Activated Macrophage Induces Epithelial-mesenchymal Transition Of Renal Tubular Epithelial Cell

Renal transplantation is the best and most effective treatment of end-stage renaldisease. In recent years, the short term survival rate of renal allograft has raisedsignificantly, but the long term survival rate is always poor. At late stage of renalallograft, patients will have chronic rejection and presented as renal fibrosis, which ischaracterized as tubular atrophy and tubulointerstitial fibrosis. Activatedmyofibroblasts which produce excessive extracellular matrix in renal interstitial havevarious sources, now it is demonstrated that a major derivation of myofibroblasts isfrom the transition of tubular epithelial cells to mesenchymal cells, that isEpithelial-Mesenchymal Transition (EMT).Experiments have shown that T lymphocytes can directly attack renal tubularbasement membrane, which presented as "tubulitis", and a large number of Tlymphocytes infiltrate in tubulointerstitial in acute rejection, so it is widely consideredthat acute cellular rejection is mainly mediated by T lymphocytes. However, recentstudies have shown that a large number of macrophages also infiltrate intubulointerstitial while acute rejection, especially in Banff1b and more severerejection. So we put our sight on the macrophages to study whether macrophages playa great role on EMT of renal tubular epithelial cell.Objective: In this experiment, we choose culture medium of macrophage J774activated by LPS to observe the ability of macrophage in inducing NRK52E cellsundergo epithelial-mesenchymal transition. We also look forward to explore themechanism of macrophage inducing EMT by testing the composition of activatedmacrophage culture medium.Method: Murine macrophage J774was simulated by LPS whose concentrationis1μg/ml，1.5μg/ml，2.5μg/ml and5μg/ml in vitro for24hours and48hours,and the levels of TNF-α and IL-6in the supernant of J774were detected by ELISA. Morphological changes were observed under a phase contrast microscope when2.5μg/ml LPS activated J774for24hours. After stimulation, macrophages were washedwith serum-free DMEM three times, DMEM from the final wash was collected andused as control medium. After washed three times, NRK52E was cultured inserum-free DMEM for another48hours, then the medium was collected and used asinduction medium. NRK52E cells were divided into control group and inducedgroup，incubation conditions of two groups were DMEM medium: control medium as1:2in the control medium and DMEM medium: induction medium as1:2in inductivemedium, both add FBS to5%. After48hours, cell morphology was observed underphase contrast microscope, at the same time, Real-Time PCR, Western Blot andImmunocytochemical staining were used to test the expression of epithelial andmesenchymal markers on the NRK52E cells. In addition, we carried out massspectrometric detection to observe the cytokines in macrophage culture mediumactivated by LPS(2.5μg/ml) for24hours.Results: It is demonstrated by ELISA that the supernatant of macrophage J774had no TNF-α and IL-6when the concentration of LPS was0in not simulated group.When J774was activated by LPS whose concentration was1μg/ml，1.5μg/ml，2.5μg/ml and5μg/ml in vitro for24hours or48hours in simulated group, we can detectthe existence of TNF-α and IL-6definitely from the J774supernatant. Above resultsshow that J774was activated by different concentration of LPS for24hours or48hours. But when J774was activated by low concentration (1μg/ml) of LPS for48hours, the content of TNF-α and IL-6decreased in the supernant of macrophage J774compared with that when J774was activated by LPS for24hours. So we select theactivated time of LPS was24hours in the next experiment. We can detect acomparatively higher level of TNF-α and IL-6in the supernant of J774when it wasactivated by LPS whose concentration was2.5μg/ml, therefore we use2.5μg/ml LPSto activate J774for24hours in the after experiments. Then we activated J774by LPS（2.5μg/ml）for24hours, and found that the morphology of macrophage J774changed from round to protruding pseudopods and fried eggs like adherent underinverted microscope. The above results show that LPS activated macrophages in vitro. We cultured NRK52E in control and inductive culture condition for48hours,compared with control group, NRK52E cells in inducement group changedmorphologically from closely linked cuboid-shaped to increased cell interval,spindle-shaped and scattered fibroblast-like cells. Results from Real-Time PCRshowed that epithelial marker CK19of NRK-52E from inducement group decreasedto29%of the control group, while mesenchyme marker α-SMA increased to5.6timesof the control group. We chose GAPDH as reference protein, E-Ca as epithelialmarker, Vimentin and α-SMA as mesenchyme markers in Western Blot experiment.Results show that the expression of E-Ca decreased significantly while Vimentin andα-SMA increased largely in inducement group compared with control group. Theresults from Immunocytochemical staining show that the linear expression of E-Ca oncell membrane which is an epithelial cell marker of NRK52E decreased ininducement group compared with control group. Results from above showed that ininducement group, epithelial markers of NRK52E decreased significantly at levels ofmRNA and protein expression compared with the corresponding control groups, whilemesenchyme markers increased largely both in the expression of mRNA and proteincompared with the corresponding control groups, which showed that NRK52Eunderwent EMT by cultured with the supernatant from activated macrophage in vitrofor48hours.At the same time, ESI mass spectrometric detection showed that activatedmacrophages released a large number of proteins, except for some enzymes related tometabolization, several assured kinds of cytokines were detected, mainly including:complement C3, Hsp90, MMP-9, Osteopontin, TGF-β, IL-6and TNF-α.Conclusion: activated macrophage culture medium induced renal epithelial cellEMT successfully, and mass spectrometric detection showed activated macrophagescan release complement C3, Hsp90, MMP-9, Osteopontin, TGF-β, IL-6and TNF-α,and macrophages induce renal tubular epithelial cell EMT by secreting thesecytokines.