Genetic Screening Of Tubular Epithelial-mesenchymal Cell Transition Induced By PAX2 Gene And Identification Of Complement Pathway Related Differential Genes

IntroductionRenal interstitial fibrosis is a nonspecific pathologic process,which is the common final pathway of chronic kidney disease(CKD).To prevent renal interstitial fibrosis is the effective way to prevent the progression of CKD.The patients with CKD,especially when progress to end-stage renal disease(ESRD)stage,become the important social and economic problems because of the huge cost.An overwhelming number of studies have done to deeply understand the mechanism of renal interstitial fibrosis and search for new therapeutic targets.PAX2(Paired Box2)gene encoding protein is a kind of DNA binding protein,which is a key factor to maintain the normal development of the embryonic kidney.In the embryonic kidney,abnormal expression of PAX2 gene can induce renal-coloboma syndrome,renal hypoplasia,vesicoureteric reflux,polycystic kidney and a series of kidney disease.PAX2 protein downregulate rapidly when kidney is mature,and reexpress in some pathological conditions,such as CKD and acute kidney injury.It is essential for diagnosis and treatment of those kidney disease to reveal the mechanism of PAX2 gene reexpression and the role in those pathological conditions.Rodent unilateral ureteral obstruction(UUO)model is a commonly used animal model for the study of CKD and renal interstitial fibrosis.Studies indicate that PAX2 reexpressed in renal tubular epithelial cell in UUO rats,and the expression was positive related with time of obstruction which illustrated that PAX2 took part in the pathogenesis of renal interstitial fibrosis.In vitro experiment results show that PAX2 gene participated in the epithelial mesenchymal transition(EMT).EMT is one of the key fibrogenic mechanisms.PAX2 might aggravate renal interstitial fibrosis by promoting EMT.But the role of PAX2 in renal interstitial fibrosis is still not clear.This study developed an over expression of PAX2 cell model by transfected NRK-52 E,and then screened the downstream gene expression of PAX2 by gene chip.Further,identified the candidate genes through signal pathway analysis.Finally,candidate genes were tested in cell model and UUO model.The aim is toinvestigate the role of PAX2 gene and its downstream gene in renal fibrosis and provide clues to explore new biological targets.Materials and Methods1.Materials(1)cellNRK-52 E cell line(rat renal tubular cell)was provided by The cell bank in Chinese academy of sciences.Lentivirus vector was provided by Genechem company.NRK-52 E cell line was transfected with LV-PAX2 and LV-con separately.Rat OneArray Plus gene chip was used to screen the two groups of cells which were transfected in 72 hours.Rat OneArray Plus gene chip was provided by Phalanx Biotech Group.(2)Animal20 Wistar rats(50-100 g,4w),male,were obtained from the Laboratory Animal Center of Shengjing Hospital,China Medical University.Animals were randomly distributed into model group(UUO group)18 rats and sham operated group 6 rats.Then UUO group rats were distributed into 3 groups by 3,7,14 days after surgery.2.Methods(1)Establishment of the PAX2 gene induced EMT cell modelNRK-52 E cells were transfected with LV-PAX2 and LV-con separately.Cell morphology and transfection efficiency were observed by inverted microscope and fluorescence microscope.PAX2 expression and EMT related markers were detected by Western Blot and Real-time PCR.Cell migration function changes were tested by cell scratch test.The best time point for PAX2 expression was 72 h after transfection.PAX2 induce EMT in NRK-52 E cells in vitro.(2)Screening of differential mRNA expression in NRK-52 E cells over expressed with PAX2 gene.The two groups transfected with LV-PAX2 and LV-con in 72 h were extracted total mRNA separately,then performed hybridization reaction by Rat OneArray Plus expression profile chip.The results were analysis by The Agilent 0.1 XDR scanning system.Further,Gene Ontology analysis and KEGG pathway analysis were used to select candidate gene.The complement cascades were selected to be the candidategenes.Further validation was performed by real time PCR and western blot.(3)Identification of complement pathway related differential genes in cell model and UUO rat kidneyBased on the results from the expression profile chip,and combined with the literature search,the complement cascades including Complement3(C3),CD55,CFH,SERPING1 and C1 S were selected to be the candidate genes.Further validation was performed by real time PCR and Western blot at the cellular level.In UUO rat kidney,the kidney morphology change observed in 3d,7d and 14 d.Renal interstitial fibrosis degree was tested by Masson staining.The complement related differential genes expression were conformed by immunohistochemical staining and Western blot.3.Statistical AnalysisExperimental data were shown as mean ± standard deviation.SPSS17.0 software was used for statistical analysis.Inter-group comparison was done by t-test,and correlation analysis between two variables was done by Pearson correlation analysis.P<0.05 was considered statistical significance.Results1.Establishment of a PAX2 gene induced EMT cell modelGreen fluorescence protein began to appear at 48 hours timepoint after trasnsfection and PAX2 protein expressed in NRK-52 E.PAX2 protein expression at 72 hours timepoint was obviously higher than that at 48 hours and 96 hours time point.PAX2 gene change the morphology of NRK-52 E cells to spindle shaped.Western blot analysis showed that transfected with PAX2 gene decreased abundance of E-cadherin protein and increased abundance of ?