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* From the Division of Pulmonary and Critical Care Medicine, Michael Reese Hospital, University of Illinois at Chicago, Chicago, IL, and Departamento de Enfermedades Respiratorias, Facultad de Medicina de la Pontificia Universidad Católica de Chile, Santiago, Chile.
Correspondence to: Jacob I. Sznajder, MD, Department of Medicine, Tarry Bldg 14-707, 303 E Superior St, Chicago, IL 60611
Mechanical ventilation is used in the treatment of patients with respiratory failure, but ventilation with high tidal volumes (HVTs) can cause volutrauma. In animal models, HVT ventilation may cause lung injury and pulmonary edema by overdistending the lung (volutrauma).1 2 The clearance of pulmonary fluid is effected mostly by active Na+ transport out of the alveoli, predominantly by the apical Na+ channels and the basolaterally located Na,K-adenosine triphosphatases (ATPases), which generate the electrochemical gradient responsible for the vectorial Na+ flux from the airspaces and water following isosmotically.3 4 5
It has been reported previously that changes of lung edema clearance paralleled Na,K-ATPase function in normal and pathologic conditions.6 7 However, it is not known whether mechanical overstretching of the lungs affects lung ability to clear edema. Therefore, we set out to test whether ventilator-associated lung injury (VALI) in rats affects the lung ability to clear edema. We studied active Na+ transport and lung liquid clearance after rats had been ventilated with HVTs and low tidal volumes (LVTs), in the isolated-perfused, fluid-filled rat lung model and tested whether the Na,K-ATPase activity in alveolar type II (ATII) cells isolated at the end of the experimental protocol was affected by mechanical ventilation.
Experimental Design and Results
A total of 70 rat lungs from pathogen-free, male, Sprague-Dawley rats weighing 280 to 320 g were studied. Rats were ventilated for 25, 40, and 60 min with the following experimental protocols: (1) low tidal volume (LVT): tidal volume of 10 mL/kg and peak airway pressure of approximately 8 cm H2O; (2) (HVT): tidal volume of 40 mL/kg and peak airway pressure of approximately 35 cm H2O; and compared with (3) control nonventilated rats.
Immediately following mechanical ventilation, rats were exsanguinated
and the heart and lung removed en bloc. To study the lung
liquid clearance and the active and passive solute movement, the
isolated-perfused fluid-filled model was used as previously
described.3
6
7
To obtain the wet/dry lung weight ratio,
the right upper lobe was ligated, excised, weighed in a tared
container, and dried in an evaporator (Speed-Vac; Savant Instruments;
Farmingdale, NY) until a constant weight. ATII cells were
isolated from the remaining lobes. Na,K-ATPase hydrolytic activity was
determined in intact cells as the rate of 32P-ATP
hydrolysis and it was calculated as the difference between test samples
(total ATPase activity) and samples assayed in the same medium, but
devoid of Na+ and K+ and in the presence of 2.5
mM ouabain (ouabain-insensitive ATPase activity). We also isolated RNA
from these ATII cells and converted it into complementary DNA using the
transcriptase reverse reaction. The resultant complementary DNAs were
amplified by polymerase chain reaction using specific primers for the
1-isoform. The amplified bands were analyzed by agarose
gel electrophoresis and quantified by densitometric scan (Eagle Eye II;
Stratagene; La Jolla, CA) and normalized against the internal control,
G3PDH.
We observed that rats ventilated with HVT for 25, 40, and
60 min had increased extravascular lung water. The wet/dry lung weight
ratio increased significantly after HVT ventilation for 25,
40, and 60 min (5.97 ± 0.27, 6.03 ± 0.21, 6.51 ± 0.2,
respectively) as compared with LVT and control
nonventilated rats (4.96 ± 0.04 and 4.86 ± 0.09, respectively).
Rats ventilated with HVT for 40 and 60 min showed decreased
active Na+ transport and lung liquid clearance
(0.26 ± 0.03 and 0.11 ± 0.08 mL/h, respectively) as compared with
LVT and control nonventilated rats (0.5 ± 0.02 mL/h).
Lung permeability to small (22Na+ and
3H-mannitol) and large (albumin) solutes increased in rats
exposed to HVT. Lung clearance in LVT
ventilated rats did not change when compared with control nonventilated
rats. The Na,K-ATPase activity decreased by approximately 50% in ATII
cells isolated from rats ventilated for 40 min with HVT as
compared with LVT and control nonventilated rats. Also, the
1 messenger RNA (mRNA) steady-state levels did not
change in rats ventilated for 40 min with LVT and
HVT as compared with control rats.
Discussion
Our data show that mechanical ventilation with HVT
decreases the ability of the lung to clear edema, and that this
impairment increased with time of ventilation. Lung edema clearance
decreased by approximately 48% and 78% after 40 min and 60 min of
HVT ventilation, respectively, without significant changes
after LVT ventilation. Lung edema clearance reduction
paralleled increased extravascular water and permeability to solutes
(Na+, mannitol, albumin) in rats exposed to HVT
ventilation for 40 and 60 min compared with LVT and control
nonventilated rats. We also studied Na,K-ATPase function in VALI to
evaluate whether the decrease in active Na+ transport and
lung edema clearance paralleled Na,K-ATPase function and we found in
rats ventilated with HVT for 40 min a decrease in
Na,K-ATPase activity of approximately 50% as compared with
LVT and control nonventilated rats. Na,K-ATPase
1-subunit mRNA did not change during mechanical
ventilation with LVT or HVT as compared with
control nonventilated rats. Although it has been reported that
mechanical ventilation with HVT increased c-fos
mRNA,8
in the present report, the changes observed in
Na,K-ATPase activity probably were not due to transcriptional
modifications.
In summary, our study demonstrates for the first time (to our knowledge) that VALI decreases active Na+ transport and lung edema clearance in association with a downregulation of Na,K-ATPase activity in ATII cells isolated from HVT ventilated rats.
Footnotes
Supported in part by the following grants: NIH HL-48129; American Heart Association 96012890; NRSA (KMR); Research and Education Foundation of the Michael Reese Medical staff; and Pontificia Universidad Católica de Chile.
References
This article has been cited by other articles:
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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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F. J. Saldias, Z. S. Azzam, K. M. Ridge, A. Yeldandi, D. H. Rutschman, D. Schraufnagel, and J. I. Sznajder Alveolar fluid reabsorption is impaired by increased left atrial pressures in rats Am J Physiol Lung Cell Mol Physiol, September 1, 2001; 281(3): L591 - L597. [Abstract] [Full Text] [PDF]
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