Mitochondrial dysfunction is an early event in high-NaCl-induced apoptosis of mIMCD3 cells.
Raising osmolality to 700 mosmol/kgH(2)O by the addition of NaCl rapidly kills most murine inner renal medullary collecting duct cells (mIMCD3), but they survive at 500 mosmol/kgH(2)O. At 300 and 500 mosmol/kgH(2)O, NADH autofluorescence is present in a mitochondria-associated, punctate perinuclear pattern. Within 45 s to 30 min at 700 mosmol/kgH(2)O, the autofluorescence spreads diffusely throughout the cell. This correlates with mitochondrial membrane depolarization, measured as decreased tetramethylrhodamine 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/kgH(2)O. General caspase activity and caspase-9 activity increase only after 6 h at 700 mosmol/kgH(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/kgH(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.
Michea
Luis
L
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1603,USA.
Combs
Christian
C
Andrews
Peter
P
Dmitrieva
Natalia
N
Burg
Maurice B
MB
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000187:drug effects* / Q000503:physiopathology*; D051379:Mice; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D009243:NAD / Q000378:metabolism; D009994:Osmolar Concentration; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012235:Rhodamines; D012965:Sodium Chloride / Q000633:toxicity*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D014508:Urea / Q000494:pharmacology; D051028:bcl-2-Associated X Protein
C494827:Bax protein, mouse; D003574:Cytochrome c Group; D005456:Fluorescent Dyes; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012235:Rhodamines; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; C401833:tetramethylrhodamine methyl ester; D009243:NAD; D012965:Sodium Chloride; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D014508:Urea; D020169:Caspases