NADPH oxidases-derived ROS downregulate TRPC6 in mesangial cells in diabetes

描述（申请人提供）：糖尿病早期，GFR异常。这种早期血流动力学表型引起糖尿病肾的后续死亡。糖尿病性高滤过是由肾传入小动脉和MCs对血管收缩剂的反应性降低共同引起的。Ca2+内流减少是糖尿病MCs收缩性降低的关键因素。然而，其潜在的机制仍然知之甚少。此外，新出现的证据暗示NADPH氧化酶-特别是nox4衍生的ROS参与糖尿病肾病的发展。然而，其潜在的机制和下游信号通路在很大程度上是未知的。本研究旨在验证TRPC6蛋白（一种新发现的Ca2+可渗透通道蛋白）参与MCs的收缩功能以及NADPH氧化酶介导的ROS对MCs中该蛋白的下调导致糖尿病高滤过的假设。三个具体目标将受到考验。(1)确定TRPC6是否在体外、离体和体内系统中调节肾小球MCs的收缩功能和Ca2+信号。(2)通过体外（培养MCs）和体内动物模型，探讨ROS介导糖尿病肾小球MCs中TRPC6蛋白表达下调的假设。(3)确定糖尿病TRPC6下调信号通路中以NADPH氧化酶为重点的ROS来源和以NF-ko为重点的下游ROS分子。从这项新研究中获得的信息将推进我们目前对糖尿病肾病发展的分子机制的理解，从而为通过干预所提出的途径来设计药物和临床治疗糖尿病提供理论依据。此外，TRPC6已被发现在多种细胞类型中发挥重要作用。然而，TRPC6通道的调控，特别是在基因转录水平上的调控，目前尚不清楚。拟议的研究将通过研究ROS是否通过NF-ko机制抑制TRPC6基因转录来解决这一重要问题。因此，该项目对ROS和TRPC6领域都很感兴趣。

DESCRIPTION (provided by applicant): At the early stage of diabetes, the GFR becomes supernormal. This early hemodynamic phenotype provokes the subsequent demise of a diabetic kidney. The diabetic hyperfiltration is derived from a combined decreased responsiveness of both the renal afferent arterioles and the MCs to vasoconstrictors. Reduced Ca2+ influx is a critical contributing factor to the hypocontractility of MCs in diabetes. However, the underlying mechanism(s) are still poorly understood. Furthermore, emerging evidence implicates NADPH oxidases-, particularly Nox4-derived ROS in the development of diabetic nephropathy. However, the underlying mechanism and downstream signaling pathway are at a large extent unknown. This proposal seeks to test the hypothesis that TRPC6 protein, a newly found Ca2+ permeable channel protein, contributes to the contractile function of MCs and downregulation of the protein in MCs by NADPH oxidases-mediated ROS results in diabetic hyperfiltration. Three specific aims will be tested. (1) Determine whether TRPC6 regulates contractile function and Ca2+ signaling of glomerular MCs in in vitro, ex vivo, and in vivo systems. (2) Explore the postulate that ROS mediate downregulation of TRPC6 protein expression in glomerular MCs by diabetes in an in vitro (cultured MCs) and in vivo animal model. (3) Determine the source of ROS, focusing on NADPH oxidases, and the molecules downstream ROS, focusing on NF-ko, in the signaling pathway of TRPC6 downregulation by diabetes. The information obtained from this novel study will advance our current understanding of the molecular mechanism for the development of diabetic nephropathy, and therefore provides a rationale for drug design and clinical treatment of diabetes by intervening in the proposed pathway. In addition, TRPC6 has been found to play an important role in a variety of cell types. However, regulation of TRPC6 channel, particularly at gene transcriptional level, is unknown currently. The proposed studies will tackle this important issue by investigating if ROS repress TRPC6 gene transcription through the NF-ko mechanism. Thus, this project is of interest to both ROS and TRPC6 fields. PUBLIC HEALTH RELEVANCE: The proposed study is to test the hypothesis that a decrease in expression level of TRPC6 channel protein leads to the diabetic hyperfiltration at early stage of Diabetes. We further propose that a reactive oxygen species-involved signaling pathway mediates the decrease in TRPC6 protein in diabetes.