Roles Of Carboxy-terminal Modulator Protein In Extracellular Matrix Accumulation In Diabetic Nephropathy

Objectives: Diabetic nephropathy(DN)is characterized by extracellular matrix(ECM)accumulation,glomerulosclerosis,epithelial-to-mesenchymal transition(EMT)of renal tubular epithelial cells and renal tubular interstitial fibrosis,which ultimately lead to a progressive loss of renal function.Especially,extracellular matrix deposit was regarded as early event of the progressive fibrosis.Collagen is the most important structural protein of extracellular matrix and over 28 different types of collagen have been identified.Among these,types 1,2,3 collagens are the most abundant.Transforming growth factor ?1(TGF-?1)and alpha smooth muscle actin(?-SMA)were proven to be key regulators of extracellular matrix metabolism in many tissues.PI3K/Akt is a multiple functional signal pathway that mediated cell proliferation,motility,differentiation,lipid metabolism and fibrosis.PI3 Ks are capable of phosphorylating the 3 position hydroxyl group of the inositol ring of phosphatidylinositol.PI3-kinases can activate protein kinase B also named as Akt to regulate diverse group of cellular functions.PI3K/Akt pathway was reported to play a key role in regulating fibrosis of diabetic nephropathy.Our previous study also revealed that PI3K/Akt pathway mediated high glucose-induced extracellular matrix accumulation and lipogenesis in human renal tubular epithelial cells by regulating the expression of TGF-?1 and sterol regulatory element binding protein-1(SREBP-1).Therefore,researches aimed at PI3K/Akt pathway became gradually the hot points to prevent the renal injury of diabetes mellitus.So far several proteins that modify Akt activation state via direct binding to Akt have been identified such as phosphatase and tensin homolog deleted on chromosometen(PTEN),SH2 domain-containing inositol phosphatase(SHIP)and carboxy-terminal modulator protein(CTMP).In our previous study,PTEN was revealed to decrease SREBP-1/fatty acid synthase(FASN)/acetyl-CoA carboxylase(ACC)pathway and lipogenesis,as well as connective tissue growth factor(CTGF)expression and ECM secretion in kidney of diabetic animals by inhibiting activated Akt.Again,CTMP,also named as thioesterase superfamily member 4(THEM4),was reported to bind to the carboxy-terminal regulatory domain of Akt and inhibit its activation.At present,it is not well known whether CTMP involved in extracellular matrix metabolism in diabetic nephropathy via inhibiting Akt activity.Therefore,in the present study in vitro cultured renal tubular cells and diabetic mice were chosen to detect the expression and effect of CTMP on TGF-?1,?-SMA and extracellular matrix deposit.Methods:1 The expression of phospho-Akt(Ser 473),CTMP,TGF-?1,?-SMA,Col ? and FN in the kidneys of STZ-induced diabetic miceMale CD1 mice at 5-6 week of age were randomly divided into 2 groups: normal control mice and diabetic mice.The diabetic mice were injected intraperitoneally with 150 mg/kg body weight STZ in 0.1 M sodium citrate solution(pH 4.5),and control group mice were only injected with 0.1 M sodium citrate solution.Both normal control mice and diabetic mice were raised for 8 week after STZ injection until they were sacrificed.The mice were weighed and housed individually for 24 h in metabolic cages to collect urine for the detection of 24 h urinary protein.Blood samples were collected from extracted eyeball for determination of the renal function(Scr and BUN)and blood glucose.Subsequently,the renal cortex was removed and immediately preserved in liquid nitrogen for extraction of protein and RNA or saved in neutral-bufferedformalin for histopathological examination.Western blot,Immunohistochemistry and real-time PCR were used to detect the expression of phospho-Akt(Ser 473),CTMP,TGF-?1,?-SMA,Col ?,FN and CTMP mRNA in the kidneys of normal mice and diabetic mice.2 Effect of high glucose on phospho-Akt(Ser 473),CTMP,TGF-?1 and ?-SMA expression and Col?,Col ? secretion in HKC cellsHKC cells were incubated at a humidified atmosphere of 5% CO2 and 37 oC in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum(FBS)and penicillin/streptomycin(100 kU/L and 100 mg/L,respectively),with a normal D-glucose concentration for 5.5 mM.Subsequently,HKC cells were respectively cultured in DMEM containing 30 mM glucose(high glucose)for 0 h,6 h,12 h,24 h and 48 h.And at each time point phospho-Akt(Ser 473),CTMP,TGF-?1 and ?