Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation

IntroductionCentral organ systems, including heart, brain, gut, andkidney, are particularly sensitive to ischemia followedby reperfusion (I/R). Renal I/R injury alone imposes asignificant burden on the healthcare system because itnot only restricts function and availability of donorkidneys for transplantation, but is also a serious com-plication of circulatory shock.

More specifically, inflammation is considered to be amajor cause of I/R-induced tissue injury, because ther-apeutic strategies aimed at reducing inflammationoften ameliorate I/R injury (1). The local inflammato-ry reaction that follows reperfusion involves cytokinessuch as proinflammatory TNF-α, as well as recruit-ment of neutrophils. In vitro studies imply certain rolesfor the anoxia-induced release of chemokines and neu-trophil-endothelium interactions (2, 3) in the induc-tion of inflammation after I/R. Nevertheless, little isknown about the specific events that trigger thisinflammatory response in vivo.

In this study, we investigate whether another earlyconsequence of I/R, apoptotic cell death (4), con-tributes to the inflammatory response in a murinemodel of renal I/R. Our results challenge the paradigmwhich states that apoptosis represents a physiological

mode of cell deletion allowing disposal of superfluouscells with minimal tissue reaction, or even with activerestraining of inflammation (5). We show that exoge-nous IGF-1 (a growth and survival factor) and theactive caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F(ZVAD-fmk) attenuate reperfusion-induced inflam-mation, most likely because of their ability to inhibitapoptosis in vivo. These findings strongly suggest thatapoptotic cell death, either directly or indirectly, sig-nificantly contributes to I/R-induced inflammation, aswell as to the consequent tissue damage, and may pro-vide new therapeutic means to treat I/R injury.

MethodsAntibodies and reagents. Anti-murine neutrophil mAbGr-1 was obtained from PharMingen (San Diego, Cal-ifornia, USA); peroxidase-conjugated goat anti-rat andgoat anti-rabbit IgGs were obtained from The JacksonLaboratory (Bar Harbor, Maine, USA); peroxidase-con-jugated sheep anti-digoxigenin, digoxigenin 11-dUTP,and terminal deoxynucleotidyl transferase (TdT) wereobtained from Boehringer Mannheim GmbH(Mannheim, Germany); peroxidase-conjugated rabbitanti-sheep IgG was obtained from DAKO A/S(Glostrup, Denmark); the rabbit antiserum SA 2846

The Journal of Clinical Investigation | September 1999 | Volume 104 | Number 5 541

Inhibition of apoptosis induced by ischemia-reperfusionprevents inflammation

Marc A.R.C. Daemen,1 Cornelis van ‘t Veer,1 Geertrui Denecker,2 Vincent H. Heemskerk,1

Tim G.A.M. Wolfs,1 Matthias Clauss,3 Peter Vandenabeele,2 and Wim A. Buurman1

1Department of General Surgery, University of Maastricht, 6200 MD Maastricht, the Netherlands2Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent,B-9000 Ghent, Belgium

3Department of Molecular Cell Biology, Max-Planck-Institut für Physiologische und Klinische Forshung, 61231 Bad Nauheim, Germany

Address correspondence to: W.A. Buurman, Department of General Surgery, University of Maastricht, PO Box 616, 6200 MD Maastricht, the Netherlands. Phone: 31-4-33-88-14-63; Fax: 31-4-33-88-41-54; E-mail: [email protected].

Received for publication April 1, 1999, and accepted in revised form July 13, 1999.

Ischemia followed by reperfusion leads to severe organ injury and dysfunction. Inflammation is con-sidered to be the most important cause of tissue injury in organs subjected to ischemia. The mecha-nism that triggers inflammation and organ injury after ischemia remains to be elucidated, althoughdifferent causes have been postulated. We investigated the role of apoptosis in the induction of inflam-mation and organ damage after renal ischemia. Using a murine model, we demonstrate a relationshipbetween apoptosis and subsequent inflammation. At the time of reperfusion, administration of theantiapoptotic agents IGF-1 and ZVAD-fmk (a caspase inactivator) prevented the early onset of not onlyrenal apoptosis, but also inflammation and tissue injury. Conversely, when the antiapoptotic agentswere administered after onset of apoptosis, these protective effects were completely abrogated. Thepresence of apoptosis was directly correlated with posttranslational processing of the endothelialmonocyte-activating polypeptide II (EMAP-II), which may explain apoptosis-induced influx andsequestration of leukocytes in the reperfused kidney. These results strongly suggest that apoptosis isa crucial event that can initiate reperfusion-induced inflammation and subsequent tissue injury. Thenewly described pathophysiological insights provide important opportunities to effectively preventclinical manifestations of reperfusion injury in the kidney, and potentially in other organs.

J. Clin. Invest. 104:541–549 (1999).

was generated (Eurogentec, Seraing, Belgium) againsthuman recombinant mature endothelial monocyte-activating polypeptide II (EMAP-II) (23 kDa) and rec-ognized murine EMAP-II; Taq polymerase wasobtained from Perkin-Elmer Corp. (Norwalk, Con-necticut, USA); LY294002 was obtained from Cal-biochem-Novabiochem Corp. (San Diego, California,USA); recombinant human IGF-1 was kindly providedby H.P. Guler (Chiron Corp., Emeryville, California,USA); ZVAD-fmk and ZFA-fmk were obtained fromEnzyme Systems Products Inc. (Livermore, California,USA); Ac-YVAD-amc and Ac-DEVD-amc wereobtained from the Peptide Institute (Osaka, Japan). Allother reagents were purchased from Sigma ChemicalCo. (St. Louis, Missouri, USA).

