Effect Of Pyridoxamine And Telmisartan On Fibrosis Of Human Proximal Tubular Epithelial Cells

Objective The aim of this study was to explore the effect and mechanism of single or combined application of pyridoxamine and telmisartan on fibrosis of human proximal tubular epithelial cells (HK-2) induced by angiotensin Ⅱ (Ang Ⅱ) and their contribution to interstitial fibrosis of hypertensive renal injury.Methods Ang Ⅱ-induced HK-2cells were treated with pyridoxamine (0.1mmol/L, lmmol/L) and telmisartan (10"5mol/L) alternatively or in combination (1mmol/L pyridoxamine+10-5mol/L telmisartan), and diphenyleneiodonium chloride (DPI,10-5mol/L) as well (P0.1, PI, T, TP and DPI groups respectively). Cell proliferation was evaluated by3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis was detected by AnnexinV-FITC/PI double staining assay. Intracellular reactive oxygen species (ROS) was measured by flow cytometry. The real-time PCR was applied for estimating the mRNA expression of RAGE, TGF-β1, MCP-1and MMP-9. The RAGE, TGF-β1and CTGF protein expression were analyzed by Western Blot.Results1. Compared with the control group, Ang Ⅱ inhibited the proliferation of HK-2cells (P<0.01).The cell growth-inhibitory effect of Ang Ⅱ were reduced in T, P0.1, PI and TP group (all P<0.01). Among them, P0.1(P<0.05) and PI (P<0.01) group exhibited significant proliferative effects than T group, the proliferative effect of TP group was superior to T or PI group(P<0.01).2. Flow cytometry showed that Ang Ⅱ induced cell apoptosis in HK-2cells. Elevated total, early and advanced apoptosis rate were observed in Ang Ⅱ group compared with the control group (P<0.01). Compared with the Ang Ⅱ group, reduced total apoptosis rate were found in T, P0.1and P1groups respectively (P<0.01), but without significant differences among them(P>0.05). The effect of P0.1and P1groups were both superior to T group in reducing early cells apoptosis(both P<0.01).The cells early apoptosis rate of TP group was lower than T or P1group (P<0.01)3. Compared with the control group, Ang Ⅱ significantly enhanced intercellular ROS level (P<0.01).The intracellular ROS level were decreased in T, P0.1, P1, TP and DPI groups respectively (all P<0.01). The ROS level of P0.1and PI groups were lower than T group (both P<0.01), which is more lower in TP group(P<0.01).4.The RAGE, TGF-β1, MCP-1and MMP-9mRNA levels were up-regulated by Ang Ⅱ compared with the control group(P<0.01), which were all down-regulated by the intervention of T, P0.1, P1, TP and DPI respectively (all P<0.01).The mRNA levels of P0.1(P<0.05or P<0.01) and PI groups (P<0.01) were lower than T group while the levels of TP group were significantly lower than T or P1group(P<0.01).5. The RAGE, TGF-β1and CTGF protein expressions were up-regulated by Ang Ⅱ compared with the control group (P<0.01), which were all down-regulated by the intervention of T, P0.1, P1, TP and DPI respectively(all/><0.01).The protein expressions of P0.1and P1groups were lower than T group (P<0.05or P<0.01), the expressions of TP group were significantly lower than T or P1group(P<0.01).Conclusion Both pyridoxamine and telmisartan exhibit protective effects on alleviating interstitial fibrosis in HK-2cells by reducing cell growth-inhibitory effect of Ang II, inhibiting cell apoptosis, decreasing intracellular ROS levels and down-regulating inflammatory and fibrogenic factors expression. Combined use of pyridoxamine and telmisartan is superior to the single application of either one. The effects may be related to oxidative stress alleviation and AGEs-RAGE inhibition.