Effection Of Active Vitamin D3on Nephrein、Podocin、WT1Expression In Podocytes Of Diabetic Rats

Objective:We build diabetic Sprague Dawley rat model induced bystreptozotocin in this study,administrating Active vitamin D3to intervention.Using immunohistochemistry,reverse transcription PCR,light microscopymethod,to observe the expression of nephrin, podocin and WT1onpodocyte.TO explore whether active vitamin D can improve the diabetic ratpodocytes consider over-function barrier,result in antiproteinuric effect, delaythe progression of renal disease,and observation of the early application ofcalcium and phosphorus metabolism.Thus it provide the experimental theorybasis of the early prevention of diabetic nephropathy and to look for newtreatment strategies.Nephrin is a transmembrane protein in the podocyte which was foundfirstly,belonging to a member of the immunoglobulin superfamily,specificallyexpressed in podocytes.Nephrin can induce Akt pathway and several targetprotein phosphorylation,protecting and reducing podocytes toapoptosis.Podocin connects Nephrin by the CD-2AP to form complex,andwhich show a hairpin-like structure inserted into the cell membrane,there isa enhanced effect to nephrin signal transduction.Abnormal expression of themmay affect the glomerular filtration barrier permeability,resulting in thegeneration of proteinuria.Both of them play a key role in the maintenance ofthe glomerular filtration barrier integrity. Wilms tumor suppressor gene (WT1)has a regulatory role for a large number of gene expression,that is one ofspecific markers of podocyte,and play an important role in the maintenance ofnormal structure and function of podocyte.Method:The clean health male SD rats,altogether36,weight about200±20g.After three days of adaptive feeding,24rats selecting randomly were given streptozotocin for building diabetic model which were randomlydivided into diabetes model group (DN group,n=12),diabetes+vitamin D3group (DT group,n=12),the left12rats were classified as normal controlgroup (NC group,n=12). DT group in the diabetic model were gave calcitriol0.03ug/(kg d) dose for gavage,at the same time,NC group and DN groupwere gived the same amount of saline for gavage. At the end of8week and12week,we collected the hypercalcemia,hyperphosphatemia,Creatinine,and renaltissue samples,and measured24h urinary protein and urine creatinine.Weobserved rat renal histopathological changes with hematoxylin-eosin (HE) inthe light microscope.We observed expression of nephrin, podocin and WT1by immunochemistry and RT-PCR on kidney tissues,All the datas wereexpressed as mean value±SD.One-way ANOVA was used to compare meanvalues among groups.Two independent samples t-test was used to comparebetween the two groups,and used non-parametric tests which does not meetthe normality and homogeneity of variance data.Analysis were carried outusing SPSS13.0,P-value of<0.05was considered to be statistically significant.Results:1General situation The rats of DN group and DT injected withSTZ appeared polydipsia,polyphagia,polyuria and gradually emaciation,andReduced activity after2to4days.These symptoms were most significant inDN group.2Biochemical test results24h urinary protein excretion,serumcreatinine,serum calcium,and serum phosphorus.By the end of8and12week,The urinary protein excretion,and serum creatinine of DN groupincreased significantly than NC group(P<0.05),DT group lower than DNgroup(P<0.05).At the8th weeks,calcium,phosphorus of NC group is reducedcompared to the DN group,this change was more pronounced at12weeks(P<0.05). DT group was difference compared with the DN group at8weeks(P>0.05), and serum calcium levels than DN group, but lower than theNC group at12weeks.3Histochemical changes Hematoxylin-eosin (HE) staining results.The light microscope HE staining revealed that,The tissue of rats in NC group showed no obvious pathological changes throughout theexperiment.Compared with the same period in the NC group,experiments ofthe12th week,the diabetic glomerular volume increases,diffuse mesangialmatrix increase,and mesangial cells increased significantly.Renalinterstitialand tubular had no significantly changed on pathomorphology.4Immunohistochemistry The expression decreased in group DN,groupDT compared with group NC in renal tissue of rats with nephrin, podocin andWT1at the end of8and12week, and the difference were moresignificant(P<0.05) at12weekend.The expression of each factor wereincreased in DT group than in DN group at8weeks and12weeks,but lowerthan the same period in NC group.5RT-PCR Compared with the same period in normal rats,expression ofnephrin, podocin and WT-1were reduced in renal tissue of diabetic rats,andthe changes were more significantly(P<0.05)at the end of12week. Theexpression of each factor were increased in DT group than in DN group t8weeks and12weeks, but lower than the same period in NC group (P <0.05).Conclusion:1In diabetic nephropathy,the expression of nephrin,podocinand WT1decrease,and was negatively correlated with the level ofproteinuria.So it suggests that these factors play a role in the development ofdiabetic nephropathy.2Active vitamin D can significantly reduce the generation of proteinuriaof diabetic rats,delaying the occurrence of diabetic nephropathy.Themechanism may be that,raising protein expression of nephrin and podocin andWT1.Diabetic rats early application of active vitamin D3can delay theoccurrence and progression of diabetic nephropathy,improving the diabetic ratglomerular consider over barrier dysfunction.