Original Report: Laboratory Investigation

Am J Nephrol 2004;24:165–177DOI: 10.1159/000076547

Nephrology American Journal of

EUK-134 Reduces Renal Dysfunction and InjuryCaused by Oxidative and Nitrosative Stress ofthe Kidney

Prabal K. Chatterjeea Nimesh S.A. Patela Espen O. Kvalea

Paul A.J. Brownb Keith N. Stewartc Helder Mota-Filiped Martyn A. Sharpee

Rosanna Di Paolaf Salvatore Cuzzocreaf Christoph Thiemermanna

aDepartment of Experimental Medicine, Nephrology and Critical Care, William Harvey Research Institute,Queen Mary University of London, London, bDepartments of Pathology and cMedicine and Therapeutics,University of Aberdeen, Aberdeen, UK; dLaboratory of Pharmacology, Faculty of Pharmacy, University of Lisbon,Lisbon, Portugal; eDepartment of Neurochemistry, Institute of Neurology, University College London, London, UK,and fDepartment of Clinical and Experimental Medicine and Pharmacology, University of Messina, Messina, Italy

Received: May 5, 2003Accepted: December 9, 2003Published online: January 29, 2004

Prof. Christoph ThiemermannDepartment of Experimental Medicine, Nephrology and Critical CareWilliam Harvey Research Institute, Queen Mary University of LondonCharterhouse Square, London, EC1M 6BQ (UK)Tel. +44 20 7882 6118, Fax +44 20 251 1685, E-Mail c.thiemermann@qmul.ac.uk

ABCFax + 41 61 306 12 34E-Mail karger@karger.chwww.karger.com

© 2004 S. Karger AG, Basel0250–8095/04/0242–0165$21.00/0

Accessible online at:www.karger.com/ajn

Key WordsKidney W Proximal tubule W Ischemia W Reperfusion injury W

Oxidative stress W Nitrosative stress W Superoxidedismutase W Catalase W EUK-134

AbstractBackground/Aims: Oxidative and nitrosative stress playsimportant roles in the pathogenesis of renal ischemia/reperfusion (I/R) injury. Here we investigate the effect ofEUK-134, a synthetic superoxide dismutase and catalasemimetic, (i) on renal dysfunction and injury caused by I/Rin vivo and (ii) on proximal tubular cell (PTC) injury anddeath caused by oxidative and nitrosative stress. Meth-

ods: Rats, subjected to bilateral renal ischemia (45 min)followed by reperfusion (6 h), were administered EUK-134 (0.3 and 3 mg/kg, i.v.) prior to and during reperfu-sion, after which biochemical and histological indicatorsof renal dysfunction and injury were measured. Theexpression of poly(ADP-ribose) (PAR) and inducible nit-ric oxide (NO) synthase (iNOS) and nitrotyrosine forma-tion were determined immunohistochemically and usedas indicators of oxidative and nitrosative stress. Primarycultures of rat PTCs, isolated and cultured from the kid-

ney cortex, were incubated with hydrogen peroxide(H2O2; 1 mM for 2 h) in the presence of increasing con-centrations of EUK-134 (1–100 ÌM) after which PTC injuryand death were measured. The effects of EUK-134 onserum levels of NO in rats subjected to renal I/R or on NOproduction by PTCs incubated with interferon-Á (IFN-Á,100 IU/ml) and bacterial lipopolysaccharide (LPS, 10 Ìg/ml) in combination for 24 h were also measured. Results:

EUK-134 produced a significant reduction in renal dys-function and injury caused by I/R. Specifically, serum cre-atinine levels, an indicator of renal dysfunction, werereduced from 227 B 11 (n = 12, I/R only) to 146 B 9 ÌM(n = 12, I/R +3 mg/kg EUK-134). Urinary N-acetyl-ß-D-glu-cosaminidase activity, an indicator of tubular damage,was reduced from 42 B 5 (n = 12, I/R only) to 22 B 3 IU/l(n = 12, I/R +3 mg/kg EUK-134). EUK-134 significantlyreduced renal injury caused by oxidative stress in vivo(reduction in PAR formation), and in vitro EUK-134 re-duced PTC injury and death caused by H2O2. However,EUK-134 also reduced nitrosative stress caused by I/R invivo (reduction of iNOS expression and nitrotyrosine for-mation), which was reflected by a significant reduction inserum NO levels in rats subjected to renal I/R. Specifical-ly, serum NO levels were reduced from 57 B 12 (n = 12,

166 Am J Nephrol 2004;24:165–177 Chatterjee/Patel/Kvale/Brown/Stewart/Mota-Filipe/Sharpe/Di Paola/Cuzzocrea/Thiemermann

I/R only) to 23 B 3 mM (n = 12, I/R +3 mg/kg EUK-134). Invitro, EUK-134 significantly reduced NO production byPTCs incubated with IFN-Á/LPS. Conclusion: We proposethat EUK-134 reduces renal I/R injury not only via reduc-tion of oxidative stress, but also by reducing nitrosativestress caused by renal I/R.

Copyright © 2004 S. Karger AG, Basel

Introduction

Despite significant advances in critical care medicine,ischemic acute renal failure (ARF) remains a major clini-cal problem, producing considerable morbidity and mor-tality, which has not decreased significantly over the last50 years [1]. Renal ischemia, whether caused by shock orduring surgery or transplantation, is a major cause ofARF, initiating a complex and interrelated sequence ofevents resulting in injury to and eventual death of renalcells [2]. The prognosis is complicated by the fact thatreperfusion, although essential for the survival of isch-emic renal tissue, causes additional damage (reperfusioninjury) [3] which contributes to the renal dysfunction andinjury associated with ischemia/reperfusion (I/R) of thekidney. Within the kidney, the proximal tubule (PT)appears to be particularly susceptible to I/R injury, lead-ing to acute tubular necrosis (ATN) which plays a pivotalpart in the pathogenesis of ARF [4]. Previous interven-tions against ARF have proved to be largely negative anddialysis still remains the only effective therapy [1, 5]. Thedevelopment of novel therapies for the reduction of renaldysfunction and injury caused by I/R have, therefore,become topics of intense research interest [1, 5].

