Deletion of the kinase domain in death-associated protein kinase attenuates tubular cell apoptosis in renal ischemia-reperfusion injury.
Death-associated protein kinase (DAPK) is a calcium/calmodulin-dependent serine/threonine kinase localized to renal tubular epithelial cells. To elucidate the contribution of DAPK activity to apoptosis in renal ischemia-reperfusion (IR) injury, wild-type (WT) mice and DAPK-mutant mice, which express a DAPK deletion mutant that lacks a portion of the kinase domain, were subjected to renal pedicle clamping and reperfusion. After IR, DAPK activity was elevated in WT kidneys but not in mutant kidneys (1785.7 +/- 54.1 pmol/min/mg versus 160.7 +/- 60.6 pmol/min/mg). Furthermore, there were more TUNEL-positive nuclei and activated caspase 3-positive cells in WT kidneys than in mutant kidneys after IR (24.0 +/- 5.9 nuclei or 9.4 +/- 0.6 cells per high-power field [HPF] versus 6.3 +/- 2.2 nuclei or 4.4 +/- 0.7 cells/HPF at 40 h after ischemia). In addition, the increase in p53-positive tubule cells after IR was greater in WT kidney than in mutant kidneys (9.9 +/- 1.4 cells/HPF versus 0.8 +/- 0.4 cells/HPF), which is consistent with the theory that DAPK activity stabilizes p53 protein. Finally, serum creatinine levels after IR were higher in WT mice than in mutant mice (2.54 +/- 0.34 mg/dl versus 0.87 +/- 0.24 mg/dl at 40 h after ischemia). Thus, these results indicate that deletion of the kinase domain from DAPK molecule can attenuate tubular cell apoptosis and renal dysfunction after IR injury.
Kishino
Masanori
M
Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan.
Yukawa
Kazunori
K
Hoshino
Katsuaki
K
Kimura
Akihiko
A
Shirasawa
Nobuyuki
N
Otani
Haruhisa
H
Tanaka
Tetsuji
T
Owada-Makabe
Kyoko
K
Tsubota
Yuji
Y
Maeda
Masanobu
M
Ichinose
Masakazu
M
Takeda
Kiyoshi
K
Akira
Shizuo
S
Mune
Masatoshi
M
D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D064550:Death-Associated Protein Kinases; D004789:Enzyme Activation; D017353:Gene Deletion; D006720:Homozygote; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007668:Kidney / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009154:Mutation; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D015427:Reperfusion Injury; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases