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INTRAVENOUS GLUTAMINE ADMINISTRATION REDUCES LUNG AND DISTAL ORGAN INJURY IN MALNOURISHED RATS WITH SEPSIS Gisele Pena de Oliveira,* Johnatas Dutra Silva,* Carla Cristina de Arau´jo,* Luiz Felipe Mancilha Prota,† Soraia Carvalho Abreu,* Caroline Madeira,‡ Marcelo Marcos Morales,† Christina Maeda Takiya,§ Bruno Lourenc¸o Diaz,|| Vera Luiza Capelozzi,¶ Rogerio Panizzutti,‡ Paolo Pelosi,** and Patricia Rieken Maceˆdo Rocco* Laboratories of *Pulmonary Investigation and † Cellular and Molecular Physiology, Carlos Chagas Filho Biophysics Institute, ‡ Laboratory of Frontiers in Neuroscience, Institute of Biomedical Sciences, and Laboratories of §Cellular Pathology and ||Inflammation, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro; ¶ Department of Pathology, School of Medicine, University of Sa˜o Paulo, Sa˜o Paulo, Brazil; and **IRCCS AOU San Martino-IST, Department of Surgical Sciences and Integrated Diagnostics, University of Genoa, Genoa, Italy Received 15 Jul 2013; first review completed 30 Jul 2013; accepted in final form 26 Nov 2013 ABSTRACT—Malnutrition is a risk factor for infection, compromising immune response. Glutamine (Gln) protects the lungs and distal organs in well-nourished septic and nonseptic conditions; however, no study to date has analyzed the effects of Gln in the presence of sepsis and malnutrition. In the present work, we tested the hypothesis that early therapy with intravenous Gln prevents lung and distal organ damage in septic malnourished rats. Protein-energy malnutrition was induced in male Wistar rats for 4 weeks. At the end of 4 weeks, malnourished animals were assigned to a sepsis-inducing cecal ligation and puncture group or a sham surgery group. One hour after surgery, animals were given saline (Sal) or L-alanyl-L-glutamine (Gln) intravenously. In addition, a control group (C) was set up with rats fed ad libitum, not submitted to surgery or treatment. Forty-eight hours after surgery, in malnutrition-sham rats, Gln therapy lessened neutrophil lung infiltration and apoptosis in lung and liver. In malnutritionYcecal ligation and puncture rats, Gln therapy yielded (a) reduced static lung elastance, alveolar collapse, inflammation (neutrophil infiltration, interleukin 6), and collagen deposition; (b) repair of types I and II epithelial cells; (c) no significant changes in heat shock protein 70 expression or heat shock factor 1 phosphorylation; (d) a greater number of M1 and M2 macrophages in lung tissue; and (e) less apoptosis in the lung, kidney, small intestine, and liver. In conclusion, early therapy with intravenous Gln reduced inflammation, fibrosis, and apoptosis, minimizing lung and distal organ injury, in septic malnourished rats. These beneficial effects may be associated with macrophage activation in the lung. KEYWORDS—Apoptosis, macrophage phenotype, lung injury, lung mechanics, morphometry ABBREVIATIONS—CLP V cecal ligation and puncture; Est; L V static lung elastance; GSH V reduced glutathione; HSF V heat shock factor; HSP V heat shock protein; ICU V intensive care unit; IL V interleukin; IFN V interferon; JNK V c-Jun N-terminal kinase; M1 V macrophage phenotype 1; M2 V macrophage phenotype 2; MCP V macrophage chemoattractant protein; NF-.B V nuclear transcription factor .B; Pes V esophageal pressure; Sal V saline

plasma concentrations of glutamine (Gln) (5), the most abundant nonessential amino acid. Glutamine plays a relevant role in immune function, wound repair, and maintenance of intestinal mucosal integrity. In patients admitted to an intensive care unit (ICU), low plasma concentrations of Gln are an independent risk factor for post-ICU mortality (6). Nevertheless, a recent randomized controlled trial in a mixed population of severe critically ill patients showed that Gln supplementation was associated with increased mortality at 6 months (7). The population enrolled in that trial (7) was severely ill compared with the populations of previous studies (8). Experimental studies report that intravenous (i.v.) administration of Gln protects the lung and distal organs during sepsis and endotoxemia (9, 10), with beneficial immune effects that have been explained by modulation of leukocyte function (11), inhibition of nuclear transcription factor .B (NF-.B) (12), increase in heat shock factor 1 (HSF-1) phosphorylation, and expression of heat shock protein (HSP) 70 (13), a protective protein associated with physiological stress, apoptosis, and antioxidant capacity (14). These mechanisms have been previously evaluated

INTRODUCTION Malnutrition is an important risk factor for infection, sepsis, and death (1, 2). It compromises immune response by reducing cell-mediated immunity, phagocyte function, secretory antibody response (3), and antibody affinity and by affecting the complement system and cytokine production (4). Furthermore, the increase in catabolic reactions associated with malnutrition affects muscle proteins and leads to decreased storage and Address reprint requests to Patricia Rieken Maceˆdo Rocco, MD, PhD, Laboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Centro de Cieˆncias da Sau´de, Avenida Carlos Chagas Filho, s/n, Bloco G014, Ilha do Funda˜o, 21941-902, Rio de Janeiro, Brazil. E-mail: [email protected]. Source of funding: Centers of Excellence Program (PRONEX-FAPERJ), Brazilian Council for Scientific and Technological Development (CNPq), Rio de Janeiro State Research Foundation (FAPERJ), Sa˜o Paulo State Research Foundation (FAPESP), National Institute of Science and Technology for Drugs and Medicine (INCT-INOFAR), Coordination for the Improvement of Higher Level Personnel (CAPES), and the European Community_s Seventh Framework Programme (TARKINAID, FP7-2007Y2013). The authors have no conflicts of interest to declare. DOI: 10.1097/SHK.0000000000000102 Copyright Ó 2014 by the Shock Society

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SHOCK MARCH 2014 in well-nourished septic (9, 10, 13) and nonseptic (11, 14) conditions, but not in the presence of malnutrition associated or not with sepsis. In the present study, we hypothesized that a single i.v. dose of Gln may promote improvement of lung morphofunction as well as curtail liver, kidney, and small intestine villus injury in septic malnourished rats. Thus, we analyzed the effects of Gln on survival rate, lung mechanics and histology, inflammatory and remodeling processes, and apoptosis in the lung and distal organs of malnourished rats with and without sepsis. To compare changes induced by malnutrition associated with sepsis, we set up a control group of healthy animals fed ad libitum, not submitted to surgery or treatment. Furthermore, the possible mechanisms of Gln action were investigated, focusing on (a) HSP70 expression and HSF-1 phosphorylation, (b) polarized M1 macrophages (phagocytosis, antigen processing and presentation, and T-cell activation) (15) and M2 macrophages (expression of anti-inflammatory cytokines and tissue inhibitors of metalloproteinase) (16), and (c) levels of interleukin 6 (IL-6), interferon + (IFN-+), and macrophage chemoattractant protein 1 (MCP-1) in lung parenchyma. MATERIALS AND METHODS This study was approved by the Animal Research Ethics Committee of the Federal University of Rio de Janeiro Health Sciences Center (CEUA-CCS, IBCCF 019). All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the US National Academy of Sciences.

