Klotho alleviates toxic effect of indoxyl sulfate on vascular endothelial cells and its mechanism / 中华肾脏病杂志

ABSTRACT

Objective To investigate the effect of klotho on the human vein umbilical endothelial cells (HUVECs) injury induced by indoxyl sulfate (IS) and to explore its mechanism and the role of endoplasmic reticulum stress (ERS) in this process.Methods (1) The cell vitalities of HUVECs incubated with different concentration of IS (5,25,50 mg/L) for 48 h and with 50 mg/L IS fordifferent time points (12,24,48 h) were measured by CCK-8 assay.(2) HUVECs were incubated with 50 mg/L IS and different concentration of klotho (0,1,10,100 μg/L) for 48 h and their cell viabilities were measured by CCK-8 assay.(3) HUVECs were divided into four groupscontrol group,IS group (50mg/L IS),klotho group (50 mg/L IS+ 100 μg/L klotho) and Compound C group (50 mg/L IS+100 μg/L klotho+ 10 μmol/L Compound C).The cell vitality and the apoptosis of HUVECs were evaluated by CCK-8 assay and flow cytometry,respectively.The mRNA and protein expressions of GRP78 and CHOP were measured by real-time PCR and Western blotting.The phosphorylation level of AMPK was tested by Western blotting.Results IS inhibited cell vitality in the time-dependent and concentration-dependent manner.The cell viability of HUVECs with 50 mg/L IS was lower than normal control (P＜0.05).The inhibited cell vitality induced by IS was partly restored by klotho in concentration-dependent manner.The cell viability was higher in 100 μg/L klotho+50 mg/L IS group than 50 mg/L IS group (P ＜ 0.05).Compared with control group,the expressions of GRP78 and CHOP and cell apoptosis increased,however,the level of phosphorylated AMPK (p-AMPK) decreased in IS group (all P ＜ 0.05).Compared with IS group,the expressions of GRP78 and CHOP and cell apoptosis decreased and the level of p-AMPK increased in klotho group (all P ＜ 0.05).Furthermore,the above effects of klotho could be partly blocked by Compound C.The above indexes showed statistical differences between Compound C group and klotho group.Conclusions IS can inhibit the HUVECs cell vitality,and induce ERS and cell apoptosis.Klotho protein could antagonize the above effects,probably through activating AMPK pathway and reducing ERS-mediated cell apoptosis.