Genistein suppresses renal fibrosis in chronic kidney disease through regulation of the CCAR2/SIRT1/p53 signaling axis

• Genistein has a significant effect against renal fibrosis. • Genistein binds to the CCAR2 protein via tryptophan at position 108. • Genistein inhibits CCAR2 from activating Sirt1 activity, thereby ameliorating renal fibrosis. Chronic kidney disease (CKD), a major global health burden, is characterized by progressive renal fibrosis, for which effective therapies are lacking. While the natural isoflavone genistein (Gen) has been documented to possess broad anti-fibrotic properties, its specific function and underlying mechanism in renal fibrosis remain largely undefined. In this study, we demonstrate that Gen administration significantly attenuates renal fibrosis in both UUO and adenine-induced murine CKD models. To elucidate the molecular mechanism, we employed a strategy using biotin-conjugated Gen as a bait for pull-down assays, followed by mass spectrometry analysis, which successfully identified CCAR2 as a novel direct cellular target of Gen. This specific interaction was further validated by CETSA, molecular docking and Co-IP, which confirmed the binding site at 108 Tryptophan. Functional investigations revealed that CCAR2 intrinsically functions as a promoter of renal fibrogenesis, and its overexpression exacerbated the fibrotic response, whereas its genetic knockdown or pharmacological inhibition by Gen potently alleviated fibrosis in vitro and in vivo settings. Mechanistically, our data indicate that CCAR2 acts as an endogenous inhibitor of SIRT1 deacetylase activity, consequently leading to increased acetylation and transcriptional activation of the downstream effector p53. Critically, genistein, by directly binding to CCAR2, sterically relieves its inhibitory effect on SIRT1, thereby restoring SIRT1 activity and subsequently suppressing p53-mediated pro-fibrotic signaling, ultimately conferring robust protection against renal fibrosis. Collectively, our findings reveal that genistein ameliorates renal fibrosis through precise targeting of the newly identified CCAR2/SIRT1/p53 signaling pathway, thereby offering a promising therapeutic strategy for patients with fibrotic CKD.