Effect Of SHIP On Extracellular Matrix Deposition In Diabetic Kidney Diseases

Objectives:Diabetic kidney disease(DKD)is one of the most serious complications in diabetic patients,which may be the most detrimental regarding patients' life quality and life expectancy among all diabetes-related complications.Although glomerular injury is believed to initiate kidney damage in DKD,more emerging evidence suggests that renal tubular injury plays a key role in the development of DKD.The main morphological changes of DKD include the epithelial-to-mesenchymal transition(EMT)known as the transition of tubular epithelial cells into fibroblasts,the excessive deposition of extracellular matrix(ECM)in renal tubulointerstitium,subsequently leading to interstitial fibrosis with a progressive,irreversible decline in renal function.Many factors are reported to be involved in the renal tubular injury of diabetic kidney disease,and high glucose has been shown to be an initiating factor to induce functional and structural injury in renal tubular epithelial cells,accompanied with ECM accumulation and interstitial fibrosisMore and more experimental studies focused on the molecular mechanism involved in renal extracellular matrix deposit.Cell signal pathways have been identified as mediators of these events such as P38 MAPK,ERK1/2,and JAK/STAT.In the previous studies,PI3K/Akt pathway was reported to play a pivotal role in regulating renal the epithelial-tomesenchymal transition and extracellular matrix homeostasis.The activation of PI3 K results in the addition of a phosphate group to its lipid substrate phosphatidylinositol-4,5-bisphosphate,PI(4,5)P2,to form phosphatidylinositol-3,4,5-trisphosphate,PI(3,4,5)P3,in the cell membrane.PI(3,4,5)P3 initiates a variety of signaling cascades by interacting with pleckstrin homology(PH)domain-containing proteins,most notably the serine/threonine kinase Akt.SH2 domain-containing inositol 5'-phosphatase(SHIP)is commonly viewed as opposing the activation of the PI3K/Akt pathway by removing specific phosphate groups from PI(3,4,5)P3.SHIP attacks its substrate PI(3,4,5)P3 at the 5 position of the inositol ring,generating PI(3,4)P2,which functions as an allosteric activator of SHIP to inhibit Akt activation.In human leukemia cell line K562,SHIP gene deletion or mutation leading a loss of 5' phosphatase activity,activated PI3K/Akt pathway which reduce etoposide sensitivity and promote the cell proliferation.Latest studies show that SHIP plays an important role in ECM accumulation and fibrosis through the negative regulation of the PI3K/Akt pathway.The mice with miR-155 knockout or topical antagomir-155 treatment increased SHIP protein level and inhibited Akt signaling in skin upon bleomycin induced fibrosis.Together with the finding,it was also suggested that increased collagen deposition was easily seen in the subepithelial areas of the airways of SHIP(-/-)mice,which was accompanied by a significantly increased production of TGF-?1.However,it is not known whether SHIP,known as a negative regulator of PI3K/Akt pathway,involved in renal ECM accumulation and fibrosis in diabetic kidney disease.In the present study,we detected the expression and effect of SHIP on extracellular matrix secretion and fibrosis of diabetic kidney diseases through diabetic mice model and in vitro-cultured renal tubular cells(HK2 cells).The results of the present research also supply the novel content and targets for prevention or treatment of diabetic renal tubular interstitial fibrosis.Methods:1 The expression of SHIP in the kidney of STZ-induced diabetic miceThe male CD1 mice were randomly grouped as control mice and diabetic mice.Diabetic mice models were induced by an intraperitoneal injection of streptozotocin at a dose of 150 mg/kg body weight(sodium citrate solution,pH 4.4)and control mice received the same amount of sodium citrate solution.Those animals with a blood glucose level exceeding 16.7 mM were defined as diabetic after 72 h of the injection.Blood was obtained from tail vein and blood glucose was monitored every week.At the age of 16 weeks,all animals were sacrificed after serum and 24 h urine collected.The renal cortex was removed and harvested for further analysis.One portion of the renal tissues was fixed in 4% paraformaldehyde for HE staining,Masson staining and immunohistochemical examinations.The remaining renal cortex were snap frozen in liquid nitrogen and kept at-80 °C for the extraction of protein and mRNA.Immunohistochemistry and Western blot were used to detected the expressions of SHIP,phospho-Akt(Ser 473),phospho-Akt(Thr 308),Akt,TGF-?1,E-cadherin,?