EP1 receptor mediates Adriamycin-induced podocyte injury through activating p38 MAPK / 中华肾脏病杂志

ABSTRACT

Objective To investigate the effect of prostaglandin E2 receptor 1 (EP1) on Adriamycin (ADR)-induced glomerular podocytes injury and its possible mechanism.Methods (1) In vivo experiments6-8 weeks old male Balb/c mice were randomly divided into four groupsControl group;ADR group;EP1 agonist 17-phenyl PGE2+ADR group;EP1 antagonist SC-19220+ADR group.The mouse model of nephrotic syndrome was induced by injection of ADR (10 mg/kg) into tail vein,and then EP1 agonist (1 μg/g) and antagonist (25 μg/g) were administered respectively.Six weeks later,all mice were sacrificed and urine,blood and kidney tissues were collected.Detecting urine protein,blood chemistry,changes of renal pathology and podocyte-related proteins,electron microscopy changes of podocytes.(2) In vitro experimentsPodocytes were cultured in vitro and divided into different groupsControl group;ADR group (0.2 μmol/L);EP1 agonist (0.1,1,10 μmol/L)+ADR (0.2 μmol/L) group;antagonist (0.1,0.5,1 μmol/L)+ ADR (0.2 μmol/L) group.The proliferation of podocytes was measured by CCK-8.Expression of PGE2 in podocytes was detected by ELISA.Indirect immunofluorescence was used to determine the localization of podocyte-related proteins nephrin,podocin and CD2AP.Expression of nephrin,podocin,CD2AP,COX2 in podocytes was detected by Western blotting and Real-time quantitative PCR.p38 MAPK or phospho-p38 MAPK was measured by Western blotting as well.Flow cytometry was used to detect cell apoptosis.Results (1) In vivo experimentsCompared with control group,obvious proteinuria,blood biochemical changes and renal pathological changes were observed in ADR group,proteinuria,blood biochemical and renal pathological changes were more serious in mice dealt with agonist,while antagonist could reduce ADR-induced injury (all P ＜ 0.05).Results of immunohistochemistry showed that the expression of podocyterelated proteins nephrin,podocin and CD2AP in ADR group were significantly lower than those in control group,and EP1 agonist could further inhibit expression of these proteins,while antagonist could reverse this inhibitory effects (P ＜ 0.05).Electron microscopic results showed that mice in ADR group appeared foot enlargement and fusion,and the agonist group further aggravated the injury,while antagonist intervention could inhibit the injury of podocytes.(2) In vitro experimentsCompared with control group,expression of PGE2 and COX2 were increased;mRNA and protein expression of nephrin,podocin,CD2AP were decreased,p38 MAPK activity and podocytes apoptosis were increased in ADR group (P ＜ 0.05);Agonist could aggravate podocytes damage (P ＜ 0.05),while Antagonist could downregulate the expression of PGE2 and COX2,promote the expression of nephrin,podocin and CD2AP,and inhibit the activity of p38 MAPK and podocytes apoptosis (P ＜ 0.05).The addition of p38 MAPK inhibitor(10 μmol/L) could reduce the inhibitory effect of EP1 agonist on the expression of podocyterelated proteins nephrin,podocin and CD2AP (P ＜ 0.05).Conclusions EP1 receptor may activate the p38 MAPK signaling pathway to inhibit podocytes-related proteins nephrin,podocin and CD2AP,as well as mediate the ADR induced podocyte injury.Inhibition of EP1 receptor however have a protective effect.