【作者】 孙亮亮；

【导师】 刘志民； 石勇铨； 邹俊杰；

【作者基本信息】 第二军医大学 ， 内科学， 2008， 博士

【摘要】 肾小球脏层上皮细胞即足细胞(podocyte),是维持肾小球滤过屏障结构和功能正常的主要细胞之一。大量形态学研究证实,许多肾小球疾病可见足细胞结构异常,包括足突增宽、融合、消失以及足细胞减少。足细胞是终末分化细胞,无增殖、再分化能力,基因突变、免疫因素、血流动力学异常、高糖、高脂及氧化应激均能导致足细胞异常、凋亡和数量减少。糖尿病肾病(diabetic nephropathy,DN)是糖尿病的严重慢性并发症,是西方国家终末期肾病(end stage renal disease,ESRD)的主要病因。随着饮食结构和生活方式的改变,肥胖的发病率逐年增多,由其导致的肥胖相关性肾病(obesity-relatedglomerulopathy,ORG)也越来越受到研究者的重视。两者早期均以微量白蛋白尿为主要表现,有共同的发病基础。足细胞数量减少、足突形态改变、足突蛋白的丢失被认为是ORG和DN发病早期重要的改变,并且其在蛋白尿的发生、发展中起到了关键性的作用。统一学说和共同土壤学说提出,氧化应激是糖尿病、肥胖等炎症性疾病组织损伤的共同病理生理学机制。氧化应激活化的MAPK家族信号转导通路对肾小球固有细胞增殖、凋亡的调控已成为目前研究的焦点。本研究采用多种研究手段针对ORG、DN中足细胞损伤的形式、与蛋白尿的相关性以及氧化应激对足细胞凋亡的调控进行了探讨,并使用通络方剂、a硫辛酸等抗氧化剂对糖尿病肾病进行了干预研究,发现足细胞数量、密度和足突蛋白Nephrin的减少在肥胖、糖尿病肾损害中有重要的意义,氧化应激诱导MAPK家族的JNK/SAPK蛋白磷酸化可进一步激活凋亡相关蛋白caspase-3信号通路的表达改变,使足细胞凋亡、数量减少、足突裂孔膜蛋白表达异常,足突融合、增宽、微绒毛化改变,从而导致肾小球滤过屏障受损,引起蛋白尿的发生。通络方剂对糖尿病肾病足细胞损伤有一定的保护作用。研究目的:研究足细胞损伤与和肥胖、糖尿病肾病的相关性,探讨氧化应激诱导的足细胞凋亡在肥胖相关性肾病和糖尿病肾病中的意义。研究对象及方法:以SD大鼠为研究对象。设正常对照组(以下简称CON组)大鼠18只;高热量饮食(40%热量来自脂肪)诱导的食源性肥胖模型(以下简称DIO模型)大鼠22只;高热量饮食基础上腹腔注射35mg/kg链脲佐菌素诱导的非遗传性2型糖尿病模型(以下简称DM模型)大鼠19只。课题分成四个部分进行:第一部分:采用免疫组织化学方法对足细胞核蛋白WT-1标记染色,运用体视学体视框/分合法(disector/fractionator)结合Image-ProPlus 6.0图像分析软件计数足细胞数量,测量肾小球体积,计算足细胞密度,Spearman相关性分析足细胞数量和密度与上述两种动物模型蛋白尿的关系。第二部分:透射电镜观察第12周时足细胞超微结构的改变;western blot检测三组动物不同时相点肾皮质足突蛋白nephrin的表达变化,研究足细胞形态改变和nephrin表达变化在ORG和DN发病中的意义。第三部分:检测三组动物不同时相点肾皮质氧化产物MDA含量和SOD活力,评估两种模型肾脏氧化应激程度;western blot同时间检测p-JNK蛋白和足细胞凋亡相关蛋白cleaved caspase-3在肾皮质中的表达,讨论JNK/SAPK信号通路对ORG和DN足细胞凋亡的调控。第四部分:以抗氧化剂a硫辛酸(100mg/kg/d)为对照,观察通络方剂(0.4g/kg/d)对DM模型大鼠足细胞损伤的保护作用。结果:1.肾小球足细胞计数与肥胖、糖尿病大鼠蛋白尿的相关性分析:①与CON组相比,DIO模型大鼠体重和胰岛素持续升高;4周、8周、12周三个时相点尿白蛋白肌酐比值逐渐升高(p＜0.05);随着肾小球体积增大足细胞密度逐渐减小;足细胞密度与尿白蛋白肌酐比值呈负相关(r=-0.825,R~2=0.6811,p＜0.05);②与CON组相比,DM模型大鼠血糖持续保持高水平;4周、8周、12周三个时相点24小时尿蛋白定量和尿白蛋白肌酐比值均逐渐升高,肾小球足细胞数量和密度均逐渐减小(p＜0.05);足细胞计数与24小时尿蛋白定量呈负相关(r=-0.828,R~2=0.6842,p＜0.05)。2.足突蛋白Nephrin在肥胖、糖尿病大鼠肾脏的表达及足细胞形态学观察:①与CON组相比,第4周、8周、12周,DIO模型和DM模型大鼠肾皮质nephrin/β-actin蛋白光密度比值逐渐减小,差异有统计学意义(p＜0.05);②第12周时透射电镜观察,DIO模型大鼠GBM节段性增厚,足细胞内线粒体肿胀、空泡化,可见脂滴和蛋白小体形成,足突增宽、融合并有微绒毛化改变;DM模型大鼠GBM均匀增厚,系膜区增宽,足突融合、微绒毛化较DIO组更加明显,足细胞胞浆内可见自噬小体前体,足细胞核内可见凋亡小体形成。3.氧化应激JNK/SAPK信号通路对肥胖、糖尿病大鼠肾小球足细胞凋亡的调控:①与CON组相比,第4周、8周、12周,DIO模型和DM模型大鼠肾皮质MDA含量逐渐增多,SOD活力逐渐下降,差异有统计学意义(p＜0.05);②与CON组相比,第4周、8周、12周DIO模型和DM模型大鼠肾皮质磷酸化JNK蛋白的表达量逐渐增多,cleaved caspase-3蛋白的表达与磷酸化JNK一致,而总JNK蛋白表达在两模型组的各个时相点均无差异。4.通络方剂等抗氧化剂对糖尿病大鼠足细胞损伤的保护作用:通络方剂(0.4g/kg/d)与抗氧化剂a-硫辛酸(100mg/kg/d)分别干预DM模型大鼠12周后,两组大鼠24小时尿蛋白定量明显低于未治疗组,足细胞计数明显高于未治疗组,差异有统计学意义;与DM对照组相比,两种药物干预组大鼠肾皮质MDA含量减少,SOD活力升高,磷酸化JNK和cleaved caspase-3蛋白表达均降低(p＜0.05)。两干预组之间上述指标相比差异无统计学意义(p＞0.05)。结论:1.高热量饮食可成功诱导食源性肥胖大鼠模型,DIO大鼠具有肥胖、高血脂、高胰岛素血症等特征,模型稳定;高热量饮食+35mg/kg的STZ可成功诱导非遗传性2型糖尿病模型,DM大鼠具有高血糖、高血脂、胰岛素水平不低的特征,模型稳定。2.DIO模型大鼠足细胞相对密度减小是足细胞受损的主要形式;DM模型大鼠足细胞绝对数量减少是其足细胞受损的主要形式;足细胞损伤与蛋白尿的发生均密切相关;足突增宽、融合微绒毛化、足细胞凋亡是这两种动物模型中足细胞主要的形态学改变。足突裂孔膜Nephrin蛋白表达下调是判断早期肾小球滤过屏障受损的重要指标,在ORG和DN的发病中有重要意义。3.DIO模型和DM模型大鼠肾皮质氧化应激水平明显升高,氧化应激激活JNK/SAPK通路,其下游信号通路活化caspase-3蛋白诱导足细胞凋亡。4.通络方剂具有抗氧化作用,对DM模型大鼠肾小球足细胞的损伤有保护作用,这种保护作用可能是通过减轻糖尿病肾皮质氧化应激实现。更多还原

【Abstract】 Glomerular epithelial cell(podocyte) is major cell in maintaining the structure and function of glomerular filtration barrier.Large morphological study confirmed that podocyte structural abnormalities accompanied many glomerular diseases,including foot protrusion widened,integration,as well as cells reduction.Podocyte differentiation is the end,no proliferation,differentiation capacity,gene mutations,immune factors, hemodynamic abnormalities,high-sugar,high-fat and oxidative stress can lead to abnormal cells,apoptosis and reduce the number.Diabetic nephropathy(DN) is a serious chronic