calpain inhibitor-1 reduces renal ischemia/reperfusion injury in the rat

Kidney International, Vol. 59 (2001), pp. 2073–2083

Calpain inhibitor-1 reduces renal ischemia/reperfusion injuryin the rat

PRABAL K. CHATTERJEE, PAUL A.J. BROWN, SALVATORE CUZZOCREA, KAI ZACHAROWSKI,KEITH N. STEWART, HELDER MOTA-FILIPE, MICHELLE C. MCDONALD,and CHRISTOPH THIEMERMANN

Department of Experimental Medicine and Nephrology, The William Harvey Research Institute, and the Royal London Schoolof Medicine and Dentistry, London, England, and Department of Pathology, University Medical Buildings, Aberdeen, Scotland,United Kingdom; Institute of Pharmacology, University of Messina School of Medicine, Messina, Italy; and Laboratory ofPharmacology, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal

Calpain inhibitor-1 reduces renal ischemia/reperfusion injury Renal ischemia is one of the most common causes ofin the rat. acute renal failure (ARF), initiating a complex and inter-

Background. Activation of the cysteine protease calpain has related sequence of events, resulting in injury to and thebeen implicated in renal ischemia/reperfusion (I/R) injury. The

eventual death of renal cells [1, 2], with proximal tubuleaim of this study was to investigate the effects of calpain inhibi-(PT) cells demonstrating particular susceptibility [2, 3].tor-1 (Cal I-1) in an in vivo model of renal I/R injury.Although reperfusion is essential for the survival of isch-Methods. Male Wistar rats were administered Cal I-1 (10

mg/kg, IP) 30 minutes before undergoing bilateral renal ischemic tissue, there is good evidence that reperfusion itselfemia (45 minutes) followed by reperfusion (6 hours). Plasma causes additional cellular injury (reperfusion injury) [3],concentrations of urea, creatinine, Na1, g-glutamyl transferase which has been attributed to the generation of reactive(gGT), aspartate aminotransferase (AST) and urinary Na1,

oxygen species (ROS), adenosine 59-triphosphate (ATP)glutathione S-transferase (GST), and N-acetyl-b-d-glucosami-depletion, neutrophil infiltration, phospholipase activa-nidase (NAG) were measured for the assessment of renal dys-

function and I/R injury. Creatinine clearance (CCr) and frac- tion and membrane lipid alterations, cytoskeletal dys-tional excretion of Na1 (FENa) were used as indicators of function, and intracellular Ca21 accumulation [3–5]. Nu-glomerular and tubular function, respectively. Kidney myeloper- merous studies have demonstrated a role for Ca21 inoxidase (MPO) activity and malondialdehyde (MDA) levels the pathophysiology of renal injury associated with bothwere measured for assessment of neutrophil infiltration and

hypoxia and ischemia/reperfusion (I/R) [5, 6]. Intracellu-lipid peroxidation, respectively. Renal sections were used forlar accumulation of Ca21 under ischemic conditions re-histologic grading of renal injury and for immunohistochemical

localization of inducible nitric oxide synthase (iNOS) and sults in the activation of Ca21-dependent enzymes suchcyclooxygenase-2 (COX-2). as calpain and nitric oxide synthase (NOS), which are

Results. Cal I-1 significantly reduced I/R-mediated increases implicated in the pathophysiology of ARF [6, 7].in urea, creatinine, gGT, AST, NAG, and FENa and significantly Calpains are nonlysosomal neutral cysteine proteasesimproved CCr. Cal I-1 also significantly reduced kidney MPO

that are activated by Ca21 and that appear to be involvedactivity and MDA levels. Cal I-1 also reduced histologic evi-in Ca21 signaling in mammalian cells [8]. To date, two majordence of I/R-mediated renal damage and caused a substantial

reduction in the expression of iNOS and COX-2, both of which isoforms have been identified: calpain I (or m-calpain) andinvolve activation of nuclear factor-kB (NF-kB). calpain II (or m-calpain), which require low (mmol/L)

Conclusions. These results suggest that Cal I-1 reduces the and high (mmol/L) Ca21 concentrations for activation,renal dysfunction and injury associated with I/R of the kidney. respectively [8]. Following activation, calpain selectivelyWe suggest that the mechanism could involve the inhibition

cleaves a specific subset of cellular proteins, includingof I/R-mediated activation of NF-kB.cytoskeletal and membrane proteins, several enzymes,and transcription factors [9], including nuclear factor-kB(NF-kB) [10], which is involved in the expression ofKey words: kidney injury, inducible nitric oxide synthase, COX-2, nu-

clear factor-kB, acute renal failure. including inducible NOS (iNOS) [11] and cyclooxygen-ase-2 (COX-2) [12].Received for publication August 11, 2000