Renal ischemia-reperfusion injury model. Male Swissmice weighing 20–25 g were obtained from CharlesRiver Breeding Laboratories (Heidelberg, Germany)and were housed individually in standard laboratorycages, with ad libitum access to food and water. Thestudies were approved by the Institutional AnimalCare Committee of the University of Maastricht. Atthe start of the experiments, mice were anesthetizedwith sodium pentobarbital (50 mg/kg, intraperi-toneally). A rectal probe (Cole-Parmer Instrument Co.,Vernon Hills, Illinois, USA) to monitor body tempera-ture was inserted, and body temperature was main-tained at 39°C by a heating lamp until animals recov-ered from anesthesia. Ischemia was induced byclamping the left renal pedicle for 45 minutes using anontraumatic vascular clamp through a midlineabdominal incision. After clamp removal, the left kid-ney was inspected for restoration of blood flow. Next,the contralateral kidney was removed and stored forfurther analysis. The abdomen was closed in 2 layers,and 0.25% bupivacaine was applied topically for post-operative pain management. Finally, to maintain fluidbalance and volume status, mice were supplementedwith 1 mL prewarmed (37°C) PBS subcutaneously.

The animals were sacrificed at different time points,ranging from just before reperfusion through 24hours after reperfusion. At the time of sacrifice, bloodwas collected by orbital puncture, and the left kidneywas harvested for further analysis.

At the time of reperfusion, mice were administered100 µg recombinant human IGF-1 subcutaneously (n= 10), 30 µg ZVAD-fmk in 1% DMSO intraperitoneally(n = 10), or 20 µg recombinant human EGF dissolvedin 0.3 mL PBS subcutaneously (n = 10). In separategroups, 15 µg LY294002 in 0.3 mL PBS containing 1%DMSO was administered intraperitoneally with (n =10) or without (n = 10) IGF-1 treatment. Also in sepa-rate groups, IGF-1 (n = 10) or ZVAD-fmk (n = 10) wereadministered under similar conditions except after 2hours of reperfusion. When compared with PBS treat-ment, neither the solvent DMSO nor treatment with 30µg Z-Phe-Ala-Ch2F (ZFA-fmk) in 1% DMSO (as a neg-ative control for the caspase-inhibiting properties ofZVAD-fmk) affected the outcome of performed exper-iments (data not shown). Therefore, the control groupwas treated subcutaneously with a vehicle consisting of0.3 mL PBS (n = 12). A sham-operated group (n = 12)was subjected to the same surgical procedure withoutclamping the renal pedicle, and then was treated withPBS and sacrificed at corresponding time points. Thedosages selected were based on pilot experiments withrespect to ZVAD-fmk and LY294002 (data not shown)and on studies in which IGF-1 or EGF was found toattenuate renal reperfusion injury (6).

Assays for myeloperoxidase and blood urea nitrogen. Toquantify the extent of renal neutrophil accumulation,renal myeloperoxidase (MPO) content was determined.Snap-frozen samples were thawed, homogenized for 30seconds using a tissue homogenizer (T25B; IKA-WerkeGmbH, Staufen, Germany) at 20,000 g in 0.5% hexade-cyltrimethylammonium bromide dissolved in 50 mMPBS (pH 6.0), and then made up to concentrations of0.17 g renal tissue/mL. After a 2-hour heat incubation in

542 The Journal of Clinical Investigation | September 1999 | Volume 104 | Number 5

Figure 1Representative light micrographsshowing evident in situ detection ofDNA nick ends by TUNEL histology(a–c). The tubular epithelial cells ofthe outer medulla from biopsiesobtained after 2 hours of reperfusionshowed positive staining (a), in con-trast to kidneys obtained from sham-

operated mice (b) (×250). The presence of apoptotic bodies couldbe detected extracellularly and extruded into the tubular lumen after2 hours of reperfusion (c) (×1,000). Toluidine blue stains of 1-µmsections, obtained from kidneys that were fixed in situ after 3 hoursof reperfusion and subsequently embedded in plastic (d and e),revealed the presence of dense condensation of nuclear chromatinand nuclear fragmentation in apoptotic tubular epithelial cells(arrow) in the outer medulla (d), whereas morphological attributesof tubular epithelial cell apoptosis were generally absent in sham-operated mice (e) (×1,000).

a 60°C water bath to verify MPO heat resistance, sam-ples were frozen and thawed to release cellular MPO.Next, samples were spun at 11,000 g for 5 minutes, andsupernatants were then collected. Enzymatic detectionof MPO was performed in a 96-well plate (Corning-Costar Corp., Cambridge, Massachusetts, USA). Assaymixtures consisted of 40 µL H2O2 (final concentration:0.3 mM) in 80 mM PBS (pH 5.4) and 40 µL in 0.5% hexa-decyltrimethylammonium bromide in 50 mM PBS (pH6.0) containing diluted sample. The reaction was initiat-ed by addition of 20 µL tetramethylbenzidine (final con-centration: 1.6 mM) in DMSO and stopped after 15 min-utes by the addition of 50 µL/well of 1 M H2SO4.Subsequently, OD was determined at 450 nm. All sam-ples were assayed in triplicate. MPO activity was calcu-lated per milligram of renal tissue by comparing OD ofsample wells with a titration curve of horseradish perox-idase. The obtained relative MPO activities were stan-dardized with respect to wet/dry ratios of the assayedrenal tissue. The activities are presented relative to theamount of MPO present in the contralateral kidney,which was harvested immediately after reperfusion andnormalized with respect to the MPO increase at day 1 inPBS-treated mice subjected to I/R. Blood urea nitrogen(BUN) content was measured in serum obtained by

orbital puncture at the time of sacrifice using a BUNUnimate 5 kit in a Cobas Fara autoanalyzer (Hoffmann-La Roche Ltd., Basel, Switzerland).

