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J Appl Physiol 85: 1572-1577, 1998;
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Vol. 85, Issue 4, 1572-1577, October 1998

SPECIAL COMMUNICATION
Time course of active and passive liquid and solute movement in the isolated perfused rat lung model

F. J. Saldías1, A. Comellas2, C. Guerrero2, K. M. Ridge2, D. H. Rutschman2, and J. I. Sznajder2

1 Departamento de Enfermedades Respiratorias, Pontificia Universidad Católica de Chile, Santiago, Chile; and 2 Division of Pulmonary and Critical Care Medicine, Michael Reese Hospital, University of Illinois at Chicago, Chicago, Illinois 60616

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The isolated perfused rat lung model (IL) is used to study alveolar epithelial transport properties. Most of the previous studies have been done over a short period of time and have not used the same preparation as a control and intervention group. We evaluated whether the IL preparation could be used for a prolonged period of time (5 h) and studied the rates of active Na+ transport, lung liquid clearance, and passive movement of solutes. Active Na+ transport and lung liquid clearance were stable from 1 to 5 h. The passive movement of small solutes (Na+, mannitol) did not change significantly, and albumin movement increased slightly at the fifth hour. Total RNA isolated from IL after 5 h was intact, and the Na+-K+-ATPase activity in alveolar type II cells isolated at the end of 5-h experiments was equal to Na+-K+-ATPase function from freshly isolated alveolar type II cells. Finally, we measured the stimulatory effect of the beta -adrenergic-agonist terbutaline and the inhibitory effect of the Na+-K+-ATPase-antagonist ouabain by using the same animal as a control. Accordingly, the isolated perfused lung model is functionally stable for at least 5 h, and it could be utilized to evaluate the effect of different interventions by using the same preparation.

isolated perfused lung; active sodium transport; lung edema clearance; lung permeability

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE ISOLATED PERFUSED, fluid-filled rat lung model (IL) is utilized to study lung liquid clearance and passive movement of small (i.e., Na+, sucrose, mannitol) and large solutes (i.e., albumin, dextran) across the alveolar-capillary membrane (1, 4, 8, 12, 15, 16, 18, 20, 21, 25). Variables such as temperature, pH, hydrostatic and osmotic pressures, and concentration of electrolytes can be tightly controlled in this ex vivo model (22). Although lungs are not normally filled with liquid, it has been shown that this model is appropriate for the study of lung edema clearance because it reproduces conditions observed in patients with pulmonary edema, where a liquid-liquid interface occurs (24, 26).

Active and passive movements of Na+ across the alveolar epithelium have been studied in the isolated perfused lung model (1, 4, 8, 12, 15, 16, 18, 20, 21, 25), intact animals (11), and cultured alveolar type II (AT2) cells (5, 13, 17). Na+ enters predominantly via the apical Na+ channels, and it is actively transported out of alveolar epithelial cells by basolaterally located Na+-K+-ATPase (1, 4, 5, 8, 11-13, 18, 22). Water moves passively, following the ionic gradient generated by active Na+ transport, from the alveolar space into the interstitial and pulmonary vascular compartments, probably through water channels (aquaporins) located in alveolar epithelial cells (6).

It has been previously reported that beta -adrenergic agonists stimulate active Na+ transport and lung edema clearance in mammals (3, 9, 14, 20, 23). The stimulatory effect of beta -adrenergic agonists has been demonstrated in several species (sheep, dog, rat, mice, guinea pig), including human lungs (3, 9, 11, 14, 19-21, 23). Most of the previous studies employing the IL have been conducted over a short period of time (~30-60 min) and have not used the same preparation as an internal control to evaluate the effects of different interventions.

The main purposes of this study were 1) to determine whether the isolated rat lung model is functionally stable for a prolonged period of time (up to 5 h) compared with the usual 60-min experiments; 2) to examine whether active Na+ transport and lung permeability to small (Na+, mannitol) and large (albumin) solutes are stable for at least 5 h; and 3) to test whether the same isolated lung preparation could be used to compare the rate of edema clearance of basal conditions to the effects of stimulatory or inhibitory agents.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Pathogen-free, male Sprague-Dawley rats weighing 280-360 g were purchased from Harlan Sprague Dawley (Indianapolis, IN). A total of 42 rat lungs were studied. All animals were provided food and water ad libitum and were maintained on a 12:12-h light-dark cycle. All animals received care in compliance with the Principles of Laboratory Animal Care formulated by the Institute of Laboratory Animal Resources and the Guide for the Care and Use of Laboratory Animals [DHEW Publication No. (NIH) 86-23, Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20892]. Terbutaline and ouabain were purchased from Sigma Chemical (St. Louis, MO).

