Departments of *Internal Medicine, Physiology, Legal Medicine, Anatomy, and ¶Gynecology and Obstetrics, Wakayama Medical University, Wakayama, Japan; and #Department of Host Defense, Research Institute for Microbial Disease, Osaka University, Osaka, Japan.
Correspondence to Dr. Masatoshi Mune, Department of Internal Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-8509, Japan. Phone: 81-73-441-0619; Fax: 81-73-441-2877; E-mail: mmune{at}wakayama-med.ac.jp
ABSTRACT. Death-associated protein kinase (DAPK) is a calcium/calmodulin-dependentserine/threonine kinase localized to renal tubular epithelialcells. To elucidate the contribution of DAPK activity to apoptosisin renal ischemia-reperfusion (IR) injury, wild-type (WT) miceand DAPK-mutant mice, which express a DAPK deletion mutant thatlacks a portion of the kinase domain, were subjected to renalpedicle clamping and reperfusion. After IR, DAPK activity waselevated 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 caspase3-positive cells in WT kidneys than in mutant kidneys afterIR (24.0 ± 5.9 nuclei or 9.4 ± 0.6 cells per high-powerfield [HPF] versus 6.3 ± 2.2 nuclei or 4.4 ± 0.7cells/HPF at 40 h after ischemia). In addition, the increasein p53-positive tubule cells after IR was greater in WT kidneythan in mutant kidneys (9.9 ± 1.4 cells/HPF versus 0.8± 0.4 cells/HPF), which is consistent with the theorythat DAPK activity stabilizes p53 protein. Finally, serum creatininelevels 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 hafter ischemia). Thus, these results indicate that deletionof the kinase domain from DAPK molecule can attenuate tubularcell apoptosis and renal dysfunction after IR injury.
Ischemic acute renal failure (ARF) represents an important clinicalproblem with high mortality rates (1). Histochemical and molecularstudy of renal ischemia-reperfusion (IR) (2–4) has characterizedseveral factors associated with renal IR injuries, includingreactive oxygen species (5), nitric oxide (6–8), cellsurface molecules (8–12), transcription factor NF-B (13),growth factors (14), cytokines (15–18), and phospholipidmessengers (8,19,20). These inflammatory components induce tubulecell necrosis or apoptosis and result in ARF (21,22). Mild orshort-duration ischemia results primarily in apoptosis ratherthan necrosis (2,23). This represents a mechanism that couldpotentially be interrupted to prevent ARF (24). Thus, characterizationof the molecular mechanisms underlying apoptosis in this casemay be of therapeutic benefit.
Intracellular mediators, such as calpain and p53, have beenimplicated in renal cell apoptosis in IR injuries (25,26). Furthermore,the mitogen-activated protein family of kinases, such as JNKand p38, likely play crucial roles in the response of renaltubule cells to ischemic and toxic stresses associated withARF (3,24). However, the exact mechanism by which these kinasesmediate apoptosis of renal tubule cells in ischemia-inducedARF remains unclear (3,24).
Death-associated protein kinase (DAPK) is a calcium/calmodulin-dependentkinase that phosphorylates serine/threonine residues on proteins(27,28). DAPK modulates cell death induced by IFN- (27), TNF-(29), Fas (29), and detachment from the extracellular matrix(30). DAPK has been also implicated in TGF--induced apoptosisin several cultured cell lines in which Smad proteins mediatetranscriptional activation of DAPK (31). Because TGF-1 is acrucial participant in tubular cell apoptosis in chronic progressivekidney disease (32), DAPK may also play a role in injury afterrenal IR. Indeed, our immunohistochemical analyses have confirmedthe localization of DAPK to renal tubular epithelial cells inhuman and rodents (33). The goal of the study presented herewas to investigate whether DAPK activity contributes to renalcell apoptosis in renal IR injury.
Animals (Generation of DAPK-Mutant Mice)
Mice expressing a mutant DAPK lacking a portion of the kinasedomain (amino acids 22 to 95) were generated by gene targetingin E14.1 embryonic stem (ES) cells (33). Briefly, a targetingvector was designed to replace the exon encoding amino acids22 to 95 of mature DAPK with the neomycin resistance gene. Thevector was introduced into E14.1 ES cells by electroporation.The clones resistant to G418 and ganciclovir were screened byPCR and confirmed by Southern blot analysis. The mutant ES cellswere injected into blastocysts (C57BL/6) and transferred tothe uteri of pseudopregnant mice to generate chimeras. Chimeraswere bred to C57BL/6 mice for germ-line transmission of themutant allele. Pairs of the resulting heterozygous mice weresubsequently bred to gain homozygous DAPK-mutant mice. Wild-type(WT) littermates were used as controls. Animals were housedin the animal facilities of Wakayama Medical University. Ourinstitutional Animal Ethics Review Committee approved all experimentalprotocols.