Animal preparation and experimental protocol Daily food intake was estimated over 3 days in 52 male Wistar rats (weight 200Y250 g, aged 2 months). The average daily food intake was È27 g. A malnutrition group (n = 46) was set up to receive water ad libitum and 9 g food per day, corresponding to one-third of the estimated daily food consumption as previously reported (17). A control (C) group (n = 6), receiving water and food ad libitum (containing 56% carbohydrate, 23% protein, 4.5% fat, 6% fiber, and 10.5% ash and minerals; Bio-Tec chow; Biobase Alimentac¸a˜o ´ guas Frias, SC, Brazil), was used to monitor the changes in the Animal Ltda, A malnutrition group parameters (17). This group was not submitted to surgery or treatment with Gln. All rats were housed in individual microisolator cages (filter tops) in a controlled environment, specifically designated room to prevent the spread of infectious agents. Animals were handled uniformly and maintained without physical activities on a 12:12-h light-dark cycle at normal room temperature (È25-C). Body weight was measured three times a week, and the physical condition of the animals was observed daily. After 4 weeks, the animals in the malnutrition group were assigned to undergo sham (n = 20) or cecal ligation and puncture (CLP) (n = 26) surgery. Briefly, the rats were anesthetized with ketamine (80 mg/kg body weight) and xylazine (5 mg/kg body weight) intraperitoneally (i.p.). In the malnutritionCLP groups, a midline laparotomy was performed, the cecum was carefully isolated to avoid damage to blood vessels, and a 3.0 cotton ligature was placed around the cecum just below the ileocecal valve to avoid bowel obstruction. After that, the cecum was punctured twice with an 18-gauge needle (9, 18). In the malnutrition-sham groups, a laparotomy was performed without cecal ligation and perforation. A layered closure of the abdominal cavity was performed with 3.0 silk sutures, followed by fluid resuscitation (5 mL/100 g body weight of prewarmed sterile saline [Sal], subcutaneously [s.c.]). All animals received tramadol (0.05 mg/kg body weight, s.c., every 24 h) for postoperative analgesia. After wound closure and resuscitation, rats were returned to their cages, where they received water and food ad libitum. All manipulations described were performed by the same investigator to ensure consistency. Five animals, three in the malnutrition-sham group and two in the malnutrition-CLP group, died during anesthesia or surgical procedure. These deaths may have been associated with nutritional status. In addition, in the malnutrition group, five rats died due to CLP-induced sepsis. Therefore, 17 and 19 animals were studied in the malnutrition-sham and malnutrition-CLP groups, respectively.

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One hour after surgery, malnutrition-sham and malnutrition-CLP rats were assigned to receive i.v. Sal (È1 mL, Sal) or Gln (0.75 g/kg body weight, È1 mL, Gln), after induction of inhaled anesthesia with sevoflurane. Glutamine was administered as L-alanyl-L-Gln dipeptide (Dipeptiven 20%; Fresenius Kabi Brazil, LTDA Campinas, Sa˜o Paulo, Brazil) (9). All parameters were analyzed 48 h after sham or CLP surgery based on pilot studies in well-nourished and malnourished animals. We evaluated the time course of changes in lung mechanics and histology and observed diffuse alveolar damage and impairment of lung mechanics at 48 h. Thus, at this moment, rats were sedated (diazepam 5 mg, i.p.), anesthetized (thiopental sodium 20 mg/kg, i.p.), tracheotomized, paralyzed (pancuronium bromide 1 mg/kg, i.v.), and ventilated with a constantflow ventilator (Samay VR15; Universidad de la Republica, Montevideo, Uruguay) set to the following parameters: tidal volume = 8 mL/kg, respiratory rate = 100 breaths/min, inspiratory to expiratory ratio = 1:2, zero end-expiratory pressure, and fraction of inspired oxygen = 0.21. After 5 min of ventilation, lung mechanics were computed. All animals underwent laparotomy and blood collection from the abdominal aorta, which was then sectioned (as was the vena cava) to induce euthanasia by terminal bleeding. The lungs, liver, kidney, and small intestine were removed and prepared for histological analysis.

Plasma Gln and alanine levels L-Glutamine and L-alanine (Ala) levels in plasma were measured by highperformance liquid chromatography as previously described (19).

Lung mechanics Airflow and tracheal pressure and esophageal pressure (Pes) were measured. Tidal volume was calculated by numerical integration of the flow signal. Changes in Pes, which reflect chest wall pressure, were measured with a 30-cmlong water-filled catheter (PE205) with side holes at the tip connected to a SCIREQ differential pressure transducer (SC-24; SCIREQ, Montreal, Quebec, Canada). Transpulmonary pressure was computed as the difference between tracheal pressure and Pes. Airways were occluded at end-inspiration until transpulmonary plateau pressure was reached (at the end of 5 s), and static lung elastance (Est,L) was calculated as $transpulmonary pressure ($P,L = difference between end-inspiratory and end-expiratory transpulmonary pressure) divided by tidal volume (9). All signals were filtered (100 Hz), amplified in a four-channel conditioner (SC-24 SCIREQ), sampled at 200 Hz with a 12-bit analog-to-digital converter (DT2801A; Data Translation, Marlboro, Mass), and stored in a microcomputer. All data were collected using LABDAT software (RHT-InfoData, Montreal, Quebec, Canada) (9). Mechanical measurements were performed 10 to 15 times in each animal and analyzed using ANADAT data analysis software (RHT-InfoData).

Histology Lungs were fixed in 3% buffered formaldehyde, embedded in paraffin, and stained with hematoxylin-eosin. The slides were coded and examined only at the end of all measurements. The volume fractions of the lung occupied by collapsed alveoli (alveoli with rough or plicate walls), hyperinflated structures (alveolar ducts, alveolar sacs, or alveoli, all wider than 120 Hm), or normal pulmonary areas (those not presenting overdistended or plicate walls) were determined by the pointcounting technique (9) at a magnification of 200 across 10 random, noncoincident microscopic fields. The number of total cells, neutrophils, and mononuclear cells, as well as the amount of pulmonary tissue, were determined in each sample also by the point-counting technique (9) across 10 random, noncoincident microscopic fields at 1,000 magnification. Data were reported as the fraction area of pulmonary tissue. Collagen (Picrosirius-polarization method) (17) and elastic fibers (Weigert resorcin fuchsin method with oxidation) (17) were quantified in the alveolar septa at 400 magnification. The area occupied by fibers was determined by digital densitometric recognition and divided by the area of each studied septum, to avoid bias due to septal edema or alveolar collapse. The results were expressed as the fractional area occupied by elastic and collagen fibers in the alveolar septa.

