2024/10/12 更新

写真a

ウチモト カエデ
内許 玉楓
UCHIMOTO KAEDE
所属
教育研究院 医歯学系 医学系列 助教
医歯学総合研究科 腎研究センター 助教
職名
助教
通称等の別名
張 瑩
外部リンク

学位

  • 医学博士 ( 2007年3月   新潟大学 )

研究キーワード

  • 腎臓病学

  • 実験病理学

研究分野

  • ライフサイエンス / 腎臓内科学

経歴(researchmap)

  • 新潟大学   医歯学総合研究科 腎研究センター   助教

    2016年4月 - 現在

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  • 新潟大学   医歯学総合研究科 腎研究施設   助教

    2015年9月 - 2016年3月

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経歴

  • 新潟大学   医歯学総合研究科 腎研究センター   助教

    2016年4月 - 現在

  • 新潟大学   医歯学総合研究科 腎研究センター   助教

    2015年9月 - 2016年3月

学歴

  • 新潟大学   医学系研究科

    2003年4月 - 2007年3月

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    国名: 日本国

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  • ハルビン医科大学   医学部

    - 2002年8月

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    国名: 中華人民共和国

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取得資格

  • アイエルツ

 

論文

  • 14-3-3 Proteins stabilize actin and vimentin filaments to maintain processes in renal glomerular podocyte. 国際誌

    Hidenori Yasuda, Yoshiyasu Fukusumi, Ying Zhang, Hiroshi Kawachi

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology   37 ( 10 )   e23168   2023年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1096/fj.202300865R

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  • 微小変化型ネフローゼ症候群におけるポドサイト傷害

    河内 裕, 福住好恭, 内許玉楓

    腎臓内科   15 ( 6 )   644 - 651   2022年6月

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  • Th17 Cells Participate in Thy1.1 Glomerulonephritis Which Is Ameliorated by Tacrolimus. 査読 国際誌

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

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

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1159/000524111

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  • Tacrolimus ameliorates podocyte injury by restoring FK506 binding protein 12 (FKBP12) at actin cytoskeleton. 査読 国際誌

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

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology   35 ( 11 )   e21983   2021年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1096/fj.202101052R

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  • プライマリ―カルチャー樹立法

    内許玉楓, 河内裕

    腎と透析   91 ( 5 )   965 - 969   2021年11月

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  • Synbindin Downregulation Participates in Slit Diaphragm Dysfunction. 査読 国際誌

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

    American journal of nephrology   52 ( 8 )   1 - 10   2021年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1159/000517975

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  • Nephrin-Ephrin-B1-Na+/H+ Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury. 査読 国際誌

    Yoshiyasu Fukusumi, Hidenori Yasuda, Ying Zhang, Hiroshi Kawachi

    The American journal of pathology   191 ( 7 )   1209 - 1226   2021年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    DOI: 10.1016/j.ajpath.2021.04.004

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  • Xanthine oxidoreductase inhibitor topiroxostat ameliorates podocyte injury by inhibiting the reduction of nephrin and podoplanin. 査読

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

    Nefrologia   41 ( 5 )   539 - 547   2021年3月

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    Background
    Topiroxostat, an inhibitor of xanthine oxidoreductase (XOR) was shown to reduce urinary albumin excretion of hyperuricemic patients with chronic kidney disease. However, its pharmacological mechanism is not well understood. In this study, we examined the effects of topiroxostat on glomerular podocytes. Podocyte is characterized by foot process and a unique cell-cell junction slit diaphragm functioning as a final barrier to prevent proteinuria.

    Methods
    The effects of topiroxostat on the expressions of podocyte functional molecules were analysed in db/db mice, a diabetic nephropathy model, anti-nephrin antibody-induced rat podocyte injury model and cultured podocytes treated with adriamycin.

    Results
    Topiroxostat treatment ameliorated albuminuria in db/db mice. The expression of desmin, a podocyte injury marker was increased, and nephrin and podocin, key molecules of slit diaphragm, and podoplanin, an essential molecule in maintaining foot process were downregulated in db/db mice. Topiroxostat treatment prevented the alterations in the expressions of these molecules in db/db mice. XOR activity in kidney was increased in rats with anti-nephrin antibody-induced podocyte injury. Topiroxostat treatment reduced XOR activity and restored the decreased expression of nephrin, podocin and podoplanin in the podocyte injury. Furthermore, topiroxostat enhanced the expression of podoplanin in injured human cultured podocytes.

    Conclusions
    Podocyte injury was evident in db/db mice. Topiroxostat ameliorated albuminuria in diabetic nephropathy model by preventing podocyte injury. Increase of XOR activity in kidney contributes to development of podocyte injury caused by stimulation to slit diaphragm. Topiroxostat has an effect to stabilize slit diaphragm and foot processes by inhibiting the reduction of nephrin, podocin and podoplanin.

    DOI: 10.1016/j.nefroe.2021.11.007

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  • Par-6-ephrin-B1 interaction is regulated by nephrin mediated signal and is crucial in maintaining slit diaphragm of podocyte 査読 国際誌

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

    American Journal of Pathology   in press ( 2 )   333 - 346   2020年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.ajpath.2019.10.015

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  • Nephrin-binding Ephrin-B1 at the slit diaphragm controls podocyte function through the JNK pathway 査読 国際誌

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

    Journal of the American Society of Nephrology   29 ( 5 )   1462 - 1474   2018年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:American Society of Nephrology  

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

    DOI: 10.1681/ASN.2017090993

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  • Why are they missing? : Bioinformatics characterization of missing human proteins 査読

    Amr Elguoshy, Sameh Magdeldin, Bo Xu, Yoshitoshi Hirao, Ying Zhang, Naohiko Kinoshita, Yusuke Takisawa, Masaaki Nameta, Keiko Yamamoto, Ali El-Refy, Fawzy El-Fiky, Tadashi Yamamoto

    JOURNAL OF PROTEOMICS   149   7 - 14   2016年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    NeXtProt is a web-based protein knowledge platform that supports research on human proteins. NeXtProt (release 2015-04-28) lists 20,060 proteins, among them, 3373 canonical proteins (16.8%) lack credible experimental evidence at protein level (PE2:PE5). Therefore, they are considered as "missing proteins". A comprehensive bio-informatic workflow has been proposed to analyze these "missing" proteins. The aims of current study were to analyze physicochemical properties, existence and distribution of the tryptic cleavage sites, and to pinpoint the signature peptides of the missing proteins. Our findings showed that 23.7% of missing proteins were hydrophobic proteins possessing transmembrane domains (TMD). Also, forty missing entries generate tryptic peptides were either out of mass detection range (>30 aa) or mapped to different proteins (<9 aa). Additionally, 21% of missing entries didn't generate any unique tryptic peptides. In silico endopeptidase combination strategy increased the possibility of missing proteins identification. Coherently, using both mature protein database and signal peptidome database could be a promising option to identify some missing proteins by targeting their unique N-terminal tryptic peptide from mature protein database and or C-terminus tryptic peptide from signal peptidome database. In conclusion, Identification of missing protein requires additional consideration during sample preparation, extraction, digestion and data analysis to increase its incidence of identification. (C) 2016 Published by Elsevier B.V.

    DOI: 10.1016/j.jprot.2016.08.005

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  • レニン-アンジオテンシン-アルドステロン系(RAAS)阻害薬 査読

    河内裕, 福住好恭, 張瑩

    腎と透析   81 ( 1 )   2016年7月

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  • Comprehensive data analysis of human ureter proteome. 査読

    Magdeldin S, Hirao Y, El Guoshy A, Xu B, Zhang Y, Fujinaka H, Yamamoto K, Yates JR, Yamamoto T

    Data Brief.   6   853 - 857   2016年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Avian Podocytes, Which Lack Nephrin, Use Adherens Junction Proteins at Intercellular Junctions 査読 国際誌

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

    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY   64 ( 1 )   67 - 76   2016年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SAGE PUBLICATIONS LTD  

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

    DOI: 10.1369/0022155415611708

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  • A proteomic glimpse into human ureter proteome 査読

    Sameh Magdeldin, Yoshitoshi Hirao, Amr Elguoshy, Bo Xu, Ying Zhang, Hidehiko Fujinaka, Keiko Yamamoto, John R. Yates, Tadashi Yamamoto

    PROTEOMICS   16 ( 1 )   80 - 84   2016年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    Urine has evolved as one of the most important biofluids in clinical proteomics due to its noninvasive sampling and its stability. Yet, it is used in clinical diagnostics of several disorders by detecting changes in its components including urinary protein/polypeptide profile. Despite the fact that majority of proteins detected in urine are primarily originated from the urogenital (UG) tract, determining its precise source within the UG tract remains elusive. In this article, we performed a comprehensive analysis of ureter proteome to assemble the first unbiased ureter dataset. Next, we compared these data to urine, urinary exosome, and kidney mass spectrometric datasets. Our result concluded that among 2217 nonredundant ureter proteins, 751 protein candidates (33.8%) were detected in urine as urinary protein/polypeptide or exosomal protein. On the other hand, comparing ureter protein hits (48) that are not shown in corresponding databases to urinary bladder and prostate human protein atlas databases pinpointed 21 proteins that might be unique to ureter tissue. In conclusion, this finding offers future perspectives for possible identification of ureter disease-associated biomarkers such as ureter carcinoma. In addition, the ureter proteomic dataset published in this article will provide a valuable resource for researchers working in the field of urology and urine biomarker discovery. All MS data have been deposited in the ProteomeXchange with identifier PXD002620 ().

