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Am J Physiol Lung Cell Mol Physiol 293: L859-L869, 2007. First published July 6, 2007; doi:10.1152/ajplung.00008.2007
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EDITORIAL FOCUS

Nitric oxide-dependent inhibition of alveolar fluid clearance in hydrostatic lung edema

Stephanie M. Kaestle,1 Christian A. Reich,2 Ning Yin,1 Helmut Habazettl,1,3 Jorg Weimann,2,4 and Wolfgang M. Kuebler1,3

1Institute of Physiology and 2Department of Anesthesiology and Intensive Care Medicine, Charité-Universitaetsmedizin Berlin; 3Department of Anesthesiology, German Heart Institute Berlin, Germany; and 4Department of Anesthesiology, Vrije University Medical Center, Amsterdam, The Netherlands

Submitted 5 January 2007 ; accepted in final form 2 July 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Formation of cardiogenic pulmonary edema in acute left heart failure is traditionally attributed to increased fluid filtration from pulmonary capillaries and subsequent alveolar flooding. Here, we demonstrate that hydrostatic edema formation at moderately elevated vascular pressures is predominantly caused by an inhibition of alveolar fluid reabsorption, which is mediated by endothelial-derived nitric oxide (NO). In isolated rat lungs, we quantified fluid fluxes into and out of the alveolar space and endothelial NO production by a two-compartmental double-indicator dilution technique and in situ fluorescence imaging, respectively. Elevation of hydrostatic pressure induced Ca2+-dependent endothelial NO production and caused a net fluid shift into the alveolar space, which was predominantly attributable to impaired fluid reabsorption. Inhibition of NO production or soluble guanylate cyclase reconstituted alveolar fluid reabsorption, whereas fluid clearance was blocked by exogenous NO donors or cGMP analogs. In isolated mouse lungs, hydrostatic edema formation was attenuated by NO synthase inhibition. Similarly, edema formation was decreased in isolated mouse lungs of endothelial NO synthase-deficient mice. Chronic heart failure results in endothelial dysfunction and preservation of alveolar fluid reabsorption. These findings identify impaired alveolar fluid clearance as an important mechanism in the pathogenesis of hydrostatic lung edema. This effect is mediated by endothelial-derived NO acting as an intercompartmental signaling molecule at the alveolo-capillary barrier.

alveolar fluid reabsorption; epithelial sodium channel; alveolar epithelium; congestive heart failure

HYDROSTATIC LUNG EDEMA results from increased fluid filtration across the lung microvascular barrier due to elevated hydrostatic or reduced oncotic pressures in lung microvessels (18). Initially, the edema is confined to the interstitial space and limited by the powerful lymphatic drainage (39), a low interstitial compliance (38, 39), and the tight alveolar membrane (50). When these protective mechanisms are finally exceeded, fluid enters the air space, resulting in alveolar flooding and impaired gas exchange (5).

Alveolar fluid influx is counteracted by alveolar fluid clearance, an active reabsorption process in the alveolar space and the distal airways by which epithelial cells remove fluid back into the pulmonary interstitium and vasculature. Epithelial reabsorption of alveolar fluid is chiefly attributable to active Na+ transport via Na+ transporters in the apical plasma membrane and the basolaterally located Na+-K+-ATPase (24, 34). In patients with severe hydrostatic lung edema, alveolar fluid reabsorption was found to be impaired or submaximal in 62% of cases, indicating that impaired fluid clearance may contribute pivotally to edema formation (52). Since endothelial cells are the primary sensors of hemodynamic forces in lung capillaries, this finding furthermore suggests the existence of intercellular signaling mechanisms that communicate the hydrostatic stress from the capillary to the alveolar compartment and limit the epithelial fluid reabsorption capacity.

Endothelial-derived nitric oxide (NO) may constitute such a potential link between lung hydrostatic stress and alveolar fluid reabsorption. Recently, we reported that elevation of lung capillary pressure activates endothelial NO synthase (eNOS) via a phosphatidylinositol-3-OH kinase (PI3K)-dependent mechanism (29). At the thin blood-gas barrier, endothelial-derived NO can rapidly diffuse to adjacent alveolar epithelial cells and thus serve as an intercompartmental cross-talk signal. Of note, NO has been shown to modulate active Na+ transport in alveolar type II epithelial cells, but both stimulation and inhibition of transport characteristics have been reported depending on the amount of NO and the period of exposure. On the one hand, exogenous NO donors or cyclic GMP (cGMP) analogs reduce the opening probability of the apical Na+-permeant cation channel in isolated type II pneumocytes (27), decrease Na+ transport across confluent type II monolayers (17), and reduce amiloride-sensitive fluid clearance in vivo (41). Transgenic lack or inhibition of the inducible NO synthase (iNOS), on the other hand, also reduces amiloride-sensitive Na+ transport and fluid clearance (19, 20), presumably via a posttranscriptional downregulation of the {alpha}- and {gamma}-subunits of the epithelial Na+ channel (20).

Chronic congestive heart failure (CHF) induces lung endothelial dysfunction (40) and thus reduces NO bioavailability in the air spaces (1, 49). Notably, CHF patients are also protected from hydrostatic lung edema (11, 12), and regulation of fluid reabsorption by NO may establish a causative link between both findings.

