Characterization of TRPC6 As A Cause for Focal and Segmental Glomerulosclerosis

描述(由申请人提供)：我们先前已经发现该瞬时受体电位阳离子通道6(TRPC6)基因的一个点突变，导致一种常染色体显性形式的家族性局灶性和节段性肾小球硬化(FSGS)。这种P112Q突变导致TRPC6介导的钙信号在血管紧张素II等激动剂的作用下发生显著变化。然而，TRPC6P112Q突变导致FSGS典型疾病表型的确切机制尚不清楚。我们假设，单个TRPC6P112Q等位基因的存在会导致一种足以促进肾脏疾病发展的中间表型，并且TRPC6P112Q蛋白可能会放大由血管紧张素II等配体触发的损伤信号，这些配体在促进肾脏损伤和蛋白尿方面发挥关键作用。为了验证这些假设，我们提出了以下具体目标：(1)定义FSGS2家族中一个独特的人类细胞系资源中TRPC6P112Q突变的杂合性所赋予的中间表型。这些细胞系将被用来确定这种突变对钙信号、TRPC6运输、细胞生长和细胞凋亡的影响。(2)利用小鼠模型建立FSGS基因TRPC6P112Q突变的因果关系。将特定目标1的体外工作扩展到体内环境，我们将建立TRPC6P112Q杂合突变的敲入小鼠模型。(3)确定TRPC6P112Q突变仅在足细胞中表达是否足以引起蛋白尿。TRPC6在足细胞中表达，足细胞功能异常在FSGS的发病机制中起重要作用。我们认为TRPC6P112Q蛋白在足细胞中的表达扰乱了关键的细胞功能，我们将通过建立一个仅在足细胞中表达突变的TRPC6P112Q蛋白的转基因小鼠来测试这一点。我们预计这些动物会出现明显的蛋白尿和FSGS。(4)明确TRPC6缺乏对肾脏的影响。我们假设TRPC6P112Q突变导致一种功能获得表型的过度钙流动，从而导致肾小球损伤。这种突变可能改变了TRPC6维持正常肾小球功能所必需的一些关键功能。为了区分这些可能性，将在一组-/-TRPC6小鼠身上检查肾脏结构和功能。我们最初的假设认为，TRPC6的缺失可能会增强对肾脏损伤和蛋白尿的抵抗力。我们将在诱导性肾脏疾病模型中测试这一点。

DESCRIPTION (provided by applicant): We have previously identified a point mutation in thes Transient Receptor Potential Cation Channel 6 (TRPC6) gene causing an autosomal dominant form of familial focal and segmental glomerulosclerosis (FSGS). This P112Q substitution causes a marked alteration in TRPC6-mediated calcium signals in response to agonists such as angiotensin II. However, the precise mechanism by which the TRPC6P112Q mutation leads to the prototypical disease phenotype of FSGS is not clear. We hypothesize that the presence of a single TRPC6P112Q allele causes an intermediate phenotype which is sufficient to promote the development of renal disease and that the TRPC6P112Q protein may amplify injurious signals triggered by ligands such as angiotensin II that play a key role in promoting kidney injury and proteinuria. To test these hypotheses, we propose the following specific aims: (1) To define the intermediate phenotype conferred by heterozygosity for the TRPC6P112Q mutation in a unique resource of human cell lines from the FSGS2 family. These cell lines will be used to define the consequences of this mutation on calcium signaling, TRPC6 trafficking, cell growth and apoptosis. (2) To establish causality of the TRPC6P112Q mutation for FSGS using a mouse model. Extending the in vitro work in specific aim 1 to the in vivo setting, we will develop a knock-in mouse model of the heterozygous TRPC6P112Q mutation. (3) To determine whether expression of the TRPC6P112Q mutation only in podocytes is sufficient to cause proteinuria. TRPC6 is expressed in podocytes and abnormalities of podocyte function are essential to the pathogenesis of FSGS. We suggest that expression of the TRPC6P112Q protein in podocytes disrupts key cellular functions and we will test this by generating a transgenic mouse line expressing the mutant TRPC6P112Q protein only in podocytes. We anticipate that these animals will develop overt proteinuria and FSGS. (4) To define the consequences of TRPC6 deficiency in the kidney. We have hypothesized that the TRPC6P112Q mutation causes a gain-of-function phenotype of exaggerated calcium flux that generates glomerular injury. It is possible that the mutation alters some critical function of TRPC6 necessary for maintaining normal glomerular function. To distinguish these possibilities, kidney structure and function will be examined in a line of -/-TRPC6 mice. Our original hypothesis suggests that the absence of TRPC6 may confer resistance to kidney injury and proteinuria. We will test this in models of induced renal disease.