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Effect of N-acetylcysteine (NAC) on acute lung injury and acute kidney injury in hemorrhagic shock
Resuscitation 84 (2013) 121–127
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Experimental paper
Effect of N-acetylcysteine (NAC) on acute lung injury and acute kidney injury in hemorrhagic shock夽,夽夽 Jin Hee Lee a , You Hwan Jo a , Kyuseok Kim a,∗∗ , Jae Hyuk Lee a , Kwang Pil Rim b , Woon Yong Kwon c , Gil Joon Suh c , Joong Eui Rhee a,∗ a
Department of Emergency Medicine, Seoul National University Bundang Hospital, Gyeonggi, Republic of Korea Department of Emergency Medicine, Seoul Medical Center, Seoul, Republic of Korea c Department of Emergency Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 25 October 2011 Received in revised form 10 April 2012 Accepted 27 May 2012
Keywords: Hemorrhagic shock N-acetylcysteine Acute lung injury Acute kidney injury
a b s t r a c t Aim of the study: N-acetylcysteine (NAC) has been investigated to attenuate organ injury in various experimental and clinical studies. However, results in hemorrhagic shock (HS) were controversial. We determined the effects of continuous administration of NAC on acute lung injury (ALI) and acute kidney injury (AKI) in HS model. Methods: Twenty male Sprague-Dawley rats were used. Pressure controlled HS model defined by mean arterial pressure (MAP) 40 ± 2 mmHg for 90 min followed by resuscitation and observation was used. Rats (n = 10 per group) were randomized into 2 groups with NAC or dextrose. Intravenous NAC was given continuously from 15 min after induction of HS to the end of observation period (2 h). We measured serum IL-6, nitrite/nitrate concentration. NF-B p65 DNA binding activity, expressions of cytoplasmic phosphorylated IB-␣ (p-IB-␣) and IB-␣, malondialdehyde (MDA) and histopathological injury scores in lung and kidney were also evaluated. Results: MAP did not show any difference during the study period. NAC decreased histopathologic scores in both lung and kidney. Lung and kidney MDA levels were significantly lower in the NAC group compared to control group. Serum nitrite/nitrate and IL-6 were also significantly lower in the NAC group. The levels of lung cytoplasmic p-IB-␣ expression was mitigated by NAC, and NF-B p65 DNA binding activity was also significantly decreased in the NAC group. Conclusions: Continuous infusion of NAC attenuated inflammatory response and acute lung and kidney injury after hemorrhagic shock in rats. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Ischemic and reperfusion injury that results from hemorrhagic shock (HS) induce the development of systemic inflammatory response syndrome and subsequent multiple organ failure including acute lung injury (ALI) and acute kidney injury (AKI). In patients with multiple organ failure, inflammatory cytokines and reactive
夽 A Spanish translated version of the summary of this article appears as Appendix in the final online version at http://dx.doi.org/10.1016/j.resuscitation.2012.05.017. 夽夽 This study was presented in 40th Society of Critical Care Medicine Conference and awarded as top 10 abstract. ∗ Corresponding author at: Department of Emergency Medicine, Seoul National University Bundang Hospital, 166 Gumi-ro, Bundang-gu, Seongnam-si, Gyeonggi-do 463-707, Republic of Korea. Tel.: +82 31 787 7571; fax: +82 31 787 4081. ∗∗ Corresponding author at: Department of Emergency Medicine, Seoul National University Bundang Hospital, 166 Gumi-ro, Bundang-gu, Seongnam-si, Gyeonggi-do 463-707, Republic of Korea. Tel.: +82 31 787 7579; fax: +82 31 787 4081. E-mail addresses: [email protected] (K. Kim), [email protected] (J.E. Rhee). 0300-9572/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.resuscitation.2012.05.017
oxygen species are mobilized into the systemic circulation and marginate to end organs, causing direct local cytotoxic cellular effects.1 N-acetylcysteine (NAC) is a reactive oxygen species (ROS) scavenger2 which elicits beneficial effects on inflammation process, such as suppression of cytokine expression/release, and inhibition of nuclear factor kappa B (NF-B).3–5 NAC effects on ALI or AKI has been evaluated in various experimental conditions including endotoxemia,6,7 fat embolism,8 cardiopulmonary bypass model,5 ischemic renal failure9 and hemorrhagic shock.10 NAC showed beneficial effect in most studies except in HS model.10 In that study, NAC was administrated by oral route for 3 days before the experiment. The plasma half-life of NAC is relatively short and no NAC is detectable 10–12 h after oral administration.11 Besides, recently it was reported that higher dose and continuous infusion of NAC are required to achieve acute antioxidant and antiinflammatory effects.12,13 Considering the dose and route of NAC in that study, the effect of NAC could not be maximized in previous study.10
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In this context, we hypothesized that continuous administration of high dose NAC would reduce ALI and AKI in HS model. 2. Methods This study was approved by the Institutional Animal Care and Use Committee of our hospital in accordance with policies and the animal protection laws. 2.1. General animals preparation Male Sprague-Dawley rats weighing 320–380 g were used. The rats were housed in a controlled environment with free access to food and water before the experiment. The rats were anesthetized with 90 mg/kg ketamine and 10 mg/kg xylazine administered intraperitoneally and maintained with inhaled isoflurane. The animals underwent tracheostomy with 14 gauze angiocath and were ventilated using a small animal ventilator (Harvard Apparatus, MA, US, tidal volume 2.5 mL, respiration rate 80/min). A sterile cut down was performed in both groin, and a PE 50 was inserted into the left femoral vein (for fluid infusion and medication), left femoral artery (for blood withdrawal and transfusion) and right femoral artery (for pressure monitoring and sampling). All procedure was performed under a heating lamp to maintain the body temperature of 36–38 ◦ C, which was measured with an indwelling rectal thermometer. Arterial blood gas analyses were performed using Stat Profile® Critical Care Xpress (Nova Biomedical Corporation, Waltham, MA, USA), and minute ventilation was adjusted to keep the PaCO2 between 30 mmHg and 40 mmHg after we checked baseline ABGA. Baseline measurements included blood pressure, heart rate, pH, PaO2 , PaCO2 , HCO3 − , and base excess. 