INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Mechanical ventilation is used in the care of patients with acute respiratory failure; however, it may also cause or worsen lung injury (1). Mechanical ventilation with high tidal volumes (VT) for short periods of time may cause lung injury and pulmonary edema in rats by overdistending the lung (volutrauma) (4). Regardless of its pathogenesis, once edema is established, the clearance of pulmonary fluid is effected mostly by active Na⁺ transport out of the alveoli (5). Although other mechanisms may have a role, the clearance of pulmonary edema appears to be mainly contributed by the apical Na⁺ channels and the basolaterally located Na,K-ATPases, which generate the electrochemical gradient responsible for the vectorial Na⁺ flux from the airspace, with water movement following isosmotically (8).

The Na,K-ATPase maintains cellular electrochemical gradients through active countertransport of Na⁺ and K⁺ across the plasma membrane coupled to the hydrolysis of ATP (11). The catalytic Na,K-ATPase -subunit contains the binding sites for ATP and the -subunit is thought to be responsible for integrating the -subunit into the plasma membrane and conferring normal activity to the enzyme complex (12).

It has been previously reported that changes of lung edema clearance paralleled Na,K-ATPase function in normal and pathological conditions (15, 16). Ventilating rats with high VT causes lung injury and pulmonary edema formation (1, 3, 4). However, it is not known whether mechanical overstretching of the lungs affects its ability to clear edema. Therefore, we set out to test whether ventilator-associated lung injury (VALI) in rats affects the lung's ability to clear edema. In the isolated- perfused fluid-filled rat lung model we studied active Na⁺ transport and lung edema clearance after rats had been ventilated with high, moderate, and low VT. We also determined 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.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

A total of 85 rat lungs were studied. Pathogen-free, male, Sprague-Dawley rats weighing 280 to 320 g were purchased from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). All animals were provided food and water ad libitum and maintained on a 12 h:12 h light-dark cycle. IgG, Na₂ATP, and ouabain were obtained from Sigma (St. Louis, MO). [-³²P]ATP was purchased from Amersham (Arlington Heights, IL).

Mechanical Ventilation

Adult rats were ventilated with a rodent ventilator (Model 683; Harvard Apparatus, South Natick, MA) for 25, 40, and 60 min with the following experimental protocols: (1) low VT: VT of 10 ml/kg and peak airway pressure (Pao) of ~ 8 cm H₂O; (2) moderate VT: VT of 30 ml/kg and peak Pao of ~ 20 cm H₂O; (3) high VT: VT of 40 ml/kg and peak Pao of ~ 35 cm H₂O and compared with (4) control nonventilated rats.

Wet-to-Dry Weight Ratio

Rats were anesthetized with pentobarbital, the chest was opened by midsternal incision, and rats were exsanguinated. The heart and lungs were removed en bloc. The right upper lobe was ligated, excised, and weighed in a tared container. The right upper lobe was then dried in a Speed-Vac evaporator (Savant Instruments, Farmingdale, NY) until a constant weight was obtained and then the wet-to-dry lung weight ratio was calculated.

Isolated Lungs

The protocols with isolated lung preparation were studied as previously described (7, 10, 15). Briefly, rats were anesthetized with 50 mg/ kg body weight of pentobarbital. A tracheotomy was performed and the rats were mechanically ventilated with low, moderate, and high VT. After the experimental protocol the thorax was opened and 400 U heparin injected in the right ventricle. The pulmonary artery and left atrium were cannulated, and the pulmonary circulation was flushed of remaining blood by perfusing with buffered salt albumin (BSA) solution containing 135.5 mM Na⁺, 119.1 mM Cl, 25 mM HCO₃, 4.1 mM K⁺, 2.8 mM Mg⁺², 2.5 mM Ca⁺², 0.8 mM SO₄², 8.3 mM glucose, 3% bovine albumin, and osmolality of 300 mOsm/kg H₂O. The solution was maintained at pH ~ 7.40 by bubbling a mixture of 5% CO₂ and 95% O₂ as needed. Two sequential bronchoalveolar lavages (BAL) were performed with 3 ml of BSA solution containing 0.1 mg/ ml Evans blue dye (EBD) (Sigma), 0.02 µCi/ml ²²Na⁺ (DuPont-NEN, Boston, MA), and 0.12 µCi/ml [³H]mannitol (DuPont). The lungs were then instilled with the volume necessary to leave 5 ml in the alveolar space. Finally, the lungs were immersed in a "pleural bath" reservoir containing 100 ml BSA solution maintained at 37° C. This allowed us to follow markers that had moved across the pleura or were drained by the lung lymphatics.

