| Renal tubulointerstitial fibrosis (TIF) is a common pathway of almost any advanced chronic renal diseases'progressing to end stage renal disease,including chronic allograft dysfunction. Emerging evidences suggest the fuctions of tubular epithelial myofibroblast transdifferentiation (TEMT) in tubulointerstitial fibrosis'development. Myofibroblast produced by EMT is considered the key factor in chronic renal fibrosis. Tubular epithelial cell can take place epithelial to mesenchymal transition (EMT) by many factors including injury, inflammation, growth factors and so on. TGF-β1 plays an important role in tubular epithelial myofibroblast transdifferentiation.Object: we observe the effect of TGF-β1 on EMT of tubular epithelial cell in vitro.It may be useful for the further investigation of the relation between EMT and chronic renal fibrosis.Methods: Human proximal tubular epithelial cell lines (HK-2) were divided into four groups: negative control, treated with TGF-β1 (1ng/ml), TGF-β1 (5ng/ml), and TGF-β1(10ng/ml) at different point 24h, 48h,and 72h. The proteins for pan-cytokeratin, E-cadherin,α-SMA, and S100A4 in HK-2 cells were measured by immunocytochemical staining.The mRNA for keratin-19, vimentin,α-SMA in HK-2 cells is measured by RT-PCR.Results:In our study, HK-2 cells cultured in different concentration of TGF-β1 lost the typical cobblestone pattern of an epithelial monolayer, and displayed a spindle-shape, fibroblast-like morphology.The longer HK-2 cells were induced by TGF-β1, the more spindle-shape cells appeared. Immunocytochemical staining showed:pan-cytokeratin ,an epithelial marker that was essential for the structural integrity of renal epithelium was positive .There were few changes of the expression of pan-cytokeratin during the whole 72 hours in TGF-β1 treated groups.The expression of E-cadherin which was a marker of HK-2 decreased in the periods of 48 hours in all TGF-β1 treated groups compared with non TGF-β1 treated group.Untill 72 hours HK-2 cells induced by TGF-β1 did not express E-cadherin.At the same time, HK-2 cells incubated in TGF-β1 expressedα-SMA-positive particles in the cytoplasm,especially in HK-2 cells treated by TGF-β1 at the dose of 5ng/ml .After 72 hours, more and moreα-SMA-positive particles were in HK-2 cells'cytoplasm .And there were no appearantly differences in the expression ofα-SMA-positive particles between all HK-2 cells induced by different doses of TGF-β1.The immunocytochemical staining result of S100A4 was similar toα-SMA .The expression of S100A4 in HK-2 cells with TGF-β1 treatment was stronger than in HK-2 cells without TGF-β1 treatment in 24 hours.And it was stronger after 48 hours and 72 hours in TGF-β1 treatment group.To make sure the results of pronteins'immunocytochemical staining,we used RT-PCR measured mRNA expression of keratin-19,vimentin, andα-SMA after HK-2 incubated with different doses of TGF-β1 for 48 hours .Our results showed:the mRNA expression of vimentin, andα-SMA in TGF-β1 treatment group was more than non TGF-β1 treatment group, and the mRNA expression of vimentin, andα-SMA in 5ng/ml TGF-β1 group was the most . The mRNA of keratin-19 in HK-2 cells with TGF-β1 treatment was also more than non TGF-β1 treatment group. The results of RT-PCR are compatible with the results of immunocytochemical staining. These datas illustrated that there was dose-dependent effect between TEMT and the concentration of TGF-β1.Conclusion: HK-2 induced by TGF-β1 may take place EMT in vitro. There was dose-dependent effect between TEMT and the concentration of TGF-β1.Although the mesechymal markerα-SMA and S100A4 appeared , some epithelial maker such as pan-cytokeratin in HK-2 cells still expressed.
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