2.2. Experimental protocol HS was initiated with a pressure-controlled hemorrhage of mean arterial pressure (MAP) to 40 ± 2 mmHg. And shock was maintained for 90 min. After shock period, shock was resuscitated by reinfusion of the shed blood for 10 min followed by a period of observation for 2 h. Rats (n = 10 per group) were randomized into 2 groups with NAC diluted with 5% dextrose (NAC group) or 5% dextrose alone (control group). NAC-mixed with 5% dextrose (30 mg/mL, NAC group) or 5% dextrose without NAC was continuously administrated intravenously at a rate of 0.5 mL/100 g/h (150 mg/kg/h of NAC) from 15 min after induction of hemorrhagic shock to the end of observation period. The total amount of NAC was 487.5 mg/kg during 195 min. 2.3. Tissue preparation At the end of the observation period, the animals were euthanized with blood withdrawal via arterial line and the lung and kidney were quickly removed. The right lower lobe of lung was fixed with 10% formaldehyde solution for pathologic specimen preparation, whereas the rest of lung was cut into small pieces, snap frozen in liquid nitrogen, and stored at −70 ◦ C. The kidney was divided two parts coronally. One was fixed with 10% formaldehyde solution for histologic specimen preparation, another was cut into small pieces, snap frozen in liquid nitrogen, and stored at −70 ◦ C. 2.4. Acute lung injury (ALI) and acute kidney injury (AKI) scoring The lung was stained with hematoxylin and eosin (H&E) and the kidney with H&E and Periodic Acid-Schiff staining. The ALI and
AKI scoring were scored by one pathologist (blinded to treatment assignment). In brief, the ALI was in the following four categories: alveolar congestion, hemorrhage, infiltration of neutrophils in air space or vessel wall, and thickness of alveolar wall/hyaline membrane formation. The severity of each category was graded from 0 (minimal) to 4 (maximum), and the total score was calculated.14 Kidney damage was scored by grading glomerular, tubular, and interstitial changes. Glomerular damage (sclerotic changes such as matrix expansion, the narrowing or disappearance of the Bowman’s space, the adhesion of the capillary tuft to the Bowman’s capsule, the capillary collapse and the thickening of the glomerular basement membrane) was evaluated as: 0, absent; 1, <25% of glomeruli affected; 2, 25–50% glomeruli affected; 3, >50% of glomeruli affected. The grading for tubular changes (intracellular vacuolization) was scaled as: 0, absent; 1, <25% of tubules injured; 2, 25–50% of tubules injured; 3, >50% of tubules injured. The presence of interstitial inflammation was judged as: 0, absent; 1, mild; 2, moderate; 3, severe.15 2.5. Cytokines Plasma concentrations of IL-6 was determined with an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN). 2.6. Nitrite/nitrate assay Plasma nitrite/nitrate concentration was expressed as total sum of plasma nitrite (NO2 − ) and nitrate (NO3 − ) by using the Griess method kit.16 The nitrite/nitrate concentration was read at 543 nm with a spectrophotometer. Sodium nitrite was used as the standard. Values were expressed as micromoles per liter. 2.7. Malondialdehyde (MDA) concentration in lung and kidney tissues The extent of lipid peroxidation within the lungs and kidneys was determined by measuring the concentration of MDA, a byproduct of lipid peroxidation, using the Ohkawa method with the thiobarbituric acid.17 The level of MDA was presented as nanomoles per gram of tissue. 2.8. Assay of NF-B activity NF-B activity was measured as the nuclear translocation and DNA binding of the p65 subunit in 2.5 g nuclear extracts from tissues, using a commercially available ELISA (TransAM NF-B p65, Active Mitif, Carlsbad, CA). A TransAM ELISA-based assay NF-B kit (Active Motif) was used to detect the activation of the p65 (Rel A) of NF-B in the tissue. NF-B-specific oligonucleotide was immobilized to a 96-well plate. The tissue nuclear extract (20 g) was added to the plate and incubated for 1 h at room temperature. After washes, a primary antibody identifying activated p65 was added and incubated for another 1 h. An anti-IgG horse radish peroxidase conjugate was then added to the plate, and the color was developed according to the manufacturer’s instruction. The optical density value at 450 nm was measured on a plate reader. 2.9. Western blot assay To determine the lung expressions of cytoplasmic phosphorylated IB-␣ (p-IB-␣) and IB-␣, we performed Western blotting. In brief, cytoplasmic extracts (40 g per lane) were run on 12% sodium dodecyl sulphate–polyacrylamide gels, and then transferred to polyvinylidene difluoride membranes (Amersham
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Table 1 Baseline characteristics.
Body weight (g) Baseline ABGA pH pCO2 (mmHg) pO2 (mmHg) HCO3 − (mmol/L) Base excess (mmol/L) Lactate (mmol/L) Preparation time Tracheostomy time (min) Vascular access time (min) Initial vital signs Mean arterial pressure (mmHg) Heart rate (beat/min) Body temperature (◦ C) Hemorrhagic volume (mL)
Control group (n = 10)
NAC group (n = 10)
p
351.8 ± 1.9
343.0 ± 3.9
0.06
7.49 26 81 19.7 −3.9 1.3
± ± ± ± ± ±
0.02 2 4 0.6 0.6 0.2
2.0 ± 0.3 14.1 ± 0.9 83.1 282.3 36.4 8.1
± ± ± ±
6.2 15.1 0.4 0.4
7.50 22 93 18.4 −3.4 1.7
± ± ± ± ± ±
0.01 3 6 2.6 0.8 0.4
1.7 ± 0.3 13.1 ± 0.8 78.1 291.8 36.1 7.9
± ± ± ±
3.8 6.6 0.3 0.5
0.88 0.31 0.13 0.63 0.66 0.38 0.46 0.40 0.50 0.57 0.43 0.74
International, Buckinghamshire, UK). The following primary antibodies (Cell Signaling, Beverly, CA) were used for immunoblotting: rabbit antirat-p-IB-␣, IB-␣ antibodies. As secondary antibodies, antirabbit immunoglobulin G (Enzo Life Sciences International, PA, USA) coupled with peroxidase and diluted 1:5000 in trisbuffered saline–Tween were used. Protein bands were detected by an ECL enhanced chemiluminescence system (Amersham International, Buckinghamshire, UK). Optical densities were quantified by a computer-assisted densitometric analysis of the exposed films (Lap Work Software; Seoulin Bioscience, Seoul, Republic of Korea). All blots were normalized against -actin to control for protein loading. For -actin measurements, the mentioned Western blot method was applied using a specific mouse monoclonal anti-actin antibody (Sigma–Aldrich, St. Louis, MO, USA).
