Apoptosis induced by hypertonicity in Madin Darley canine kidney cells: protective effect of betaine.
BACKGROUND
In mammals, the renal medulla is in a hypertonic environment related to the renal concentrating mechanism. Renal cells accumulate osmolytes such as betaine to protect cells from the perturbing effect of high concentration of electrolytes. Hypertonicity-induced cell death and the effect of betaine were investigated in Madin Darby canine kidney (MDCK) cells.


METHODS
Cell viability was detected by 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide assay. DNA fragmentation was determined by FACS analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining and agarose gel electrophoresis. Activities of caspase-1, -3, -8, and -9 were measured.


RESULTS
When the cells were exposed to 700 mOsm medium for 24 h, 40% of the cells were detached. TUNEL staining showed that about 20% of detached cells were apoptotic, indicating that both necrosis and apoptosis contributed to the hypertonicity-induced cell death in MDCK cells. DNA laddering was demonstrated in hypertonic cells. Caspase-3, -8, and -9 activities of the adherent cells exposed to 700 mOsm for 24 h increased approximately 20-, 3-, and 4-fold the value of isotonic cells, respectively. However, there was no significant change in caspase-1 activity. Addition of 1 mM betaine into the medium protected the cells against the hypertonicity-induced cytotoxicity and apoptosis. Betaine prevented the induction of caspase-3, -8, and -9 activities after hypertonic exposure to about 50%.


CONCLUSIONS
The present study demonstrates that (i) apoptosis is involved in the hypertonicity-induced cell death in MDCK cells; (ii) caspase-3, -8, and -9 may contribute to the apoptosis; and (iii) betaine has protective effect on the hypertonicity-induced apoptosis.
School of Allied Health Sciences, Department of Internal Medicine and Therapeutics and. Faculty of Health and Sport Science, Osaka University School of Medicine, Suita, Osaka, Japan.
M
M
Horio
A
A
Ito
Y
Y
Matsuoka
T
T
Moriyama
Y
Y
Orita
M
M
Takenaka
E
E
Imai
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001622:Betaine / Q000494:pharmacology*; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D004285:Dogs; D004790:Enzyme Induction / Q000187:drug effects; D006982:Hypertonic Solutions / Q000494:pharmacology*; D007668:Kidney / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*
D061945:Caspase Inhibitors; D006982:Hypertonic Solutions; D001622:Betaine; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases