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Abstract: PO0875

Exocyst Inactivation in Urothelial Cells Disrupts Autophagy and Upregulates the Fibroblast Growth Factor-Inducible 14 (Fn14) Receptor

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 500 Development, Stem Cells, and Regenerative Medicine

Authors

  • Ortega, Michael, University of Hawai'i at Manoa John A Burns School of Medicine, Honolulu, Hawaii, United States
  • Villiger, Ross, University of Hawai'i at Manoa John A Burns School of Medicine, Honolulu, Hawaii, United States
  • Harrison-Chau, Malia H., University of Hawai'i at Manoa John A Burns School of Medicine, Honolulu, Hawaii, United States
  • Lieu, Suzanna Kiki, University of Hawai'i at Manoa John A Burns School of Medicine, Honolulu, Hawaii, United States
  • Tamashiro, Kadee-Kalia, University of Hawai'i at Manoa John A Burns School of Medicine, Honolulu, Hawaii, United States
  • Lee, Amanda J., University of Hawai'i at Manoa John A Burns School of Medicine, Honolulu, Hawaii, United States
  • Patwardhan, Geetika Yi, University of Hawai'i at Manoa John A Burns School of Medicine, Honolulu, Hawaii, United States
  • Fogelgren, Ben, University of Hawai'i at Manoa John A Burns School of Medicine, Honolulu, Hawaii, United States
Background

Despite their prevalence, the etiology of congenital ureter obstructions in infants is poorly understood, with little evidence identifying genetic or environmental causes. We previously reported a unique mouse model of in utero ureteropelvic junction obstruction (UPJO) in which ureter urothelial cells with deleted Exoc5 gene failed to differentiate into a stratified epithelium and underwent cell death. This resulted in bilateral UPJOs, hydronephrosis, and neonatal lethality. Here, we investigated the urothelial cells prior to cell death to identify the disrupted cell processes necessary for urothelial differentiation and ureter development.

Methods

Gene expression profiling was performed on E16.5 ureters of Exoc5FL/FL;Ksp-Cre mice and control littermates, with validation using real time qPCR and immunohistochemistry. Follow up investigations utilized an ex vivo ureter explant organ culture model, where mouse embryonic ureters were isolated at E15.5 and maintained in culture for 72 hours. Additionally, we used primary human urothelial cells (pHUCs) and immortalized SV-HUC1 cells for advanced studies on exocyst regulation of autophagy, which was measured through immunoblotting and immunostaining of p62, LC3I/II, and autophagy-related genes (ATGs).

Results

Analysis of gene profiling data from E16.5 Exoc5FL/FL;Ksp-Cre ureters revealed that metabolic pathways were significantly downregulated and NF-kB signaling was significantly upregulated, indicating cell stress. The highest upregulated gene was Fn14 (>30-fold), a member of the TNF receptor subfamily that binds the ligand TWEAK. Fn14 is upregulated in damaged tissues and can activate non-canonical NF-kB signaling and cell death via multiple pathways. Using ureter explants and cell line models, we found exocyst is critical for urothelial autophagy, which when disrupted, led to a high Fn14 increase and cell death.

Conclusion

From our data, we propose that autophagy is necessary for urothelial differentiation during ureter development, and irregular autophagy may trigger urothelial cell death through Fn14 signaling. This disruption of autophagy during a critical stage in ureter development may contribute to UPJOs in humans.

Funding

  • NIDDK Support