Fig. 1. Mean arterial pressure during experiment. There was no significant difference between two groups (p > 0.05). Data are expressed as mean ± SD. NAC, N-acetylcysteine.
3.3. Malondialdehyde (MDA) The lung MDA content was significantly lower in the NAC group than control group (231.9 (129.7–275.2) vs. 337.7 (264.5–473.1) nmol/g, NAC vs. control, p = 0.01). The kidney MDA content in the NAC group was also lower than control group (60.5 (39.0–84.1) vs. 134.3 (114.4–136.8) nmol/g, NAC vs. control, p = 0.002) (Fig. 3). 3.4. Cytokines and nitrite/nitrate
2.10. Statistical analysis The values were expressed as mean ± SD or median (interquartile range). Data were analyzed using the SPSS 15.0 statistical package (SPSS, Inc., Chicago, IL, USA). The Student t-test was used to compare the differences between the two groups. The significance of differences in the repeated measured values between groups, such as mean arterial pressure (MAP), heart rate (HR) was analyzed using a repeated-measures analysis of variance. If needed, Mann–Whitney U-test was used. A p value of <0.05 was considered statistically significant.
3. Results
In the NAC group, the IL-6 levels were significantly lower compared to control group (227.1 ± 96.5 vs. 530.3 ± 308.5 pg/mL, p = 0.01) (Fig. 4). Plasma concentrations of nitrite/nitrate were lower in the NAC group (16.5 ± 2.1 vs. 19.3 ± 2.3 mol/L, p = 0.01) (Fig. 4). 3.5. Cytoplasmic p-IB-˛ and IB-˛ expressions The lung cytoplasmic p-IB-␣ expression in NAC group was lower than in the control group (2.48 (2.25–2.83) vs. 3.44 (2.93–4.81), p = 0.02). However, there was no significant difference of the expression of lung cytoplasmic IB-␣ between groups (1.00 (0.95–1.19) vs. 1.26 (0.89–1.44), NAC vs. control, respectively) (Fig. 5A).
3.1. Basic characteristics 3.6. NF-B activity All the animals examined in the study were alive during the experiments. Baseline characteristics including body weights, ABGA, and withdrawn blood amount was comparable between both groups (p > 0.05) (Table 1). MAP did not differ between the control and the NAC group at baseline, during shock state, reperfusion and observation period (p > 0.05) (Fig. 1).
The levels of lung NF-B p65 DNA binding activity were more decreased in the NAC group than in the control group (0.10 ± 0.02 vs. 0.23 ± 0.13 pg/mL, p = 0.04). But, there was no significant difference of the levels of kidney NF-B activity between groups (p = 0.97) (Fig. 5B). 4. Discussion
3.2. Histological injury ALI and AKI score were significantly lower in the NAC group (7.0 (5.0–8.25) vs. 8.5 (7.0–11.0), p = 0.04; 1.0 (1.0–2.0) vs. 2.0 (1.8–3.0), p = 0.04, respectively) (Fig. 2).
This study demonstrated that continuous infusion of high dose NAC attenuated ALI and AKI in a rat model of HS. Oxidative stress, systemic inflammatory response, and histological injury were significantly lower in rats receiving NAC than in those receiving
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Fig. 2. Acute lung injury score (A) and histologic findings (B and C) and acute kidney injury score (D) and histologic findings (E and F) in the control and N-acetylcysteine (NAC) group. Acute lung injury score (A) and acute kidney injury score (D) in the NAC group were significantly lower than the control group. H&E staining for lung and H&E and Periodic Acid-Schiff staining for kidney; original magnification, 40×. *p < 0.05 compared with the control group.
placebo. To the best of our knowledge, this is the first study evaluating the effect of continuous infusion of NAC on ALI and AKI in HS model. There are several studies that antioxidants such as allopurinol, tempol, desferrioxamine, or fluvastatin have shown beneficial effects in HS.18–21 NAC could have beneficial effects in HS as a wellknown anti-oxidant, but a recently published study failed to show the beneficial effect of NAC. However, the above mentioned study may have limitations in the dosage, timing, and route of treatment
of NAC since low dose NAC was administered by oral route for 3 days before the experiment.10 The plasma concentrations of orally administrated NAC peak after 60 min and quickly decrease after 90 min,22 and the plasma half-life is estimated to be about 2.5 h and no NAC is detectable 10–12 h after oral administration.11 However, infusion of NAC results in a peak concentration within 15 min with a half-life of 5.7 h.23 Continuous NAC infusion recovered the decreased total glutathione content and glutathione peroxidase activity, but not by the bolus administration of NAC in I/R rat
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Fig. 3. Malondialdehyde (MDA) level indicating lipid peroxidation. The lung and kidney MDA content were significantly lower in the N-acetylcysteine (NAC) group than in the Control. *p < 0.05 compared with the control group.
Fig. 4. Plasma interleukin-6 (IL-6) and nitrate/nitrate concentration. IL-6 and nitrate/nitrate concentration was significantly lower in the N-acetylcysteine (NAC). *p < 0.05 compared with the control group. Data are expressed as mean ± SD.
