HIGHLIGHTED TOPICS
Lung Edema Clearance: 20 Years of Progress
Invited Review: Active fluid clearance from the distal air spaces of the lung

¹ University of California, San Francisco, California 94143-0624; ² Department of Physiology, INSERM 4426; Faculté de Médecine, Xavier Bichat Université, Paris 7; and ³ EA3512, IFR02, Faculté Xavier Bichat, 75018 Paris, France

	ABSTRACT

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Active ion transport drives iso-osmolar alveolar fluid clearance, a hypothesis originally suggested by in vivo studies in sheep 20 yr ago. Over the last two decades, remarkable progress has been made in establishing a critical role for active sodium transport as a primary mechanism that drives fluid clearance from the distal air spaces of the lung. The rate of fluid transport can be increased in most species, including the human lung, by cAMP stimulation. Catecholamine-independent mechanisms, including hormones, growth factors, and cytokines, can also upregulate epithelial fluid clearance in the lung. The new insights into the role of the distal lung epithelium in actively regulating lung fluid balance has important implications for the resolution of clinical pulmonary edema.

pulmonary edema; lung fluid balance; acute lung injury; active ion transport; alveolar epithelium; pulmonary epithelium; -adrenergic agonists

	INTRODUCTION

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THIS ARTICLE WILL BRIEFLY REVIEW evidence that active ion transport is the primary mechanism responsible for the removal of fluid from the distal air spaces of the intact adult lung. The first section will consider the mechanisms for active fluid clearance from several species, providing an historical perspective on how the evidence was accumulated by using a variety of experimental preparations, including studies of the ex vivo human lung. The second section will discuss the important discovery that active fluid clearance can be upregulated by cAMP stimulation. This section will also briefly consider evidence that chloride and cystic fibrosis transmembrane conductance regulator (CFTR) may play a role in cAMP-mediated fluid transport across the distal pulmonary epithelia. The third section will briefly consider some of the cAMP-independent mechanisms that can increase the rate of alveolar fluid clearance, including hormonal factors, cytokines, and growth factors.

	BASAL FLUID CLEARANCE FROM THE DISTAL AIR SPACES OF THE LUNG

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A substantial number of innovative experimental methods have been used to study fluid and protein transport from the distal air spaces of the intact lung, including isolated perfused lung preparations, in situ lung preparations, surface fluorescence methods, and intact lung preparations in living animals for short and extended time periods (54, 78).

In vivo studies in sheep. The first in vivo evidence that active ion transport could account for the removal of alveolar edema fluid across the distal pulmonary epithelium was obtained in studies of anesthetized, ventilated sheep (55). In those studies, the critical discovery was that isoosmolar fluid clearance of salt and water occurred in the face of a rising concentration of protein in the distal air spaces of the lung, whether the instilled solution was autologous serum, autologous plasma, or an isoosmolar protein solution. The initial protein concentration of the instilled protein solution was the same as the circulating plasma. After 4 h, the concentration of the protein had increased from ~6.5 to 8.4 g/100 ml, whereas the plasma protein concentration was unchanged. In longer term studies in unanesthetized, spontaneously breathing sheep, alveolar protein concentrations increased to remarkably high levels. After 12 and 24 h, alveolar protein concentration increased to 10.2 and 12.9 g/100 ml, respectively (52). The overall rise in protein concentration was 40 cmH₂O greater than the protein osmotic pressure in the vascular or interstitial spaces of the lung. The concentration of protein in the lung lymph draining the lung interstitium declined, providing further evidence that protein-free fluid was being reabsorbed from the distal air spaces into the lung interstitium (10, 55). Also, morphological studies showed that the interstitial fluid did not contain the Evans blue dye-labeled alveolar protein (51) (Fig. 1). On balance, these data provided convincing physiological and morphological evidence that active ion transport must be responsible for fluid clearance.

View larger version (139K): [in this window] [in a new window]   Fig. 1.   Color photograph of a sample of frozen lung tissue taken from the lower lobe of a sheep 4 h after 100 ml of Evans blue-labeled autologous serum had been instilled into the air spaces with a fiberoptic bronchoscope. Note the prominent fluid cuffs (arrows) around pulmonary arterioles (PA) (1-2 mm), which have little or no blue staining in contrast to the blue staining of the bronchi (BR) and alveolar ducts. Because Evans blue binds avidly to albumin, these morphological data indicate that very little of the protein instilled with the serum into the air spaces crossed the alveolar and distal epithelial airway barrier by 4 h. A few small lymphatics (L) are present within the fluid cuffs, and they appear to be clear also. Reprinted with permission from W. B. Saunders (52).

