Experimental Research On The Effect Of Activin A On Tubulointerstitial Fibrosis In Diabetic Nephropathy

BackgroundThe population of diabetic patients is dramatically increasing in the world.The development of the various complications of diabetes contributes to the high morbidity and mortality.Diabetic nephropathy (DN) is one of the most common chronic complications of diabetes mellitus and the main cause of end-stage renal disease(ESRD).In the past,DN was chiefly recognized as a kind of glomerular disease. However,a recent study has indicated that tubular injury and progressive interstitial fibrosis are more closely related to DN prognosis.So the research on the mechanisms of the intiation and development of diabetic tubulointerstitial injuries will be benefit for development of an accurate treatment.The core of tubulointerstitial injuries in DN is the increased accumulation of extracellular matrix(ECM) in the injured region. Myofibroblasts are the main source of increased ECM deposition seen in tubulointerstitium.It has been demonstrated that not only interstitial fibroblasts,but also tubular epithelial cells can transform into myofibroblasts in the presense of high glucose levels. alpha-Smooth-muscle actin(α-SMA),absent in normal tubular epithelial and fibroblasts,is a common mark used to localize myofibroblasts in vivo and in vitro.A variety of cytokines and growth factors are involved in ECM production and degradation,among which transforming growth factor beta(TGF-β) superfamily members play an important role.According to the differences of molecular structure and biology,the TGF-βsuperfamily can be divided into three main families:TGF-β,bone morphogenetic protein(BMP) and activin.Some studies have discovered that,in the early phase of DN,TGF-βexhibits upregulation in tubular epithelia and facilitates the transdifferentiation of tubular epithelia cells into myofibroblasts.In vitro cultured tubular epithelial cells,both high glucose and advanced glycation endproducts (AGEs) may promote ECM formation through a TGF-βdependent way.The roles of BMP-7 and TGF-βare exactly opposite.Endogenous BMP-7 may keep tubular epithelial cells from producing ECM.In an experimental DN model,the expression of BMP-7 and its receptor is obviously downregulated.Activin,a kind of glycoprotein,was first discovered in the gonads and named for its specifically facilitating anterior pituitary gland to synthesize and release follicle stimulating hormone(FSH).Activin consists of twoβ-subunits which are combined by disulfide bond to form a dimeric protein.Based on the constructive differences ofβ-subunit, activin is divided into three forms:activin A(βAβA), activin B(βBβB) and activin AB(βAβB).Among them,the studies on activin A are the most.Similar to other members of the TGF-βsuperfamily,activin A mediates its biological effects mainly through the Smad signal pathway.Follistatin is a single-chain polypeptide isolated from ovarian fluid,which inhibits pituitary FSH secretion. This protein binds to activin with high affinity and blocks its actions.Follistatin and activin together compose a balanced system of automatic control to maintain the normal growth and metabolism of tissues and organs.The underlying pathogenic link between activin A and the development of tubulointerstitial injury has not widely been investigated yet and the effect of activin A on tubulointerstitial fibrosis in DN is still waiting for elucidation.ObjectiveThe purpose of this study is to investigated the expression of activin A /Smad signal on tubulointerstitium of streptozotocin(STZ)-induced diabetic rats and high glucose-cultured HK-2 ceils and its relation withα-SMA and fibronectin.Methods1.Animal experimentMale Wistar rats were randomized into a normal control group(NC) and diabetes mellitus group(DM).After fasting for 12 h,DM rats received an i.p.injection of STZ(60 mg/kg),and NC rats received a corresponding amount of citrate buffer.Blood samples were collected from the tail vein 72 h after administration to determine random blood glucose(BG).The rats with random BG more than 16.7 mmol/L were regarded as DM rats.The rats were given free access to tap water and standard chow.Six rats were respectively sacrificed 4,8,12 and 16 weeks after model establishment in each group.One day before execution,rats were weighed,and then put in individual metabolic cages for the exact collection of 24h urine samples.Blood samples were withdrawn from the abdominal aorta and the left kidneys were removed and dissected into two parts.One part was fixed in 10% paraformaldehyde for pathological and immunohistochemical detection, and another part was reserved in liquid nitrogen for polymerase chain reaction(PCR) assay.Renal hypertrophy index was monitored by left kidney weight/body weight(KW/BW,mg/g).The urine albumin excretion rate(AER) was measured by radioimmunoassay according to the manufacturer' s suggestions.BG,Urine and serum creatinine concentration were measured with a multiparametric analyzer(model 7150;HITACHI).Creatinine clearance rate(Ccr) was calculated as urinary creatinine×urine volume/serum creatinine,and was expressed as mL/min·per kg.The morphology of tubulointerstitium was observed by light microscopy.Immunohistochemistry was applied to analyze the protein expression of activinβA,follistatin,P-Smad2/3,α-SMA and fibronectin and real-time PCR was used to detect the mRNA expression of activinβA,follistatin,α-SMA and fibronectin in renal tissue.2.Cell cultureHK-2 cells,were maintained in Dulbecco' s modified Eagle' s medium(DMEM) which was supplemented