Reverse Transcription (RT)-PCR
Total RNA was prepared from kidney tissue with the SV TotalRNA Isolation System (Promega, Madison, WI). Total RNA fromkidney tissues was transcribed by reverse transcriptase withrandom primers to make template cDNA. The PCR primers were:5'-CACCATGACTGTGTTCAGGCAGGA-3' (sense primer) and 5'-GATCCAGGGGTGCTGCAAACT-3'(antisense primer 1) or 5'-GACGTCATAGCTGGACAAGGA-3' (antisenseprimer 2). Amplification by PCR with KOD-Plus-DNA polymerase(Toyobo, Japan) was performed in 25-µl reaction mixtureswith a GeneAmp PCR System thermocycler (Perkin-Elmer AppliedBiosystems, Foster City, CA). An initial step was performedat 94°C for 2 min. The PCR cycles were as follows: denaturationat 94°C for 15 s, annealing at 57°C for 30 s, and extensionat 68°C for 1 min 15 s for 30 cycles, followed by a finalextension step at 68°C for 7 min.
Western Blotting
Identical amounts of proteins quantified by a Coomassie blueassay were loaded in the lanes of a 7.5% Tris-HCl gel, resolvedby electrophoresis, and then transferred to a polyvinylidenefluoride filter membrane. DAPK was detected with the ECL Westernblotting system (Amersham Pharmacia Biotech, Buckinghamshire,UK) with an anti-DAPK antibody (Sigma, St. Louis, MO).
IR Model (Induction of IR)
Under anesthesia with pentobarbital sodium (50 mg/kg), an abdominalincision was made. Ischemia was induced by clamping the leftrenal pedicle for 30 min with a vascular clamp (Roboz, Gaithersburg,MD) while mice were kept at constant temperature (37°C)and well hydrated. Then the clamp was removed, and the kidneywas allowed to perfuse. At 16, 40, and 120 h after ischemia,animals were anesthetized, and the renal tissues were removed.
To assess the effect of IR on renal function, surgery was performedin the same manner, except for clamping of both renal pedicles.At 40 h after ischemia, mice were killed, and serum and kidneysamples were obtained. Sham operation was performed in bothWT and mutant mice by manipulation of the renal pedicles withoutclamping. The sham-operated kidneys were used as controls ineach animal. Five animals from each respective genotype wereused for each experiment.
DAPK Kinase Assay
Soluble homogenates from mouse kidney regions were preparedby dousing tissue in T-PER Tissue Protein Extraction Reagent(Pierce, Rockford, IL) containing protease inhibitors, -Complete(Roche, Penzberg, Germany). WT DAPK and mutant DAPK moleculeswere immunoprecipitated from tissue extracts with Seize X ProteinG Immunoprecipitation Kit (Pierce). Initially, anti-DAPK monoclonalantibodies (Sigma) bound to ImmunoPure Immobilized Protein Gwere cross-linked with disuccinimidyl suberate. Soluble homogenateswere mixed and incubated with the antibody-coupled gel at 4°Cfor 3 h. Then, immunoprecipitated DAPK molecules were eluted,assessed by Western blotting, and quantified. The CoomassiePlus assay (Pierce) was used to assure equal amounts of DAPKin the kinase assay.
The kinase activities of DAPK and mutant DAPK were quantifiedessentially as described by others (34), with some modifications.The DAPK molecules eluted from the gel were incubated with 200µM ATP and [32P]ATP (2.5 µCi per reaction) in assaybuffer (20 mM HEPES pH 7.5, 1 mM DTT, 2 mM MgCl2, 2 mM MnCl2,75 mM NaCl, 1 mM sodium orthovanadate, 1 mM okadaic acid, 1x-Complete) for 10 min at 25°C, either with the biotinylatedpeptide substrate biotin-(C)6-KKRPQRRYSNVF (20 µM), orwithout peptide substrate for determination of background phosphorylation.All reactions were terminated by adding guanidine hydrochloridesolution (2.5 M), and samples were spotted onto SAM2 BiotinCapture Membrane (Promega, Madison, WI). The SAM2 Biotin CaptureMembrane was washed once for 30 s with 2 M NaCl, then threetimes for 2 min each with 2 M NaCl, then four times for 2 mineach with 2 M NaCl in 1% H3PO4, and then twice for 30 s eachwith deionized water. Phosphate transfer was measured by scintillationcounting.
