Bilirubin activates transcription of HIF-1α in human proximal tubular cells cultured in the physiologic oxygen content

Abstract

The expression of hypoxia-inducible factor (HIF) is influenced by reactive oxygen species (ROS). Effect of bilirubin on HIF-1 expression in proximal tubular cells was investigated under physiological oxygen concentration, which is relative hypoxic condition mimicking oxygen content in the medulla of renal tissue. The human kidney (HK2) cells were cultured in 5% oxygen with or without bilirubin. HIF-1α protein expression was increased by bilirubin treatment at 0.01-0.2 mg/dL concentration. The messenger RNA expression of HIF-1α was increased by 1.69±0.05 folds in the cells cultured with 0.1 mg/dL bilirubin, compared to the control cells. The inhibitors of PI3K/mTOR, PI3K/AKT, and ERK 1/2 pathways did not attenuate increased HIF-1α expression by bilirubin. HIF-1α expression decreased by 10 µM exogenous hydrogen peroxide (H2O2); scavenger of ROS with or without bilirubin in the HK2 cells increased HIF-1α concentration more than that in the cells without bilirubin. Exogenous H2O2 decreased the phosphorylation of P70S6 kinase, which was completely reversed by bilirubin treatment. Knockdown of NOX4 gene by small interfering RNA (siRNA) increased HIF-1α mRNA expression. In coonclusion, bilirubin enhances HIF-1α transcription as well as the up-regulation of HIF-1α protein translation through the attenuation of ROS and subunits of NADPH oxidase.

Keywords: Bilirubin; HIF-1; Proximal Tubular Cell; ROS.

Fig. 1

Bilirubin increased HIF-1α protein expression. (A-D) Western blotting and relative ratio of HIF-1α protein to actin normalized to control. The vertical bar indicates 95% CI of the mean value. ^*P < 0.001, ^†P = 0.025, ^‡P = 0.020, ^§P = 0.015 as compared to Con. Con, control samples of human HK2 cells cultured under 5% oxygen condition for 1 hr.

Fig. 2

Bilirubin effect on the expression of HIF-1α protein and mRNA. (A) Western blotting with the proteins from cells cultured with (0.1 mg/dL) or without bilirubin or actinomycin-D (0.15 µg/mL before bilirubin treatment). (B) The relative ratio of HIF-1α protein to actin normalized to control in western blotting. (C) RT-PCR for mRNA expression of HIF-1α, GLUT-1, and actin. (D) The relative ratio of HIF-1α mRNA to actin normalized to control in RT-PCR. (E) The relative ratio of GLUT-1 mRNA to actin normalized to control in RT-PCR. The vertical bar indicates 95% CI of the mean value. ^*P = 0.008 compared to Con; ^†P = 0.001 compared to Bil. BiI, Bilirubin 0.1 mg/dL was added to culture media with each indicated concentration for 1 hr; Bil+AD, Actinomycin-D (0.15 µg/mL) was added 15 min before bilirubin treatment; Con, Control samples of human HK2 cells cultured under 5% oxygen condition for 1 hr; N, Control samples of human HK2 cells cultured under 21% oxygen condition for 1 hr.

Fig. 3

Bilirubin effect on the expression of HIF-1α protein in presence of inhibitors for various pathways. (A) Western blotting with the proteins from cells cultured with (0.1 mg/dL) or without bilirubin and with wortmannin (100 nM), Ly294002 (20 µM), rapamycin (10 nM), or miltefosine (5 µM). (B) The relative ratio of HIF-1α protein to actin normalized to control in western blotting. The vertical bar indicates 95% CI of the mean value. ^*P < 0.001, ^†P = 0.001, ^‡P = 0.041 compared to Con. There is no difference in protein expression between cells treated with bilirubin and cells treatment with bilirubin and Ly294002 (P = 0.060), wortmannin, rapamycin, or miltefosine (P = 0.138). BiI, Bilirubin (0.1 mg/dL) was added to culture media with each indicated concentration for 1 hr; Con, Control samples of human HK2 cells cultured under 5% oxygen condition for 1 hr; Wortmannin, Ly294002, rapamycin, or miltefosine was added 15 min before bilirubin treatment. Wm, wortmannin; Ly, Ly294002; Rm, rapamycin; Mf, miltefosine.

Fig. 4

The effect of H₂O₂ on proteins expressions. (A) H₂O₂ production determined using H2DCF-DA. (B, C, E) Western blotting with the proteins from cells cultured with (10 µM) or without H₂O₂, bilirubin (0.1 mg/dL), catalase (1,000 U/mL), or DPI (10 µM). (D) The relative ratio of HIF-1α protein to actin normalized to control in western blotting. (F) The relative ratios of phospho-P70S6K to P70S6K and phospho-ERK1/2 to ERK1/2 protein normalized to control in western blotting. The vertical bar indicates 95% CI of the mean value. ^*,†P < 0.001 compared to N, ^‡P = 0.017 compared to Con, ^§,∥P < 0.001 compared to Con, ^¶P < 0.001 compared to H₂O₂, ^**P = 0.003 and ^††P = 0.209 compared to Con, ^‡‡P = 0.014 compared to H₂O₂. Ca, catalase; DPI, diphenyleneiodonium. Human HK2 cells cultured under 5% oxygen condition for 1 hr.

Fig. 5

The effect of bilirubin on the expression of NADPH oxidase subunits. (A) Western blotting with the proteins from cells cultured with (0.1 mg/dL) or without bilirubin. (B-D) The relative ratio of proteins of NADPH oxidase subunits to actin normalized to control in western blotting. (E) RT-PCR from cells cultured with (0.1 mg/dL) or without bilirubin. (F-H) The relative ratios of mRNAs of NADPH oxidase subunits to actin normalized to control in RT-PCR. Human HK2 cells cultured under 5% oxygen condition for 1 hr. The vertical bar indicates 95% CI of the mean value.

Fig. 6

The effect of siRNA Nox4 on the expression of HIF-1α. (A) RT-PCR from cells cultured with (0.1 mg/dL) or without bilirubin and siRNA Nox4. (B) The relative ratios of mRNAs of HIF-1α to actin normalized to control in RT-PCR. Human HK2 cells cultured under 5% oxygen condition for 1 hr. The vertical bar indicates 95% CI of the mean value. ^*P < 0.001 compared to Con, ^†P = 0.024 compared to Con, ^‡P = 0.008 compared to Bil.

Fig. 7

The apoptotic signals according to bilirubin treatment. (A) Western blotting from cells cultured with (0.1 mg/dL) or without bilirubin. (B) The relative ratios of proteins normalized to control in western blotting. Human HK2 cells cultured under 5% oxygen condition for 1 hr. The vertical bar indicates 95% CI of the mean value.