Other studies in the intact lung have supported the hypothesis that removal of alveolar fluid required active transport processes. Elimination of ventilation to one lung did not change the rate of fluid clearance in sheep, thus ruling out changes in transpulmonary airway pressure as a major determinant of fluid clearance in the normal lung (71). Also, if active ion transport were responsible for fluid clearance, then fluid clearance should be temperature dependent. In an in situ perfused goat lung preparation, the rate of fluid clearance progressively declined as temperature was lowered from 37 to 18°C (84). Similar results were obtained in perfused rat lungs (68) in which hypothermia inhibited sodium and fluid transport.

Pharmacological inhibition of in vivo fluid absorption. Overall, the process of transepithelial sodium transport can be considered a two-step process: sodium that enters the apical membrane of alveolar cells is pumped out at the basolateral membrane by Na⁺-K⁺- ATPase. Additional evidence for active ion transport was obtained in intact animals with the use of amiloride, an inhibitor of sodium uptake by the apical membrane of alveolar epithelium and distal airway epithelium. Amiloride inhibited 40-90% of basal fluid clearance in sheep, rabbits, rats, guinea pigs, and mice (5, 6, 10, 16, 22, 30, 41, 60, 70, 85, 96). Amiloride also inhibited sodium uptake in distal airway epithelium from sheep and guinea pigs (1, 3).

The importance of the sodium pump for alveolar liquid clearance has consistently been demonstrated in various species, showing that pump inhibition (with ouabain) reduces liquid clearance. This is the case in isolated rodent lungs (5, 38) or intact animals (41), in in situ sheep lungs (71), and in the resected human lung (70). Several humoral factors increase alveolar liquid clearance by increasing the abundance of the enzyme incorporated in cell plasma membrane (11, 12, 39). Overexpression of the beta-subunit that controls Na⁺-K⁺-ATPase heterodimer assembly in the endoplasmic reticulum and intracellular trafficking of these heterodimers to the basolateral plasma membrane in rat lungs by adenovirus gene transfer increases lung liquid clearance (23).

Human lung studies of active fluid clearance. All of these observations in several animal species were substantiated and extended to the human lung with studies of fluid clearance in an ex vivo human lung preparation. The results provided evidence that fluid clearance from the distal air spaces occurred with a rise in air space protein concentration, an effect that was inhibited by hypothermia or blockade of Na⁺-K⁺- ATPase with ouabain (Fig. 2A). The ouabain effect in the human lung was less than in animal studies (71, 84) probably because ouabain could be delivered only from the apical side of the epithelium (70). Also, amiloride inhibited ~40% of basal fluid clearance in the human lung (Fig. 2A). Although water channels (aquaporins) are present in the lung, there is no evidence that these channels regulate fluid clearance (49, 53, 54). As will be discussed in the next section, fluid clearance can be markedly increased in the human lung with cAMP, an effect that occurs in most but not all species.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Alveolar fluid clearance measured in an ex vivo lung preparation at 37°C over 4 h. A: amiloride (103 M) and ouabain (103 M) in the instilled solution (i.e., apical exposure) decrease fluid clearance by 40-50%. Hypothermia to 23°C inhibited nearly 90% of the clearance. B: terbutaline (104 M) doubled fluid clearance, an effect that was inhibited with propranolol (104 M) or amiloride (104 M). Data adapted from Ref. 68.

