J Clin Pharmacol xxxx;xx:x-x 1365J Clin Pharmacol 2012;52:1365-1372 1365

Acute kidney injury (AKI) has long been recog-nized to be a severe disorder.1 Using a broad

consensus of experts, the Acute Dialysis Quality Initiative group developed a standardized scale to define acute renal failure (ARF). The acronym RIFLE stands for the increasing severity classes for this disease entity. They are as follows: risk (R), injury (I), failure (F), loss (L), and end-stage kidney disease (E).2 Based on the RIFLE score, the rate of AKI varies from 10.8% to 100% in the intensive care unit (ICU) setting.1-6 The most common etiology of ARF in ICUs is acute tubular necrosis, which accounts for 80% of

Reactive oxygen species and inflammation have been implicated in renal tubule cell injury. However, there is some controversy concerning whether antioxidants might attenuate oxidative damage and inflammation in humans after hypotension in the setting of critical illness. This study was a prospective, randomized, double-blinded, placebo-controlled study that included patients with hypotension. Patients were randomized to receive either N-acetylcysteine (NAC; 50 mg/kg by 4 hours followed by 100 mg/kg/d for 48 hours diluted in 5% glucose) and deferoxamine (DFX; at a single dose of 1000 mg diluted in 5% glucose) or placebo. The primary study outcome was the serum levels of markers of oxidative damage and inflammatory response. Secondary outcomes included the incidence of acute renal failure, serum creatinine at hospital discharge, intensive care unit length of stay, and

length of hospital stay. Thirty patients were enrolled in the study. The use of NAC plus DFX decreased the oxida-tive damage parameters but not plasma interleukin-6 levels. In contrast, plasma nitrite levels increased 24 hours after NAC plus DFX administration. On analysis of secondary outcomes, it was observed that creatinine lev-els at hospital discharge were lower in patients receiving NAC plus DFX when compared with placebo. NAC plus DFX administration was able to decrease plasma markers of oxidative damage and creatinine levels at hospital discharge.

Keywords: Renal failure; antioxidants; hypotension; shock

Journal of Clinical Pharmacology, 2012;52:1365-1372© 2012 The Author(s)

From Laboratório de Fisiopatologia Experimental and Instituto Nacional Translacional em Medicina (INCT-TM), Programa de Pós-Graduação em Ciências da Saúde, Unidade Acadêmica de Ciências da Saúde, Universidade do Extremo Sul Catarinense, SC, Brazil (Dr Fraga, Ms Tomasi, Ms Biff, Ms Topanotti, Ms Felisberto, Ms Vuolo, Dr Petronilho, Dr Dal-Pizzol, Dr Ritter), and Unidade de Terapia Intensiva, Hospital São José, Criciúma, SC, Brazil (Dr Fraga, Dr Dal-Pizzol, Dr Ritter). Submitted for publication March 17, 2011; revised version accepted July 3, 2011. Address for correspondence: Felipe Dal Pizzol, MD, PhD, Laboratório de Fisiopatologia Experimental, PPGCS, UNASAU, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil; e-mail: piz@unesc .net.DOI: 10.1177/0091270011418657

The Effects of N-Acetylcysteine and Deferoxamine on Plasma Cytokine and Oxidative Damage Parameters

in Critically Ill Patients With Prolonged Hypotension: A Randomized

Controlled Trial

Cassiana Mazon Fraga, MD, Cristiane Damiani Tomasi, MSc, Daiane Biff, MSc, Maria Fernanda Locks Topanotti, BSc, Francine Felisberto, BSc,

Francieli Vuolo, BSc, Fabricia Petronilho, PhD, Felipe Dal-Pizzol, MD, PhD, and Cristiane Ritter, MD, PhD

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FRAgA ET AL

cases.2 Hypotension, as a result of sepsis, heart failure, or severe hypovolemia, is a common cause of acute tubular necrosis.7

