by inhibiting P-selectin-dependent leukocyte infiltration in glomeruli. Sulphated oligosaccharides may be beneficial for the therapy of mesangial proliferative glomerulonephritis. Copyright © 2002 John Wiley &amp
Sons, Ltd.

DOI： 10.1002/path.1209

Fractalkine expression and the recruitment of CX(3)CR1(+) cells in the prolonged mesangial proliferative glomerulonephritis Reviewed

Y Ito, H Kawachi, Y Morioka, T Nakatsue, H Koike, Y Ikezumi, A Oyanagi, Y Natori, Y Natori, T Nakamura, F Gejyo, F Shimizu

KIDNEY INTERNATIONAL 61 ( 6 ) 2044 - 2057 2002.6

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL PUBLISHING INC

Background. We established the reversible and the prolonged models of mesangial proliferative glomerulonephritis (GN) with anti-Thy 1 antibody 1-22-3. However, the essential factors leading to the prolonged glomerular alterations have not been identified.
Methods. The expressions of several chemokines and cytokines were compared in the reversible and the prolonged models. Expression of fractalkine and the number of the fractalkine receptor CX3 CR1-positive cells in the glomeruli in the prolonged model were significantly higher than those in the reversible model. Then, the localization of fractalkine and the characteristics of CX3 CR1(+) cells were analyzed in glomeruli. To elucidate the significance of the fractalkine expression, we analyzed the expression in the model treated with angiotensin II receptor antagonist, candesartan.
Results. Immunostaining of fractalkine was detected on endothelial cells on the fifth day, and fractalkine staining also was detected in the mesangial area on day 14. Major parts of the CX3 CR1(+) cells in the glomeruli were macrophages, especially ED3(+) cells. Candesartan treatment ameliorated the glomerular morphological findings at six weeks after disease induction. Although the treatment did not ameliorate the morphological finding at two weeks, decreased expression of fractalkine and CX3 CR1(+) were already detected at two weeks in rats treated with candesartan.
Conclusions. Fractalkine expression and the recruitment of CX3 CR1(+) cells in glomeruli might play an important role in the development of the prolonged disease. These expressions could be predictors of the prolonged disease of the mesangial proliferative glomerulonephritis.

mAb 5-1-6 nephropathy and nephrin Invited Reviewed

H Kawachi, H Koike, F Shimizu

MICROSCOPY RESEARCH AND TECHNIQUE 57 ( 4 ) 236 - 240 2002.5

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

It is well established that the glomerular capillary wall consists of three layers: endothelial cell, glomerular basement membrane, and the slit diaphragm bridging foot processes of glomerular epithelial cell. Which structure in the glomerular capillary wall represents the primary filter for retaining plasma proteins is not clearly elucidated. An anti-slit diaphragm monoclonal antibody (mAb) 5-1-6 causes massive proteinuria in rats by single intravenous injection, which clearly indicates that the slit diaphragm plays a critical role for maintaining the barrier function of the glomerular capillary wall. Recently, we concluded that mAb 5-1-6 recognized a rat homolog of nephrin, a gene product of NPHS1. The expression of nephrin decreased in puromycin aminonucleoside nephropathy and adriamycin nephropathy as well as mAb 5-1-6-induced nephropathy, which suggested that nephrin was involved in the development of proteinuria in these proteinuric states, In mAb 5-1-6 nephropathy, the slit diaphragm was maintained morphologically normal, although nephrin expression dramatically decreased. The finding suggested that nephrin was not a sole component of the slit diaphragm. To better understand the structure of the slit diaphragm, it is particularly important to identify other components that build up the structure of the slit diaphragm together with nephrin. (C) 2002 Wiley-Liss, Inc.

DOI： 10.1002/jemt.10081

Kidney-targeted naked DNA transfer by retrograde renal vein injection in rats Reviewed

Hiroki Maruyama, Noboru Higuchi, Yuji Nishikawa, Hiroyuki Hirahara, Noriaki Iino, Shigemi Kameda, Hiroshi Kawachi, Eishin Yaoita, Fumitake Gejyo, Jun-Ichi Miyazaki

Human Gene Therapy 13 ( 3 ) 455 - 468 2002.2

Language：English Publishing type：Research paper (scientific journal)

Kidney-targeted gene transfer is expected to revolutionize the treatment of renal diseases. Previous gene transfer methods using nonviral vectors administered via renal arterial, pelvic, or ureteric routes into the glomerulus, tubules, or interstitial fibroblasts have resulted in low-level expression for &lt
1 month. The peritubular capillaries (PTC) network is one of the main targets of kidney transplant rejection and of progressive tubulointerstitial fibrosis, which typifies all progressive renal diseases. To access the PTC, we retrogradely injected a lacZ expression plasmid in Ringer's solution into the renal vein of rats. We detected lacZ expression exclusively in the interstitial fibroblasts near the PTC of the injected kidney by immunoelectron microscopic analysis. Nephrotoxicity attributable to gene transfer was not apparent. We then used a rat erythropoietin (Epo) expression plasmid vector, pCAGGS-Epo, in a reporter assay. We obtained maximal Epo expression when the DNA solution was injected within 5 sec, and with a volume of 1.0 ml. We observed a dose-response relationship between serum Epo levels and the amount of injected DNA up to 100 μg. We detected the transgene-derived Epo mRNA by reverse transcription polymerase chain reaction only in the kidneys injected with pCAGGS-Epo. After an injection of 100 μg of pCAGGS-Epo, the serum Epo levels peaked at 208.3 ± 71.8 mU/ml at week 5, and gradually decreased to 116.2 ± 38.7 mU/ml at week 24. A similar pattern was obtained using smaller doses of plasmid, 2 μg or 30 μg of pCAGGS-Epo. Transgene-derived Epo secretion resulted in significant erythropoiesis. This novel technique is simple and safe, allowing high-level and long-term stable gene expression specific to the fibroblasts near the PTC, and should have therapeutic value for future applications in humans.

DOI： 10.1089/10430340252792585

Rapidly progressive, pauci-immune diffuse crescentic glomerulonephritis in an infant Reviewed

Hiroshi Tanaka, Shinobu Waga, Koichi Suzuki, Tohru Nakahata, Hiroshi Kawachi, Fujio Shimizu, Etsuro Ito

Pediatric Nephrology 17 ( 9 ) 730 - 732 2002

Language：English Publishing type：Research paper (scientific journal)

A Japanese male infant presented with nephrotic syndrome at 41 days. His renal function progressively deteriorated, and he died at 4 months of the age. An open renal biopsy revealed diffuse crescentic glomerulonephritis (CrGN) without immune complex deposition, which is not characteristic of the congenital nephrotic syndrome (CNS). Examination for nephrin antigen using rabbit anti-nephrin extra- and intracellular site antibodies was positive. These clinical observations suggest that the patient had a unique histological variant of CNS. This is the first report of rapidly progressive, pauci-immune diffuse CrGN in infancy.

DOI： 10.1007/s00467-002-0938-7

Molecular structure and function of the slit diaphragm: expression of nephrin in proteinuric states and in developing glomeruli Invited Reviewed

H Kawachi, H Koike, F Shimizu

NEPHROLOGY DIALYSIS TRANSPLANTATION 17 ( 9 ) 20 - 22 2002

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Nephrin is identified as a product of the gene mutated in a patient with congenital nephrotic syndrome of the Finnish type. To analyse the function of nephrin and the relationship of nephrin and the target antigen of nephritogenic anti-rat slit diaphragm monoclonal antibody (mAb) 5-1-6, the rat homologue of nephrin was cloned. The deduced amino acid sequence of rat nephrin shows 82.2% homology to human nephrin. Signal sequences of rat nephrin are 36 amino acids, whereas those of human nephrin are reported to be 22 amino acids. The localization of rat nephrin always coincided with that of mAb 5-1-6 antigen. The specific anti-nephrin antibody recognized the mAb 5-1-6-antigen complex. mAb 5-1-6 reacted with COS cells transfected with rat nephrin cDNA. These results demonstrate that nephritogenic mAb 5-1-6 identifies the extracellular domain of rat nephrin, thereby documenting that nephrin is a functional protein of the slit diaphragm. The staining pattern of nephrin shifted from a linear-like pattern to a discontinuous coarse granular pattern not only in mAb 5-1-6-induced nephropathy, but also in puromycin aminonucleoside nephropathy, adriamycin nephropathy and passive Heymann nephropathy. Rat nephrin is detected first on the basal and lateral side below the junctional complex at the S-shaped body stage. With the interdigitation of foot processes, nephrin becomes concentrated in the slit pore, and finally restricted in the site of the slit diaphragm bridging two adjacent foot processes.

FK506 ameliorates proteinuria and glomerular lesions induced by anti-Thy 1.1 monoclonal antibody 1-22-3 Reviewed

Yohei Ikezumi, Katsue Kanno, Hiroko Koike, Masayuki Tomita, Makoto Uchiyama, Fujio Shimizu, Hiroshi Kawachi

Kidney International 61 ( 4 ) 1339 - 1350 2002

Language：English Publishing type：Research paper (scientific journal) Publisher：Blackwell Publishing Inc.

Background. We have previously reported that CD4 T lymphocytes and their cytokines contribute to development of Thy 1.1 glomerulonephritis (GN). FK506 is reported to suppress the production of Th1 cytokines. The aims of this study were to elucidate the role of Th1 cytokines on mesangial alteration and to examine whether FK506 is available for therapy of mesangial proliferative GN. Methods. The effects of daily treatments of FK506 from day -5 and from day +1 of Thy 1.1 GN induction on glomerular alterations were analyzed. Results. FK506 treatment with 1.0 and 0.3 mg/kg body weight (BW) daily from day 1 to day 4 significantly reduced the glomerular expression of mRNA for interferon-γ (IFN-γ 1.0 mg/kg BW FK506, 32.4% to the placebo group, P &lt
0.01) and IL-2 (55.6%, P &lt
0.01) on day 5. FK506 treatment from day -5 of GN induction reduced proteinuria and glomerular alteration in a dose-dependent manner. Although no side effects were detected in rats with 0.3 mg/kg BW of FK506 treatment from day +1, the treatment also ameliorated proteinuria (day 14, 3.7 ± 0.89 vs. 19.8 ± 12.3 mg/100 g BW/day P &lt
0.05) and glomerular alterations [total cell number, 63.1 ± 3.1 vs. 80.2 ± 7.4, P &lt
0.01
matrix expansion, 0.90 ± 0.30 vs. 1.34 ± 0.27, P &lt
0.05
α-smooth muscle actin (αSMA) expression
1.20 ± 0.12 vs. 1.96 ± 0.29, P &lt
0.01] on day 14. Conclusion. Th1 cytokines may play an important role in the development of mesangial proliferative glomerulonephritis, and could be targets for therapy. FK506 might be available for clinical use.

DOI： 10.1046/j.1523-1755.2002.00259.x

Nephrin expression in the post-natal developing kidney in normotensive and hypertensive rats Reviewed

Fabrice Bonnet, Christos Tikellis, Hiroshi Kawachi, Wendy C. Burns, Peter J. Wookey, Zemin Cao, Mark E. Cooper

Clinical and Experimental Hypertension 24 ( 5 ) 371 - 381 2002

Language：English Publishing type：Research paper (scientific journal)

Nephrin is a slit diaphragm protein and its expression in the developing kidney is largely unknown. In this study, we explored the expression of nephrin in the developmental kidney in spontaneously hypertensive (SHR) and in Wistar-Kyoto (WKY) rats at different time points, from day 5 after birth to adulthood. Real time RT-PCR, in situ hybridization and immunohistochemistry were used to assess and quantify gene and protein expression of nephrin in the kidney. SHR had hypertension at week 10 and albuminuria at week 20. Nephrin expression in both SHR and WKY increased from day 5 to adulthood. Furthermore, both gene and protein expression of nephrin were significantly lower in SHR after birth when compared to WKY at the same age. These findings suggest that both in normotensive and hypertensive rats, nephrin expression increased from birth to the adult age and that down-regulation of nephrin in SHR evident from the early developmental kidney to adulthood may contribute to the development of albuminuria in adult SHR.

