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* From the Section of Pulmonary and Critical Care Medicine, Michael Reese Hospital, and University of Illinois at Chicago, Chicago, IL.
Correspondence to: Phillip Factor, DO, FCCP, Pulmonary and Critical Care Medicine, Michael Reese Hospital and Medical Center, 2929 S Ellis, 314 Kunstader, Chicago, IL 60616; e-mail: PFACT{at}AOL.COM
Rats exposed to acute hyperoxia develop a lung injury characterized by pulmonary edema and decreased ability to clear lung liquid.1 2 Accumulating evidence indicates that active Na+ transport is an important contributor to lung edema clearance.3 4 5 In the alveolus, Na,K-adenosine triphosphatases (ATPases) have been shown to be responsible for active Na+ transport across the alveolar epithelium.3 5 6 These pumps works in concert with other epithelial transport proteins, including water and apical Na+ channels, to generate a transepithelial Na+ gradient that causes the movement of water out of the alveolar airspace.6
Na,K-ATPases are transmembrane heterodimers that are composed of
and ß subunits.7
The subunit cleaves high-energy
phosphate bonds and exchanges intracellular Na+ for
extracellular K+. The smaller ß subunit is a glycosylated
transmembrane molecule that controls /ß heterodimer assembly and
insertion into the plasma membrane; its presence is required for normal
Na,K-ATPase function. Alveolar type II epithelial cells and whole rat
lungs express the 1 and ß1 subunits of
this multigene family and the levels of their respective messenger RNAs
and protein change in response to stimuli such as
hyperoxia.2
8
9
It has been reported that rats exposed to
acute hyperoxia have parallel changes in active Na+
transport and lung edema clearance.2
8
9
Specifically,
decreased Na,K-ATPase expression is associated with decreased lung
liquid clearance. Similar changes have been reported for an
amiloride-sensitive apical Na+ channel.10
As a
result of these studies, we hypothesized that adenoviral-mediated
overexpression of Na,K-ATPase subunit proteins could improve lung
liquid clearance during hyperoxic lung injury.
We have observed previously that adenoviral-mediated transfer of
Na,K-ATPase subunit genes increases Na,K-ATPase messenger RNA and
protein expression in rat and human alveolar epithelial
cells.11
12
13
Na,K-ATPase function was increased by up to
250% following gene transfer in these in vitro experiments.
We have also reported recently that tracheal instillation of
4 x 109 plaque forming units of an adenovirus expressing
a Na,K-ATPase ß1 subunit gene (adß1)
significantly increased alveolar ß1 protein expression
and improved lung liquid clearance in normal rats.14
Instillation of an otherwise identical virus that expresses a rat
1 complementary DNA (cDNA) (ad1) did not
alter lung liquid clearance. These prior results indicate that
Na,K-ATPase function and lung liquid clearance can be improved via
Na,K-ATPase subunit overexpression and that the ß1
subunit may be rate limiting in the rat alveolar epithelium.
Our prior studies used replication-deficient human type 5
adenoviruses containing individual cDNAs for the 1 and
ß1 subunits of rat Na,K-ATPase.13
These
vectors employ the immediate-early cytomegalovirus promoter and the
SV40 t intron polyadenylation signal. They were generated via
homologous recombination of shuttle vectors containing Na,K-ATPase
subunit cDNAs with a plasmid that contains the entire adenovirus type 5
genome (pJM17). Following homologous recombination, they were serially
plaque-purified and expanded in 293 cells.
To study the effect of Na,K-ATPase overexpression on hyperoxic
lung injury, we used 270- to 300-g adult, male Sprague-Dawley rats.
Sedated, spontaneously breathing rats were intubated and given
4 x 109 plaque forming units of the above-described
adenoviruses. Virus was administered in four 200-µL aliquots of a
vehicle composed of 50% diluent/50% surfactant (Survanta, Abbott
Laboratories; Columbus, OH). Vehicle was delivered at 5-min intervals
interspersed with 90° rotation of the animals between instillations.
Immediately prior to each instillation, the thorax was compressed to
force expiration. Following endotracheal instillation, compression was
released, allowing the animals to take a forceful inspiration that
facilitated distal dispersion of vehicle. Rats were allowed to recover
for 7 days to allow resolution of adenoviral-related host responses.
