Faculty Profiles - SAITO Akihiko

Papers - SAITO Akihiko

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Fanconi syndrome with acute proximal tubular injury induced by a dietary supplement containing beni-koji: a case series report. International journal

Masato Habuka, Michihiro Hosojima, Yusuke Yata, Kenshiro Kurumada, Moeri Yamagiwa, Masataka Yonezawa, Masanori Sudo, Hideyuki Kabasawa, Asa Ogawa, Hitomi Hama, Yumi Ito, Naofumi Imai, Suguru Yamamoto, Akihiko Saito, Shin Goto

BMC nephrology 25 ( 1 ) 446 - 446 2024.12

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

BACKGROUND: Fanconi syndrome is a disorder of the proximal tubule that leads to malabsorption of various electrolytes and substances and is a common consequence of drug-induced nephrotoxicity. However, cases of dietary supplement-induced Fanconi syndrome are rare, and detailed reports on the evaluation of renal histology in patients with this syndrome are lacking. CASE PRESENTATION: We present two cases of dietary supplement-induced Fanconi syndrome that was confirmed by kidney biopsy. Based on their medical history and laboratory and histological findings, both cases were diagnosed as acute proximal tubular injury caused by ingestion of a lipid-lowering dietary supplement containing beni-koji leading to Fanconi syndrome. After discontinuation of the dietary supplement and correction of dehydration and electrolyte imbalance, renal function completely recovered in one case but progressed to chronic kidney disease in the other. CONCLUSIONS: Clinicians should consider dietary supplement-induced Fanconi syndrome as a differential diagnosis in patients who become ill while taking a dietary supplement. Kidney biopsy is useful for diagnosing acute tubular injury with Fanconi syndrome and investigating its pathogenesis. Patients who have developed dietary supplement-induced Fanconi syndrome require long-term monitoring to detect and prevent progression to chronic kidney disease.

DOI： 10.1186/s12882-024-03903-5

Development of sandwich enzyme-linked immunosorbent assays quantifying mouse urinary megalin, a novel proximal tubular biomarker. International journal

Rina Sofuku, Sayaka Miyazaki, Michihiro Hosojima, Sawako Goto, Kazuya Takemoto, Hideyuki Kabasawa, Taeko Endo, Koichi Komochi, Nanako Sugita, Hiroyuki Aoki, Ryota Kobayashi, Ichiei Narita, Akihiko Saito

Journal of immunological methods 534 113763 - 113763 2024.11

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

Megalin, a type I transmembrane protein, serves as a multi-ligand endocytic receptor in the apical membrane of proximal tubules. Its ectodomain and full-length forms are excreted into human urine, with the former being more abundant. We previously developed two types of sandwich enzyme-linked immunosorbent assays (ELISAs) utilizing monoclonal antibodies that target the amino-terminal ligand-binding domain-I and the carboxyl-terminal cytoplasmic region of human megalin, respectively. The former, termed "A-megalin" ELISA, primarily identifies ectodomains of megalin, whereas the latter, "C-megalin" ELISA, specifically recognizes full-length megalin originating from urinary extracellular vesicles. This study developed novel sandwich ELISAs to assess mouse urinary A-megalin and C-megalin, thereby facilitating studies involving these biomarkers in mouse disease models. Immunoblotting and immunohistochemistry of monoclonal antibodies against human megalin were performed to assess their compatibility with mouse megalin in novel sandwich ELISAs, which were constructed and validated using human assay protocols. Immunoblot analysis of megalin in urinary extracellular vesicles and supernatant was performed to investigate the ratio of ectodomain to full-length forms in mouse urine. Stable measurements having a precision and accuracy within 15 % were achieved in the measurement of quality control samples. A-megalin and C-megalin were detectable in the urine of C57BL/6 mice, whereas most urine samples from kidney-specific conditional megalin-knockout mice were below detection limits. Ectodomain forms of megalin were at least approximately 70 times more abundant than the full-length form, even in mouse urine. In conclusion, we successfully developed sandwich ELISAs for assessing mouse urinary A-megalin and C-megalin to evaluate primarily ectodomain and full-length forms of megalin, respectively.

DOI： 10.1016/j.jim.2024.113763

Changes in daily intake of nutrients and foods including confectionery after the initiation of empagliflozin in Japanese patients with type 2 diabetes: a pilot study. International journal

Toshiko Murayama, Michihiro Hosojima, Hideyuki Kabasawa, Takahiro Tanaka, Nobutaka Kitamura, Mai Tanaka, Shoji Kuwahara, Yoshiki Suzuki, Ichiei Narita, Akihiko Saito

BMC nutrition 10 ( 1 ) 95 - 95 2024.7

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

INTRODUCTION: It is unclear how dietary intake changes after sodium-glucose cotransporter 2 inhibitor (SGLT2i) treatment is started in patients with type 2 diabetes. METHODS: We performed a non-controlled, open-label study that enrolled 51 patients with type 2 diabetes. The patients were newly administered empagliflozin, and their dietary habits were examined using a self-administered diet history questionnaire at the beginning of the study and after 24 weeks. We investigated the association of changes in HbA1c and body weight with changes in energy, nutrient, and food group intakes. RESULTS: At 24 weeks after the start of the study, HbA1c improved significantly and body weight decreased. In the food group, only the intake of confectionery increased, and there were no significant differences in the association between changes in HbA1c and body weight and changes in energy, nutrient, and food group intakes after 24 weeks. However, a significant negative correlation was found between change in HbA1c after 4 weeks and change in energy intake after 24 weeks, and principal component analysis showed an association between change in HbA1c levels after 4 weeks and change in energy intake and some food group intakes including confectionery after 24 weeks. CONCLUSION: In this study, after 24 weeks of treatment with empagliflozin, only intake of confectionery increased. Early assessment by dietitians after initiation of SGLT2i treatment might be important because our data suggested that the reduction in blood glucose levels after the start of empagliflozin was associated with a subsequent increase in energy intake. TRIAL REGISTRATION: University Hospital Medical Information Network-Clinical Trials Registry (UMIN-CTR) on September 5, 2016 (registration ID, UMIN000002309|| http://www.umin.ac.jp/ctr/ ).

DOI： 10.1186/s40795-024-00902-5

メガリン機能抑制を介したSGLT2阻害薬による腎保護機序

竹本和矢, 細島康宏, 後藤佐和子, 蒲澤秀門, 杉田奈々子, 遠藤多恵子, 青木弘行, 小餅耕一, 成田一衛, 斎藤亮彦

日本腎臓学会誌 66 ( 4 ) 581 - 581 2024.6

Cryo-EM structures elucidate the multiligand receptor nature of megalin. International journal

Sawako Goto, Akihisa Tsutsumi, Yongchan Lee, Michihiro Hosojima, Hideyuki Kabasawa, Koichi Komochi, Satoru Nagatoishi, Kazuya Takemoto, Kouhei Tsumoto, Tomohiro Nishizawa, Masahide Kikkawa, Akihiko Saito

Proceedings of the National Academy of Sciences of the United States of America 121 ( 22 ) e2318859121 2024.5

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

Megalin (low-density lipoprotein receptor-related protein 2) is a giant glycoprotein of about 600 kDa, mediating the endocytosis of more than 60 ligands, including those of proteins, peptides, and drug compounds [S. Goto, M. Hosojima, H. Kabasawa, A. Saito, Int. J. Biochem. Cell Biol. 157, 106393 (2023)]. It is expressed predominantly in renal proximal tubule epithelial cells, as well as in the brain, lungs, eyes, inner ear, thyroid gland, and placenta. Megalin is also known to mediate the endocytosis of toxic compounds, particularly those that cause renal and hearing disorders [Y. Hori et al., J. Am. Soc. Nephrol. 28, 1783-1791 (2017)]. Genetic megalin deficiency causes Donnai-Barrow syndrome/facio-oculo-acoustico-renal syndrome in humans. However, it is not known how megalin interacts with such a wide variety of ligands and plays pathological roles in various organs. In this study, we elucidated the dimeric architecture of megalin, purified from rat kidneys, using cryoelectron microscopy. The maps revealed the densities of endogenous ligands bound to various regions throughout the dimer, elucidating the multiligand receptor nature of megalin. We also determined the structure of megalin in complex with receptor-associated protein, a molecular chaperone for megalin. The results will facilitate further studies on the pathophysiology of megalin-dependent multiligand endocytic pathways in multiple organs and will also be useful for the development of megalin-targeted drugs for renal and hearing disorders, Alzheimer's disease [B. V. Zlokovic et al., Proc. Natl. Acad. Sci. U.S.A. 93, 4229-4234 (1996)], and other illnesses.

DOI： 10.1073/pnas.2318859121

Megalin-related mechanism of hemolysis-induced acute kidney injury and the therapeutic strategy. International journal

Sawako Goto, Michihiro Hosojima, Hideyuki Kabasawa, Kaho Arai, Kazuya Takemoto, Hiroyuki Aoki, Koichi Komochi, Ryota Kobayashi, Nanako Sugita, Taeko Endo, Ryohei Kaseda, Yutaka Yoshida, Ichiei Narita, Yoshiaki Hirayama, Akihiko Saito

The Journal of pathology 2024.5

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

Hemolysis-induced acute kidney injury (AKI) is attributed to heme-mediated proximal tubule epithelial cell (PTEC) injury and tubular cast formation due to intratubular protein condensation. Megalin is a multiligand endocytic receptor for proteins, peptides, and drugs in PTECs and mediates the uptake of free hemoglobin and the heme-scavenging protein α1-microglobulin. However, understanding of how megalin is involved in the development of hemolysis-induced AKI remains elusive. Here, we investigated the megalin-related pathogenesis of hemolysis-induced AKI and a therapeutic strategy using cilastatin, a megalin blocker. A phenylhydrazine-induced hemolysis model developed in kidney-specific mosaic megalin knockout (MegKO) mice confirmed megalin-dependent PTEC injury revealed by the co-expression of kidney injury molecule-1 (KIM-1). In the hemolysis model in kidney-specific conditional MegKO mice, the uptake of hemoglobin and α1-microglobulin as well as KIM-1 expression in PTECs was suppressed, but tubular cast formation was augmented, likely due to the nonselective inhibition of protein reabsorption in PTECs. Quartz crystal microbalance analysis revealed that cilastatin suppressed the binding of megalin with hemoglobin and α1-microglobulin. Cilastatin also inhibited the specific uptake of fluorescent hemoglobin by megalin-expressing rat yolk sac tumor-derived L2 cells. In a mouse model of hemolysis-induced AKI, repeated cilastatin administration suppressed PTEC injury by inhibiting the uptake of hemoglobin and α1-microglobulin and also prevented cast formation. Hemopexin, another heme-scavenging protein, was also found to be a novel ligand of megalin, and its binding to megalin and uptake by PTECs in the hemolysis model were suppressed by cilastatin. Mass spectrometry-based semiquantitative analysis of urinary proteins in cilastatin-treated C57BL/6J mice indicated that cilastatin suppressed the reabsorption of a limited number of megalin ligands in PTECs, including α1-microglobulin and hemopexin. Collectively, cilastatin-mediated selective megalin blockade is an effective therapeutic strategy to prevent both heme-mediated PTEC injury and cast formation in hemolysis-induced AKI. © 2024 The Pathological Society of Great Britain and Ireland.

DOI： 10.1002/path.6284

The structure of the rat vitamin B12 transporter TC and its complex with glutathionylcobalamin. International journal

Marcel Bokhove, Takashi Kawamura, Hideo Okumura, Sawako Goto, Yoshiaki Kawano, Stefan Werner, Franziska Jarczowski, Victor Klimyuk, Akihiko Saito, Takashi Kumasaka

The Journal of biological chemistry 300 ( 5 ) 107289 - 107289 2024.5

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

Vitamin B12 (cobalamin or Cbl) functions as a cofactor in two important enzymatic processes in human cells, and life is not sustainable without it. B12 is obtained from food and travels from the stomach, through the intestine, and into the bloodstream by three B12-transporting proteins: salivary haptocorrin (HC), gastric intrinsic factor, and transcobalamin (TC), which all bind B12 with high affinity and require proteolytic degradation to liberate Cbl. After intracellular delivery of dietary B12, Cbl in the aquo/hydroxocobalamin form can coordinate various nucleophiles, for example, GSH, giving rise to glutathionylcobalamin (GSCbl), a naturally occurring form of vitamin B12. Currently, there is no data showing whether GSCbl is recognized and transported in the human body. Our crystallographic data shows for the first time the complex between a vitamin B12 transporter and GSCbl, which compared to aquo/hydroxocobalamin, binds TC equally well. Furthermore, sequence analysis and structural comparisons show that TC recognizes and transports GSCbl and that the residues involved are conserved among TCs from different organisms. Interestingly, haptocorrin and intrinsic factor are not structurally tailored to bind GSCbl. This study provides new insights into the interactions between TC and Cbl.

DOI： 10.1016/j.jbc.2024.107289

Megalin is involved in angiotensinogen-induced, angiotensin II-mediated ERK1/2 signaling to activate Na+-H+ exchanger 3 in proximal tubules. Reviewed International journal

Sawako Goto, Yutaka Yoshida, Michihiro Hosojima, Shoji Kuwahara, Hideyuki Kabasawa, Hiroyuki Aoki, Tomomichi Iida, Ryuhei Sawada, Daisuke Ugamura, Yuta Yoshizawa, Kazuya Takemoto, Koichi Komochi, Ryota Kobayashi, Ryohei Kaseda, Eishin Yaoita, Satoru Nagatoishi, Ichiei Narita, Kouhei Tsumoto, Akihiko Saito

Journal of hypertension 2023.9

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

BACKGROUND: Kidney angiotensin (Ang) II is produced mainly from liver-derived, glomerular-filtered angiotensinogen (AGT). Podocyte injury has been reported to increase the kidney Ang II content and induce Na+ retention depending on the function of megalin, a proximal tubular endocytosis receptor. However, how megalin regulates the renal content and action of Ang II remains elusive. METHODS: We used a mass spectrometry-based, parallel reaction-monitoring assay to quantitate Ang II in plasma, urine, and kidney homogenate of kidney-specific conditional megalin knockout (MegKO) and control (Ctl) mice. We also evaluated the pathophysiological changes in both mouse genotypes under the basal condition and under the condition of increased glomerular filtration of AGT induced by administration of recombinant mouse AGT (rec-mAGT). RESULTS: Under the basal condition, plasma and kidney Ang II levels were comparable in the two mouse groups. Ang II was detected abundantly in fresh spot urine in conditional MegKO mice. Megalin was also found to mediate the uptake of intravenously administered fluorescent Ang II by PTECs. Administration of rec-mAGT increased kidney Ang II, exerted renal extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, activated proximal tubular Na+-H+ exchanger 3 (NHE3), and decreased urinary Na+ excretion in Ctl mice, whereas these changes were suppressed but urinary Ang II was increased in conditional MegKO mice. CONCLUSION: Increased glomerular filtration of AGT is likely to augment Ang II production in the proximal tubular lumen. Thus, megalin-dependent Ang II uptake should be involved in the ERK1/2 signaling that activates proximal tubular NHE3 in vivo, thereby causing Na+ retention.

DOI： 10.1097/HJH.0000000000003555

ステロイド糖尿病の経過中に両側非定型大腿骨不全骨折を生じた1例

渡邉和樹, 黒澤陽一, 長谷川絵理子, 若松彩子, 蒲澤秀門, 細島康宏, 佐藤弘恵, 中枝武司, 鈴木芳樹, 斎藤亮彦, 成田一衛

糖尿病 66 ( 7 ) 567 - 567 2023.7

【尿細管機能異常の臨床】腎障害における尿細管慢性腎臓病と尿細管機能

細島康宏, 蒲澤秀門, 後藤佐和子, 斎藤亮彦

腎臓内科 17 ( 5 ) 505 - 512 2023.5

3ヵ月間の生活習慣改善が血清レプチンとアルブミン尿に与える影響肥満の効果修飾について

蒲澤佳子, 細島康宏, 伊藤由美, 成田一衛, 斎藤亮彦

日本腎臓学会誌 65 ( 3 ) 318 - 318 2023.5

SGLT2阻害薬使用時におけるメガリンおよびメガリンリガンドの尿中排泄量の推移

細島康宏, 蒲澤秀門, 田中崇裕, 北村信隆, 後藤佐和子, 鈴木芳樹, 成田一衛, 斎藤亮彦

日本腎臓学会誌 65 ( 3 ) 319 - 319 2023.5

メガリンが関与する造影剤腎症の発症メカニズムの解明とその予防法の開発

後藤佐和子, 細島康宏, 蒲澤秀門, 忰田亮平, 成田一衛, 斎藤亮彦

日本腎臓学会誌 65 ( 3 ) 248 - 248 2023.5

シスプラチンを含むがん化学療法におけるシラスタチンの急性腎障害予防効果を検討するための薬物動態試験

蒲澤秀門, 細島康宏, 後藤佐和子, 田中崇裕, 北村信隆, 成田一衛, 斎藤亮彦

日本腎臓学会誌 65 ( 3 ) 274 - 274 2023.5

The endocytosis receptor megalin: From bench to bedside. International journal

Sawako Goto, Michihiro Hosojima, Hideyuki Kabasawa, Akihiko Saito

The international journal of biochemistry & cell biology 157 106393 - 106393 2023.4

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

The large (∼600 kDa) endocytosis receptor megalin/low-density lipoprotein receptor-related protein 2 is highly expressed at the apical membrane of proximal tubular epithelial cells (PTECs). Megalin plays an important role in the endocytosis of various ligands via interactions with intracellular adaptor proteins, which mediate the trafficking of megalin in PTECs. Megalin mediates the retrieval of essential substances, including carrier-bound vitamins and elements, and impairment of the endocytic process may result in the loss of those substances. In addition, megalin reabsorbs nephrotoxic substances such as antimicrobial (colistin, vancomycin, and gentamicin) or anticancer (cisplatin) drugs and advanced glycation end product-modified or fatty acid-containing albumin. The megalin-mediated uptake of these nephrotoxic ligands causes metabolic overload in PTECs and leads to kidney injury. Blockade or suppression of the megalin-mediated endocytosis of nephrotoxic substances may represent a novel therapeutic strategy for drug-induced nephrotoxicity or metabolic kidney disease. Megalin reabsorbs urinary biomarker proteins such as albumin, α1-microglobulin, β2-microglobulin, and liver-type fatty acid-binding protein; thus, the above-mentioned megalin-targeted therapy may have an effect on the urinary excretion of these biomarkers. We have previously established a sandwich enzyme-linked immunosorbent assay to measure the ectodomain (A-megalin) and full-length (C-megalin) forms of urinary megalin using monoclonal antibodies against the amino- and carboxyl-terminals of megalin, respectively, and reported their clinical usefulness. In addition, there have been reports of patients with novel pathological anti-brush border autoantibodies targeting megalin in the kidney. Even with these breakthroughs in the characterization of megalin, a large number of issues remain to be addressed in future research.

DOI： 10.1016/j.biocel.2023.106393

臨床現場における糖尿病性腎症のマネージメント CKDの食事療法における低たんぱく質ご飯の有用性

細島康宏, 蒲澤秀門, 鈴木芳樹, 斎藤亮彦, 成田一衛

糖尿病 66 ( Suppl.1 ) S - 43 2023.4

SGLT2阻害薬使用時における尿中メガリンおよびメガリンリガンド分子の動態

細島康宏, 蒲澤秀門, 田中崇裕, 北村信隆, 後藤佐和子, 鈴木芳樹, 成田一衛, 斎藤亮彦

糖尿病 66 ( Suppl.1 ) S - 286 2023.4

血液透析中の食事摂取状況と新型コロナウイルス感染症(COVID-19)流行の影響に関する連続横断研究

蒲澤秀門, 細島康宏, 斎藤亮彦, 鈴木芳樹, 成田一衛

日本病態栄養学会誌 26 ( Suppl. ) S - 55 2023.1

A case of idiopathic nodular glomerulosclerosis successfully treated by intensive blockade of the renin-angiotensin-aldosterone system.

Hiroki Yamaguchi, Michihiro Hosojima, Hideyuki Kabasawa, Yumi Ito, Yoshiki Suzuki, Akihiko Saito, Masaaki Arakawa, Ichiei Narita

CEN case reports 2022.12

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

Idiopathic nodular glomerulosclerosis has a poor renal prognosis and is characterized by diffuse nodular glomerulosclerotic lesions in the absence of diabetic mellitus. Here, we report the case of a 69-year-old woman with no smoking history who developed renal dysfunction and proteinuria in the absence of overt diabetes or obesity. A biopsy specimen showed nodular mesangial sclerosis with arteriolar hyalinosis and severe large-vessel arteriosclerosis, leading to a diagnosis of idiopathic nodular glomerulosclerosis. Addition of esaxerenone to her existing renin-angiotensin-aldosterone inhibitor therapy led to a rapid decrease in the proteinuria levels and the maintenance of renal function without any complications for more than a year. The results suggest that intensive renin-angiotensin-aldosterone blockade might be an effective treatment for idiopathic nodular glomerulosclerosis.

DOI： 10.1007/s13730-022-00766-3

Efficacy of Low-Protein Rice for Dietary Protein Restriction in CKD Patients: A Multicenter, Randomized, Controlled Study. International journal

Michihiro Hosojima, Hideyuki Kabasawa, Ryohei Kaseda, Tomomi Ishikawa-Tanaka, Yoshitsugu Obi, Toshiko Murayama, Shoji Kuwahara, Yoshiki Suzuki, Ichiei Narita, Akihiko Saito

Kidney360 3 ( 11 ) 1861 - 1870 2022.11

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

BACKGROUND: The benefits of dietary protein restriction in CKD remain unclear, largely due to inadequate adherence in most clinical trials. We examined whether low-protein rice (LPR) previously developed to reduce the protein content of rice, a major staple food, would help improve adherence to dietary protein restriction. METHODS: This open-label, multicenter, randomized, controlled trial evaluated the efficacy of LPR use for reducing dietary protein intake (DPI) in patients with CKD stages G3aA2-G4. Participants were randomly assigned in a 1:1 ratio to an LPR or control group and were followed up for 24 weeks. Both groups received regular counseling by dietitians to help achieve a target DPI of 0.7 g/kg ideal body weight (IBW) per day. The amount of protein in LPR is about 4% of that in ordinary rice, and the participants in the LPR group were instructed to consume LPR with at least two meals per day. The primary outcome was estimated dietary protein intake (eDPI) determined using the Maroni formula. The secondary outcomes included creatinine clearance (CCr) and urinary protein on the basis of 24-hour urine collection. RESULTS: In total, 51 patients were randomized to either the LPR group or the control group. At baseline, mean age was 62.5 years, 70% were men, mean CCr was 52.0 ml/min, and mean eDPI was 0.99 g/kg IBW per day. At 24 weeks, mean eDPI decreased to 0.80 g/kg IBW per day in the LPR group and to 0.91 g/kg IBW per day in the control group, giving a between-group difference of 0.11 g/kg IBW per day (95% confidence interval, 0.03 to 0.19 g/kg IBW per day; P=0.006). There was no significant between-group difference in CCr, but urinary protein was lower at 24 weeks in the LPR group than in the control group. CONCLUSIONS: LPR is a feasible tool for efficiently reducing DPI in patients with CKD. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Randomized, Multicenter, Controlled Study for the Efficacy of Low-Protein Rice Diet in Patients with Chronic Kidney Disease, UMIN000015630.

DOI： 10.34067/KID.0002982022

Urinary A- and C-megalin predict progression of diabetic kidney disease: an exploratory retrospective cohort study. International journal

Tomomichi Iida, Michihiro Hosojima, Hideyuki Kabasawa, Keiko Yamamoto-Kabasawa, Sawako Goto, Takahiro Tanaka, Nobutaka Kitamura, Mitsutaka Nakada, Shino Itoh, Shinya Ogasawara, Ryohei Kaseda, Yoshiki Suzuki, Ichiei Narita, Akihiko Saito

Journal of diabetes and its complications 36 ( 11 ) 108312 - 108312 2022.9

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

AIMS: Megalin, a proximal tubular endocytosis receptor, is excreted in urine in two forms: ectodomain (A-megalin) and full-length (C-megalin). We explored whether urinary megalin levels can be used as independent prognostic biomarkers in the progression of diabetic kidney disease (DKD). METHODS: The associations between baseline urinary A-megalin/creatinine (Cr) and/or C-megalin/Cr levels and the subsequent estimated glomerular filtration rate (eGFR) slope were analyzed using a generalized estimating equation. Patients were categorized into higher or lower groups based on the optimal cutoff values, obtained from a receiver operating characteristic curve, of the two forms of urinary megalin. RESULTS: We retrospectively analyzed 188 patients with type 2 diabetes. The eGFR slopes of the higher A-megalin/Cr and higher C-megalin/Cr groups were - 0.904 and -0.749 ml/min/1.73 m2/year steeper than those of the lower groups, respectively. Moreover, the eGFR slope was -1.888 ml/min/1.73 m2/year steeper in the group with both higher A- and higher C-megalin/Cr than in the other group. These results remained significant when adjusted for known urinary biomarkers (albumin, α1-microglobulin, β2-microglobulin, and N-acetyl-β-d-glucosaminidase). CONCLUSIONS: Urinary A- and C-megalin/Cr levels are likely to be prognostic biomarkers in the progression of DKD independent of other urinary biomarkers.