Here, we investigated the effect of hydrostatic stress in lung capillaries on alveolar fluid reabsorption and its mediation by NO in isolated mouse and rat lungs and in lungs from rats with CHF. Our findings identify a novel cross talk between the capillary and the alveolar compartment that may constitute an important mechanism in the pathogenesis of hydrostatic lung edema.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male Sprague-Dawley rats of 348 ± 5 g body wt and male C57BL/6 (wild-type) and eNOS knockout mice (eNOS–/–) of identical background (all 25 ± 1 g body wt) were obtained from Charles River Laboratories (Charles River Wiga, Sulzfeld, Germany). All experiments were performed in accordance with the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health publication no. 86-23, rev. 1985). The study was approved by the animal care and use committee of the local government authorities.

Fluorescent probes and drugs. Texas red dextran (TRD; 70 kDa) and the NO-sensitive dye 4-amino-5-methylamino-2'-7'-difluorofluorescein diacetate (DAF-FM DA) were obtained from Molecular Probes (Eugene, OR). 8-BrcGMP was from Calbiochem (Bad-Soden, Germany). All other probes and chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany).

Supracoronary aortic banding. CHF was induced by supracoronary aortic banding 8 wk before investigations as previously described (22). In brief, juvenile male Sprague-Dawley rats (90 ± 10 g body wt) were anesthetized by intraperitoneal injections of ketamine (87 mg/kg body wt) and xylazine (13 mg/kg body wt). Following left infraclavicular hemithoracotomy, the supracoronary aorta was banded by implantation of a titanium clip with an internal diameter of 0.8 mm (Weck Closure Systems, Research Triangle Park, NC). Wounds were surgically closed, and postoperative analgesia was provided with 4 mg/kg body wt caprofen subcutaneously (Rimadyl; Pfizer, Karlsruhe, Germany) for 3 days.

Isolated perfused lung preparations. Experimental procedures have been described previously (7, 48). For isolated perfusion, lungs from anesthetized Sprague-Dawley rats were continuously pump-perfused at 14 ml/min and 37°C. Perfusate was either autologous heparinized blood (for fluorescence imaging experiments) or 100 ml of BSA solution (for 2-compartmental double-indicator dilution technique) consisting of 137 mmol/l NaCl, 1 mmol/l CaCl2, 2.68 mmol/l KCl, 1 mmol/l MgCl2, and 3% BSA at pH 7.4 and osmolarity of 300 ± 5 mOsmol/l. Lungs were constantly inflated with a gas mixture of 21% O2, 74% N2, and 5% CO2 at a positive airway pressure of 5 cmH2O. At baseline, left atrial pressure (PLA) was adjusted to 5 cmH2O yielding pulmonary artery pressures (PPA) of 12 ± 1 cmH2O. PPA and PLA were recorded continuously (DASYlab 32; Datalog, Moenchengladbach, Germany).

Lungs of anesthetized mice were excised, placed in a 37°C water-jacketed chamber (Type 839; Hugo Sachs Elektronik, March-Hugstetten, Germany), and constantly inflated with 21% O2, 5% CO2, and 74% N2 at 5 cmH2O. Lungs were continuously pump-perfused at 1.25 ml/min with BSA solution in a nonrecirculating system at 37°C and a baseline PLA of 2 cmH2O.

In situ fluorescence imaging of endothelial NO production. Isolated perfused rat lungs were positioned under an upright microscope (Axiotechvario 100HD; Zeiss, Jena, Germany) on a custom-built stage and superfused with normal saline at 37°C to prevent drying or cooling of the lung surface. A microcatheter (Ref. 800/110/100; SIMS Portex, Kent, UK) was advanced via the left atrium and wedged in a pulmonary vein draining a capillary area on the lung surface. Via the microcatheter, membrane-permeant DAF-FM DA (5 µmol/l), which deesterifies intracellularly to cell-impermeant, NO-sensitive DAF-FM, was infused for 20 min as previously described (29). Weakly fluorescent DAF-FM is converted in a NO-dependent, irreversible reaction to an intensely fluorescent benzotriazole derivative with fluorescence intensity linearly reflecting NO concentration (26). Fluorescence of DAF-FM-loaded endothelial cells was excited at 480 nm by monochromatic illumination (Polychrome IV; T.I.L.L. Photonics, Martinsried, Germany), collected through an approchromat objective (UAPO x40 W2/340; Olympus, Hamburg, Germany) and dichroic and emission filters (FT 510, LP520; Zeiss) by a charge-coupled device camera (Sensicam; PCO, Kehlheim, Germany), and subjected to digital image analysis (TILLvisION 4.01; T.I.L.L. Photonics). Single venular capillaries were viewed at a focal plane corresponding to maximum diameter (14–30 µm). Exposure time for each single image was limited to 5 ms. Fluorescence images obtained in 10-s intervals were background-corrected, and fluorescence intensity (F) was expressed relative to its individual baseline (F0). Since the conversion of DAF-FM to the benzotriazole derivative is irreversible, the ratio F/F0 reflects NO production integrated over time.

