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(The FASEB Journal. 2001;15:171-186.)
© 2001 FASEB

Calpain inhibitor I reduces the activation of nuclear factor-{kappa}B and organ injury/dysfunction in hemorrhagic shock

MICHELLE C. McDONALD*, HELDER MOTA-FILIPE*, ANDREW PAUL{dagger}, SALVATORE CUZZOCREA{ddagger}, MAHA ABDELRAHMAN*, STEVEN HARWOOD*, ROBIN PLEVIN{dagger}, PRABAL K. CHATTERJEE*, MUHAMMAD M. YAQOOB* and CHRISTOPH THIEMERMANN*1

* Department of Experimental Medicine and Nephrology, William Harvey Research Institute, St. Bartholomew’s and The Royal London School of Medicine and Dentistry, London EC1M 6BQ, U.K.;
{dagger} Department of Physiology and Pharmacology, University of Strathclyde, SIBS, Glasgow, G40NR, Scotland; and
{ddagger} Institute of Pharmacology, School of Medicine, University of Messina, Messina 98123, Italy

1Correspondence: Department of Experimental Medicine and Nephrology, William Harvey Research Institute, St. Bartholomew’s and The Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, U.K. E-mail: c.thiemermann{at}mds.qmw.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There is limited evidence that inhibition of the activity of the cytosolic cysteine protease calpain reduces ischemia/reperfusion injury. The multiple organ injury associated with hemorrhagic shock is due at least in part to ischemia (during hemorrhage) and reperfusion (during resuscitation) of target organs. Here we investigate the effects of calpain inhibitor I on the organ injury (kidney, liver, pancreas, lung, intestine) and dysfunction (kidney) associated with hemorrhagic shock in the anesthetized rat. Hemorrhage and resuscitation with shed blood resulted in an increase in calpain activity (heart), activation of NF-{kappa}B (kidney), expression of iNOS and COX-2 (kidney), and the development of multiple organ injury and dysfunction, all of which were attenuated by calpain inhibitor I (10 mg/kg i.p.), administered 30 min prior to hemorrhage. Chymostatin, a serine protease inhibitor that does not prevent the activation of NF-{kappa}B, had no effect on the organ injury/failure caused by hemorrhagic shock. Pretreatment (for 1 h) of murine macrophages or rat aortic smooth muscle cells (activated with endotoxin) with calpain inhibitor I attenuated the binding of activated NF-{kappa}B to DNA and the degradation of I{kappa}B{alpha}, I{kappa}Bß, and I{kappa}B{varepsilon}. Selective inhibition of iNOS activity with L-NIL reduced the circulatory failure and liver injury, while selective inhibition of COX-2 activity with SC58635 reduced the renal dysfunction and liver injury caused by hemorrhagic shock. Thus, we provide evidence that the mechanisms by which calpain inhibitor I reduces the circulatory failure as well as the organ injury and dysfunction in hemorrhagic shock include 1) inhibition of calpain activity, 2) inhibition of the activation of NF-{kappa}B and thus prevention of the expression of NF{kappa}B-dependent genes, 3) prevention of the expression of iNOS, and 4) prevention of the expression of COX-2. Inhibition of calpain activity may represent a novel therapeutic approach for the therapy of hemorrhagic shock.—McDonald, M. C., Mota-Filipe, H., Paul, A., Cuzzocrea, S., Abdelrahman, M., Harwood, S., Plevin, R., Chatterjee, P. K., Yaqoob, M. M., Thiemermann, C. Calpain inhibitor I reduces the activation of nuclear factor-{kappa}B and organ injury/dysfunction in hemorrhagic shock.


Key Words: calpain • cyclo-oxygenase • endotoxin • hemorrhage • multiple organ failure • nitric oxide • reperfusion injury


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE NEUTRAL PROTEASE calpain is one of many intracellular proteins, the activity of which depends on intracellular calcium levels. Several isoforms of calpain have been identified, including calpain I (or µ-calpain) and calpain II (or m-calpain), which require low and high micromolar concentrations of calcium for their activation, respectively (see refs 1 , 2 ). After activation by calcium, calpain cleaves a specific subset of cellular proteins including cytoskeletal proteins, membrane receptors, calmodulin binding proteins, G-proteins, protein kinase C (and other enzymes involved in signal transduction), and many transcription factors including nuclear factor-{kappa}B (NF-{kappa}B) (3) . For instance, calpain inhibitor I reduces the degradation of I{kappa}B (I{kappa}B{alpha} or I{kappa}Bß) in the proteasome and hence prevents the translocation of NF-{kappa}B from the cytosol into the nucleus (4 5 6 7 8) . Thus, calpain inhibitor I prevents the expression (e.g., after exposure to endotoxin) of many NF-{kappa}B-dependent genes, including those for inducible nitric oxide synthase (iNOS) (9 10 11 12) and cyclooxygenase-2 (COX-2) (13 , 14) .

One common cause of circulatory shock is the severe blood loss associated with trauma. Despite improvements in intensive care medicine, the mortality of hemorrhagic shock remains very high (15 , 16) . Thus, there is still a great need for new approaches to improve therapy and outcome for patients with hemorrhagic shock (16) . In clinical practice, hemorrhagic shock leads to a delayed vascular decompensation (resulting in severe hypotension) and (in ~ 25% of all patients) the dysfunction or failure of several important organs including lung, kidney, gut, liver, and brain (17) . There is evidence that ischemia (due to reduced blood and oxygen supply during hemorrhage) and reperfusion (during resuscitation) play an important role in the pathophysiology of the multiple organ dysfunction syndrome (MODS) in hemorrhagic shock (see ref 18 ). The activity of calpain(s) is regulated not only by the intracellular levels of calcium, but also by endogenous activators and inhibitors (e.g., calpastatin) (19) . Ischemia and reperfusion lead to an increase in the intracellular levels of calcium and the activation of calpain (20) and to a decline in calpastatin activity (21) . Similarly, tissue trauma leads to a substantial increase in calpain activity (22) . There is evidence that inhibition of calpain I activity reduces the injury associated with ischemia/reperfusion of the brain (23 24 25 26) , liver (27 , 28) , and heart (20 , 29 30 31 32 33) .

