| Background/purpose:Diabetic nephropathy is one of the most important microvascular complications in diabetes mellitus and its pathogenesis remains far from clear.Evidences show that renal oxidative stress may play an important role in the development of diabetic nephropathy,and glomerular podocytes are the main injury target in diabetic kidney. In the present study,we investigated mitochondrial oxidative stress in renal cortex as well as its effect on the ultrastructure and function of podocytes in experimental diabetic rodents.Meanwhile,whether renal damage from oxidative stress could be prevented by mitochondrial targeting antioxidantα-lipoic acid would have answer in our work..Methods:1.Experimental diabetic model was induced by single injection of STZ(streptozotocin)into tail vein in rats,followed by randomly division into three groups:NC,DM andα-lipoic acid treated DM(LA group).2.At 2w,4w,8w,12w,renal biochemical indexes and histology in each group were determined;indexes related to mitochondrial oxidative stress in renal cortex of rats were assayed by colorimetry.Glomerular podocytes were quantitified via indirect immunofluorescence technique.The mRNA and protein expression of podocyte slit diaphragm structural protein nephrin were detected by real time PCR and western blot accordingly.Image J software was used to perform quantitative analysis.3.Images were analyzed by Image J,and all the data were expressed as(?)±s,and analyzed by SPSS 11.0.Results:1.Diabetic rats induced successfully by STZ developed classic renal damage along with the development of diabetes.2.Indexes of oxidative stress in mitochondria from rat renal cortex in each groups: there were no significant changes of mitochondrial oxidative stress indexes in NC group,while in the other two group:1)A significant increase in MDA level was found in DM group from 4w after DM induction.Mitochondrial MDA level in rats of LA group reduced greatly from 4w, which was higher than the one in NC group(P＜0.01).2)The content of GSH declined gradually with the progression of disease in DM group,Treatment withα-lipoic acid substantially prevent GSH decrease in diabetic rats.3)Mn-SOD activity in rats in DM groups was markedly reduced from 2w,and decreased as diabetes went on in comparison with NC group.In contrast, Mn-SOD activity in rats of LA group was ameliorated from 8w(P＜0.01).4)The activity of GSH-Px in DM group was reduced at 2w,compared to NC group, but began to increase from 4w.GSH-Px activity in rats in LA group was lower than the one in DM and NC group at 4w,and higher than NC group,but lower than DM group at 8w and 12w.5)CAT activity in rats in DM group was significantly reduced than the one in NC group from 4w.No significant difference could be seen between LA and DM group(P＞0.05).3.There was equal and continuous PCX positive signal along the wall of glomerular vessels in rats in NC group,while in DM group,the PCX signal was distributed discontinuously and unevenly.Treatment withα-lipoic acid could improve it.4.Compared to NC group,the expression of nephrin mRNA and protein was significantly reduced in DM group from 2w to 12w;Treatment withα-lipoic acid could increase the mRNA and protein expression from 4w.5.Podocytes could be seldom found in urine of rats in NC group.Numerous podocytes were found during the observation period as time went by and significantly increased compared to NC group at the same time point(P＜0.01). No significant difference between LA and DM group(P＞0.05).6.analysis of correlation and regression1)umA,uTP,BUN and CRE in diabetic rats were positively correlated with MDA and GSH-Px,and negatively associated with Mn-SOD,GSH and CAT(P＜0.01). The improvement of proteinuria and renal function in rats of LA group was linear correlated with the change of MDA and GSH.2)VG,GBM,density of mesangial matrix,and fibrosis of renal tubular in diabetic rats were positively correlated with MDA and GSH-Px,and inversely associated with Mn-SOD,GSH and CAT(P＜0.01).The amelioration of kidney pathology showed linear correlation with the change of MDA,GSH and Mn-SOD by LA.3)MDA and GSH-Px were inversely correlated with PCX influorescence density,the gene and protein expression of nephrin in diabetic rats,and positively associated with podocytes in urinary sediments(P＜0.01);GSH,Mn-SOD and CAT were positively correlated with PCX influorescence density,the gene and protein expression of nephrin,and inversely associated with urinary podocytes(P＜0.01).4)Proteinuria and renal function in diabetic rats were negatively correlated with PCX influorescence density,the gene and protein expression of nephrin,and positively associated with podocytes in urinary sediments(P＜0.01).5)PCX influorescence density,the gene and protein expression of nephrin were ??negatively correlated with VG,GBM,density of mesangial matrix,and fibrosis of renal tubular,and positively associated with podocytes in urinary sediments(P＜0.01).Conclusion:1.Obvious oxidative stress injury and impairment of defense ability existed simultaneously in mitochondria in diabetic renal cortex.2.Oxidative stress is significantly correlated with proteinuria and renal dysfunction.3.Mitochondrial oxidative stress in renal cortex is markedly associated with the effacement and broadened of foot process,reduced podocyte number and nephrin expression.4.Podocyte detached into urinary space is contributed to the reduction of glomerular podocyte number,and associated with increased oxidative stress.5.Treatment with antioxidant LA can partly reduce proteinuria in STZ-induced diabetes and delay the progression of DN,resulted from decreased MDA content and increased Mn-SOD activity and GSH content,leading to protect podocytes against oxidative injury.6.Podocytes in the urine was not an indicator for L A effect on DN in present study.
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