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

描述(申请人提供)：糖尿病早期，肾小球滤过率异常。这种早期的血流动力学表型导致糖尿病肾脏随后的死亡。糖尿病高滤过是由于肾传入小动脉和肾小动脉系膜细胞对血管收缩药的反应性降低所致。钙离子内流减少是糖尿病患者肾小球系膜细胞收缩功能减退的一个重要因素。然而，其背后的机制(S)仍然知之甚少。此外，新的证据表明NADPH氧化酶，特别是NOX4衍生的ROS与糖尿病肾病的发生有关。然而，其潜在的机制和下游的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透性通道蛋白TRPC6蛋白参与系膜细胞收缩功能的假设，以及NADPH氧化酶介导的ROS下调系膜系膜细胞蛋白表达导致糖尿病高滤过的假说。将测试三个具体目标。(1)确定TRPC6在体外、体外和体内系统中是否调节肾小球系膜细胞的收缩功能和钙信号。(2)通过体外培养的肾小球系膜细胞和体内动物模型，探讨ROS介导糖尿病大鼠肾小球系膜细胞TRPC6蛋白表达下调的可能机制。(3)确定糖尿病下调TRPC6信号通路中ROS的来源，重点是NADPH氧化酶，以及ROS下游的分子，重点是NF-Ko。从这一新颖的研究中获得的信息将促进我们目前对糖尿病肾病发生的分子机制的理解，从而为通过干预所提出的途径来进行糖尿病的药物设计和临床治疗提供理论基础。此外，已发现TRPC6在多种细胞类型中发挥重要作用。然而，目前对TRPC6通道的调控，尤其是在基因转录水平上的调控尚不清楚。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，该项目是区域研究组织和TRPC6领域都感兴趣的。 公共卫生相关性：这项拟议的研究旨在验证TRPC6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，在糖尿病中，TRPC6蛋白的减少可能是通过一条与活性氧有关的信号通路来实现的。

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.