Calpain inhibitor-1 (Cal I-1) is a cell-permeable pep-and in revised form November 30, 2000Accepted for publication December 26, 2000 tide aldehyde that blocks the active site of calpain

[13, 14]. Along with other inhibitors of calpain such as 2001 by the International Society of Nephrology

2073

Chatterjee et al: Cal I-1 reduces renal I/R injury2074

E64 or PD150606 and the naturally occurring calpain surgically for renal I/R as described previously [29].Briefly, anesthetized rats were placed onto a thermostati-inhibitor protein calpastatin, Cal I-1 has been used to

investigate the role of the excessive activation of calpain cally controlled heating mat (Harvard Apparatus Ltd.,Kent, UK), and body temperature was maintained at 38 6in the pathophysiology of a variety of disorders, including

cataract, restenosis, arthritis, stroke, and myocardial in- 18C by means of a rectal probe attached to a homeother-mic blanket. A tracheotomy was performed to maintainfarction [13, 14]. Several studies have demonstrated that

calpain activation is implicated in the pathophysiology airway patency and to facilitate spontaneous respiration.The right carotid artery was cannulated (PP50, I.D. 0.58of I/R injury in several organs, including the brain [15],

heart [16], and liver [17]. Interestingly, I/R has also been mm; Portex, Kent, UK) and connected to a pressuretransducer (Senso-Nor 840, Horten, Norway) for thereported to attenuate calpastatin activity [18]. In the

kidney, there is evidence from in vitro studies using rat measurement of mean arterial blood pressure (MAP)and derivation of the heart rate (HR) from the pulseand rabbit PT suspensions that calpain activation is in-

volved in the cellular injury/death associated with hyp- waveform, which were displayed on a data-acquisitionsystem (MacLab 8e, AD Instruments, Hastings, UK)oxia [19, 20] and exposure to toxic agents [21, 22], and

that calpain inhibitors may be beneficial under these installed on an Apple Macintosh computer. MAP andHR were monitored for the duration of each experiment.conditions [19, 20, 22]. Although there is some evidence

that calpain is activated in the ischemic rat kidney in The jugular vein was cannulated (PP25, I.D. 0.40 mm;Portex) for the administration of drugs. A midline lapa-vivo [23], the beneficial actions of calpain inhibitors in

animal models of renal I/R remain unclear [24]. rotomy was performed and the bladder was cannulated(PP90, I.D. 0.76 mm; Portex). Both kidneys were located,Several studies have demonstrated that Cal I-1 can

reduce the proteolytic cleavage of IkB-a/b from its com- and the renal pedicles, containing the artery, vein, andnerve supplying each kidney, were carefully isolated.plex with the NF-kB/Rel A unit and, hence, inhibit the

activation of NF-kB and its subsequent translocationRenal ischemia/reperfusionfrom the cytosol into the nucleus [25–27]. Thus, Cal I-1

can reduce the expression (for example, after exposure As described previously [29], rats were allowed tostabilize for 30 minutes before they were subjected toto endotoxin or inflammatory cytokines) of many NF-kB–

dependent genes, including iNOS [26–28] and COX-2 bilateral renal occlusion for 45 minutes using artery clipsto clamp the renal pedicles. Reperfusion commenced once[28]. However, to date, such an effect has not been dem-

onstrated in renal tissues in vitro or in vivo. the artery clips were removed (control animals). Occlusionwas verified visually by change in the color of the kidneysTherefore, the aim of this study was to investigate the

effects of Cal I-1 on renal dysfunction/injury caused by to a paler shade and reperfusion by a blush. Other ratswere subjected to sham operation (sham operated) whererenal I/R in the anesthetized rat. In order to gain a better

insight into the mechanism of action of Cal I-1, we also identical surgical procedures to control animals wereused, except that they did not undergo bilateral renalinvestigated the effects of Cal I-1 on the expression of

iNOS and COX-2 in kidneys of rats subjected to renal clamping and were maintained under anesthesia for theduration of the experiment. At the end of all experi-I/R. Furthermore, the actions of Cal I-1 were compared

with that of the serine protease inhibitor chymostatin. ments, animals were killed by an overdose of sodiumthiopentone.

METHODS Experimental protocolAnimal preparation Upon completion of surgical procedures, the animals

were randomly allocated into seven groups.In vivo studies were carried out using 56 male Wistarrats (Tuck, Rayleigh, Essex, UK) weighing 200 to 250 g, (1) I/R control group. These animals underwent renal

ischemia for 45 minutes followed by reperfusion for sixthat received a standard diet and water ad libitum andwere cared for in accordance with both the Home Office hours (N 5 12).

