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TRANSLATIONAL PHYSIOLOGY
1 expression, and epithelial barrier dysfunction in the alcoholic rat lung
1Atlanta Veterans Affairs Medical Center Pulmonary Section, Decatur; 2Division of Pulmonary, Allergy, and Critical Care Medicine, and 3Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia
Submitted 29 April 2005 ; accepted in final form 17 May 2005
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ABSTRACT |
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1 (TGF-1), and alveolar epithelial barrier dysfunction, even though the lung appears grossly normal. However, during endotoxemia, ethanol-fed rats release more activated TGF-1 into the alveolar space where it can exacerbate epithelial barrier dysfunction and lung edema. Ethanol ingestion activates the renin-angiotensin system, and angiotensin II is capable of inducing oxidative stress and TGF-1 expression. We determined that lisinopril, an angiotensin-converting enzyme inhibitor that decreases angiotensin II formation, limited lung glutathione depletion, and treatment with either lisinopril or losartan, a selective angiotensin II type 1 receptor blocker, normalized TGF-1 expression. The glutathione precursor procysteine also prevented TGF-1 expression, suggesting that TGF-1 may be induced indirectly by angiotensin II-mediated oxidative stress and glutathione depletion. Importantly, lisinopril treatment normalized barrier function in alveolar epithelial cell monolayers from ethanol-fed rats, and treatment with either lisinopril or losartan normalized alveolar epithelial barrier function in ethanol-fed rats in vivo, as reflected by lung liquid clearance of an intratracheal saline challenge, even during endotoxemia. In parallel, lisinopril treatment limited TGF-1 protein release into the alveolar space during endotoxemia. Together, these results suggest that angiotensin II mediates oxidative stress and the consequent TGF-1 expression and alveolar epithelial barrier dysfunction that characterize the alcoholic lung. acute respiratory distress syndrome; epithelium; angiotensin-converting enzyme; alcohol abuse
75,000–150,000 individuals per year in the United States alone (19, 37). In the two epidemiological studies linking alcohol abuse and ARDS (25, 27), 50% of all individuals with ARDS were alcoholics, indicating that alcohol abuse causes tens of thousands of cases of ARDS per year. A cardinal feature of ARDS is alveolar epithelial cell dysfunction, including disruption of the alveolar epithelial barrier and flooding of the alveolar space with proteinaceous fluid rich in cytokines. Although incremental improvements in supportive care have improved survival in selected individuals (1), the overall mortality from ARDS remains unacceptably high at 40–60% (37), and there are no effective pharmacological therapies to complement our current supportive care. The recently identified association between alcohol abuse and ARDS has prompted laboratory investigations that have generated new insights into the pathophysiology of acute lung injury, particularly in this highly vulnerable subgroup. To study the fundamental mechanisms underlying the epidemiological association between alcohol abuse and ARDS, we developed a rat model of ethanol-mediated susceptibility to acute lung injury and determined that chronic ethanol ingestion produces multiple defects in alveolar epithelial function. For example, alveolar epithelial type II cells isolated from ethanol-fed rats formed more permeable monolayers (16) and had abnormal surfactant synthesis and secretion (17) in vitro. In parallel, chronic ethanol ingestion in rats impaired alveolar epithelial barrier function in vivo, as reflected by decreased alveolar liquid clearance and increased protein flux (16), and increased sepsis-mediated acute lung injury in vivo (33). Another remarkable and previously unrecognized finding was that chronic ethanol ingestion produces significant oxidative stress, as reflected by markedly decreased glutathione levels within the alveolar epithelial lining fluid and in alveolar epithelial cells (5, 6, 16, 17, 22). The critical role of glutathione depletion in ethanol-induced lung dysfunction is supported by multiple studies in which glutathione supplementation of the ethanol diet restores alveolar epithelial function in vitro and in vivo (15–17) and decreases sepsis-mediated surfactant dysfunction and acute lung injury in vivo (33). The potential clinical relevance of these findings in our rat model is reflected by our observation that otherwise healthy human alcoholic subjects have comparably decreased levels of glutathione in their lung lavage fluid (26).