Two-compartmental double-indicator dilution technique. Fluid fluxes into and out of the alveolar space were quantified by a double-indicator dilution technique. A high-molecular-weight fluorescence tracer, TRD, was instilled into the alveolar space for determination of alveolar net fluid shift while a low-molecular-weight tracer, Na+ fluorescein (NaF), was added to the perfusate to allow for differentiation between alveolar fluid influx and alveolar fluid reabsorption. At time –10 min, 0 min, and 60 min, samples were drawn from both compartments, and alveolar fluid reabsorption, alveolar fluid influx, and net fluid shift were calculated assuming a two-compartmental model. Physiological assumptions and mathematical algorithms underlying this model are presented and discussed in an online supplement to this article, available online at the AJP-Lung web site.

Experimental protocols. 1) PLA elevation in rats: in fluorescence imaging experiments, baseline recordings were obtained at PLA of 5 cmH2O over 15 min. Then, PLA was raised to 15 cmH2O for 30 min by adjusting the height of the venous outflow reservoir. In double-indicator dilution studies, lungs were perfused at baseline PLA of 5 cmH2O from time–10 to time 0. At time 0, PLA was raised to 10 or 15 cmH2O, respectively, for 60 min. 2) PLA elevation in mice: to avoid stress failure of lung capillaries, PLA levels were adapted to the lower hydrostatic pressure gradients in mouse lungs. Baseline pressure was set to 2 cmH2O as previously described (48), and hydrostatic stress was applied by tripling this value, i.e., by elevation of PLA to 6 cmH2O for 25 min. Lung wet/dry weight ratios were determined by use of the microwave drying technique (44). 3) Loss of compartmentalization: in isolated-perfused rat lungs, decompartmentalization at the alveolo-capillary membrane was induced by three to five consecutive ventilatory cycles at peak inspiratory pressures of >30 cmH2O. 4) Ca2+-free and Ca2+-rich conditions: isolated rat lungs were constantly perfused with BSA solution containing nominally zero or 1.0 mmol/l Ca2+, respectively. 5) Pharmacological interventions: the NO synthase inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME; 250 µmol/l), the PI3K inhibitor LY-294002 (10 µmol/l), and the exogenous NO donors S-nitroso glutathione (GSNO; 100 µmol/l) and (Z)-1-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate (papa NONOate; 100 µmol/l), respectively, were added to the perfusate. The soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazoloquinoxalin-1-one (ODQ; 100 µmol/l), the cGMP analog 8-BrcGMP (1 mmol/l), L-NAME (250 µmol/l), the sodium channel inhibitor amiloride (10 µmol/l), the Na+-K+-ATPase inhibitor ouabain (100 µmol/l), and dopamine (100 µmol/l) were administered with the alveolar tracer solution into the air space.

Statistical analysis. All data are given as means ± SE. Different groups were compared by the Mann-Whitney U-test by using SigmaStat software (SigmaStat 3.10; Systat Software, Erkrath, Germany). Linear and nonlinear regression analyses were performed by using GraphPad Prism software (version 4.0; GraphPad Software, San Diego, CA). Statistical significance was assumed at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Two-compartmental double-indicator dilution technique. A double-indicator dilution technique was used to differentiate between alveolar fluid influx and reabsorption kinetics in the isolated perfused rat lung. The model is based on the assumption that the distribution of the high-molecular-weight tracer TRD is confined to the alveolar compartment, whereas the low-molecular-weight tracer NaF reflects fluid fluxes into and out of the alveolar space due to its rapid convective transport. Within the investigated hydrostatic pressure range of 5–15 cmH2O, TRD was strictly confined to the alveolar compartment as demonstrated by its absence in the perfusate (Fig. 1 A). In contrast, decompartmentalization at the alveolo-capillary barrier resulted in a marked translocation of the dye into the vascular space independent of the applied vascular pressure (Fig. 1A).


Figure 1
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  		Fig. 1. Pressure-dependent alveolar fluid shifts determined by 2-compartmental double-indicator dilution technique in isolated-perfused rat lungs. A: in intact lungs ({circ}), Texas red dextran (TRD) is not detectable in the perfusate at left atrial pressure (PLA) levels of 5–15 cmH2O. After decompartmentalization of the alveolo-capillary barrier, TRD fluorescence is markedly elevated (bullet). Fluorescence intensities are expressed as difference from baseline. N = 4 each, *P < 0.05 vs. controls without decompartmentalization. B and C: group data of alveolar fluid reabsorption (circles), alveolar fluid influx (triangles), and net fluid shift (squares) determined at PLA of 5, 10, and 15 cmH2O in isolated perfused rat lungs by 2-compartmental double-indicator dilution technique. B: alveolar fluid reabsorption decreased exponentially with higher PLA as described by the curve fit y = 1.4 – 0.025 x e0.32 x (r2 >0.999), whereas alveolar fluid influx increased linearly with 0.61 ml/h per 5 cmH2O (r2 = 0.998). The difference between fluid influx and fluid reabsorption (gray area, B) reflects the net fluid shift into the alveolar space (C). C: increase of net fluid shift with elevation of PLA is described by the exponential curve fit y = –0.73 + 0.11 x e0.25 x (r2 >0.999). All data are n = 7 each, *P < 0.05 vs. PLA = 5 cmH2O.