Influence of passive factors on fluid clearance. Several passive factors may influence the rate of alveolar liquid clearance, including the degree of interstitial hydration, the exchange surface area, lung volume, and epithelial barrier permeability. The extent of lung interstitial edema is important because it is a major determinant of fluid movement from the interstitium to the air spaces during pulmonary edema (86). When interstitial fluid exceeds 30-50% normal interstitial volume, edema fluid overflows into the alveoli. Thus alveolar liquid clearance may be impaired if there is not adequate removal of interstitial edema fluid. For example, in the absence of perfusion, or optimal function of lung lymphatics, the liquid pumped out from alveoli by the epithelium may accumulate in the interstitium, flow back, and impair alveolar liquid clearance (30, 38). Clearly, the greater the exchange surface area, the faster clearance may be. Liquid instilled by micropuncture in a peripheral alveolus very rapidly spreads from one acinus to another (93). This spreading may increase the exchange surface area during the early phase of pulmonary edema when only a few distal air spaces fill with fluid and oppose complete air space flooding. The redistribution of alveolar liquid over a larger exchange surface area may also explain the increased clearance that occurs during partial liquid ventilation with perfluorocarbon (67). For yet unclear reasons, increasing lung volume within the physiological range may impair alveolar clearance (91), although the data generated in this experimental model have not been reproduced by other investigators. Epithelial passive (paracellular) permeability may increase by stretching the lung. Because a high reflection coefficient of the epithelial barrier is theoretically essential for transepithelial sodium transport to drive water, the effect of altering passive permeability on alveolar liquid clearance is an important issue. In normal lungs, large (×20) increases in small solute paracellular permeability and albumin permeability produced by diverse polycationic agents has no adverse effect on alveolar liquid clearance and, thus, do not appreciably affect the sodium reflection coefficient (81). Similarly, a significant increase in alveolar epithelial permeability to proteins does not necessarily decrease the ability of alveolar epithelium to clear luminal liquid after bleomycin lung injury (26) or during the resolution of oleic acid-induced edema (95). Therefore, an appreciable decrease in epithelial barrier integrity may coexist with a normal fluid clearance rate.

Species differences in basal fluid clearance. Important species differences in the basal rates of fluid clearance have been identified (53). To normalize for differences in lung size or the available surface area, different instilled volumes were used ranging from 1.5 to 13.0 ml/kg. The slowest fluid clearance was measured in dogs (9, 33), intermediate rates of fluid clearance in sheep and goats (10, 52, 55, 84), and the highest basal fluid clearance rates were measured in rabbits, guinea pigs, rats, and mice (30, 31, 38, 41, 60). The basal rate of fluid clearance in the human lung has been difficult to estimate, but on the basis of the isolated, nonperfused human lung model, basal fluid clearance rates appear to be intermediate to fast (70). In fact, the fluid clearance in the ex vivo human lung is approximately half of the rate in the ex vivo rat lung (69). The explanation for the species differences is not apparent, although it may be related to the number or activity of sodium or chloride channels or the density of Na⁺-K⁺-ATPases in alveolar epithelium in different species. However, morphometric studies (17) have shown no significant difference in the number of alveolar type II cells in different species. It is also possible that the contribution of the distal airway epithelium may not be uniform in all species.

Most of the studies were focused on the specific role of sodium as the key transported ion in the active removal of salt and water from the distal air spaces of the lung. Amiloride, a well known inhibitor of apical sodium channels, inhibited a large fraction of fluid clearance. The discovery of epithelial sodium channel (ENaC) added further support to the critical importance of sodium, particularly since knockout of the alpha subunit of ENaC prevented normal clearance of alveolar lung fluid in newborn mice, resulting in their death apparently from respiratory failure (37). Amiloride is a good inhibitor of ENaC, so it is reasonable to assume that amiloride blocks ENaC, although it is not necessarily specific for ENaC alone. For example, higher doses can also inhibit Na⁺-K⁺-ATPase.

What accounts for the amiloride-insensitive fractions of alveolar fluid clearance, an important issue given that >50% of basal fluid clearance is insensitive to amiloride in the human lung, as well as in rat, sheep, and guinea pig lungs? There may be transport by nonspecific cation channels or other undiscovered sodium-selective channels. There is some evidence that the amiloride-insensitive fraction of 8-Br-cyclic 3'5'-guanosine monophosphate (8-Br-cGMP), which stimulated short-circuit current and sodium uptake in rat tracheal epithelia, was inhibited by dichlorobenzamyl or l-cis-diltiazem, both inhibitors of cyclic nucleotide-gated cation channels (CNG) (83). In subsequent studies, dichlorobenzamyl inhibited a significant fraction of lung fluid absorption in sheep (44). The CNG1 channel has been localized to distal lung epithelium and alveolar epithelial cells (20). In a rat study, l-cis-diltiazem inhibited a fraction of terbutaline or cGMP-stimulated fluid clearance that was not inhibited by amiloride, suggesting that part of the amiloride-insensitive fraction fluid clearance in the distal lung could be mediated through CNG channels, activated by cGMP (61). Also, there are data that iNOS (/) mice have normal rates of basal fluid clearance, but none of it is inhibited by amiloride (36). In rats, there is some evidence that the amiloride-insensitive part of fluid clearance may be mediated through the sodium-glucose cotransporter (7). The amiloride-insensitive part of fluid clearance is abolished when sodium is replaced by choline in the instillate or when phloridzin, a specific inhibitor of the sodium-glucose cotransport, is added to the instillate. In the rabbit, inhibition of sodium-glucose transport with phloridzin had no effect (85). Thus the importance of sodium-glucose cotransport for fluid clearance from the air spaces remains uncertain.