Perfusion of the lungs was performed with 90 ml of the same BSA solution containing 0.16 mg/ml fluorescein isothiocyanate-tagged albumin (FITC-albumin; Sigma). The perfusate was pumped from a lower reservoir to an upper reservoir by a peristaltic pump and from there flowed through the pulmonary artery and exited via the left atrium. Left atrial and pulmonary artery pressures were maintained at 0 and 12 cm H₂O and recorded via a pressure transducer with a zero reference point at the level of the left atrium. Pulmonary artery pressure and left atrial pressure were recorded continuously with a multichannel recorder (Gould 3000 Oscillograph Recorder; Gould Inc., Cleveland, OH).

Samples were drawn from the three reservoirs: airspace instillate, "pleural bath," and perfusate at 10 and 70 min after starting the experimental protocol. To ensure homogeneous sampling from the airspaces, 2 ml of instillate were aspirated and reintroduced into the air-spaces three times before removing each sample. This has been shown to provide a reproducible mixed sample in our laboratory (7, 10, 15). All samples were centrifuged at 1,000 × g for 15 min. Colorimetric analysis of the supernatant for EBD (absorbance at 620 nm) was performed in a Hitachi model U2000 spectrophotometer (Hitachi Inst., San Jose, CA). Analysis of FITC-albumin (excitation 487 nm and emission 520 nm) was performed in a Perkin-Elmer fluorescence spectrometer (model LS-3B; Perkin-Elmer, Oakbrook, IL). ²²Na⁺ and [³H]mannitol were measured in a betacounter (Packard Tricarb, Downers Grove, IL).

Calculations

The alveolar lining fluid volume (VELF) was calculated by instilling 3 ml of fluid (V₀) containing a known concentration of albumin (EBD)₀ tagged by Evans blue dye into the airspace. After brief mixing, a sample was removed and the Evans blue dye concentration at time t (EBD)_t was determined. The mass of Evans blue tagged albumin is the same in the instillate [V₀(EBD)₀] and in the lung after initial mixing [(V₀ + VELF)(EBD)_t]. Equating the two yields:

(1)

or

(2)

Similarly, the alveolar fluid volume at time t (V_t) is estimated by:

(3)

The movement of sodium from the alveolar space during a defined period of time is assumed to be accompanied by isotonic water flux and is given by: J_Na,net = J_Na,out  J_Na,in, where J_Na,net is the net or active Na⁺ transport, J_Na,out is the total or unidirectional Na⁺ outflux from the rat airspace, and J_Na,in is the passive bidirectional flux of Na⁺ between the airspace and the other compartments. The volume flux J = J_Na,net/[Na⁺] is the average rate of change in the volume and is given by:

(4)

As described by Rutschman and coworkers (7), the passive movement of ²²Na⁺, J_Na,in, is given by:

(5)

where C_x is the ²²Na⁺ concentration at time x and [Na⁺] is the constant Na⁺ concentration in the BSA solution. Similarly, the volume flux of mannitol (typically expressed as permeability of surface area PA) is given by:

(6)

where M_x is the [³H]mannitol mass at time x.

Albumin flux from the pulmonary circulation into the alveolar space was determined from the fraction of FITC-albumin that appeared in the alveolar space during the experimental protocol. These calculations were carried out for each sampling period.

