J Appl Physiol 93: 1860–1866, 2002.
First published ; 10.1152/japplphysiol.00022.2002.
highlighted topics
Lung Edema Clearance: 20 Years of Progress
Invited Review: Lung edema clearance:
role of Na⫹-K⫹-ATPase
J. I. SZNAJDER,1 P. FACTOR,1,2 AND D. H. INGBAR3
Pulmonary and Critical Care Medicine, Northwestern University, Chicago 60611; 2Evanston
Northwestern Healthcare, Evanston, Illinois 60201; and 3Pulmonary, Allergy and Critical Care,
Departments of Medicine and Pediatrics, University of Minnesota, Minneapolis, Minnesota 55455
1
Sznajder, J. I., P. Factor, and D. H. Ingbar. Invited Review:
Lung edema clearance: role of Na⫹-K⫹-ATPase. J Appl Physiol 93:
1860–1866, 2002. 10.1152/japplphysiol.00022.2002.—Acute hypoxemic
respiratory failure is a consequence of edema accumulation due to
elevation of pulmonary capillary pressures and/or increases in permeability of the alveolocapillary barrier. It has been recognized that lung
edema clearance is distinct from edema accumulation and is largely
effected by active Na⫹ transport out of the alveoli rather than reversal of
the Starling forces, which control liquid flux from the pulmonary circulation into the alveolus. The alveolar epithelial Na⫹-K⫹-ATPase has an
important role in regulating cell integrity and homeostasis. In the last 15
yr, Na⫹-K⫹-ATPase has been localized to the alveolar epithelium and its
contribution to lung edema clearance has been appreciated. The importance of the alveolar epithelial Na⫹-K⫹-ATPase function is reflected in
the changes in the lung’s ability to clear edema when the Na⫹-K⫹ATPase is inhibited or increased. An important focus of the ongoing
research is the study of the mechanisms of Na⫹-K⫹-ATPase regulation in
the alveolar epithelium during lung injury and how to accelerate lung
edema clearance by modulating Na⫹-K⫹-ATPase activity.
acute respiratory distress syndrome; alveolar epithelium; ion transport
occurs as the result of
active Na transport across the alveolar epithelium via
apical amiloride-sensitive Na⫹ channels and basolateral Na⫹-K⫹-ATPases (19, 41, 57). This active vectorial
Na⫹ flux produces a transepithelial osmotic gradient
that causes water to passively move from the air spaces
to the alveolar interstitium. In some models of acute
lung injury and in patients with acute respiratory
distress syndrome (ARDS), the lung’s ability to clear
edema is impaired (3, 14, 16, 27, 49, 65, 72, 83, 89).
Parallel studies in alveolar epithelial cells suggest that
this impairment may be due to decreased function of
epithelial Na⫹-K⫹-ATPase (13, 40, 49, 65). For example, severe lung injury in rats exposed to acute hyperRESOLUTION OF PULMONARY EDEMA
⫹
Address for reprint requests and other correspondence: J. I. Sznajder, Pulmonary and Critical Care Medicine, Northwestern Univ.,
300 E. Superior, Tarry 14-707, Chicago, IL 60611.
1860
oxia (100% O2 for 64 h) reduces active Na⫹ transport
in lungs and alveolar epithelial cells. However, moderate hyperoxic injury may lead to increased Na⫹-K⫹ATPase mRNA levels, with variable functional effects
(16, 35, 62). Similarly, subacute hyperoxia (85% O2 for
7 days) is associated with proliferation of alveolar epithelial cells, upregulation of Na⫹-K⫹-ATPase in the
alveolar epithelium, and increased lung edema clearance (63). Hypoxia also can inhibit the function of both
alveolar epithelial Na⫹-K⫹-ATPase and the Na⫹ channel (17, 54, 55, 90). These observations suggest a paradigm in which during the acute phases of lung injury
active Na⫹ transport may be impaired while edema
accumulates due to changes in permeability of the
alveolocapillary barrier. This acute phase is followed
by a proliferative response associated with increased
edema clearance (6, 63). Support for this model has
been demonstrated in studies of patients with lung
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injury and ARDS in whom impairment of the lung’s
ability to clear edema correlated with worse outcomes
(14, 38). There is also ample experimental evidence
from healthy animal models and during lung injury
that upregulating the Na⫹-K⫹-ATPases increases active Na⫹ transport across the alveolar epithelium and
thus edema clearance (4, 8, 24, 27, 71, 75, 76).
