L-ARGININE ATTENUATES ACUTE LUNG INJURY AFTER SMOKE... : Shock (original) (raw)
INTRODUCTION
Acute airway injury caused by smoke inhalation results in airway complications, such as cast formation and poor gas exchange, which have the potential to be fatal to exposed patients (1, 2). L-Arginine (L-Arg), a nutritionally nonessential amino acid, is the critical substrate for NO production through enzymatic oxidation by NOS (3). It has been shown that the conversion from Arg to citrulline is significantly increased, and Arg levels are depleted in severely burned patients (4). Arginine has also been linked to acute lung injury (ALI) through a compromised Na+ exportation mechanism that clears fluid from the damaged air spaces (5). NOS exists in three isoforms-two constitutive forms that are Ca2+-calmodulin dependent, and an inducible form (iNOS) that is Ca2+-independent and up-regulated in response to inflammatory cytokines. With the induction of iNOS, the Arg substrate pool decreases to the extent of being the limiting reagent for the production of NO (4). In response to the lack of substrate, iNOS begins to oxidize oxygen molecules, thereby forming superoxide species (6). The role of iNOS in the inflammatory response and in ALI has previously been described in burn and smoke inhalation (B + S) injuries (7). In addition, NO has been shown to alter inflammatory cytokine expression in bronchoalveolar lavage samples, aggravating ALI (8). We hypothesized that the manipulation of NO synthesis by supplementation of Arg would attenuate the inflammatory sequelae resulting from B + S injury, thus providing a potential therapeutic treatment of ALI.
MATERIALS AND METHODS
This study was approved by the Animal Care and Use Committee of the University of Texas Medical Branch. All the animals were handled according to the guidelines established by the American Physiology Society and the National Institutes of Health.
Animal model
For this study, we used a previously described sheep model of B + S injury (9) to investigate the pathophysiological aspects of organ failure. Briefly, 17 adult female sheep (30 - 40 kg) were surgically prepared for study under endotracheal halothane anesthesia (10). After 5 days of recovery, the sheep were again anesthetized with halothane, and inhalation injury was induced with a modified bee smoker to deliver four series of 12 breaths of cotton smoke (11). Immediately thereafter, a third-degree, 40% total body surface area cutaneous burn injury was induced to the anesthetized sheep (9). After the combination injury, all animals were maintained on mechanical ventilation (Servo Ventilator 900C, Siemens-Elema, Sweden) throughout the 48-h experimental period. Ventilation was performed with a positive end-expiratory pressure of 5 cm water and a tidal volume of 15 mL·kg−1 (sheep lung is more compliant than humans). During the first 3 h after injury, oxygen concentration in the inspired air was maintained at 100%, and the respiratory rate was maintained at 30 per minute. Both values were then adjusted according to blood gas analysis (arterial oxygen and carbon dioxide). Hemodynamic variables were monitored, and the systemic vascular resistance index (SVRI) and pulmonary vascular resistance index were calculated. All the animals were resuscitated using lactated Ringer solution according to the formula (4 mL·kg−1 per percent burned body surface for the first 24 h followed by 2mL·kg−1 per percent burned body surface for the second 24 h). However, in the first 8 h after injury, sheep received half of the fluid that was given during the first 24 h.
Before injury, sheep were divided randomly into three groups: sham (the noninjured, nontreated; n = 6), control (injured and treated with saline; n = 6), and L-Arg (injured and treated with L-Arg; n = 5). After randomization, baseline data were obtained, and standardized B + S injuries were induced. L-Arg (Sigma, St. Louis, Mo) was dissolved in 0.9% NaCl with a total volume of 1,000 mL and continuously infused (57 mg·kg−1·h−1) for 48 h starting 1 h postinjury. Control group received same amount of saline. The study protocol is shown in Figure 1.
Experimental protocol. L-Arginine infusion was started 1 h after smoke inhalation injury. Fluid resuscitation using Ringer lactate was started immediately after the insult.
