Glomerular Disease Mechanisms mediated by Podocyte TRPC6

项目描述（申请人提供）：本项目旨在研究足细胞中功能失调的离子通道TRPC6的致病机制。TRPC6是与Ca2+信号进入足细胞足突相关的狭口隔膜的一部分，TRPC6突变或诱导野生型TRPC6蛋白的表达分别可引起遗传性和获得性蛋白尿疾病。因此，TRPC6的（天）调节可能影响数百万肾小球疾病患者。我们已经生成了新的初步数据，证明TRPC6和synaptopodin之间具有独特的功能相互作用，产生调节回路，协调生理足细胞功能，但在TRPC6介导的Ca2+信号失调的情况下引发足细胞损伤。我们提议验证我们的中心假设，即TRPC6和synaptopodin在动态足细胞肌动蛋白细胞骨架的调控中合作。当synaptopodin与TRPC6结合并调节其膜表达时，TRPC6介导的Ca2+内流通过Ca2+敏感酶钙调磷酸酶和蛋白激酶A （PKA）决定了synaptopodin的稳定性。根据我们的新数据，TRPC6通道活性的增加通过钙调磷酸酶的激活破坏了正常的足细胞肌动蛋白细胞骨架动力学，进而导致突触蛋白的降解，从而导致蛋白尿肾病。此外，TRPC6介导的Ca2+内流到足细胞的减少导致PKA活性降低，从而减少保护性突触蛋白磷酸化及其随后的降解。正常的Ca2+转运TRPC6维持生理突触素水平，允许足细胞足过程系统和肾屏障的动态调节。特异性目标1将讨论TRPC6如何调节突触肽介导的肌动蛋白细胞骨架动力学。Specific Aim 2试图定义synaptopodin如何影响TRPC6通道的活性和定位。在Specific Aim 3中，我们将研究TRPC6缺乏和TRPC6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TRPC6失调导致足细胞损伤的重要下游机制。我们的发现可能对了解TRPC6相关的人类肾脏疾病（包括局灶节段性肾小球硬化（FSGS））的病理生物学有广泛的意义，并促进抗蛋白尿药物的开发，这些药物干扰TRPC6及其对足细胞的细胞作用。

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.