Apoptosis assays. Histological aspects of apoptosiswere studied by standard TdT-mediated dUTP nickend labeling (TUNEL histology). To this end, 8-µmfrozen sections were fixed for 30 minutes in 4%buffered formaldehyde, washed in PBS, pretreated for10 minutes with PBS containing 0.6% H2O2, and thenwashed again. DNA fragments were elongated andlabeled with TdT and digoxigenin 11-dUTP. Subse-quently, sections were treated with sheep anti-digoxi-genin antibodies and washed. Finally, peroxidase-labeled rabbit anti-sheep antibodies, preincubated for30 minutes in 10% murine serum, were applied and,after washing, developed with diaminobenzidine sub-strate. Sections that were pretreated with DNase 1 tonick all DNA served as positive controls.

To quantify renal caspase activity, fluorogenic sub-strates were used with predominant caspase-1– and cas-pase-3–like affinities. Samples were homogenized,snap-frozen, and then stored at –70°C in a buffer con-taining 200 mM NaCl, 10 mM Tris-HCl (pH 7.0), 5 mMEDTA, 10% glycerol, 1 mM PMSF, 0.1 µM aprotinin,1.0 µM leupeptin, and 5 mM oxidized glutathione.


Figure 2Renal caspase-like activities were determined kinetically in homogenates of tissue obtained after 24 hours (a and c) and 2 hours (b and d)of renal reperfusion in a fluorogenic substrate assay in which Ac-YVAD-amc (caspase-1–like) (a and b) or Ac-DEVD-amc (caspase-3–like) (c and d) served as substrates. Note that these data may represent a more generalized form of caspase activation, as outlined in the maintext. Data are expressed as the increase in fluorescence as a function of time, normalized against data obtained from the sham-operatedgroup. The groups that indicate t = 2 on the x-axis received the indicated treatment after 2 hours of reperfusion. All other groups were treat-ed at the time of reperfusion. *P < 0.05, **P < 0.01 vs. control-treated animals. #P < 0.05, ##P < 0.01 vs. contralateral control kidney. Thedata shown are mean ± SEM.

Renal lysates (containing 40 µg total protein) wereincubated with 50 µM of the fluorogenic substrates Ac-YVAD-amc (caspase-1–like) or Ac-DEVD-amc (caspase-3–like) in a cell-free system buffer containing 10 mMHEPES (pH 7.4), 220 mM mannitol, 68 mM sucrose, 2mM NaCl, 2.5 mM KH2PO4, 0.5 mM EGTA, 2 mMMgCl2, 5 mM pyruvate, 0.1 mM PMSF, and 1 mM DTT.The release of fluorescent 7-amino-4-methylcoumarinwas measured for 1 hour at 2-minute intervals by spec-trofluorometry (Cytofluor; PerSeptive Biosystems,Framingham, Massachusetts, USA). Data are expressedas the increase in fluorescence as a function of time,and then normalized against the data obtained fromthe sham-operated group.

The presence of internucleosomal DNA cleavage inkidneys was investigated with a commercially availableligase-mediated PCR (LM-PCR) assay kit (Apoalert;CLONTECH Laboratories Inc., Palo Alto, California,USA), enabling semiquantitative measurement of theextent of apoptosis. In brief, DNA was isolated from tis-sue samples (frozen previously at –70°C) using a com-mercially available DNA purification kit (Wizard;Promega Corp., Madison, Wisconsin, USA) accordingto the manufacturer’s instructions. DNA purity andconcentration were determined by electrophoresisthrough a 0.8% agarose gel containing ethidium bro-mide, followed by observation under ultraviolet illu-mination, as well as by measuring absorbance at260/280 nm. Dephosphorylated adaptors were ligatedto 5′ phosphorylated blunt ends with T4 DNA ligase(during 16 hours at 16°C); they then served as primersin LM-PCR under the following conditions: hot start(72°C for 8 minutes, Taq polymerase added after 3 min-utes), 25 cycles (94°C for 15 seconds, and 72°C for 180

seconds), and postcycling (72°C for 15 minutes). Toconfirm that equal amounts of DNA were used forPCR, an internal control that consisted of DNA ampli-fication with En-2 primer pairs was performed. Ampli-fied DNA was subjected to gel electrophoresis on a 1.2%agarose gel containing ethidium bromide.

Histology. Histological aspects of apoptosis werestudied to verify that I/R also induced morphologi-cal characteristics of apoptosis. Kidneys were fixed insitu by perfusion with 2.5% glutaraldehyde in phos-phate buffer through a left ventricular cardiaccatheter in anesthetized mice after 3 hours of reper-fusion. After perfusion fixation, renal specimens con-taining outer medulla segments were fixed in 2.5%glutaraldehyde overnight. Next, segments were post-fixed in 1% osmium tetroxide, dehydrated in gradedalcohols, and then embedded in Epon 812 plastic.Finally, 1-µm sections were stained with toluidineblue for light microscopic examination.

From animals in the different treatment groups,specimens of harvested kidneys were immediatelyfrozen and stored at –70°C. Next, 5-µm sections werestained for neutrophils with mAb Gr-1, using peroxi-dase-labeled goat anti-rat IgG as the secondary detec-tion mAb and 3-amino-9-ethylcarbazole as a chro-mogen followed by a hematoxylin counterstain. Toblock nonspecific peroxidase activity, sections were pre-treated for 10 minutes with PBS containing 0.03%H2O2. No significant staining was detected in slidesincubated with control mAb instead of the primarydetecting mAb.

