Investigation of renal fibrosis molecular mechanisms focusing on membrane proteins

• Project/Area Number: 18K08233
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 53040:Nephrology-related
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: YUI Naofumi 東京医科歯科大学, 大学院医歯学総合研究科, 非常勤講師 (00633976)
• Project Period (FY): 2018-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2020: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2019: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2018: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
• Keywords: 腎臓線維化 / 細胞骨格 / ミオシン / アクチン / 細胞膜輸送体 / 慢性腎臓病 / 非筋肉型ミオシン / レンチウイルス / ノックダウン / 線維芽細胞 / 筋線維芽細胞 / α平滑筋アクチン

Outline of Final Research Achievements

In this study, We investigated what kind of molecules are accumulated in plasma membrane of renal fibroblasts during its differentiation into myofibroblast. We performed cell surface biotinylation assays with or without TGF-b1 stimulation using NRK-49F rat renal fibroblast. The collected molecules are analyzed using silver-staining and LC-MS. We identified MYH-9 as a membrane protein of renal fibroblast during its differentiation. Using its specific inhibitor, blebbistatin, and lentivirus-mediated gene knockdown, its importance on aSMA production, Smad2/3 nucleus transportation are clearly demonstrated. Upon differentiation into myofibroblast, MYH-9 accumulated in plasma membrane and nucleus. By inhibition using blebbistation, MYH-9 accumulation on plasma membrane and nucleus was blocked, and TGF-b1 induced aSMA production, Smad2/3 nuclear acccumulation were suppressed.

Academic Significance and Societal Importance of the Research Achievements

本邦では慢性腎臓病は8人に1人が罹患しているとされ国民的疾患概念である。この進行を緩やかにし腎不全の発症を抑制することは今後の医療の最も重要な課題の一つと言える。そのためには原因疾患は何であれ共通する病態生理である腎臓線維化を治療できるようにすることが必要となる。腎臓線維化がどのような機序で進展してゆくのかを解明することが重要な一歩になる。今回腎臓の線維芽細胞が活性化する際にどのような分子が細胞膜を貫通する形で集積するかを分子生物学的手法を用いて研究しMYH-9という分子を同定し機能を解析しその重要性を細胞生物学的に示すことが出来た。今後のこの領域の研究の方向性に貢献しうるものと考える。

Research Products

• [Journal Article] LRBA is essential for urinary concentration and body water homeostasis (2022)
  • Author(s): Hara Y, Ando F, Oikawa D, Ichimura K, Yanagawa H, Sakamaki Y, Nanamatsu A, Fujiki T, Mori S, Suzuki S, Yui N, Mandai S, Susa K, Mori T, Sohara E, Rai T, Takahashi M, Sasaki S, Kagechika H, Tokunaga F, Uchida S.
  • Journal Title: Proceedings of the National Academy of Sciences Volume: 119 Issue: 30
  • DOI: 10.1073/pnas.2202125119
  • Peer Reviewed / Open Access
• [Journal Article] Chlorpromazine Induces Basolateral Aquaporin-2 Accumulation via F-Actin Depolymerization and Blockade of Endocytosis in Renal Epithelial Cells. (2020)
  • Author(s): Yui N, Bouley R, Terlouw A, Cheung PW, Brown D.
  • Journal Title: Cells Volume: 23 Issue: 4 Pages: 1057-1057
  • DOI: 10.3390/cells9041057
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] AKAPs-PKA disruptors increase AQP2 activity independently of vasopressin in a model of nephrogenic diabetes insipidus (2018)
  • Author(s): Ando Fumiaki、Mori Shuichi、Yui Naofumi、Morimoto Tetsuji、Nomura Naohiro、Sohara Eisei、Rai Tatemitsu、Sasaki Sei、Kondo Yoshiaki、Kagechika Hiroyuki、Uchida Shinichi
  • Journal Title: Nature Communications Volume: 9 Issue: 1 Pages: 1411-1411
  • DOI: 10.1038/s41467-018-03771-2
  • Peer Reviewed / Open Access
• [Presentation] 腎臓線維芽細胞活性化における非筋肉型ミオシン 2A の重要性 (2020)
  • Author(s): 油井 直史，名和 眞希子，内田 信一.
  • Organizer: 日本腎臓学会学術総会
• [Presentation] Derivatives of FMP-API-1/27 Robustly Activate AQP2 Water Channels Independently of Vasopressin (2019)
  • Author(s): Ando, Fumiaki, Yui, Naofumi, Mandai, Shintaro, Isobe, Kiyoshi, Mori, Takayasu, Susa, Koichiro, Nomura, Naohiro, Sohara, Eisei, Rai, Tatemitsu, Uchida, Shinichi,
  • Organizer: American Society of Nephrology
  • Int'l Joint Research
• [Presentation] Epithelial Depolarization Induces AQP2 Up-regulation via Non-Canonical Ser-269 Phospho-Regulation Independently of Any Hormonal and Chemical Stimulation (2018)
  • Author(s): Yui Naofumi、Ando Fumiaki、Sasaki Sei、Uchida Shinichi
  • Organizer: American Society of Nephrology
  • Int'l Joint Research
• [Presentation] AKAPs-PKA Disruptors Robustly Increase AQP2 Activity Independently of Vasopressin (2018)
  • Author(s): Ando Fumiaki、Yui Naofumi、Nomura Naohiro、Rai Tatemitsu、Sasaki Sei、Uchida Shinichi
  • Organizer: American Society of Nephrology
  • Int'l Joint Research
• [Presentation] Phosphorylation of Ser261 and Dephosphorylation of Ser269 Is Important for Urinary Excretion of AQP2 (2018)
  • Author(s): Sasaki Sei、Sakai Masaki、Mizumura Hiroki、Matsumoto Tomoki、Noda Yumi、Yui Naofumi、Ishibashi Kenichi
  • Organizer: American Society of Nephrology
  • Int'l Joint Research