Tissue Collection and Morphologic Analysis
The kidneys were fixed in 4% buffered paraformaldehyde (pH 7.4).Specimens were then cut into 4-µm thin sections and stainedwith periodic acid-Schiff.
Assessment of Renal Function
Serum BUN and creatinine levels were measured as markers ofrenal function with Fujifilm DRI-CHEM 3500V autoanalyzer (FujiPhoto Film, Tokyo, Japan).
Apoptosis Assays (TUNEL)
Apoptotic cells in kidney tissues on slides were visualizedwith the In situ Apoptosis Detection Kit (Takara Bio, Kyoto,Japan) following the protocol provided by the manufacturer.Initially, paraffin-embedded tissue sections were hydrated.To permeabilize the sections by enzyme solution efficiently,tissues were treated with a buffer containing proteinase K (400µg/ml) for 5 min at room temperature. After washing withPBS, endogenous peroxidase was blocked by incubation of slidesin a solution of 3% H2O2 in water at room temperature for 5min. After washing with PBS, tissues were then incubated witha reaction buffer containing terminal deoxynucleotidyl transferase(TdT) and FITC-labeled substrate for 90 min at 37°C. Tissueswere further incubated with anti-FITC HRP conjugate (TakaraBio) for 30 min at 37°C and reacted with diaminobenzidinesolution for 10 min at room temperature to visualize positivenuclei. After counterstaining with hematoxylin, cells on slideswere examined by light microscopy.
Immunohistochemical Analysis (Detection of Activated Caspase 3 and p53)
To directly detect activated caspase 3 or p53 in IR kidneys,tissue sections were treated in 10 mM sodium citrate buffer(pH 6.0) and placed in a microwave oven for antigen unmasking.After blocking with 5% goat serum in PBS, tissue sections wereincubated with cleaved caspase 3 (Asp175) antibody (Cell SignalingTechnology, Beverly, MA) or with p53 antibody (Cell SignalingTechnology) at 4°C overnight. Sections were then incubatedwith dextran polymer conjugated with secondary antibodies torabbit Ig and peroxidase (DakoCytomation, Kyoto, Japan), andactivated caspase 3 or p53 was visualized in situ by the diaminobenzidinedetection procedure.
Assessment of Immunohistochemical Characteristics and Statistical Analyses
Over 20 pictures from three immunostained sections per eachkidney were taken at a magnification of 400x (high-power field[HPF]) by a light microscope equipped with a 3CCD camera (HV-C20S;Nikon, Tokyo, Japan) and stored in a Macintosh computer connectedto a 3CCD camera. The pictures were then analyzed by countingthe number of TUNEL-positive nuclei and cleaved caspase 3 orp53 positive cells in a blinded manner by two independent investigators.Data are expressed as mean values ± SE. Comparison betweenexperimental groups was performed by ANOVA followed by the Scheffétest for post hoc analysis. A P value of less than 0.05 wasconsidered statistically significant.
DAPK-Mutant Mice
Analysis of mRNA expressed in mutant kidneys demonstrated transcriptsthat encode a mutant DAPK protein lacking amino acids 22 to95 of the kinase domain (Figure 1, A and B). Western blottinganalysis of tissue from mutant mice demonstrated the mutantDAPK with a smaller molecular weight (Figure 1C). BUN and creatininelevels were similar when comparing WT mice to mutant mice. (BUN,27.88 ± 1.28 mg/dl versus 29.43 ± 3.64 mg/dl;creatinine, 0.48 ± 0.05 mg/dl versus 0.45 ± 0.03mg/dl).
Figure 1. Targeted disruption of the mouse death-associated protein kinase (DAPK) gene deleted 74 amino acids from the catalytic kinase domain. (A) Reverse transcription (RT)-PCR from DAPK-mutant kidneys detects a shorter transcript than that from wild-type (WT) DAPK mRNA (RT-PCR1). RT-PCR that uses another antisense primer also generates a shorter product from DAPK-mutant kidneys than that from wild type (RT-PCR2). These imply the existence of an alternative splicing product in the mutant kidney. WT, wild-type; MT, mutant. (B) DNA sequencing of the RT-PCR products from both genotypes revealed that the mRNA in mutant kidneys encoded a mutant DAPK molecule lacking amino acids 22 to 95. WT DAPK, WT DAPK protein expressed in WT kidneys. MT DAPK, mutant DAPK protein in mutant kidneys. (C) Kidney tissue extracts from WT and mutant mice were separated by SDS-PAGE, blotted and probed with a monoclonal antibody to DAPK. Western blotting detected a mutant DAPK in the mutant kidneys that was smaller than WT DAPK.
