The exocyst in ureter development and congenital obstructions
Biography
Overview
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?TGFb?1?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? ureter? explant? model? to? test? if? urine? contributes? to? the? pathogenic? fibroproliferative? response.?? Finally,?we?will?block?TGFb?1-?SMAD2/3?signaling?in?vivo?to?determine?if?we?can?ameliorate?the?UPJO?phenotype? in?Sec10FL/FL;?Ksp-?Cre?mice.??The?anticipated?outcome?of?this?proposal?is?identifying?the?molecular?mechanisms? that?cause?prenatal?UPJO?in?this?mouse?model,?which?will?have?a?high?impact?on?our?understanding?of?human? ureter?development?and?CON,?and?will?lay?the?groundwork?for?development?of?novel?therapies.?? ??
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