The Rho GTPases Cdc42 and Rac1 in diabetic Nephropathy

描述(由申请人提供)：项目目标 Rho GTP酶CDc42和rac1是细胞内的信号分子，最著名的是调节肌动蛋白细胞骨架。然而，调节细胞反应的其他作用也已被描述。在非吞噬细胞中，rac1对NADPH氧化酶的激活很重要，而Cdc42似乎通过竞争性抑制抑制了NADPH氧化酶产生的活性氧(ROS)。大量证据表明，NADPH氧化酶是糖尿病/葡萄糖诱导的ROS损伤的主要因素，最近的研究表明，在糖尿病肾病中，足细胞过度产生NADPH介导的ROS导致足细胞凋亡、耗竭和疾病进展。因此，研究调节足细胞NADPH氧化酶活性的机制可以揭示糖尿病损伤的新分子靶点和改进的治疗方法。 这项建议包括初步数据显示，在糖尿病人的肾小球和两种糖尿病肾病的小鼠模型中，CDC42的表达减少。利用人类和小鼠全基因组基因表达谱构建了一个保守的肾小球转录网络，我们发现CDC42是最重要的调控节点之一。此外，我们还培育并鉴定了足细胞中有条件缺失CDc42或rac1的小鼠。在这些小鼠中的发现强调了Rho GTP酶在足细胞生物学和肾小球健康中的重要性。 我们推测，足细胞中Rac1的缺失将减少足细胞中ROS的生成和细胞的凋亡，从而导致小鼠糖尿病肾病的改善。相反，CDC42的缺失将加剧足细胞凋亡和糖尿病肾病。具体地说，我们将：(1)利用RNAi技术研究高糖环境下体外培养的人足细胞ROS诱导和凋亡中CDc42和rac1的作用，以及(2)在足细胞特异性CDc42或rac1失活的糖尿病肾病小鼠模型中，研究在体足细胞中Rho GTP酶CDc42和rac1的作用。我们的第三(3)个目标将是利用Kretzler实验室的系统生物学专业知识来定义足细胞特异性CDC42或rac1失活的糖尿病小鼠的肾小球转录网络，并与野生型糖尿病小鼠和人类的肾小球转录网络进行比较。 糖尿病肾病是美国终末期肾病的主要原因，对人类的痛苦和巨大的经济负担有很大的影响。从机制上理解CDC42和rac1在糖尿病肾病中的作用将揭示新的疾病途径和治疗靶点。 公共卫生相关性：Rho GTPase rac1是NADPH氧化酶的必需成分，而Cdc42可以竞争性地抑制rac1介导的NADPH氧化酶的激活。我们将利用体外、体内和系统生物学的方法，研究CDC42和rac1在高血糖、NADPH氧化酶、氧化损伤、足细胞死亡和糖尿病肾病进展的机制环节中的作用。糖尿病肾病是美国终末期肾病的主要原因，从机制上了解CDC42和rac1在糖尿病肾病中的作用将揭示新的疾病途径和治疗靶点。

DESCRIPTION (provided by applicant): Project Aims The Rho GTPases Cdc42 and Rac1 are intracellular signaling molecules best known for regulating the actin cytoskeleton. However, additional roles modulating cellular responses have also been described. Rac1 is important for the activation of NADPH oxidase in nonphagocytic cells and Cdc42 appears to inhibit the generation of reactive oxygen species (ROS) by NADPH oxidase through competitive inhibition. Numerous lines of evidence reveal NADPH oxidase as a major contributor to diabetes/glucose-induced ROS-mediated injury and recent publications indicate excessive NADPH-mediated ROS production in the podocyte results in podocyte apoptosis, depletion, and disease progression in diabetic nephropathy. Thus, investigating mechanisms that regulate NADPH oxidase activation in podocytes could unveil new molecular targets and improved therapies in diabetes-induced injury. This proposal includes preliminary data showing that Cdc42 expression is reduced in the glomeruli of diabetic humans and in two mouse models of diabetic nephropathy. Constructing a conserved glomerular transcriptional network using genome-wide gene expression profiling from humans and mice, we find Cdc42 to be one of the most important regulatory nodes. Furthermore, we have generated and characterized mice with conditional deletions of either Cdc42 or Rac1 in the podocyte. Findings in these mice underscore the importance of Rho GTPases in podocyte biology and glomerular health. We hypothesize that a loss of Rac1 in podocytes will reduce ROS generation and apoptosis in the podocyte and result in amelioration of murine diabetic nephropathy. In contrast, a loss of Cdc42 will exacerbate podocyte apoptosis and diabetic nephropathy. Specifically we will: (1) Investigate the role Cdc42 and Rac1 on ROS induction and apoptosis in human podocytes IN VITRO in a high glucose environment using RNAi technology, and (2) Investigate the role of the Rho GTPases Cdc42 and Rac1 in podocytes IN VIVO in a mouse model of diabetic nephropathy with podocyte specific inactivation of Cdc42 or Rac1. Our third (3) aim will be to use the Systems Biology expertise of the Kretzler laboratory to define the glomerular transcriptional networks in diabetic mice with podocyte specific inactivation of Cdc42 or Rac1, and compare to glomerular transcriptional networks in wild-type diabetic mice and humans. Diabetic nephropathy is the leading cause of end-stage renal disease in the United States and has a large impact on human suffering and a substantial economic burden. Providing a mechanistic understanding of the role of Cdc42 and Rac1 in diabetic nephropathy will reveal novel disease pathways and therapeutic targets. PUBLIC HEALTH RELEVANCE: The Rho GTPase Rac1 is a required component of NADPH oxidase, and Cdc42 can competitively inhibit Rac1-mediated NADPH oxidase activation. We will investigate the role of Cdc42 and Rac1 in the mechanistic link of hyperglycemia, NADPH oxidase, oxidative injury, podocyte death, and the progression of diabetic nephropathy using in vitro, in vivo, and systems biology approaches. Diabetic nephropathy is the leading cause of end-stage renal disease in the United States, and providing a mechanistic understanding of the role of Cdc42 and Rac1 in diabetic nephropathy will reveal novel disease pathways and therapeutic targets.