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1 Internal Medicine, University of Michigan, Ann Arbor, MI, USA; Internal Medicine, University Hospital Essen, Essen, Germany
2 Internal Medicine, University Hospital Essen, Essen, Germany
3 Internal Medicine, VA Ann Arbor Healthcare System, Ann Arbor, MI, USA; Internal Medicine, University of Michigan, Ann Arbor, MI, USA
* To whom correspondence should be addressed. E-mail: wnberg{at}umich.edu.
Proximal tubules develop a severe energetic deficit during hypoxia/reoxygenation (H/R) that previous studies using fluorescent potentiometric probes have suggested is characterized by sustained, partial mitochondrial de-energization. To validate the primary occurrence of mitochondrial de-energization in the process, optimize approaches for estimating changes of mitochondrial membrane potential (DeltaPsi(m)) in the system, and clarify the mechanisms for the defect, we further investigated the behavior of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazocarbocyanine iodide (JC-1) in these cells and introduce a more dynamic and quantitative approach employing safranine O for use with the tubule system. Although use of JC-1 can be complicated by decreases of the plasma membrane potential that limit cellular uptake of JC-1 and such behavior was demonstrated in ouabain-treated tubules, changes of DeltaPsi(m) entirely accounted for the decreases in formation of red fluorescent JC-1 aggregates and of the ratio of red/green fluorescence observed after H/R. The red JC-1 aggregates did not readily dissociate when tubules were de-energized after JC-1 uptake, making it unsuitable for dynamic studies of energization. Safranine O uptake by digitonin-permeabilized tubules required very small amounts of tubules, permitted measurements of DeltaPsi(m) for relatively prolonged periods after the end of the experimental maneuvers, was rapidly reversible during de-energization, and allowed for direct assessment of both substrate-dependent, electron transport-mediated DeltaPsi(m) and ATP hydrolysis-supported DeltaPsi(m). Both types of energization measured using safranine O in tubules permeabilized after H/R were impaired, but combining substrates and ATP substantially restored DeltaPsi(m).
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