 
Pressure-dependent fluid shifts across the alveolo-capillary barrier. In isolated perfused rat lungs, the net fluid shift into the alveolar space was determined from the kinetics of the alveolar TRD concentration. At PLA of 5 cmH2O, net fluid shift was close to 0 ml/h (Fig. 1C) since fluid influx into and fluid reabsorption out of the alveolar space were in balance (Fig. 1B). With higher PLA, net fluid shift increased exponentially indicating progressive alveolar flooding (Fig. 1C). This effect was not only attributable to increased fluid filtration but also to a marked reduction in alveolar fluid reabsorption. Although alveolar fluid influx increased with higher PLA, yet without reaching significance (P < 0.09), this effect contributed only ~30% to the total increase in net fluid shift. The predominant cause of the net fluid shift was an exponential decrease of alveolar fluid reabsorption that even became negative at PLA of 15 cmH2O.

Endothelial-derived NO regulates alveolar fluid reabsorption. To investigate the role of NO in hydrostatic edema formation, we determined wet/dry weight ratios in isolated mouse lungs following 25 min of perfusion at control PLA of 2 cmH2O or at elevated PLA of 6 cmH2O. Whereas PLA elevation resulted in massive edema formation in control lungs as indicated by a 2.5-fold increase in wet/dry weight ratios, this effect was markedly attenuated in the presence of the NO synthase inhibitor L-NAME (Fig. 2). A similar reduction in wet/dry weight ratios was detected in mice deficient of endothelial NO synthase. Capillary pressures increased identically in control and treatment groups (Fig. 2B), suggesting that differences in hydrostatic edema formation were attributable to NO production by eNOS. Neither L-NAME nor eNOS deficiency had an effect on lung wet/dry weight ratio at physiological PLA of 2 cmH2O.


Figure 2
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  		Fig. 2. Lung wet/dry weight ratios (A) and calculated capillary pressures (Pcap; B) in wild-type control mice, wild-type mice treated with L-NAME, and eNOS–/– mice determined after 25-min isolated lung perfusion at either baseline PLA of 2 cmH2O (filled bars) or at elevated PLA of 6 cmH2O (open bars), respectively. All data are n = 4 each, *P < 0.05 vs. baseline PLA, #P < 0.05 vs. wild-type control.

 
The finding that eNOS-derived NO causes lung edema at increased hydrostatic pressure, but not at baseline PLA, suggests that eNOS activity may be pressure-regulated. As demonstrated by stable DAF-FM fluorescence in isolated perfused rat lungs (Fig. 3, A and B), endothelial NO production was below the detection limit at baseline PLA. Elevation of PLA increased DAF-FM fluorescence in the capillary wall, which was blocked by L-NAME (Fig. 3, A and B). As we discuss later, this finding indicates pressure-induced endothelial NO formation that coincided with impaired alveolar fluid reabsorption.


Figure 3
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  		Fig. 3. Nitric oxide (NO) dependence of alveolar fluid reabsorption. A and B: capillary NO response to increased vascular pressure as determined by in situ fluorescence imaging. A: representative images of 4-amino-5-methylamino-2'-7'-difluorofluorescein (DAF-FM)-loaded endothelial cells in situ show pressure-induced endothelial NO production in lung venular capillaries. Images were obtained at baseline PLA of 5 cmH2O after 0 min (left) and 15 min (middle), and after 30 min of PLA elevation to 15 cmH2O (right). Vessel margins are shown by dotted lines. Note increased endothelial fluorescence at elevated PLA. B: representative fluorescence profile in a single DAF-FM-loaded endothelial cell of a lung venular capillary. DAF-FM fluorescence intensity was determined over 15 min at baseline PLA and during 30 min of PLA elevation to 15 cmH2O. Fluorescence intensity (F) is expressed relative to its individual baseline (F0). Increase of DAF-FM fluorescence at elevated PLA was blocked by L-NAME. Replicated in n = 5 each. C and D: alveolar fluid fluxes determined by the 2-compartmental double-indicator dilution technique. Both S-nitroso glutathione (GSNO) and papa NONOate inhibited alveolar fluid reabsorption at baseline PLA but had no significant effect on alveolar fluid influx (C). Inhibition of NO synthase by addition of L-NAME to the perfusate reconstituted alveolar fluid reabsorption at elevated PLA of 15 cmH2O but did not affect alveolar fluid influx (D). Effects after alveolar instillation of L-NAME were less pronounced (gray bars). All data are n = 7 each, *P < 0.05 vs. control.

 
Next, we studied the effects of NO on alveolar fluid reabsorption in the isolated perfused rat lung. At baseline PLA, lung perfusion with the exogenous NO donors GSNO or papa NONOate, respectively, completely blocked alveolar fluid reabsorption, but had no significant effect on alveolar fluid influx (Fig. 3C). At PLA of 15 cmH2O, lung perfusion with L-NAME fully reconstituted alveolar fluid reabsorption to baseline values (Fig. 3D). The recovery of alveolar fluid clearance was less pronounced when L-NAME was administered via the alveolar space.