Western blot analysis. Frozen samples of harvested kid-neys were homogenized in a buffer containing 200 mMNaCl, 10 mM Tris-HCl (pH 7.0), 5 mM EDTA, 10%glycerol, 1 mM PMSF, and 0.1 µM aprotinin. After cen-trifugation at 15,000 g, protein in the supernatants wasquantified by the Bradford method and boiled inLaemmli buffer. Then, 35 µg of protein per lane waselectrophoresed in 15% SDS-PAGE. Proteins weretransferred to nitrocellulose membranes, which weresubsequently blocked with PBS/5% nonfat dry milkand then incubated for 3 hours with EMAP-II anti-serum (SA 2846; diluted 1:2,000 in PBS/0.1% Tween-20). After washing, the membranes were incubated for2 hours with peroxidase-labeled goat anti-rabbit IgG(diluted 1:2,000 in PBS/0.1% Tween 20), washed, andthen developed by enhanced chemiluminescence.

Statistics. Data are expressed as the mean ± SEM,and statistical analysis was performed by Student’s ttest. P values less than 0.05 were considered statisti-cally significant.

ResultsRenal I/R induces apoptosis and subsequent inflammation. Todetermine the extent of apoptosis, and to excludenecrosis or mechanisms independent from apoptosisas main contributors to DNA fragmentation, differenttechniques were used. Kidneys were investigated byTUNEL histology to localize DNA fragmentation after


Figure 3The extent of renal apoptosis in different treatment groups is alsoreflected by the presence of fragmented DNA (as a result of internu-cleosomal DNA cleavage), amplified by LM-PCR and then viewed onethidium bromide–stained gel. M, molecular weight markers (range:100–2,000 bp).

reperfusion. For morphological assessment of apopto-sis, toluidine blue histology was performed using plas-tic-embedded tissues. Also, caspase-like activities andinternucleosomal DNA cleavage were measured inhomogenates of kidneys obtained after reperfusion.

TUNEL-positive nuclei, restricted mainly to the tubu-lar epithelial cells of the outer medulla, were identifiedin biopsies obtained after as little as 2 hours of reper-fusion (Figure 1, a and b). At this time point, TUNELhistology revealed the extracellular presence of apop-totic bodies, which were often extruded into the tubu-lar lumen (Figure 1c). In a separate group of mice, sac-rificed after 3 hours of reperfusion, toluidineblue–stained 1-µm sections showed dense condensa-tion of nuclear chromatin and nuclear fragmentationin apoptotic tubular epithelial cells (Figure 1, d and e).In addition, increased renal caspase-1– and caspase-3–like activities (Figure 2, b and d), as well as apparentinternucleosomal DNA cleavage (Figure 3), wereobserved after 2 hours of reperfusion.

As documented previously (4), after 1 day of reperfu-sion the number of tubular epithelial cells containingTUNEL-positive nuclei had increased, and TUNEL-positive nuclei of infiltrating immune cells wereobserved (data not shown). Also at this time, increasedrenal caspase-1– (Figure 2a) and caspase-3–like (Figure2c) activities were detected. Together with the observedoccurrence of internucleosomal DNA cleavage (Figure3), these findings suggest the presence of ongoing renalapoptosis starting as early as 2 hours after reperfusion.

IGF-1 abrogates early apoptosis, impaired renal function, andI/R-induced inflammation. We first investigated the rela-tion between I/R-induced apoptosis and inflammationby using IGF-1, because pilot experiments revealed thatthis agent prevented I/R-induced apoptosis in murinekidneys. Administration of IGF-1 at the time of reper-fusion — as opposed to administration of PBS — pre-vented apoptosis, as indicated by a decreased number ofTUNEL-positive nuclei (data not shown), decreased cas-pase-like activities (Figure 2), and the absence of appar-ent internucleosomal DNA cleavage (Figure 3). Thesedata reveal the antiapoptotic potential of IGF-1 in anearly phase after renal ischemia.

Next, we studied whether abrogation of early apopto-sis affects the later consequences of renal I/R. IGF-1administration at the time of reperfusion prevented theI/R-induced impairment of renal function, as reflectedby increased BUN content at day 1 (Figure 4). Moreover,IGF-1 treatment limited I/R-induced inflammation atthat time point by reducing the renal influx of neu-trophils, as reflected by the presence of Gr-1–positivecells (Figure 5, a and b) and the increased renal MPOactivity (Figure 5c). Taken together, these findings indi-cate that exogenous IGF-1 prevents I/R-induced apop-tosis, impairment of renal function, and inflammation.

Protection from I/R-induced apoptosis by IGF-1 is mediatedby phosphatidylinositol 3-kinase. Phosphatidylinositol 3-kinase (PI 3-kinase), an important component of signaltransduction by insulin family receptors, has been

reported to mediate antiapoptotic signaling by IGF-1in vitro (7). To investigate more closely the mechanismsby which IGF-1 attenuates reperfusion-induced apop-tosis and inflammation, we treated mice with a combi-nation of IGF-1 and LY294002 (a selective PI 3-kinaseinhibitor) (8). In contrast to IGF-1 treatment, com-bined administration of IGF-1 and LY294002 at thetime of reperfusion failed to prevent apoptosis. Ani-mals treated with this combination exhibited an abro-gation of IGF-1–induced inhibition of apoptosis,resulting in increased numbers of TUNEL-positivenuclei (data not shown), significant caspase-like activ-ity (Figure 2), as well as apparent internucleosomalDNA cleavage (Figure 3) at day 1 after I/R. Moreover, atthis time point, they exhibited decreased renal function(Figure 4) and an increased renal neutrophil influx(Figure 5c) when compared with IGF-1–treated ani-mals. LY294002 treatment, compared with PBS treat-ment, did not result in significant differences in any ofthe parameters evaluated (Figures 2–5). These datademonstrate that in vivo antiapoptotic signaling byIGF-1 is mediated by the PI 3-kinase pathway.