Transmission electron microscopy Three slices were cut from three different segments of the left lung, thus reducing eventual analysis bias. Two pathologists performed a semiquantitative analysis of at least 20 different images per animal. The following parameters were analyzed: (a) type I and II epithelial cell injury, (b) denudation of basement membrane, (c) endothelial cell damage, (d) septal rupture, (e) cell apoptosis, and (f) necrosis. Pathologic findings were graded on a five-point, semiquantitative, severity-based scoring system as follows: 0 = normal lung parenchyma, 1 = changes in 1% to 25%, 2 = changes in 26% to 50%, 3 = changes in 51% to 75%, and 4 = changes in 76% to 100% of examined tissue (9).

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Apoptosis assays Vcaspase 3 immunohistochemical analysis Apoptotic cells of lung, kidney, liver, and small intestine were quantified using immunohistochemistry for caspase 3. Initially, all tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin. Thin sections were obtained and stained with hematoxylin-eosin. Additional subserial sections from all paraffin blocks were used for immunohistochemistry with antiYcaspase 3 antibody (Santa Cruz Biotechnology, Santa Cruz, Calif). Immunohistochemistry was performed according to manufacturer instructions. Sections were visualized by treating the slides with diaminobenzidine tetrahydrochloride. To determine the levels of apoptotic marker expression, lung, kidney, liver, and small intestine tissue was assessed in 10 fields by the point-counting technique, using a 100-point grid of known area (62,500 mm2 at 440 magnification) attached to the eyepiece of the microscope. At 400 magnification, the alveolar septal area in each field was calculated according to the number of points overlaying connective tissue as a proportion of the total grid area. Then, the number of positive cells within the alveolar septa area and distal organ tissue was counted. The cell fractional area was determined as the number of positive cells in each field divided by the tissue area.

HSP70 expression and HSF-1 phosphorylation in lung tissue Expressions of HSP70 and phosphorylated HSF-1 proteins in whole lungs of control and malnutrition animals were assessed and compared by Western blotting. Heat shock protein 70 and HSF-1 proteins were also measured in another group of animals, which was fed ad libitum (well nourished, n = 20) and then randomly divided into sham (n = 10) or CLP (n = 10) subgroups. One hour after surgical procedure, well-nourished animals were assigned to receive i.v. Sal (È1 mL, Sal) or Gln (0.75 g/kg body weight, È1 mL, Gln), following the protocol previously described for the malnutrition group. Immunodetection of HSP70 was performed using a specific mouse monoclonal antibody, C92 (1:1,000 dilution; StressGen, Victoria, British Columbia, Canada). A rat polyclonal antiYHSF-1 antibody (1:1,000 dilution; StressGen) was used to detect HSF-1 phosphorylation. A mouse monoclonal antibody to "-actin (Santa Cruz Biotechnology) was used as an internal control for comparison in all experimental groups. The use of "-actin as an internal control, in malnutrition and sepsis conditions, is well accepted in literature (6, 14). Sequential incubation was performed with an alkaline phosphataseYcoupled antiYmouse secondary antibody (Santa Cruz Biotechnology) for HSP70 and "-actin (1:4,000) and a goat antiYrat immunoglobulin G (GE Healthcare, Buckinghamshire, England) (1:5,000) for HSF-1. Protein bands were detected using Amersham ECL Plus Western blotting detection reagents (GE Healthcare, Buckinghamshire, England). Heat shock protein 70 and HSF-1 bands for each given sample were analyzed by densitometry with the Scion Image 4.0 software (Scion Corporation, Frederick, Md) and normalized by dividing the corresponding "-actin values in the same lane.

Immunohistochemistry for neutrophils and total, M1, and M2 macrophages Immunohistochemistry for neutrophils, as well as total, M1, and M2 macrophages in lung tissue, was done using rabbit polyclonal antibody to myeloperoxidase (catalog Ab9535, 1:500 dilution; Abcam, Cambridge, Mass); mouse antiYrat CD68 monoclonal antibody (catalog MCA341R, 1:100 dilution; AbD Serotec, Bio-Rad Laboratories, Hercules, Calif); mouse antiYrat inducible nitric oxide synthase (catalog RB 9242, 1:250 dilution; Neomarkers, Lab Vision, Fremont, Calif); and mouse antiYrat arginase I polyclonal antibody (Santa Cruz Biotechnology, Inc, catalog SC-20150, 1:100 dilution), respectively. Paraffin-embedded tissue sections (4 Hm) were dewaxed and rehydrated and underwent heat-mediated antigen retrieval for myeloperoxidase in distilled water, in a steamer, for 10 min, and for CD68 in citric acid buffer 10 mM (pH 6.0), in a steamer, for 30 min. Antigen retrieval for arginase I was performed in citric acid buffer 10 mM (pH 6.0), in a steamer for 15 min, and for inducible nitric oxide synthase, in Tris-EDTA (pH 9.0), in a steamer, for 15 min. Endogenous peroxidase activity was inhibited in 70% hydrogen peroxide solution in methanol. Nonspecific immunoglobulin binding was blocked by 10% bovine serum albumin in phosphate Sal buffer (pH 7.4), before primary antibody incubation. Antibodies were revealed with biotinylated secondary antibody (LSAB2 System-HRP for use on rat specimens; Dako, Glostrup, Denmark), followed by streptavidin-conjugated horseradish peroxidase (LSAB2 System-HRP for use on rat specimens; Dako). Detection was done with peroxide/DAB (Liquid DAB + substrate chromogen system; Dako). Slides were counterstained with Giemsa solution. Analysis was performed in 30 images of high-power fields (400) per slide, manually selected using a light microscope (Nikon Eclipse 400; Nikon Instruments, Tokyo, Japan), and captured with an Evolution VF Color Cooled 12-bit digital camera (Media Cybernetics, Silver Spring, Md). The areas occupied by nucleated macrophages and cells with positive staining for the phenotype marker in each tissue area were then measured and divided by tissue

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area using (Image-Pro Plus 6.3 for Windows, Media Cybernetics, Silver Spring, Md) software, a 400 magnification, and expressed as fractional area occupied by positive cells.

Enzyme-linked immunosorbent assay Interleukin 6, IFN-+, and MCP-1 were quantified by enzyme-linked immunosorbent assay (Duo Set; R&D Systems, Minneapolis, Minn), according to manufacturer protocols.