Measurement of plasma NO2/NO3 and L-Arg levels
The concentration of nitrogen oxides (NOx) (total amount of NO metabolites) in plasma was measured with a chemiluminescent NO analyzer (Antek Model 7020, Antek Instruments, Houston, Tex) as previously described (12). Serum concentrations of L-Arg were analyzed using gas chromatography mass spectrometry technique by an isotopic dilution method as described before (13). Briefly, 10 μL of [U-13C6]Arg (4.13 μM) was added to 100 μL of each plasma sample. The amino acids were extracted using the ion-exchange column and prepared as methyl ester trifluroacetyl derivative. The samples were then analyzed using on-column injection with an HP 5980 series II Gas Chromatograph coupled to an HP 5980 series II Mass Spectrometer (Hewlett-Packard, Palo Alto, Calif). Selective ion monitoring of Arg was conducted on the [M-20]− ion using negative chemical ionization with methane as the reagent gas. Arginine was measured at m/z 326 (M + 6) and m/z 320 (M + 0). The measurement was correct by linear regression analysis on the data from standard samples containing various known amount of natural Arg against a fixed amount, the internal standard [U-13C6]Arg.
Lung histopathology and lung wet-to-dry weight ratio
A lung slice was removed from the right lower lobe of each sheep and was histologically scored by a pathologist unaware of animal groupings (14). Changes in parenchyma were graded on a scale of 0 to 4 (0, normal; 4, severe) for congestion, edema, inflammation, and hemorrhage. Airway obstruction by cast formation was also evaluated as previously described (9, 14).
The remaining lower half of the right lower lobe was used for determination of wet-to-dry weight ratio, which was calculated for use as an index of lung water content (15).
Immunohistochemistry of nitrotyrosine
Lung tissues were fixed in formalin, processed to paraffin, and sectioned at 4 μm for immunohistochemical procedures. A rabbit polyclonal antibody against nitrotyrosine (Upstate Biotechnology, Lake Placid, NY) was used at a dose of 0.1 μg·mL−1. Immunohistochemistry was performed with protease digestion antibody incubation overnight at 4°C. Antigen detection was performed with the Vectastain Elite peroxidase kit with DAB chromogen (Vector Laboratories, Inc, Burlingame, Calif) and counterstained with hematoxylin. Appropriate controls were run with rabbit immunoglobulin at the same concentration of 0.1 μg·mL−1. Slides were qualitatively evaluated according to staining intensity, using a scale of 0 to 3, in the tissue and specific cells. A score of 0 represented no staining compared with controls, 1 represented light staining, 2 represented moderate staining, and 3 represented strong staining.
Statistical analysis
Statistical analysis was performed using the statistical software package StatView 5.0 (SAS Institute, Cary, NC). Data are expressed as mean ± SEM. ANOVA with Tukey post hoc test or repeated-measures ANOVA was used to determine the statistical differences between the treatment groups. Appropriate intergroup comparisons at individual time points were made using the Student Newman-Keuls test for unpaired data. In the histological study, a nonparametric Mann-Whitney U test was performed. A P value less than 0.05 was considered statistically significant.
RESULTS
All animals survived the 48-h study period after the combined B+ S injury. Body weights of saline-treated controls and L-Arg group were similar (36.5 ± 1.5 and 37.1 ± 1.5 kg, respectively). The arterial carboxyhemoglobin levels immediately after smoke exposure were 58.8% ± 4.9% in control and 61.2% ± 5.0% in the L-Arg group. There was no statistical difference (P = 0.79) between these values, indicating that both control and treated animals received similar smoke inhalation injuries. The arterial carboxyhemoglobin level was 3.12 ± 0.5 in sham animals after the fake smoke procedure.