Naⴙ-Kⴙ-ATPase STRUCTURE AND BIOCHEMISTRY
The Na⫹ pump consists of two major subunits, ␣ and
, which typically form a heterodimer in the plasma
membrane enzyme (79). In some tissues, there is a
third ␥-subunit that modifies the functional activity of
the enzyme, but its significance in lung is undefined.
The essential role of Na⫹-K⫹-ATPase in cellular function has been recognized for more than 30 yr; more
recently, its importance in the lung has been reported
(6, 12, 27, 44, 62, 63). The ␣- and -subunits have
multiple isoforms; to date, four ␣- and five -isoforms
have been described (10, 61). These isoforms are expressed in a tissue-specific and developmentally regulated manner. The ␣- and -subunits have ⬎70% homology between isoforms within each subunit; there
also is significant homology of the Na⫹-K⫹-ATPase
coding and promoter DNA sequences across species
(50–52).
The Na⫹-K⫹-ATPase ␣-subunit. The catalytic ␣-subunit exchanges intracellular Na⫹ for extracellular K⫹
in a 3:2 ratio and contains ouabain binding and phosphorylation sites. It has many transmembrane domains and forms the cationic pore. Transcriptional
regulation studies of the rat Na⫹-K⫹-ATPase ␣1-subunit have identified a major transcription initiation
site 262 bp upstream from the translation initiation
site that is preceded by a TATA box at position ⫺32.
Included in this 5⬘-flanking region are two highly conserved SP1 transcription factor-binding sites, two glucocorticoid response element half-consensus sequences
(96), a consensus cAMP response element, and a positive regulatory region located at ⫺155/⫺49 bp from the
transcription initiation site. An “Atp1a1” regulatory
element (or ARE) at ⫺94/⫺69 bp binds both common
and at least seven cell type-specific transcription factors, which could account for differential, cell-specific
expression of this subunit (81). The human ␣1-gene
promoter has a TATA box, five SP1-like elements (77),
and three potential thyroid hormone response elements (29).
In the lungs, the ␣2-subunit is expressed in alveolar
epithelial cells and appears to have a role in alveolar
fluid clearance (5). Polymorphisms of the human ␣2subunit have been linked to increased susceptibility to
seizures (15), but lung abnormalities have not been
associated with polymorphisms of any of the subunits
or isoforms. Polymorphisms at one ␣2-locus decreases
cardiorespiratory endurance (maximal oxygen consumption) with training by as much as 40% (68).
The Na⫹-K⫹-ATPase -subunit. The smaller -subunit has a single transmembrane-spanning domain
J Appl Physiol • VOL
and, unlike the ␣-subunit, is glycosylated (59). The
precise function of the -subunit is controversial, but it
appears to have a role in the assembly and trafficking
of the Na⫹ pump heterodimer to the correct domain of
the cell membrane, as well as membrane-associated
half-life. In different cell types and tissues, the relative
quantities of ␣ and  mRNA and protein are variable.
In rat alveolar type II cells and in the rat lung, the
quantities of -subunit seem to limit the functional
activity of the Na⫹ pump enzyme (5, 27). Transcriptional regulation of the 1-subunit gene is less defined
than for ␣1, but it is likely to be as or more important
in the lung (37). Genomic clones of the rat 1-subunit
promoter contain a potential TATA box at position
⫺31, four GC-rich boxes, and two sites with half consensus sequences for thyroid hormone response elements (53). Intron I of the rat 1-gene has a positive
effect on basal transcription (C. H. Wendt and D. H.
Ingbar, unpublished observations), but the specific regulatory elements involved are not defined. In the 5⬘upstream region, two major and three minor transcription initiation sites have been identified. There is also
a positive regulatory region (⫺650 to ⫺630 bp) that is
required for mineralocorticoid receptor or glucocorticoid receptor activation (21). The NH2-terminal region
of the mineralocorticoid receptor inhibits GC stimulation of Na⫹-K⫹-ATPase 1-subunit transcription (46).
Hyperoxia stimulates transcription of this subunit
through an increase in the binding of SP1 transcription
factor to the proximal promoter region (93).
