cAMP/PKA signal activation prevents chemical-induced podocyte injury / 中华肾脏病杂志

ABSTRACT

Objective To investigate the role of activated cylic AMP(cAMP) signaling in chemical-induced podocyte injury.Methods Eight-weeks-old male BalB/C mice were randomly divided into three groupscontrol group,Adriamycin (ADR) group and Forskolin+ADR group.ADR nephropathy models were established by tail intravenous injection,and part of them were injected Forskolin,an agonist of adenylate cyclase,intraperitoneally.Phosphorylation of cAMP response element binding protein (CREB) was detected by laser confocal microscopy,morphology of foot processes were determined with transmission electron microscope,and WT-1 expression in glomeruli were detected by immunohistochemistry.Conditionally immortalized podocytes were treated with puromycin aminonucleoside (PAN),Exchange protein directly activated by cAMP (Epac) agonist 8-pCPT-2-O-Me-cAMP (2Me),protein kinase A (PKA) antagonist H89 and its agonist pCPT-cAMP(pCPT).Western blot was used to detect the expression levels of Epac,caspase3 and cleaved caspase3.PKA activity was assayed using cAMP-dependent protein kinase detection system.Cell viability was determined by a cell count kit and podocyte apoptosis was estimated by TUNEL staining.Mitochondrial membrane potential was evaluated by JC-1 staining.Results (1)Compared with ADR group,the urine albumin decreased significantly (P ＜ 0.05) among Forskolin + ADR group and the WT-1 positive cells per glomerulus increased obviously (P ＜ 0.05).(2)PAN decreased podocyte number in a time-dependent manner (P ＜ 0.05),pre-treatment with pCPT obviously inhibited PAN induced podocyte decrease (P ＜0.05),but H89 prevented the effect of pCPT in a dose-dependent manner (P ＜ 0.05).(3)JC-1 staining showed that the percentage of podocyte with green fluorescence for control,PAN and pCPT+PAN group were (12.67±2.15)％,(31.35±4.60)％ and (16.96 ± 2.51)％ respectively (P ＜ 0.05),and pretreatment with H89 inhibited the effect of pCPT (P ＜ 0.05).(4) PAN promoted podocyte apoptosis and cleaved caspase3 expression (P ＜ 0.05),and pretreatment with pCPT significantly prevented PAN-induced podocyte apoptosis and cleaved caspase3 expression (P ＜ 0.05).Conclusions cAMP signaling activation ameliorated podocyte injury in ADR nice and PAN-induced podocyte apoptosis,and cAMP/ PKA pathway may mediate these processes.