Effect Of IL-17 On Epithelial-Myofibroblast Transdifferentiation In HK-2 Cells In Vitro.

Objective:Renal interstitial fibrosis is the common pathological basis for various causes of renal diseases ultimately progressing to end-stage renal failure.It has reported that Tubular epithelial-mesenchymal transdifferentiation plays an important role in renal interstitial fibrosis,and interleukin-17 signaling has been implicated in some tissue fibrosis.The aim of this study was to investigate the effect of IL-17 on EMT,cell proliferation and secretion in HK-2 cells,providing new theoretical and experimental evidence for clinical strategies of anti-fibrosis.Methods:1.To determine the optimal stimulation of IL-17A,the time and dose-dependent experiments were performed.The HK-2 cells was cultured and stimulated by different concentrations of IL-17A(0,20,40,80,160,320 ng/ml)for different times(24,48,72h),then the cell proliferation and secretion of type Ⅰ collagen and type Ⅲ collagen were measured by CCK-8 assay or ELISA.2.To exam the effect of IL-17A on EMT,HK-2 cells were respectively stimulated with normal control group,IL-17A(80ng/ml),IL-17A(80ng/ml)+ TGF-β1 antibody(2μg/ml),TGF-β1(10ng/ml),TGF-β1 antibody(2μg/ml)for 72h,then the expression of E-cadherin,α-SMA,type I collagen and type Ⅲ collagen protein were measured by western blotting or immunohistochemistry,the expression of E-cadherin and α-SMA mRNA were measured by Real time RT-PCR.3.For the mechanisms of IL-17A on EMT in HK-2 cells,cells were stimulated respectively with normal control group,IL-17A(80ng/ml),IL-17A(80ng/ml)+ BMP-7(50ng/ml),TGF-β1(10ng/ml)+BMP-7(50ng/ml),TGF-β1(10ng/ml),then the expression of E-cadherin,α-SMA,type Ⅰ collagen and type Ⅲ collagen protein were measured by western blotting,the expression of a-SMA and Smad3 mRNA were measured by RT-PC.R.Results:1.①CCK-8 results suggested:the dose within the range of 20～80 ng/ml,IL-17A induced cell proliferation,however cell proliferation began to decrease with IL-17A over 160 ng/ml(P<0.05).The time-dependent test suggested that cell proliferation increased with the prolongation of IL-17A treatment(24h,48h,and 72h).②Results of cell culture supernatants by ELISA:Within dose range of 0～320ng/ml,IL-17A promoted the secretion of Col Ⅰ and Col Ⅲ in HK-2 cells.With the time of IL-17A treatment prolonged(24,48,72h),secretion of Col Ⅰand Col Ⅲ increased.2.Real time RT-PCR,Western blotting and Immunohistochemistry results suggested:IL-17A and TGF-β1 significantly decreased the expression of E-cadherin protein and mRNA(P<0.05),but increased the expression of α-SMA,Col Ⅰ and Col Ⅲ(P<0.05),compared with normal control group.The expression of E-cadherin significantly recovered(P<0.05)by TGF-β1 antibody antagonism,however the expression of positive EMT markers a-SMA,Col Ⅰ and Col Ⅲ significantly attenuated(P<0.05).3.①Western blotting results suggested:IL-17A and TGF-β1 could promote the expression of α-SMA,Col I and Col III(P<0.05)in HK-2 cells,and IL-17A performed better auxo-action.BMP-7 could suppress the expression of a-SMA,Col I and Col III by IL-17A or TGF-β1 induction in HK-2 cells(P<0.05).② The RT-PCR results suggested:IL-17A and TGF-β1 both could promote the expression ofα-SMA and Smad3 mRNA(P<0.05),however BMP-7 may attenuate the effect by IL-17A and TGF-β1(P<0.05).Conclusion:① IL-17A may promote the HK-2 proliferation and secretion of extracellular matrix on a time-and dose-dependent manner in vitro.②IL-17 may induce HK-2 cells’ EMT by a TGF-betal-dependent pathway.③BMP-7 could attenuate EMT induced by IL-17A and TGF-β1 through depressing Smad3 signaling pathway.