Adenovirus-mediated Anti-sense ERK2 Gene Therapy Inhibits Tubular Epithelial-Mesenchymal Transition Both In Vitro And In Vivo And Ameliorates Chronic Allograft Nephropathy In A Rat Model

Objective: To investigate the ERK2 signal pass way on Epithelial Mesenchymal Transition in vitro and in vivo and the effects of Adanti-ERK2 attenuated CAN in kidney allografts was well examined.Methods: (1) The Adanti-ERK2 and Ad-LacZ were generated. (2)The renal tubular epithelial cells were transfected by Ad-antiERK2 with multiplicity of infection (MOI) 50, 100 and 200 for 1h, followed by the green fluorescence observation 2 days later. The live cells ratio was detected 24h and 48h later. (3)The cultured renal tubular epithelial cells were divided into four groups: control group (no gene transfer); Ad-LacZ group (transfected with Ad-LacZ); CTGF-treated group (treated with CTGF at a final concentration of 5 ng/ml); and Adanti-ERK2 group (treated with CTGF at a final concentration of 5 ng/ml and Adanti-ERK2 with moderate MOI). The cells were collected at 72h time points. Immunohistochemistry was used to detect the E-Cadherin, Vimentin andα-SMA; Western-blot was used to detect the ERK2 in cells. Boyden Chamber was used to detect the migration of the renal tubular epithelial cells at 1d, 3d and 5d. (4)Male Lewis (LEW, RT11) rat received male Fisher (F344, RT11v1) renal allograft. The recipients were divided into three groups: control group; Ad-LacZ group and Adanti-ERK2 group. All recipients were sacrificed for grafts and serum at 24 weeks after transplantation. Morphometric analysis was used to determine the fibrosis of grafts. Immunohistochemistry was used to detect the expression of E-Cadherin, Vimentin,α-SMA and TβRⅠ. The infiltration of CD4~+ T, CD8~+ T lymphocyte and ED-1~+ monocytes was well detected in the same way. ELISA was used to detect TGF-β1 in serum.Results: (1) Over 95% renal tubular epithelial cells expressed green fluorescence aftertransfected by Ad-antiERK2 (MOI 100) for 2 days. The live cell ratio at 2 and 4 days had nodifference from those of the control group. So the moderate MOI was 100. (2) Comparedwith CTGF-treated group, there were more E-Cadherin, and less Vimentin expressed in thecell of Adanti-ERK2 group. Theα-SMA was detected in the cells of CTGF-treated group.For the expression of E-Cadherin, Vimentin,α-SMA, there were no diference between theAdanti-ERK2 group with the control and the Ad-LacZ groups. (3)On the first two days, themigration of renal tubular epithelial cells in the four groups was no difference, there were nocells migrated through the pores to the opposite side of the transwell filters. Since from 3d,the transformed cells began to migrate through the pores to the opposite side of filters in theCTGF-treated group. In the 5d, compared with CTGF-treated group, there were markedlyless transformed cells in the opposite side of filters in Adanti-ERK2 group. (4)The grafts incontrol group and Ad-LacZ group showed with CAN. There were less E-Cadherin in renaltubular epithelial cells in control group but more Vimentin andα-SMA. In Adanti-ERK2group, the fibrosis was ameliorated and less T lymphocytes and ED-1~+ monocytes infiltratedin the interstitium. The expression of E-Cadherin was no markedly difference inAdanti-ERK2 group and in normal rats. (5)Compared with the control groups, theexpression of TGF-β1 and TβR I in Adanti-ERK2 group were down-regulated.Conclusions: Epithelial-Mesenchymal transition (EMT) plays an important role in the progress of chronic allograft nephropathy (CAN). In vitro, Adanti-ERK2 gene therapy can prevent CTGF induing Epithelial Mesenchymal Transition of renal tubular epithelial cells. In vivo, Adanti-ERK2 gene therapy protects renal allograft and attenuates graft fibrosis, which may be correlated with the decreased renal tubular epithelial mesenchymal transition, the decreased infiltration of CD4~+ T lymphocyte, CD8~+ T lymphocyte and ED-1~+ monocytes in renal interstitium, and the down-regulated TGF-(31 expression. In conclusion, our results suggest Adanti-ERK2 gene therapy inhibits tubular EMT both in vitro and in vivo, and attenuates CAN in kidney allografts. It is possible to develop elaborate molecular drug(s) to treat CAN in the future, as the ERK signaling pathway may be a promising novel efficient therapeutic target.