Pax8 Maintains the Epithelial Phenotype of Proximal Tubule Cells via Distal Regulatory Elements

Background: Pax2 and Pax8 are homologous DNA-binding proteins that orchestrate kidney development, regulate urine concentration in the kidney medulla, and dictate sensitivity to ischemic injury in the kidney cortex. However, the targets of Pax proteins that mediate these functions remain poorly defined. The goal of this project was to identify genes regulated by Pax proteins in the proximal tubule. Methods: A line of Pax8-conditional, Pax2-null immortalized proximal tubule epithelial cells with a constitutive driver of tamoxifen-dependent Cre (Rosa26-CreER) were derived from adult transgenic mice. A line with spontaneous loss of Pax2 was selected to focus on the effects of Pax8, which is expressed at higher levels in the proximal tubule. Pax8 can be depleted from these cells by addition of 4-OH tamoxifen (4-OHT) to the culture medium. RNA and chromatin samples were collected before and after genetic depletion of Pax8 and submitted for RNA sequencing and chromatin-immunoprecipitation sequencing (ChIP-seq) for Pax8 and histone H3 lysine 4 trimethylation (H3K4me3). Results: Pax8 protein was depleted by 2 days after 4-OHT exposure. By day 3, cells lost cobblestone epithelial morphology and organization of cell-cell junction proteins. Cell proliferation also slowed. RNA sequencing showed upregulated genes were associated with epithelial-to-mesenchymal transition and inflammatory responses. Downregulated genes were associated with oxidative metabolism. These expression changes mimicked those observed in vivo after proximal-tubule-selective depletion of Pax8. ChIP-seq was used to assess the correlation between gene expression and Pax8 chromatin binding. Promoters bound by Pax8 showed a small but significant reduction in H3K4me3 after Pax8 depletion relative to those lacking Pax8. However, differential gene expression was most strongly enriched with genes associated with distal Pax8 binding sites, between 3-100 kb from the transcription start site. These sites had low H3K4me3 signal. Conclusion: Pax8 is necessary for the maintenance of differentiated epithelial phenotype in proximal tubule epithelial cells. Differential gene expression induced by Pax8 depletion was strongly associated with Pax8 binding at distal regulatory elements with low H3K4me3. This finding suggests Pax8 maintains gene expression of proximal tubules cells via binding at enhancer elements. Funding: NIDDK Support