Renovascular disease induces functionally relevant, locus-specific alterations to DNA methylation and hydroxymethylation in swine scattered tubular-like cells

Background: Scattered tubular-like cells (STCs) are dedifferentiated renal tubular cells that repair other damaged kidney cells. STCs may be damaged and rendered ineffective by renovascular disease (RVD), but the underlying mechanisms remain unknown. We hypothesized that RVD induces changes in methylated (5mC) and hydroxymethylated (5hmC) DNA and modulates the transcriptomic profile and functional properties of swine STCs.

Methods: CD24+/CD133+ STCs were harvested from pig kidneys after 10 weeks of RVD or sham (n=6 each) and their 5mC and 5hmC profiles of individual peaks were examined by immunoprecipitation sequencing (MeDIP-/hMeDIP-seq, respectively, n=3 each). Integrated (MeDIP/hMeDIPseq/mRNA-seq) analysis was performed followed by functional analysis of overlapping differentially expressed (DE) genes. STC-protective effects were assessed in vitro before and after epigenetic (Bobcat339) modulation.

Results: MeDIP-seq analysis identified 1,362 hyper-methylated and 1,432 hypo-methylated peaks in RVD-STCs compared to Normal-STCs, which correlated with 80 upregulated and 55 downregulated genes in RVD-STCs. hMeDIP-seq revealed 1,447 hyper-hydroxymethylated and 765 hypo-hydroxymethylated peaks in RVD-STCs versus Normal-STCs, which correlated with 80 genes upregulated and 53 downregulated in RVD-STCs. Overlapping upregulated genes were mainly implicated in the regulation of oxidative phosphorylation, apoptosis, and lipid metabolism (e.g., STAT6), whereas overlapping downregulated genes were mainly involved in cell proliferation. Importantly, RVD increased STAT6 protein expression and impaired the proliferative capacity of STCs, which were partially reversed by treatment with Bobcat339, which also enhanced the ability of RVD-STCs to promote the viability of injured tubular epithelial cells.

Conclusions: Renal ischemia induces locus-specific epigenetic alterations, associated with transcriptomic changes and impaired reparative function of swine STCs. These observations may contribute to develop novel approaches to preserve the reparative capacity of STCs in individuals with RVD.