Mechanisms of proteinuria induced by RhoA GTPases

描述（由申请人提供）： 遗传学研究强调了肾小球上皮细胞（足细胞）在肾小球生物学中的重要性。肾病综合征的几种家族性形式是由肾小球足细胞专门表达的蛋白质突变引起的。它们相互交错的足突覆盖肾小球基底膜（GBM）的外表面，并在维持肾小球滤过屏障的完整性方面发挥关键作用。越来越多的文献表明，这一功能可能是由属于Rho GT3家族的小GTP酶调节的。在这方面，RhoA可以通过促进足细胞表型来稳定肾小球结构，足细胞表型抑制蛋白尿和足突（FP）消失。在这种情况下，一些基础水平的RhoA活性将是有益的。相反，炎症过程诱导的高水平RhoA活性可能会导致足细胞损伤，这可能是通过抑制促生存途径，进而增强足细胞凋亡。由于足细胞在出生后不能有效地复制，足细胞凋亡可能导致功能足细胞数量减少，越来越多的证据表明，这可能是促进肾小球硬化的最终共同途径。基于这些观察结果，我们假设基础Rho活性的抑制通过使肾小球结构不稳定和促进FP消失和肾小球选择性透过性的变化而对肾小球滤过屏障功能有害。相反，高水平的Rho活性也通过引起足细胞耗竭对肾小球功能具有不利影响。为了研究这些假设，提出了两个具体的目标。在具体目标#1中，我们将研究Rho依赖性信号级联在促进足细胞损伤中的作用，通过创建转基因（TG）小鼠，其使用诱导型启动子系统在肾小球足细胞中特异性表达组成型激活的RhoA GT3。然后，我们将确定Rho激活是否通过增强体内足细胞凋亡引起肾小球损伤，以及确定介导凋亡效应的信号通路。在具体目标#2中，我们将通过使用TG技术在肾小球足细胞中表达显性负性Rho GTdR，并确定基础条件下RhoA抑制是否引起足细胞表型的变化，从而促进蛋白尿，来确定基础Rho活性是否在维持肾小球滤过屏障的完整性中起重要作用。如果所提出的实验是成功的，这些研究将提供重要的见解Rho依赖性信号级联在调节足细胞功能的作用。 公共卫生相关性： 肾脏疾病是一种严重且昂贵的医疗问题，影响着美国超过250万人。这项拨款申请的目的是确定是否涉及肾脏疾病发病机制的多种激素系统激活促进肾脏损伤的最终共同信号通路。如果研究成功，这种信号通路将成为开发预防肾衰竭药物的重要治疗靶点。

DESCRIPTION (provided by applicant): Genetic studies have highlighted the importance of glomerular epithelial cells (podocytes) in glomerular biology. Several familial forms of nephrotic syndrome are caused by mutations in proteins expressed exclusively by glomerular podocytes. Their interdigitating foot processes cover the exterior surface of the glomerular basement membrane (GBM) and play a key role in maintaining the integrity of the glomerular filtration barrier. A growing literature suggests that this function may be regulated by small GTPases belonging to the Rho GTPase family. In this regard, RhoA may stabilize the glomerular architecture by promoting a podocyte phenotype that inhibits proteinuria and foot process (FP) effacement. In this scenario, some basal level of RhoA activity would be beneficial. In contrast, high levels of RhoA activity induced by inflammatory processes may cause podocyte injury, perhaps by inhibiting prosurvival pathways and, in turn, enhancing podocyte apoptosis. Because of the inability of the podocyte to effectively replicate postnatally, podocyte apoptosis may cause a decrease in the number of functional podocytes, which, accumulating evidence suggests, may be a final common pathway promoting glomerulosclerosis. Based on these observations, we hypothesized that inhibition of basal Rho activity is detrimental to glomerular filtration barrier function by destabilizing the glomerular architecture and promoting FP effacement and changes in glomerular permselectivity. In contrast, high levels of Rho activity also have adverse effects on glomerular function by causing podocyte depletion. To investigate these hypotheses, two specific aims are proposed. In specific aim #1, we will investigate the role of Rho-dependent signaling cascades in promoting podocyte injury by creating transgenic (TG) mice that express a constitutively activate RhoA GTPase specifically in glomerular podocytes using an inducible promoter system. We will then determine if Rho activation causes glomerular injury by enhancing podocyte apoptosis in vivo as well as determine the signaling pathways mediating the apoptotic effect. In specific aim #2, we will determine if basal Rho activity plays an important role in maintaining the integrity of the glomerular filtration barrier by expressing a dominant negative Rho GTPase in glomerular podocytes using TG technologies and determining if RhoA inhibition under basal conditions causes a change in the podocyte phenotype that promotes proteinuria. If the proposed experiments are successful, the studies will provide important insights into the role of Rho-dependent signaling cascades in regulating podocyte function. PUBLIC HEALTH RELEVANCE: Kidney disease is a significant and expensive medical problem affecting over 2.5 million people in the United States of America. The goal of this grant application is determine if multiple hormonal systems implicated in the pathogenesis of kidney disease activate a final common signaling pathway that promotes kidney damage. If the studies are successful, this signaling pathway would be an important therapeutic target for the development of drugs to prevent kidney failure.