After recovery, they were placed in a thermally controlled
environmental chamber and exposed to >95% O2 for 64
h. Lung liquid clearance was then measured using a fluid-filled,
isolated lung model that allows measurement of permeability for solutes
and lung liquid clearance. This model employs the instillation of
22Na+, 3H-mannitol and Evan's blue
albumin into the airspace compartment.2
4
The vascular
compartment is continually perfused at fixed left atrial and pulmonary
artery pressures with a buffered solution containing fluorescein
isothiocyanate-albumin. The lungs are placed in a "pleural bath";
pH and temperature are held constant over a 60-min experimental period.
Changes in concentration of Evan's blue albumin are used to calculate
alveolar liquid clearance. Movement of the tracers between compartments
is used to measure alveolar permeability. Separate survival studies
were conducted by exposing rats to > 95% O2 for up to 14
days. Between 15 and 20 rats per group were studied; mortality was
recorded at 12-h intervals. In all hyperoxia studies, the
adß1 and ad1 groups were contrasted to
untreated room air (room air control) and hyperoxic animals (hyperoxic
control), vehicle without adenovirus (surfactant), and a similar virus
containing no cDNA (adNull).
Lung liquid clearance in the room air control, hyperoxic control,
and surfactant animals was 0.51 ± 0.06, 0.28 ± 0.10 mL/h, and
0.34 ± 0.05 mL/h, respectively. Liquid clearance was increased
3.3-fold in the adß1 infected animals (0.93 ± 0.13
mL/h, p < 0.001 vs surfactant). Clearance was minimally increased in
the adNull (0.56 ± 0.15 mL/h, p < 0.02 vs surfactant) but not in
the ad1 animals (0.37 ± 0.12 mL/h). As compared to
room air controls, alveolar permeability for small solutes
(Na+ and mannitol) was minimally increased in all hyperoxic
animals. No difference in permeability for albumin was noted among the
hyperoxic animals.
All of the adß1 rats exposed to hyperoxia survived to the
end of the 14-day experimental period (p < 0.0002 vs hyperoxic
control). There were no significant differences in LD50
values among the non-adß1 groups. Overall survival for
each of the groups was 100%, 31%, 25%, 25%, and 17% for
adß1, hyperoxic control, surfactant, adNull, and
ad1, respectively.
These results show that adenoviral-mediated overexpression of a Na,K-ATPase ß1 subunit enhances lung edema clearance following acute hyperoxia. This increase occurred despite the increased alveolar permeability for small solutes. Overexpression of this subunit also markedly improved survival from a lung injury associated with a high mortality. These data suggest that the requisite mechanisms necessary for lung liquid clearance in the alveolar epithelium following hyperoxia can be restored via augmentation of a key component, the Na,K-ATPase ß1 subunit. They also support our previous data that shows that the ß1 subunit may be rate limiting in the alveolar epithelium. Our findings support the concept that Na,K-ATPase function and maintenance of lung edema clearance are important contributors to alveolar function in the presence of acute lung injury. Conceivably, gene transfer of Na,K-ATPase subunit genes may eventually prove useful for the treatment of acute lung injury.
Footnotes
Supported by the Research and Education Foundation of Michael Reese Hospital, American Heart Association of Metropolitan Chicago, American Lung Association of Chicago, and HL-48129.
References
1 subunit increases Na,K-ATPase function in A549 cells. Am J Respir Cell Mol Biol 18,741-749This article has been cited by other articles:
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  C. Sartori and M.A. Matthay Alveolar epithelial fluid transport in acute lung injury: new insights Eur. Respir. J., November 1, 2002; 20(5): 1299 - 1313. [Abstract] [Full Text] [PDF]
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 J. L. Fisher and S. S. Margulies Na+-K+-ATPase activity in alveolar epithelial cells increases with cyclic stretch Am J Physiol Lung Cell Mol Physiol, October 1, 2002; 283(4): L737 - L746. [Abstract] [Full Text] [PDF]
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A. Dagenais, C. Denis, M.-F. Vives, S. Girouard, C. Masse, T. Nguyen, T. Yamagata, C. Grygorczyk, R. Kothary, and Y. Berthiaume Modulation of {alpha}-ENaC and {alpha}1-Na+-K+-ATPase by cAMP and dexamethasone in alveolar epithelial cells Am J Physiol Lung Cell Mol Physiol, July 1, 2001; 281(1): L217 - L230. [Abstract] [Full Text] [PDF]
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 U. Thome, L. Chen, P. Factor, V. Dumasius, B. Freeman, J. Iasha Sznajder, and S. Matalon Na,K-ATPase Gene Transfer Mitigates an Oxidant-Induced Decrease of Active Sodium Transport in Rat Fetal ATII Cells Am. J. Respir. Cell Mol. Biol., March 1, 2001; 24(3): 245 - 252. [Abstract] [Full Text] [PDF]
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