Here we investigate the effects of calpain inhibitor I on the organ injury and dysfunction caused by severe hemorrhage and resuscitation in the anesthetized rat. In particular, we investigate the effects of calpain inhibitor I on the renal dysfunction, liver injury, pancreatic injury, intestinal injury, and lung injury associated with hemorrhagic shock. To gain a better insight into the mechanism(s) of action of calpain inhibitor I, we have also investigated 1) the effects of calpain inhibitor I on the activation of NF-{kappa}B in cultured macrophages (binding of NF-{kappa}B to DNA, degradation of I{kappa}B{alpha} or I{kappa}Bß) and rat aortic smooth muscle cells (binding NF-{kappa}B to DNA, degradation of I{kappa}B{alpha}, I{kappa}Bß, and I{kappa}B{varepsilon}) challenged with endotoxin; 2) whether hemorrhage and resuscitation lead to a) an increase in calpain activity (heart) and/or b) the nuclear translocation of p65 (e.g., activation of NF-{kappa}B) in the kidney in vivo; and 3) whether calpain inhibitor I inhibits a) calpain activity, b) the activation of NF-{kappa}B, c) the expression of iNOS and COX-2 protein (kidney) in rats with hemorrhagic shock. Having found that calpain inhibitor I prevents the expression of iNOS and COX-2 protein, we have also investigated whether selective inhibition of either iNOS activity with L-N6-(L-iminoethyl)lysine dihydrochloride (L-NIL) or COX-2 activity with SC58635 attenuates the circulatory failure or the organ injury/dysfunction associated with hemorrhagic shock.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The protocols described below were performed under the guidelines of the Institutional Animal Research Committee, and the care of the animals was in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication No. 85–23, revised 1996).

Surgical procedure
This study was carried out on 131 male Wistar rats (Tuck, Rayleigh, Essex, U.K.) weighing 250–320 g receiving a standard diet and water ad libitum. All animals were anesthetized with thiopentone sodium [120 mg/kg intraperitoneally (i.p.)] and anesthesia was maintained by supplementary injections of thiopentone sodium as required. The trachea was cannulated to facilitate respiration and rectal temperature was maintained at 37°C with a homeothermic blanket. The right femoral artery was catheterized and connected to a pressure transducer (Senso-Nor 840, Senso-Nor, Horten, Norway) to measure phasic and mean arterial blood pressure (MAP) and heart rate (HR), which were displayed on a data acquisition system (MacLab 8e, AD Instruments, Hastings, U.K.) installed on an Apple Macintosh computer. The right carotid artery was cannulated to bleed the animals (see below). The jugular vein was cannulated for the administration of drugs. The bladder was also cannulated to facilitate urine flow. Upon completion of the surgical procedure, cardiovascular parameters were allowed to stabilize for 15 min. Then blood was withdrawn from the catheter placed in the carotid artery in order to achieve a fall in MAP to 50 mmHg within 10 min. Thereafter, MAP was maintained at 50 mmHg for a total period of 90 min by either withdrawal (during the compensation period) or reinjection of blood. At 90 min after initiation of hemorrhage, the shed blood was reinjected into the animal. At the same time, an equivalent volume of Ringers lactate solution was administered over 10 min.

Evaluation of the effects of calpain inhibitor I on the circulatory failure and MODS: experimental design
Five experimental groups were used for these experiments, as follows. 1) Hemorrhage control group: At 30 min prior to hemorrhage, animals were pretreated with saline [1 ml/kg intravenous (i.v.) bolus, n=7]. 2) Hemorrhage calpain inhibitor I group: At 30 min prior to hemorrhage, animals were pretreated with calpain inhibitor I (10 mg/kg i.p., n=7). 3) Hemorrhage chymostatin croup: At 30 min prior to hemorrhage, animals were pretreated with chymostatin (10 mg/kg i.p., n=9). 4) Sham control group: Rats were subjected to the same surgical procedure without causing a hemorrhage (n=5). 5) Sham calpain inhibitor I group: Rats were subjected to the same surgical procedure without causing a hemorrhage, but received calpain inhibitor I (dose regimen as above, n=5).

Evaluation of the effects of the inhibition of iNOS or COX-2 activity on the circulatory failure and MODS: experimental design
The following eight experimental groups were used. 1) Hemorrhage control group: Upon resuscitation with the shed blood, control rats were treated with saline (1 ml/kg i.v. bolus, followed by 1 ml/kg/h i.v., n=12). 2) Hemorrhage L-NIL group: 5 min prior to resuscitation, animals were treated with the selective iNOS inhibitor L-NIL (3 mg/kg i.v., followed by 3 mg · kg-1 · h-1 i.v., n=10). 3) Hemorrhage DMSO: 30 min before hemorrhage, animals were pretreated with DMSO (1 ml/kg, 50% v/v i.p., n=9). 4) Hemorrhage SC58635 group: 30 min before hemorrhage, animals were pretreated with the selective COX-2 inhibitor SC58635 (3 mg/kg i.p., n=8). 5) Sham saline group: Rats were subjected to the same surgical procedure without causing a hemorrhage, but received saline (n=8). 6) Sham L-NIL group: Rats were subjected to the same surgical procedure without causing a hemorrhage, but received L-NIL (dose regimen as above, n=7). 7) Sham DMSO group: Rats were subjected to the same surgical procedure without causing a hemorrhage, but were pretreated with DMSO (n=8). 8) Sham SC58635 group: Rats were subjected to the same surgical procedure without causing a hemorrhage, but received SC58635 (dose regimen as above, n=3).

Quantification of organ function and injury
Four hours after resuscitation (end of the experiment), 1.5 ml of blood was collected into a serum gel S/1.3 tube (Sarstedt, Germany) from the catheter placed in the right carotid artery. The blood sample was centrifuged (1610 g for 3 min at room temperature) to separate serum. All serum samples were analyzed within 24 h by a contract laboratory for veterinary clinical chemistry (Vetlab Services, Sussex, U.K.). The following marker enzymes were measured in the serum as biochemical indicators of multiple organ injury/dysfunction: 1) liver injury was assessed by measuring the rise in serum levels of alanine aminotransferase (ALT, a specific marker for hepatic parenchymal injury) and aspartate aminotransferase (AST, a nonspecific marker for hepatic injury) (34 , 35) ; 2) renal dysfunction was assessed by measuring the rises in serum levels of creatinine (an indicator of reduced glomerular filtration rate, and hence renal dysfunction) and urea (an indicator of impaired excretory function of the kidney and/or increased catabolism). (36) ; 3) the serum level of lipase was determined as an indicator of pancreatic injury (37) .

Light microscopy
Organ (lung, intestine, kidney) biopsies were taken at the end of the experiment. The biopsies were fixed for 1 wk in buffered formaldehyde solution [10% w/v in phosphate-buffered saline (PBS) 0.01 M, pH 7.4] at room temperature, dehydrated by graded ethanol, and embedded in Paraplast (Sherwood Medical, Rahway, N.J.). Sections (7 µm thick) were deparaffinized with xylene, stained with trichromic Van Gieson, and studied using light microscopy (Dialux 22 Leitz).