Renal I/R results in both oxidative and nitrosativestress which contributes to injury to PT cells (PTCs) andATN, renal dysfunction and injury, and ultimately toischemic ARF [6, 7]. Generation of reactive oxygen spe-cies (ROS) such as superoxide and hydrogen peroxidecontributes to PTC and renal I/R injury via their ability tocause cellular damage via interactions with key cellularcomponents such as lipids, proteins and DNA [6, 8]. Fur-thermore, high levels of nitric oxide (NO) produced byinducible NO synthase (iNOS) are implicated in PTCinjury and renal dysfunction/injury associated with renalI/R, ATN and ARF [7, 9]. Additionally, superoxide an-ions also react with NO to form peroxynitrite [10], whichcauses injury via oxidant injury and protein tyrosinenitration [11]. Specifically in the kidney, generation ofperoxynitrite has been implicated in the pathophysiologyof both renal I/R and hypoxia-re-oxygenation injury of

PTCs [7, 9, 12, 13]. Together, production of NO and itsmetabolite peroxynitrite results in nitrosative stresswhich causes injury via, amongst other mechanisms, ini-tiation of lipid peroxidation and direct inhibition of mito-chondrial respiratory chain enzymes [11, 14].

Several in vivo and in vitro investigations have dem-onstrated that interventions that reduce oxidative andnitrosative stress will ameliorate renal I/R injury [15, 16].Antioxidant enzymes that degrade ROS such as catalaseor superoxide dismutase (SOD) have been used with somedegree of success [15, 17]. Although such interventionshave shown promise, the potential benefits of the sys-temic administration of agents such as catalase and SODhave been limited due to problems with delivery or stabil-ity [2, 18, 19]. Specifically, SOD does not permeate bio-logical membranes and is therefore not able to reduce thedetrimental effects of superoxide produced within the cell(e.g. via uncoupling of the mitochondrial respiratorychain). Furthermore, superoxide produced within cellsdoes not leave the cell in which it has been generated, butremains potentially injurious to intracellular structures.Similar problems have been faced using catalase againstrenal I/R injury, due to its large size (MW F250,000)which limits its glomerular filtration and thereby restrictsits bioavailability [2, 18]. Thus, an increase in theamounts of extracellular SOD or catalase does not neces-sarily attenuate the effects of superoxide or hydroxyl radi-cal generated by intracellular sources, and limited distri-bution of SOD into extracellular spaces or compartmentsprevents it from effectively reaching areas of significantI/R injury, e.g. the PT [19]. Finally, the half-life of plasmaelimination of native SOD is minute due to its high sus-ceptibility to proteolytic digestion and renal handling,and further problems have been faced due to the instabili-ty of SOD in aqueous solutions [20].

To overcome these limitations, small molecules whichhave the catalytic activity of both SOD and catalase, suchas the manganese-salen (Mn-salen) complexes EUK-134[manganese 3-methoxy N,N)-bis(salicyclidene)ethylene-diamine chloride] and EUK-8 [manganese N,N)-bis(sali-cylidene)ethylenediamine chloride] have been developed[21]. These agents, which remove both superoxide andhydrogen peroxide, have been shown to exert beneficialeffects in several in vivo and in vitro animal models ofhuman disease including Alzheimer’s disease [22], Par-kinson’s disease [23], motor neuron disease [24], multiplesclerosis [25], excitotoxic neuronal injury [26], stroke [27]and I/R of the heart [28]. Furthermore EUK-8 reducedacute lung injury caused by endotoxic shock in pigs [29],and both EUK-8 and EUK-134 reduced renal dysfunction

EUK-134 Reduces Renal I/R Injury Am J Nephrol 2004;24:165–177 167

Table 1. Experimental groups studied

Group studied Numberof rats

Protocol

I/R + saline 12 Administered saline only (2 ml/kg i.v. bolus 5 min before I/R followed by2 ml/kg/h i.v. throughout)

0.3 Pre-Isc 12 0.3 mg/kg EUK-134 i.v. 5 min prior to I/R followed by 0.3 mg/kg/h i.v.throughout I/R

0.3 Post-Isc 12 0.3 mg/kg EUK-134 i.v. 5 min prior to reperfusion followed by 0.3 mg/kg/h i.v.throughout reperfusion

3 Post-Isc 12 3 mg/kg EUK-134 i.v. 5 min prior to reperfusion followed by 3 mg/kg/h i.v.during reperfusion

Sham + saline 12 Administered saline only (2 ml/kg i.v. bolus followed by 2 ml/kg/h)

Sham + EUK-1340.3 Pre-I/R

4 0.3 mg/kg EUK-134 i.v. 5 min prior to I/R followed by 0.3 mg/kg/h i.v. infusionthroughout I/R

Sham + EUK-1340.3 Post-I/R

4 0.3 mg/kg EUK-134 i.v. bolus 5 min prior to reperfusion followed by 0.3 mg/kg i.v.throughout reperfusion

Sham + EUK-1343 Post-I/R

4 3 mg/kg EUK-134 i.v. bolus 5 min prior to reperfusion followed by 3 mg/kg i.v.infusion throughout reperfusion

Separate groups of rats were subjected to either 45 min of renal ischemia followed by 6 h of reperfusion or shamoperation and treated either with saline (vehicle for EUK-134) or EUK-134.

in rodent models of hemorrhagic and endotoxic shock[30–32]. EUK-134 therefore has the potential to be usefulin conditions associated with I/R injury of the kidney andassociated ischemic ARF. In 1996, Gianello et al. [33]observed that EUK-134 could reduce renal dysfunctioncaused by left renal artery clamping of uninephrectom-ized rats, although no mechanisms for the reno-protectionwere reported. Several of the studies listed above providesome evidence that one beneficial mode of action ofEUK-8 and EUK-134 is via reduction of oxidative stress.However, recent evidence now suggests that these Mn-sa-lens can also reduce nitrosative stress via the catalyticbreakdown of peroxynitrite and NO [34].

