Chrysophanol ameliorates renal interstitial fibrosis by inhibiting the TGF-β/Smad signaling pathway

Renal interstitial fibrosis (RIF) is a major pathological feature of chronic kidney disease at middle and end stages. Chrysophanol (CP), 1,8-dihydroxy-3-methyl-9,10-anthraquinone, is an anthraquinone isolated from Rheum palmatum L. with a variety of pharmacological activities including the suppression of RIF. However, the effect of CP on renal fibrosis and its potential mechanism have not been elucidated. We conducted a comprehensive study by determining the expression levels of fibrotic markers and proteins including TGF-β1, α-SMA, and Smad3 related to transforming growth factor-beta/Smad (TGF-β/Smad) pathway in unilateral ureteral obstruction (UUO) mice and TGF-β1-stimulated HK-2 cells with the treatment of CP using western blotting and RT-qPCR analyses. Using small interfering RNA and co-immunoprecipitation, we evaluated the influences of CP on the interactions between Smad3 and Smad7 proteins and also on TGF-β RI and TGF-βR II. We found that CP administration significantly ameliorated UUO-induced kidney damage by reversing abnormal serum and urine biochemical parameters and decreasing the production of fibrotic markers including collagen I, collagen III, fibronectin, and α-SMA. Our results showed that TGF-β1 and phospho-Smad3 (p-Smad3) expression was significantly down-regulated and Smad7 expression was up-regulated by CP in UUO mice compared to the model group; however, the expression of Smad2, Smad4, and TGF-β receptors was not affected. Furthermore, CP modulated these fibrotic markers as well as p-Smad3 and Smad7 in TGF-β1-induced HK-2 cells. The inhibitory effect of CP was markedly reduced in TGF-β1-treated HK-2 cells transfected with Smad3 siRNA. Additionally, co-immunoprecipitation analysis indicated that CP blocked the interaction between Smad3 and TGF-β receptor I to suppress p-Smad3 expression. These findings demonstrated that CP alleviated RIF by inhibiting Smad3 phosphorylation, which provides a molecular basis for a new drug candidate for the treatment of RIF.