Fig. 5. (A) Cytoplasmic inhibitor B-␣ (IB-␣) and phosphorylated inhibitor B-␣ (p-IB-␣) in lung. (B) The levels of nuclear factor-B (NF-B) p65 DNA binding. The lung cytoplasmic p-IB-␣ expression in the N-acetylcystein (NAC) group was lower than in the control, but, there was no significant difference of the expression of IB-␣ between groups. The levels of lung NF-B p65 DNA binding activity were significantly lower in the N-acetylcysteine (NAC) group, but, there was no significant difference of the levels of kidney NF-B. *p < 0.05 compared with the control group.
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model.12 Considering the pharmacokinetic properties, the effect of NAC on oxidative stress and inflammatory response is determined by both the dosage and duration of administration. In vivo, higher doses (at least greater than 150 mg/kg) are required to achieve acute antioxidant and anti-inflammatory effects.13 In the present study, we used continuous high dose intravenous infusion of NAC (150 mg/kg/h) in HS model. The protective role of NAC is reflected by the lower plasma levels of IL-6 and NO. Concomitantly, we demonstrated an additional modulatory role of NAC on tissue MDA contents, cytoplasmic p-IB-␣ expressions, and the levels of NF-B activity. IL-6, the major proinflammatory cytokine, was significantly decreased in the NAC group. IL-6 is the important cytokine in the early inflammatory response to trauma, and it has been used as a marker to show the severity of the inflammatory response.24 TNF-␣ and IL-1 secretion are stimulated by a number of different physiological stresses such as hemorrhage, hypoxia, and ischemia/reperfusion. These cytokines can induce secretion of a variety of proinflammatory cytokines such as IL-6.24 In this study, we have shown that post-treatment with NAC attenuated the IL-6, indicating its protective action against HSinduced organ damage by reducing proinflammatory cytokine synthesis. Nitrite/nitrate have been implicated as a culprit in various I/R injuries.25 In this study, plasma concentrations of nitrite and nitrate were lower in the NAC group. Our data suggest that the beneficial effects of NAC on ALI and AKI might be associated with the attenuation of nitrite/nitrate production. ROS have been known to play an important role in the pathogenesis of various injuries including global I/R injury and MDA, an important decomposition product of lipid peroxides, is an indirect measure of free radical activity.26 We have shown that tissue MDA of the NAC group was significantly less than that of the control group. This result partly supports that the antioxidant effect of NAC might play a role in the beneficial effects of NAC in HS model. ROS had also been implicated in the activation or modulation of a number of important intracellular signaling pathways, e.g., the NF-B. However, the role of ROS in terms of initiating the NF-B signaling pathway might not be universal, and it could be dependent on the proinflammatory stimulus and the different cell types involved.27 The lung has been supposed as the central target organ for systemic inflammatory mediators released after trauma, and serves as an important component of SIRS.28 The levels of lung NF-B activity and the lung cytoplasmic p-IB-␣ expression were decreased in NAC group. Therefore, we suggest that NAC may decrease ROS formation and suppress ROS-dependent IB-␣ phosphorylation in cytoplasm during I/R injury. Our study has some limitations. First, there was not enough severe injury for kidney. Histological kidney injury scores were low in both groups (1.0 (1.0–2.0) vs. 2.0 (1.8–3.0), NAC vs. control, respectively). We assume, that there were no differences of the activities of kidney NF-B between groups because of injury severity. Second, we could not confirm the survival benefit of NAC because we sacrificed the animals to obtain tissue for pathologic specimen within a definite period.
5. Conclusion This study demonstrated that continuous infusion of NAC attenuated inflammatory response and acute lung and kidney injury after hemorrhagic shock in rats. Therefore, NAC could be considered as an adjunctive therapy in ALI and AKI after hemorrhagic shock.
Conflict of interest statement We have no conflict of interest and any copyright constraints. Source of support This study was supported by grant no. 03-2012-022 from the SNUBH Research Fund. References 1. Dewar D, Moore FA, Moore EE, Balogh Z. Postinjury multiple organ failure. Injury 2009;40:912–8. 2. Aruoma OI, Halliwell B, Hoey BM, Butler J. The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Radic Biol Med 1989;6:593–7. 3. Tsuji F, Miyake Y, Aono H, Kawashima Y, Mita S. Effects of bucillamine and Nacetyl-l-cysteine on cytokine production and collagen-induced arthritis (CIA). Clin Exp Immunol 1999;115:26–31. 4. Verhasselt V, Vanden Berghe W, Vanderheyde N, Willems F, Haegeman G, Goldman M. N-acetyl-l-cysteine inhibits primary human T cell responses at the dendritic cell level: association with NF-kappaB inhibition. J Immunol 1999;162:2569–74. 5. Zhu J, Yin R, Shao H, Dong G, Luo L, Jing H. N-acetylcysteine to ameliorate acute renal injury in a rat cardiopulmonary bypass model. J Thorac Cardiovasc Surg 2007;133:696–703. 6. Modig J, Sandin R. Haematological, physiological and survival data in a porcine model of adult respiratory distress syndrome induced by endotoxaemia. Effects of treatment with N-acetylcysteine. Acta Chir Scand 1988;154: 169–77. 7. Kao SJ, Wang D, Lin HI, Chen HI. N-acetylcysteine abrogates acute lung injury induced by endotoxin. Clin Exp Pharmacol Physiol 2006;33:33–40. 8. Liu DD, Kao SJ, Chen HI. N-acetylcysteine attenuates acute lung injury induced by fat embolism. Crit Care Med 2008;36:565–71. 9. DiMari J, Megyesi J, Udvarhelyi N, Price P, Davis R, Safirstein R. N-acetyl cysteine ameliorates ischemic renal failure. Am J Physiol 1997;272:F292–8. 10. Alkan A, Eroglu F, Eroglu E, Ergin C, Cerci C, Alsancak G. Protective effects of Nacetylcysteine and erdosteine on hemorrhagic shock-induced acute lung injury. Eur J Emerg Med 2006;13:281–5. 11. De Caro L, Ghizzi A, Costa R, Longo A, Ventresca GP, Lodola E. Pharmacokinetics and bioavailability of oral acetylcysteine in healthy volunteers. Arzneimittelforschung 1989;39:382–6. 12. Abe M, Takiguchi Y, Ichimaru S, Tsuchiya K, Wada K. Comparison of the protective effect of N-acetylcysteine by different treatments on rat myocardial ischemia-reperfusion injury. J Pharmacol Sci 2008;106:571–7. 