| Background and objectiveDiabetic kidney disease(DKD)has become more and more common,and it is indicated that renal tubules play an important role in the generation of DKD.Tubulointerstitial fibrosis is one of the most common pathological changes in renal tubules during the course of DKD.The earliest detectable clinical manifestation of DKD is Microalbuminuria,and albumin has been found to be a key factor in promoting tubulointerstitial fibrosis.Therefore,it is important to explore the pathophysiological mechanism of renal tubular epithelial cell injury induced by albumin during the development of DKD.One of the important characteristics of cell senescence is cell cycle arrest.The results showed that the renal tubular epithelial cells were blocked in g 2/M phase after injury,which resulted in the increase of pro-fibrotic factors and promoted renal fibrosis.Therefore,delaying cell senescence may be one of the potential important methods to inhibit DKD and improve renal fibrosis.Sodium-glucose co-transporter 2(SGLT2)inhibitors are newly developed antidiabetic drugs in recent years,and they have additional renal protective effects.It has been found that inhibition of SGLT2 can inhibit the expression of senescence markers induced by high glucose in renal tubular epithelial cells,but the mechanism is still not clear.Therefore,we investigated whether albumin could induce premature senescence of renal tubular epithelial cells by interfering with renal tubular epithelial cells(HK-2)to mimic the microenvironment of proteinuria in vitro using albumin(BSA);And whether its possible mechanisms are associated with albumin-induced tubulointerstitial fibrosis;to further explore whether the SGLT2 inhibitor dapagliflozin delays interstitial fibrosis by improving tubular epithelial cell senescence,then play a role in renal protection.Methods1.The proximal tubular epithelial cells(HK-2)were cultured with different concentrations of albumin for 72 hours to simulate the microenvironment of proteinuria in vivo.The cell morphology and density were observed under the light microscope,then the Galactosidases activity was observed under the microscope afterβ-Galactosidases staining,the integrality of nuclear membrane was observed by immunofluorescence staining,and the mRNA expression of LaminB1 and IL-1α,IL-6 and IL-8,components of senescence-associated secretory phenotype(SASP),were detected by RT-PCR,the expression of γ-H2AX,a marker of DNA damage,was observed by Western blot.2.HK-2 cells were cultured with different concentrations of albumin for 72 hours.The specific antibody p-PH3Ser10 was used to observe whether the cells were arrested in G 2/M phase by immunofluorescence staining,the expression of p21,CDK1 and CDC25C were detected by Western blot and RT-PCR,and the expression of p21,CDK1 and CDC25C were detected by Western blot.3.After HK-2 cells were cultured with albumin for 72 hours,the expression of p21 in HK-2 cells was down-regulated by si-p21,the progression of G 2/M phase and the expression of pro-fibrotic factor mRNA and protein in HK-2 cells were observed and detected by immunofluorescence staining,RT-PCR and Western blot,futhermore,the phosphorylation of CDK1 and CDC25C was detected by Western blot,and HK-2 cells were cultured with different concentrations of dapagliflozin and albumin for 72 hours,the effect of different concentrations of dapagliflozin on the G2M phase of cells was observed by immunofluorescence staining,the expression of p21,CDK1,CDC25C and pro-fibrotic factor were detected by Western blot in order to understand the effect of different concentrations of dapagliflozin on cells.Results1.After treated with BSA at different concentrations for 72 h,the density of HK-2 cells decreased with the increase of albumin concentration,especially in BSA(20 mg/ml)group,classical senescence tests(SA-β-Gal staining)showed that BSA induced higher levels of SA-β-Gal activity compared with controls.At the same time,20 m g/ml BSA could promote the expression of il-1α,IL-8 and IL-6 in the senescence-associated secretory phenotype(SASP)of HK-2 cells.In addition,.we observed that the expression of laminB1 in the nuclear membrane of HK-2 cells was significantly decreased after 72 h of BSA(20 mg/ml)intervention,whereas the expression of y-H2AX,a DNA double-strand break marker,was increased by 1.5-fold by Western blot.These results suggest that albumin can induce renal tubular epithelial cell senescence.2.Albumin-induced G 2/m arrest of renal tubular epithelial cells(HK-2)leads to interstitial fibrosis,we found that the number of p-H3(+)HK-2 cells increased with the increase of BSA concentration by immunofluorescence staining,which indicated that the G 2/M phase arrest of albumin-induced senescence of HK-2 cells occurred Western blot showed that the expression of CTGF and α-SMA was increased in BSA treated group at 20 mg/ml.These data suggest that BSA has the ability to induce the production of pro-fibrotic factors in HK-2 cells.Compared with the control group,the expression of p21 mRNA and protein in BSA(20mg/ml)group increased by 59%and 3.2-fold,respectively.We further decreased the expression of p21 by siRNA-p21 transfection,and found that the expression of p-H3(+)cells was significantly decreased in HK-2 cells transfected with BSA combined with Sirna-p21.At the same time,Western blot results showed that after siRNA-p21 transfection,the expression of p-H3(+)cells was significantly decreased,the expression of CTGF and α-SMA in HK-2 cells in BSA group was significantly decreased.The increased phosphorylation of CDC25C and CDK1 is a sign of cell cycle arrest in G 2/M phase.Our study showed that the phosphorylation level of CDC25C and Cdk1 increased significantly with the increase of BSA concentration.We then transfected HK-2 cells treated with 20 mg/ml BSA using siRNA-p21,which showed reduced levels of CDK1 and CDC25C phosphorylation.3.Dapagliflozin improved interstitial fibrosis by inhibiting G2M arrest of renal tubular epithelial cells.We used different concentrations of dapagliflozin combined with BSA to intervene HK-2 cells,the results showed that dapagliflozin can reduce the expression of TGF-βl and CTGF in renal tubular epithelial cells,and the results of immunofluorescence staining also showed that it can improve the G2M arrest of renal tubular epithelial cells Furthermore,Western blot analysis showed that the expression of p21 in HK-2 cells was inhibited by different concentrations of dapagliflozin,the phosphorylation levels of CDK1 and CDC25C also decreased significantly.ConclusionThe renal tubular epithelial cells are senescent induced by albumin,and the G 2/M phase arrest of the senescent tubular epithelial cells results in the increase of pro-fibrotic factors By inhibiting the expression of p21,the expression of p21 was reduced,and the expression of p21 was inhibited by dapagliflozin,by altering the phosphorylation levels of Cdkl and CDC25C,the cell cycle is affected and cells arrested in the G 2/M phase are reduced,thus reducing the production of pro-fibrotic factors and exerting an anti-fibrotic effect;... |
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