REGULATION OF ALVEOLAR EPITHELIAL
Naⴙ-Kⴙ-ATPase
Short-term regulatory mechanisms. Several reports
have suggested that basal Na⫹-K⫹-ATPase activity in
intact cells is one-third of its maximal capacity (78).
Thus recruitment of this reserve capacity represents a
mechanism by which cellular Na⫹-K⫹-ATPase activity
can be rapidly upregulated. Short-term increases in
Na⫹-K⫹-ATPase function can be regulated via three
pathways: 1) changes in the number of molecules in the
cell plasma membrane, 2) changes in the catalytic
property of enzymes already present at the plasma
membrane, and 3) changes in enzyme affinity for Na⫹.
Recent data indicate that Na⫹-K⫹-ATPase activity can
be rapidly increased via at least two mechanisms.
First, both dopamine and -adrenergic agonists increase lung edema clearance within 1 h (1, 4, 6, 27).
The -adrenergic agonists increase the pump’s affinity
for Na⫹ and recruit Na⫹ pump subunit proteins to the
basolateral plasma membrane from intracellular endosomal compartments (30) (see Fig. 1). In lung alveolar
epithelial cells, activation of G protein-coupled receptors, via either dopaminergic or adrenergic stimuli,
rapidly (30 s to 15 min) increases Na⫹-K⫹-ATPase
activity by insertion of Na⫹ pump proteins from intracellular compartments into the plasma membrane (4,
48, 69) (see Fig. 2). These effects are dependent on a
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Fig. 1. Schematic representation of the pathway of
Na⫹-K⫹-ATPase traffic from the plasma membrane
to intracellular endosomal and lysosomal compartments and recruitment back of the Na⫹ pumps into
the basolateral membranes on cathecholomine stimulation in the alveolar epithelium.
dynamic interaction between protein-transporting vesicles, microtubulae, and the actin cytoskeleton as pretreatment with colchicine, brefeldine, or phallacidin
prevents this recruitment. Interestingly, the shortterm regulation of Na⫹-K⫹-ATPase in alveolar type 2
epithelial cells by dopamine has been associated with
D1a- but not D2-receptor stimulation. These highly
regulated processes occur via simultaneous, phosphorylation events regulated by novel protein kinases and
dephosphorylation events regulated by protein phosphatase 2A (48, 69). A second, rapid mechanism by
which -adrenergic agonists stimulate transepithelial
Na⫹ transport and Na⫹-K⫹-ATPase is via cAMP-dependent activation of apical Cl⫺ channels in alveolar
epithelial cells (42).
Long-term regulatory mechanisms. Long-term regulation of Na⫹-K⫹-ATPase occurs via transcriptional
and posttranscriptional mechanisms, including changes
in membrane enzyme-specific activity, and increases in
plasma membrane Na⫹ pump proteins due to trafficking of heterodimers to the plasma membrane from
intracellular pools, translation, protein degradation
Fig. 2. Schematic representation of the dopaminergic-receptor (D2R) and 2-adrenergic-receptor (2-AR) pathways leading to transcriptional
and posttranscriptional regulation of the Na⫹
pump protein in alveolar epithelial cells. ERK,
extracellular regulated kinase; mTOR, mammalian target of rapamycin; PKA, protein kinase A;
PKC, protein Kinase C.
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rates, mRNA stability, and transcription (reviewed in
Refs. 9, 41, 86).