Activation of eNOS is regulated by Ca2+/calmodulin signaling (15) and by phosphorylation of its Ser1177 residue via the PI3K/Akt pathway (13). To further analyze the role of eNOS and its upstream regulation in the control of alveolar fluid reabsorption, we blocked endothelial Ca2+ signaling or PI3K by perfusing lungs with either Ca2+-free solution or the inhibitor LY-294002, respectively, as previously described (29, 30). Ca2+-free perfusion completely blocked the pressure-induced formation of NO in lung capillary endothelial cells (Fig. 4A), whereas perfusion with a Ca2+-rich buffer had no inhibitory effect (Fig. 4B). In lungs perfused with either Ca2+-free solution or the PI3K inhibitor LY-294002, intact alveolar fluid reabsorption was preserved at elevated PLA of 15 cmH2O, whereas no significant effect on alveolar fluid influx was detectable (Fig. 4C). These findings support the notion that pressure-induced Ca2+- and PI3K-dependent endothelial NO production promotes hydrostatic edema formation by impairing alveolar fluid reabsorption.


Figure 4
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  		Fig. 4. Effects of upstream inhibition of endothelial NO production or blood perfusion on alveolar fluid reabsorption. A and B: capillary NO response to increased vascular pressure in lungs perfused with Ca2+-free buffer as determined by in situ fluorescence imaging. A: representative fluorescence images of DAF-FM-loaded endothelial cells obtained at baseline PLA and after 30 min of PLA elevation to 15 cmH2O in Ca2+-free perfused isolated rat lungs show lack of NO response. B: representative fluorescence tracings of single endothelial cells in lungs perfused with Ca2+-rich and Ca2+-free buffer during 15 min at baseline PLA and over 30 min of pressure elevation to 15 cmH2O. Fluorescence intensity (F) is expressed relative to its individual baseline (F0). Replicated in n = 5 each. C and D: alveolar fluid fluxes determined by the 2-compartmental double-indicator dilution technique. Perfusion of isolated lungs with Ca2+-free buffer or with the PI3K inhibitor LY-294002 increased alveolar fluid reabsorption at elevated PLA of 15 cmH2O but did not change alveolar fluid influx (C). In blood-perfused isolated lungs, PLA elevation continues to inhibit alveolar fluid reabsorption, yet with a lesser attenuation compared with buffer-perfused lungs (D). All data are n = 7 each, *P < 0.05 vs. control.

 
Since oxyhemoglobin within red blood cells is a potent endogenous scavenger of NO and may thus limit or even neutralize the effects of endothelial-derived NO in vivo, we determined the effects of hydrostatic stress on alveolar fluid reabsorption in lungs perfused with autologous blood. In the presence of red blood cells, PLA elevation again reduced alveolar fluid reabsorption, yet to a lesser extent compared with buffer-perfused rat lungs (Fig. 4D). These data are in line with the finding that inhibition of alveolar fluid reabsorption was NO dependent and extend the relevance of this mechanism to the blood-perfused lung.

To mimic downstream signaling effects of endothelial-derived NO in intact alveolar epithelium, we instilled lungs with the cell-permeable cGMP-analog 8-BrcGMP. At baseline PLA of 5 cmH2O, 8-BrcGMP reduced alveolar fluid clearance, thus evoking a similar response as PLA elevation (Fig. 5A). In contrast, inhibition of cGMP formation by the soluble guanylate cyclase inhibitor ODQ rescued intact alveolar fluid clearance at elevated PLA (Fig. 5B). A similar preservation of fluid clearance was achieved by alveolar instillation of dopamine (Fig. 5C), which increases epithelial Na+ transport, presumably by increasing basolateral surface expression of Na+-K+-ATPase (4).


Figure 5
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  		Fig. 5. Regulation of alveolar fluid reabsorption determined by the 2-compartmental double-indicator dilution technique. Alveolar instillation of the cGMP analog 8-BrcGMP at baseline PLA of 5 cmH2O reduced alveolar fluid reabsorption (A). Alveolar instillation of the soluble guanylate cyclase inhibitor ODQ (B) or the catecholamine dopamine (C) preserved alveolar fluid reabsorption at elevated PLA of 15 cmH2O. None of these pharmacological interventions affected alveolar fluid influx. All data are n = 7 each, *P < 0.05 vs. control.

 
To further analyze the mechanisms by which NO may impair alveolar fluid reabsorption, we assessed 1) whether inhibitors of the apical Na+ channel or the basolateral Na+-K+-ATPase may mimic the effects of hydrostatic stress and 2) whether these blockers may evoke an additive inhibitory effect on alveolar fluid reabsorption at elevated PLA. At baseline PLA of 5 cmH2O, both the Na+-K+-ATPase inhibitor ouabain and the Na+ channel blocker amiloride reduced alveolar fluid reabsorption to or even below ~0 ml/h, respectively (Fig. 6A). At elevated PLA of 15 cmH2O, amiloride did not further reduce alveolar fluid reabsorption, whereas ouabain reset fluid clearance to ~0 ml/h, thus effectively increasing it compared with control values (Fig. 6B). As we discuss later, this finding indicates that hydrostatic stress impairs fluid reabsorption by inhibition of apical Na+ channels, whereas the Na+-K+- ATPase contributes to active alveolar fluid secretion during hydrostatic stress.