Kinase Activity of DAPK during Renal IR
To investigate the effect of IR on DAPK activity, WT and mutantDAPK was isolated from experimental and sham-operated kidneyfrom all animals at various time points. In WT kidneys, DAPKactivity was significantly increased and peaked at 16 h afterischemia, indicating that IR stimuli enhanced DAPK catalyticactivity. In contrast, DAPK activity was not significantly increasedin mutant kidneys after IR (Figure 2).
Figure 2. Mutant death-associated protein kinase (DAPK) molecules were not activated in response to ischemia-reperfusion (IR) stimuli. DAPK catalytic activity was significantly increased in wild-type (WT) kidneys at 16 h after IR. In contrast, mutant DAPK molecules were not significantly activated in mutant kidneys after renal IR (*P < 0.05). DAPK kinase activity was more than 11-fold higher in WT kidneys than that in mutant kidneys at 16 h after ischemia (1785.7 ± 54.1 versus 160.7 ± 60.6 pmol/min/mg).
Histologic Evaluation of Kidneys with IR Injury
Extensive tubular injuries with obstructing granular cast formationand epithelial cell sloughing was present in WT kidneys at 40h after ischemia (Figure 3), which is consistent with previousreports (2,6–12,14,15). The mutant kidneys after IR displayedsimilar histopathology, as represented by tubular damage associatedwith debris and cast formation (Figure 3). However, small condensedand fragmented nuclei representing apoptosis were more prominentin WT kidneys as compared with mutant kidneys (Figure 3, C and D).
Figure 3. Effects of renal ischemia-reperfusion (IR) on histopathology in death-associated protein kinase (DAPK)-mutant and wild-type (WT) mice. Light microscopy of kidney 40 h after renal IR in WT mice displaying extensive tubular damage with cast formation and many apoptotic bodies (A, original magnification, x100; C, original magnification, x400). Arrows indicate the site of apoptotic bodies. A comparable tissue section from DAPK-mutant mice shows similar tubular epithelial cell injuries (B, original magnification, x100; D, original magnification, x400).
Significant Attenuation of IR-induced Apoptosis in Mutant Kidneys
To assess the direct contribution of DAPK activity to renalcell apoptosis in IR injuries, TUNEL assays were performed onkidneys from both genotypes at 16, 40, and 120 h after reperfusion.WT kidneys after IR showed an extensive amount of TUNEL-positivestaining (Figure 4A, a and c). In contrast, mutant kidneys afterIR had fewer TUNEL-positive cells (Figure 4A, b and d). Thus,renal cell apoptosis at 16 and 40 h after ischemia was increasedin WT kidneys when compared with mutant kidneys (3.8 ±1.2 versus 0.5 ± 0.1 nuclei/HPF at 16 h; 24.0 ±5.9 versus 6.3 ± 2.2 nuclei/HPF at 40 h; Figure 4B).
Figure 4. Renal cell apoptosis after renal ischemia-reperfusion (IR). (A) Wild-type (WT) kidneys 40 h after IR display extensive amount of TUNEL-positive tubular epithelial cells (a, original magnification, x100; c, original magnification, x400). However, death-associated protein kinase (DAPK)-mutant kidneys after IR show a significant reduction in TUNEL-positive tubular epithelial cells (b, original magnification, x100; d, original magnification, x400). (B) Attenuation of renal cell apoptosis in DAPK-mutant mice. A significant attenuation of renal cell apoptosis was noted in kidneys of mutant mice (MT) after IR as compared with kidneys from WT mice (WT) 16 and 40 h after renal IR (*P < 0.05).
