Research Of Expression Of Smad2 And Smad4 And Smad7 In Kidney Of Diabetic Rats

BackgroundDiabetic nephropathy(DN)is one of the major diabetic complications.The early stage DN is characterized by renal hypertrophy and the accumulation of extra cellular matrix(ECM)in the glomerulus and tubulointerstitium.These changes are related to the subsequent development of renal fibrosis.The mechanisms of the development and progression of early-stage DN still remain elusive;however,the function of the transforming growth factor-beta(TGF-β)and its downstream Smad signaling pathway is very important.The Smad signaling pathway can be activated by hyperglycemia and advanced glycation end products(AGEs) and angiotensinⅡetc.in the DN.TGF-β/Smad signal transferring contributes to ECM production and degradation.Many studies have demonstrated that reduce Smad signaling pathway can decrease ECM accumulation.TGF-βand Smad2/Smad3/Smad4 induce collagen synthesis by stimulating tubular epithelial cells.The increasing of Smad7 expression can suppress the activation of Smad2 and Smad3 and Smad4, decrease the expression of collagenⅠand collagenⅡand accumulation of fibroblasts.Furthermore,the activation of TGF-β/Smad signal transferring stimulates epithelial-to-mesenchymal transition,contributes to the interstitium fibrosis of kidney.The kidney will keep inflammation station while its injured in DN,Smad signaling transfer pathway attends the inflamed reaction of kidney,and the excess expression of Smad7 may have important anti-inflame function.The study is designed to investigate the expression of Smad2 and Smad4 and Smad7 in the kidney tissue of STZ-induced diabetic rats and discuss the function of Smads signaling pathway in the mechanisms of DN.Materials and methodsThe 6-week old male SD rats buy from Medical College of Hangzhou Normal University.The average weight of 24 rats is xxx, Selecte 12 rats to make DN model by random and then inject STZ with 55mg/kg into abdomen of these rats.Measure the blood at the tail vein after 72 hours,the glycemia of one rat is less than 16.7mmol/L and the rat is gotten rid of the experiment.The glycemia of other 11 rats are more than 16.7mmol/L.The models are successful and these rats form the diabetic rats group(DM).Other 12 rats form the normal rats group(CG). The diabetic rats don't use any medicine to decrease glycemia.At the end of 12-week,collect the 24-hour-volume of urine to measure 24-hour UAE. The next day the rats are weighed,then killed to collected vascular blood and gain the kidney of two sides to be weighed.The serum is separated from blood to measure GLU and BUN and SCr and TP.The kidney tissues are fixed and cut into 3urn-slices.The emiquantitative analysis is used to analyze Smad2 and Smad4 and Smad7 protein of part of slices of the DN rats' kidney by Immunohistochemistry.Other slices are HE dyed to observe the kidney's histological structure of two group rats under microscope.Statistics:The software named SPSS11.5 is used to analyze the experimental data.The data of every group conform to normal distribution by single sample K-S analysis.The t-test is used to compare two groups.It has statistical meaning when P less than 0.01.Pearson's product-moment correlation analysis is used to analyze the relative data. Immunohistochemistry is analyzed by IPP(Image pro plus,Media cybernetics)image analysis software.Select 5 windows of every piece of glass to measure the average light density and the results are showed by Mean±sd.Results1.Change of weight and weight of kidney and ratio of weight / kidney weight and 24-hour-volume of UAE:After 12 weeks,the weight of the rats of DM decrease clearly compared to the rats of CG(P<0.01).The ratio between weight and weight of kidney and UAE of DM are high then those of CG(P<0.01). The weights of kidney of the two groups are not so clear.2.Change of GLU and BUN and SCr and TP of two groups:12 weeks later,the GLU and BUN and SCrof DM increased obviously compared to CG(P<0.01).But the difference of TP isn't so clear between two groups.3.Comparison of expression of Smad2 and Smad4 and Smad7 protein in kidney of the rats:The expression of Smad4 and Smad7 in kidney of DM apparently increased comparing to CG(P<0.01),however the difference of expression of Smad2 isn't so clear between two groups.4.Change of kidney tissues histological structure of two groups of the rats:HE dyed and observed under microscope we can find that the kidney histological structure of CG is normal and can't fmd pathological change of DN.But the pathological change of DN appeared at the STZ-induced DN rats apparently.ConclusionSmad signaling pathway is activated and contributes to the development and progression of diabetic nephropathy.