diabetic complications,the major cause of end-stage renal disease(ESRD) in western countries.With diet and lifestyle changes,the incidence of obesity increasing year by year,by the result of obesity-related nephropathy has received increasing attention to researchers.Microalbuminuria is early performance in ORG and DN.Decrease in the number of cells,the foot protrusion morphological changes,the loss of foot protrusion protein is considered ORG and the incidence of early DN important change,and proteinuria in the occurrence and development play a crucial role.Unified theory proposed oxidative stress is common pathophysiology mechanism in diabetes,obesity and other inflammatory diseases. Oxidative stress-activated MAPK signal transduction pathway family on the inherent glomerular cell proliferation and apoptosis has become the focus of the present study.This study used a variety of research tools on ORG,DN podocyte injury in the form of proteinuria associated with oxidative stress,as well as the regulation of apoptosis were discussed.And the use of Tong Xin Luo,a lipoic acid and other antioxidants on the intervention of diabetic nephropathy study.We found that the number of cells,density and foot protrusion of nephrin reduction in obesity,diabetes kidney damage in significance. Oxidative stress-induced MAPK family of JNK/SAPK protein phosphorylation could be further activated cells apoptosis-related protein signaling pathway of caspase-3 expression changed so that adequate apoptosis,reduction in the number foot hole sudden abnormal expression of membrane proteins,foot protrusion integration widened,the microvilli of change,resulting in glomerular filtration barrier damage caused proteinuria occurred.Tong Xin Luo have some protection effection on podocytes injury in diabetic nephropathy.Methods:SD rats have been studied.Control group(CON,n=18);high calorie diet(40% calories from fat) induced obesity model(DIO,n=22);high-calorie diet on the basis of celiac injection of 35mg/kgSTZ-induced non-genetic model of type 2 diabetes mellitus (DM,n=19).research is divided into four parts:PartⅠ:By immunohistochemical methods on protein WT-1 marker staining using stereological dissector/fractionator methods in Image-Pro image analysis software counts the number of podocytes,the measurement of glomerular volume,the density of podocytes, and Spearman correlation analysis between PN and proteinuria.PartⅡ:TEM observation of the first 12 weeks cells ultrastructural changes;western blot test three groups of animals at the same time do not point renal cortex foot protrusion nephrin expression of protein on cells nephrin expression patterns change and changes in the incidence ORG and DN The significance.PartⅢ:Detection of the three groups of renal cortex oxidation products MDA content and SOD,to assess the degree of oxidative stress;at the same time western blot p-foot JNK protein and apoptosis-related protein cleaved caspase-3 in the cortex.Discuss JNK/SAPK signaling on the ORG and DN and the regulation of apoptosis.PartⅣ:Observation Tong Xin Luo(0.4g/kg/d) and a-lipoic acid protection in DN and podocytes injury.Results:1.Podocyte cell count and obesity,proteinuria in diabetic rats Correlation Analysis:①compared with CON group,DIO rats sustained weight and insulin increased 4 weeks,8 weeks,12 weeks,three points urinary albumin creatinine ratio gradually increased (p＜0.05);glomerular volume increased with the foot cell density decreases gradually foot cell density and urinary albumin creatinine ratio was negatively correlated(r=-0.825,R~2 =0.6811,p＜0.05);②compared with CON group,DM rats sustained high blood sugar 4 weeks,8 weeks,12 weeks three time points 24-hour urine protein and urinary albumin creatinine ratio gradually increased,podocyte the volume and density of cells gradually decreased(p＜0.05);podocyte cell count and 24-hour urine protein was negatively correlated(r=-0.828,R~2=0.6842,p＜0.05).2.Nephrin expression in obese,diabetic rat kidney and morphological observation:①compared with the CON,in DIO model and DM the model of rat renal cortex,nephrin/β-actin protein optical density ratio decreases,the difference was significant(p＜0.05);②TEM observation:DIO rats GBM segmental thickening,swelling of mitochondria within cells,vacuoles,and that lipid and protein body formation,foot protrusion widened, integration and microvilli of the change;DM rats GBM uniform thickness,mesangial area widened,foot process integration,microvilli of the more obvious than DIO group,within the cytoplasm of cells that the body macrophage precursors,adequate apoptotic nuclei body formation.3.JNK/SAPK signaling pathway on the regulation of apoptosis of obese,diabetic rats podocytes:①compared with the CON,four weeks,eight weeks,12 weeks,DIO model and the model of rat renal cortex DM content of MDA gradually increased,SOD activity has been gradually declining,a statistically significant difference(p＜0.05);②compared with the CON,four weeks,eight weeks,12 weeks and DM DIO rat model of renal cortex p-JNK protein was gradually increased,cleaved caspase-3 expression and p-JNK consistent,and T-JNK protein expression in the model group various time points no difference.4.Tong Xin Luo and other antioxidants role in the protection of podocyte injury in diabetic rats:Tong Xin Luo(0.4g/kg/d) and a-lipoic acid(100mg/kg/d) respectively intervention DM rats after 12 weeks,the rats were 24-hour urine protein was significantly lower than that of the treatment group enough cell count was significantly higher than that of the treatment group,there were significant differences with DM compared to the control group,two drug intervention group rat renal cortex MDA content less,the higher the activity of SOD,p-JNK and cleaved caspase-3 protein expression were lower(p＜0.05). Between these two indicators of drug group compared the difference was not statistically significant(p＜0.05).Conclusion:1.Successful high calorie diet-induced obese rats,DIO rats with obesity, hyperlipidemia,hyperinsulinemia,and other characteristics of model stability;high-calorie diet+35 mg/kg STZ-induced can be successful nongenetic 2 diabetic model,DM rats with high blood sugar,high blood fat,low insulin level is not the characteristics of model stability.2.DIO model cells is reduced relative density cells damaged the main form;DM model cells absolute decrease in the number of cells is the main form of damage;two cells the occurrence of injury and proteinuria are closely related;microvilli foot process, apoptosis is adequate model of the two major morphological changes of cells.Nephrin foot protrusion hole membrane protein expression is reduced glomerular filtration barrier early judgement an important indicator of damage in the DN ORG and in the pathogenesis of important significance.3.DIO model and the model DM rat kidney serious degree of oxidative stress and oxidative stress signaling pathway may lead through JNK/SAPK/caspase-3 cell apoptosis.4.Tong Xin Luo antioxidant role of the DM model cells injury protection,such protection may be by reducing oxidative stress in diabetic kidney cortex to achieve.更多还原