他克莫司通过减少肾小球足细胞凋亡降低2型糖尿病大鼠尿白蛋白

Tacrolimus reduces urine albumin through decreasing glomerular podocyte apoptosis in type 2 diabetic rats

目的探讨他克莫司降低2型糖尿病(T2DM)大鼠尿白蛋白的可能机制。方法高脂高糖饲料喂养8周联合低剂量链脲佐菌素腹腔注射构建T2DM大鼠模型,随机选取后分为糖尿病模型组(DM组,n=10)和他克莫司(FK506)治疗组(FK组,n=10),另设正常对照组(n=10)。FK组给予FK506灌胃干预8周,DM组、正常对照组给予等量枸橼酸缓冲液灌胃8周。干预前后测定各组大鼠肾肥大指数(肾质量/体质量,KM/BM)、收缩压、24h尿白蛋白/尿肌酐,空腹血糖、内生肌酐清除率(CCr),总胆固醇、三酰甘油、丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)和血白细胞;光镜、电镜观察肾脏病理改变;免疫组织化学方法测定nephrin蛋白表达;TUNEL法检测足细胞凋亡;Western blot法测定cleaved-caspase-3的表达和bax/bcl-2。结果与正常对照组相比,DM大鼠KM/BM、收缩压、空腹血糖、CCr、总胆固醇、三酰甘油及24h尿白蛋白/尿肌酐明显升高(均P<0.05),经FK506干预后大鼠KM/BM、24h尿白蛋白/尿肌酐明显降低(均P<0.05),而对收缩压、空腹血糖、CCr、ALT、AST和血白细胞的影响无统计学意义;光镜下,DM大鼠肾小球体积增大,系膜细胞增生,基底膜增厚;FK506干预后病理改变明显减轻;电镜下,DM组肾小球基底膜显著增厚,足突紊乱、融合率明显升高[(73.50±3.60)%比(4.20±1.91)%,P<0.05];经FK506干预后上述病变有所减轻,足突融合率下降[(33.80±1.93)%比(73.50±3.60)%,P<0.05];免疫组化结果显示,DM组肾组织nephrin蛋白表达较正常对照组下降,而FK506干预后nephrin蛋白表达明显恢复(P<0.05);TUNEL法检测发现DM大鼠足细胞凋亡明显增多(P<0.05),而FK506干预后足细胞凋亡明显减少(P<0.05);Western blot结果显示:DM组肾组织cleaved-caspase-3蛋白表达和bax/bcl-2较正常对照组上升(P<0.05),而FK组cleaved-caspase-3蛋白表达和bax/bcl-2明显降低(P<0.05)。结论他克莫司可以通过减少糖尿病肾病大鼠足细胞的凋亡而发挥降低24h尿白蛋白,延缓肾功能减退的作用。

Objective To investigate the potential mechanism of urine albumin reduction by the application of tacrolimus in rats with type 2diabetic mellitus（T2DM）. Methods The T2 DM rat models were established by feeding them with high fat and high sugar food for 8weeks combining with the intraperitoneal injection of low-dose streptozotocin. These model rats were randomly selected and then divided into the diabetic mellitus group（DM group,n=10）and FK506 treatment group（FK group,n=10）. The normal control group（n=10）was also established.The rats in the FK group were exposed to FK506 by intragastric administration for 8weeks,while the rats in the DM group and the normal control group were given the same amount of citrate buffer solution by intragastric administration for 8 weeks. Kidney hypertrophy index（kidney mass/body mass,KM/BM）,systolic blood pressure,24-hour urinary albumin/urine creatinine,fasting blood glucose,endogenous creatinine clearance rate（CCr）,total cholesterol,triglyceride,alanine transaminase（ALT）,aspartate aminotransferase（AST）and white blood cells were determined before and after the intervention in all groups. Renal pathological changes were observed by light and electron microscopy,nephrin protein expression was detected by immunohistochemical method,and the podocyte apoptosis was observed by TUNEL assay. The expression of cleaved-caspase-3and the ratio of bax/bcl-2 were detected by Western blot. Results Compared with the normal control group,KM/BM,systolic blood pressure,fasting blood glucose,CCr,total cholesterol,triglyceride and urinary albumin/urine creatinine in the DM group were significantly increased（P〈0.05）. Compared with the DM group,KM/BM and urinary albumin/urine creatinine were decreased in FK group（P〈0.05）,while there were no significant differences in systolic blood pressure,fasting blood glucose,CCr,ALT,AST and white blood cells between these two groups. While the increased glomerular volume,mesangial cell proliferation and thickened basement membrane were observed by light microscopy in the DM group,these aboved pathological changes were markedly attenuated in the FK group. Under an electron microscope,the glomerular basement membrane was significantly thickened,and foot processes were in disorder and the fusion rate was significantly increased in the DM group compared to the normal control group[（73.50±3.60）% vs（4.20±1.91）%,P〈0.05]. Compared to the DM group,these aboved pathological changes of FK group attenuated,and the fusion rate was decreased significantly[（33.80±1.93）% vs（73.50±3.60）%,P〈0.05]. Immunohistochemical assay showed that the expression of nephrin protein was significantly decreased in the DM group compared with the normal control group,which was increased after treatment with FK506（P〈0.05）. TUNEL assay showed that the number of podocyte apoptosis was significantly increased in the DM group compared with the normal control group（P〈0.05）,which was significantly decreased after treatment with FK506（P〈0.05）. Western blot showed that the expression of cleaved-caspase-3and the ratio of bax to bcl-2increased in the DM group when was compared with the normal control group（P〈0.05）,which were significantly decreased in the FK group（P〈0.05）. Conclusion Tacrolimus could reduce urine albumin and delay the progression of nephropathy by reducing podocyte apoptosis in T2 DM rats.