Klotho suppresses cyst growth in ADPKD through the regulation of ferroptosis and reversing DNA methylation

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive cyst growth, inflammation, fibrosis, and renal failure. Klotho is a renoprotective protein with anti-proliferative, anti-inflammatory, and antioxidant properties, but its mechanistic role in ADPKD remains poorly defined. METHODS: Klotho expression was analyzed in human ADPKD kidneys, public bulk and single-cell RNA-seq datasets, Pkd1 mutant renal epithelial cells, and Pkd1 mouse models. The therapeutic effects of recombinant Klotho were evaluated in late-onset Pkd1fl/fl: tamoxifen-Cre mice. Molecular mechanisms were investigated using cell viability assays, Western blotting, RT-qPCR, immunohistochemistry, ferroptosis assays, DNA methylation analysis, chromatin immunoprecipitation assay. RESULTS: Klotho expression was markedly reduced in human ADPKD kidneys and Pkd1-deficient cells and mice, particularly in distal nephron segments. Recombinant Klotho treatment significantly reduced cyst burden, kidney enlargement, and blood urea nitrogen levels, while decreasing epithelial proliferation and increasing apoptosis in cyst-lining cells. Mechanistically, Klotho suppressed PKD-associated signaling pathways, including AKT, STAT3, and Rb, reduced NF-κB-mediated inflammation via downregulation of Smyd2, and attenuated TGF-β-Smad-dependent renal fibrosis. Klotho also inhibited ferroptosis by upregulating GPX-4 and reducing lipid peroxidation. Epigenetic analyses revealed DNMT1-mediated hypermethylation of the Klotho promoter and gene body in ADPKD kidneys, contributing to Klotho silencing. Inhibition of DNMT1 restored Klotho expression, and recombinant Klotho reduced DNMT1 levels, suggesting a negative feedback loop. CONCLUSIONS: Klotho is epigenetically repressed in ADPKD and functions as a central regulator of cyst growth, inflammation, fibrosis, and ferroptosis. Restoration of Klotho signaling markedly attenuates disease progression in Pkd1 mutant mice, identifying the Klotho-DNMT1 axis as a promising therapeutic target for ADPKD.