The Mechanism Research Of PEA3 In Tubulointerstitial Fibrosis

Chronic kidney disease(CKD)is one of the world’s most important public health problems.The most serious consequence of CKD is caused by the loss of kidney function,eventually leading to end-stage renal disease(ESRD).While countries nephrology scholars had done a lot of work,effective clinically treatment methods in tubulointerstitial fibrosis is still lacking.Therefore,to explore the methods to delay and block the progression of tubulointerstitial fibrosis is important.PEA3 belongs to the ETS family of transcription factors.The study found that PEA3 family transcription factor expressed in numerous organs both during embryonic development and in adults,such as breast,salivary gland and kidney.Our group found that the expression of PEA3 significantly improved in the process of embryonic development,and PEA3 can transformate from mesenchymal cells to normal renal tissue structure and form a tube-like structure epithelial cells and terminally differentiate into tubular and glomerular by inducing the expression of WT1.Using the technology of cell interference and over-expression,we found that PEA3 can block TGF-β1 induced tubular epithelial cells epithelial-mesenchymal transdifferentiation,which means PEA3 plays a protective role in the process of kidney damage.Therefore,we hypothesized that PEA3 overexpressed in vivo suppress the renal tubular interstitial fibrosis induced by unilateral ureteral obstruction.Hydrodynamic gene injection is a method of exogenous gene overexpression in vivo by rapid and large injection of plasmid DNA in vein.Therefore,we used hydrodynamic gene injection techniques,rapid and large injection of plasmid DNA through the tail vein of mice,to preparation of plasmid overexpressing mice model.Objective:Investigate the expression of PEA3 in the kidney of mice after the PEA3 plasmid DNA injected in tail vein by hydrodynamic gene delivery method.UUO(Unilateral ureteral obstruction)or sham surgery was performed to investigate the role of PEA3 in renal tubulointerstitial fibrosis,and further exploration was performed in the effect of protein levels of PGC-1α(Peroxisome proliferator-activated receptor gamma coactivator 1-alpha)and TFAM(Mitochondrial transcription factor A)after PEA3 overexpression in vivo.Methods:C56BL/6J male mice divided into four group:control group(Sham + Vehi),PEA3 overexpression group(Sham + PEA3),UUO model group(UUO + Vehi),and PEA3 intervention group(UUO + PEA3).Plasmids were administered by tail vein at 1 and 7 days at a dose of 50mg in 2ml saline,with animals receiving 2.5mg/kg of PEA3 plasmid and 2.5mg/kg control plasmid.Animals were euthanized after 14 days.Using quantitative real-time PCR(RT-PCR)and Western blot to determined the expression of PEA3.Plasmids were administered by tail vein at-7 and-1 days at a dose of 50mg in 2ml saline,with animals receiving 2.5mg/kg of PEA3 plasmid and 2.5mg/kg control plasmid.UUO or Sham surgery was performed on day 0 and animals were euthanized 7 days post-surgery.Applied Masson staining to observe the degree of renal tubulointerstitial fibrosis;Using quantitative real-time PCR(RT-PCR)and Western blot to determined the expression of PEA3,as well as,the expression ofα-SMA,Collagen Ⅲ,TGF-β1,E-cadherin,PGC-la and TFAM in kidney tissue.Results:① Compared with Sham+Vehi group,the mRNA and protein level of PE A3 was increased in Sham+PEA3 group by hydrodynamic gene injection(increased about 7.47,1.54 times,respectively,P<0.05).Compared with UUO +Vehi,the mRNA and protein level of PEA3 in UUO + PEAS group also significantly increased(increased about 3.33,1.93 times,respectively).②Masson staining revealed renal interstitial collagen deposited,and inflammatory cell infiltrated,tubular atrophied in UUO+Vehi group compared with Sham+Vehi group,While compared with UUO+Vehi group,UUO + PEA3 group reduced the renal tubulointerstitial fibrosis.Compared with Sham+Vehi group,the mRNA expression of Vimentin,TGF-β1,Fibronectin and a-SMA significantly increased in UUO+Vehi group(increased about 6.05,4.63,12.04,4.48 times,respectively);This result can be reversed in PEA3 over-expression group(decreased approximately 74%,69%,65%and 58%,respectively).Compared with Vehi group PEA3 overexpression UUO group,After PEA3 overexpression that can significantly blocked the expression of these genes.The protein expression of Vimentin,Fibronectin,Collagen Ⅲ and a-SMA significantly increased(increased about 2.82,8.02,3.96 and 2.43 times,respectively)and E-cadherin is significantly decreased(decreased about 55%)in UUO+Vehi group compared with Sham+Vehi.PEA3 overexpression reversal expression of these proteins,Which Vimentin,Fibronectin,Collagen Ⅲ and a-SMA protein expression reduced by 56%,79%,63%and 64%,E-cadherin protein expression increased 2.31 times.③ Detecting the protein expression of PGC-la and TFAM found that those protein expression was significantly lower in UUO+Vehi group compared with Sham+Vehi group(reduced 72%and 73%,respectively);While PEA3 overexpression can significantly inhibit UUO induced PGC-1α and TFAM downregulation(increased approximately 4.72 and 2.68 times of the UUO+PEA3 group,respectively).Conclusions:PEA3 inhibited renal tubulointerstitial fibrosis of obstructive nephropathy mice and possibly by upregulating the expression of PGC-1α and TFAM protein expression.