14-3-3ζ-Mediated Stimulation of Oxidative Phosphorylation Exacerbates Oxidative Damage Under Hypothermic Oxygenated Conditions in Human Renal Tubular Cells (HK-2).
Cellular survival and death are at least partially regulated by the phosphorylation of proteins. A chaperon protein, 14-3-3ζ, regulates the activity of many proteins by covering the phosphorylation site within a 14-3-3 binding motif. Therefore, regulation of 14-3-3ζ activity may affect the fate of cells subjected to cold preservation and/or hypothermic oxygenated conditions. The present study assessed whether 14-3-3ζ protects cells from hypothermic oxygenation-induced injury and clarified its role in mitochondrial functions. Human renal tubular cell line HK-2 or 14-3-3ζ-overexpressed HK-2 (ζHK-2) cells were subjected to 72 hours of normoxic cold preservation in UW solution with or without antioxidants and hydroperoxides. Cellular death, adenosine triphosphate (ATP) content, and MTT catabolism were evaluated. Deferoxamine treatment reduced cellular death and augmented ATP content in both cell types. These indices were higher in ζHK-2, regardless of deferoxamine treatment. Exposure to hydroperoxides did not affect cellular death in either cell type, whereas hydroperoxide supplementation significantly reduced ATP content, except for low-dose hydrogen peroxide in HK-2 cells. MTT assay at normal state showed higher values in ζHK-2 cells, whereas it was impaired by hydroperoxides in both cell types. These results suggest that accumulation of hydroperoxides as a byproduct of the augmented oxidative phosphorylation by 14-3-3ζ overexpression causes mitochondrial dysfunction. In conclusion, despite possessing many potentially protective functions, 14-3-3ζ exacerbates cellular injury under hypothermic oxygenated conditions. 14-3-3ζ accelerates mitochondrial functions together with iron-dependent oxidative damage. Although further investigations are necessary, upregulation of 14-3-3ζ could be a method to maintain mitochondrial function under hypothermic oxygenated conditions, as shown in hypothermic machine preservation of renal grafts, when appropriate antioxidant treatment is administered.
Department of Transplant Surgery, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, Japan.
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Fukai
Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, Japan.
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Kobayashi
Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, Japan.
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Ishikawa
Department of Gastroenterological Surgery I, Hokkaido University Hospital, Sapporo, Hokkaido, Japan.
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Wakayama
Department of Gastroenterological Surgery I, Hokkaido University Hospital, Sapporo, Hokkaido, Japan. Electronic address: shingoshimada1979@true.ocn.ne.jp.
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Shimada
Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, Japan.
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Umemoto
Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, Japan.
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Ohtani
Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, Japan.
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Fujiyoshi
Department of Transplant Surgery, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, Japan.
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Yamashita
Division of Organ Transplantation, Central Clinical Facilities, Hokkaido University Hospital, Sapporo, Hokkaido, Japan.
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Shimamura
Department of Gastroenterological Surgery I, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, Japan.
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Taketomi
D048948:14-3-3 Proteins / Q000378:metabolism / Q000502:physiology*; D000241:Adenosine / Q000494:pharmacology; D000493:Allopurinol / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D002314:Cardioplegic Solutions / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods; D003676:Deferoxamine / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D008928:Mitochondria / Q000378:metabolism / Q000502:physiology; D009926:Organ Preservation / Q000379:methods; D019188:Organ Preservation Solutions / Q000494:pharmacology; D010085:Oxidative Phosphorylation; D018384:Oxidative Stress / Q000502:physiology; D011887:Raffinose / Q000494:pharmacology; D017262:Siderophores / Q000494:pharmacology
D048948:14-3-3 Proteins; D000975:Antioxidants; D002314:Cardioplegic Solutions; D007328:Insulin; D019188:Organ Preservation Solutions; D017262:Siderophores; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D005978:Glutathione; D003676:Deferoxamine; D000241:Adenosine; D011887:Raffinose