with 10%fetal bovine serum(FBS), penicillin(100 U/mL) and streptomycin(100μg/mL) and cultured in an atmosphere of 5%CO₂ and 95%air at 37℃.When 80%confluent,the cells were incubated in a serum-free medium for 24 h and then exposed to the following experimental conditions for different time periods (12,24 and 48 h):5 mmol/L glucose(normal glucose group,NG), 5 mmol/L glucose plus 25 mmol/L mannitol(osmotic control,mannitol group,MG),25 mmol/L glucose(high glucose group,HG),25 mmol/L glucose plus 100 ng/mL rh-follistatin(100 ng/mL rh-follistatin intervention group,HG + FS100) or 25 mmol/L glucose plus 500 ng/mL rh-follistatin(500 ng/mL rh-follistatin intervention group, HG + FS500).Then,the cells and cellular supernatant were collected for further detection.ActivinβA and P-Smad2/3 expression were determined by Western blotting analysis.TGF-β,α-SMA and fibronectin released into media were quantitatively analyzed by enzyme-linked immunosorbent assay(ELISA).Results1.Animal experiment1.1 General biochemical parametersAt each time point,BG in the DM group rats was higher than that in the NC group.In the DM group,KW/BW,AER,and Ccr began to rise from 8 weeks and showed a gradually increasing trend.There were statistically significant differences between DM and NC groups and between 8 and 16 weeks in the DM group(P＜0.01,respectively).1.2 Pathological changes of tubulointerstitiumThe PAS stain showed that at each time point,the renal tubular epithelial cells in the NC group rats were intact,and there were neither interstitial oedema nor interstitial fibrosis.Vacuolar degeneration in tubular epithelial cells,tubular hypertrophy, dilation and atrophy,inflammatory cell infiltration and renal interstitial fibrosis occurred from 8 weeks and worsened over time in the DM group rats.By semiquantitative analysis,the tubulointerstitial injury index was gradually elevated in the DM group from 8 to 16 weeks.1.3 Immunohistochemistry staining for activinβA,follistatin,P-Smad2/3,α-SMA and fibronectin in tubulointerstitiumThe immunohistochemical results demonstrated that activinβA staining was prominent in tubular epithelial cells in the DM group rats,but no staining was detected in the NC group rats.By semiquantitative analysis,significantly elevated activinβA staining was observed in the DM group rats at 4 weeks and reached peak values at 12 weeks.By contrast,follistatin staining was abundant in tubular epithelial cells in the NC group rats.However,in the DM group rats, its staining began to decrease gradually from 4 weeks to 16 weeks.P-Smad2/3 and fibronectin showed slight staining in tubulointerstitium in the NC group rats.But in the DM group rats,an abruptly increased staining could be detected at 8 weeks and lasted until 16 weeks.In the NC group rats,immunostaining forα-SMA was essentially confined to the media of arteries and arterioles.By contrast,in the DM group rats,besides the vascular wall,the expression ofα-SMA was detected in the tubular epitheliar cells and interstitium from 8 weeks.1.4 6ene expression of activinβA,follistatin,α-SMA and fibronectin in renal tissueCompared with NC group rats,ActivinβA mRNA was markedly elevated to 3.3-fold higher at 4 weeks and 7.7-fold at 12 weeks,and then decreased slightly at 16 weeks in the DM group rats.On the contrary, follistatin mRNA was decreased 32.5%at 4 weeks and 52.9%at 12 weeks in the DM group rats.No significant change inα-SMA and fibronectin mRNA was detected at 4 weeks in the DM group rats,but a gradually increase was found at 8 weeks and lasted until the end of the study.2.Cell culture2.1 ActivinβA and P-Smad2/3 expression in HK-2 cells and the intervention of follistatinWestern blotting results demonstrated that ActivinβA exhibited very slight activation in the NG group,but its production was enhanced in the HG group after 12 h culture and increased gradually until study completion.Follistatin blocked its production in a dosedependent fashion.Treatment with 500 ng/mh rh-follistatin reduced activinβA production to NG group levels after 48 h culture(P＞0.05). P-Smad2/3 was significantly increased after 24 h culture in the HG group compared with the NG group and follistatin treatment decreased its overexpression in the HG group.The fall averaged 26%in the HG + FSIO0 group and 41%in the HG + FS500 group after 48 h culture. There were no statistical differences in the expression of activinβA and P-Smad2/3 between the MG and NG groups(p＞0.05).2.2 TGF-β,α-SMA and fibronectin protein in cellular supernatant and the intervention of follistatinResults of ELISA disclosed that TGF-βwas markedly higher in the HG group than that in the NG group throughout the 48 h experimental periods and both doses of follistatin failed to modify the overexpression of TGF-βin HG.Compared with the NG group,α-SMA and fibronectin in the HG group were obviously upregulated after 24h culture(p＜0.01).Follistatin reduced their overexpression in a dosedependent manner(p＜0.01).There were no statistical differences in the expression of TGFβ,α-SMA and fibronectin between the MG and NG groups(p＞0.05).Conclusion1.Activin A/Smad pathway activation,transdifferentiation and fibronectin production are involved in renal tubulointerstitial injuries of STZ-induced diabetic rats.2.High glucose can activate Activin A/Smad pathway,induce tubular epithelial cells-myofibroblast transdiffrentiation and promote fibronectin production in HK-2 cells.3.Inhibition of high glucose stimulated transdifferentiation and fibronectin production in HK-2 cells by follistatin is associated with blocking of Activin A/Smad pathway.