More recently, we determined that transforming growth factor-1 (TGF-1) is induced by chronic ethanol ingestion and could mediate alveolar epithelial barrier dysfunction in the alcoholic lung during endotoxemia (4). Specifically, chronic ethanol ingestion increased gene and protein expression for TGF-1 in the rat lung (4). The majority of the TGF-1 protein in the alcoholic lung tissue is in the latent form but is released as activated TGF-1 into the alveolar air space during endotoxemia, and the lavage fluid from these rats induces permeability in naive alveolar epithelial monolayer via TGF-1-specific effects (4). Together, these studies indicate that increased expression of TGF-1 protein in the chronic alcoholic lung leads to increased release of activated TGF-1 into the alveolar air space during acute inflammation where it can further impair epithelial barrier function and promote alveolar protein leak. However, chronic low-level activation of TGF-1 protein in the alcoholic lung could also contribute to the chronic alveolar epithelial barrier dysfunction that we have identified in both the experimental rat model (16) as well as in otherwise healthy alcoholic human subjects (8). Therefore, glutathione depletion and excessive TGF-1 expression are important features of a chronic alcoholic phenotype characterized by alveolar epithelial dysfunction as well as an acute phenotype that is highly susceptible to acute edematous injury during sepsis. Now the question is, how does chronic ethanol ingestion create this susceptible phenotype?
Chronic ethanol ingestion increases plasma levels of angiotensin II in rodents (40) and in humans (21, 30), and it has been postulated that activation of the renin-angiotensin system may explain the association between alcohol abuse and hypertension in humans (39). One potential mechanism is direct conversion of angiotensinogen to angiotensin I by metabolites of ethanol (32), thereby bypassing renin, which is the usual regulatory step in the renin-angiotensin system. Although clearly important for normal homeostatic functions such as salt and water balance and vascular tone, angiotensin II is implicated in diverse pathophysiological conditions including vascular and myocardial injury (10, 31), in part due to specific activation of apoptosis pathways (18). The relevance of these pathways to the alcoholic lung is suggested by reports that alveolar epithelial type II cells have receptors for angiotensin II (11). Angiotensin II induces apoptosis in human and rat type II cells in vitro (34–36) and causes alveolar epithelial cell injury in rabbits in vivo (13). Relevant to our recent study on ethanol-induced TGF-1 expression in the alcoholic rat lung (4), angiotensin II induces TGF- expression and lung collagen deposition in the lungs of bleomycin-treated rats (23) and is a potent inducer of TGF-1 expression in other tissues such as the kidney (14, 20, 28).
Together, these observations suggest that the renin-angiotensin system, through angiotensin II, could play a causative role in mediating the glutathione depletion and excessive TGF-1 expression that we have identified as being potentially important in ethanol-induced susceptibility to acute lung injury. In this current study, we examined the role of angiotensin II in ethanol-mediated glutathione depletion, TGF-1 expression, and alveolar epithelial barrier function in a model of chronic ethanol ingestion in rats.
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MATERIALS AND METHODS |
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5–10%) compared with control-fed rats at the end of the 6-wk dietary protocol, regardless of whether or not they received lisinopril or losartan. However, body sizes, as reflected by length from nose to tail or width from paw to paw when outstretched, were not affected by the ethanol diet. In parallel, lung sizes, as reflected by baseline dry and wet weights, were not affected by the ethanol diet. Determination of lung lavage glutathione concentrations. We used a variation of the high-performance liquid chromatography method presented by Martin and White (24) as we have published in this model previously (15, 17) as well in alcoholic human subjects (8, 26). GSH levels were normalized to the levels of secretory IgA in the lavage fluid as we have reported previously (8). In our experience, ethanol ingestion does not affect secretory IgA levels in the lung lavage fluid, and there is less variability with this correction than with the urea dilution method we used in our original study in the rat model (17).