DOI： 10.1081/CEH-120004798

Podocyte injuries exacerbate mesangial proliferative glomerulonephritis Reviewed

Y Morioka, H Koike, Y Ikezumi, Y Ito, A Oyanagi, F Gejyo, F Shimizu, H Kawachi

KIDNEY INTERNATIONAL 60 ( 6 ) 2192 - 2204 2001.12

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL SCIENCE INC

Background. From the observations of morphology seen in early phases of the experimental models of the irreversible mesangial proliferative glomerulonephritis, we hypothesized that podocyte injury is one of the important factors in bringing upon irreversible glomerular alterations. To verify this hypothesis, we investigated whether podocyte injury induced by puromycin aminonucleoside (PAN) injection affects the mesangial alterations of anti-Thy 1.1 glomerulonephritis.
Methods. Female Wistar rats were injected with 0.5 mg monoclonal antibody (mAb) 1-22-3 five days after the injection of 10 mg or 5 mg/100 g body weight (BW) of puromycin aminonucleoside (PAN), and sacrificed at 7 days or 8 weeks after the mAb 1-22-3 injection.
Results. Consecutive injections of 10 mg/100 g BW of PAN and mAb 1-22-3 caused the irreversible mesangial alteration with persistent proteinuria (at week 8, proteinuria 100.3 +/- 57.8 mg/24 h, matrix score 1.13 +/- 0.52, collagen type I score 2.04 +/- 0.53, mRNA for collagen type 1227 +/- 79% to the group with a single injection of 1-22-3). Although single injection of 5 mg/100 g BW of PAN was not capable of inducing abnormal proteinuria, consecutive injections of 5 mg/100 g BW of PAN and mAb 1-22-3 also caused irreversible mesangial alteration and persistent proteinuria.
Conclusions. Podocyte injury might be an important factor that exacerbates mesangial proliferation and mesangial matrix expansion. The irreversible mesangial alterations caused by consecutive injections of PAN and mAb 1-22-3 may be a novel model that could be used to analyze the mechanism of progressive mesangial alteration.

DOI： 10.1046/j.1523-1755.2001.00047.x

正常および病的状態下の糸球体濾過障壁. Invited

河内裕

腎と透析 51 602 - 606 2001.12

糸球体濾過障壁を構成する蛋白と機能 Invited

河内裕

腎と透析 50 825 - 830 2001.12

Irbesartan normalizes the deficiency in glomerular nephrin expression in a model of diabetes and hypertension. Reviewed

Bonnet F, Cooper ME, Kawachi H, Allen TJ, Boner G, Gao Z

Diabetologia 44 874 - 877 2001.12

腎糸球体上皮細胞足突起間スリット膜の構造と機能. Invited

河内裕

医学のあゆみ 198 691 - 695 2001.12

Crescent-forming mechanism in an irreversible Thy-1 model in rats Reviewed

A Oyanagi, M Orikasa, H Kawachi, Y Ito, H Koike, F Gejo, F Shimizu

NEPHRON 89 ( 4 ) 439 - 447 2001.12

Language：English Publishing type：Research paper (scientific journal) Publisher：KARGER

Background. The crescent-forming mechanism has not yet been fully clarified and a cell which constitutes a crescent still remains controversial. This study was undertaken to analyze the crescent-forming mechanism in an irreversible Thy-1 model by applying a new marker-recognizing monoclonal antibody (mAb) OS-3. Methods: An irreversible Thy-1 model was induced by an intravenous injection of 500 mug of anti-Thy-1 mAb 1-22-3 to unilaterally nephrectomized Wistar rats. Seven rats were sacrificed 3, 7 and 14 days after the mAb injection respectively and the renal tissues were examined histologically and immunohistochemically. Results: Inflammatory cells were demonstrated mostly in the interstitium, but they were located within advanced cellular crescents in later stages. OS-3, which stained parietal glomerular epithelial cell (PGEC) only partly in a normal rat kidney section, reacted to PGEC more extensively at day 3 and also with cellular crescents at day 7. During the course of this model the podocytes lost their characteristic to be stained by anti-podocalyxcin Ab and obtained a new marker of a diseased state, i.e. to be positively stained by OS-3. Conclusion: Glomerular epithelial cells, but not inflammatory cells, are suggested to directly participate in the crescent formation in early stages, and podocytes with phenotypic changes might be partly involved in the formation of the crescents. Copyright (C) 2001 S. Karger AG, Basel.

DOI： 10.1159/000046117

Isotretinoin alleviates renal damage in rat chronic glomerulonephritis Reviewed

Matthias Schaier, Ingo Lehrke, Kerstin Schade, Christian Morath, Fujio Shimizu, Hiroshi Kawachi, Hermann-Josef Grone, Eberhard Ritz, Jürgen Wagner

Kidney International 60 ( 6 ) 2222 - 2234 2001

Language：English Publishing type：Research paper (scientific journal)

Background. Retinoids, derivatives of vitamin A, have strong anti-inflammatory and antiproliferative properties. We previously demonstrated that the pan-agonists all-transretinoic acid (RA) and isotretinoin (13-cis RA) alleviate renal damage in rat acute glomerulonephritis (GN) induced by anti-Thy-1.1 mAb OX-7. Methods. The present study examined the effects of low dose and high dose treatment with isotretinoin in the chronic glomerulonephritis model, Thy-GN. Thy-GN was induced by a single intravenous injection of monoclonal antibody (mAb) 1-22-3 in uninephrectomized Wistar rats (N = 7 to 10 per group). Control and nephritic groups were treated with vehicle (veh), low dose isotretinoin (2 mg/kg body wt), or high dose isotretinoin (10 mg/kg body wt). The experiment was terminated 60 days after induction of Thy-GN. Results. In animals with Thy-GN, isotretinoin abrogated the increase in blood pressure and significantly reduced albuminuria. Glomerulosclerosis index, glomerular and interstitial cell counts, as well as the area of the interstitial space were significantly lower in nephritic rats treated with low and high dose isotretinoin compared to vehicle-treated nephritic controls. Treatment with isotretinoin also significantly reduced the number of glomerular and interstitial macrophages. The increase of transforming growth factor (TGF)-β1, TGF receptor II and preproendothelin-1 gene expression in vehicle-treated nephritic rats was significantly attenuated by isotretinoin. Conclusions. Treatment with isotretinoin significantly reduces glomerular and interstitial damage in rats with chronic glomerulonephritis as indicated by different functional and histological markers. Retinoids may provide a novel therapeutic option for the treatment of glomerulonephritis.

DOI： 10.1046/j.1523-1755.2001.00056.x

Glomerular CD8+ cells predict progression of childhood IgA nephropathy Reviewed

T. Watanabe, H. Kawachi, Y. Ikezumi, T. Yanagihara, Y. Oda, F. Shimizu

Pediatric Nephrology 16 ( 7 ) 561 - 567 2001

Language：English Publishing type：Research paper (scientific journal)

The aim of this study was to evaluate whether the infiltrating T-lymphocyte can be a predictor in the disease progression of IgA nephropathy (IgAN). Twenty children with IgAN, followed for more than 5 years, were divided into progressive (n=5) and non-progressive groups (n=15). We assessed glomerular and interstitial infiltration of T-lymphocytes (CD4+ and CD8+ cells) and expression of α-smooth muscle actin (α-SMA) and transforming growth factor- β (TGF- β) using an indirect immunofluorescence method on the renal biopsies. We analyzed their relationship to the degree of proteinuria, histological changes, and prognosis. The number of CD8+ cells in glomeruli and in interstitium was higher in the progressive group than in the non-progressive group. The glomerular α-SMA staining was more intensive in the progressive group than in the non-progressive group. Urinary protein and the degree of histological changes were also higher in the progressive group than in the non-progressive group. Among these markers, the number of glomerular CD8+ cells was the most apparent difference between the two groups. In conclusion, these results indicate that the number of glomerular CD8+ cells is the most sensitive predictor of disease progression in childhood IgAN.

DOI： 10.1007/s004670100605

Caveolae in mesangial cells and caveolin expression in mesangial proliferative glomerulonephritis Reviewed

O. Tamai, N. Oka, T. Kikuchi, Y. Koda, M. Soejima, Y. Wada, M. Fujisawa, K. Tamaki, H. Kawachi, F. Shimizu, H. Kimura, T. Imaizumi, S. Okuda

Kidney International 59 ( 2 ) 471 - 480 2001

Language：English Publishing type：Research paper (scientific journal) Publisher：Blackwell Publishing Inc.

Background. Caveolae are plasma membrane invaginations that have a diameter of 40 to 60 nm. Recent evidences have demonstrated that caveolae contain a variety of signal transduction molecules. Caveolin is a marker protein of caveolae and has been proposed to play a negative regulatory role in signal transduction. The aim of this study was to investigate the behavior of caveolae and caveolin in experimental glomerulonephritis, the localization of both platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) receptors in the caveolae membrane, and the regulation of caveolin expression in cultured mesangial cells. Methocis. The expression of caveolin-1 was examined by immunoblotting and immunohistology using anti-caveolin antibody in anti-Thy-1 nephritis. The caveolae membrane fraction of mesangial cells was isolated by sucrose gradient method and expression of PDGF receptor and TGF-β receptor were detected by immunoblotting. The effects of mitogens such as phorbol 12-myristate 13-acetate (PMA) and PDGF on the expression of eaveolin-1 protein and mRNA were also examined in cultured mesangial cells. Results. Caveolin-1 was mainly expressed in glomeruli and was significantly up-regulated in anti-Thy-1 nephritis rat kidney. In cultured mesangial cells, the membrane invaginations of caveolae were revealed by electron microscopy. PDGF receptors abounded in the caveolae membrane and rapidly changed their subcellular distribution after ligand stimulation. In contrast, TGF-β receptors abounded in the non-caveolae membrane and did not change after ligand stimulation. Decreases in caveolin-1 protein, which were associated with increases in mRNA expression after the exposure of PMA or PDGF-BB, suggested an increased turnover of caveolin-1 in mesangial cells stimulated by mitogens. Conclusion. To our knowledge, this electron microscopical study is the first to demonstrate the presence of caveolae in cultured mesangial cells. Caveolae integrate PDGF receptors, and caveolin-1 may play a role in the pathogenesis of the mesangial proliferative glomerular diseases through PDGF signaling.

DOI： 10.1046/j.1523-1755.2001.059002471.x

FAT is a component of glomerular slit diaphragms Reviewed

Tsutomu Inoue, Eishin Yaoita, Hidetake Kurihara, Fujio Shimizu, Tatsuo Sakai, Tatsuya Kobayashi, Kazufumi Ohshiro, Hiroshi Kawachi, Hirokazu Okada, Hiromichi Suzuki, Itaru Kihara, Tadashi Yamamoto

Kidney International 59 ( 3 ) 1003 - 1012 2001

Language：English Publishing type：Research paper (scientific journal)

Background. Slit diaphragms are intercellular junctions of podocytes of the renal glomerulus. The molecular composition of slit diaphragms is still elusive. Slit diaphragms are characterized by the presence of a wide intercellular space. The morphological feature is shared by desmosomes and adherens junctions, which contain members of the cadherin superfamily. Thus, we have hypothesized that some components of slit diaphragms belong to the cadherin superfamily. Consequently, we have isolated cDNA encoding FAT from reverse-transcribed (RT) glomerular cDNA by homology polymerase chain reaction (PCR) using primers based on conserved sequences in cadherin molecules. FAT is a novel member of the cadherin superfamily with 34 tandem cadherin-like extracellular repeats, and it closely resembles the Drosophila tumor suppressor fat. Methods. Expression of FAT was examined in glomeruli of the adult rat kidney by the ribonuclease protection assay and in situ hybridization. To localize the FAT protein in podocytes minutely, we prepared affinity-purified antibody against FAT by immunizing rabbits against an oligopeptide corresponding to the C-terminal 20 amino acids. Results. Expression of FAT mRNA was detected in total RNA from glomeruli. In situ hybridization revealed significant signals in podocytes. Western blot analysis using solubilized glomeruli showed a single band, in which the molecular weight was more than 500 kD. Immunostaining of cultured epithelial cells from rat kidney (NRK52E) revealed FAT accumulation in cell-cell contact sites. In the glomerulus, FAT staining was observed distinctly along glomerular capillary walls. Double-label immunostaining using monoclonal antibody against slit diaphragms (mAb 5-1-6) showed identical localization of anti-FAT antibody and mAb 5-1-6. Furthermore, the double-label immunogold technique with ultrathin cryosections demonstrated that gold particles for FAT cytoplasmic domain were located at the base of slit diaphragms labeled by mAb 5-1-6 and that the cytoplasmic domain of FAT colocalized with ZO-1, a cytoplasmic component associated with slit diaphragms. Conclusion. The molecular structure of FAT and its colocalization with 5-1-6 antigen and ZO-1 indicate that FAT is a component of slit diaphragms.

