| Diabetic nephropathy(DN)is a major cause of CKD,and its incidence is increasing each year.The pathological changes in DN include glomerular basement membrane(GBM)thickening,mesangial expansion,nodular glomerular sclerosis,and tubulointerstitial fibrosis.Among all the pathological changes in DN,interstitial fibrosis and tubular atrophy remain independent risk factors for renal survival.E1A binding protein P300(EP300,also known as p300)is a lysine acetyltransferase and a master regulator of gene transcription that promotes gene transcription in different cell types and is critical for cellular functions such as proliferation,apoptosis,and differentiation.A Chinese cohort showed that EP300 gene polymorphism correlated with the development and advancement of diabetic kidney disease by the TaqMan probe method.In the animal TFDB 3.0 database,it was predicted that EP300,which is a transcription factor,is the upstream regulator of hypoxia-inducible factor 2α.Vascular endothelial growth factor A(VEGFA)is a downstream target of HIF-2α,and HIF-2α activity is positively correlated with VEGFA expression.Studies have shown that HIF2α could promote renal fibrosis in UUO mice.Based on the above studies,we hypothesized that EP300 could participate in the process of renal tubulointerstitial fibrosis in diabetic nephropathy by regulating the expression of HIF2α.In this study,we investigated the role of EP300 in promoting HIF2α expression in renal tubulointerstitial fibrosis in DN,in vivo and in vitro.Part Ⅰ The expression level of EP300 and HIF2α in the renal tubulointerstitial of patients with DNObjectiveThe clinical data of diabetic nephropathy patients and control group were analyzed and the expression of EP300 and HIF2α in renal tissue of diabetic nephropathy patients were observed.MethodsTissue samples were obtained from 10 patients(renal puncture tissues)with a pathological diagnosis of DN who underwent renal biopsy at the Department of Nephrology and 5 patients(tissues adjacent to carcinoma)with a pathological diagnosis of kidney cancer who underwent nephrectomy without diabetic mellitus at the Department of Urology,First Affiliated Hospital of Zhengzhou University,China,between October 2021 and December 2021.Renal biopsy tissues were collected from patients as Diabetic Nephropathy group(DN),and paracancer tissue as Normal Control group(NC).Immunohistochemistry and immunofluorescence methods were used to observe the expression differences and localization of EP300 and HIF2α,respectively.At the same time,the clinical data of the two groups of patients were collected and the difference of data was analyzed by SPSS 26.0 software.Results1.Age and fasting plasma glucose were not different between the DN and normal control(NC)groups(P>0.05).HbA1c,serum creatinine,urea nitrogen,serum albumin and hemoglobin showed significant differences between the DN and NC groups(P<0.05).Of the 10 patients with diabetic nephropathy,7(70%)were pathologically diagnosed as stage DN-Ⅲ and 3 patients(30%)as stage DN-Ⅱ.2.The protein expression levels of VEGFA,α-SMA,and fibronectin,which reflect the degree of fibrosis,were upregulated in the renal tubules of DN patients,and E-cadherin was downregulated.3.The expression of HIF2α was increased in DN patients compared to NCs.Furthermore,EP300 was expressed in human renal tissues and was significantly upregulated in DN samples.4.EP300 and HIF2α were expressed in renal tubular tissues and were strongly expressed in DN patients compared to control samples.Conclusionsl.The renal tubular fibrosis protein secretion increased in diabetic nephropathy patients,and the fibrosis trend was obvious.2.The expressions of EP300 and HIF2α were increased in the renal tissues of diabetic nephropathy patients,and were significantly upregulated in the renal tubulointerstitial.Part Ⅱ Study on the mechanism of EP300 regulating HIF2α in human renal tubular epithelial cells(HK-2)ObjectiveTo investigate the expression and localization of EP300 in HK-2 cells and the specific regulatory sites of EP300 on HIF2α after high glucose stimulation.Relationship between the expression of EP300,HIF2α,VEGFA,α-SMA,Fibronectin and E-cadherin in HK-2 cells and the time and concentration of high glucose stimulation.To investigate the regulation of EP300 on HIF2α and its effect on cell fibrosis in HK-2 cells.To investigate the effect of inhibition of HIF2α on EP300 induced cell mesenchymal transition.MethodsAfter high glucose stimulated human renal tubular epithelial cells(HK-2)in vitro,the expression of EP300 and HIF2α was detected by fluorescence.Western Blot method was used to detect the expression level of EP300 in cytoplasmic protein and cytonuclear protein.Normal cells were collected and DNA binding to EP300 protein was pulled using CHIP kit and EP300 CHIP class antibody.NovaSeq 6000 sequence analysis was performed to verify the binding site of EP300 protein and HIF2α gene sequence.Whether there was the binding of EP300 and HIF2α at protein level was verified by COIP and Western blot.To investigate the concentration and