(2) I/R Cal I-1 group. These animals were adminis-Guidance in the Operation of the Animals (Scientific Pro-cedures) Act 1986, published by H.M.S.O. (London, UK) tered Cal I-1 (10 mg/kg IP) 30 minutes prior to I/R as

described previously (N 5 12). Cal I-1 was dissolved inand the Institutional Animal Research Committee Guidefor the Care and Use of Laboratory Animals published by 50% (vol/vol) EtOH and 50% (vol/vol) saline. We have

previously shown this dose and method of administrationthe U.S. National Institutes of Health (NIH publicationnumber 85-23, revised 1996). All animals were anesthe- of Cal I-1 to (a) inhibit an increase in calpain activity [28],

(b) reduce the expression of iNOS and COX-2 [28, 30],tized with sodium thiopentone (Intravalt Sodium, 120mg/kg IP; Rhone Merieux Ltd., Essex, UK), and anesthe- and (c) to attenuate the activation of NF-kB [30] associ-

ated with endotoxic and hemorrhagic shock in the ratsia was maintained by supplementary intravenous infu-sions of sodium thiopentone. Animals were prepared [28, 30].

Chatterjee et al: Cal I-1 reduces renal I/R injury 2075

(3) I/R chymostatin group. These animals were admin- described method [35]. Briefly, at the end of the experi-ments, kidney tissue was weighed and homogenized inistered chymostatin (10 mg/kg IP) 30 minutes prior toa solution containing 0.5% (wt/vol) hexadecyltrimethy-I/R (N 5 6). Chymostatin was dissolved in 50% (vol/lammonium bromide dissolved in 10 mmol/L potassiumvol) EtOH and 50% (vol/vol) saline.phosphate buffer (pH 7.4) and centrifuged for 30 minutes(4) I/R vehicle group. These animals were adminis-at 20,000 3 g at 48C. An aliquot of supernatant was thentered the vehicle for Cal I-1 and chymostatin [50%removed and added to a reaction mixture containing 1.6EtOH/50% saline (vol/vol) IP] 30 minutes prior to I/Rmmol/L tetramethylbenzidine and 0.1 mmol/L hydrogen(N 5 6).peroxide (H2O2). The rate of change in absorbance was(5) Sham group. Sham-operated rats were subjectedmeasured spectrophotometrically at 650 nm. MPO activ-to identical surgical procedures described previously inity was defined as the quantity of enzyme required tothis article, except for renal I/R, and they were main-degrade 1 mmol of H2O2 at 378C and was expressed intained under anesthesia for the duration of the experi-mIU/100 mg wet tissue.ment (that is, 45 minutes 1 6 hours, N 5 12).

(6) Sham Cal I-1 group. These were identical to theDetermination of malondialdehye levels

sham-operated animals except for the pretreatment withLevels of malondialdehyde (MDA) in kidneys wereCal I-1 (10 mg/kg IP) 30 minutes prior to surgery (N 5 4).

determined as an indicator of lipid peroxidation follow-(7) Sham vehicle group. These were identical to sham-ing a protocol described previously [36]. Briefly, kidneyoperated animals except they were pretreated with vehi-tissue was weighed and homogenized in a 1.15% (wt/cle [50% EtOH/50% saline (vol/vol) IP] 30 minutes priorvol) KCl solution. A 100 mL aliquot of homogenate wasto the surgery (N 5 4).then removed and added to a reaction mixture con-All animals in these seven groups received a continu-taining 200 mL 8.1% (wt/vol) lauryl sulfate, 1.5 mL 20%ous infusion of 0.9% (wt/vol) saline (4 mL/kg/h, IV)(vol/vol) acetic acid, 1.5 mL 0.8% (wt/vol) thiobarbituricthroughout the I/R period.acid, and 700 mL distilled water. Samples were thenboiled for 1 hour at 958C and centrifuged at 3000 3 gMeasurement of biochemical parametersfor 10 minutes. The absorbance of the supernatant wasAt the end of the reperfusion period, blood (1 mL)measured spectrophotometrically at 650 nm. MDA lev-samples were collected via the carotid artery. The sam-els were expressed as mmol/L MDA/100 mg wet tissue.ples were centrifuged (6000 r.p.m. for 3 minutes) to sepa-

rate plasma. All plasma samples were analyzed for bio- Histologic evaluationchemical parameters within 24 hours after collection At postmortem, a 5 mm section of kidney was removed(Vetlab Services, Sussex, UK). Plasma concentrations and placed in formalin and processed through to wax.of urea and creatinine were measured as indicators of Five micrometer sections were cut and stained with he-impaired glomerular function [31]. Plasma concentra- matoxylin and eosin. Histologic assessment of tubulartions of g-glutamyl transferase (gGT) and aspartate ami- necrosis was determined semiquantitatively using anotransferase (AST), enzymes that are both located in method modified from McWhinnie et al [37]. Randomthe PT [32], were used as indicators of renal reperfusion cortical fields were observed using a 320 objective. Ainjury (Discussion section). graticule grid (25 squares) was used to determine the