Reactive oxygen species (ROS) have been impli-cated as important effectors of tubular cell injury in both ischemic and toxic acute tubular necrosis.8-11 Ischemia-reperfusion injury to the kidney also induces an inflammatory response, which worsens and prolongs tubular and endothelial injury by increasing renal expression of many proinflamma-tory cytokines and adhesion molecules that stimu-late the production of vasoactive mediators and the activation of leukocytes. Activated leukocytes can directly injure both endothelial and tubular cells by releasing a number of potent oxidants, including superoxide and nitric oxide.12,13

N-acetylcysteine (NAC) is a thiol-containing com-pound with antioxidant, anti-inflammatory, and microcirculatory effects.14 NAC acts as an antioxi-dant either directly by scavenging for ROS or indi-rectly by replenishing depleted cellular glutathione stores. Its anti-inflammatory properties include its inhibition of both neutrophil activation and leuko-cyte-endothelial cell adhesion and its attenuation of cytokine release. It likely does this by modulating the gene expression of these mediators at the tran-scriptional level.15 NAC has some utility in the pre-vention of intravenous contrast-induced nephropathy (CIN).7,16 NAC has also been studied in several dis-eases that involve oxidative stress, including sepsis, with controversial results.7,15-21 Some of these limita-tions could be secondary to the fact that NAC at high concentrations, probably because of its capacity to reduce iron to the catalytically active form, partici-pates in the generation of hydroxyl radicals via the Fenton reaction, thus increasing oxidative stress.19,22 In this context, the use of deferoxamine (DFX), an iron chelator, seems to improve NAC’s effects in sev-eral different animal models of disease.23-25 In addi-tion, the NAC doses used in clinical research vary between studies, and there is some doubt concern-ing whether the doses used can attenuate oxidative damage and inflammation in humans.18,19

Thus, we hypothesized that the combination of NAC and DFX would decrease oxidative stress and inflammatory markers, therefore attenuating AKI in patients with hypotension admitted to an ICU.

METHODs

Patient selection. This randomized, double-blinded, placebo-controlled study was approved by the Hospital

São José ethics committee. This study is regis-tered at clinical trials.gov with the protocol number NCT00870883.

All patients admitted to a 20-bed ICU were screened daily. Patients were eligible for inclusion if they had new-onset hypotension of at least 30 min-utes’ duration, which was defined as a mean arterial blood pressure lower than 60 mm Hg or a require-ment for vasopressor medications (ie, dopamine at a dosage greater than 10 µg/kg/min or norepinephrine or epinephrine at any concentration; inotropes, such as dobutamine were not included).7 Patients who had a qualifying episode of hypotension within the previous 48 hours were excluded. These determina-tions were made by examining bedside vital signs monitors and flow sheets. The first dose of NAC plus DFX or placebo had to be given within 12 hours of meeting the inclusion criterion. Patients were excluded if they met any of the following exclusion criteria: age younger than 18 years, participating in another research study, known allergy to NAC or DFX,26,27 receiving high-dose NAC for acetaminophen overdose, receiving NAC for CIN prophylaxis in the past 7 days, pregnant or lactating, received activated charcoal in previous 48 hours, serum creatinine higher than 3.5 mg/dL or already on dialysis, advanced malignancy, or patients with the need for extracor-poreal circulation in the previous 48 hours. Informed consent and authorization were obtained from patients or their surrogate medical decision makers between March 2009 and December 2009.

Study protocol. Patients were randomized by a research nurse to receive NAC and DFX or placebo. The NAC dosage was 50 mg/kg by 4 hours followed by 100 mg/kg/d for 48 hours diluted in 5% glucose.15 DFX was administered at a single dose of 1000 mg diluted in 5% glucose and was administered intrave-nously at a rate of 15 mg/kg/h. The DFX administra-tion regimen was based on preclinical data from our group demonstrating that a single dose is sufficient to improve NAC’s effects in several different animal models of acute disease.18,23-25 In acute iron overload, the maximum DFX dose that can be given in 24 hours is 80 mg/kg. However, because shock is not associated with acute iron overload, we decided to use a lower DFX dose (1.000 mg/24 hours) because of safety issues. Patients in the placebo arm received the same volume and infusion rate of 5% glucose without the addition of NAC and DFX. The research nurse was not involved in the study in any other way and was responsible for the preparation of the infu-sion in the hospital pharmacy. After the preparation

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PHARMACODyNAMICS 1367

of the infusion, vials were covered, and the research nurse began the patient’s infusion. Because NAC has a particular odor, the installation and removal of the vials were performed only by the research nurse so as to keep other members of the ICU staff blind to the participant’s group. If any member of the research team or ICU staff detected an adverse drug reaction, this knowledge was communicated to the research nurse who was authorized to open the concealment and submit data to the 2 independent researchers responsible for the monitoring of the trial.

