Molecular Biology Study On Effects Of Grape Seed Proanthocyanidin Extracts On Kidney In Diabetic Rats

Background and Objective With the increasing of living standard,morbidity and complications of diabetes mellitus rise sharply in the world.Now patients of diabetes are about 1,710,000,000 throughout the world.Diabetic nephropathy(DN) is emerging as the leading cause of end-stage renal failure in developed country and the second cause in our country.United State Renal Disease Statistic(USRDS)indicated that exceed 30%of the end-stage renal failure were induced by DN.DN threatens severely the life and health of mankind,it is an important problem that is followed with interest by all over the world.The structural changes of kidney in diabetes consist of glomerular and tubuloepithelial hypertrophy,followed by thickening of glomerular and tubular basement membranes and progressive accumulation of extracellular matrix proteins in the mesangium and the interstitium.This contributes to glomerulosclerosis and tubulointerstitial fibrosis.Overexpression of cytokines and growth factors play a central role in the initiation and progression of DN,for example:transforming growth factorβ1(TGF-β1),connective tissue growth factor(CTGF),growth hormone,insulin-like growth factor(IGF)and vascular endothelial growth factor(VEGF).Recent studies have suggested that TGF-β1 could be the most important cytokine in the nephrosclerosis. There are many intrinsic cells in the kidney,especially mesangial cells that can synthetize and secrete TGF-β1,and with special receptors of TGF-β1.TGF-β1 is one effector molecule that has been studied extensively as a major mediator of the hypertrophic and prosclerotic changes in diabetic kidney disease.TGF-β1 stimulates the synthesis of key extracellular matrix molecules including typeⅠcollagen,typeⅢcollagen,typeⅣcollagen,typeⅤcollagen,typeⅥcollagen,fibronectin,and laminin.TGF-β1 also decreases matrix degradation by inhibiting proteases as well as activating protease inhibitors(e.g.,plasminogen activator inhibitor-l).TGF-β1 promotes cell-matrix interactions by upregulating integrins,the cell surface receptors for matrix.In addition,high ambient glucose increases TGF-β1 mRNA and protein level in cultured proximal tubular cells and glomerular epithelial and mesangial cells.In experimental and human diabetes mellitus,several reports describe overexpression of TGF-β1 in the glomeruli and tubulointerstitium.The data presented here strongly support the consensus that the TGF-βsystem mediates the renal hypertrophy, glomerulosclerosis,and tubulointerstitial fibrosis of diabetic kidney disease.Inhibiting renal TGF-β1 activity can partially reverse the glomerular basement membrane(GBM) thickening and mesangial matrix expansion,attenuate the nephrosclerosis.But TGF-β1 can regulate immune and inhibit proliferation,inhibiting TGF-β1 completely by gene knock-out,the animal can't live.People have to search for more special pathway. CTGF is a cysteine-rich member of a new family of growth regulators.It is an important factor in the pathogenesis of mesangial matrix accumulation and progressive glomerulosclerosis in DN.In development of progressive extracellular matrix accumulation,CTGF may act as a downstream mediator of TGF-β1.Some mediators upregulates the expression and bioactivity of CTGF in renal intrinsic cell.These mediators encompass the following:high glucose concentration,early and advanced products of nonenzymatic glycation of proteins,mechanical tension and TGF-β.CTGF acts on IGF,VEGE,TGF-β,Bone morphogenetic proteins(BMPs),and affects their signaling pathways.Some experts suggesting that CTGF blockade could be a new therapeutic target against DN.Several lines of evidence suggest that advanced glycation end products(AGEs)may be involved in the development of diabetic glomerular lesions.AGEs mediate their effects through two different pathways via a receptor-independent AGEs cross-link formation pathway and a receptor-dependent pathway where AGEs bind to specific cell surface-associated receptors,such as Receptor for advanced glycation end product(RAGE).AGEs modification indeed alters the structure and function of matrix tissue proteins and,more interestingly, AGE-modified proteins stimulate a variety of cellular responses via a specific cell-surface receptor on several cell types,including glomerular cells.RAGE is one of AGE-specific cellular receptors.RAGE has been proposed to play an important role in the development of DN.AGEs can activate RAGE gene through nuclear transcription factor-k B(NF-kB),AGEs accumulation may involve upregulation and activation of RAGE.The AGE-RAGE interaction is associated with the pathogenesis of DN. Except these growth factors that cause fibrosis,there is anti-fibrotic growth factor: Bone morphogenetic proteins-7(BMP-7)that expresses lower in DN and can inhibit fibrosis of experimental DN.BMP-7 has a