DNA Methyltransferase Inhibitor Alleviates Kidney Inflammation and Injury by Reversing DNA Hypermethylation-Associated Klotho Suppression in Diabetic db/db Mice

Background: Klotho is a well-known anti-aging gene with anti-inflammatory effects. The renal expression of klotho is reduced both in patients with diabetic kidney disease (DKD) and diabetic mice. However, its mechanism is unclear. Studies have indicated elevated DNA methylation levels in the klotho promoter region of the kidney in DKD patients. Since DNA methylation can suppress gene expression, we speculate whether demethylation treatment can alleviate renal inflammation and improve DKD by upregulating renal klotho expression? Methods: Male diabetic db/db (C57BLKS/J-LepRdb/LepRdb) mice and age-matched wild-type (BKS) mice were utilized in the study. At 8 weeks of age, db/db mice were given intraperitoneal injection of the DNA methyltransferase inhibitor 5-azacytidine (1 mg/kg body weight or 2 mg/kg body weight) or saline as control every other day. After 12 weeks of treatment, the blood, urine and kidney samples were collected for measurements. Results: The renal expression of klotho in db/db mice is significantly reduced, along with an increase in DNA methylation levels in the promoter region. 5-azacytidine reduced the renal klotho DNA methylation level and increased klotho expression in db/db mice. This indicates that DNA methylation is involved in the downregulation of klotho in the kidney of DKD. In db/db mice, the renal expression levels of NF-κB and its downstream inflammation-related genes are elevated in db/db mice.5-azacytidine alleviated the renal expression of these inflammatory mediators. It also alleviated the activation of kidney inflammasome and macrophage infiltration. KEGG enrichment analysis of the RNA sequencing results showed that the differentially expressed genes were mainly enriched in NF-κB and TNF signaling pathways. In addition, 5-azacytidine concentration-dependently reduced serum levels of glucose and improved systemic insulin sensitivity. The urinary albumin excretion and serum creatinine in db/db mice were markedly decreased. This was accompanied by alleviation of glomerular hypertrophy, mesangial matrix expansion, glomerular basement membrane thickness and the extent of foot process effacement. Conclusion: DNA methyltransferase inhibitor could alleviate renal inflammation and improve DKD by reversing DNA hypermethylation-associated klotho suppression in diabetic db/db mice.