Activated Protein C Epigenetically Regulates DNMT1 Expression and the Sustained Hyperglycemic Memory in Diabetic Kidney Disease by Targeting miR-148a-3p

Background: Activated protein C (aPC) reverses the sustained expression of the senescence-associated cell cycle inhibitor p21 and hence the hyperglycemic memory in diabetic kidney disease (DKD) by regulating DNA methyltransferase-1 (DNMT1). How aPC regulates DNMT1 in the context of DKD remains unknown. Methods: We conducted in silico analyses to identify the top microRNA (miRNA) regulators of DNMT1. The expression of the target miRNAs was studied in kidneys of a murine DKD model and in control and glucose-stimulated kidney tubular cells in vitro. Mechanistic relevance of the identified miRNA for the effects of aPC was studied in vitro and in vivo using specific locked nucleic acid. Results: miR-148a-3p was identified in silico as a top regulator of DNMT1 expression. Increased expression of miR-148a-3p in the kidneys of wild-type diabetic mice was confirmed. This was associated with DNMT1 suppression. miR-148a-3p expression was reduced in diabetic APChigh mice, that have increased plasma levels of aPC, which has been shown to increase DNMT1 expression. High glucose treatment induced miR-148a-3p expression in kidney tubular cells and the expression remained sustained despite glucose reduction. Exposure to aPC markedly reduced miR-148a-3p expression in vitro. Treatment of diabetic mice with a locked nucleic acid targeting miR-148a-3p markedly reduced albuminuria and increased DNMT1 expression in the kidneys preventing sustained p21 expression and the associated renal senescence and injury. Conclusion: Our data identify miR-148a-3p as an epigenetic regulator targeted by aPC to restore DNMT1 expression and hence to reverse the sustained p21-associated senescence phenotype and the hyperglycemic memory in DKD. This identifies an epigenetic mechanism targeted by aPC. Funding: Government Support – Non-U.S.