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Abstract: SA-PO571

PHD Inhibition Reduces Reactive Oxidative Stress in In Vitro Ischemia Model

Session Information

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Ito, Marie, the University of Tokyo graduate school of medicine, Tokyo, Bunkyo-ku, Japan
  • Tanaka, Tetsuhiro, the University of Tokyo School of Medicine, Tokyo, Japan
  • Wakashima, Takeshi, Central Pharmaceutical Research Institute, Japan Tobacco Inc., Osaka, Japan
  • Nangaku, Masaomi, the University of Tokyo School of Medicine, Tokyo, Japan
  • Fukui, Kenji, Central Pharmaceutical Research Institute, Japan Tobacco Inc., Osaka, Japan
Background

Hypoxia-inducible factor (HIF) is widely accepted to mediate the protection by hypoxic preconditioning in both in vitro and in vivo ischemia models but the underlying mechanism remains unknown. Prolyl hydroxylase domain proteins (PHDs) serve as main HIF regulator via hydroxylation of HIF-α leading to its degradation. Several PHD inhibitors, including enarodustat are now under clinical trials as treatment for renal anemia and the beneficial effect might go far beyond.

Methods

We investigated the role of PHD inhibition in oxygen-glucose deprived (OGD) model of human renal tubular cells (HK2) as an in vitro model of renal ischemia. Enarodustat or siRNA knockdown of PHD isoforms was used to study the impact of PHD inhibition on cytoprotection and ROS. Cell viability and ROS were measured using the Muse™ Count & Viability Assay Kit and Oxidative Stress Kit according to the manufacturer’s instructions. mRNAs for antioxidative genes (GPX1, catalase, SOD1, SOD2, HO-1 and NQO1) were quantified by real-time PCR. Specific effects of HIF isoforms and respective antioxidative genes were studied using siRNA knockdown series. The role of autophagy was evaluated with Atg7-KO cells.

Results

Enarodustat treatment and siRNA knockdown of PHD2, but not of PHD1 or PHD3, significantly increased cell viability and reduced reactive oxygen species (ROS) levels by HIF stabilization. These effects were offset by simultaneous knockdown of HIF-α isoforms. HIF-1α alone could confer protection in both viability and ROS levels, while the contribution of HIF2 was only evident in combination with HIF1, with a larger role in viability. As candidate mechanisms for ROS elimination, we examined the role of autophagy and antioxidants. Autophagy reduces ROS by degrading damaged mitochondria in several models. However, in our OGD model cell death and ROS were not increased in Atg7-KO cells. Next we measured the mRNA levels of antioxidants and among them superoxide dismutase 2 (SOD2) and heme oxygenase-1 (HO-1) are significantly increased in enarodustat-treated cells. While SOD2 knockdown had little effect, HO-1 knockdown partially abrogated the cytoprotection and ROS reduction by PHD inhibition.

Conclusion

HIF stabilization by PHD inhibition increased cell viability and decreased ROS levels, and HO-1 partially mediated the effect.