CP‐25 Inhibits Methotrexate‐Induced Apoptosis in Renal Tubular Epithelial Cells

Abstract

ObjectiveMethotrexate (MTX), known as nephrotoxic drug, frequently causes renal injury via inducing apoptotic death in human renal tubular epithelial cells(RTEC). Paeoniflorin‐6′‐O‐benzene sulfonate (CP‐25) is active compound obtained by modifying the structure of Paeoniflorin. We have previously showed CP‐25 inhibited chronic inflammation and effectively improved symptoms of arthritis and Sjogren syndrome in experimental animal models. This study was to investigate effects of CP‐25 on MTX‐induced apoptosis in RTEC lines in vitro and the related mechanisms.MethodsNRK‐52E and NK‐2 were treated with various concentration of MTX in the presence or absence of CP‐25. After treatment, the proliferation of cells were measured by MTT assay, and apoptotic death of the cells were detected by Annexin V/PI dual staining. The expression of apoptotic related protein were measured by western blotting.ResultsTreatment of MTX significantly inhibited proliferation of NK‐2, NRK‐52E in dose‐and time‐dependant manners. Moreover, MTX could significantly induce apoptotic death in NK‐2 and NRK‐52E cells. Further study revealed that MTX exposure to the cells could activate intrinsic and extrinsic apoptotic signal pathways, which was evidenced by increased expression of Bax, cyt‐c, clea‐caspase‐3, clea‐caspase‐8, and clea‐caspase‐9, and decreased expression of Bcl‐2 and XIAP. Interestingly, we found that co‐treatment of MTX with CP‐25 could significantly reduce MTX‐induced apoptotic death in the cells. As expected, CP‐25 inhibited MTX‐induced activation of intrinsic and extrinsic apoptotic pathways via regulating abnormal expression of apoptosis‐related proteins. Finally, we found MTX exposure to the cells significantly increased nuclear translocation of NF‐κB coupled with increase in phosphorylated Iκ‐B, which was significantly inhibited by CP‐25.ConclusionCP‐25 could inhibit MTX‐induced pro‐apoptotic effects on RTEC. The underlying mechanism is associated with inhibitory effects of CP‐25 on the activation of intrinsic/extrinsic apoptotic pathways and NF‐κB nuclear translocation. Our data indicate CP‐25 maybe a promising therapeutic drug for the treatment of MTX‐induced renal damage.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.