-SMA,Col 1 and Col 3 proteins in renal cortex;The level of TGF-?1 mRNA was detected by Real-time PCR.2 Effects of SHIP expression on ECM production in in vitro-cultured HK2 cellsThe human renal proximal tubular epithelial cell line(HK2 cells)were maintained in a mixture of Dulbecco's modified Eagle's medium/Nutrient Mixture F-12(DMEM-F12,3:1)medium containing 10% fetal bovine serum(FBS)and 1% penicillin-streptomycin in a 95% humidity and 5% CO2 atmosphere at 37 °C.?In order to determine the effect of high glucose on the SHIP expression and ECM secretion: HK2 cells were divided into 5 groups.The serum-deprived HK2 cells were respectively stimulated by 30 mM glucose for 0 h,12 h,24 h,36 h and 48 h.The protein expression of SHIP was detected by immunofluorescence.The expression of SHIP,phospho-Akt(Ser 473),phospho-Akt(Thr 308),Akt,TGF-?1,E-cadherin and ?-SMA was respectively evaluated by Western blot.Moreover,the Col 3 level in the medium supernatant was measured with an ELISA.?To investigate the direct effect of SHIP knockdown on extracellular matrix production:HK2 cells in 6-well plates grew for 24 hours in the absence of antibiotics and were transfected with negative control siRNA or specific SHIP siRNA using Lipofectamine? RNAiMAX.HK2 cells were randomly grouped: normal control group,negative control group and SHIP siRNA group.After transfection recovery of 48 hours,Western blot was used to detect the expression of SHIP,phospho-Akt(Ser 473),phospho-Akt(Thr 308),Akt,TGF-?1,E-cadherin and ?-SMA.ELISA was used to detected the level of secreted Col 3.?To determine the influence of SHIP overexpression on extracellular matrix secretion: HK2 cells transient transfection with M90 or M90-SHIP was performed according to the manufacturer's instruction of FuGENE?R6 Transfection Reagent.HK2 cells were randomly divided into 4 groups: normal glucose group(NG),high glucose group(HG),high glucose plus control vector transfection group(HG+M90)and high glucose plus SHIP vector transfection group(HG+M90-SHIP).At 24 h after transfection,HK2 cells received high glucose treatment for 48 h followed by the relative detections.Detection methods and indicators consistent with part ?.?LY294002,known as the classic inhibitor of PI3K/Akt pathway,was added in high glucose-stimulated HK2 cells to evaluate the relationship between PI3K/Akt pathway and extracellular matrix production: HK2 cells were randomly grouped as normal glucose group(NG),high glucose group(HG),high glucose plus DMSO group(HG+DMSO)and high glucose plus 30 ?M LY294002 group(HG+LY294002).?SB451542,as a specific inhibitor of TGF-?1,was used to explore whether the SHIP-dependent changes in ECM were mediated by TGF-?1 pathway in SHIP siRNA-transfected HK2 cells.Cells were randomly grouped as a normal glucose group(NG),a normal glucose with SHIP siRNA transfection group(NG+SHIP siRNA)and a normal glucose with SHIP si RNA and 3 ?M SB431542 group(NG+SHIP si RNA+SB431542).Detection methods and indicators consistent with part?.3 Effects of intraperitoneal injection of rAd-INPP5 D vector on ECM accumulation in kidneys of diabetic miceAt 8 weeks after STZ injection,the diabetic mice were randomly divided into three groups(eight mice per group): diabetic mice group(DM group),diabetic mice receiving rAd-EGFP vector group(DM+rAd-EGFP group)and diabetic mice receiving rAd-INPP5 D vector group(DM+rAd-INPP5 D group).Healthy CD1 mice with same ages were used as normal controls(Control group).The r Ad-EGFP vector or rAd-INPP5 D vector(5×108 pfu)were injected intraperitoneally once every ten days from 8 weeks after STZ injection.8 weeks later,all animals were sacrificed after serum and 24 h urine collected.The renal cortex was removed and harvested for further analysis.One portion of the renal tissues was fixed in 4% paraformaldehyde for Masson staining and immunohistochemical examinations.The remaining renal cortex were snap frozen in liquid nitrogen and kept at-80 °C for the extraction of protein and mRNA.Immunohistochemistry and Western blot were used to detected the expressions of SHIP,phospho-Akt(Ser 473),phospho-Akt(Thr 308),Akt,TGF-?1,E-cadherin,?