Measuring m in isolated tubules

ABOUT 30% OF INTRACELLULAR volume is made up of mitochondria, particularly in cells demanding higher energy such as cardiac myocytes and renal proximal tubules, where they serve as important “powerhouses” (3, 6). Mitochondria are integral to the maintenance of normal cellular physiology and play a central role in the processes involved in apoptosis (4–6). Changes in mitochondrial function contribute to the development of apoptosis (5). Hence, this organelle has been the focus of numerous investigators, particularly in research to improve techniques to study early changes in mitochondrial function in the cell biology of apoptosis. Assessment of mitochondrial membrane potential ( m) represents one such index of mitochondrial function and a key process controlling the ultimate fate of a cell. The proton motive force across the inner mitochondrial membrane that drives oxidative phosphorylation is generated by the active pumping of protons from the mitochondrial matrix to the intermembrane space by the electron transport chain and is composed of the pH gradient ( 10%) and the membrane potential (90%). Early studies focused on measurement of m for an understanding of the mechanism of oxidative phosphorylation and mitochondrial ion transport and was calculated from the distribution of K or Rb across the mitochondrial membrane in the presence of the ionophore valinomycin (9). This method had the drawback of requiring an artificial system, prompting several investigators to develop spectroscopic or fluorescent probes sensitive to changes in m. Several probes have since been developed to monitor changes in m, including 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolylcarbocyanine iodide (JC-1), rhodamine 123, safranin O, as well as others (8). The most common probe used to measure changes in m is the cationic dye JC-1. The basis of this technique is that accumulation of JC-1 in mitochondria is dependent on membrane potential, wherein low m causes the dye to remain in a monomeric form and exhibit a green color with very low amounts in the mitochondria. On the other hand, high m results in a greater accumulation in the mitochondria, where the dye forms J-aggregates and shifts the fluorescence to red (10, 12). The use of JC-1 for measuring m has some limitations. For example, the red aggregates of JC-1 do not readily dissociate during deenergization after JC-1 uptake, rendering it unsuitable for dynamic studies to evaluate tubular cell energization (2). In addition, changes in JC-1 can be affected by plasma membrane potential ( p), which limits uptake of JC-1 by the cell (11). The lack of ATP will decrease Na pump activity and would thereby reduce p and consequently decrease mitochondrial uptake independently of changes in m (13, 14). Furthermore, the relationship between changes in JC-1 fluorescence and m is not linear (11). Another caveat is the heterogeneity in membrane potential across mitochondria within cells as well as regional heterogeneity within a single mitochondrion (12). However, this is potentially relevant to most probes used for measuring m. In this issue, Feldkamp and colleagues (2) have investigated the basis for mitochondrial deenergization observed in freshly isolated renal proximal tubules during hypoxia-reoxygenation injury. Tubules subjected to hypoxia-reoxygenation show impaired recovery of ATP levels and mitochondrial energization that is improved by the addition of intermediates from the citric acid cycle during reoxygenation (15). The studies by Feldkamp and colleagues (2) have validated the use of JC-1 as a marker for mitochondrial deenergization in tubules after hypoxiareoxygenation and show that plasma membrane uptake of the dye is not a major limiting factor for mitochondrial signal. Comparing changes in JC-1 and safranin O as measures for m, they demonstrate the superior efficacy of safranin O as a probe in isolated renal tubules. The technique using safranin O required very small numbers of tubules, permitted measurements of m for relatively prolonged periods, and was rapidly reversible during mitochondrial deenergization. Furthermore, safranin O allowed for direct assessment of both substrate-dependent, electron transport-mediated m and ATP hydrolysis-supported m. The technique was more versatile, provided quantitative assessment, and was dynamic, allowing assessment of repeated cycles of mitochondrial energization, deenergization, and reenergization. Safranin belongs to a family of dyes based on phenazine, used in the textile industry and as a biological stain for cartilage, mucin, and mast cell granules on tissues. Akerman and Wikstrom (1) first used safranin, a positively charged dye, to estimate m because it underwent large spectral shifts in energized mitochondria. These workers showed that the induction of a diffusion potential of potassium or hydrogen ions across the membrane of isolated rat liver mitochondria gave rise to a spectral shift in safranin due to stacking, which was identical to that observed on energization of the mitochondria by respiration or ATP hydrolysis and suggested that safranin could be a useful probe for changes in m. Freshly isolated renal proximal tubules serve as a good “ex vivo” model for ischemic acute renal failure (13) and have provided important insights into the cell biology of renal injury. Successful application of probes for the measurement of m in such model systems, as reported in the article by Feldkamp and colleagues (2), will provide critical and pertinent information for the study of mitochondrial function in the pathophysiology of renal tubular injury after hypoxia-reoxygenation. The studies are also broadly applicable to studies of mitochondrial function and apoptosis in multiple models utilizing kidney and other cells (7).