Characterization of TRPC6 As A Cause for Focal and Segmental Glomerulosclerosis

描述（由申请人提供）：我们之前已经发现瞬时受体电位阳离子通道6 （TRPC6）基因的点突变导致家族性局灶性和节段性肾小球硬化（FSGS）的常染色体显性形式。这种P112Q取代导致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改变。然而，TRPC6P112Q突变导致FSGS原型疾病表型的确切机制尚不清楚。我们假设单个TRPC6P112Q等位基因的存在导致一种足以促进肾脏疾病发展的中间表型，TRPC6P112Q蛋白可能放大由配体（如血管紧张素II）触发的损伤信号，这些配体在促进肾损伤和蛋白尿中起关键作用。为了验证这些假设，我们提出了以下具体目标：(1)确定来自FSGS2家族的独特人类细胞系资源中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、细胞生长和凋亡的影响。(2)利用小鼠模型建立TRPC6P112Q突变与FSGS的因果关系。将特异性目标1的体外工作扩展到体内环境，我们将开发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，足细胞功能异常对FSGS的发病至关重要。我们认为足细胞中TRPC6P112Q蛋白的表达会破坏关键的细胞功能，我们将通过产生仅在足细胞中表达突变TRPC6P112Q蛋白的转基因小鼠来验证这一点。我们预计这些动物会出现明显的蛋白尿和FSGS。(4)明确肾虚TRPC6的后果。我们假设TRPC6P112Q突变导致过度钙通量的功能获得表型，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-/- trpc6小鼠中检查肾脏结构和功能。我们最初的假设表明，TRPC6的缺失可能导致对肾损伤和蛋白尿的抵抗。我们将在肾脏疾病模型中进行试验。

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.