摘要
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.
出处
《中南大学学报（医学版）》
CAS
CSCD
北大核心
2010年第3期222-229,共8页
Journal of Central South University ：Medical Science
基金
中南大学创新性课题(YD09202)~~
关键词
高糖
醛固酮
安体舒通
肾小管上皮细胞转分化
high glucose
aldosterone
spironolactone
epithelial-mesenchymal transition