This study investigates the effects of EUK-134 on therenal injury and dysfunction caused by I/R. Having dis-covered that EUK-134 can reduce renal dysfunction andinjury caused by I/R, we have carried out further studiesaimed at elucidating the mechanisms by which EUK-134protects the kidney against I/R injury. Specifically, wehave investigated the effects of EUK-134 on the oxidativeand nitrosative stress caused by renal I/R via assessmentof immunohistochemical markers. Finally, we have usedprimary cultures of rat PTCs to investigate the effect ofEUK-134 on (i) the oxidative stress caused by hydrogen

peroxide, and (ii) NO production by PTCs subsequent toincubation with interferon-Á (IFN-Á) and bacterial lipo-polysaccharide (LPS).

Methods

In vivo Experimental DesignIn vivo studies were carried out on 72 male Wistar rats (Tuck,

Rayleigh, Essex, UK). Rats received a standard diet and water adlibitum and were cared for in accordance with both the Home OfficeGuidance in the Operation of the Animals (Scientific Procedures)Act 1986, published by Her Majesty’s Stationery Office, London,UK, and the Guiding Principles in the Care and Use of Animals pub-lished by the American Physiological Society. After anesthesia (sodi-um thiopentone) and basic surgical procedures (e.g. cannulation ofthe trachea, carotid artery, jugular vein and bladder), rats were ran-domly allocated into 8 groups as described in table 1. Rats were sub-jected to renal I/R which involved bilateral renal ischemia for 45 minfollowed by reperfusion for 6 h as described in detail previously [35],whereas rats used as sham-operated controls were not subjected torenal I/R.

Measurement of Biochemical ParametersDuring the reperfusion period, blood (1 ml) and urine samples

were collected and used for the measurement of biochemical and uri-nary indicators of renal function and injury as described previously

168 Am J Nephrol 2004;24:165–177 Chatterjee/Patel/Kvale/Brown/Stewart/Mota-Filipe/Sharpe/Di Paola/Cuzzocrea/Thiemermann

[35]. Serum urea and creatinine levels were used as indicators ofimpaired glomerular function [35]. Urine samples were collectedduring the reperfusion period and the volume of urine producedrecorded. Urine concentrations of creatinine and Na+ were measuredand used in conjunction with serum creatinine and Na+ levels andurine flow to estimate creatinine clearance and fractional excretionof Na+, which were used as respective markers of glomerular andtubular dysfunction [35]. Aspartate aminotransferase (AST) and Á-glutamyltransferase (Á-GT), enzymes which are both located in thekidney and, specifically, within the PT and which are released duringreperfusion, were measured and used as indicators of reperfusioninjury [35]. Additionally, urinary N-acetyl-ß-D-glucosaminidase(NAG) activity, an indicator of tubular damage and possibly tubularfunction, was also measured [35].

Histological EvaluationAt the end of each experiment, one kidney from each rat was

removed and stored in 10% (w/v) neutral buffered formaldehyde pre-pared using phosphate-buffered saline (PBS, 0.01 M, pH 7.4). Renalsections were prepared as described previously and used for theassessment of tubular injury caused by renal I/R [15, 16]. Briefly, 100intersections were examined for each kidney and a score from 0 to 3was given for each tubular profile involving an intersection: 0 = nor-mal histology; 1 = tubular cell swelling, brush border loss, nuclearcondensation, with up to 1/3 of tubular profile showing nuclear loss;2 = as for score 1, but 11/3 and !2/3 of tubular profile showednuclear loss, and 3 = 12/3 of tubular profile showed nuclear loss. Thetotal score for each kidney was calculated by addition of all 100scores with a maximum score of 300 and expressed as ‘histologicalscore (out of 300)’.

Immunohistochemical Analysis of PAR, iNOS andNitrotyrosineImmunohistochemical localization of PAR, iNOS and nitrotyro-

sine in rat kidney sections was performed as described previously[15, 16]. Briefly, sections were incubated overnight at 4°C with pri-mary anti-PAR, anti-iNOS and anti-nitrotyrosine antibodies (1:500v/v in PBS; DBA, Milan, Italy). Separate sections were also incu-bated with control solutions consisting of PBS alone or a 1:500 dilu-tion of nonspecific purified rabbit IgG (DBA). Specific labeling wasdetected using a biotin-conjugated goat anti-rabbit IgG (DBA) andavidin-biotin peroxidase (DBA). Samples were then viewed under alight microscope.

Isolation and Culture of Rat Proximal Tubular CellsPrimary cultures of PTCs were prepared from the kidneys

obtained from 12 male Wistar rats (250–350 g) using collagenasedigestion, differential sieving and Percoll density centrifugation asdescribed previously [15, 16]. Isolated PTCs were cultured in mini-mum essential medium (MEM) containing 10% (v/v) fetal calf serumin a humidified 5% CO2/95% air atmosphere at 37°C. Growthmedium was changed every 48 h until the cells reached confluence,which took between 7 and 10 days, and primary cultures were used assoon as possible after the cells reached confluence.

