醛固酮及其拮抗剂安体舒通对高糖刺激下肾小管上皮细胞转分化的影响

Effect of aldosterone and its antagonist spironolactone on epithelial-mesenchymal transition of mormal rat kidney epithelial cells in high glucose

目的:通过观察高糖环境下,醛固酮(aldosterone,Aldo)及其拮抗剂安体舒通(spironolac-tone,SP)对大鼠肾小管上皮细胞(normal rat kidney epithelial cell,NRK-52E)转分化(epithelial-mesenchymaltransition,EMT)的影响,探讨Aldo及SP在糖尿病肾病(diabetic nephropathy,DN)肾保护作用中的机制。方法:用无血清DMEM(Dulbecco's modification of Eagle's medium Dulbecco)同步化培养NRK-52E细胞系12h后分为6组。LG组:采用低糖(1000mg/L)DMEM;HG组:采用高糖(4500mg/L)DMEM培养;10nmol/L Aldo组、50nmol/L Aldo组、100nmol/L Aldo组:分别采用高糖DMEM加10,50,100nmol/L Al-do培养;SP组采用高糖(4500mg/L)DMEM加10-7mol/L SP培养。采用细胞免疫化学、RT-PCR和Western免疫印迹方法检测各组细胞E-cadherin和α-SMA mRNA的表达情况。结果:RT-PCR结果表明,与LG组比较,HG组E-cadherinmRNA表达明显降低(P<0.01),α-SMAmRNA表达明显升高(P<0.05);50nmol/L Aldo组、100nmol/L Aldo组E-cadherinmRNA表达明显低于高糖组,而α-SMAmRNA表达明显高于高糖组(P<0.05),两者与Aldo呈浓度依赖关系(r=-0.70,P<0.05;r=0.67,P<0.05);SP组E-cadherinmRNA明显高于HG组,而α-SMAmRNA表达低于HG组(P<0.01)。细胞免疫化学和West-ern免疫印迹检测表明,与低糖组比较,HG组E-cadherin蛋白表达明显减低,而α-SMA表达明显升高(P<0.01);10nmol/L Aldo组、50nmol/L Aldo组、100nmol/L Aldo组E-cadherin蛋白表达明显低于HG组,而α-SMA蛋白表达明显高于HG组(P<0.05),与Aldo呈浓度依赖关系(r=-0.83,P<0.05;r=0.81,P<0.05);而SP组E-cadherin蛋白表达明显高于高糖组,α-SMA蛋白则明显低于HG组(P<0.05)。结论:Aldo能促进高糖环境下肾小管上皮细胞EMT的发生,致DN肾间质纤维化,而使用SP可抑制高糖诱导肾小管上皮细胞的EMT,这可能是其阻遏肾间质纤维化的重要机制。

Objective To determine the effect of aldosterone and its antagonist,spironolactone on epithelial-mesenchymal transition（EMT）of normal rat kidney epithelial cells（NRK-52E）in a high glucose milieu,and to explore the mechanism of renoprotection in diabetic nephropathy（DN）in rats involving aldosterone and spironolacton.Methods NRK-52E cells were simultaneously cultured in the serum-free Dulbecco＇s modification of Eagle＇s medium Dulbecco（DMEM）for 12 hours.Then the low glucose（LG）group was cultured in LG（1000 mg/L）DMEM：The high glucose（HG）group was cultured in high glucose（4 500 mg/L）DMEM.The aldosterone（Aldo）groups were cultured in high glucose DMEM with the addition of 10,50 and 100 nmol/L aldosterone respectively.The SP group was cultured in high glucose（4 500 mg/L）DMEM plus 10-7mol/L spironolactone.Immunohistochemistry,RT-PCR and Western blot were used to detect E-cadherin and α smooth muscle actin（α-SMA）mRNA expression.Results RT-PCR showed that compared with the LG Group,E-cadherin mRNA expression in the HG group was significantly lower,and α-SMA mRNA expression was significantly increased（P〈0.05）.E-cadherin mRNA expression in the 50 nmol/L Aldo group and 100 nmol/L Aldo group was significantly lower than that in the HG group,while the expression of α-SMA mRNA was significantly increased in the HG group（P〈0.05）,with a dose-dependent relationship with aldosterone（r=-0.70,P〈0.05;r=0.67,P〈0.05）.E-cadherin mRNA in the SP group was significantly higher,while α-SMA mRNA expression was lower than that in the HG group（P〈0.01）.Immunohistochemistry and Western blot showed that compared with the LG group,E-cadherin protein expression was significantly reduced,and α-SMA expression was significantly increased in the HG group（P〈0.01）.In the 10 nmol/L Aldo,50 nmol/L Aldo,and the 100 nmol/L Aldo groups,E-cadherin protein expression was significantly lower,and α-SMA protein expression was significantly higher than that in the HG group（P〈0.05）,with a dose-dependent relationship with aldosterone（r=-0.83,P〈0.05;r=0.81,P〈0.05）.In the SP group,E-cadherin protein expression was higher,and α-SMA protein level was lower than that in the HG group（P〈0.05）.Conclusion Aldosterone can promote EMT of tubular epithelial cells in a high sugar milieu,leading to renal interstitial fibrosis in Diabetic nephropathy.Spironolactone can inhibit high glucose-induced renal tubular epithelial cells EMT,which may be an important mechanism for the inhibition of renal interstitial fibrosis.