Induction of endotoxemia in vivo.
In selected experiments, rats were given endotoxin (Salmonella typhimurium lipopolysaccharide, 2 mg/kg intraperitoneally) 2 h before either determination of lung liquid clearance in vivo or determination of TGF-1 and total protein concentrations in lung lavage fluid.
Lung tissue preparation for determination of TGF-1.
In selected experiments, rats were anesthetized with pentobarbital sodium (60 mg/kg intraperitoneally), and a tracheostomy cannula was placed and secured with a 2–0 ligature. The chest cavity was opened, and 100 units of heparin were injected into the right ventricle and allowed to circulate for 1 min. A perfusion catheter was placed in the pulmonary outflow tract, and, after transection of the left atrium, the lungs were perfused blood-free with saline, excised free from other tissues, and stored at –70°C for later analysis of TGF-1 protein levels by ELISA.
ELISA for determination of TGF-1 protein levels.
For the tissue TGF-1 assays, frozen rat lung tissue (0.5 g) was combined with 2 ml of cold acid-ethanol (93% ethanol + 2% concentrated HCl), 85 µg/ml PMSF, and 5 µg/ml pepstatin A. This mixture was then homogenized for 1–2 min with a polytron homogenizer. The samples were extracted overnight at 4°C by gentle rocking followed by centrifugation at 10,000 g for 10 min. The pellets were then discarded, and the supernatants were dialyzed against 4 mM HCl using 3.5 kDa cut-off dialysis tubing. Samples were again centrifuged at 13,000 g for 10 min, and the supernatants were stored at –70°C until assayed. This preparation by necessity activates any latent TGF-1 in the tissue because the commercial ELISA kit employed only detects the active form of TGF-1; therefore, the measured levels represent total TGF-1 in the tissue (latent + active). Levels of TGF-1 in the lung lavage fluid and the prepared lung tissue were determined with a commercial ELISA kit (R&D Systems, Minneapolis, MN). Absorbance was read at 450 nm quantitated against a standard curve. The amount of TGF-1 in the lung lavage fluid and lung tissue was then expressed per milligram of protein in each sample. For the lung lavage fluid TGF-1 assays, the lavage supernatants were not acidified before performing the ELISA, as we had previously determined that TGF-1 protein levels in the lavage fluid as determined by the commercial assay were not affected by acidification, indicating that all of the TGF-1 protein released into the alveolar space was in the free or active form (4).
Determination of lung liquid clearance in vivo. As published previously (29), following induction of anesthesia, a tracheostomy cannula was placed, a saline challenge (2 ml) was given intratracheally, and the rats were mechanically ventilated with a Harvard rodent ventilator (tidal volume of 7 ml/kg at a rate of 60/min) for 30 min. The lungs were then removed en bloc, and the right lung was isolated and its bronchus was tied with a suture. The bronchus was then cut distal to the suture, and the right lung weight was determined at baseline (wet wt) and then after desiccation overnight at 70°C (dry wt). The ratio of the wet weight to the dry weight was calculated and expressed (wet:dry) for each experimental determination and used as a marker of lung liquid clearance. Specifically, lung liquid clearance was inversely proportional to the wet:dry ratio such that a relatively lower wet:dry ratio reflected a relatively greater lung liquid clearance and vice versa.
Determination of alveolar epithelial lining fluid protein.
After the right lung was isolated and its bronchus was ligated as described above, saline was instilled into the left lung (5 ml via the tracheostomy tube x3). The recovered lavage fluid (12 ± 1 ml in all cases) was centrifuged at 1,500 g for 10 min, and the supernatant was stored at –80°C for subsequent determinations of total protein and TGF-1 protein. Total protein levels in the lung lavage fluid were performed as we have published previously (29) using a bicinchoninic acid assay.
