G Protein Regulation of Glomerular Epithelial Cells

G蛋白对肾小球上皮细胞的调节

• 批准号: 6707294
• 负责人: BRADLEY M DENKER
• 金额: $ 15.22万
• 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/6707294
• 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）：足细胞是一种高度专业的上皮细胞，沿着肾小球毛细管基底膜形成多个脚步。脚部过程之间的“连接”（缝隙隔膜）对于选择性肾小球渗透性至关重要。足细胞损伤是肾小球疾病的标志，在糖尿病性肾病和其他慢性肾脏疾病原因的早期很突出。缝隙隔膜的丧失导致肾小球血流动力学，蛋白尿和进行性肾衰竭的改变。狭缝隔膜具有典型上皮细胞连接的许多特征，但对这些结构的调节方式知之甚少。根据我们在MDCK细胞中的工作，我们假设G蛋白可能是调节缝隙隔膜结构/功能的关键分子。通过与ZO-1相互作用的GA12可能会调节Podocytes中的RHO和/或SRC途径以及肌动蛋白细胞骨架。在AIM 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）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。 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.在AIM 2中，将开发动物模型来表征这些信号通路。将使用两种方法； Q229LA12的活性GA12（Q229L）和转基因足细胞比表达的“敲击”。 “敲击”的靶向构建体几乎是完整的，小鼠足球素（NPHS2）启动子将用于将活化的GA12靶向足细胞。将对动物进行生化研究，以了解肾脏疾病的证据（蛋白尿，肌酐），并通过各个年龄的光和电子显微镜在形态上进行研究。培养的足细胞研究的结果将确定未来的转基因模型（Rho，SRC）。这些研究将揭示对调节足细胞结构和功能的新见解。动物模型将将这些发现扩展到体内系统，在这些系统中，可以测试常见足细胞疾病（糖尿病等）的新型治疗策略。