PROJECT SUMMARY Congenital obstructive nephropathy (CON), the most common cause of chronic kidney disease and end stage renal disease in children, is caused by obstruction of the urinary tract during fetal development. The most common form of CON is ureteropelvic junction obstruction (UPJO), when the blockage occurs where the renal pelvis connects to the ureter. Despite the high medical burden, we have a poor understanding of the molecular and genetic causes of UPJOs, with very few non-surgical animal models. Using in vitro cell models, we have shown the eight-protein exocyst trafficking complex to be important for mechanisms of epithelial morphogenesis. To facilitate in vivo studies of the exocyst during mammalian development, we have generated a novel conditional knockout mouse for the exocyst subunit Sec10 using Cre-lox transgenic technology. Targeted deletion of Sec10 in ureteric bud-derived epithelia, using our floxed-Sec10 (Sec10FL) and the Ksp-Cre mouse strains, caused in utero bilateral UPJOs with hydronephrosis, complete anuria, and neonatal lethality. Preliminary studies revealed Sec10FL/FL;;Ksp-Cre knockout ureter urothelium failed to differentiate a superficial layer between gestational day 16.5 (E16.5) and E17.5. This led to urothelial cell death and a leaky urothelial barrier against urine, with an increase in TGFb1 expression and mesenchymal cell proliferation. By E18.5, the ureter lumen at the UPJ was obliterated due to stromal remodeling and overgrowth of fibroblastic cells. Based on these findings, we hypothesize that exocyst trafficking is necessary to establish a functional urothelium in embryonic ureters, and failure of the urothelial barrier activates a pathogenic wound healing response that rapidly occludes the ureter lumen. We will test this hypothesis through the following Aims: (1) Understand how defects in exocyst-dependent membrane trafficking lead to arrested urothelial differentiation and cell death. Our preliminary data show E-cadherin fails to traffic to cell-cell junctions in the E16.5 Sec10FL/FL;;Ksp-Cre urothelial cells. Here, we will determine if Sec10 deletion causes disrupted trafficking of other key cell-cell junction proteins and how this perturbs the mechanism of urothelial barrier formation and the dynamics of stratification. We will also test if deletion of E-cadherin in the embryonic ureter is sufficient to recapitulate the UPJO phenotype. (2) Identify the pathogenic mechanism driving the mesenchymal expansion responsible for UPJOs in Sec10FL/FL;;Ksp-Cre ureters. We will test if urothelial cell death at critical stages of ureter development is sufficient to cause the UPJO phenotype. We will utilize a novel ex vivo ur...