DNMT inhibitor ameliorates hyperglycemia and renal inflammation partially via reversing hypermethylation-induced Klotho suppression in diabetic mice

In diabetic kidney disease (DKD), DNA hypermethylation-mediated silencing of protective genes constitutes a key mechanism of metabolic memory, driving persistent renal injury. Klotho suppression critically exacerbates renal inflammation and injury. Our preliminary studies documented Klotho promoter hypermethylation in DKD kidneys. However, direct evidence linking its sustained downregulation to this epigenetic aberration and demethylation’s therapeutic benefits remains unestablished. This study investigates whether demethylation treatment-mediated reversal of Klotho promoter hypermethylation restores its expression to alleviate renal inflammation and injury in DKD. Diabetic db/db mice and age-matched wild-type controls were used. At 8 weeks, db/db mice received intraperitoneal injections of the DNA methyltransferase (DNMT) inhibitor 5-Azacytidine (5-Aza) or phosphate-buffered saline (PBS) three times weekly. After 12 weeks, samples were collected for analysis. HK-2 cells were utilized for in vitro validation. Demethylation treatment with 5-Aza reduced Klotho promoter hypermethylation and upregulated Klotho expression in db/db mouse kidneys and HK-2 cells. 5-Aza dose-dependently reduced fasting blood glucose by up to 72%, proteinuria, and serum creatinine levels, while improving renal pathology (glomerular hypertrophy, mesangial expansion, basement membrane thickening, podocyte injury). RNA sequencing demonstrated suppression of multiple inflammatory signaling pathways in kidneys of 5-Aza-treated db/db mice. Furthermore, 5-Aza substantially attenuated renal macrophage infiltration and inflammasome activation in db/db mice, concurrently reducing systemic inflammation and improving insulin resistance. Our study demonstrates that DNA hypermethylation drives Klotho downregulation in DKD. DNMT inhibitor alleviates hyperglycemia, renal inflammation and injury in db/db mice by reversing hypermethylation-mediated Klotho suppression. Targeting aberrant DNA methylation represents a novel therapeutic strategy for DKD.