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

项目摘要 糖尿病早期，肾小球滤过率异常。这是早期的血流动力学表型 引发糖尿病肾脏随后的死亡。糖尿病高滤过是由联合 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。还原的钙离子 内流是糖尿病患者肾小球系膜细胞收缩功能减退的关键因素。然而，潜在的 机制(S)仍然知之甚少。此外，新出现的证据表明NADPH氧化酶-， 尤其是NOX4衍生的ROS在糖尿病肾病的发生发展中起重要作用。然而，潜在的 其作用机制和下游信号通路在很大程度上尚不清楚。这项提议旨在测试 一种新发现的钙离子通透性通道蛋白TRPC6蛋白参与收缩的假说 巨噬细胞功能及NADPH氧化酶介导的ROS下调巨噬细胞蛋白表达 糖尿病高滤过。将测试三个具体目标。(1)确定TRPC6是否调节收缩 肾小球系膜细胞在体外、体外和体内系统中的功能和钙信号。(2)探索假设 ROS介导糖尿病大鼠肾小球系膜细胞TRPC6蛋白表达下调 (培养的MC)和活体动物模型。(3)确定ROS的来源，重点是NADPH氧化酶，以及 在TRPC6的信号通路中，ROS下游的分子主要集中在核因子-B，通过 糖尿病。从这项新颖的研究中获得的信息将促进我们目前对 糖尿病肾病发生发展的分子机制，为药物治疗提供理论依据 通过对所提出的途径进行干预来设计和临床治疗糖尿病。此外，TRPC6已经被 发现在各种类型的细胞中起着重要作用。然而，对TRPC6频道的监管，特别是在 基因转录水平，目前尚不清楚。拟议的研究将通过以下方式解决这一重要问题 探讨ROS是否通过核因子-B机制抑制TRPC6基因转录。因此，这个项目是 对ROS和TRPC6油田都感兴趣。

Project Summary 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-¿B, 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-¿B mechanism. Thus, this project is of interest to both ROS and TRPC6 fields.