In vitro Experimental DesignOxidative Stress Caused by Hydrogen PeroxideWe have previously reported that exposure of rat PTCs to hydro-

gen peroxide (H2O2) causes a concentration-dependent (10 ÌM–10 mM ) and time-dependent (1–6 h) increase in cellular injury and

death [15]. To investigate the effects of EUK-134 on cellular injuryand death caused by H2O2, PTCs were pre-incubated (10 min at37°C) with EUK-134 (1–100 ÌM ) before incubation with H2O2(1 mM for 2 h) after which time cellular injury and cell death pro-duced by H2O2 were assessed as described below.

Nitrosative Stress Caused by Nitric OxideConfluent primary cultures of PTCs were incubated with 100 IU/

ml IFN-Á and 10 Ìg/ml LPS in combination for 24 h in the absence orpresence of increasing concentrations of EUK-134 (1–100 ÌM ).EUK-134 was added to incubation medium (MEM) 10 min beforethe addition of IFN-Á and LPS. Upon completion of incubations(24 h after the addition of IFN-Á/LPS), NO levels in incubationmedium were measured as nitrite (NO2)/nitrate (NO3) after conver-sion of NO3 to NO2 using NO3 reductase as described below andpreviously by Chatterjee et al. [16].

Measurement of Cellular Injury (MTT Assay)As previously described [15], cellular injury was determined indi-

rectly by measurement of the mitochondrial-dependent conversionof MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide] into a formazan which, unlike the tetrazolium salt, can be mea-sured spectrophotometrically at 550 nm. Briefly, PTCs were incu-bated with MTT (0.2 mg/ml in PBS for 1 h at 37 °C) after which theMTT solution was removed by aspiration. The cells were then solubi-lized in DMSO and an aliquot was transferred into a 96-well plateafter which the reduction of MTT to formazan was quantified spec-trophotometrically by measurement of absorbance at 550 nm usingan Anthos Labtec microplate reader (Labtec, Uckfield, Sussex, UK).Results are expressed as a percentage of the mitochondrial respira-tion measured in control cells not exposed to H2O2, which was takenas 100%.

Measurement of Cytotoxicity (LDH Assay)Cell death was determined by measurement of lactate dehydroge-

nase (LDH) released into the incubation medium due to the loss ofmembrane integrity using a commercially available cytotoxicity de-tection kit (Roche Diagnostics Ltd., Lewes, East Sussex, UK) as pre-viously described [15]. Briefly, an aliquot of incubation medium wastransferred into a 96-well plate and reaction buffer added to each welland the mixture was incubated for 30 min at room temperature. Thereaction was stopped by the addition of 2 M hydrochloric acid andabsorbance measured spectrophotometrically at 492 nm using anAnthos Labtec microplate reader (Labtec). Results are expressed aspercentage of the total LDH released from control cells (i.e. those notexposed to H2O2) which were incubated with 1% (w/v) Triton X-100for 30 min.

Measurement of Nitrite/Nitrate ConcentrationsConcentrations of NO2/NO3, the primary oxidation products of

NO subsequent to reaction with oxygen, were measured and used asan indicator of NO synthesis. NO2/NO3 levels in rat serum and incu-bation medium from PTCs were measured as previously describedafter enzymatic conversion of NO3 to NO2 using NO3 reductase[16].

MaterialsUnless otherwise stated, all compounds used in this study were

purchased from Sigma-Aldrich Company Ltd. (Poole, Dorset, UK).All solutions used in vivo were prepared using non-pyrogenic saline

EUK-134 Reduces Renal I/R Injury Am J Nephrol 2004;24:165–177 169

(0.9% [w/v] NaCl; Baxter Healthcare Ltd., Thetford, Norfolk, UK).Sodium thiopentone was obtained from Rhône Mérieux (Harlow,Essex, UK). EUK-134, which was kindly supplied by Dr. MartynSharpe, Department of Neurochemistry, Institute of Neurology, Uni-versity College London, UK, was synthesized as described in detailpreviously [34].

Statistical AnalysisAll values described in the text and figures are expressed as mean

B standard error of the mean (SEM) for the number of observations.For in vivo studies, each data point represents biochemical measure-ments obtained from 4–12 separate rats. For histological scoring,each data point represents analysis of kidneys taken from 4–8 indi-vidual rats. For immunohistochemical analysis, the figures shownare representative of at least 3 experiments performed on differentexperimental days. For in vitro studies involving PTC, measure-ments were taken from cultures obtained from 12 separate isolationsusing both kidneys from 12 separate rats. For the measurement ofPTC injury, death and NO2/NO3 levels in incubation medium,experiments were performed in triplicate. Statistical analysis was car-ried out using GraphPad Prism/Instat 1.1 (GraphPad Software, SanDiego, Calif., USA). Data were analyzed using one-way ANOVA fol-lowed by Dunnett’s post hoc test and a p value of !0.05 was consid-ered to be significant.

Results

In vivo StudiesThe mean B SEM for the weights of the rats used in in

vivo experiments was 267 B 3 g (n = 72). When comparedto rats subjected to sham operation, renal I/R or adminis-tration of EUK-134 did not have a significant effect oneither mean arterial blood pressure or heart rate through-out the experimental period (data not shown). EUK-134,when administered to rats subjected to sham operation orrenal I/R, did not have a significant effect on biochemical,urinary or histochemical markers of renal dysfunction orinjury when compared to rats administered saline only(data not shown). Furthermore, when compared to ratsused as shams, renal I/R (in the presence or absence ofEUK-134) did not have a significant effect on urine flow(0.015 B 0.002 ml/min, n = 72).