-SMA protein in cells at 72 hours timepoint.2.2.Screening of differential mRNA expression in NRK-52 E cells over expressed with PAX2 geneDifferential mRNA expression in NRK-52 E cells over expressed with PAX2 gene and conrol cells were screening by Rat OneArray Plus expression profile chip.Standard selection criteria to identify differentially expressed genes are as follows: log2 |Foldchange| ? 1 and P< 0.05.The number of differentially expressed genes for each comparison is 298 upregulated and 293 downregulated.KEGG pathway analysis of differentially expressed genes showed the involved pathway include cytokine and receptors,extracellular matrix and receptor,MAPK,local adhesion,cancer,complement and coagulation cascades etc.Gene Ontology(GO)analysis showed the involved genesets include hydrolase activity acting on ester bonds,phosphoric ester hydrolase activity,receptor activity,phosphoprotein phosphatase activity,phosphoric monoester hydrolase activity structural molecule activity,substrate specific channel activity extracellular matrix structural constituent,protein tyrosine phosphatase activity and transmembrane receptor activity etc.Complement cascades were selected to be the candidate genes.The involved genes include C3,CD55,CFH,C1 S and SERPING1.C3 was upregulated and the other ones were downregulated(n=3,P<0.05).3.3.Identification of complement pathway related differential genes in cell model and UUO rat kidneyReal time PCR showed that: PAX2 induced C3 increased(n=3,P < 0.05),CD55 and CFH decreased(n=3,P < 0.05)in cells.C1 S and SERPING1 had no statistical difference.Western blot showed that:PAX2 induced C3 increased(n=3,P < 0.05),CD55 and CFH decreased(n=3,P < 0.05)in cells which were consistent with the Real time PCR results.The kidney appeared as grayish-white in color,lacklustre,rough surface,obvious grainy,visible hydronephrosis,thinner renal parenchyma,dilated renal pelvis,compressed calyceal papilla,flat and blunt fornix papilla,some of which became concave shape;rat kidney of sham group was dark-red color with smooth surface and intact renal capsule that was not attached to renal parenchyma.Clear boundary between cortex and medulla could be observed in rat kidney of sham group.Masson staining showed appearance of renal fibrosis after urinary obstruction.Tubular ectasia,analosis and collapse of lumens appeared,renal interstitium widened and collagen fibers were deposited.It was proved that the UUO model was successfully built in this experiment.?-SMA protein increased gradually with the extension of obstruction time andmainly expressed in the renal tubule.Pearson correlation analysis was performed on?-SMA protein expression level and PAX2 protein expression level.Results showed that: ?-SMA protein level was positively correlated with PAX2 protein level(r=0.985,P<0.05).E-cadherin protein decreased gradually with the extension of obstruction time and mainly expressed in the renal tubule.Pearson correlation analysis was performed on E-cadherin protein expression level and PAX2 protein expression level.Results showed that: E-cadherin protein level was negatively correlated with PAX2 protein level(r=—0.981,P<0.05).PAX2 protein increased gradually with the extension of obstruction time and mainly expressed in the dilated renal tubule nuclei.Pearson correlation analysis was performed on PAX2 protein expression level and relative area of renal interstitium.Results showed that: PAX2 protein level was positively correlated with the level of renal interstitial fibrosis(r=0.983,P<0.05).C3 protein increased gradually with the extension of obstruction time and mainly expressed in the renal tubule.Pearson correlation analysis was performed on C3 protein expression level and PAX2 protein expression level.Results showed that: C3 protein level was positively correlated with PAX2 protein level(r=0.985,P<0.05).CFH protein decreased gradually with the extension of obstruction time and mainly expressed in the renal tubule.Pearson correlation analysis was performed on CFH protein expression level and PAX2 protein expression level.Results showed that: CFH protein level was negatively correlated with PAX2 protein level(r=—0.985,P<0.05)CD55 protein decreased gradually with the extension of obstruction time and mainly expressed in the renal tubule.Pearson correlation analysis was performed on CD55 protein expression level and PAX2 protein expression level.Results showed that: CD55 protein level was negatively correlated with PAX2 protein level(r=—0.991,P<0.05).Conclusions1.Established a cell model of PAX2 gene induced EMT in NRK-52 E cells in vitro successfully,and 72 h after transfection is the best time point.2.The cytological level,PAX2 gene influence cytokine and receptors,extracellular matrix and receptor,MAPK,local adhesion,cancer and complement signaling pathwaysetc.3.The cytological level,PAX2 gene increased the expression of C3,and reduced the CD55 and CFH expression.In UUO rat kidney,the alternative complement pathway related genes C3 was upregulated and CD55,CFH were downregulated over obstructive time.C3 may promote renal interstitial fibrosis while CD55 and CFH may prevent that.PAX2 may participate the pathological process of renal interstitial fibrosis through regulate the alternative complement pathway related genes.