Characterization of TRPC6 As A Cause for Focal and Segmental Glomerulosclerosis

TRPC6 作为局灶性和节段性肾小球硬化病因的特征

• 批准号: 7084165
• 负责人: MICHELLE P. WINN
• 金额: $ 33.13万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7084165
• 关键词: apoptosis; calcium flux; cell growth regulation; cell line; disease /disorder etiology; disease /disorder model; gene expression; gene mutation; genetically modified animals; glomerulosclerosis; intracellular transport; kidney function; laboratory mouse; model design /development; podocyte; protein deficiency; protein structure function; protein transport; proteinuria

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.

描述（由申请人提供）：我们先前已经鉴定了瞬时受体电位阳离子通道6（TRPC 6）基因中的一个点突变，该点突变导致常染色体显性形式的家族性局灶性节段性肾小球硬化症（FSGS）。这种P112 Q取代引起TRPC 6介导的钙信号响应于激动剂如血管紧张素II的显著改变。然而，TRPC 6 P112 Q突变导致FSGS原型疾病表型的确切机制尚不清楚。我们假设，一个单一的TRPC 6P 112 Q等位基因的存在导致一个中间表型，这是足以促进肾脏疾病的发展，TRPC 6P 112 Q蛋白可能会放大由配体，如血管紧张素II，在促进肾损伤和蛋白尿发挥关键作用触发的有害信号。为了验证这些假设，我们提出了以下具体目标：（1）在来自FSGS 2家族的独特人类细胞系资源中定义TRPC 6P 112 Q突变的杂合性所赋予的中间表型。这些细胞系将用于确定这种突变对钙信号传导、TRPC 6运输、细胞生长和凋亡的影响。(2)使用小鼠模型确定TRPC 6P 112 Q突变与FSGS的因果关系。将特定目标1中的体外工作扩展到体内环境，我们将开发杂合TRPC 6P 112 Q突变的敲入小鼠模型。(3)确定仅在足细胞中表达TRPC 6P 112 Q突变是否足以引起蛋白尿。TRPC 6在足细胞中表达，足细胞功能异常在FSGS的发病机制中至关重要。我们认为TRPC 6P 112 Q蛋白在足细胞中的表达破坏了关键的细胞功能，我们将通过产生仅在足细胞中表达突变TRPC 6P 112 Q蛋白的转基因小鼠系来测试这一点。我们预计这些动物将出现明显的蛋白尿和FSGS。(4)明确肾脏TRPC 6缺乏的后果。我们假设TRPC 6P 112 Q突变导致功能获得性表型的钙流量增加，从而导致肾小球损伤。这是可能的，突变改变了一些关键功能的TRPC 6维持正常肾小球功能所必需的。为了区分这些可能性，将在一系列-/-TRPC 6小鼠中检查肾脏结构和功能。我们最初的假设表明，TRPC 6的缺乏可能赋予抵抗肾损伤和蛋白尿。我们将在诱发性肾病模型中对此进行测试。