Thick ascending limb of Henle (TAL) necrosis in the isolated perfused kidney is an important model of renal hypoxia, but physiologic and metabolic correlation with this morphologic damage has been inadequate. More precise estimation of TAL adenine nucleotides in this model was obtained in the present study by high pressure liquid chromatography analysis of biopsy samples from the inner stripe of the outer medulla during perfusion. The inner stripe, which is the zone where TAL are concentrated, showed ATP depletion and low adenylate energy charge (AEC) early in perfusion prior to the appearance of TAL necrosis. Free water clearance (CH2O) was used as an estimate of TAL transport activity; the CH2O observed during 90 minute perfusions was found to be predictive of the extent of TAL necrosis in each experiment. The results support the idea that there is significant medullary hypoxia in the isolated perfused kidney and that TAL solute transport is a determinant of injury in this model. In further studies, the effects of ouabain (10(-3) M), furosemide (10(-4) M) or acidosis (pH 7.0 rather than the usual pH 7.4) on TAL transport activity and adenine nucleotide levels were compared. All three maneuvers have been shown previously to reduce TAL injury in the isolated perfused kidney. Addition of ouabain or furosemide reduced CH2O and TAL necrosis in parallel while acidosis had no effect on CH2O during perfusion. Both ouabain and furosemide attenuated ATP depletion and resulted in higher AEC while acidosis had no effect on these indices of cellular hypoxia. Therefore, the mechanism of cytoprotection by acidosis appears distinct from that of ouabain or furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)