Epigenetic Studies in Pkd1 and Pkd2 Conditional Knockout Reveal Mechanism of Suppression of Cholesterol Biosynthesis and Altered Mitochondrial Function

Background: ADPKD caused by mutations in PKD1 or PKD2, presents with remarkably similar phenotypes despite differences in gene function, subcellular localization and temporal expression. The pathogenesis of ADPKD and phenocopy convergence remains unclear. Recent studies report important roles of lipids including cholesterol on function and targeting of PC2. Mitochondrial dysfunction and metabolic reprogramming play important roles in cystogenesis. We have found function of polycystins in ER is vital for mitochondrial health. Mitochondria metabolites regulate the activities of DNA demethylases. We examined the role of ER-mitochondria connection in DNA methylation shifts in ADPKD. Methods: DNA meth array performed on proximal tubules from Pkd1- and Pkd2-cKO mice at precystic stages. Expression studies were done by qRT-PCR, western blotting, and IHC. Total cholesterol content was quantified by fluorometric assay. Filipin staining and confocal imaging were used to assess intracellular cholesterol. Results: DNA methylation analysis revealed global methylation shifts in Pkd1 and Pkd2-cKO vs control. Among these is marked hypermethylation at promoter of 7-dehydrocholesterol reductase gene (Dhcr7) coding an ER-resident enzyme crucial in cholesterol biosynthesis. Dhcr7 transcript and protein levels were also downregulated. Total cholesterol levels were significantly reduced in kidney lysates from cystic Pkd1- and Pkd2-cKO. Filipin staining confirmed a reduction in intracellular cholesterol in cultured proximal tubule cells from Pkd1-cKO mice. Gene ontogeny analysis of differentially methylated genes revealed enrichment in pathways related to mitochondrial function, fatty-acid oxidation, and branched-chain amino acid metabolism. Importantly, the global methylation shifts and candidate gene methylation changes were all reversed by genetic correction of ER Ca release defects. Conclusion: ER-mediated mitochondria dysfunction in ADPKD drives epigenetic rewiring including DNA methylation shifts. The mitochondria-led DNA methylation shifts amplify mitochondrial dysfunction, metabolic reprogramming, and promotes cystogenesis. Cholesterol derivative is believed crucial for PC2 channel activity; the reduction in cholesterol synthesis in Pkd1-cKO may be a mechanism for phenocopy between PKD1 and PKD2 mutations. Funding: Private Foundation Support