ATII Cells Isolation and Na,K-ATPase Activity

ATII cells were isolated from control, low VT, moderate VT, and high VT rats ventilated for 40 min, as previously described (15). Briefly, the lungs were perfused via the pulmonary artery, lavaged, and digested with elastase, 30 U/ml (Worthington Biochemical, Lakewood, NJ) for 20 min at 37° C. The tissue was minced and filtered through sterile gauze and 70-µm nylon mesh. The crude cell suspension was purified by differential adherence to IgG-pretreated dishes, and Na,K-ATPase activity was determined in intact cells as described before (15). Protein was quantified by Bradford assay (19) and 10 µg protein were transferred to the Na,K-ATPase assay medium (final volume 100 µl) containing in mM: NaCl 50, KCl 5, MgCl₂ 10, ethyleneglycol-bis-(-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) 1, TRIS-HCl 50, Na₂ATP 7, and [-³²P]ATP in tracer amounts (3.3 nCi/µl). Cells were transiently exposed to a thermic shock (10 min at 20° C) to render membranes permeable to ATP. The samples were then incubated at 37° C for 15 min, and the reaction was terminated by addition of TCA/charcoal (5%/10% wt/vol) suspension and rapid cooling to 4° C. After separating the charcoal phase (12,000 × g for 5 min) containing the unhydrolyzed nucleotide, the liberated ³²P was counted in an aliquot (200 µl) from the supernatant. Na,K-ATPase activity 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). Nonspecific ATP hydrolysis was determined in samples in the absence of enzyme. The specific activity of the enzyme is expressed in nmol Pi/mg protein/min.

Total RNA Isolation and Reverse Transcriptase-Polymerase Chain Reaction Analysis

Total cellular RNA was isolated from ATII cells from control rats and rats exposed to low, moderate, and high VT for 40 min, using RNeasy total RNA kit (Qiagen Inc., Santa Clarita, CA), as described by the manufacturer, based on the method by Chomczynski and Sacchi (20). RNA was quantified by measurement of absorbance at 260 nm. The reverse transcriptase (RT) reaction was performed using the Superscript Preamplification System (GIBCO-BRL, Gaithersburg, MD) following the manufacturer's instructions. Briefly, 1 µg of total RNA was converted into complementary DNA (cDNA), after denaturing at 70° C for 15 min, by incubation with a buffer containing oligo-dT primers, the RT enzyme, and deoxyribonucleoside triphosphates (dNTPs) mix for 50 min at 42° C. The RT enzyme was then inactivated by incubation at 70° C for 15 min and the RNA removed by incubation with ribonuclease H (RNase H) for 20 min at 37° C. The resultant cDNAs were amplified by polymerase chain reaction (PCR) using a Perkin Elmer 4800 Thermal Cycler (Perkin-Elmer-Cetus, Norwalk, CT). Specific primers for the Na,K-ATPase 1-subunit and rat glyceraldehyde 3-phosphate dehydrogenase (G3PDH) (CLONTECH, Palo Alto, CA) were used (Table 1). The concentration of MgCl₂ was 1.5 mM for each primer pair. For the 1 isoform, amplification was performed as follows: 94° C × 2 min (initial denaturalization), 21 cycles of 94° C × 1 min, 53° C × 1 min 30 s, and 72° C × 2 min, followed by a final extension at 72° C × 7 min. For the G3PDH, amplification was performed as follows: 94° C × 2 min (initial denaturalization), 21 cycles of 94° C × 45 s, 60° C × 45 s, and 72° C × 2 min, followed by a final extension at 72° C × 7 min. 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.

Data Analysis

One-way analysis of variance was used, followed by a multiple comparison test (Tukey) when the F statistic indicated significance. Results were considered significant when p < 0.05. Data are presented as mean values ± SEM.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Rats ventilated with high VT for 25, 40, and 60 min had increased extravascular lung water. The wet/dry lung weight ratio, a gravimetric estimate of pulmonary edema, increased significantly after high VT ventilation for 25, 40, and 60 min compared with low VT, moderate VT, and control nonventilated rats (Table 2). Also, the epithelial lining fluid (ELF) volume estimated by the EBD dilution in the first BAL increased significantly after 60 min of high VT ventilation compared with other groups (Figure 1).

View larger version (17K): [in this window] [in a new window]   Figure 1.   ELF volume increased in lungs from rats ventilated with high VT (HVT) for 60 min. Bars represent means ± SEM. *p < 0.05 compared with low VT (LVT), moderate VT (MVT), and control nonventilated rats. CT = control group.