Before antioxidant or placebo administration (D0), 24 (D1), 48 (D2), and 72 (D3) hours after study entry, blood was drawn (unless patient was dis-charged from the ICU) and serum was obtained to posterior determination of oxidative and inflamma-tory parameters.

Data collection. After entry into the study, demo-graphic information was collected for each patient. Preexisting medical problems were determined. The patient’s baseline creatinine for the study was con-sidered to be the most recent creatinine measured before entry into the study. The median (interquar-tile range) time to baseline creatinine measure and study enrollment was 24 (12-36) hours. The Acute Physiology and Chronic Health Evaluation II and Sequential Organ Failure Assessment (SOFA) scores were computed for the 24-hour period preceding the administration of the first dose of study medication. After entry into the study, SOFA scores were com-puted daily while patients remained in the ICU. During the duration of the hospitalization, serum creatinine values were recorded. The primary study outcome was the serum levels of markers of oxida-tive damage and inflammatory response. The sec-ondary outcomes were the incidence of ARF, ICU length of stay, and length of hospital stay.

Thiobarbituric acid reactive species (TBARS). As an index of oxidative stress, we used the formation of TBARS during an acid-heating reaction, as previ-ously described.28 Briefly, the samples were mixed with 1 mL of trichloroacetic acid 10% and 1 mL of thiobarbituric acid 0.67%. They were then heated in a boiling water bath for 15 minutes with the addition of butylated hydroxytoluene. TBARS was determined by the absorbance at 535 nm using 1,1,3,3-tetrameth-oxypropane as an external standard. Results are expressed as malondialdehyde equivalents per mil-ligram of protein.

Measurement of protein carbonyls. The oxidative damage to proteins was assessed by determining the

number of carbonyl groups on the proteins. This assessment was based on the reaction with dinitro-phenylhydrazine, as previously described.29 Briefly, proteins were precipitated by the addition of 20% trichloroacetic acid and redissolved in dinitrophe-nylhydrazine; the absorbance was read at 370 nm.

Measurement of total nitrite concentration. The total concentration of NO3

– and NO2– was determined in

thawed serum supernatant using a modified Griess reaction method.30 Triplicate samples of serum were incubated for a minimum of 3 hours at 20°C with glucose-6-phosphate (500 µmol/L), glucose-6-phosphate dehy-drogenase (160 U/L), NADPH (1 µmol/L), and nitrate reductase (20 U/L) in phosphate buffer (80 mmol/L, pH 7.5). The Griess reaction was then initiated by the addition of sulfanilamide to a final concentration of 0.5% (wt/vol), orthophosphoric acid (1.25%, vol/vol), and N-(1 naphthyl) ethylenediamine hydrochloride (0.05%, wt/vol). After a further incubation at 20°C for 10 minutes, the absorbance of each sample mixture was measured at 540 nm and corrected for opacity by measuring the absorbance at 750 nm. The corrected absorbance was interpolated on a standard curve of absorbance plotted versus concentration to determine the concentration of NO2

- in the sample. As all of the NO3

– had already been reduced to NO2- via nitrate

reductase, this represented the combined concentra-tion of NO3

- and NO2- in the serum. Results of the

NO2- assay were expressed as NO2

- concentration in micromoles per liter of serum.

Interleukin-6 measurements. The interleukin-6 (IL-6) concentration, which is a general marker of proin-flammatory response, was determined by capture enzyme-linked immunosorbent assay (ELISA) using ELISA kits (R&D Systems, Minneapolis, Minnesota), as indicated by the manufacturer. This assay yields an intra-assay reproducibility of 4.2%, an interassay reproducibility of 6.4%, and a minimum detectable dose of less than 0.70 pg/mL.

