Anaerobic and aerobic pathways for salvage of proximal tubules from hypoxia-induced mitochondrial injury

Abstract

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also rescued the tubules, and its benefit, like that of alpha-KG + malate, persisted after the extra substrate was withdrawn.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.

Energetic Determinants of Tyrosine Phosphorylation of Focal Adhesion Proteins during Hypoxia/Reoxygenation of Kidney Proximal Tubules

“…For staining with the carbocyanine dye, 5,5Ј,6,6Ј-tetrachloro-1,1Ј,3,3Ј-tetraethylbenzimidazocarbocyanine iodide (JC-1; Molecular Probes, Eugene, OR), 34,35,39 an aliquot from a 1000ϫ stock solution in dimethyl sulfoxide was mixed with an equal volume of serum, dispersed as an intermediate 100ϫ stock solution in phosphate-buff-ered saline (PBS), and then added to a final concentration of 5 g/ml in the tubule suspension at the end of the desired experimental period. The suspension was regassed with O 2 /CO 2 and incubated in the dark for an additional 15 minutes at 37°C, then tubules were washed three times in an ice-cold solution containing (in mmol/L): 110 NaCl, 25 NaHEPES, pH 7.2, 1.25 CaCl 2 , 1.0 MgCl 2 , 1.0 KH 2 PO 4 , 3.5 KCl, 5.0 glycine, and 5% polyethylene glycol (average molecular weight 8000).…”

“…Similar degrees of recovery could be documented in tubules that had been provided with anaerobic mitochondrial substrates only during reoxygenation. 34,35 These observations suggest that the low levels of additional ATP generation during hypoxia or early reoxygenation made possible by the substrates were sufficient for utilization to maintain or repair structure by cells that were otherwise committed to continuing ATP depletion and a lethal outcome. 35 In the present studies we have assessed the impact of the tubule energetic deficit and its modification by protective substrates on the alterations of cytoskeletal and focal adhesion protein distribution and tyrosine phosphorylation during hypoxia/reoxygenation.…”

“…30 -32 We have found that, despite glycine cytoprotection, freshly isolated, kidney proximal tubule cells develop a profound mitochondrial functional deficit during hypoxia/reoxygenation that is characterized by incomplete recovery of energization during reoxygenation, impaired respiration for substrates dependent on function of electron transport complex I, partial de-energization, and persistence of mitochondrial matrix condensation. [33][34][35] The mitochondrial lesion can be substantially ameliorated and recovery of cell ATP strikingly enhanced by supplementing the tubules with ␣-ketoglutarate plus aspartate (␣KG/ASP), ␣-ketoglutarate plus malate (␣KG/MAL), or other specific citric acid cycle metabolites during either hypoxia or reoxygenation. 34,35 The substrates promote mitochondrial pathways of anaerobic metabolism to increase ATP production by substrate level phosphorylation and energization by anaerobic respiration in electron transport complexes I and II 34,35 and provide succinate to bypass the complex I block when aerobic metabolism resumes.…”

“…[33][34][35] The mitochondrial lesion can be substantially ameliorated and recovery of cell ATP strikingly enhanced by supplementing the tubules with ␣-ketoglutarate plus aspartate (␣KG/ASP), ␣-ketoglutarate plus malate (␣KG/MAL), or other specific citric acid cycle metabolites during either hypoxia or reoxygenation. 34,35 The substrates promote mitochondrial pathways of anaerobic metabolism to increase ATP production by substrate level phosphorylation and energization by anaerobic respiration in electron transport complexes I and II 34,35 and provide succinate to bypass the complex I block when aerobic metabolism resumes. 35 Two related observations in these initial studies 34,35 spurred us to further investigate the relationships between anaerobic ATP generated in mitochondria and its utilization by cells to effect repair and survival.…”

“…34,35 The substrates promote mitochondrial pathways of anaerobic metabolism to increase ATP production by substrate level phosphorylation and energization by anaerobic respiration in electron transport complexes I and II 34,35 and provide succinate to bypass the complex I block when aerobic metabolism resumes. 35 Two related observations in these initial studies 34,35 spurred us to further investigate the relationships between anaerobic ATP generated in mitochondria and its utilization by cells to effect repair and survival. First, we were struck by the relatively small yields of energy by anaerobic metabolism of mitochondrial substrates in proximal tubules as assessed by absolute levels of ATP attained during hypoxia, ie, increases from 4.3% of oxygenated control levels in tubules without protective substrates at the end of 60 minutes hypoxia to 6.9% with the substrates.…”