Isolated Lungs

The isolated lung preparation was performed as previously described (15, 16, 18, 25). Briefly, rats were anesthetized with 50 mg/kg body weight of pentobarbital. A tracheotomy was performed, and the rats were mechanically ventilated with a tidal volume of 2.5 ml, peak airway pressure of 8-10 cmH2O, and 100% O2 for 5 min. The chest was opened via a median sternotomy, after which 400 U of heparin sodium were injected into the right ventricle. After exsanguination, the heart and lungs were removed en bloc. The pulmonary artery and left atrium were catheterized, and the pulmonary circulation was flushed of remaining blood by perfusing with buffered salt albumin (BSA) solution containing (in mM) 135.5 Na+, 119.1 Cl-, 25 HCO-3, 4.1 K+, 2.8 Mg2+, 2.5 Ca2+, 0.8 SO2-4, and 8.3 glucose, as well as 3% bovine albumin, and an osmolality of 300 mosmol/kgH2O. The solution was maintained at pH 7.40 by bubbling a mixture of 5% CO2-95% O2 as needed. Two sequential bronchoalveolar lavages were performed with 3 ml of BSA solution containing 0.1 mg/ml Evans blue dye (EBD; Sigma Chemical), 0.02 µCi/ml 22Na+ (DuPont-New England Nuclear, Boston, MA), and 0.12 µCi/ml [3H]mannitol (DuPont-New England Nuclear). The lungs were then instilled with the volume necessary to leave 5 ml of BSA solution in the alveolar space. Finally, the lungs were immersed in a "pleural bath" reservoir containing 100 ml of BSA solution maintained at 37°C. This allowed us to follow markers that had moved across the pleural membrane 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-tagged albumin (FITC-albumin, Sigma Chemical). The perfusate was pumped from a lower reservoir to an upper reservoir by a peristaltic pump, and from there it flowed through the pulmonary artery and exited via the left atrium. Left atrial and pulmonary arterial pressures were maintained at 0 and 12 cmH2O, respectively, and were continuously recorded via a pressure transducer with a zero reference point at the level of the left atrium.

Samples were drawn from the three reservoirs: air-space instillate, pleural bath, and perfusate at 0, 60, and 120 min, and, in some cases, at 180, 240, and 300 min, depending on the experimental protocol. To ensure homogeneous sampling from the air spaces, 2 ml of instillate were aspirated and reintroduced into the air space three times before each sample was removed. This has been shown in our laboratory (15, 16, 18, 25) to provide a reproducibly mixed sample. 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 spectrophotometer (model U2000, Hitachi Instruments, 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 beta counter (Packard Tricarb, Downers Grove, IL).

Specific Protocols

Group A. To evaluate the functional stability of the preparation, we studied active Na+ transport and lung liquid clearance over 5 h in a control group (n = 18).

Group B. We measured lung liquid clearance and epithelial permeability in basal conditions during the first hour and the stimulatory effect of terbutaline (10-5 M) perfused through the pulmonary circulation during the second hour in the same preparation (n = 6).

Group C. We determined lung liquid clearance and passive movement of solutes in basal conditions in the first hour and the inhibitory effect of ouabain (5 × 10-4 M) added to the perfusate during the second hour in the same preparation (n = 6).

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 EBD into the air space. After a brief mixing, a sample was removed and the EBD concentration at time t, [EBD]t, was determined. The mass of EBD-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
V<SUB>0</SUB>(EBD)<SUB>0</SUB> = (EBD)<SUB><IT>t</IT></SUB>(V<SUB>0</SUB> + V<SUB>ELF</SUB>) (1)
or
V<SUB>ELF</SUB> = V<SUB>0</SUB>(EBD)<SUB>0</SUB>/(EBD)<SUB><IT>t</IT></SUB> − V<SUB>0</SUB> (2)
Similarly, the alveolar fluid volume at time t (Vt) is estimated by
V<SUB><IT>t</IT></SUB> = V<SUB>0</SUB>(EBD)<SUB>0</SUB>/(EBD)<SUB><IT>t</IT></SUB> (3)
The movement of Na+ from the alveolar space during a defined period of time is assumed to be accompanied by isotonic water flux and is given by JNa,net = JNa,out - JNa,in, where JNa,net is the net or active Na+ transport, JNa,out is the total or unidirectional Na+ outflux from the rat air space, and JNa,in is the passive bidirectional flux of Na+ between the air space and the other compartments. Because Na+ concentration ([Na+]) remains constant in all compartments, the net volume flux J = JNa,net/[Na+] is the average rate of change in the volume and is given by
<IT>J</IT> = (V<SUB><IT>t</IT></SUB> − V<SUB>0</SUB>)/<IT>t</IT> (4)
As described in Ref. 18, the passive movement of 22Na+, JNa,in, is given by
<IT>J</IT><SUB>Na,in</SUB> = [Na<SUP>+</SUP>]<IT>J</IT>(ln C<SUB><IT>t</IT></SUB> − ln C<SUB>0</SUB>)/(ln V<SUB><IT>t</IT></SUB> − ln V<SUB>0</SUB>) (5)
where Cx 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
<IT>PA</IT> = <IT>J</IT>(ln M<SUB><IT>t</IT></SUB> − ln M<SUB>0</SUB>)/(ln V<SUB><IT>t</IT></SUB> − ln V<SUB>0</SUB>) (6)
where Mx 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.

