肾素抑制剂对高糖诱导足细胞凋亡与mTOR表达的影响

发布时间：2018-05-07 04:17

本文选题：高糖 + 足细胞　；参考：《武汉大学》2014年博士论文

【摘要】：背景慢性肾脏病(chronic kidney disease,CKD)是严重威胁人民身体健康且消耗巨大社会资源的常见疾病,最近的调查显示国内CKD患病率约为10.8%,由于其致病因素广泛,其发病率一直居高不下,且有逐年上升的趋势,其高死亡率、高致残率、高社会成本支出、低知晓率也同样令人关注。糖尿病肾病是慢性肾脏病的主要病因之一,在全球有约3亿糖尿病病人,其中约30%发展为糖尿病肾病,恩施地区也属于糖尿病高发区,根据恩施土家族苗族自治州血液净化统计资料,透析病人中糖尿病肾病占14.9%。肾脏血流动力学变化、肾脏微血管病变、小球系膜细胞及基质增生等曾被认为是糖尿病肾病的主要病理生理变化,近年随着对足细胞的深入研究,认为足细胞损伤、足细胞功能改变、足细胞过度凋亡与糖尿病肾病的蛋白尿、肾小球硬化密切相关,足细胞受损是糖尿病肾病发展的关键,因此有学者认为糖尿病肾病的本质就是足细胞病。肾素血管紧张素系统(RAS)的激活与糖尿病肾病的发生与发展关系密切,在RAS与糖尿病肾病足细胞受损之间的复杂网络关系中,PI3K-AKT-mTOR信号传导途径起着重要作用,mTOR的活化能减少足细胞自噬,促进足细胞凋亡。虽然糖尿病肾病时观察到了足细胞mTOR信号通路的活化及足细胞损伤,以及阻断mTOR信号系统带来的益处,但足细胞mTOR信号通路具体调控过程还在探索中,糖尿病时高糖能否直接活化足细胞mTOR信号通路、导致足细胞凋亡目前也不清楚。肾脏局部的RAS系统除了通过影响肾脏的血流动力学外,还影响尿蛋白排泄、系膜基质增生、小管间质纤维化,而足细胞作为糖尿病肾病病变的核心,不仅是RAS引起损伤作用的靶目标,亦是局部RAS的重要来源,RAS激活对足细胞本身的影响目前尚未完全明了,特别是对足细胞凋亡、足细胞mTOR信号通路的影响研究更少。目的观察高糖诱导足细胞mTOR表达及凋亡改变以及肾素抑制剂对其的影响。方法1.将培养的成熟足细胞分为对照组、高渗组、高糖组,对照组置于含5mmol/L葡萄糖的RPMI1640培养液,高渗组为含5mmol/L葡萄糖、25mmol/L甘露醇的RPMI1640培养液,高糖组为含30mmol/L葡萄糖的RPMI1640培养液。各组足细胞培养24小时后应用RT-PCR测定各组mTOR mRNA表达,Western blot测定各组足细胞mTOR蛋白表达,TUNEL法测定各组足细胞凋亡率。2.将培养足细胞分为对照组、无义转染组、siRNA转染组,转染组加入转染试剂(Lipofecter旨质体)、mTOR siRNA进行转染,无义对照组加入转染试剂、对照(无义)siRNA,空白对照组仅加转染试剂(空脂质体)。按转染试剂盒说明书转染24小时,将转染成功后的足细胞置于含30mmol/L葡萄糖RPMI1640培养液继续培养24小时。通过RT-PCR测定各组mTOR mRNA表达,Western blot测定各组足细胞mTOR蛋白表达,TUNEL法测定各组足细胞凋亡率。3.将培养的足细胞分为对照组、低剂量Aliskiren组(含10nmol/L的Aliskiren)、中剂量Aliskiren组(含20nmol/L的Aliskiren)、高剂量Aliskiren组(含30nmol/L的Aliskiren),在30mmol/L葡萄糖RPMI1640培养液培养24小时后应用RT-PCR测定各组mTOR mRNA表达,Western blot测定各组足细胞mTOR蛋白表达,TUNEL法测定各组足细胞凋亡。结果在高糖刺激下,足细胞表达mTOR mRNA较高渗组及对照组显著上调(P0.01),mTOR蛋白表达也显著上调(P0.01),足细胞凋亡高于高渗组及对照组(P0.01)。应用siRNA技术沉默mTOR基因表达后,足细胞mTOR mRNA及mTOR蛋白均较对照组和无义转染组显著下调(P0.01),转染组在高糖刺激下足细胞凋亡率显著下降(P0.05)。给予肾素抑制剂Aliskiren干预后,足细胞mTOR蛋白表达较对照组下调(P0.01),足细胞凋亡也较对照组下降(P0.01),随着干预剂量的增加,其mTOR蛋白表达下调更加显著,三组剂量组间有统计学差异(P0.05)。结论高糖可能通过上调足细胞mTOR表达诱导其凋亡；肾素抑制剂对足细胞的保护作用可能与其下调足细胞mTOR表达,减少高糖刺激下的足细胞凋亡有关。目的观察肾素抑制剂Aliskiren对糖尿病肾病大鼠肾脏mTOR表达、尿液足细胞排泄的影响。方法40只Wistar大鼠采用腹腔内一次性注射STZ制作糖尿病模型,随机分糖尿病组及Aliskiren治疗组,另20只正常Wistar大鼠作为对照组,Aliskiren治疗组每日给予Aliskiren10mg/kg一次性灌胃,8周后留取尿液计数各组大鼠尿液足细胞排泄数；取大鼠肾皮质,RT-PCR测定各组大鼠肾脏mTORmRNA表达,Western blot测定mTOR蛋白表达。结果糖尿病大鼠尿液足细胞排泄显著高于对照组(P0.01),应用Aliskiren干预8周后,糖尿病大鼠足细胞排泄明显减少(P0.01)；糖尿病大鼠肾组织mTOR表达显著高于对照组(P0.01),Aliskiren治疗后大鼠肾组织mTOR表达较糖尿病组显著下调(P0.01)。结论肾素抑制剂能减少糖尿病大鼠足细胞排泄,减少尿蛋白,其肾脏保护机制可能与下调肾组织mTOR表达有关。
