safranine-t and Hypoxia

Kidney proximal tubules develop a severe but highly reversible energetic deficit due to nonesterified fatty acid (NEFA)-induced dissipation of mitochondrial membrane potential (DeltaPsi(m)) during reoxygenation after severe hypoxia. To assess the mechanism for this behavior, we have compared the efficacies of different NEFA for inducing mitochondrial deenergization in permeabilized tubules measured using safranin O uptake and studied the modification of NEFA-induced deenergization by inhibitors of the ADP/ATP carrier and glutamate using both normoxic tubules treated with exogenous NEFA and tubules deenergized during hypoxia-reoxygenation (H/R). Among the long-chain NEFA that accumulate during H/R of isolated tubules and ischemia-reperfusion of the kidney in vivo, oleate, linoleate, and arachidonate had strong effects to dissipate DeltaPsi(m) that were slightly greater than palmitate, while stearate was inactive at concentrations reached in the cells. This behavior correlates well with the protonophoric effects of each NEFA. Inhibition of the ADP/ATP carrier with either carboxyatractyloside or bongkrekic acid or addition of glutamate to compete for the aspartate/glutamate carrier improved DeltaPsi(m) in the presence of exogenous oleate and after H/R. Effects on the two carriers were additive and restored safranin O uptake to as much as 80% of normal under both conditions. The data strongly support NEFA cycling across the inner mitochondrial membrane using anion carriers as the main mechanism for NEFA-induced deenergization in this system and provide the first evidence for a contribution of this process to pathophysiological events that impact importantly on energetics of intact cells.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Carrier Proteins; Dose-Response Relationship, Drug; Energy Metabolism; Fatty Acids; Female; Glutamic Acid; Hypoxia; Indicators and Reagents; Kidney Tubules, Proximal; Membrane Potential, Mitochondrial; Oleic Acid; Oxygen; Phenazines; Rabbits

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 (DeltaPsim) 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 DeltaPsim 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 DeltaPsim 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 DeltaPsim, and ATP hydrolysis-supported DeltaPsim. Both types of energization measured using safranin O in tubules permeabilized after H/R were impaired, but combining substrates and ATP substantially restored DeltaPsim.

Topics: Acute Kidney Injury; Adenosine Triphosphate; Animals; Benzimidazoles; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane Permeability; Coloring Agents; Energy Metabolism; Enzyme Inhibitors; Female; Fluorescent Dyes; Hypoxia; Ionophores; Kidney Tubules, Proximal; Membrane Potentials; Mitochondria; Ouabain; Phenazines; Proton-Translocating ATPases; Rabbits

Isolated kidney proximal tubules subjected to hypoxia/reoxygenation (H/R) have incomplete recovery of mitochondrial membrane potential (DeltaPsi(m)) that can be improved, but not normalized, by ATP in permeabilized cells as measured by safranin O uptake. In these studies, the mechanisms for the decreased DeltaPsi(m) in the tubules after H/R are further investigated and impairment of the function of the mitochondrial F(1)F(O)-ATPase is assessed. Normoxic control tubules had a small ATP-dependent component to DeltaPsi(m), but it required low micromolar levels of ATP, not the millimolar levels needed to support DeltaPsi(m) in tubules de-energized with rotenone or after H/R. Micromolar levels of ATP did not improve DeltaPsi(m) after either mild or severe H/R injury. The dependence of DeltaPsi(m) on millimolar levels of ATP after H/R decreased over time during reoxygenation. ATP hydrolysis by the oligomycin-sensitive, mitochondrial F(1)F(O)-ATPase was well preserved after H/R as long as Mg(2+) was available, indicating that function of both the F(1)F(O)-ATPase and of the adenine nucleotide translocase, which delivers nucleotides to it, are largely intact. However, ATP hydrolysis by the ATPase did not restore DeltaPsi(m) as much as expected from the rate of ATP utilization. These findings, taken together with the observation that substrate-supported generation of DeltaPsi(m) is impaired despite intact electron transport, make it likely that uncoupling plays a major role in the mitochondrial dysfunction in proximal tubules during H/R.

Topics: Adenosine Triphosphate; Animals; Energy Metabolism; Female; Hypoxia; Indicators and Reagents; Kidney Tubules, Proximal; Membrane Potentials; Mitochondria; Mitochondrial ADP, ATP Translocases; Oxygen; Phenazines; Proton-Translocating ATPases; Rabbits