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Abstract: PO1988

Fate-Mapping Supports a Linear Model of Urothelial Formation and Regeneration

Session Information

Category: Pediatric Nephrology

  • 1700 Pediatric Nephrology

Authors

  • Grounds, Kelly, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States
  • Li, Birong, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States
  • Becknell, Brian, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States
  • Jackson, Ashley R., Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States
Background

Urothelium is a highly specialized, slow turnover epithelium that lines the kidney, ureter, bladder and proximal urethra. Bladder urothelium contains several cell types organized into basal (B), intermediate (I) and superficial (S) cell layers. The progenitor responsible for urothelial repair has been the focus of many investigations, with strong support for both B and I cell contenders. We have previously demonstrated that keratin 5+ (K5) urothelial cells (UCs) are context specific progenitors in the kidney. Here, we mapped the fate of K5-UCs across development and following cyclophosphamide (CYC)-induced urothelial injury in the bladder.

Methods

Using tamoxifen (TMX)-inducible Krt5CreERT2;RosazsGreen mice, we permanently labeled K5-UCs with zsGreen (zsGreenK5) across development and evaluated their capacity to form I and S cells during homeostasis or following CYC-induced urothelial injury. Immunofluorescence microscopy was used to determine whether zsGreenK5-UCs were K5+(B-cells), Uroplakin+ (Upk; I and S-cells), or K20+(S-cells). Organoid forming assays were used to evaluate progenitor capacity in vitro.

Results

Baseline analysis of our Cre;LoxP strategy confirmed that zsGreenK5 is specifically expressed in basal K5-UCs 24h after TMX administration at all induction stages. The fate of zsGreenK5-UCs varied, with neonatal (postnatal day [P]1, P7) stages giving rise to adult (P42) I and S cells, the juvenile (P14) stage giving rise to I but not S cells, and adult (P35, P42) stages not escaping the B cell layer. CYC-induced urothelial injury did not engage adult zsGreenK5-UCs for repair, whereas neonatal and juvenile zsGreenK5-UCs gave rise to I and S cells following CYC treatment. Organoid forming assays confirmed that zsGreenK5-UCs could form organoids that express B and I cell markers, and neonatal UCs formed larger organoids than adult UCs.

Conclusion

We show that precise temporal populations of K5-UCs form I cells during homeostasis which in turn are engaged as adults for S cell formation in response to injury. We believe that these findings unite B and I cell progenitor models, by temporally linking a linear progression of B→I→S cell formation. A more complete understanding of the role of discrete urothelial cell populations will enable precise control of urothelial cell differentiation and will inform targeted tissue regeneration strategies.

Funding

  • NIDDK Support