Mechanisms And Drug Intervention Research On Oxidative Stress Induced Injury Of Podocyte In Obesity-related Glomerulopathy, Diabetic Nephropathy

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.