DOI： 10.1016/j.jdiacomp.2022.108312

Intradialytic parenteral nutrition using a standard amino acid solution not for renal failure in maintenance hemodialysis patients with malnutrition: a multicenter pilot study

Ryota Yasukawa, Michihiro Hosojima, Hideyuki Kabasawa, Aya Takeyama, Daisuke Ugamura, Yoshiki Suzuki, Akihiko Saito, Ichiei Narita

RENAL REPLACEMENT THERAPY 8 ( 1 ) 2022.9

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

Background Standard amino acid solutions have recently been removed from the contraindications for use in dialysis patients in Japan. However, the details of their safety and efficacy in these patients are still not known. In this study, we investigated the safety and efficacy of intradialytic parenteral nutrition (IDPN) using ENEFLUID (R) injection containing standard amino acids, glucose, electrolytes, fats, and water-soluble vitamins in maintenance hemodialysis patients with malnutrition. Methods This clinical trial was designed as a multicenter, prospective, non-randomized, open-label, single-arm, observational pilot study. The participants were patients on maintenance hemodialysis who were in the nutritional high-risk group according to the Nutritional Risk Index for Japanese Hemodialysis Patients. One bag of ENEFLUID (R) injection was administered during every hemodialysis session for 12 weeks. The primary endpoint was change in serum transthyretin levels between before and after the 12-week period. As safety endpoints, we evaluated changes in body fluid volume and blood biochemical tests, including blood urea nitrogen and electrolytes, as well as blood glucose variability using flash glucose monitoring (FGM). Results The mean age and body mass index of the 13 participants were 79.0 +/- 10.7 years and 18.0 +/- 1.7 kg/m(2), respectively. No significant changes were observed in nutritional parameters, including serum transthyretin, between before and after the start of the study. After IDPN initiation, there was no obvious fluid overload or significant changes in blood biochemical tests, including electrolytes, and the treatment could be safely continued for 12 weeks. In the FGM analysis, asymptomatic hypoglycemia during hemodialysis was observed at the beginning of the study, but there was a trend toward improvement after 12 weeks (area over the curve < 70 mg/dl per dialysis session: 747.5 +/- 1333.9 to 21.6 +/- 54.3, P = 0.09). Conclusions IDPN using ENEFLUID (R) injection can be safely continued, although it does not significantly improve markers of nutritional status. It also showed the potential to ameliorate asymptomatic hypoglycemia during hemodialysis sessions. More detailed studies of the improvement in nutritional indicators are needed. Trial registration: This study was registered with the University Hospital Medical Information Network-Clinical Trials Registry (UMIN-CTR) on May 9, 2021 (registration ID, UMIN000044051).

DOI： 10.1186/s41100-022-00432-5

An exploratory clinical trial on the efficacy and safety of glucagon-like peptide-1 receptor agonist dulaglutide in patients with type 2 diabetes on maintenance hemodialysis

Daisuke Ugamura, Michihiro Hosojima, Hideyuki Kabasawa, Naohito Tanabe, Yuta Yoshizawa, Yoshiki Suzuki, Akihiko Saito, Ichiei Narita

RENAL REPLACEMENT THERAPY 8 ( 1 ) 2022.6

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

Background Dulaglutide is a once-weekly glucagon-like peptide-1 (GLP-1) receptor agonist approved for the treatment of type 2 diabetes mellitus (T2DM). However, the efficacy and safety of dulaglutide remain unclear in insulin-treated patients with T2DM on maintenance hemodialysis (HD). Methods Dulaglutide treatment was initiated, and the insulin dose was adjusted according to the needs of individual participants. Primary outcomes were changes in the mean and standard deviation (SD) of blood glucose (BG) levels and mean amplitude of glycemic excursions (MAGE) evaluated by continuous glucose monitoring (CGM) for six days, glycated albumin (GA), glycated hemoglobin (HbA1c), pre-dialysis blood glucose levels, and daily total insulin dose from the baseline over 24 weeks. Secondary outcomes were changes in treatment satisfaction and QOL levels from the baseline, measured by using the Diabetes Treatment Satisfaction Questionnaire (DTSQ) and the Diabetes Therapy-Related Quality of Life questionnaire (DTR-QOL) scores. Results The analysis was performed on the 12 participants who completed the study. The GA level (median - 1.8 [interquartile range - 6.6, - 0.3] %; p = 0.026) and daily total insulin dose (- 15.0 [- 24.5, - 9.4] U/day; p = 0.002) significantly decreased without increasing hypoglycemia (area over the glucose curve < 70 mg/dL: 0.0 [- 0.2, 0.0] mg center dot 24 h/dl; p = 0.917). Four patients successfully withdrew from insulin therapy. The levels of HbA1c, SD of BG, and MAGE showed a decreasing tendency, but no significant improvement. Regarding treatment satisfaction and QOL, the total scores of DTSQ (8.0 [0.3, 12.5]; p = 0.041) and DTR-QOL (15.5 [- 1.8, 42.0]; p = 0.023) significantly improved. Conclusion Dulaglutide may help improve glycemic control, treatment satisfaction, and QOL without increasing hypoglycemia in insulin-treated patients with T2DM on maintenance HD. Trial registration This study was registered with the University Hospital Medical Information Network-Clinical Trials Registry (UMIN-CTR) on October 11, 2016 (registration ID, UMIN000024283).

DOI： 10.1186/s41100-022-00409-4

2型糖尿病患者における尿中メガリン測定による腎予後予測

飯田倫理, 細島康宏, 蒲澤秀門, 蒲澤佳子, 後藤佐和子, 田中崇裕, 北村信隆, 忰田亮平, 鈴木芳樹, 成田一衛, 斎藤亮彦

日本腎臓学会誌 64 ( 3 ) 240 - 240 2022.5

2型糖尿病患者における尿中メガリンの腎予後予測能の検討

飯田倫理, 細島康宏, 蒲澤秀門, 蒲澤佳子, 後藤佐和子, 田中崇裕, 北村信隆, 忰田亮平, 鈴木芳樹, 成田一衛, 斎藤亮彦

糖尿病 65 ( Suppl.1 ) S - 150 2022.4

Association of metabolic syndrome traits with urinary biomarkers in Japanese adults. International journal

Keiko Kabasawa, Michihiro Hosojima, Yumi Ito, Kazuo Matsushima, Junta Tanaka, Masanori Hara, Kazutoshi Nakamura, Ichiei Narita, Akihiko Saito

Diabetology & metabolic syndrome 14 ( 1 ) 9 - 9 2022.1

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

BACKGROUND: Although metabolic syndrome traits are risk factors for chronic kidney disease, few studies have examined their association with urinary biomarkers. METHODS: Urinary biomarkers, including A-megalin, C-megalin, podocalyxin, albumin, α1-microglobulin, β2-microglobulin, and N-acetyl-β-D-glucosaminidase, were cross-sectionally assessed in 347 individuals (52.7% men) with a urine albumin-to-creatinine ratio (ACR) < 300 mg/g in a health checkup. Metabolic syndrome traits were adopted from the National Cholesterol Education Program (third revision) of the Adult Treatment Panel criteria modified for Asians. RESULTS: Participants had a mean body mass index, estimated glomerular filtration rate (eGFR), and median ACR of 23.0 kg/m2, 74.8 mL/min/1.73 m2, and 7.5 mg/g, respectively. In age- and sex-adjusted logistic regression analysis, A-megalin and albumin were significantly associated with the clustering number of metabolic syndrome traits (3 or more). After further adjustment with eGFR, higher quartiles of A-megalin and albumin were each independently associated with the clustering number of metabolic syndrome traits (adjusted odds ratio for A-megalin: 1.30 per quartile, 95% CI 1.03-1.64; albumin: 1.42 per quartile, 95% CI 1.12-1.79). CONCLUSIONS: Both urinary A-megalin and albumin are associated with the clustering number of metabolic syndrome traits. Further research on urinary A-megalin is warranted to examine its role as a potential marker of kidney damage from metabolic risk factors.

DOI： 10.1186/s13098-021-00779-5

Urinary FABP1 is a biomarker for impaired proximal tubular protein reabsorption and is synergistically enhanced by concurrent liver injury. International journal

Ryo Kawakami, Miki Matsui, Ayumu Konno, Ryosuke Kaneko, Shreya Shrestha, Suman Shrestha, Hiroaki Sunaga, Hirofumi Hanaoka, Sawako Goto, Michihiro Hosojima, Hideyuki Kabasawa, Masaru Obokata, Norimichi Koitabashi, Hiroki Matsui, Tsutomu Sasaki, Akihiko Saito, Motoko Yanagita, Hirokazu Hirai, Masahiko Kurabayashi, Tatsuya Iso

The Journal of pathology 255 ( 4 ) 362 - 373 2021.12

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

Urinary fatty acid binding protein 1 (FABP1, also known as liver-type FABP) has been implicated as a biomarker of acute kidney injury (AKI) in humans. However, the precise biological mechanisms underlying its elevation remain elusive. Here, we show that urinary FABP1 primarily reflects impaired protein reabsorption in proximal tubule epithelial cells (PTECs). Bilateral nephrectomy resulted in a marked increase in serum FABP1 levels, suggesting that the kidney is an essential organ for removing serum FABP1. Injected recombinant FABP1 was filtered through the glomeruli and robustly reabsorbed via the apical membrane of PTECs. Urinary FABP1 was significantly elevated in mice devoid of megalin, a giant endocytic receptor for protein reabsorption. Elevation of urinary FABP1 was also observed in patients with Dent disease, a rare genetic disease characterized by defective megalin function in PTECs. Urinary FABP1 levels were exponentially increased following acetaminophen overdose, with both nephrotoxicity and hepatotoxicity observed. FABP1-deficient mice with liver-specific overexpression of FABP1 showed a massive increase in urinary FABP1 levels upon acetaminophen injection, indicating that urinary FABP1 is liver-derived. Lastly, we employed transgenic mice expressing diphtheria toxin receptor (DT-R) either in a hepatocyte- or in a PTEC-specific manner, or both. Upon administration of diphtheria toxin (DT), massive excretion of urinary FABP1 was induced in mice with both kidney and liver injury, while mice with either injury type showed marginal excretion. Collectively, our data demonstrated that intact PTECs have a considerable capacity to reabsorb liver-derived FABP1 through a megalin-mediated mechanism. Thus, urinary FABP1, which is synergistically enhanced by concurrent liver injury, is a biomarker for impaired protein reabsorption in AKI. These findings address the use of urinary FABP1 as a biomarker of histologically injured PTECs that secrete FABP1 into primary urine, and suggest the use of this biomarker to simultaneously monitor impaired tubular reabsorption and liver function. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

DOI： 10.1002/path.5775

CKD・DKD診療の新展開高齢CKD・DKD診療の現状

細島康宏, 蒲澤秀門, 鈴木芳樹, 斎藤亮彦, 成田一衛

日本腎臓学会誌 63 ( 6-E ) 665 - 665 2021.9

Rice Endosperm Protein Improves the Anti-Inflammatory Effects of High-Density Lipoprotein and Produces Lower Atherosclerotic Lesion Accelerated by the Renal Mass Reduction than Casein in a Mouse Model. International journal

Ryohei Kaseda, Michihiro Hosojima, Shoji Kuwahara, Hideyuki Kabasawa, Hiroyuki Aoki, Yuki Higuchi, Valentina Kon, Ichiei Narita, Akihiko Saito

Journal of the American College of Nutrition 41 ( 7 ) 1 - 11 2021.8

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

Chronic kidney disease (CKD) impairs the anti-inflammatory effects of high-density lipoprotein (HDL) and increases cardiovascular mortality. Though the potential role of dietary interventions to manage HDL is well studied, the clinical trials aimed to increase HDL levels have failed to reduce cardiovascular risk, rendering HDL function to be explored as a more relevant clinical parameter. This study investigates the effects of rice endosperm protein (REP), a plant-based protein, on the anti-inflammatory properties of HDL and renal injury-driven atherosclerosis in comparison with casein, an animal protein. Ten-week-old apolipoprotein E-deficient hyperlipidemic mice underwent uninephrectomy. The mice (n = 6 each) were pair-fed a normal casein-based diet or a REP-based diet (both with 20.0% protein content) for seven weeks. Atherosclerotic lesions were detected by en face Sudan IV staining of the aorta. The number and sizes of the atherosclerotic lesions were significantly lower in the REP-based diet-fed group than the casein-based diet-fed group (p = 0.038). However, the REP-based diet neither elicited an ameliorative effect on kidney function or histology nor impacted the cholesterol profiles. Furthermore, HDL from the REP-based diet-fed mice significantly suppressed the inflammatory cytokine response of human umbilical vein endothelial cells than that from the casein-based diet-fed mice (MCP-1, p = 0.010; IL-6, p = 0.011; IL-1β, p = 0.028). The REP-based diet has a higher potential to lessen the atherosclerotic lesions accelerated by renal mass reduction than a casein-based diet, which could be associated with the anti-inflammatory effects of HDL.

DOI： 10.1080/07315724.2021.1950584

Measurement of Plasma Glucagon Levels Using Mass Spectrometry in Patients with Type 2 Diabetes on Maintenance Hemodialysis. International journal

Yuta Yoshizawa, Michihiro Hosojima, Hideyuki Kabasawa, Naohito Tanabe, Atsushi Miyachi, Hitoshi Hamajima, Eri Mieno, Masaki Kobayashi, Tadahiro Kitamura, Ichiei Narita, Akihiko Saito

Kidney & blood pressure research 46 ( 5 ) 1 - 5 2021.8

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

BACKGROUND: Recently, attention has been focused on the effect of glucagon on blood glucose variability. The dynamics of glucagon have attracted attention as a new target in the treatment of diabetes patients. However, the dynamics of glucagon in hemodialysis (HD) patients with type 2 diabetes mellitus (T2DM) remain unclear. OBJECTIVES: The aim of this study was to assess the dynamics of glucagon in HD patients with T2DM. MATERIALS AND METHODS: We measured plasma glucagon in HD patients with T2DM by liquid chromatography-high-resolution mass spectrometry (LC-HRMS), sandwich enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). The glucagon levels measured by each method were compared. We used the glucagon levels determined by our developed LC-HRMS method as the standard in this study. RESULTS: Plasma glucagon levels measured by LC-HRMS before HD were significantly higher than those measured after HD. Plasma glucagon levels measured using sandwich ELISA had a significantly higher correlation with those measured using LC-HRMS compared with RIA. CONCLUSIONS: This was the first study to assess glucagon levels in HD patients with T2DM using LC-HRMS, which is considered a highly accurate method. Sandwich ELISA was shown to measure glucagon levels accurately as well.

DOI： 10.1159/000518027

糸球体結節性病変と係蹄壁へのIgG線状沈着を呈した境界型糖尿病の1例

山口浩毅, 細島康宏, 蒲澤秀門, 後藤佐和子, 後藤慧, 伊藤由美, 今井直史, 金子佳賢, 鈴木芳樹, 齋藤亮彦, 成田一衛

糖尿病 64 ( 7 ) 411 - 411 2021.7

糸球体結節性病変と係蹄壁へのIgG線状沈着を呈した境界型糖尿病の1例

山口浩毅, 細島康宏, 蒲澤秀門, 後藤佐和子, 後藤慧, 伊藤由美, 今井直史, 金子佳賢, 鈴木芳樹, 齋藤亮彦, 成田一衛

糖尿病 64 ( 7 ) 411 - 411 2021.7

進行期CKD患者における栄養指導の実際(横断研究) REACH-J-CKDコホート研究より

細島康宏, 蒲澤秀門, 村山稔子, 若杉三奈子, 鈴木芳樹, 斎藤亮彦, 岡田浩一, 和田隆志, 山縣邦弘, 柏原直樹, 成田一衛

日本腎臓学会誌 63 ( 4 ) 461 - 461 2021.6

進行期CKD患者における栄養指導の実際(横断研究) REACH-J-CKDコホート研究より

細島康宏, 蒲澤秀門, 村山稔子, 若杉三奈子, 鈴木芳樹, 斎藤亮彦, 岡田浩一, 和田隆志, 山縣邦弘, 柏原直樹, 成田一衛

日本腎臓学会誌 63 ( 4 ) 461 - 461 2021.6

透析患者における経腸栄養と静脈栄養 up to date 維持血液透析患者における透析時静脈栄養(IDPN)の実際

細島康宏, 蒲澤秀門, 鈴木芳樹, 斎藤亮彦, 成田一衛

日本透析医学会雑誌 54 ( Suppl.1 ) 217 - 217 2021.5

小児尿中メガリン基準値の設定

金子昌弘, 山田剛史, 後藤佐和子, 蒲澤秀門, 細島康宏, 斎藤亮彦

日本小児腎臓病学会雑誌 34 ( 1Suppl. ) 136 - 136 2021.5

2型糖尿病患者におけるCOVID-19の流行が食事習慣に及ぼす影響に関する自記式食事歴法質問票(DHQ)を用いた検討

蒲澤秀門, 細島康宏, 村山稔子, 桑原頌治, 鈴木芳樹, 成田一衛, 斎藤亮彦

糖尿病 64 ( Suppl.1 ) I - 5 2021.5

小児尿中メガリン基準値の設定

金子昌弘, 山田剛史, 後藤佐和子, 蒲澤秀門, 細島康宏, 斎藤亮彦

日本小児腎臓病学会雑誌 34 ( 1Suppl. ) 136 - 136 2021.5

腎臓における脂肪酸処理の正しく評価されていないメカニズム脂肪酸再吸収のための優れた機能(Unappreciated mechanisms of fatty acids handling in the kidney: Remarkable capability for fatty acid reabsorption)

川上亮, 大日方英, 花岡宏史, 金井彩香, 中野大介, 齋藤亮彦, 西山成, 柳田素子, 倉林正彦, 磯達也

日本内分泌学会雑誌 96 ( 4 ) 1116 - 1116 2021.4

Effects of the Once-Weekly DPP4 Inhibitor Omarigliptin on Glycemic Control in Patients with Type 2 Diabetes Mellitus on Maintenance Hemodialysis: A 24-Week Open-Label, Multicenter Randomized Controlled Study

Yuta Yoshizawa, Michihiro Hosojima, Hideyuki Kabasawa, Naohito Tanabe, Daisuke Ugamura, Yutaka Koda, Hisaki Shimada, Tetsuya Takasawa, Takahito Ito, Tadahiro Kitamura, Masaki Kobayashi, Yoshiki Suzuki, Ichiei Narita, Akihiko Saito

DIABETES THERAPY 12 ( 3 ) 655 - 667 2021.3

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

Introduction: Dipeptidyl peptidase 4 (DPP4) inhibitors are widely used in patients with type 2 diabetes mellitus (T2DM) on maintenance hemodialysis (HD), but the efficacy of the once-weekly DPP4 inhibitor omarigliptin is not known.Methods: This prospective, randomized, open-label, parallel-group, non-inferiority/superiority, once-daily DPP4 inhibitor linagliptin-controlled, multicenter study examined glycemic control and safety of omarigliptin (UMIN000024284). Sample size was calculated to confirm non-inferiority in terms of changes in glycated hemoglobin (HbA1c). We enrolled 33 patients with T2DM on maintenance HD who had been treated with linagliptin for at least 3 months. The patients were randomized to receive omarigliptin (12.5 mg/week; n = 16) or linagliptin (5 mg/day; n = 17). Primary endpoints were changes in HbA1c and glycoalbumin (GA) over 24 weeks.Results: Differences in the mean change in primary endpoint values between the omarigliptin and linagliptin groups were - 0.61% [- 1.14, - 0.09] for HbA1c, with a two-tailed upper 95% limit (i.e., one-tailed 97.5% upper limit) of 0.25%, below the non-inferiority limit, and - 1.67% [- 4.23, + 0.88] for GA, with a two-tailed upper 95% limit of 0.75%, above the non-inferiority limit. At 24 weeks, the omarigliptin group showed significantly greater reduction in HbA1c than the linagliptin group (- 0.2% +/- 0.6% vs. 0.4% +/- 0.8%, two-tailed p = 0.024) and significantly greater reduction in blood glucose after a single HD session (- 18.4 +/- 31.4 mg/dL vs. 25.2 +/- 59.5 mg/dL, respectively, two-tailed p = 0.019). No subjects in the omarigliptin group developed hypoglycemia.Conclusions: Our data showed that omarigliptin was non-inferior to linagliptin in glycemic control. Omarigliptin is feasible for glycemic control in patients with T2DM on maintenance HD.

DOI： 10.1007/s13300-020-00991-y

Effects of the Once-Weekly DPP4 Inhibitor Omarigliptin on Glycemic Control in Patients with Type 2 Diabetes Mellitus on Maintenance Hemodialysis: A 24-Week Open-Label, Multicenter Randomized Controlled Study. International journal

Yuta Yoshizawa, Michihiro Hosojima, Hideyuki Kabasawa, Naohito Tanabe, Daisuke Ugamura, Yutaka Koda, Hisaki Shimada, Tetsuya Takasawa, Takahito Ito, Tadahiro Kitamura, Masaki Kobayashi, Yoshiki Suzuki, Ichiei Narita, Akihiko Saito

Diabetes therapy : research, treatment and education of diabetes and related disorders 12 ( 3 ) 655 - 667 2021.3

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

INTRODUCTION: Dipeptidyl peptidase 4 (DPP4) inhibitors are widely used in patients with type 2 diabetes mellitus (T2DM) on maintenance hemodialysis (HD), but the efficacy of the once-weekly DPP4 inhibitor omarigliptin is not known. METHODS: This prospective, randomized, open-label, parallel-group, non-inferiority/superiority, once-daily DPP4 inhibitor linagliptin-controlled, multicenter study examined glycemic control and safety of omarigliptin (UMIN000024284). Sample size was calculated to confirm non-inferiority in terms of changes in glycated hemoglobin (HbA1c). We enrolled 33 patients with T2DM on maintenance HD who had been treated with linagliptin for at least 3 months. The patients were randomized to receive omarigliptin (12.5 mg/week; n = 16) or linagliptin (5 mg/day; n = 17). Primary endpoints were changes in HbA1c and glycoalbumin (GA) over 24 weeks. RESULTS: Differences in the mean change in primary endpoint values between the omarigliptin and linagliptin groups were - 0.61% [- 1.14, - 0.09] for HbA1c, with a two-tailed upper 95% limit (i.e., one-tailed 97.5% upper limit) of 0.25%, below the non-inferiority limit, and - 1.67% [- 4.23, + 0.88] for GA, with a two-tailed upper 95% limit of 0.75%, above the non-inferiority limit. At 24 weeks, the omarigliptin group showed significantly greater reduction in HbA1c than the linagliptin group (- 0.2% ± 0.6% vs. 0.4% ± 0.8%, two-tailed p = 0.024) and significantly greater reduction in blood glucose after a single HD session (- 18.4 ± 31.4 mg/dL vs. 25.2 ± 59.5 mg/dL, respectively, two-tailed p = 0.019). No subjects in the omarigliptin group developed hypoglycemia. CONCLUSIONS: Our data showed that omarigliptin was non-inferior to linagliptin in glycemic control. Omarigliptin is feasible for glycemic control in patients with T2DM on maintenance HD. CLINICAL TRIALS REGISTRATION: UMIN000024284.

DOI： 10.1007/s13300-020-00991-y

Combination therapy of cisplatin with cilastatin enables an increased dose of cisplatin, enhancing its antitumor effect by suppression of nephrotoxicity. International journal

Masashi Arita, Satoshi Watanabe, Nobumasa Aoki, Shoji Kuwahara, Ryo Suzuki, Sawako Goto, Yuko Abe, Miho Takahashi, Miyuki Sato, Satoshi Hokari, Aya Ohtsubo, Satoshi Shoji, Koichiro Nozaki, Kosuke Ichikawa, Rie Kondo, Masachika Hayashi, Yasuyoshi Ohshima, Hideyuki Kabasawa, Michihiro Hosojima, Toshiyuki Koya, Akihiko Saito, Toshiaki Kikuchi

Scientific reports 11 ( 1 ) 750 - 750 2021.1

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

Cisplatin, one of the most active anticancer agents, is widely used in standard chemotherapy for various cancers. Cisplatin is more poorly tolerated than other chemotherapeutic drugs, and the main dose-limiting toxicity of cisplatin is its nephrotoxicity, which is dose-dependent. Although less toxic methods of cisplatin administration have been established, cisplatin-induced nephrotoxicity remains an unsolved problem. Megalin is an endocytic receptor expressed at the apical membrane of proximal tubules. We previously demonstrated that nephrotoxic drugs, including cisplatin, are reabsorbed through megalin and cause proximal tubular cell injury. We further found that cilastatin blocked the binding of cisplatin to megalin in vitro. In this study, we investigated whether cilastatin could reduce cisplatin-induced nephrotoxicity without influencing the antitumor effects of cisplatin. Nephrotoxicity was decreased or absent in mice treated with cisplatin and cilastatin, as determined by kidney injury molecule-1 staining and the blood urea nitrogen content. Combined with cilastatin, a twofold dose of cisplatin was used to successfully treat the mice, which enhanced the antitumor effects of cisplatin but reduced its nephrotoxicity. These findings suggest that we can increase the dose of cisplatin when combined with cilastatin and improve the outcome of cancer patients.

DOI： 10.1038/s41598-020-80853-6

Effects of DPP-4 Inhibitors on Blood Glucose Variability in Japanese Patients with Type 2 Diabetes on Maintenance Hemodialysis: A Prospective Observational Exploratory Study. International journal

Tomomi Ishikawa-Tanaka, Michihiro Hosojima, Hideyuki Kabasawa, Ryohei Kaseda, Ryota Yasukawa, Yusuke Yata, Shoji Kuwahara, Emiko Kono, Takuma Takata, Noriaki Iino, Takahiro Tanaka, Nobutaka Kitamura, Yoshiki Suzuki, Akihiko Saito, Ichiei Narita

Diabetes therapy : research, treatment and education of diabetes and related disorders 11 ( 12 ) 2845 - 2861 2020.12

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

INTRODUCTION: The precise blood glucose (BG) profile of hemodialysis patients is unclear, as is the effectiveness of dipeptidyl peptidase-4 (DPP-4) inhibitors in hemodialysis patients with type 2 diabetes. Here, we used continuous glucose monitoring (CGM) to evaluate BG variability in these patients and to assess the efficacy of DPP-4 inhibitors, particularly during hemodialysis sessions and at nighttime (UMIN000012638). METHODS: We examined BG profiles using CGM in 31 maintenance hemodialysis patients with type 2 diabetes. Differences between patients with and without DPP-4 inhibitors (n = 15 and 16, respectively) were analyzed using a linear mixed-effects model to assess changes in glucose levels in 5-min intervals. RESULTS: The model revealed that DPP-4 inhibitor use was significantly associated with suppression of a rapid drop in glucose levels, both with and without adjustment for BG levels at the start of hemodialysis. Moreover, the model revealed that the two groups differed significantly in the pattern of changes in BG levels from 0:00 to 6:55 am. DPP-4 inhibitors suppressed the tendency for subsequent nocturnal hypoglycemia. CONCLUSIONS: This prospective observational exploratory study showed that DPP-4 inhibitors could suppress BG variability during hemodialysis sessions as well as subsequent nocturnal changes in patients with type 2 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov identifier, UMIN000012638.

