Adrenomedullin (adm) Induced By Hypoxia In Rat Proximal Tubular Epithelial Cell Apoptosis And Its Mechanism

Objective To study the effect and mechanism of adrenomedullin(ADM) in rat renal tubular epithelial cells(NRK-52E) after hypoxia/reoxygenation(H/R).Methods NRK-52E cells were cultivated in vitro and randomly alloted to 4 groups: control group; H/R group; empty plasmid group; ADM group. NRK-52E cells were transfected by transfection complex comprising optimal proportion of pcDNA3.1/AMD plasmid and Fugene HD reagenis. Cells were counted by trypanblau and cell survival rate was computed. The concentration of lactate dehydrogenase (LDH) was detected by spectrophotometric method to evaluate cell vitality. The apoptotic rate of NRK-52E cells was measured by flow eytometery(FCM). The mRNA expressions of ADM,Bax,Bcl-2,Fas,Bcl-2 were detected by Semi-quantitative RT-PCR, and caspase-3,8,9 protein expressions were measured detected by Western blotting.Results 1)The expressions of ADM mRNA significantly increased in H/R group than in controls (p< 0.05), and in ADM group than in H/R group by RT-PCR; 2)Compared with the control group, the living cell count and cell survival rate significantly decreased and the LDH concentration in media significantly enhanced in H/R group (p<0.05).However, the living cell count and cell survival rate significantly increased in ADM group than in H/R group (p<0.05).LDH concentration in media significantly decreased in ADM group than in H/R group (p＜0.05); 3) Endogenous apoptotic:Compared with the control group, the levels of Bax,Bcl-2,Caspase-3 and Caspase-9 significantly increased in H/R group (p<0.05). Bax/Bcl-2 was increased (p<0.05). Compared with H/R group, the expressions of Bax,Caspase-3 and Caspase-9 significantly reduced and the up-regulation of Bcl-2 significantly increased (p<0.05), therefore the Bax/Bcl-2 was significantly decreased (p<0.05) in ADM group; 4) Exogenous apoptotic:Compared with the control group, the expressions of Fas and Caspase-8 were significantly up-regulated in H/R group(p<0.05). Compared with H/R group, the expressions of Fas and Caspase-8 were significantly down-regulated in H/R group (p<0.05); 5)The cell apoptotic rate was significantly increased in H/R group(p<0.05) than in control, Compared with H/R group, The cell apoptotic rate was significantly increased in ADM group.The above indexes had no differences between the empty plasmid group and the H/R group (p<0.05).Conclusion 1) H/R could lead to NRK-52E cells apoptosis by increasing the activity of Caspase-3,8,9, Bax, Bcl-2; 2) The regulation of up-regulating the expression of Bcl-2 and down-regulating the expression of Caspase-3,8,9, Bax and Fas may be the possible protective mechanism of ADM against apoptosis.