Interleukin 6 Induces Phenotypic Change Of Renal Tubular Epithelial Cell In Vitro

BackgroundChronic renal interstitial fibrosis caused by transplant rejection has been the main cause of chronic renal dysfunction in allograft kidney,the main pathological changes of which are renal tubular atrophy accompanied with interstitial fibrosis.It is characterized by activation of myofibroblast and amounts of extracellular matrix secreted.Myofibroblasts related to renal fibrosis is currently considered to have a variety of sources,including: activation of renal intrinsic fibroblasts,bone marrow cell source and renal tubular epithelial cell,vascular endothelial cell or perivascular cell transition.Among them,epithelial-mesenchymal transition(EMT)of renal tubular epithelial cells is one of the significant cause of chronic fibrosis.Acute rejection of renal allograft is diverse in histomorphology,mainly characterized by infiltration of inflammatory cells(immune cells),renal interstitial inflammation,tubulitis and arterial endometritis.Among them,renal interstitial inflammation and tubulitis are more common.Particularly,the severity of tubulitis can directly reflect the severity of acute rejection.In recent years,the possible correlationship between the infiltration macrophages and chronic fibrosis in renal allografts has attracted more and more attention.It has been reported that the more infiltrated macrophages in acute rejection,the worse status of allograft kidneywould be after 1,2 and 4 years,suggesting that macrophage infiltration may be closely related to chronic renal allograft fibrosis.Our previous work showed that rat renal tubular epithelial cell NRK52 E appeared significant decrease in the expression of epithelial markers and increase in mesenchymal markers when co-cultured with activated macrophages,i.e.,EMT;mass spectrometry analysis showed that the activated macrophages secreted a variety of cytokines,including transforming growth factor-?1,interleukin-6,complement C3,heat shock protein 90,matrix metalloproteinase 9,osteopontin and tumor necrosis factor-?.As a recognized fibrosis factor,TGF-?1 induces EMT of renal tubular cells,and thus participates in fibrosis.It is unclear whether other factors secreted by activated macrophages also have the effect to induce EMT of renal tubular cells and contribute to the process of renal fibrosis.In this study,we decided to determine whether IL-6 has the ability to induce phenotypic change and then EMT in NRK53 E cells in vitro.PurposeThe renal tubular epithelial cell line NRK52 E is used to detect the ability of IL-6 to induce phenotypic change of NRK52 E from epithelial phenotype to mesenchymal phenotype in vitro.MethodsChoice: E-cadherin(E-Ca)and cytokeratin-19(CK19)as markers of epithelial cells,alpha-smooth muscle actin(?-SMA)and Vimentin as mesenchymal cell markers.With the final IL-6 concentration of 25ng/ml,50ng/ml and 100ng/ml in medium DMEM,NRK52 E cells were cultured for 24 hours,48 hours and 72 hours,equal volume of IL-6 solvent as control,the morphological changes of NRK52 E cells of control group and induction groups were observed in different inducted time by inverted microscope.The Real-Time qPCR method was used to detect the expression of mRNA of epithelial cell and mesenchymal cell markers.Similarly,NRK52 E cells were cultured for 24,48 and 72 hours with the final concentration of 50ng/ml IL-6,and the protein expressions of epithelial makers and mesenchymal markers were detected by Western Blot method.Finally,NRK52 E cells were cultured for 72 hours with 50ng/ml IL-6,and immunocytochemistry and immunofluorescence staining were used to detect and verify the expression of epithelial markers and mesenchymal markers.ResultsMorphological observation: NRK52 E cells in the control group remained adherent,and showed a typical cobblestone paving stone-like,and close connection between cells during culturing process.After 24 hours,cells in the inducted groups showed short spindle-like,with increasing of IL-6 concentration,the spindle shaped were more obvious;48 and 72 hours later,cells were visible fusiform,suggesting that IL-6 can make NRK52 E cells undergo morphological change in vitro,from the typical cobblestone morphology of epithelial cells into short spindle and spindle,besides,with the increasing of IL-6 concentration and time,cells undergo spindle changes more obviously.Real-Time qPCR results: NRK52 E cells were cultured for 24 and 48 hours in different concentration of IL-6,compared with the control group,the epithelial markers of induced cells CK19 and E-Ca,expressions of mRNA had no significant difference,while mesenchymal markers ?-SMA and vimentin mRNA expressions in inducted groups after 24 hours,appeared the transient below control group,and after 48 hours,?-SMA,vimentin mRNA expressions rapidly returned to the control level and even appeared that mRNA expression of ?-SMA was significantly higher than that of the control in the concentration of 50ng/ml IL-6.When continued to be cultured after 72 hours,epithelial markers CK19,E-Ca mRNA expressions decreased significantly,while mesenchymal markers ?-SMA,vimentin mRNA expressions were significantly higher than control group,indicating that NRK52 E cells were cultured for 72 hours,the cells showed epithelial phenotype change d to mesenchymal phenotype.Western Blot Results: with the concentration of 50ng/ml IL-6 induced NRK52 E 24 hours and 48 hours in vitro,the protein expressions of epithelial markers,CK19 and E-Ca,and mesenchymal markers,vimentin had no obvious changes,but the protein expression of a-SMA displayed: after 24 hours,it appeared a significantly lower than control group,when cultured for 48 hours,it was quickly restored,even showed its expression was significantly higher than control group,the difference was statistically significant.After 72 hours,CK19 and E-Ca protein expressions were lower than control group,while the protein expressions of mesenchymal markers,?-SMA and vimentin were significantly increased.Western Blot results are consistent with the Real-time qPCR.Immunocytochemical staining showed that NRK52 E cells induced by 50ng/ml IL-6 for 72 h in vitro,the control cells saw the epithelial marker E-Ca strongly positive staining,clear linear distribution in the cell membrane,while mesenchymal marker vimentin was negative.In the inducted group,the staining of E-Ca was only weakly positive.The positive staining of vimentin was also found in the same time.Immunofluorescence staining results were consistent with the results of immunocytochemical staining,that is,after IL-6 inducing 72 hours,it showed the induced cells of epithelial marker E-Ca immunofluorescence staining was reduced,while there was a clear mesenchymal marker vimentin intensively positive staining.Immunocytochemistry and immunofluorescence staining further confirmed that after IL-6 inducing NRK52 E cells 72 hours,they appeared obvious phenotypic changes.In conclusion,with morphology,mRNA expression levels and protein expression levels,suggesting that IL-6 can induce NRK52 E cells in vitro from epithelial phenotype to mesenchymal phenotype or phenotypic change.ConclusionIL-6 can successfully induce renal tubule epithelial cell line NRK52 E cells from epithelial phenotype to mesenchymal phenotype or phentypic change in vitro.