Original Contribution

Exposure to low- vs iso-osmolar contrast agents reducesNADPH-dependent reactive oxygen species generation in a cellularmodel of renal injury

Giuseppe Stefano Netti a, Clelia Prattichizzo a, Eustacchio Montemurno b, Simona Simone c,Cesira Cafiero c, Federica Rascio b, Giovanni Stallone b, Elena Ranieri a,Giuseppe Grandaliano b, Loreto Gesualdo c,n

a Department of Medical and Surgical Sciences, Section of Clinical Pathology, University of Foggia, Foggia 71122, Italyb Department of Medical and Surgical Sciences, Section of Nephrology, University of Foggia, Foggia 71122, Italyc Department of Emergency and Organ Transplantation, Section of Nephrology, University of Bari "Aldo Moro," Bari 70124, Italy

a r t i c l e i n f o

Article history:Received 5 August 2013Received in revised form13 November 2013Accepted 15 November 2013Available online 1 December 2013

Keywords:Contrast-induced nephropathyLow-osmolar contrast mediumIso-osmolar contrast mediumReactive oxygen speciesNADPH oxidaseFree radicals

a b s t r a c t

Contrast-induced nephropathy represents the third cause of hospital-acquired acute renal failure.This study investigated the effects of low- vs iso-osmolar contrast medium (CM) exposure on NADPH-dependent reactive oxygen species (ROS) generation by tubular cells. X-ray attenuation of iohexol,iopamidol, and iodixanol was assessed at equimolar iodine concentrations and their effects on humanrenal proximal tubular cells (PTCs) were evaluated with equally attenuating solutions of each CM.Cytotoxicity, apoptosis, and necrosis were investigated by trypan blue exclusion, MTT assay, and annexinV/propidium iodide assay, respectively. ROS production was assessed by DCF assay, NADPH oxidaseactivity by the lucigenin-enhanced chemiluminescence method, and Nox4 expression by immunoblot.Yielding the same X-ray attenuation, CM cytotoxicity was assessed in PTCs at equimolar iodineconcentrations. More necrosis was present after incubation with iohexol and iopamidol than afterincubation with equal concentrations of iodixanol. Iohexol and iodixanol at low iodine concentrationsinduced less cytotoxicity than iopamidol. Moreover, both iohexol and iopamidol induced more apoptosisthan iodixanol, with a dose-dependent effect. ROS generation was significantly higher with iopamidoland iohexol compared to iodixanol. NADPH oxidase activity and Nox4 protein expression significantlyincreased after exposure to iopamidol and iohexol, with a dose-dependent effect, compared withiodixanol. CM-induced Nox4 expression and activity depended upon Src activation. In conclusion, atangiographic concentrations, iodixanol induces fewer cytotoxic effects on cultured tubular cells thaniohexol and iopamidol along with a lower induction of Nox4-dependent ROS generation. This enzymemay, thus, represent a potential therapeutic target to prevent iodinated CM-related oxidative stress.

& 2013 Elsevier Inc. All rights reserved.

Acute kidney injury (AKI) is a frequent and devastating pro-blem in hospitalized adults with increasing rates of mortality andmorbidity [1]. Several studies in large adult cohorts have shownthat contrast-induced AKI (CI-AKI), formerly known as contrast-induced nephropathy, is the third most common cause of hospital-acquired AKI, accounting for 4–10% of cases [2]. The incidence ofCI-AKI in the general population does not exceed 2%; however, inhigh-risk patients it can rise to more than 50% [3].

CI-AKI is defined as a sudden decrease in renal function after ivadministration of contrast medium (CM) in the absence of any

other cause [4] and is significantly associate with increased cost,hospital stay, and in-hospital and long-term morbidity and mor-tality [5–7].

The pathogenesis of CI-AKI is poorly understood. CM osmolarityhas been suggested to play a key role. Low-osmolar nonionic CMshave fewer nephrotoxic effects than high-osmolar ionic contrastmedia [8]. In addition, in high-risk patients the iso-osmolar dimeriodixanol has been recently shown to be associated with fewernephrotoxic effects than low-osmolar monomeric CMs [9]. However,these results are still debated [10], and even iso-osmolar contrastagents have been reported to cause nephrotoxicity [11].