time dependence of EP300,HIF2α,VEGFA,α-SMA,Fibronectin and E-cadherin on high glucose stimulation.HK-2 was stimulated with different glucose concentrations and treatment time,and the protein and mRNA expression levels of each indicator were detected by Western Blot and fluorescence quantitative PCR.The cells were infected with 1/2 medium method,EP300 was interfered or overexpressed by adenovirus,respectively,and the silencing and overexpression efficiency were detected by Western Blot and fluorescence quantitative PCR.Using adenovirus to silence or overexpression EP300 level,the expression of every indicator(EP300、HIF2α、VEGFA、α-SMA and E-cadherin)was observed in HK-2 stimulated high glucose or normal HK-2 cells.After EP00 was overexpressed in normal HK-2 cells and HIF2αactivity was inhibited by PT2385,the effects of HIF2α on EP300 upstream and downstream VEGFA and α-SMA expression levels were observed.Results1.EP300 protein expression was mainly localized in the nucleus in HK-2 cells,whereas HIF2α protein expression was observed in the cytoplasm and nucleus in HK-2 cells(green staining).When HK-2 cells were stimulated with high glucose in vitro,EP300 was strongly expressed in the nucleus,and HIF2α was upregulated primarily in the cytoplasm.2.The ChIP-seq results showed that the EP300 protein binds to HIF2α DNA sequences and positively regulates HIF2α gene expression.The EP300 protein regulates HIF2α expression at the gene level but not the protein level.3.The expression levels of EP300,HIF2α,VEGFA,α-SMA,Fibronectin and E-cadherin were concentration-dependent and time-dependent on glucose.4.After EP300 expression was silenced in high glucose stimulation group,HIF2α expression was down-regulated,fibrosis markers were also down-regulated,and E-cadherin was up-regulated,which could alleviate the fibrosis level of HK-2 cells.5.Overexpression of EP300 in normal group can increase HIF2α expression level,up-regulate fibrosis index and down-regulate E-cadherin,which can induce hK-2 cell mesenchymal transition.6.On the basis of EP300 overexpression in normal HK-2 cells,inhibition of HIF2α activity by PT2385 can down-regulate the expression levels of VEGFA andα-SMA,but did not affect EP300 level.Conclusions1.In HK-2 cells cultured in vitro,EP300 mainly activated HIF2α transcription level in the nucleus,and played a positive role in regulating the increase of HIF2αexpression level.2.The expression levels of EP300 and HIF2α in HK-2 cells were affected by glucose concentration and treatment time,similar to the fibrosis levels of cells.3.EP300 participated in the fibrosis process of HK-2 cells induced by high glucose by regulating HIF2α.Part Ⅲ Effect of EP300 silencing on renal tubular fibrosis in Diabetic nephropathy C57BL/6J miceObjectiveTo verify after inhibition of EP300,the expression of downstream HIF2α and the level of renal tubular fibrosis in diabetic nephropathy mice.MethodsThe diabetic nephropathy model of C57BL/6L mice was established and purified EP300 interfering adenovirus was injected in situ.Changes in body weight,blood glucose and urinary protein concentration of mice were monitored during modeling.Renal tissue changes were observed using HE,PAS and Masson staining.The mice were divided into 4 groups:normal control mouse group(NC),diabetic nephropathy model mouse group(DN),diabetic nephropathy model+EP300 injection silenced adenovirus group(DN+Sh-EP300),diabetic nephropathy model+empty Vector control group(DN+Vector).The expressions of EP300,HIF2α,VEGFA,α-SMA and E-cadherin were detected by Western Blot and fluorescence quantitative PCR.The expression of each indicator in the renal tubule region was detected by immunofluorescence,and the expression difference between the lentivirus injection group and the model group was observed.Results1.Fasting blood glucose(FBS)continued to rise in mice 3 months after modeling,and the weight of DN group was significantly lower than that of NC group at modeling end point(P<0.05),urinary protein concentration was significantly higher than that of NC group(P<0.05).HE staining showed that the renal tubule matrix increased in DN group,some tubules atrophied,and eosinophils infiltrated renal interstitium.PAS and Masson staining showed that the basement membrane of renal tubules was thickened and blue stained collagen fibers were increased in DN group.2.Compared with NC group,the expression of EP300,HIF2α,VEGFA andα-SMA was up-regulated in DN group,while the expression of E-cadherin was down-regulated.After inhibition of EP300,compared with DN group,the expression level of HIF2α in renal tissue was significantly decreased,the expression of fibrosis markers was also decreased,and E-cadherin was up-regulated.3.Immunofluorescence showed that the expression of EP300,HIF2α,VEGFA and α-SMA in renal tubules was up-regulated,while the expression of E-cadherin was down-regulated.Conclusions1.Inhibition of EP300 could down-regulate the expression of HIF2α and delay renal tubulointerstitial fibrosis in DN mice。... |
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