Plasma concentration of L-Arg
Plasma concentration of L-Arg was analyzed at baseline and several times during the study. As shown in Figure 2A, L-Arg concentration dropped to less than 50% in the injured control group. The L-Arg-supplemented group showed a marked increase (~3-fold increase compared with baseline value) in plasma concentration of L-Arg at 3, 6, and 12 h. The concentration then gradually decreased but remained significantly higher than the baseline levels. There was a significant difference in the plasma concentration of L-Arg between the L-Arg and control groups throughout the experimental period (Fig. 2A).
Plasma concentrations of Arg (A) and pulmonary gas exchange. Pulmonary gas exchange was evaluated by the Pao2/Fio2 ratio (B) and the pulmonary shunt fraction (C) calculated by a standard formula. The data are expressed as the mean ± SEM. *P < 0.05 vs. control. † P < 0.05 vs. baseline.
Pao2/Fio2 ratio and pulmonary shunt fraction (Qs/Qt)
The Pao2/Fio2 (Fig. 2B) ratio slightly increased after the sham animals were placed on a ventilator, but was markedly depressed in control animals after the combined B + S injury. In the injured control animals, Pao2/Fio2 ratio began to decrease 3 h after the insult and steadily worsened to reach the criteria of clinically defined adult respiratory distress syndrome (Pao2/Fio2 ratio < 200) between 24 and 48 h. Treatment with L-Arg delayed the decline in Pao2/Fio2 ratio by approximately 24 to 36 h. L-Arg-treated animals had significantly higher Pao2/Fio2 ratios than controls, with a mean ratio maintained more than 200 for the first 42 h of the experimental period (Fig. 2B).
Pulmonary shunt fraction (Fig. 2C) remained unchanged in sham animals, whereas it significantly increased in control animals after B + S injury. Treatment with L-Arg resulted in a pulmonary shunt fraction significantly lower than the untreated control animals at 36 and 48 h after insult (Fig. 2C).
Lung wet-to-dry weight ratio
Lung water content (Fig. 3A) was evaluated by measuring lung wet-to-dry weight ratios at 48 h. Control animals showed a significant increase in lung wet-to-dry weight ratio as compared with sham animals. L-Arg supplementation resulted in a lower mean value for wet-to-dry weight ratio than the untreated control, although this difference was not statistically significant (Fig. 3A).
Lung wet-to-dry weight ratio (A), total histopathology scores (B), and nitrotyrosine stain (original magnification ×100) in the lung 48 h after B + S injury in control (C) and L-Arg (D) groups (representative of all the experiments). The data (A, B) are expressed as the mean ± SEM. *P < 0.05 vs. sham.
Histopathological changes
Effects of L-Arg on lung histopathological changes were scored for edema, congestion, inflammation, and hemorrhage (Table 1). The scores in all categories were higher in the control group as compared with the sham group. The increases in histopathology scores in each category were lower in the L-Arg group as with the control group (Table 1). However, statistical differences were not found between the control and the L-Arg groups except the scores for hemorrhage. Total histopathology scores (Fig. 3B) were calculated, resulting in higher scores (congestion, edema, inflammation, and hemorrhage) for the control versus sham groups. The score was slightly lower in L-Arg versus the control group. However, there were no significant differences between sham versuscontrol and control versus L-Arg groups (Fig. 3B).
Effect of L-Arg supplementation on histopathology scores
Nitrotyrosine staining
Nitrotyrosine staining in the control group (Fig. 3C) at 48 h after insult was moderately positive, with a mean score of 2.0. Nitrotyrosine staining was also present in injured epithelial cells, some mucous gland cells, endothelial cells, and macrophages (Fig. 3C). In the L-Arg animals, the staining was significantly less (Fig. 3D), with a mean score of 1.2 (P < 0.02 vs. control). Macrophages exhibited strong staining in both groups.
Airway obstruction
We determined airway obstruction scores in bronchi (Fig. 4A) and bronchioles (Fig. 4B) for the three groups studied. The sham group showed little evidence of airway obstruction: 5.4% and 3.3% of bronchi and bronchioles, respectively. The control group of injured animals showed high airway obstruction scores: 19.8% and 10.7% for bronchi and bronchioles, respectively. L-Arg supplementation resulted in significant reduction in the extent of airway obstruction, with only 6.4% of bronchi and 7.0% of bronchioles obstructed.
