Glomerular Disease Mechanisms mediated by Podocyte TRPC6

描述（由申请人提供）：本项目旨在描述足细胞中功能障碍的离子通道TRPC 6的致病机制。TRPC 6是将Ca 2+信号与足细胞足突相关的狭缝隔膜的一部分，突变的TRPC 6或野生型TRPC 6蛋白的诱导表达可分别引起遗传性和获得性蛋白尿疾病。因此，TRPC 6的（失调）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TRPC 6和synaptopodin之间的独特的功能相互作用，产生一个调节环，协调生理足细胞功能，但触发足细胞损伤的情况下，TRPC 6介导的Ca2+信号失调。我们建议测试我们的中心假设，TRPC6和synaptopodin合作的动态足细胞肌动蛋白细胞骨架的调节。虽然突触足蛋白结合TRPC 6并调节其膜表达，但TRPC 6介导的Ca 2+内流通过Ca 2+敏感酶钙调磷酸酶和蛋白激酶A（PKA）决定突触足蛋白的稳定性。根据我们的新数据，增加的TRPC 6通道活性通过激活钙调磷酸酶破坏正常的足细胞肌动蛋白细胞骨架动力学，这反过来又导致突触足蛋白的降解，从而引起蛋白尿性肾病。此外，减少TRPC 6介导的Ca2+流入足细胞导致PKA活性降低，从而降低保护性突触足蛋白磷酸化及其随后的降解。TRPC 6的正常Ca2+转运维持生理性突触足蛋白水平，其允许足细胞足突系统和肾屏障的动态调节。具体目标1将解决TRPC 6如何调节突触足蛋白介导的肌动蛋白细胞骨架动力学。具体目标2旨在确定突触足蛋白如何影响TRPC 6通道活性和定位。在具体目标3中，我们将研究TRPC 6缺乏和TRPC 6过度活跃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，允许足细胞损伤源于TRPC 6失调。我们的研究结果可能对理解TRPC 6相关的人类肾脏疾病（包括局灶节段性肾小球硬化症（FSGS））的病理生物学具有广泛的意义，并促进干扰TRPC 6及其对足细胞的细胞效应的抗蛋白尿药物的开发。 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TRPC 6相关的人类肾脏疾病（包括局灶节段性肾小球硬化症（FSGS））的病理生物学具有广泛的意义，并促进干扰TRPC 6通道功能的抗蛋白尿药物的开发。

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.