protective effect on the kidney,by decreasing apoptosis,maintaining and restoring the epithelial phenotype,increasing matrix degradation,decreasing lots of inflammation factors,affecting TGF-β/Smads pathway and displaying anti-fibrotic activity.Recent studies have demonstrated that injury and loss of podocyte is one central mediator of glomerulosclerosis in DN.Podocyte molecules(nephrin,podocin, CD2-associated protein)make a pivotal contribution to the maintenance of the selective filtration barrier of the normal glomerulus.A marked reduction was observed at the protein levels of nephrin and podocalyxin in DN.This suggests that the alterations in the expression of podocyte-associated molecules represent a compensatory reaction of the podocyte that results from damage associated with proteinuria.Current therapeutics include intensified glycemic control,blood pressure lowering agents,inhibiting renin-angiotensin system(RAS)and intensified lipid control,et al. Though these can attenuate proteinuria and improve renal function in diabetes and decrease mortality of DN,renal disease continues to progress relentlessly in diabetic patients,DN is an important clinical problem that need be solved urgently.Grape seed proanthocyanidin extracts(GSPE),which belong to a class of polyphenols,are widely distributed throughout the plant kingdom.GSPE have been reported to possess a wide range of biologic properties against oxidative stress.The antioxidative activities of GSPE were found to be much stronger than vitamin C or vitamin E in aqueous systems.Recent studies demonstrate that GSPE have potent antioxidant properties and should be considered a potential agent in the prevention of some diseases.GSPE has been reported to exert protective effects on various forms of cardiac disorders.The cardiovascular protective effects of GSPE are believed to be ascribed to its antioxidative properties.GSPE were showed to have preventive actions on diseases such as atherosclerosis,gastric ulcer,large bowel cancer,cataracts and diabetes.However,the effects of GSPE on DN have been little understood.The aim of this study was to determine the effect of GSPE on kidney in diabetic rats.Material and Methods 45 rats injected with streptozotocin(STZ)were randomly divided into diabetes group(DM),small dosage GSPE treated group(T1) and big dosage GSPE treated group(T2).Other 12 normal rats were used as control group(C),12 normal rats were administrated GSPE at a dosage 250mg·kg^-1·d^-1as treated control group(CT).The rats in T1 group were administrated GSPE at a dosage 250mg·kg^-1·d^-1,the rats in T2 group were administrated GSPE at a dosage 500mg·kg^-1·d^-1,and animals in other groups were treated with normal saline.The fasting plasma glucose(FPG),hemoglobin Alc(HbA_1C),systolic blood pressure,24h urinary protein,serum creatinine(Scr),urinary creatinine,serum AGEs and ratio of kidney weight to body weight were measured in each rat after 24 weeks.The renal pathological changes were examined with PAS staining,Masson staining and electron Microscope.The mRNA expression of TGF-β1,CTGF,RAGE,BMP-7,nephrin,and podocin in kidney were detected by reverse transcription-polymerase chain reaction(RT-PCR),the protein expression were detected by immunohistochemical staining respectively and Western blot,and were quantified by computer image analysis system.Then these parameters of each group are compared.Results FPG,HbA_1C,AGEs,blood pressure,24h urinary protein,Ccr and ratio of kidney weight to body weight were significantly higher in DM group compared to C group(p＜0.01).Extracellular matrix(ECM)accumulation were higher in DM group than that of C group(p＜0.01),and intrisinc cells in the kidney were damaged seriously in DM group.Expression of mRNA and protein of TGF-β1,CTGF and RAGE increased in DM group compared to C group(p＜0.05 or p＜0.01),whereas the expression of mRNA and protein of BMP-7 and nephrin was progressively down-regulated(p＜0.05)in DM group compared to C group.Diabetic rats treated with GSPE(T1 or T2 group)had less urinary protein,lower blood pressure,serum AGEs and Ccr compared to DM group(p＜0.05 or p＜0.01).The expression of mRNA and protein of BMP-7 and nephrin were significantly higher in T1 and T2 group compared to DM group(p＜0.05 or p＜0.01),whereas the expression of mRNA and protein of TGF-β1,CTGF and RAGE was progressively down-regulated in T1 and T2 group compared to DM group(p＜0.01).Between T1 and T2 group the expression of mRNA of CTGF,Ccr and ratio of kidney weight to body weight were significantly different(p＜0.05 or p＜0.01).Conclusions GSPE can decrease proteinuria,blood pressure and glomerular high filtration in diabetic rats,improve the diabetic nephropathy.GSPE has clear renal protective effects.Renoprotective mechanism of GSPE are correlated with suppression on AGEs,down-regulated overexpression of RAGE,TGF-β1,CTGF and up-regulated expression of BMP-7 and nephrin.