Statistical analysis. The Kolmogorov-Smirnov test was used to test the normality of the data, and either the Student t test or the Mann-Whitney test was used as appropriate. Data were presented as the median (25-75) due to the low number of patients included. Associations between treatment arms and subgroups of categorical variables were assessed using χ2 analyses. The a priori sample size was determined to be 30 patients; this sample size was calculated to be sufficient to determine a relative difference in plasma oxidative damage parame-ters of 20% with α = .05% and β = .20, based on the

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FRAgA ET AL

comparison of 2 means using a 2-sided test (http://www.stat.ubc.ca/~rollin/stats/ssize/n2.html). The dif-ference was set at 20% as the minimum alteration in plasma oxidative damage parameters that provides a relevant biological effect based on data obtained in animal models of sepsis. This trial was designed to determine the effect of NAC plus DFX on the inci-dence of acute renal failure (with a calculated n = 70 for each group; see www.clinicaltrials.gov). As there has been no report concerning the doses of NAC and DFX that can be used in humans, we determined that the blind would be opened when 30 patients were included to determine if the used doses were acting as effective antioxidants or whether the doses needed to be revised.

REsulTs

Forty-five patients were assessed for eligibility. Of these, 12 were excluded because they did not meet the inclusion criteria, and 3 were excluded because they were hypotensive for more than 12 hours prior to their inclusion in the study (Figure 1). Thirty patients were enrolled into the study: 15 patients in the NAC plus DFX arm and 15 patients in the pla-cebo arm. The baseline characteristics of patients in both arms were not significantly different (Table I). No patient dropped out early from the study, and no severe adverse reaction was observed during the study protocol.

The use of NAC plus DFX decreased the levels of oxidative damage markers (Table II). This effect was more pronounced for TBARS than protein carbonyls and was evident during antioxidant administration. It returned to placebo levels after the end of antioxi-dant administration. Patients in the antioxidant arm had lower plasma IL-6 levels 24 hours after the start of treatment (Table II). As observed with the oxida-tive damage parameters, IL-6 returned to placebo levels at the end of antioxidant administration.

On analysis of secondary outcomes, it was observed that only mean creatinine levels at hospital discharge were lower in patients receiving NAC plus DFX when compared with placebo (Table III). The incidence of ARF using the RIFLE criteria, length of ICU and hospital stay, and SOFA scores during drug infusion were not significantly different in the 2 arms (Table III). Analyzing the components of the SOFA score, we observed a worse cardiovascular SOFA in the NAC + DFX arm (4 [3-4] vs 2 [1-4], P = .01) at day 2 and a better renal SOFA in antioxidant-treated patients (1 [1-2] vs 1 [1-3], P = .02). Worse

cardiovascular SOFA scores were accompanied by an increase in plasma nitrite levels in the antioxi-dant arm at D1 (0.27 [0.12-0.75] vs 0.04 [0.01-0.12], P = .02), which returned to placebo levels at 48 and 72 hours (data not shown).

DIsCussION

The main result of this study was that the combina-tion of NAC plus DFX decreases markers of oxida-tive stress and inflammatory response in critically ill patients with hypotension.