Renal medullary hypoxia and direct tubular toxic effects of CM arethe major pathogenic events of CI-AKI [12,13]. A direct toxic effect ofCM on renal tubules has been shown in both clinical trials andexperimental models [14–16]. Studies in humans suggested thatreactive oxygen species (ROS) may contribute to CI-AKI [17–19].

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journal homepage: www.elsevier.com/locate/freeradbiomed

Free Radical Biology and Medicine

0891-5849/$ - see front matter & 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.freeradbiomed.2013.11.016

Abbreviations: AKI, acute kidney injury; CM, contrast medium; DPI, diphenyleneiodonium; DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate; NAC, N-acetylcys-teine; ROS, reactive oxygen species

n Corresponding author. Fax: 0039 080 5575710.E-mail address: loreto.gesualdo@uniba.it (L. Gesualdo).

Free Radical Biology and Medicine 68 (2014) 35–42

Several animal models confirmed that CI-AKI is characterized by anincreased ROS production [20,21].

Oxygen radicals are endogenously produced and levels canincrease during oxidative stress. The most common oxygen radi-cals are superoxide (O2

�), hydrogen peroxide (H2O2), and hydroxylradical (OH�). O2

� and OH� are more reactive than H2O2, which isnot a radical, but exhibits a greater membrane permeability [22].

Although in vitro observations suggest a role for oxidativestress in CM-induced tubular cell necrosis [23], the sources ofROS in CI-AKI are still largely unclear.

In this study we investigated the cytotoxic and proapoptoticeffects of the low-osmolar CMs iohexol and iopamidol versus theiso-osmolar CM iodixanol, along with their ability to generateintracellular ROS by the activation of Nox4 in human proximaltubular cells.

Materials and methods

Contrast media

Three CMs were used: nonionic low-osmolar iohexol (672mOsm/kg H2O; Omnipaque-300, GE Healthcare, Princeton, NJ,USA), nonionic low-osmolar iopamidol (616 mOsm/kg H2O; Iopa-miro-300, Bracco Imaging, Milan, Italy), and nonionic iso-osmolariodixanol (290 mOsm/kg H2O; Visipaque-320, GE Healthcare, LittleChalfont, UK). All contrast agents were used in ready-to-useformulations.

Definition of equally attenuating CM concentrations

One-milliliter syringes filled with 20, 50, 125, and 250 mgiodine(mg I)/ml concentrations of iohexol, iopamidol, and iodix-anol were exposed to the X-rays of a digital angiographic unit at arange of 70–80 kV. Digital images were recorded and equallyattenuating concentrations were determined by comparison ofmean gray scale intensity of comparable regions of interest, asquantified by NIH ImageJ software (http://rsbweb.nih.gov/ij/).

Cell culture

HK-2 cells, a proximal tubular epithelial cell line of humanorigin, were obtained from the American Type Culture Collection(Rockville, MD, USA) and grown in Dulbecco’s modified Eagle’smedium (DMEM)–F12 medium (Sigma–Aldrich, St. Louis, MO,USA) supplemented with 10% fetal bovine serum (FBS), 2 mML-glutamine, and 100 U/ml penicillin–streptomycin (all from Sigma–Aldrich) at 37 1C in a humidified atmosphere with 5% CO2.Subconfluent cells were starved for 18 h in DMEM–F12 withoutFBS and then incubated with either control medium (serum-freeDMEM–F12) or various concentrations of the contrast agentsdiluted in serum-free medium for the indicated time periods.The final osmolarity in each condition was similar to that ofserum-free medium (280 and 320 mOsm/kg).

Trypan blue exclusion test

Cell viability was assessed by using the trypan blue exclusiontest. Briefly, cells were treated for 24 h with either control mediumor iohexol, iopamidol, or iodixanol at increasing concentrations(12.5, 25, 50, 100, 150, 270, and 300 mg I/ml), detached with 0.05%trypsin in Hanks’ balanced salt solution, stained with 0.4% trypanblue solution for 3 min, and counted using a Neubauer chamber.The cell viability was calculated as total viable cells (number ofunstained cells)/total number of cells (number stained and

unstained cells) � 100. All of the samples were assayed intriplicate.