Alveolar epithelial cell isolation and formation of monolayers in vitro. As reported previously (5, 6, 16, 17), alveolar epithelial type II cells were isolated from control- and ethanol-fed rats and ethanol-fed rats whose diets had been supplemented with either lisinopril or losartan. Cells were resuspended at a density of 1 x 106 cells/ml of DMEM containing 10% serum, and 3 x 106 cells (3 ml) were plated on a 35-mm-diameter permeable microporous membrane (1-µm pore, Transwell, Corning) and cultured for a total of 8 days at 37°C in 90% air-10% CO2.
Determination of alveolar epithelial barrier function in vitro. The barrier function of the cell monolayers after 8 days in culture was determined as published previously (4) by adding [3H]inulin and [14C]sucrose (100,000 dpm) to the media covering the basolateral surfaces of the cultured cells. At multiple time intervals (15, 30, 60, or 120 min), the media covering the apical surfaces of the monolayers were removed, and the radioactivity was determined. Leak was defined as the fraction of the initial radioactivity placed on the basolateral surface that appeared on the apical surface of the monolayer after 120 min (2 h).
Statistical analysis. Values shown represent the means ± SE. Values were compared by analysis of variance and corrected by Student-Newman-Keuls test for differences between groups. A P value of <0.05 was considered significant.
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RESULTS |
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80% of the ethanol-induced GSH depletion was prevented by lisinopril treatment.
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1 protein in ethanol-fed rats.
Previously, we determined that chronic ethanol ingestion approximately doubles the expression of TGF-1 protein in the lung (4). However, the mechanism(s) by which ethanol induces TGF-1 expression was not addressed in that study. We hypothesized that TGF-1 expression was a consequence of angiotensin II-induced oxidative stress in the alcoholic lung. Therefore, in these experiments, we tested the effects of angiotensin II inhibitors, and, independently, glutathione supplementation, on TGF-1 expression in the lungs of ethanol-fed rats. As shown in Fig. 2, TGF-1 protein levels were increased (P < 0.05) in the lungs of ethanol-fed rats, as we have shown previously (4). In contrast, concomitant dietary treatment with the ACE inhibitor lisinopril or the angiotensin II type 1 receptor blocker losartan decreased TGF-1 protein levels in the lungs of ethanol-fed rats such that they were not different (P > 0.05) than levels in control-fed rat lungs (Fig. 2). In parallel, glutathione supplementation with procysteine in ethanol-fed rats also decreased lung TGF-1 expression, as reflected by TGF-1 protein levels that were also not different (P > 0.05) than those in control-fed rats (Fig. 2). Together, the results shown in Figs. 1 and 2 suggest that the renin-angiotensin system mediates lung oxidative stress during chronic ethanol ingestion, which in turn induces TGF-1 expression (see DISCUSSION).
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1 expression that characterize the alcoholic lung, including the ability of alveolar epithelial cells from ethanol-fed rats to form tight monolayers in vitro, we next examined the effects of angiotensin II inhibition on alveolar epithelial function in ethanol-fed rats in vivo. First, we determined that the baseline wet:dry ratios in control-fed and ethanol-fed rats that were not challenged with saline were identical (4.7 ± 0.7 vs. 4.7 ± 0.7). However, consistent with our previously published findings (16, 29), ethanol-fed rats had decreased (P < 0.05) lung liquid clearance in vivo compared with control-fed rats, as reflected by increased lung tissue wet:dry ratios (Fig. 4). In contrast, dietary treatment with either lisinopril or losartan improved lung liquid clearance in ethanol-fed rats in vivo to the same capacity (P > 0.05) as control-fed rats (Fig. 4).