-SMA protein expression were examined by Western blot and immunofluorescence staining respectively.Col ? and Col ? were detected using the competitive sandwich ELISA kit according to the manufacturer's instructions.Each sample was assayed in triplicate and experiment was repeated three times to lessen inter and intra-assay coefficients of variation.3 Effect of CTMP expression vector on phospho-Akt(Ser 473),CTMP,TGF-?1 and ?-SMA expression and Col?,Col ? secretion in HKC cells treated with high glucoseHKC cells were cultured under conditions as described above.In order to examine the direct effect of CTMP expression vector on PI3K/Akt pathway and collagen secretion,pYr-ads-4-CTMP expression vector was constructed and transfected into the HKC with lipofectamine 2000 in vitro.The cells were randomly divided into four groups: normal glucose group(Control),high glucose group(HG),high glucose plus pYr-adshuttle-4 vector group(HG+V vector)and high glucose plus pYr-ads-4-CTMP vector group(HG+CTMP vector).And then the cells were collected for the immunofluorescent staining and extraction of protein at 48 h after HG stimulation.The collagen secretion,phospho-Akt(Ser 473),CTMP,TGF-?1 and ?-SMA expression were analyzed.4 Effect of CTMP expression vector on phospho-Akt(Ser 473),CTMP,TGF-?1 and ?-SMA expression and extracellular matrix deposit in the kidneys of STZ-induced diabetic miceFor in vivo vector delivery experiment,mice were randomly divided into four groups: normal control mice(Control group),diabetic mice(DM group),diabetic mice receiving receiving pYr-adshuttle-4 vector(DM+V vector)and diabetic mice receiving pYr-ads-4-CTMP vector(DM+CTMP vector).pYr-ads-4-CTMP vector or pYr-adshuttle-4 vector(1 mg/kg body weight)were mixed with TransIT-EE Hydrodynamic Delivery Solution from Mirus Co.and injected into tail vein once a week from 4 week after STZ injection.4 weeks later,mice were weighed and relative analyses were performed.Results:1 CTMP was inhibited in the kidneys of diabetic mice accompanied with increased phospho-Akt(Ser 473),TGF-?1,?-SMA and extracellular matrixThe serum glucose,24-hour urine protein(Upro),serum creatinine(Scr)and serum BUN were respectively increased by 4.92,2.84,1.66 and 7.12 times in diabetic mice compared with normal mice(P<0.05).Immunohistochemistry revealed that CTMP was located in renal tubular cells of normal mice,whereas CTMP expression was reduced in diabetic mice illustrated as weakened positive signal.Image analyses testified a 63.33% decrease of CTMP in the kidneys of diabetic mice compared with those of normal mice.In line with these,Western blot presented that diabetic mice showed 37.7% decreased CTMP expression in the kidneys compared with normal mice.Furthermore,overexpression of phospho-Akt(Ser 473),TGF-?1 and ?-SMA were confirmed in cytoplasm of renal tubular cells in diabetic mice in comparison with normal mice by the method of immunohistochemistry.Also,quantitative analyses of the results of Western blot revealed that phospho-Akt(Ser 473),TGF-?1 and ?-SMA were respectively enhanced by 3.66,1.29 and 2.33 times in the kidneys of diabetic mice compared with those of normal mice.Masson staining showed that there was no significant positive-stained extracellular matrix accumulation in the kidneys of normal mice,however evident extracellular matrix accumulation was found in renal interstitial region of diabetic mice.Again,col and ? FN was chosen to be detected in order to further explicit extracellular matrix accumulation of kidneys.The Col and ?FN positive region increased in the kidneys of diabetic mice compared with normal mice.IOD of positive signal was analyzed and the results showed that Col ? and FN were respectively increased to 1.99 and 2.82 times in the kidneys of diabetic mice in comparison with those of normal mice.2 High glucose inhibited CTMP expression,and induced increased phospho-Akt(Ser 473),TGF-?1,?