The Mobility Of Normal Kidney Epithelial Cell In The Course Of Epithelial-mesenchymal Transition (EMT)

Objective The progression of chronic kidney disease (CKD) is considered to be an irreversible process that eventually leads to end-stage renal failure.The pathogenesis of CKD is characterized by progressiveloss of kidney function and relentless accumulation and deposition of extracellular matrix (ECM), leading to widespread tissue fibrosis. The renal interstitial fibrosis(RIF) is particularly interesting, because the deterioration of renal function is largely determined by the extent and severity of interstitial lesions in many forms of renal disease. Mature tubular epithelial cells in disease kidney can undergo epithelial-to-mesenchymal transition (EMT), a phenotypic conversion that is fundamentally linked to the pathogenesis of renal interstitial fibrosis. The EMT as a main cytology behavior for the renal interstitial fibrosis had been widely accepted in recent years.In view of the importance of EMT in the progress of the CKD,the full understanding of its pathological mechanism was very important.Yang etc. inferred that the EMT was proposed as an highly regulated process that consists of four key steps:(1) loss of epithelial cell adhesion; (2) de novoα-smooth muscle actin(α-SMA) expression and actin reorganization; (3) disruption of tubular basement membrane; and (4) enhanced cell migration and invasion. Of the many factors that regulate EMT in different ways, transforming growth factor-β1 (TGF-β1)is the most potent inducer that is capable of initiating and completing the entire EMT course both in vitro and in vivo.While the definited mobility changes of the normal renal kidney epithelial cell in the couse of epithelial-to-mesenchymal transition(EMT) is unknown yet. So this study aimed to evaluate the cell mobility and proliferation cycle of NRK-52E cells in the couse of EMT.Methods NRK-52E cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and treated with 5ng/ml TGF-β1 to induce EMT.The expression of EMT-related genes such as a-SMA and E-cadherin was examined by Western blotting to demonstrated the occurrence of EMT.Changes in cell mobility were assessed by transwell chamber assay and wound healing assay after treated with TGF-β1 for 4h,8h,12h,24h and 48h.And the proliferation cycle of NRK-52E cells evaluated by the Flow Cytometry.Results First, following TGF-β1 treatment,phase-contrast microscopy revealed a loss of the adherent phenotype of NRK-52E cells,decreased in cell-to-cell contact.and the induction of a myofibroblast-like state.And,NRK-52E cells showed downregulation of epithelial marker E-cadherin and increased the expression of mesenchymal marker a-SMA.And the alterations depended on the action time of TGF-β1.the proliferation cycle of NRK-52E cells showed no significant deference after treated with TGF-β1(5ng/ml).Second, the proliferation cycle of NRK-52E cells showed no significant deference after treated with TGF-β1(5ng/ml). Tird, The transwell chamber assay and wound healing assay show that compared with the control group,the cell mobility in group treated with TGF-β1 (5ng/ml) was significantly enhanced from 12h after treated with TGF-β1 (P<0.01).Conclusion These resules suggested that:The migration ability of the NRK-52E cells increased incessantly in the couse of EMT induced by TGF-β1) (5ng/ml) without the influence of cell proliferation in vitro.