Urine samples were collected during the reperfusion number of line intersects involving tubular profiles. Oneperiod, and the volume of urine produced was recorded. hundred intersections were examined for each kidney,Urine concentrations of creatinine and Na1 were mea- and a score from 0 to 3 was given for each tubular profilesured (Vetlab Services) at the end of the reperfusion involving an intersection: 0 5 normal histology (Fig. 6A);period and were used in conjunction with plasma concen- 1 5 tubular cell swelling, brush border loss, nuclear con-trations to estimate creatinine clearance (CCr) and frac- densation, with up to one third of the tubular profiletional excretion of Na1 (FENa) using standard formulae. showing nuclear loss (Fig. 6B); 2 5 same as for score 1,These were used as respective indicators of glomerular but greater than one third and less than two thirds ofand tubular function during the reperfusion period. Con- the tubular profile show nuclear loss (Fig. 6C); and 3 5centrations of urinary glutathione S-transferase (GST) greater than two thirds of the tubular profile showingand N-acetyl-b-d-glucosaminidase (NAG), specific indi- nuclear loss (Fig. 6D).cators of tubular damage [33, 34], were also measured The total score for each kidney was calculated by the(Laboratory of Pharmacology, University of Lisbon, Lis- addition of all 100 scores with a maximum score of 300.bon, Portugal).

Immunohistochemical analysis of iNOS andDetermination of myeloperoxidase activity COX-2 expression

Myeloperoxidase (MPO) activity in kidneys was used The expression of iNOS and COX-2 proteins was eval-uated in kidneys from untreated (sham-operated), con-as an indicator of neutrophil infiltration using a previously


trol, and animals administered Cal I-1 using immunohis-tochemical protocols described previously [38]. At theend of the experiment, kidneys were bisected, snap fro-zen in liquid nitrogen, and stored at 2708C. When re-quired, samples were thawed, dehydrated using gradedethanol, and embedded in Paraplast, after which 8 mmsections were cut. After deparaffinization, endogenousperoxidase was quenched using 0.3% (vol/vol) H2O2 in60% (vol/vol) methanol for 30 minutes. Sections werepermeablized using 0.1% (vol/vol) Triton X-100 in phos-phate-buffered saline (PBS; 0.01 mol/L, pH 7.4) for 20minutes. Nonspecific adsorption was minimized by incu-bating the section in 2% (vol/vol) normal goat serum(DBA, Milan, Italy) in PBS for 20 minutes. Sequentialincubation for 15 minutes with avidin and biotin (DBA)was used to block endogenous binding sites. The sectionswere then incubated overnight with 1:1000 dilution ofprimary anti-iNOS or anti-COX-2 antibody (DBA) orwith control solutions. Controls included buffer alone ornonspecific purified rabbit IgG (DBA). Specific labelingwas detected with a biotin-conjugated goat anti-rabbitIgG (DBA) and avidin-biotin peroxidase complex (DBA).

To verify the binding specificity for iNOS or COX-2(negative controls), some sections were also incubatedwith only the secondary antibody (no primary antibody).Positive controls for iNOS and COX-2 binding wereperformed on sections of kidney obtained from mice thatwere administered lipopolysaccharide (LPS; 50 mg/kg, IV).

Materials

Fig. 1. Alterations in (A) plasma urea and (B) plasma creatinine con-Unless otherwise stated, all compounds used in thiscentrations during renal ischemia/reperfusion (I/R) in the presence ofstudy were purchased from Sigma-Aldrich CompanyCal I-1 (10 mg/kg IP), chymostatin (Chymo, 10 mg/kg IP), or vehicle

Ltd. (Poole, Dorset, UK). Cal I-1 was purchased from [50% (vol/vol) EtOH/saline, IP]. *P , 0.05 vs. sham-operated group;1P , 0.05 vs. control group (45 minutes of ischemia and 6 hours orCalbiochem Novabiochem (Nottingham, UK). All solu-reperfusion).tions used for in vivo infusions were prepared using non-

pyrogenic saline [0.9% (wt/vol) NaCl; Baxter HealthcareLtd., Thetford, Norfolk, UK].