Figure 6
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  		Fig. 6. NO blocks amiloride-sensitive alveolar fluid reabsorption. At baseline PLA of 5 cmH2O, alveolar instillation of the Na+ channel inhibitor amiloride or the Na+-K+- ATPase inhibitor ouabain blocked alveolar fluid reabsorption (A). At elevated PLA of 15 cmH2O, amiloride had no further effect on alveolar fluid reabsorption, indicating that amiloride-sensitive ion channels were already blocked. In contrast, ouabain reset alveolar fluid clearance to ~0 ml/h by blocking both absorption and secretion pathways (B). Alveolar fluid influx was not significantly affected by the inhibitors. All data are n = 7 each, *P < 0.05 vs. control.

 
Alveolar fluid reabsorption in CHF. To elucidate mechanisms contributing to the partial protection of CHF patients against hydrostatic lung edema, we studied alveolar fluid reabsorption in a supracoronary aortic banding model of CHF in rats. Functional fluorescence imaging of DAF-FM-loaded endothelial cells revealed that the fluorescence response to PLA elevation is abrogated in lungs of CHF rats (Fig. 7A). Since exogenous NO donors still evoked a similar fluorescence signal as in controls (data not shown), this finding demonstrates lack of endothelial NO production. The abrogated NO response was associated with a reconstitution of alveolar fluid clearance at elevated PLA (Fig. 7B). At baseline PLA, alveolar fluid reabsorption did not differ between lungs from CHF and control rats and could still be blocked by GSNO, indicating that lack of pressure-induced NO production contributes to the preservation of alveolar fluid reabsorption in CHF lungs (Fig. 7C).


Figure 7
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  		Fig. 7. Endothelial NO production and alveolar fluid reabsorption in heart failure. A: capillary NO response to increased vascular pressure determined by in situ fluorescence imaging in lungs of rats with congestive heart failure (CHF). Representative fluorescence images of DAF-FM-loaded endothelial cells obtained at baseline PLA (left) and after 30 min of PLA elevation to 15 cmH2O (right) reveal endothelial dysfunction. Replicated in n = 5. B and C: alveolar fluid fluxes determined by the 2-compartmental double-indicator dilution technique. Alveolar fluid reabsorption at elevated PLA of 15 cmH2O is preserved in lungs of rats with CHF (B). At baseline PLA of 5 cmH2O, alveolar fluid reabsorption and fluid influx did not differ between lungs from control and heart failure rats. Administration of GSNO blocked alveolar fluid reabsorption in heart failure rats (C). All data are n = 7 each, *P < 0.05 vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Here, we demonstrate that NO-dependent impairment of alveolar fluid reabsorption aggravates acute hydrostatic lung edema. In isolated perfused rat lungs, >70% of the pressure-induced net fluid shift into the alveolar space was attributable to impaired fluid reabsorption. Endothelial-derived NO was identified as intercompartmental signaling molecule communicating the vascular pressure increase to the adjacent epithelium, since 1) PLA elevation increases endothelial NO formation, 2) NOS inhibition or genetic deficiency of eNOS attenuates hydrostatic lung edema, and 3) inhibition of endothelial NO synthase by L-NAME, Ca2+-free perfusion, or PI3K inhibition reconstitutes alveolar fluid reabsorption but does not affect alveolar fluid influx. NO-dependent inhibition of alveolar fluid reabsorption is mediated at least in part via the cGMP/guanylate cyclase pathway, because 1) the cell-permeable cGMP analog 8-BrcGMP reduces fluid reabsorption, while 2) fluid clearance is rescued by a guanylate cyclase inhibitor. In rats with congestive heart failure, lack of endothelial NO production contributes to intact alveolar fluid reabsorption, thus protecting the lungs from alveolar flooding. These findings present a novel mechanism in the pathogenesis of hydrostatic lung edema with potential therapeutic implications and characterize a new signaling pathway between the capillary and the alveolar compartments of the lung.

Hydrostatic edema and impaired alveolar fluid reabsorption. Hydrostatic lung edema was evident in isolated perfused rat lungs at PLA of 15 cmH2O as net fluid shift into the alveolar space. This finding is in accordance with previous data demonstrating alveolar flooding and increased solute flux into the alveolar space of rats at PLA of 15–20 cmH2O (23, 46). In larger mammals, alveolar edema only occurs when PLA exceeds 30 cmH2O (16, 18), suggesting that comparable fluid shifts will require considerably higher levels of hydrostatic stress in humans. Conversely, lower vascular pressures had to be applied in isolated perfused mouse lung experiments in this study to avoid rapid microvascular stress failure and excessive edema formation. The relative pressure increase compared with experiments in isolated rat lungs was conserved by tripling of the respective baseline PLA. PLA elevation from 2 to 6 cmH2O was sufficient to cause a 2.5-fold increase in wet/dry weight ratio in mice. This pressure increase will also change zonal perfusion in the lung, which may contribute to edema formation by recruitment of previously unperfused capillaries (53). The same mechanism may also add to pressure-induced net fluid shifts in the isolated rat lung. Importantly, the potential impact of capillary recruitment does not compromise our findings on the role of endothelial-derived NO in the regulation of alveolar fluid reabsorption, since all pharmacological interventions or transgenic models were compared with respective controls at similar vascular pressures. Pulmonary edema in these models was furthermore not attributable to lung injury as indicated by lack of edema at baseline pressure and absence of TRD tracer translocation into the perfusate. Of note, hydrostatic lung edema can be expected to be less pronounced in vivo when lymphatic pulmonary drainage is intact.