-SMA Col 1 and Col 3 proteins in renal cortex;The level of TGF-?1 mRNA was detected by Real-time PCR.Results:1 The expression of SHIP in the kidney of STZ-induced diabetic mice?Compared with control mice,the levels of food intake,volume of urine,Glu,Scr,BUN,TG and KI were significantly increased in diabetic group(P<0.05).?The results of HE staining and Masson staining showed that diabetic mice revealed slightly glomeruli hypertrophy,mesangium matrix expansion,renal interstitial broadening and extracellular matrix accumulation.?The results of immunohistochemistry revealed that SHIP and phospho-Akt(Ser 473,Thr 308)were mainly expressed in renal tubular cells of normal mice and diabetic mice.Additionally,the expression of SHIP was markedly reduced in the kidneys of diabetic mice,which was illustrated as a weak positive signal compared with those of normal mice.Through Image-Pro Plus analysis,SHIP was decreased by 86.7% in the kidneys of diabetic mice.Also,the expression of phospho-Akt(Ser 473,Thr 308)were up-regulated by 2.99 and 2.64 times,respectively,in the renal tubular cells of diabetic mice in comparison with those of normal mice.In line with this,Western blot also confirmed that SHIP expression was reduced by approximately 73.19% and the expression levels of phospho-Akt(Ser 473,Thr 308)were enhanced by approximately 3.40 and 3.22 times,respectively,in the renal cortex of diabetic mice in comparison with those of normal mice.? The result of immunohistochemistry revealed that the TGF-?1 protein was mainly located in the cytoplasm of renal proximal tubular cells of diabetic mice,while there was no significant staining in the kidneys of normal mice.The expression of ?-SMA protein was mainly located in injured renal proximal tubular epithelial cells of diabetic mice,not only in vascular smooth muscle cell.The expression of E-cadherin was decreased in the cytoplasm and cell membrane of renal tubular cells of diabetic mice compared with normal mice.The results of the immunohistochemistry also showed that the positive regions of Col 3 and Col 1 expression were increased in renal tubular cells and the tubulointerstitium of diabetic mice without significant expression in the kidneys of normal mice(P<0.05).Furthermore,the expression of TGF-?1 ? ?-SMA ? Col 3 and E-cadherin were detected by Western blot,and it was revealed that the TGF-?1??-SMA and Col 3 proteins were increased by 1.99,2.48 and 4.51 times,respectively and the E-cadherin expression was decreased by 67.38% in the kidneys of diabetic mice compared with those of normal mice(P<0.05).Yet,the results of real-time PCR showed that renal TGF-?1 mRNA in diabetic mice increased versus normal mice(P<0.05).2 Effects of SHIP expression on ECM production in in vitro-cultured HK2 cells?Compared with the 0 h of HG stimulation,the protein expression of SHIP began to decrease at 24 h after the stimulation of HG.The expression of SHIP was decreased by approximately 62.2% in 48 h of HG stimulation group compared with 0 h of HG stimulation group.The levels of phospho-Akt(Ser 473,Thr 308)proteins were significantly increased in time-dependent by high glucose stimulation.The expressions of phospho-Akt(Ser 473,Thr 308)were,respectively,increased by 2.91 and 3.50 times in 48 h of HG stimulation group.Also,it was found that exposure of HK2 cells to high glucose significantly increased the expression of TGF-?1??-SMA and secreted Col 3 protein by 2.08,4.96 and 1.41 times,respectively and down-regulated the protein expression of E-cadherin by 61.6%.? In normal glucose-cultured SHIP si RNA-transfected HK2 cells,SHIP siRNA caused a decrease in the expression of the SHIP protein compared with the negative control siRNA(P<0.05).The protein levels of phospho-Akt(Ser 473,Thr 308)were increased by 4.76 and 3.89 times,respectively,in the SHIP siRNA-transfected group compared with the negative control group without a difference between normal control group and negative control group.Additionally,the knockdown of SHIP significantly increased the expression of TGF-?1 ? ?-SMA and secreted Col 3 protein and down-regulated the protein expression of E-cadherin(P<0.05).? The expression of the SHIP protein level was efficiently increased in high glucose-cultured HK2 cells transfected with the M90-SHIP vector.The protein levels of phospho-Akt(Ser 473,Thr 308)were inhibited by 42.2% and 38.3%,respectively,in M90-SHIP vector-transfected HK2 cells in comparison with M90 vector-transfected cells.M90-SHIP vector can prevent high glucose-caused increased ECM production in HK2 cells.Subsequently,we found that 48.9% decreased TGF-?1,59.4% decreased ?