Immunohistochemical localization of COX-2, iNOS, and p65
The expression of COX-2 and iNOS proteins was evaluated by immunohistochemistry in the kidney of all animals as described previously (38) . In addition, we have evaluated the location of p65 as an indicator of the activation of NF-{kappa}B in vivo. Localization of p65 (Rel A) in the cytoplasm indicates that the NF-{kappa}B heterodimer is still in its ‘dormant’ form and hence located in the cytoplasm. In contrast, localization for p65 in the nucleus indicates that the NF-{kappa}B heterodimer has translocated into the nucleus and is therefore able to activate the transcription of NF-{kappa}B-dependent genes. At the end of the resuscitation period, the relevant organs were fixed in 10% (w/v) buffered formaldehyde and 8 µm sections were prepared from paraffin embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (v/v) H2O2 in 60% (v/v) methanol for 30 min. The sections were permeabilized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the section in 2% normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin. The sections were then incubated overnight with anti-iNOS antibody (1:1000 in PBS, v/v), anti-COX-2 antibody (1:500 in PBS, v/v), or anti-p65 antibody (1:500 in PBS, v/v). Controls included buffer alone or nonspecific purified rabbit immunoglobulin G (IgG). Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin–biotin peroxidase complex.

Culture and stimulation of RAW 264.7 macrophages
RAW 264.7 murine macrophages were obtained from the European Cell Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal calf serum (FCS), 2 mM glutamine, 250 IU/ml penicillin, and 250 µg/ml streptomycin at 37°C in a humidified atmosphere of air/CO2 (19:1). For experimentation, the cells were maintained in DMEM containing 10% (v/v) FCS and stimulated with lipopolysaccharide (LPS; 1 µg/ml) from Escherichia coli serotype 127:B8) as appropriate.

Culture and stimulation of rat aortic smooth muscle cells (RASMCs)
Smooth muscle cells were isolated from the thoracic aortas of male Sprague-Dawley rats (180–200 g) by digestion with collagenase and elastase as described previously (39 , 40) . RASMCs were cultured in DMEM containing 10% FCS and used as previously outlined (39 , 40) . For experimentation, the cells were grown to near confluence on 6-well culture plate (I{kappa}B degradation experiments) or 10 cm dishes (DNA binding experiments) and rendered quiescent by serum deprivation for 48 h.

Immunoblotting
Cells were incubated with vehicle, agents, or LPS, as appropriate, washed twice in ice-cold PBS, and solubilized in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (70°C) with repeated dispersion through a 21G needle. The prepared samples were then boiled for 5 min and stored at -20°C until required. Aliquots of cell samples (20–100 µg of protein) were subjected to electrophoresis on 7.5 or 11% (w/v) SDS-PAGE gels and transblotted onto nitrocellulose. The nitrocellulose membranes were incubated for 3 h in 150 mM NaCl, 20 mM Tris pH 7.4, 0.03% (v/v) Tween 20 (NATT) and incubated overnight with either I{kappa}B{alpha}/ß (0.5 µg/ml) or I{kappa}B{varepsilon} (1 µg/ml) or iNOS antibodies (1 µg/ml), as appropriate. After extensive washing, the membranes were incubated with anti-mouse or anti-rabbit horseradish peroxidase-coupled IgG for 90 min and then washed in NATT. The immunoblots were developed using the enhanced chemiluminescence (ECL) detection system (Amersham, Bucks, U.K.).

Assay of NF-{kappa}B activity: electrophoretic mobility shift assay (EMSA): preparation of nuclear extracts
Cells were grown on 6-well plates, exposed to vehicle, agents, or LPS, as appropriate, and reactions were terminated by washing cells twice with ice-cold PBS. Cells were then removed by scraping and transferred to Eppendorf tubes. The cellular material was recovered by centrifugation (13,000 rpm, 1 min) in a bench-top centrifuge and the supernatant was aspirated; the pellet was resuspended in 400 µl of Buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5 mM phenyl methyl sulfonyl fluoride (PMSF), 10 µg/ml each of leupeptin, pepstatin A, and aprotinin) and allowed to swell on ice for 15 min. Twenty-five microliters of 10% (w/v) Nonidet P-40 was added and samples were vortexed for 10 s prior to centrifugation at 13,000 rpm for 30 s. The recovered supernatant was removed and 50 µl of Buffer B (20 mM HEPES, pH 7.9, 25% (v/v) glycerol, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 10 µg/ml each of leupeptin, pepstatin A, and aprotinin) was added to the pellet (crude nuclear material) and agitated for 15 min at 4°C. The samples were then sonicated on ice in a bath-type sonicator (2x30 s) and extracted nuclear material was recovered as the supernatant after centrifugation (13,000 rpmx15 min) at 4°C. Protein content of the recovered nuclear extracts was determined using the Bradford assay.

DNA binding reaction
Nuclear extracts (5 µg) were incubated in binding buffer [10 mM Tris-HCl pH 7.5, 4% (v/v) glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 50 µg/ml poly(dI-dC).poly(dI-dC)] for 15 min prior to addition of 1 µl (~50,000 cpm) of 32P-labeled double-stranded NF-{kappa}B consensus oligonucleotide (5 `-AGT TGA GGG GAC TTT CCC AGG C-3`) (Promega, Southampton, U.K.) for 20–30 min. After incubation, 1 µl of gel loading buffer (10x; 250 mM Tris-HCl pH 7.5, 0.2% (w/v) bromphenol blue, 40% (v/v) glycerol) was added to samples and protein–DNA complexes were resolved by nondenaturing electrophoresis on 5% (w/v) acrylamide slab gels. Gels were initially prerun in (0.5x) Tris borate-EDTA buffer (TBE) for 30 min at 100V; subsequent to loading of samples, electrophoresis was maintained at 100V for 45–60 min. Gels were dried and NF-{kappa}B probe complexes visualized by autoradiography. Specificity of NF-{kappa}B probe complex formation was examined by competitive analysis performed using 25 molar excess of unlabeled double-stranded DNA oligonucleotides in the binding reaction. Unlabeled NF-{kappa}B DNA probe was used as a specific competitor and an unrelated AP-2 double-stranded consensus DNA oligonucleotide (Promega) was used as a nonspecific competitor.

Calpain activity assay
Three experimental groups were used initially: 1) animals were subjected to the surgical procedure without causing a hemorrhage (sham, n=7); 2) hemorrhage group: animals were subjected to 90 min hemorrhage (no resuscitation, n=6); 3) hemorrhage and resuscitation group: animals were pretreated with saline (1 ml/kg) and subjected to 90 min hemorrhage, followed by 4 h resuscitation (n=7). After finding that hemorrhage and resuscitation cause an increase in calpain activity, two additional experimental groups were used: 4) hemorrhage and resuscitation vehicle group—animals were pretreated with vehicle (50% ethanol i.p.) and subjected to 90 min hemorrhage, followed by 4 h resuscitation (n=7); 5) hemorrhage and resuscitation calpain inhibitor I group—30 min prior to hemorrhage, animals were pretreated with calpain inhibitor I (10 mg/kg i.p.) and subjected to 90 min of hemorrhage, followed by 4 h resuscitation (n=6).

