271 - Notch Signaling Regulates Proliferation of Renal Keratin5+ Urothelial Cells Following Urinary Tract Obstruction

Urinary Tract Obstruction (UTO) is a leading cause of pediatric chronic kidney disease (CKD). While no interventions can prevent CKD progression, heterogenous outcomes suggest differences in ability of the kidney to adapt to hydrostatic pressure and pelvicalyceal dilatation. Congenital and acquired UTO triggers protective urothelial remodeling. Basal Keratin 5 (K5)-UCs function as age-restricted progenitors, giving rise to protective apical Uroplakin (UPK)-UCs, but the molecular regulation of renal K5-UC progenitors is unknown. The Notch signaling pathway regulates progenitor proliferation and differentiation in several organ systems. Therefore, we hypothesize that the Notch signaling pathway governs renal K5-UCs during UTO.

Objective: The objective is to determine whether the Notch signaling pathway governs renal K5-UCs during UTO.

Design/Methods: We profiled the expression of members of the Notch signaling pathway using RNAscope (Jag2, Notch1, Dll1), immunohistochemical (ICH) and immunofluorescent (IF) localization (Notch1, NICD, RBPJ, Hes7). We used Megabladder mice (Mgb, congenital lower UTO), and unilateral ureteral obstruction (UUO, acquired upper UTO) to model UTO. We used K5CreERT2/+;RBPJfl/fl;R26tdT/+ mice (RBPJ-cKO) to induce disruption of Notch signaling in K5-UCs. We investigated the impact of Notch loss of function on urothelial integrity using IF (K5, UPK, Ki67, EdU-incorporation).

Results:

 Renal urothelium expresses Jag2, Dll1 (ligands), and Notch1 (receptor), but low or undetectable levels of NICD (activated Notch) during homeostasis. UTO leads to increased NICD in K5-UCs, thus, we proceeded to disrupt Notch during UTO. After validating deletion of RBPJ (loss of Notch function) in K5-UCs, we then performed UUO in RBPJ-cKO and control (RBPJfl/fl;R26tdT/+) mice. At post-operative day 2 (peak stage of UTO-induced urothelial proliferation) RBPJ-cKOs exhibited increased Ki67 and EdU in K5-UCs compared to controls.

Conclusion(s): Our study suggests renal K5-UC progenitors are regulated by the Notch signaling pathway during UTO. Further studies are warranted to determine whether increased K5-UC proliferation in RBPJ-cKOs leads to impaired renal urothelial remodeling, and whether this impacts parenchymal integrity. Nevertheless, these findings advance our understanding of renal adaptation to UTO, and implicate a potential signaling pathway with therapeutic utility for mitigating obstructive nephropathy.