DOI： 10.1007/s13300-020-00928-5

インスリン使用中の維持血液透析患者におけるデュラグルチド併用による血糖管理の有効性およびQOL改善に関する探索的臨床試験

宇賀村大亮, 細島康宏, 蒲澤秀門, 田邊直仁, 吉澤優太, 忰田亮平, 鈴木芳樹, 成田一衛, 斎藤亮彦

糖尿病 63 ( Suppl.1 ) S - 300 2020.8

Rice bran protein ameliorates diabetes, reduces fatty liver, and has renoprotective effects in Zucker Diabetic Fatty rats

Masatoshi Kubota, Reiko Watanabe, Michihiro Hosojima, Akihiko Saito, Ai Sasou, Takehiro Masumura, Yukikazu Harada, Hiroyuki Hashimoto, Shinobu Fujimura, Motoni Kadowaki

Journal of Functional Foods 70 103981 - 103981 2020.7

DKDと食事 Up to date CKD、DKD患者への食事療法腎臓専門医の立場から(たんぱく質制限の効果も含め)

細島康宏, 蒲澤秀門, 成田一衛, 鈴木芳樹, 斎藤亮彦

日本腎臓学会誌 62 ( 4 ) 228 - 228 2020.7

メガリンを介する腎内レニン-アンジオテンシン(Ang)系(RAS)の動的均衡化制御機構

後藤佐和子, 吉田豊, 細島康宏, 蒲澤秀門, 忰田亮平, 成田一衛, 斎藤亮彦

日本腎臓学会誌 62 ( 4 ) 370 - 370 2020.7

マウス尿中メガリン測定用サンドイッチELISAの開発とその応用

早福莉那, 後藤佐和子, 細島康宏, 蒲澤秀門, 忰田亮平, 成田一衛, 斎藤亮彦

日本腎臓学会誌 62 ( 4 ) 339 - 339 2020.7

The cellular model of albumin endocytosis uncovers link between membrane and nuclear proteins. Reviewed International journal

Seiya Urae, Yutaka Harita, Tomohiro Udagawa, Koji L Ode, Masami Nagahama, Yuko Kajiho, Shoichiro Kanda, Akihiko Saito, Hiroki R Ueda, Masaomi Nangaku, Akira Oka

Journal of cell science 133 ( 13 ) 2020.6

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

Cubilin (CUBN) and amnionless (AMN), expressed in kidney and intestine, form a multiligand receptor complex called CUBAM that plays a crucial role in albumin absorption. To date, the mechanism of albumin endocytosis mediated by CUBAM remains to be elucidated. Here, we established an assay to quantitatively evaluate the albumin uptake by CUBAM using cells expressing full-length CUBN, which elucidated the crucial role of C-terminal part of CUBN and endocytosis signal motifs of AMN in albumin endocytosis. We also demonstrated that nuclear valosin-containing protein-like 2 (NVL2), an interacting protein of AMN, is involved in this process. While NVL2 was mainly localized in the nucleolus in cells without AMN expression, it was translocated to the extranuclear compartment when co-expressed with AMN. NVL2 knockdown significantly impaired the internalization of the CUBN-albumin complex in cultured cells, demonstrating an involvement of NVL2 in endocytic regulation. These findings uncover a link between membrane and nucleolar proteins which is involved in endocytic processes.

DOI： 10.1242/jcs.242859

Low bone mineral density due to secondary hyperparathyroidism in the GlatmTg(CAG-A4GALT) mouse model of Fabry disease. International journal

Hiroki Maruyama, Atsumi Taguchi, Mariko Mikame, Hongmei Lu, Norihiro Tada, Muneaki Ishijima, Haruka Kaneko, Mariko Kawai, Sawako Goto, Akihiko Saito, Riuko Ohashi, Yuji Nishikawa, Satoshi Ishii

FASEB bioAdvances 2 ( 6 ) 365 - 381 2020.6

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

Low bone mineral density (BMD)-diagnosed as osteoporosis or osteopenia-has been reported as a new characteristic feature of Fabry disease; however, the mechanism underlying the development of low BMD is unknown. We previously revealed that a mouse model of Fabry disease [GlatmTg(CAG-A4GALT)] exhibits impaired functioning of medullary thick ascending limb (mTAL), leading to insufficient Ca2+ reabsorption and hypercalciuria. Here, we investigated bone metabolism in GlatmTg(CAG-A4GALT) mice without marked glomerular or proximal tubular damage. Low BMD was detected by 20 weeks of age via micro-X-ray-computed tomography. Bone histomorphometry revealed that low BMD results by accelerated bone resorption and osteomalacia. Plasma parathyroid hormone levels increased in response to low blood Ca2+-not plasma fibroblast growth factor 23 (FGF-23) elevation-by 5 weeks of age and showed progressively increased phosphaturic action. Secondary hyperparathyroidism developed by 20 weeks of age and caused hyperphosphatemia, which increased plasma FGF-23 levels with phosphaturic action. The expression of 1α-hydroxylase [synthesis of 1α,25(OH)2D3] in the kidney did not decrease, but that of 24-hydroxylase [degradation of 1α,25(OH)2D3] decreased. Vitamin D deficiency was ruled out as the cause of osteomalacia, as plasma 1α,25(OH)2D3 and 25(OH)D3 levels were maintained. Results demonstrate that secondary hyperparathyroidism due to mTAL impairment causes accelerated bone resorption and osteomalacia due to hyperphosphaturia and hypercalciuria, leading to low BMD in Fabry model mice.

DOI： 10.1096/fba.2019-00080

SGLT2阻害薬による腎保護作用機序と尿中コンパニオン診断法の検討

忰田亮平, 飯田倫理, 細島康宏, 蒲澤秀門, 後藤佐和子, 桑原頌治, 宇賀村大亮, 吉澤優太, 田中崇裕, 成田一衛, 斎藤亮彦

臨床薬理の進歩 ( 41 ) 148 - 153 2020.6

Language：Japanese Publisher：(公財)臨床薬理研究振興財団

軽度〜中等度腎機能障害を有する成人2型糖尿病患者(残存機能ネフロンに過剰代謝負荷がかかっていることが推測される)を対象に、エンパグリフロジンによる腎機能保護作用を検証するとともに尿中メガリン(およびメガリンリガンド分子)の動態を調べ、腎機能保護作用がメガリン機能に関連したものであるか検討した。さらに、尿中メガリンなどの検査がコンパニオン診断として有用であるか、探索的な検討を行った。また、SGLT2阻害薬がメガリンの発現・機能に及ぼす影響についても検討を継続した。臨床試験の被験者51名で、平均年齢は62歳であり、男性が32名であった。2名が有害反応のため脱落したが、6ヵ月後、HbA1cとBMIが減少した。現在、メガリンを含む尿中バイオマーカーの推移について解析を行っている。ダパグリフロジン、エンパグリフロジンを投与されたマウスでは尿中の糖排泄増加を認めたが血糖値に差は認められなかった。免疫組織化学染色では近位尿細管におけるメガリンの染色性が低下していることが確認された。またウエスタンブロット法において、メガリンの蛋白がvehicle群と比較して低下していることが認められた。リアルタイムRT-PCRにおいてもメガリンmRNAがvehicle群と比較して低下していることが確認された。ダパグリフロジンを添加されたIRPTCではメガリン蛋白の低下が認められ、mRNAの低下も認められた。

Rice Endosperm Protein Administration to Juvenile Mice Regulates Gut Microbiota and Suppresses the Development of High-Fat Diet-Induced Obesity and Related Disorders in Adulthood. Reviewed International journal

Yuki Higuchi, Michihiro Hosojima, Hideyuki Kabasawa, Shoji Kuwahara, Sawako Goto, Koji Toba, Ryohei Kaseda, Takahiro Tanaka, Nobutaka Kitamura, Hayato Takihara, Shujiro Okuda, Masayuki Taniguchi, Hitoshi Arao, Ichiei Narita, Akihiko Saito

Nutrients 11 ( 12 ) 2019.12

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

Obesity and related disorders, which are increasing in adults worldwide, are closely linked to childhood diet and are associated with chronic inflammation. Rice endosperm protein (REP) intake during adulthood has been reported to improve lipid metabolism and suppress the progression of diabetic kidney disease in animal models. However, the effects of REP intake during childhood on adulthood health are unclear. Therefore, we used a mouse model to experimentally investigate the preconditioning effects of REP intake during childhood on the development of obesity and related disorders in adulthood. Male C57BL/6J mice were pair-fed a normal-fat diet containing casein or REP during the juvenile period and then a high-fat diet (HFD) containing casein or REP during adulthood. Mice fed REP during the juvenile period showed better body weight, blood pressure, serum lipid profiles, lipopolysaccharide (LPS)-binding protein levels, and glucose tolerance in adulthood than those fed casein during the juvenile period. HFD-induced renal tubulo-glomerular alterations and hepatic microvesicular steatosis were less evident in REP-fed mice than in casein-fed ones. REP intake during the juvenile period improved HFD-induced dysbiosis (i.e., Escherichia genus proliferation and reduced gut microbiota diversity), thereby suppressing endotoxin-related chronic inflammation. Indeed, REP-derived peptides showed antibacterial activity against Escherichia coli, a major producer of LPS. In conclusion, REP supplementation during the juvenile period may regulate the gut microbiota and thus suppress the development of obesity and related disorders in adulthood in mice.

DOI： 10.3390/nu11122919

Correlation of prechemotherapy urinary megalin ectodomain (A-megalin) levels with the development of cisplatin-induced nephrotoxicity: a prospective observational study. Reviewed International journal

Satoshi Shoji, Michihiro Hosojima, Hideyuki Kabasawa, Rie Kondo, Satoru Miura, Satoshi Watanabe, Nobumasa Aoki, Ryohei Kaseda, Shoji Kuwahara, Naohito Tanabe, Yoshiaki Hirayama, Ichiei Narita, Toshiaki Kikuchi, Hiroshi Kagamu, Akihiko Saito

BMC cancer 19 ( 1 ) 1170 - 1170 2019.12

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

BACKGROUND: Cisplatin is a potent chemotherapeutic agent used to treat a variety of solid tumors. One of the major side effects of cisplatin is dose-limiting nephrotoxicity. We recently demonstrated that the renal uptake of cisplatin and resultant cisplatin-induced nephrotoxicity are mediated in part by megalin, an endocytic receptor in proximal tubule epithelial cells (PTECs). We also developed sandwich enzyme-linked immunosorbent assays to measure the megalin ectodomain (A-megalin) and full-length megalin (C-megalin) in urine using monoclonal antibodies against the amino- and carboxyl-termini of megalin, respectively. The present study examined the correlation of urinary megalin level with cisplatin-induced nephrotoxicity and its utility as a biomarker in patients with thoracic cancer. METHODS: This prospective observational study involved 45 chemotherapy-naïve patients scheduled to receive chemotherapy with ≥60 mg/m2 cisplatin for histologically diagnosed small cell lung cancer, non-small cell lung cancer, or malignant pleural mesothelioma. Before and after the first course of chemotherapy, we measured urinary A- and C-megalin and other markers of PTEC injury, such as N-acetyl-β-D-glucosaminidase, α1-microglobulin, β2-microglobulin, neutrophil gelatinase-associated lipocalin, and liver-type fatty acid-binding protein, and compared the values with the change in the estimated glomerular filtration rate (eGFR) and clinical risk factors for renal impairment. RESULTS: A negative correlation was found between baseline urinary A-megalin levels and change in eGFR (r = - 0.458, P = 0.002). According to Kaplan-Meier survival curves, eGFR decline was associated with the baseline urinary A-megalin quartile (P = 0.038). In addition, according to the hazard ratios (HRs) for eGFR decline > 10 mL/min/1.73 m2 calculated using a Cox proportional hazard model, the highest quartile had a significantly higher risk of eGFR decline compared with the lowest quartile (HR 7.243; 95% confidence interval 1.545-33.962). Other baseline urinary markers showed no correlation with eGFR decline. CONCLUSIONS: This is the first report demonstrating that prechemotherapy urinary A-megalin levels are correlated with the development of cisplatin-induced nephrotoxicity. This finding has clinical implications for the identification of patients at risk for cisplatin-induced nephrotoxicity and the development of possible prophylactic therapies.

DOI： 10.1186/s12885-019-6398-2

Higher estimated net endogenous acid production with lower intake of fruits and vegetables based on a dietary survey is associated with the progression of chronic kidney disease. Reviewed International journal

Koji Toba, Michihiro Hosojima, Hideyuki Kabasawa, Shoji Kuwahara, Toshiko Murayama, Keiko Yamamoto-Kabasawa, Ryohei Kaseda, Eri Wada, Reiko Watanabe, Naohito Tanabe, Yoshiki Suzuki, Ichiei Narita, Akihiko Saito

BMC nephrology 20 ( 1 ) 421 - 421 2019.11

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

BACKGROUND: Dietary acid load has been suggested to mediate the progression of chronic kidney disease (CKD). However, it is unclear what kinds of foods are actually associated with dietary acid load in patients with CKD. The self-administered diet history questionnaire (DHQ), which semi-quantitatively assesses the dietary habits of Japanese individuals through 150 question items, can estimate average daily intake of various foods and nutrients during the previous month. Using the DHQ, we investigated the association of dietary acid load with CKD progression. We also analyzed the kinds of food that significantly affect dietary acid load. METHODS: Subjects were 96 outpatients with CKD (average estimated glomerular filtration rate [eGFR], 53.0 ± 18.1 ml/min/1.73 m2) at Niigata University Hospital, who had completed the DHQ in 2011. We calculated net endogenous acid production (NEAP) from potassium and protein intake evaluated by the DHQ in order to assess dietary acid load. CKD progression was assessed by comparing eGFR between 2008 and 2014. RESULTS: NEAP was not correlated with protein intake (r = 0.088, p = 0.398), but was negatively correlated with potassium intake (r = - 0.748, p < 0.001). Reduction in eGFR from 2008 to 2014 was estimated to be significantly greater in patients with higher NEAP (NEAP > 50.1 mEq/day, n = 45) than in those with lower NEAP (NEAP ≤50.1 mEq/day, n = 50) by 5.9 (95% confidence interval [95%CI], 0.1 to 11.6) ml/min/1.73 m2. According to multiple logistic regression analysis, higher NEAP was significantly associated with lower intake of fruits (odds ratio [OR], 6.454; 95%CI, 2.19 to 19.00), green and yellow vegetables (OR, 5.18; 95%CI, 1.83 to14.66), and other vegetables (OR, 3.87; 95%CI, 1.29 to 11.62). CONCLUSIONS: Elevated NEAP could be a risk factor for CKD progression. Low intake of fruits and vegetables would increase dietary acid load and might affect the progression of renal dysfunction in Japanese CKD patients.

DOI： 10.1186/s12882-019-1591-8

Podocyte Injury Augments Intrarenal Angiotensin II Generation and Sodium Retention in a Megalin-Dependent Manner. Reviewed International journal

Masahiro Koizumi, Kohei Ueda, Fumio Niimura, Akira Nishiyama, Motoko Yanagita, Akihiko Saito, Ira Pastan, Toshiro Fujita, Masafumi Fukagawa, Taiji Matsusaka

Hypertension (Dallas, Tex. : 1979) 74 ( 3 ) 509 - 517 2019.9

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

We have previously shown that podocyte injury increases the glomerular filtration of liver-derived Agt (angiotensinogen) and the generation of intrarenal Ang II (angiotensin II) and that the filtered Agt is reabsorbed by proximal tubules in a manner dependent on megalin. In the present study, we aimed to study the role of megalin in the generation of renal Ang II and sodium handling during nephrotic syndrome. We generated proximal tubule-specific megalin KO (knockout) mice and crossed these animals with NEP25 mice, in which podocyte-specific injury can be induced by injection of the immunotoxin LMB2. Without podocyte injury, renal Agt staining was markedly diminished and urinary Agt increased in KO mice. However, renal Ang II was similar between KO and control mice on average: 117 (95% CI, 101-134) versus 101 (95% CI, 68-133) fmol/g tissue. We next tested the effect of megalin KO on intrarenal Ang II generation with podocyte injury. Control NEP25 mice showed markedly increased renal Agt staining and renal Ang II levels: 450 (336-565) fmol/g tissue. Megalin KO/NEP25 mice showed markedly diminished Agt reabsorption and attenuated renal Ang II: 199 (156-242) fmol/g tissue (P<0.001). Compared with control NEP25 mice, megalin KO/NEP25 mice excreted 5-fold more sodium in the urine. Western blot analysis showed that megalin KO decreased NHE3 and the cleaved α and γ forms of Epithelial Na Channel. These data indicate that Agt reabsorbed by proximal tubules via megalin in nephrotic syndrome is converted to Ang II, which may contribute to sodium retention and edema formation by activating NHE3 and Epithelial Na Channel.

DOI： 10.1161/HYPERTENSIONAHA.118.12352

シラスタチンによりシスプラチン腎症を抑制することでシスプラチン投与を増量できる(Cilastatin suppresses cisplatin-induced nephrotoxicity and enables to increase the dose of cisplatin for cancer therapy)

有田将史, 渡部聡, 青木信将, 高橋美帆, 庄子聡, 野嵜幸一郎, 市川紘将, 近藤利恵, 桑原頌治, 田中純太, 小屋俊之, 斎藤亮彦, 菊地利明

日本癌学会総会記事 78回 P - 1398 2019.9

2型糖尿病を合併した維持血液透析患者におけるオマリグリプチンの有用性の検討

吉澤優太, 細島康宏, 蒲澤秀門, 田邊直仁, 甲田豊, 島田久基, 高澤哲也, 伊藤孝仁, 鈴木芳樹, 成田一衛, 斎藤亮彦

糖尿病 62 ( 7 ) 419 - 419 2019.7

Association of estimated dietary acid load with albuminuria in Japanese adults: a cross-sectional study. Reviewed

Kabasawa K, Hosojima M, Takachi R, Nakamura K, Ito Y, Saito A, Sawada N, Tsugane S, Tanaka J, Narita I

BMC nephrology 20 ( 1 ) 194 2019.5

自記式食事歴質問票(DHQ)を用いたSGLT2阻害薬の効果と食事摂取状況の検討

村山稔子, 細島康宏, 蒲澤秀門, 桑原頌治, 田中崇裕, 鈴木芳樹, 成田一衛, 斎藤亮彦

糖尿病 62 ( Suppl.1 ) S - 263 2019.4

Significance of urinary C-megalin excretion in vitamin D metabolism in pre-dialysis CKD patients. Reviewed International journal

Toi N, Inaba M, Ishimura E, Tsugawa N, Imanishi Y, Emoto M, Hirayama Y, Nakatani S, Saito A, Yamada S

Scientific reports 9 ( 1 ) 2207 - 2207 2019.2

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

Serum 1,25(OH)2D and 24,25(OH)2D are decreased in CKD. Megalin in proximal tubular epithelial cells reabsorbs glomerular-filtered 25(OH)D-DBP complex to convert 25(OH)D to 1,25(OH)2D and 24,25(OH)2D. Urinary C-megalin excretion is increased via exocytosis from injured nephrons overloaded with megalin-mediated protein metabolism. This study investigated the significance of urinary C-megalin excretion in vitamin D metabolism in 153 pre-dialysis CKD patients. Urinary C-megalin was positively associated with urinary protein, β2MG and α1MG, and exhibited negative correlations with serum 25(OH)D, 1,25(OH)2D and 24,25(OH)2D. Multiple regression analysis showed that urinary C-megalin had a significantly negative association with 25(OH)D. Serum 1,25(OH)2D and 24,25(OH)2D, as well as 1,25(OH)2D/25(OH)D and 24,25(OH)2D/25(OH)D ratios, showed positive correlations with eGFR. Additionally, wholePTH was positively associated with 1,25(OH)2D/25(OH)D and 1,25(OH)2D/24,25(OH)2D, while FGF23 was positively associated with 24,25(OH)2D/25(OH)D and negatively with 1,25(OH)2D/24,25(OH)2D. Urinary C-megalin emerged as an independent factor positively associated with 1,25(OH)2D/25(OH)D and 1,25(OH)2D/24,25(OH)2D. Although 1,25(OH)2D and 24,25(OH)2D are decreased in CKD patient serum, our findings suggest that PTH and FGF23 retain their effects to regulate vitamin D metabolism even in the kidneys of these patients, while production of 1,25(OH)2D and 24,25(OH)2D from 25(OH)D is restricted due to either impairment of megalin-mediated reabsorption of the 25(OH)D-DBP complex or reduced renal mass.

DOI： 10.1038/s41598-019-38613-8

β2-Microglobulin and Neutrophil Gelatinase-Associated Lipocalin, Potential Novel Urine Biomarkers in Periodontitis: A Cross-Sectional Study in Japanese. Reviewed International journal

Mayuka Nakajima, Michihiro Hosojima, Koichi Tabeta, Sayuri Miyauchi, Miki Yamada-Hara, Naoki Takahashi, Haruna Miyazawa, Yumi Matsuda-Matsukawa, Keisuke Sato, Noriko Sugita, Yasutaka Komatsu, Tomomi Ishikawa, Kazuhiro Akiishi, Kazuhisa Yamazaki, Kiminori Kato, Akihiko Saito, Hiromasa Yoshie

International journal of dentistry 2019 1394678 - 1394678 2019

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

Objectives: Several serum biomarkers have been reported to increase in periodontitis patients as possible mediators linking periodontal inflammation to systemic diseases. However, the relationship between periodontitis and urine biomarkers is still unclear. The aim of this cross-sectional study was to investigate potential urine biomarkers of periodontitis in a Japanese population. Materials and Methods: This study included 108 male subjects, and microbiological and clinical parameters were evaluated as a periodontitis marker. The correlation between nine urine biomarkers (typically used to diagnose kidney disease) and periodontal parameters was analyzed. Based on the findings, β2-microglobulin (β2-MG) and neutrophil gelatinase-associated lipocalin (NGAL) were selected for comparison and multivariate regression analysis, and the Kruskal-Wallis test followed by Bonferroni correction was used to identify differences in their concentrations between the three periodontitis groups (severe, moderate, and no/mild periodontitis). Results: β2-MG and NGAL exhibited a significant correlation with clinical parameters of periodontitis. The prevalence of clinical parameters such as bleeding on probing and number of sites with probing depth (PD) ≥ 6 mm were greater in the β2-MG high group (≥300 μg/g creatinine) than in the normal group (P=0.017 and 0.019, respectively). Multivariate regression analysis indicated that the number of sites with PD ≥ 6 mm was independently associated with urine β2-MG. Moreover, the number of sites with the clinical attachment level (CAL) ≥ 6 mm was greater in the NGAL high group (highest quartile) (P=0.041). Multivariate regression analysis showed that the number of sites with CAL ≥ 6 mm was associated independently with urine NGAL. Finally, β2-MG was significantly higher in the severe periodontitis subjects compared to the no/mild periodontitis subjects. Conclusion: The significant association between urine β2-MG or NGAL and periodontitis was revealed. These biomarkers can potentially be used to screen for or diagnose periodontitis. This trial is registered with the UMIN Clinical Trials Registry UMIN000013485.

DOI： 10.1155/2019/1394678

Circulating FABP4 is eliminated by the kidney via glomerular filtration followed by megalin-mediated reabsorption Reviewed International journal

Shrestha S, Sunada S, Hanaoka H, Yamaguchi A, Kuwahara S, Umbarawan Y, Nakajima K, Machida T, Murakami M, Saito A, Tsushima Y, Kurabayashi M, Iso T

Scientific Reports (2018) 8 ( 16451 ) 16451 - 16451 2018.11

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

Circulating fatty acid binding protein 4 (FABP4), secreted from adipocytes, is a potential biomarker for metabolic and cardiovascular diseases. Circulating FABP4 levels are positively associated with adiposity and adrenergic stimulation, but negatively with renal function. In this study, we addressed the issue of how the kidney regulates clearance of circulating FABP4. Tracing study revealed remarkable accumulation of 125I-labeled FABP4 in the kidney. Exogenous FABP4 was exclusively detected in the apical membrane of proximal tubule epithelial cells (PTECs). Bilateral nephrectomy resulted in marked elevation of circulating FABP4 levels. Accelerated lipolysis by β-3 adrenergic stimulation led to a marked elevation in circulating FABP4 in mice with severe renal dysfunction. Megalin, an endocytic receptor expressed in PTECs, plays a major role in reabsorption of proteins filtered through glomeruli. Quartz-crystal microbalance study revealed that FABP4 binds to megalin. In kidney-specific megalin knockout mice, a large amount of FABP4 was excreted in urine while circulating FABP4 levels were significantly reduced. Our data suggest that circulating FABP4 is processed by the kidney via the glomerular filtration followed by megalin-mediated reabsorption. Thus, it is likely that circulating FABP4 levels are determined mainly by balance between secretion rate of FABP4 from adipocytes and clearance rate of the kidney.

