The G9a-TRIM21 axis exacerbates diabetic renal ischemia-reperfusion injury by inducing methylation-dependent ubiquitination and degradation of FoxO3a to promote oxidative stress and pyroptosis.

INTRODUCTION: Diabetic renal ischemia-reperfusion injury (RIRI) is a severe surgical complication with particularly poor outcomes in diabetic patients. The histone methyltransferase G9a has been implicated in various pathological processes, but its role in diabetic RIRI remains unclear. Emerging evidence suggests that non-histone protein methylation may play crucial roles in cellular responses to ischemic injury. OBJECTIVES: This study aimed to investigate the functional role of G9a in diabetic RIRI and elucidate its molecular mechanisms, with particular focus on its regulation of FoxO3a stability, oxidative stress and cellular pyroptosis. METHODS: We established diabetic RIRI models using G9a conditional knockout mice and HK-2 cells under high glucose conditions. Renal function was assessed through serum creatinine and BUN measurements. Histopathological evaluation and molecular analyses were performed to examine tissue damage and pyroptosis markers. Mechanistic studies included mass spectrometry identification of G9a-interacting proteins, co-immunoprecipitation to verify protein interactions, and ubiquitination assays to characterize post-translational modifications. Site-directed mutagenesis was employed to identify critical residues in FoxO3a regulation. RESULTS: G9a expression was significantly upregulated in diabetic RIRI models. Genetic ablation of G9a attenuated renal injury and reduced oxidative stress and pyroptosis in both in vivo and in vitro models. Mechanistically, G9a directly interacted with and methylated FoxO3a at lysine 262, which facilitated its recognition by the E3 ubiquitin ligase TRIM21. TRIM21 subsequently mediated K48-linked polyubiquitination of FoxO3a at lysine 176, leading to proteasomal degradation. This G9a-mediated FoxO3a degradation promoted oxidative stress, NLRP3 inflammasome activation and pyroptosis. Importantly, pharmacological inhibition of G9a with BIX-01294 or FoxO3a overexpression significantly ameliorated diabetic RIRI. CONCLUSION: Our study reveals a novel G9a-TRIM21-FoxO3a regulatory axis in diabetic RIRI, where G9a-mediated methylation licenses FoxO3a ubiquitination and degradation, thereby promoting pyroptosis and oxidative stress. These findings identify G9a as a potential therapeutic target for preventing or treating diabetic kidney ischemia-reperfusion injury.