Inflammation is dependent on antecedent apoptosis afterrenal I/R. Besides antiapoptotic properties, IGF-1 has awell-documented mitogenic potential that may con-tribute to the repair and recovery of ischemicallyinjured renal tissue. To address the question whetherthe mitogenic effects of IGF-1 were responsible for theobserved prevention of apoptosis and inflammation inour model, mice were treated with EGF, a powerfulmitogen for renal epithelial cells. EGF administered atthe time of reperfusion failed to prevent reperfusion-induced renal apoptosis, as reflected by increased num-bers of TUNEL-positive nuclei (data not shown), sig-nificant caspase-like activity (Figure 2), and apparentinternucleosomal DNA cleavage (Figure 3) at day 1.Hence, the absence of any antiapoptotic effect excludesthe possibility that EGF, as opposed to IGF-1, func-


Figure 4Renal function in the different experimental groups as reflected byBUN content. **P < 0.01, ***P < 0.001 vs. control-treated animals.The data shown are mean ± SEM.

tions as a survival factor in the model used. Moreover,as opposed to IGF-1, EGF did not prevent I/R-inducedimpaired renal function (Figure 4) or inflammation(Figure 5c) at day 1.

To investigate whether the anti-inflammatory effectof exogenous IGF-1 is a direct effect rather than a con-sequence of the absence of apoptosis, mice were treatedwith the tripeptide caspase inhibitor ZVAD-fmk at thetime of reperfusion, as well as after 2 hours of reperfu-sion. At the later time point, the presence of renal apop-tosis is evident (Figure 1), whereas an inflammatoryresponse, as defined by the present parameters, cannotyet be distinguished (Figure 5c). In line with a recentreport on cardiac I/R (9), we show that ZVAD-fmkadministered at the time of reperfusion completelyabrogated renal apoptosis (Figures 2 and 3). In addition,ZVAD-fmk administered at the time of reperfusion pre-vented the impairment of renal function (Figure 4) andthe development of the inflammatory response (Figure5c) at day 1. In contrast, ZVAD-fmk administered after2 hours of reperfusion not only failed to attenuate theextent of the already initiated apoptotic response (Fig-ures 2 and 3), but it also failed to attenuate subsequentimpairment of renal function (Figure 4) and the inflam-matory response (Figure 5c). Similarly, at day 1, noreversal of renal apoptosis could be detected in micethat received IGF-1 after 2 hours of reperfusion (Figures2 and 3); in addition, these animals exhibited signs ofinflammation (Figure 5c) and impaired renal function(Figure 4), as seen in PBS-treated animals. These find-ings indicate that development of inflammation isdependent on antecedent apoptosis after I/R. Sinceabrogation of apoptosis by IGF-1 or ZVAD-fmk limitsI/R-induced inflammation, apoptosis is a likely candi-date for induction of inflammation in the model used.

Posttranslational processing of EMAP-II coincides with I/R-induced apoptosis and is inhibited by IGF-1 and ZVAD-fmk.The precursor form of EMAP-II can serve as a targetsubstrate for activated caspases (10). We used Western

blotting to investigate the effect of I/R on posttransla-tional processing of EMAP-II. Figure 6 shows that I/Rresults in enhanced cleavage of the 43-kDa EMAP-IIprecursor as early as 2 hours of reperfusion. The active23-kDa cleavage product remained detectable in kid-neys subjected to 24 hours of reperfusion. Hence, theformation of functional EMAP-II coincides with themanifestation of apoptosis after renal I/R. EMAP-IIactivation was clearly inhibited in kidneys obtainedfrom sham-operated animals or from mice treated withZVAD-fmk at the time of reperfusion, after 2 hours(data not shown) or 24 hours (Figure 6) of reperfusion.At these time points, animals that were treated withIGF-1 at the time of reperfusion exhibited only low-intensity 23-kDa bands (Figure 6). Digital image analy-sis of activated EMAP-II protein band intensities of therepresentative EMAP-II Western blot (Figure 6) revealsthat the intensity of the 23-kDa band after IGF-1 treat-ment is approximately 5% of the 23-kDa band intensi-ty 24 hours after PBS treatment. These findings are inline with the other parameters measured in IGF-1–treated animals. As shown in Figure 6, the formationof 23-kDa EMAP-II also resulted in formation of bandsrepresenting a molecular mass between 23 and 43 kDa,which has been suggested to be the result of interme-diate cleavage product formation (10). These resultsdemonstrate that renal I/R induces EMAP-II activation.Both IGF-1 and the caspase inhibitor ZVAD-fmkstrongly reduce EMAP-II activation, which may explainthe observed ability of these agents to inhibit the apop-tosis-induced inflammatory response after renal I/R.

DiscussionWe hypothesized that apoptosis after I/R constitutes apotential trigger of inflammation, and we studied thispossibility by using inhibitors of the apoptotic responsein a murine model of renal I/R. Evidence for the earlyinvolvement of apoptosis is provided by the presence ofDNA fragmentation, morphological features character-


Figure 5Representative light micrographs (a and b) showing infiltrating neutrophils in areas with impaired renal morphological integrity in biop-sies obtained from mice subjected to renal I/R after 1 day of reperfusion (a). Neutrophils were stained with the anti-murine neutrophilmAb Gr-1. Neither loss of morphological integrity nor infiltrating neutrophils were observed in kidneys obtained from sham-operated mice(b) (×200). Neutrophil influx was assessed quantitatively by determination of MPO increase at 2 hours and 1 day of reperfusion (c). Val-ues are presented relative to the amount of MPO present in the contralateral kidney harvested immediately after reperfusion, and thennormalized with respect to the MPO increase at day 1 in PBS-treated mice subjected to I/R. *P < 0.05 vs. control-treated animals. The datashown are mean ± SEM.

istic for apoptosis, as well as increased caspase-1– and cas-pase-3–like activities in kidneys after 2 hours of reperfu-sion. These findings are in line with various in vivo andin vitro reports showing that renal apoptosis afterischemia is induced by hypoxia (11) and ATP depletion(12). In addition, hydrolyzation of the plasma membranecomponent sphingosine, and consequent liberation ofceramide, has been reported to initiate apoptosis after aslittle as 30 minutes of renal reperfusion (13). Further-more, during I/R-induced inflammation at day 1, upreg-ulated Fas (capable of inducing apoptosis independentof ceramide signaling) (4), TNF-α (14), and P53 (15) havebeen associated with I/R-induced apoptosis. In contrast,our in vivo findings clearly differ from data presented byUeda et al. (16), who reported that anoxia followed byreoxygenation of isolated rat proximal tubules in vitromay occur without morphological aspects of apoptosis.