DOI： 10.1046/j.1523-1755.2001.0590031003.x

Effects of anti-TGF-β type II receptor antibody on experimental glomerulonephritis Reviewed

Hirotake Kasuga, Yasuhiko Ito, Shinji Sakamoto, Hiroshi Kawachi, Fujio Shimizu, Yukio Yuzawa, Seiichi Matsuo

Kidney International 60 ( 5 ) 1745 - 1755 2001

Language：English Publishing type：Research paper (scientific journal)

Background. Renal fibrosis, characterized by the accumulation of extracellular matrix (ECM), is a common histopathological feature of progressive renal disease of diverse etiology. Interaction between transforming growth factor-β (TGF-β) and TGF-β type II receptor (TGF-βIIR) may play an important role in the ongoing fibrotic process. TGF-βIIR and TGF-β have been reported to be up-regulated in human glomerulopathies. In order to block the TGF-β system, many studies have inhibited TGF-β itself, but not its receptors. Our study explored the effects of fully human monoclonal antibody against TGF-βIIR (hTGF-βIIRAb) on experimental proliferative glomerulonephritis. Methods. hTGF-βIIRAb was generated from Xenomice. The expression of TGF-βIIR was studied by immunohistochemistry in normal and anti-Thy-1 nephritis rats. hTGF-βIIRAb or control Ab was injected intraperitoneally at day 0 and day 4 of anti-Thy-1 nephritis, and rats were sacrificed at day 7. Effects of hTGF-βIIRAb were assessed by histological and immunopathological measurements. Results. The specificity of hTGF-βIIRAb was confirmed by ELISA and Western blot analysis. By immunostaining, TGFβIIR expression was up-regulated in the proliferative lesions of anti-Thy-1 nephritis at day 7. In the hTGF-βIIRAb-treated group, the extent of mesangial expansion was less than that in the control group. By immunohistology, α-smooth muscle actin, fibronectin-EDA, and type I collagen were significantly reduced in the hTGF-βIIRAb-treated group. Conclusions. Anti-TGF-βIIR antibody ameliorated ECM accumulation in anti-Thy-1 nephritis. Our data suggest that TGF-βIIR may be one of the therapeutic targets, and that fully human monoclonal antibody against TGF-βIIR may have a new therapeutic potential for renal fibrosis.

DOI： 10.1046/j.1523-1755.2001.00990.x

Nephrin とネフローゼ

河内裕, 清水不二雄

医学のあゆみ 193 66 - 70 2000.12

Severe proteinuria, sustained for 6 months, induces tubular epithelial cell injury and cell infiltration in rats but not progressive interstitial fibrosis

Hiroshi Kikuchi, Hiroshi Kikuchi, Hiroshi Kawachi, Yumi Ito, Yumi Ito, Katsuyuki Matsui, Hitonari Nosaka, Akihiko Saito, Akihiko Saito, Michiaki Orikasa, Masaaki Arakawa, Fujio Shimizu, Fujio Shimizu

Nephrology Dialysis Transplantation 15 799 - 810 2000.1

Background. Sustained proteinuria is reported to be very harmful to the tubulointerstitium, leading to severe interstitial injury. However, it remains unclear whether sustained proteinuria itself is responsible for severe interstitial injury because, in the previously reported models, the development of factors other than proteinuria in tubulointerstitial lesions could not be excluded completely. Methods. After treatment to induce immune tolerance to mouse immunoglobulin, 20 rats were injected with anti-rat slit diaphragm monoclonal antibody (mAb) 5-1-6 twice a week for 6 months and were then sacrificed. Results. mAb 5-1-6 induced massive proteinuria in 11 rats. In nine rats with mild proteinuria, no histological alteration could be detected with light microscopy and immunofluorescence. In nephrotic rats, light microscopy showed minor glomerular abnormalities, with interstitial oedema, tubular epithelial cell degeneration and interstitial cell infiltration. Immunofluorescence revealed increased expression of vimentin and an increased number of OX1-, OX19- and ED1-positive cells. However, we could not detect any accumulation of type I and IV collagen or laminin in the tubulointerstitium. RT-PCR showed that the expression of mRNA for type I collagen was not increased, compared with that in control rats. Conclusions. We succeeded in developing a model of persistent nephrosis without severe glomerular abnormalities, nephrectomy or other manoeuvres known to induce disturbed haemodynamics, using an agent without tubulointerstitial toxicity, and considered it to be suitable for investigating the direct toxicity of proteinuria. In this model, isolated massive proteinuria induced interstitial injury. However, the degree of injury was suggested to be much less than that observed in other previously developed models.

DOI： 10.1093/ndt/15.6.799

The prevention of glomerulosclerosis in rats using traditional Chinese medicine, Sairei-to.(共著)

Ping Li, Ping Li, Hiroshi Kawachi, Yasuhito Suzuki, Akihiko Saito, Michiaki Orikasa, Zhen Sheng Shi, Fujio Shimizu, Fujio Shimizu

Nephrology 5 83 - 89 2000.1

The effects of Sairei-to on chronic irreversible lesions were examined by the administration of drugs after the initiation of irreversible renal lesions by uninephrectomy and anti-thy-1 antibody injection. Twenty-one female Wistar rats were divided into three groups. Group 1 was treated with phosphate-buffered saline (PBS) as a control and groups 2 and 3 were treated with Sairei-to 400 mg/kg bodyweight per day on a daily basis beginning on day 1 (group 2) and day 7 (group 3) to day 42 after the intravenous administration of 500 μg anti-thy-1 monoclonal antibody (MoAb) into uninephrectomized rats, respectively. Statistically significant effects of Sairei-to on proteinuria were observed on days 3, 7, 14, and 21 in group 2 and on day 21 in group 3 as compared with the PBS controls. On day 42, light microscopy showed that Sairei-to ameliorated morphological lesions (Matrix score: 133.7±50.35 for group 1 vs 40.02±24.0 for group 2, P<0.05). The weight of the kidneys in groups given Sairei-to was also lower than that in the PBS control. In addition, immunofluorescent findings showed that Sairei- to suppressed the expression of transforming growth factor-beta (TGF-β), alpha-smooth muscle actin (α-SMA) and collagen type I in glomeruli and decreased the number of ED1- and OX8-positive cells in tubulo-interstitium as well as in glomeruli. In conclusion, Sairei-to blocked the progression of renal lesions in this model even when it was administered after the initiation of the disease. In addition, earlier treatment more effectively prevented the progression of the renal lesions.

DOI： 10.1046/j.1440-1797.2000.00503.x

Renal interstitial cell infiltration in rats induced by immune reactions around proximal tubular basement membrane Reviewed

S. Qian, H. Kawachi, H. Kanazawa, M. Orikasa, H. Takamiya, F. Shimizu

Clinical and Experimental Nephrology 4 ( 3 ) 201 - 206 2000

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Japan

Background. Since it has been shown that the severity of tubulointerstitial nephritis (TIN) and, in particular, the degree of monocyte infiltration, correlate both with the degree of renal impairment at biopsy and with the risk of disease progression, attention has been focused on the development of experimental models of TIN. Methods. We induced TIN by injecting rats with a monoclonal antibody, R3b1 (which binds around the proximal tubular basement membrane (TBM) but not to glomeruli), and also by enhancing the host immune reactions to R3b1 bound around the TBM. Results. R3b1 was demonstrated to bind around the proximal TBM but not to glomeruli in vitro and also in vivo. The binding of R3b1 around the proximal TBM induced mild focal ED-1-positive cell infiltration in the interstitium. Enhanced host immune reaction to bound R3b1 resulted in a transient increase in the number of focally infiltrated ED-1-positive cells in the interstitium, although it shortened the period during which R3b1 was demonstrable around the TBM. There were no significant increases in immunostaining for vimentin and osteopontin, or collagen types I and IV, suggesting that, immunohistochemically, there was no tubular cell damage and no interstitial fibrosis, respectively. Light microscopy revealed focal interstitial cell infiltration, supporting the results obtained by immunofluorescence as ED-1-positive cell infiltration. Tubular cell atrophy, enlargement, and interstitial fibrosis were not observed. Conclusions. The enhancement of host immune reaction to mouse immunoglobulins, i.e., to monoclonal antibody R3b1 bound around the proximal TBM induced by two immunizations, resulted in an increased degree of focal ED-1-positive cell infiltration in the interstitium, but no demonstrable tubular cell injury or interstitial fibrosis. R3b1 did not induce progressive tubulointerstitial injury, even in rats preimmunized and booster-immunized with mouse IgG.

DOI： 10.1007/s101570070022

Molecular composition and function of the slit diaphragm: Nephrin, the molecule responsible for proteinuria Reviewed

Hiroshi Kawachi, Fujio Shimizu

Clinical and Experimental Nephrology 4 ( 3 ) 161 - 172 2000

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Japan

Although the role of the glomerular basement membrane has been emphasized as the barrier for retaining plasma proteins, some recent studies have clearly shown that the slit diaphragm is the structure most likely to be the main barrier in the glomerular capillary wall. Nephrin, ZO-1, P-cadherin and FAT are reported to be components of the slit diaphragm. Of these molecules, the molecular natures of ZO-1 and nephrin are relatively well investigated. ZO-1 was originally identified as a component of the tight junction. Nephrin was identified as a gene product mutated in congenital nephrotic syndrome. ZO-1 is localized at the cytoplasmic surface just at the insertion point of the slit diaphragm, whereas nephrin is located at the outer leaflet of plasma membranes. The expression of nephrin is decreased in several kinds of proteinuric states, and anti-nephrin monoclonal antibody (mAb) 5-1-6 is capable of inducing massive proteinuria, which indicates that nephrin is a functional protein in the slit diaphragm. Nephrin is expected to be a target of therapy for proteinuria. A model of nephrin assembly that forms the structure of the slit diaphragm has been proposed. However, we observed that the slit diaphragm structure was maintained in mAb 5-1-6 nephropathy, although nephrin expression was dramatically decreased. These findings suggest that nephrin is not essential for maintaining the structural integrity of the slit diaphragm. Although it has been proposed that P-cadherin is a core protein in the slit diaphragm, this hypothesis is still controversial. To better understand the structure of the slit diaphragm, it is particularly important to identify other components that build up the structure of the slit diaphragm together with nephrin and ZO-1.

DOI： 10.1007/s101570070017

Benidipine, a long-acting calcium-channel blocker, prevents the progression to end-stage renal failure in a rat mesangioproliferative glomerulonephritis Reviewed

Takamichi Nakamura, Jun-Ei Obata, Makoto Onitsuka, Yuji Shimada, Yoji Yoshida, Hiroshi Kawachi, Fujio Shimizu

Nephron 86 ( 3 ) 315 - 326 2000

Language：English Publishing type：Research paper (scientific journal) Publisher：S. Karger AG

Background: Although the renoprotective effect of calcium-channel blockers (CCBs) has been examined in several models of hypertensive nephropathy, it remains unclear. It also remains to be clarified whether CCBs prevent the progression to end-stage renal failure in chronic progressive glomerulonephritis (GN). A new rat model of progressive mesangioproliferative GN was used to study the effect of benidipine hydrochloride, a long-acting dihydropyridine CCB, on the clinical features and morphological lesions. Methods: This animal model of progressive GN was induced by a single intravenous injection of anti-Thy-1 monoclonal antibody (MoAb 1-22-3) two weeks after unilateral nephrectomy. After 10 weeks of treatment with benidipine (1, 3, and 5 mg/kg body weight, p.o.) or hydralazine (5 mg/kg body weight, p.o.), systolic blood pressure (SBP), urinary protein excretion, creatinine clearance, glomerulosclerosis index, tubulointerstitial lesion index, glomerular cross-sectional area, and glomerular expression of transforming growth factor-β (TGF-β) and α-smooth muscle actin (α-SMA) were measured. Results: Untreated rats developed hypertension, massive proteinuria, renal dysfunction, severe glomerular and tubulointerstitial injury, higher glomerular size, and marked glomerular staining for TGF-β and α-SMA, while uninephrectomized control rats did not. Each dose of benidipine and hydralazine equally reduced SBP to uninephrectomized control levels. Three and five mg/kg/day of benidipine increased creatinine clearance, ameliorated glomerular and tubulointerstitial injury, and reduced glomerular staining for TGF-β and α-SMA, but 1 mg/kg/day of benidipine and hydralazine failed. Only a dose of 5 mg/kg/day of benidipine reduced glomerular size, although it did not reduce the size to control levels. Conclusion: These results indicate that in a rat model of progressive mesangioproliferative GN, benidipine prevents the progression to end-stage renal failure in a dose-dependent manner. This renoprotective action is associated with the suppression of glomerular expression of TGF-β and α-SMA. Copyright (C) 2000 S. Karger AG, Basel.

DOI： 10.1159/000045787

An anti-CD5 monoclonal antibody ameliorates proteinuria and glomerular lesions in rat mesangioproliferative glomerulonephritis Reviewed

Yohei Ikezumi, Hiroshi Kawachi, Shinichi Toyabe, Makoto Uchiyama, Fujio Shimizu

Kidney International 58 ( 1 ) 100 - 114 2000

Language：English Publishing type：Research paper (scientific journal) Publisher：Blackwell Publishing Inc.

