Kidney failure is a major cause of morbidity and mortality in the United States and worldwide. Transcriptional regulation of podocyte diseases is not as yet well understood. Our long term goal is to define the precise mechanisms of proteinuria and glomerular disease, so as to develop new treatments in the future. The goal of this proposal is to understand the role of transcriptional factor Zinc Fingers and Homeoboxes 3 (ZHX3) in the pathogenesis of glomerular disease. In Specific Aim 1, the post-translational cleavage and phosphorylation of ZHX3 prior to migration into the nucleus will be studied. We will then study whether blocking nuclear import or export of ZHX3 causes alterations in the gene expression profile of ZHX3 target genes. In Specific Aim 2, the individual effects of both ZHX3 protein fragments on the gene expression profile in cultured cells will be studied by real time PCR.Next, interaction with other known and putative interacting proteins will be assessed. Specific factors that influence the binding of ZHX3 to the COL4A3 promoter will be studied by a combination of chromatin immunoprecipitation and site directed mutagenesis. Next, the influence of ZHX3 and interacting partners on the promoter activity of selected genes will be assessed. In Specific Aim 3, the indirect protein : protein interaction between ZHX3 and aminopeptidase A (APA) in the non-nuclear compartment will be explored in detail. During induction of proteinuria with anti-APA antibodies in mice, migration of ZHX3 into the podocyte nucleus occurs long before the onset of overt proteinuria. Proteins that interact with APA and ZHX3 will be individually identified using a combination of LC/MS analysis of anti-APA and anti-ZHX3 immunoprecipitates and ongoing yeast two-hybrid studies. Proteins that associate with both ZHX3 and APA will be knocked down in cultured GEC, and the effect of this knockdown on the anti-APA antibody induced gene expression profile in cultured GECs will be assessed. Finally, expression constructs of proteins that link APA and ZHX3 will be co-transfected in a non-epithelial cell line to reproduce co-immunoprecipitation of ZHX3 and APA. Studying the development of kidney disease and the leakage of protein in the urine in rodents will help us better understand the process of kidney disease in humans. This will lead to the development of appropriate treatment strategies in the future.