MTT assay

Measurement of cell metabolic activity was performed by the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) assay. Briefly, subconfluent cells were treated with eithercontrol medium or various concentrations of each contrast agent(20, 50, and 100 mg I/ml) diluted in serum-free DMEM–F12. Then,MTT (5 mg/ml) was added to each condition for 4 h. Thereafter,the formazan was dissolved with 0.04 M HCl in isopropyl alcoholafter blotting the culture medium. Finally, the plates were incu-bated at 37 1C for 1–2 h and the reduction of MTT was quantifiedby absorbance at a wavelength of 570 nm using a microplatereader (Victor; PerkinElmer, Waltham, MA, USA). Cell viabilityratio (%) was calculated as (Atreated group/Acontrol group) � 100. Eachassay was performed in triplicate.

Analysis of apoptotic cells

The Annexin V–FITC Apoptosis Detection Kit (Beckman Coulter,Milan, Italy) was used to assess cell apoptosis by flow cytometry.Cells were stimulated with either control medium or each contrastagent at various concentrations (20, 50, and 100 mg I/ml) for 24 h.After the double staining with annexin V–FITC and propidiumiodide (PI), the cells were analyzed with a flow cytometer (Cell LabQUANTA; Beckman Coulter). Cells were discriminated into viablecells (AnnV�/PI�), dead cells (AnnV�/PIþ), early apoptotic cells(AnnVþ/PI�), and late apoptotic cells (AnnVþ/PIþ). All of thesamples were assayed in triplicate.

Measurement of intracellular ROS production

The oxidation-sensitive fluorescent probe 2′,7′-dichlorodihy-drofluorescein diacetate (DCFH-DA; Molecular Probes, Eugene, OR,USA) was used to detect the generation of intracellular ROS aspreviously described [24,25]. To this purpose, HK-2 (1�105) cellswere incubated with various concentrations of each CM (20–50 mgI/ml) and ROS formation was assessed at various time intervals (0, 10,20, 30, and 40 min of CM exposure). To assess the inhibitory effect ofN-acetylcysteine (NAC) on ROS production, HK-2 cells were preincu-bated for 1 h with NAC (5 mM). To assess the inhibitory effect ofSU6656 on ROS production, HK-2 cells were preincubated for 12 hwith the inhibitor at 5 μM. DCF fluorescence was detected atexcitation at 495 nm and emission at 525 nm wavelengths, with amicroplate reader (Victor; PerkinElmer). Emitted fluorescence wasnormalized to the total amount of proteins. Triplicate wells weremeasured in each experimental group.

NADPH oxidase assay

NAD(P)H oxidase activity was determined by the lucigenin-enhanced chemiluminescence method as previously described [26].HK-2 cells were stimulated with various concentrations of eachCM (20–50 mg I/ml) for 40 min and then rinsed in phosphate-buffered saline (PBS), harvested in lysis buffer (20 mM KH2PO4,pH 7.0, 1 mM EGTA, 1 mM phenylmethanesulfonyl fluoride(PMSF), 8 mg/ml leupeptin) on ice, and centrifuged at 800 g at4 1C for 10 min. Homogenates were subjected to a centrifugation(800 g) at 4 1C for 10 min to remove cell debris and used imme-diately. To start the assay, 100 μl of supernatant was added to900 μl of 50 mM phosphate buffer, pH 7.0, containing 150 mMsucrose, 5 μM lucigenin, and 100 μM NADPH (Roche Diagnostics,Mannheim, Germany). Photon emission in terms of relative light

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units was measured every 30 s for 6 min in a luminometer(GloMax 20/20; Promega, Madison, WI, USA). A buffer blank wassubtracted from each reading. Superoxide production was mea-sured as the rate of relative chemiluminescence (light) units perminute per microgram of protein (RLU/min mg) and expressed asfold increase compared to untreated cells. As positive control, HK-2 cells were treated with 100 mM H2O2 for 30 min, whereasinhibition of NAD(P)H oxidase activity was performed by pretreat-ment with 1 mM DPI. Protein content was measured using theBradford method (Bio-Rad Laboratories, Hercules, CA, USA). All theexperiments were performed in triplicate.