[Abstract]:Chronic kidney disease (CKD) is a common disease which seriously threatens the health of the people and consumes huge social resources. The recent survey shows that the prevalence rate of CKD in China is about 10.8%. Because of its widespread pathogenicity, the incidence of the disease has always been high, and it has a trend of increasing year by year. The high mortality rate, high disability rate, and high rate of disability are high. Diabetes nephropathy is one of the main causes of chronic kidney disease. There are about 300 million diabetic patients around the world, about 30% of them are diabetic nephropathy, and Enshi is also a high risk area for diabetes. According to the blood purification statistics of the Enshi Tujia and Miao Autonomous Prefecture, the diabetes mellitus is dialysable. Renal hemodynamic changes in 14.9%., renal microvascular lesions, glomerular mesangial cells and matrix hyperplasia have been considered as the main pathophysiological changes in diabetic nephropathy. In recent years, with the in-depth study of podocytes, podocyte injury, podocyte function change, hyperapoptosis of podocytes and proteinuria in diabetic nephropathy have been considered. Glomerular sclerosis is closely related and podocyte damage is the key to the development of diabetic nephropathy. Therefore, some scholars believe that the essence of diabetic nephropathy is podocyte.
The activation of renin angiotensin system (RAS) is closely related to the occurrence and development of diabetic nephropathy. In the complex network relationship between RAS and diabetic nephrotic foot cell damage, the PI3K-AKT-mTOR signal transduction pathway plays an important role. The activation of mTOR can reduce the autophagy of podocyte and promote the apoptosis of the podocyte. The activation of podocyte mTOR signaling pathway and podocyte damage, as well as the benefits of blocking the mTOR signal system, are also detected, but the specific regulatory process of the mTOR signaling pathway is still explored. Whether the hyperglycemic glucose can directly activate the mTOR signal pathway of the podocyte leads to the absence of the apoptosis of the podocyte. The partial RAS system in the kidney is not known. By affecting the hemodynamics of the kidney, it also affects urinary protein excretion, mesangial matrix hyperplasia and tubulointerstitial fibrosis, and podocyte as the core of diabetic nephropathy is not only the target of RAS causing damage, but also an important source of local RAS. The effect of RAS activation on the foot cell itself is not completely clear, especially There is less research on podocyte apoptosis and podocyte mTOR signaling pathway.
