Kidney Week

Abstract: TH-PO0545

Pax8 Regulates Proximal-Tubule-Specific Identity via Distal Enhancer Elements

Session Information

• Development, Stem Cells, and Regenerative Medicine
  November 06, 2025 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Development, Stem Cells, and Regenerative Medicine

• 600 Development, Stem Cells, and Regenerative Medicine

Authors

• Orban, Steven, University of Michigan, Ann Arbor, Michigan, United States

Steven Orban, MS

• Telang, Asha Claire, University of Michigan, Ann Arbor, Michigan, United States

Asha Claire Telang

• Ference-Salo, Jenna T., University of Michigan, Ann Arbor, Michigan, United States

Jenna T. Ference-Salo

• Dressler, Greg R., University of Michigan, Ann Arbor, Michigan, United States

Greg R. Dressler, PhD

• Wan, Ma, National Institute of Environmental Health Sciences, Durham, North Carolina, United States

Ma Wan, PhD

• Delker, Don, National Institute of Environmental Health Sciences, Durham, North Carolina, United States

Don Delker, PhD

• Watts, Jason A., National Institute of Environmental Health Sciences, Durham, North Carolina, United States

Jason A. Watts, MD, PhD

• Beamish, Jeffrey A., University of Michigan, Ann Arbor, Michigan, United States

Jeffrey A. Beamish, MD, PhD

Background

Pax8 is a kidney-selective transcription factor that regulates kidney development, physiology, and recovery from injury. How Pax8 functions remains poorly defined. This project aimed to identify targets and mechanisms of Pax8 function in proximal tubule epithelial cells.

Methods

A proximal tubule epithelial cell line with conditional Pax8 and a constitutive driver of tamoxifen-dependent Cre was derived from adult transgenic mice. Pax8 was genetically depleted from these cells by addition of 4-hydroxytamoxifen to the culture medium. A derivative of this cell line was transduced with a doxycycline-inducible Pax8 expression vector, allowing for temporally-controlled rescue. RNA expression was measured using RNA sequencing (RNA-seq). Epigenetic changes associated with Pax8 were measured using chromatin-immunoprecipitation sequencing (ChIP-seq) for Pax8, transposase-accessible chromatin sequencing (ATAC-seq), and cleavage under targets and tagmentation (CUT&Tag) for histone marks (H3K4me1, H3K4me3, and H3K27ac).

Results

Loss of Pax8 protein following 4-hydroxytamoxifen administration was confirmed by western blot. Following Pax8 deletion, cells lose epithelial cobblestone morphology, upregulate epithelial-to-mesenchymal transition genes (Gene Set Enrichment Analysis, P_adj = 2x10^-4), and downregulate proximal-tubule-specific genes (P_adj = 6x10^-4). Remarkably, doxycycline-induced restoration of Pax8 expression in de-differentiated Pax8-depleted cells restored epithelial morphology and expression of downregulated genes (z-score averaged expression, P < 1x10^-4), including proximal-tubule-specific genes (P_adj = 2x10^-2). Pax8-bound gene promoters did not show changes in histone H3K4me3 or chromatin accessibility after Pax8 depletion. In contrast, Pax8-bound distal enhancer elements lost H3K4me1, H3K27ac, and chromatin accessibility. Differentially expressed genes were disproportionately associated with Pax8-bound distal regulatory elements (P < 1x10^-4).

Conclusion

Pax8 can reversibly modulate proximal-tubule-specific gene expression and epithelial morphology in cultured proximal tubule cells. Pax8 loss dynamically alters distal enhancer elements but not promoters. These observations suggest that Pax8 regulates proximal tubule phenotype via distal enhancer elements.

Funding

• NIDDK Support

Digital Object Identifier (DOI)

• doi: 10.1681/ASN.2025a313mksw