    DOI: 10.1002/pmic.201500214

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  • Datasets from label-free quantitative proteomic analysis of human glomeruli with sclerotic lesions 査読

    Ying Zhang, Bo Xu, Naohiko Kinoshita, Yutaka Yoshida, Masayuki Tasaki, Hidehiko Fujinaka, Sameh Magdeldin, Eishin Yaoita, Tadashi Yamamoto

    Data in Brief   4   180 - 185   2015年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier Inc.  

    Human glomeruli with intermediate (i-GS) and advanced (GS) sclerotic lesions as well as the normal control (Nor) were captured from laser microdissection, digested by trypsin and subjected to shotgun LC-MS/MS analysis (LTQ-Orbitrap XL). The label-free quantification was performed using the Normalized Spectral Index (SIN) to assess the relative molar concentration of each protein identified in a sample. All the experimental data are shown in this article. The data is associated to the research article submitted to Journal of Proteomics [1].

    DOI: 10.1016/j.dib.2015.05.013

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  • Complementary Protein and Peptide OFFGEL Fractionation for High-Throughput Proteomic Analysis 査読

    Sameh Magdedin, Amr Elguoshy, Yutaka Yoshida, Yoshitoshi Hirao, Bo Xu, Ying Zhang, Keiko Yamamoto, Hiroki Takimoto, Hidehiko Fujinaka, Naohiko Kinoshita, Tadashi Yamamoto

    ANALYTICAL CHEMISTRY   87 ( 16 )   8481 - 8488   2015年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER CHEMICAL SOC  

    OFFGEL fractionation of mouse kidney protein lysate and its tryptic peptide digest has been examined in this study for better understanding the differences between protein and peptide fractionation methods and attaining maximum recruitment of this modern methodology for in-depth proteomic analysis. With the same initial protein/peptide load for both fractionation methods, protein OFFGEL fractionation showed a preponderance in terms of protein identification, fractionation efficiency, and focusing resolution, while peptide OFF GEL was better in recovery, number of peptide matches, and protein coverage. This result suggests that the protein fractionation method is more suitable for shotgun analysis while peptide fractionation suits well quantitative peptide analysis [isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tags (TMT)]. Taken together, utilization of the advantages of both fractionation approaches could be attained by coupling both methods to be applied on complex biological tissue. A typical result is shown in this article by identification of 8262 confident proteins of whole mouse kidney under stringent condition. We therefore fractionation as an effective and efficient addition to both label-free and quantitative label proteomics workflow.

    DOI: 10.1021/acs.analchem.5b01911

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  • Unrestricted modification search reveals lysine methylation as major modification induced by tissue formalin fixation and paraffin embedding. 査読

    Zhang Y, Muller M, Xu B, Yoshida Y, Horlacher O, Nikitin F, Garessus S, Magdeldin S, Kinoshita N, Fujinaka H, Yaoita E, Hasegawa M, Lisacek F, Yamamoto T

    Proteomics.   15 ( 15 )   2568 - 2579   2015年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1002/pmic.201400454

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  • Label-free quantitative proteomic analysis reveals strong involvement of complement alternative and terminal pathways in human glomerular sclerotic lesions 査読

    Ying Zhang, Bo Xu, Naohiko Kinoshita, Yutaka Yoshida, Masayuki Tasaki, Hidehiko Fujinaka, Sameh Magdeldin, Eishin Yaoita, Tadashi Yamamoto

    JOURNAL OF PROTEOMICS   123   89 - 100   2015年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    Since glomerular sclerosis frequently accompanies various glomerular diseases at the end stages, it is challenging to differentiate ubiquitous biological processes underlying this pathology from those critically involved in specific diseases. Furthermore, in-depth proteomic profile of human glomerular sclerosis remains limited. In this study, human glomeruli with intermediate (i-GS) and advanced (GS) sclerotic lesions, which were excluded from specific renal diseases and assumed to be aging-related, were laser captured from macroscopically normal cortex distant from urological carcinoma, and subjected to label-free quantitative proteomic analysis. We explicate an evident increase of membrane attack complex in i-GS and GS with an up-going tendency, which is accompanied by increasing of inhibitory regulators of alternative and terminal pathways. GO annotation and IPA pathway analysis agree to these results. Proteomic findings are validated by immunohistochemical studies which indicate that alternative and terminal pathways are positively involved in the glomerular sclerosis seen in distinct renal diseases. Furthermore, proteomic analysis also demonstrates remarkable increases of complement factor B in GS and TGF-beta 1 in both GS and i-GS. Identification of complement factor B implicates that on-site activation of alternative pathway may occur in injured glomeruli and stepwise increase of TGF-beta 1 suggests its contribution to the progression of glomerulosclerosis.
    Biological significance
    This study provides in-depth quantitative proteornic profiles of human glomeruli with intermediate and advanced sclerotic lesions. It reveals that the over-expression of alternative and terminal pathway components is significantly involved in human glomerulosclerosis seen in distinct renal diseases. Proteomic identification of the increased TGF-beta 1 provides supporting evidence for the role of podocyte apoptosis leading to human glomerulosclerosis. (C) 2015 Elsevier B.V. All rights reserved.

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  • The neXtProt knowledgebase on human proteins: current status 査読

    Pascale Gaudet, Pierre-Andre Michel, Monique Zahn-Zabal, Isabelle Cusin, Paula D. Duek, Olivier Evalet, Alain Gateau, Anne Gleizes, Mario Pereira, Daniel Teixeira, Ying Zhang, Lydie Lane, Amos Bairoch

    NUCLEIC ACIDS RESEARCH   43 ( D1 )   D764 - D770   2015年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    neXtProt (http://www.nextprot.org) is a human protein-centric knowledgebase developed at the SIB Swiss Institute of Bioinformatics. Focused solely on human proteins, neXtProt aims to provide a state of the art resource for the representation of human biology by capturing a wide range of data, precise annotations, fully traceable data provenance and a web interface which enables researchers to find and view information in a comprehensive manner. Since the introductory neXtProt publication, significant advances have been made on three main aspects: the representation of proteomics data, an extended representation of human variants and the development of an advanced search capability built around semantic technologies. These changes are presented in the current neXtProt update.

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

    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年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER  

    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.
    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.
    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.
    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.

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  • Heparin increasing podocyte-specific gene expressions 査読

    Eishin Yaoita, Yutaka Yoshida, Masaaki Nameta, Ying Zhang, Hidehiko Fujinaka, Sameh Magdeldin, Bo Xu, Tadashi Yamamoto

    NEPHROLOGY   19 ( 4 )   195 - 201   2014年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    AimHeparin, a highly sulfated glycosaminoglycan, has been shown to have a renoprotective effect on renal diseases, but its mechanisms remain to be elucidated. In this study, we examined the effect of heparin on podocytes by using primary cultured podocytes positive for podocyte-specific markers including podocin and podocalyxin.
    MethodsPodocytes were cultured from highly purified glomeruli isolated by the method with renal perfusion with magnetic beads and digestion of collagenase. Podocyte-specific gene expressions and proteins were examined by real-time polymerase chain reaction (PCR), Western blotting and immunofluorescence microscopy.
    ResultsReal-time PCR showed that addition of heparin to the culture media significantly upregulated most of the podocyte-specific genes in a dose-dependent and time-dependent manner. Western blotting showed a marked increase in protein levels of nephrin and podocin. Podocin localization at cell-cell contact sites became conspicuous in the presence of heparin. The effect of heparin was observed even in culture media deprived of bovine foetal serum. Heparan sulfate, less sulfated than heparin, and hyaluronan did not show such effects, but sulfated dextran did markedly.
    ConclusionHeparin acts on cultured podocytes to increase podocyte-specific gene expressions. A high degree of sulfation is crucial for the effect of heparin.
    Summary at a Glance This study characterises the rapid downregulation of podocyte specific genes in primary cultures of podocytes isolated from rat glomeruli. However, the addition of heparin sulphate promoted nephrin and podocin expression in cultured podocytes, which may explain the protective effect of heparin sulphate in models of proteinuria and glomerulosclerosis.

