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

描述（申请人提供）：在糖尿病的早期阶段，GFR变得异常。这种早期血液动力学表型引起糖尿病肾脏的随后死亡。糖尿病性高滤过是由于肾传入小动脉和MC对血管收缩剂的反应性降低所致。Ca 2+内流减少是糖尿病MC收缩功能低下的关键因素。然而，对潜在的机制仍然知之甚少。此外，新出现的证据表明NADPH氧化酶-，特别是Nox 4-衍生的ROS在糖尿病肾病的发展。然而，潜在的机制和下游信号通路在很大程度上是未知的。本研究旨在验证一种新发现的钙离子通道蛋白TRPC 6对MCs的收缩功能有贡献的假说，并通过NADPH氧化酶介导的ROS下调MCs中的TRPC 6蛋白导致糖尿病超滤。将测试三个具体目标。(1)确定TRPC 6是否在体外、离体和体内系统中调节肾小球MC的收缩功能和Ca 2+信号传导。(2)探索ROS介导糖尿病在体外（培养的MC）和体内动物模型中肾小球MC中TRPC 6蛋白表达下调的假设。(3)确定糖尿病TRPC 6下调信号通路中ROS的来源，重点是NADPH氧化酶，以及ROS下游分子，重点是NF-κ B。从这项新的研究中获得的信息将促进我们目前对糖尿病肾病发展的分子机制的理解，因此为药物设计和通过干预拟议途径进行糖尿病临床治疗提供了理论基础。此外，已发现TRPC 6在多种细胞类型中发挥重要作用。然而，TRPC 6通道的调控，特别是在基因转录水平，目前是未知的。拟议的研究将通过调查ROS是否通过NF-κ B机制抑制TRPC 6基因转录来解决这一重要问题。因此，该项目对ROS和TRPC 6领域都很感兴趣。 公共卫生相关性：本研究旨在验证TRPC 6通道蛋白表达水平降低导致糖尿病早期高滤过的假设。我们进一步提出活性氧参与的信号通路介导了糖尿病中TRPC 6蛋白的减少。

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.