Statistical analysis Data were tested for normal distribution using the Kolmogorov-Smirnov test with Lilliefors correction, whereas the Levene median test was used to evaluate the homogeneity of variances. To compare differences in body weight (a) at initial, between control and malnutrition groups; (b) at the end of 4 weeks between control and malnutrition groups; and (c) at initial and 4 weeks in malnutrition group, Student t test was used. Kaplan-Meier survival curve and logrank test were used to compare survival data. Forty-eight hours after surgery, differences among the control, malnutrition-sham-Sal, malnutrition-sham-Gln, malnutrition-CLP-Sal, and malnutrition-CLP-Gln groups were assessed by oneway analysis of variance (ANOVA) followed by Tukey test. Differences in HSP70 expression and HSF-1 phosphorylation in well-nourished sham-Sal, wellnourished sham-Gln, well-nourished CLP-Sal, and well-nourished CLP-Gln were compared by two-way ANOVA followed by Tukey test. Nonparametric data were analyzed using ANOVA on ranks followed by Dunn post hoc test. Parametric data were expressed as mean T SEM, whereas nonparametric data were expressed as median (interquartile range). The SigmaStat 3.1 statistical software package (Jandel Corporation, San Raphael, Calif) was used for analyses. The number of animals was calculated based on previous data reported for the variables analyzed, with a power of 0.8 and ! of 0.05.

RESULTS Body weight and survival rate in experimental groups

According to the previous study of our laboratory describing the present model of malnutrition (17), a reduction of approximately 30% in body weight was recorded in malnourished animals at the end of the 4-week malnutrition protocol compared with initial (Fig. 1). In addition, our data revealed that the control group gained weight during the 4 weeks of protocol, whereas the malnutrition group lost weight, producing a difference of 55% at the end of the 4-week malnutrition protocol (Fig. 1). The initial body weight of the control and malnutrition groups was similar (Fig. 1). The survival rate after 48 h was 100% in control, the malnutrition-sham-Sal, and malnutrition-sham-Gln groups, showing that our malnutrition protocol did not cause death. However, when sepsis was induced, survival was 71% (4 of 14 animals died) in the malnutrition-CLP-Sal group, and 90%

FIG. 1. Body weight at baseline and weekly after the start of a malnutrition protocol. In the malnutrition group (n = 36), food intake was reduced to one-third of estimated daily consumption, whereas control (C) animals (n = 6) received food ad libitum during 4 weeks. Values are means T SEM. White bars represent the well-nourished group, and gray bars represent the malnourished group.

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did not change significantly at 48 h after either sepsis induction (1,186.07 T 22.32 and 2,042.70 T 96.08 Hmol/L, in malnutritionsham-Sal vs. 1,156.92 T 112.49 and 1,620.26 T 183.43 Hmol/L, in malnutrition-CLP-Sal, P = 0.833 and P = 0.1, respectively) or Gln administration (1,076.24 T 44.32 and 1,805.83 T 167.32 Hmol/L, in malnutrition-sham-Gln vs. malnutritionsham-Sal, and 1,255.59 T 166.78 and 1,620.76 T 149.90 Hmol/L, in malnutrition-CLP-Gln versus malnutrition-CLP-Sal, P = 0.444 and P = 0.538, respectively). In malnourished animals, Gln protected lung against dysfunction induced by sepsis FIG. 2. Static lung elastance in malnourished animals 48 h after surgery. Sepsis was induced by CLP surgery, and malnutritionYsham-operated animals were used as a control. One hour after surgery, malnutrition-sham and malnutrition-CLP animals were treated with i.v. Sal or glutamine (Gln). Bars represent means T SEM. Malnutrition-sham-Sal (n = 10), malnutrition-shamGln (n = 7), malnutrition-CLP-Sal (n = 10); malnutrition-CLP-Gln (n = 9). **Significantly different from malnutrition-sham-Sal group (P G 0.05). † Significantly different from malnutrition-CLP-Sal group (P G 0.05).

(1 of 10 animals died) in the malnutrition-CLP-Gln group (P = 0.44). Cecal ligation and puncture led to a mortality rate of 30% in well-nourished animals. Plasma Gln and alanine levels did not change after sepsis or Gln administration

Plasma Gln depletion is demonstrated to be an independent predictor of mortality in ICU patients (6), and it may be an indicator of an insufficient availability of endogenous Gln. To investigate Gln deficiency in our experimental groups, we analyzed plasma L-Gln concentration. Because Gln was administered as L-alanyl-L-Gln dipeptide,we also measured plasma L-alanine levels. In the control group, basal L-Gln and L-alanine were 854.53 T 27.21 and 1,095.73 T 12.38 Hmol/L respectively. No differences in plasma L-Gln and L-alanine levels were found between control and malnutrition. Plasma L-Gln and L-alanine

To investigate the effects of Gln in lung function during malnutrition (malnutrition-sham) and malnutrition associated to sepsis (malnutrition-CLP), we analyzed lung mechanics. Our investigation revealed that, in malnutrition-sham animals, Gln did not impact Est,L (Fig. 2). When sepsis was associated with malnutrition (malnutrition-CLP-Sal), Est,L was higher in this group compared with the malnutrition-sham-Sal group (Fig. 2). In the malnutrition-CLP group, Gln administration resulted in lower Est,L in comparison to malnutrition-CLP-Sal, protecting against lung dysfunction induced by sepsis (Fig. 2). Gln did not alter malnutrition damage, but protected to morphological additional changes induced by sepsis

To support our functional data, we evaluated the morphometry of lung parenchyma. According to a previous study (17), our model of malnutrition led to the development of emphysemalike lesions (hyperinflated areas) and a significantly higher number of collapsed areas in malnutrition groups compared with control (Table 1; Fig. 3). In the malnutrition-sham group, the use of i.v. Gln did not modify these lesions (malnutritionsham-Gln) (Table 1; Fig. 3). Additional damage was induced by sepsis, such as demonstrated by greater alveolar collapse in malnutrition-CLP-Sal group compared with malnutrition-shamSal animals (Table 1; Fig. 3). In the malnutrition-CLP group,

TABLE 1. Lung morphometry, tissue cellularity, and elastic and collagen fibers in control (C) and malnutrition groups Malnutrition Sham Parameters Normal, % Collapse, %