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  • Deep Proteome Mapping of Mouse Kidney Based on OFFGel Prefractionation Reveals Remarkable Protein Post- Translational Modifications 査読

    Sameh Magdeldin, Keiko Yamamoto, Yutaka Yoshida, Bo Xu, Ying Zhang, Hidehiko Fujinaka, Eishin Yaoita, John R. Yates, Tadashi Yamamoto

    JOURNAL OF PROTEOME RESEARCH   13 ( 3 )   1636 - 1646   2014年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER CHEMICAL SOC  

    Performing a comprehensive nonbiased proteome analysis is an extraordinary challenge due to sample complexity and wide dynamic range, especially in eukaryotic tissues. Thus, prefractionation steps conducted prior to mass spectrometric analysis are critically important to reduce complex biological matrices and allow in-depth analysis. Here we demonstrated the use of OFFGel prefractionation to identify more low abundant and hydrophobic proteins than in a nonfractionated sample. Moreover, OFFGel prefractionation of a kidney protein sample was able to unveil protein functional relevance by detecting PTMs, especially when prefractionation was augmented with a targeted enrichment strategy such as TiO2 phospho-enrichment. The OFFGel-TiO2 combination used in this study was comparable to other global phosphoproteomics approaches (SCX-TiO2 ERLIC-TiO2, or HILIC-TiO2). The detailed mouse kidney proteome with the phosphopeptide enrichment presented here serves as a useful platform for a better understanding of how the renal protein modification machinery works and, ultimately, will contribute to our understanding of pathological processes as well as normal physiological renal functions.

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  • 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) 査読

    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年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Springer-Verlag Tokyo  

    DOI: 10.1007/s10157-013-0846-0

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  • Basics and recent advances of two dimensional- polyacrylamide gel electrophoresis.

    Magdeldin Sameh, Enany Shymaa, Yoshida Yutaka, Xu Bo, Zhang Ying, Zureena Zam, Lokamani Ilambarthi, Yaoita Eishin, Yamamoto Tadashi

    Clin Proteomics   11 ( 1 )   16 - 16   2014年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Gel- based proteomics is one of the most versatile methods for fractionating protein complexes. Among these methods, two dimensional- polyacrylamide gel electrophoresis (2-DE) represents a mainstay orthogonal approach, which is popularly used to simultaneously fractionate, identify, and quantify proteins when coupled with mass spectrometric identification or other immunological tests. Although 2-DE was first introduced more than three decades ago, several challenges and limitations to its utility still exist. This review discusses the principles of 2-DE as well as both recent methodological advances and new applications.

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  • Two dimensional electrophoresis of the exo-proteome produced from community acquired methicillin resistant Staphylococcus aureus belonging to clonal complex 80

    Shymaa Enany, Yutaka Yoshida, Sameh Magdeldin, Xu Bo, Ying Zhang, Mohamed Enany, Tadashi Yamamoto

    Microbiological Research   168 ( 8 )   504 - 511   2013年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Two-dimensional electrophoresis (2DE) combined with mass spectrometry was used to characterize the exo-proteome secreted by two strains (ER13 and ER21) representing community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) belonging to clonal complex 80 (CC80). Common spots were detected between the 2 gels using the Progenesis SameSpots software. Two hundred and fifty-one and 312 spots from the exo-proteome of ER13 and ER21 were resolved, respectively. 2DE overlap comparison showed that 59 spots were shared. LC-MS/MS analysis identified 57 proteins from these spots comprising about 21% extracellular, 48% cytoplasmic, 2% cytoplasmic membrane, 2% cell wall, and 26% with unknown localization. The identified proteins were classified with respect to their Gene Ontology (GO) annotation as ~24% virulence determinants and toxins, ~17% involved in carbohydrate metabolism, ~14% involved in environmental stress, and ~12% associated with cell division. The identification of the enterotoxin B from the exo-products of both strains used in our study, as belonging to CC80 was interesting. © 2013 Elsevier GmbH.

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  • Profiling of kidney vascular endothelial cell plasma membrane proteins by liquid chromatography-tandem mass spectrometry 査読

    Zan Liu, Bo Xu, Masaaki Nameta, Ying Zhang, Sameh Magdeldin, Yutaka Yoshida, Keiko Yamamoto, Hidehiko Fujinaka, Eishin Yaoita, Masayuki Tasaki, Yuki Nakagawa, Kazuhide Saito, Kota Takahashi, Tadashi Yamamoto

    CLINICAL AND EXPERIMENTAL NEPHROLOGY   17 ( 3 )   327 - 337   2013年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER  

    Vascular endothelial cells (VECs) play crucial roles in physiological and pathologic conditions in tissues and organs. Most of these roles are related to VEC plasma membrane proteins. In the kidney, VECs are closely associated with structures and functions; however, plasma membrane proteins in kidney VECs remain to be fully elucidated.
    Rat kidneys were perfused with cationic colloidal silica nanoparticles (CCSN) to label the VEC plasma membrane. The CCSN-labeled plasma membrane fraction was collected by gradient ultracentrifugation. The VEC plasma membrane or whole-kidney lysate proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and digested with trypsin in gels for liquid chromatography-tandem mass spectrometry. Enrichment analysis was then performed.
    The VEC plasma membrane proteins were purified by the CCSN method with high yield (approximately 20 mu g from 1 g of rat kidney). By Mascot search, 582 proteins were identified in the VEC plasma membrane fraction, and 1,205 proteins were identified in the kidney lysate. In addition to 16 VEC marker proteins such as integrin beta-1 and intercellular adhesion molecule-2 (ICAM-2), 8 novel proteins such as Deltex 3-like protein and phosphatidylinositol binding clathrin assembly protein (PICALM) were identified. As expected, many key functions of plasma membranes in general and of endothelial cells in particular (i.e., leukocyte adhesion) were significantly overrepresented in the proteome of CCSN-labeled kidney VEC fraction.
    The CCSN method is a reliable technique for isolation of VEC plasma membrane from the kidney, and proteomic analysis followed by bioinformatics revealed the characteristics of in vivo VECs in the kidney.

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  • Profiling and annotation of human kidney glomerulus proteome. 査読

    Cui Z, Yoshida Y, Xu B, Zhang Y, Nameta M, Magdeldin S, Makiguchi T, Ikoma T, Fujinaka H, Yaoita E, Yamamoto T

    Proteome science   11 ( 1 )   13   2013年4月

  • Extensive proteomic profiling of the secretome of European community acquired methicillin resistant Staphylococcus aureus clone 査読

    Shymaa Enany, Yutaka Yoshida, Sameh Magdeldin, Ying Zhang, Xu Bo, Tadashi Yamamoto

    Peptides   37 ( 1 )   128 - 137   2012年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    European community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) clone remains a striking pathogenic clone spreading in European and Mediterranean countries. Since analysis of the secretome produced from this clone by proteomics could provide a comprehensive picture of both core exoproteins as well as virulence factors, we applied two proteomic approaches, pre-fractionation of proteins on SDS-PAGE followed by in-gel trypsin digestion, and in-solution trypsin-digestion followed by off-line SCX fractionation, both of which were coupled with LC-MS/MS analyses. A total of 174 distinct proteins were identified with a high-confidence. Functional classification of these identified proteins resulted in16.09% of protein synthesis, 13.79% of virulence, 6.89% of toxin, and 17.24% of unknown function. Prediction of their cellular localizations revealed 18.39% in extracellular space, 36.20% in cytoplasm, 5.17% in cytoplasmic membranes, 6.89% in cell wall, 1.14% in multiple localizations, and 32.18% in unknown localization. Among them, 52% proteins were predicted to be secreted through signal peptide-independent pathways. Most notably, the expression of some proteins such as enterotoxins U and B were identified for the first time in this clone. © 2012 Elsevier Inc.

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  • Proteomic approach to human kidney glomerulus prepared by laser microdissection from frozen biopsy specimens: exploration of proteome after removal of blood-derived proteins. 査読

    Yoshida Y, Nameta M, Kuwano M, Zhang Y, Bo X, Magdeldin S, Cui Z, Fujinaka H, Yaoita E, Tomonaga T, Yamamoto T

    Proteomics. Clinical applications   6 ( 7月8日 )   412 - 417   2012年8月

  • Murine colon proteome and characterization of the protein pathways 査読

    Sameh Magdeldin, Yutaka Yoshida, Huiping Li, Yoshitaka Maeda, Munesuke Yokoyama, Shymaa Enany, Ying Zhang, Bo Xu, Hidehiko Fujinaka, Eishin Yaoita, Sei Sasaki, Tadashi Yamamoto

    BIODATA MINING   5   2012年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:BIOMED CENTRAL LTD  

    Background: Most of the current proteomic researches focus on proteome alteration due to pathological disorders (i.e.: colorectal cancer) rather than normal healthy state when mentioning colon. As a result, there are lacks of information regarding normal whole tissue-colon proteome.
    Results: We report here a detailed murine (mouse) whole tissue-colon protein reference dataset composed of 1237 confident protein (FDR < 2) with comprehensive insight on its peptide properties, cellular and subcellular localization, functional network GO annotation analysis, and its relative abundances. The presented dataset includes wide spectra of pI and Mw ranged from 3-12 and 4-600 KDa, respectively. Gravy index scoring predicted 19.5% membranous and 80.5% globularly located proteins. GO hierarchies and functional network analysis illustrated proteins function together with their relevance and implication of several candidates in malignancy such as Mitogen-activated protein kinase (Mapk8, 9) in colorectal cancer, Fibroblast growth factor receptor (Fgfr 2), Glutathione S-transferase (Gstp1) in prostate cancer, and Cell division control protein (Cdc42), Ras-related protein (Rac1,2) in pancreatic cancer. Protein abundances calculated with 3 different algorithms (NSAF, PAF and emPAI) provide a relative quantification under normal condition as guidance.
    Conclusions: This highly confidence colon proteome catalogue will not only serve as a useful reference for further experiments characterizing differentially expressed proteins induced from diseased conditions, but also will aid in better understanding the ontology and functional absorptive mechanism of the colon as well.