RESULTSStatistical analysis

Effect of Cal I-1 and chymostatin on ischemia/All values described in the text and figures are ex- reperfusion-mediated glomerular dysfunction

pressed as mean 6 SEM for N observations. For in vivo Animals that underwent renal I/R exhibited significantstudies, each data point represents biochemical measure- increases in the plasma concentrations of urea and creati-ments obtained from 6 to 12 separate animals. For his- nine compared with sham-operated animals (Fig. 1) andtologic scoring, each data point represents analysis of a significant reduction in CCr (Fig. 2A), suggesting a sig-kidneys taken from 6 to 12 individual animals. For immu- nificant degree of glomerular dysfunction.nohistochemical analysis, the figures shown are represen- Pretreatment of rats with Cal I-1 prior to I/R producedtative of at least three experiments performed on differ- relatively small, but significant, reductions in the plasmaent experimental days. Statistical analysis was carried levels of urea and creatinine (Fig. 1) and increased CCr

out using GraphPad Prism/InStat (GraphPad Software, significantly (Fig. 2A). The serine protease inhibitor chy-Inc., San Diego, CA, USA). Data were analyzed using mostatin, administered prior to I/R, did not have a sig-one-way analysis of variance (ANOVA) followed by nificant effect on the increased plasma urea and creati-Dunnett’s post hoc test. A P value of less than 0.05 was nine concentrations or the reduced CCr associated with

I/R (Figs. 1 and 2A). The administration of vehicle forconsidered to be significant (NS, nonsignificant).


Fig. 2. Effect of renal I/R on (A) creatinine clearance and (B) fractionalFig. 3. Alterations in (A) urinary glutathione S-transferase (GST) andexcretion of Na in the presence of calpain inhibitor-1 (Cal I-1) (10(B) urinary N-acetyl-b-D-glucosaminidase (NAG) levels during renalmg/kg IP), chymostatin (Chymo, 10 mg/kg IP), or vehicle [50% (vol/I/R in the presence of calpain inhibitor-1 (Cal I-1, 10 mg/kg IP), chymo-vol) EtOH/saline, IP]. *P , 0.05 vs. sham-operated group; 1P , 0.05statin (Chymo, 10 mg/kg IP), or vehicle [50% (vol/vol) EtOH/saline,vs. control group (45 minutes of ischemia and 6 hours of reperfusion).IP]. *P , 0.05 vs. sham-operated group; 1P , 0.05 vs. control group(45 minutes of ischemia and 6 hours of reperfusion).

Cal I-1 and chymostatin [50% (vol/vol) EtOH] to ratsprior to I/R did not result in any significant alterations

improvement in tubular function (Fig. 2B). The adminis-on plasma levels of urea or creatinine or in CCr comparedtration of chymostatin or vehicle did not produce signifi-with control animals (Figs. 1 and 2A). The administrationcant alterations in FENa compared with control animals,of Cal I-1 or vehicle to sham-operated rats did not resultand administration of Cal I-1 to sham-operated rats didin any alteration in plasma levels of urea or creatininenot alter FENa in comparison with values obtained fromor in CCr in comparison with sham-operated animals ad-sham-operated animals administered saline only (Fig. 2B).ministered saline only (data not shown).

Renal I/R produced a significant increase in the uri-Effects of Cal I-1 and chymostatin on ischemia/ nary concentrations of both GST and NAG (Fig. 3),reperfusion-mediated tubular dysfunction/injury suggesting significant tubular injury. The administration

of Cal I-1 significantly reduced both urinary GST andFractional excretion of sodium (FENa), calculated us-NAG concentrations, suggesting attenuation of tubularing plasma and urinary concentrations of Na1 in associa-injury. The administration of chymostatin or vehicle didtion with urine production (urine flow, mL/min), wasnot produce significant alterations in urinary GST orused as an indicator of tubular function. I/R producedNAG concentrations compared with control animalsa significant increase in FENa, suggesting tubular dysfunc-(Fig. 3). The administration of Cal I-1 or vehicle to thetion (Fig. 2B); however, administration of Cal I-1 prior to

I/R produced a significant reduction in FENa, suggesting sham-operated animals did not alter GST or NAG con-


Fig. 5. Effect of renal I/R on kidney (A) myeloperoxidase (MPO)activity and (B) malondialdehyde (MDA) levels in the presence of calpainFig. 4. Alterations in (A) plasma g-glutamyl transferase (gGT) andinhibitor-1 (Cal I-1, 10 mg/kg IP), chymostatin (Chymo, 10 mg/kg IP),(B) plasma aspartate aminotransferase (AST) concentrations duringor vehicle [50% (vol/vol) EtOH/saline]. *P , 0.05 vs. sham-operatedrenal I/R in the presence of calpain inhibitor-1 (Cal I-1, 10 mg/kg IP),group; 1P , 0.05 vs. control group (45 minutes of ischemia and 6 hourschymostatin (Chymo, 10 mg/kg IP), or vehicle [50% (vol/vol) EtOH/of reperfusion).saline, IP]. *P , 0.05 vs. sham-operated group; 1P , 0.05 vs. control

group (45 minutes of ischemia and 6 hours of reperfusion).