Another important issue is whether chloride transport occurs by a paracellular or a transcellular route. The evidence is incomplete presently, but there is some evidence that chloride may be transported by a transcellular route, although the best evidence for transcellular transport by a known chloride channel is cAMP-mediated transport by CFTR (see UPREGULATION OF FLUID CLEARANCE BY CAMP STIMULATION) (24).

	UPREGULATION OF FLUID CLEARANCE BY CAMP STIMULATION

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The observation that -adrenergic agonists caused lung liquid absorption in fetal lambs near delivery (92) suggested that activation of this pathway may play a role in the regulation of alveolar liquid clearance in adult lungs. Subtle, but important interspecies differences have been found that concern the adrenergic receptors that mediate the response and their possible interplay, the ions involved, and the magnitude of ion and water movements.

Rats. Most of the studies on the effects of catecholamines on ion and alveolar liquid transport have been done in rats by using either in vivo preparations or isolated lungs. Rats are a convenient model because of the clear-cut stimulation of alveolar liquid absorption produced by -adrenergic agonists in this species (29, 41, 69, 79). The general -adrenergic (isoproterenol) or -2 (terbutaline) agonists approximately double the rate of alveolar liquid clearance that, expressed as percent initial liquid volume, reaches about 20-30%/h. In keeping with the classical notion that -adrenergic agonists work through an increase in intracellular cAMP, stimulation of the same magnitude is achieved with a cell-permeable, lipid-soluble analog of cAMP given together with a phosphodiesterase inhibitor (32, 79). The signaling pathway may involve, other than protein kinase A, subsequent activation of a mitogen-activated protein kinase extracellular signaling-related kinase pathway (65).

The increase in liquid clearance occurs at least in part because of transepithelial sodium transport through sodium channels because it is inhibited by amiloride in rats (79). This may occur because of insertion of sodium channels in apical alveolar cell membrane (14, 57) and an increase in their open probability (47). Incorporation of Na⁺-K⁺-ATPase units in alveolar epithelium participate in the increase in sodium transport (74). In fact, increased plasma membrane incorporation of alpha subunits of the enzyme in response to isoproterenol has been observed in cultured rat alveolar type II cells (11). One study suggested that -adrenergic stimulation of sodium transport was mediated by ₂ adrenergic receptors in rats because the stimulation observed with dobutamine (a ₂ and ₁ agonist) was inhibited by a specific ₂ antagonist (90). However, more recent data indicate that a ₁ agonist, denopamine, stimulates clearance in rats (72), although stimulation of ₁ receptors by a high dose of terbutaline had an inhibitory effect by unknown mechanisms (73).

Potassium and chloride (Cl) movements have been studied by measuring unidirectional 86-Rb and 36-Cl fluxes across the alveolar epithelium in isolated rat lungs. The -adrenergic agonist isoproterenol increases potassium absorption in addition to stimulating sodium and liquid absorption. Luminal (but not basolateral) ouabain decreases potassium absorption but does not affect liquid clearance. The same phenomenon was observed with large (10³ M) concentration of a cAMP analog. This result suggested that a ouabain-sensitive pump (such as K/H ATPase) may be present in the apical membrane of alveolar cells (80). Apical potassium channels are also probably present in alveolar cells because barium, a nonselective blocker of these channels, increased the potassium absorption rate (probably by eliminating a potassium backflux). However, these potassium movements are limited, and their inhibition does not appreciably affect liquid clearance (80).