13. Sadowska AM, Manuel YKB, De Backer WA. Antioxidant and anti-inflammatory efficacy of NAC in the treatment of COPD: discordant in vitro and in vivo doseeffects: a review. Pulm Pharmacol Ther 2007;20:9–22. 14. Nishina K, Mikawa K, Takao Y, Obara H. The efficacy of fluorocarbon, surfactant, and their combination for improving acute lung injury induced by intratracheal acidified infant formula. Anesth Analg 2005;100:964–71. 15. Parlakpinar H, Acet A, Gul M, Altinoz E, Esrefoglu M, Colak C. Protective effects of melatonin on renal failure in pinealectomized rats. Int J Urol 2007;14: 743–8. 16. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 1982;126:131–8. 17. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351–8. 18. Mannion D, Fitzpatrick GJ, Feeley M. Role of xanthine oxidase inhibition in survival from hemorrhagic shock. Circ Shock 1994;42:39–43. 19. Mota-Filipe H, McDonald MC, Cuzzocrea S, Thiemermann C. A membranepermeable radical scavenger reduces the organ injury in hemorrhagic shock. Shock 1999;12:255–61. 20. Sanan S, Sharma G, Malhotra R, Sanan DP, Jain P, Vadhera P. Protection by desferrioxamine against histopathological changes of the liver in the post-oligaemic phase of clinical haemorrhagic shock in dogs: correlation with improved survival rate and recovery. Free Radic Res Commun 1989;6: 29–38. 21. Lee CC, Lee RP, Subeq YM, Lee CJ, Chen TM, Hsu BG. Fluvastatin attenuates severe hemorrhagic shock-induced organ damage in rats. Resuscitation 2009;80:372–8. 22. Allegra L, Dal Sasso M, Bovio C, Massoni C, Fonti E, Braga PC. Human neutrophil oxidative bursts and their in vitro modulation by different N-acetylcysteine concentrations. Arzneimittelforschung 2002;52:669–76. 23. Prescott LF, Donovan JW, Jarvie DR, Proudfoot AT. The disposition and kinetics of intravenous N-acetylcysteine in patients with paracetamol overdosage. Eur J Clin Pharmacol 1989;37:501–6. 24. Lenz A, Franklin GA, Cheadle WG. Systemic inflammation after trauma. Injury 2007;38:1336–45. 25. Szabo C. The pathophysiological role of peroxynitrite in shock, inflammation, and ischemia-reperfusion injury. Shock 1996;6:79–88.
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Contents lists available at SciVerse ScienceDirect
Resuscitation journal homepage: www.elsevier.com/locate/resuscitation
Experimental paper
Effect of N-acetylcysteine (NAC) on acute lung injury and acute kidney injury in hemorrhagic shock夽,夽夽 Jin Hee Lee a , You Hwan Jo a , Kyuseok Kim a,∗∗ , Jae Hyuk Lee a , Kwang Pil Rim b , Woon Yong Kwon c , Gil Joon Suh c , Joong Eui Rhee a,∗ a
Department of Emergency Medicine, Seoul National University Bundang Hospital, Gyeonggi, Republic of Korea Department of Emergency Medicine, Seoul Medical Center, Seoul, Republic of Korea c Department of Emergency Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 25 October 2011 Received in revised form 10 April 2012 Accepted 27 May 2012
Keywords: Hemorrhagic shock N-acetylcysteine Acute lung injury Acute kidney injury
a b s t r a c t Aim of the study: N-acetylcysteine (NAC) has been investigated to attenuate organ injury in various experimental and clinical studies. However, results in hemorrhagic shock (HS) were controversial. We determined the effects of continuous administration of NAC on acute lung injury (ALI) and acute kidney injury (AKI) in HS model. Methods: Twenty male Sprague-Dawley rats were used. Pressure controlled HS model defined by mean arterial pressure (MAP) 40 ± 2 mmHg for 90 min followed by resuscitation and observation was used. Rats (n = 10 per group) were randomized into 2 groups with NAC or dextrose. Intravenous NAC was given continuously from 15 min after induction of HS to the end of observation period (2 h). We measured serum IL-6, nitrite/nitrate concentration. NF-B p65 DNA binding activity, expressions of cytoplasmic phosphorylated IB-␣ (p-IB-␣) and IB-␣, malondialdehyde (MDA) and histopathological injury scores in lung and kidney were also evaluated. Results: MAP did not show any difference during the study period. NAC decreased histopathologic scores in both lung and kidney. Lung and kidney MDA levels were significantly lower in the NAC group compared to control group. Serum nitrite/nitrate and IL-6 were also significantly lower in the NAC group. The levels of lung cytoplasmic p-IB-␣ expression was mitigated by NAC, and NF-B p65 DNA binding activity was also significantly decreased in the NAC group. Conclusions: Continuous infusion of NAC attenuated inflammatory response and acute lung and kidney injury after hemorrhagic shock in rats. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Ischemic and reperfusion injury that results from hemorrhagic shock (HS) induce the development of systemic inflammatory response syndrome and subsequent multiple organ failure including acute lung injury (ALI) and acute kidney injury (AKI). In patients with multiple organ failure, inflammatory cytokines and reactive
夽 A Spanish translated version of the summary of this article appears as Appendix in the final online version at http://dx.doi.org/10.1016/j.resuscitation.2012.05.017. 夽夽 This study was presented in 40th Society of Critical Care Medicine Conference and awarded as top 10 abstract. ∗ Corresponding author at: Department of Emergency Medicine, Seoul National University Bundang Hospital, 166 Gumi-ro, Bundang-gu, Seongnam-si, Gyeonggi-do 463-707, Republic of Korea. Tel.: +82 31 787 7571; fax: +82 31 787 4081. ∗∗ Corresponding author at: Department of Emergency Medicine, Seoul National University Bundang Hospital, 166 Gumi-ro, Bundang-gu, Seongnam-si, Gyeonggi-do 463-707, Republic of Korea. Tel.: +82 31 787 7579; fax: +82 31 787 4081. E-mail addresses: [email protected] (K. Kim), [email protected] (J.E. Rhee). 0300-9572/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.resuscitation.2012.05.017
oxygen species are mobilized into the systemic circulation and marginate to end organs, causing direct local cytotoxic cellular effects.1 N-acetylcysteine (NAC) is a reactive oxygen species (ROS) scavenger2 which elicits beneficial effects on inflammation process, such as suppression of cytokine expression/release, and inhibition of nuclear factor kappa B (NF-B).3–5 NAC effects on ALI or AKI has been evaluated in various experimental conditions including endotoxemia,6,7 fat embolism,8 cardiopulmonary bypass model,5 ischemic renal failure9 and hemorrhagic shock.10 NAC showed beneficial effect in most studies except in HS model.10 In that study, NAC was administrated by oral route for 3 days before the experiment. The plasma half-life of NAC is relatively short and no NAC is detectable 10–12 h after oral administration.11 Besides, recently it was reported that higher dose and continuous infusion of NAC are required to achieve acute antioxidant and antiinflammatory effects.12,13 Considering the dose and route of NAC in that study, the effect of NAC could not be maximized in previous study.10