DOI： 10.1038/s41598-018-34902-w

週1回のGLP-1受容体作動薬エキセナチド使用中に腸管気腫症の増悪を来した1例

石川友美, 細島康宏, 佐藤健, 塩谷基, 蒲澤秀門, 忰田亮平, 中枝武司, 和田庸子, 鈴木芳樹, 斎藤亮彦, 成田一衛

糖尿病 61 ( 9 ) 624 - 624 2018.9

Increased serum PCSK9, a potential biomarker to screen for periodontitis, and decreased total bilirubin associated with probing depth in a Japanese community survey Reviewed

K. Tabeta, M. Hosojima, M. Nakajima, S. Miyauchi, H. Miyazawa, N. Takahashi, Y. Matsuda, N. Sugita, Y. Komatsu, K. Sato, T. Ishikawa, K. Akiishi, K. Yamazaki, K. Kato, A. Saito, H. Yoshie

Journal of Periodontal Research 53 ( 3 ) 446 - 456 2018.6

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

Background and Objectives: Previous reports suggest that several serum biomarkers play roles in the pathogenesis, inflammatory response, and oxidative stress in periodontitis caused by bacterial infections, linking chronic periodontitis to atherosclerotic vascular disease (ASVD). The aim of this preliminary study was to investigate, in a Japanese cross-sectional community survey, potential serum biomarkers of periodontitis that are associated with ASVD and chronic periodontitis. Material and Methods: The study cohort included a total of 108 male subjects who underwent annual health examinations. Serum biomarkers (high-sensitivity C-reactive protein [hs-CRP], proprotein convertase subtilisin/kexin type 9 [PCSK9], interleukin-6, tumor necrosis factor-α, soluble CD14, myeloperoxidase, matrix metalloproteinase-3, adiponectin, total bilirubin [TBIL], and serum lipids) were analyzed to determine their association (if any) with periodontal parameters. Aortic stiffness was evaluated using the brachial-ankle aortic pulse wave velocity (PWV) index and the cardio-ankle vascular index (CAVI). Results: The concentrations of PCSK9 and hs-CRP were increased (P =.001 and.042, respectively), and the concentration of TBIL was decreased (P =.046), in subjects with periodontal disease (determined as a probing depth of ≥4 mm in at least one site) compared with periodontally healthy subjects. The ratio of low-density lipoprotein cholesterol (LDL-C) to high-density lipoprotein cholesterol and the concentrations of triglycerides, remnant-like particles-cholesterol, and oxidized LDL were elevated in subjects with periodontal disease compared with periodontally healthy subjects (P =.038,.007,.002, and.049, respectively). Multivariate regression analyses indicated that the number of sites with a pocket depth of ≥4 mm was associated with the concentration of PCSK9 and inversely associated with the concentration of TBIL independently (standardized β =.243, P =.040
standardized β = −.443, P =.0002
respectively). Analysis of receiver operating characteristic curves of PCSK9 indicated moderate accuracy for predicting the presence of disease sites (probing depth ≥ 4 mm) (area under the curve = 0.740). No significance in the values of PWV and CAVI was observed between subjects with periodontal disease and periodontally healthy subjects. Conclusion: In Japanese male subjects, the concentrations of serum PCSK9 and TBIL were correlated with periodontal parameters. Moreover, PCSK9 could be a candidate biomarker for diagnosing chronic periodontitis, and may also have potential to evaluate the risk for periodontitis to cause ASVD. Longitudinal studies of larger populations are necessary to confirm the exact association of periodontitis with increased serum PCSK9 and decreased TBIL.

DOI： 10.1111/jre.12533

幼少期における米(胚乳)タンパク質の摂取が成熟期の高脂肪食負荷による肥満および肥満関連腎症に及ぼす影響

樋口裕樹, 細島康宏, 蒲澤秀門, 桑原頌治, 忰田亮平, 成田一衛, 斎藤亮彦

糖尿病 61 ( Suppl.1 ) S - 241 2018.4

Bardoxolone methylによる腎近位尿細管のメガリン調節機構の検討

吉澤優太, 桑原頌治, 細島康宏, 蒲澤秀門, 忰田亮平, 宇賀村大亮, 後藤佐和子, 飯田倫理, 鈴木芳樹, 成田一衛, 斎藤亮彦

糖尿病 61 ( Suppl.1 ) S - 383 2018.4

SGLT2阻害薬ダパグリフロジンの腎近位尿細管細胞における血糖値非依存的なメガリン発現抑制作用

飯田倫理, 桑原頌治, 細島康宏, 石川友美, 後藤佐和子, 蒲澤秀門, 忰田亮平, 鈴木芳樹, 成田一衛, 斎藤亮彦

糖尿病 61 ( Suppl.1 ) S - 382 2018.4

Urinary C-megalin for screening of renal scarring in children after febrile urinary tract infection Reviewed

Sohsaku Yamanouchi, Takahisa Kimata, Jiro Kino, Tetsuya Kitao, Chikushi Suruda, Shoji Tsuji, Hiroyuki Kurosawa, Yoshiaki Hirayama, Akihiko Saito, Kazunari Kaneko

Pediatric Research 83 ( 3 ) 662 - 668 2018.3

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

BackgroundFebrile urinary tract infection (fUTI) in children may cause renal scarring. This study aimed to investigate the usefulness of urinary biomarkers for diagnosing renal scarring after fUTI.MethodsThirty-seven children (median age: 1.36 years, range: 0.52-12.17 years, 25 boys) with a history of fUTI, who underwent renal scintigraphy for 4 months or longer after the last episode of fUTI, were analyzed. A spot urine sample was obtained on the day of renal scintigraphy to measure levels of total protein, N-acetyl-β-D-glucosaminidase (NAG), β 2 -microglobulin (BMG), neutrophil gelatinase-associated lipocalin (NGAL), liver-type fatty acid binding protein (L-FABP), and C-megalin (full-length megalin). Results were corrected for urinary creatinine (Cr) and compared between the group with renal scarring (n=23) and that without scarring (n=14). Urinary levels of C-megalin were also measured in healthy control subjects.ResultsNo significant differences in total protein, NGAL, L-FABP, NAG, and BMG levels were found between the groups. However, C-megalin levels were significantly higher in the renal scarring group than in the non-renal scarring group and healthy controls (P&lt
0.001). A cutoff value of 6.5 pmol/nmol of urinary C-megalin/Cr yielded 73.9% of specificity and 92.9% of sensitivity.ConclusionUrinary C-megalin is useful for diagnosing renal scarring caused by fUTI.

DOI： 10.1038/pr.2017.276

Amnionless-mediated glycosylation is crucial for cell surface targeting of cubilin in renal and intestinal cells. Reviewed International journal

Tomohiro Udagawa, Yutaka Harita, Kenichiro Miura, Jun Mitsui, Koji L Ode, Shinichi Morishita, Seiya Urae, Shoichiro Kanda, Yuko Kajiho, Haruko Tsurumi, Hiroki R Ueda, Shoji Tsuji, Akihiko Saito, Akira Oka

Scientific reports 8 ( 1 ) 2351 - 2351 2018.2

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

Mutations in either cubilin (CUBN) or amnionless (AMN) genes cause Imerslund-Gräsbeck syndrome (IGS), a hereditary disease characterised by anaemia attributed to selective intestinal malabsorption of cobalamin and low-molecular weight proteinuria. Although cubilin protein does not have a transmembrane segment, it functions as a multi-ligand receptor by binding to the transmembrane protein, amnionless. We established a system to quantitatively analyse membrane targeting of the protein complex in cultured renal and intestinal cells and analysed the pathogenic mechanisms of mutations found in IGS patients. A novel CUBN mutation, several previously reported CUBN missense mutations and all previously reported AMN missense mutations resulted in endoplasmic reticulum (ER) retention and completely inhibited amnionless-dependent plasma membrane expression of cubilin. The ER retention of cubilin and amnionless was confirmed in renal proximal tubular cells of a patient with IGS. Notably, the interaction between cubilin and amnionless was not sufficient, but amnionless-mediated glycosylation of cubilin was necessary for their surface expression. Quantitative mass spectrometry and mutagenesis demonstrated that N-linked glycosylation of at least 4 residues of cubilin protein was required for its surface targeting. These results delineated the molecular mechanisms of membrane trafficking of cubilin in renal and intestinal cells.

DOI： 10.1038/s41598-018-20731-4

Urinary Iron Excretion is Associated with Urinary Full-Length Megalin and Renal Oxidative Stress in Chronic Kidney Disease. Reviewed

Nakatani S, Nakatani A, Ishimura E, Toi N, Tsuda A, Mori K, Emoto M, Hirayama Y, Saito A, Inaba M

Kidney & blood pressure research 43 ( 2 ) 458 - 470 2018

[Exosomal biomarkers for kidney disease.] Reviewed

Hosojima M, Kabasawa H, Kuwahara S, Saito A

Clinical calcium 28 ( 2 ) 252 - 259 2018

A Randomized, Double-Blind, Crossover Pilot Trial of Rice Endosperm Protein Supplementation in Maintenance Hemodialysis Patients Reviewed

Michihiro Hosojima, Hisaki Shimada, Yoshitsugu Obi, Shoji Kuwahara, Ryohei Kaseda, Hideyuki Kabasawa, Hazuki Kondo, Mikio Fujii, Reiko Watanabe, Yoshiki Suzuki, Motoni Kadowaki, Shigeru Miyazaki, Akihiko Saito

SCIENTIFIC REPORTS 7 ( 1 ) 18003 2017.12

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

In maintenance hemodialysis (MHD) patients, low protein intake is associated with protein-energy wasting, a risk factor that affects outcome. However, increased protein intake may lead to hyperphosphatemia and hyperkalemia, which are also mortality risk factors. Here, we evaluated the safety and effects of purified rice endosperm protein (REP), which contains less phosphorus and potassium than soy and casein proteins, as a supplemental protein source for MHD patients. This randomized, double-blind, placebo-controlled, crossover pilot study of REP supplementation (5 g/day x 4 weeks) was carried out in 50 Japanese adult MHD patients (1 dropped out); the primary outcome was the change in the urea kinetic-based normalized protein catabolic rate (nPCR), an indicator of protein intake in MHD patients. Intention-to-treat analyses of 24 patients in the REP-first group and 25 in the placebo-first group showed that REP supplementation increased nPCR significantly by 0.07 g/kg/day (95% confidence interval, 0.03-0.11), whereas changes in serum phosphorus and potassium concentrations were not different from the placebo. REP supplementation did not show a significant effect on other nutritional or metabolic parameters and no specific complications. In conclusion, purified REP with efficient bioavailability may be safe and useful for dietary supplementation in MHD patients.

DOI： 10.1038/s41598-017-18340-8

Megalin Blockade with Cilastatin Suppresses Drug-Induced Nephrotoxicity Reviewed

Yoshihisa Hori, Nobumasa Aoki, Shoji Kuwahara, Michihiro Hosojima, Ryohei Kaseda, Sawako Goto, Tomomichi Iida, Shankhajit De, Hideyuki Kabasawa, Reika Kaneko, Hiroyuki Aoki, Yoshinari Tanabe, Hiroshi Kagamu, Ichiei Narita, Toshiaki Kikuchi, Akihiko Saito

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 28 ( 6 ) 1783 - 1791 2017.6

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

Nephrotoxicity induced by antimicrobial or anticancer drugs is a serious clinical problem. Megalin, an endocytic receptor expressed at the apical membranes of proximal tubules, mediates the nephrotoxicity of aminoglycosides and colistin, key antimicrobials for multidrug-resistant organisms. The mechanisms underlying the nephrotoxicity induced by vancomycin, an antimicrobial for methicillin-resistant Staphylococcus aureus, and cisplatin, an important anticancer drug, are unknown, although the nephrotoxicity of these drugs and gentamicin, an aminoglycoside, is suppressed experimentally with cilastatin. In the clinical setting, cilastatin has been used safely to suppress dehydropeptidase-I-mediated renal metabolism of imipenem, a carbapenem antimicrobial, and thereby limit tubular injury. Here, we tested the hypothesis that cilastatin also blocks megalin-mediated uptake of vancomycin, cisplatin, colistin, and aminoglycosides, thereby limiting the nephrotoxicity of these drugs. Quartz crystal microbalance analysis showed that megalin also binds vancomycin and cisplatin and that cilastatin competes with megalin for binding to gentamicin, colistin, vancomycin, and cisplatin. In kidney specific mosaic megalin knockout mice treated with colistin, vancomycin, or cisplatin, the megalin-replete proximal tubule epithelial cells exhibited signs of injury, whereas the megalin-deficient cells did not. Furthermore, concomitant cilastatin administration suppressed colistin-induced nephrotoxicity in C57BL/6J mice. Notably, cilastatin did not inhibit the antibacterial activity of gentamicin, colistin, or vancomycin in vitro, just as cilastatin did not affect the anticancer activity of cisplatin in previous studies. In conclusion, megalin blockade with cilastatin efficiently suppresses the nephrotoxicity induced by gentamicin, colistin, vancomycin, or cisplatin. Cilastatin may be a promising agent for inhibiting various forms of drug-induced nephrotoxicity mediated via megalin in the clinical setting.

DOI： 10.1681/ASN.2016060606

Differences in the adsorption of nafamostat mesilate between polyester-polymer alloy and polysulfone membranes Reviewed

Sawako Goto, Susumu Ookawara, Akihiko Saito

JOURNAL OF ARTIFICIAL ORGANS 20 ( 2 ) 138 - 144 2017.6

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

We previously experienced severe clot formation in a polyester-polymer alloy (PEPA) dialyzer and hemodialysis (HD) circuit with nafamostat mesilate (NM) as an anticoagulant. The possibility of NM adsorption to the PEPA membrane was taken into consideration, but there was not enough information. In the present study, we evaluated differences in the adsorption of NM between a PEPA membrane (FDX-120 GW, Nikkiso, Tokyo, Japan) and two different polysulfone membranes (FX-140, Fresenius Medical Care, Tokyo, Japan; NV-15U, Toray Medical, Tokyo, Japan). We calculated the NM concentration by measuring absorbance at 241 nm using a spectrometer. NM adsorption was evaluated in three ways. First, we evaluated NM adsorption to hollow fibers. Then, we passed an NM solution through dialyzers and evaluated its adsorption in a single-pass examination. Finally, we circulated an NM solution in an HD circuit using a blood pump and evaluated NM adsorption. In all the experiments, NM adsorption to the PEPA membrane was greater than that to the polysulfone membranes examined. In the blood pump experiment, the estimated adsorption quantities of NM to the PEPA membrane and the FX-140 and NV-15U polysulfone membranes were 12.0 +/- 0.1, 1.0 +/- 0.1, and 4.1 +/- 0.4 mg/m(2), respectively. NM adsorption was confirmed, especially in the early phase, and the PEPA membrane adsorbed greater amounts of NM than the polysulfone membranes. We should pay attention to the choice of dialyzer as well as the appropriate dose of NM administration during the preparation of HD circuits.

DOI： 10.1007/s10047-016-0937-2

Exocytosis-Mediated Urinary Full-Length Megalin Excretion Is Linked With the Pathogenesis of Diabetic Nephropathy Reviewed

Shankhajit De, Shoji Kuwahara, Michihiro Hosojima, Tomomi Ishikawa, Ryohei Kaseda, Piyali Sarkar, Yusuke Yoshioka, Hideyuki Kabasawa, Tomomichi Iida, Sawako Goto, Koji Toba, Yuki Higuchi, Yoshiki Suzuki, Masanori Hara, Hiroyuki Kurosawa, Ichiei Narita, Yoshiaki Hirayama, Takahiro Ochiya, Akihiko Saito

DIABETES 66 ( 5 ) 1391 - 1404 2017.5

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER DIABETES ASSOC

Efficient biomarkers for diabetic nephropathy (DN) have not been established. Using ELISA, we found previously that urinary levels of full-length megalin (C-megalin), a multiligand endocytic receptor in proximal tubules, was positively correlated with DN progression in patients with type 2 diabetes mellitus (T2DM). Here, we found that urinary extracellular vesicle (UEV) excretion and C-megalin content in UEVs or in their exosomal fraction increased along with the progression of the albuminuric stages in patients with T2DM. Cultured immortalized rat proximal tubule cells (IRPTCs) treated with fatty acid-free BSA or advanced glycation end product-modified BSA (AGE-BSA), endocytic ligands of megalin, increased EV excretion, and their C-megalin content. C-megalin excretion from IRPTCs via extracellular vesicles was significantly blocked by an exosome-specific inhibitor, GW4869, indicating that this excretion is mainly exocytosis-mediated. AGE-BSA treatment of IRPTCs caused apparent lysosomal dysfunction, which stimulated multivesicular body formation, resulting in increased exosomal C-megalin excretion. In a high-fat diet-induced, megalin-mediated kidney injury model in mice, urinary C-megalin excretion also increased via UEVs. Collectively, exocytosis-mediated urinary C-megalin excretion is associated with the development and progression of DN in patients with T2DM, particularly due to megalin-mediated lysosomal dysfunction in proximal tubules, and hence it could be a candidate biomarker linked with DN pathogenesis.

DOI： 10.2337/db16-1031

Decreased urinary excretion of the ectodomain form of megalin (A-megalin) in children with OCRL gene mutations Reviewed

Chikushi Suruda, Shoji Tsuji, Sohsaku Yamanouchi, Takahisa Kimata, Nguyen Thanh Huan, Hiroyuki Kurosawa, Yoshiaki Hirayama, Hiroyasu Tsukaguchi, Akihiko Saito, Kazunari Kaneko

PEDIATRIC NEPHROLOGY 32 ( 4 ) 621 - 625 2017.4

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

The oculocerebrorenal syndrome of Lowe gene (OCRL) is located on chromosome Xq25-26 and encodes an inositol polyphosphate-5-phosphatase (OCRL-1). Mutations in this gene cause Lowe syndrome (LS) or type 2 Dent disease, of which low-molecular-weight (LMW) proteinuria is a characteristic feature. Megalin is considered to play an important role in the development of renal tubular proteinuria. Two forms of megalin are excreted into the urine: full-length megalin (C-megalin) and megalin ectodomain (A-megalin). We have explored the role of megalin in the development of LMW proteinuria in patients with OCRL mutations by determining urinary megalin fractions.
We measured A- and C-megalin in spot urine samples from five male patients with OCRL mutations (median age 9 years), using sandwich enzyme-linked immunosorbent assays, and adjusted the obtained values for excreted creatinine. The results were compared with those of 50 control subjects and one patient with type 1 Dent disease (T1D).
All patients demonstrated normal levels of urinary C-megalin. However, patients with OCRL mutations or T1D showed abnormally low levels of urinary A-megalin, with the exception of one 5-year-old boy with LS, who was the youngest patient enrolled in the study.
Decreased excretion of urinary A-megalin in four out of five patients with OCRL mutations suggests that LMW proteinuria may be caused by impaired megalin recycling within the proximal tubular cells. Homologous enzymes, similar to inositol polyphosphate-5-phosphatase B in mice, may help to compensate for defective OCRL-1 function during early childhood.

DOI： 10.1007/s00467-016-3535-x

The Assessment of the Efficacy of Dipeptidyl Peptidase-4 Inhibitors in Patients with Glucocorticoid-induced Diabetes by Continuous Glucose Monitoring Reviewed

Yusuke Yata, Michihiro Hosojima, Hideyuki Kabasawa, Tomomi Ishikawa, Ryohei Kaseda, Noriaki Iino, Yoshiki Suzuki, Akihiko Saito, Ichiei Narita

INTERNAL MEDICINE 56 ( 19 ) 2555 - 2562 2017

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN SOC INTERNAL MEDICINE

Objective The administration of glucocorticoids usually causes a mild increase in fasting glucose levels and a greater dose-dependent increase in postprandial values in patients without pre-existing diabetes mellitus. Patients with persistent hyperglycemia due to glucocorticoid therapy sometimes require insulin therapy, which might result in increased weight gain and more episodes of hypoglycemia, some of which are severe. On the other hand, scant evidence is available on the efficacy of oral hypoglycemic agents in treating glucocorticoid-induced diabetes. In this study, we evaluated the efficacy of dipeptidyl peptidase (DPP)-4 inhibitors in patients with glucocorticoid-induced diabetes by continuous glucose monitoring (CGM).
Methods We examined the glycemic profiles using CGM at baseline and 1-4 weeks after initiating DPP-4 inhibitor treatment in patients with newly developed glucocorticoid-induced diabetes.
Results Eleven patients who had been diagnosed with kidney disease or other diseases with renal involvement were recruited for the present retrospective study. After starting DPP-4 inhibitors, the mean and standard deviation (SD) of the glucose level, and the mean amplitude of glycemic excursion (MAGE) were significantly improved in comparison to baseline. Furthermore, the area over the curve (AOC) for the glucose levels <70 mg/dL was not increased in comparison to baseline after the initiation of DPP-4 inhibitor treatment. The results indicate that the treatment of patients with glucocorticoid-induced diabetes using DPP-4 inhibitors can minimize the risk of hypoglycemia and reduce glucose variability.
Conclusion DPP-4 inhibitors are potentially useful for blood glucose control in patients with glucocorticoid-induced diabetes.

DOI： 10.2169/internalmedicine.8296-16

BENEFICIAL EFFECTS OF RICE ENDOSPERM PROTEIN ON CHRONIC KIDNEY DISEASE-MINERAL AND BONE DISORDER (CKD-MBD) IN ZUCKER DIABETIC FATTY RATS Reviewed

Reiko Watanabe, Masatoshi Kubota, Shogo Sugaki, Michihiro Hosojima, Akihiko Saito, Mikio Fujii, Ichiro Sasagawa, Shinobu Fujimura, Motoni Kadowaki

ANNALS OF NUTRITION AND METABOLISM 71 1218 - 1218 2017

Glucose Variability Based on Continuous Glucose Monitoring Assessment Is Associated with Postoperative Complications after Cardiovascular Surgery Reviewed

Hiroki Sato, Michihiro Hosojima, Tomomi Ishikawa, Kenji Aoki, Takeshi Okamoto, Akihiko Saito, Masanori Tsuchida

ANNALS OF THORACIC AND CARDIOVASCULAR SURGERY 23 ( 5 ) 239 - 247 2017

Language：English Publishing type：Research paper (scientific journal) Publisher：MEDICAL TRIBUNE INC

Purpose: This purpose of this prospective study was to use a continuous glucose monitoring (CGM) system to evaluate the suitability of our institution's glucose management protocol after cardiovascular surgery and to clarify the impact of glycemic variability on postoperative complications.
Methods: In all, 76 patients who underwent elective cardiovascular surgery and were monitored perioperatively using a CGM system were evaluated. Postoperative glucose management consisted of continuous intravenous insulin infusion (CIII) in the intensive care unit, and subcutaneous insulin injections (SQII) after oral food intake started. CIII and subcutaneous injections were initiated when blood glucose level exceeded 150 mg/dL. CGM data were used to analyze perioperative glycemic variability and association with postoperative complications.
Results: Target glucose levels (71-180 mg/dL) were achieved during 97.1 +/- 5.5% and 86.4 +/- 19.0% of the continuous insulin infusion and subcutaneous injection periods, respectively. Major postoperative complications were surgical site infections, found in 6.6% of total patients, and atrial fibrillation, found in 44% of patients with off-pump coronary artery bypass grafting. High glycemic variability during SQII was associated with increased risk for both complications.
Conclusion: Data analysis revealed that our glucose management protocol during CIII was adequate. However, the management protocol during SQII required improvement.

DOI： 10.5761/atcs.oa.17-00045

Rice endosperm protein slows progression of fatty liver and diabetic nephropathy in Zucker diabetic fatty rats Reviewed

Masatoshi Kubota, Reiko Watanabe, Miki Yamaguchi, Michihiro Hosojima, Akihiko Saito, Mikio Fujii, Shinobu Fujimura, Motoni Kadowaki

BRITISH JOURNAL OF NUTRITION 116 ( 8 ) 1326 - 1335 2016.10

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

We previously reported that rice endosperm protein (REP) has renoprotective effects in Goto-Kakizaki rats, a non-obese diabetic model. However, whether these effects occur in obese diabetes remains unclear. This study aimed to clarify the effects of REP on obese diabetes, especially on fatty liver and diabetic nephropathy, using the obese diabetic model Zucker diabetic fatty (ZDF) rats. In total, 7-week-old male ZDF rats were fed diets containing 20 % REP or casein (C) for 8 weeks. Changes in fasting blood glucose levels and urinary markers were monitored during the experimental period. Hepatic lipids and metabolites were measured and renal glomeruli were observed morphologically. HbA1c levels were significantly lower in rats fed REP, compared with C (P < 005). Compared with C in the liver, REP prevented lipid accumulation (total lipid, TAG and total cholesterol, P < 001). Liver metabolome analysis indicated that levels of metabolites associated with glycolysis, the pentose phosphate pathway and carnitine metabolism were significantly greater in the REP group than in the C group (P < 005), suggesting activation of both glucose catabolism and fatty acid oxidation. The metabolite increases promoted by REP may contribute to suppression of liver lipid accumulation. Urinary excretion of albumin and N-acetyl--d-glucosaminidase was significantly reduced in rats fed REP for 8 weeks (P < 001). In addition, there was a distinct suppression of mesangial matrix expansion and glomerular hypertrophy in response to REP (P < 001). Thus, REP had preventive effects on obese diabetes, fatty liver and diabetic nephropathy.

DOI： 10.1017/S0007114516003512

Megalin-Mediated Tubuloglomerular Alterations in High-Fat Diet-Induced Kidney Disease Reviewed

Shoji Kuwahara, Michihiro Hosojima, Reika Kaneko, Hiroyuki Aoki, Daisuke Nakano, Taiji Sasagawa, Hideyuki Kabasawa, Ryohei Kaseda, Ryota Yasukawa, Tomomi Ishikawa, Akiyo Suzuki, Hiroyoshi Sato, Shun Kageyama, Takahiro Tanaka, Nobutaka Kitamura, Ichiei Narita, Masaaki Komatsu, Akira Nishiyama, Akihiko Saito

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 27 ( 7 ) 1996 - 2008 2016.7

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

Obesity, an important risk factor for metabolic syndrome (MetS) and cardiovascular disease, is often complicated by CKD, which further increases cardiovascular risk and causes ESRD. To elucidate the mechanism underlying this relationship, we investigated the role of the endocytic receptor megalin in proximal tubule epithelial cells (PTECs). We studied a high-fat diet (HFD)-induced obesity/MetS model using kidney-specific mosaic megalin knockout (KO) mice. Compared with control littermates fed a normal-fat diet, control litter mates fed an HFD for 12 weeks showed autolysosomal dysfunction with autophagy impairment and increased expression of hypertrophy, lipid peroxidation, and senescence markers in PTECs of the S2 segment, peritubular capillary rarefaction with localized interstitial fibrosis, and glomerular hypertrophy with mesangial expansion. These were ameliorated in HFD-fed megalin KO mice, even though these mice had the same levels of obesity, dyslipidemia, and hyperglycemia as HFD-fed control mice. Intravital renal imaging of HFD-fed wild-type mice also demonstrated the accumulation of autofluorescent lipofuscin-like substances in PTECs of the S2 segment, accompanied by focal narrowing of tubular lumens and peritubular capillaries. In cultured PTECs, fatty acid-rich albumin induced the increased expression of genes encoding PDGF-B and monocyte chemoattractant protein-1 via megalin, with large (auto)lysosome formation, compared with fatty acid-depleted albumin. Collectively, the megalin-mediated endocytic handling of glomerular-filtered (lipo)toxic substances appears to be involved primarily in hypertrophic and senescent PTEC injury with autophagy impairment, causing peritubular capillary damage and retrograde glomerular alterations in HFD-induced kidney disease. Megalin could be a therapeutic target for obesity/MetS-related CKD, independently of weight, dyslipidemia, and hyperglycemia modification.

