| Background: Diabetic nephropathy(DN)is a common and serious complication of diabetes mellitus(DM).The pathological findings of which mainly include glomerulosclerosis and tubulointerstitial fibrosis.With the increase in the incidence of diabetes mellitus,diabetic nephropathy has been found to become one of the main causes of chronic renal failure,yet so far its pathogenesis is not fully understood,and treatment is not adequately effective.Phosphatase and a tensin homolog deleted from chromosome ten(PTEN),which is a tumor suppressor gene with lipid phosphatase and protein phosphatase activities,is a key negative regulator of PI3K/AKT/mTOR signaling.Studies have demonstrated that autophagy was inhibited and fibrosis was aggravated in diabetic rats with interaction by PTEN.However,given the fact that its regulatory mechanisms are still not clear,the downregulation of PTEN protein throughout the process may be affected by a variety of factors.Research findings have revealed that the activation of the Notch signaling pathway leads to the downregulation of PTEN,which further initiates the development of gastric cancer.Hes1,on the same basis,can induce colon cancer cells EMT and cytoskeleton remodeling.It has been suggested that decreasing PTEN protein levels may be related to the regulation of Notch1 and Hes1.Notch1 may regulate PTEN protein expression through the downstream target gene Hes1,causing the anti-fibrosis effect of PTEN.However,during the process of DN tubulointerstitial fibrosis,it is unclear whether Notch1 regulates autophagy through PTEN and promotes fibrosis.Studies regarding the regulation of autophagy by Notch1/Hes1/PTEN signaling and the mechanisms of renal tubulointerstitial fibrosis in DN have not been reported.Thus,the present study is necessary to shed light on the regulating mechanisms of PTEN protein downregulation,autophagy inhibition and fibrosis exacerbation,such that new therapeutic targets for the prevention and treatment of renal tubulointerstitial fibrosis in diabetic nephropathy can be developed.Objective: To observe the relationship between the changes in Notch1 protein levels,PTEN and autophagy in renal tubular epithelial cells of diabetic renal tissue and high glucose-cultured NRK-52 e cells and to clarify their association with renal tubulointerstitial fibrosis.To elucidate the regulatory mechanism of Notch1 on PTEN,autophagy and renal tubulointerstitial fibrosis in HG-treated NRK-52 e cells.To observe changes in Hes1 expression levels in diabetic mice and high glucose-treated NRK-52 e cells and to explore the regulation mechanism of Notch1-mediated PTEN via interactions with Hes1.Methods:1.To observe the relationship between the changes in Notch1 protein levels,PTEN and autophagy in renal tubular epithelial cells of diabetic renal tissue and high glucose-cultured NRK-52 e cells and to clarify their association with renal tubulointerstitial fibrosis.(1)In vivo experiments utilized C57BL/KsJ db/db male mice and age-matched db/m mice.The mice were randomly assigned to two groups: 1)the db/m normal control group and 2)the db/db normal experimental group.Biochemical indexes were used to detect blood glucose,glycosylated hemoglobin,plasma creatinine,triglyceride and cholesterol.Pathological changes in renal tissue were observed using HE and Masson staining.Immunohistochemical staining revealed Notch1,PTEN,LC3,Collagen-I and Collagen-III protein expression and localization in renal tissue.Western blotting was used to detect the protein levels of Notch1,PTEN,p-Akt(Thr308),Akt,p-mTOR(Ser2448),mTOR,LC3,P62,Collagen-I and Collagen-III.Real-time PCR was employed to determine the mRNA expression levels of Notch1 and PTEN.(2)The in vitro experiments utilized NRK-52 e cells.These cells were divided into the following three groups: Normal glucose(NG)group,High glucose(HG)group and Mannitol(MA)group.Western blotting was used to determine the protein levels of Notch1,PTEN,LC3,P62,Collagen-I and Collagen-III.Real-time PCR was employed to detect the mRNA expression levels of Notch1 and PTEN,and western blotting was employed to detect the protein levels of p-Akt(Thr308),Akt,p-mTOR(Ser2448)and mTOR in NG-and HG-treated NRK-52 e cells.2.To elucidate the regulatory mechanism of Notch1 on PTEN,autophagy and renal tubulointerstitial fibrosis in high glucose-treated NRK-52 e cells.Using siNotch1 to knock down the expression of Notch1,NRK-52 e cells were divided into four groups: the NG group,the NG+siNotch1 group,the HG group,and the HG+siNotch1 group.Notch1,PTEN,LC3,P62,Collagen-I and Collagen-III protein levels were determined using western blotting.The cells were