Immunoblot

HK-2 cells were stimulated with various concentrations of eachCM (20–50 mg I/ml) for 24 h, and to assess the inhibitory effect ofSU6656, HK-2 cells were preincubated for 12 h with the inhibitorat 5 μM and then rinsed in PBS and lysed with RIPA buffer (1 mMPMSF, 5 mM EDTA, 1 mM sodium orthovanadate, 150 mM sodiumchloride, 8 mg/ml leupeptin, 1.5% Nonidet P-40, 20 mM Tris–HCl,pH 7.4). The lysates were centrifuged at 10,000 g at 4 1C for 10 min.Protein concentration was determined by the Bradford method(Bio-Rad Laboratories). Proteins were subjected to SDS–PAGE on

Fig. 1. Comparison of cell viability, apoptosis, and necrosis on HK-2 after 24-h incubation with different contrast agents. (A) At trypan blue testing, after 24-h incubation,there were more viable cells after incubation with iodixanol than with the other CMs. Data are reported as mean percentage of viable cells 7 SD, from at least threeindependent experiments. (B) After 24-h incubation, iohexol and iodixanol at low iodine concentrations (20, 50, and 100 mg I/ml) induced comparable inhibition of MTTconversion with a dose-dependent effect, whereas iopamidol showed a marked cytotoxic effect, compared with iodixanol. Data are reported as mean percentage ofundamaged control cells 7 SD, for at least three independent experiments. (C) After annexin V–FITC/PI staining, both iohexol and iopamidol induced more apoptosis thaniodixanol, with a dose-dependent effect, as assessed by flow cytometry. The plots are representative of at least three independent experiments.

G.S. Netti et al. / Free Radical Biology and Medicine 68 (2014) 35–42 37

an 8% gel and then electrotransferred onto a polyvinylidenedifluoride membrane (Millipore, Bedford, MA, USA). The filterwas blocked with 5% nonfat milk powder in PBS containing 0.1%Tween 20 (TBS). The blot was then incubated with anti-Nox4antibody (1:100; AbCam, Cambridge, UK). The membranes werewashed in TBS and incubated with horseradish peroxidase-conjugated antibodies (goat anti-mouse IgG 1:50,000; Bio-RadLaboratories). Membranes were stripped and immunoblotted withanti-β-actin antibody (1:10,000 in TBS) and the signal wasdetected using the enhanced chemiluminescence system (ECL;Amersham, Piscataway, NJ, USA). The film was acquired using aUVP Chemidoc-IT scanner (UVP, LLC, Upland, CA, USA) andquantified by NIH ImageJ software. β-Actin band intensity wasused for equal loading control and for normalization.

Statistical analysis

The results of the quantitative variables were expressed asmeans 7 SD, and those of the qualitative variables wereexpressed as proportions. Differences between groups were ana-lyzed by unpaired t-test analysis or Mann–Whitney U test, asappropriate. Correlation between two variables was ascertained byPearson and Spearman's correlation test, as appropriate. All testswere two-tailed. A p value o0.05 was considered statisticallysignificant. Statistical analyses were performed using the SPSS 17.0software (SPSS, Inc., Chicago, IL, USA).

Results

X-ray attenuation does not differ among contrast agents

We first compared the radiopacity of the three CMs selected forthis study: low-osmolar CMs iohexol and iopamidol and iso-osmolar CM iodixanol. To this aim 1-ml syringes were filled withdilutions of ready-to-use 250, 125, 50, and 20 mg I/ml concentra-tions of each CM and X-ray attenuation was measured at 70–80 kV(data not shown). Quantitative analysis of digitally recordedimages did not show statistically significant differences amongX-ray attenuation of the different CMs at the concentrationsexamined (data not shown).

