Prostaglandin E2 Receptor EP1 Mediates Adriamycin-induced Podocyte In Jury Through Activating P38MAPK Pathway

Objective To investigate the effect of prostaglandin E2 receptor 1(EP1)on Adriamycin(ADR)-induced glomerular podocyte injury and its possible mechanism.Methods In vivo1.6-8 weeks old male Balb/c mice were randomly divided into four groups:(1)control group;(2)ADR group;(3)EP1 agonist 17-phenyl PGE2 + ADR group;(4)EP1 antagonist SC-19220 + ADR group.2.The mouse model of nephrotic syndrome was induced by injection of ADR(10 mg /kg)into tail vein.3.EP1 agonist 17-phenyl PGE2(1?g/g)and antagonist SC-19220(25?g/g)were administered respectively.4.Six weeks later,all mice were sacrificed and urine,blood and kidney tissue were collected.Detecting urinary albumin,blood biochemical,changes of renal pathology,podocyte-associated proteins and electron microscopy changes of podocytes.In vitro1.Mouse glomerular podocytes were cultured in vitro.2.Cell grouping:(1)control group;(2)ADR group(0.2?mol/L);(3)EP1 agonist 17-phenyl PGE2(0.1,1,10?mol/L)+ ADR(0.2?mol/L)group;(4)EP1 antagonist SC-19220(0.1,0.5,1?mol/L)+ADR(0.2?mol/ L)group.3.Cell proliferation was detected by CCK-8.4.Expression of PGE2 in podocytes was detected by ELISA.5.Indirect immunofluorescence was used to determine the localization of podocyte-associated proteins nephrin,podocin and CD2 AP.6.Expression of nephrin,podocin,CD2 AP,cyclooxygenase 2(COX2)in podocytes was detected by Western blotting and Real-time q PCR.7.The activity of P38 MAPK was measured by Western blotting.8.Flow cytometry was used to detect cell apoptosis.Results In vivo1.Compared with control group,obvious proteinuria,blood biochemical changes and renal pathological changes were observed in ADR group(P<0.05),mice treated with 17-phenyl PGE2 appeared more serious kidney damage(P<0.05),while SC-19220 can reduce ADR-induced injury(P<0.05).2.Results of immunohistochemistry showed that,the expression of podocyte-associated proteins nephrin,podocin and CD2 AP in ADR group were significantly lower than those in control group(P<0.05),and 17-phenyl PGE2 could further inhibit expression of these proteins(P<0.05),while SC-19220 could reverse this inhibitory effects caused by ADR(P<0.05).3.Results of electron microscopy showed that,podocytes in ADR group showed significant foot process disorder and fusion,this performance was aggravated after agonist intervention.While antagonist intervention could reduce podocyte injury and inhibit foot process fusion.In vitro1.The proliferation of podocytes in ADR group was lower than control group(P<0.05);while 17-phenyl PGE2 could further decrease theproliferation of podocytes(P<0.05);SC-19220 could promote podocytes proliferation in a dose-dependent manner(P<0.05).2.Compared with control group,expression of PGE2 ? COX2 were increased in ADR group(P<0.05);Compared with ADR group,17-phenyl PGE2 could further increase the expression of PGE2?COX2(P<0.05);while treatment of SC-19220 could inhibit PGE2?COX2 expression(P<0.05).3.Compared with control group,nephrin,podocin,CD2 AP m RNA expression were decreased in ADR group(P<0.05);Compared with ADR group,17-phenyl PGE2 intervention could further decrease the expression of nephrin,podocin,CD2 AP m RNA in a dose-dependent manner(P<0.05);while SC-19220 could increase expression of nephrin,podocin,CD2 AP m RNA(P<0.05).4.Compared with control group,the expression of nephrin,podocin,CD2 AP protein were decreased in ADR group(P<0.05);Compared with ADR group,17-phenyl PGE2 intervention could further decrease the expression of nephrin,podocin,CD2 AP protein in a dose-dependent manner(P<0.05);while SC-19220 could increase expression of nephrin,podocin,CD2 AP protein(P<0.05).5.Compared with control group,p38 MAPK activity and podocytes apoptosis were increased in ADR group(P<0.05);17-phenyl PGE2 could further increase p38 MAPK activity and podocytes apoptosis(P<0.05);While SC-19220 could inhibit the activity of p38 MAPK and podocytes apoptosis in a dose-dependent manner(P<0.05).Addition of p38 MAPK inhibitor(10?mol/L)can reduce the inhibitory effect of EP1 agonist on the expression of podocyte-related proteins nephrin,podocin and CD2AP(P<0.05).Conclusions EP1 receptor may activate the p38 MAPK signaling pathway to inhibit podocyte-associated proteins nephrin,podocin and CD2 AP,and mediate the ADR-induced podocyte injury,while inhibitioOf EP1 receptor have a protective effect.