Effect of EUK-134 on Renal DysfunctionCaused by I/RRats, which underwent renal I/R only (I/R + saline),

exhibited significant increases in the serum concentra-tions of urea and creatinine compared to sham-operatedrats (sham + saline) (fig. 1), suggesting a significant degreeof renal (glomerular) dysfunction. When compared to ratssubjected to renal I/R only (I/R + saline), administrationof 0.3 mg/kg EUK-134 prior to I/R followed by 0.3 mg/kg/h throughout I/R produced a modest, but significant

Fig. 1. Effect of EUK-134 on glomerular dysfunction caused by renalI/R. Alterations in serum urea (A) and creatinine (B) in rats subjectedto sham operation and administered saline or subjected to bilateralrenal I/R and administered EUK-134 as described in table 1. Dataare expressed as mean B SEM of the number of observations. * p !0.05 vs. I/R + saline.

reduction in serum levels of urea and creatinine (fig. 1).Administration of 0.3 and 3 mg/kg EUK-134 prior toreperfusion followed by 0.3 or 3 mg/kg/h throughoutreperfusion produced a significant, dose-dependent re-duction in serum urea and creatinine levels (fig. 1).

Effect of EUK-134 on Glomerular and TubularDysfunction Caused by I/RRenal I/R produced a significant reduction in creati-

nine clearance when compared to sham-operated rats(fig. 2A), indicating a marked reduction in glomerular fil-tration rate. This was reflected by a significant increase in

170 Am J Nephrol 2004;24:165–177 Chatterjee/Patel/Kvale/Brown/Stewart/Mota-Filipe/Sharpe/Di Paola/Cuzzocrea/Thiemermann

Fig. 2. Effect of EUK-134 on glomerular and tubular dysfunctioncaused by renal I/R. Alterations in creatinine clearance (A) and frac-tional excretion of Na+ (B) in rats subjected to sham operation andadministered saline or subjected to bilateral renal I/R and adminis-tered EUK-134 as described in table 1. Data are expressed as meanB SEM of n observations. * p ! 0.05 vs. I/R + saline.

Fig. 3. Effect of EUK-134 on reperfusion-injury caused by renal I/R.Alterations in serum aspartate aminotransferase (AST) (A) and Á-glutamyltransferase (Á-GT) levels (B) in rats subjected to sham opera-tion and administered saline or subjected to bilateral renal I/R andadministered EUK-134 as described in table 1. Data are expressed asmean B SEM of n observations. * p ! 0.05 vs. I/R + saline.

the fractional excretion of Na+ caused by renal I/R, indi-cating marked tubular dysfunction (fig. 2B). When com-pared to rats subjected to renal I/R only, administrationof 0.3 mg/kg EUK-134 prior to I/R followed by 0.3 mg/kg/h throughout I/R produced a significant improvementin creatinine clearance and a significant reduction in thefractional excretion of Na+ (fig. 2). Administration of 0.3and 3 mg/kg EUK-134 prior to reperfusion followed by0.3 or 3 mg/kg/h throughout reperfusion produced a sig-nificant, dose-dependent improvement in creatinineclearance (fig. 2A) and a dose-dependent reduction infractional excretion of Na+ (fig. 2B).

Effect of EUK-134 on Reperfusion Injury Caused byRenal I/ROn comparison with values obtained from sham-oper-

ated rats, renal I/R produced significant increases in serumconcentrations of AST and Á-GT, suggesting significantreperfusion injury (fig. 3). When compared to rats sub-jected to renal I/R only, administration of 0.3 mg/kg EUK-134 prior to I/R followed by 0.3 mg/kg/h throughout I/Rproduced significant reductions in serum levels of AST andÁ-GT (fig. 3). Administration of 0.3 and 3 mg/kg EUK-134prior to reperfusion followed by 0.3 or 3 mg/kg/h through-out the reperfusion period produced dose-dependent re-

EUK-134 Reduces Renal I/R Injury Am J Nephrol 2004;24:165–177 171

ductions in serum levels of AST and Á-GT (fig. 3), suggest-ing a significant reduction in reperfusion injury.

Effect of EUK-134 on Tubular Injury Caused byRenal I/RRenal I/R caused a significant increase in urinary

NAG activity suggesting marked tubular injury (fig. 4A).This was reflected by the increased histological score mea-sured from kidneys obtained from rats subjected to renalI/R (fig. 4B). Administration of 0.3 mg/kg EUK-134 priorto I/R followed by 0.3 mg/kg/h throughout I/R produced asignificant reduction of both NAG enzymuria and histo-logical scoring of tubular injury (fig. 4). Administration of0.3 and 3 mg/kg EUK-134 prior to reperfusion followedby 0.3 or 3 mg/kg/h throughout the reperfusion periodproduced a dose-dependent reduction in urinary NAGactivity (fig. 4A) which was reflected by a significant anddose-dependent reduction in the histological scoring oftubular injury (fig. 4B).

Isolation of Rat PTCs and Primary CultureThe mean B SEM for the weights of the rats used in in

vitro experiments was 287 B 4 g (n = 12). Isolation andprimary culture of rat PTCs produced confluent mono-layers exhibiting typical ‘cobblestone’ morphology associ-ated with epithelial cells that did not exhibit any evidenceof fibroblast contamination.

Effects of EUK-134 on Oxidative Stress in vitroand in vivoIncubation of rat PTCs with H2O2 (1 mM for 2 h)

caused a substantial (F80%) reduction in mitochondrialrespiration compared to control (untreated) cells (fig. 5A).Pretreatment of rat PTCs with EUK-134 caused a concen-tration-dependent reduction in the impairment in mito-chondrial respiration caused by H2O2 (fig. 5A). Incuba-tion with H2O2 (1 mM for 2 h) also caused a significantincrease in LDH release from rat PT cells, compared tocontrol (untreated) cells (fig. 5B). Pretreatment withEUK-134 caused a concentration-dependent reduction inLDH release caused by H2O2 (fig. 5B).