-SMA,43.7% decreased secreted Col 3 and 2.11 times increased E-cadherin were proven in M90-SHIP vector-transfected HK2 cells in comparison with M90 vector-transfected cells.?The high glucose-induced up-regulation of phospho-Akt(Ser 473,Thr 308)could be reduced by 73.4% and 54.3%,respectively,in response to the treatment with LY294002.The blocked of the PI3K/Akt pathway with LY294002 effectively attenuated the high glucose-induced increased ECM production in HK2 cells.The expression of TGF-?1 and ?-SMA were decreased by 65.9% and 65.7%,respectively,and the expression of E-cadherin was increased by 1.87 times in high glucose and LY294002-cultured HK2 cells compared with high glucose and DMSO-treated cells.?Compared with those cells in the absence of SB431542,the presence of SB431542 inhibited the levels of ?-SMA and secreted Col 3 by 37.4% and 51.2%,respectively,and increased the level of E-cadherin by 1.99 times in SHIP siRNA-transfected HK2 cells.3 Effects of intraperitoneal injection of rAd-INPP5 D vector on ECM accumulation in kidneys of diabetic mice? Treatment of diabetic mice with intraperitoneal injection of rAd-INPP5 D vector attenuated the increases in Scr,BUN and TG associated with diabetes(P<0.05).However,there was no evident alteration of these parameters in diabetic mice receiving rAd-EGFP vector.In addition,diabetic mice with or without receiving rAd-EGFP vector also showed increase in food intake,urine volume,blood glucose and KI,for which intraperitoneal injection of rAd-INPP5 D vector had no effect(P>0.05).?Histological sections of kidneys from diabetic mice revealed marked ECM accumulation in tubulointerstitium.The diabetic mice with the injection of rAd-INPP5 D vector exhibited a remarkable amelioration on these morphological changes.?The results of immunohistochemistry confirmed that SHIP expression was increased and phospho-Akt(Ser 473,Thr 308)expressions were decreased in renal tubular cells of diabetic mice receiving rAd-INPP5 D vector(P<0.05).Image analyses of brown yellow-stained positive signals revealed that SHIP was increased by 2.91 times with rAd-INPP5 D vector injection in kidneys of diabetic mice.In line with these,Western blot also confirmed that the expression of SHIP protein was enhanced by 3.36 times and the expression of phospho-Akt(Ser 473,Thr 308)were respectively inhibited by 65.26% and 70.38% in rAd-INPP5 D vector-injected diabetic mice in comparison with rAd-EGFP vector-injected diabetic mice.? The result of immunohistochemistry revealed that the expression levels of TGF-?1 in the cytoplasm of renal proximal tubular cells and Col 1 and Col 3 in renal tubular cells and the tubulointerstitium were decreased and the level of E-cadherin in the cytoplasm and cell membrane of renal tubular cells was increased by rAd-INPP5 D vector injection in kidneys of diabetic mice(P<0.05).Furthermore,the expression of TGF-?1,E-cadherin,?-SMA and Col 1 were detected by Western blot.It was revealed that 47.66% decreased TGF-?1,44.52% decreased ?-SMA,59.13% decreased Col 1 and 2.48 times increased E-cadherin were proven in rAd-INPP5 D vector-injected diabetic mice in comparison with rAd-EGFP vector-injected diabetic mice.The results of Real-time PCR showed that renal TGF-?1 mRNA in diabetic mice with or without receiving rAd-EGFP vector increased versus normal mice.However,renal TGF-?1 mRNA was lowered in diabetic mice receiving rAd-INPP5 D vector compared with those receiving rAd-EGFP vector(P<0.05).Conclusions:1 Decrease in SHIP found in the kidneys of diabetic mouse model and high glucose-cultured HK2 cells is concomitant with the activation of PI3K/Akt signal pathway and extracellular matrix deposition.These findings suggest that the SHIP/PI3K/Akt pathway may mediate renal tubular epithelial cell extracellular matrix deposition of diabetic kidney disease.2 Downregulation of SHIP by siRNA in normal glucose-cultured HK2 cells lead to extracellular matrix secretion through promoting activation of PI3K/Akt signal pathway.These results suggested that SHIP serves as a protective determinant in renal tubular epithelial cell.High glucose maybe induce HK2 cells extracellular matrix secretion by reduce the expression of SHIP.3 Overexpression of SHIP or LY294002 prevent high glucose-induced extracellular matrix deposition in the kidneys of diabetic mice and cultured HK2 cells by inhibiting the activation of PI3K/Akt signal pathway,which suggests that SHIP provides a treatment for diabetic kidney diseases via inhibiting PI3K/Akt pathway.4 SB431542 inhibits extracellular matrix secretion in SHIP siRNAtransfected HK2 cells,indicating that SHIP-dependent changes in the extracellular matrix secretion were really mediated by TGF-?1 pathway.