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.