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  • 凍結腎生検試料からLaser microdissectionにより調製した糸球体のプロテオーム解析:血液由来タンパク質の除去

    吉田 豊, 行田 正晃, 許 波, 張 瑩, 桑野 昌喜, 朝長 毅, 山本 格

    日本プロテオーム学会大会要旨集   2012   91 - 91   2012年

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    記述言語:日本語   出版者・発行元:日本プロテオーム学会(日本ヒトプロテオーム機構)  

    DOI: 10.14889/jhupo.2012.0.91.0

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  • Reno-protective effects of Eicosapentaenoic Acid (EPA) against PAN induced nephrosis in WKY rats 査読

    Ismail Tamer Ahmed, Mohamed Mohamed Soliman, Hossam Fouad Attia, Munoz Cuellar Lino, Xu Bo, Ying Zhang, Tadashi Yamamoto

    Asian Journal of Biochemistry   7 ( 1 )   16 - 26   2012年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Eicosapentaenoic Acid (EPA) is an omega-3 fatty acid (polyunsaturated fatty acid) that has pleiotropic effects as hypolipidemic and anti-inflammatory actions. Podocytes injury in the renal glomeruli has been proposed as the crucial mechanism in the development of focal segmental glomerulosclerosis or nephrosis. The effect of EPA on Puromycin aminonucleoside (PAN) induced nephrosis was tested. EPA was administered daily for 28 days at a dose of 1 g kg-1 b.wt. then PAN was injected intravenously at a dose of 6 mg/100 g of body weight followed by EPA for 6 days. PAN nephrosis induced increase in proteinuria, lipid profiles, podocytes proteins expression and immunolocalization. EPA induced decrease in proteinuria and lipid profiles induced by PAN nephrosis. Also, EPA induced significant down-regulation in expression of connexin 43 and synaptopodin. Moreover, EPA induced 50% decrease in glomerular cell adhesion induced by PAN nephrosis. Immunoflerusecnce shows expression of desmin and connexin 43 in rat glomeruli that increased by PAN and decreased by EPA. These findings collectively showed that EPA has reno-protective effect during inflammation. © 2012 Academic Journals Inc.

    DOI: 10.3923/ajb.2012.16.26

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  • Comparison of human glomerulus proteomic profiles obtained from low quantities of samples by different mass spectrometry with the comprehensive database

    Ying Zhang, Yutaka Yoshida, Bo Xu, Sameh Magdeldin, Hidehiko Fujinaka, Zan Liu, Masahito Miyamoto, Eishin Yaoita, Tadashi Yamamoto

    Proteome Science   9   2011年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Background: We have previously constructed an in-depth human glomerulus proteome database from a large amount of sample for understanding renal disease pathogenesis and aiding the biomarker exploration. However, it is usually a challenge for clinical research to get enough tissues for large-scale proteomic characterization. Therefore, in this study, we focused on high-confidence proteomics analysis on small amounts of human glomeruli comparable to those obtained from biopsies using different mass spectrometers and compared these results to the comprehensive database.Results: One microgram of human glomerular protein digest was analyzed each on five LC- combined mass spectrometers (LIT-TOF, LTQ-Orbitrap, Q-TOF, LIT and MALDI-TOF/TOF) yielding 139, 185, 94, 255 and 108 proteins respectively identified with strict criteria to ensure high confidence (&gt
    99%) and low false discovery rate (FDR) (&lt
    1%). An integrated profile of 332 distinct glomerular proteins was subsequently generated without discerned bias due to protein physicochemical properties (pI and MW), of which around 60% were detected commonly by more than two LC-MS/MS platforms. Comparative analysis with the comprehensive database demonstrated 14 proteins uniquely identified in this study and more than 70% of identified proteins in small datasets were concentrated to the top abundant 500 in the comprehensive database which consists of 2775 non-redundant proteins.Conclusion: This study showed representative human glomerulus proteomic profiles obtained from biopsies through analysis of comparable amounts of samples by different mass spectrometry. Our results implicated that high abundant proteins are more likely to be reproducibly identified in multiple mass spectrometers runs and different mass spectrometers. Furthermore, many podocyte essential proteins such as nephrin, podocin, podocalyxin and synaptopodin were also identified from the small samples in this study. Bioinformatic enrichment analysis results extended our understanding of the major glomerular proteins about their subcellular distributions and functions. The present study indicated that the proteins localized in certain cellular compartments, such as actin cytoskeleton, mitochondrial matrix, cell surface, basolateral plasma membrane, contractile fiber, proteinaceous extracellular matrix and adherens junction, represent high abundant glomerular proteins and these subcellular structures are also highly significantly over-represented in the glomerulus compared to the whole human background. © 2011 Zhang et al
    licensee BioMed Central Ltd.

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  • Usage of electrostatic eliminator reduces human keratin contamination significantly in gel-based proteomics analysis 査読

    Bo Xu, Ying Zhang, Zongjiang Zhao, Yutaka Yoshida, Sameh Magdeldin, Hidehiko Fujinaka, Tamer Ahmed Ismail, Eishin Yaoita, Tadashi Yamamoto

    JOURNAL OF PROTEOMICS   74 ( 7 )   1022 - 1029   2011年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    In the field of bottom-up proteomics, heavy contamination of human keratins could hinder the comprehensive protein identification, especially for the detection of low abundance proteins. In this study, we examined the keratin contamination in the four major experimental procedures in gel-based proteomic analysis including gel preparation, gel electrophoresis, gel staining, and in-gel digestion. We found that in-gel digestion procedure might be of importance corresponding to keratin contaminants compared to the other three ones. The human keratin contamination was reduced significantly by using an electrostatic eliminator during in-gel digestion, suggesting that static electricity built up on insulated experimental materials might be one of the essential causes of keratin contamination. We herein proposed a series of methods for improving experimental conditions and sample treatment in order to minimize the keratin contamination in an economical and practical way. (C) 2011 Elsevier B.V. All rights reserved.

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  • Novel expression of claudin-5 in glomerular podocytes 査読

    Ryo Koda, Linning Zhao, Eishin Yaoita, Yutaka Yoshida, Sachiko Tsukita, Atsushi Tamura, Masaaki Nameta, Ying Zhang, Hidehiko Fujinaka, Sameh Magdeldin, Bo Xu, Ichiei Narita, Tadashi Yamamoto

    CELL AND TISSUE RESEARCH   343 ( 3 )   637 - 648   2011年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER  

    Tight junctions are the main intercellular junctions of podocytes of the renal glomerulus under nephrotic conditions. Their requisite components, claudins, still remain to be identified. We have measured the mRNA levels of claudin subtypes by quantitative real-time PCR using isolated rat glomeruli. Claudin-5 was found to be expressed most abundantly in glomeruli. Mass spectrometric analysis of membrane preparation from isolated glomeruli also confirmed only claudin-5 expression without any detection of other claudin subtypes. In situ hybridization and immunolocalization studies revealed that claudin-5 was localized mainly in glomeruli where podocytes were the only cells expressing claudin-5. Claudin-5 protein was observed on the entire surface of podocytes including apical and basal domains of the plasma membrane in the normal condition and was inclined to be concentrated on tight junctions in puromycin aminonucleoside nephrosis. Total protein levels of claudin-5 in isolated glomeruli were not significantly upregulated in the nephrosis. These findings suggest that claudin-5 is a main claudin expressed in podocytes and that the formation of tight junctions in the nephrosis may be due to local recruitment of claudin-5 rather than due to total upregulation of the claudin protein levels.

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  • "All and None" refining strategy; Fishing your correct protein from proteomics ocean 査読

    Sameh Magdeldin, Yutaka Yoshida, Ying Zhang, Bo Xu, Eishin Yaoita, Tadashi Yamamoto

    Journal of Proteomics and Bioinformatics   4 ( 6 )   123 - 124   2011年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We have developed an easy-to use methodology for refining large extensible markup language (XML) - based proteomics dataset with a high stringent and simple approach using VBA- coded plug-in. A methodology we term it (All and None). Selections of targeted candidates differentially significant between compared groups were selected based on its appearance or absence followed by peptide screening with a novel and simple approach. By testing the reliability and efficiency of this method, All and None was confirmed to be an applicable process for initial screening of biological biomarkers in complex specimens and tissue extract. © 2011 Magdeldin S, et al.

