| Background:Obstructive nephropathy is an important cause of chronic kidney disease that ultimately progresses to tubulointerstitial fibrosis and end-stage renal failure. In such cases, renal damage progresses even after the ureteral obstruction is relieved, and most patients with obstructive nephropathy are identified only when they have already reached end-stage renal failure. To date, there is no effective treatment available to prevent tubulointerstitial fibrosis and the progressive loss of renal function. Lefty is a new member of the TGF-βprotein superfamily. And it is one of the key embryonic signals that drive the development of an asymmetric body plan by inhibition of TGF-β1/Smads signaling pathway. Moreover, our previous study has shown that only the expression of Lefty mRNA was downregulated significantly (↓13.55 folds) in the kidneys of patients with severe ureteral obstruction using gene microarray analyses. In this study, we construct eukaryotic expression vector pcDNA3.1/Hygro(+)-LeftyA and establish the cell line that can stably express LeftyA to study the effects and the mechanisms of LeftyA on TGF-β1 induced renal epithelial fibrosis.Objective:Constructing eukaryotic expression vector pcDNA3.1/Hygro(+)-LeftyA and establish the cell line that can stably express LeftyA To study the effects and mechanisms of LeftyA on epithelial to mesenchymal transition (EMT) of human proximal tubular epithelial cells induced by TGF-β1.Methods:The Coding sequence (CDS) of LeftyA was gained from pCMV-SPORT6 and then was cloned into eukaryotic expression vector pcDNA3.1/ Hygro(+).The recombinant of pcDNA3.1/Hygro(+)-LeftyA was transfectd into HK-2 cells. After screening culture by Hygromycin B, stable transfected cell line was established and the expression of LeftyA was identified by Western blot and RT-PCR assay. HK-2 cells and cells expressing LeftyA protein were stimulated by TGF-β1 respectively. Then the morphological changes were observed by inverted phase contrast microscope, the expression of E-cadherin, a-SMA, p-Smad2/3, connective tissue growth factor (CTGF), Col I and LeftyA mRNA and protein was detected by RT-PCR, Western blot respectively to investigate the effects and mechanisms of LeftyA on EMT of HK-2 cells induced by TGF-β1.Results:The CDS fragment of LeftyA, which was amplified by PCR, was inserted into pcDNA3.1/Hygro(+) vector by restriction endonuclease reaction and ligation reaction. Restriction endonuclease reaction and gene array confirmed its integrity. HK-2 cell line stably expressing LeftyA can be established by transfection with LipofectamineTM 2000 and selection by Hygromycin B. The expression of LeftyA in such cell line was confirmed by Western blot and RT-PCR assay.The expression of E-cadherin mRNA and protein were markedly decreased in HK-2 cells induced by TGF-β1 and the expression of a-SMA mRNA and protein were dramatically increased. Western blot analysis demonstrated that the protein level of LeftyA was progressively reduced in a time-dependent manner in response to TGF-β1 treatment in HK-2 cells. Forced expression of exogenous LeftyA led to a severe blockage of TGF-β1-induced E-cadherin suppression, a-SMA and Col I induction.And the results showed that stimulation with TGF-β1 led to Smad2/3 signaling pathway activation, including p-Smad2/3 expression and its nuclear translocation. CTGF and Col I mRNA expression can also be induced by TGF-β1. Overexpression of LeftyA efficiently blocked p-Smad2/3 activation and CTGF expression induced by TGF-β1.Conclusion:The construction of pcDNA3.1/Hygro(+)-LeftyA and the establishment of HK-2 cell line stably expressing LeftyA were proved to be feasible. And overexpression of LeftyA can suppress EMT induced by TGF-β1 through inhibition of TGF-β1/Smads signaling and CTGF expression during the process of renal fibrosis, which suggests a protective role for LeftyA in TGF-β1-induced tubular EMT and renal fibrosis. |
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