The administration of Cal I-1 or vehicle to the sham-operated animals did not alter plasma concentrations ofcentrations in comparison with values obtained from

sham-operated animals administered saline only (data gGT or AST in comparison with values obtained fromsham-operated animals administered saline only (datanot shown).not shown).

Effects of Cal I-1 and chymostatin on ischemia/reperfusion-mediated reperfusion injury Effects of Cal I-1 and chymostatin on kidney MPO

activity and MDA levelsRenal I/R produced significant increases in the plasmaconcentrations of gGT and AST in comparison with val- Rats subjected to renal I/R exhibited a substantial

increase in kidney MPO activity and MDA levels, sug-ues obtained from sham-operated animals (Fig. 4).Plasma concentrations of gGT and AST, which were gesting increased neutrophil infiltration and lipid peroxi-

dation, respectively (Fig. 5). However, pretreatment ofused as markers of reperfusion injury, were significantlyreduced subsequent to pretreatment with Cal I-1 prior rats with Cal I-1 prior to I/R produced a significant reduc-

tion of MPO activity in comparison with the activityto I/R (Fig. 4). The administration of chymostatin orvehicle did not produce significant alterations in plasma obtained from control rat kidneys (Fig. 5A). Similarly,

pretreatment of rats with Cal I-1 produced a significantlevels of gGT or AST in comparison with control animals(Fig. 4). reduction of the MDA levels associated with I/R (Fig.


Fig. 6. Histologic evaluation of renal I/R. Histology of a normal proximal tubule (PT) (A) is compared with tubules revealing brush border loss,nuclear condensation, and cytoplasmic swelling and loss of nuclei in up to one third of the tubular profile (B). More severe cellular loss, withbetween one third and two thirds of the tubular profile denuded of nuclei (C), and greater than two thirds of nuclei lost (D) are also represented.HandE, 3700. (E) Effect of renal I/R on total severity score in the presence of calpain inhibitor-1 (Cal I-1, 10 mg/kg IP), chymostatin (Chymo,10 mg/kg IP), or vehicle [50% (vol/vol) EtOH/saline]. *P , 0.05 vs. sham-operated group; 1P , 0.05 vs. control group (45 minutes of ischemiaand 6 hours of reperfusion).

5B). The administration of chymostatin or vehicle prior performed on sections of kidney obtained from micesubjected to endotoxemia, demonstrated marked posi-to I/R did not have a significant effect on MPO activitytive staining for iNOS protein (Fig. 7D). No positiveor MDA levels in comparison with values obtained fromstaining for iNOS protein was observed in kidney sec-control animals (Fig. 5).tions from rats subjected to renal I/R, which were incu-

Effects of Cal I-1 and chymostatin on I/R-mediated bated with the secondary antibody only (no primary anti-renal histopathology body–negative control; Fig. 7E).

In comparison with kidney sections obtained fromIn comparison with the normal tubular histology ob-sham-operated rats (Fig. 8A), sections prepared from ratserved in kidneys taken from sham-operated rats (Fig.kidneys subjected to I/R demonstrated marked staining6A), animals that underwent renal I/R demonstrated thefor COX-2 (Fig. 8B). Kidneys obtained from rats admin-recognized features of severe acute tubular damage (Fig.istered Cal I-1 demonstrated markedly reduced staining6D) [39]. These features included brush border loss, nu-for COX-2 upon comparison with kidneys obtained fromclear condensation, cytoplasmic swelling, and a consis-control animals (Fig. 8C), suggesting a reduction in thetent loss of significant numbers of nuclei from tubularexpression of COX-2 subsequent to pretreatment withprofiles (Fig. 6D).Cal I-1 prior to I/R. Staining for endogenous biotin wasWhen compared with the total severity score mea-not detected in any sections. The positive control forsured from kidneys obtained from sham-operated ani-COX-2 staining, performed on sections of kidney ob-mals, I/R produced a significant increase in total severitytained from mice subjected to endotoxemia, demon-score, which was significantly reduced by administrationstrated marked positive staining for COX-2 protein (Fig.of Cal I-1 prior to I/R (Fig. 6E). The administration of8D). In contrast, no positive staining for COX-2 proteinchymostatin or vehicle to rats prior to I/R did not havecould be observed in kidney sections from rats subjecteda significant effect on total severity score (Fig. 6E).to renal I/R that were incubated with the secondaryantibody only (no primary antibody–negative control;Immunohistochemical localization of iNOS andFig. 8E).COX-2 formation

When compared with kidney sections obtained fromDISCUSSIONsham-operated rats (Fig. 7A), immunohistochemical

analysis of sections obtained from rats subjected to renal There is good evidence from both in vivo and in vitroI/R revealed positive staining for iNOS (Fig. 7B). In studies that calpain activation plays an important role incontrast, substantially reduced staining was observed in the pathophysiology of renal injury mediated by hypoxiathe kidney sections obtained from rats administered Cal and I/R [23, 40]. We demonstrate here, to our knowledge

for the first time, that the administration of Cal I-1 priorI-1 (Fig. 7C). The positive control for iNOS staining,