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  • Differential Proteomic Shotgun Analysis Elucidates Involvement of Water Channel Aquaporin 8 in Presence of alpha-Amylase in the Colon 査読

    Sameh Magdeldin, Huiping Li, Yutaka Yoshida, Ichiro Satokata, Yoshitaka Maeda, Munesuke Yokoyama, Shymaa Enany, Ying Zhang, Bo Xu, Hidehiko Fujinaka, Eishin Yaoita, Tadashi Yamamoto

    JOURNAL OF PROTEOME RESEARCH   9 ( 12 )   6635 - 6646   2010年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER CHEMICAL SOC  

    Aquaporin (AQP) family plays a pivotal role in fluid secretion and absorption especially in the digestive system and secretory glands Within this family AQP8 was reported to be widely expressed in the epithelia of the digestive tract, liver, and pancreas In two parallel experimental platforms with different analytical and comparative approaches, in gel tryptic digestion with macro-embedded spreadsheet analysis and in-solution tryptic digestion with LC MS alignment based approach we compared wild type and AQP8 knockout mice colon proteomes Shared result between both experiments revealed down-regulation of alpha-amylase 2 in AQP8-deleted mice model Verification on both transcriptional and translational levels confirmed the involvement of AQP8 in alpha-amylase 2 regulation Given the profound role of AQP8 as a water and solutes transporter, it might be important in modulating a-amylase 2 synthesis by colonic epithelial cells as well Here, we also proved the capability of our coupled approaches for selecting the most reliable and significant candidates, an applicable process for initial screening of biological biomarkers in complex specimens and tissue extracts

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  • Comparison of two dimensional electrophoresis mouse colon proteomes before and after knocking out Aquaporin 8 査読

    Sameh Magdeldin, Huiping Li, Yutaka Yoshida, Shymaa Enany, Ying Zhang, Bo Xu, Hidehiko Fujinaka, Eishin Yaoita, Tadashi Yamamoto

    JOURNAL OF PROTEOMICS   73 ( 10 )   2031 - 2040   2010年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    Aquaporin (AQP) family plays a fundamental role in transmembrane water and small solutes movement. Within this family, aquaporin 8 (AQP8), showed to be widely distributed in the digestive system especially colon. To investigate the possible protein alterations involved in AQP8 regulation and trafficking, we extensively compared between wild type and AQP8 knockout mouse colon using semi-quantitative fluorescence- stained two dimensional gel electrophoresis (2-DE) coupled with nano LC-Ms/Ms. Our analysis revealed identification and regulation of 21 proteins, most notably, actin-related family which suggests its possible involvement in regulating AQP8 secretory vesicles migration to be integrated as a cell membrane protein. (C) 2010 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.jprot.2010.06.010

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  • Glomerular proteins related to slit diaphragm and matrix adhesion in the foot processes are highly tyrosine phosphorylated in the normal rat kidney. 査読

    Zhang Y, Yoshida Y, Nameta M, Xu B, Taguchi I, Ikeda T, Fujinaka H, Magdeldin S, Tsukaguchi H, Harita Y, Yaoita E, Yamamoto T

    Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association   25 ( 6 )   1785 - 1795   2010年6月

  • Dietary supplementation with arachidonic acid but not eicosapentaenoic or docosahexaenoic acids alter lipids metabolism in C57BL/6J mice

    Sameh Magdeldin, Yaser Elewa, Takako Ikeda, Junko Ikei, Ying Zhang, Bo Xu, Masaaki Nameta, Hidehiko Fujinaka, Yutaka Yoshida, Eishin Yaoita, Tadashi Yamamoto

    GENERAL PHYSIOLOGY AND BIOPHYSICS   28 ( 3 )   266 - 275   2009年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:GENERAL PHYSIOL AND BIOPHYSICS  

    In order to investigate the effects of dietary supplementation rich in omega 3 and omega 6 fatty acids, we set up an experiment of twenty four C57BL/6J male mice segregated into 3 groups: normal diet (ND), omega 3 polyunsaturated fatty acid (n-3 PUFA,) and omega 6 (n-6 PUFA). At the end of the experiment that lasted for I month, food consumption of ND and n-3 PUFA were similar while it decreased in n-6 PUFA group. Total cholesterol, triglycerides, free fatty acids, and phospholipids profiles were increased in n-6 PUFA. LDL decreased in n-3 PUFA while increased in n-6 PUFA fed mice comparing to control group. On the other hand, there was no difference between treatments in HDL and glucose levels. Expression of leptin (ob) gene transcripts in epididymal fat were significantly elevated in n-6 PUFA mice compared to ND and n-3 PUFA groups while hypothalamic ob receptor A (obRa) mRNA did not changed in response to diet regimes. Transmission and scanning electron microscopy showed different degrees in fatty changes in the liver of both PUFA groups including lipid droplet infiltration and Ito cells with over accumulated lipids. In conclusion, under PUFA dietary supplementation, the hyperlipidemic status and elevated ob expression of n-6 PUFA but not n-3 PUFA fed mice suggests altered lipid metabolism between PUFA groups and/or different endocrine involvement. Moreover, the coincidently structural changes observed in liver of this group direct us to call for further studies to investigate the anti-obesity effect and safety of these PUFA under high supplementation condition.

    DOI: 10.4149/gpb_2009_03_266

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  • Identification and characterization of major proteins carrying ABO blood group antigens in the human kidney. 国際誌

    Masayuki Tasaki, Yutaka Yoshida, Masahito Miyamoto, Masaaki Nameta, Lino M Cuellar, Bo Xu, Ying Zhang, Eishin Yaoita, Yuki Nakagawa, Kazuhide Saito, Tadashi Yamamoto, Kota Takahashi

    Transplantation   87 ( 8 )   1125 - 33   2009年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    BACKGROUND: It is generally admitted that ABO(H) blood group antigens are linked to lipids and proteins. Although glycolipids carrying ABO antigens have been well characterized in human kidneys, glycoproteins carrying ABO antigens are largely unknown, and their molecular properties remain to be elucidated. METHODS: All the blood group A antigen-linked proteins in human kidney could be solubilized and captured on immobilized Helix pomatia lectin that recognizes A antigens. These proteins were separated on SDS-PAGE gels. The gel pieces containing protein bands immunoreactive with anti-A antibody were excised, in-gel digested with trypsin, and analyzed by nanoLC tandem mass spectrometer. Protein candidates that carry ABO antigens were confirmed by immunoprecipitation and double-labeled immunofluorescense microscopy. RESULTS: All the glycoproteins carrying ABO antigens were found to be Asn-linked glycoproteins, and presented as multiple bands on SDS-PAGE with molecular masses ranging from 60 to 270 kDa. The protein bands were subjected for mass spectrometric analysis, which identified 121 distinct proteins with high confidence. Of the identified proteins, 55 N-glycosylated, membrane proteins were selected as glycoprotein candidates that carry ABO antigens. Among them, most abundantly expressed proteins as estimated by the number of peptide matches in the MS spectrometric analysis, such as platelet endothelial cell adhesion molecule 1, plasmalemmal vesicle-associated protein, and von Willebrand factor, were further characterized. CONCLUSIONS: Several glycoproteins were identified that represented major glycoproteins carrying ABO antigens in the human kidney, which exhibited distinct features in localization to most of vascular endothelial cells.

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  • ヒト腎組織に発現するABO血液型をもつタンパク質の解析と特性 プロテオミクスを用いて

    田崎 正行, 吉田 豊, 田口 いづみ, 張 瑩, 行田 正晃, 中川 由紀, 斎藤 和英, 高橋 公太, 山本 格

    日本腎臓学会誌   51 ( 3 )   369 - 369   2009年4月

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    記述言語:日本語   出版者・発行元:(一社)日本腎臓学会  

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  • Mesangial Cells Connected by the N-Cadherin-Catenin System in the Rat Kidney 査読

    Masaaki Nameta, Eishin Yaoita, Nobutaka Kato, Linning Zhao, Ying Zhang, Hidehiko Fujinaka, Bo Xu, Yutaka Yoshida, Tadashi Yamamoto

    NEPHRON EXPERIMENTAL NEPHROLOGY   112 ( 4 )   E92 - E98   2009年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:KARGER  

    Background/Aims: Mesangial cells bear the tensional forces generated in the glomerular capillary wall. Not only mesangial cells per se, but also their intercellular junctions should be physically stable against the tensional forces; this prompted the search for actin filament- reinforced adherens junctions of mesangial cells. We previously reported alpha- and beta-catenins localized at the cell-cell contact sites of mesangial cells in the rat. Classical cadherin expressed by mesangial cells, however, remains to be elucidated. Methods: Expression of classical cadherins, especially N-cadherin, was examined in rat glomeruli by ribonuclease protection assay, Western blot analysis and immunofluorescence and immuno-electron microscopy. Results: Ribonuclease protection assay detected significant expression of N-cadherin in rat glomeruli. Western blot analysis showed that rabbit and murine antibodies against N-cadherin reacted with a specific band in isolated glomeruli. Immunofluorescence microscopy revealed that both antibodies reacted only with the mesangium in glomeruli. Immunoelectron microscopy demonstrated that the immunogold particles for N-cadherin were found predominantly at cell-cell contact sites of mesangial cells where actin filaments concentrated. Conclusion: N-cadherin interconnects mesangial cells, suggesting that the cadherin-catenin-actin filament system in the mesangium may play a role in the counteraction of the hydraulic pressure gradient across the capillary wall. Copyright (C) 2009 S. Karger AG, Basel