C 92.69 T 0.72 7.31 T 0.72

Sal 50.98 T 3.25*

CLP Gln 55.22 T 6.59*

Sal

Gln †

51.56 T 1.81*‡

†

42.78 T 1.10*

25.07 T 0.65*

23.87 T 3.03*

34.48 T 0.98*

27.68 T 0.87*‡§

0T0

23.95 T 3.05*

20.91 T 2.61*

22.64 T 0.86*

20.76 T 1.23*

Total cells, %

44.38 T 2.04

55.34 T 1.73

47.26 T 1.42*

53.09 T 1.05

43.22 T 1.05‡

Neutrophils, %

3.23 T 0.45

15.58 T 0.57*

41.15 T 2.32

39.76 T 1.43

Hyperinflation, %

Mononuclear cells, %

7.01 T 0.77*† 40.37 T 1.01

12.49 T 0.94*† 40.61 T 0.76

5.31 T 0.44ठ39.77 T 1.17

Collagen fiber, %

2.58 T 0.19

1.49 T 0.18*

1.56 T 0.31*

3.47 T 0.37†

1.70 T 0.30‡

Elastic fiber, %

5.94 T 0.49

3.45 T 0.29*

4.25 T 0.28*

3.44 T 0.29*

4.09 T 0.34*

Values are means T SEM. All values were computed in 10 random, noncoincident fields per rat. Fraction area of total cells, neutrophils, and mononuclear cells. Malnourished rats received one-third of their estimated daily food consumption during 4 weeks (malnutrition), whereas control animals were fed ad libitum. Sepsis was then induced by CLP surgery. MalnutritionYsham-operated animals were used as controls for CLP animals. One hour after surgery, malnutrition-sham and malnutrition-CLP animals were treated with i.v. Sal or Gln. Control (n = 6), malnutrition-sham-Sal (n = 10), malnutrition-sham-Gln (n = 7), malnutrition-CLP-Sal (n = 10), and malnutrition-CLP-Gln (n = 9). *Significantly different from the control group (P G 0.05). † Significantly different from the malnutrition-sham-Sal group (P G 0.05). ‡ Significantly different from the malnutrition-CLP-Sal group (P G 0.05). § Significantly different from the malnutrition-sham-Gln group (P G 0.05).

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FIG. 3. Representative photomicrographs (light microscopy) of lung parenchyma of control (C), malnutrition-sham-Sal, malnutrition-sham-Gln, malnutrition-CLP-Sal, and malnutrition-CLP-Gln animals 48 h after surgery. Asterisks denote emphysema-like lesions (hyperinflated structures). Arrows show alveolar collapse. Note greater alveolar collapse in malnutrition-CLP-Sal group. Original magnification 200. Hematoxylin-eosin stain. Bars = 100 Hm.

Gln led to decreased areas of alveolar collapse. No difference was found among the groups in area of hyperinflation (Table 1; Fig. 3). Gln modulated remodeling process minimizing collagen fiber deposition in malnutrition-CLP animals

Extensive remodeling is a damaging consequence of lung injury induced by sepsis that compromises lung function. To investigate the effect of Gln on remodeling in malnutrition (malnutrition-sham group) and malnutrition-associated sepsis (malnutrition-CLP group), we measured total collagen fiber deposition in pulmonary septa. In malnutrition-sham groups, collagen fiber content was lower compared with control, but similar among malnutrition groups, demonstrating no effect

of an i.v. dose of Gln in malnutrition without sepsis, at 48 h (Table 1). In sepsis-associated malnutrition (malnutrition-CLPSal), collagen fiber content was significantly higher compared with malnutrition-sham-Sal animals (Table 1). In this case, the use of Gln yielded less collagen fiber (malnutrition-CLP-Gln group). Malnutrition groups presented lower elastic fiber content in comparison to control; however, no significant differences were observed among malnutrition groups (Table 1). In malnourished animals, Gln protected lung against ultrastructural damage induced by sepsis

To evaluate the role of Gln to avoid pulmonary ultrastructural damage during malnutrition and malnutrition associated with sepsis, epithelial types I and II lesions, denudation of

TABLE 2. Semiquantitative analysis of lung electron microscopy in control (C) and malnutrition groups Malnutrition Sham Parameters

C

Type I lesion

0 (0Y0)

2 (1.75Y2.25)*

2 (1Y2)*

3 (2.75Y3.25)*

2 (1Y2)*†

Type II lesion

0 (0Y0)

2 (2Y2.25)*

2 (1Y2.25)*

4 (3Y4)*‡

2 (1.75Y2)*†

Denudation of basement membrane

0 (0Y0)

Sal

CLP

1 (1Y2)*

Gln

1 (1Y2)*

Sal

Gln

‡

3 (2.75Y4)*

1 (1Y2)*†

Endothelial damage

0 (0Y0)

2 (1.75Y2.25)*

2 (1Y2)*

3 (2.75Y4)*

2 (1.75Y3)*

Septal rupture

0 (0Y0)

2 (2Y2.25)*

2 (1.75Y2.25)*

3 (2Y3.25)*

2 (1.75Y2.25)*

Apoptosis

0 (0Y1)

2 (1.75Y2)*

1 (1Y2)*

3.5 (3Y4)*‡

1 (1Y2)*

‡

Necrosis

0 (0Y0)

1 (1Y2)*

3 (2Y3)*

2 (1.75Y2)*† 1 (1Y2)*†

Values are median (25th percentileY75th percentile) of five rats in each group in types I and II lesion, denudation of basement membrane, endothelial damage, and septal rupture. Lung tissue scoring was done independently by two investigators. Malnutrition rats received one-third of their estimated daily food consumption during 4 weeks, whereas the control animals were fed ad libitum. Sepsis was then induced by CLP surgery. MalnutritionYshamoperated animals were used as controls for CLP animals. One hour after surgery, malnutrition-sham and malnutrition-CLP animals were treated with i.v. Sal or Gln. *Significantly different from the control group (P G 0.05). † Significantly different from the malnutrition-CLP-Sal group (P G 0.05). ‡ Significantly different from the malnutrition-sham-Sal group (P G 0.05).

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FIG. 4. Representative photomicrographs (electron microscopy) of lung parenchyma of control (C), malnutrition-sham-Sal, malnutrition-sham-Gln, malnutrition-CLP-Sal, and malnutrition-CLP-Gln animals 48 h after surgery. Malnutrition-sham-Sal and malnutrition-CLP-Sal animals exhibit edema and type II pneumocyte injury. Malnutrition-sham-Gln and malnutrition-CLP-Gln animals exhibit less damage. Note the integrity of lung parenchyma in the control group. Alveolar space (A), type II pneumocytes (PII), macrophage (M6), endothelial cell (EN), neutrophil (N), fibroblast (F). Original magnification 3,700. Bars = 5 Hm.

basement membrane, endothelial damage, and septal rupture were analyzed by electron microscopy. Greater lung damage was found in malnutrition groups compared with the control group. In addition, all parameters evaluated were similar in malnutrition-sham-Sal and malnutrition-sham-Gln groups, revealing no effect of Gln on malnutrition without sepsis. The degree of alveolar epithelial type II cell damage, basement membrane denudation, apoptosis, and necrosis was significantly higher in malnutrition-CLP-Sal compared with malnutrition-sham-Sal animals (Table 2). In malnutritionCLP, Gln reduced the degree of injury in alveolar epithelial type I and II cell, basement membrane denudation, apoptosis, and necrosis (Table 2; Fig. 4).