Reduction of oxidative stress in vitro was reflected invivo by the ability of EUK-134 to reduce formation ofPAR in the kidneys of rats subjected to renal I/R (fig. 5C–E). PAR formation was used as an indicator of the activityof PAR polymerase (PARP) and therefore, as an indicatorof ROS formation and oxidative stress [36]. In compari-son to renal sections obtained from sham-operated ratswhich were administered saline only (fig. 5C), immuno-histochemical analysis of renal sections obtained from

Fig. 4. Effect of EUK-134 on tubular injury caused by renal I/R.Alterations in urinary N-acetyl-ß-D-glucosaminidase activity (A) andhistological scoring of tubular injury (B) in rats subjected to shamoperation and administered saline or subjected to bilateral renal I/Rand administered EUK-134 as described in table 1. Data areexpressed as mean B SEM of n observations. * p ! 0.05 vs. I/R +saline.

rats subjected to renal I/R revealed positive staining forPAR (fig. 5D). In contrast, substantially reduced stainingwas observed in the kidney sections obtained from ratsthat were administered EUK-134 (fig. 5E). No evidenceof staining for PAR was observed in kidney sectionsobtained from sham-operated rats that were administeredEUK-134 (data not shown).

Effect of EUK-134 on Nitrosative Stress in vitroand in vivoIncubation of primary cultures of rat PTCs with IFN-Á

and LPS in combination for 24 h produced a significant

172 Am J Nephrol 2004;24:165–177 Chatterjee/Patel/Kvale/Brown/Stewart/Mota-Filipe/Sharpe/Di Paola/Cuzzocrea/Thiemermann

Fig. 5. Effect of EUK-134 on renal oxidative stress in vivo and invitro. PTCs were incubated with H2O2 (1 mM for 2 h) in the absenceor presence of increasing concentrations of EUK-134 (1–100 ÌM )after which the MTT assay was used to measure mitochondrial respi-ration (impairment of which was used as an indicator of cellular dys-function; A) and lactate dehydrogenase (LDH) release from PTC wasmeasured and used as an indicator of cell death (B). Data areexpressed as mean B SEM of data obtained from 12 rats, * p ! 0.05when compared with PTCs treated with H2O2 only (H2O2 only, solidcolumn). C–E PAR formation in rat kidney sections obtained fromrats subjected to sham operation (C), I/R only (D) and I/R+EUK-134(3 mg/kg EUK-134 i.v. prior to reperfusion followed by 3 mg/kg/h i.v.throughout the reperfusion period; E). Original magnification !125.Figures are representative of at least 3 experiments performed ondifferent days.

increase in NO production as determined by measure-ment of NO2/NO3 levels (fig. 6A). Incubation of PTCswith IFN-Á and LPS in the presence of increasing concen-trations of EUK-134 (1–100 ÌM) resulted in a significant,concentration-dependent inhibition of NO production(fig. 6A).

The ability of EUK-134 to reduce NO production byPTCs exposed to IFN-Á and LPS in vitro was reflected in

vivo. Renal I/R resulted in a significant increase in theserum levels of NO2/NO3 that was used as an indicator ofNO formation in vivo (fig. 6B). Administration of 0.3 mg/kg EUK-134 prior to I/R followed by 0.3 mg/kg/hthroughout I/R produced a significant reduction in serumlevels of NO2/NO3 (fig. 6B). Administration of 0.3 and3 mg/kg EUK-134 prior to reperfusion followed by 0.3 or3 mg/kg/h throughout the reperfusion period produced a

EUK-134 Reduces Renal I/R Injury Am J Nephrol 2004;24:165–177 173

Fig. 6. Effect of EUK-134 on renal nitrosative stress in vitro and invivo. A PTCs were incubated with incubation medium only (un-treated) or with 100 ÌM EUK-134 only (EUK-134 only) or a combina-tion of IFN-Á (100 IU/ml) and LPS (10 Ìg/ml; CK only) for 24 h. PTcells were also incubated with IFN-Á and LPS in the presence ofincreasing concentrations of EUK-134 (1–100 ÌM ). NO2/NO3 levelswere measured in the incubation medium using the Griess assay. Dataare expressed as mean B SEM. * p ! 0.05 vs. CK only group.B Alterations in serum NO2/NO3 levels in rats subjected to sham oper-ation and administered saline or subjected to bilateral renal I/R andadministered EUK-134 as described in table 1. Data are expressed asmean B SEM of n observations. * p ! 0.05 vs. I/R + saline. C–E iNOSexpression in rat kidney sections obtained from rats subjected to shamoperation (C), I/R only (D) and I/R+EUK-134 (3 mg/kg EUK-134 i.v.prior to reperfusion followed by 3 mg/kg/h i.v. throughout reperfusion;E). Original magnification !125–250. Figures are representative of atleast 3 experiments performed on different days.

174 Am J Nephrol 2004;24:165–177 Chatterjee/Patel/Kvale/Brown/Stewart/Mota-Filipe/Sharpe/Di Paola/Cuzzocrea/Thiemermann

significant, dose-dependent reduction in serum NO2/NO3

levels (fig. 6B).Once it was established that EUK-134 could reduce

plasma NO levels in vivo, we investigated whether EUK-134 could modulate iNOS expression caused by renalI/R (fig. 6C–E). When compared to kidney sections ob-tained from sham-operated rats administered saline only(fig. 6C), immunohistochemical analysis of sections ob-tained from rats subjected to renal I/R revealed positivestaining for iNOS (fig. 6D). In contrast, reduced stainingwas observed in kidney sections obtained from rats sub-jected to renal I/R but which were administered EUK-134(fig. 6E). iNOS expression was absent in the kidneys ofrats subjected to sham operation which were adminis-tered EUK-134 (data not shown).