The calpain activity assay used is based on those described by Sasaki et al. (41) for measurement in purified porcine kidney and by Edelstein et al. (42) in rat proximal tubules. Briefly, whole rat hearts (derived from the experimental groups shown above) were rapidly removed, and snap frozen in liquid N2, and stored at -80°C. The assay buffer consisted of 63.2 mM imadazole-HCl containing 10 mM of 2-mercaptoethanol (pH 7.3). Calcium-free buffer was prepared in assay buffer containing 20 mM EGTA and 25 mM EDTA (pH 7.3). Calcium buffer consisted of assay buffer with 1.25 mM CaCl2 added (pH 7.3). Freshly thawed hearts were weighed before the addition of chilled calcium-free buffer (5 ml/g tissue). Tissue was disrupted with a sintered glass homogenizer prior to centrifugation (14000 g, 4°C, 30 min, Sorval RMC 14). The assay was then performed on this supernatant after it was diluted fivefold in calcium-free buffer. To four tubes containing 500 µl of diluted supernatant, one pair had 1.5 ml of calcium-free buffer added; the other pair had the same volume of calcium buffer. After a 10 min preincubation shaking in water bath at 37°C, 10 µl of the substrate N-succinyl-Leu-Tyr-7-amino-4-methyl coumarin (10 mM in DMSO) was added to all tubes. After a further 30 min incubation period, fluorescence was detected at 380 excitation and 460 nm emission. Calpain activity was determined as the difference between the calcium-dependent fluorescence and the non-calcium-dependent fluorescence. A 7-amino-4-methyl coumarin (AMC) standard curve was constructed for each assay containing the same concentration of added DMSO as the samples. Calpain activity was expressed as nanomoles of AMC released per minute of incubation time per minute of total protein. Protein was determined by Bradford assay.

Materials
Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Company Ltd. (Poole, Dorset, U.K.). Thiopentone sodium (Intraval Sodium) was obtained from Rhône Mérieux Ltd. (Harlow, Essex, U.K.). Biotin blocking kit, biotin-conjugated goat anti-rabbit IgG, primary anti-iNOS, anti-COX-2, and avidin–biotin peroxidase complex were obtained from DBA (Milan, Italy). Calpain inhibitor I was purchased from Calbiochem Novabiochem (Nottingham, U.K.). A polyclonal antibody to iNOS was purchased from Affiniti Research Products Ltd. (Exeter, England). Antibodies to I{kappa}B{alpha}, I{kappa}Bß, I{kappa}B{varepsilon}, and p65 were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). ECL detection reagents were purchased from Amersham International (Little Chalfont, Bucks., U.K.) and all cell culture reagents were supplied by Life Technologies, Inc. (Paisley, Scotland, U.K.). L-N6-(L-iminoethyl)lysine dihydrochloride was obtained from Alexis Corporation (Nottingham, U.K.). All other chemicals were of the highest commercial grade available. AMC was from ICN Pharmaceuticals Ltd. (Basingstoke, Hampshire, U.K.). All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl; Baxter Healthcare Ltd., Thetford, Norfolk, U.K.).

Statistical evaluation
All data are presented as means ± SE of n observations, where n represents the number of animals or blood samples studied. For repeated measurements (hemodynamics), a 2-factorial analysis of variance (ANOVA) was performed. Data without repeated measurements (multiple organ injury/failure) were analyzed by 1-factorial ANOVA, followed by a Dunnett’s test for multiple comparisons. For comparison of two groups (calpain activity), statistical analysis was performed by unpaired Student’s t test. A P value of less than 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of calpain inhibitor I on the delayed vascular decompensation (circulatory failure) caused by hemorrhage and resuscitation
Baseline values of MAP in all groups of animals ranged from 108 ± 5 to 129 ± 6 mmHg and were not significantly different between groups (Table 1 ). In sham-operated rats (no hemorrhage), administration of calpain inhibitor I did not affect MAP (Table 1) . In rats subjected to hemorrhage, resuscitation with shed blood led to an immediate increase in blood pressure from ~ 50 mmHg to 103 ± 5 mmHg. Thereafter, there was a progressive decline in MAP to ~65 mmHg at the end of the experiment (Table 1) . The MAP of rats treated with calpain inhibitor was higher (at the end of the resuscitation period) than in the control group. However, the observed effect of calpain inhibitor I on blood pressure was small and not statistically significant (Table 1 , P>0.05). The protease inhibitor chymostatin had no effect on the fall in MAP associated with hemorrhage and resuscitation (Table 1) .


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  		Table 1. Alterations in mean arterial pressure (MAP) and heart rate (HR)

Baseline values of HR in all groups of animals ranged from 369 ± 10 to 384 ± 11 beats/min (bpm) and were not significantly different between groups (Table 1) . In control animals, administration of calpain inhibitor I did not result in any significant alterations in HR. Hemorrhagic shock also did not cause a significant alteration in HR (Table 1 , P>0.05). Similarly, calpain inhibitor I had no significant effect on HR (Table 1) . The protease inhibitor chymostatin had no effect on HR in rats subjected to hemorrhage and resuscitation (Table 1) .

Effects of calpain inhibitor I on the multiple organ dysfunction syndrome caused by hemorrhage in the rat
Effects on renal dysfunction
In sham-operated animals, administration of saline or calpain inhibitor I did not result in any significant alterations in the serum levels of urea (Fig. 1A ) or creatinine (Fig. 1B ). When compared with sham-operated rats, hemorrhage/resuscitation resulted in significant rises in the serum levels of urea and creatinine, demonstrating the development of renal dysfunction. Pretreatment of rats subjected to hemorrhage and resuscitation with calpain inhibitor I abolished the renal dysfunction caused by hemorrhage (Fig. 1) . The serine protease inhibitor chymostatin caused a small but significant reduction of the rise in the serum levels of creatinine, but did not significantly affect the increase in the serum levels of urea (Fig. 1) .



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  		Figure 1. Serum levels of A) urea and B) creatinine in rats subjected to the surgical procedure without causing a hemorrhage and treated with either saline (sham, open column, n=5) or calpain inhibitor I (sham+Cal-I, n=5). Rats subjected to hemorrhagic shock were treated with either saline (HS, n=7), calpain inhibitor I (HS+Cal-I, n=7), or chymostatin (HS+Chym, n=9). *P<0.05 when compared with HS by ANOVA, followed by Dunnett’s post hoc test.

Effects on liver injury
In sham-operated rats, administration of saline or calpain inhibitor I did not result in any significant alterations in the serum levels of AST (Fig. 2A ) and ALT (Fig. 2B ). When compared with sham-operated rats, hemorrhage/resuscitation resulted in significant rises in the serum levels of AST and ALT, demonstrating the development of hepatocellular injury. Pretreatment of rats subjected to hemorrhage and resuscitation with calpain inhibitor I abolished the liver injury caused by hemorrhage (Fig. 2) . In contrast, chymostatin did not reduce the hepatocellular injury caused by hemorrhagic shock (Fig. 2) .