Glutamine contributed to reduce cell apoptosis in malnutrition-sham and malnutrition-CLP groups

One important mechanism of action related to the benefits of Gln use is the modulation of apoptosis during sepsis (20).Thus, we analyzed cell apoptosis rate not only in the lung, but also in kidney, liver, and small intestine, because we wanted to know about distal organ injury induced by our models of malnutrition and sepsis and the protective effects of Gln on that. The immunohistochemistry for caspase 3 revealed that, in malnourished group without sepsis (malnutrition-sham) rats, Gln led to reduced cell apoptosis in lung and liver. The induction of sepsis enhanced apoptosis rate in lung and distal organ tissue (malnutrition-sham-Sal versus malnutrition-CLP-Sal)

TABLE 3. Cell apoptosis in control (C) and malnutrition groups Malnutrition Sham Organs Lung

C 0T0

Sal 10 T 0.9*

CLP Gln

Sal †

Gln †

5.7 T 0.2*

19.6 T 2.2†‡§

26.1 T 2.5

Liver

0T0

5.5 T 0.9*

3.5 T 0.68*

15.9 T 2.1*

13.5 T 0.5*†§

Kidney

0T0

9.1 T 1.1*

8.3 T 0.8*

32.8 T 3.5*†

18.3 T 0.9*†§

10.4 T 1.3*

†

Small intestine

0T0

18.3 T 3.5*

†

†

33.1 T 1.8*

20.6 T 2.8*§

Values are mean T SEM of four rats in each group. Quantitative immunohistochemistry of caspase 3. Malnutrition rats received one-third of their estimated daily food consumption during 4 weeks, whereas the control animals were fed ad libitum. Sepsis was then induced by CLP surgery. MalnutritionYsham-operated animals were used as controls for CLP animals. One hour after surgery, malnutrition-sham and malnutrition-CLP animals were treated with i.v. Sal or Gln. *Significantly different from the control group (P G 0.05). † Significantly different from the malnutrition-sham-Sal group (P G 0.05). ‡ Significantly different from the malnutrition-sham-Gln group (P G 0.05). § Significantly different from the malnutrition-CLP-Sal group (P G 0.05).

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FIG. 5. Relative densitometry of HSP70 and HSF-1 protein expression in lung tissue of well-nourished animals and malnutrition groups 48 h after surgery. A, Malnutrition rats received one-third of their estimated daily food consumption during 4 weeks, whereas control (C) animals were fed ad libitum. Sepsis was induced by CLP surgery, and malnutritionYsham-operated animals were used as a control to CLP. One hour after surgery, malnutrition-sham and malnutrition-CLP animals were treated with i.v. Sal or Gln. Bars represent means T SEM of four animals in each group. *Significantly different from control group (P G 0.05). B, Well-nourished rats were fed ad libitum. Sepsis was induced by CLP surgery, and well-nourished sham-operated animals were used as controls for CLP. One hour after surgery, well-nourished sham and well-nourished CLP animals were treated with i.v. Sal or Gln. Bars represent means T SEM of four to five animals in each group.

(Table 3). In malnutrition-CLP rats, Gln reduced apoptosis in lung, liver, kidney, and small intestine (Table 3). Gln did not modify HSP70 and HSF-1 phosphorylation

Enhanced expression of HSP70 has been considered, for some authors, the central component of Gln’s beneficial effects during sepsis in well-nourished animals (13). The phosphorylation and nuclear translocation of the transcriptional factor HSF-1 seem to be one of the key mechanisms involved in the increased expression of HSP70 (13). Forty-eight hours after sepsis induction, the malnutrition-CLP-Sal group presented lower expression of HSF-1 and HSP70 compared with the control group. However, we did not observe changes in HSP70 expression or HSF-1 phosphorylation in the malnutrition-sham and malnutrition-CLP groups (Fig. 5A). In well-nourished rats, no significant differences in HSF-1 and HSP70 expressions in lung tissue were observed among Sham and well-nourished CLP groups with Gln treatment (Fig. 5B). Gln changed inflammatory response in sepsis associated to malnutrition

Macrophages metabolize Gln at a high rate. Because our malnutrition and sepsis protocols could lead to a state of Gln

deficiency, we analyzed macrophage activation. In malnutritionsham animals, Gln did not change total macrophage in the subpopulations M1 and M2 (Figs. 6 and 7). In malnutrition associated to sepsis, Gln increased the total number of macrophages, as well as the number of M1 and M2 macrophages, in lung tissue (Figs. 6 and 7). We also evaluated neutrophil recruitment by the pointcounting technique. In malnutrition-sham rats, Gln use reduced neutrophil infiltration in lung tissue (Table 1). Neutrophil infiltration in lung parenchyma was lower in malnutrition-CLPSal compared with malnutrition-sham-Sal animals. In malnutrition associated to sepsis (malnutrition-CLP), Gln reduced neutrophil infiltration (Table 1). The number of mononuclear cells was similar in all groups (Table 1). Because we found no differences in neutrophil recruitment after sepsis induction (malnutrition-sham-Sal versus malnutrition-CLP-Sal) using the point-counting technique, neutrophil recruitment in lung tissue was also analyzed using immunohistochemistry for myeloperoxidase. All malnutrition groups showed increased neutrophil infiltration compared with the control group. Neutrophils reduced in malnutrition-sham-Gln and malnutrition-CLP-Gln groups compared with malnutrition-sham-Sal and malnutritionCLP-Sal groups, respectively (Figs. 6 and 7). The number of

FIG. 6. Immunohistochemistry of neutrophils, total macrophages (M6), and M1 and M2 macrophage phenotypes in lung tissue of malnourished animals, 48 h after surgery. Malnutrition rats received one-third of their estimated daily food consumption during 4 weeks, whereas control (C) animals were fed ad libitum. Sepsis was induced by CLP surgery, and malnutritionYsham-operated animals were used as a control to CLP. One hour after surgery, malnutritionsham and malnutrition-CLP animals were treated with i.v. Sal or Gln. Bars represent means T SEM of four animals in each group. *Significantly different from Control group (PG 0.05). **Significantly different from malnutrition-sham-Sal group (P G 0.05). #Significantly different from malnutrition-sham-Gln group (P G 0.05). † Significantly different from malnutrition-CLP-Sal group (P G 0.05).

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FIG. 7. Representative photomicrographs (light microscopy) of immunohistochemical staining for neutrophils, total macrophage (M6), and M1 and M2 macrophage phenotypes in control, malnutrition-sham-Sal, malnutrition-sham-Gln, malnutrition-CLP-Sal, and malnutrition-CLP-Gln animals, 48 h after surgery. A, Control (C). B, Malnutrition-sham-Sal. C, Malnutrition-sham-Gln. D, Malnutrition-CLP-Sal. E, Malnutrition-CLP-Gln group. Arrows indicate positive cells. Note the higher number of neutrophils, M1, M2, and total macrophages in the CLP-Gln group. Original magnification 400. Giemsa stain. Bars = 50 Hm.