The present findings show that mice treated with asingle dose of IGF-1 at the time of reperfusion exhibit-ed abrogation of apoptosis, as demonstrated bydecreased numbers of TUNEL-positive nuclei,decreased renal caspase-like activities, and the absenceof apparent internucleosomal DNA cleavage. Theseresults confirm that IGF-1 is a potent inhibitor of reper-fusion-induced apoptosis. In addition, ZVAD-fmk, abroad spectrum inhibitor of activated caspases, abro-gated I/R-induced apoptosis, whereas ZFA-fmk, ahomologous peptide lacking the caspase-inhibitingproperties of ZVAD-fmk, failed to prevent apoptosis.Hence, inhibition of apoptosis prevented the loss of kid-ney function and the development of an inflammatoryresponse. In contrast, administration of antiapoptoticIGF-1 or ZVAD-fmk after 2 hours of reperfusion (afterthe onset of apoptosis) failed to prevent loss of kidneyfunction and inflammation. Few studies have addressedthe potential of IGF-1 in ischemia-induced apoptosis(17, 18). The latter report (18) assumed a distinct “anti-neutrophil property” apart from the antiapoptoticeffects of IGF-1. In contrast, our findings clearly defineapoptosis as an event arising proximal to the onset ofI/R-induced inflammation.

We investigated whether mechanisms other than theinhibition of apoptosis contributed to the anti-inflam-matory and renal function–improving effects of IGF-1and ZVAD-fmk. Caspase-1– and caspase-3–like activitiesduring I/R-induced apoptosis were significantly pre-vented by IGF-1 or ZVAD-fmk administered at the timeof reperfusion. Whereas caspase-3 activation reflectsmany forms of apoptosis (19), a functional role for cas-pase-1 during apoptosis appears to be restricted to celldeath induced by specific agents such as FasL (20) andgranzyme B (21). Caspase-1, also referred to as IL-1–con-verting enzyme (ICE), is involved in, but not essential to,the maturation of IL-1 and IL-18 (22, 23). Therefore,activated caspase-1–induced release of IL-1 and/or IL-18may contribute to the inflammatory response after I/R(23). However, inhibition of endogenous IL-1 (ref. 24 andour own unpublished observations) and IL-18 (25) onlyminimally decreased renal influx of neutrophils and loss

of kidney function after 1 day of reperfusion when com-pared with administration of IGF-1 or ZVAD-fmk (datanot shown). Finally, it has to be taken into account thatthe measured renal caspase-like activities are likely torepresent a more generalized form of caspase activation,because enzymatic activities of caspase-1 and caspase-3on Ac-YVAD-amc and Ac-DEVD-amc have been shownto be not fully specific (26).

The anabolic effect attributed to IGF-1, putativelyleading to enhanced tissue repair, may add to the ther-apeutic effect of IGF-1 (6). Moreover, IGF-1–inducedrenal nitric oxide (NO) can improve kidney function bydirectly enhancing renal circulation (27), as well as bycontributing to limitation of apoptosis through inhibi-tion of caspases (28). However, direct anti-inflammato-ry effects of IGF-1 have not been reported previously.The present findings show that neither treatment withEGF, a renotropic growth factor with anabolic effectssimilar to IGF-1 (29), nor treatment with IGF-1 orZVAD-fmk after 2 hours of reperfusion attenuated theloss of kidney function or inflammation.

Similar to in vitro observations (7, 30), we furthershowed, by coadministrating the PI 3-kinase inhibitorLY294002, that PI 3-kinase constitutes an essentialcomponent of IGF-1 receptor–mediated, antiapoptoticsignaling in vivo. Taken together, these results demon-strate that the anti-inflammatory effects of IGF-1 andZVAD-fmk are distinctively mediated by antiapoptoticsignaling rather than by inhibition of IL-1 and/or IL-18maturation, anabolic stimulation, or NO-inducedenhanced kidney function. Nevertheless, despite thelack of an apparent functional role for activated cas-pase-1 in our model, our findings do not definitely ruleout the possibility that apoptosis and inflammation are2 largely independent processes that share thus farunknown upstream molecular mechanisms.

The present findings demonstrate that inhibition ofapoptosis after renal I/R prevents subsequent inflam-mation. This apparently shows apoptosis to be critical


Figure 6Posttranslational processing of the EMAP-II protein is induced in par-allel with apoptosis after as little as 2 hours of reperfusion. Westernblots were performed with protein isolated from kidneys in mice thatwere sacrificed after different periods of reperfusion. Incubation withthe EMAP-II antiserum SA 2846 revealed constitutive expression ofinactive pro–EMAP-II, which resulted in a 43-kDa band. After 2 hoursof reperfusion, the presence of 23-kDa mature EMAP-II wasobserved, which was even more apparent after 24 hours of reperfu-sion. Antiapoptotic treatment at the time of reperfusion with IGF-1or ZVAD-fmk evidently inhibited I/R-induced EMAP-II maturation.