Airway obstruction (A) in bronchi and bronchioles (B). Data are expressed as the mean ± SEM. *P < 0.05 vs. control; † P < 0.05 vs. sham.
Hematocrit and urine output
During the course of the 48-h experiment, untreated control animals showed a significant increase in hematocrit-a 1.5- and almost 2.0-fold increase at 24 and 48 h, respectively (Fig. 5A). This was paralleled by an 80% decrease in urinary output over the last 24 h of the experimental period despite fluid resuscitation (Fig. 5B). This rise in hematocrit and decrease in urinary output were blunted with the administration of L-Arg. Hematocrit and urine levels for L-Arg-treated animals closely resembled those of sham animals.
Hematocrit (A) and urine output (B). Data are expressed as the mean ± SEM. *P < 0.05 vs. control; † P < 0.05 vs. sham; ‡ P < 0.05 vs. baseline values.
Hemodynamics
MAP rose throughout the study in both the control and sham groups. Paired t test results showed that MAP was significantly higher in the control group at 42 and 48 h compared with baseline values. The L-Arg-treated group showed no elevation in MAP and maintained the baseline level during the experiment (Fig. 6A). Systemic vascular resistance index (Fig. 6B) increased in the initial 6 h and maintained elevated levels in the control group. The L-Arg group, however, showed a slight decrease in SVRI after an initial 12-h elevation period. Systemic vascular resistance index in the L-Arg group was statistically lower as compared with the control at 30 and 42 h after the insult. Pulmonary vascular resistance index (Fig. 6C) rose in both the control and L-Arg groups. However, the elevation was less marked in the L-Arg group, and there was a significant difference between the groups at 24 h.
Hemodynamics and changes in plasma levels of nitrate and nitrite (NOx; D). The changes in MAP (A), SVRI (B), and pulmonary vascular resistance index (PVRI; C) are presented. Data are expressed as the mean ± SEM. *P < 0.05 vs. control.
Plasma NOx levels
In sham animals, plasma NOx increased initially after 3 h but then decreased to the baseline levels. In the control group, plasma NOx gradually increased and peaked at 48 h. The L-Arg-treated group showed an increase in plasma NOx that peaked at 12 h and sustained this level for the duration of the 48-h study. However, the L-Arg treatment did not significantly affect the increased plasma NOx levels seen in control animals (Fig. 6D).
DISCUSSION
Burn patients have reduced synthesis and increased metabolism of L-Arg. NOS and NO are also increased after a combination of B + S injury (7). As we show in this article, plasma Arg level is severely decreased after B + S injury in sheep. In our previous study, Arg metabolism in the lung was increased more than 3-fold after B + S injury (16). Simultaneously, plasma Arg levels decreased to less than half of the baseline values after B + S injury. These changes were partially reversed by the administration of l-_NG_-monomethyL-Arginine (16), suggesting that the depletion of Arg was due, in part, to the result of NO formation. In addition, studies using a combination of Arg and ornithine tracers also suggested a significantly higher rate of Arg catabolism by its conversion to ornithine and subsequent oxidation (17). Increased rate of Arg catabolism was also observed in hypotensive septic patients (18). Hence, there is an Arg-depleted state after B + S injury.