Iohexol and iopamidol induce more cytotoxic effect on human renaltubular cells compared to iodixanol

In an attempt to assess cell cytotoxicity at a wide range ofconcentrations, human renal tubular cells (HK-2) were exposedto increased concentrations of the three CMs for 24 h. At trypanblue testing there were more viable cells after incubation withiodixanol compared to the other CMs (93.974.0% vs 72.778.8%for iohexol and 93.974.0% vs 33.3712.5% for iopamidolat 150 mg I/ml, p o 0.05; 93.676.8% vs 14.478.3% for iohexoland 93.676.8% vs 0% for iopamidol at 270 mg I/ml, p o 0.05;82.8710.2% vs 10.278.6% for iohexol and 82.8710.2% vs 0% foriopamidol at 300 mg I/ml, p o 0.05; Fig. 1A). Intriguingly, cellcytotoxicity of both iohexol and iopamidol increased in a dose-dependent manner, whereas iodixanol did not significantly affectcell viability at higher doses, compared to lower doses.

Subsequent tests were performed at CM concentrations thatare more likely to be reached at the renal level during angio-graphic procedures (20–100 mg I/ml). After 24-h incubation atlower iodine concentrations (20, 50, and 100 mg I/ml), iohexol andiodixanol induced comparable inhibition of MTT conversion with adose-dependent effect (53.6714.3 vs 54.970.7% at 20 mg I/ml,

44.0715.0 vs 36.875.5% at 50 mg I/ml, and 31.677.3 vs43.176.9% at 100 mg I/ml of undamaged control cells for iohexoland iodixanol, respectively; p ¼ ns), whereas iopamidol showed amarked cytotoxic effect, compared with iodixanol (26.074.8 vs54.970.7% at 20 mg I/ml, 23.276.7 vs 36.875.5% at 50 mg I/ml,and 22.576.0 vs 43.176.9% at 100 mg I/ml of undamaged controlcells for iohexol and iodixanol, respectively; p o 0.05; Fig. 1B).

Both iohexol and iopamidol induced more apoptosis thaniodixanol, according to annexin V–FITC/PI staining, with a dose-dependent effect (iodixanol vs iopamidol, for early apoptosis,22.475.0 vs 30.375.4% at 20 mg I/ml, 27.576.4 vs 44.877.2%at 50 mg I/ml, and 30.074.5 vs 44.675.3% at 100 mg I/ml,respectively; for late apoptosis/necrosis, 6.572.7 vs 15.374.9%at 20 mg I/ml, 8.573.5 vs 17.376.3% at 50 mg I/ml, and 12.873.6vs 23.274.7% at 100 mg I/ml, respectively; p o 0.05 at eachconcentration; Fig. 1C).

ROS generation is markedly increased after exposure to iohexoland iopamidol compared to iodixanol

To investigate the possible mechanisms underlying the directtoxicity of iodinated CM on human renal tubular cells, wemeasured the intracellular ROS levels in HK-2 cells stimulatedwith the three CMs at 20 and 50 mg I/ml. ROS generation wassignificantly increased in HK-2 cells treated with iopamidol andiohexol compared with iodixanol, with a time-dependent effect(after 40 min 21,26372124 and 898571601 vs 44307801 meanfluorescence intensity (MFI)/μg of proteins at 20 mg I/ml, p o0.05; 984471124 and 81137101 vs 40767601 MFI/μg of pro-teins at 50 mg I/ml, p o 0.05; Fig. 2A and B). In this setting ROSproduction was abolished by preincubation with a ROS scavenger,NAC (5 mM), as explored under all the experimental conditions(Fig. 2A and B).

