Multi-omics and functional analyses link tubular ZDHHC12-mediated SMAD3 palmitoylation to renal fibrosis after ischemia–reperfusion injury

Renal ischemia–reperfusion injury (IRI) causes acute tubular damage and can progress to renal fibrosis, but the molecular regulators linking early injury to fibrotic remodeling remain unclear. Here, we integrated mouse renal RNA sequencing, GSVA, four public GEO datasets, machine-learning-based feature selection, single-cell RNA sequencing, spatial transcriptomics, human kidney specimens, and functional validation to investigate palmitoylation in IRI-associated fibrosis. Transcriptomic analysis showed time-dependent changes after IRI, with early metabolic disruption followed by inflammatory and extracellular matrix-related responses. GSVA revealed increased palmitoylation scores at days 1 and 3 after reperfusion. Integrated bioinformatic analysis prioritized Zdhhc12 as a candidate gene, and validation experiments showed sustained ZDHHC12 upregulation from day 1 to day 14 after IRI. Single-cell and spatial analyses identified proximal tubular cells as a major site of increased palmitoylation activity and ZDHHC12 expression. ZDHHC12 was also elevated in human post-ischemia/reperfusion renal tissues and TGF-β1-stimulated renal tubular epithelial cells. Functionally, ZDHHC12 knockdown reduced TGF-β1-induced Collagen I and α-SMA expression in vitro, whereas renal tubule-specific ZDHHC12 knockdown alleviated tubular injury, inflammation, renal dysfunction, and fibrosis in vivo. Mechanistically, IRI and profibrotic stimulation were associated with palmitic acid accumulation, increased global S-palmitoylation, and enhanced SMAD3 palmitoylation. ZDHHC12 knockdown suppressed SMAD3 palmitoylation, whereas ZDHHC12 overexpression promoted SMAD3 palmitoylation and profibrotic signaling, which were attenuated by 2-bromopalmitate. Site-directed mutagenesis and in silico analysis further identified Cys421 as a critical palmitoylation site of SMAD3, linking ZDHHC12-mediated palmitoylation to fibrotic activation. These findings identify ZDHHC12-mediated SMAD3 Cys421 palmitoylation as a mechanism linking renal IRI to fibrosis.