In designing this study, we aimed to use NAC, an inexpensive and well-tolerated therapy, which acts as an oxidant scavenger and is widely used in the clinical and preclinical setting for inflammatory or ischemia-reperfusion–associated diseases.7,18-20 In this context, the use of NAC to prevent contrast-induced nephropathy is probably the best example of the relevance of NAC in the clinical setting.16,17 In contrast, the administration of NAC is a matter of debate for several other clinical indications. Komisarof et al7 used NAC to prevent AKI in patients with hypotension and could not find any benefit in its use. Similarly, Spapen15 recently showed that NAC did not protect septic patients from renal dys-function. These limitations of NAC could be attrib-utable to several factors. The oxidative metabolism of NAC can generate thiyl free radicals and can reduce Fe (III) ions, making them able to participate in the generation of hydroxyl radical via the Fenton reaction. These free radicals are increasingly consid-ered to be intermediates in processes that may be involved in the development of the biological dam-age resulting from oxidative stress.18,22,24,31 In vitro, NAC increases hydroxyl radical generation in a system with Fe (III)–citrate and hydrogen peroxide by reducing ferric iron to its catalytic active Fe (II) form.31,32 In this context, Sprong et al19 demon-strated that high doses of NAC increase mortality in animals that have received endotoxin. Because of this interaction of NAC and iron, we demonstrated that the combination of NAC and an iron chelator decreases oxidative stress and limits neutrophil infiltration and mitochondrial dysfunction in ani-mal models of sepsis, acute lung injury, acute hepatic failure, and gentamicin-induced nephro-toxicity.22-25 In addition, Buculu et al33 demon-strated that the combination of NAC plus DFX has beneficial effects in an animal model of nephrotic syndrome. Thus, we demonstrated that these ben-eficial effects could be translated to the clinical

EFFECTS OF NAC AND DFX ON PLASMA CyTOkINE

PHARMACODyNAMICS 1369

Figure 1 Flow chart of patients.

setting via the ability of this combination to decrease oxidative damage and the inflammatory response in critically ill patients.

The second limitation of NAC use in the clinical setting is the lack of solid evidence concerning the antioxidant and anti-inflammatory effects of NAC administration in humans with different pathologic conditions.22 In addition, the range of NAC dosing across the clinical and experimental literature varies widely, and no optimal dose for any indication has

been established. Thus, our primary objective was to demonstrate that the antioxidants we used were able to decrease oxidative damage and inflammatory markers in humans with hypotension. The pre-sented results support a decrease in plasma oxida-tive damage parameters and a decrease in plasma IL-6 levels. Because these alterations are relevant to the development of AKI after hypotension, we believe that the antioxidants used could have a role in the prevention of this condition.

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Table II Primary Outcomes

N-Acetylcysteine Plus Deferoxamine Placebo P Value

Protein carbonyls, nmol/mg protein, D0 0.43 (0.23-0.82) 0.61 (0.20-1.30) .69Protein carbonyls, nmol/mg protein, D1 0.25 (0.09-0.74) 0.54 (0.36-1.38) .01Protein carbonyls, nmol/mg protein, D2 0.84 (0.18-1.03) 1.09 (0.08-1.60) .31Protein carbonyls, nmol/mg protein, D3 0.45 (0.07-0.90) 0.68 (0.16-1.02) .37TBARS, nmol/mg protein × 10-3, D0 0.3 (0.1-0.3) 0.3 (0.1-0.6) .51TBARS, nmol/mg protein × 10-3, D1 0.2 (0.1-0.3) 0.5 (0.2-0.6) <.01TBARS, nmol/mg protein × 10-3 , D2 0.3 (0.1-0.4) 0.6 (0.2-0.7) .02TBARS, nmol/mg protein × 10-3, D3 0.1 (0.1-0.2) 0.2 (0.1-0.5) .96IL-6, pg/mL, D0 158 (123-231) 111 (68-193) .26IL-6, pg/mL, D1 61 (37-123) 225 (76-341) .03IL-6, pg/mL, D2 70 (26-347) 181 (43-321) .54IL-6, pg/mL, D3 76 (56-223) 247 (134-842) .11

Abbreviations: IL-6, interleukin-6; TBARS, thiobarbituric acid reactive species.D0, n = 15 each group; D1, n = 15 each group; D2, n = 11 placebo group, n = 12 antioxidant group; D3, n = 10 placebo group, n = 11 antioxidant group.