iNOS catalyzes production of large amounts of NO from Arg. iNOS is composed of a reductase domain and an oxidase domain (19). Induction of the high-output iNOS usually occurs in an oxidative environment, and, thus, high levels of NO have the opportunity to react with superoxide, leading to peroxynitrite formation and cell toxicity. Moreover, generation of NO is dependent on the availability of sufficient amounts of substrate and/or cofactors, in particular, BH4. Relative deficiency of L-Arg and BH4 leads to an "uncoupling" of NOS activity and the production of superoxide (O−2) anion instead of NO. It should be noted that the continuous high level of Ca2+/CaM-independent activity of iNOS may predispose to depletion of substrate and cofactors and "convert" iNOS into a predominantly O−2-generating enzyme. Although all NOS enzymes may potentially generate O−2, iNOS is the most likely to produce O−2_in vivo_. These inproperties may define the roles of iNOS in host immunity, enabling its participation in antimicrobial and antitumor activities; however, it also contributes to the formation of peroxynitrite. The latter has been shown to be cytotoxic, causing damage to cellular DNA and increasing vascular permeability; it is possible that peroxynitrite can cause organ failure such as ALI or renal failure (20, 21). Therefore, we hypothesized that the restoration of Arg concentrations would decrease the possibility of peroxynitrite being formed, thus possibly ameliorating ALI after B + S injury.
In the present study, we started a constant infusion of L-Arg (i.v.) 1 h after the injury and continued this infusion for the duration of the 48-h study. The dosage we used for this study was 57 mg·kg−1·h−1. In another group of sheep, we conducted a preliminary dose response study in which we infused L-Arg at a rate of 19 mg·kg−1·h−1 (n = 4) and found that we could maintain the baseline Arg levels during the first 24 h after insult but could not maintain them after this period (data not shown). We also noted that these preliminary animals showed no beneficial effects with Arg supplementation. Plasma concentrations of Arg still decreased at this dose (112 μM at baseline and 59 μM at 48 h). The dose was then doubled to 38 mg·kg−1·h−1 (n = 1), but there was still no evidence of any beneficial effects. At this dose, plasma concentrations of Arg still decreased, especially between 24 and 48 h. In view of these results, we increased the dose up to 57 mg·g−1·h−1 and found that this dose resulted in maintenance of the plasma Arg concentrations at a level equal to or more than normal during the 48-h period after B + S injury. We therefore decided to continue the present study with this dosage.
In the present study, we noted two major physiological benefits of L-Arg administration-the improvement of both lung and renal function.
Before we started the experiment, we had a concern if L-Arg supplementation would augment the NO formation to the point that hypotension or shock would occur. However, as shown in Figure 6, SVRI decreased in the L-Arg group to some extent, but MAP was maintained at baseline levels with no hypotension. In fact, plasma NOx levels were not further increased by L-Arg treatment, especially at second the 24 h. The plasma NOx levels slightly tended to increase at the initial 24 h in L-Arg group. Although the exact mechanism is not yet clear, it is possible that due to the negative feedback effect of the produced NO on iNOS induction (8), the initially increased NO production might ameliorate the further increase in NO production.
With regard to gas exchange, a mismatching of ventilation/perfusion can result in a failure in maintaining adequate pulmonary function. This is especially true in case of smoke inhalation injury, in which a large portion of airways are obstructed by mucus, shed epithelial cells, and migrated neutrophils. As shown in Figure 4, airway obstruction was significantly reduced after L-Arg administration. We have previously reported that pulmonary epithelium expresses iNOS (7). In the present study, nitrotyrosine stain was also positive in pulmonary epithelial cells and in some mucous gland cells, endothelial cells, and macrophages (Fig. 3). L-Arg supplementation significantly inhibited the accumulation of nitrotyrosine in the lung, suggesting that the L-Arg treatment inhibited the peroxynitrite formation through a mechanism as discussed above.