for the induction of inflammation in the present model.Our results also demonstrate that I/R induces activationof EMAP-II. Inactive pro–EMAP-II may serve as a targetsubstrate for activated caspases (10), which we presentlyshow to be upregulated as a consequence of I/R. The evi-dent formation of mature EMAP-II coinciding with elab-oration of I/R-induced apoptosis, as well as the observeddecrease in EMAP-II activation after IGF-1 or ZVAD-fmktreatment, is in line with a recent report of Knies et al.(10). This study showed that the release of matureEMAP-II is directly linked to apoptosis and can be abro-gated in vitro by caspase inhibition. Mature EMAP-II isa potent chemokine that stimulates migration of poly-morphonuclear neutrophils and monocytes in vivo,release of tissue factor on endothelial cells (31), andTNF-α from monocytes (32). Its peptide sequenceexhibits strong regional homology with other proin-flammatory mediators such as human von Willebrandfactor antigen II (33) and IL-8 (34). Furthermore, it wasrecently demonstrated that mediators of apoptosis cancleave aminoacyl-tRNA synthetase, which subsequentlyleads to release of proinflammatory cleavage productssimilar to EMAP-II, like the NH2-terminal catalyticdomain of tyrosyl tRNA synthetase (35).

The present data suggest that EMAP-II maturation,as a consequence of I/R-induced apoptosis, con-tributes to recruitment of inflammatory cells to thereperfused kidney. However, more studies are neededto determine the functional role of EMAP-II in I/R andto elucidate other mechanisms by which apoptosisinduces an inflammatory response. The absence ofsufficient phagocytosis of apoptotic cells has beenassociated with further degeneration, and ultimatelynecrosis, of apoptotic cells, a process termed second-ary necrosis (36, 37). This secondary necrosis has beensuggested to occur when the extent of necrosis over-whelms the phagocytic capacity of the tissue (38), andit appears to be another important link between apop-tosis and inflammation. It is tempting to concludethat phagocytosis cannot deal sufficiently with suddenwidespread apoptosis within an organ subjected toprolonged ischemia, which ultimately leads to sec-ondary necrosis and inflammation.

Many documented attempts to directly reduce the I/R-induced inflammatory response in various models haveunmasked inflammation as a significant cause of subse-quent tissue injury and dysfunction (1, 14). The presentstudies indicate that early apoptosis is a crucial event inthe process that initiates inflammation and subsequenttissue injury. This is because abrogation of early I/R-induced apoptosis prevents the development of inflam-mation and organ dysfunction. This concept of apopto-sis-induced inflammation after I/R puts forth new,important opportunities to effectively prevent clinicalmanifestations of reperfusion injury. Such therapies mayinclude, prevention of complications arising from the useof ischemically damaged donor organs, cardiopulmonarybypass surgery, aortic cross-clamping, and circulatoryshock. Agents such as IGF-1 (which can be safely admin-

istered to humans) and ZVAD-fmk, as well as new anti-apoptotic substances currently under investigation, mayprovide new therapeutic means to treat these conditions.

AcknowledgmentsWe thank F. le Noble (Department of Physiology, Uni-versity of Maastricht) and P. Frederik and P. Bomans(both from Electron Microscopy Unit, University ofMaastricht) for expert technical assistance. This studywas supported by grant C98.1719 from the Dutch Kid-ney Foundation, and by grant PL962107 from theEuropean Community Biotechnology program.

1. Vedder, N.B., et al. 1990. Inhibition of leukocyte adherence by anti-CD18monoclonal antibody attenuates reperfusion injury in the rabbit ear.Proc. Natl. Acad. Sci. USA. 87:2643–2646.

2. Shreeniwas, R., et al. 1992. Hypoxia-mediated induction of endothelialcell interleukin-1 alpha. An autocrine mechanism promoting expressionof leukocyte adhesion molecules on the vessel surface. J. Clin. Invest.90:2333–2339.

3. Metinko, A.P., Kunkel, S.L., Standiford, T.J., and Strieter, R.M. 1992.Anoxia-hyperoxia induces monocyte-derived interleukin-8. J. Clin. Invest.90:791–798.

4. Nogae, S., et al. 1998. Induction of apoptosis in ischemia-reperfusion modelof mouse kidney: possible involvement of Fas. J. Am. Soc. Nephrol. 9:620–631.

5. Fadok, V.A., et al. 1998. Macrophages that have ingested apoptotic cellsin vitro inhibit proinflammatory cytokine production throughautocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF.J. Clin. Invest. 101:890–898.

6. Goes, N., Urmson, J., Vincent, D., Ramassar, V., and Halloran, P.F. 1996.Effect of recombinant human insulin-like growth factor-1 on the inflam-matory response to acute renal injury. J. Am. Soc. Nephrol. 7:710–720.

7. Kulik, G., Klippel, A., and Weber, M.J. 1997. Antiapoptotic signalling bythe insulin-like growth factor I receptor, phosphatidylinositol 3-kinase,and Akt. Mol. Cell. Biol. 17:1595–1606.

8. Vlahos, C.J., Matter, W.F., Hui, K.Y., and Brown, R.F. 1994. A specificinhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269:5241–5248.

9. Yaoita, H., Ogawa, K., Maehara, K., and Maruyama, Y. 1998. Attenuationof ischemia/reperfusion injury in rats by a caspase inhibitor. Circulation.97:276–281.

10. Knies, U.E., et al. 1998. Regulation of endothelial monocyte-activatingpolypeptide II release by apoptosis. Proc. Natl. Acad. Sci. USA.95:12322–12327.

11. Beeri, R., et al. 1995. Rapid DNA fragmentation from hypoxia along thethick ascending limb of rat kidneys. Kidney Int. 47:1806–1810.

12. Lieberthal, W., Menza, S.A., and Levine, J.S. 1998. Graded ATP depletioncan cause necrosis or apoptosis of cultured mouse proximal tubularcells. Am. J. Physiol. 43:F315–F327.

