G Protein Regulation of Glomerular Epithelial Cells

G蛋白对肾小球上皮细胞的调节

• 批准号: 6844857
• 负责人: BRADLEY M DENKER
• 金额: $ 15.59万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6844857
• 关键词: G protein; MDCK cell; basement membrane; cell component structure /function; chronic renal failure; diabetic nephropathy; hemodynamics; immunoelectron microscopy; immunofluorescence technique; kidney cell; podocyte; protein protein interaction; renal glomerulus; tight junctions; tissue /cell culture; transfection; urinary bladder epithelium; western blottings

DESCRIPTION (provided by applicant): The podocyte is a highly specialized epithelial cell that forms multiple foot processes along the glomerular capillary basement membrane. The "junction" between foot processes (slit diaphragm) is critical to selective glomerular permeability. Podocyte injury is a hallmark of glomerular disease and is prominent early in the course of diabetic nephropathy and other causes of chronic kidney disease. Loss of slit diaphragms results in altered glomerular hemodynamics, proteinuria and progressive renal failure. The slit diaphragm shares many features of typical epithelial cell junctions, yet little is known about how these structures are regulated. Based upon our work in MDCK cells, we hypothesize that G proteins are likely to be critical molecules for regulating slit diaphragm structure/function. Ga12 through interactions with ZO-1 is likely to regulate Rho and/or Src pathways and the actin cytoskeleton in podocytes. In Aim 1, the interaction of ZO-1 and Ga12 will be studied in mouse glomeruli and cultured podocytes by confocal and immunoelectron microscopy and by coprecipitations (immuno- and GST fusion proteins). Cultured podocytes will be used to define signaling from Ga12 to Rho and/or Src kinase pathways. A combination of inhibitors and stable podocyte cell lines expressing active and inactive mutants of Ga12, Rho, and Src will be established to characterize functional effects on the barrier (paracellular flux assays) and changes in structure (by confocal localization of signaling (Ga12, Rho, Src) and slit diaphragm (ZO-t, actin, nephrin)) proteins. In Aim 2, animal models will be developed to characterize these signaling pathways in-vivo. Two approaches will be utilized; A "knock in" of active Ga12 (Q229L) and transgenic podocyte specific expression of Q229La12. A targeting construct for the "knock in" is nearly complete, and the mouse podocin (NPHS2) promoter wil be used for targeting activated Ga12 to the podocyte. Animals will be studied biochemically for evidence of renal disease (proteinuria, creatinine) and morphologically by light and electron microscopy at various ages. Results from studies in cultured podocytes will determine future transgenic models (Rho, Src). These studies will reveal novel insights into regulation of podocyte structure and function. The animal models will extend these findings to in-vivo systems where novel treatment strategies for common podocyte diseases (diabetes and others) can be tested.

描述（由申请人提供）：足细胞是一种高度特化的上皮细胞，沿着肾小球毛细血管基底膜形成多个足突。足突（裂隙隔膜）之间的“连接处”对于选择性肾小球通透性至关重要。足细胞损伤是肾小球疾病的一个标志，并且在糖尿病肾病和其他慢性肾病病因的病程早期尤为突出。裂隙隔膜的丧失会导致肾小球血流动力学改变、蛋白尿和进行性肾衰竭。狭缝隔膜具有典型上皮细胞连接的许多特征，但人们对这些结构是如何调节的知之甚少。根据我们在 MDCK 细胞中的工作，我们假设 G 蛋白可能是调节狭缝隔膜结构/功能的关键分子。 Ga12 通过与 ZO-1 相互作用可能调节 Rho 和/或 Src 通路以及足细胞中的肌动蛋白细胞骨架。在目标 1 中，将通过共聚焦和免疫电子显微镜以及共沉淀（免疫和 GST 融合蛋白）在小鼠肾小球和培养的足细胞中研究 ZO-1 和 Ga12 的相互作用。培养的足细胞将用于定义从 Ga12 到 Rho 和/或 Src 激酶途径的信号传导。将建立表达 Ga12、Rho 和 Src 活性和非活性突变体的抑制剂和稳定足细胞细胞系的组合，以表征对屏障的功能影响（细胞旁通量测定）和结构变化（通过信号传导的共聚焦定位（Ga12、Rho） ，Src）和狭缝隔膜（ZO-t，肌动蛋白，去氧肾上腺素））蛋白质。在目标 2 中，将开发动物模型来表征这些体内信号通路。将使用两种方法；活性 Ga12 (Q229L) 的“敲入”和 Q229La12 转基因足细胞的特异性表达。 “敲入”的靶向构建体已接近完成，小鼠足细胞蛋白 (NPHS2) 启动子将用于将活化的 Ga12 靶向足细胞。对不同年龄的动物进行生化研究以获取肾脏疾病的证据（蛋白尿、肌酐），并通过光学和电子显微镜进行形态学研究。培养足细胞的研究结果将决定未来的转基因模型（Rho，Src）。这些研究将揭示足细胞结构和功能调节的新见解。动物模型将把这些发现扩展到体内系统，可以测试常见足细胞疾病（糖尿病等）的新治疗策略。