The effect of tubular epithelial cells activated by aldosterone on renal interstitial fibroblasts in co-culture system

Abstract

Local aldosterone (ALDO) could be synthesized by human proximal tubular epithelial cell lines (HKC) after the stimulation of endothelin-1 (ET-1) in vitro. T to observe the effect of tubular epithelial cells activated by aldosterone (ALDO) on renal interstitial fibroblasts in co-culture system. (1) Human Proximal tubular epithelial cells of the line HKC were stimulated with ALDO at different concentrations and times, then reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were performed to detect TGF-beta1 expression; (2) HKC were co-stimulated with ET-1 (10(-9) mol/L) and spironolactone at different concentrations and times to evaluate the influence of endogenous ALDO on TGF-beta1 expression; (3) HKC which were activated by 10(-7) mol/L ALDO for 12 h, and hRIFs were co-cultured for 48 h with or without anti-TGF-beta1 antibody (1.0, 2.0 microg/ml) in the media, then the production of type I collagen (Col-I) in the cell layer of hRIFs was detected by ELISA. (1) After stimulation with ALDO, the expression of TGF-beta1 by HKC was up-regulated in a dose-and time-dependent manner. With 10(-9) or 10(-7) mol/L ALDO stimulation (mRNA determination at 12 h and protein at 48 h), the expression of TGF-beta1 was significantly increased (vs 0 mol/L ALDO, P < 0.05 or 0.01). With 10(-7) mol/L ALDO stimulated at different times (mRNA determination at 8 h, 12 h and 16 h and protein at 12 h, 24 h and 48 h), the expression of TGF-beta1 was also significantly increased (vs 0 h, P < 0.05 or 0.01). (2) After co-stimulation with ET-1 and spironolatone, the expression of TGF-beta1 by HKC was down-regulated in a dose-and-time dependent manner along with spironolatone. The expression of TGF-beta1 mRNA and protein was decreased in the 10(-9) or 10(-7) mol/L spironolatone groups compared with 0 mol/L group (P < 0.05 or 0.01). The expression of TGF-beta1 (mRNA and protein) was significantly decreased in 10(-9) mol/L ET-1 and 10(-7) mol/L spironolatone co-stimulation group compared with 10(-9) mol/L ET-1 stimulated group. (3) The production of Col-I by hRIFs which were co-cultured with activated HKC by ALDO, was significantly increased (vs control group in which hRIFs co-cultured with normal HKC, P < 0.01), and this effect was partially inhibited by 1.0 or 2.0 microg/ml anti-TGF-beta1 antibody (P < 0.05). The expression of TGF-beta1 in HKC cells can not only be up-regulated by exogenous ALDO, but also can be up-regulated by endogenous ALDO in an autocrine manner. The HKC cells activated by ALDO can promote the synthesis of Col-I in hRIFs by a "cross talking" way, and this effect is partially mediated by TGF-beta1.