| Objective: Diabetic kidney disease(DKD)is not only a chronic complication of diabetes,but also is the main cause of chronic kidney disease,which seriously affects the quality of life and survival rate of patients.Renal tubular epithelial cell interstitial transdifferentiation occurs during the occurrence and development of DKD,and the exact mechanism has not been fully elucidated.F-box and WD repeat domain-containing 7(FBXW7)is an important component of the largest E3 ubiquitin ligase family Skp1-Cullin1-F-box(SCF)complex.In this study,diabetic mice and human renal tubular epithelial cells cultured in vitro human renal tubular cell lines(HK2),were used to investigate the effect of FBXW7 on the transdifferentiation of renal tubular epithelial cells.Methods:1.Western blot and immunohistochemistry were used to detect the expression of FBXW7 protein in kidneys of diabetic mice.Male CD1 mice were randomly divided into normal control group,2months DM group and 4 months DM group.Type Ⅰdiabetic mice were constructed successfully using a single high intraperitoneal dose(150mg/kg)injection of streptozotocin(STZ)randomized to blood glucose ≥16.7.The mice were killed after feeding for 2 months and 4 months respectively.The expression of FBXW7 in kidneys of control mice and diabetic mice by western blot and immunohistochemistry.2.Western blot and immunofluorescence were used to detect effect of high glucose on FBXW7 expression in HK2 cells.HK2 cells were cultured in DMEM-F12 medium containing 10%serum and 1% penicillin in a 5% CO2 incubator at 37 ℃.Cells with high glucose were cultured for 0h,6h,12h,24h,48h and 72h.Western blot wasused to detect the expression of FBXW7 protein.Immunofluorescence was used to investigate the expression of FBXW7.3.Western blot and immunofluorescence were used to detect the effect of FBXW7 on EMT of HK2 cells.(1)The effect of overexpression of FBXW7 on HK2 cells cultured with normal glucose.HK2 cells were randomly divided into three groups:untransfected group,pCMV3 empty vector control group and pCMV3-FBXW7-GFP plasmid group.The expression of FBXW7,α-SMA,E-cadherin and fibronectin was detected by western blot.(2)The effect of down-regulation of FBXW7 gene on HK2 cells induced by high glucose.Plasmids p Genesil-1-FBXW7 and p Genesil-1 were transfected into HK2 cells by High Gene transfection reagent and stimulated with high glucose for 6 h or 48 h.Western blot was used to detect the expression of FBXW7,E-cadherin,α-SMA and fibronectin.The expression of TGF-β1 and α-SMA protein was detected by immunofluorescence.4.Detection of the effect of FBXW7 on KLF5(Sp/Krüpple-like factor 5)protein expression(1)The interaction between FBXW7 and KLF5 in HK2 cells was detected under physiological conditions.Plasmids pCMV3-FBXW7-GFP and pcDNA3.1-Flag-KLF5 were co-transfected into HK2 cells,and the cell lysis was collected 48 hours later.Flag-Beads was used to bind the Flag protein on overexpression vector.The interaction between FBXW7 and KLF5 were detected by western blot.(2)The effect of overexpressed FBXW7 on KLF5 of HK2 cells was detected.HK2 cells were randomly divided into three groups: untransfected group,pCMV3 empty vector control group and pCMV3-FBXW7-GFP recombinant plasmid expression group.The expression of KLF5 was detected by western blot and immunofluorescence.(3)Detection of the effect of FBXW7 on KLF5 protein expression..HK2 cells were randomly divided into three groups: pcDNA3.1-FLAG-KLF5 group,pcDNA3.1-FLAG-KLF5+pcDNA3.1-Ub-HA group andpcDNA3.1-FLAG-KLF5+pcDNA3.1-Ub-HA+pCMV3-FBXW7-GFP group.The expression of KLF5 protein was detected by western blot.(4)The effect of MG132 on the down regulation of KLF5 induced by FBXW7 overexpression was detected.HK2 cells were randomly divided into pCMV3-FBXW7-GFP group and pCMV3-FBXW7-GFP+MG132 group.The expression of KLF5 protein was detected by western blot after MG132 treatment for 12 hours.5.Western blot and immunofluorescence were used to detect the effect of up-regulation of KLF5 on EMT in HK2 cells induced by overexpression of FBXW7(1)The effect of overexpression of KLF5 on EMT in HK2 cells induced by high glucose.The cells were randomly divided into high glucose+pcDNA3.1 and high glucose+pcDNA3-FLAG-KLF5 group.The expression of E-cadherin,α-SMA,fibronectin and TGF-β1 protein detected by western blot.The expression of α-SMA was detected by immunofluorescence.(2)The effect of up-regulation of KLF5 on the EMT of HK2 cells induced by overexpression of FBXW7 gene.The cells were randomly divided into non-transfection group,empty vector control group,pCMV3-FBXW7-GFP group and pCMV3-FBXW7-GFP+pcDNA3-FLAG-KLF5 group.The expression of KLF5,E-cadherin,α-SMA,fibronectin and TGF-β1 was detected by western blot.The expression of E-cadherin and α-SMA was detected by immunofluorescence.Results:1.The expression of FBXW7 in renal tubular epithelial cells of diabetic mice.The immunohistochemical results showed that the expression of FBXW7 in the kidney of diabetic group was higher than that of normal control group.The western blot results showed that the expression of FBXW7 protein in renal cortex of diabetic mice was higher than that of normal