Enoxolone Alleviates Renal Fibrosis in Diabetic Kidney Disease by Inhibiting the Smad3-Smad4 Complex.

Introduction: This study aimed to explore the ameliorative effects of enoxolone on renal fibrosis in diabetic kidney disease (DKD) and elucidate its underlying molecular mechanisms, thereby proposing a novel candidate drug and providing a theoretical foundation for clinical anti-fibrotic therapy in DKD. Methods: We analyzed the single-cell transcriptome dataset GSE209781 and integrated it with the connectivity map database to identify potential therapeutic compounds for DKD. Molecular docking and surface plasmon resonance techniques were employed to verify the binding interaction between enoxolone and Smad3. In vitro experiments involved human renal tubular epithelial HK2 cells and primary mouse tubular epithelial cells derived from Smad3-knockout (Smad3-KO) mice, which were stimulated with high glucose and advanced glycation end-products (HG+AGEs). Co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), quantitative real-time polymerase chain reaction (qPCR), and Western blot analyses were conducted to assess the effects of enoxolone on the Smad3-Smad4 interaction, binding of Smad3 to the COL1A1 promoter, and expression of fibrotic genes. An in vivo streptozotocin-induced DKD mouse model with adeno-associated virus-mediated Smad3 overexpression was established to evaluate renal function, fibrosis, and associated molecular changes. Results: Enoxolone was identified as a key candidate capable of reversing collagen expression disorder in proximal tubular epithelial cells treated with high glucose and AGEs. It binds directly to the Arg292 site in the MH2 domain of Smad3 with a high affinity. Enoxolone significantly inhibited the formation of the Smad3-Smad4 complex, reduced the enrichment of Smad3 on the COL1A1 promoter, and downregulated COL1A1 expression. In DKD mice, enoxolone reduced blood urea nitrogen, serum creatinine, and renal hydroxyproline levels and alleviated collagen deposition and tubular injury, whereas Smad3 overexpression reversed these effects. Conclusion: Enoxolone targets Arg292 of Smad3, inhibiting Smad3-Smad4 complex formation and downstream pro-fibrotic gene transcription, thereby attenuating renal fibrosis in DKD. Enoxolone has emerged as a promising anti-fibrotic candidate for the treatment of DKD.