transfected with autophagic double-standard adenovirus(mRFP-GFP-LC3).Sixteen hours after transfection,were re-transfected with siNotch1 for forty-eight hours,after which the cell nuclei were dyed with 4’,6-diamidino-2-phenylindole(DAPI)and immediately analyzed using confocal microscopy.Positive controls were treated with the autophagy inducer,rapamycin(100 nM),for 48 hours.The mRFP-GFP-LC3 puncta formation and localization of mRFP-GFP-LC3 were observed.Notch1,Collagen-I and Collagen-III protein expression levels were determined using immunofluorescence.3.To observe changes in Hes1 expression levels in diabetic mice and high glucose-treated NRK-52 e cells and to explore the regulation mechanism of Notch1-mediated PTEN via interactions with Hes1.(1)In vivo experiments utilized C57BL/KsJ db/db male mice and age-matched db/m mice.The mice were randomly assigned to two groups: 1)the db/m normal control group,and 2)the db/db normal experimental group.Hes1 expression levels in db/db normal kidney tissues were determined using western blotting and real-time PCR.Immunohistochemical staining revealed Hes1 protein expression and localization in kidney tissue.(2)In vitro experiments utilized NRK-52 e cells.These cells were divided into the following three groups: the NG group,the HG group and the MA group.Western blotting was used to determine Hes1 protein levels.Real-time PCR was employed to detect Hes1 mRNA expression levels in the NG and HG groups.Using pHAGE-Hes1 plasmid transfected cells with normal glucose treated NRK-52 e cells.These cells were divided into two groups: the Vector group and the pHAGE-Hes1 group.Real-time PCR was used to detect mRNA expression of PTEN.Furthermore,the pGL3-PTEN-promoter plasmid was constructed and subsequently cotransfected into cells with the pHAGE-Hes1 plasmid.The pHAGE-Vector was used as a control for the pGL3-PTEN-promoter plasmid.Luciferase activity was measured at 48 h after transfection.Cotransfection with pEF hICN1.CMV.GFP-Notch1 plasmids and siHes1 was performed to study the effects of Notch1 on PTEN expression by Hes1.First,pEF hICN1.CMV.GFP-Notch1 was used to upregulate Notch1 and Hes1 expression levels,respectively,and siHes1 was used to inhibit Hes1 expression.The NRK-52 e cells were divided into four groups: 1)control group,2)pNotch1 group,3)pNotch1+si-Ctrl group and 4)pNotch1+siHes1 group under NG treatment conditions.The NRK-52 e cells were also divided into these four groups under HG conditions.Western blotting analyses that the levels of Notch1,Hes1 and PTEN protein expression.We also confirmed these results with immunofluorescence analysis.Results:1.The relationship between the changes in Notch1 protein levels,PTEN and autophagy in renal tubular epithelial cells of diabetic renal tissue and high glucose-cultured NRK-52 e cells and to clarify their association with renal tubulointerstitial fibrosis.Increased levels of blood glucose,glycosylated hemoglobin,plasma creatinine,triglyceride and cholesterol levels were found in the db/db group compared with the db/m group(P<0.01).Through HE staining renal tubular epithelial cells showed vacuolar degeneration,renal tubular expansion and invasion of inflammatory cells in db/db group.The db/m group Masson staining found cellulosic substances deposited in the of the glomerular capillaries and in db/db group.Western blotting analysis results indicated that protein expression levels of PTEN and LC3 decreased in db/db mice in comparison with the db/m group(P < 0.01,P < 0.05).In addition,increased protein expression levels of Notch1,p-mTOR(Ser2448),p-Akt(Thr308),P62,Collagen-I and Collagen-III were found in db/db mice compared with db/m group(P < 0.01),but no significant change was found in the combined expression of mTOR and Akt proteins.The db/db group displayed increased Notch1 mRNA levels and decreased PTEN mRNA levels by real-time PCR(P < 0.05).Immunohistochemical staining results showed differences in Notch1,PTEN,LC3,Collagen-I and Collagen-III expression levels between db/m and db/db mice,and the accumulation points of PTEN and LC3 in IHC was significantly reduced in the tubules and glomeruli in the db/db group.In contrast,the accumulation of Notch1,Collagen-I and Collagen-III in IHC was significantly elevated in the tubules and glomeruli as against the db/m group.The expression of Notch1 protein was negatively correlated with the expression of PTEN protein relative to the db/m group(r=﹣0.685,P<0.05).