Mitochondrial dysfunction is an early event in high-NaCl-induced apoptosis of mIMCD3 cells

Raising osmolality to 700 mosmol/kg[H.sub.2]O by the addition of NaCl rapidly kills most murine inner renal medullary collecting duct cells (mIMCD3), but they survive at 500 mosmol/kg[H.sub.2]O. At 300 and 500 mosmol/kg[H.sub.2]O, NADH autofluorescence is present in a mitochondria-associated, punctate perinuclear pattern. Within 45 s to 30 min at 700 mosmol/kg[H.sub.2]O, the autofluorescence spreads diffusely throughout the cell. This correlates with mitochondrial membrane depolarization, measured as decreased tetramethyl-rhodamine methyl ester perchlorate (TMRM) fluorescence. Mitochondrial dysfunction should increase the cellular ADP/ATP ratio. In agreement, this ratio increases within 1-6 h. Mitochondrial morphology (transmission electron microscopy) is unaffected, but nuclear hypercondensation becomes evident. Progressive apoptosis occurs beginning 1 h after osmolality is raised to 700, but not to 500, mosmol/kg[H.sub.2]O. General caspase activity and caspase-9 activity increase only after 6 h at 700 mosmol/kg[H.sub.2]O. The mitochondrial Bcl-2/Bax ratio decreases within 1-3 h, but no cytochrome c release is evident. The mitochondria contain little p53 at any osmolality. Adding urea to 700 mosmol/kg[H.sub.2]O does not change NADH or TMRM fluorescence. We conclude that extreme acute hypertonicity causes a mitochondrial dysfunction involved in the initiation of apoptosis. caspase; endonuclease G; cytochrome c; mitochondria membrane potential; murine inner medullary collecting duct; sodium chloride