-SMA,secreted Col?and secreted Col ?in HKC cellsCTMP protein expression decreased with the stimulation of high glucose and reached its nadir at 24 h after stimulation as shown in the results of Western blot.In line with these,immunofluorescence also revealed that CTMP was mainly located in the cytoplasm of HKC cells and decreased with the treatment of high glucose.On the contrary,the expression of phospho-Akt(Ser473),TGF-?1 and ?-SMA gradually increased in HKC cells after high glucose stimulation and peaked at 24 h without any significant change in Akt expression.Again,we investigated the role of CTMP in regulating secreted Col?and secreted Col ? using the method of ELISA.As supposed,high glucose significantly enhanced the secretions of Col? and Col ? at 48 h in HKC cells.3 CTMP expression vector reversed high glucose-induced decreased CTMP,increased phospho-Akt(Ser 473),TGF-?1,?-SMA,secreted Col?and secreted Col ? in HKC cellsThe results of Western blot revealed that 1.98 times enhanced CTMP,35.11% decreased phospho-Akt(Ser 473),76.53% decreased TGF-?1 and 51.59% decreased ?-SMA were proven in high glucose medium-cultured pYr-ads-4-CTMP vector-transfected HKC cells compared with cells transfected with pYr-adshuttle-4 vector.Moreover,there was no significant difference in Akt protein expression among cells of all four groups.Again,ELISA results revealed that high glucose significantly enhanced the secretions of Col?and Col ?in HKC cells(P<0.05).However,the transfection of pYr-ads-4-CTMP vector reduced secreted Col?and Col ?by 32.31% and 51.83% in comparison with the transfection of pYr-adshuttle-4 vector in HKC cells.4 The delivery of pYr-ads-4-CTMP vector increased CTMP expression and decreased phospho-Akt(Ser 473),TGF-?1,?-SMA and extracellular matrix deposit in the kidneys of diabetic miceThe in vivo delivery of pYr-ads-4-CTMP vector markedly reduced 24-hour Upro,serum creatinine(Scr)and serum BUN in diabetic mice(P<0.05).There was no evident alteration of these parameters between diabetic mice and diabetic mice delivered with pYr-adshuttle-4 vector.The results of real-time PCR revealed that CTMP mRNA decreased in diabetic mice compared with normal mice.Again,CTMP mRNA expression was evidently increased in diabetic mice delivered by pYr-ads-4-CTMP vector.Moreover,Western blot analyses confirmed that pYr-ads-4-CTMP vector not pYr-adshuttle-4 vector enhanced renal CTMP protein accompanied with a 60% decrease in phospho-Akt(Ser 473)in diabetic mice(P<0.05).Supposedly,the results of immunohistochemistry also presented that increase of CTMP and decrease of phospho-Akt(Ser 473)were confirmed in renal tubular cells of diabetic mice receiving pYr-ads-4-CTMP vector.Analyses of positive region revealed that CTMP was increased by 9.43 times and phospho-Akt(Ser 473)was decreased by 54.33% in the kidneys of diabetic mice receiving p Yr-ads-4-CTMP vector compared with those of diabetic mice receiving pYr-adshuttle-4 vector.Immunohistochemistry revealed that strong renal expressions of TGF-?1 and ?-SMA were effectively reversed in diabetic mice by delivery of pYr-ads-4-CTMP vector via tail vein.Likewise,Western blot showed that TGF-?1 and ?-SMA were respectively decreased by 76.50% and 24.37% in diabetic mice delivered with p Yr-ads-4-CTMP vector in comparison with diabetic mice delivered with pYr-adshuttle-4 vector.In addition,the results of Masson staining showed extracellular matrix deposit was in local region of renal interstitium of diabetic mice,which was alleviated with treatment with pYr-ads-4-CTMP vector.Similarly,Col ? and FN expression were confirmed to be increased in the kidneys of diabetic mice that were effectively prevented in diabetic mice receiving pYr-ads-4-CTMP vector.Conclusions:1 CTMP decreases in kidney of diabetes mellitus accompanied with increased phospho-Akt(Ser 473),TGF-?1,?-SMA and extracellular matrix.2 High glucose decreases CTMP expression and increases phospho-Akt,TGF-?1,?-SMA expression as well as Col?and Col ? secretion in HKC cells.3 Upregulation of CTMP reversed high glucose-induced decreased CTMP,increased phospho-Akt(Ser 473),TGF-?1,?-SMA,secreted Col?and secreted Col ? in HKC cells.4 Upregulation of CTMP prevented extracellular matrix deposit of diabetic nephropathy by regulating the phosphorylation of Akt and inhibiting TGF-?1,?-SMA expression in diabetic mice.