Novel Reciprocal Regulation Between DNA Methylation and NF-kB Activation in Maladaptive Kidney Repair

Background: Chronic inflammation is key to maladaptive kidney repair and renal fibrosis after AKI, leading to the progression to CKD. However, it remains unclear how this persistent, low level of inflammation is maintained. Methods: Cultured mouse kidney proximal tubule cells were treated with RLDC in vitro, mice were subjected to RLDC in vivo. 5-aza (a pharmacologic inhibitor of DNA methyltransferase-DNMT) and JSH23 (inhibitor of NF-kB) were used initially to prove the involvement of DNA methylation and NF-kB in these models. We further established kidney proximal tubule-specific Dnmt3a (PT-Dnmt3a-KO) or p65 knockout (PT-p65-KO) mouse models to study maladaptive kidney repair after RLDC treatment. snRNA-Seq was performed to identify potential targets of DNA methylation. Results: DNMT3a was induced during maladaptive kidney repair after RLDC treatment, accompanied by increases in DNA methylation. Inhibition of DNA methylation with 5-aza suppressed renal fibrosis and CKD progression. Knockout of Dnmt3a from kidney proximal tubules prevented renal fibrosis, promoted kidney repair, and improved renal function recovery in post-RLDC mice. Both 5-aza and Dnmt3a deficiency alleviated p65 expression, NF-kB activation and chronic inflammation in post-RLDC kidneys. snRNA-Seq revealed the DNA methylation of PDLIM2, an E3 ligase for p65 ubiquitination and degradation. Moreover, inhibition of DNA methylation with 5-aza or DNMT3a knockout preserved PDLIM2 and reduced p65 in RLDC-treated mice, indicating the regulation of NF-kB by DNA methylation via PDLIM2. The inhibition of NF-kB with JSH23 or knockout of proximal tubule p65 decreased DNMT3a expression and DNA methylation, alleviated renal fibrosis, improved renal function, and suppressed CKD progression after RLDC treatment. Chromatin immunoprecipitation assay further detected the binding of p65 to DNMT3a gene promoter after RLDC-treatment, suggesting transcriptional regulation of DNMT3a and associated DNA methylation. Conclusion: The results unveil a novel reciprocal regulation between DNA methylation and NF-kB activation that is responsible for the chronic, persistent inflammation during maladaptive kidney repair. On one hand, DNMT3a-mediated hypermethylation represses PDLIM2 expression, leading to p65 accumulation for NF-kB activation and the induction of pro-inflammatory factors. On the other hand, NF-kB induces DNMT3 transcriptionally. Funding: NIDDK Support, Veterans Affairs Support