【作者】 王彤；

【导师】 马瑞霞；

【作者基本信息】 青岛大学 ， 内科学（肾病）（专业学位）， 2018， 硕士

【摘要】 背景:众所周知,糖尿病肾病(diabetic nephropathy,DN)已成为终末期肾病(end stage renal disease,ESRD)最常见的原因之一,约30%-40%DN患者可发展为ESRD,且近年来有上升趋势。近些年对于DN的研究已经取得很大进展,但DN的发病率仍持续升高,且治疗效果差,给患者及其社会带来沉重的经济负担,因此,急需新的靶向治疗药物以缓解DN的进展,DN在临床上主要表现为持续性的蛋白尿及进行性肾功能恶化,而足细胞作为肾小球滤过屏障的重要组成部分,DN早期即伴随着足细胞损伤及蛋白尿的形成。近年来,足细胞病变被认为是DN发病机制的中心环节,也是蛋白尿产生的重要原因。研究显示,正常足细胞具有较高水平的自噬,而大量的体内外实验显示DN中肾脏足细胞自噬水平呈下调状态,以上实验结果均提示DN中足细胞自噬水平的抑制在足细胞病变中起重要作用。并且有研究发现他克莫司可恢复心肌细胞的自噬作用,但对DN肾脏足细胞的研究甚少。目的:本实验通过构建2型糖尿病(type 2 diabetic mellitus,T2DM)大鼠模型,探讨他克莫司对DN大鼠肾脏足细胞损伤和自噬的影响,进一步揭示自噬在DN肾脏足细胞保护机制。方法:随机选取40只清洁级的健康8周龄雄性wistar大鼠,在其中随机选取10只大鼠作为对照组(NC组),剩余30只大鼠给予高糖高脂喂养8周后,给予一次性腹腔注射小剂量链脲佐菌素(Streptozotocin,STZ,30mg/kg)构建T2DM模型大鼠,造模成功22只,选取状态较好的20只造模大鼠,将其随机分为糖尿病组(DM组)和他克莫司治疗组(TAC组),后者给予FK506(0.5mg/kg·d)干预8周。观察各组大鼠的血糖、血脂、肝肾功、血常规等一般指标,即肾肥大指数(kidney weight/body weight,KW/BW)、血压(systolic blood pressure,SBP)、空腹血糖(fasting blood glucose,FBG)、胆固醇(cholesterol,CH)、甘油三酯(triglycerides,TG)、谷丙转氨酶(alanine transaminase,ALT)、谷草转氨酶(aspartate aminotransferase,AST)、血肌酐(serum creatinine,Scr)、血白细胞(white blood cell,WBC)、尿肌酐(urine creatinine,UCr)、24小时尿白蛋白(urinary albumin,UAL),并根据公式测定内生肌酐清除率(creatinine clearance rate,Ccr)、24小时尿白蛋白排泄率(urinary albumin excretion rate,UAE),光镜、电镜观察肾脏病理和足细胞超微结构改变,免疫组织化学检测nephrin蛋白表达,Western-blot测定自噬标志蛋白微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)的表达。结果:1)本研究选取30只wistar大鼠进行造模,造模成功22只,造模成功率73.33%。2)与NC组大鼠相比,DM组大鼠KW/BW、FBG、SBP、CH、TG、Ccr、UAE明显升高(P<0.05),TAC组大鼠KW/BW、Ccr、UAE明显低于DM组(P<0.05),其他指标无明显差异。2)光镜下DM组大鼠肾小球体积增大,肾小球系膜细胞及系膜区基质增多,TAC组上述病理变化有不同程度恢复。3)电镜下DM组大鼠基底膜不均匀增高,足细胞数量减少,足突融合、消失,FK506干预后上述病变明显减轻。4)与NC组相比,DM组大鼠肾组织nephrin及LC3-Ⅱ表达减弱(P<0.05),而TAC组大鼠较DM组nephrin及LC3-Ⅱ表达增强(P<0.05)。5)Pearson相关性分析提示,LC3-Ⅱ/GADPH含量与nephrin蛋白表达量成正相关(r=0.949,p<0.05)。结论:他克莫司可降低DN大鼠尿蛋白,同时减轻足细胞的损伤,他克莫司可能通过上调T2DM大鼠肾脏足细胞的自噬作用降低尿白蛋白排泄率、延缓DN肾功能损伤的进展。更多还原