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  		Figure 2. Serum levels of A) AST and B) ALT in rats subjected to the surgical procedure without causing a hemorrhage and treated with either saline (sham, open column, n=5) or calpain inhibitor I (sham+Cal-I, n=5). Rats subjected to hemorrhagic shock were treated with either saline (HS, n=7), calpain inhibitor I (HS+Cal-I, n=7), or chymostatin (HS+Chym, n=9). *P<0.05 when compared with HS by ANOVA, followed by Dunnett’s post hoc test.

Effects on pancreatic injury
In sham-operated rats administration of saline or calpain, inhibitor I did not result in any significant alterations in the serum levels of lipase (Fig. 3 ). When compared with sham-operated rats, hemorrhage/resuscitation resulted in significant rises in the serum levels of lipase, demonstrating the development of pancreatic injury. Pretreatment of rats subjected to hemorrhage and resuscitation with calpain inhibitor I abolished the pancreatic injury caused by hemorrhage (Fig. 3) . In contrast, chymostatin did not significantly reduce the pancreatic injury caused by hemorrhagic shock (Fig. 3) .



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  		Figure 3. Serum levels of lipase in rats subjected to the surgical procedure without causing a hemorrhage and treated with either saline (sham, open column, n=5) or calpain inhibitor I (sham+Cal-I, n=5). Rats subjected to hemorrhagic shock were treated with either saline (HS, n=7), calpain inhibitor I (sham+Cal-I, n=7), or chymostatin (HS+Chym, n=9). *P<0.05 when compared with HS by ANOVA, followed by Dunnett’s post hoc test.

Effects of calpain inhibitor I on the injury (histological evaluation) of the kidney, lung, liver, and intestine of rats subjected to hemorrhage and resuscitation
When compared to organs obtained from sham-operated rats that had not been subjected to hemorrhage and resuscitation, the kidneys, lungs, and the intestine of rats subjected to hemorrhage and resuscitation showed substantial histological alterations consistent with shock-induced organ injury. Most notably, the degree of organ injury was reduced in the lungs (Fig. 4 ), kidneys (Fig. 5 ), and the intestine (Fig. 6 ) of rats pretreated with calpain inhibitor I and subjected to hemorrhage and resuscitation.



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  		Figure 4. Representative histological sections of a lung obtained from rats subjected to A) sham operation (no hemorrhage), B) hemorrhage for 1.5 h and resuscitation with shed blood (for 4 h), and C) hemorrhage for 1.5 h that were pretreated with calpain inhibitor I. Please note that hemorrhage and resuscitation resulted in the following histological signs of tissue injury and inflammation: changes in the architecture of the alveoli, extravasation of red blood cells, and infiltration of inflammatory cells. These changes in morphology were less pronounced in rats that had been pretreated with calpain inhibitor I.



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  		Figure 5. Representative histological sections of a kidney obtained from rats subjected to A) sham operation (no hemorrhage), B) hemorrhage for 1.5 h and resuscitation with shed blood (for 4 h), and C) hemorrhage for 1.5 h that were pretreated with calpain inhibitor I. Please note that hemorrhage and resuscitation resulted in necrosis and vacuolization of tubular cells, the degree of which was reduced by calpain inhibitor I.



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  		Figure 6. Representative histological sections of the intestine obtained from rats subjected to A) sham operation (no hemorrhage), B) hemorrhage for 1.5 h and resuscitation with shed blood (for 4 h), and C) hemorrhage for 1.5 h that were pretreated with calpain inhibitor I. Please note that hemorrhage and resuscitation resulted in the following histological signs of tissue injury and inflammation in the distal ileum: changes in the architecture of the villi and infiltration of inflammatory cells. These changes in morphology were less pronounced in rats that had been pretreated with calpain inhibitor I.

Effects of calpain inhibitor I on the activation of NF-{kappa}B in the kidney of rats subjected to hemorrhage and resuscitation
In the kidneys of sham-operated animals, the staining for p65 was limited to the cytoplasm of renal cells (Fig. 7 ). In the kidneys of rats subjected to hemorrhage and resuscitation, there was staining for p65 in the nuclei of renal cells indicating translocation of NF-{kappa}B to the nucleus. In rats subjected to hemorrhage and resuscitation that had been pretreated with calpain inhibitor I, there was less staining for p65 in the nuclei of renal cells (Fig. 7) .



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  		Figure 7. Representative sections of kidneys obtained from rats subjected to A) sham operation (no hemorrhage), B) hemorrhage for 1.5 h and resuscitation with shed blood (for 4 h), and C) hemorrhage for 1.5 h that were pretreated with calpain inhibitor I. In the sections obtained from sham-operated rats (A), positive staining (brown) for p65 was limited to the cytoplasm (arrow). In the sections obtained from rats subjected to hemorrhage and resuscitation (B), positive staining (brown) for p65 was observed in the nuclei of renal cells (arrow). Please note that the staining for p65 observed in renal sections obtained from HS rats treated with calpain inhibitor I was largely limited to the cytoplasm (arrow).

Effects of calpain inhibitor I on the expression of iNOS and COX-2 in the kidney of rats subjected to hemorrhage and resuscitation
When compared to organs obtained from sham-operated rats that had not been subjected to hemorrhage and resuscitation, the kidneys of rats subjected to hemorrhage and resuscitation showed a marked staining for iNOS (Fig. 8A , B ) and COX-2 protein (Fig. 9A , B ). In contrast, the degree of staining for iNOS (Fig. 8C ) and COX-2 protein (Fig. 9C ) was markedly reduced in rats that had been pretreated with calpain inhibitor I prior to the onset of hemorrhage.



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  		Figure 8. Representative histological sections of a kidney obtained from rats subjected to A) sham operation (no hemorrhage), B) hemorrhage for 1.5 h and resuscitation with shed blood (for 4 h), and C) hemorrhage for 1.5 h that were pretreated with calpain inhibitor I. When compared to sham-operated animals (no staining), hemorrhage and resuscitation B) result in substantial renal injury as well as positive (brown) staining for inducible nitric oxide synthase (iNOS, determined by immunohistochemistry), indicating the expression of iNOS protein. Please note that calpain inhibitor I largely attenuated these pathological alterations associated with hemorrhage and resuscitation (C).



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  		Figure 9. Representative histological sections of a kidney obtained from rats subjected to A) sham operation (no hemorrhage), B) hemorrhage for 1.5 h and resuscitation with shed blood (for 4 h), and C) hemorrhage for 1.5 h that were pretreated with calpain inhibitor I. When compared to sham-operated animals (no staining), hemorrhage and resuscitation (B) result in substantial renal injury as well as positive (brown) staining for cyclo-oxygenase-2 (COX-2, determined by immunohistochemistry), indicating the expression of COX-2 protein. Please note that calpain inhibitor I largely attenuated these pathological alterations associated with hemorrhage and resuscitation (C).

