Glomerular Disease Mechanisms mediated by Podocyte TRPC6

描述(申请人提供)：该项目旨在描述足细胞中功能失调的离子通道TRPC6的致病机制。TRPC6是连接钙信号进入足细胞足突的缝隙横隔膜的一部分，突变的TRPC6或野生型TRPC6蛋白的诱导表达分别可导致遗传性和获得性蛋白尿疾病。因此，TRPC6的(dys-)调节可能影响数百万肾小球疾病患者。我们已经产生了新的初步数据，证明了TRPC6和突触素之间独特的功能相互作用产生了一个调节环，该环协调生理足细胞功能，但在TRPC6介导的钙信号失调的情况下触发足细胞损伤。我们建议检验我们的中心假设，即TRPC6和突触素在动态足细胞肌动蛋白细胞骨架的调节中协同作用。突触素与TRPC6结合并调节其膜表达，TRPC6介导的钙内流通过钙敏感酶钙调神经磷酸酶和蛋白激酶A(PKA)决定突触素的稳定性。根据我们的新数据，TRPC6通道活性的增加通过激活钙调神经磷酸酶而扰乱正常足细胞肌动蛋白的细胞骨架动力学，进而导致突触素的降解，从而导致蛋白尿性肾病。此外，TRPC6介导的钙内流减少导致PKA活性降低，从而减少保护性突触素的磷酸化，从而导致其随后的降解。TRPC6的正常钙转运维持生理突触素水平，允许动态调节足细胞足突系统和肾屏障。具体目标1将阐述TRPC6如何调节突触素介导的肌动蛋白细胞骨架动力学。具体目标2试图确定突触素如何影响TRPC6通道的活性和定位。在具体目标3中，我们将在体内研究TRPC6缺乏和TRPC6过度活动对足细胞肌动蛋白细胞骨架动力学和肾小球屏障功能的影响。我们的工作将阐明一个重要的下游机制，即允许由调节失调的TRPC6引起的足细胞损伤。我们的发现可能对理解包括局灶节段性肾小球硬化(FSGS)在内的TRPC6相关人类肾脏疾病的病理生物学有广泛的意义，并促进干扰TRPC6的抗蛋白尿药的开发及其对足细胞的细胞效应。 公共卫生相关性：这项拨款提案的广泛、长期目标是促进我们对足细胞生物学和导致蛋白尿和肾病综合征的分子机制的理解，这些分子机制源于足细胞损伤。如果我们的假设是正确的，我们的工作可能对理解包括局灶节段性肾小球硬化(FSGS)在内的TRPC6相关人类肾脏疾病的病理生物学具有广泛的意义，并促进干扰TRPC6通道功能的抗蛋白尿药的开发。

DESCRIPTION (provided by applicant): This project aims to delineate disease causing mechanisms of dysfunctional ion channel TRPC6 in podocytes. TRPC6 is part of the slit diaphragm relating Ca2+ signals into podocyte foot processes and mutated TRPC6 or induced expression of wild type TRPC6 protein can cause hereditary and acquired proteinuric diseases, respectively. Thus, (dys-) regulation of TRPC6 likely affects millions of patients with glomerular disease. We have generated novel preliminary data demonstrating a unique functional interaction between TRPC6 and synaptopodin producing a regulatory loop, that coordinates physiological podocyte function but triggers podocyte injury in the case of dysregulated TRPC6 mediated Ca2+ signals. We propose to test our central hypothesis that TRPC6 and synaptopodin cooperate in the regulation of the dynamic podocyte actin cytoskeleton. While synaptopodin binds to TRPC6 and regulates its membrane expression, TRPC6 mediated Ca2+ influx determines the stability of synaptopodin through Ca2+ sensitive enzymes calcineurin and protein kinase A (PKA). According to our novel data, increased TRPC6 channel activity disrupts normal podocyte actin cytoskeletal dynamics via the activation of calcineurin that in turn leads to the degradation of synaptopodin thereby causing proteinuric kidney disease. In addition, diminished TRPC6 mediated Ca2+ influx into podocytes leads to reduced activity of PKA and thus reduced protective synaptopodin phosphorylation with its subsequent degradation. Normal Ca2+ transport of TRPC6 maintains physiological synaptopodin levels that allow a dynamic regulation of the podocyte foot process system and kidney barrier. Specific Aim 1 will address how TRPC6 regulates synaptopodin-mediated actin cytoskeletal dynamics. Specific Aim 2 seeks to define how synaptopodin affects TRPC6 channel activity and localization. In Specific Aim 3, we will study the consequences of TRPC6 deficiency and TRPC6 hyperactivity on podocyte actin cytoskeletal dynamics and glomerular barrier function in vivo. Our work will clarify an important downstream mechanism that permits podocyte injury originating from dysregulated TRPC6. Our findings may have broad implications for the understanding of the pathobiology of TRPC6-related human kidney diseases including Focal Segmental Glomerulosclerosis (FSGS) and promote the development of anti-proteinuric drugs interfering with TRPC6 and its cellular effects on podocytes. PUBLIC HEALTH RELEVANCE: The broad, long-term goal of this grant proposal is to advance our understanding of podocyte biology and the molecular mechanisms leading to proteinuria and nephrotic syndrome that originate from podocyte injury. If our hypothesis is correct, our work may have broad implications for the understanding of the pathobiology of TRPC6-related human kidney diseases including Focal Segmental Glomerulosclerosis (FSGS) and promote the development of anti-proteinuric drugs interfering with TRPC6 channel function.