    DOI: 10.1159/000224797

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  • Claudin-6 localized in tight junctions of rat podocytes 査読

    Linning Zhao, Eishin Yaoita, Masaaki Nameta, Ying Zhang, Lino Munoz Cuellar, Hidehiko Fujinaka, Bo Xu, Yutaka Yoshida, Katsuyoshi Hatakeyama, Tadashi Yamamoto

    AMERICAN JOURNAL OF PHYSIOLOGY-REGULATORY INTEGRATIVE AND COMPARATIVE PHYSIOLOGY   294 ( 6 )   R1856 - R1862   2008年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER PHYSIOLOGICAL SOC  

    Tight junctions rarely exist in podocytes of the normal renal glomerulus, whereas they are the main intercellular junctions of podocytes in nephrosis and in the early stage of development. Claudins have been identified as tight junction-specific integral membrane proteins. Those of podocytes, however, remain to be elucidated. In the present study, we investigated the expression and localization of claudin-6 in the rat kidney, especially in podocytes. Western blot analysis and RT-PCR revealed that the neonatal kidney expressed much higher levels of claudin-6 than the adult kidney. Immunofluorescence microscopy showed intense claudin-6 staining in most of the tubules and glomeruli in neonates. The staining in tubules declined distinctly in adults, whereas staining in glomeruli was well preserved during development. Claudin-6 in glomeruli was distributed along the glomerular capillary wall and colocalized with zonula occludens-1. The staining became conspicuous after kidney perfusion with protamine sulfate (PS) to increase tight junctions in podocytes. Immunoelectron microscopy showed that immunogold particles for claudin-6 were accumulated at close cell-cell contact sites of podocytes in PS-perfused kidneys, whereas a very limited number of immunogold particles were detected, mainly on the basal cell membrane and occasionally at the slit diaphragm and close cell-cell contact sites in normal control kidneys. In puromycin aminonucleoside nephrosis, immunogold particles were also found mainly at cell-contact sites of podocytes. These findings indicate that claudin-6 is a transmembrane protein of tight junctions in podocytes during development and under pathological conditions.

    DOI: 10.1152/ajpregu.00862.2007

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  • In-depth proteomic profiling of the normal human kidney glomerulus using two-dimensional protein prefractionation in combination with liquid chromatography-tandem mass spectrometry. 国際誌

    Masahito Miyamoto, Yutaka Yoshida, Izumi Taguchi, Yoshimi Nagasaka, Masayuki Tasaki, Ying Zhang, Bo Xu, Masaaki Nameta, Hiroshi Sezaki, Lino M Cuellar, Tetsuo Osawa, Hideo Morishita, Shigeki Sekiyama, Eishin Yaoita, Kenjiro Kimura, Tadashi Yamamoto

    Journal of proteome research   6 ( 9 )   3680 - 90   2007年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The kidney glomerulus plays a pivotal role in ultrafiltration of plasma into urine and also is the locus of kidney disease progressing to chronic renal failure. We have focused proteomic analysis on the glomerulus that is most proximal to the disease locus. In the present study, we aimed to provide a confident, in-depth profiling of the glomerulus proteome. The glomeruli were highly purified from the kidney cortex from a male, 68-year-old patient who underwent nephroureterectomy due to ureter carcinoma. The patient was normal in clinical examinations including serum creatinine and urea levels and liver function, and did not receive any chemotherapy and radiotherapy. The cortical tissue was histologically normal, and no significant deposition of immunoglobulins and complement C3 was observed. We employed a novel strategy of protein separation using 1D (SDS-PAGE) and 2D (solution-phase IEF in combination with SDS-PAGE) prefractionation prior to the shotgun analysis with LC-MS/MS. The protein prefractionation produced 90 fractions, and eventually provided a confident set of identified proteins consisting of 6686 unique proteins (3679 proteins with two or more peptide matches and 3007 proteins with one peptide match), representing 2966 distinct genes. All the identified proteins were annotated and classified in terms of molecular function and biological process, compiled into 1D and 2D protein arrays, consisting of 15 and 75 sections, corresponding to the protein fractions which were defined by MW and pI range, and deposited on a Web-based database (http://www.hkupp.org). The most remarkable feature of the glomerulus proteome was a high incidence of identification of cytoskeleton-related proteins, presumably reflecting the well-developed, cytoskeletal organization of glomerular cells related to their physiological functions.

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  • 正常ラットの腎臓におけるチロシンリン酸化タンパク質の局在 査読

    Zhang Y, Yoshida Y, Xu B, Nameta M, Miyamoto M, Yaoita E, Yamamoto T

    2007年3月

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    記述言語:英語   掲載種別:学位論文(博士)  

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  • Localization of tyrosine-phosphorylated proteins in the normal rat kidney

    Ying Zhang, Yutaka Yoshida, Bo Xu, Masaaki Nameta, Masahito Miyamoto, Eishin Yaoita, Tadashi Yamamoto

    Acta Medica et Biologica   55 ( 1 )   1 - 7   2007年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Protein tyrosine phosphorylation is responsible for activating or repressing the activity of proteins involved in numerous signal transduction pathways in a living organism. Using immunochemical methods with two well characterized monoclonal anti-phosphotyrosine antibodies, we examined the distribution and localization of tyrosine-phosphorylated proteins expressed in the three major compartments of the normal rat kidney: the glomerulus, cortex, and medulla. Western blotting analysis revealed that tyrosine-phosphorylated proteins were predominantly expressed in the glomerulus, compared with the cortex and medulla. The intensity of tyrosine-phosphorylated protein bands was augmented by using orthovanadate - an inhibitor of protein tyrosine phosphatase - during sample preparation, and the bands were undetectable when the antibodies were absorbed with phosphotyrosine but not with phosphoserine or phosphothreonine. Immunofluorescence microscopy indicated that the phosphotyrosine staining was intense along glomerular capillary walls and sparse along parts of the tubuli. Immunoelectron microscopy further showed that phosphotyrosine immunoreactivity was predominantly localized at the basal membranes of podocyte foot processes. In addition, some immunogold labeling was also observed at cell-matrix attachment sites of glomerular endothelial cells and mesangial cells as well as at basal membranes of epithelial cells of proximal tubules, distal tubules, and collecting ducts. In conclusion, tyrosine-phosphorylated proteins are predominantly and constitutively expressed in the normal rat glomerulus, especially in the basal membranes of podocyte foot processes, suggesting tyrosine-phosphorylated proteins could play an important role in maintaining the unique organization of foot processes and in the glomerular ultrafiltration there.

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  • Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database

    Yutaka Yoshida, Kenji Miyazaki, Junichi Kamiie, Masao Sato, Seiji Okuizumi, Akihisa Kenmochi, Ken'ichi Kamijo, Takuji Nabetani, Akira Tsugita, Bo Xu, Ying Zhang, Eishin Yaoita, Tetsuo Osawa, Tadashi Yamamoto

    Proteomics   5 ( 4 )   1083 - 1096   2005年3月

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    記述言語:英語   掲載種別:研究論文(国際会議プロシーディングス)  

    To contribute to physiology and pathophysiology of the glomerulus of human kidney, we have launched a proteomic study of human glomerulus, and compiled a profile of proteins expressed in the glomerulus of normal human kidney by two-dimensional gel electrophoresis (2-DE) and identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kidney cortices with normal appearance were obtained from patients under surgical nephrectomy due to renal tumor, and glomeruli were highly purified by a standard sieving method followed by picking-up under a phase-contrast microscope. The glomerular proteins were separated by 2-DE with 24 cm immobilized pH gradient strips in the 3-10 range in the first dimension and 26 × 20 cm sodium dodecyl sulfate polyacrylamide electrophoresis gels of 12.5% in the second dimension. Gels were silver-stained, and valid spots were processed for identification through an integrated robotic system that consisted of a spot picker, an in-gel digester, and a MALDI-TOF MS and/or a LC-MS/MS. From 2-DE gel images of glomeruli of four subjects with no apparent pathologic manifestations, a synthetic gel image of normal glomerular proteins was created. The synthetic gel image contained 1713 valid spots, of which 1559 spots were commonly observed in the respective 2-DE gels. Among the 1559 spots, 347 protein spots, representing 212 proteins, have so far been identified, and used for the construction of an extensible markup language (XML)-based database. The database is deposited on a web site (http://www.sw.nec.co.jp/bio/rd/hgldb/index. html) in a form accessible to researchers to contribute to proteomic studies of human glomerulus in health and disease. © 2005 WILEY-VCH Verlag GmbH &amp
    Co. KGaA, Weinheim.

