Abstract | BACKGROUND: Cell death and survival pathways are critical determinants of epithelial cell fate after ischemia. Forkhead proteins have been implicated in the regulation of cellular survival. METHODS AND RESULTS: We have found that none of the forkhead family of proteins, FKHR, is phosphorylated after ischemia/reperfusion in the rat kidney. The time course of phosphorylation is similar to the time course of activation of the forkhead protein kinase Akt/ protein kinase B (PKB), with maximal phosphorylation at 24 to 48 hours postreperfusion when the process of regeneration peaks. Extracellular signal-regulated kinase (ERK)1/2 activation has also been implicated as prosurvival in the injured kidney. ERK1/2 were phosphorylated in postischemic kidneys at 5, 30, and 90 minutes of reperfusion, with phosphorylation decreased by 24 and 48 hours. Immunocytochemical analysis revealed increased phospho-ERK1/2 in the thick ascending limb and isolated cells of the S3 segment, which have lost apical actin staining. To understand the relationship between forkhead phosphorylation, Akt, and ERK1/2, an in vitro model of injury was employed. After 40 minutes of chemical anoxia followed by dextrose addition for 20 minutes to replete adenosine triphosphate ( ATP) levels, FKHR and FKHRL1 are phosphorylated. The levels of phospho-Akt are increased for at least 120 minutes after dextrose addition with a maximum at 20 minutes. Phosphorylation of Akt, FKHR, and FKHRL1 are phosphatidylinositol 3-kinase (PI 3-kinase) dependent since phosphorylation is reduced by the PI 3-kinase inhibitors, wortmannin, or LY294002. Inhibition of mitogen-activated protein kinase ( MAPK)/ERK kinase (MEK1/2), the upstream activator of ERK1/2, has no effect on forkhead protein phosphorylation after chemical anoxia/ dextrose addition. CONCLUSION: We conclude that PI 3-kinase and Akt are activated after renal ischemia/reperfusion and that Akt phosphorylation leads to phosphorylation of FKHR and FKHRL1, which may affect epithelial cell fate in acute renal failure.
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Authors | Michele Andreucci, Ashour Michael, Cornelis Kramers, Kwon Moo Park, Ang Chen, Tilman Matthaeus, Alessandro Alessandrini, Syed Haq, Thomas Force, Joseph V Bonventre |
Journal | Kidney international
(Kidney Int)
Vol. 64
Issue 4
Pg. 1189-98
(Oct 2003)
ISSN: 0085-2538 [Print] United States |
PMID | 12969136
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)
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Chemical References |
- Proto-Oncogene Proteins
- Phalloidine
- Adenosine Triphosphate
- Akt1 protein, rat
- Protein Serine-Threonine Kinases
- Proto-Oncogene Proteins c-akt
- Mitogen-Activated Protein Kinases
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Topics |
- Adenosine Triphosphate
(deficiency, metabolism)
- Animals
- Hypoxia
(chemically induced, metabolism)
- Immunohistochemistry
- Kidney
(metabolism)
- LLC-PK1 Cells
- Mitogen-Activated Protein Kinases
(metabolism)
- Phalloidine
(metabolism)
- Phosphorylation
- Protein Serine-Threonine Kinases
- Proto-Oncogene Proteins
(metabolism)
- Proto-Oncogene Proteins c-akt
- Rats
- Rats, Sprague-Dawley
- Renal Circulation
- Reperfusion Injury
(metabolism)
- Swine
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