Faculty Profiles - KAWACHI Hiroshi

Papers - KAWACHI Hiroshi

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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

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