Table I Baseline Characteristics of the Patients

N-Acetylcysteine Plus Deferoxamine (n = 15) Placebo (n = 15) P Value

Age, y 55 (27-59) 53 (42-68) .31Female gender, n (%) 7 (47) 4 (27) .25Race/ethnicity, n (%) .76 White 9 (60) 10 (67) Black 6 (40) 5 (33)Baseline creatinine, time zero, mg/dL 1.1 (0.8-1.6) 1.1 (0.7-1.4) .25APACHE II 20 (12-28) 22 (16-29) .50SOFA score 11 (9-13) 10 (8-11) .27Time to receive first dose, min 420 (240-660) 420 (180-720) .75Etiology of hypotension, n (%) .73 Sepsis 8 (53) 9 (60) Hypovolemia 5 (33) 5 (33) Cardiogenic 2 (14) 1 (7)Comorbidities Diabetes, n (%) 3 (20) 3 (20) 1.0 Heart failure, n (%) 3 (20) 2 (13) .68

Abbreviations: APACHE II, Acute Physiology and Chronic Health Evaluation II; SOFA, Sequential Organ Failure Assessment.

Although we did not demonstrate a significant improvement in the RIFLE score in the antioxidant-treated group, we observed significantly lower creati-nine levels at hospital discharge in the treated group and an improvement in renal SOFA during antioxidant administration. Because of the small sample size, these results could be secondary to type I error; thus, a large clinical trial must be designed to determine the effects of NAC plus DFX on clinically relevant outcomes. Retrospectively calculating power to these secondary outcomes, it was determined that for creatinine level at hospital discharge, the sample size of 30 patients

provided a power >0.8. To no other clinical outcome, 30 patients provided a power >0.8. In addition, one pub-lished study suggested a direct effect of NAC on plasma creatinine concentration, rather than an improved glo-merular filtration rate.34 However, this finding was not supported by a recently published study.35

In addition, the use of NAC is associated with hypotension; thus, it could worsen renal function-ing in several conditions.36 Although we did not observe significant differences in the SOFA score during antioxidant administration, treated individuals presented with worse cardiovascular SOFA scores

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PHARMACODyNAMICS 1371

Table III Secondary Outcomes

N-Acetylcysteine Plus Deferoxamine (n = 15) Placebo (n = 15) P Value

Worst RIFLE during ICU stay, n (%) None 6 (40) 2 (13) .23 Risk 2 (13) 1 (7) Injury 3 (20) 3 (20) Failure 4 (27) 9 (60)SOFA score Day 2 ICU stay 12 (7-14) 9 (6-12) .18 Day 3 ICU stay 11 (6-14) 10 (6-11) .59Creatinine level hospital discharge, mg/dL 0.7 (0.5-1.3) 1.5 (0.8-3.8) .01ICU length of stay, d 5 (4-19) 10 (3-17) .81Hospital length of stay, d 10 (6-23) 14 (7-20) .43

during this period. Thus, it seems that the use of DFX did not prevent this detrimental effect of NAC. Some studies suggest that NAC increases endothe-lial nitric oxide, resulting in vasodilatation and low-ered blood pressure.19,36 This is reinforced by the fact that NAC plus DFX increased nitrite levels in our study. Despite this, it seemed that the poor cardio-vascular SOFA scores in the antioxidant group were not related to worsening renal function, as this fact is of minor importance, at least to our patients.

Some limitations of our study must be mentioned. First, small sample size included in the study may have compromised our results concerning clinically relevant outcomes, such as the RIFLE score, the need for renal replacement therapy, and mortality. Thus, the study is underpowered to determine the rele-vance of the clinical outcomes described here, including creatinine at hospital discharge. Second, we evaluated systemic levels of inflammation and oxidative stress but not kidney-related markers of oxidative damage and inflammation. Thus, the measure of these markers directly in the kidneys could provide different insights on the effects of the NAC + DFX combination. This limitation could not be adequately addressed primarily because of the ethical and medical limitations imposed by perform-ing renal biopsies on unstable critically ill patients. In addition, the markers of oxidative damage that we used are not state of the art, and IL-6 is far from rep-resenting the complexity of the inflammatory response. Despite this, we believe that given our study aims (ie, to determine whether the antioxidants studied could interfere with general markers of oxidative damage and the inflammatory response), the measured parameters were able to give us a reasonable idea concerning antioxidant efficacy.

CONClusION

NAC + DFX administration was able to decrease plasma markers of oxidative damage and plasma IL-6. A large study must be performed to determine whether these effects are relevant to the prevention of kidney failure after hypotension in critically ill patients.