Fig. 7. Immunohistochemical localization of inducible nitric oxide syn-thase (iNOS) in rat kidney following I/R. (A) Kidney section from asham animal (original magnification, OM, 380) and (B) control group(renal I/R only, OM 332), with typical areas of iNOS immunoreactivityindicated by arrows. In comparison, the iNOS immunoreactivity ofkidneys from rats treated with Cal I-1 (10 mg/kg IP) (C) was markedlyreduced (OM 380). (D) Positive control for iNOS immunoreactivity:Kidney sections were prepared from mice subjected to endotoxemia(LPS, 50 mg/kg IV). Typical areas of iNOS immunoreactivity are indi-cated by arrows (OM 380). (E) Negative control for iNOS immunoreac-tivity: Kidney sections were prepared from rats subjected to I/R onlyand incubated with secondary antibody only (no primary antibody, OM380). Figures are representative of at least three experiments performedon different days.

to renal I/R significantly reduces the renal dysfunction merular and tubular dysfunction [41]. In this study, themoderate but significant increases in the plasma concen-and injury caused by I/R of the rat kidney. This conclu-

sion is supported by four key findings: In a rat model of trations of urea and creatinine subsequent to I/R suggestthe impairment of glomerular function [31], and thisrenal I/R, Cal I-1 significantly reduced the I/R-mediated

increases in (1) the plasma levels of urea and creatinine, was reflected by a significant reduction in CCr observedsubsequent to I/R. However, I/R also resulted in signifi-(2) the FENa and urinary concentrations of GST and

NAG, (3) the plasma levels of gGT and AST, and (4) cant increases in urinary NAG and GST concentrations,which can be regarded as markers for tubular injury andMPO activity and the levels of MDA in the kidney. Cal

I-1 also significantly reduced the histologic evidence of possibly tubular function [33, 34]. This was reflected bya significant increase in FENa. Taken together, theseI/R-mediated tubular injury and substantially reduced

the immunohistochemical evidence of iNOS and COX-2 markers suggest a significant reduction in tubular func-tion and increased tubular injury in this model of renalexpression, suggesting that the protective mechanism

provided by Cal I-1 may involve, to some degree, the I/R. Although there are recognized limitations in the useclearance techniques to measure CCr and FENa in renalinhibition of the activation of NF-kB.

Ischemia/reperfusion of the kidney causes both glo- models that involve extensive tubular injury, the compar-


Fig. 8. Immunohistochemical localization of cyclooxygenase-2 (COX-2)in rat kidney following I/R. (A) Kidney section from Sham animal (OM380). (B) Control group (renal I/R only, OM 332) with typical areasof COX-2 immunoreactivity indicated by arrows. COX-2 immunoreac-tivity of kidneys from rats treated with Cal I-1 (C) was markedly reducedin comparison with staining obtained from the control group (OM 380).(D) Positive control for COX-2 immunoreactivity: Kidney sections wereprepared from mice subjected to endotoxemia (LPS, 50 mg/kg IV).Typical areas of COX-2 immunoreactivity are indicated by arrows (OM380). (E) Negative control for COX-2 immunoreactivity: Kidney sec-tions were prepared from rats subjected to I/R only and incubated withsecondary antibody only (no primary antibody, OM 3125). Figures arerepresentative of at least three experiments performed on different days.

atively larger increase in the markers of tubular dysfunc- demonstrated that gGT is significantly elevated subse-quent to short-term ischemia of the rat kidney [45].tion/injury (FENa, NAG, GST) suggests that there is a

greater dysfunction and injury to the tubules rather than Therefore, it is certainly feasible that the increasedplasma levels measured subsequent to renal I/R origi-the glomeruli in this model of renal I/R. Marked tubular

damage in models of I/R (such as the one used here) nated from the kidney after extensive damage to thetubular architecture. Renal I/R also caused a significanthas been demonstrated previously [42] and was reflected,

in this study, in the histopathological analysis of kidneys increase in both MPO activity, indicating neutrophil ac-cumulation, and in MDA levels, indicating increasedsubsequent to I/R obtained from control animals.

