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 mostcommonformofCONisureteropelvicjunctionobstruction(UPJO),whentheblockageoccurswherethe renal pelvis connects to the ureter. Despite the high medical burden, we have a poor understanding of the molecularandgeneticcausesofUPJOs,withveryfewnon-surgicalanimalmodels.Usinginvitrocellmodels, 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 deathandaleakyurothelialbarrieragainsturine,withanincreaseinTGFb?1expressionandmesenchymalcell proliferation.ByE18.5,theureterlumenattheUPJwasobliteratedduetostromalremodelingandovergrowth offibroblasticcells.Basedonthesefindings,wehypothesizethatexocysttraffickingisnecessarytoestablish afunctionalurotheliuminembryonicureters,andfailureoftheurothelialbarrieractivatesapathogenicwound 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 barrierformationandthedynamicsofstratification.WewillalsotestifdeletionofE-cadherinintheembryonic ureterissufficienttorecapitulatetheUPJOphenotype.(2)Identifythepathogenicmechanismdrivingthe mesenchymal expansion responsible for UPJOs in Sec10FL/FL;?Ksp-Cre ureters. We will test if urothelial celldeathatcriticalstagesofureterdevelopmentissufficienttocausetheUPJOphenotype.Wewillutilizea novel ex vivo ureter explant model to test if urine contributes to the pathogenic fibroproliferative response. Finally,wewillblockTGFb?1-SMAD2/3signalinginvivotodetermineifwecanamelioratetheUPJOphenotype inSec10FL/FL;?Ksp-Cremice.Theanticipatedoutcomeofthisproposalisidentifyingthemolecularmechanisms thatcauseprenatalUPJOinthismousemodel,whichwillhaveahighimpactonourunderstandingofhuman ureterdevelopmentandCON,andwilllaythegroundworkfordevelopmentofnoveltherapies.
Congenitalobstructionsoftheurinarytractarethemostcommoncausesofkidneydiseaseininfantsand children.WehavediscoveredthatmutationofSec10,akeysubunitoftheexocysttraffickingcomplex,results in lethal prenatal ureter obstructions in mice. This was due to disrupted maturation and subsequent degenerationoftheepithelialcellsthatlinetheureter.Withthisnewanduniquemousemodelofhumanureter obstructions,wecandeterminethecriticalroleoftheexocystinureterdevelopment.Thisproposalwilllaythe groundworkforidentificationofnewcandidategenesandpotentialtherapiesforthisdeadlypediatricdisease.