cAMP-induced changes in Cl movement across the alveolar epithelium have not been completely clarified. Rat pneumocytes could secrete Cl since CFTR channels are probably present in their apical membrane (43) and they display NaK2Cl cotransport activity (15). cAMP activates Cl channels in rabbit pneumocytes, which results in Cl secretion (47). However, Jiang et al. (42, 43) presented evidence that the opening of this Cl channels after -adrenergic agonist stimulation resulted in Cl absorption. Cl absorption through pneumocyte monolayers has also been reported in response to terbutaline (46). Other investigators suggested that this Cl absorption could increase the electrochemical gradient for Na absorption and account for the stimulatory effects of -adrenergic agonists on Na and liquid absorption (42). There are new data on the role of cAMP-mediated Cl absorption in mouse and human lung (see below) (24).

In isolated rat lungs, basal unidirectional ³⁶Cl fluxes in the absorption and secretion directions are identical (77). Both increased about twofold during stimulation of liquid absorption with a cAMP analog. Inhibition of sodium and liquid absorption with amiloride decreased Cl flux in the absorption direction but did not change Cl flux in the other direction. Apical application of diphenylamine-2-carboxylate (DPC) (a Cl-channel blocker) or basolateral bumetanide (which inhibits NaK2Cl cotransport) did not affect Cl flux during cAMP stimulation. It is worth noting that, in contrast to what was found in cultured rabbit pneumocytes (58), terbutaline- and cAMP-stimulated responses in adult rat alveolar cell monolayers were unaffected by basolateral bumetanide treatment but were sensitive to Cl-channel blockers such as DPC (62). In isolated lungs, cAMP stimulation resulted in enhanced Cl and liquid absorption from rat air spaces. But the lack of effect of DPC did not support the conclusion that cAMP-activated Cl channels are implicated in the increase in sodium transport (77), although DPC is not specific for CFTR. Further studies of isolated rat type II cells may clarify these issues in the future.

Mice. Mouse alveolar liquid absorption is remarkably fast, about twice that of rats, and is similarly doubled by -adrenergic agonists (2, 30, 38). Some caution should be taken when using mouse preparations. Their high rate of alveolar liquid absorption makes them unsuitable for use as unperfused ex vivo lungs beyond short-term studies because of the rapid shift of liquid from the distal air spaces to the interstitium (30, 38), an effect that causes backflux of interstitial fluid to the air spaces. Selective human -2 receptor expression in the alveolar type II cells of a transgenic mouse (56) results in a 40% increase in alveolar liquid clearance. This increase is the consequence of endogenous catecholamine secretion because it is abolished by adrenalectomy. In keeping with the observations made in rat alveolar cells (11), activation of the -adrenergic-cAMP-protein kinase A pathway results in an increase in ₂ Na⁺-K⁺-ATPase subunit expression (56). Surprisingly, no stimulation of alveolar transepithelial sodium transport by terbutaline was observed in hamsters (32) for reasons that are not clear at this time. New data suggests that cAMP-dependent transport in mice depends on CFTR for Cl uptake (24).

Rabbits. -Adrenergic agonists do not stimulate alveolar liquid clearance in rabbits (21, 85), despite the presence of -adrenergic receptors on rabbit pneumocytes (25). Increasing cAMP in rabbit lungs with forskolin results in a slight but nonsignificant increase of alveolar liquid clearance (58). The lack of stimulation of liquid absorption with -adrenergic agonists may be the consequence of Cl secretion that occurs simultaneously with stimulation of sodium absorption. There are cAMP-activated (CFTR or CFTR-like) channels in rabbit pneumocytes through which this secretion might take place. Rabbits that were administered amiloride and forskolin had a greater fluid clearance rate than those administered amiloride, suggesting that the small increase in liquid clearance after instillation of forskolin may be due to sodium absorption through amiloride-insensitive pathways.

Guinea pigs. There is a high basal alveolar liquid clearance rate (38%/h) in guinea pigs. Epinephrine and isoproterenol increase alveolar liquid clearance up to 50%/h, whereas terbutaline (a ₂-adrenergic agonist) had no effect (60). The stimulation of clearance by epinephrine or isoproterenol was mediated by ₁-receptors because the ₁-adrenergic inhibitor atenolol suppressed the response, and the ₁-agonist denopamine increased liquid clearance (72). Interestingly, isoproterenol and epinephrine, but not terbutaline, increased cAMP in lung tissue. Liquid clearance in guinea pigs is mediated only partly through amiloride-sensitive sodium channels because amiloride decreased clearance by 30-40% only, which contrasts with rats and mice.