The alveolar epithelium rather than the endothelial layer constitutes the critical membrane limiting the transport of fluid and small solutes across the alveolar septal barrier (14, 50). Here, we identified impaired fluid reabsorption as fundamental cause for hydrostatic lung edema formation following moderate elevation of PLA. At elevated PLA, alveolar fluid clearance even decreased to negative values, which is in accordance with published data by Saldias and coworkers (46) obtained in an elegant multi-indicator radioisotope model. As we discuss below, this negativation of fluid reabsorption may indicate stimulation of active fluid secretion into the alveolar space.

Intercompartmental signaling by NO. Impairment of alveolar fluid reabsorption at elevated PLA requires the transduction of a hemodynamic stimulus into an epithelial response. Lung endothelial cells may act as mechanosensors and generate a signal that is transmitted to the alveolar epithelium. Here, we identify endothelial-derived NO as intercompartmental signal mediating epithelial responses to hydrostatic stress. In situ fluorescence imaging of DAF-FM-loaded lung capillaries demonstrated that PLA elevation increased NO formation in the vascular wall. By colocalization with a fluorescently tagged AcLDL marker, we have previously identified the DAF-FM-loaded cells within the capillary wall as microvascular endothelial cells (29). More importantly, we have shown that pressure-induced NO formation is mediated via PI3K and that PI3K-dependent phosphorylation of its downstream targets Akt and eNOS was confined to lung microvascular endothelial cells (29). These data clearly identify the endothelium as the predominant source of NO in acute hydrostatic stress. This finding is further supported by data from the present study demonstrating that hydrostatic edema is significantly attenuated in eNOS–/– mice and that blocking of all NOS isoforms by L-NAME does not further reduce lung wet/dry weight ratio.

Inhibition of the endothelial NO response by L-NAME, Ca2+-free perfusion (28), or PI3K inhibition (29) reconstituted alveolar fluid reabsorption in rat lungs, confirming the hypothesis that fluid reabsorption at elevated PLA was impaired by endothelial NO production. This notion is further supported by the finding that reconstitution of fluid reabsorption was less pronounced when L-NAME was administered via the alveolar rather than the capillary route. In contrast, administration of GSNO, which can release NO via reduction, or homolysis blocked alveolar fluid clearance at baseline hydrostatic pressure, and this effect was replicated by the structurally different NO donor papa NONOate. Hence, the pressure-induced inhibition of alveolar fluid reabsorption could be reproduced by exogenous NO donors. These effects were not attributable to changes in lung hydrostatic pressures or total vascular surface area, since none of the pharmacological interventions caused significant alterations in lung hemodynamics or alveolar fluid influx rates.

In lung microvessels in vivo, hemoglobin may act as physiological sink of NO. Hence, NO scavenging by hemoglobin is likely to explain the fact that the inhibitory effect of hydrostatic stress on alveolar fluid reabsorption was less pronounced in blood compared with buffer-perfused rat lungs. Yet, NO-dependent attenuation of alveolar fluid clearance still constitutes an important pathophysiological mechanism since a considerable inhibition persisted even in the presence of red blood cells. The fact that blood perfusion only partially diminished the effect of hydrostatic pressure on alveolar fluid clearance is consistent with the notion that flowing red blood cells are relatively weak scavengers of endothelial NO, presumably due to diffusion limitations for the reaction of NO with red cell-encapsulated hemoglobin (33) and the rheological formation of a skimmed plasma layer (32).

Regulation of epithelial Na+ transport. Alveolar fluid reabsorption is primarily driven by active transport of ions, in particular Na+, across the alveolar epithelium (36, 37). Alveolar transepithelial Na+ transport occurs predominantly via sodium uptake by amiloride-sensitive Na+ channels at the apical membrane and Na+-K+-ATPases at the basolateral membrane (17, 35). In isolated alveolar type II cells, NO donors such as GSNO or S-nitroso-N-acetylpenicillamine have been shown to reduce the open probability of a Na+-permeant cation channel (27). In confluent monolayers of rat type II epithelial cells, spermine NONOate and papa NONOate decreased vectorial Na+ transport by inhibiting both the amiloride-sensitive Na+ channels and the Na+-K+-ATPase (17). The cell-permeable cGMP analog 8-BrcGMP mimicked the inhibitory effect of NO donors in single cell patch-clamp studies (27, 31), yet not in confluent monolayers (17). Dopamine may directly counteract the inhibitory effects of NO, since it increases the open probability of the amiloride-sensitive sodium channel and enhances the plasma membrane expression of Na+-K+-ATPase in alveolar epithelial cells (4, 21). The present findings from isolated lungs are essentially in accordance with these findings and indicate that NO impairs alveolar fluid reabsorption at least in part by inhibiting amiloride-sensitive Na+ transport via the soluble guanylate cyclase/cGMP pathway. Whereas both inhibition of amiloride-sensitive Na+ channels and Na+-K+-ATPase blocked alveolar fluid reabsorption at baseline, only ouabain but not amiloride affected alveolar fluid reabsorption in hydrostatic stress, indicating that amiloride-sensitive Na+ transport was already blocked. The fact that ouabain reversed negative alveolar fluid reabsorption to ~0 ml/h at elevated PLA supports the notion that pressure-induced fluid secretion was a consequence of active cellular transport processes, e.g., epithelial Cl– secretion (42) rather than a result of focal lung injury or stress failure. NO-dependent regulation of additional alveolar epithelial ion channels such as CFTR channels may potentially further contribute to impaired alveolar fluid reabsorption and fluid secretion in hydrostatic stress (8).