control group,and the expression of FBXW7 in renal tissue of 2-month and 4-month diabeticmice increased by 1.43 and 4.2 times(P<0.05).2.Effect of high glucose on the expression of FBXW7 protein in HK2 cells.Western blotting demonstrated that the expression of FBXW7 was increased after a short time of high glucose stimulation,with the peak appearing at 6 h,which was 1.45 times higher than that in high glucose stimulation 0 h group(P<0.05).FBXW7 expression was also detected with immunofluorescence,and the results showed that FBXW7 was primarily localized in the cytoplasm of HK2 cells.Compared with cells treated with high glucose for 0h,the positive signals(indicated in green fluorescence)of FBXW7 were evidently increased in cells treated with high glucose for 6 h.3.Effect of FBXW7 on EMT of HK2 cells(1)The effect of overexpression of FBXW7 on EMT of HK2 cells.Western blot results showed that the expression of FBXW7 protein in HK2 cells transfected with pCMV3-FBXW7-GFP group was 2.00 times higher than pCMV3 empty vector control group(P<0.05).E-cadherin expression decreased by 55.6%,α-SMA and fibronectin expression increased by 3.63 times and 1.41 times in pCMV3-FBXW7-GFP-transfected HK2 cells compared to pCMV3-transected cells(P<0.05).(2)Effect of down-regulation of FBXW7 gene on EMT of HK2 cells induced by high glucose.The results of western blot showed that the FBXW7 expression was reduced by 79.3% in p Genesil-1-FBXW7-transfected HK2 cells compared with p Genesil-1-transfected HK2 cells(P<0.05).The level ofα-SMA and fibronectin were decreased by 54.7% and 76.8% respectively,and the level of E-cadherin was increased by 1.25 times in high glucose-treated HK2 cells transfected with p Genesil-1-FBXW7 compared with those transfected with p Genesil-1(P<0.05).The results of immunofluorescence detection showed that the fluorescence intensity of TGF-β1 and α-SMA expression in HK2 cells in p Genesil-1-FBXW7 group was lower than that in p Genesil-1 group.4.Effect of FBXW7 on KLF5 protein expression(1)Co-IP experiments showed that Flag-beads did not bind to FBXW7 in the control group,indicating non-specific binding between beads and FBXW7.After Flag-beads binding KLF5,FBXW7 was also in the binding protein.The results suggested that KLF5 and FBXW7 interact under physiological conditions.(2)The effect of overexpression of FBXW7 on KLF5 of HK2 cells.Western blot results showed that FBXW7 overexpression resulted in a marked reduction of endogenous KLF5 protein.Compared with pcDNA3.1transfection,KLF5 expression was reduced by 58.1% with pcDNA3.1-FBXW7-GFP transfection(P<0.05).In addition,immunofluorescence detection also revealed that KLF5 expression was significantly inhibited by pcDNA3.1-FBXW7-GFP plasmid transfection in HK2 cells.(3)The change of ubiquitin level modification of KLF5 by FBXW7.The results of western blot were illustrated that overexpression of Ub promoted the degradation of KLF5 protein.Moreover,compared with only the Ub plasmid transfection group,the expressions of KLF5 was decreased by 45% in the FBXW7-plus-Ub plasmid transfection group(P<0.05).(4)The effect of MG132 on the down-regulation of KLF5 induced by FBXW7 overexpression.The results of western blot showed that the expression of KLF5 protein was increased by 1.73 times in pCMV3-FBXW7-GFP+MG132 group than that in pCMV3-FBXW7-GFP group(P<0.05).5.Effect of KLF5 on EMT of HK2 cells(1)Effect of overexpression of KLF5 on transdifferentiation of HK2 cells induced by high glucose.The results of western blot were illustrated that KLF5 expression was effectively enhanced by pcDNA3.1-FLAG-KLF5 transfection in high glucose-cultured HK2 cells.Analysis revealed a 2.03 times increase in KLF5 protein compared to the pcDNA3.1 control plasmid group(P<0.05).The expression of E-cadherin was increased by 98% and the expressions of TGF-β1,α-SMA and fibronectin were respectively decreased by 63.4%,64.1% and 72.7% in high glucose-treated HK2 cells transfected withpcDNA3.1-FLAG-KLF5 compared with those transfected with pcDNA3.1(P<0.05).Also,the results of immunofluorescence were similar to the results of western blot.KLF5 plasmid transfection inhibited high glucose-inducedα-SMA overexpression in HK2 cells.(2)Effect of up-regulation of KLF5 on transdifferentiation of HK2 cells induced by overexpression of FBXW7 gene.Western blot results showed that compared with pCMV3-FBXW7-GFP group,the expression of TGF-β1,α-SMA and fibronectin in FBXW7+KLF5 plasmid transfection group respectively decreased by 82.3%,64.5% and 51% and the expression of E-cadherin was increased by 91.5% in the FBXW7-plus-KLF5 plasmid transfection group(P<0.05).In addition,immunofluorescence detection also revealed that α-SMA expression was significantly inhibited by pcDNA3.1-FLAG-KLF5 plasmid transfection in pCMV3-FBXW7-GFPpretransfected HK2 cells.Conclusions:1.The expression of FBXW7 in the kidney of diabetic mice and in high glucose-treated HK2 cells were increased.2.Inhibition of FBXW7 expression can reverse EMT formation in HK2 cells induced by high glucose.3.KLF5 can reverse the EMT of HK2 cells caused by overexpression of FBXW7. |
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