Effect of EUK-134 on Nitrotyrosine Formation duringRenal I/RThe formation of nitrotyrosine was used as another

indicator of nitrosative stress [15, 16] (fig. 7). When com-pared to kidney sections obtained from sham-operatedrats administered saline only (fig. 7A), immunohisto-chemical analysis of sections obtained from rats subjectedto renal I/R revealed positive staining for nitrotyrosine(fig. 7B). In contrast, reduced staining was observed inkidney sections obtained from rats subjected to renal I/Rbut which were administered EUK-134 (fig. 7C). Nitroty-rosine expression was absent in the kidneys of rats sub-jected to sham operation that were administered EUK-134 (data not shown).

Discussion

We show here that bilateral renal artery occlusion (for45 min) followed by reperfusion (for 6 h) in the anesthe-tized rat results in a pronounced fall in renal function asindicated by a significant rise in serum levels of urea andcreatinine. Renal dysfunction was reflected by a markedfall in creatinine clearance (indicating reduction in glo-merular filtration rate) and was associated with a markedincrease in fractional excretion of Na+ indicating dysfunc-tion of the PT. Tubular injury, as indicated by increasedurinary NAG activity, was prevalent. Moreover, charac-teristic histological signs of marked tubular injury wereobserved in keeping with the observation that the S3 seg-ment of the PT is particularly susceptible to renal I/Rinjury [37]. All these data, together with evidence ofincreased oxidative and nitrosative stress, confirm a rec-ognized pattern of renal dysfunction and injury caused by

Fig. 7. Effect of EUK-134 on nitrotyrosine formation in rat kidney.Nitrotyrosine formation in rat kidney sections obtained from ratssubjected to sham operation (A), I/R only (B) and I/R+EUK-134(3 mg/kg EUK-134 i.v. prior to reperfusion followed by 3 mg/kg/hi.v. throughout the reperfusion period; C). Original magnification!250. Figures are representative of at least 3 experiments performedon different days.

EUK-134 Reduces Renal I/R Injury Am J Nephrol 2004;24:165–177 175

I/R of the kidney [2–4, 6, 7, 9] which involves both glo-merular and tubular dysfunction [38].

The findings of the current study can be summarizedas follows: (i) EUK-134 reduces the development of glo-merular and tubular dysfunction caused by renal I/R;(ii) EUK-134 reduces the biochemical and histological evi-dence of tubular injury; (iii) EUK-134 reduces oxidativestress both in vitro and in vivo, and (iv) EUK-134 attenu-ates nitrosative stress in vivo and in vitro. The in vivoeffects of EUK-134 against the renal dysfunction and inju-ry caused by I/R of the rat kidney, although modest, werenevertheless significant. Administration of 3 mg/kg EUK-134 upon reperfusion provided a similar level of reno-pro-tection to that obtained when EUK-134 was administeredat a lower dose (0.3 mg/kg) prior to I/R. Although this sug-gests that EUK-134 has a greater effect when administeredprior to I/R, which may not be ideal for the therapy ofestablished ARF, the results presented here suggest thatEUK-134 or similar complexes possess therapeutic poten-tial in situations where renal ischemia can be predicted(e.g. during renal transplantation or abdominal surgeryinvolving renal artery clamping). It is also likely that whenused clinically, plasma concentrations of EUK-134 wouldreach effective concentrations. In this study, based on themolecular weight of EUK-134 (436) and the average weightof the rats (267 g), the dose of EUK-134 that provided max-imal reno-protection in vivo (3 mg/kg) corresponded to theconcentration of EUK-134 that provided the greatest pro-tection against PTC injury and death in vitro (100 ÌM).However, final plasma and renal concentrations of EUK-134 in vivo, which will be modulated by its breakdown andexcretion, remain to be determined directly.

It should also be borne in mind that the ARF modelused in this study, although highly reproducible, does noteffectively mimic ARF in humans. Specifically, clinicalARF is multifactorial and often associated with multipleorgan failure and sepsis [1]. Therefore the results present-ed here using a model of renal dysfunction and injurycaused by I/R of the rat kidney may not necessarily bereflected in humans suffering ischemic ARF. In fact, themultifactorial causes of ARF are one of the reasons whycurrent therapies against ARF, although showing promisein the laboratory setting, have been largely ineffectiveagainst ischemic ARF in humans [5]. However, we haverecently reported that EUK-134 can also reduce renal dys-function in animal models of both hemorrhagic and endo-toxic shock [30, 31] and therefore, exploration of theeffects of EUK-134 (or similar compounds) against isch-emic ARF in humans would certainly be interesting. How-ever, the effectiveness of EUK-134 against other forms of

renal failure (e.g. other experimental models of ischemicand nephrotoxic ARF and chronic renal failure) remainsto be determined, and the beneficial effects reported heremay not necessarily apply to other forms of renal failure.