DOI： 10.1681/ASN.2015020190

Beneficial effects of rice endosperm protein intake in Japanese men with risk factors for metabolic syndrome: a randomized, crossover clinical trial

Michihiro Hosojima, Ryohei Kaseda, Hazuki Kondo, Mikio Fujii, Masatoshi Kubota, Reiko Watanabe, Naohito Tanabe, Motoni Kadowaki, Yoshiki Suzuki, Akihiko Saito

BMC Nutrition 2 ( 1 ) 2016.5

Protective effect of composite earthworm powder against diabetic complications via increased fibrinolytic function and improvement of lipid metabolism in ZDF rats Reviewed

Tomoko Kawakami, Airi Fujikawa, Yohei Ishiyama, Michihiro Hosojima, Akihiko Saito, Masatoshi Kubota, Shinobu Fujimura, Motoni Kadowaki

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 80 ( 10 ) 1980 - 1989 2016

Language：English Publishing type：Research paper (scientific journal) Publisher：TAYLOR & FRANCIS LTD

Thrombosis is the leading cause of mortality globally. It is not only a complication but also a risk factor for progression of diabetes. However, alternative oral therapies and prophylaxis with less adverse effect for thrombosis have not been well studied. In this study, composite powder containing earthworm (CEP) was used and its fibrinolytic activity was measured. CEP was found to have a high urokinase-type plasminogen activator like activity in an in vitro assay. It also had significantly shortened euglobulin clot lysis time (ECLT) at 4 and 24h after ingestion in Sprague Dawley rats. Zucker Diabetic Fatty rats were used to assess the effect of CEP on diabetes and diabetic nephropathy. After 10weeks of feeding, CEP significantly shortened ECLT and attenuated HbA1c, hepatic lipid accumulation, and urinary albumin excretion and improved glomerular mesangial matrix score. Therefore, CEP may have beneficial effects on diabetes and diabetic nephropathy.

DOI： 10.1080/09168451.2016.1166932

Benefits of a 12-week lifestyle modification program including diet and combined aerobic and resistance exercise on albuminuria in diabetic and non-diabetic Japanese populations Reviewed

Keiko Yamamoto-Kabasawa, Michihiro Hosojima, Yusuke Yata, Mariko Saito, Noriko Tanaka, Junta Tanaka, Naohito Tanabe, Ichiei Narita, Masaaki Arakawa, Akihiko Saito

CLINICAL AND EXPERIMENTAL NEPHROLOGY 19 ( 6 ) 1079 - 1089 2015.12

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

Background Albuminuria is a biomarker for chronic kidney disease and an independent predictor of cardiovascular and all-cause mortality. A recent meta-analysis concluded that these risks increase with urinary albumin concentration, even when below the microalbuminuria threshold. Thus, minimizing urinary albumin may be a valuable therapeutic goal regardless of disease status.
Methods We investigated the benefits and safety of a 12-week lifestyle modification program including diet and combined aerobic and resistance exercise for reducing albuminuria in 295 normoalbuminuric or microalbuminuric Japanese adults, including 30 with type 2 diabetes mellitus (T2DM), 104 with metabolic syndrome (MS), and 145 with hypertension (HT).
Results In the study population, the urinary albumin:creatinine ratio (UACR) was reduced significantly (Delta UACR -3.8 +/- A 16.8 mg/g, P < 0.001) with no change in estimated glomerular filtration rate (eGFR) (Delta eGFR -0.4 +/- A 7.4 mL/min/1.73 m(2), P = 0.343). The reduction in UACR was associated with decreased fasting plasma glucose (P < 0.05). The UACR was also reduced in the T2DM, MS, and HT groups with no change in eGFR. Reduced UACR was associated with decreased fasting plasma glucose in the MS group and decreased systolic blood pressure in the HT group. The UACR was also reduced in 46 subjects using renin-angiotensin system inhibitors with no change in eGFR. Conclusions Our 12-week lifestyle modification program reduced UACR, maintained eGFR, and improved multiple fitness findings in Japanese subjects including T2DM, MS, and HT patients.

DOI： 10.1007/s10157-015-1103-5

Reduction of Albuminuria by Sitagliptin Is Associated with Improvement of Renal Proximal Tubular Function in Patients with Type 2 Diabetes Reviewed

Michihiro Hosojima, Taiji Sasagawa, Hideyuki Kabasawa, Tomomi Ishikawa, Ryohei Kaseda, Yoshiki Suzuki, Ichiei Narita, Hiroyuki Kurosawa, Yoshiaki Hirayama, Akihiko Saito

DIABETES 64 A151 - A151 2015.6

Drainage patterns of the superficial middle cerebral vein: Effects on perioperative managements of petroclival meningioma. Reviewed

Fukuda M, Saito A, Takao T, Hiraishi T, Yajima N, Fujii Y

Surgical neurology international 6 130 2015

Significance of Urinary Full-Length Megalin in Patients with IgA Nephropathy Reviewed

Takuto Seki, Katsuhiko Asanuma, Rin Asao, Kanae Nonaka, Yu Sasaki, Juan Alejandro Oliva Trejo, Hiroyuki Kurosawa, Yoshiaki Hirayama, Satoshi Horikoshi, Yasuhiko Tomino, Akihiko Saito

PLOS ONE 9 ( 12 ) e114400 2014.12

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

Background and Objectives: Megalin is highly expressed at the apical membranes of proximal tubular epithelial cells. A urinary full-length megalin (C-megalin) assay is linked to the severity of diabetic nephropathy in type 2 diabetes. This study examined the relationship between levels of urinary C-megalin and histological findings in adult patients with IgA nephropathy (IgAN).
Design, Setting, Participants, & Measurements: Urine samples voided in the morning on the day of renal biopsy were obtained from 73 patients with IgAN (29 men and 44 women; mean age, 33 years) and 5 patients with membranous nephropathy (MN). Renal pathologic variables were analyzed using the Oxford classification of IgAN, the Shigematsu classification and the Clinical Guidelines of IgAN in Japan. The levels of urinary C-megalin were measured by sandwich ELISA.
Results: Histological analysis based on the Oxford classification revealed that the levels of urinary C-megalin were correlated with mesangial hypercellularity in IgAN patients (OR=1.76, 95% CI: 1.04-3.27, P<0.05). There was a significant correlation between the levels of urinary C-megalin and the severity of chronic extracapillary abnormalities according to the Shigematsu classification in IgAN patients (beta=0.33, P=0.008). The levels of urinary C-megalin were significantly higher in all risk levels of IgAN patients requiring dialysis using the Clinical Guidelines of IgAN in Japan than in the control group. The levels of urinary C-megalin were significantly higher in the high risk and very high risk grades than in the low risk grade (P<0.05). The levels of urinary C-megalin were significantly higher in MN patients compared to the control group.
Conclusions: The levels of urinary C-megalin are associated with histological abnormalities in adult IgAN patients. There is a possibility that urinary C-megalin is

DOI： 10.1371/journal.pone.0114400

The endocytic receptor megalin and its associated proteins in proximal tubule epithelial cells Reviewed

Shankhajit De, Shoji Kuwahara, Akihiko Saito

Membranes 4 ( 3 ) 333 - 335 2014.7

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

Receptor-mediated endocytosis in renal proximal tubule epithelial cells (PTECs) is important for the reabsorption and metabolization of proteins and other substances, including carrier-bound vitamins and trace elements, in glomerular filtrates. Impairment of this endocytic process results in the loss of such substances and development of proteinuria, which is an important clinical indicator of kidney diseases and is also a risk marker for cardiovascular disease. Megalin, a member of the low-density lipoprotein receptor gene family, is a multiligand receptor expressed in the apical membrane of PTECs and plays a central role in the endocytic process. Megalin interacts with various intracellular adaptor proteins for intracellular trafficking and cooperatively functions with other membrane molecules, including the cubilin-amnionless complex. Evidence suggests that megalin and the cubilin-amnionless complex are involved in the uptake of toxic substances into PTECs, which leads to the development of kidney disease. Studies of megalin and its associated molecules will be useful for future development of novel strategies for the diagnosis and treatment of kidney diseases. © 2014 by the authors
licensee MDPI, Basel, Switzerland.

DOI： 10.3390/membranes4030333

RELATIONSHIP BETWEEN LEVELS OF URINARY FULL-LENGTH MEGALIN AND HISTOLOGICAL FINDINGS IN PATIENTS WITH IgA NEPHROPATHY Reviewed

Seki Takuto, Asanuma Katsuhiko, Asao Rin, Nonaka Kanae, Kodama Fumiko, Sasaki Yu, Akiba-Takagi Miyuki, Hosoe-Nagai Yoshiko, Kurosawa Hiroyuki, Hirayama Yoshiaki, Horikoshi Satoshi, Saito Akihiko, Tomino Yasuhiko

NEPHROLOGY 19 146 - 146 2014.5

Keap1 inhibition attenuates glomerulosclerosis Reviewed

Yoichi Miyazaki, Akihiro Shimizu, Ira Pastan, Keiko Taguchi, Eriko Naganuma, Takafumi Suzuki, Tatsuo Hosoya, Takashi Yokoo, Akihiko Saito, Toshio Miyata, Masayuki Yamamoto, Taiji Matsusaka

NEPHROLOGY DIALYSIS TRANSPLANTATION 29 ( 4 ) 783 - 791 2014.4

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

NFE2-related factor 2 (Nrf2) is a master regulatory transcription factor for antioxidant genes. Inhibition of its adaptor protein, Kelch-like ECH-associated protein 1 (Keap1), activates Nrf2. Podocyte injury triggers the progressive deterioration of glomerular damage toward glomerulosclerosis. We examined whether modulation of the Keap1-Nrf2 system has an impact on this process.
Methods. Nrf2 null-mutant (KO) and Keap1 hypomorphic knockdown (KD) mice were crossed with NEP25 mice, in which podocyte-specific injury can be induced by an immunotoxin.
Results. Thiobarbituric acid reactive substances, 8-hydroxy-deoxyguanosine and phosphorylated JNK were increased in the injured NEP25 kidney. Real-time PCR revealed that Keap1 KD upregulated Nrf2 target genes, including Gclc, Gclm, Gstp1, Gstp2 and Nqo1 in the glomerulus. However, podocyte injury did not upregulate these genes in Keap1 wild-type mice, nor did it further increase the expression of those genes in Keap1 KD mice. Three weeks after the induction of podocyte injury, glomerulosclerosis was considerably more attenuated in Keap1 KD mice than in control mice (median sclerosis index, 0.27 versus 3.03, on a 0-4 scale). Keap1 KD mice also showed considerably preserved nephrin staining (median index, 6.76 versus 0.91, on a 0-8 scale) and decreased glomeruli containing desmin-positive injured podocytes (median percentage, 24.5% versus 85.8%), along with a decrease in mRNAs for Fn1, Tgfb1, Col4a4 and Col1a2.
Conclusions. Thus, podocyte injury cannot effectively activate Nrf2, but Nrf2 activation by Keap1 knockdown attenuates glomerulosclerosis. These results indicate that the Nrf2-Keap1 system is a promising drug target for the treatment of chronic kidney diseases.

DOI： 10.1093/ndt/gfu002

Upregulation of prolactin receptor in proximal tubular cells was induced in cardiac dysfunction model mice Reviewed

Yohei Tsuchida, Yoshikatsu Kaneko, Tadashi Otsuka, Kei Goto, Akihiko Saito, Keiko Yamamoto, Tadashi Yamamoto, Ichiei Narita

CLINICAL AND EXPERIMENTAL NEPHROLOGY 18 ( 1 ) 65 - 74 2014.2

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

In order to clarify the interaction between cardiac dysfunction and sodium homeostasis in the kidney, we used a murine model of cardiac dysfunction and investigated the effect on sodium transporters in renal tubular cells.
Cardiac function was deteriorated by abdominal aortic banding, and the gene expression of sodium transporters in the kidneys was evaluated by real-time RT-PCR and compared with that in the kidneys of control mice.
Gene expression of all three variants of the murine prolactin receptor was enhanced by aortic banding. Upregulated prolactin receptor was distributed in the proximal tubular cells of the pars recta in the deep inner cortex and the outer stripe of the outer medulla. Prolactin has been reported to be a natriuretic hormone that inhibits proximal tubular Na+/K+-ATPase activity, resulting in reduced sodium reabsorption and the acceleration of natriuresis. Inhibition of endogenous prolactin secretion by bromocriptine administration decreased the urine sodium excretion in both aortic banding and control mice. On the other hand, excess exogenous prolactin administration enhanced urine potassium excretion in aortic banding mice. Furthermore, a high-sodium diet accelerated urinary sodium excretion, which was also significantly decreased by inhibition of endogenous prolactin secretion in aortic banding mice.
We reported that the prolactin receptor was upregulated by aortic banding treatment. Prolactin-prolactin receptor interaction in the proximal tubular cells of the pars recta should involve a different mechanism of kaliuresis other than inhibition of Na+/K+-ATPase.

DOI： 10.1007/s10157-013-0820-x

Erratum to: Decreased urinary calbindin 1 levels in proteinuric rats and humans with distal nephron segment injuries (Clinical and Experimental Nephrology DOI: 10.1007/s10157-013-0835-3) Reviewed

Tomoko Iida, Hidehiko Fujinaka, Bo Xu, Ying Zhang, Sameh Magdeldin, Masaaki Nameta, Zan Liu, Yutaka Yoshida, Eishin Yaoita, Shuichi Tomizawa, Akihiko Saito, Tadashi Yamamoto

Clinical and Experimental Nephrology 18 ( 3 ) 444 2014

Decreased urinary calbindin 1 levels in proteinuric rats and humans with distal nephron segment injuries Reviewed

Tomoko Iida, Hidehiko Fujinaka, Bo Xu, Ying Zhang, Sameh Magdeldin, Masaaki Nameta, Zan Liu, Yutaka Yoshida, Eishin Yaoita, Shuichi Tomizawa, Akihiko Saito, Tadashi Yamamoto

Clinical and Experimental Nephrology 18 ( 3 ) 432 - 443 2014

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer-Verlag Tokyo

Background: Several proteins have been proposed as new urinary biomarkers of kidney injuries, but they are not always capable of identifying the kidney nephron segment that has been injured. Since calbindin 1 protein is exclusively localized in the kidney distal nephron segment, it is presumed that its expression is altered during distal nephron segment injuries, resulting in changes in its urinary excretion. Methods: Calbindin 1 expression in normal rat kidneys was compared with that in the kidneys of rats that had suffered distal nephron segment injuries (unilateral ureteral obstruction [UUO] or anti-glomerular basement membrane glomerulonephritis [anti-GBM GN]) using immunohistochemical examinations and real-time polymerase chain reaction. The urinary calbindin 1 protein concentration of normal rats was also compared with that of anti-GBM GN rats and of cisplatin nephropathy rats using Western blotting. We also compared the kidney and urinary calbindin 1 protein concentrations of normal human subjects with those of proteinuric patients [immunoglobulin (Ig)A nephropathy
IgAN] with distal nephron segment injuries. Results: Calbindin 1 mRNA expression in the renal cortices and calbindin 1 protein expression in the kidney distal nephron segments were decreased in the UUO and anti-GBM GN rat kidneys. The urinary calbindin 1 protein levels of the anti-GBM GN rats were also markedly decreased, whereas those of the cisplatin nephropathy rats were slightly decreased. The human IgAN patients displayed decreased renal calbindin 1 protein expression in their dilated distal tubules, and some patients displayed decreased urinary calbindin 1 levels. Conclusion: Since it has been demonstrated that decreased urinary calbindin 1 levels are indicative of decreased calbindin 1 kidney expression due to distal nephron segment injuries, calbindin 1 might be a useful urinary biomarker for identifying distal nephron segment injuries. © 2013 Japanese Society of Nephrology.

DOI： 10.1007/s10157-013-0835-3

Successful removal of a huge hypervascular tentorial cavernous angioma after preoperative endovascular embolization: Case report Reviewed

Junichi Yoshimura, Yoshihiro Tsukamoto, Masakazu Sano, Hitoshi Hasegawa, Kazuhiko Nishino, Akihiko Saito, Masafumi Fukuda, Kouichirou Okamoto, Yukihiko Fujii

Journal of Neurosurgery: Pediatrics 14 ( 1 ) 43 - 47 2014

Language：English Publishing type：Research paper (scientific journal) Publisher：American Association of Neurological Surgeons

The authors report a rare case of a huge hypervascular tentorial cavernous angioma treated with preoperative endovascular embolization, followed by successful gross-total removal. A 15-year-old girl presented with scintillation, diplopia, and papilledema. Computed tomography and MRI studies revealed a huge irregularly shaped tumor located in the right occipital and suboccipital regions. The tumor, which had both intra- and extradural components, showed marked enhancement and invasion of the overlying occipital bone. Angiography revealed marked tumor stain, with blood supply mainly from a large branch of the left posterior meningeal artery. Therefore, this lesion was diagnosed as a tentorium-based extraaxial tumor. For differential diagnosis, meningioma, hemangiopericytoma, and malignant skull tumor were considered. Tumor feeders were endovascularly embolized with particles of polyvinyl alcohol. On the following day, the tumor was safely gross totally removed with minimum blood loss. Histopathological examination confirmed the diagnosis of cavernous angioma. To date, there have been no reports of tentorium-based cavernous angiomas endovascularly embolized preoperatively. A tentorial cavernous angioma is most likely to show massive intraoperative bleeding. Therefore, preoperative embolization appears to be quite useful for safe maximum resection. Hence, the authors assert that the differential diagnosis of tentorium-based tumors should include tentorial cavernous angioma, for which preoperative endovascular embolization should be considered. ©AANS, 2014.

DOI： 10.3171/2014.4.PEDS13628

Rice protein ameliorates the progression of diabetic nephropathy in Goto-Kakizaki rats with high-sucrose feeding Reviewed

Masatoshi Kubota, Reiko Watanabe, Hideyuki Kabasawa, Noriaki Iino, Akihiko Saito, Takehisa Kumagai, Shinobu Fujimura, Motoni Kadowaki

BRITISH JOURNAL OF NUTRITION 110 ( 7 ) 1211 - 1219 2013.10

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

The effect of rice protein (RP) on diabetic nephropathy in non-obese, spontaneous type 2 diabetic Goto-Kakizaki (GK) rats was investigated. GK rats at 7 weeks of age were fed 20% RP or casein (C) in standard or high-sucrose diets for 10 weeks. Plasma total cholesterol, TAG, alkaline phosphatase (ALP), adiponectin, creatinine and urinary albumin excretion (UAE) were measured and renal histology was evaluated. Compared with C, RP lowered plasma TAG and improved plasma adiponectin levels in GK rats fed the standard diet (P<0.05), and also lowered total cholesterol and ALP in high-sucrose-fed GK rats (P<0.05). RP markedly suppressed the sharp increase in UAE when GK rats were fed high-sucrose diets (P<0.05), and prevented glomerular mesangial matrix expansion in the deep renal cortex near the corticomedullary junction (P<0.05). These results strongly indicate that dietary RP can ameliorate the progression of diabetic nephropathy at an early stage compared with C.

DOI： 10.1017/S0007114513000354

Rice Protein Ameliorates Progression of Diabetic Nephropathy in Goto-Kakizaki Rats with High-Sucrose Feeding Reviewed

Kubota M, Watanabe R, Kabasawa H, Iino N, Saito A, Kumagai T, Fujimura S, Kadowaki M

British Journal of Nutrition 110 ( 7 ) 1211 - 1219 2013.7

GFR ESTIMATION BY URINARY SOLUBLE MEGALIN

Akira Hiwatashi, Masahiro Hagiwara, Syuichi Tsuruoka, Joichi Usui, Kei Nagai, Hirayasu Kai, Naoki Morito, Chie Saito, Keigyo Yoh, Michihiro Hosojima, Akihiko Saito, Kunihiro Yamagata

NEPHROLOGY DIALYSIS TRANSPLANTATION 28 ( 1 ) 145 - 145 2013.5

Dietary alkali-extracted rice protein improves some diabetes-related markers in obese type 2 diabetic rats Reviewed

Watanabe Reiko, Kubota Masatoshi, Yamaguchi Miki, Saito Akihiko, Fujii Mikio, Fujimura Shinobu, Kadowaki Motoni

FASEB JOURNAL 27 2013.4

Protective effects of alkali-extracted rice protein on diabetic nephropathy in obese type 2 diabetic rats Reviewed

Masatoshi Kubota, Reiko Watanabe, Miki Yamaguchi, Michihiro Hosojima, Akihiko Saito, Mikio Fujii, Shinobu Fujimura, Motoni Kadowaki

FASEB JOURNAL 27 2013.4

Intraoperative monitoring of motor evoked potentials during glioma removal Reviewed

Masafumi Fukuda, Makoto Oishi, Tetsuro Takao, Tetsuya Hiraishi, Tsutomu Kobayashi, Hiroshi Aoki, Ryosuke Ogura, Akihiko Saito, Yukihiko Fujii

Neurological Surgery 41 ( 3 ) 219 - 227 2013.3

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

Objective : To determine whether motor evoked potentials (MEPs) provide reliable monitoring of the motor system during resection of gliomas in or adjacent to the motor cortex or pyramidal tract. Materials and methods : MEP recording was performed during 64 operations in 55 patients harboring gliomas. Intraoperative MEP findings were classified into 3 groups : Group A was defined as having no significant MEP changes, Group B as having reversible MEP changes (≥50% amplitude decrease or loss), and Group C as having irreversible changes. Postoperative motor function was evaluated according to the presence/absence of deterioration immediately after surgery and 1 month later, as compared to preoperative motor status Results : Immediately after surgery, 13 of 39 (33%) patients in Group A, 6 of 17 (35%) in Group B, and 7 of 8 (88%) in Group C experienced deterioration of motor function. One month after surgery, 4 of 39 (10%) patients in Group A, 3 of 17 (18%) in Group B, and 4 of 8 (50%) showed deterioration of motor function Both immediately (χ2=8.3, p&lt
0.05) and 1 month (χ2=6.9. p&lt
0.05) after surgery, MEP alterations correlated significantly with postoperative deterioration of motor function. Despite MEPs being stable throughout surgery (Group A), there were some patients with deterioration of motor function initially appearing to represent false negative monitoring. However, these deteriorations were confirmed to have been caused by secondary hemorrhage, venous return dysfunction, postoperative convulsion, or resection of the supplementary motor area. Conclusions : MEP monitoring provides reliable information on the motor system during glioma surgery. Although false negative MEP results may exist in some patients, most data were not influenced by intraoperative manipulation but rather were attributable to secondary postoperative events.

Urinary podocalyxin is an early marker for podocyte injury in patients with diabetes: Establishment of a highly sensitive ELISA to detect urinary podocalyxin Reviewed

M. Hara, K. Yamagata, Y. Tomino, A. Saito, Y. Hirayama, S. Ogasawara, H. Kurosawa, S. Sekine, K. Yan

Diabetologia 55 ( 11 ) 2913 - 2919 2012.11

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

Aims/objective Nephropathy, a major complication of diabetes, is the leading cause of end-stage renal disease. Recent studies have demonstrated that podocyte injury is involved in the onset of and progression to renal insufficiency. Here, we describe a novel, highly sensitive ELISA for detecting urinary podocalyxin, a glycoconjugate on the podocyte apical surface that indicates podocyte injury, particularly in the early phase of diabetic nephropathy. Methods Urine samples from patients with glomerular diseases (n=142) and type 2 diabetes (n=71) were used to quantify urinary podocalyxin by ELISA. Urine samples were obtained from 69 healthy controls for whom laboratory data were within normal values. Podocalyxin was detected in urine by immunofluorescence, immunoelectron microscopy and western blotting. Results Morphologically, urinary podocalyxin was present as a vesicular structure
western blotting showed it as a positive band at 165-170 kDa. Levels of urinary podocalyxin were elevated in patients with various glomerular diseases and patients with diabetes. In patients with diabetes, urinary podocalyxin was higher than the cut-off value in 53.8% patients at the normoalbuminuric stage, 64.7% at the microalbuminuric stage and 66.7% at the macroalbuminuric stage. Positive correlations were observed between urinary podocalyxin levels and HbA1c, urinary β2 microglobulin, α1 microglobulin and urinary N-acetyl-β-D-glucosaminidase, although urinary podocalyxin levels were not correlated with other laboratory markers such as blood pressure, lipid level, serum creatinine, estimated GFR or proteinuria. Conclusions/interpretation Urinary podocalyxin may be a useful biomarker for detecting early podocyte injury in patients with diabetes. © The Author(s) 2012.

DOI： 10.1007/s00125-012-2661-7

Liver Angiotensinogen Is the Primary Source of Renal Angiotensin II Reviewed

Taiji Matsusaka, Fumio Niimura, Akihiro Shimizu, Ira Pastan, Akihiko Saito, Hiroyuki Kobori, Akira Nishiyama, Iekuni Ichikawa

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 23 ( 7 ) 1181 - 1189 2012.7

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

Angiotensin II content in the kidney is much higher than in the plasma, and it increases more in kidney diseases through an uncertain mechanism. Because the kidney abundantly expresses angiotensinogen mRNA, transcriptional dysregulation of angiotensinogen within the kidney is one potential cause of increased renal angiotensin II in the setting of disease. Here, we observed that kidney-specific angiotensinogen knockout mice had levels of renal angiotensinogen protein and angiotensin II that were similar to those levels of control mice. In contrast, liver-specific knockout of angiotensinogen nearly abolished plasma and renal angiotensinogen protein and renal tissue angiotensin II. Immunohistochemical analysis in mosaic proximal tubules of megalin knockout mice revealed that angiotensinogen protein was incorporated selectively in megalin-intact cells of the proximal tubule, indicating that the proximal tubule reabsorbs filtered angiotensinogen through megalin. Disruption of the filtration barrier in a transgenic mouse model of podocyte-selective injury increased renal angiotensin II content and markedly increased both tubular and urinary angiotensinogen protein without an increase in renal renin activity, supporting the dependency of renal angiotensin II generation on filtered angiotensinogen. Taken together, these data suggest that liver-derived angiotensinogen is the primary source of renal angiotensinogen protein and angiotensin II. Furthermore, an abnormal increase in the permeability of the glomerular capillary wall to angiotensinogen, which characterizes proteinuric kidney diseases, enhances the synthesis of renal angiotensin II.

