Kidney proximal tubules exhibit decreased
ATP and reduced, but not absent, mitochondrial membrane potential (Deltapsi(m)) during reoxygenation after severe
hypoxia. This energetic deficit, which plays a pivotal role in overall cellular recovery, cannot be explained by loss of mitochondrial membrane integrity, decreased electron transport, or compromised F1F0-ATPase and
adenine nucleotide translocase activities. Addition of
oleate to permeabilized tubules produced concentration-dependent decreases of Deltapsi(m) measured by
safranin O uptake (threshold for
oleate = 0.25 microM, 1.6 nmol/mg
protein; maximal effect = 4 microM, 26 nmol/mg) that were reversed by delipidated BSA (
dBSA). Cell nonesterified
fatty acid (
NEFA) levels increased from <1 to 17.4 nmol/mg
protein during 60- min
hypoxia and remained elevated at 7.6 nmol/mg after 60 min reoxygenation, at which time
ATP had recovered to only 10% of control values.
Safranin O uptake in reoxygenated tubules, which was decreased 85% after 60-min
hypoxia, was normalized by
dBSA, which improved
ATP synthesis as well.
dBSA also almost completely normalized Deltapsi(m) when the duration of
hypoxia was increased to 120 min. In intact tubules, the protective substrate combination of
alpha-ketoglutarate +
malate (alpha-KG/MAL) increased
ATP three- to fourfold, limited
NEFA accumulation during
hypoxia by 50%, and lowered
NEFA during reoxygenation. Notably,
dBSA also improved
ATP recovery when added to intact tubules during reoxygenation and was additive to the effect of alpha-KG/MAL. We conclude that
NEFA overload is the primary cause of energetic failure of reoxygenated proximal tubules and lowering
NEFA substantially contributes to the benefit from supplementation with alpha-KG/MAL.