CAJ | 학술논문

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(spironolactone,SP)对大鼠肾小管上皮细胞(normal rat kidney epithelial cell,NRK-52E)转分化(epithelial-mesenchymal transition,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12 h后分为6组.LG组:采用低糖(1 000 mg/L) DMEM;HG组:采用高糖(4 500 mg/L)DMEM培养;10 nmol/L Aldo组、50 nmol/L Aldo组、100 nmol/L Aldo组:分别采用高糖DMEM加10,50,100 nmol/L Aldo培养;SP组采用高糖(4 500 mg/L)DMEM加10~(-7) mol/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-cadherin mRNA表达明显降低(P＜0.01),α-SMA mRNA表达明显升高(P＜0.05);50 nmol/L Aldo组、100 nmol/L Aldo组E-cadherin mRNA表达明显低于高糖组,而α-SMA mRNA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-cadherin mRNA明显高于HG组,而α-SMA mRNA表达低于HG组(P＜0.01).细胞免疫化学和Western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);10 nmol/L Aldo组、50 nmol/L Aldo组、100 nmol/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.
목적:통과관찰고당배경하,철고동(aldosterone, Aldo)급기길항제안체서통(spironolactone,SP)대대서신소관상피세포(normal rat kidney epithelial cell,NRK-52E)전분화(epithelial-mesenchymal transition,EMT)적영향,탐토Aldo급SP재당뇨병신병(diabetic nephropathy,DN)신보호작용중적궤제.방법:용무혈청DMEM(Dulbecco's modification of Eagle's medium Dulbecco)동보화배양NRK-52E세포계12 h후분위6조.LG조:채용저당(1 000 mg/L) DMEM;HG조:채용고당(4 500 mg/L)DMEM배양;10 nmol/L Aldo조、50 nmol/L Aldo조、100 nmol/L Aldo조:분별채용고당DMEM가10,50,100 nmol/L Aldo배양;SP조채용고당(4 500 mg/L)DMEM가10~(-7) mol/L SP배양.채용세포면역화학、RT-PCR화Western면역인적방법검측각조세포E-cadherin화α-SMA mRNA적표체정황.결과:RT-PCR결과표명,여LG조비교,HG조E-cadherin mRNA표체명현강저(P＜0.01),α-SMA mRNA표체명현승고(P＜0.05);50 nmol/L Aldo조、100 nmol/L Aldo조E-cadherin mRNA표체명현저우고당조,이α-SMA mRNA표체명현고우고당조(P＜0.05),량자여Aldo정농도의뢰관계(r=-0.70,P＜0.05;r=0.67, P＜0.05);SP조E-cadherin mRNA명현고우HG조,이α-SMA mRNA표체저우HG조(P＜0.01).세포면역화학화Western면역인적검측표명,여저당조비교,HG조E-cadherin단백표체명현감저,이α-SMA표체명현승고(P＜0.01);10 nmol/L Aldo조、50 nmol/L Aldo조、100 nmol/L Aldo조E-cadherin단백표체명현저우HG조,이α-SMA단백표체명현고우HG조(P＜0.05),여Aldo정농도의뢰관계(r=-0.83,P＜0.05;r=0.81, P＜0.05);이SP조E-cadherin단백표체명현고우고당조,α-SMA단백칙명현저우HG조(P＜0.05).결론:Aldo능촉진고당배경하신소관상피세포EMT적발생,치DN신간질섬유화,이사용SP가억제고당유도신소관상피세포적EMT,저가능시기조알신간질섬유화적중요궤제.
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~(-7)mol/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.