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Am J Physiol Renal Physiol 288: F1092-F1102, 2005. First published December 29, 2004; doi:10.1152/ajprenal.00443.2004
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EDITORIAL FOCUS

Assessment of mitochondrial membrane potential in proximal tubules after hypoxia-reoxygenation

Thorsten Feldkamp,1,2 Andreas Kribben,2 and Joel M. Weinberg1

1Division of Nephrology, Department of Internal Medicine, Veterans Affairs Ann Arbor Healthcare System and University of Michigan, Ann Arbor, Michigan; and 2Division of Nephrology and Hypertension, Department of Internal Medicine, University Hospital Essen, Essen, Germany

Submitted 9 December 2004 ; accepted in final form 25 December 2004

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 deenergization. To validate the primary occurrence of mitochondrial deenergization in the process, optimize approaches for estimating changes in mitochondrial membrane potential ({Delta}{Psi}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 safranin O for use with the tubule system. Although use of JC-1 can be complicated by decreases in the plasma membrane potential that limit cellular uptake of JC-1 and such behavior was demonstrated in ouabain-treated tubules, changes in {Delta}{Psi}m entirely accounted for the decreases in the formation of red fluorescent JC-1 aggregates and in the ratio of red/green fluorescence observed after H/R. The red JC-1 aggregates did not readily dissociate when tubules were deenergized after JC-1 uptake, making it unsuitable for dynamic studies of energization. Safranin O uptake by digitonin-permeabilized tubules required very small numbers of tubules, permitted measurements of {Delta}{Psi}m for relatively prolonged periods after the end of the experimental maneuvers, was rapidly reversible during deenergization, and allowed for direct assessment of both substrate-dependent, electron transport-mediated {Delta}{Psi}m, and ATP hydrolysis-supported {Delta}{Psi}m. Both types of energization measured using safranin O in tubules permeabilized after H/R were impaired, but combining substrates and ATP substantially restored {Delta}{Psi}m.

acute renal failure; ATP; kidney


Address for reprint requests and other correspondence: J. M. Weinberg, Nephrology Div., Dept. of Internal Medicine, Rm. 1560, MSRB II, Univ. of Michigan Medical Ctr., Ann Arbor, MI 48109-0676 (E-mail: wnberg{at}umich.edu)




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