Preservation of complex I function during hypoxia-reoxygenation-induced mitochondrial injury in proximal tubules

Abstract

Inhibition of complex I has been considered to be an important contributor to mitochondrial dysfunction in tissues subjected to ischemia-reperfusion. We have investigated the role of complex I in a severe energetic deficit that develops in kidney proximal tubules subjected to hypoxia-reoxygenation and is strongly ameliorated by supplementation with specific citric acid cycle metabolites, including succinate and the combination of -ketoglutarate plus malate. NADH: ubiquinone reductase activity in the tubules was decreased by only 26% during 60-min hypoxia and did not change further during 60-min reoxygenation. During titration of complex I activity with rotenone, progressive reduction of NAD+ to NADH was detected at >20% complex I inhibition, but substantial decreases in ATP levels and mitochondrial membrane potential did not occur until >70% inhibition. NAD+ was reduced to NADH during hypoxia, but the NADH formed was fully reoxidized during reoxygenation, consistent with the conclusion that complex I function was not limiting for recovery. Extensive degradation of cytosolic and mitochondrial NAD(H) pools occurred during either hypoxia or severe electron transport inhibition by rotenone, with patterns of metabolite accumulation consistent with catabolism by both NAD+ glycohydrolase and pyrophosphatase. This degradation was strongly blocked by alpha-ketoglutarate plus malate. The data demonstrate surprisingly little sensitivity of these cells to inhibition of complex I and high levels of resistance to development of complex I dysfunction during hypoxia-reoxygenation and indicate that events upstream of complex I are important for the energetic deficit. The work provides new insight into fundamental aspects of mitochondrial pathophysiology in proximal tubules during acute renal failure.

F 1 F O-ATPase Activity and ATP Dependence of Mitochondrial Energization in Proximal Tubules after Hypoxia/Reoxygenation

“…Proximal tubules were prepared from kidney cortex of female New Zealand white rabbits by collagenase digestion and centrifugation on self-forming Percoll gradients as described (1)(2)(3)(4)11).…”

“…The energetic deficit occurs despite preserved electron transport (4) and minimal cytochrome c release (2). ATP recovery can be substantially improved by supplementing tubules with citric acid cycle metabolites such as ␣-ketoglutarate and malate individually and in combination (1)(2)(3).…”

“…Mitochondrial membrane potential (⌬⌿ m ), as assessed by 5,5Ј,6,6Ј-tetrachloro-1,1Ј,3,3Ј-tetraethylbenzimidazocarbocyanine iodide (JC-1) uptake is consistently decreased, but not absent, in affected tubules and is restored by the protective citric acid cycle metabolites (1,2,4,5). However, the nonlinearity of formation of JC-1 red aggregates relative to ⌬⌿ m and their slow and incomplete dissociation back to monomers during de-energization limits the use of JC-1 to dynamically follow and manipulate the changes of ⌬⌿ m to gain more mechanistic information about the factors contributing to them (5).…”

Insight into SUCNR1 (GPR91) structure and function

“…Succinate is a citric acid cycle intermediate that accumulates in mitochondria, cytosol and outside the cell in case of oxygen deprivation due to the imbalance between energy demand and oxygen supply (Feldkamp et al, 2004;Hébert, 2004;Peti-Peterdi et al, 2013;Toma et al, 2008). It activates SUCNR1 signaling pathways for the detection of local stress, including ischemia, hypoxia, toxicity, and hyperglycemia.…”

Orphan detectors of metabolism

“…TCA-cycle intermediates such as Ȋ-ketoglutarate and succinate have also been shown to reduce the mitochondrial injury,in kidneys and other tissues, that is caused by successive decreases and increases in oxygen concentrations 12 ; such intermediates are also used in kidney (and liver) preservation solutions for organ transplantation 13 . What role, if any, might the TCA receptors have here?…”