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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.

F 1 F O-ATPase Activity and ATP Dependence of Mitochondrial Energization in Proximal Tubules after Hypoxia/Reoxygenation