Although EUK-134 is now well recognized as a SOD/catalase mimetic [21], it is intriguing to consider the mech-anisms by which EUK-134 can protect the kidney againstI/R. The mechanism for the dismutation of superoxide byEUK-134 involves the reduction of manganese(III) tomanganese(II) by superoxide, which is subsequently oxi-dized to oxygen. Mn(II) is subsequently oxidized back toMn(III) by another molecule of superoxide yielding hydro-gen peroxide; a mechanism very similar to that of SOD[34]. The mechanism by which EUK-134 acts as a catalasemimic involves the oxidation of the Mn-salen complex tooxoMn-salen by H2O2 releasing H2O. The oxoMn-salen isthen further reduced by H2O2 to regenerate the Mn-salenalong with the generation of H2O and O2 [34]. We reporthere that EUK-134 can reduce the renal cell injury anddeath caused by oxidative stress. The contribution of ROSand oxidative stress of the kidney subjected to I/R hasbeen known for many years [8, 38]. ROS may be producedfrom several sources within the reperfused kidney includ-ing mitochondrial generation, oxidation of hypoxanthine,from arachidonic acid metabolism [6, 8, 18] and evenfrom peroxynitrite [39]. Under physiological conditions,several endogenous protective mechanisms are presentwhich counteract the formation of ROS, e.g. antioxidantenzymes such as glutathione peroxidase, SOD and cata-lase, and antioxidative agents such as ascorbate (vitaminC) and ·-tocopherol (vitamin E) [8, 40]. However, duringreperfusion of previously ischemic tissues, these endoge-nous protective mechanisms are overwhelmed therebyallowing ROS to exert their detrimental effects [41]. Addi-tionally, levels of renal protective enzymes such as SODare rapidly depleted in the kidney subjected to I/R [42].Furthermore, ROS produce strand breaks in DNA leadingto PARP activation and subsequent depletion of its sub-strate NAD+. As NAD+ functions as a cofactor in glycoly-sis and the tricarboxylic acid cycle, its depletion leads to arapid fall in intracellular ATP – a phenomenon termed the‘PARP suicide hypothesis’ [36]. There is now good evi-dence that PARP activation plays an important role in theinjury and dysfunction caused by I/R and inflammation ofthe kidney [36, 43, 44]. The results obtained from thisstudy suggest that EUK-134 reduces oxidative stress, sub-sequent DNA damage and PARP activation during I/R ofthe kidney. Several studies have demonstrated the benefi-cial effects of various interventions against renal injurycaused by ROS, which act either by reducing the genera-

176 Am J Nephrol 2004;24:165–177 Chatterjee/Patel/Kvale/Brown/Stewart/Mota-Filipe/Sharpe/Di Paola/Cuzzocrea/Thiemermann

tion of, or the effects of, ROS. Such therapeutic strategiesinclude administration of agents which inhibit oxidativedamage caused by iron (e.g. desferrioxamine) [15] oradministration of ROS scavengers such as TEMPOL andTEMPONE [15, 45]. In 1996, Gianello et al. [33] reportedthat EUK-134 reduced renal dysfunction caused by leftrenal artery clamping of uninephrectomized rats, how-ever, mechanisms of reno-protection were not evaluated inthat study. In contrast, we demonstrate here that EUK-134 provides significant protection against renal I/R inju-ry caused by the oxidative stress, which plays a major rolein the development of ARF [6, 38, 41, 46].

Recent evidence now suggests that EUK-134 can alsoreduce nitrosative stress via the catalytic breakdown ofperoxynitrite and NO to the respective benign speciesNO2 and NO3 [34]. In a recent study by Sharpe et al. [34],in which the interactions of EUK-134 with NO andperoxynitrite were studied, it was reported that in thepresence of per-species (e.g. H2O2, peroxynitrite, perace-tate and persulfate), Mn-salen complexes are oxidized tooxoMn-salen which are potent oxidants which rapidlyoxidize NO to NO2 and NO2 to NO3 [34]. Production ofNO and its metabolite peroxynitrite during renal I/Rresults in renal injury and dysfunction (nitrosative stress)[11, 14] and reduction of the generation or presence ofNO can provide reno-protection. Specifically, inhibitionof iNOS expression (e.g. using inhibitors of nuclear factor-ÎB activation), iNOS activity (e.g. using inhibitors ofiNOS activity), absence of iNOS itself (using iNOS knock-out mice) or scavenging of NO (e.g. using hemoglobin)can ameliorate or prevent NO-mediated renal injury [16,47–50]. Furthermore, reduced generation or presence ofNO will provide beneficial effects via attenuation ofperoxynitrite formation [12] and reno-protective effectsof reduced peroxynitrite against renal I/R injury havebeen confirmed using the peroxynitrite scavenger ebselen[13]. We report here that EUK-134 can reduce nitrosativestress caused by renal I/R in vivo and in vitro. Specifical-ly, as the Griess assay used in this study measures the

presence of NO2/NO3 in biological fluids, our results indi-cate that EUK-134 not only reduces the presence of NObut also the generation of NO itself. This is supported byour finding that EUK-134 reduced the expression ofiNOS in the kidneys of rats subjected to renal I/R. Fur-thermore, reduction of the generation or presence of NOresults in a marked attenuation of peroxynitrite forma-tion, thereby reducing nitrosative stress. This is supportedby reports that peroxynitrite formation can be attenuatedby a reduction in NO generation using inhibitors of iNOSactivity such as L-N6-(1-iminoethyl)lysine [12]. Further-more, peroxynitrite also causes DNA strand breaks result-ing in PARP activation [36]. In this study, renal I/Rcaused an increase in the immunological evidence of theformation of peroxynitrite, which was identified as in-creased nitration of proteins. We report here that EUK-134 reduces evidence of nitrotyrosine formation duringrenal I/R injury and propose that EUK-134 attenuates theformation of peroxynitrite thereby contributing to a re-duction in nitrosative stress.

In conclusion, we report here for the first time thatEUK-134 reduces the renal dysfunction and injury causedby I/R of the kidney of the rat via reduction of both oxida-tive and nitrosative stress. As both oxidative and nitrosa-tive stress are associated with renal I/R injury and are sig-nificant factors in the development of ischemic ARF, wepropose that EUK-134 may be of therapeutic potential insituations where the kidney is exposed to ischemia fol-lowed by reperfusion (e.g. during renal transplantation).

Acknowledgements

The authors would like to thank Dr. Rui Pinto, University of Lis-bon, Portugal, and the Clınica Médica é Diagnostico Dr. JoaquimChaves, Lisbon, Portugal, for measurement of urinary NAG activi-ties. P.K.C. was funded by the National Kidney Research Fund(Project Grant R41/2/2000). This work was, in part, supported bythe Clınica Médica é Diagnostico Dr Joaquim Chaves, Lisbon,Portugal.

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