NADPH oxidase activity is increased after exposure to iohexoland iopamidol compared to iodixanol

To investigate the role of NADPH oxidase in ROS generation inthis setting, we tested its activity in HK-2 cells stimulated with thethree CMs at 20 and 50 mg I/ml. NADPH-dependent superoxidegeneration, assessed by the lucigenin-enhanced chemilumines-cence method, was significantly higher after exposure to iohexoland iopamidol, with a dose-dependent effect, compared to iodix-anol (5634.97582.3 and 5447.97799.5 vs 3759.67495.9 RLU/min μg at 20 mg I/ml, p o 0.05; 6717.67811.9 and 6254.87616.6vs 3932.27583.6 RLU/min μg at 50 mg I/ml, p o 0.05; Fig. 3).

Low-osmolar contrast agents induce expression of Nox4

Immunoblotting analysis showed a detectable expression ofNox4, the renal isoform of NADPH oxidase, in HK-2 cells culturedunder normal growth conditions (Fig. 4). Nox4 protein expressionwas significantly induced after exposure to iohexol and iopamidol,with a dose-dependent effect, compared to iodixanol (p o 0.01 vsiohexol and iopamidol at 20 mg I/ml and p o 0.05 vs iohexol andiopamidol at 50 mg I/ml; Fig. 4). Moreover, at both 20 and 50 mg I/ml, a direct correlation was observed between Nox4 expressionand activity (R2 ¼ 0.9614, p o 0.05, at 20 mg I/ml and R2 ¼0.9427, p o 0.05, at 50 mg I/ml).

Src inhibition markedly reduces Nox4 activity and expression

To investigate the possible mechanisms inducing expression ofNox4, we treated HK-2 cells with a Src inhibitor (SU6656). Afterpretreatment with SU6656 at 10 μM for 12 h, HK-2 cells were

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stimulated with the three CMs and the intracellular ROS levelswere assessed with DCF assay. ROS generation was significantlydecreased in HK-2 cells preincubated with SU6656 (Fig. 5A).Immunoblotting analysis showed a detectable expression ofNox4 in HK-2 cells cultured after exposure to CMs, whereasNox4 expressionwas significantly reduced after pretreatment withSU6656 (Fig. 5B).

Discussion

In this study we demonstrate that the lower cytotoxic andproapoptotic effects of the iso-osmolar CM iodixanol, compared tothe low-osmolar CMs iohexol and iopamidol, on human kidneycells are mediated by a differential activation of the NADPHoxidase isoenzyme Nox4, which leads to intracellular ROS genera-tion and cell apoptosis, and that these events are mediated by CM-induced Src activation.

The pathogenesis of contrast-induced nephropathy is poorlyunderstood. Classically the osmolality of CM has been consideredto play an important role in this setting. First-generation CMs wereionic monomers containing a benzene ring with three iodineatoms, with an osmolarity between 1500 and 1800 mOsm/kg(high-osmolar CMs). To minimize the strong nephrotoxicityrelated to high osmolarity, nonionic monomers were developed,reducing osmolarity by half (low-osmolar CMs) but still exhibitingmore than double the osmolarity of plasma. Third-generationagents are dimers almost iso-osmolar to plasma (iso-osmolarCMs) but with increased viscosity, which, in turn, results inproblematic injection through small vascular catheters. Never-theless, some reports suggest that even third-generation CMspresent a nephrotoxic potential [11].

Thus, it is not yet elucidated whether iso-osmolar CMs reallyexert fewer nephrotoxic effects than low-osmolar CMs. Further-more it is conceivable that the differences observed in the clinicalsetting are due not only to differences in osmolarity. Variations inrenal blood flow, leading to hypoxia of the renal medulla, anddirect cytotoxic effects of CMs on renal cells may contribute to theCI-AKI pathogenesis [12,13].

In our in vitro study both iso-osmolar and low-osmolarCM were tested at very low concentrations, which does not

Fig. 2. Intracellular ROS production after exposure to different contrast agents.Intracellular ROS levels were investigated by DCFH-DA in HK-2 cells treatedwith iohexol, iopamidol, and iodixanol at (A) 20 and (B) 50 mg I/ml. ROSgeneration was significantly increased in HK-2 cells treated with iopamidoland iohexol compared with iodixanol with a time-dependent effect. Preincuba-tion with a ROS scavenger, NAC (5 mM), significantly inhibited ROS productionin all the experimental groups at the concentrations explored (dotted lines).Data are representative of at least three experiments and are expressed asmeans 7 SD.