Background: Increased numbers of lymphocytes have been identified in biopsy specimens of human mesangial proliferative glomerulonephritis (GN). However, the causal relationship between infiltrating T lymphocytes and mesangial changes in mesangial proliferative GN has not been previously evaluated. In this study, we elucidated the role of lymphocytes in the development of mesangial proliferative GN. Method: Immunohistological and flow cytometric analyses as well as a reverse transcription-polymerase chain reaction (RTPCR) studies were performed in monoclonal antibody (mAb) 1-22-3- induced Thy 1.1 GN. To elucidate the role of these lymphocytes, depletion studies were carried out using anti-CD8 mAb (OX-8), which depletes both CD8+ T lymphocytes and natural killer (NK) cells and anti-CD5 mAb (OX-19), which depletes both CD4+ and CD8+ T lymphocytes. Results: Immunofluorescence (IF) studies revealed that NK cells and CD4+ T lymphocytes were recruited into glomeruli. Glomerular mRNA expression for interferon-γ interleukin-2 (IL-2), IL-10, and perforin increased after induction of GN. Increased expressions of several chemokines, which have the potential to attract lymphocytes, were also detected. Anti-CD8 mAb treatment completely prevented the recruitment of NK cells: however, it had no protective effect on proteinuria and mesangial injury. By contrast, anti-CD5 mAb treatment suppressed the recruitment of CD4+ T lymphocytes into glomeruli and reduced proteinuria (60.4 ± 25.7 vs. 120.0 ± 32.3 mg/day, P &lt
0.05) and mesangial changes evaluated by total number of cells in glomeruli (63.2 ± 6.0 vs. 81.4 ± 5.9, P &lt
0.01) and α- smooth muscle actin staining score (1.4 ± 0.2 vs. 2.2 ± 0.4, P &lt
0.01) on day 14 after induction of GN. mRNA expression for IL-2 was significantly reduced by OX-19 treatment. Conclusion: T lymphocytes participate in the development of mesangial proliferative GN.

DOI： 10.1046/j.1523-1755.2000.00145.x

Cloning of rat nephrin: Expression in developing glomeruli and in proteinuric states Reviewed

Hiroshi Kawachi, Hiroko Koike, Hidetake Kurihara, Eishin Yaoita, Michiaki Orikasa, Michael A. Shia, Tatsuo Sakai, Tadashi Yamamoto, David J. Salant, Fujio Shimizu

Kidney International 57 ( 5 ) 1949 - 1961 2000

Language：English Publishing type：Research paper (scientific journal) Publisher：Blackwell Publishing Inc.

Background. Nephrin is identified as a product of the gene mutated in a patient with congenital nephrotic syndrome of the Finnish type. However, its precise localization and function are not yet fully clarified. Methods. To clone the rat homologue of nephrin, polymerase chain reaction (PCR) was employed. To elucidate the localization and expression of nephrin, immunohistological analysis with a specific antirat nephrin antibody, reverse transcription-PCR, and RNase protection assay were performed. Results. Amino acid sequences of rat and human nephrin are highly homologous (82.2% identity). The domain structure of nephrin is also highly conserved between rats and humans. The rat nephrin was detected only in kidney glomeruli along glomerular capillary walls, and its localization was always identical to that of the anti-slit diaphragm monoclonal antibody (mAb) 5-1-6-recognized antigen in normal matured and fetal rat glomeruli and in the glomeruli of proteinuric states. The nephrin staining pattern was clearly distinguished from that of zonula occludens-1 (ZO-1), α3-integrin, or podocalyxin. mRNA expression for nephrin was first detected in the fetal rat kidneys at 18.5 embryonic days. Nephrin mRNA expression decreased just after injection of mab 5-1-6 (47.4%) or puromycin aminonucleoside (51.2%), and the staining pattern of nephrin shifted from a linear to a granular pattern in both proteinuric states. Conclusions. Nephrin is localized in slit diaphragm in the matured glomeruli and is identical with mAb 5-1-6 antigen. Nephrin is involved in the development of proteinuria not only in mab 5-1-6 nephropathy, but also in puromycin aminonucleoside nephropathy.

DOI： 10.1046/j.1523-1755.2000.00044.x

Analysis of cytokine production in the colon of nude mice with experimental colitis induced by adoptive transfer of immunocompetent cells from mice infected with a murine retrovirus Reviewed

Hidehisa Suriki, Kenji Suzuki, Yasuyuki Baba, Katsuhiko Hasegawa, Rintaro Narisawa, Yoshiaki Okada, Toshiaki Mizuochi, Hiroshi Kawachi, Fujio Shimizu, Hitoshi Asakura

Clinical Immunology 97 ( 1 ) 33 - 42 2000

Language：English Publishing type：Research paper (scientific journal) Publisher：Academic Press Inc.

LP-BM5 murine leukemia virus (MuLV) is known to induce murine AIDS (MAIDS). We have shown that Sjogren's syndrome (SjS)-like exocrinopathy can be induced in mice with MAIDS and that adoptive transfer of spleen cells from MAIDS mice can induce inflammatory bowel disease-like colitis as well as SjS-like exocrinopathy in nude mice. To assess the role of interferon (IFN)-γ and interleukin (IL)-10 in the pathogenesis of our experimental model, we tried to identify the cells producing these cytokines and their localization in the colitis lesions in situ. Expression of mRNA for IFN-γ and IL-10 was assessed by RT-PCR, and protein expression of these cytokines was also analyzed in frozen sections of colon by double-color-staining immunofluorescence (IF). An increase of IFN-γ and IL-10 mRNA was detected in the colon of mice with colitis, but not in that of control mice. Double-color IF showed that Mac-1+ cells were positive for IFN-γ or IL-10 and that most CD4+ T cells were positive for IL-10, although the population of IFN-γ-positive CD4+ T cells was low. In our experimental colitis model, Mac-1+ macrophages that produce both IFN-γ and IL-10 might play a crucial role in the pathogenesis of colitis in combination with CD4+ T cells. (C) 2000 Academic Press.

DOI： 10.1006/clim.2000.4909

Localization of Smad6 and Smad7 in the rat kidney and their regulated expression in the anti-Thy-1 nephritis Reviewed

Keiko Uchida, Kosaku Nitta, Hideo Kobayashi, Hiroshi Kawachi, Fujio Shimizu, Wako Yumura, Hiroshi Nihei

Molecular Cell Biology Research Communications 4 ( 2 ) 98 - 105 2000

Language：English Publishing type：Research paper (scientific journal) Publisher：Academic Press Inc.

Under normal conditions, kidney expresses Smad6 and Smad7 most abundantly among the organs of the body. To understand the physiological roles of these Smad expressions in the kidney, we first identified the sites of Smad6 and Smad7 expression in the rat kidney by in situ hybridization. The expression of Smad7 in the rat kidney was only observed in the glomeruli, while Smad6 was expressed in both the glomeruli and thick ascending limb of Henle's loop. In order to investigate whether Smad6 and 7 are also involved in the negative feedback loop of TGF-β signaling in vivo, we examined the changes of mRNA levels of these Smads in the glomeruli of rat anti-Thy1 (1-22-3) nephritis, a model where the expression of TGF-β in the glomeruli has been shown to be most up-regulated from day 4 to 14 after the antibody injection. Unexpectedly, 7 days after injection, the levels of Smad6 and Smad7 did not increase but rather decreased to ∼ 70% of the levels on day 0. During that period, Smad7 immunostaining was observed in the glomerular endothelial cells (GEN) where Smad3 immunostaining was also observed. This suggested that Smad7 expression was not augmented by the TGF-β signal in GEN in vivo in anti-Thy-1 nephritis. The absence of up-regulation of these inhibitory Smads may be involved in the pathogenesis of anti-Thy-1 nephritis. © 2000 Academic Press.

DOI： 10.1006/mcbr.2000.0258

Nephritogenic mAb 5-1-6 is directed art the extracellular domain of rat nephrin Reviewed

PS Topham, H Kawachi, SA Haydar, S Chugh, TA Addona, KB Charron, LB Holzman, M Shia, F Shimizu, DJ Salant

JOURNAL OF CLINICAL INVESTIGATION 104 ( 11 ) 1559 - 1566 1999.12

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC CLINICAL INVESTIGATION INC

mAb 5-1-6 identifies an antigen on rat podocyte slit-diaphragms and induces severe proteinuria when injected into rats. Nephrin, an Ig-like transmembrane protein that is mutated in congenital nephrotic syndrome of the Finnish type, has been localized to the slit-diaphragm on human podocytes. Here we document that the mAb 5-1-6 antigen is rat nephrin. After incubation of rat glomeruli with this mAb, the antibody/antigen complex was chemically cross-linked, extracted, and immunoprecipitated, prior to Western analysis. By mass spectrometry and 2D gel electrophoresis, we identified several peptides with complete identity to human nephrin. In addition, the 185-kDa protein immunoprecipitated by mAb 5-1-6 from rat glomerular extracts reacts with a rabbit anti-mouse nephrin antibody. Finally, nephrin and the mAb 5-1-6 antigen have identical glomerular localization patterns on immunofluorescence of rat kidney. These results demonstrate that the nephritogenic mAb 5-1-6 identifies the extracellular domain of nephrin, thereby documenting the importance of the slit-diaphragm and its component, nephrin, in the regulation of glomerular permselectivity.

Expression of the molecule detectable by anti-propolypeptide of Von Willebrand factor antibody in rat mesangial cells in anti-Thy 1.1 mAb 1-22-3 induced glomerulonephritis: A marker of injured mesangial cells Reviewed

Minako Wakasugi, Hiroshi Kawachi, Saori Omori, Junichi Takagi, Shinichi Nishi, Masaaki Arakawa, Fujio Shimizu

Nephron 82 ( 4 ) 338 - 347 1999

Language：English Publishing type：Research paper (scientific journal)

We have previously reported that propolypeptide of von Willebrand factor (pp-vWF) binds to collagen with an affinity comparable to that of mature vWF, inhibits collagen-induced platelet aggregation, is cross-linked with laminin, and also serves as a ligand for very-late antigen 4 integrin. These observations from in vitro experiments suggest that pp-vWF is incorporated in the extracellular matrix and affects the cell-matrix interaction and that pp-vWF functions in leukocyte recruitment to inflammatory and vascular injury sites. We, therefore, hypothesize that pp-vWF might be involved in the induction and/or progression of mesangial proliferative glomerulonephritis. To test this hypothesis, we examined the kinetics of the immunostaining of the molecule detectable by an affinity-purified anti-pp-vWF antibody in rat glomeruli in monoclonal antibody 1-22-3 induced glomerulonephritis, Immunostaining by pp-vWF antibody was observed in the nuclear rim of mesangial cells in monoclonal antibody 1-22-3 induced glomerulonephritis. Positive staining first appeared on day 10 after monoclonal antibody injection, when mesangial cell proliferation and mesangial matrix expansion had already begun. Staining was still detected on day 56, when morphologic alterations observed by light microscopy had been normalized. The pp-vWF antibody recognized molecule appeared later than α-smooth muscle actin or collagen type I. Positive staining was not detected in cultured mesangial cells. It should be noted that the positive staining by pp-vWF antibody in mesangial cells was still detected in previously injured glomeruli that have almost recovered normal morphology. These observations indicate that positive staining by pp-vWF antibody could be a very useful marker for identifying a past episode of injury in mesangial cells.

DOI： 10.1159/000045449

Blocking angiotensin II ameliorates proteinuria and glomerular lesions in progressive mesangioproliferative glomerulonephritis Reviewed

Takamichi Nakamura, Jun-Ei Obata, Hideaki Kimura, Shinichi Ohno, Yoji Yoshida, Hiroshi Kawachi, Fujio Shimizu

Kidney International 55 ( 3 ) 877 - 889 1999

Language：English Publishing type：Research paper (scientific journal) Publisher：Blackwell Publishing Inc.

Background. The renin-angiotensin system is thought to be involved in the progression of glomerulonephritis (GN) into end-stage renal failure (ESRF) because of the observed renoprotective effects of angiotensin- converting enzyme inhibitors (ACEIs). However, ACEIs have pharmacological effects other than ACE inhibition that may help lower blood pressure and preserve glomerular structure. We previously reported a new animal model of progressive glomerulosclerosis induced by a single intravenous injection of an anti-Thy-1 monoclonal antibody, MoAb 1-22-3, in uninephrectomized rats. Using this new model of progressive GN, we examined the hypothesis that ACEIs prevent the progression to ESRF by modulating the effects of angiotensin II (Ang II) on the production of transforming growth factor-β (TGF-β) and extracellular matrix components. Methods. We studied the effect of an ACEI (cilazapril) and an Ang II type 1 receptor antagonist (candesartan) on the clinical features and morphological lesions in the rat model previously reported. After 10 weeks of treatment with equihypotensive doses of cilazapril, cilazapril plus Hoe 140 (a bradykinin receptor B2 antagonist), candesartan, and hydralazine, we examined systolic blood pressure, urinary protein excretion, creatinine clearance, the glomerulosclerosis index, and the tubulointerstitial lesion index. We performed a semiquantitative evaluation of glomerular immunostaining for TGF-β and collagen types I and III by immunofluorescence study and of these cortical mRNA levels by Northern blot analysis. Results. Untreated rats developed massive proteinuria, renal dysfunction, and severe glomerular and tubulointerstitial injury, whereas uninephrectomized control rats did not. There was a significant increase in the levels of glomerular protein and cortical mRNA for TGF-β and collagen types I and III in untreated rats. Cilazapril and candesartan prevented massive proteinuria, increased creatinine clearance, and ameliorated glomerular and tubulointerstitial injury. These drugs also reduced levels of glomerular protein and cortical mRNA for TGF-β and collagen types I and III. Hoe 140 failed to blunt the renoprotective effect of cilazapril. Hydralazine did not exhibit a renoprotective effect. Conclusion. These results indicate that ACEIs prevent the progression to ESRF by modulating the effects of Ang II via Ang II type 1 receptor on the production of TGF-β and collagen types I and III, as well as on intrarenal hemodynamics, but not by either increasing bradykinin activity or reducing blood pressure in this rat model of mesangial proliferative GN.

DOI： 10.1046/j.1523-1755.1999.055003877.x

Role of mesangial cell damage in progressive renal disease Reviewed

F Shimizu, H Kawachi, M Orikasa

KIDNEY & BLOOD PRESSURE RESEARCH 22 ( 1-2 ) 5 - 12 1999

Language：English Publishing type：Research paper (scientific journal) Publisher：KARGER

Mesangial cell proliferation and matrix accumulation are considered to contribute to the development of glomerulosclerosis. To investigate the pathological role played by mesangial cell damage in progressive renal disease appropriate progressive models initiated by a mesangial cell injury should be developed, Monoclonal antibody (mAb) 1-22-3 (IgG3) recognizes a critical epitope of the Thy-1.1 molecule on the mesangial cell surface, binding of which induces more severe mesangial cell injury than in the case of OX-7, a commercially obtainable anti-Thy-1.1 mAb, The mAb 1-22-3 has enabled us to develop irreversible models of renal damage induced by two consecutive injections, by a single injection into unilaterally nephrectomized rats, or by a single simultaneous injection with another mAb, in addition to the corresponding reversible model. Detailed examinations using the combination of both types of models, all of which are initiated by an immune mechanism directed against the identical epitope on the Thy-1.1 molecule, are expected to provide new insights into mechanisms of irreversible renal injury initiated by mesangial cell damage and also to develop novel and rational therapeutic approaches to progressive, primarily mesangial diseases.

DOI： 10.1159/000025903

単クローン抗体により惹起されるモデルを用いた糸球体腎炎の発症, 進展機序の解析.

河内裕

新潟医学会雑誌 112 219 - 225 1998.12

.Effect of Sairei-to on irreversible glomerular sclerotic lesions in rats. Reviewed

Li,P, Kawachi,H, Orikasa,M, Shi,Z.S, Shimizu,F

Nephrology 4 49 - 59 1998.12

腎とアポトーシス Reviewed

河内裕, 清水不二雄

医学のあゆみ 187 486 - 489 1998.12

Altered anionic GBM components in monoclonal antibody against slit diaphragm-injected proteinuric rats Reviewed

Yoshihide Fujigaki, Mitsumasa Nagase, Sumi Hidaka, Katsuyuki Matsui, Mikiko Shirai, Hitonari Nosaka, Hiroshi Kawachi, Fujio Shimizu, Akira Hishida

Kidney International 54 ( 5 ) 1491 - 1500 1998

Language：English Publishing type：Research paper (scientific journal) Publisher：Blackwell Publishing Inc.

Background. We previously reported that monoclonal antibody (mAb) 5-1-6 bound to renal filtration slits induces massive proteinuria without causing ultrastructural changes in the glomerulus. This study evaluated the underlying mechanisms of the increase in glomerular permeability. Methods. The distribution of endogenous albumin and IgG in the glomerular basement membrane (GBM) was studied in in situ drip-fixed glomeruli of Munich-Wistar rats by use of immunogold immunocytochemistry in the presence and absence of mAb 5-1-6. The density of foot process glycocalyx components was estimated by labeling with Limax fluvus lectin- or Helix pomatia lectin-gold complexes. Anionic sites in the GBM were examined by labeling with cationic gold at pH 2.0 or 7.4. Carboxyl groups, which also furnish all anionic charge to the GBM, were examined by specific biotinylation and colloidal gold probe methods. In addition, the infusion-staining of anionic sites was performed by use of ruthenium red in both Munich-Wistar and Wistar rats. Results. The urinary excretion of albumin and IgG was increased markedly in the treated rats, indicating a non-selective barrier defect. In the control rats, albumin and IgG molecules were mainly located along the inner half of the GBM, and to a lesser degree in the lamina rara externa. In the treated rats, the albumin and IgG moieties were more equally distributed throughout the width of the GBM. Newly appearing, small dense peaks at the outer side of the GBM were evident, indicating a barrier function of outer zone of the GBM and/or epithelial cell layer. No intergroup differences in the density of lectin binding sites on foot processes were seen. The reduction in the number of ruthenium red-positive anionic sites and cationic gold (pH 2.0)-labeled anionic sites in the lamina rara externa was significant in the treated rats at day 3, indicating a possible alteration of charged proteoglycan in the lamina rara externa. No such changes were seen with cationic gold (pH 7.4)- labeled anionic sites in the GBM. The density of labeled carboxyl groups was significantly reduced in the treated rats relative to the controls. Conclusions. These results show that the injection of mAb 5-1-6 induced a perturbation of the charge- and probably the size-selective glomerular filtration barrier. The observed reduction in the levels of various negatively charged substances resulted in massive proteinuria, implying that alteration of target antigens can affect the integrity of the GBM constituents maintaining the normal barrier function.

DOI： 10.1046/j.1523-1755.1998.00131.x

Comparative nephritogenicity of two monoclonal antibodies that recognize different epitopes of rat Thy-1.1 molecule Reviewed

H. Nakayama, T. Oite, H. Kawachi, T. Morioka, H. Kobayashi, M. Orikasa, M. Arakawa, F. Shimizu

Nephron 78 ( 4 ) 453 - 463 1998

Language：English Publishing type：Research paper (scientific journal)

The pathophysiological role of the Thy-1.1 molecule expressed on rat mesangial cells with regard to mesangial cell dysfunction and injury remains unknown. The mechanism of Thy-1.1-associated injury has now been investigated with two monoclonal antibodies, 1-22-3 and OX7, that recognize different epitopes of Thy-1.1. Mesangiolysis and mesangial cell proliferation were more marked in rats injected with 1-22-3 than in those treated with OX7. Immunostaining for rat complement component C3 and also C9 was similar in the kidneys of rats 1 h after injection of either antibody. Alpha smooth muscle actin was first detected 3 days after injection of 1-22-3 and peaked on day 5
type I collagen staining showed a mesangial pattern on days 5 and 10. The staining for alpha smooth muscle actin and type I collagen was less intense in OX7-treated rats than in the 1-22-3-injected rats. The amounts of mRNAs encoding collagen types I and III peaked 5 days after injection of 1-22-3 and 10 days after injection of OX7. Rats injected with 1-22-3 developed proteinuria that was already marked on day 1 and peaked at 150 mg/day on day 3, whereas OX7 induced a low grade of proteinuria with large interindividual variability on day 3. Immunostaining for rat C3 in the normal rat kidneys, incubated in vitro with 1-22-3 or OX7 followed by incubation with normal rat fresh serum as a complement source, as well as the levels of serum complement activity, CH50, 30 min after injection of 1-22-3 or OX7 were similar, suggesting that the difference in the nephritogenicity of these two antibodies is not attributable to a difference in their complement-fixing activities, but rather may result from the difference in epitope specificities. The epitope recognized by 1-22-3 thus appears to be important in the initiation and progression of antibody-induced nephritis.

DOI： 10.1159/000044975

Immunohistochemical identification of type B intercalated cells in the rat kidney by a monoclonal antibody Reviewed

Haruko Yanase, Michiaki Orikasa, Fujio Shimizu, Hiroshi Kawachi, Toshihiko Iwanaga

Archives of Histology and Cytology 61 ( 2 ) 151 - 161 1998

Language：English Publishing type：Research paper (scientific journal) Publisher：Japan Society of Histological Documentation

We recently produced monoclonal antibodies using macrophagic cells derived from cultured rat glomeruli as the antigen. One of the antibodies, named OS-3, was found to detect a cell population scattered in collecting ducts of the rat kidney as well as macrophages in various tissues. The present study deals with the cellular and subcellular localization of immunoreactivities with OS-3 in the kidney and other organs of rats. Double immunostaining using OS-3 and an antiserum against either calbindin or epidermal cytokeratin showed that OS-3-immunoreactive cells exist exclusively in both the connecting segment and cortical collecting duct, and differ from calbindin- or cytokeratinpositive epithelial cells. Ultrastructurally, OS-3- immunoreactive cells appeared spherical in shape with few cytoplasmic microprojections on the narrow apical surface. Their relatively dark cytoplasm contained numerous mitochondria and a developed tubulo-vesicular system. The intense immunoreactivity was selectively localized in the basolateral membrane exhibiting shallow but complicated infoldings. Distribution and ultrastructural properties of the OS-3-immunoreactive cells showed that they were type B intercalated cells, which are engaged in the regulation of the acid-base balance mainly by secreting HCO3 -. Another positive staining with OS-3 was found in the macula densa and some epithelial cells of Bowman's capsule, the former monitoring Cl- concentrations in the urine. Immunoblotting of extracts from the rat kidney demonstrated a protein band immunoreactive to OS-3 at a molecular weight of 43 kDa. Aside from the kidney, a specific and intense immunoreactivity with OS-3 was also found in the epithelial cells of the pancreatic excretory duct and in the secretory cells of the salivary, pyloric and duodenal glands, all of which are HCO3 - secreting cells. These immunohistochemical findings imply that OS-3 is useful for the detection of type B intercalated cells and recognizes a functional molecule involved in the production/secretion of HCO3 - or transport of Cl- .

DOI： 10.1679/aohc.61.151

Quantification of apoptotic cells using isolated glomeruli Reviewed

S Omori, T Morioka, M Wakasugi, H Kawachi, M Arakawa, F Shimizu, T Oite

NEPHRON 77 ( 4 ) 474 - 478 1997.12

Language：English Publishing type：Research paper (scientific journal) Publisher：KARGER

In order to detect apoptotic cells in the kidney, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method is used in tissue sections. But the number of apoptotic cells per glomerulus in several experimental models of nephritides is about 1 per single tissue section even at peak levels. In this study, we have reported that the TUNEL method and immunostaining of cell-specific markers to a whole isolated glomerulus in combination with laser scan microscopy are potentially useful methods for the analysis of cell turnover within glomeruli.

Slit diaphragm-reactive nephritogenic MAb 5-1-6 alters expression of ZO-1 in rat podocytes Reviewed

H Kawachi, H Kurihara, PS Topham, D Brown, MA Shia, M Orikasa, F Shimizu, DJ Salant

AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY 273 ( 6 ) F984 - F993 1997.12

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER PHYSIOLOGICAL SOC

Monoclonal antibody (MAb) 5-1-6 identifies a 51-kDa protein (p51) on rat podocyte foot processes and causes severe complement-and leukocyte-independent proteinuria when injected into rats. In the studies reported here, we used various immunohistological techniques to define the precise location of p51 and its relationship to ZO-1, a known component of the podocyte slit diaphragm in adult rat glomeruli. Our results demonstrate that p51 and ZO-1 lie close to each other on opposite sides of the podocyte plasma membrane at the point of insertion of the slit diaphragm: ZO-1 on the cytoplasmic face and p51 on the slit diaphragm and adjoining outer leaflet of the plasma membrane bordering the filtration slits. In addition to their geographic proximity, there appears to be a relationship between p51 and ZO-1.After MAb 5-1-6 injection, there was a progressive decline in stainable ZO-1 in the podocytes of heavily proteinuric rats. In addition, Western blot analysis of glomerular lysates showed that the decline in staining was due to a loss of immunoreactive ZO-1 rather than redistribution or diffusion of the protein. Simultaneously, the distribution of glomerular-bound MAb 5-1-6 became more clumped, apparently because of partial endocytosis into a lysosomal compartment, while the slit diaphragms remained morphologically intact. These findings suggest that MAb 5-1-6 alters the molecular composition of the slit diaphragm and thereby affects the glomerular permeability barrier.