Transcriptional regulation of the Na⫹ pump subunit
genes is an important component of the multifaceted
response to growth hormones, hormonal stimulation,
hyperoxia, and cellular stress. The triggers for increased Na⫹-K⫹-ATPase expression in the lung just
before birth and the specific transcription factors and
signaling pathways that initiate transcription in response to stress or stimulation are being defined. Unequal amounts of ␣ and  mRNA and protein concentrations are present in many tissues, although the final
Na⫹ pump ␣- and -subunit stoichiometry is 1:1. Because the subunit genes are on different chromosomes,
transcription may be independently regulated. Increased transcription of the Na⫹-K⫹-ATPase subunit
genes in the lung may be mediated by hormones such
as dexamethasone, insulin, and aldosterone (5, 37, 39,
41, 43, 45, 47, 94, 95). Aldosterone increases both
transcription and plasma membrane insertion of preformed pump molecules (25, 64). Both functional enzyme activity and gene transcription are increased by
low intracellular K⫹ concentration or high Na⫹ concentration or by various hormones, including thyroid hormone (59), and in the lung by aldosterone (64) and
glucocorticoids (41). Corticosteroids, dexamethasone,
3,5,3⬘-triiodothyronine (T3), and aldosterone, as well as
keratinocyte growth factor and epidermal growth factor, increase Na⫹ reabsorption in mammalian lungs
(11, 18, 27, 28, 31–34, 58, 66, 73, 74, 84, 85). Similar to
steroids and growth factors, the commonly used drugs
dopamine (via D2 receptors) and -adrenergic agonists
can activate Na⫹-K⫹-ATPase gene transcription and
translation in alveolar epithelial cells (37) (67). Dopaminergic D2-receptor-mediated stimulation of Na⫹-K⫹ATPase mRNA and protein synthesis occurs via mitogen-activated protein kinases and a Ras-Raf-mitogenactivated protein kinase kinase pathway (37). For
example, terbutaline stimulated rat alveolar epithelial
cell Na⫹-K⫹-ATPase function after several days (60). A
more recent study reported that -adrenergic stimulation of serum-starved alveolar epithelial cells regulated Na⫹-K⫹-ATPase translation via extracellular
regulated kinase-rapamycin pathways independent of
changes in Na⫹-K⫹-ATPase transcription (67).
Translation of Na⫹-K⫹-ATPase mRNA is an important locus of regulation in a variety of settings. For
example, similar increases in steady-state levels of
mRNA result in different activity levels of the Na⫹
pump, indicating that posttranscriptional steps play a
role in the regulation of Na⫹-K⫹-ATPase (36, 67). In
vitro studies of translation demonstrated that untranslated mRNA regions can affect subunit translation.
The mRNA for ␣1 is translated less efficiently than that
for 1 because of ␣1 mRNA’s 3⬘ untranslated mRNA
region being extremely GC rich and folded in a complex
fashion and because translational efficiency may be
altered by glucocorticoids (22).
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OVEREXPRESSION OF Naⴙ-Kⴙ-ATPase IN THE
ALVEOLAR EPITHELIUM
In several models of lung injury and most ARDS
patients, lung edema clearance is impaired (7, 82, 91,
92). Thus methods that improve lung edema clearance
might offer a therapeutic option for these patients with
acute respiratory failure. For many years, it was believed that the Na⫹ channel was the locus of control for
Na⫹ reabsorption, but recent data indicate that upregulation of Na⫹-K⫹-ATPase alone is sufficient to
increase alveolar fluid clearance (2, 26, 27). Many of
the agents discussed above can stimulate fluid clearance by regulating the alveolar epithelial Na⫹-K⫹ATPase, including dopamine, -adrenergic agonists,
glucocorticoids, T3, keratinocyte growth factor, and epidermal growth factor. Because in several models of
lung injury models and in many ARDS patients lung
edema clearance is impaired, a clinical goal is the
augmentation of fluid clearance in patients with decreased or normal levels edema clearance, through
increased Na⫹-K⫹-ATPase and/or Na⫹ channel function. The proof-of-concept experiments that augmentation of Na⫹-K⫹-ATPase is a valid approach and can be
physiologically beneficial are based on gene transfer
experiments. The two gene transfer approaches have
been direct transfer of Na⫹-K⫹-ATPase genes and
overexpression of the -adrenergic receptor gene to
promote the increase of both Na⫹ channels and Na⫹K⫹-ATPase.
Adenoviral-mediated gene transfer has been utilized
to transduce the alveolar epithelium of rats to study
the role of alveolar Na⫹-K⫹-ATPase in lung edema
clearance (2, 26, 27). First-generation (E1a⫺/E3⫺), replication-incompetent human type 5 adenoviruses that
express rat Na⫹-K⫹-ATPase ␣1- or 1-subunit cDNA
were used to transduce lung epithelial cells. Overexpression of a 1-subunit, but not an ␣1-subunit, increased Na⫹-K⫹-ATPase function in adult rat alveolar
epithelial cells and rat fetal distal lung epithelial cells
(87). Conversely, Na⫹-K⫹-ATPase function in a human
lung cell line (A549) was increased only after overexpression of an ␣1-subunit gene (28). These studies were
extended to in vivo models by transducing the alveolar
epithelium of normal adult rats using a surfactantbased delivery system that increased alveolar fluid
reabsorption by ⬎100% in rat lungs overexpressing a
1-subunit gene.