Fig. 3. NADPH oxidase activity after exposure to different contrast agents.NADPH oxidase activity was measured by lucigenin-enhanced chemilumines-cence in HK-2 cells treated with iohexol, iopamidol, and iodixanol at 20 and50 mg I/ml for 40 min. NADPH oxidase activity was significantly higher afterexposure to iohexol and iopamidol, with a dose-dependent effect, compared toiodixanol. As positive control, HK-2 cells were treated with 100 mM H2O2.Preincubation with the NADPH oxidase inhibitor DPI (10 mM) significantlydecreased NADPH oxidase activity. ROS production is expressed as meanfluorescence normalized to the total amount of proteins 7 SD. Data arerepresentative of at least three experiments.

Fig. 4. Exposure to contrast agents differentially induces Nox4. Nox4 proteinexpression was analyzed by immunoblotting in HK-2 cells treated with iohexol,iopamidol, and iodixanol at 20 and 50 mg I/ml. Nox4 protein expression wassignificantly higher after exposure to iohexol and iopamidol, with a dose-depen-dent effect, compared to iodixanol. As positive control, HK-2 cells were treated with100 mM H2O2. Preincubation with the NADPH oxidase inhibitor DPI (10 mM)significantly decreased Nox4 protein expression. Nox4 protein expression wasnormalized to β-actin band intensity. Data are represented as mean Nox4/β-actinratio 7 SD and are representative of at least three experiments.

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significantly change total osmolarity, which ranges between280 and 320 mOsm/kg. Thus it is conceivable that differentproapoptotic effects are involved in direct CM toxicity on renaltubular cells.

Moreover, from a clinical standpoint it should be appropriate tocompare contrast agent concentrations that yield the same X-rayattenuation. Thus, under our experimental conditions the examinediso-osmolar and low-osmolar CM showed the same X-ray opacity.

Researchers in early in vitro studies found evidence for directrenal tubular cell toxic effects of CM [15]. In vitro experimentsremain the only approach to examine the direct cytotoxic effects ofCMs on renal cells in the absence of confounding variables, whichcan be found in vivo (e.g., hypoxia due to hemodynamic changes),although the interaction between CM and renal cells is static incell cultures and transient in the clinical setting [27]. The finding offewer in vivo nephrotoxic effects of low-osmolar contrast media,compared with those of high-osmolar ionic compounds, correlateswith their fewer in vitro cytotoxic effects [28].

Understanding of molecular mechanisms underlying CI-AKIonset may therefore have important clinical advantages. Bothclinical trials and animal models suggested a toxic effect of CMson tubular cells [14–16]. Romano et al., in an experimental study[23], observed that CM induces a dose- and time-dependent renalcell apoptosis through the activation of the intrinsic pathway.

Studies in humans indicate that ROS contribute to the devel-opment of CI-AKI [17–19]. Indeed, several experimental animalmodels showed that CI-AKI is accompanied by the increasedproduction of oxygen radicals [19]. In particular, O2

� may rapidlyscavenge nitric oxide (NO), leading to a blunted NO activity in therenal microvasculature and to an increase in oxygen consumption.The resulting tissue hypoxia might hamper the endothelial–epithelial structure and function [22]. Moreover ROS activatestress kinases, including JNK and p38 MAP kinase [29–31], whichmay in turn induce apoptosis.

Apoptosis is an evolutionarily conserved mechanism of elim-ination of undesired cells [32]. The extrinsic pathway is primed bythe activation of various death receptors expressed on the cellsurface. On the other hand, the intrinsic pathway is triggered byseveral intracellular and extracellular stresses whose signals con-verge on the mitochondria [33,34].

Moreover, ROS act as important mediators of inflammatoryprocesses. They stimulate the secretion of profibrotic factors,cytokines, and growth factors and stimulate proliferation, migra-tion, and apoptosis [35–38]. Several enzymatic systems accountfor cellular ROS formation, including mitochondria, NO synthases,and cytochrome P450 enzymes. NADPH oxidases of the Nox familyare enzymes known to generate the most prominent sources ofROS [39]. So far, seven different Nox homologues have beencharacterized, which vary in their expression pattern, subunitcomposition, activity, and type of ROS production [38].

