Effect of Bilirubin on the Expression of Hypoxia-induced factor 1α (HIF-1α) in Renal Proximal Tubular Cell Line

Abstract

Introduction: Renal dysfunction has a common process of tubular cell death and tubulointerstitial inflammation followed by fibrosis, whereas initiation of such dysfunction is likely to have multi-hit characteristics. Renal tubulointerstitial hypoxia causes fibrogenesis and also stimulates infiltration and maturation of immune cells to generate inflammation. Hypoxia-inducible factor (HIF) is influenced by the level of reactive oxygen species (ROS) and plays an integral role in the body's response to hypoxia. Bilirubin has been reported to be an effective antioxidant, and has the beneficial effects on renal diseases. However, the exact mechanisms controlling HIF expression under hypoxia are not resolved. The aim of this study is to elucidate the effect of bilirubin on HIF-1 expression under hypoxia and the possible mechanism of HIF-1 regulation by bilirubin in human proximal tubular cells in vitro. Methods: The human proximal tubular kidney (HK2) cells treated with actinomycin D were cultured for 15 h in 5% oxygen with or without bilirubin. HIF-1α messenger RNA (mRNA) and protein expression were measured in the cells cultured with 0.1 mg/dL bilirubin and compared to the control cells. Next, in order to elucidate the molecular mechanism of bilirubin on HIF system, the changes of ROS and HIF system were measured after treating the cell with the inhibitors of PI3K/mTOR, PI3K/AKT, and ERK 1/2 pathways which affect the transcription and translation of HIF-1α expression. Then, HIF-1α expression and phosphorylation of P70S6 kinase were measured after treating the cell with 10μM exogenous hydrogen peroxide (H2O2) to investigate the effect of NADPH oxydase system which is another major source of ROS generation in mitochondria possibly contributing to HIF system. Finally, NOX4 gene, which is also known to affect the expression of HIF system, was blocked by small interfering RNA (siRNA) and then HIF-1α mRNA expression was evaluated. Results: The messenger RNA (mRNA) expression of HIF-1α that was suppressed by actinomycin D was increased by 1.69 ± 0.05 folds in the cells cultured with 0.1 mg/dL bilirubin, compared to the control cells. The inhibitor (miltefosin) of PI3K/AKT pathway attenuated HIF-1α expression increased by bilirubin whereas the inhibitors of PI3K/mTOR and ERK 1/2 pathways did not. HIF-1α expression was decreased by 10μM exogenous 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. Conclusions: Bilirubin enhanced HIF-1α mRNA transcription as well as the upregulation of HIF-1α protein translation through 1) inhibition of oxidaive stress, at least partly by downregulating subunits of NADPH oxidase and 2) activation of PI3K/AKT pathway in HK2 cells cultured under hypoxia. These findings suggest that bilirubin could be a cytoprotective molecule with antioxidative properties in renal tubular injury. * This work is published in J Korean Med Sci. Sep 2014

29(Suppl 2): S146?S154.