Abstract: TH-PO423

Inhibition of Glycolysis Attenuates Ischemia-Reperfusion Injury via Metabolic and Redox Changes in Proximal Tubular Cells

Session Information

Category: Nutrition, Inflammation, and Metabolism

  • 1401 Nutrition, Inflammation, Metabolism

Authors

  • Shinohara, Akinari, The University of Tokyo School of Medicine, Tokyo, Japan
  • Kawakami, Takahisa, The University of Tokyo School of Medicine, Tokyo, Japan
  • Nangaku, Masaomi, The University of Tokyo School of Medicine, Tokyo, Japan
Background

Proximal tubular cells utilize fatty acid oxidation (FAO) more than glycolysis for ATP production in the physiological state. However, it remains largely unknown whether their metabolism affects pathophysiology of renal diseases. In this study, we investigated effects of glycolysis inhibition with 2DG, a representative glycolysis inhibitor, on murine renal ischemia-reperfusion injury (IRI), to which proximal tubular cell (PTC) injury by oxidative stress is central.

Methods

Eight-week-old male C57BL/6J mice were treated with 500 mg/kg 2DG or vehicle by i.p. 24 hours before bilateral IR, and renal injury was evaluated on day 1. We also examined effects of 2DG on PTCs, using HK-2 in vitro. HK-2 cells were treated with 5 mM 2DG for 6 hours and exposed to oxidative stress with 4 mM hydrogen peroxide. Cytotoxicity was measured with generation of reactive oxygen species (ROS) assessed by flow cytometry using dihydroethidium and LDH assay.

Results

Glycolysis inhibition by 2DG ameliorated renal dysfunction on day 1: serum creatinine was 1.4 ± 0.4 mg/dL in the vehicle group and 0.9 ± 0.1 mg/dL in the 2DG group. The reduced IRI was also demonstrated by a decrease in histological tubular injury score and mRNA expression of Kim-1 in the 2DG group. 2DG-preconditioned kidney cortex showed far less phospho-AMPK in immunoblot, indicating increased ATP in PTCs, which can suppress cell injury and death in IRI. We investigated its mechanism and found that 2DG increased free fatty acid in serum and PPARα expression in the kidney, suggesting that activated FAO promoted ATP production in PTCs. In vitro, treatment with 2DG suppressed generation of ROS and cell death. We focused on pentose phosphorylation pathway (PPP), because glycolysis inhibition can promote PPP as a bypass of glucose metabolism, and its key function is generation of reducing equivalents of NADPH. Indeed, PPP enzymes, including glucose-6-phosphate dehydrogenase and transketolase, were up-regulated, and NADPH/NADP ratio was increased by 2DG. We also found that glutathione peroxidases, key antioxidative enzymes, were induced by 2DG.

Conclusion

In conclusion, glycolysis inhibition ameliorated renal IRI via an ATP increase by enhanced FAO and a favorable redox state by promoted PPP in PTCs, implicating the importance of PTC metabolism in renal diseases.