DOI： 10.1681/ASN.2011121159

Significance of Urinary Full-Length and Ectodomain Forms of Megalin in Patients With Type 2 Diabetes Reviewed

Shinya Ogasawara, Michihiro Hosojima, Ryohei Kaseda, Hideyuki Kabasawa, Keiko Yamamoto-Kabasawa, Hiroyuki Kurosawa, Hiroyoshi Sato, Noriaki Iino, Tetsuro Takeda, Yoshiki Suzuki, Ichie Narita, Kunihiro Yamagata, Yasuhiko Tomino, Fumitake Gejyo, Yoshiaki Hirayama, Sakari Sekine, Akihiko Saito

DIABETES CARE 35 ( 5 ) 1112 - 1118 2012.5

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER DIABETES ASSOC

OBJECTIVE-Megalin, an endocytic receptor in proximal tubule cells, is involved in the mechanisms of albuminuria in diabetic nephropathy (DN). To develop efficient novel biomarkers associated with the pathogenesis of DN, we investigated urinary megalin excretion in type 2 diabetes.
RESEARCH DESIGN AND METHODS-Sandwich enzyme-linked immunosorbent assay systems were established with monoclonal antibodies against the NH2 (amino [A]-megalin assay) and CO OH (C-megalin assay) termini of megalin to analyze urinary forms of megalin in 68 patients with type 2 diabetes.
RESULTS-The A-megalin assay mainly detected a megalin ectodomain form in the soluble urinary fraction, whereas the C-megalin assay identified a full-length form in both soluble and insoluble fractions. Urinary C-megalin levels were significantly high in patients with normoalbuminuria, were elevated in line with increased albuminuria, and showed a better association with estimated glomerular filtration rate (eGFR) (<60 mL/min/1.73 m(2)) than did urinary albumin. In contrast, urinary A-megalin levels were increased in patients with normo- and microalbuminuria but not in those with macroalbuminuria. Urinary C-megalin levels were also positively associated with plasma inorganic phosphate and negatively with hemoglobin levels in those showing no features of bleeding and not taking vitamin D analogs, phosphate binders, or erythropoiesis-stimulating agents.
CONCLUSIONS-Urinary full-length megalin excretion as measured by the C-megalin assay is well associated with reduced eGFR and linked to the severity of DN, phosphate dysregulation, and anemia, whereas urinary excretion of megalin ectodomain as measured by the A-megalin assay may be associated with distinctive mechanisms of earlier DN in type 2 diabetes.

DOI： 10.2337/dc11-1684

New beneficial effects of alkali-extracted rice protein in type 2 diabetic Goto-Kakizaki rats Reviewed

Reiko Watanabe, Masatoshi Kubota, Risa Ito, Akihiko Saito, Mikio Fujii, Motoni Kadowaki

FASEB JOURNAL 26 2012.4

Effect of alkali-extracted rice protein on the renal function in type 2 diabetic rats Reviewed

Reiko Watanabe, Masatoshi Kubota, Akihiko Saito, Takehisa Kumagai, Motoni Kadowaki

AMINO ACIDS 41 S68 - S68 2011.7

Role of Megalin and Cubilin in the Metabolism of Vitamin D-3 Reviewed

Ryohei Kaseda, Michihiro Hosojima, Hiroyoshi Sato, Akihiko Saito

THERAPEUTIC APHERESIS AND DIALYSIS 15 14 - 17 2011.6

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

Vitamin D deficiency is associated with various medical conditions including musculoskeletal disorders, infection, metabolic diseases, and cardiovascular disease. Megalin and cubilin, endocytic receptors in proximal tubule cells, are involved in the reabsorption of vitamin D binding protein from glomerular filtrates and the subsequent intracellular conversion of 25-hydroxyvitamin D-3 to biologically active 1 alpha,25-dihydroxyvitamin D-3. Dysfunction of these receptors, which is commonly found in patients with diabetic nephropathy, even at early stages, may explain why vitamin D deficiency is often complicated in these patients. Therapeutic strategies to protect the functions of these receptors from injury could be used to prevent vitamin D deficiency and its related disorders.

DOI： 10.1111/j.1744-9987.2011.00920.x

Megalin is downregulated via LPS-TNF-alpha-ERK1/2 signaling pathway in proximal tubule cells Reviewed

Aya Takeyama, Hiroyoshi Sato, Taeko Soma-Nagae, Hideyuki Kabasawa, Akiyo Suzuki, Keiko Yamamoto-Kabasawa, Michihiro Hosojima, Reika Kaneko, Fumie Higuchi, Ryohei Kaseda, Shinya Ogasawara, Ichiei Narita, Akihiko Saito

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 407 ( 1 ) 108 - 112 2011.4

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

Expression and function of megalin, an endocytic receptor in proximal tubule cells (PTCs), are reduced in diabetic nephropathy, involved in the development of proteinuria/albuminuria. Lipopolysaccharide (LPS) is chronically increased in diabetic sera, by the mechanism called metabolic endotoxemia. We investigated low-level LPS-mediated signaling that regulates megalin expression in immortalized rat PTCs (IRPTCs). Incubation of the cells with LPS (10 ng/ml) for 48 h suppressed megalin protein expression and its endocytic function. TNF-alpha mRNA expression was increased by LPS treatment, and knockdown of the mRNA with siRNA inhibited LPS-mediated downregulation of megalin mRNA expression at the 24-h time point. Incubation of IRPTCs with exogenous TNF-alpha also suppressed megalin mRNA and protein expression at the 24- and 48-h time points, respectively. MEK1 inhibitor PD98059 competed partially but significantly TNF-alpha-mediated downregulation of megalin mRNA expression. Collectively, low-level LPS-mediated TNF-alpha-ERK1/2 signaling pathway is involved in downregulation of megalin expression in IRPTCs. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2011.02.118

Proximal tubule cell hypothesis for cardiorenal syndrome in diabetes. Reviewed

Saito A, Kaseda R, Hosojima M, Sato H

International journal of nephrology 2011 957164 2010.12

Clinicopathological characteristics of patients with IgG4-related tubulointerstitial nephritis Reviewed

Takako Saeki, Shinichi Nishi, Naofumi Imai, Tomoyuki Ito, Hajime Yamazaki, Mitsuhiro Kawano, Motohisa Yamamoto, Hiroki Takahashi, Shoko Matsui, Shinji Nakada, Tomoki Origuchi, Akira Hirabayashi, Noriyuki Homma, Yutaka Tsubata, Takuma Takata, Yoko Wada, Akihiko Saito, Sachiko Fukase, Kunihiro Ishioka, Kana Miyazaki, Yasufumi Masaki, Hisanori Umehara, Susumu Sugai, Ichiei Narita

KIDNEY INTERNATIONAL 78 ( 10 ) 1016 - 1023 2010.11

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

IgG4-related disease is a recently recognized multi-organ disorder characterized by high levels of serum IgG4 and dense infiltration of IgG4-positive cells into several organs. Although the pancreas was the first organ recognized to be affected by IgG4-related disorder in the syndrome of autoimmune pancreatitis, we present here clinicopathological features of 23 patients diagnosed as having renal parenchymal lesions. These injuries were associated with a high level of serum IgG4 and abundant IgG4-positive plasma cell infiltration into the renal interstitium with fibrosis. In all patients, tubulointerstitial nephritis was the major finding. Although 14 of the 23 patients did not have any pancreatic lesions, their clinicopathological features were quite uniform and similar to those shown in autoimmune pancreatitis. These included predominance in middle-aged to elderly men, frequent association with IgG4-related conditions in other organs, high levels of serum IgG and IgG4, a high frequency of hypocomplementemia, a high serum IgE level, a patchy and diffuse lesion distribution, a swirling fibrosis in the renal pathology, and a good response to corticosteroids. Thus, we suggest that renal parenchymal lesions actually develop in association with IgG4-related disease, for which we propose the term 'IgG4-related tubulointerstitial nephritis.'

DOI： 10.1038/ki.2010.271

Molecular Mechanisms of Receptor-Mediated Endocytosis in the Renal Proximal Tubular Epithelium Reviewed

Akihiko Saito, Hiroyoshi Sato, Noriaki Iino, Tetsuro Takeda

JOURNAL OF BIOMEDICINE AND BIOTECHNOLOGY 2010 403272 2010

Language：English Publisher：HINDAWI PUBLISHING CORPORATION

Receptor-mediated endocytosis is a pivotal function of renal proximal tubule epithelial cells (PTECs) to reabsorb and metabolize substantial amounts of proteins and other substances in glomerular filtrates. The function accounts for the conservation of nutrients, including carrier-bound vitamins and trace elements, filtered by glomeruli. Impairment of the process results in a loss of such substances and development of proteinuria, an important clinical sign of kidney disease and a risk marker for cardiovascular disease. Megalin is a multiligand endocytic receptor expressed at clathrin-coated pits of PTEC, playing a central role in the process. Megalin cooperates with various membrane molecules and interacts with many intracellular adaptor proteins for endocytic trafficking. Megalin is also involved in signaling pathways in the cells. Megalin-mediated endocytic overload leads to damage of PTEC. Further studies are needed to elucidate the mechanism of megalin-mediated endocytosis and develop strategies for preventing the damage of PTEC.

DOI： 10.1155/2010/403272

Megalin and nonmuscle myosin heavy chain IIA interact with the adaptor protein Disabled-2 in proximal tubule cells Reviewed

Kiyoko Hosaka, Tetsuro Takeda, Noriaki Iino, Michihiro Hosojima, Hiroyoshi Sato, Ryohei Kaseda, Keiko Yamamoto, Asako Kobayashi, Fumitake Gejyo, Akihiko Saito

KIDNEY INTERNATIONAL 75 ( 12 ) 1308 - 1315 2009.6

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

Megalin plays a critical role in the endocytosis of albumin and other filtered low-molecular-weight proteins. Here we studied the interaction between megalin and Disabled-2 (Dab2), an adaptor protein that binds to the cytoplasmic domain of megalin and appears to control its trafficking. We co-immunoprecipitated megalin and Dab2 from cultured proximal tubule cells and identified the proteins by liquid chromatography and tandem mass spectrometry. We found two proteins associated with the megalin/Dab2 complex, nonmuscle myosin heavy chain IIA (NMHC-IIA) and beta-actin. Subcellular fractionation followed by sucrose velocity gradient separation showed that megalin, Dab2, and NMHC-IIA existed as a complex in the same endosomal fractions. In vitro pull-down assays demonstrated that NMHC-IIA was bound to the carboxyl-terminal region of Dab2, but not to megalin's cytoplasmic domain. We then transfected COS-7 cells with plasmids that induced the expression of Dab2, NMHC-IIA, and the megalin minireceptor, a truncated form of megalin. Co-immunoprecipitation studies showed that the minireceptor and NMHC-IIA co-immunoprecipitated only with Dab2. Furthermore, the uptake of (125)I-lactoferrin, an endocytic ligand of megalin, by rat yolk sac-derived megalin-expressing L2 cells was inhibited by blebbistatin, a specific inhibitor of nonmuscle myosin II. Our study shows that NMHC-IIA is functionally linked to megalin by interaction with Dab2 and is likely involved in megalin-mediated endocytosis in proximal tubule cells. Kidney International (2009) 75, 1308-1315; doi:10.1038/ki.2009.85; published online 1 April 2009

DOI： 10.1038/ki.2009.85

Regulation of Megalin Expression in Cultured Proximal Tubule Cells by Angiotensin II Type 1A Receptor- and Insulin-Mediated Signaling Cross Talk Reviewed

Michihiro Hosojima, Hiroyoshi Sato, Keiko Yamamoto, Ryohei Kaseda, Taeko Soma, Asako Kobayashi, Akiyo Suzuki, Hideyuki Kabasawa, Aya Takeyama, Kenji Ikuyama, Noriaki Iino, Akira Nishiyama, Thomas J. Thekkumkara, Tetsuro Takeda, Yoshiki Suzuki, Fumitake Gejyo, Akihiko Saito

ENDOCRINOLOGY 150 ( 2 ) 871 - 878 2009.2

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

Impairment of proximal tubular endocytosis of glomerular-filtered proteins including albumin results in the development of proteinuria/albuminuria in patients with chronic kidney disease. However, the mechanisms regulating the proximal tubular function are largely unknown. This study aimed to investigate the role of angiotensin II type 1A receptor (AT(1A)R)- and insulin-mediated signaling pathways in regulating the expression of megalin, a multiligand endocytic receptor in proximal tubule cells (PTCs). Opossum kidney PTC-derived OK cells that stably express rat AT(1A)R but are deficient in endogenous angiotensin II receptors (AT(1A)R-OK cells) were used for this study. Treatment of the cells with angiotensin II suppressed mRNA and protein expression of megalin at 3- and 24-h incubation time points, respectively. Cellular uptake and degradation of albumin and receptor-associated protein, megalin's endocytic ligands were suppressed 24 h after angiotensin II treatment. The AT(1A)R-mediated decrease in megalin expression was partially prevented by ERK inhibitors. Insulin competed with the AT(1A)R-mediated ERK activation and decrease in megalin expression. Inhibitors of phosphatidylinositol 3- kinase (PI3K), a major component of insulin signaling, also suppressed megalin expression, and activation of the insulin receptor substrate (IRS)/PI3K system was prevented by angiotensin II. Collectively the AT(1A)R-mediated ERK signaling is involved in suppressing megalin expression in the OK cell line, and insulin competes with this pathway. Conversely, the insulin-IRS/PI3K signaling, with which angiotensin II competes, tends to stimulate megalin expression. In conclusion, there is AT(1A)R- and insulin-mediated competitive signaling cross talk to regulate megalin expression in cultured PTCs. (Endocrinology 150: 871 - 878, 2009)

DOI： 10.1210/en.2008-0886

Megalin contributes to the early injury of proximal tubule cells during nonselective proteinuria Reviewed

Yaeko Motoyoshi, Taiji Matsusaka, Akihiko Saito, Ira Pastan, Thomas E. Willnow, Shuki Mizutani, Iekuni Ichikawa

KIDNEY INTERNATIONAL 74 ( 10 ) 1262 - 1269 2008.11

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

Megalin, a member of the LDL receptor family, is expressed on the apical membrane of proximal tubules and serves as an endocytic scavenger of filtered proteins and hence might contribute to the tubule injury as a consequence of glomerular disease. To study its role, we crossed megalin knockout mosaic mice (lacking megalin expression in 60% of proximal tubule cells) with NEP25 mice (a transgenic line expressing human CD25 in the podocyte). Treatment of this transgenic mouse with the immunotoxin causes nephrotic syndrome, focal segmental glomerulosclerosis and tubule-interstitial injury. Following this treatment, the double transgenic mice had massive non-selective proteinuria and mild glomerular and tubular injury. Comparison of megalin-containing to megalin-deficient proximal tubule cells within each kidney showed that albumin, immunoglobulin light chain, IgA and IgG were preferentially accumulated in proximal tubule cells expressing megalin. Tubule injury markers such as heme-oxygenase-1, monocyte chemoattractant protein-1 and cellular apoptosis were also preferentially found in these megalin-expressing cells. These results show that megalin plays a pivotal role in the reabsorption of small to large molecular size proteins and provides direct in vivo evidence that reabsorption of filtered proteins triggers events leading to tubule injury.

DOI： 10.1038/ki.2008.405

Anemia is a predictive factor for the development of CKD only in man, but not in woman, of general Japanese population Reviewed

Noriaki Iino, Ichiei Narita, Akihiko Saito, Fumitake Gejyo

NEPHROLOGY 13 A34 - A34 2008.5

Alterations in serum phosphate levels predict the long-term response to intravenous calcitriol therapy in dialysis patients with secondary hyperparathyroidism Reviewed

Kiyoko Hosaka, Junichiro James Kazama, Suguru Yamamoto, Yumi Ito, Noriaki Iino, Hiroki Maruyama, Akihiko Saito, Ichiei Narita, Fumitake Gejyo

JOURNAL OF BONE AND MINERAL METABOLISM 26 ( 2 ) 185 - 190 2008.3

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

Calcitriol therapy is a central strategy for the treatment of uremic secondary hyperparathyroidism. Although indiscriminate use of calcitriol may lead to worse outcomes, it is difficult to make a decision to discontinue calcitriol therapy when its parathyroid suppression effect remains unsatisfactory. In this study, intravenous calcitriol was administered to 120 chronic hemodialysis patients. Therapy continued for 48 weeks or until plasma intact parathyroid hormone (iPTH) levels decreased to below 300 pg/ml or until the development of any significant adverse effect. Of the 120 patients, the treatment goal was achieved in 47 patients during the first 4 weeks, in 10 during the next 4 weeks, and in 22 patients thereafter. Logistic regression analysis and stepwise regression analysis revealed that iPTH levels were the only significant predictor of the response to calcitriol therapy at weeks 0 and 4. Besides iPTH, the inorganic phosphate (P) levels were another significant predictor of the ultimate response to calcitriol therapy at week 8. The point of best discrimination for successful treatment was P = 6.0 mg/dl at week 8, or P level at week 8/pretreatment P level = 1.0. In conclusion, the P level at week 8 is a predictor of the response to calcitriol therapy for uremic secondary hyperparathyroidism. Changes in treatment are recommended if patients show unsatisfactory parathyroid suppression with a hyperphosphatemic tendency.

DOI： 10.1007/s00774-007-0809-1

Localization of megalin in rat vestibular dark cells and endolymphatic sac epithelial cells Reviewed

Maki Arai, Kunihiro Mizuta, Akihiko Saito, Yasuyuki Hashimoto, Satoshi Iwasaki, Takahiro Watanabe, Hiroyuki Mineta

ACTA OTO-LARYNGOLOGICA 128 ( 6 ) 627 - 633 2008

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

Conclusion: Megalin immunoreactivity was observed in kidney proximal tubule cells, vestibular dark cells, and epithelial cells of the endolymphatic sac. Endocytic mechanisms appear to differ between the endolymphatic sac and proximal tubule cells. We speculate that megalin is secreted by a certain type of cell into the endolymphatic space, and is then absorbed from the endolymphatic space by another type of cell to maintain endolymphatic sac homeostasis. Objectives: We previously detected megalin immunoreactivity in the rat cochlear duct. Megalin may be involved in endocytosis in the vestibular organ and endolymphatic sac. To examine this possibility, we extended our immunocytochemical investigation to the rat inner ear cells with special attention to vestibular dark cells and endolymphatic sac. Materials and methods: We observed immunoreactivity of megalin under light and electron microscopy. The primary antibody was rabbit polyclonal antibody that had been raised against rat immunoaffmity-purified megalin. Results: The luminal membrane and subapical area of dark cells in the semicircular canal were immunolabeled. The stainable substance in the endolymphatic space was strongly stained. The cytoplasm of epithelial cells was also stained in various patterns.

DOI： 10.1080/00016480701668531

Role of megalin, a proximal tubular endocytic receptor, in calcium and phosphate homeostasis Reviewed

Akihiko Saito, Noriaki Iino, Tetsuro Takeda, Fumitake Gejyo

THERAPEUTIC APHERESIS AND DIALYSIS 11 S23 - S26 2007.10

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

Megalin is expressed at the apical membranes of proximal tubule cells, acting as an endocytic receptor for a variety of ligands filtered by glomeruli. Megalin, also known as a Ca(2+)-binding receptor, is thought to be involved in systemic and intrarenal calcium and phosphate homeostasis. The complex of 25( OH) D(3) and vitamin D-binding protein is endocytosed via megalin into proximal tubule cells, leading to the activation of 25( OH) D3 to 1, 25( OH) D3 in the cells. Megalin knockout mice revealed impaired osteogenesis due to vitamin D deficiency. Megalin is also involved in the metabolism of parathyroid hormone and the regulation of the sodium phosphate cotransporter NaPi-IIa. Decreased expression of megalin may be associated with the pathogenesis of hyperphosphaturia observed in patients with Dent's disease. Further studies will elucidate more detailed roles of megalin in pathological states and the mechanisms for interacting with other molecules for the endocytic functions.

DOI： 10.1111/j.1744-9987.2007.00514.x

Tubulointerstitial nephritis associated with IgG4-related systemic disease Reviewed

Takako Saeki, Akihiko Saito, Hajime Yamazaki, Iwao Emura, Naofumi Imai, Mitsuhiro Ueno, Shinichi Nishi, Syoji Miyamura, Fumitake Gejyo

Clinical and Experimental Nephrology 11 ( 2 ) 168 - 173 2007.6

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

We report three patients with tubulointerstitial nephritis (TIN) with high serum IgG4 concentrations. None of the patients had notable pancreatic lesions when the TIN developed, although one had a history of autoimmune pancreatitis (AIP). Nevertheless, the clinicopathological findings were quite similar to those of AIP. They were all middle-aged to elderly men. Sialadenitis and lymphadenopathy were often evident. Serum total IgG and IgG4 concentrations were elevated and hypocomplementemia was observed. Although antinuclear antibodies were positive, anti-Ro and anti-La antibodies were negative. Renal biopsy showed dense lymphoplasmacytic infiltration with fibrosis in the renal interstitium, and the infiltrated plasma cells had strong immunoreactivity for IgG4. Furthermore, lymphoplasmacytic infiltration and abundant IgG4-positive plasma cells were observed in the salivary glands of a patient. Steroid therapy was effective for TIN in all three patients. The present findings support the recently proposed concept of IgG4-related systemic disease, and suggest that IgG4 is associated not only with AIP but also with other systemic lymphoplasmacytic diseases, including TIN. The conditions responsible for the pathogenesis of TIN need to be considered, irrespective of the presence of AIP. © 2007 Japanese Society of Nephrology.

DOI： 10.1007/s10157-007-0464-9

Megalin-mediated endocytosis of cystatin C in proximal tubule cells Reviewed

Ryohei Kaseda, Noriaki Iino, Michihiro Hosojima, Tetsuro Takeda, Kiyoko Hosaka, Asako Kobayashi, Keiko Yamamoto, Akiyo Suzuki, Ayaka Kasai, Yoshiki Suzuki, Fumitake Gejyo, Akihiko Saito

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 357 ( 4 ) 1130 - 1134 2007.6

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

Serum levels of cystatin C, an endogenous cysteine protemase inhibitor, are often used as an indicator of glomerular filtration rate. Although it is known that cystatin C is filtered by glomeruli and metabolized in proximal tubule cells (PTC), the precise molecular mechanism underlying this process is undetermined. Using quartz-crystal microbalance analyses, we demonstrate that cystatin C binds directly to megalin, an endocytic receptor in PTC, in a Ca(+)-dependent manner. We also find that cystatin C is endocytosed specifically via megalin in rat yolk sac epithelium-derived L2 cells which share a variety of characteristics with PTC. Finally, it? vivo studies using kidney-specific megalin knockout mice provide evidence that megalin mediates proximal tubular uptake of cystatin C. We conclude that megalin is an endocytic receptor of cystatin C in PTC. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.04.072

Functional characterization of a novel missense CLCN5 mutation causing alterations in proximal tubular endocytic machinery in Dent's disease Reviewed

Atsuhito Tanuma, Hiroyoshi Sato, Tetsuro Takeda, Michihiro Hosojima, Hiroaki Obayashi, Hitomi Hama, Noriaki Iino, Kiyoko Hosaka, Ryohei Kaseda, Naofumi Imai, Mitsuhiro Ueno, Maya Yamazaki, Kenji Sakimura, Fumitake Gejyo, Akihiko Saito

NEPHRON PHYSIOLOGY 107 ( 4 ) P87 - P97 2007

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

Background/Aims: Mutations of the endosomal chloride/ proton exchanger gene, CLCN5, cause Dent's disease, an X-linked recessive proximal tubular disorder. The renal endocytic system was found to be affected in clcn5 knockout mice. However, the impaired endocytic machinery of Dent's disease patients has not been thoroughly investigated. Methods: The CLCN5 gene was sequenced in a Japanese patient with Dent's disease and his family. The loss-of-function phenotype of the missense CLCN5 mutation was investigated by gene expression in Xenopus oocytes and CHO cells. Immunohistochemical analysis was performed on kidney biopsy specimens for endocytic machinery proteins, megalin, cubilin, and disabled-2 (Dab2) in proximal tubules. Results: Genomic analysis revealed a novel G-to-A transition at the first nucleotide of the 333rd codon of CLCN5, causing a substitution of glycine with arginine. Inefficient expression of the mutant gene in Xenopus oocytes resulted in abolished chloride currents. Impaired N-glycosylation of the mutant protein was evident in the DNA-transfected CHO cells. Proximal tubular expression of megalin, cubilin, and Dab2 was markedly reduced and irregular staining in some portions was observed in the patient compared with controls. Conclusions: A novel G333R CLCN5 mutation caused defective expression of megalin, cubilin, and Dab2 in a patient with Dent's disease. Copyright (c) 2007 S. Karger AG, Basel.

DOI： 10.1159/000111253

Renal lesions in IgG4-related systemic disease Reviewed

Takako Saeki, Shinichi Nishi, Tomoyuki Ito, Hajime Yamazaki, Syoji Miyamura, Iwao Emura, Naofumi Imai, Mitsuhiro Ueno, Akihiko Saito, Fumitake Gejyo

INTERNAL MEDICINE 46 ( 17 ) 1365 - 1371 2007

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN SOC INTERNAL MEDICINE

Objective Recently, a new concept of IgG4-related systemic disease including autoimmune pancreatitis, characterized by a high serum IgG4 level and tissue infiltration by IgG4-positive plasma cells, has been proposed. Our aim was to investigate the renal involvement in this condition.
Patients and Methods We investigated the results of laboratory and imaging studies of the kidneys in 7 patients with IgG4-related systemic disease, and examined the renal histology in four of them. All patients showed elevated serum IgG4 levels, and 4 had autoimmune pancreatitis. The other three patients showed involvement of various extrapancreatic organs (lymphadenopathy, sialadenitis or renal insufficiency), and abundant IgG4-positive plasma cell infiltration was confirmed in their affected tissues.
Results Six of the 7 patients showed some renal abnormalities. In one patient, hydronephrosis was observed accompanied by retroperitoneal fibrosis. Another patient showed multiple low-density areas in both kidneys by computed tomography, and gallium citrate scintigraphy showed gallium-67 accumulation in both kidneys, although renal function was normal. Four patients had tubulointerstitial nephritis. In two of them, the tubulointerstitial nephritis was diffuse. In one patient, marked diffuse but patchily distributed lymphoplasmacytic infiltration of the renal interstitium was observed. In another patient, computed tomography showed a tumor-like low-density mass; open biopsy of the mass showed aggregates of lymphocytes and plasma cells in the renal interstitium.
Conclusion Renal parenchymal lesions in IgG4-related systemic disease are due to dense lymphoplasmacytic infiltration of the renal interstitium, and the lesions vary from diffuse tubulointerstitial nephritis to tumor-like masses according to the distribution patterns of the infiltrating cells.