13. Zager, R.A., Iwata, M., Conrad, D.S., Burkhart, K.M., and Igarashi, Y.1997. Altered ceramide and sphingosine expression during the induc-tion phase of ischemic acute renal failure. Kidney Int. 52:60–70.

14. Daemen, M.A.R.C., Van de Ven, W.C.M., Heineman, E., and Buurman,W.A. 1999. Pro- and anti-inflammatory mechanisms in renal reperfusioninjury in mice. Modulation by endogenous tumor necrosis factor alphaand interleukin-10. Transplantation. 67:792–800.

15. Raafat, A.M., et al. 1997. Calcium blockade reduces renal apoptosis dur-ing ischemia reperfusion. Shock. 8:186–192.

16. Ueda, N., Walker, P.D., Hsu, S.M., and Shah, S.V. 1995. Activation of a15-kDa endonuclease in hypoxia/reoxygenation injury without mor-phologic features of apoptosis. Proc. Natl. Acad. Sci. USA. 92:7202–7206.

17. Hirschberg, R., and Ding, H. 1998. Mechanisms of insulin-like growthfactor-I-induced accelerated recovery in experimental ischemic acuterenal failure. Miner. Electrolyte Metab. 24:211–219.

18. Buerke, M., et al. 1995. Cardioprotective effect of insulin-like growth fac-tor I in myocardial ischemia followed by reperfusion. Proc. Natl. Acad. Sci.USA. 92:8031–8035.

19. Lazebnik, Y.A., Kaufmann, S.H., Desnoyers, S., Poirier, G.G., and Earn-shaw, W.C. 1994. Cleavage of poly(ADP-ribose) polymerase by a pro-teinase with properties like ICE. Nature. 371:346–347.

20. Los, M., et al. 1995. Requirement of an ICE/CED-3 protease forFas/APO-1-mediated apoptosis. Nature. 375:81–83.

21. Shi, L., et al. 1996. Activation of an interleukin 1 converting enzyme-dependent apoptosis pathway by granzyme B. Proc. Natl. Acad. Sci. USA.93:11002–11007.

22. Ghayur, T., et al. 1997. Caspase-1 processes IFN-gamma-inducing factorand regulates LPS-induced IFN-gamma production. Nature. 386:619–623.


23. Miwa, K., et al. 1998. Caspase 1-independent IL-1 beta release andinflammation induced by the apoptosis inducer Fas ligand. Nat. Med.4:1287–1292.

24. Haq, M., Norman, J., Saba, S.R., Ramirez, G., and Rabb, H. 1998. Role ofIL-1 in renal ischemic reperfusion injury. J. Am. Soc. Nephrol. 9:614–619.

25. Daemen, M.A.R.C., Van ‘t Veer, C., Wolfs, T.G.A.M., and Buurman, W.A.1999. Ischemia-reperfusion induced IFN-gamma upregulation: involve-ment of IL-12 and IL-18. J. Immunol. 162:5506–5510.

26. Van de Craen, M., Declercq, W., Van den Brande, I., Fiers, W., and Van-denabeele, P. 1999. The proteolytic procaspase activation network: an invitro analysis. Cell Death Differ. In press.

27. Noguchi, S., et al. 1993. Insulin-like growth factor-I ameliorates tran-sient ischemia-induced acute renal failure in rats. J. Pharmacol. Exp. Ther.267:919–926.

28. Li, J., Billiar, T.R., Talanian, R.V., and Kim, Y.M. 1997. Nitric oxidereversibly inhibits seven members of the caspase family via S-nitrosyla-tion. Biochem. Biophys. Res. Commun. 240:419–424.

29. Norman, J., et al. 1987. EGF-induced mitogenesis in proximal tubularcells: potentiation by angiotensin II. Am. J. Physiol. 253:F299–F309.

30. Parrizas, M., Saltiel, A.R., and LeRoith, D. 1997. Insulin-like growth fac-tor 1 inhibits apoptosis using the phosphatidylinositol 3′-kinase andmitogen-activated protein kinase pathways. J. Biol. Chem. 272:154–161.

31. Kao, J., et al. 1994. A peptide derived from the amino terminus ofendothelial-monocyte-activating polypeptide II modulates mononuclear

and polymorphonuclear leukocyte functions, defines an apparentlynovel cellular interaction site, and induces an acute inflammatoryresponse. J. Biol. Chem. 269:9774–9782.

32. Kao, J., et al. 1994. Characterization of a novel tumor-derived cytokine.Endothelial-monocyte activating polypeptide II. J. Biol. Chem.269:25106–25119.

33. Fay, P.J., et al. 1986. Propolypeptide of von Willebrand factor circulates inblood and is identical to von Willebrand antigen II. Science. 232:995–998.

34. Clark, L.I., Schumacher, C., Baggiolini, M., and Moser, B. 1991. Struc-ture-activity relationships of interleukin-8 determined using chemical-ly synthesized analogs. Critical role of NH2-terminal residues and evi-dence for uncoupling of neutrophil chemotaxis, exocytosis, and receptorbinding activities. J. Biol. Chem. 266:23128–23134.

35. Wkasugi, K., and Schimmel, P. 1999. Two distinct cytokines releasedfrom a human aminoacyl-tRNA synthetase. Science. 284:147–150.

36. Lieberthal, W., Triaca, V., and Levine, J. 1996. Mechanisms of deathinduced by cisplatin in proximal tubular epithelial cells: apoptosis vs.necrosis. Am. J. Physiol. 270:F700–F708.

37. Lieberthal, W., and Levine, J.S. 1996. Mechanisms of apoptosis and itspotential role in renal tubular epithelial cell injury. Am. J. Physiol.271:F477–F488.

38. Schwartzman, R.A., and Cidlowski, J.A. 1993. Apoptosis: the biochem-istry and molecular biology of programmed cell death. Endocr. Rev.14:133–151.


Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation

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