Objective To observe the changes of mTOR expression and apoptosis in podocytes induced by high glucose and the effects of renin inhibitors on them.
Methods 1. the cultured mature podocytes were divided into control group, hypertonic group, high glucose group and control group in RPMI1640 culture containing 5mmol/L glucose. Hypertonic group was RPMI1640 culture solution containing 5mmol/L glucose, 25mmol/L mannitol, and high sugar group was RPMI1640 culture medium containing 30mmol/L glucose. After 24 hours of culture, RT-PCR was used in each group. The expression of mTOR mRNA in each group, Western blot was used to determine the expression of mTOR protein in each group of podocytes. TUNEL method was used to determine the apoptotic rate of foot cells in each group. The podocytes were divided into control group, no sense transfection group, siRNA transfection group, the transfection group was added to the transfection reagent (Lipofecter plastid), mTOR siRNA transfected, the non sense control group was added to the transfection reagent, and the control (Wu Yi) s IRNA, the blank control group only added the transfection reagent (air liposome). After transfection of the transfection kit for 24 hours, the transfected podocyte was placed in the culture solution containing 30mmol/L glucose RPMI1640 for 24 hours. The expression of mTOR mRNA in each group was determined by RT-PCR, Western blot was used to determine the expression of mTOR protein in each group, and the TUNEL method was used to determine each group. The apoptotic rate of.3. was divided into the control group, the low dose Aliskiren group (including 10nmol/L Aliskiren), the medium dose Aliskiren group (Aliskiren containing 20nmol/L), the high dose Aliskiren group (30nmol/L Aliskiren), and the determination of the expression in the 30mmol/L glucose RPMI1640 culture 24 hours after the culture. N blot was used to detect the expression of mTOR protein in podocytes of each group, and podocyte apoptosis was detected by TUNEL.
Results under the high glucose stimulation, the expression of mTOR mRNA in the hypertonic group and the control group increased significantly (P0.01), and the expression of mTOR protein was also significantly up (P0.01). The apoptosis of the Poda was higher than that in the hypertonic group and the control group (P0.01). The mTOR mRNA and mTOR protein of the podocytes were significantly lower than those of the control group and the non sense transfection group after the siRNA technique was silent on the expression of mTOR gene. The apoptosis rate of foot cells decreased significantly (P0.05) in the transfected group under the stimulation of high glucose (P0.01). The expression of mTOR protein in the poddine was lower than that of the control group (P0.01), and the apoptosis of the Poda was also lower than that of the control group (P0.01). The expression of mTOR protein decreased significantly with the increase of intervention dose. There were three groups of dose groups. Statistical difference (P0.05).
Conclusion high glucose may induce apoptosis by up regulating the expression of mTOR in podocyte, and the protective effect of renin inhibitor on podocyte may be related to the down-regulation of mTOR expression in podocyte and the reduction of podocyte apoptosis under high glucose stimulation.
Objective To observe the effect of renin inhibitor Aliskiren on mTOR expression and urine podocyte excretion in diabetic nephropathy rats.
Methods 40 Wistar rats were treated by intraperitoneal injection of STZ to make diabetic model, randomly divided into diabetes group and Aliskiren treatment group, the other 20 normal Wistar rats as control group. Aliskiren treatment group was given Aliskiren10mg/kg once every day, after 8 weeks, the urine podocyte excretion number of rats in each group was collected; rats were collected. In the renal cortex, RT-PCR was used to detect the mTORmRNA expression in the kidneys of each group, and the expression of mTOR protein was detected by Western blot.
Results the excretion of urine podocyte in diabetic rats was significantly higher than that of the control group (P0.01). After 8 weeks of Aliskiren intervention, the excretion of podocyte in diabetic rats decreased significantly (P0.01), and the expression of mTOR in renal tissue of diabetic rats was significantly higher than that of the control group (P0.01). The expression of mTOR in renal tissue of rats was significantly lower than that of the diabetic group after Aliskiren treatment (P0.01).
Conclusion renin inhibitor can reduce podocyte excretion and decrease urinary protein in diabetic rats, and its renal protective mechanism may be related to downregulation of mTOR expression in renal tissue.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692

【参考文献】