Effects Of Valsartan On The Expression Of JNK Pathway And Caspase-3 In Renal Tissue Of Diabetic Rats

Objective: With the growing of diabebes mellitus population,diabetic nephropathy has a high morbidity,has become the main cause to progress to end-stage renal disease. Accompany with the progression of diabetes,the activation of RAS increased,oxidised NAD(P)H can stimuli increase the production of endovascular reactive oxygen species through angiotensinⅡbond with angiotensin receptorⅠ, superoxide anion O2-,for instance,leading to the decrease of bioactive NO and glutathione peroxidase,And then it can make inactive NO transform into toxic peroxynitrite lead to the activation of JNK pathway, significantly high Angiotensinogen expression in glomerular mesangial cells,proximal tubule cells,can do damage to kidney.With the elevation of caspase-3 in diabetes,the apoptotic rate in glomerular podocytes mesangial cells tubules epithelial cells and mesenchymal cells also increased.AngiotensinⅡtype 1 receptor blocker inhibits can block the vicious cycle of RAS and ROS producing,improve insulin sensiti- vity,ameliorate insulin resistance, inhibit the formation and gather of AGE,decrease urinary angiotensinogen concentration,reduction the expected incidence of diabetes.This experiment was conducted to investigate the effection of valsartan to cell apoptosis mediated by JNK pathway and casepase-3 and the possible mechanism in the early stage of diabetic nephropathy,to provide theoretical support for the clinical treatment.Methods:Male SPF wistar rats,were divided to 3 group: normal control group(n=10),diabetic group(n=17),valsartan-treated group(n=17).The rats of diabetic group and valsartan-treated group received a signal intraperitoneal injection of STZ at a dose of 60mg/kg and the rats of normal control group only received an injection of the same volume of sodium citrate.The diabetic model was considered to be successful when the blood glucose was equal or greater than 16.7mmol/l after 72 hours of STZ injection.The rats of valsartan-treated group and normal control group were received valsartan (10mg/kg·d) by gastric perfusion and diabetic group were perfused with the same volume of water.All rats were allowed free access to food and water during the experiment.Collect blood and urine samples,Dispose of half of rats of every group separately used ether on 4,8 weeks, measured renal weight/body weight ratio,the abdominal cavity was opened to find inferior vena cava and take blood so that measure blood glucose,urea nitrogen,serum creatinine, serum uric acid, triglyceride, cholesterol, serum albumin,HDL,LDL and tested 24-hour proteinuria excretion. Renal tissues was measured JNK, p-jnk and caspase-3 via immunohistochemical staining, detected MKK7,JNK, p-jnk,casapse-3 via Western blotting;and measured cell apoptotic rate via flow cytometry assays.Analyze data by using SPSS13.0.Results1. Immunohistochemical staining:①The expression of JNK, p-jnk,caspase-3 increased significantly between diabetic group and valsartan- treated group with no significant differences in intergroup (P>0.05),higher than that of normal control group(P<0.05) at 4 weeks.At 8 weeks,the positive areas are increased significantly between diabetic group and valsartan-treated group,than that of normal control group(P<0.05);the positive areas of valsartan-treated group is lower than that of diabetic group(P<0.05).2 Western bloting:There are no significant differences of MKK7,JNK, p-jnk and caspase-3 among three group at 4 weeks(P>0.05). At 8 weeks Comparison with diabetic group,the amount of MKK7,p-jnk,caspase-3 in valsartan-treated group and normal control group are significantly lower(P<0.05),and the expression level is higher in valsartan group than that of normal control group (P<0.05),the expression of JNK is lower between normal control group and valsartan-treated group with no significant differences in intergroup,than that of diabetic group.3 Flow cytometry assays: There were no significant differences of renal apoptotic rate between normal control group and valsartan-treated group(P>0.05),also no significant differences between diabetic group and valsartan-treated group(P>0.05),the apoptotic rate in diabetic group is higher than that of normal control group(P<0.05) at 4 weeks.Comparison with diabetic group,the value of kidney apoptotic rate is lower between normal control group and valsartan-treated group(P<0.05);the mean value of valsartan-treated group is higher than that of normal control group at 8 weeks(P<0.05).4 The correlation analysis of MKK7,JNK,p-jnk,caspase-3 with apoptotic: The correlation coefficient of MKK7,JNK,p-jnk,caspase-3 with apoptotic separately are r=0.908, 0.838, 0.870,0.935,( P<0.05).5 Proteinuria:The amount of 24-hour proteinuria excretion is no significant differences in each group at 4 weeks(P>0.05);At 8 weeks ,the amount of 24-hour proteinuria excretion between diabetic group and valsartan-treated group,higher than that of normal control group(P<0.05), the amount of proteinuria excretion of valsartan-treated group is lower than that of diabetic group (P<0.05).6. Renal mass/body mass ratio: At 4,8 weeks there are no statistical differences between diabetic group and valsartan-treated group (P>0.05),but both higher than that of normal control group(P<0.01);Body weight:The body weight of normal control group at 8 weeks is heavier than the other groups(P>0.05).7 Blood Glucose:At 4,8 weeks,blood glucose is equal or greater than 16.7mmol/l between diabetic group and valsartan-treated group, higher significantly than that of normal control group.8 Blood biochemical tests:No significant differences are found among each group in the results of urea nitrogen,serum creatinine,serum uric acid, triglyceride,cholesterol,serum albumin,HDL and LDL tests among each group.Conclusion: A variety of cellular stresses can stimulate enhance activation of JNK pathway in diabetes,and then induce a cascade of signaling events,lead to higher expression of MKK7,JNK,p-jnk and caspase-3 which is pivotal factor.Valsartan treatment is beneficial to kidney through down- regulate the activation of JNK pathway and caspase-3,reduce apoptotic rate,diabetic group,lower proteinuria quantity.