Role Of Podocyte And The Associated Molcules In Pathogenesis Of Diabetic Nephropathy And Effects Of Losartan

BACKGROUD AND OBJICTIVE:Diabetic nephropathy(DN)is a major chronic complication of diabetes mellitus.But the exact pathogenesis of diabetic nephropathy has not been fully elucidated so far.Proteinuria is one of the most common clinical hallmarks of diabetic nephropathy,which has been believed to be a dangerous factor that leads to renal damage.The occurrence of proteinuria is associated with the glomerular filtration barrier.The structural and function obstacle of glomerular filtration barrier composed of podocyte and related protein has played an important role in the forming of proteinuria of diabetic nephropthy,and has been focused on recently.In the present study,diabetes rat model was established,and podocyte number and podocyte density and the expression of nephrin, WT1,PCX in the renal tissues of experimental diabetic rats as well as the excretion of nephrin in urine were observed,to investigate the changes and its significance of podocyte and the associated protein in DN proteinuria occurrence and development.In addition,the possible machanism for losartan to reduce proteinuria and protect renal function is also investigated,in order to provide theoretical basis for the pathogenesis, diagnosis,prevention and treatment of DN.METHODS:Diabetic rats model was induced by a single intraperitoneal injection of streptozotocin(STZ).The diabetic rats were randomly divided into diabetic group without treatment(DM group),early losartan-treated diabetic group(DL1 group,treated with losartan 30mg/L in the beginning of the study),mid-term losartan-treated diabetic group(DL2 group, treated with losartan 30mg/L in the 4th week after the beginning of study), and the normal rats were served as a normal control group(NC group). Blood glucose(BG),Blood Urea Nitrogen(BUN),Serum Creatinine(Scr), Urinary Creatinine(Ucr)and 24-hours urinary albumin were measured in the 2nd,4th,6th,8th and 12th week and Ccr was calculated after the onset of diabetes in NC group and DM group.The renal cortical tissues were obtained and used for optics microscopy.The expression values of nephrin,WT1,PCX in kidney were measured with immunohistochemical method and Western-Blot analysis.The excretions of nephrin in urine were measured with Western-Blot analysis.DL1,DL2 group were dealed and observed the same with DM group.RESULTS:1.Compared with NC group,BG increased significantly in DM group and DL1,DL2 groups(P＜0.01);no statistic differences of BG were found between DM group and DL1,DL2 groups.2.Urinary albumin(mg/L)increased gradually till the 12th weeks in DM group.It was as high as 72.21±8.24 in the 4th week,significantly higher than in NC group by 6.87±0.67(P＜0.05),no statistic differences between DM group and NC group were found at the 2nd week,showing that diabetic nephropathy appeared at the 4th week after the onset of diabetes.Blood Urea Nitrogen(BUN)(mmol/L)increased gradually till the 12th week in DM group.No statistic differences were found in NC group at the 2nd week.It is as high as 10.24±0.46 at four weeks, significantly higher than in NC group 5.34±0.23(P＜0.05). Ccr(ml/min·Kg)dropped gradually in DM group,and in the 6th week it reached 3.27±0.26,which had relatively statistic differences(P＜0.05) compared with NC group(6.78±0.37).It showed that albuminuria appear earlier than BUN and Ccr in DM group.The urine albumin in DL1,DL2 groups were lower than in DM group[(98.54±22.37), (112.76±21.74)vs(211.35±18.23),P＜0.05].The BUN were lower than in DM group[(12.27±1.64),(13.09±1.58)vs(15.33±1.76),P＜0.05].The Ccr were higher than in DM group[(3.26±0.68), (2.76±0.74)vs(1.93±0.37),P＜0.05].But,there were no statistic differences between DL1 and DL2 group.3.The podocyte density(WT1 posotive cell area/glomeruli area%) was decreased(17.6±0.93)at the 2nd week in DM group compared with that in NC group(P＜0.05).The podocyte number(n/1000um~2 glomeruli area)in DM group was lower than in NC group[(13.6±2.04)vs (18.2±2.13),P＜0.05]at the 4th week.Immunohistochemical and Western-Blot analysis showed the expression of nephrin,WT1,PCX was obviously downregulated in the 2nd week in DM group.They were lower than in NC group,parting for nephrin[Immunohistochemical analysis (50.07±1.83)vs(55.24±1.96),P＜0.05;Western-Blot analysis (0.36±0.121)vs(0.54±0.137),P＜0.05];WT1[Immunohistochemical analysis(19.37±2.09)vs(25.67±2.01),P＜0.05;Western-Blot analysis (0.14±0.048)vs(0.23±0.059),P＜0.05];PCX[Immunohistochemical analysis(68.19±1.86)vs(71.35±1.67,P＜0.05;Western-Blot analysis (0.51±0.186)vs(0.77±0.205),P＜0.05].The podocyte density and podocyte number of glomeruli,the expression of nephrin,WT1,PCX were further decreasing along with the progress of time till the 12th week. There were statistic differences between different weeks(P＜0.05).The podocyte density and podocyte number of glomeruli,the expression of nephrin,WT1,PCX were upregulated after being treated with losartan. Each index in DL1 group was 14.3±0.98,11.4±1.97,36.67±2.05, 14.53±2.36,46.74±1.98 respectively.In DL2 group it was 13.5±1.14, 10.9±2.03,34.83±1.98,13.48±2.24,44.29±1.83,compared with each corresponding index of DM group(7.2±0.96,5.0±2.01,9.93±1.95, 3.56±1.76,11.35±1.67),P＜0.05.DL1 group were more upregulated than DL2 group,but there were no statistic differences between them(P＞0.05).4.Nephrin were not appeared in urine of NC group rats.It could have had appeared in urine of diabetic rats,at the earlist in the 2nd week,and right now,urine albumin did not increas obviously yet.Urine albumin was increased obviously,nephrin in urine were also increased gradually, after reaching the peak at the 8th week,then it decreased gradually again.5.Related analysis shows that urinary albumin excrection was negatively correlated with podocyte density and podocyte number of glomeruli,the expression of nephrin,WT1,PCX respectively(r=-0.736, P＜0.01;r=-0.578,P＜0.05;r=-0.863,P＜0.01;r=-0.758,P＜0.01; r=-0.776,P＜0.01)CONCLUSION:1.The podocyte density and podocyte number of glomeruli,the expression of nephrin,WT1,PCX were downregulated in renal of diabetic rats early in the 2nd week.They were early damage index of DN. Podocyte and its related molecular nephrin,WT1,PCX have participated in the occurrence and development of proteinuria in diabetic nephropthy.2.Losartan may reduce urine protein and delay DN progress through delaying or restrainting the lose of podocyte,maitaining normal podocyte quantity,as well as restrainting downregulation of expression of nephrin, WT1,PCX in glomeruli of diabetic rats.For the treatment of diabetic nephropathy that has occurred,losartan also had effects.3.In the early stage of diabetic nephropathy,nephrin could excrete in urine.Urine nephrin may be as an early diagnoses index,which will be helpful for inspection and judgement of progress of DN.