Creation of an in vivo cytosensor using engineered mesangial cells. Reviewed

Kitamura M, Kawachi H

J Clin Invest 100 1394 - 1399 1997.12

Suppressive effects of Sairei-to on monoclonal antibody 1-22-3-induced glomerulonephritis: Analysis of effective components Reviewed

Ping Li, Hiroshi Kawachi, Tetsuo Morioka, Michiaki Orikasa, Takashi Oite, Zhen Sheng Shi, Fujio Shimizu

Pathology International 47 ( 7 ) 430 - 435 1997

Language：English Publishing type：Research paper (scientific journal)

The effects of traditional Chinese medicine (Sairei-to) on experimental glomerulonephritis induced in rats by monoclonal antibody (mAb) 1-22-3 injection was examined. The level of proteinuria in the Sairei-to-treated group was significantly lower than that in the PBS treated group. This suppressive effect was caused by the major component of Sairei-to, Syo- saiko-to but not by another component, Gorei-san. The suppressive effect of Syo-saiko-to was identified in its components (Bupleuri radix, Pinelliae tuber and Zingiberis rhizoma), but not in the other combined components (Ginseng radix and Zizyphi fructus). Further study revealed that the suppressive effects of the combined components were mainly derived from Bupleuri radix. It was demonstrated that the actual active ingredient is probably Salkosaponin-d. Light microscopy revealed that Sairei-to and its effective components suppressed the proliferation of mesangial cells end mesangial matrix expansion. Semiquantitative morphological studies of glomerular lesions on the eighth day showed that Syo-saiko-to and its combined components (Bupleuri radix, Zingiberis rhizoma and Pinelliae tuber) suppressed mesangial matrix expansion significantly compared with phosphate- buffered saline control groups (matrix score: 28.0±19.1 vs 102.3±14.1
30.9±30.1 vs 102.3±14.1, P&lt
0.005, respectively). It was concluded that Salkosaponin-d, as well as Bupleuri radix, Syo-saiko-to and Sairei-to can suppress proteinuria and morphological changes in the rat glomeruionephritis model induced by mAb 1-22-3.

DOI： 10.1111/j.1440-1827.1997.tb04520.x

Slit diaphragm-reactive nephritogenic MAb 5-1-6 alters expression of ZO-1 in rat podocytes Reviewed

Hiroshi Kawachi, Hidetake Kurihara, Peter S. Topham, Dennis Brown, Michael A. Shia, Michiaki Orikasa, Fujio Shimizu, David J. Salant

American Journal of Physiology 273 ( 6 ) F984 - F993 1997

Language：English Publishing type：Research paper (scientific journal)

Monoclonal antibody (MAb) 5-1-6 identifies a 51-kDa protein (p51) on rat podocyte foot processes and causes severe complement- and leukocyte-independent proteinuria when injected into rats. In the studies reported here, we used various immunohistological techniques to define the precise location of p51 and its relationship to ZO-1, a known component of the podocyte slit diaphragm in adult rat glomeruli. Our results demonstrate that p51 and ZO-1 lie close to each other on opposite sides of the podocyte plasma membrane at the point of insertion of the slit diaphragm: ZO-1 on the cytoplasmic face and p51 on the slit diaphragm and adjoining outer leaflet of the plasma membrane bordering the filtration slits. In addition to their geographic proximity, there appears to be a relationship between p51 and ZO-1. After MAb 5-1-6 injection, there was a progressive decline in stainable ZO-1 in the podocytes of heavily proteinuric rats. In addition, Western blot analysis of glomerular lysates showed that the decline in staining was due to a loss of immunoreactive ZO-1 rather than redistribution or diffusion of the protein. Simultaneously, the distribution of glomerular-bound MAb 5-1-6 became more clumped, apparently because of partial endocytosis into a lysosomal compartment, while the slit diaphragms remained morphologically intact. These findings suggest that MAb 5-1-6 alters the molecular composition of the slit diaphragm and thereby affects the glomerular permeability barrier. glomerular epithelial cell
foot process
tight junction
proteinuria
51-kilodalton protein Copyright © 1997 the American Physiological Society.

Quantification of apoptotic cells using isolated glomeruli Reviewed

Saori Omori, T. Morioka, M. Wakasugi, H. Kawachi, M. Arakawa, F. Shimizu, T. Oite

Nephron 77 ( 4 ) 474 - 478 1997

Language：English Publishing type：Research paper (scientific journal)

In order to detect apoptotic cells in the kidney, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method is used in tissue sections. But the number of apoptotic cells per glomerulus in several experimental models of nephritides is about 1 per single tissue section even at peak levels. In this study, we have reported that the TUNEL method and immunostaining of cell-specific markers to a whole isolated glomerulus in combination with laser scan microscopy are potentially useful methods for the analysis of cell turnover within glomeruli. © 1997 S. Karger AG, Basel.

DOI： 10.1159/000190327

Candesartan prevents the progression of mesangioproliferative nephritis in rats Reviewed

T. Nakamura, J. E. Obata, M. Onizuka, H. Kimura, S. Ohno, Y. Yoshida, H. Kawachi, F. Shimizu

Kidney International, Supplement 51 ( 63 ) S226 - S228 1997

Language：English Publishing type：Research paper (international conference proceedings)

We previously reported a new animal model of progressive glomerulonephritis induced by a single intravenous injection of the anti- Thy-1 monoclonal antibody MoAb 1-22-3 into uninephrectomized rats (Clin Exp Immunol 102:181-185, 1995). We examined the effects of angiotensin II (Ang II) receptor antagonist (candesartan) on the clinical features and morphological lesions of this new model. By 10 weeks after induction of nephritis, untreated rats had developed hypertension, massive proteinuria, renal dysfunction, and severe glomerular injury, while uninephrectomized control rats had not. There was a significant increase in levels of glomerular protein and cortical mRNA for transforming growth factor-β (TGF- β) and type I and type III collagens in untreated nephritic rats. Ten week treatments with candesartan and hydralazine significantly reduced blood pressure (BP) to an equal extent. Candesartan, but not hydralazine, prevented proteinuria, normalized renal function, and ameliorated glomerular injury. Candesartan also reduced levels of glomerular protein and cortical mRNA for TGF-β and type I and type III collagens, while hydaralazine did not. These findings suggest that candesartan prevents progression to end-stage renal failure by modulating the effects of Ang II at least in part on the production of TGF-β and type I and type III collagens, and not merely on systemic BP.

Detailed analysis of phenotypes of macrophages infiltrating glomeruli in rat anti-thy1 nephritis Reviewed

H. Kobayashi, M. Orikasa, M. Naito, K. Kawasaki, T. Oite, H. Kawachi, A. Yamashita, M. Takeya, H. Kobayashi, F. Shimizu

Nephron 77 ( 3 ) 333 - 339 1997

Language：English Publishing type：Research paper (scientific journal)

A phenotypic analysis of infiltrating macrophages in rat anti-Thy1 glomerulonephntis induced by monoclonal antibody (mAb) 1-22-3 was carried out using recently reported macrophage-specific mAbs. This was combined with a more detailed quantitative analysis, counting positive cells in isolated glomeruli, to obtain more information on the roles played by macrophages in glomerulonephritis. In normal glomeruli a small number of ED1- or OX-3(anti-Ia)-positive cells but almost no ED2-, TRPM-3- or Mar-3-positive cells were observed. ED1-positive cells increased from 2 h and peaked between days 3 and 7 after mAb injection. TRPM-3-positive cells increased from day 3 and peaked on day 7, later than ED1. The numbers of OX-3-positive cells changed in parallel with those of ED1-positive cells. Mar-3, which stained blood monocytes and ED2, which is an indicator of tissue-fixed resident macrophages, did not react with glomerular infiltrating macrophages. In a double staining study, about 40% of ED1- or OX-3-positive cells costained with TRPM-3 on day 3 and the percentage increased on day 7, but hardly any cells were positive for TRPM-3 alone. This results in two different phenotypes (ED1+, ED2−, OX-3+, Mar-3−, TRPM-3− and ED1+, ED2−, OX-3+, Mar-3−, TRPM-3+) of infiltrating macrophages. We conclude that in rat anti-Thyl glomerulonephntis, monocytes/ macrophages may infiltrate the mesangium, rapidly changing their phenotype (Mar-3+ to Mar-3−) and resulting in a gradual shift to TRPM-3-positive, activated macrophages. © 1997 S. Karger AG, Basel.

DOI： 10.1159/000190297

Roles of TGF-β and latent TGF-β-binding protein in glomerulosclerosis induced by two consecutive injections of monoclonal antibody 1-22-3 in Rats Reviewed

Masaru Nakayama, Seiya Okuda, Kiyoshi Tamaki, Fujio Shimizu, Hiroshi Kawachi, Takashi Ando, Taihei Yanagida, Masatoshi Fujisliima

Nephron 76 ( 1 ) 82 - 89 1997

Language：English Publishing type：Research paper (scientific journal)

The present study demonstrated the elevated synthesis and gene expressions of transforming growth factor β (TGF-β) or latent TGF-β binding protein (LTBP) in an irreversible glomerulosclerosis rat model induced by two consecutive injections of monoclonal antibody (MoAb) 1-22-3. The rats were intravenously injected with 500 #x03BC
g of MoAb 1-22-3 either once or twice at an interval of 2 weeks. The rats were sacrificed at 24 h, 1 week, 2 weeks or 16 weeks after the last injection. At 24 h, the mesangiolytic changes in the rats with two injections of MoAb 1-22-3 were similar to those in the rats with one injection. The glomerular matrix score in the rats with two injections was significantly higher than that in the rats with one injection at weeks 1 2 or 16. An increased LTBP localization in the glomeruli of the rats at week 1 after either one or two injections was detected in the segmentally expanded mesangial matrix. Moreover, LTBP in the glomeruli of rats at week 1 after two injections appeared to be more strongly stained in the enlarged mesangial matrix than that in the rats after one injection. A TGF-β bioassay using mink lung epithelial cells revealed that the total TGF-β in the glomerular culture conditioned medium in the rats at week 1 after two injections was significantly larger than that in the rats after one injection. A Northern blotting analysis of the glomeruli showed that both the expressions of TGF-β and LTBP mRNA in the rats after two injections were higher than those in the rats after one injection. These findings suggested that the elevated TGF-β or LTBP may thus be related to the irreversible glomerulosclerosis that was induced by two injections of MoAb 1-22-3 into rats. © 1997 S. Karger AG, Basel.

DOI： 10.1159/000190145

Structural continuity of filtration slit (slit diaphragm) to plasma membrane of podocyte Reviewed

Y Fujigaki, T Morioka, K Matsui, H Kawachi, M Orikasa, T Oite, F Shimizu, Batsford, SR, A Vogt

KIDNEY INTERNATIONAL 50 ( 1 ) 54 - 62 1996.7

Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

Murine monocional antibody 5-1-6 was reported to bind to the slit membrane and closely related structures in rat renal glomeruli; it induced heavy, reversible proteinuria and appeared to redistribute onto the plasma membrane of epithelial cells after binding at the original target sites. This phenomenon of antigenic movement has not been analyzed in detail to date. In addition to normal kidneys we also studied localization of the antigen recognized by monoclonal antibody 5-1-6 in protamine sulfate-perfused rat kidneys, in which slit diaphragms are known to be functionally modified. Isolated glomeruli as well as ultrathin kidney cryosections were labeled by the immunogold technique to clarify the relation between this antigen and the slit diaphragm. Sequential localization of injected monoclonal antibody was visualized using a post-embedding immunogold method in rats 2 hours to 12 daps after injection of antibody. Ultrastructural immunoqold labeling demonstrated that under normal conditions antigenic molecules were expressed mainly in the area beneath the slit diaphragms. Occasionally labeling was found at the base of the foot process, facing the glomerular basement membrane. After protamine sulfate treatment antigenic sites were dislocated due to the lifting and disruption of slit diaphragms, indicating that this antigen is associated with slit diaphragms. Injected antibody was localized at the filtration slits at 2 hours, and by 12 hours it had moved onto the apical plasma cell membrane of foot process. In addition, from 3 days onwards patch or cap-like formation on the plasma cell membrane of podocytes was seen. Possible shedding of antibody from podocyte cell surface membrane was occasionally encountered, but internalization of antibody was a minor event. Elution experiments in isolated glomeruli al day 3 indicated that antigen and antibody were both localized on the podocyte cell surface membrane, suggesting redistribution of immune complexes. In conclusion, filtration slits (slit diaphragms) and the apical membrane of foot process of podocytes demonstrate structural continuity as revealed by the movement of the antigen recognized by monoclonal antibody 5-1-6 as antigen-antibody complexes.

