Na-P-i cotransporter type I activity causes a transient intracellular alkalinization during ATP depletion in rabbit medullary thick ascending limb cells

Abstract

The cellular pathophysiology of renal ischemia-reperfusion injury was investigated in primary cell cultures from rabbit medullary thick ascending limb (MTAL). Metabolic inhibition (MI) was achieved with cyanide and 2-deoxy-glucose. Sixty minutes of MI caused a profound but reversible decrease in intracellular concentration of ATP ([ATP](i)). Intracellular pH (pH(i)) first decreased after initiation of MI, followed by a transient alkalinization. When [ATP](i) reached its lowest value (<1% of control), the cells slowly acidified to reach a stable pH(i) of 6.92 after 50 min of MI. In the presence of EIPA (10 mu mol/L), the pattern of changes in pH(i) was unchanged and acidification was not increased, indicating that the Na+/H+ exchangers were inactive during ATP depletion. When inorganic phosphate (P-i) or Na+ was omitted from the apical solutions during MI, the transient alkalinization was no longer observed and the cytosol slowly acidified. Experiments on Na+-dependent alkalinizations revealed the presence of a Na-P-i cotransporter in the apical cell membrane. With indirect immunofluorescence, the Na-P-i cotransporter expressed in these primary cell cultures could be identified as Na-P-i type I. Although the exact physiological role of Na-P-i type I still is unresolved, these experiments demonstrate that apical Na-P-i type I activity is increased at the onset of ATP depletion in MTAL cells.