The Rho GTPases Cdc42 and Rac1 in diabetic Nephropathy

描述（由申请人提供）：项目目标 Rho GTP酶Cdc 42和Rac 1是细胞内信号传导分子，以调节肌动蛋白细胞骨架而闻名。然而，还描述了调节细胞反应的其他作用。Rac 1对非吞噬细胞中NADPH氧化酶的激活是重要的，Cdc 42似乎通过竞争性抑制抑制NADPH氧化酶产生活性氧（ROS）。大量证据表明NADPH氧化酶是糖尿病/葡萄糖诱导的ROS介导的损伤的主要贡献者，最近的出版物表明足细胞中NADPH介导的ROS过度产生导致足细胞凋亡、消耗和糖尿病肾病的疾病进展。因此，研究足细胞中调节NADPH氧化酶活化的机制可以揭示新的分子靶点并改善糖尿病诱导的损伤的治疗。 该提案包括初步数据，表明Cdc 42表达减少的肾小球糖尿病人类和两个小鼠模型的糖尿病肾病。利用人类和小鼠全基因组基因表达谱构建保守的肾小球转录网络，我们发现Cdc 42是最重要的调控节点之一。此外，我们已经产生并表征了足细胞中Cdc 42或Rac 1条件性缺失的小鼠。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Rac 1在足细胞中的缺失将减少足细胞中ROS的产生和细胞凋亡，并导致小鼠糖尿病肾病的改善。相反，Cdc 42的缺失将加剧足细胞凋亡和糖尿病肾病。具体而言，我们将：（1）使用RNAi技术研究Cdc 42和Rac 1在高糖环境中体外人足细胞中对ROS诱导和凋亡的作用，和（2）研究Rho GTP酶Cdc 42和Rac 1在具有Cdc 42或Rac 1的足细胞特异性失活的糖尿病肾病小鼠模型中体内足细胞中的作用。我们的第三（3）个目标是利用Kretzler实验室的系统生物学专业知识来定义糖尿病小鼠中的肾小球转录网络，其中Cdc 42或Rac 1具有足细胞特异性失活，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。提供Cdc 42和Rac 1在糖尿病肾病中作用的机制理解将揭示新的疾病途径和治疗靶点。 公共卫生相关性：Rho GTdR Rac 1是NADPH氧化酶的必需组分，Cdc 42可竞争性抑制Rac 1介导的NADPH氧化酶活化。我们将研究Cdc 42和Rac 1在高血糖，NADPH氧化酶，氧化损伤，足细胞死亡，糖尿病肾病的进展，使用体外，体内和系统生物学方法的机制联系的作用。糖尿病肾病是美国终末期肾病的主要原因，对Cdc 42和Rac 1在糖尿病肾病中作用的机制理解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.