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In this context, we hypothesized that continuous administration of high dose NAC would reduce ALI and AKI in HS model. 2. Methods This study was approved by the Institutional Animal Care and Use Committee of our hospital in accordance with policies and the animal protection laws. 2.1. General animals preparation Male Sprague-Dawley rats weighing 320–380 g were used. The rats were housed in a controlled environment with free access to food and water before the experiment. The rats were anesthetized with 90 mg/kg ketamine and 10 mg/kg xylazine administered intraperitoneally and maintained with inhaled isoflurane. The animals underwent tracheostomy with 14 gauze angiocath and were ventilated using a small animal ventilator (Harvard Apparatus, MA, US, tidal volume 2.5 mL, respiration rate 80/min). A sterile cut down was performed in both groin, and a PE 50 was inserted into the left femoral vein (for fluid infusion and medication), left femoral artery (for blood withdrawal and transfusion) and right femoral artery (for pressure monitoring and sampling). All procedure was performed under a heating lamp to maintain the body temperature of 36–38 ◦ C, which was measured with an indwelling rectal thermometer. Arterial blood gas analyses were performed using Stat Profile® Critical Care Xpress (Nova Biomedical Corporation, Waltham, MA, USA), and minute ventilation was adjusted to keep the PaCO2 between 30 mmHg and 40 mmHg after we checked baseline ABGA. Baseline measurements included blood pressure, heart rate, pH, PaO2 , PaCO2 , HCO3 − , and base excess. 2.2. Experimental protocol HS was initiated with a pressure-controlled hemorrhage of mean arterial pressure (MAP) to 40 ± 2 mmHg. And shock was maintained for 90 min. After shock period, shock was resuscitated by reinfusion of the shed blood for 10 min followed by a period of observation for 2 h. Rats (n = 10 per group) were randomized into 2 groups with NAC diluted with 5% dextrose (NAC group) or 5% dextrose alone (control group). NAC-mixed with 5% dextrose (30 mg/mL, NAC group) or 5% dextrose without NAC was continuously administrated intravenously at a rate of 0.5 mL/100 g/h (150 mg/kg/h of NAC) from 15 min after induction of hemorrhagic shock to the end of observation period. The total amount of NAC was 487.5 mg/kg during 195 min. 2.3. Tissue preparation At the end of the observation period, the animals were euthanized with blood withdrawal via arterial line and the lung and kidney were quickly removed. The right lower lobe of lung was fixed with 10% formaldehyde solution for pathologic specimen preparation, whereas the rest of lung was cut into small pieces, snap frozen in liquid nitrogen, and stored at −70 ◦ C. The kidney was divided two parts coronally. One was fixed with 10% formaldehyde solution for histologic specimen preparation, another was cut into small pieces, snap frozen in liquid nitrogen, and stored at −70 ◦ C. 2.4. Acute lung injury (ALI) and acute kidney injury (AKI) scoring The lung was stained with hematoxylin and eosin (H&E) and the kidney with H&E and Periodic Acid-Schiff staining. The ALI and
AKI scoring were scored by one pathologist (blinded to treatment assignment). In brief, the ALI was in the following four categories: alveolar congestion, hemorrhage, infiltration of neutrophils in air space or vessel wall, and thickness of alveolar wall/hyaline membrane formation. The severity of each category was graded from 0 (minimal) to 4 (maximum), and the total score was calculated.14 Kidney damage was scored by grading glomerular, tubular, and interstitial changes. Glomerular damage (sclerotic changes such as matrix expansion, the narrowing or disappearance of the Bowman’s space, the adhesion of the capillary tuft to the Bowman’s capsule, the capillary collapse and the thickening of the glomerular basement membrane) was evaluated as: 0, absent; 1, <25% of glomeruli affected; 2, 25–50% glomeruli affected; 3, >50% of glomeruli affected. The grading for tubular changes (intracellular vacuolization) was scaled as: 0, absent; 1, <25% of tubules injured; 2, 25–50% of tubules injured; 3, >50% of tubules injured. The presence of interstitial inflammation was judged as: 0, absent; 1, mild; 2, moderate; 3, severe.15 2.5. Cytokines Plasma concentrations of IL-6 was determined with an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN). 2.6. Nitrite/nitrate assay Plasma nitrite/nitrate concentration was expressed as total sum of plasma nitrite (NO2 − ) and nitrate (NO3 − ) by using the Griess method kit.16 The nitrite/nitrate concentration was read at 543 nm with a spectrophotometer. Sodium nitrite was used as the standard. Values were expressed as micromoles per liter. 2.7. Malondialdehyde (MDA) concentration in lung and kidney tissues The extent of lipid peroxidation within the lungs and kidneys was determined by measuring the concentration of MDA, a byproduct of lipid peroxidation, using the Ohkawa method with the thiobarbituric acid.17 The level of MDA was presented as nanomoles per gram of tissue. 2.8. Assay of NF-B activity NF-B activity was measured as the nuclear translocation and DNA binding of the p65 subunit in 2.5 g nuclear extracts from tissues, using a commercially available ELISA (TransAM NF-B p65, Active Mitif, Carlsbad, CA). A TransAM ELISA-based assay NF-B kit (Active Motif) was used to detect the activation of the p65 (Rel A) of NF-B in the tissue. NF-B-specific oligonucleotide was immobilized to a 96-well plate. The tissue nuclear extract (20 g) was added to the plate and incubated for 1 h at room temperature. After washes, a primary antibody identifying activated p65 was added and incubated for another 1 h. An anti-IgG horse radish peroxidase conjugate was then added to the plate, and the color was developed according to the manufacturer’s instruction. The optical density value at 450 nm was measured on a plate reader. 2.9. Western blot assay To determine the lung expressions of cytoplasmic phosphorylated IB-␣ (p-IB-␣) and IB-␣, we performed Western blotting. In brief, cytoplasmic extracts (40 g per lane) were run on 12% sodium dodecyl sulphate–polyacrylamide gels, and then transferred to polyvinylidene difluoride membranes (Amersham
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Table 1 Baseline characteristics.
