Egr1 Regulates,the Progress Of Diabetic Kidney Disease By Intrarenal Renin-angiotensin System

BacgroundDiabetic kidney disease(DKD)is one of the most important microvascular complications of diabetes mellitus(DM).The limitations of RAS blockers for treating DKD are they not only can not effectively inhibit the activation of local RAS system in the kidney,but also lead to increased local renin feedback.Therefore,it may be a new pathway to treat DKD by looking for an effective and complete inhibition of the intrarenal RAS system.Early growth response protein 1(Egr1)is one of the important transcription factors in the progression of DKD.Bioinformatics analysis showed that:RAS system components including AGT,renin,ACE,AT1R promoters have Egrl binding sites.Therefore,our group intends to explore a scientific hypothesis which is Egrl can promote the progression of DKD by upregulating intrarenal RAS system(AGT,renin,ACE,AT1R)and knockdown of Egrl can completely inhibit the intrarenal RAS system and delay the DKD progression.It may provide a new way to DKD treatment.Methods1 Animal experiments(1)The first part:C57BL/6 mice were induced to DKD model by using STZ and high fat diet(HFD).Mice were sacrificed at 12 weeks after the establishment of the DM model.(2)The second part:Egr1 knockdown and Egr1 overexpression DKD mice model were induced by injecting the corresponding plasmids into tail vein every week from 12 to 16 weeks.2 Cell cultureHuman renal tubular epithelial cell line(HK2)was cultured in RPMI-1640 medium(2.0g/L glucose)+ 10%FBS.Mouse mesangial cells(SV40 MES 13)and 293T cells were cultured in DMEM medium(1.0g/L glucose)+ 5%FBS.Environment of incubator is 37 ? with 5%CO2.Passages of growth,liquid change,decking or other experimental operations were done according to the cell growth situation.3 Cell transfectionThe day before cell transfection,the cells were inoculated into a cell culture plate at an average cell density of 60-80%.After being starved for 24 hours in a serum-free medium,the cells were transfected with lipo3000 to achieve gene overexpression or knockdown.4 RT-qPCRTotal RNA of mouse kidney cortices and cells was extracted with Trizol.Total RNA was transcripted to cDNA by one step method.SYBR method was used for real-time fluorescence quantitative PCR reaction.?-actin was as an internal control and the target gene expression was calculated by 2-??Ct method.5 Western BlotTissue and cell total protein was extracted with RIPA.10%SDS-PAGE gel was prepared.The sample volume was 20-50?g per well.Samples were separated by electrophoresis and transferred to PVDF membranes.PVDF membrane was blocked with 5%non-fat milk or BSA,incubated with primary antibody(4? shaker overnight),incubated with fluorescent secondary antibody(1h at room temperature)and then washed with TBST.PVDF membranes were showed in the Odyssey near infrared imaging system.Gel-Pro analyze was used for results analysis.6 renal histopathology and immunohistochemistryThe kidneys were removed,embedded in paraffin and sectioned to pieces(3?m).PAS staining and Masson staining were used to observei the morphological structure of renal tissues.Immunhistochemistry(IHC)was used to analyze the expression of Egr1,RAS and fibrosis indexes.7 Chromatin immune coprecipitationThe cells were divided into control group,TGF-?1(2h)and TGF-?1(48h)stimulation groups.According to ChIP-IT(?)ExPress kit(Active Motif,Bedford,MA)Cruz Biotechnology),control IgG and Egr1 antibody were used to cross-link protein-DNA.Enrichment effects were detected by RT-qPCR.Enrichment multiples were calculated using the standard curve method.8 dual luciferase reporter genesThe ACE luciferase plasmid(GV23 8-ACE)was constructed and cotransfected with different concentrations of Egr1 plasmid(pENTER-Egr1).Firefly and ranilla fluorescence were detected with the dual luciferase reporter gene assay kit.9 laser confocalKidney tissue was co-stained with Egr1 and AGT antibodies,Egrl and renin antibodies.Leica TCS-SL confocal microscope was used to observe co-localization.10 enzyme-linked immunosorbent assaysBlood glucose,glycosylated hemoglobin(HbAlc),creatinine,blood lipids,urinary albumin and kidney Ang? were measured by enzyme-linked immunosorbent assay kit.11 statistical analysisSPSS21.0 was used for statistical analysis.The data was expressed as mean ±standard deviation(X±SD).Two independent sample t-test was used to compare normal distribution data.Data of non-normal distribution was tested using Mann-Whitney U test.One-way ANOVA was used to compare data of multiple groups.Levene homogeneity of variance test was used first.If the variance was homogeneous,the LSD method was further used for multiple comparisons between groups.In contract,F-test correction was selected.If there was a significant difference between the groups,Dunnett's T3 method was used for multiple comparisons.P<0.05 was considered as statistically significant differences.Results1 24h urine albumin in DM group increased significantly compared with control group.The mRNA and protein expressions of Egrl,AGT,renin,ACE and AT1R increased aslo.2 The mRNA and protein expression levels of Egrl in TGF-?1-treated HK-2 cells had transient increase.The mRNA and protein expressions of AGT,renin,ACE and AT1R increased aslo.HK2 cells were transfected with pENTER-Egrl Plasmid(0 ug,1 ug,2 ug,4 ug),the mRNA and protein expressions of AGT,renin,ACE and AT1R increased following.When Egr1 was knocked down in TGF-?1-treated HK-2 cells,the mRNA and protein expressions of AGT,renin,ACE and ATIR decreased following.3 Confocal laser scanning showed that Egrl co-localized with AGT in kidney cortices.4 The promoters of AGT,renin,.ACE and AT1R gene exist Egrl binding sites by chromatin immunoprecipitation experiments.The enrichment efficiency of AGT,renin,ACE and ATIR increased after TGF-?1 treating.5 After transfection with pENTER-Egrl plasmid(0.2,0.4,0.6,0.8 and 1.0 ug)in 293T cells respectively,the firefly luciferase fold activity increased.6 Urine albumin and serum creatinine increased significantly in Egrl overexpression DKD mouse model.The mRNA and protein expressions of AGT,renin,ACE and AT1R increased aslo.7 24h urine albumin significantly decreased in Egrl knockdown DKD mouse model compared with Control group.AGT,renin,ACE and AT1R mRNA and protein decreased following.Masson staining showed that the stained collagen fibers reduced in the kidneys of DKD mice after Egrl knockdown.In addition,fibrosis factor(FN and a-SMA)decreased.protein expression of CTGF and TNF-? decreased also.In conclusion1 RAS components(AGT,renin,ACE,AT1R)are the targets of Egrl.2 Downregulation of Egrl can delay the progression of DKD,one of the possible mechanisms is to inhibit the activation of intrarenal RAS.