    DOI: 10.1002/pmic.200401075

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MISC

  • ER陽性HER2陰性乳癌の術前化学療法における化学療法感受性予測バイオマーカーの探索

    長谷川 美樹, 神林 智寿子, 若井 俊文, 朝長 毅, 山本 格, 吉田 豊, 白水 崇, 佐藤 信昭, 張 瑩, 小山 諭, 牧野 春彦, 永橋 昌幸, 金子 耕司

    日本プロテオーム学会大会要旨集   2015 ( 0 )   144 - 144   2015年

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    記述言語:日本語   出版者・発行元:日本プロテオーム学会(日本ヒトプロテオーム機構)  

    DOI: 10.14889/jhupo.2015.0.144.0

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  • Comparative proteomic analysis of the liver in a murine model of non-alcoholic steatohepatitis

    Masaaki Takamura, Bo Xu, Satoshi Yamagiwa, Yasunobu Matsuda, Shuichiro Shimada, Ying Zhang, Yutaka Yoshida, Eishin Yaoita, Minoru Nomoto, Tadashi Yamamoto, Yutaka Aoyagi

    HEPATOLOGY   56   855A - 855A   2012年10月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:WILEY-BLACKWELL  

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  • Glomerular proteins related to slit diaphragm and matrix adhesion in the foot processes are highly tyrosine phosphorylated in the normal rat kidney (vol 25, pg 1785, 2010)

    Ying Zhang, Yutaka Yoshida, Masaaki Nameta, Bo Xu, Izumi Taguchi, Takako Ikeda, Hidehiko Fujinaka, Sameh Magdeldin, Hiroyasu Tsukaguchi, Yutaka Harita, Eishin Yaoita, Tadashi Yamamoto

    NEPHROLOGY DIALYSIS TRANSPLANTATION   27 ( 6 )   2606 - 2606   2012年6月

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    記述言語:英語   出版者・発行元:OXFORD UNIV PRESS  

    DOI: 10.1093/ndt/gfs155

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  • 実験的NASHモデルマウス肝組織のプロテオーム解析

    高村 昌昭, 許 波, 嶋田 修一郎, 張 瑩, 吉田 豊, 矢尾板 永信, 山際 訓, 松田 康伸, 野本 実, 山本 格, 青柳 豊

    日本消化器病学会雑誌   109 ( 臨増総会 )   A325 - A325   2012年3月

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    記述言語:日本語   出版者・発行元:(一財)日本消化器病学会  

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  • Human kidney glomerulus proteome and biomarker discovery of kidney diseases

    Yutaka Yoshida, Masahito Miyamoto, Izumi Taguchi, Bo Xu, Ying Zhang, Eishin Yaoita, Hidehiko Fujinaka, Tadashi Yamamoto

    PROTEOMICS CLINICAL APPLICATIONS   2 ( 3 )   420 - 427   2008年3月

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    記述言語:英語   掲載種別:書評論文,書評,文献紹介等   出版者・発行元:WILEY-V C H VERLAG GMBH  

    The kidney glomerulus is the site of plasma filtration and production of primary urine in the kidney. The structure not only plays a pivotal role in ultrafiltration of plasma into urine but also is the locus of kidney diseases progressing to chronic renal failure. Patients afflicted with these glomerular diseases frequently progress to irreversible loss of renal function and inevitably require replacement therapies. The diagnosis and treatment of glomerular diseases are now based on clinical manifestations, urinary protein excretion level, and renal pathology of needle biopsy specimens. The molecular mechanisms underlying the progression of glomerular diseases are still obscure despite a great number of clinical and experimental studies. Proteomics is a particularly promising approach for the discovery of proteins relevant to physiological and pathophysiological processes, and has been recently employed in nephrology. Although until now most efforts of proteomic analysis have been conducted with urine, the biological fluid that is easily collected without invasive procedures, proteomic analysis of the glomerulus, the tissue most proximal to the disease loci, is the most straightforward approach. In this review, we attempt to outline the current status of clinical proteomics of the glomerulus and provide a perspective of protein biomarker discovery of glomerular diseases.

    DOI: 10.1002/prca.200780016

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講演・口頭発表等

  • TRPM4の発現、機能低下とその結果生じるTRPC6機能亢進、Ca2+流入の増加はポドサイト傷害の重要な初期変化である

    内許玉楓, 福住好恭, 安田英紀, 常 國慶, 萱場 睦, 河内 裕

    第67回 日本腎臓学会学術総会  2024年6月 

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  • 抗ネフリン抗体検出簡便法の確立:免疫組織学 的手法との感度比較

    萱場 睦, 内許玉楓, 永井 隆, 福住好恭, 常 国慶, 河内 裕

    第67回 日本腎臓学会学術総会  2024年6月 

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  • 14-3-3 Proteins Stabilize Vimentin and Actin Filaments to Maintain Primary and Foot Processes in Podocyte(14-3-3蛋白質はビメンチンとアクチンフィラメントを安定化させ、ポドサイトの一次突起と足突起を維持する)

    安田英紀, 福住好恭, 内許玉楓, 河内 裕

    アメリカ腎臓学会(国際学会)  2023年11月 

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  • Downregulation of TRPM4 and Consequent Increase in TRPC6 Activity Are Critical Initiation Events Leading to Podocyte Injury(TRPM4の発現、機能低下とその結果生じるTRPC6機能亢進はポドサイト傷害の重要な初期変化である)

    内許玉楓, 福住好恭, 安田英紀, 常 国慶, 萱場 睦, 河内 裕

    アメリカ腎臓学会(国際学会)  2023年11月 

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  • Nephrin-Ephrin-B1-Par6 complex is crucial for slit diaphragm in podocytes: Ephrin-B1 suppresses tight junction formation by interfering with Par6-Cdc42 binding

    福住好恭, 張瑩(内許玉楓), 安田英紀, 河内裕

    アメリカ腎臓学会(国際学会)  2022年11月 

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  • Ephrin-B1はPar-6-Cdc42結合と阻害しTJ形成の抑制、スリット膜維持に働く

    福住好恭, 安田英紀, 張瑩(内許玉楓), 河内裕

    第65回日本腎臓学会  2022年6月 

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  • Neurexin 1α containing splice site 4 interacts with nephrin and contributes to maintenance of the integrity of podocyte slit diaphragm

    福住好恭, 安田英紀, 張瑩(内許玉楓), 河内裕

    アメリカ腎臓学会(国際学会)  2021年11月 

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  • NHERF2はスリット膜の分子複合体とポドサイト頂部の分子複合体を連結し、ポドサイトの細胞骨格を維持する

    福住好恭, 安田英紀, 張瑩(内許玉楓, 河内裕

    第64回日本腎臓学会  2021年6月 

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  • スプライスサイト4を持つNeurexin 1αバリアントはスリット膜構造の維持に重要な役割を果たす

    福住好恭, 安田英紀, 張瑩(内許玉楓), 河内裕

    第64回日本腎臓学会  2021年6月 

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  • PDZ蛋白質NHERF2はEphrin-B1とEzrinを連結させ、スリット膜機能維持に重要な役割を果たす

    福住好恭, 安田英紀, 張瑩(内許玉楓), 河内裕

    第63回日本腎臓学会  2020年6月 

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  • メサンギウム増殖性腎炎におけるTh17細胞の関与について

    渡辺秀平, 安田英紀, 張瑩(内許玉楓), 福住好恭, 河内裕

    第62回日本腎臓学会  2019年6月 

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  • キサンチン酸化還元酵素阻害薬トピロキソスタットのポドサイト保護作用

    張瑩(内許玉楓), 福住好恭, 中村敬志, 芦澤直樹, 河内裕

    第61回日本腎臓学会  2018年6月 

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  • TRPM4 is expressed at the apical surface of podocyte just above slit diaphragm, and its altered expression is involved in podocyte injury

    張瑩(内許玉楓), 福住好恭, 河内裕

    アメリカ腎臓学会(国際学会)  2017年11月 

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  • スリット膜特異的障害モデルにおける発現上昇遺伝子の検討:次世代シーケンサを用いたRNA-seq解析

    張瑩(内許玉楓), 福住好恭, 河内裕

    第60回日本腎臓学会  2017年6月 

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  • RNA-seq Based Differential Expression Analysis in Rats with Slit Diaphragm Specific Dysfunction: The Glomerular Expression Profiles of Nephropathy Induced by Anti-nephrin Antibody

    張瑩(内許玉楓), 福住好恭, 河内裕

    アメリカ腎臓学会  2016年11月 

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  • 次世代シーケンサー解析による新規ネフリン 関連分子、スリット膜機能分子の同定

    張 瑩(内許玉楓), 福住 好恭, 河内 裕

    第59回日本腎臓学会  2016年6月 

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    記述言語:日本語   会議種別:ポスター発表  

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共同研究・競争的資金等の研究

  • スリット膜機能維持におけるNeurexin-シナプス小胞関連分子相互作用の役割

    研究課題/領域番号:24K11405

    2024年4月 - 2028年3月

    制度名:科学研究費助成事業

    研究種目:基盤研究(C)

    提供機関:日本学術振興会

    福住好恭, 内許玉楓, 永井 隆, 河内 裕

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    配分額:4680000円 ( 直接経費:3600000円 、 間接経費:1080000円 )