neutrophils was similar in malnutrition-sham-Sal and malnutritionCLP-Sal animals (Figs. 6 and 7). In addition, studies demonstrated that Gln may attenuate the release of proinflammatory cytokines after sepsis (12). Thus, we evaluated the effects of Gln in cytokines expression (IL-6, MCP-1, and IFN-+) in control as well as in malnutrition-sham and malnutrition-CLP animals. Interleukin 6 and MCP-1 levels were higher in malnutrition-CLP-Sal animals than in the control group. In malnutrition-sham, Gln presented no effect in cytokines expression. When sepsis was associated to malnutrition (malnutrition-CLP-Sal), IL-6 levels were significantly lower in malnutrition-CLP rats treated with Gln compared with malnutrition-CLP-Sal rats. Interferon + level was similar in all groups (Table 4). DISCUSSION In the present study, we investigated the effects of early therapy with administration of a single i.v. dose of Gln (0.75 g/kg body weight) on the lungs and distal organs of sham-operated malnourished rats and in malnourished rats with CLP-induced

sepsis. In malnutrition-sham animals, Gln decreased neutrophil lung infiltration and the number of apoptotic cells in lung and liver samples. In malnutrition-CLP-induced sepsis, Gln reduced Est,L, alveolar collapse, inflammation (neutrophil infiltration and IL-6), and collagen deposition. It also led to less cell apoptosis in lung, kidney, liver, and small intestine; improvement in the repair of alveolar type I and II epithelial cells; and an increased number of M1 and M2 macrophages in lung tissue. There were no changes in HSP70 expression or HSF-1 phosphorylation. Therefore, in septic malnourished rats, early therapy with i.v. Gln minimized lung and distal organ injury. This effect may be associated with macrophage activation in the lung, but not with changes in HSP70 expression or HSF-1 phosphorylation. The experimental model of malnutrition used in this study, which was previously described by our group (17), is characterized by balanced food restriction (nutrient load reduction associated with proportional decrement in micronutrients). It thus promotes a gradual reduction in body weight along with structural and functional damage to the lung parenchyma (17). In addition, we observed atrophy of abdominal and limb

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TABLE 4. Cytokines levels in lung tissue in control (C) and malnutrition groups Malnutrition Sham Lung Tissue

CLP

C

Sal

Gln

6.7 T 1.7

25.5 T 6.9

25.9 T 8.2

MCP-1

61.3 T 10.9

96.6 T 16.3

59.5 T 17.1

121.5 T 6.6*

INF-+

32.8 T 12.1

35.7 T 4.4

38.2 T 13.9

50.2 T 10.7

IL-6

Sal 47.6 T 15.1*

Gln 12.9 T 3.3† 107.9 T 23.1 34.8 T 4.8

Values are mean T SEM of six rats in each group. Levels of IL-6, MCP-1, and IFN-+ in lung tissue (pg/mL). Malnutrition rats received one-third of their estimated daily food consumption during 4 weeks, whereas control animals were fed ad libitum. Sepsis was then induced by CLP surgery. MalnutritionYsham-operated animals were used as controls for CLP animals. One hour after surgery, malnutrition-sham and malnutrition-CLP animals were treated with i.v. Sal or Gln. *Significantly different from the control group (P G 0.05). † Significantly different from the malnutrition-CLP-Sal group (P G 0.05).

muscles, as well as pronounced fur loss. Because malnutrition increases susceptibility to infection (3, 4, 21), rats were housed in microisolator cages (filter tops) to prevent a possible bias caused by spread of infectious agents. A criterion-standard model for sepsis (CLP surgery) was also used (18). L-Alanyl-L-Gln dipeptide was chosen as a source of Gln administration because dipeptides are more soluble and stable than free Gln (22), and because alanine is not recognized for any protective effects in sepsis and does not enhance HSP70 expression. In addition, recent studies found similar protective effects on mesenteric (23) microcirculation with i.v. free Gln and L-alanyl-L-Gln administration in experimental endotoxemia. The dose used in the present study (0.75 g/kg body weight, i.v.) is safe and effective against sepsis-induced CLP in well-nourished animals (9). In addition, it has been found to markedly enhance HSP expression in multiple organs (24), a possible mechanism of Gln action in sepsis. Based on previous studies showing the benefits of early use (9, 10, 13), Gln was administered 1 h after sepsis induction on the basis of previous studies using this early treatment (9, 10, 13) and to design a translational study. Saline, instead of isonitrogenous amino acid, was used as a control for Gln administration (9). Glutamine was injected intravenously because malnutrition may impair the estimation of Gln absorption through the intestinal mucosa (25). Finally, analysis of Gln and Ala plasma levels did not reveal any difference between the experimental groups. We believe that the concentrations of these amino acids may have increased early after Gln injection and later returned to baseline levels. A recent clinical trial in critically ill patients with multiple organ failure found that median baseline plasma Gln levels were within normal limits (7). Additional experiments are required to investigate the dynamics of Gln levels in blood in our experimental model of combined malnutrition and sepsis. In the present study, we used a model of sepsis induced by CLP. The mortality rate in malnutrition-CLP-Sal rats was 29%, similar to the 30% observed in well-nourished CLPSal animals (9). We had expected mortality rate to be higher in malnutrition-CLP animals; however, malnutrition status activates endogenous survival factors, such as peroxisome proliferativeYactivated receptor + coactivator 1!, which may reduce systemic inflammation (26). We used malnutritionYsham-operated animals as control for malnutrition-CLP-operated animals. In malnutrition-sham group, we observed neutrophil infiltration as well as activation

of macrophages. This might have been caused by the inflammatory response to the surgical procedure or anesthesia per se (27). In malnutrition-sham group, Gln contributed to reduce neutrophil infiltration and apoptosis in lung and liver, but did not affect the parenchymal damage due to malnutrition. We cannot exclude that daily doses of Gln and a higher period of treatment could show a repair in malnutrition injury in the lung. In septic malnourished animals, Gln minimized alveolar epithelial cell damage, which may contribute to reduce collagen fiber deposition. In this line, extensive lesion of alveolar type II epithelial cells and lung cell apoptosis cause loss of cellular attachment to the underlying basement membrane and exposure of the alveolar epithelial basement membrane to inflammatory products, resulting in alveolar collapse, activation of fibroblast proliferation, and collagen production, leading to fibrosis (28). In addition, reduction of lung fibrosis by Gln may be associated with decreased inflammation. In our study, Gln reduced neutrophil infiltration and IL-6 levels in lung tissue. This reduction in inflammation and the improvement in pulmonary tissue healing were associated with a decrease in alveolar collapse and improvement in lung mechanics. Interestingly, in malnourished animals, sepsis did not increase neutrophil infiltration in lung tissue, which may be explained by bone marrow hypoplasia, interruption of cell maturation, prominent lymphopenia with depletion of lymphoid lineage, and changes in cellular development associated with malnutrition (4). However, Gln decreased neutrophil infiltration in both nonseptic and septic undernourished animals, which may be explained by a Gln-induced curtailing of the interaction between leukocytes and the endothelium during sepsis (23). In rats with intraabdominal sepsis, parenteral nutrition supplemented with Gln has been reported to replenish serum levels of IL-6 and IL-10 (29). In a model of acute kidney injury induced by sepsis, i.v. Gln administration led to a decrease in phosphorylated NF-.B p65 protein expression, reducing kidney injury (30). We did not investigate phosphorylated NF-.B, but our analysis of inflammatory mediators, which showed lower levels of IL-6 in CLPGln, suggested that this mechanism may be involved in the beneficial effects of Gln in septic malnourished animals. Previous studies have also explained the beneficial effects of Gln in septic well-nourished animals by increased HSF-1 phosphorylation (8) and enhancement of HSP70 expression (13). Heat shock protein 70 is a protective protein associated with physiological stress and involved in apoptosis as well as