DOI： 10.2169/internalmedicine.46.0183

Functional characterization of a novel missense CLCN5 mutation causing alterations in proximal tubular endocytic machinery in Dent's disease. Reviewed

Tanuma A, Sato H, Takeda T, Hosojima M, Obayashi H, Hama H, Iino N, Hosaka K, Kaseda R, Imai N, Ueno M, Yamazaki M, Sakimura K, Gejyo F, Saito A

Nephron. Physiology 107 ( 4 ) p87 - 97 2007

Current clinical aspects of dialysis-related amyloidosis in chronic dialysis patients Reviewed

Akihiko Saito, Fumitake Gejyo

THERAPEUTIC APHERESIS AND DIALYSIS 10 ( 4 ) 316 - 320 2006.8

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

The current understanding of dialysis-related amyloidosis has evolved over the past two decades. In the early 1980s, several researchers found amyloid deposits in the synovia of carpal tunnel syndrome (CTS), which have been recognized as a complication of chronic hemodialysis. The enigma was resolved in 1985, when beta(2)-microglobulin (beta(2)-m) with a molecular weight of 12 000 Da was identified as the major constitutional protein of this amyloid. Amyloid fibrils of this type that contain the sub-unit protein of human leukocyte antigens (HLA), beta(2)-m, deposit predominantly in osteoarticular tissues, inducing musculoskeletal symptoms such as CTS, polyarthralgia, bone cyst showing radiolucency at X-ray examination and destructive spondyloarthropathy. In addition, extra articular symptoms such as ischemic colitis, megaloglossia, and heart failure, that is, systemic involvement occasionally occur. We confirmed that the prevalence of CTS increases with duration of dialysis. Most patients with CTS associated with beta(2)-m amyloid deposits have undergone hemodialysis for 10 years or more. Up to 50% of patients had developed this complication after 20 years and the percentage was even higher after 25 years. General categories of therapeutic approaches for amyloidosis include prevention of onset or progression, symptomatic therapy (conservative treatment, orthopedic procedures, and physiotherapy), and renal transplantation. It is critical to elucidate the detail mechanisms of the amyloid fibril formation, and establish its radical treatment. It is also important to develop novel therapies such as cell implantation to compensate for normal kidney functions of uremic toxin protein metabolism.

DOI： 10.1111/j.1744-9987.2006.00383.x

[Megalin and cubilin: protein reabsorption in proximal tubule]. Reviewed

Takeda T, Saito A, Gejyo F

Nihon rinsho. Japanese journal of clinical medicine 64 Suppl 2 332 - 338 2006.2

Lymphoplasmacytic infiltration of multiple organs with immunoreactivity for IgG4: IgG4-related systemic disease Reviewed

Takako Saeki, Akihiko Saito, Tohru Hiura, Hajime Yamazaki, Iwao Emura, Mitsuhiro Ueno, Syoji Miyamura, Fumitake Gejyo

INTERNAL MEDICINE 45 ( 3 ) 163 - 167 2006

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN SOC INTERNAL MEDICINE

We report three elderly men with high serum IgG4 concentrations and multiple lymphoplasmacytic inflammation of the salivary glands, lymph nodes, pancreas, and renal interstitium. The infiltrating plasma cells had strong immunoreactivity for IgG4, even in patients without pancreatic lesions. These cases show that IgG4 is associated not only with autoimmune pancreatitis, but also with other systemic lymphoplasmacytic disease.

DOI： 10.2169/internalmedicine.45.1431

Role of megalin, a proximal tubular endocytic receptor, in the pathogenesis of diabetic and metabolic syndrome-related nephropathies: protein metabolic overload hypothesis Reviewed

A Saito, T Takeda, H Hama, Y Oyama, K Hosaka, A Tanuma, R Kaseda, M Ueno, S Nishi, S Ogasawara, F Gondaira, Y Suzuki, F Gejyo

NEPHROLOGY 10 S26 - S31 2005.10

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

Megalin is an endocytic receptor on the apical membranes of proximal tubule cells (PTC) in the kidney, and is involved in the reabsorption and metabolism of various proteins that have been filtered by glomeruli. Patients with diabetes, especially type 2 diabetes, or metabolic syndrome are likely to have elevated serum levels of advanced glycation end products, liver-type fatty acid binding protein, angiotensin 11, insulin and leptin, and renal metabolism of these proteins is potentially overloaded. Some of these proteins are themselves nephrotoxic, while others are carriers of nephrotoxic molecules. Megalin is involved in the proximal tubular uptake of these proteins. We hypothesize that megalin-mediated metabolic overload in PTC leads to compensatory cellular hypertrophy and sustained Na(+) reabsorption, causing systemic hypertension and glomerular hyperfiltration via tubuloglomerular feedback, and named this as 'protein metabolic overload hypothesis'. Impaired metabolism of bioactive proteins such as angiotensin 11 and insulin in PTC may enhance hypertrophy of PTC and/or Na(+) reabsorption. Sleep apnoea syndrome, a frequent complication of diabetes and metabolic syndrome, may cause renal hypoxia and result in relative overload of protein metabolism in the kidneys. The development of strategies to identify patients with diabetes or metabolic syndrome who are at high risk for renal metabolic overload would allow intensive treatment of these patients in an effort to prevent the development of nephropathy. Further studies on the intracellular molecular signalling associated with megalin-mediated metabolic pathways may lead to the development of novel strategies for the treatment of nephropathies related to diabetes and metabolic syndrome.

DOI： 10.1111/j.1440-1797.2005.00453.x

Evidence for megalin-mediated proximal tubular uptake of L-FABP, a carrier of potentially nephrotoxic molecules Reviewed

Y Oyama, T Takeda, H Hama, A Tanuma, N Iino, K Sato, R Kaseda, ML Ma, T Yamamoto, H Fujii, JJ Kazama, S Odani, Y Terada, K Mizuta, F Gejyo, A Saito

LABORATORY INVESTIGATION 85 ( 4 ) 522 - 531 2005.4

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

Liver-type fatty acid binding protein (L-FABP) binds with high affinity to hydrophobic molecules including free fatty acid, bile acid and bilirubin, which are potentially nephrotoxic, and is involved in their metabolism mainly in hepatocytes. L-FABP is released into the circulation, and patients with liver damage have an elevated plasma L-FABP level. L-FABP is also present in renal tubules; however, the precise localization of L-FABP and its potential role in the renal tubules are not known. In this study, we examined the cellular and subcellular localization of L-FABP in the rat kidney and tried to determine from where the L-FABP in kidney tissues had originated. Immunohistochemical studies of kidney sections localized L-FABP in the lysosomes of proximal tubule cells (PTC). In rats with carbon tetrachloride (CCl(4))-induced acute liver injury, we detected high levels of L-FABP in the circulation and in the kidney compared with those in the control rat by immunoblotting, while reverse transcription-polymerase chain reaction showed that the level of L-FABP mRNA expression in the kidney of CCl(4)-treated rats was low and did not differ from that in the control rat. When (35)S-L-FABP was intravenously administered to rats, the kidneys took up (35)S-L-FABP more preferentially than the liver and heart, and histoautoradiography of kidney sections revealed that (35)S-L-FABP was internalized via the apical domains of PTC. Quartz-crystal microbalance analysis revealed that L-FABP bound to megalin, a multiligand endocytotic receptor on PTC, in a Ca(2+)-dependent manner. Degradation assays using megalin-expressing rat yolk sac tumor-derived L2 cells demonstrated that megalin mediated the cellular uptake and catabolism of (125)I-L-FABP. In conclusion, circulatory L-FABP was found to be filtered by glomeruli and internalized by PTC probably via megalin-mediated endocytosis. These results suggest a novel renal uptake pathway for L-FABP, a carrier of hydrophobic molecules, some of which may exert nephrotoxic effects.

DOI： 10.1038/labinvest.3700240

Significance of proximal ribular metabolism of advanced glycation end products in kidney diseases Reviewed

A Saito, T Takeda, K Sato, H Hama, A Tanuma, R Kaseda, Y Suzuki, F Gejyo

MAILLARD REACTION: CHEMISTRY AT THE INTERFACE OF NUTRITION, AGING, AND DISEASE 1043 637 - 643 2005

Language：English Publishing type：Research paper (scientific journal) Publisher：NEW YORK ACAD SCIENCES

Advanced glycation end products (AGEs) are formed by the nonenzymatic Maillard reaction between sugars and proteins. Low-molecular weight AGES are filtered by renal glomeruli and then reabsorbed and metabolized by proximal tubule cells (PTCs). High-molecular weight AGES are also delivered to PTCs in proteinuric states. In patients with diabetes, AGE generation is increased, and the actions of AGES on PTCs are likely involved in the pathogenesis of diabetic nephropathy. In patients with chronic renal failure (CRF), reduced renal metabolism of AGEs likely accounts for the accumulation of AGEs in serum, leading to uremic complications including dialysis-related amyloidosis. AGE precursors such as reactive carbonyl compounds also accumulate in the sera of patients with CRF. It is likely that PTCs take up AGEs and AGE precursors via specific endocytotic receptors or transporters. Megalin is a multiligand endocytotic receptor that is abundantly expressed on PTCs. There is evidence that megalin is involved in the cellular uptake and degradation of AGES. We previously reported a cell therapy model involving implantation of megalin-expressing cells into experimental mice with renal failure for elimination of uremic toxin proteins. Further studies are needed to clarify the molecular mechanisms of the metabolism of AGEs and their precursors to develop a strategy

DOI： 10.1196/annals.1333.072

Evidence indicating that renal tubular metabolism of leptin is mediated by megalin but not by the leptin receptors

H Hama, A Saito, T Takeda, A Tanuma, YS Xie, K Sato, JJ Kazama, F Gejyo

ENDOCRINOLOGY 145 ( 8 ) 3935 - 3940 2004.8

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

Leptin is secreted by adipocytes and is a circulating factor that regulates food intake and energy expenditure. Its serum level is elevated in patients with renal failure and has been suggested to be associated with malnutritional factors in these patients. Leptin has been suggested to be primarily metabolized by the kidneys, although the precise molecular mechanisms are not known. The purpose of this study was to determine the nephron segments and potential receptors involved in renal leptin metabolism. To determine the segment involved in leptin uptake, we performed histoautoradiography of kidney sections obtained from rats that had been injected iv with I-125-leptin. The ability of megalin, a multiligand endocytic receptor in the proximal tubules, to bind and endocytose leptin was examined by ligand blotting analysis, quartz-crystal microbalance, and degradation assays using megalin-expressing rat yolk sac L2 cells. Immunohistochemistry was performed to localize leptin receptors (LEP-R) in the rat kidney using two antibodies that recognize different epitopes on the LEP-R proteins. Circulating I-125-leptin was filtered by glomeruli and internalized by proximal convoluted tubules. Megalin bound leptin in the presence of Ca2+ and mediated its cellular internalization and degradation. On immunohistochemistry, LEP-R were localized in the proximal straight tubules, loops of Henle, distal tubules, and collecting ducts. In conclusion, circulating leptin was filtered by glomeruli and taken up by proximal convoluted tubules, where megalin likely mediates its binding and uptake. The localization of LEP-R suggests that they are not primarily involved in leptin metabolism in the proximal tubules.

DOI： 10.1210/en.2004-0074

Differentiation-induced cultured podocytes express endocytically active megalin, a Heymann nephritis antigen

H Yamazaki, A Saito, H Ooi, N Kobayashi, P Mundel, F Gejyo

NEPHRON EXPERIMENTAL NEPHROLOGY 96 ( 2 ) E52 - E58 2004

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

Background/Aims: Megalin is a multiligand endocytic receptor expressed in a number of epithelia. In the Lewis rat kidney, podocytes, as well as proximal tubule cells, express megalin that acts as a pathogenic antigen for Heymann nephritis (HN), an experimental model of membranous nephropathy. To obtain a tool to investigate the molecular mechanisms of megalin-mediated endocytosis and the pathogenesis of HN, we examined whether a differentiation-inducible mouse podocyte cell line expressed endocytically active megalin. Methods: Immunofluorescence and immunoprecipitation analyses with an anti-rat megalin antibody were carried out to investigate whether megalin was expressed in the differentiated and undifferentiated podocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed to elucidate whether the cells synthesize megalin mRNA. I-125-labeled receptor-associated protein (RAP), an endocytic ligand for megalin, was used for cellular internalization and degradation assays. Results: Immunofluorescence, immunoprecipitation and RT-PCR analyses revealed that megalin was synthesized in both differentiated and undifferentiated cells and localized to the cell surfaces. Effective endocytosis of RAP via megalin was shown under the differentiated condition. Conclusion: Endocytically active megalin is expressed in differentiation-induced cultured podocytes. This cell line could be a useful tool for studies on megalin-mediated endocytosis and the pathogenesis of HN. Copyright (C) 2004 S. Karger AG, Basel.

DOI： 10.1159/000076404

Different type and localization of CD44 on surface membrane of regenerative renal tubular epithelial cells in vivo

YS Xie, S Nishi, S Fukase, H Nakamura, XM Chen, N Imai, M Sakatsume, A Saito, M Ueno, Narita, I, T Yamamoto, F Gejyo

AMERICAN JOURNAL OF NEPHROLOGY 24 ( 2 ) 188 - 197 2004

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

Background: CD44 is a transmembrane glycoprotein comprising an extracellular domain, a transmembrane domain, and a cytoplasmic tail. Previous studies demonstrated that CD44 was generally restricted to lateral- basal plasma membrane ( PM) of epithelial cells, whether it localized on apical PM in vivo has not been clarified. Methods: In this study, we used a gentamicin-induced acute tubular necrosis (ATN) and spontaneous recovery model in rats and two distinct antibodies, an anti-rat distal extracellular domain (OX49) of standard CD44 ( CD44-OX49) and an anti-rat CD44 cytoplasmic tail (CD44CPT), to survey the localization of CD44-OX49 and CD44CPT on the PM in renal tubular epithelial cells in different recovery stages after ATN with immunohistochemistry and immunoelectron-microscopic examinations. Results: CD44-OX49 was localized not only on the lateral-basal PM in tubular epithelial cells, but also on the apical surface membrane in PCNA-positive newly regenerative tubular epithelial cells in early recovery stages after ATN. However, CD44CPT was only localized on the lateral-basal PM. The immunoelectron-microscopic results showed that CD44-OX49 localization was changed from the apical to lateral to basal surface membrane in renal tubular epithelial cells during the recovery process after ATN, finally disappearing from basal PM when normal polarized epithelial cells formed. Conclusions: These results suggest that there were two types of CD44 including CD44 without a cytoplasmic tail localizing on the apical surface membrane related to newly regenerative epithelial cells, and CD44 with a cytoplasmic tail localizing on the lateral-basal PM related to establishment of tubular epithelial cell polarity after ATN in vivo. Copyright (C) 2004 S. Karger AG, Basel.

DOI： 10.1159/000076758

Bioengineered implantation of megalin-expressing cells: A potential intracorporeal therapeutic model for uremic toxin protein clearance in renal failure Reviewed

A Saito, JJ Kazama, N Iino, KJ Cho, N Sato, H Yamazaki, Y Oyama, T Takeda, RA Orlando, F Shimizu, Y Tabata, F Gejyo

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 14 ( 8 ) 2025 - 2032 2003.8

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

Patients who have renal failure and are on dialysis therapy experience serious complications caused by low-molecular-weight uremic toxin proteins normally filtered by glomeruli and metabolized by proximal tubule cells (PTC). Dialysis-related amyloidosis is one such complication induced by systemic deposition of amyloid proteins derived from 12-kD beta(2)-microglobulin (beta(2)-M). Despite the use of high-flux membrane hemodialysis devices and direct absorbent columns, the removal of beta(2)-M is suboptimal, because the effects are transient and insufficient. Megalin is expressed in the apical membranes of PTC and recognized as a multiligand endocytic receptor that binds numerous low-molecular-weight proteins, including, beta(2)-M. This study tested the feasibility of an intra corporeal therapeutic model of continuous beta(2)-m removal using megalin-expressing cell implantation. By cell association and degradation assays, rat yolk sac-derived L2 cells were identified to internalize and degrade beta(2)-M via megalin. The cells were effectively implanted within the subcutaneous tissues of nude mice using a type I collagen scaffold and a method inducing local angiogenesis. After nephrectomy and intraperitoneal injection with I-125-beta(2)-m, it was found that the implanted cells took up the labeled ligand, efficiently removing it from the blood. Bioengineered implantation of megalin-expressing cells may represent a new supportive therapy for dialysis patients to compensate for the loss of renal protein metabolism and remove uremic toxin proteins.

DOI： 10.1097/01.ASN.0000078804.98322.4A

Infection-associated hemophagocytic syndrome in a diabetic patient undergoing chronic hemodialysis Reviewed

Yoko Wada, Maki Sato, Akihiko Saito, Fumitake Gejyo

Clinical and Experimental Nephrology 7 ( 2 ) 163 - 166 2003.6

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

Hemophagocytic syndrome (HPS) is an uncommon but severe illness associated with a variety of infections, malignant tumors, and autoimmune diseases. We report a case of infection-associated HPS in a patient receiving chronic hemodialysis. Peptostreptococcus-induced sepsis and abscess formation in the left iliopsoas muscle led to the onset of infection-associated HPS in this patient. The patient had diabetes mellitus and end-stage renal disease, and it is likely that immunological dysfunctions from these disorders played a part in the onset of both severe bacterial infections and HPS.

DOI： 10.1007/s10157-003-0230-6

Role of megalin in endocytosis of advanced glycation end products: Implications for a novel protein binding to both megalin and advanced glycation end products Reviewed

A Saito, R Nagai, A Tanuma, H Hama, KJ Cho, T Takeda, Y Yoshida, T Toda, F Shimizu, S Horiuchi, F Gejyo

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 14 ( 5 ) 1123 - 1131 2003.5

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

Advanced glycation end products (AGE) are filtered by glomeruli and reabsorbed and metabolized by proximal tubule cells (PTC). In renal failure, decreased renal AGE metabolism likely accounts for the accumulation in serum that is related to uremic complications. In diabetes, AGE generation is increased, and the handling mechanisms in PTC are likely associated with the pathogenesis of tubulointerstitial injury. It is therefore important to clarify the mechanisms of the AGE metabolism to develop a strategy for removing AGE in uremia and to elucidate the pathogenesis of diabetic nephropathy. To this end, this study focused on the molecular analysis of megalin, a multi-ligand endocytic receptor, in PTC. AGE uptake analysis was performed using the rat yolk sac-derived L2 cell line system established for the analysis of megalin's endocytic functions. The cells mediated specific internalization and degradation of AGE, which were significantly blocked by anti-megalin IgG, indicating that megalin is involved in the cellular processes. However, cell surface AGE-binding assays and ligand blot analysis revealed no evidence that megalin is a direct AGE receptor. Affinity chromatography and ligand blot analysis originally revealed that 200-kD and 400-kD proteins in the cells bind to AGE and the 200-kD protein to megalin in a Ca2+-dependent manner. The binding of megalin with the 200-kD protein was suppressed by receptor-associated protein (RAP), a ligand for megalin. In conclusion, megalin functions for endocytosis of AGE via an indirect mechanism. L2 cells express novel AGE-binding proteins, one of which may interact with megalin.

DOI： 10.1097/01.ASN.0000062962.51879.F8

IgA腎症の進展におけるACE及びAngiotensinogen(AGT)遺伝子多型の検討

後藤眞, 成田一衛, 斉藤徳子, 坂爪実, 飯野則昭, 斉藤亮彦, 西慎一, 上野光博, 下条文武

日本腎臓学会誌 43 ( 3 ) 270 - 270 2001.4

メサンギウム増殖性糸球体腎炎(MesPGN)におけるLRP遺伝子多型の検討

齋藤徳子, 成田一衛, 後藤眞, 坂爪実, 斎藤亮彦, 大森健太郎, 上野光博, 下条文武

日本腎臓学会誌 43 ( 3 ) 271 - 271 2001.4

IgA腎症の進展におけるレニン・アンギオテンシン系遺伝子多型の検討

後藤眞, 成田一衛, 斉藤徳子, 坂爪実, 飯野則昭, 斉藤亮彦, 西慎一, 上野光博, 今井直史, 下条文武

日本内科学会雑誌 90 ( 臨増 ) 211 - 211 2001.2

Association of Gene Polymorphism of Polymeric Immunoglobulin Receptor and IgA Nephropathy

Ichiei Narita, Daisuke Kondo, Shin Goto, Noriko Saito, Yasuo Watanabe, Hajime Yamazaki, Minoru Sakatsume, Akihiko Saito, Fumitake Gejyo

Internal Medicine 40 ( 9 ) 867 - 872 2001

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

Objective In order to explore the possibility that genetic predisposition to dysfunction of mucosal immunity and the IgA processing pathway plays a role in the pathogenesis of mesangial IgA1 deposition in IgAN, we examined the possible association of the gene polymorphism of plgR in the patients with and without IgAN. Subjects and Methods Genomic DNA of peripheral blood cells was isolated from 372 individuals including 172 histologically confirmed IgAN patients. Segments of the pIgR gene were PCR amplified and restriction fragment length polymorphism was determined as Al and A2 with and without Pvu II site, respectively. Results The pIgR genotype distribution was significantly different between the patients with IgAN and those without IgAN. Allele frequency of A2 was higher in IgAN than in other renal diseases (Al and A2
0.516 and 0.484 in IgAN, 0.641 and 0.359 in others, X2=9.84, P=0.0017, Odds ratio=1.71). Moreover, the subjects with A2A2 genotype were associated with a relatively low level of serum IgA only in the patients with IgAN but not in other renal diseases. The difference of allele frequencies was more remarkable in the patients with a serum IgA level of less than 300 mg/dl (Al and A2
0.439 and 0.561 in IgAN, 0.702 and 0.298 in others, X2=12.44, P=0.0004, Odds ratio=3.01). Conclusion This is the first demonstration of the pIgR gene polymorphisms in IgAN which are associated with its clinical phenotype. Gene polymorphisms of pIgR may be candidate genetic markers of susceptibility to IgAN. (Internal Medicine 40: 867-872, 2001). © 2001, The Japanese Society of Internal Medicine. All rights reserved.

DOI： 10.2169/internalmedicine.40.867

Expression of osteopontin in gentamicin-induced acute tubular necrosis and its recovery process

Yuansheng Xie, Shinichi Nishi, Seitaro Iguchi, Naofumi Imai, Minoru Sakatsume, Akihiko Saito, Mika Ikegame, Noriaki Iino, Hisaki Shimada, Mitsuhiro Ueno, H. Kawashima, M. Arakawa, Fumitake Gejyo

Kidney International 59 ( 3 ) 959 - 974 2001

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

Background. There is controversy regarding the exact localization and roles of osteopontin (OPN), a multipotential chemokine, in renal injury. There is little information on the expression and role of OPN in gentamicin-induced acute tubular necrosis (ATN) and its recovery process. Methods. A severe ATN model was made using male Wistar rats by injecting gentamicin (150 mg/kg/day) for five days and limiting the provision of water. The expression and localization of OPN mRNA and protein, ED1 as a macrophage marker, proliferating cellular nuclear antigen (PCNA), CD44 as an OPN receptor, megalin as a proximal tubule marker, and their relationships to each other were examined from the early tubular necrotic period to the late recovery period by Northern blotting, in situ hybridization, and double immunohistochemical staining. Results. In the gentamicin group, OPN mRNA and protein were expressed in only the PCNA-positive proliferating cortical distal tubules, not in the necrotic proximal tubules, until day 6 after the first administration, but were found markedly in PCNA-positive regenerative proximal and distal tubules on days 10, 15, and 30. The localization of PCNA-positive cells was almost always accompanied with the up-regulated expression of OPN using quantitative analysis (P &lt
0.01). CD44 expression was markedly up-regulated in the renal cortical tubular epithelium from days 6 to 30. In the control group, no expression of OPN and CD44 in the cortical area was found throughout the experimental period. Conclusions. These results suggested that OPN is related to the proliferation and regeneration of tubular epithelial cells after tubular damage.

DOI： 10.1046/j.1523-1755.2001.059003959.x

IgA腎症におけるpIgR,TGF-β1,MCP-1及びRAA系遺伝子多型の検討

後藤眞, 成田一衛, 斉藤徳子, 山崎肇, 坂爪実, 島田久基, 斉藤亮彦, 西慎一, 上野光博, 下条文武

日本内科学会雑誌 89 ( 臨増 ) 144 - 144 2000.2

The prevention of glomerulosclerosis in rats using traditional Chinese medicine, Sairei-to

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

Nephrology 5 ( 1-2 ) 83 - 89 2000

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

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&lt
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

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

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

Nephrology Dialysis Transplantation 15 ( 6 ) 799 - 810 2000

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

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

Down-regulation of interferon-γ signaling by gene transfer of Stat1 mutant in mesangial cells

Minoru Sakatsume, Ichiei Narita, Hajime Yamazaki, Akihiko Saito, Yoichi Nakagawa, Hideyuki Kuriyama, Ryozo Kuwano, Fumitake Gejyo, Masaaki Arakawa

Kidney International 57 ( 2 ) 455 - 463 2000

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

Background. Interferon-γ (IFN-γ) is secreted by T lymphocytes and natural killer (NK) cells in the cellular immunity-mediated inflammatory lesion, including endocapillary or extracapillary proliferative glomerulonephritis. It induces and/or enhances multiple histocompatibility complex (MHC) class I and II, intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthase (iNOS), and Fc receptor expression in renal resident cells, resulting in the initiation and promotion of inflammation. Recently, the signaling mechanism of IFN-γ has been investigated, and it appears that Stat1α is essential for signaling. We investigated Stat1α activation by IFN-γ in mesangial cells and attempted to regulate the signal transduction by gene transfer. Methods. Western blot with anti-Stat1 and antiphosphotyrosine after immunoprecipitation of Stat1 and Northern blot for detection of Stat1 mRNA were performed. The dominant negative form of Flag- tagged Stat1 was expressed in cultured rat mesangial cells. Flag was immunostained in transfectants, and luciferase reporter assay was carried out to measure the transcriptional activity of Stat1α. The expression of IFN-γ- inducible genes such as MHC class II (Ia-Aα) and MHC class II transactivator (CIITA) was detected by reverse transcriptase-polymerase chain reaction (RT- PCR). Results. Stat1α was tyrosine phosphorylated and activated by IFN-γ in mesangial cells, and the mRNA and protein level of Stat1α increased upon stimulation by IFN-γ. Overexpression of Stat1-mutant lacking 35 C-terminal amino acids strongly suppressed the IFN-γ-induced signal transduction and inhibited the expression of MHC class II and CIITA genes in mesangial cells. Conclusions. Stat1α is a critical molecule in the signal transduction of IFN-γ in mesangial cells. The inhibition of an endogenous function of Stat1α by gene transfer of the Stat1 mutant may be a new method to reduce the inflammatory effects of IFN-γ in localized inflammation of the kidney.