C.M.Z., F.P., F.D.-P., and C.R. conceived the study, participated in the design of the study, and drafted the article. F.D.-P. had full access to all of the data in the study and takes full responsibility for the integrity of all of the data and the accuracy of the data analysis, including and especially any adverse effects. D.B., C.D.T., M.F.L.T., F.F., and F.V. participated in the design of the study and the collection of data and performed experimental analyses.

Financial disclosure: This research was supported by grants from CNPq (C.R., and F.D.-P.), FAPESC (F.D.-P.), and UNESC (C.R., and F.D.-P.). C.R., and F.D.-P. are CNPq Research Fellows. L.S.C. and C.D.T. are holders of a FAPESC studentship.

Trial registration: Clinicaltrials.gov NCT00870883.

REfERENCEs

1. Hoste EAJ, Schurgers M. Epidemiology of acute kidney injury: how big is the problem? Crit Care Med. 2008;36:S146-S151.2. Kellum JA. Acute kidney injury. Crit Care Med. 2008;36: S141-S145.3. Hoste EA, Clermont G, Kersten A, et al. RIFLE criteria for acute kidney injury are associated with hospital mortality in critically ill patients: a cohort analysis. Crit Care. 2006;10:R73.4. Cruz DN, Bolgan I, Perazella MA, et al. North East Italian Prospective Hospital Renal Outcome Survey on Acute Kidney Injury (NEiPHROS-AKI): targeting the problem with the RIFLE criteria. Clin J Am Soc Nephrol. 2007;2:418-425.5. Uchino S, Kellun JA, Bellomo R, et al. Acute renal failure in critically ill patients. JAMA. 2005;294:813-818.6. Maccariello E, Soares M, Valente C, et al. RIFLE classification in patients with acute kidney injury in need of renal replacement therapy. Intensive Care Med. 2007;33:597-605.

1372 • J Clin Pharmacol 2012;52:1365-1372

FRAgA ET AL

7. Komisarof JA, Gilkey GM, Peters DM, et al. N-acetylcysteine for patients with prolonged hypotension as prophylaxis for acute renal failure. Crit Care Med. 2007;35:435-441.8. Laurent B, Ardaillou R. Reative oxygen species: production and role in the kidney. Am J Physiol. 1986;251:F765-F776.9. Ueda N, Shah S. Endonuclease induced damage and cell death in oxidant injury to renal tubular epithelial cells. J Clin Invest. 1992;90:2593-2597.10. Beckman JS, Koppenol WH. Nitric oxide, superoxide and peroxynitrite: the good, the bad the ugly. Am J Physiol. 1996;271: C1424-C1437.11. McCord J. Oxygen derived free radicals in postischemic tissue injury. N Engl J Med. 1985;312:159-163.12. Takada M, Nadeau KC, Shaw GD, et al. The cytokine adhesion molecule cascade on ischemia/reperfusion injury of the rat kid-ney: inhibition by a soluble P selectin ligand. J Clin Invest. 1999;99:2682-2690.13. Rabb H, O’Meara YM, Maderna P, et al. Leucocytes, cell adhe-sion molecules and ischemic renal failure. kidney Int. 1997;51: 1463-1468.14. Atkuri KR, Mantovani JJ, Herzenberg LA, Herzenberg LA. N-acetylcysteine: a safe antidote for cysteine/glutathione defi-ciency. Curr Opin Pharmacol. 2007;7:355-359.

15. Spapen HD, Diltoer MW, Nguyen DN, et al. Effects of N-acetylcysteine on microalbuminuria and organ failure in acute severe sepsis. Chest. 205;127:1413-1419.

16. Kelly AM, Dwamena B, Cronin P, et al. Meta-analysis: effec-tiveness of drugs for preventing contrast induced nephropathy. Ann Intern Med. 2008;148:284-294.

17. Duong MH, Mackenzie TA, Malenka DJ. N-acetylcysteine prophylaxis significantly reduces the risk of radiocontrast nephropathy: comprehensive meta-analysis. Catheter Cardiovasc Interv. 2005;64:471-479.