Effects of calpain inhibitor I on the activation of NF-{kappa}B and the expression of iNOS protein in macrophages stimulated with endotoxin
Pretreatment of RAW 264.7 macrophages with calpain inhibitor I (1–100 µM, 60 min) prior to exposure of cells to LPS (1 µg/ml) for 2 h resulted in concentration-dependent inhibition of LPS-stimulated NF-{kappa}B DNA binding activity (Fig. 10A ). Pretreatment of macrophages with a maximal concentration of calpain inhibitor I (100 µM, 60 min) also resulted in the inhibition of LPS-stimulated I{kappa}B{alpha} and I{kappa}Bß degradation (Fig. 10B ) at both 30 min and 2 h, respectively, times when LPS-stimulated I{kappa}B isoform degradation is maximal (A. Paul and R. Plevin, unpublished results). Pretreatment with increasing concentrations of calpain inhibitor I also resulted in concentration-dependent inhibition of LPS-stimulated iNOS expression (Fig. 10C ) as assessed by Western blotting.



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  		Figure 10. The effect of calpain inhibitor I on LPS-stimulated NF{kappa}B DNA binding activity, I{kappa}B degradation, and iNOS expression in RAW 264.7 macrophages. A) Cells were pretreated with vehicle (C) or increasing concentrations of calpain inhibitor I (Cal I-1;1–100 µM) for 1 h prior to exposure to LPS (1 µg/ml) for 24 h. Subsequent to preparation of nuclear extracts, NF{kappa}B DNA binding activity was determined by EMSA as described in Materials and Methods. The positions of NF{kappa}B protein–DNA complexes and nonspecific DNA binding complexes (n.s.) are indicated. B) Cells were pretreated with vehicle (C) or Cal I-1 (100 µM) as indicated for 1 h prior to stimulation with LPS (1 µg/ml; L) for 30 min or 2 h and whole cell extracts were prepared. Cellular I{kappa}B{alpha} and I{kappa}Bß contents were then determined by Western blotting as described in Materials and Methods. C) Cells were pretreated with vehicle (C) or increasing concentrations of Cal I-1 (1–100 µM) for 1 h prior to exposure to LPS (1 µg/ml) for 12 h. Whole cell extracts were then prepared and assayed for iNOS expression as outlined in Materials and Methods. All experiments shown are representative of at least 3 others.

Effects of calpain inhibitor I on the activation of NF-{kappa}B in RASMCs stimulated with endotoxin
Pretreatment of RASMCs with calpain inhibitor I (100 µM, 60 min) resulted in the partial inhibition of the LPS-stimulated degradation of I{kappa}B{alpha}, I{kappa}Bß, and I{kappa}B{varepsilon} (Fig. 11 ) at 30 min and 2 h, respectively, times when LPS-stimulated I{kappa}B isoform degradation are maximal (A. Paul, S. Wilson, and R. Plevin, unpublished results). In parallel, pretreatment of RASMCs with calpain inhibitor I (100 µM, 60 min) prior to exposure of cells to LPS (100 µg/ml) for 2 h also resulted in partial inhibition of LPS-stimulated NF-{kappa}B–DNA binding activity (data not shown).



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  		Figure 11. The effect of calpain inhibitor I on LPS-stimulated I{kappa}B degradation in RASMCs. Quiescent RASMCs were pretreated with vehicle (C) or calpain inhibitor I (Cal I-1; 100 µM) for 1 h as indicated prior to exposure to LPS (L; 100 µg/ml) for either 30 min or 2 h, and whole cell extracts were prepared. Cellular I{kappa}B{alpha} (A), I{kappa}Bß (B), and I{kappa}B{varepsilon} (C) content were then determined by Western blotting as described in Materials and Methods. All experiments shown are representative of at least 3 others.

Effects of calpain inhibitor I on the increase in calpain I activity in rats subjected to hemorrhage and resuscitation
When compared to sham-operated rats, hemorrhage followed by resuscitation (for 4 h) resulted in a significant increase in tissue calpain activity (Fig. 12A ). In contrast, hemorrhage alone resulted in a small increase in calpain activity, which was not significant (Fig. 12A ). When compared to rats that had been pretreated with the vehicle for calpain inhibitor I (ethanol 50%, 1 ml/kg i.p.), pretreatment of rats with calpain inhibitor I significantly attenuated the increase in calpain activity caused by hemorrhage and resuscitation (Fig. 12B ).



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  		Figure 12. A) Determination in tissue calpain activity (expressed in nmol AMC/min/mg tissue) in hearts obtained from rats subjected to the surgical procedure without causing a hemorrhage and treated with either saline (sham, n=8) or in rats subjected to hemorrhage for 90 min (H, n=6) or hemorrhage and resuscitation (H/R, n=7). *P<0.05 when compared with sham by ANOVA, followed by Dunnett’s post hoc test. B) Determination in tissue calpain activity in hearts obtained from rats subjected to hemorrhage and resuscitation treated with either DMSO (H/R vehicle, n=7) or calpain inhibitor I (H/R Cal I, n=6). *P<0.05 when compared with H/R vehicle by unpaired Student’s t test.

Effects of the selective iNOS inhibitor L-NIL on the delayed vascular decompensation (circulatory failure) caused by hemorrhage
In rats subjected to hemorrhage, resuscitation with shed blood led to an immediate increase in blood pressure from ~45 mmHg to 118 ± 4 mm Hg. Thereafter, there was a progressive decline in MAP to ~65 mm Hg at the end of the experiment (Fig. 13a ). The selective iNOS inhibitor L-NIL significantly attenuated the delayed fall in MAP associated with hemorrhage and resuscitation (Fig. 13a , P <0.05). In sham-operated rats, neither administration of saline nor administration of the iNOS inhibitor L-NIL had any significant effect on MAP (Fig. 13a ) or HR (Table 2 ). Hemorrhagic shock did not cause a significant alteration in heart rate (Table 2 , P>0.05).



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  		Figure 13. a) Mean arterial blood pressure (MAP) and serum levels of b) urea, c) creatinine, d) AST, e) ALT, and f) lipase in rats subjected to the surgical procedure without causing a hemorrhage and treated with either saline (sham, n=8) or L-NIL (sham+L-NIL, n=7). Rats subjected to hemorrhagic shock were treated with either saline (HS, n=12) or L-NIL (HS+L-NIL, n=10). *P<0.05 when compared with HS by ANOVA, followed by Dunnett’s post hoc test.