DOI： 10.1046/j.1523-1755.2000.00865.x

Ultrastructural localization of megalin in the rat cochlear duct

K Mizuta, A Saito, T Watanabe, M Nagura, M Arakawa, F Shimizu, T Hoshino

HEARING RESEARCH 129 ( 1-2 ) 83 - 91 1999.3

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

Megalin is an endocytic receptor predominantly expressed in the kidney proximal tubule cells. In the present study, localization of megalin was examined using a post-embedding immunogold method in the rat cochlear duct. Marginal cells of the stria vascularis were labeled on the apical surface, but not on the basolateral surface. This localization pattern resembles kidney proximal tubule cells. Immunoreactivity was also detected on various other cells, including epithelial cells of the spiral prominence and epithelial cells of Reissner's membrane. In contrast, virtually no gold particles were seen on intermediate cells and basal cells of the stria vascularis, mesothelial cells of Reissner's membrane or fibrocytes in the lateral wall. Also unlabeled were cells in the tympanic wall of the cochlear duct, including sensory cells and supporting cells of the organ of Corti. The present findings show the involvement of megalin in endocytosis of marginal cells and are suggestive of different uptake mechanisms for aminoglycosides in the kidney proximal tubule cells and in the cochlear sensory cells. (C) 1999 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0378-5955(98)00221-4

All four putative ligand-binding domains in megalin contain pathogenic epitopes capable of inducing passive Heymann nephritis

H Yamazaki, R Ullrich, M Exner, A Saito, RA Orlando, D Kerjaschki, MG Farquhar

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 9 ( 9 ) 1638 - 1644 1998.9

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

Megalin (gp330) is the main target antigen involved in the induction of Heymann nephritis (HN), a rat model of human membranous nephropathy. Its large extracellular region contains four putative ligand-binding domains separated by spacer regions. Previously, it was reported that the second ligand-binding domain (LBD II) of megalin is involved in the pathogenesis of passive HN because it is capable of binding antibodies in vivo and initiating formation of immune deposits (ID). This study explores the possibility that pathogenic epitopes might also be present in the other putative ligand-binding domains. Recombinant fragments of ligand-binding domains (LBD)I through IV expressed in a baculovirus system were used to generate polyclonal domain-specific antibodies. Antibodies raised against each of the recombinant megalin fragments reacted preferentially with its respective antigen and with whole megalin by immunoblotting. Each of the antibodies also gave a characteristic brush-border staining for megalin by indirect immunofluorescence on rat kidney. When rats were injected with the domain-specific antibodies to test their ability to produce passive HN, glomerular ID were present in kidneys of all injected animals. The staining pattern in glomeruli of rats injected with LED I, III, or IV was similar to that obtained with antibodies to LED II. It is concluded that passive HN can be induced with antibodies against LED I, III, and IV, as well as LED II, and that each of the ligand-binding domains contains a pathogenic epitope. These findings provide further evidence for the multiple epitope model of HN.

Identification of genes specifically expressed in chronic and progressive glomerulosclerosis Reviewed

Narita, I, H Nakayama, S Goto, T Takeda, M Sakatsume, A Saito, Y Nakagawa, M Arakawa

KIDNEY INTERNATIONAL S215 - S217 1997.12

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

To identify genes expressed predominantly in the kidney with chronic and progressive glomerulosclerosis but not in acute and transient form of glomerulonephritis, we induced progressive glomerulosclerosis in rats by applying unilateral nephrectomy prior to injection of monoclonal anti-Thy1.1 antibody (OX-7), which cause acute and transient glomerulonephritis with single injection. In rats with nephrectomy and OX-7 injection (Nx group), proteinuria increased with time and mesangial expansion accompanied with interstitial fibrosis was recognized, whereas transient proteinuria and mesangiolysis followed by mesangial hypercellularity were seen in rats with sham operation and OX-7 injection (Sham group). Four weeks after the induction of glomerulonephritis, mRNAs were isolated from kidney cortex of both groups and used for cDNA synthesis. By subtraction hybridization of cDNAs from Nx with excess amount of those from Sham, we isolated and sequenced several genes expressed specifically in the Nx group. These included genes, which contain identical sequences with serine protease inhibitors, cytokine receptors, osteopontin as well as genes with unknown function. These genes may play important roles in the process which promotes acute glomerular damage advance to chronic and progressive glomerulosclerosis.

Identification of the second cluster of ligand-binding repeats in megalin as a site for receptor-ligand interactions

RA Orlando, M Exner, RP Czekay, H Yamazaki, A Saito, R Ullrich, D Kerjaschki, MG Farquhar

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 94 ( 6 ) 2368 - 2373 1997.3

Language：English Publishing type：Research paper (scientific journal) Publisher：NATL ACAD SCIENCES

Megalin is a large cell surface receptor that mediates the binding and internalization of a number of structurally and functionally distinct ligands from the lipoprotein and protease:protease inhibitor families, To begin to address how megalin is able to bind ligands with unique structurally properties, we have mapped a binding site for apolipoprotein E (apoE)-beta very low density lipoprotein (beta VLDL), lipoprotein lipase, aprotinin, lactoferrin, and the receptor-associated protein (RAP) within the primary sequence of the receptor, RAP is known to inhibit the binding of all ligands to megalin, We identified a ligand-binding site on megalin by raising mAb against purified megalin, selected for a mAb whose binding to megalin is inhibited by RAP, and mapped the epitope for this mAb, mAb AC10 inhibited the binding of apoE-beta VLDL, lipoprotein Lipase, aprotinin, and lactoferrin to megalin in a concentration-dependent manner. When cDNA fragments encoding the four cysteine-rich ligand-binding repeats in megalin were expressed in a baculovirus system and immunoblotted with AC10, it recognized only the second cluster of ligand-binding repeats. The location of the epitope recognized by mAb AC10 within this domain was pinpointed to amino acids 1111-1210. From these studies we conclude that the binding of apoE-beta VLDL, lactoferrin, aprotinin, lipoprotein lipase, and RAP to megalin is either competitively or sterically inhibited by mAb AC10 suggesting that these ligands bind to the same or closely overlapping sites within the second cluster of ligand-binding repeats.

DOI： 10.1073/pnas.94.6.2368

Identification of genes specifically expressed in chronic and progressive glomerulosclerosis

I. Narita, H. Nakayama, S. Goto, T. Takeda, M. Sakatsume, A. Saito, Y. Nakagawa, M. Arakawa

Kidney International, Supplement 51 ( 63 ) S215 - S217 1997

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

To identify genes expressed predominantly in the kidney with chronic and progressive glomerulosclerosis but not in acute and transient form of glomerulonephritis, we induced progressive glomerulosclerosis in rats by applying unilateral nephrectomy prior to injection of monoclonal anti-Thy1.1 antibody (OX-7), which cause acute and transient glomerulonephritis with single injection. In rats with nephrectomy and OX-7 injection (Nx group), proteinuria increased with time and mesangial expansion accompanied with interstitial fibrosis was recognized, whereas transient proteinuria and mesangiolysis followed by mesangial hypercellularity were seen in rats with sham operation and OX-7 injection (Sham group). Four weeks after the induction of glomerulonephritis, mRNAs were isolated from kidney cortex of both groups and used for cDNA synthesis. By subtraction hybridization of cDNAs from Nx with excess amount of those from Sham, we isolated and sequenced several genes expressed specifically in the Nx group. These included genes, which contain identical sequences with serine protease inhibitors, cytokine receptors, osteopontin as well as genes with unknown function. These genes may play important roles in the process which promotes acute glomerular damage advance to chronic and progressive glomerulosclerosis.

Molecular Genetics of Renal Diseases

Akihiko Saito, Hajime Yamazaki, Yoichi Nakagawa, Masaaki Arakawa

Internal Medicine 36 ( 2 ) 81 - 86 1997

Language：English Publishing type：Research paper (scientific journal) Publisher：Japanese Society of Internal Medicine

Recent progress in molecular genetics has strongly influenced nephrology. Gene mutations have been identified which cause various monogenic hereditary renal diseases including Alport syndrome, autosomal dominant polycystic kidney disease, and tubular transporter disorders. The data obtained will be useful not only to develop new methods of diagnosis and treatment of such particular diseases but also to expand the knowledge of renal physiology and pathophysiology. In addition, genetic factors have been investigated which are involved in the development and progression of more common renal diseases such as IgA nephropathy and diabetic nephropathy. There have also been great advances in molecular studies of experimental renal diseases such as Heymann nephritis and in the use of transgenic and knockout mice. In this review we focused on the important achievements made recently in the field of molecular nephrology.

DOI： 10.2169/internalmedicine.36.81

Mapping rat megalin: The second cluster of ligand binding repeats contains a 46-amino acid pathogenic epitope involved in the formation of immune deposits in Heymann nephritis

A Saito, H Yamazaki, K Rader, A Nakatani, R Ullrich, D Kerjaschki, RA Orlando, MG Farquhar

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 93 ( 16 ) 8601 - 8605 1996.8

Language：English Publishing type：Research paper (scientific journal) Publisher：NATL ACAD SCIENCES

Megalin (gp330), an epithelial endocytic receptor, is a major target antigen of Heymann nephritis (HN), an autoimmune disease in rats. To elucidate the mechanisms of HN, we have mapped a pathogenic epitope in megalin that binds anti-megalin antibodies. We focused our attention on four clusters of cysteine-rich, low density lipoprotein receptor (LDLR) ligand binding repeats in the extracellular domain of megalin because they represent putative ligand binding regions and therefore would be expected to be exposed in vivo and to be able to bind circulating antibodies. Rat megalin cDNA fragments I through IV encoding the first through fourth clusters of ligand-binding repeats, respectively, were expressed in a baculovirus system. All four expression products were detected by immunoblotting with two antisera capable of inducing passive HN (pHN). When antibodies eluted from glomeruli of rats with pHN were used for immunoblotting, only the expression product encoded by fragment II was detected. This indicates that the second cluster of LDLR ligand binding repeats is directly involved in binding anti-megalin antibodies and in the induction of pHN. To narrow the major epitope in this domain, fragment II was used to prepare proteins sequentially truncated from the C- and N-terminal ends by in vitro translation. Analysis of the truncated translation products by immunoprecipitation with anti-megalin IgG revealed that the fifth ligand-binding repeat (amino acids 1160-1205) contains the major epitope recognized. This suggests that a 46-amino acid sequence in the second cluster of LDLR ligand binding repeats contains a major pathogenic epitope that plays a key role in pHN. Identification of this epitope will facilitate studies on the pathogenesis of HN.

DOI： 10.1073/pnas.93.16.8601

THE HEYMANN NEPHRITIS ANTIGENIC COMPLEX - MEGALIN (GP330) AND RAP

MG FARQUHAR, A SAITO, D KERJASCHKI, RA ORLANDO

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 6 ( 1 ) 35 - 47 1995.7

Language：English Publisher：WILLIAMS & WILKINS

Heymann nephritis (HN) has been extensively studied as a model of human membranous nephropathy since it was first described by Heymann in 1959, HN was induced in active form by the immunization of rats with antigens derived from the proximal tubule brush border, resulting in subepithelial glomerular immune deposits. HN was also induced passively by the injection of antibrush border antibodies into normal rats, A breakthrough in the understanding of the pathogenesis of HN was made in the 1970s, when it was established that the disease was due to the binding of circulating antibodies to glomerular components. This in turn led to a search to identify the endogenous antigen(s), In 1982, gp330 (now called megalin), a glycoprotein located in clathrin-coated pits of glomerular and proximal tubular epithelia, was identified as a target antigen, In 1990, a second protein (44 kd), now known as RAP (for receptor associated protein), that binds to megalin was also shown to be a target antigen. Both molecules have been cloned and sequenced, and their role in normal epithelial cells has been explored. It has come to light that megalin (gp330) is a member of the low-density lipoprotein receptor gene family and functions as a multiligand receptor for the uptake of a variety of macromolecules (plasminogen, protease: protease inhibitor complexes, apolipoprotein E-enriched very low-density lipoproteins, lactoferrin, among others). RAP associates with megalin and appears to function as a chaperone assisting in the folding of megalin in the endoplasmic reticulum and its transport to the cell surface. This review considers what is now known about the structure, function, and trafficking of megalin and RAP and the role of these two molecules in the pathogenesis of HN.

No linkage to the COL4A3 gene locus in Japanese thin basement membrane disease families

Hajime YAMAZAKI, Yoichi NAKAGAWA, Akihiko SAITO, Shinichi NISHI, Shiminoru SAKATSUME, Tetsuro TAKEDA, Yuichiro MARUYAMA, Sojiro OGINO, Shiro MARUYAMA, Toshio MOCHIZUKI, Stephen T REEDERS, Masaaki ARAKAWA

Nephrology 1 ( 4 ) 315 - 321 1995

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

Summary: Patients with thin basement membrane disease (TBMD) exhibit persistent haematuria with a diffuse thinning of the glomerular basement membrane (GBM), especially of the lamina densa. It appears to be an autosomal dominant trait. It has been reported that the Goodpasture epitope, which is located in the non‐collagenous domain of type IV collagen α 3 chain, may be reduced in patients with TBMD. We speculated that the candidate gene for TBMD could be the type IV collagen α 3 chain gene (COL4A3), which is present closely to type IV collagen α 4 chain gene (COL4A4) on chromosome 2q35–37. We conducted a linkage analysis to investigate the relationship between familial TBMD and COL4A3 gene, using COL4A3 cDNA polymorphism and a (CA)n microsatellite marker located in the COL4A3 gene. We examined 32 individuals from four Japanese families with TBMD. There were no associations between the patients with haematuria and certain alleles of the two markers in the pedigrees of three families. It has been reported that type IV collagen α 1 chain gene (COL4A1) and α 2 chain gene (COL4A2) are not involved in TBMD, and that α 5 chain gene (COL4A5) and a 6 chain gene (COL4A6) map to chromosome X. In conclusion, our findings suggested that familial TBMD is not caused by the genetic abnormalities of type IV collagen genes isolated thus far. Copyright © 1995, Wiley Blackwell. All rights reserved

DOI： 10.1111/j.1440-1797.1995.tb00046.x

Mesangial cell-matrix interactions: Modulation of matrix expression in mesangial cells by extracellular matrices

M. Sakatsume, A. Saito, T. Takeda, H. Yamazaki, I. Narita, S. I. Nishi, Y. Nakagawa, M. Arakawa

Experimental Nephrology 3 ( 6 ) 362 - 368 1995

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

Aberrant extracellular matrices (ECMs) observed in mesangial areas during the progression of glomerulonephropathy could be produced by mesangial cells (MCs), interacting with altered ECM components, under the influence of various mediators. We studied the interaction between MCs and ECMs, especially focusing on matrix expression of MCs in contact with ECMs. We analyzed mRNA expression and protein synthesis of ECM components by cultured rat MCs which were plated on different types of ECMs, basement membrane-type matrix (BMM), type IV collagen and type I collagen. The level of mRNA for ECM components (α1 type I collagen, α1 type IV collagen, fribronectin and laminin B2) increased in MCs on the two types of collagen compared with BMM. The relative increase of α1 type I collagen mRNA expression in MCs on type I collagen was greater than that of α1 type IV collagen expression. At the protein level, type I collagen production by MCs on coated type I collagen showed a 2.2-fold increase relative to that on BMM. MCs on type IV collagen produced it at an intermediate level. The phenomena were not associated with differences of proliferative activity, but were accompanied by minor morphological differences. These results indicate that ECMs can modulate the phenotype of MCs in terms of matrix expression and suggest that MC-matrix interaction may in part contribute to abnormal ECM accumulation in diseased glomeruli.

COMPLETE CLONING AND SEQUENCING OF RAT GP330 MEGALIN, A DISTINCTIVE MEMBER OF THE LOW-DENSITY-LIPOPROTEIN RECEPTOR GENE FAMILY

A SAITO, S PIETROMONACO, AKC LOO, MG FARQUHAR

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 91 ( 21 ) 9725 - 9729 1994.10

Language：English Publishing type：Research paper (scientific journal) Publisher：NATL ACAD SCIENCES

We completed the cDNA cloning and sequencing of gp330, the major kidney glomerular antigen for rat Heymann nephritis. The deduced 4660-aa sequence, expected to constitute a mature protein of M(r) 516,715, consists of a probable N-terminal signal peptide sequence (25 aa), an extracellular region (4400 aa), a single transmembrane domain (22 aa), and a C-terminal cytoplasmic tail (213 aa), The extracellular region contains three types of cysteine-rich repeats characteristic of the low density lipoprotein receptor (LDLR) gene family-36 LDLR ligand-binding repeats forming four clusters of putative ligand-binding domains, 16 growth factor repeats separated by 8 YWTD spacer regions, and 1 C-terminal epidermal growth factor repeat. The cytoplasmic tail contains two copies of the (FX)NPXY moth, which represents a signal for coated pit-mediated internalization and an additional similar moth. The overall structure of gp330 is similar to that of the LDLR-related protein (LRP)/alpha(2)-macroglobulin receptor and shows even greater similarity to the Caenorhabditis elegans protein, reported as a homologue of LRP. However, gp330 differs from these proteins in (i) the cysteine-rich repeat arrangements found in the extreme extracellular N- and C-terminal regions, (ii) the distribution pattern of cysteine residues in the YWTD spacer regions, (iii) the location of the RX(K/R)R consensus recognition sequence of furin, a precursor processing endoprotease, and (iv) the length and structure of the cytoplasmic tail. We suggest the name megalin (from Greek mega) for gp330, the largest plasma membrane protein identified so far in vertebrates. The cloned cDNA will be useful for studies on the physiological functions of gp330/megalin and for determining its role in Heymann nephritis.

DOI： 10.1073/pnas.91.21.9725

A DELETION MUTATION IN THE 3' END OF THE ALPHA-5(IV) COLLAGEN GENE IN JUVENILE-ONSET ALPORT SYNDROME

A SAITO, M SAKATSUME, H YAMAZAKI, F OGATA, Y HIRASAWA, M ARAKAWA

JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY 4 ( 9 ) 1649 - 1653 1994.3

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

Alport syndrome is a hereditary progressive glomerular basement membrane disorder in which juvenile-or adult-onset renal failure is often accompanied by sensorineural deafness and ocular abnormalities. Recently, mutations have been found in the type IV collagen alpha 5 chain gene in patients with X-linked Alport syndrome. This study searched for gene mutations in seven unrelated Japanese patients by the use of conventional Southern blot analysis with cDNA probes for the carboxyl-terminal noncollagenous domain that is encoded by exons 46 to 51. A deletion mutation was found in a patient who developed juvenile-onset (age 15) ESRD with typical ultrastructural glomerular basement membrane destruction and sensorineural hearing loss but no characteristic ocular abnormalities. His mother showed hematuria and proteinuria with nomal renal function, suggesting that she may be the heterozygous carrier. Exon-specific polymerase chain reaction amplified the coding sequence of exon 48 but not exons 49 to 51. Analysis with pulsed-field gel electrophoresis revealed that the deletion is approximately 10 kb in length and does not involve the CpG island, which is located in the 3' distal site of the gene. Identification of this novel deletion causing juvenile-type Alport syndrome would contribute to elucidating the mechanisms of renal failure progression in the syndrome.

Alternative splicing in the α5(IV) collagen gene in human kidney and skin tissues

Akihiko Saito, Minoru Sakatsume, Hajime Yamazaki, Masaaki Arakawa

The Japanese Journal of Nephrology 36 ( 1 ) 19 - 24 1994

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

Type IV collagen is a complex molecule composed of three α chains that can be of types αl(IV) to α5(IV). Each α chain contains an amino-terminal collagenous domain and a carboxyl-terminal noncol-lagenous (NC) domain consisting of 229 amino acids, which is involved in intermolecular cross-linking to construct the basement membrane network. Specific localization of α5(IV) collagen in glomerular basement membranes (GBM) in the kidney indicates its crucial role for that construction. In human kidney cortex and skin RNA, we found an alternative splicing transcript which skips exon 50 of the α5(IV) collagen gene using reverse transcription-polymerase chain reaction and direct sequencing of the PCR products. This alternative splicing introduces a stop codon at the first codon of exon 51, resulting in an mRNA encoding a protein lacking the 84 NC domain carboxylterminal amino acid residues encoded by exons 50 and 51. Recently, gene mutations affecting the α5(IV) collagen NC domain have been reported in patients with X-linked Alport syndrome who display GBM destruction and progressive renal failure, which are usually milder in female patients. Therefore, the alternative splicing, if its truncated products are dominantly expressed in glomeruli, may cause GBM impairment and renal failure progression. © 1994, Japanese Society of Nephrology. All rights reserved.

DOI： 10.14842/jpnjnephrol1959.36.19

Clinical Significance of Urinary Methylguanidine in Non-Dialyzed Patients with Chronic Renal Failure —Prediction of Progression Rate of Renal Dysfunction—

Yutaka Koda, Akihiko Saito, Katsunobu Shinohara, Ran Ei, Shigeru Miyazaki, Yasuko Yuasa, Shinji Sakai, Masashi Suzuki, Sachio Takahashi, Yoshihei Hirasawa, Motoo Nakajima

Japanese Journal of Nephrology 35 ( 3 ) 203 - 208 1993

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

To clarify a clinical significance of urinary methylguanidine (U-MG, dl) in non-dialyzed patients with chronic renal failure, we measured U-MG, urinry creatinine (U-Cr, mg/ dl) and serum creatinine (S-Cr, mg/dl), concurrently and continually in 36 out-patients whose S-Cr was over 4. 0 mg/dl. Fresh urine sample was obtained and U-MG was measured by an enzymic method. © 1993, Japanese Society of Nephrology. All rights reserved.

DOI： 10.14842/jpnjnephrol1959.35.203

Molecular genetics of Alport syndrome

H. Yamazaki, A. Saito, Y. Nakagawa, M. Arakawa

Nippon rinsho. Japanese journal of clinical medicine 50 3021 - 3026 1992.12

Alport syndrome is a hereditary glomerulonephritis in which progressive loss of kidney function is often accompanied by sensorineural deafness. Ultrastructural studies in glomerular basement membranes (GBM) of Alport syndrome patients implicate an altered GBM protein structure as the cause of nephritis. The product of COL4A5, the alpha 5 (IV) collagen chain, is a specific component of GBM of the kidney. Various mutations in the COL4A5 gene have been identified in X-linked dominant Alport syndrome, and these aberrations of the alpha 5 (IV) can account for at least a part of X-linked Alport syndrome.

LONG-DISTANCE RESTRICTION MAPPING OF THE PROXIMAL LONG ARM OF HUMAN CHROMOSOME-21 WITH NOT-I LINKING CLONES

H ICHIKAWA, K SHIMIZU, A SAITO, D WANG, R OLIVA, H KOBAYASHI, Y KANEKO, H MIYOSHI, CL SMITH, CR CANTOR, M OHKI

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 89 ( 1 ) 23 - 27 1992.1

Language：English Publishing type：Research paper (scientific journal) Publisher：NATL ACAD PRESS

Human chromosome 21 is the smallest of the 22 autosomes and 2 sex chromosomes. Hybridization of the human repetitive sequence Alu to pulsed-field gel-fractionated Not I-digested genomic DNA from a human-mouse hybrid cell line containing chromosome 21 as the sole human component identified chromosome 21 Not I restriction fragments. A Not I restriction map of regions of the chromosome was constructed, by identifying neighboring Alu bands with Not I linking clones. This approach simplifies the task of physical mapping and avoids ambiguities in Not I fragment assignments that arise from gel-to-gel mobility variations. A contiguous map was constructed with six Not I linking clones that covers at least the proximal one-third of the long arm of chromosome 21 and spans 20 megabases. A more detailed restriction map revealed 11 likely CpG islands in this region and localized 11 additional DNA markers.

DOI： 10.1073/pnas.89.1.23

Single dialysis system

T. Kawasaki, A. Saito, H. Yamazaki, H. Ichikawa

Nippon rinsho. Japanese journal of clinical medicine 49 Suppl 196 - 204 1991.12

Central dialysate supply system

T. Kawasaki, A. Saito, H. Yamazaki, H. Ichikawa

Nippon rinsho. Japanese journal of clinical medicine 49 Suppl 205 - 211 1991.12

Construction and characterization of a NotI linking library of human chromosome 21

Akihiko Saito, Jose P. Abad, Denan Wang, Misao Ohki, Charles R. Cantor, Cassandra L. Smith

Genomics 10 ( 3 ) 618 - 630 1991

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

Effective procedures have been developed for constructing NotI linking libraries starting from chromosome-specific genomic libraries. Fifteen different single copy and two rDNA NotI linking clones from human chromosome 21 were identified in two libraries. Their chromosomal origin was confirmed, and regional location established using hybrid cell panels. Hybridization experiments with these probes revealed pairs of genomic NotI fragments, each ranging in size from &lt
0.05 to 4.0 Mb. Many fragments displayed cell type variation. The total size of the NolI fragments detected in a human fibroblast cell line (GM6167) and mouse hybrid cell containing chromosome 21 as its only human component (WAV17) were approximately 32 and 34 Mb, respectively. If these fragments were all nonoverlapping, this would correspond to about 70% of the 50-Mb content estimated for the whole chromosome. The linking clones will be enormously useful in the subsequent construction of a NotI restriction map of this chromosome. Characterization of these clones indicates the presence of numerous additional sites for other enzymes that recognize sequences containing CpG. Thus most NotI linking clones appear to derive from CpG islands and probably identify the 5′ end of genes. © 1991.

DOI： 10.1016/0888-7543(91)90444-J

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