Kidney proximal tubule cells developed severe energy deficits during hypoxia͞reoxygenation not attributable to cellular disruption, lack of purine precursors, the mitochondrial permeability transition, or loss of cytochrome c. Reoxygenated cells showed decreased respiration with complex I substrates, but minimal or no impairment with electron donors at complexes II and IV. This was accompanied by diminished mitochondrial membrane potential (⌬⌿ m). The energy deficit, respiratory inhibition, and loss of ⌬⌿m were strongly ameliorated by provision of ␣-ketoglutarate plus aspartate (␣KG͞ASP) supplements during either hypoxia or only during reoxygenation. Measurements of 13 C-labeled metabolites in [3-13 C]aspartate-treated cells indicated the operation of anaerobic pathways of ␣KG͞ASP metabolism to generate ATP, yielding succinate as end product. Anaerobic metabolism of ␣KG͞ASP also mitigated the loss of ⌬⌿ m that occurred during hypoxia before reoxygenation. Rotenone, but not antimycin or oligomycin, prevented this effect, indicating that electron transport in complex I, rather than F 1F0-ATPase activity, had been responsible for maintenance of ⌬⌿ m by the substrates. Thus, tubule cells subjected to hypoxia͞reoxygenation can have persistent energy deficits associated with complex I dysfunction for substantial periods of time before onset of the mitochondrial permeability transition and͞or loss of cytochrome c. The lesion can be prevented or reversed by citric acid cycle metabolites that anaerobically generate ATP by intramitochondrial substrate-level phosphorylation and maintain ⌬⌿ m via electron transport in complex I. Utilization of these anaerobic pathways of mitochondrial energy metabolism known to be present in other mammalian tissues may provide strategies to limit mitochondrial dysfunction and allow cellular repair before the onset of irreversible injury by ischemia or hypoxia. S tructural, biochemical, and functional abnormalities of mitochondria are widely believed to be important pathogenetic factors that underlie ischemic or hypoxic cell injury (1). Two defects recently have gained credence and captured attention. One is characterized by pore formation in the inner mitochondrial membrane, deenergization, and high-amplitude swelling (mitochondrial permeability transition or MPT) (1-4). The second involves leakage of cytochrome c from the intermembrane space into the cytosol (5). Cytochrome c leakage may follow the MPT or occur independently. There is general agreement that these dramatic alterations result in cell death by necrosis and apoptosis in diverse types of cell injury, including those caused by hypoxia and ischemia (1, 3-5). However, the proximate events that lead to the MPT and loss of cytochrome c are unclear and are subjects of ongoing investigation.We have reported that cells in freshly isolated kidney proximal tubules exhibit profound functional deficits of their mitochondria when they are reoxygenated after hypoxic incubation (6). The defect is characterized by failure of oxidative phosphor...

We have further examined the mechanisms for a severe mitochondrial energetic deficit, deenergization, and impaired respiration in complex I that develop in kidney proximal tubules during hypoxia-reoxygenation, and their prevention and reversal by supplementation with alpha-ketoglutarate (alpha-KG) + aspartate. The abnormalities preceded the mitochondrial permeability transition and cytochrome c loss. Anaerobic metabolism of alpha-KG + aspartate generated ATP and maintained mitochondrial membrane potential. Other citric-acid cycle intermediates that can promote anaerobic metabolism (malate and fumarate) were also effective singly or in combination with alpha-KG. Succinate, the end product of these anaerobic pathways that can bypass complex I, was not protective when provided only during hypoxia. However, during reoxygenation, succinate also rescued the tubules, and its benefit, like that of alpha-KG + malate, persisted after the extra substrate was withdrawn. Thus proximal tubules can be salvaged from hypoxia-reoxygenation mitochondrial injury by both anaerobic metabolism of citric-acid cycle intermediates and aerobic metabolism of succinate. These results bear on the understanding of a fundamental mode of mitochondrial dysfunction during tubule injury and on strategies to prevent and reverse it.