BHMT Prevents Renal Ischemia/Reperfusion Injury via Suppressing ROS-induced Apoptosis by Targeting NOX4

Ischemia/reperfusion injury (IRI) remains a major contributor to acute kidney injury (AKI), primarily characterized by excessive reactive oxygen species (ROS) production leading to cellular apoptosis. Through gene expression omnibus (GEO) analysis, we identified betaine-homocysteine S-methyltransferase (BHMT) as significantly downregulated in both ischemia/reperfusion (I/R) and hypoxia/reoxygenation (H/R) models. Our study employed RT-qPCR and western blot analyses to assess RNA and protein expression, while cellular injury was evaluated through MTT assay, flow cytometry, and ROS-related assays. BHMT overexpression significantly attenuated ROS generation and cellular apoptosis in both H/R and I/R conditions. Mechanistic investigations revealed that BHMT enhanced S-adenosylmethionine (SAM) synthesis, subsequently increasing DNA methyltransferase (DNMT) activity. This enhancement promoted DNMT1/DNMT3B-mediated methylation of the NADPH oxidase 4 (NOX4) promoter, effectively suppressing NOX4 transcription and expression. Rescue experiments confirmed that BHMT's protective effects against H/R-induced cell apoptosis and ROS generation were mediated through NOX4 downregulation. Our findings demonstrate that BHMT ameliorates renal IRI by suppressing ROS-stimulated apoptosis via NOX4 regulation, suggesting its potential as both a biomarker and therapeutic target for IRI-induced AKI.