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  		Table 2. Alterations in heart rate (HR)

Effects of the selective iNOS inhibitor L-NIL on the multiple organ dysfunction syndrome caused by hemorrhage in the rat
When compared with sham-operated rats, hemorrhage/resuscitation resulted in significant rises in the serum levels of urea, creatinine (renal dysfunction), AST, ALT (liver injury), and lipase (pancreatic injury, Fig. 13b , c , d , e , f ). Treatment of rats subjected to hemorrhage and resuscitation with the selective iNOS inhibitor L-NIL attenuated the rise in the serum levels of ALT (but not of any of the other parameters measured) caused by hemorrhage and resuscitation (Fig. 13b , c , d , e , f ). In sham-operated rats, neither administration of saline nor administration of the selective iNOS inhibitor L-NIL had any effect on the biochemical indicators of organ injury/dysfunction (Fig. 13b , c , d , e , f ).

Effects of the selective COX-2 inhibitor SC58635 on the delayed vascular decompensation (circulatory failure) caused hemorrhage
In rats subjected to hemorrhage (pretreated with DMSO, vehicle for SC58635), resuscitation with shed blood led to an immediate increase in blood pressure from ~45 mmHg to 109 ± 4 mm Hg. Thereafter, there was a progressive decline in MAP to ~70 mm Hg at the end of the experiment (Fig. 14a ). The selective COX-2 inhibitor SC58635 did not affect the delayed fall in MAP associated with hemorrhage and resuscitation (Fig. 14a , P <0.05). In sham-operated rats, neither administration of DMSO (vehicle) nor administration of the selective COX-2 inhibitor SC58635 had any significant effect on MAP (Fig. 14a ) or HR (Table 3 ). Hemorrhagic shock did not cause a significant alteration in heart rate (Table 2 , P>0.05).



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  		Figure 14. a) Mean arterial blood pressure (MAP) and serum levels of b) urea, c) creatinine, d) AST, e) ALT, and f) lipase in rats subjected to the surgical procedure without causing a hemorrhage and treated with either DMSO (sham DMSO, n=8) or SC58635 (sham+SC58635, n=3). Rats subjected to hemorrhagic shock were treated with either DMSO (HS-DMSO, n=9) or SC58635 (HS+SC58635, n=8). *P<0.05 when compared with HS by ANOVA, followed by Dunnett’s post hoc test.


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  		Table 3. Alterations in heart rate (HR)

Effects of the selective COX-2 inhibitor SC58635 on the multiple organ dysfunction syndrome caused by hemorrhage in the rat
When compared with sham-operated rats, hemorrhage/resuscitation (pretreated with DMSO, vehicle for SC58635) resulted in significant rises in the serum levels of urea, creatinine (renal dysfunction), AST, ALT (liver injury), and lipase (pancreatic injury, Fig. 14b , c , d , e , f ). Treatment of rats subjected to hemorrhage and resuscitation with the selective COX-2 inhibitor SC58635 attenuated the rise in the serum levels of creatinine and ALT (but not of any of the other parameters measured) caused by hemorrhage and resuscitation (Fig. 14b , c , d , e , f ). In sham-operated rats, neither administration of DMSO nor administration of the selective COX-2 inhibitor SC58635 had any effect on the biochemical indicators of organ injury/dysfunction (Fig. 14b , c , d , e , f ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The progression of shock to multiple organ failure (or MODS) is associated with an increase in mortality such that with the number of organs failing (from one to four), mortality progressively increases from 30% (in the absence of MODS) to 100% (34) . Hemorrhage for 90 min followed by resuscitation with shed blood (for 4 h) resulted in a substantial increase in the serum levels of urea and creatinine, indicating the development of renal dysfunction. Hemorrhage and resuscitation also caused an increase in the serum levels of the transaminases AST and ALT, indicating the development of hepatocellular injury. Hemorrhage and resuscitation were also associated with an increase in the serum levels of lipase, and thus pancreatic injury. We also demonstrate that the model of hemorrhagic shock used here causes a substantial degree of tissue injury to the lung, kidney, and intestine. In addition, we have recently reported that the protocol of severe hemorrhage and resuscitation used here leads to histological signs of liver injury as well as the nitrosylation of proteins (secondary to the formation of peroxynitrite) in the lung, kidney, intestine, and liver (43) . This study provides the first evidence that pretreatment of rats with calpain inhibitor I attenuates the 1) renal dysfunction and injury, 2) liver injury, 3) pancreatic injury, 4) intestinal injury, and 5) lung injury caused by hemorrhage and resuscitation. The ensuing paragraphs discuss the potential mechanism(s) by which calpain inhibitor I reduces the organ injury and dysfunction in hemorrhagic shock.

Inhibition of protease activity
One could argue that some of the effects of calpain inhibitor I are due to the ability of this agent to inhibit the activity of serine proteases. This is unlikely, however, as chymostatin, a potent inhibitor of serine proteases (44) , did not affect the liver (rise in serum levels of AST and ALT) or pancreatic injury (rise in serum lipase) caused by hemorrhage and resuscitation. Although chymostatin did not affect the rise in the serum levels of urea, chymostatin caused a small reduction in the serum levels of creatinine. Taken together, these results support the view that an inhibition of protease activity is unlikely to account for the beneficial effects of calpain inhibitor I observed in this study.

Inhibition of the activation of NF-{kappa}B
This study demonstrates that calpain inhibitor I attenuates the activation of NF{kappa}B (the binding of activated NF-{kappa}B to DNA) caused by endotoxin in cultured macrophages in a concentration-dependent fashion. In these cells, calpain inhibitor I also prevented the degradation of I{kappa}B{alpha} and I{kappa}Bß demonstrating that calpain inhibitor I indeed reduces the proteolytic cleavage of this inhibitor protein in the proteasome. We believe that the ability of calpain inhibitor I to prevent the activation of NF-{kappa}B is not limited to macrophages. To support this hypothesis, we show that calpain inhibitor I also attenuates the activation of NF-{kappa}B (the binding of activated NF-{kappa}B to DNA) caused by endotoxin in cultured RASMCs in a concentration-dependent fashion. In these cells, calpain inhibitor I also prevented the degradation of I{kappa}B{alpha}, I{kappa}Bß, and I{kappa}B{varepsilon} demonstrating that calpain inhibitor I indeed reduces the proteolytic cleavage of this inhibitor protein in the proteasome of RASMCs. We show here for the first time that hemorrhage, followed by 4 h of resuscitation (but not hemorrhage alone), leads to the translocation of p65 (and therefore of the heterodimer) from the cytosol to the nucleus of renal cells. This finding suggests that the protocol of hemorrhage and resuscitation used in our study leads to the activation of NF-{kappa}B in the kidney. This conclusion is also supported by our finding that hemorrhage and resuscitation lead to the expression of proteins (iNOS and COX-2; see below), the genes of which are regulated by NF-{kappa}B.