Sheep. Terbutaline or epinephrine increased alveolar liquid clearance in anesthetized, ventilated adult animals by ~100% over baseline values (10). Physiological monitoring showed that the increased clearance was not explained by changes in pulmonary hemodynamics, pulmonary blood flow, or lung lymph flow. The increase in fluid clearance was inhibited by propranolol (a beta blocker) or amiloride (10). In subsequent studies, a cAMP analog together with a phosphodiesterase inhibitor increased clearance (8).

Dogs. Alveolar liquid clearance in dogs is 50% lower than in sheep, although cAMP stimulation with terbutaline administration in the alveolar instillate doubled the volume of liquid cleared from the lungs and alveolar spaces over 4 h (9). Despite a lower basal rate, clearance is thus similarly stimulated by -adrenergic agonists in dogs as in several other species (50). The very low rate of clearance in dogs is not explained at this time.

Human lungs and clinical relevance. Studies in human lungs using an ex vivo, nonperfused preparation demonstrated a marked stimulation with a cAMP agonist (Fig. 2B). Unstimulated alveolar fluid clearance was twice as fast in rat lungs as in human lungs (6 vs. 3%/h) (69). This finding suggested that the ex vivo lung preparation might underestimate the rate of in vivo alveolar fluid absorption. Indeed, in patients with alveolar proteinosis, alveolar liquid clearance may be as high as 54%/h (29). Several studies have shown that -adrenergic administration accelerates liquid clearance during experimental pulmonary edema (29, 50, 75, 76). Alveolar liquid clearance is increased by aerosolized salmeterol in sheep (29) during pulmonary edema. Relatively low doses (10⁶ M) of this long-acting, lipophilic agonist increases clearance in the human lung (69), suggesting that aerosolized -adrenergics may be a useful adjunct to accelerate the resolution of pulmonary edema.

	UPREGULATION OF FLUID CLEARANCE BY CATECHOLAMINE-INDEPENDENT MECHANISMS

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Several catecholamine-independent mechanisms can upregulate fluid transport across the distal air spaces of the lung, including hormones, growth factors, and proinflammatory cytokines (Table 1).

View this table: [in this window] [in a new window]   Table 1.   Mechanisms for catecholamine independent upregulation of epithelial fluid clearance

Glucocorticoids and mineralocorticoids. Glucocorticoid hormones regulate sodium uptake and fluid transport in both adult and fetal lungs (39, 66, 89). In adult guinea pig lungs, endogenous plasma levels of cortisol are important for maintaining normal lung liquid balance and distal air space liquid clearance (59). Dexamethasone, a more potent glucocorticoid agonist than cortisol, also regulates transepithelial sodium transport by increasing in vivo lung fluid clearance in anesthetized, ventilated rats (27). Molecular studies have reported that dexamethasone increased the activity and expression of both sodium channels and Na⁺-K⁺- ATPase by acting at both transcriptional and posttranscriptional levels. In vivo experiments have demonstrated a differential regulation of ENaC subunits by corticosteroids: - but neither - nor -ENaC mRNA levels increase within 8 h of treatment. In vitro, dexamethasone has been shown to increase mRNA of the three subunits of ENaC in studies using primary cultures of fetal and adult lung cells (18, 66, 89), but only - and -ENaC mRNA in the human alveolar epithelial cell line A549 (48). This latter observation is in contrast with the recent evaluation of the human -ENaC promoter showing the presence of glucocorticoid-responsive element in the 5'-flanking region in humans (82) and in rats (18, 64). Dexamethasone treatment increases the activity and expression of Na⁺-K⁺- ATPase in fetal and adult rat lungs (4, 18, 39). In adult alveolar type II cells, dexamethasone increased ₁ but not ₁ Na⁺-K⁺-ATPase mRNA (4, 18), whereas both ₁ and ₁ Na⁺-K⁺-ATPase proteins were increased, which suggests that posttranslational events may be involved in the regulation of Na⁺-K⁺-ATPase by glucocorticoids (4).