As shown in Figure 2, control rat lungs cleared approximately 10% of the instilled volume in 1 h (0.50 ± 0.02 ml/h). Rats ventilated with high VT for 40 and 60 min had decreased active Na⁺ transport and lung edema clearance (0.26 ± 0.03 and 0.11 ± 0.08 ml/h, respectively) compared with low VT, moderate VT, and control nonventilated rats.

View larger version (44K): [in this window] [in a new window]   Figure 2.   Lung edema clearance decreased in lungs from rats ventilated with high VT for 40 and 60 min. Bars represent means ± SEM. *p < 0.05, ***p < 0.001 compared with low VT, moderate VT, and control nonventilated rats. CT = control group. Abbreviations as in Figure 1.

The lung permeability for small solutes (²²Na⁺ and [³H]mannitol) increased in rats ventilated with high VT for 40 and 60 min compared with low VT, moderate VT and control nonventilated rats (Figures 3A and 3B). The albumin movement from the pulmonary circulation into the airspace also increased in rats exposed to high VT for 60 min (Figure 4). The Na⁺ concentration in the instillate, perfusate, and "pleural bath" was constant at approximately 140 mmol/L and flow rates did not change in any of the experimental groups (Table 2).

View larger version (22K): [in this window] [in a new window]   Figure 3.   Passive solute movement increased in lungs from rats ventilated with high VT. (A) Passive 22Na+ movement and (B) passive [3H]mannitol movement. Bars represent means ± SEM. *p < 0.05, ***p < 0.01 compared with low VT, moderate VT, and control nonventilated rats. Abbreviations as in Figure 1.

View larger version (15K): [in this window] [in a new window]   Figure 4.   Effects of mechanical ventilation on albumin flux from the pulmonary circulation into the rat alveolar spaces. Bars represent means ± SEM. *p < 0.05 compared with control nonventilated rats. Abbreviations as in Figure 1.

We studied the Na,K-ATPase function in ATII cells isolated from rats ventilated with low, moderate, and high VT for 40 min and compared them with control nonventilated rats. This time period was chosen because the decrease in lung clearance was significant compared with control rats and there were no significant changes in the lung permeability to albumin. As shown in Figure 5, the Na,K-ATPase activity decreased by ~ 25% and ~ 50% in ATII cells isolated from rats ventilated with moderate VT and high VT, respectively, compared with low VT and control nonventilated rats.

View larger version (11K): [in this window] [in a new window]   Figure 5.   Effect of mechanical ventilation on Na,K-ATPase activity. Na,K-ATPase activity decreased in ATII cells isolated from rats after moderate and high VT ventilation for 40 min. *p < 0.05 compared with low VT and control nonventilated rats. Bars represent means ± SEM. Abbreviations as in Figure 1.

We also studied by RT-PCR whether the Na,K-ATPase 1 messenger RNA (mRNA) was modulated by the different protocols of ventilation. As shown in Figure 6, the 1 mRNA steady-state levels did not change in rats ventilated for 40 min with low, moderate, and high VT compared with control rats.

View larger version (19K): [in this window] [in a new window]   Figure 6.   Effect of mechanical ventilation on Na,K-ATPase 1-subunit mRNA. Total RNA was isolated from ATII cells from control rats and rats ventilated with low, moderate, and high VT for 40 min. (A) Representative cDNA amplification using RT-PCR and specific primers for 1 Na,K-ATPase isoforms. (B) Quantitative densitometric scans of four different experiments. Densitometric values were normalized to the G3PDH internal control. cDNA abundance in control rats was arbitrarily defined as 1. Data are presented as means ± SEM. Abbreviations as in Figure 1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Mechanical ventilation with high VT has been shown to increase vascular filtration pressures, capillary stress fracture of the endothelium, epithelium, and basement membrane, and to cause pulmonary edema (1). In the present study we assessed the ability of the lung to clear edema in rats ventilated with high, moderate, and low VT compared with control nonventilated rats. Our data show that mechanical ventilation of rats with high VT decreased the ability of the lung to clear edema, and that this impairment worsened with time of ventilation.