8-BrcGMP attenuated alveolar fluid reabsorption at baseline PLA, whereas the soluble guanylate cyclase inhibitor ODQ reconstituted fluid clearance at elevated PLA. Data by Guo and coworkers (17) indicate that additional mechanisms such as direct S-nitrosylation may contribute to the inhibitory effect of NO on amiloride-sensitive Na+ channels (45). Yet, since inhibitors of S-nitrosylation such as N-ethylmaleimide also decrease basal Na+ currents in alveolar epithelial cells and accordingly result in rapid edema formation (data not shown), the evaluation of these mechanisms remains subject to future studies.

Alveolar fluid reabsorption in CHF. Both clinical and experimental studies demonstrate that hydrostatic edema formation is attenuated in lungs exposed to chronic hydrostatic stress (11, 51). Recently, considerable research has focused on structural and functional adaptations that may limit alveolar fluid influx from lung microvessels in CHF (2, 25). Here, we propose preservation of intact alveolar fluid reabsorption as an additional protective mechanism. Whereas elevation of PLA to 15 cmH2O resulted in a considerable net fluid shift into the alveolar space of control rats, this was completely prevented in lungs of rats with CHF. In accordance with recent data by Azzam and coworkers (3), the protection was predominantly caused by a restoration of alveolar fluid reabsorption, whereas alveolar fluid influx did not differ from controls at PLA of 15 cmH2O. Whereas the study by Azzam et al. identified increased abundance and activity of Na+-K+-ATPase at the alveolar epithelial plasma membrane as an underlying mechanism, our data demonstrate that, in addition, lung endothelial dysfunction contributes to this protective effect. In situ fluorescence imaging revealed that chronic pressure stress results in deficient endogenous NO production in lung microvessels. Inflammatory upregulation of iNOS was absent in this model (unpublished data). These results are in accordance with our previous finding that plasma concentrations of cGMP are markedly reduced in CHF rats (22). They are furthermore consistent with experimental and clinical data demonstrating a diminished NO-dependent lung vasodilatory response, an attenuated vasoconstrictive response to NO inhibitors, and reduced NO concentrations in the exhalate in heart failure (9, 43, 49). Supplementation of NO by an exogenous NO donor abolished the relative protection in CHF lungs, resulting in a similar reduction of alveolar fluid reabsorption as in controls. Thus endothelial dysfunction in CHF may constitute a protective mechanism that preserves intact alveolar fluid clearance and prevents the formation of excessive alveolar edema.

Clinical significance. The present study was conducted in isolated perfused lungs from small rodents that are more sensitive to hydrostatic stress than larger mammals and lack physiological protection mechanisms such as lymphatic drainage. Yet, clinical data support the potential relevance of this novel pathomechanism in humans. In a previous study of 65 patients diagnosed with severe hydrostatic lung edema, alveolar fluid clearance was severely impaired in 25% and submaximal in 37%, thus constituting a potentially relevant pathogenetic factor in almost 2/3 of patients (52). In agreement with a central role for NO in this process, submaximal or impaired alveolar fluid clearance was associated with significantly higher concentrations of nitrate and nitrite in edema fluid from patients with either hydrostatic lung edema or acute lung injury (54). While the potential relevance of impaired fluid clearance due to therapeutic delivery of exogenous NO donors or inhaled NO may frequently be obscured by hemodynamic effects that promote the resolution of lung edema (10, 47), inhaled NO has been shown to cause pulmonary edema in patients with NYHA III CHF (6). Hence, inhibition of alveolar fluid reabsorption by endothelial-derived NO may constitute a relevant and so far unrecognized mechanism in the pathogenesis of hydrostatic lung edema.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the Deutsche Forschungsgemeinschaft-Graduate School 865 "Mechanisms of Vascular Regulation" and the Kaiserin-Friedrich Foundation, Berlin, Germany.


   ACKNOWLEDGMENTS
 
We thank Ursula Hilse and Sylvia May for excellent technical assistance and Manfred Lambertz for mathematical support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. M. Kuebler, Institute of Physiology Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin Arnimallee 22, 14195 Berlin, Germany (e-mail: wolfgang.kuebler{at}charite.de)

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.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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