Prevention of the expression of iNOS
Activation of the transcription factor NF-{kappa}B plays an important role in the expression of iNOS (9 , 10 , 37 , 45 , 46) . An enhanced formation of NO by iNOS may contribute to the circulatory failure and the organ dysfunction associated with hemorrhagic shock (47 , 48) . We demonstrate here that the prevention by calpain inhibitor I of the activation of NF-{kappa}B is associated with a concentration-dependent reduction in the expression of iNOS protein in macrophages activated with endotoxin. Most notably, we demonstrate that calpain inhibitor I abolishes the expression of iNOS protein in the kidney of rats subjected to severe hemorrhage and resuscitation. Thus, it is possible that the prevention by calpain inhibitor I of the expression of iNOS may contribute to the beneficial effects of calpain inhibitor I in hemorrhagic shock. To elucidate the contribution of an enhanced formation of NO by iNOS to the circulatory failure or the multiple organ injury and dysfunction caused by hemorrhage and resuscitation in the rat, we have investigated the effects of a selective inhibitor of iNOS activity (L-NIL) in this model. We document that L-NIL attenuates the delayed fall in blood pressure as well as the rise in serum levels of ALT caused by hemorrhagic shock. These findings support the conclusion that an enhanced formation of NO by iNOS contributes to the circulatory failure and to the liver injury caused by hemorrhagic shock in the rat. One could argue that the dose of L-NIL used in this study did not cause a maximal inhibition of iNOS activity. This is unlikely, however, as the dose of L-NIL used here abolishes iNOS activity in Wistar rats challenged with endotoxin (49) . Taken together, these results support the view that an enhanced formation of NO from iNOS contributes to, but does not account for, the circulatory failure and multiple organ injury/dysfunction caused by hemorrhagic shock.

Prevention of the expression of COX-2
The promotor region of the murine and human COX-2 genes contain binding sites for NF-{kappa}B (50 , 51) . The expression of the COX-2 gene is activated by oxidant stress (52) , and reactive oxygen intermediates cause the activation of NF-{kappa}B (53) , suggesting that NF-{kappa}B is one of the transcription factors involved. The increase in prostaglandin formation (COX activity) by murine osteoblasts (cell line MC3T3-E1) involves the activation of NF-{kappa}B (13) . Although severe hemorrhage leads to an enhanced formation of prostaglandins, it is unclear whether this is due to induction of COX-2 (54) . We report here for the first time that hemorrhage and resuscitation lead to the expression of COX-2 protein. We also demonstrate that calpain inhibitor I attenuates the expression of COX-2 protein (in the kidney) caused by hemorrhagic shock in the rat. We document that the selective COX-2 inhibitor SC 58635 (55) attenuates the rise in the serum levels of creatinine and ALT caused by hemorrhagic shock. These findings support the view that an enhanced formation of arachidonic acid metabolites by COX-2 contributes to the renal dysfunction and the liver injury caused by hemorrhagic shock in the rat. It should be emphasized that the dose of SC58635 used in this study abolishes COX-2 activity in Wistar rats challenged with endotoxin (55) .

Inhibition of calpain activity by calpain inhibitor I
Organ ischemia and reperfusion (during resuscitation) occur during episodes of hemorrhage and resuscitation and contribute to organ injury (18) . Both ischemia/reperfusion and tissue trauma lead to the activation of calpain (20 , 22) . We report here for the first time that hemorrhage and resuscitation, but not hemorrhage alone, lead to a significant increase in tissue calpain activity. We also document that the dose of calpain inhibitor I used in our study abolishes the rise in calpain activity caused by hemorrhage and resuscitation. Thus, it is possible that prevention of calpain activity contributes to the beneficial effects of calpain inhibitor I in hemorrhagic shock. There is now good evidence that the inhibition of calpain I activity also reduces the injury associated with ischemia/reperfusion of the brain (23 24 25 26) , liver (27 , 28) , and heart (20 , 29 30 31 32) . The mechanism by which inhibitors of calpain activity protect tissues/organs against reperfusion injury is not entirely clear. Calpain acts on several substrates, causing proteolytic modifications of proteins that result in changes in their biochemical and morphological parameters, which are highly likely to be implicated in the pathological processes associated with ischemia/reperfusion injury. Activation of calpain results in the proteolysis of several cellular proteins, associated mostly with the cellular membrane, including cytoskeletal proteins (e.g., spectrin, fodrin, and microtubule-associated proteins), membrane proteins (e.g., growth factor receptors, adhesion molecules, and ion transporters), enzymes (kinases, phosphatases, and phospholipases), as well as cytokines and transcription factors (see ref 2 ). Although many of these are implicated in mechanisms contributing to ischemia/reperfusion injury, the exact role of calpain activation in postischemic tissues has not been clearly defined. In the brain, ischemia of hippocampal neurons triggers the proteolysis of cytoskeletal spectrin (a preferred substrate of calpain, therefore often used as one indicator of calpain activation) and the inhibition of this proteolysis protects neurons against cytotoxicity (56 , 57) . Hypoxia of rat cardiac myocytes results in increased calpain activation (indicated by increased accumulation of spectrin breakdown products), which is inhibited by calpain inhibitor I and E64 (58 , 59) . The detrimental effects of calpain activation in the heart have been suggested to be secondary to the proteolysis of cytoskeletal structures (58 , 59) . It has been proposed that the activation of calpain in livers subjected to ischemia/reperfusion injury leads to tissue injury due to 1) degradation of vital cell membrane and cytoskeletal structure proteins, 2) activation of protein kinase C, and 3) initiation of apoptosis (27) . Taken together, the exact role of calpain I in the pathophysiology of reperfusion injury is not clear. Similarly, the mechanism of the protective effect of calpain inhibitor I against the injury arising from organ ischemia is unclear and warrants further investigation.

In conclusion, this study demonstrates for the first time that calpain inhibitor I, but not the serine protease inhibitor chymostatin, attenuates the 1) renal dysfunction and injury; 2) hepatocellular injury, 3) lung injury, 4) intestinal injury, 5) pancreatic injury caused by severe hemorrhage and resuscitation in the anesthetized rat. In this study, we also provide the first evidence that hemorrhage and resuscitation lead to an increase in calpain activity as well as activation of the transcription factor NF-{kappa}B. We provide evidence that the mechanisms by which calpain inhibitor reduces the circulatory failure as well as the organ injury and dysfunction in hemorrhagic shock include 1) inhibition of calpain activity, 2) inhibition of the activation of NF-{kappa}B, and thus prevention of the expression of NF-{kappa}B-dependent genes, 3) prevention of the expression of iNOS, and 4) prevention of the expression of COX-2. Our results support the view that calpain inhibitor I may be useful in the therapy of hemorrhagic shock.


   ACKNOWLEDGMENTS
 
H.M.F. was funded by a postdoctoral grant provided by the Portuguese Fundação para a Ciência e Tecnologia (Praxis XXI/BPD/16333/98). M.C.M. and P.K.C. are recipients of a Ph.D. studentship/fellowship provided by the Joint Research Board of St. Bartholomew’s Hospital Medical College (G7Z4/XMLA). C.T. is a Senior Fellow of the British Heart Foundation (FS 96/018).

Received for publication June 16, 2000. Revision received July 10, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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  4. Baeuerle, P