Lung edema clearance decreased by ~ 48% and ~ 78% at 40 min and 60 min of high VT ventilation, respectively, without changes after ventilation with low and moderate VT compared with control rats. After 60 min of high VT, lung edema clearance reduction paralleled increases in permeability to solutes and ELF volume compared with low VT, moderate VT, and control nonventilated rats. The permeability to the small solute Na⁺ increased in rats exposed to high VT ventilation for 40 and 60 min, and permeability for mannitol and albumin only increased after 60 min of high VT ventilation (Figures 3 and 4). The changes in albumin permeability were modest and of similar magnitude to studies in rats with hyperoxic lung injury, where the isolated-perfused fluid-filled lung model has been shown to be accurate for measurement of liquid and solute flux across the alveolocapillary membrane (10, 15, 17).

Previous studies suggest a direct relationship between Na, K-ATPase activity and lung edema clearance. For example, Barnard and coworkers (21) reported that the increase of lung edema clearance by dopamine was mediated via a modulation of active Na⁺ transport by the Na,K-ATPase, and Saldías and coworkers (16) demonstrated that the -adrenergic agonist isoproterenol stimulated lung edema clearance by upregulating both the apical Na⁺ channels and the Na,K-ATPase in the alveolar epithelium. Also, changes in lung edema clearance during acute and subacute hyperoxia paralleled changes in Na,K-ATPase function (15, 17). In a preliminary study we observed, in ATII cells isolated from four rats, that Na,K-ATPase activity increased after 25 min of high VT ventilation to a peak Pao of 35 cm H₂O; however, we did not assess the ability of the lungs to clear edema (22). In the present report, we assessed Na,K-ATPase activity in ATII cells to evaluate whether the decrease in active Na⁺ transport and lung edema clearance paralleled Na,K-ATPase function.

Our data show that in ATII cells harvested from rats ventilated with moderate VT and high VT for 40 min, Na,K-ATPase activity decreased by ~ 25% and ~ 50%, respectively, compared with low VT and control nonventilated rats. Lung edema clearance was decreased only after ventilation of rats with high VT. Under normal physiological conditions the Na,K-ATPase functions at ~ 20 to 50% of its maximal velocity rate (max) (23). In the present report we measured Na,K-ATPase activity using max conditions, therefore changes in activity would reflect changes in the number of functional Na-pumps and not turnover rate. Thus, we reasoned that the reduced Na,K-ATPase activity in both moderate VT and high VT reflects a reduction in the number of functional pumps in ATII cells compared with control rats. We speculate that the remaining pumps in the moderate VT condition were functioning above the basal/resting rate and therefore able to maintain a normal rate of lung edema clearance. In contrast, the larger decrease in Na⁺-pump activity (max) reflecting reduced number of functional pumps in the high VT alveolar epithelial cells was not able to maintain a normal rate of lung edema clearance (see Figures 2 and 5). Although we reason that the lung clearance-Na,K-ATPase function association is probably a cause-effect relationship (i.e., high VT ventilation downregulates alveolar epithelial Na,K-ATPase, which decreases vectorial Na⁺ flux and thus lung ability to clear edema), the previous and our present studies do not unequivocally prove it. Also, it is possible that other systems that we did not study in the present report may affect the resolution of edema clearance such as Na⁺/H⁺ exchanger, Na⁺-HCO₃, Na⁺/glucose cotransporters, sodium channels, or aquaporins (8, 24 -27).

Na,K-ATPase 1 mRNA did not change during mechanical ventilation with low, moderate, or high VT compared with control nonventilated rats. Although it has been reported that mechanical ventilation with high VT in rats increased c-fos mRNA (28), in the present report the changes observed in Na,K-ATPase activity do not appear to be due to transcriptional modifications of Na,K-ATPase.

VALI is associated with significant endothelial and epithelial damage, the structural counterpart of alterations in lung permeability (1). These alterations in permeability are probably caused by the previously described mechanisms such as the stretched pore phenomenon, capillary stress failure, surfactant dysfunction, increased chemokines and metalloproteinases with loss of the structural integrity of heparin sulfate-containing proteoglycans (29). Our study demonstrates that ventilator-associated lung injury not only increases lung permeability to small and large solutes but also decreases active Na⁺ transport and lung edema clearance in rats ventilated with high VT. Ventilation of rats with low (10 ml/kg) or moderate (30 ml/kg) VT does not affect lung ability to clear edema. These findings may have clinical implications in the treatment of patients with hypoxemic respiratory failure.