Effect of pro-inflammatory interleukin-17A on epithelial cell phenotype inversion in HK-2 cells in vitro

Abstract

Renal interstitial fibrosis (RIF) is a pathological change common to a variety of chronic renal diseases, ultimately progressing to end-stage renal failure. It is believed that epithelial cell phenotype inversion plays an important role in RIF, which is characterized by expression of the mesenchymal maker α-SMA, loss of the epithelial maker E-cadherin, and enhanced secretion of extracellular matrix. IL-17, a newly discovered pro-inflammatory cytokine, has recently been reported to play an important role in tissue fibrosis, involving pulmonary, liver, intestine and skin tissues. This study aimed to investigate whether IL-17A, a member of the IL-17 family, can induce epithelial cell phenotype inversion, and to explore the molecular mechanism of this phenotype inversion, in vitro. HK-2 cells were cultured and incubated with IL-17A. Cell proliferation was measured by CCK-8 assay, and the secretion of types I and III collagen was detected by ELISA in dose-dependent and time-dependent experiments. To find out whether IL-17A can induce epithelial cell phenotype inversion, HK-2 cells were stimulated with 80 ng/mL of IL-17A and 10 ng/mL of TGF-β1 as a positive control, for 72 h. To explore the potential signaling pathway, anti-TGF-β1 antibody was added before IL-17A treatment. At the same time, anti-TGF-β1 antibody alone was added to the medium as the negative control group. The expression of types I and III collagen, α-SMA and E-cadherin proteins, and mRNA was measured by real-time PCR, western blotting and immuno-histochemistry. IL-17A promoted the proliferation of HK-2 cells and secretion of types I and III collagen in a dose-dependent and time-dependent manner. Compared with the normal control, IL-17A could stimulate the expression of α-SMA, types I and III collagen, and suppressed the expression of E-cadherin in HK-2 cells. Incubation of IL-17A with TGF-β1 antibody decreased significantly the expression of α-SMA, but increased the expression of E-cadherin in HK-2 cells. Our results suggest that IL-17A might promote the proliferation of HK-2 cells and secretion of extracellular matrix, and induce epithelial cell phenotype inversion via a TGF-β1-dependent pathway. Blocking the pro-inflammatory cytokine IL-17A might be a potential target for the treatment of fibrotic kidney disease.