Wuling San ameliorates renal fibrosis through modulating S1PR2 activation and suppressing the RhoA/ROCK pathway in adenine-induced chronic kidney disease

Ethnopharmacological relevance: Wuling San (WLS), a classical formula from Treatise on Febrile Diseases, has significant efficacy in ameliorating the clinical symptoms of chronic kidney disease (CKD) with fluid retention, yet its molecular mechanisms remain unclear.

Aim of the study: This study aimed to evaluate the anti-fibrotic effect and molecular mechanisms of WLS on CKD.

Materials and methods: An adenine-induced CKD model was established to evaluate the effect of WLS. Nephroseq database analysis and the experiments in renal tubular specific conditional knockout of S1PR2 (S1PR2-CKO) mice were performed to explore the relationship between S1PR2 expression and renal fibrosis progression. Network pharmacology analysis predicted the potential molecular targets underlying the efficacy of WLS. Renal function and histopathological changes were examined. The expression of inflammatory factors and epithelial mesenchymal transition (EMT) related fibrosis markers were determined by qRT-PCR, immunohistochemistry, immunofluorescence, and Western blot. In vitro, TGF-β1 induced mRTECs were pretreated with the S1PR2 Agonist CYM-5520 or antagonist JTE-013 to further investigate the mechanisms of WLS via S1PR2.

Results: WLS improved renal damages in Ade-induced CKD mice. Through network pharmacology, Nephroseq database analysis, and S1PR2-CKO mice studies, we found that S1PR2 not only is critically involved in renal fibrosis progression, but also is a potential therapeutic target of WLS. Both in vivo and vitro, WLS enhanced S1PR2, cAMP and E-cadherin expression, while suppressed RhoA, ROCK and Vimentin expression. These results confirmed that WLS ameliorates fibrosis by activating S1PR2 and inhibiting the RhoA/ROCK-mediated EMT process.

Conclusion: WLS exerted the improvement on CKD via activating S1PR2 and regulating RhoA/ROCK pathway to inhibit EMT and against renal fibrosis. This study elucidates the therapeutic effect of WLS on CKD and its possible mechanism, and provides novel targets for the CKD treatment.