Body weight (g) Baseline ABGA pH pCO2 (mmHg) pO2 (mmHg) HCO3 − (mmol/L) Base excess (mmol/L) Lactate (mmol/L) Preparation time Tracheostomy time (min) Vascular access time (min) Initial vital signs Mean arterial pressure (mmHg) Heart rate (beat/min) Body temperature (◦ C) Hemorrhagic volume (mL)
Control group (n = 10)
NAC group (n = 10)
p
351.8 ± 1.9
343.0 ± 3.9
0.06
7.49 26 81 19.7 −3.9 1.3
± ± ± ± ± ±
0.02 2 4 0.6 0.6 0.2
2.0 ± 0.3 14.1 ± 0.9 83.1 282.3 36.4 8.1
± ± ± ±
6.2 15.1 0.4 0.4
7.50 22 93 18.4 −3.4 1.7
± ± ± ± ± ±
0.01 3 6 2.6 0.8 0.4
1.7 ± 0.3 13.1 ± 0.8 78.1 291.8 36.1 7.9
± ± ± ±
3.8 6.6 0.3 0.5
0.88 0.31 0.13 0.63 0.66 0.38 0.46 0.40 0.50 0.57 0.43 0.74
International, Buckinghamshire, UK). The following primary antibodies (Cell Signaling, Beverly, CA) were used for immunoblotting: rabbit antirat-p-IB-␣, IB-␣ antibodies. As secondary antibodies, antirabbit immunoglobulin G (Enzo Life Sciences International, PA, USA) coupled with peroxidase and diluted 1:5000 in trisbuffered saline–Tween were used. Protein bands were detected by an ECL enhanced chemiluminescence system (Amersham International, Buckinghamshire, UK). Optical densities were quantified by a computer-assisted densitometric analysis of the exposed films (Lap Work Software; Seoulin Bioscience, Seoul, Republic of Korea). All blots were normalized against -actin to control for protein loading. For -actin measurements, the mentioned Western blot method was applied using a specific mouse monoclonal anti-actin antibody (Sigma–Aldrich, St. Louis, MO, USA).
Fig. 1. Mean arterial pressure during experiment. There was no significant difference between two groups (p > 0.05). Data are expressed as mean ± SD. NAC, N-acetylcysteine.
3.3. Malondialdehyde (MDA) The lung MDA content was significantly lower in the NAC group than control group (231.9 (129.7–275.2) vs. 337.7 (264.5–473.1) nmol/g, NAC vs. control, p = 0.01). The kidney MDA content in the NAC group was also lower than control group (60.5 (39.0–84.1) vs. 134.3 (114.4–136.8) nmol/g, NAC vs. control, p = 0.002) (Fig. 3). 3.4. Cytokines and nitrite/nitrate
2.10. Statistical analysis The values were expressed as mean ± SD or median (interquartile range). Data were analyzed using the SPSS 15.0 statistical package (SPSS, Inc., Chicago, IL, USA). The Student t-test was used to compare the differences between the two groups. The significance of differences in the repeated measured values between groups, such as mean arterial pressure (MAP), heart rate (HR) was analyzed using a repeated-measures analysis of variance. If needed, Mann–Whitney U-test was used. A p value of <0.05 was considered statistically significant.
3. Results
In the NAC group, the IL-6 levels were significantly lower compared to control group (227.1 ± 96.5 vs. 530.3 ± 308.5 pg/mL, p = 0.01) (Fig. 4). Plasma concentrations of nitrite/nitrate were lower in the NAC group (16.5 ± 2.1 vs. 19.3 ± 2.3 mol/L, p = 0.01) (Fig. 4). 3.5. Cytoplasmic p-IB-˛ and IB-˛ expressions The lung cytoplasmic p-IB-␣ expression in NAC group was lower than in the control group (2.48 (2.25–2.83) vs. 3.44 (2.93–4.81), p = 0.02). However, there was no significant difference of the expression of lung cytoplasmic IB-␣ between groups (1.00 (0.95–1.19) vs. 1.26 (0.89–1.44), NAC vs. control, respectively) (Fig. 5A).
3.1. Basic characteristics 3.6. NF-B activity All the animals examined in the study were alive during the experiments. Baseline characteristics including body weights, ABGA, and withdrawn blood amount was comparable between both groups (p > 0.05) (Table 1). MAP did not differ between the control and the NAC group at baseline, during shock state, reperfusion and observation period (p > 0.05) (Fig. 1).
The levels of lung NF-B p65 DNA binding activity were more decreased in the NAC group than in the control group (0.10 ± 0.02 vs. 0.23 ± 0.13 pg/mL, p = 0.04). But, there was no significant difference of the levels of kidney NF-B activity between groups (p = 0.97) (Fig. 5B). 4. Discussion
3.2. Histological injury ALI and AKI score were significantly lower in the NAC group (7.0 (5.0–8.25) vs. 8.5 (7.0–11.0), p = 0.04; 1.0 (1.0–2.0) vs. 2.0 (1.8–3.0), p = 0.04, respectively) (Fig. 2).
