Pirfenidone Elevates GLIS1 by Disrupting the USP7/DNMT1 Complex and Alleviates Renal Fibrosis in Diabetic Kidney Disease Through ROS Reduction and TGF‐β1/Smad Signaling Inhibition

Renal fibrosis is a pathological feature of advanced-stage diabetic kidney disease (DKD). Here, we investigated the effect of Pirfenidone (PFD) on renal fibrosis under diabetic conditions and its molecular mechanism. Sirius red staining and immunohistochemistry were used for histological analysis in rat kidney. Levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were detected by kits. Lipid hydroperoxide, malondialdehyde (MDA), glutathione peroxidase (GPX), and superoxide dismutase (SOD) were measured by commercial kits. Immunofluorescence staining was used to evaluate the expression and colocalization of USP7 and DNMT1. Mitochondrial and cytoplasmic reactive oxygen species (ROS) levels were detected. GLIS1, DNMT1, and USP7 mRNA were measured by RT-qPCR. Protein levels of DNMT1, USP7, fibronectin, p-Smad2, Smad2, p-Smad3, and Smad3 were detected by Western blot. Methylation of the GLIS1 promoter was assessed utilizing methylation-specific polymerase chain reaction (MSP). Chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) verified molecular interactions. PFD alleviated renal injury and fibrosis in DKD rats by suppressing TGF-β1/Smad signaling. PFD attenuated renal oxidative stress and mitochondrial dysfunction in DKD rats. GLIS1 knockdown reversed PFD's protective effects in high glucose (HG)-exposed HK-2 cells, abolishing its improvements in oxidative stress, mitochondrial function, and fibrosis. PFD attenuated HG-induced GLIS1 promoter hypermethylation by suppressing DNMT1 binding and activity, thereby restoring GLIS1 expression. PFD suppressed DNMT1 activity by disrupting USP7/DNMT1 complex. Importantly, the USP7/DNMT1 axis mediated the regulatory effect on GLIS1, which subsequently counteracted PFD's protective effects on HG-stimulated HK-2 cells. PFD upregulates GLIS1 by disrupting the USP7/DNMT1 complex, which subsequently suppresses the ROS/TGF-β1/Smad pathway to alleviate renal fibrosis in DKD. This study identifies the USP7/DNMT1/GLIS1 epigenetic axis as a critical upstream mechanism driving the protective effects of PFD, providing new therapeutic targets for DKD treatment.