(2)Western blotting was used to determine Notch1,PTEN,LC3,P62,Collagen-I and Collagen-III expression levels in normal glucose,high glucose and mannitol-treated NRK-52 e cells,showing significant upregulation of Notch1,P62,Collagen-I and Collagen-III in HG and significant downregulation of LC3 and PTEN in HG(P < 0.05,P < 0.01).Furthermore,except for the interference of hyperosmosis,no significant difference was observed between MA,which served as an osmotic control for HG,and NG.Under NG and HG conditions,p-mTOR(Ser2448)and p-Akt(Thr308)protein expression levels increased compared to the NG group(P < 0.01);however,combined mTOR and Akt protein expression levels did not change significantly.Compared with the NG group,while Notch1 mRNA levels had increased(P < 0.01),while PTEN mRNA levels were lower in the HG group(P < 0.05).2.The regulatory mechanism of Notch1 on PTEN,autophagy and renal tubulointerstitial fibrosis in high glucose-treated NRK-52 e cells.Western blotting confirmed the effects of Notch1 on PTEN protein levels,autophagy and fibrosis.Similarly,as compared with control cells under NG conditions,PTEN protein levels were reduced and the indices of fibrosis(Collagen-I and III)were increased by inhibiting autophagy in control cells under HG conditions(P < 0.05).These results revealed that autophagy was inhibited and exacerbated fibrosis in renal tubular epithelial cells.In addition,PTEN levels and autophagy recovered and fibrosis was relieved in siNotch1-transfected cells compared to the control group under HG conditions(P < 0.05,P < 0.01).Changes in autophagic flow were investigated using confocal immunofluorescence microscopy,after which we determined whether autophagosomes and autolysosomes were restored in the HG+siNotch1 group,compared to the control group under HG conditions and rapamycin treatment conditions(P < 0.01),and the autophagosomes and autolysosomes in he HG group were significantly elevated compared to the NG group.Immunofluorescence staining results showed significant increases in the accumulation of Notch1,Collagen-I and III compared to HG-treated NRK-52 e cells.Meanwhile,Notch1,Collagen-I and III expression levels were also diminished in HG+siNotch1-treated NRK-52 e cells.3.Changes in Hes1 expression levels in diabetic mice and high glucose-treated NRK-52 e cells and to explore the regulation mechanism of Notch1-mediated PTEN via interactions with Hes1.Western blotting results indicated that Hes1 protein expression increased in db/db mice compared to the db/m group(P < 0.05).Immunohistochemistry staining results showed differences in Hes1 expression between db/m and db/db mice,and the accumulation of Hes1 in IHC was significantly elevated in tubules and glomeruli compared to the db/m group.Hes1 mRNA levels were elevated in the db/db group(P < 0.01).Notch1 protein expression correlated positively with Hes1 protein expression(r=0.698,P<0.05).(2)Western blotting assays were performed to confirm the effects of Hes1 in normal glucose,high glucose and mannitol-treated NRK-52 e cells,and the results showed significant Hes1 upregulation under HG conditions(P < 0.05).Furthermore,mannitol served as an osmotic control for HG.No significant difference was observed between MA and NG groups,excepting hyperosmosis interference.Hes1 mRNA levels were higher in the HG group than the NG group(P < 0.05).We transfected cells with p HAGE-Hes1 plasmids and examined the effects of the overexpression of PTEN mRNA and the mRNA levels of PTEN mitigated by Hes1 overexpression(P < 0.05).Luciferase activity in the pHAGE-Vector + pGL3-PTEN-promoter plasmid group was increased in comparison with the pHAGE-Hes1 plasmid + pGL3-PTEN-promoter plasmid group(P < 0.01).Western blotting analyses revealed that Notch1 and Hes1 levels were upregulated(P < 0.05,P < 0.01)and PTEN protein expression was decreased in the HG group compare to the NG group(P < 0.05).In addition,compared to the effects of single transfection with pEF hICN1.CMV.GFP-Notch1 plasmids under HG treatment conditions,cotransfection with pEF hICN1.CMV.GFP-Notch1 plasmids and siHes1 inhibited Hes1 expression(P < 0.05)and restored PTEN expression(P < 0.05).The effects on protein levels were the same as those observed with IF.Conclusion:Previous study in our group suggested that the suppression of PTEN-related autophagy in renal epithelial cell was involved in the development of renal fibrosis.The results in the project demonstrate that hyperglycemic condition stimulates the expression of Notch1 in renal tubular epithelial cell.Notch1 downregulates PTEN via transcriptional factor Hes1.Suppression in PTEN decreases autophagy and promotes the expression of ECM in renal tubular epithelial cell,and eventually contributes to the development of renal tubulointerstitial fibrosis. |
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