【Abstract】 Background: It is well known that diabetic nephropathy(DN)has become one of the most common causes of end stage renal disease(ESRD).About the patients with 30%-40%DN can develop into ESRD,and there is an increasing trend.In recent years,great progress has been made in the study of DN,but the incidence of DN continues to increase,and the therapeutic effect is poor.Therefore,new targeted drugs are urgently needed to alleviate the progression of DN.The main clinical manifestations of DN are persistent proteinuria and progressive deterioration of renal function.As an important part of glomerular filtration barrier,podocyte is associated with podocyte injury and proteinuria in the early stage of DN.In recent years,podocyte lesion is considered to be the central part of the pathogenesis of DN.Studies have shown that normal podocytes have a high level of autophagy,while a large number of experiments have shown that autophagy levels of podocytes in DN are down-regulated in vivo and in vitro.These results suggest that the inhibition of podocyte autophagy in DN may play an important role in the pathogenesis of podocytes,and tacrolimus has been found to restore the autophagy of cardiacmyocytes.Objective: In this study,the rat model of type 2 diabetes mellitus(T2DM)was established to investigate the effects of tacrolimus on podocyte injury and autophagy,and to further reveal the mechanism of autophagy in DN podocyte protection.Method: Forty healthy 8-week-old male wistar rats were randomly selected.Among them,10 rats were randomly selected as normal control group.The remaining 30 rats were fed with high sugar and high fat for 8 weeks.Then T2 DM model rats were established by intraperitoneal injection of small dose streptozotocin(STZ,30mg/kg).22 rats were successfully established into T2 DM,20 rats in good condition were randomly divided into diabetic mellitus group(DM group)and tacrolimus treatment group(FK506,0.5mg/kg d)for 8 weeks.The usual parameters were measured,including renal hypertrophy index(kidney weight/body weight,KW / BW),systolic blood pressure(SBP),fasting blood glucose(FBG),serum creatinine(Scr),total cholesterol(CH),serum triglycerides(TG),Alanine transaminase(ALT),Aspartate aminotransferase(AST),and white blood cell(WBC),urinary creatinine(UCr),urinary albumin(UAL).The creatinine clearance rate(Ccr)and urinary albumin excretion rate of 24 hours(UAE)were determined according to the formula.The changes of renal pathology and ultrastructure of podocyte were observed by the light and electron microscopy.The expression of nephrin and LC3-Ⅱ were determined by immunohistochemistry and Western-blot.Result: 1.22 of 30 rats were successfully established into T2 DM,and the successful rate was 73.33%.2.Compared with the NC group,KW/BW,SBP,FBG,TG,TC,UAE,Ccr of DM rats were significantly increased(P <0.05),and the KW/BW,UAE and Ccr were decreased in TAC group(P<0.05),while other parameters were no significant difference(P>0.05).3.Increased glomerular volume,mesangial cell proliferation,accumulation of mesangial matrix were shown by light microscope in DM group,but the above pathological changes were restored in TAC group.4.Under electron microscope,basement membrane thickening,the foot process became disorder and fusion in DM group,while these changes were significantly reduced in TAC group.5.Compared with the NC group,the expression of nephrin and LC3-Ⅱ were decreased in DM group(P<0.05),and both of parameters were restored after treatment of FK506(P<0.05).6.The Pearson correlation analysis showed that the renal LC3-Ⅱ/GADPH level was positively correlated with nephrin expression intensity(r=0.949,p<0.05).Conclusions: Tarcolimus can reduce proteinuria and podocyte damage of the rats with DN.The potential mechanism is that tarcolimus may enhance podocyte autophagy in type 2 diabetic model rats,leading to attenuate podocyte injury and delay progression of DN.更多还原