| PARTⅠRecombinant Adenovirus-Mediated Decorin Gene Transfection Has The Effect of Anti-proteinuria and Anti-Fibrosis on STZ-induced Diabetic RatsBackgroundAccumulating evidence suggests that TGFβ1 is a key mediator in diabetic nephropathy.One of the approaches down-regulating TGFβ1 expression is to use proteoglycanⅡ(decorin),a natural inhibitor of TGFβ1.In the present study,a recombinant adenovirus expressing decorin(AD-DCN)was utilized to investigate the effects of decorin over-expression on kidney of streptozocin(STZ)induced diabetic rats.Material and MethodsAD-DCN was constructed according to our previous research and confirmed to express intact and active rat decorin.Recombinant adenovirus vector of LacZ gene (AD-lacZ)was constructed in the same way,used as transfection control.Diabetic Sprague-Dawley rats was induced by once intraperitoneal injection of STZ(60mg/kg).8 weeks after the diabetic model had succeeded,diabetic rats were divided into three groups randomly:diabetic group treated with PBS 0.6ml by intraperitoneal injection at8th and 12thweek;AD-DCN group and AD-lacz group treated with AD-DCN or AD-lacZ(0.6ml,1×109pFU/ml)respectively,by intraperitoneal injection at 8thand 12thweek.Moreover,12 SD rats were treated with PBS 0.6ml by intraperitoneal injection at 8thand 12thweek as normal control.At the 10th,12thand 16thweek,which was 2,4,8 weeks after the first injection of AD-DCN respectively,four rats in every group were scarified.Body weight and blood glucose concentration was monitored.Urinary albumin excretion(UAER)was expressed as the ratio of urinary albumin to urinary creatinine(ug/mg).Blood samples were analyzed for glucose,GPT,creatinine(enzymatic assay)on Hitachi -7170 automated chemistry analyzer.Left kidney were weighed and kept at -80℃for Reverse Transcription Polymerase Chain Reaction(RT-PCR),and Westen-Blot. Kidney hypertrophy index(KHI)was estimated by comparing the weight of right kidney to body weight.The right kidney was fixed in neutral buffered formalin, embedded in paraffin,sectioned,stained for periodic acid schiff(PAS),Masson and performed immumohistochemistry for typeⅣcollagen and TGFβ1.Results1.Diabetic rats,including diabetic group,AD-DCN group and AD-laeZ group,had high levels of serum glucose and slow gain in body weight compared to normal control.There was no difference of body weight and serum glucose among three groups.The level of GPT and serum cretinine as well as the mortality in four groups had no statistical difference.2.UAER increased in diabetic rats.Adenovirus itself had no effect on albuminuria, while exogenous decorin attenuated albuminuria.There were no difference of KHI among diabetic group,AD-DCN group and AD-lacZ group.These groups had higher KHI than normal control. 3.PAS staining and Masson staining suggested ECM over-expression in kidney of diabetic group and AD-laeZ group.AD-DCN inhibited abnormal deposition of ECM.4.TGFβ1 and typeⅣcollagen expression was stronger in kidney of diabetic rats than that of normal control,which was reduced by AD-DCN rather than AD-lacZ.ConclusionsSTZ-induced diabetic rats had ECM over-deposition and albuminuria increment. Adenovirus itself had no effect on these change.Decorin gene transfection mediated by a recombinant adenovirus had anti-fibrotic and anti-proteinuric effect on STZ-induced diabetic rats,without blood glucose improvement.The renal protective effect of deeorin might be due to the down-regulation of TGFβ1 expression. BackgroundIn previous study,we found that AD-DCN could prevent abnormal deposition of ECM in kidney and albuminuria of diabetic rats through TGFβ1 down-regulation.But the mechanism of renal protection of DCN is still unknown.TGFβ1 is known as the common mediator in final stage of kidney fibrosis.P-Smad2 and 3,the active form of Smad2 and 3,are important factors in TGFβ1/Smads signal system.So we detected the levels of DCN,TGFβ1,Smad2,3 and p-Smad2,3 by RT-PCR and Western-Blot,in order to investigate the effect of DCN on TGFβ1/Smads signal system in diabetic kidney.Material and MethodsAll renal tissue was acquired as part 1.Total RNA was extracted from renal tissue using Trizol Reagent.One microgram of each RNA sample were reverse transcribed by RevertAidTMH Minus M-MuLV Reverse Transcriptase according to the manufacturer's instruction.The PCR products(5ul)were separated by 1.5%(w/v) agarose gel electrophoresis and then visualized by ethidium bromide staining.The final results were measured in image density view(IDV)with Bio-Rad digital image system.The target gene was estimated by the ratio of target gene IDV toβ-actin IDV.The protein levels of DCN,TGFβ1,Smad2,3 and p-Smad2,3 were detected by Western-Blot,using total protein extracted from 100mg renal tissue.Results1.The expression of DCN mRNA and protein were elevated in diabetic group and AD-lacZ group.AD-DCN further increased DCN mRNA by 20 percent and DCN protein by 100 percent compared to diabetic group at the 10thweek.The increment of DCN protein lasted until the 16thweek.2.AD-DCN attenuated TGFβ1 mRNA and protein over-expression in diabetic renal, but it was only found at the 10thweek.3.The ratio of p-Smad2,3 to Smad2,3 was elevated in diabetic group and AD-lacZ group.AD-DCN decreased this ratio at all time points in accordance with the change of DCN protein.ConclusionsAD-DCN could elevate the levels of DCN protein,attenuate expression of TGFβ1 and persistently prevent activation of Smad2,3 in diabetic kidney. BackgroundIn the progress of diabetic nephropahy,number of cells increases at early stage and decreases at end stage,which is accompanied by cell apoptosis.Bax and Bcl-2 are a pair of contrl genes in apoptosis.The former causes apoptosis and the latter inhibits apoptosis.The ratio of Bax to Bcl-2 is important to apoptosis.In this study,we want to investigate the effect of AD-DCN on apoptosis and expression of Bax/Bcl-2 in the kidney of diabetic rats.Materials and methodsWe measured bax and bcl-2 by immunohistochemistry.Apoptosis was evaluated by means of terminal deoxynucleotidyl transferase mediated d-UTP nick end labelling (TUNEL).Index of apoptosis was measured by the ratio of positive cell to total cells under a high-power microscope perspective(×400).Pearson assay or Spearman assay in Spssl 3.0 was used for correlation analysis.Results1.More cells underwent apoptosis in diabetic kidney compared to normal control, appearing in renal distal convoluted tubules,the proximal tubules and renal glomerular.The index of apoptosis gradually increased from 10thweek to 16thweek. AD-DCN reduced the index of apoptosis.2.The expression of Bax and Bcl-2 increased in diabetic kidney,but the level of Bax was higher than that of Bcl-2.As a result,the ratio of Bax to Bcl-2 elevated.AD-DCN decreased the level of Bax while elevated that of Bcl-2,thus reduced the ratio of Bax to Bcl-2.3.There was positive correlation between the ratio of Bax to Bcl-2 and index of apoptosis.The latter was also correlated with albuminuria. Conclusions1.The index of apoptosis and the ratio of Bax to Bcl-2 increased in kidneys of diabetic rats.2.AD-DCN reduced the ratio of Bax to Bcl-2 and inhibited cell apoptosis in kidneys of diabetic rats.3.The index of apoptosis correlated with the ratio of Bax to Bcl-2 and albuminuria... |
PDF Full Text Request