PROGRESSIVE RENAL LESIONS INDUCED BY ADMINISTRATION OF MONOCLONAL-ANTIBODY-1-22-3 TO UNILATERALLY NEPHRECTOMIZED RATS Reviewed

QL CHENG, M ORIKASA, T MORIOKA, H KAWACHI, XM CHEN, T OITE, F SHIMIZU

CLINICAL AND EXPERIMENTAL IMMUNOLOGY 102 ( 1 ) 181 - 185 1995.10

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL SCIENCE LTD

A new animal model of progressive glomerulosclerosis was developed by administering a single i.v, injection of MoAb 1-22-3 to unilaterally nephrectomized rats. Renal morphological analysis revealed that glomerular lesions characterized by mesangial cell proliferation and mesangial matrix expansion were induced in about 95% of the glomeruli. Approximately 20% of the glomeruli of the unilaterally nephrectomized rats showed sclerosis or segmental sclerosis by week 6 after MoAb injection and crescent formation was observed in some glomeruli (ca 4%). Cellular infiltration was also noted in some parts of the interstitium. Increased expression of transforming growth factor-beta (TGF-beta) was observed in the unilaterally nephrectomized rats treated with MoAb 1-22-3, but we could not demonstrate pathological involvement of platelet-derived growth factor (PDGF), even though early-stage mesangial cell proliferation was observed. The mechanism of mesangial cell proliferation in this model remains to be elucidated. The relatively short period of time needed to induce the sclerotic changes is considered to be a great advantage of this model for clarifying the mechanisms involved in the chronic progression of mesangial proliferative glomerulonephritis.

Strain variation in susceptibility to the development of monoclonal antibody 5-1-6-induced proteinuria in rats Reviewed

D. Gollner, H. Kawachi, T. Oite, M. Oka, M. Nagase, F. Shimizu

Clinical and Experimental Immunology 101 ( 2 ) 341 - 345 1995

Language：English Publishing type：Research paper (scientific journal) Publisher：Blackwell Publishing Ltd

Susceptibility to the development of MoAb 5-1-6-induced proteinuria was investigated in four different rat strains, i.e. Brown-Norway (BN), Lewis (LEW), Sprague-Dawley (SD) and Wistar. An intravenous injection of 5 mg of MoAb 5-1-6 to female 7-week-old rats of a given strain induced massive proteinuria in BN, LEW and Wistar rats. However, SD rats developed almost no proteinuria. A similar tendency was observed in the second experiment, in which the injected dose of MoAb was adjusted according to the body weight of each rat (3 mg/100 g body weight). Immunofluorescence (IF) and immunoelectron microscopy (IEM) revealed no differences between the binding patterns of the MoAbs to normal rat kidneys derived from each strain. Quantitative study using 125I-labelled MoAb showed that there was no significant difference in the amount of antibody bound to the kidney 1 h and 5 days after injection between two rat strains, LEW and SD. Localization of 5-1-6 in vivo and its kinetics were investigated. In IF a linear-like pattern along capillary walls was observed 2 h after injection in both LEW and SD strains. This linear-like pattern was shifted to a granular pattern in proteinuric LEW rats 6 days after injection, whereas it remained linear-like in non-proteinuric SD rats. IEM confirmed this difference in the localization of injected MoAb 6 days after injection to LEW and SD rats also at the ultrastructural level. We conclude that there is a clear-cut strain difference in the development of proteinuria induced by MoAb 5-1-6. SD rats were less susceptible to MoAb-induced glomerular injury than BN, LEW and Wistar rats. Although the exact reason for strain variation in susceptibility to MoAb-induced proteinuria remains to be clarified, the movement of bound MoAb, presumably together with corresponding antigenic molecule along the glomerular epithelial cell surface followed by endocytosis into the epithelial cell, seems to be closely related to the induction of proteinuria.

DOI： 10.1111/j.1365-2249.1995.tb08361.x

Visceral epithelial cells in rat glomerular cell culture Reviewed

E. Yaoita, T. Yamamoto, N. Takashima, K. Kawasaki, H. Kawachi, F. Shimizu, I. Kihara

European Journal of Cell Biology 67 ( 2 ) 136 - 144 1995

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier GmbH

The purpose of this study was to identify and characterize visceral epithelial cells (VECs) of renal glomeruli in culture. Such cells have been described variably as regular, polygonal cells showing a high rate of replication and a cobblestone-like appearance at confluency and also as irregular, arborized and often multinucleated cells showing a very limited proliferative capacity. We examined early outgrowths from the glomeruli by immunofluorescence microscopy using antibodies specific for VECs (anti podocalyxin(1A), anti-pp44 and 5-1-6), for endothelial cells (anti-von Willebrand factor (vWF) and RECA-1) and for mesangial cells (anti-Thy-1). 1A and anti-pp44 reacted with several types of irregular, arborized cells, but never with regular, polygonal cells. 5-1-6 did not react with any of the cells. Neither the 1A- nor anti-pp44-positive cells (1A/pp44(+) cells) stained with anti-vWF, RECA-1 or anti-Thy-1. However, all the 1A/pp44(+) cells expressed desmin and vimentin but not cytokeratin. These results show that the 1A/pp44(+) cells are derived from VECs, supporting the idea that most polygonal cells in glomerular cultures are of parietal epithelial origin.

WATER AND PROTEIN PERMEABILITY IS REGULATED BY THE GLOMERULAR EPITHELIAL SLIT DIAPHRAGM Reviewed

RC BLANTZ, FB GABBAI, O PETERSON, CB WILSON, KIHARA, I, H KAWACHI, F SHIMIZU, T YAMAMOTO

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 4 ( 12 ) 1957 - 1964 1994.6

Language：English Publishing type：Research paper (scientific journal) Publisher：WILLIAMS & WILKINS

The glomerular barriers to water and macromolecular movement were examined 2 and 24 h after the administration of a monoclonal antibody (mAb) specific to an antigen located on the epithelial slit diaphragm and the external aspect of the glomerular basement membrane. By micropuncture techniques 2 h after mAb administration, single-nephron GFR (SNGFR) and plasma flow were unchanged but the glomerular capillary hydrostatic pressure gradient and glomerular capillary hydrostatic pressure were increased and the glomerular ultrafiltration coefficient (LpA) decreased to values that were 50% of the normal control. There was no increase in urinary protein excretion at 2 h. However, at 24 h after mAb, nephron plasma flow (SNPF) and SNGFR increased and the glomerular ultrafiltration coefficient returned to values indistinguishable from the normal control. At 24 h, there was a marked increase in protein excretion. The administration of meclofenamate decreased values for SNGFR and SNPF to normal. Immunoglobulin G was exclusively bound to glomerular capillary walls in a linear or continuous fashion at 2 h, but in a discontinuous, granular pattern at 24 h. These studies suggest that after mAb, important limiting glomerular barriers for hydraulic conductivity and protein excretion reside on the epithelial aspect of the glomerular capillary basement membrane, specifically at the level of the slit diaphragm. Studies also suggest that alterations in glomerular capillary hydraulic conductivity can be effectively separated from increases in macromolecular permeability.

EFFECT OF TRADITIONAL CHINESE MEDICINE (SAIREI-TO) ON MONOCLONAL ANTIBODY-INDUCED PROTEINURIA IN RATS Reviewed

H KAWACHI, N TAKASHIMA, M ORIKASA, T OITE, F SHIMIZU

PATHOLOGY INTERNATIONAL 44 ( 5 ) 339 - 344 1994.5

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL SCIENCE

The effects of traditional Chinese medicine (Sairei-to) on monoclonal antibody (mAb) inducing proteinuria were examined. Four hundred, 200 and 100 mg/kg body weight (BW) of Sairei-to and phosphate-buffered saline (PBS) as a control were injected intraperitoneally into four groups of female Wistar rats every day from 5 days before intravenous injection of mAb to the end of the experimental period. The amount of urinary protein excretion was significantly suppressed in roughly a dose-dependent manner. For example, 116.6 +/- 89.7 mg/day of proteinuria was observed in control groups compared to 4.2 +/- 15.2 mg/day in the 400 mg/kg BW of Sairei-to treated group 2 days after mAb injection (P < 0.01).
Statistically significant (P < 0.01) differences were again observed in a repeat experiment (122.1 +/- 53.7 vs 10.2 +/- 10.1 mg/day on the 2nd day) without affecting the glomerular filtration rate.
No significant difference was recognized between the total amount of mAb bound to glomeruli 1 h after mAb injection in Sairei-to-treated and non-treated rats, indicating that Sairei-to pretreatment has no effects on the number or quality of antigenic molecules.
The effect of Sairei-to on a non-immunological model of proteinuria was also examined. No significant reduction of proteinuria by similar Sairei-to treatment was observed in aminonucleoside of puromycin nephropathy.
The authors conclude that mAb-induced proteinuria in rats is significantly suppressed by the traditional Chinese medicine, Sairei-to.

Effect of chlorpromazine on kinetics of injected monoclonal antibody in MoAb-induced glomerular injury Reviewed

N. Takashima, H. Kawachi, T. Oite, S. Nishi, M. Arakawa, F. Shimizu

Clinical and Experimental Immunology 91 ( 1 ) 135 - 140 1993

Language：English Publishing type：Research paper (scientific journal)

The effect of chlorpromazine, one of several calmodulin antagonists that inhibit cytoskeletal movement, on the local kinetics of injected proteinuria-inducing MoAb 5-1-6 was examined to test the hypothesis that proteinuria is inhibited if the antigen recognized by MoAb 5-1-6 or injected MoAb remains on the surface of epithelial foot processes. MoAb 5-1-6 was injected into both chlorpromazine-treated (5 mg/100 g body weight) and untreated rats. As a positive control for the chlorpromazine treatment, anti-Fx1A serum was also injected into other chlorpromazine-treated and untreated rats. Chlorpromazine inhibited neither the change in localization of injected MoAb 5-1-6 nor proteinuria, although it showed an inhibitory effect on redistribution of immune complex and the fixation of complement in passive Heymann glomerulonephritis induced by injection of anti-Fx1A serum. We conclude that the kinetics of bound MoAb 5-1-6 are regulated by a system different from that operating in passive Heymann glomerulonephritis.

Quantitative study of mesangial injury with proteinuria induced by monoclonal antibody 1-22-3 Reviewed

H. Kawachi, T. Oite, F. Shimizu

Clinical and Experimental Immunology 92 ( 2 ) 342 - 346 1993

Language：English Publishing type：Research paper (scientific journal)

Murine MoAb 1-22-3 has already been reported to bind to the mesangial cell surface and to cause transient proteinuria and mesangial morphological changes characterized by mesangiolysis, subsequent mesangial cell proliferation and mesangial matrix increase by a single i.v. injection. In this study, MoAb-induced glomerulopathy was quantitatively analysed. No correlation between the severity of mesangial morphological changes and the degree of proteinuria was detected (r = 0.190). The minimum dose injected to induce abnormal proteinuria was 25 μg. This dose corresponded to 1.79 μg/2 kidneys 30 min after MoAb injection. The highest average level of proteinuria was observed in rats injected with 500 μg of MoAb, and less proteinuria was observed in rats injected with 10.0, 5.0 and 2.0 mg. Although the amounts of kidney-fixing MoAb and the subsequent deposition of rat C3 in the high-dose-injected group were larger than in the 500 μg injected group, the numbers of infiltrating inflammatory cells were the same in both groups. No correlations between the degrees of such mediators and proteinuria were observed.

Nephrotoxic serum nephritis in nude rats: The roles of host immune reactions in the accelerated type Reviewed

T. Sato, H. Kawachi, T. Morioka, N. Takashima, T. Saeki, T. Oite, N. L. Sato, M. Takeuchi, F. Shimizu

Clinical and Experimental Immunology 91 ( 1 ) 131 - 134 1993

Language：English Publishing type：Research paper (scientific journal)

By immunization with rabbit immunoglobulins and the injection of a subnephritogenic dose of rabbit nephrotoxic serum (NTS), accelerated-type nephrotoxic serum nephritis (NTN) was induced in heterozygous (rnu/+) rats but not in athymic nude (rnu/rnu) rats. By transferring rat antibody against rabbit immunoglobulins, marked proteinuria was induced also in nude rats (202.0 ± 98.4 mg/day on day 3) as in rnu/+ rats (122.6 ± 35.3 mg/day on day 3). No marked differences in histological findings could be found between both groups. The most marked increase in the number of intraglomerular infiltrating cells was observed in heterozygous rats indicating that the presence of thymus-derived cells leads to the accumulation of more cells in glomeruli. We conclude that humoral immunity alone is enough to accelerate the pathogenic mechanism which induces glomerular injury with heavy proteinuria in this model.