To further examine the extent of apoptosis after IR, immunohistochemicalanalyses were performed on kidneys from both genotypes withantibodies detecting endogenous levels of activated caspase3 (Figure 5). As shown in Figure 5B, the cleaved caspase 3 immunoreactivitywas significantly increased in renal tubule cells in postischemicWT kidneys. Although renal tubule cells with activated caspase3 was increased in mutant IR kidneys, the intensity was stillgreater in postischemic WT kidneys than in mutant kidneys (3.7± 0.6 versus 0.5 ± 0.1 cells/HPF at 16 h; 9.4± 0.6 versus 4.4 ± 0.7 cells/HPF at 40 h; 1.4± 0.2 versus 0.6 ± 0.1 cells/HPF at 120 h).
Figure 5. Activated caspase 3 during renal ischemia-reperfusion (IR). (A) Renal tubule cells with the cleaved caspase 3 immunoreactivity were detected in wild-type (WT) kidneys 40 h after IR (WT/IRI). In contrast, there were much fewer renal tubule cells with activated caspase 3 in death-associated protein kinase (DAPK)-mutant kidneys at 40 h after ischemia (MT/IRI). (B) Renal tubule cells with activated caspase 3 were significantly increased in postischemic WT kidneys. Although renal tubule cells with cleaved caspase 3 immunoreactivity were increased in mutant kidneys after IR, the increase was significantly less than that of WT kidneys after IR (*P < 0.05).
Detection of p53 in Renal Tubule Cells after IR
After IR, the amount of tubules cells expressing immunoreactivep53 was increased in WT kidneys when compared with mutant kidneys(9.9 ± 1.4 versus 0.8 ± 0.4 cells/HPF) (Figures 6 and 7).
Figure 6. p53 detected in kidneys at 40 h after renal ischemia-reperfusion (IR). Renal cells showing detectable p53 signals were evident in wild-type (WT) kidneys 40 h after IR (A, original magnification, x100; C, original magnification, x400). However, there were far fewer renal cells with p53 signals in death-associated protein kinase (DAPK)-mutant kidneys 40 h after IR (B, original magnification, x100; D, original magnification, x400).
Figure 7. p53 positive renal cells did not increase in death-associated protein kinase (DAPK)-mutant kidneys after ischemia-reperfusion (IR). A significant increase in renal cells with p53 signals was observed in wild-type (WT) kidneys at 40 h after IR. However, there were significantly fewer renal tubule cells with p53 in DAPK-mutant kidneys (MT) at 40 h after renal IR when compared with WT kidneys after IR (*P < 0.05).
Effects of the Deletion of DAPK Activity on Renal Function
To examine if the reduction in apoptosis in mutant kidneys hadany improvement on renal function after IR injuries, serum creatininelevels were measured in wild and mutant mice at 40 h after IR.After IR, mean creatinine was significantly higher in WT micethan in mutant mice (2.54 ± 0.34 versus 0.87 ±0.24 mg/dl) (Figure 8).
Figure 8. Death-associated protein kinase (DAPK)-mutant mice are functionally protected from renal ischemia-reperfusion (IR) injury as compared with wild-type (WT) mice. DAPK-mutant mice (MT) show significantly reduced serum creatinine levels as compared with WT at 40 h after renal IR (*P < 0.05).
The major findings of this study were (1) that deletion of thekinase domain of DAPK by gene targeting technology successfullyinhibited the activation of DAPK in response to renal IR, and(2) that the inactivation of DAPK catalytic activity was associatedwith reduced apoptosis and protected renal function in responseto IR. These data indicate that DAPK kinase activity plays acritical role in apoptosis and development of ARF after IR injury.
Studies have demonstrated that DAPK activity mediates apoptoticcell death of cultured cells in vitro (27–30,31,35,36)and that primary cultured cells from DAPK null knockout micehave some resistance to apoptotic stimuli (35,37). Furthermore,variants of DAPK may also prevent apoptosis in mice (38). However,a recent experiment measuring the catalytic activity of DAPKin an animal model of brain hypoxic-ischemic injury suggestedthat DAPK may play a role in neuronal development or recoveryfrom injury rather than directly in apoptosis (39). Thus, asidefrom its role in cancer (40–43), there are no studiesthat demonstrate that DAPK play a direct role in the modulationof apoptosis.
In the study presented here, kinase domain mutant DAPK moleculeswere not activated in response to IR stimuli. This is consistentwith previous findings that DAPK catalytic activity is dependenton the kinase domain (27,28,30,35,36). Furthermore, the domaindeleted from mutant DAPK protein contains a conserved lysine-42essential for DAPK catalytic activity (28), reinforcing theimportance of the lysine residue for full activation of DAPKactivity in IR kidneys in vivo. Thus, the mutant mice used inthe study presented here serve as good experimental models tocharacterize the role of DAPK catalytic activity in the apoptoticprocess in vivo.
