Xanthine oxidase inhibitor ameliorates postischemic renal injury in mice by promoting resynthesis of adenine nucleotides
Kentaro Fujii, Akiko Kubo, Kazutoshi Miyashita, Masaaki Sato, Aika Hagiwara, Hiroyuki Inoue, Masaki Ryuzaki, Masanori Tamaki, Takako Hishiki, Noriyo Hayakawa, Yasuaki Kabe, Hiroshi Itoh, Makoto Suematsu
Kentaro Fujii, Akiko Kubo, Kazutoshi Miyashita, Masaaki Sato, Aika Hagiwara, Hiroyuki Inoue, Masaki Ryuzaki, Masanori Tamaki, Takako Hishiki, Noriyo Hayakawa, Yasuaki Kabe, Hiroshi Itoh, Makoto Suematsu
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Research Article Nephrology Therapeutics

Xanthine oxidase inhibitor ameliorates postischemic renal injury in mice by promoting resynthesis of adenine nucleotides

Abstract

Although oxidative stress plays central roles in postischemic renal injury, region-specific alterations in energy and redox metabolism caused by short-duration ischemia remain unknown. Imaging mass spectrometry enabled us to reveal spatial heterogeneity of energy and redox metabolites in the postischemic murine kidney. After 10-minute ischemia and 24-hour reperfusion (10mIR), in the cortex and outer stripes of the outer medulla, ATP substantially decreased, but not in the inner stripes of the outer medulla and inner medulla. 10mIR caused renal injury with elevation of fractional excretion of sodium, although histological damage by oxidative stress was limited. Ischemia-induced NADH elevation in the cortex indicated prolonged production of reactive oxygen species by xanthine oxidase (XOD). However, consumption of reduced glutathione after reperfusion suggested the amelioration of oxidative stress. An XOD inhibitor, febuxostat, which blocks the degradation pathway of adenine nucleotides, promoted ATP recovery and exerted renoprotective effects in the postischemic kidney. Because effects of febuxostat were canceled by silencing of the hypoxanthine phosphoribosyl transferase 1 gene in cultured tubular cells, mechanisms for the renoprotective effects appear to involve the purine salvage pathway, which uses hypoxanthine to resynthesize adenine nucleotides, including ATP. These findings suggest a novel therapeutic approach for acute ischemia/reperfusion renal injury with febuxostat through salvaging high-energy adenine nucleotides.

Authors

Kentaro Fujii, Akiko Kubo, Kazutoshi Miyashita, Masaaki Sato, Aika Hagiwara, Hiroyuki Inoue, Masaki Ryuzaki, Masanori Tamaki, Takako Hishiki, Noriyo Hayakawa, Yasuaki Kabe, Hiroshi Itoh, Makoto Suematsu

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Figure 8

Febuxostat promoted resynthesis of adenine nucleotides via the HPRT1-dependent purine salvage pathway and suppressed the renal injury marker genes.

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Febuxostat promoted resynthesis of adenine nucleotides via the HPRT1-dep...
(A) Scheme of the nucleoside salvage pathway. (B) Scheme of the present study. HK-2 cells were exposed to hypoxia (5% CO2, 1% O2) for 12 hours followed by 16 hours of reoxygenation (5% CO2, 21% O2). During hypoxia, the culture medium was replaced with DMEM containing no glucose (OGD). Transfection of shRNA against human HPRT1 and nontarget shRNA into HK-2 cells was performed 24 hours prior to OGD. (C) The contents of ATP were measured from cell lysates prepared after 16 hours of reoxygenation. Data are shown as relative values against nontarget shRNA, febuxostat-negative. (D) The contents of H2O2 were measured after 16 hours of reoxygenation. Data was shown as a relative value against nontarget shRNA, febuxostat-negative. (E) Gene expression of CASP3 and KIM-1 in HK-2 cells, which were exposed to OGD for 12 hours followed by 16 hours of reoxygenation and transfected with control shRNA. Data are shown as relative value against febuxostat-negative. (F) Gene expression of CASP3 and KIM-1 in HK-2 cells, which were exposed to OGD for 12 hours followed by 16 hours of reoxygenation and transfected with shRNA specific for the human HPRT1 gene. Data are shown as relative value against febuxostat-negative. **P < 0.01 febuxostat-negative vs. febuxostat-positive in control cells. #P < 0.05 febuxostat-negative vs. febuxostat-positive in HPRT1-knockdown (HPRT1 KD) cells. NS, not significant (P > 0.05). n = 16 for each group. Differences in mean values were statistically analyzed by Student’s 2-tailed t test.