NRK52E Cells Were Induced To Undergo Epithelial-mesenchymal Transition By Thymus Cells And Effects On The Cells Migration

Renal allograft has developed into the best treatment for a variety of advanced renaldisease. In the past few decades, significant improvement has been achieved in theshort-term survival rate of renal allograft rather than the long-term survival rate. Severechronic renal dysfunction or loss of function is characterized pathologically bytubulointerstitial fibrosis. It is reported that activated fibroblasts that produce excessiveextracellular matrix in renal interstitial mainly derived from the transition of tubularepithelial cells to mesenchymal cells in the chronic renal allograft fibrosis. The clinicalobservations demonstrate that there is an association between the number and/or severity ofacute rejection episodes and decreased probability of long-term graft survival. An importantdiagnostic criterion for the majority of acute rejection is the infiltration of inflammatory cellsthat are mainly T lymphocytes into renal tubular epithelial cells. Now，the specificmechanisms of acute rejection in chronic renal fibrosis is not clear.Objective: To observe the ability of rat thymocyte o induce NRK52E cells undergoepithelial-mesenchymal transition and their effect on cell migration. We look forward toproviding experimental evidence for the mechanism of chronic renal fibrosis after acuterejection.Methods: Thymocytes were simulated by ConA (final concentration of5μg/ml) andPHA (final concentration of10μg/ml) in vitro for24hours. Morphological changes wereobserved under a phase contrast microscope, the distribution of the cell cycle of thymocyteswere analyzed by flow cytometry, and IFN-γ and IL-2secretions were detected byenzyme-linked immunosorbent assay, to ensure that the thymocytes were activated. Afterstimulation, thymocytes were washed with serum-free DMEM three times, and cultured inserum-free DMEM for another48hours. After48hours the medium was collected and usedas induction medium. DMEM from the final wash was collected and used as control medium.NRK52E cells were divided into control group and induced group，incubation conditions oftwo groups were45%DMEM medium+45%in the control medium to+10%FBS and45%in DMEM medium+45%induction medium+10%FBS. After24hours, cell morphologywas observed under phase contrast microscopy, and RT-PCR, immunocytochemical staining and immunofluorescence staining were used to test the expression of epithelial andmesenchymal markers on the NRK52e cells. In addition, we carried out the cell scratchexperiment to observe the change of cell migration ability after the induction。Result: With the stimulation of ConA and PHA for24hours, mean diameters of thymuscells increased from14.00±1.2μm to20.36±2.1μm (P <0.05), accompanied by thepercentage of cells in G2/S phase increased from19.5%±3.3to31.3%±5.2%of the (P <0.05). After48hours，the concentration of IFN-γ was791±26.7pg/ml in the medium ofinduced group compared with633.5±18.3pg/ml in control group. The concentration of IL-2were5531.79±40.2pg/ml、5267.45±31.7pg/ml、5387.1±89.2pg/ml at2448and72hours inthe medium of induced group, while4352.65±67.1pg/ml、4241.5±56.2pg/ml and4673.5±87.9pg/ml (P <0.05) in the medium of control group. After inducement, NRK52Ecells changed morphologically from cuboid-shaped and clustered cells to spindle-shaped andscattered fibroblast-like cells The result of RT-PCR showed that the expression of E-cadherinwas down-regulated, with the relative mRNA expression decreased from0.764±0.032to0.214±0.025(P <0.05); and the expression of α-smooth muscle actin was up-regulated,with the relative mRNA expression decreased from0.56±0.031to0.647±0.021(P <0.05).Immunocytochemical staining and immunofluorescence staining showed that the expressionof E-cadherin and cytokeratin-19was reduced accompanied by increased expression ofα-smooth muscle actin and vimentin. The cell scratch experiment revealed that cellmigration ability was increased,with he relative migration rate increased from62.1±6.0to83.0±2.8(P <0.05).Conclusion: Rat thymus cells could induce renal tubular epithelial cells in vitroundergo epithelial-mesenchymal transition and after transition mobile ability of epithelialcells was promoted.