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SHOCK MARCH 2014 redox balance (10, 14). In this context, the effects of HSP70 and reduced glutathione (GSH) can be partly attributed to the suppression of apoptosis (20). Reduced HSP70 is associated with increased caspase activity in Gln deficiency (20). Supporting these data, it was demonstrated that HSP70 alters the interaction between caspases 8, 9, and 3, disrupting these protein complexes and promoting survival (31). In a model of acute kidney injury induced by glycerol injection, Gln administration prevented apoptosis by enhancing HSP70 and GSH levels. Heat shock protein 70 strongly associates with c-Jun N-terminal kinase (JNK), thereby inhibiting JNK phosphorylation, while increasing cellular GSH levels to reduce oxidative stress and sensitivity to apoptotic triggers (32). The stress-activated protein kinase/JNK pathway is involved in the apoptosis process increased by Fas stimulation. JNK/stressactivated protein kinase induction by the Fas ligand is mediated by apoptosis signal-regulating kinase 1, a critical kinase protein in apoptosis, which is activated after Fas ligand in the absence of Gln (33). Moreover, it is recognized that Gln deprivation triggers intracellular events, which rapidly target the mitochondria, activating the intrinsic pathway of apoptosis, partly via induction of oxidative stress and reduced levels of Bcl-2, a prosurvival protein (34). In this context, Bcl-2 and GSH were upregulated in T cells after Gln treatment, promoting redistribution of GSH to the nucleus, thereby altering nuclear redox and blocking caspase activity (35). However, in well-nourished and malnutrition-sham and -CLP animals, no influence of Gln on HSF-1 phosphorylation and HSP70 expression was found in the present study. This suggests that increased GSH levels or direct inhibition of NF-KB transcription factor induced by i.v. Gln may have played a role in reducing cell apoptosis in our malnourished nonseptic and septic animals. We could not discard the possibility that HSF-1 phosphorylation and HSP70 expression were increased in other organs or early after Gln administration, developing protective effects at 48 h before sepsis induction, because increased expression of HSP70 with the use of Gln was observed before 48 h (13, 24). The M1 and M2 macrophage phenotypes were also measured in this study. Our data suggest that in malnourished animals with sepsis, the reduction in inflammation was likely due to the activation of macrophage phenotype M1, triggering the production of reactive oxygen and nitrogen species that facilitate the killing of microbial pathogens (15). Conversely, the reduced collagen production could be attributed to the activation of macrophage phenotype M2, which regulates wound healing and tissue repair (16). Interestingly, the increase in M1 was paralleled by an increase in M2, suggesting a balance between the proinflammatory and anti-inflammatory capabilities of these phenotypes. The effects of Gln on macrophage polarization are not clear. This was, to the best of our knowledge, the first study to investigate the role of macrophages in the modulation of inflammatory response, fibrogenesis, and apoptosis in the lung as induced by i.v. Gln in malnourished rats with sepsis. It is recognized that pathogen-associated and damage-associated molecular patterns also act in synergy with natural killer cellYderived IFN-+ to polarize macrophages toward the M1 phenotype. In contrast, M2 polarization is a

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programmed response facilitated by signal transducer and activator of transcription 6 activating cytokines IL-4 and IL-13 while inhibiting the transcription of many M1-associated genes (16). However, no differences in IFN-+ were found, and IL-4 and IL-13 were not analyzed. Clinical implications

Even though i.v. Gln led to beneficial effects in experimental sepsis, clinical studies have reported controversial results in critically ill patients (7, 8). Differences among clinical studies may be attributed to the dose, timing, and route of Gln administration, as well as to the association between enteral and parenteral nutrition. In a recent randomized controlled trial (7), the effects of Gln and/or antioxidant supplementation were investigated in a heterogeneous population of severely (Acute Physiology and Chronic Health Evaluation score ,26) critically ill patients. The authors included mechanically ventilated patients admitted to an ICU with failure of two or more organs related to their acute illness. They reported a significant increase in in-hospital mortality and mortality at 6 months among patients who received Gln as compared with patients who did not receive Gln. Comorbidities, including malnutrition, were not clearly reported in the group of septic patients. Because hospital mortality has been reported to be higher in undernourished compared with well-nourished patients (2), we expect that Gln could be more beneficial in less severe sepsis. Thus, our study proposed the use of Gln in the presence of malnourishment and less severe sepsis. This study has several limitations: (a) we used specific models of malnutrition and sepsis, developed by reducing overall nutrient intake and by CLP surgery, respectively; thus, our results may not be extrapolated to other malnutrition and sepsis models in small or large animals; (b) we did not analyze biochemical markers of inflammatory activity and did not conduct body mass analysis to evaluate malnutrition status; (c) the expression of only one HSP was evaluated and only in the lung; in addition; we did not analyze HSP70 and HSF-1 expression early in the course of sepsis; (d) we compared the effects of Gln versus Sal and not against an isonitrogenous amino acid control; (e) we analyzed cytokines 48 h after the CLP procedure, and consequently, the levels of IFN-+, IL-6, and MCP-1 were unknown before this time point; (f) because Gln was administered early after the onset of sepsis, we were unable to draw conclusions regarding the effects of Gln later in the course of sepsis; (g) we did not perform serial analyses of blood samples to evaluate plasma levels of Gln and Ala after L-alanyl-L-Gln administration; (h) to inject Sal or Gln, animals were anesthetized with sevoflurane, which may lead to immunomodulation (27); (i) even though immunofluorescence from labeled cell typeYspecific antibodies allows the quantification of cells and extracellular matrix components, electron microscopy analysis was used to evaluate epithelial and endothelial cell or alveolar-capillary membrane damage; and (j) we cannot exclude that CLP animals were underresuscitated because the amount of fluid given after surgery was similar. On the other hand, different fluid management among CLP groups may also affect lung injury and inflammatory response.

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In conclusion, our data suggest that early therapy with i.v. Gln may prevent additional lung and distal organ damage in malnourished rats with sepsis. These effects seem to be triggered, at least in part, by antiapoptotic activity and macrophage phenotype modulation induced by Gln. ACKNOWLEDGMENT The authors thank Ms. Carolina M. L. Barbosa for her help with Western Blot analysis; Mr. Andre´ Benedito da Silva, Mrs. Ana Carolina Vargas, and Mrs. Giselle Reis for animal care; Mrs. Ana Lucia Neves da Silva for her help with microscopy; Mrs. Moira Elizabeth Schottler and Ms. Claudia Buchweitz for their assistance in editing the manuscript; and MAQUET for supporting the authors technically.

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