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1 expression and liquid clearance in the lungs of ethanol-fed rats, we predicted that it would improve lung liquid clearance and decrease protein leak in ethanol-fed rats during endotoxemia. We first determined that lung liquid clearance was even more impaired (P < 0.05) in endotoxemic, ethanol-fed rats relative to lung liquid clearance in endotoxemic, control-fed rats (Fig. 5), whereas endotoxemia had no significant effect (P > 0.05) on the ability of control-fed rats to clear a lung liquid challenge (wet:dry ratios of 6.8 ± 0.4 for control-fed rats in Fig. 5 vs. 6.7 ± 0.2 for control-fed rats in Fig. 4). Importantly, treatment with either lisinopril or losartan improved (P < 0.05) lung liquid clearance in ethanol-fed rats, even following endotoxemia, such that their liquid clearance was the same (P > 0.05) as endotoxemic, control-fed rats (Fig. 5).
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1 and total protein accumulation in the alveolar air space during endotoxemia in vivo.
Previously, we determined that chronic ethanol ingestion induces TGF-1 expression in lung tissue and that it, during endotoxemia, markedly increases the release of activated TGF-1 into the alveolar space where it promotes epithelial permeability (4). Therefore, as angiotensin II appears to mediate glutathione depletion (Fig. 1) and consequent TGF-1 expression (Fig. 2), we predicted that lisinopril treatment would decrease the release of TGF-1 into the air space of ethanol-fed rats during endotoxemia. For these experiments, rats were made endotoxemic but were not subjected to a saline challenge. As shown in Fig. 6A, ethanol-fed rats released more than three times as much TGF-1 protein into the alveolar space during endotoxemia as control-fed rats (P < 0.05). In contrast, rats fed the ethanol diet supplemented with lisinopril released the same (P > 0.05) amount of TGF-1 protein into the alveolar space during endotoxemia as control-fed rats (Fig. 6A). As TGF-1 protein levels in the lavage fluids were corrected for total protein levels, we needed to ensure that the observed differences shown in Fig. 6A were relatively specific for TGF-1 and not simply a reflection of total protein accumulation in the air space. Therefore, we also determined total protein concentrations in the lung lavage fluids of these endotoxemic rats. Lung lavage fluid recovery was not significantly different among the treatment groups (not shown), so protein concentrations were expressed per volume of lavage fluid. As shown in Fig. 6B, total protein levels in the lavage fluids of endotoxemic, ethanol-fed rats were increased (P < 0.05) compared with endotoxemic, control-fed rats. However, this increase in total protein was modest (30%) and therefore could not account for the >300% increase in TGF-1 protein levels shown in Fig. 6A. In parallel with its effects on TGF-1 protein, lisinopril treatment decreased (P < 0.05) total protein levels in the alveolar space in endotoxemic, ethanol-fed rats (Fig. 6B). Overall, the results in Fig. 6 are consistent with the results in Fig. 2 and suggest that angiotensin II not only induces the expression of TGF-1 protein in the alcoholic lung but ultimately leads to changes in the lung that promote the release of TGF-1 protein into the alveolar air space during an acute inflammatory stress such as endotoxemia.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1 expression in ethanol-induced alveolar epithelial dysfunction, and this current study unifies these pathophysiological pathways by revealing a common mechanism by which ethanol ingestion produces oxidative stress and induces TGF-1 expression within the lung. Specifically, we determined that dietary treatment with the ACE inhibitor lisinopril, which blocks ACE-dependent formation of angiotensin II, almost completely preserved glutathione levels within the alveolar space of ethanol-fed rats and prevented excess TGF-1 expression in the lung tissue. In parallel, losartan, a specific angiotensin II type 1 receptor blocker, also normalized ethanol-induced TGF-1 expression, further evidence that this induction is mediated by angiotensin II and not by some other product of ACE activity. In addition, dietary treatment with the glutathione precursor procysteine normalized TGF-1 expression in the lung tissue, suggesting that angiotensin II-induced glutathione depletion is required for angiotensin II-mediated induction of TGF-1. Furthermore, ACE inhibition decreased the release of TGF-1 into the alveolar space during endotoxemia, which could reflect either less TGF-1 in the lung tissue or some other barrier-protective effect of ACE inhibition in the alcoholic lung. Finally, and perhaps most importantly, treatment with either