18. Barichello T, Machado RA, Constantino L, et al. Antioxidant treatment prevented late memory impairment in an animal model of sepsis. Crit Care Med. 2007;35:2186-2190.

19. Sprong RC, Winkelhuyzen-Janssen ML, Aarman CJM, et al. Low dose N-acetylcysteine protects rats against endotoxin medi-ated oxidative stress, but high dose increase mortality. Am J Respir Crit Care Med. 1998;157:1283-1293.

20. Burns KEA, Chu MWA, Novick RJ, et al. Perioperative N-acetylcysteine to prevent renal dysfunction in high risk patients undergoing CABG surgery: a randomized controlled trial. JAMA. 2005;294:342-350.21. Peake SL, Szakmany T, Heil P. N-acetylcysteine depresses cardiac performance in patients with septic shock. Crit Care Med. 1996;29:391-395.

22. Ritter C, Andrades M, Moreira JCF, et al. Antioxidants and sepsis: can we find the ideal approach?Crit Care Med. 2004;32: 1445-1446.23. Ritter C, Cunha AA, Echer IC, et al. Effects of N-acetylcysteine plus deferoxamine in lipopolysaccharide-induced acute lung injury in the rat. Crit Care Med. 2006;34:471-477.24. Ritter C, Andrades ME, Reinke A, et al. Treatment with N-acetylcysteine plus deferoxamine protects rats against oxidative stress and improves survival in sepsis. Crit Care Med. 2004;32: 342-349.25. Petronilho F, Constantino L, Souza B, et al. Efficacy of the combination of N- acetylcysteine and desferrioxamine in the pre-vention and treatment of gentamicin-induced acute renal failure in male wistar rats. Nephrol Dial Transplant. 2009;24:2077-2082.26. Zyoud SH, Awang R, Sulaiman SA, Al-Jabi SW. Effects of delay in infusion of N-acetylcysteine on appearance of adverse drug reactions after acetaminophen overdose: a retrospective study. Pharmacoepidemiol Drug Saf. 2010;19:1064-1070.27. Somech R, Seaban-Adel K, Atkinson A, Kirby-Allen M. Evaluation and management of pediatric patients with anaphylac-toid reactions to deferoxamine mesylate. Ann Allergy Asthma Immunol. 2007;99:575-576.28. Draper HH, Hadley M. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 1990;186:421-431.29. Levine RL, Garland D, Oliver CN. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 1990;186:464-478.30. Green IC, Wagner DA, Glowski J, Skipper PL, Wishnok IS, Tannenbaum SB. Analysis of nitrate, nitrite and [15N] nitrate in biological fluids. Anal Biochem. 1982;126:131-138.31. Sajewicz W. Effect of thiol drugs on tert-butyl hydroperoxide induced luminol chemiluminescence in human erythrocytes, erythrocyte lysate, and erythrocyte membranes. Chem Biol Interact. 2010;186:144-151.32. Sagrista ML, Garcia AF, Madariaga MA, et al. Antioxidant and pro oxidant effect of the thiolic compounds N-acetyl-L-cysteine and glutathione against free radical induced lipid peroxidation. Free Radic Res. 2002;36:329-340.33. Buculu F, Oktenli C, Kenar L, et al. Efficacy of deferoxamine, N-acetylcysteine and selenium treatments in rats with adriamycin induced nephritic syndrome. J Nephrol. 2008;21:576-583.34. Hoffmann U, Fischereder M, Kruger B, et al. The value of N-acetylcysteine in the prevention of radiocontrast agent induced nephropathy seems questionable. J Am Soc Nephrol. 2004;15:407-410.35. Haase M, Haase-Fielitz A, Ratnaike S, et al. N-acetylcysteine does not artifactually lower plasma creatinine concentration. Nephrol Dial Transplant. 2008;23:1581-1587.36. Peake SL, Moran JL, Leppard PI. N-acetylcysteine depresses cardiac performance in patients with septic shock. Crit Care Med. 1996;24:1302-1310.

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