5-5--6-6--tetrachloro-1-1--3-3--tetraethylbenzimidazolocarbocyanine and malic-acid

The mitochondrial membrane potential measured in isolated rat kidney mitochondria and in digitonin-permeabilized MDCK type II cells pre-energized with succinate, glutamate, and/or malate was reduced by micromolar diclofenac dose-dependently. However, ATP biosynthesis from glutamate/malate was significantly more compromised compared to that from succinate. Inhibition of the malate-aspartate shuttle by diclofenac with a resultant decrease in the ability of mitochondria to generate NAD(P)H was demonstrated. Diclofenac however had no effect on the activities of NADH dehydrogenase, glutamate dehydrogenase, and malate dehydrogenase. In conclusion, decreased NAD(P)H production due to an inhibition of the entry of malate and glutamate via the malate-aspartate shuttle explained the more pronounced decreased rate of ATP biosynthesis from glutamate and malate by diclofenac. This drug, therefore affects the bioavailability of two major respiratory complex I substrates which would normally contribute substantially to supplying the reducing equivalents for mitochondrial electron transport for generation of ATP in the renal cell.

Topics: Acute Kidney Injury; Adenosine Triphosphate; Animals; Aspartic Acid; Benzimidazoles; Carbocyanines; Cells, Cultured; Diclofenac; Dogs; Glutamate Dehydrogenase; Kidney; Malate Dehydrogenase; Malates; Membrane Potentials; Mitochondria; Mitochondrial Membranes; NADH Dehydrogenase; Oligomycins; Rats

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also rescued the tubules, and its benefit, like that of alpha-KG + malate, persisted after the extra substrate was withdrawn.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

Topics: Adenosine Triphosphate; Aerobiosis; Anaerobiosis; Animals; Aspartic Acid; Benzimidazoles; Carbocyanines; Cell Hypoxia; Citric Acid Cycle; Energy Metabolism; Female; Fluorescent Dyes; Fumarates; Ketoglutaric Acids; Kidney Tubules, Proximal; Malates; Membrane Potentials; Mitochondria; Oxygen; Rabbits; Rhodamines; Substrate Specificity