Hyperosmolality activates Akt and regulates apoptosis in renal tubular cells.
BACKGROUND
The novel serine-threonine kinase Akt is a critical enzyme in cell survival. We investigated the roles of the Akt pathway and apoptotic signals in (1) Madin-Darby canine kidney (MDCK) cells in a hyperosmotic condition in vitro and (2) in the inner medulla of dehydrated rat in vivo.


METHODS
The in vivo experiments were performed in 24- and 48-hour water-restricted rats. Hyperosmolality-stimulated Akt phosphorylation was examined in MDCK cells. Phosphatidylinositol 3-kinase (PI3-K) inhibitors, the dominant-negative mutant of PI3-K, the dominant-negative mutant of Akt, and the dominant-active form of Akt were used to examine the roles of the PI3-K/Akt pathways in renal tubular cell apoptosis.


RESULTS
The amount of phosphorylated Akt protein was increased in the inner medulla of dehydrated rats. Hyperosmolality induced by the addition of NaCl, urea, and raffinose phosphorylated Akt in MDCK cells in an osmolality-dependent manner. PI3-K inhibitors and the dominant-negative mutant of PI3-K inhibited the hyperosmolality-induced phosphorylation of Akt. Raising the media osmolality from a normal level to 500 or 600 mOsm/kg H2O final osmolality elicited apoptotic changes such as nucleosomal laddering of DNA and an increment of caspase-3 activity and increased activity in the cell death enzyme-linked immunosorbent assay. Dominant-active Akt prevented the mild hyperosmolality-induced apoptosis, while inhibition of the PI3-K/Akt pathways promoted apoptosis.


CONCLUSIONS
The Akt pathway is activated by hyperosmolality in vitro and in vivo, and activation of Akt prevents the mild hyperosmolality-induced apoptotic changes in MDCK cells. PI3-K/Akt pathways are involved in a hypertonic condition that confers the balance between cell survival and apoptosis.
Terada
Y
Y
Homeostasis Medicine and Nephrology, Tokyo Medical and Dental University, Tokyo, Japan. yterada.kid@tmd.ac.jp
Inoshita
S
S
Hanada
S
S
Shimamura
H
H
Kuwahara
M
M
Ogawa
W
W
Kasuga
M
M
Sasaki
S
S
Marumo
F
F
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D003681:Dehydration / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007679:Kidney Medulla / Q000166:cytology / Q000378:metabolism; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008297:Male; D009025:Morpholines / Q000494:pharmacology; D016296:Mutagenesis / Q000502:physiology; D009997:Osmotic Pressure; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017207:Rats, Sprague-Dawley; D013552:Swine; D014162:Transfection; D014868:Water Deprivation / Q000502:physiology; D014882:Water-Electrolyte Balance / Q000502:physiology*; D000077191:Wortmannin
D000730:Androstadienes; D053148:Caspase 3; D020169:Caspases; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D000077191:Wortmannin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C494920:Akt1 protein, rat; C505410:Casp3 protein, rat