In the study presented here, apoptosis was assessed by the TUNELmethod and by immunohistochemical detection of activated caspase3 and p53. Each apoptosis parameter displayed a similar patternin renal IR peaking at 40 h after ischemia, although TUNEL countsexceeded other parameters, possibly because of the inclusionof necrotic cells. In contrast, DAPK catalytic activity peakedat 16 h after ischemia and rapidly declined thereafter. Theactivation pattern of DAPK preceding apoptosis may indicatethat DAPK is an integrator of signaling to a pro-apoptosis executioner,such as caspase 3, in the apoptosis pathway (29,35). Indeed,the function of DAPK downstream from death receptors (16,29)may explain account for the early activation of DAPK in responseto IR. In fact, the activation pattern of DAPK in renal IR isreminiscent of changes of DAPK activities in ceramide-inducedapoptosis of PC12 cells (44). DAPK catalytic activities weretransiently increased 10 min after exposure of ceramide anddecreased at 90 min, with apoptosis observed at 12 h. Thus,the activation pattern of DAPK may explain why there is a significantdifference in kinase activity between genotypes only in postischemickidneys.
The apoptosis cell number in mutant kidneys after IR was significantlyless than that in WT kidneys after IR (Figures 4 and 5), indicatingthat DAPK catalytic activity mediates at least a portion ofapoptosis seen in renal IR injuries. DAPK has been shown tomodulate apoptosis induced by IFN-, TNF-, Fas, cellular detachmentfrom extracellular matrix, and ceramide (27,29,30,37). Amongthese stimuli, TNF- has been implicated in renal IR injuries(16–18). Thus, renal IR may include TNF-, leading to activationof DAPK catalytic activity via the TNF receptor 1 and resultingin renal cell apoptosis. However, IR-induced apoptosis in mutantkidneys was not completely inhibited, which implies that otherkinases, such as the mitogen-activated protein kinases familyand DAPK family members, are also involved in this process (45–48).
Experiments that used DAPK-deficient rodent embryonic fibroblastsin culture showed that DAPK activity results in stabilizationof p53 protein in the context of a stress signal (35,49). Thep53 protein is an important mediator of renal cell apoptosisafter IR, and p53 inhibitors prevent renal cell apoptosis andrenal dysfunction (26). In the study presented here, there weresignificantly fewer renal tubule cells with p53 signal in DAPK-mutantkidneys after IR when compared with WT kidneys after IR (Figures 7 and 8), which is consistent with previous reports showingstabilization of p53 by DAPK in vitro (35). Thus, IR may resultin apoptosis via a cascade involving TNF-, DAPK activation,p53 stabilization, and p53-mediated transcription of apoptosis-inducinggenes (49).
The degree of renal dysfunction after IR correlates with theextent of apoptosis (3,22,24,26,50). In the study presentedhere, serum creatinine levels after IR were lower in DAPK-mutantmice than in WT mice (Figure 8). This is consistent with theprinciple that inhibition of IR-induced renal cell apoptosisprevents renal dysfunction. Furthermore, these results suggestthat agents that modulate DAPK activity are attractive candidatesto produce clinical therapeutic benefits in patients with acuteischemic renal failure.
Acknowledgments
We thank Chiaki Yamashita for her technical assistance.
Footnotes
Masanori Kishino, Kazunori Yukawa, and Katsuaki Hoshino contributedequally to this work.
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Received for publication July 2, 2003.
Accepted for publication April 19, 2004.
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Top
Abstract
Introduction
B (13), growth factors (14), cytokines (15–18), and phospholipid messengers (8,19,20). These inflammatory components induce tubule cell necrosis or apoptosis and result in ARF (21,22). Mild or short-duration ischemia results primarily in apoptosis rather than necrosis (2,23). This represents a mechanism that could potentially be interrupted to prevent ARF (24). Thus, characterization of the molecular mechanisms underlying apoptosis in this case may be of therapeutic benefit. 
(27), TNF-
(29), Fas (29), and detachment from the extracellular matrix (30). DAPK has been also implicated in TGF-
-induced apoptosis in several cultured cell lines in which Smad proteins mediate transcriptional activation of DAPK (31). Because TGF-
). 
), indicating that DAPK catalytic activity mediates at least a portion of apoptosis seen in renal IR injuries. DAPK has been shown to modulate apoptosis induced by IFN-