The lung is a potential target organ for aldosterone because it expresses mineralocorticoid receptors and the enzyme 11--hydroxysteroid dehydrogenase that converts the glucocorticoid corticosterone into receptor-inactive 11-dehydrocorticosterone, thereby allowing preferential access of aldosterone to mineralocorticoid receptors (87). In vivo, aerosolized delivery of aldosterone resulted after 24 h in a 50% increase in alveolar fluid clearance in rats (63). Similarly, rats treated with a low-sodium diet that developed a hyperaldosteronism have an increase in alveolar fluid clearance related to a stimulation of amiloride-sensitive component (87). In alveolar type II cells, aldosterone upregulates ₁ Na⁺-K⁺-ATPase mRNA and increased amounts of ₁ and ₁ Na⁺-K⁺-ATPase proteins, suggesting that aldosterone regulates the ₁ subunit at the transcriptional/translational level, whereas the ₁ subunit is probably recruited from the intracellular pool to the basolateral membrane (63). There is no direct evidence that aldosterone regulates ENaC expression in alveolar epithelial cells. However, there is a higher frequency of highly selective channels in cells cultured in the presence of aldosterone than in cells in the absence of aldosterone (40), suggesting that aldosterone also regulates ENaC activity and/or expression.

Growth factors. Several studies have reported that growth factors can upregulate sodium uptake and net fluid transport across the distal air spaces of the lung by several different mechanisms. Keratinocyte growth factor (KGF) is a potent mitogen for alveolar epithelial type II cells. Administration of KGF (5 mg/kg) into the distal air spaces of the rat lung upregulated alveolar fluid clearance by 66% over baseline levels (49, 94), an effect that was sustained for 120 h. There was a good correlation between the increased number of alveolar type II cells and the effect of KGF on alveolar fluid clearance (94). Furthermore, the addition of a ₂-adrenergic agonist further upregulated fluid clearance in rats so that the combination of KGF treatment plus terbutaline resulted in a net clearance of 50% of the instilled fluid in 1 h in rats compared with control rats with clearance rates of 23%/h. Other investigators have shown that KGF can enhance sodium and fluid transport in normal and injured rat lungs (34, 35). In vitro studies of the effect of KGF in cultured alveolar epithelial cells indicated that KGF might also work by enhancing the expression of ₁ Na⁺-K⁺-ATPase subunits (13), thereby enhancing sodium transport independent of an effect on the number of alveolar type II cells.

Epidermal growth factor (EGF) administered by aerosolization induced in the rat a marked increase in alveolar fluid clearance 48 h later with an increase in active sodium transport. Molecular studies in alveolar type II cells have reported that EGF upregulates Na⁺-K⁺-ATPase expression but did not change ENaC expression (12). These transcriptional effects were paralleled by an increase in benzamil-inhibitable short-circuit current (19). Subsequently, the same research group reported that EGF decreased ENaC mRNA levels with an increase in the density of nonselective cation channel expression (45). Because EGF has mitogenic properties, it is possible that some of the EGF effect in vivo could have occurred by stimulating type II cell proliferation, although there was no evidence for a mitogenic effect in rat studies (88).

Transforming growth factor- can also upregulate alveolar fluid clearance rapidly apparently by direct membrane effects. One study in rats demonstrated a marked increased in alveolar fluid clearance within 1 h of instillation into the living rat lung (28). There was no increase in cAMP levels, and the effect was not inhibited by -adrenergic blockade. However, genistein prevented the effect, suggesting that the effect might be mediated by tyrosine kinase activity.

In summary, considerable evidence has been generated to support the conclusion that active ion transport is primarily responsible for isoosmolar fluid clearance across the distal lung epithelium. Both catecholamine-dependent and -independent pathways can upregulate fluid transport across the distal lung epithelium, potentially providing important mechanisms to accelerate the resolution of alveolar edema under pathological conditions.

	ACKNOWLEDGEMENTS

The authors appreciate the assistance of Rebecca Cleff in the preparation of this manuscript.

	FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grants HL-51854 and HL-51856.

Address for reprint requests and other correspondence: M. A. Matthay, Univ. of California, 505 Parnassus Ave., M-917, San Francisco, CA 94143-0624 (E-mail: mmatt{at}itsa.ucsf.edu).

10.1152/japplphysiol.01210.2001

Received 9 December 2001; accepted in final form 8 April 2002.

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