lisinopril or losartan preserved alveolar epithelial barrier function in the alcoholic lung both at baseline and following endotoxemia, as reflected by lung liquid clearance of an intratracheal saline challenge. Together, this study provides new evidence that angiotensin II mediates glutathione depletion, increased TGF-1 expression, and alveolar epithelial barrier dysfunction in the alcoholic lung. Our surrogate marker of alveolar epithelial barrier function in vivo, namely lung liquid clearance of an intratracheal saline challenge, is an integrated function that depends on active sodium transport as well as a relatively impermeable epithelium that prevents back leak of the reabsorbed fluid. Our previous work suggests that the primary defect in the alcoholic lung is increased permeability of the epithelium, whereas active sodium transport may actually be increased as a compensatory response (16). Therefore, we have used lung liquid clearance as an index of alveolar epithelial barrier function with or without endotoxemia in vivo in a more recent study (29). This is a relatively simple and reproducible index of epithelial barrier function that correlates with our previous studies in which alveolar fluid clearance and protein flux in vivo were measured with radiolabeled albumin (16). We cannot exclude the possibility that ethanol ingestion also perturbed lung lymphatic drainage and/or cardiovascular function in response to the intratracheal saline challenge and that angiotensin II inhibition somehow reversed such defects. To our knowledge, these potential mechanisms have not been examined in comparable models. However, the evidence from this study and our previous work strongly argues that ethanol-mediated susceptibility to acute edematous injury involves alveolar epithelial dysfunction.
Alcohol abuse increases the risk of developing ARDS more than threefold during septic shock (27). This epidemiological association, which was first identified less than a decade ago (25), established alcohol abuse as the first comorbid factor identified (and to date, the only factor) that independently increases the risk of ARDS. To study the mechanisms underlying this association, we developed a rat model of ethanol-induced susceptibility to acute lung injury (17, 33) and determined that within 4–6 wk of chronic ethanol ingestion, the lung shows signs of significant oxidative stress as reflected by profound depletion of the antioxidant glutathione within the alveolar epithelium and associated lining fluid (5, 17). In parallel, alveolar epithelial function, as reflected by barrier function and active fluid transport, is impaired by chronic ethanol ingestion (16). Importantly, dietary supplementation with glutathione precursors prevents glutathione depletion as well as alveolar epithelial dysfunction in ethanol-fed rats (15–17). Recently, we determined that ethanol-induced glutathione depletion is associated with a twofold increase in the expression of TGF-1 in the lung tissue (4). However, it remains predominantly (but not completely) in a latent or inactive form until an acute stress, such as endotoxemia, releases and activates TGF-1 within the alveolar space where it is capable of inducing an acute permeability defect in the alveolar epithelium (4). In this study, lisinopril prevented the exaggerated release of activated TGF-1 that is associated with (and may contribute to) impaired alveolar epithelial barrier function in the alcoholic lung during endotoxemia. Together, these findings argue that ethanol-induced oxidative stress and TGF-1 expression are mediated not by ethanol directly, but rather indirectly, through the actions of angiotensin II. Furthermore, when combined with our previous studies, our current findings suggest the following sequence
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If this scheme is correct, it still leaves open the question as to whether TGF-1 contributes to the alveolar epithelial defects that characterize the chronic alcoholic lung in addition to mediating acute epithelial barrier disruption during sepsis. Although we did not detect TGF-1 in the lung lavage fluid of ethanol-fed rats in the absence of endotoxemia (4), we cannot exclude the possibility that low-level but chronic activation of TGF-1 in the adjoining matrix space interferes with the normally tight alveolar epithelium. Alternatively, angiotensin II could perturb alveolar epithelial barrier function by a mechanism(s) independent of TGF-1, and the consequences of the excess TGF-1 expression may only be relevant in the context of sepsis or other acute inflammatory stresses that release and activate TGF-1 to pathophysiological levels. Regardless, this scheme provides a framework to design future studies and is consistent