Divalency of the monoclonal antibody 5-1-6 is required for induction of proteinuria in rats Reviewed

M. Narisawa, H. Kawachi, T. Oite, F. Shimizu

Clinical and Experimental Immunology 92 ( 3 ) 522 - 526 1993

Language：English Publishing type：Research paper (scientific journal)

A single i.v. injection of 3 mg of the F(ab')2 fragment of MoAb 5-1-6 into rats induced immediate proteinuria (128.1 ± 80.7 mg/24 h on day 1) which lasted 1-2 days. In contrast, rats administered 10 mg of the corresponding Fab fragment did not develop abnormal proteinuria even though an equivalent dose of the intact MoAb 5-1-6 far exceeded the nephritogenic dose. The total kidney binding of 125I-Fab fragment was 209.5 ± 34.3 μg/2 kidneys. This exceeded that obtained by injection of 3 mg MoAb 5-1-6 IgG1 (58.9 ± 12.5 μg/2 kidneys at 1 h) and was similar to that obtained following injection of 3 mg F(ab')2 fragment (235.3 ± 16.9 μg/2 kidneys). Immunofluorescence (IF) showed a linear pattern along the glomerular capillary wall at 1 h after the administration of MoAb 5-1-6 IgG1, F(ab')2 or Fab fragment. On day 5, fine to coarse granules were observed scattered in F(ab')2-injected rat glomeruli, whereas granules were densely localized in Fab-injected rat glomeruli. Complement-depleted rats injected with 3 mg of MoAb 5-1-6 IgG1 developed proteinuria with the same time course as non-depleted rats. This observation, together with the ability of F(ab')2 to induce proteinuria, indicates that proteinuria induced by MoAb 5-1-6 is complement-independent. This study suggests that MoAb 5-1-6-induced proteinuria is initiated by cross-linking of the epitopes by divalent MoAb 5-1-6 and is independent of complement activity.

EPITOPE-SPECIFIC INDUCTION OF MESANGIAL LESIONS WITH PROTEINURIA BY A MOAB AGAINST MESANGIAL CELL-SURFACE ANTIGEN Reviewed

H KAWACHI, M ORIKASA, K MATSUI, T IWANAGA, S TOYABE, T OITE, F SHIMIZU

CLINICAL AND EXPERIMENTAL IMMUNOLOGY 88 ( 3 ) 399 - 404 1992.6

Language：English Publishing type：Research paper (scientific journal) Publisher：BLACKWELL SCIENCE LTD

A MoAb 1-22-3 (IgG3) was produced in mice immunized with rat glomeruli. A blotting study indicated the antigen molecule recognized by this MoAb has a molecular weight of about 25 kD, which is the same as that of the Thy 1.1 molecule. This MoAb is capable of inducing the morphological changes similar to those induced by anti-thymocyte serum or the anti-Thy 1.1 MoAb, ER4 and massive proteinuria in rats by a single i.v. injection. Proteinuria started immediately after MoAb 1-22-3 injection and peaked on day 5. Reaction products were detected by immunoelectron microscopy in vitro on the limited mesangial cell surface facing endothelial cells and in vivo in partially lysed mesangial cells 30 min after injection. Unlike the proteinuria-inducing MoAb ER4, reactivity of MoAb 1-22-3 was detected neither on endothelial cells, epithelial cells, nor along the glomerular basement membrane in vivo and in vitro. There is a difference in reactivity toward guinea-pig and rabbit materials between MoAb 1-22-3 and the commercial anti-Thy 1.1 MoAb (OX7). The antigenic determinant of MoAb 1-22-3 is concluded to be a new epitope and only the binding of MoAb 1-22-3 to this epitope proved to lead to an abnormal increase of glomerular capillary wall permeability.

Mesangial sclerotic change with persistent proteinuria in rats after two consecutive injections of monoclonal antibody 1-22-3 Reviewed

H. Kawachi, T. Iwanaga, S. Toyabe, T. Oite, F. Shimizu

Clinical and Experimental Immunology 90 ( 1 ) 129 - 134 1992

Language：English Publishing type：Research paper (scientific journal)

Irreversible mesangial changes with persistent proteinuria were induced in rats given two consecutive injections 2 weeks apart of a MoAb 1-22-3 to rat mesangial cell. The characteristics of the resulting lesions were investigated and compared with those of the reversible change induced by a single injection. At 24 h after the second injection, mesangiolytic changes similar to those after a single injection were evident. The accumulation of macrophage-like cells in glomeruli observed at 1 week after the first injection was not evident during the experimental period after the second injection. Hypercellularity with the characteristics of intrinsic mesangial cell and increased mesangial matrix were already present 1 week after the second injection. And mesangial sclerotic change progressed up to 6 months. Deposition of collagen type I and type III and accumulation of collagen fibril at the ultrastructural level were evident in rats 6 months after the second injection. Proteinuria started immediately and continued for more than 6 months after the second injection. The mesangial sclerotic change with persistent proteinuria described here is considered to be a better model for investigating the mechanism of chronic progression of human mesangial proliferative glomerulonephritis.

A monoclonal antibody (1G10) recognizes a novel human mesangial antigen Reviewed

S. Kagami, K. Okada, M. Funai, K. Matui, T. Oite, H. Kawachi, F. Shimizu, Y. Kuroda

Kidney International 42 ( 3 ) 700 - 709 1992

Language：English Publishing type：Research paper (scientific journal)

We have identified a unique mesangial matrix protein of the human glomerulus by using a monoclonal antibody, 1G10, generated against cultured human glomerular cells. By immunofluorescence, the antigen recognized by 1G10 (1G10 antigen) is present in mesangium and smooth muscle tissue and cannot be detected in any other tissue examined. Immunoelectron microscopy of glomeruli indicated that 1G10 antigen is present exclusively in the mesangial matrix at the endothelial-mesangial interface. The 1G10 antigen is also expressed by cultured mesangial cells, but not by cultured glomerular epithelial cells, umbilical endothelial cells or fibroblasts. 1G10 did not react with the mesangial matrix proteins [fibronectin (FN), laminin (LAM), collagen types I, III, IV, V, and VI (Col I, III, IV, V, VI), heparin sulfate proteoglycan (HSPG), or thrombospondin (TS)] present under normal and diseased states or smooth muscle antigens (myosin, actin), but did react with a 4 M urea extract of renal cortex and a 0.3% deoxycholate extract of isolated glomeruli. Two dimensional immunoblot analysis using the urea extract demonstrated the binding of 1G10 to an ~200 KDa polypeptide with pI 6.0. On one dimensional immunoblot this band did not show cross react with polyclonal antisera to FN, LAM, Col IV, V, VI, HSPG or TS. This mesangial matrix component is trypsin and periodate sensitive, suggesting that it has the character of glycoprotein. In renal biopsy specimens from patients with mesangial proliferative glomerulonephritis (GN) and membranoproliferative GN, the expression of the 1G10 antigen increased along with mesangial hypercellularity or increased accumulation of mesangial matrix, but decreased in completely sclerosed glomeruli. No significant changes in 1G10 antigen expression was observed in membranous GN or minimal change nephrosis compared to normal glomeruli. This study suggests that the 1G10 antigen may not only be a useful marker for the clinical assessment of GN, but may also serve as a potential tool for the study of the pathogenesis of glomerular diseases characterized by cellular proliferation and mesangial matrix expansion.

DOI： 10.1038/ki.1992.337

Quantitative studies of monoclonal antibody 5‐1‐6‐induced proteinuric state in rats Reviewed

H. KAWACHI, K. MATSUI, M. ORIKASA, T. MORIOKA, T. OITE, F. SHIMIZU

Clinical & Experimental Immunology 87 ( 2 ) 215 - 219 1992

Language：English Publishing type：Research paper (scientific journal)

Murine monoclonal antibody (MoAb) 5‐1‐6 was already reported to bind to epithelial cell fool processes and to cause proteinuria in rats. In vivo kinetics of the injected MoAb 5‐1‐6, relationship between the quantity of kidney‐binding antibody and proteinuria. and changes in the amount of antigenic molecule recognized by this MoAb in the proteinuric state were studied. The amount of total kidney‐binding antibody (TK Ab) as determined I h after a 2 mg administration was 50.8 ± 10.4 μg/2 kidneys, and TKAb declined to 1.9 ± 0.4 at day 15. The minimum dose of MoAb required to induce proteinuria was 125 μg as the injected dose. This dose corresponded to 12‐8 μg of TKAb at I h and 0.34 μg of TKAb at day 5. The amount of MoAb 5‐1 ‐6 binding to isolated normal glomeruli was also shown to exceed 147‐7 μg/76000 glomeruli, indicating proteinuria to be induced provided more than 8.7% (= 12.8/147.7) of the critical epitopes is specifically occupied by MoAb. The total amount of MoAb 5‐1‐6 bound to glomeruli in vivo and in vitro was assayed with [125I]‐labelled anti‐mouse IgG. The amount of [125I] anti‐mouse IgG bound to glomeruli was 6.93 ± 0.45 μg/10 000 glomeruli from rat 5 days after this MoAb injection and 26.58 ± 0.66 μg/10000 control glomeruli, indicating the decrease in the number of MoAb 5‐1‐6‐rccognizcd antigen molecules in glomeruli isolated from the rat in proteinuric state induced by this MoAb. Thus, the MoAb 5‐1‐6‐recognized molecule itself may principally function to regulate the permeability of the glomerular capillary wall and the decrease of the molecule may lead to proteinuria. Copyright © 1992, Wiley Blackwell. All rights reserved

DOI： 10.1111/j.1365-2249.1992.tb02977.x

Altered localization of antigen recognized by proteinuriainducing monoclonal antibody in experimental nephrosis Reviewed

Teruo Okasora, Mitsumasa Nagase, Hiroshi Kawachi, Katsuyuki Matsui, Michiaki Orikasa, Tetsuo Morioka, Ichiroh Yamazaki, Takashi Oite, Fujio Shimizu

Virchows Archiv B Cell Pathology Including Molecular Pathology 60 ( 1 ) 41 - 46 1991.12

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer-Verlag

Change in the localization of the antigen recognized by the proteinuria-inducing monoclonal antibody (MA) 5-1-6 in experimental nephrosis was studied by indirect and biotin-avidin immunofluorescence, and immunoperoxidase at light and electron microscopical levels. The proteinuric state was induced by the administration of the aminonucleoside of puromycin (PAN) or adriamycin. The antigen decreased in quantity and/or its distribution changed with an increase in the amount of protein excreted in both experimental models. Recovery from the alterations observed during the development and proteinuria appeared to occur when PAN-induced proteinuria subsided. This antigenic molecule may thus be essential for maintaining the normal permselectivity of glomerular capillary walls. © 1991 Springer-Verlag.

DOI： 10.1007/BF02899526

Nephritogenicity of anti-Engelbreth-Holm-Swarm sarcoma antibody Reviewed

H. Kawachi, T. Nakamura, T. Kazama, E. Yaoita, T. Morioka, K. Matsui, T. Oite, F. Shimizu

Nephron 47 ( 2 ) 150 - 154 1987

Language：English Publishing type：Research paper (scientific journal) Publisher：S. Karger AG

We have examined the antibody activity and nephritogenicity of anti-Engelbreth-Holm-Swarm (EHS) sarcoma antiserum in order to analyze the pathogenesis of the EHS nephropathy which has already been reported by us. An increased amount of urine protein was not recognized in rats injected with a large quantity of anti-EHS sarcoma antiserum. In addition, rats immunized with rabbit IgG before anti-EHS sarcoma antiserum injection developed no abnormal proteinuria, despite positive fluorescent staining for rat IgG as well as rabbit IgG in the glomeruli. From these results we concluded that nephritogenic antibodies could not be produced in rabbits by immunization with EHS sarcoma, which could induce the EHS nephropathy in an active model.

DOI： 10.1159/000184479

Studies on the 'linear pattern' in renal glomeruli demonstrated with immunofluorescence Reviewed

H. Kawachi, T. Oite, F. Shimizu

Nephron 39 ( 1 ) 36 - 39 1985

Language：English Publishing type：Research paper (scientific journal)

We have experimentally induced the 'linear pattern' in immunofluorescence: the linear deposition of endogenous immunoglobulin (Ig) along the glomerular basement membrane (GBM) in rats injected with both protamine and nephrotoxic serum. This Ig was demonstrated to have no specific antibody activity against GBM or rabbit serum. Our findings could be of value in the analysis of the mechanism and pathological meaning of the 'linear pattern' of endogenous Ig in immunofluorescence.

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