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  • Ephrin-ネフリン-NRX複合体の機能解析によるスリット膜安定化機構の解明

    研究課題/領域番号:22H03086

    2022年4月 - 2025年3月

    制度名:科学研究費助成事業 基盤研究(B)

    研究種目:基盤研究(B)

    提供機関:日本学術振興会

    河内 裕, 成田 一衛, 金子 佳賢, 松井 克之, 葛谷 聡, 福住 好恭, 内許 玉楓, 安田 英紀

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    配分額:17290000円 ( 直接経費:13300000円 、 間接経費:3990000円 )

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  • 腎糸球体上皮細胞スリット膜におけるNeurexinの役割の解明

    研究課題/領域番号:20K08587

    2020年4月 - 2024年3月

    制度名:科学研究費助成事業 基盤研究(C)

    研究種目:基盤研究(C)

    提供機関:日本学術振興会

    福住 好恭, 内許 玉楓, 安田 英紀, 河内 裕

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    配分額:4160000円 ( 直接経費:3200000円 、 間接経費:960000円 )

    我々はシナプス前末端に存在するneurexin1がポドサイトスリット膜部に発現していること、ポドサイトで発現しているneurexin1は神経組織と異なる特異的なバリアント(splice site (SS)4(+))であることを示した。しかし、neurexin1のスリット膜での役割の詳細は不明である。
    Neurexin1のスリット膜のバリア維持機能における役割を明らかにするため、本年度(令和2年度)はneurexin1αKOマウスを作製し、ポドサイト病変、スリット膜機能分子の発現を解析した。また、neurexin1αとスリット膜機能分子nephrinとの相互作用解析を行った。
    まず、ラット糸球体可溶化材料を用いたウエスタンブロット解析から、ポドサイトで発現しているneurexin1は主に長鎖のneurexin1αであることを示した。
    次に、neurexin1αKOマウスの解析で、若齢期の10週齢では野生型マウスと比較して顕著な変化は観察されなかったが、成体期の20週齢でnephrin、podocinの著明な発現低下、及び有意な病的蛋白尿を観察した。
    相互作用解析では、ラット糸球体可溶化材料を用いた免疫沈降解析によりneurexin1αとnephrinの結合性を認めた。また、HEK293細胞を用いた強制発現系の解析で、神経組織で発現しているneurexin1α-SS4(-)はnephrinとの結合性が認められなかったが、ポドサイトでの発現型であるneurexin1α-SS4(+)はnephrinと結合性を有した。
    以上より、neurexin1αはnephrinと結合性を有するスリット膜構成分子であること、neurexin1αの発現低下により病的蛋白尿が誘導されることが示され、ポドサイト特異的neurexin1α-SS4(+)はスリット膜の機能維持に重要であることを明らかにした。

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  • ポドサイト機能維持におけるCa2+活性化型陽イオンチャネルTRPM4の役割

    研究課題/領域番号:19K08720

    2019年4月 - 2023年3月

    制度名:科学研究費助成事業 基盤研究(C)

    研究種目:基盤研究(C)

    提供機関:日本学術振興会

    内許 玉楓, 河内 裕, 福住 好恭

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    配分額:4160000円 ( 直接経費:3200000円 、 間接経費:960000円 )

    初年度(令和元年度)のポドサイトにおけるTRPM4 のsplice variantの検討で、ポドサイトにおけるTRPM4b(全長)とTRPM4a(exon 3/4欠損)、exon 4単独欠損した分子型の存在を見出した。ネフローゼ症候群モデルでは、TRPM4b 、TRPM4aの発現の増加を観察したが、exon 4単独欠損分子の発現に変化はなかった。一方ですべての分子型に共通する部分を認識するプライマーでの検討では、発現の低下が見られた。これらの観察結果は、ポドサイトには病態形成に関わる別の分子型が存在することを示唆している。本年度(令和2年度)はTRPM4の新たな分子型の探索を行い、exon 12単独欠損、exon 15/16ダブル欠損の2つの分子型の存在を確認した。exon 12はTRPM4の立体構造の維持に関与しているドメインを、exon 15とexon 16はシグナル伝達分子Phosphatidylinositol 4,5-bisphosphate(PIP2)と結合するドメインを有している。今回同定した分子型の機能解析はTRPM4のポドサイトにおける固有の機能の解明につながると考えられる。
    ポドサイトにおけるTRPM4の機能を解明するため、ヒトの培養ポドサイトを用いたノックダウン(KD)系での検討を行った。突起形成率、突起長など細胞の形態に著変はなく、スリット膜機能分子であるネフリンの発現にも変化がなかったが、TRPC6の発現が低下していることを確認した。この結果は、TRPC6はTRPM4と相互作用があることを示唆していると考えられる。

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  • 脳腎連関:シナプス関連分子を標的とした新規蛋白尿抑制治療薬の開発

    研究課題/領域番号:19H03673

    2019年4月 - 2022年3月

    制度名:科学研究費助成事業 基盤研究(B)

    研究種目:基盤研究(B)

    提供機関:日本学術振興会

    河内 裕, 葛谷 聡, 福住 好恭, 松井 克之, 内許 玉楓, 安田 英紀

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    配分額:17160000円 ( 直接経費:13200000円 、 間接経費:3960000円 )

    各種シナプス関連分子(SV2B, Neurexin, Ephrin-B1)がポドサイトに発現していること、その分子機能の低下が蛋白尿発症に関与していることを明らかにしてきた。Ephrin-B1は細胞外部でNephrinと結合していることを報告しているが、Ephrin-B1の細胞質部での結合分子は不明であった。今年度は、Ephrin-B1並びにその裏打ち分子として同定したNHERF2についての検討を中心に行った。次世代シーケンサを用いた探索でNHERF2がEphrin-B1の関連分子であることを見出し、免疫沈降法による検討でNHERF2はそのPDZ領域でEphrin-B1のC末部と結合していることを明らかにした。また、NHERF2はEzrinを介して細胞骨格と連結していることを明らかにし、Nephrin-Ephrin-B1-NHERF2-Ezrin-Actinの新たな分子連関機構を同定し、NHERF2のノックダウン系を用いた検討で、この分子連関がポドサイト機能維持に極めて重要な役割を果たしていることを明らかにした。またスリット膜細胞外部のNephrinが刺激を受けると、Nephrin, Ephrin-B1はリン酸化、NHERF2、Ezrinは脱リン酸化し、その結果この分子連関が瓦解し、各分子がバラバラになることを確認した。また、この際NHERF2はスリット膜が感知した刺激を細胞膜頂部に伝える役割を果たしていることも明らかにした。この報告はスリット膜刺激が細胞頂部に伝わる経路についての最初の報告である。
    Ephrin-B1の機能解析に並行して、臨床応用に向けた研究を進め、Ephrin-B1はスリット膜障害時、尿中に漏出し、スリット膜障害に起因する病態把握のための重要な尿中診断マーカーとなることを明らかにした。

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  • 次世代シーケンサー解析によるネフローゼ症候群の新規治療標的分子の探索

    2016年4月 - 2019年3月

    制度名:科学研究費助成事業

    研究種目:若手研究(B)

    提供機関:日本学術振興会

    張 瑩

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    担当区分:研究代表者  資金種別:競争的資金

    ネフローゼ症候群の蛋白尿発症に関わる分子機構の全体像は未解明で、現在まで蛋白尿に対する有効な治療法がない。本研究の目的は、ネフローゼ症候群の病態モデルを用い、次世代シーケンサー解析による腎臓糸球体のすべてのmRNA 配列の解読と発現量の測定を行った上で、ネフローゼ症候群の糸球体における発現変動遺伝子を同定し、ネフローゼ症候群に対する新規治療の標的候補分子を決定することである。

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  • 糸球体と尿のプロテオーム解析による糖尿病性腎症のバイオマーカーの探索

    2015年4月 - 2015年8月

    制度名:科学研究費助成事業

    研究種目:若手研究(B)

    提供機関:日本学術振興会

    張 瑩

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    担当区分:研究代表者  資金種別:競争的資金

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  • On-siteプロテオミクスによるIgA腎症・膜性腎症の抗原同定と発症機序解明

    研究課題/領域番号:22659165

    2010年 - 2012年

    制度名:科学研究費助成事業

    研究種目:挑戦的萌芽研究

    提供機関:日本学術振興会

    許 波, 山本 格, 張 エイ

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    配分額:3300000円 ( 直接経費:3000000円 、 間接経費:300000円 )

    IgA腎症と膜性腎症は免疫複合体の形成などに深く関わっていると考えられているが、その抗原についてはまだ不明な点が多い状況である。本研究はIgA腎症・膜性腎症の症例の10μm厚さの切片を特別な前処理を行った後、レーザーマイクロダイセックション(LMD)を用いて、切片から糸球体を切り出した。当研究室で開発したOn-siteプロテオミクス法で解析し、バイオインフォマテクス解析や疾患パスウェ解析に介して、正常糸球体検体と発現差異のあるタンパク質群はIgA・膜性腎症の発生機序への関わりを探求することを試みした。

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