with other evidence that the pathophysiological consequences of chronic ethanol ingestion could be mediated at least in part by the renin-angiotensin system. Angiotensin II is a pluripotent vasoactive peptide that is increased in the lungs of individuals with ARDS (38). It is formed by the sequential conversion of angiotensinogen to angiotensin I and then to angiotensin II, the latter conversion catalyzed primarily, although not exclusively, by the ACE. Chronic ethanol ingestion increases plasma levels of angiotensin II in rats (40), and it has been postulated that activation of the renin-angiotensin system may explain the association between alcohol abuse and hypertension in humans (39, 40). Although a mechanism is not known, it has been shown that acetaldehyde, the primary metabolite of ethanol, can convert angiotensinogen to angiotensin I in rat plasma in vitro (32). The biological effects of angiotensin II depend on its interaction with specific angiotensin II receptors, and at least seven subtypes have been identified. Of the angiotensin II receptors, the type 1 receptor has been best characterized. The majority of the well-known effects of angiotensin II, such as vasoconstriction, sodium retention, and tissue hypertrophy and hyperplasia, are mediated via the type 1 receptor (2). The type 1 receptor blockers, including losartan (used in this experimental study), are now in widespread clinical use in the treatment of cardiovascular diseases (9). Cellular responses to angiotensin II are remarkably diverse and include activation of the NADPH oxidase complex and generation of reactive oxygen species (41). As noted earlier, angiotensin II induces apoptosis in human and rat type II cell apoptosis in vitro (34–36) and causes alveolar epithelial cell injury in rabbits in vivo (13). It is important to emphasize that most studies, including ours, rely on the use of ACE inhibitors and/or angiotensin II receptor blockers to indirectly evaluate the pathophysiological effects of angiotensin II. This is in part due to the fact that it is relatively difficult to measure angiotensin II levels, particularly in relevant microenvironments such as the pulmonary interstitium. Furthermore, the actions of angiotensin II depend not only on ambient levels but also on the relative expression of its receptor subtypes in target tissues. For example, we determined previously that chronic ethanol ingestion shifts the expression of angiotensin II receptors from predominantly the type 1 to the type 2 receptor on the membranes of alveolar epithelial type II cells and that this is associated with a proapoptotic phenotype in these cells (3). Interestingly, there is evolving evidence that some of the beneficial effects of ACE inhibitors can be attributed to alterations in ACE signaling that are independent of angiotensin II formation per se (12). However, we used both an ACE inhibitor, and, in the experiments in which lung tissue TGF-1 expression and lung liquid clearance were determined, an AT1 receptor inhibitor as well. As the effects of lisinopril and losartan were comparable, these findings argue that ethanol-mediated lung dysfunction is yet another circumstance in which angiotensin II plays a pathophysiological role.
In summary, we report that ethanol-induced glutathione depletion and TGF-1 expression in the lungs of ethanol-fed rats are mediated by the renin-angiotensin system, most likely by the actions of angiotensin II. The functional consequences of this angiotensin II activity include decreased alveolar epithelial barrier function that is exacerbated during an acute inflammatory stress such as endotoxemia. Therefore, although the metabolic consequences of chronic ethanol ingestion are quite complex, ethanol-induced amplification of the renin-angiotensin system appears to be the major cause of the alveolar epithelial oxidative stress and TGF-1-mediated barrier disruption that characterize the alcoholic lung. These observations provide a previously unrecognized mechanism by which chronic ethanol ingestion renders the lung susceptible to acute edematous injury. The potential impact of these findings on our understanding of the pathophysiology of acute lung injury in the setting of alcohol abuse is enormous. Pharmacological blockade of the actions of angiotensin II in other conditions, such as congestive heart failure and glomerular diseases, has dramatically improved survival in many individuals. Clearly, additional studies are necessary before we can place alcohol-mediated susceptibility to acute lung injury on the list of serious human diseases for which inhibition of the renin-angiotensin system is a therapeutic target.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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