G Protein Regulation of Glomerular Epithelial Cells

描述(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(裂隙横隔膜)之间的“交界处”对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，在糖尿病肾病和其他慢性肾脏疾病的病程早期突出。裂隙横隔膜的丢失会导致肾小球血流动力学改变、蛋白尿和进行性肾功能衰竭。裂隙横隔膜有许多典型上皮细胞连接的特征，但人们对这些结构是如何调节的知之甚少。基于我们在MDCK细胞上的工作，我们假设G蛋白可能是调节裂隙横隔膜结构/功能的关键分子。GA12通过与ZO-1的相互作用可能调节足细胞中的Rho和/或Src通路以及肌动蛋白细胞骨架。在目标1中，将用共聚焦显微镜、免疫电子显微镜和共沉淀法(免疫和GST融合蛋白)研究ZO-1和GA12在小鼠肾小球和培养的足细胞中的相互作用。培养的足细胞将被用来定义从GA12到Rho和/或Src激酶的信号通路。将建立一组抑制剂和稳定的足细胞系，表达GA12、Rho和Src的活性和非活性突变体，以表征对屏障(细胞旁通量分析)和结构变化(通过共聚焦定位信号(GA12、Rho、Src)和狭缝横隔膜(ZO-t、肌动蛋白、neaffin))蛋白的功能影响。在目标2中，将开发动物模型来表征体内的这些信号通路。将使用两种方法：活性GA12(Q229L)的“敲入”和Q229La12的转基因足细胞特异性表达。“敲入”的靶向构建接近完成，小鼠足细胞蛋白(NPHS2)启动子将被用来将激活的GA12靶向足细胞。对不同年龄的动物进行生物化学研究，以寻找肾脏疾病(蛋白尿、肌酐)的证据，并通过光镜和电子显微镜进行形态观察。培养足细胞的研究结果将决定未来的转基因模型(Rho，Src)。这些研究将揭示对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，在那里可以测试常见足细胞疾病(糖尿病和其他)的新治疗策略。

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.