Renal I/R also produced significant increases in lipid peroxidation, and taken together, increased oxida-tive stress.plasma concentrations of gGT and AST, both of which

are regarded as nonspecific markers of extensive cellular In this study, Cal I-1 produced a significant reductionof renal dysfunction and injury mediated by I/R of thisdisruption or necrosis [31] and were used in this study

as markers of renal I/R. Both enzymes are present within kidney. Cal I-1 appears to provide a greater beneficialaction against tubular, rather than glomerular, dysfunc-the PT [32] and can be released into the urine subsequent

to renal injury [43, 44]. Furthermore, a recent study has tion. This is supported by our findings that compared


with rats subjected to I/R only, Cal I-1 produced rela- expression of iNOS or COX-2 protein (data not shown).tively small, but significant, decreases in plasma urea and Although this is the first report to our knowledge of suchcreatinine levels. This moderate effect on glomerular an effect of Cal I-1 in renal tissues, the mechanism(s)function was reflected by the small, but also significant, by which Cal I-1, but not chymostatin, potentially inhibitsincrease in CCr. In contrast, Cal I-1 had a marked effect the activation of NF-kB and subsequently the attenua-on markers of tubular dysfunction (FENa) and injury (uri- tion of iNOS (and COX-2) expression certainly warrantsnary NAG and GST). This was further supported by further investigation.the renal histopathology that revealed the recognized In conclusion, this study demonstrates that Cal I-1features of severe acute tubular damage in rat kidneys reduces the renal dysfunction and injury caused by renalsubjected to I/R only, which were markedly reduced by I/R in the anesthetized rat. Cal I-1 also reduces the I/R-Cal I-1. mediated expression of iNOS and COX-2 in the rat kid-

Calpain inhibitor-1 readily permeates cell membranes ney. Thus, we speculate that the inhibition by Cal I-1 ofand subsequently inhibits calpain activation [13, 14]. Al- the activation of NF-kB may contribute to the beneficialthough not measured in this study, one potential mecha- effects of Cal I-1 in I/R injury of the rat kidney.nism of action by which Cal I-1 could produce its benefi-cial effects is via the inhibition of cysteine protease ACKNOWLEDGMENTS(calpain) activity. However, this is unlikely to provide a

P.K.C. and M.C.McD. are funded by the Joint Research Board ofsignificant mechanism of protection in this model of renal St. Bartholomew’s Hospital (Grants XMLA/G7Z4). K.Z. is supported

by the Deutsche Herzstishung, H.M.F. by the Portuguese FundacaoI/R as (1) other cysteine protease inhibitors such as leu-para a Ciencia e Technologia (Praxis XXI/BPD/16333/98), and C.T.peptin and calpeptin do not protect against toxicant-by a British Heart Foundation Senior Research Fellowship (FS/96018).

mediated renal cell death and ischemic ARF, respec- This work was supported in part by the Clinica Medica e Diagnostico,Dr. Joaquim Chaves, Lisbon, Portugal. Parts of this study were pre-tively [22, 24] and (2) serine protease inhibitors such assented to the British Pharmacological Society meeting (Cambridge,antipain have also been shown not to reduce ischemia-UK) and were subsequently published (abstract; Chatterjee et al, Br

mediated ARF [22]. In this study, chymostatin, another J Pharmacol 129:34P, 2000).potent inhibitor of serine proteases such as chymotrypsin

Reprint requests to Prabal K. Chatterjee, Ph.D., Department of Ex-and papain [46], did not affect I/R-mediated renal dys-perimental Medicine and Nephrology, The William Harvey Researchfunction and injury. This is in keeping previous reports Institute, St. Bartholomew’s and the Royal London School of Medicine

from our laboratory that chymostatin, at the same dose and Dentistry, Charterhouse Square, London, EC1M 6BQ, England,United Kingdom.as that used in this study, does not protect against theE-mail: [email protected] failure and multiple organ dysfunction medi-

ated by endotoxic [28] or hemorrhagic shock [30]. Takentogether, this supports the view that an inhibition of APPENDIXprotease activity is unlikely to account for the beneficial

Abbreviations used in this article are: ARF, acute renal failure; AST,action of Cal I-1 observed in this study. aspartate aminotransferase; ATP, adenosine 59-triphosphate; Cal I-1,

calpain inhibitor-1; CCr, creatinine clearance; COX-2, cyclooxygenase-2;Another putative mechanism for the beneficial actionsFENa, fractional excretion of sodium; gGT, gamma-glutamyl transferase;observed with Cal I-1 in this study is related to its abilityGST, glutathione S-transferase; HR, heart rate; iNOS, inducible nitric

to attenuate the activation of NF-kB [25–28, 30]. NF-kB oxide synthase; I/R, ischemia/reperfusion; MAP, mean arterial pressure;MDA, malondialdehyde; MPO, myeloperoxidase; NAG, N-acetyl-is a member of a family of dimers belonging to the Rel/b-d-glucosaminidase; NF-kB, nuclear factor-kB; NOS, nitric oxide syn-NF-kB family of polypeptides, and the most frequentlythase; PT, proximal tubule; ROS, reactive oxygen species.

observed form of NF-kB is a dimer composed of twoDNA-binding proteins, namely NF-kB (or p50) and

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