Among them, Nox4 is the main kidney isoform, characterizedby the generation of H2O2 [40–42]. Owing to its constitutiveactivity Nox4 contributes to basal cellular ROS formation [41]. Anincreasing body of evidence links Nox4 to renal pathology. Indeed,this NADPH oxidase has been suggested as a major source of ROSin diabetic nephropathy [43,44] and mediates the activation offibroblasts into myofibroblasts, a key step in renal fibrogenesis [45].To date, no study has characterized the contribution of Nox4 to thepathogenesis of CI-AKI.

It is conceivable that, as well as in other clinical settings,polymorphisms that affect key pro- and antioxidant enzymesmight alter the susceptibility to developing oxidative stress-mediated injury and, in turn, CI-AKI. To date polymorphisms ofNADPH oxidase genes have been related to higher susceptibility toAKI [46] and to diabetic nephropathy secondary to type 1 and type2 diabetes mellitus [47,48], but no studies have been performed toassess a possible association between gene polymorphisms ofNox4 and allied systems and susceptibility to developing CI-AKI.

Recently a possible mechanism involved in Nox4 activation hasbeen described in vascular smooth muscle cells [49], suggestingthat the cytoplasmic tyrosine kinase Src may modulate Nox4activity. This observation led us to consider whether Src could beinvolved in Nox4 expression and activation in our setting. Indeed,the inhibition of this tyrosine kinase significantly reduced Nox4-dependent ROS generation. It is still unclear how CMs may activatedistinct intracellular signaling pathways because, to date, theyhave not been reported to interact with any specific cell-surfacereceptor.

Our study demonstrates that Nox4 activation may represent akey mechanism in the induction of cell toxicity and apoptosis afterCM exposure. Our data, although in vitro, broaden potentialdiagnostic and therapeutic strategies to prevent CM cytotoxicityon renal cells. Although tubular cell Nox4 activity is not directlymeasurable, it represents an intriguing potential target to inhibit

Fig. 5. Src inhibition markedly reduces Nox4 activity and expression. (A) IntracellularROS levels were investigated by DCFH-DA in HK-2 cells treated with iohexol,iopamidol, and iodixanol at 50 mg I/ml. Preincubation with SU6656 (5 μM) signifi-cantly decreased ROS production in all the experimental groups at the exploredconcentrations. Data are representative of at least three experiments and areexpressed as means 7 SD. (B) Nox4 protein expression was analyzed by immuno-blotting in HK-2 cells treated with iohexol, iopamidol, and iodixanol at 50 mg I/ml.Preincubation with SU6656 (5 mM) significantly decreased Nox4 protein expression inall the experimental groups. Nox4 protein expression was normalized to β-actin bandintensity. Data are represented as mean Nox4/β-actin ratio 7 SD and are representa-tive of at least three experiments.

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direct CM damage and to prevent activation of intracellular path-ways leading to apoptosis and/or necrosis of renal tubular cells.

Undoubtedly, our data warrant further in vivo studies toconfirm the role of Nox4 as a potential therapeutic target. How-ever, here we demonstrate for the first time that differentcytotoxic and proapoptotic effects of iodinated CM are associatedwith the activation of the NADPH oxidase isoenzyme Nox4, whichleads to intracellular ROS generation and cell apoptosis.

Conclusion

The pivotal role of NADPH oxidase in the upregulation of ROS ina cellular model of contrast-induced nephropathy makes thisenzyme a potential target for therapeutic intervention in iodinatedcontrast medium-related oxidative stress.

Acknowledgments

This work was funded by MERIT Program RBNE08BNL7 and byPRIN2008 Program 2008M9WSJX_002, both from the ItalianMinistry of Education and University (granted to G.S.N.) and byan unrestricted grant from GE Healthcare (granted to L.G.). C.P. isthe recipient of a MERIT Program fellowship.

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