离子通道蛋白TRPC6基因突变致钙信号紊乱参与足细胞损伤的分子机制

It was reported that TRPC6 genetic mutation could lead to podocyte injury and massive proteinuria. Molecular mechanism of mutagenesis induced podocyte injury is unclear. In preliminary studies, functional changes of TRPC6 were determined for the first time in podocytes of conditionally immortalized mouse cell line (MPC5). The patch-clamp technique for current recording was utilized in the whole-cell mode to determinate the channel function of TRPC6. It was found that TRPC6 ion channel could be activated excessively with increasing of Ca2+ concentration in cultured podocytes. However, the relevant molecular mechanisms of TRPC6 ion channel have not been reported. Therefore, we put forward research hypothesis, TRPC6 ion channel activation induced by IP3 receptor activated, and calcium signaling of TRPC6-mutant may be involved in the regulation of calcium signaling network. Moreover, screening of small molecular compounds to close TRPC6 channels may block signaling pathway related to podocyte injury. In this studies, we used inducible mouse podocytes transfected with genetic mutation of TRPC6 as a model. Application of the patch clamp verify that the TRPC6 channel activation mechanism. Analysis of Ca2+ related genes and signal pathways. Using whole-cell patch clamp technique to identify the therapeutic targets and screening novel pharmacological TRPC6 inhibitors as calcium-channel blockers. Furthermore, generation of podocyte specific TRPC6-mutant transgenic mices associated with FSGS. Sepecial calcium-channel blockers were detected in inducing TRPC6 down-regulated expression, retroconversion effects involve decreasing TRPC6 expression via positive feedback signaling pathway, receptor function and enzyme activity also been studied. In summary, providing the precise target for clinical treatment of proteinuria. The future development of effective calcium channel blocker will explore new avenues in the clinical treatment.

国内外研究证实，仅编码TRPC6基因发生突变即可导致足细胞损伤和蛋白尿，但该突变致足细胞损伤的分子机制不清。本课题前期研究首次在小鼠足细胞上应用电生理膜片钳技术，建立了检测TRPC6通道功能的实验方法，证实TRPC6主要通过调控钙离子内流而影响足细胞功能，但TRPC6的门控机制不清，IP3支路可能参与激活机制。因此我们假设：突变TRPC6通过诱导IP3受体活化使通道激活；启动的钙信号转导可能存在钙信号网络参与调节；小分子化合物封闭TRPC6通道可能阻断足细胞损伤相关分子-信号通路。本研究从钙信号转导这一全新角度，建立足细胞转染TRPC6突变体，应用膜片钳证实TRPC6通道激活机理及抑制剂的阻断作用；分析与Ca2+相关的功能基因及信号通路；通过TRPC6突变体转基因小鼠，验证钙信号相关信号分子、受体及酶的活化状态及抑制剂作用，以揭示突变TRPC6致足细胞损伤分子机制，探索新的蛋白尿治疗靶点。

肾小球滤过屏障结构和/或功能受损，导致大量蛋白从血液中漏出，被认为是蛋白尿性肾病发生的主要原因；但确切的分子机制仍未阐明，更影响了进一步研发治疗蛋白尿的分子靶点。近年来国内外学者认为肾小球滤过屏障最外侧的足细胞（podocyte）足突之间的裂孔隔膜（ slit diaphragm，SD）， 是影响肾小球滤过屏障功能的最关键结构之一，在通过对一些先天性/遗传性肾病综合征的研究先后证实SD是由许多关键蛋白如nephrin、podocin、CD2AP、neph1-3、P-cadherin 和FAT1等组成的功能复合体。大量临床研究以及动物实验证实，上述任一分子的基因异常已足以导致足细胞足突融合和大量蛋白尿的发生，但导致蛋白尿发生的分子机制远未阐明。. 本研究从钙信号转导这一全新角度，建立足细胞转染TRPC6 突变体的体外细胞模型；并绘制了IP3 启动的TRPC6 钙信号传递网络，分析其中与Ca2+相关的功能基因及信号通路；利用分子机制指导靶向药物应用，采用膜片钳技术检测小分子化合物对TRPC6 通道的阻滞作用；通过TRPC6 突变体转基因小鼠，验证通道阻滞剂对钙信号转导通路、受体及酶活性的影响；证实突变TRPC6 致足细胞损伤的分子机制。. 本研究揭示了TRPC6 门控机制，初步明确TRPC6 钙信号转导主要通路。本研究基于前期我们在体外细胞模型探讨的TRPC6 离子通道通过介导Ca2+内流诱导足细胞损伤的初步结果，以离子通道蛋白TRPC6 基因多态性为切入点，证实TRPC6 是通过与IP3 受体直接作用而被激活；并证实突变TRPC6 的钙信号转导存在钙信号网络参与调节，从与以往国内外研究不同的全新角度，阐明足细胞损伤过程中参与调控的钙信号网络及其分子调控机制，为蛋白尿发生的分子机制提供新的理论依据。. 本研究利用分子机制指导靶向药物应用：首次尝试应用小分子化合物8009-5364 和2910-0498 作为功能获得性突变TRPC6 通道的阻滞剂，研究其对钙信号网络中转导通路，受体及酶活性存在抑制效应；本课题是利用分子机制指导靶向药物应用，构建靶向制剂开辟治疗新途径的全新尝试。