As described above, adult rats exposed to 100% O2
develop acute lung injury characterized by increased
alveolar permeability, edema accumulation, and impairment of lung liquid clearance (16, 20, 65). A recent
study reported the results of adenoviral-mediated overexpression of a Na⫹-K⫹-ATPase 1-subunit gene in the
alveolar epithelium of adult rats before exposure to
hyperoxia (100% O2 for 64 h). Rats overexpressing the
Na⫹-K⫹-ATPase 1-subunit gene in the alveolar epithelium tolerated hyperoxia better, had no pleural effusions, and had lung liquid clearance rates that were
300% greater than hyperoxic controls or rats infected
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with an ␣1-subunit-expressing virus. In addition, 1subunit overexpression was associated with 100% survival through 14 days of hyperoxia, suggesting that
augmentation of lung liquid clearance may confer protection from a severe experimental lung injury. Similarly, Stern et al. (80) reported that mice (C57BL/6)
transduced with a chicken ␣3-gene fused to a 1 cDNA
have increased whole lung Na⫹-K⫹-ATPase activity
and less thiourea-induced edema than controls treated
with a plasmid vector that encoded an irrelevant
cDNA. Recently, in a model of increased left atrial
pressures, it was reported that the lung’s ability to
clear edema was decreased by 50% as left atrial pressure was increased from 0 to 15 cmH2O in isolated rat
lungs (3, 72). Overexpression of Na⫹-K⫹-ATPase 1subunit 7 days before measurement of lung liquid
clearance improved clearance in this model of increased hydrostatic pulmonary circulation pressures
(2).
-Adrenergic-receptor overexpression. -Adrenergic
agonists increase active Na⫹ transport in alveolar epithelial cells and normal and injured animal lungs by
increasing the function of both apical Na⫹ entry pathways via the epithelial Na⫹ channels and Na⫹-K⫹ATPases. These effects result from the stimulation of
both 1- and 2-adrenergic receptors (70, 75, 88), leading to upregulation of Na⫹ channels and Na⫹-K⫹ATPases in the lung epithelium (8, 56, 60, 67). Overexpression of a 2-adrenergic receptor in rat alveoli
with recombinant adenovirus that expresses a human
2-adrenergic-receptor cDNA increased lung liquid
clearance by ⬃100% compared with sham-infected
rats. The increased lung liquid clearance was associated with increased abundance in peripheral lung of
both ␣1-subunit of the epithelial Na⫹ channels in apical membrane fractions and Na⫹-K⫹-ATPase protein
abundance in basolateral cell membranes (23).
SUMMARY
Alveolar epithelial Na⫹-K⫹-ATPases are highly regulated proteins that contribute substantively to the
active Na⫹ transport necessary to maintain a dry alveolar air space. A growing body of research indicates
that downregulation of alveolar Na⫹-K⫹-ATPases is
associated with pulmonary edema in experimental
models of lung injury as well as in patients with highand low-pressure pulmonary edema. Thus methods
that counterbalance the inhibition of edema clearance
during lung injury and improve the lungs ability to
clear pulmonary edema are needed. As such, mechanisms that increase Na⫹-K⫹-ATPase function, (i.e.,
activation of dopaminergic or adrenergic receptors, corticosteroids, gene transfer) represent the rationale for
investigation toward the development of therapeutic
strategies to regulate the Na⫹-K⫹-ATPase function
and increase edema clearance. During these first 20 yr
since the demonstration of Matthay et al. (58a) that
alveolar edema is cleared by active Na⫹ transport, the
importance of alveolar Na⫹-K⫹-ATPases has been
clearly established. The mechanisms responsible for
J Appl Physiol • VOL
regulating the Na⫹ pump are now being actively studied. New experimental data are broadening our understanding of the importance of this crucial protein to
lung biology and pathophysiology.
This research was supported in part by National Heart, Lung, and
Blood Institute Grants HL-48129, HL-50152, HL-65161, and HL66211 and a grant from the Evanston Northwestern Healthcare
Research Institute.
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