Nitrotyrosine, a stable end product of peroxynitrite oxidation, is considered to be a useful marker of NO-dependent damage in vivo. Because NOx is only an indicator for enhanced NO production, protein-associated nitrotyrosine might be a more suitable marker for damage induced by reactive nitrogen intermediates derived from NO. Furthermore, most proteins have a longer half-life than NOx. Peroxynitrite is an oxidative radical known to damage DNA. When a DNA single-strand break occurs, a repairing enzyme called poly (ADP-ribose) polymerase (PARP) is activated (22, 23). Although PARP repairs DNA single-strand breaks, the process requires a significant amount of nicotinamide adenine dinucleotide and adenosine triphosphate; as a result, energy depletion occurs in these cells, leading to cell death. Hence, necrotic pulmonary epithelial cells are shed, leading to the formation of airway casts (24). Airway obstruction after B + S injury is also caused by exuded plasma within the interstitium that moves into the alveolar space with the damaged epithelial cells. With the presence of tissue factors in the alveolar space (25), for instance on the surface of epithelial cells or alveolar macrophages, exuded plasma forms a clot in the airway. In the present study, L-Arg supplementation attenuated the airway obstruction significantly. This can be explained by the inhibition of endothelial and epithelial apoptosis as the results of reduced PARP activation. Further studies are required to explore this mechanism in more detail.
The beneficial effects of L-Arg administration in burns or other diseased condition have also been reported in several studies. Nakanishi et al. (26) demonstrated that intracoronary infusion of L-Arg after ischemic injury to the myocardium, before reperfusion, decreased the area of myocardial infarction. Although infusion of Arg without ischemic insult did not cause a dilatation of coronary vessels, it did enhance the vasodilatory response of these vessels to endothelial-dependent vasodilatory agents. Garcia and Horton and Horton et al. (27, 28) also reported that using Arg-containing resuscitation fluid for burn injury improved cardiac performance and prevented gut bacterial translocation. Hofford et al. (29) showed that after endotoxin challenge, plasma Arg concentrations were also decreased to a great extent in the nonsurviving sheep models. Thus, Arg supplementation caused an increase in the vasodilatory response of pulmonary vessels after endotoxin insult. In addition, as shown in the present study, plasma NOx in the L-Arg group increased in the initial 12 h after injury but was lower than that of the control group later on, suggesting that the initially increased NO production from the induced iNOS may have a negative feedback effect on nuclear factor-κB and ameliorate the inflammatory response; hence, it may act as an antiinflammatory agent (8).
Asymmetric dimethylarginine (ADMA) is known as an endogenous NOS inhibitor. Asymmetric dimethylarginine is synthesized and released by endothelial cells, and the amounts released are sufficient to inhibit both endothelial NOS and iNOS (30). Asymmetric dimethylarginine enters the cell through a y+ cationic amino acid transporter, and it competes with L-Arg for transport (30). There is a possibility that supplemented Arg might also promote the formation of ADMA (31) that inhibits further NO formation and inflammatory reactions. It has been known that oxidative stress induces ADMA synthesis and leads to endothelial NOS uncoupling (32). We are now further exploring the effect of ARG supplementation on ADMA concentration and the related regulatory role on the pathophysiology related to NO production.
Of particular interest, NO has been shown to modulate immune response in multiple trauma, including burn injury (33, 34). It prevents overexpansion of TH1 cells, which are involved in persistent and uncontrolled inflammation and promotes expression of TH2 cytokine. It was reported that iNOS gene-deficient mice had TH1/TH2 balance shift toward TH1 and were more susceptible to LPS-induced mortality than wild-type mice (35). On the other hand, inhibition of excessive NO had beneficial effects in various pathological conditions such as sepsis or burn (36). The results of these studies suggest that the balanced inflammatory responses to pathological stimulus are important in outcome of various menaces. Although we were not able to show a direct link between immune responses and Arg deficiency in the present model, we do not exclude a possible modulatory effect of L-Arg as a substrate for NO synthase on the cytokines responsible for the immune reactions.
CONCLUSION
Supplementation with Arg after B + S injury had a positive effect on pulmonary and, possibly, kidney function. We believe that Arg supplementation can provide a potential treatment strategy for patients with ALI, especially under the pathological condition, which is accompanied with Arg depletion and iNOS activation. The mechanism is related to the effect of Arg supplementation on reduced free radical formation.
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Keywords:
NO; ARDS; airway obstruction; thermal injury; arginine
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