This study demonstrated that continuous infusion of high dose NAC attenuated ALI and AKI in a rat model of HS. Oxidative stress, systemic inflammatory response, and histological injury were significantly lower in rats receiving NAC than in those receiving
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Fig. 2. Acute lung injury score (A) and histologic findings (B and C) and acute kidney injury score (D) and histologic findings (E and F) in the control and N-acetylcysteine (NAC) group. Acute lung injury score (A) and acute kidney injury score (D) in the NAC group were significantly lower than the control group. H&E staining for lung and H&E and Periodic Acid-Schiff staining for kidney; original magnification, 40×. *p < 0.05 compared with the control group.
placebo. To the best of our knowledge, this is the first study evaluating the effect of continuous infusion of NAC on ALI and AKI in HS model. There are several studies that antioxidants such as allopurinol, tempol, desferrioxamine, or fluvastatin have shown beneficial effects in HS.18–21 NAC could have beneficial effects in HS as a wellknown anti-oxidant, but a recently published study failed to show the beneficial effect of NAC. However, the above mentioned study may have limitations in the dosage, timing, and route of treatment
of NAC since low dose NAC was administered by oral route for 3 days before the experiment.10 The plasma concentrations of orally administrated NAC peak after 60 min and quickly decrease after 90 min,22 and the plasma half-life is estimated to be about 2.5 h and no NAC is detectable 10–12 h after oral administration.11 However, infusion of NAC results in a peak concentration within 15 min with a half-life of 5.7 h.23 Continuous NAC infusion recovered the decreased total glutathione content and glutathione peroxidase activity, but not by the bolus administration of NAC in I/R rat
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Fig. 3. Malondialdehyde (MDA) level indicating lipid peroxidation. The lung and kidney MDA content were significantly lower in the N-acetylcysteine (NAC) group than in the Control. *p < 0.05 compared with the control group.
Fig. 4. Plasma interleukin-6 (IL-6) and nitrate/nitrate concentration. IL-6 and nitrate/nitrate concentration was significantly lower in the N-acetylcysteine (NAC). *p < 0.05 compared with the control group. Data are expressed as mean ± SD.
Fig. 5. (A) Cytoplasmic inhibitor B-␣ (IB-␣) and phosphorylated inhibitor B-␣ (p-IB-␣) in lung. (B) The levels of nuclear factor-B (NF-B) p65 DNA binding. The lung cytoplasmic p-IB-␣ expression in the N-acetylcystein (NAC) group was lower than in the control, but, there was no significant difference of the expression of IB-␣ between groups. The levels of lung NF-B p65 DNA binding activity were significantly lower in the N-acetylcysteine (NAC) group, but, there was no significant difference of the levels of kidney NF-B. *p < 0.05 compared with the control group.
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model.12 Considering the pharmacokinetic properties, the effect of NAC on oxidative stress and inflammatory response is determined by both the dosage and duration of administration. In vivo, higher doses (at least greater than 150 mg/kg) are required to achieve acute antioxidant and anti-inflammatory effects.13 In the present study, we used continuous high dose intravenous infusion of NAC (150 mg/kg/h) in HS model. The protective role of NAC is reflected by the lower plasma levels of IL-6 and NO. Concomitantly, we demonstrated an additional modulatory role of NAC on tissue MDA contents, cytoplasmic p-IB-␣ expressions, and the levels of NF-B activity. IL-6, the major proinflammatory cytokine, was significantly decreased in the NAC group. IL-6 is the important cytokine in the early inflammatory response to trauma, and it has been used as a marker to show the severity of the inflammatory response.24 TNF-␣ and IL-1 secretion are stimulated by a number of different physiological stresses such as hemorrhage, hypoxia, and ischemia/reperfusion. These cytokines can induce secretion of a variety of proinflammatory cytokines such as IL-6.24 In this study, we have shown that post-treatment with NAC attenuated the IL-6, indicating its protective action against HSinduced organ damage by reducing proinflammatory cytokine synthesis. Nitrite/nitrate have been implicated as a culprit in various I/R injuries.25 In this study, plasma concentrations of nitrite and nitrate were lower in the NAC group. Our data suggest that the beneficial effects of NAC on ALI and AKI might be associated with the attenuation of nitrite/nitrate production. ROS have been known to play an important role in the pathogenesis of various injuries including global I/R injury and MDA, an important decomposition product of lipid peroxides, is an indirect measure of free radical activity.26 We have shown that tissue MDA of the NAC group was significantly less than that of the control group. This result partly supports that the antioxidant effect of NAC might play a role in the beneficial effects of NAC in HS model. ROS had also been implicated in the activation or modulation of a number of important intracellular signaling pathways, e.g., the NF-B. However, the role of ROS in terms of initiating the NF-B signaling pathway might not be universal, and it could be dependent on the proinflammatory stimulus and the different cell types involved.27 The lung has been supposed as the central target organ for systemic inflammatory mediators released after trauma, and serves as an important component of SIRS.28 The levels of lung NF-B activity and the lung cytoplasmic p-IB-␣ expression were decreased in NAC group. Therefore, we suggest that NAC may decrease ROS formation and suppress ROS-dependent IB-␣ phosphorylation in cytoplasm during I/R injury. Our study has some limitations. First, there was not enough severe injury for kidney. Histological kidney injury scores were low in both groups (1.0 (1.0–2.0) vs. 2.0 (1.8–3.0), NAC vs. control, respectively). We assume, that there were no differences of the activities of kidney NF-B between groups because of injury severity. Second, we could not confirm the survival benefit of NAC because we sacrificed the animals to obtain tissue for pathologic specimen within a definite period.
5. Conclusion This study demonstrated that continuous infusion of NAC attenuated inflammatory response and acute lung and kidney injury after hemorrhagic shock in rats. Therefore, NAC could be considered as an adjunctive therapy in ALI and AKI after hemorrhagic shock.
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