Total RNA Isolation in Rat Lung Tissue

To evaluate lung tissue integrity, total RNA was isolated at the end of 5-h IL experiments and in control rat lungs by using the RNeasy total RNA protocol (Qiagen), as described by the manufacturer, on the basis of the method by Chomczyn-ski and Sacchi (2). RNA was quantified by measurement of absorbance at 260 nm. RNA analysis was performed as follows. Total RNA was denaturalized in formamide and formaldehyde by heating at 60°C for 5 min. Then, the RNA samples (10 µg each) containing 1 µl of ethidium bromide (10 mg/ml) were size fractionated on 2.2 M formaldehyde-1.2% agarose gels by electrophoresis at 80 V for 4 h. The presence and integrity of the 28S and 18S ribosomal RNAs were viewed under an ultraviolet illuminator.

AT2 Cell Isolation and Na+-K+-ATPase Activity

To determine whether the Na+-K+-ATPase function was preserved after the 5-h experiments, AT2 cells were isolated at the end of isolated perfused, fluid-filled lung experiments and in control rat lungs. AT2 cell isolation was done as previously described (15, 16, 20). Briefly, the lungs were perfused via the pulmonary artery, lavaged, and digested with elastase, 10 U/ml (Worthington Biochemical) 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 immunoglobulin G-pretreated dishes. AT2 cells were homogenized in homogenization buffer, and basolateral membranes (BLM) were obtained as described by Hammond et al. (10). After several centrifugations to discard the nuclear and mitochondrial pellet, the remaining supernatant was spun at 48,000 g for 30 min. Finally, the BLM fraction was recovered after the membrane pellet was centrifuged in a Percoll gradient (16%) at 48,000 g for 30 min. Na+-K+-ATPase activity was determined in BLM of AT2 cells as the rate of [32P]ATP hydrolysis in suspended cells in buffer containing (in mM) 5 KCl, 10 MgCl2, 1 EGTA, 50 Tris · HCl, and 3 Na2ATP (grade II, Sigma Chemical), as well as [32P]ATP (Amersham, Arlington Heights, IL) in trace amounts. NaCl was added to a final concentration of 100 mM to examine activity at the concentration at which the Na+-K+ pump normally operates at maximal transport rate (>70 mM Na+). Cells were transiently permeated by thermic shock to make them permeable to [32P]ATP. The reaction was carried out for 15 min at 37°C and terminated by placement of cells on ice and addition of a solution containing 5% trichloracetic acid and 10% charcoal to absorb nonhydrolyzed [32P]ATP. The ouabain-insensitive ATPase activity was determined in buffer lacking Na+ and K+ but including 1 mM ouabain. Nonspecific ATP hydrolysis was determined in samples in the absence of cells. The liberated 32Pi was quantified by liquid scintillation counting (Packard Tricarb) and expressed as nanomoles Pi per milligram protein per minute.

Data Analysis

Data are presented as means ± SE, with n representing the number of animals in each experimental group. When comparisons were made between two experimental groups, a paired t-test was used. When multiple comparisons were made, a one-way analysis of variance was used, followed by a multiple-comparison test (Tukey's) when the F-statistic indicated significance. Results were considered significant when P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The unidirectional Na+ flux (JNa,out), which includes both active and passive Na+ movement out of the alveolar space, was stable for the 5 h of the experimental protocol (Fig. 1). Lung liquid clearance rates were of similar magnitude over the 5-h time period (Fig. 2). The lungs of control rats instilled with 5 ml of BSA solution cleared ~8% of the instilled volume each hour. The passive movement of small solutes (22Na+, mannitol) and flow rates across the pulmonary circulation did not change significantly over 5 h (Table 1).


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  		Fig. 1.   Unidirectional Na+ flux did not increase considerably over 5 h in isolated perfused, fluid-filled rat lung model. Active and passive Na+ transports were stable for at least 5 h. Bars, means ± SE.


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  		Fig. 2.   Lung liquid clearance and active Na+ transport did not change significantly over 5 h in isolated perfused, fluid-filled rat lung model. Experimental design is functionally stable for at least 5 h in adult rats. Bars, means ± SE.

                              
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  		Table 1.   Perfusate flow rates and passive sodium and mannitol fluxes in isolated perfused rat lungs

The movement of protein tracers across the alveolar epithelial barrier was similar to the previously reported rates (11, 15, 16, 18, 25). EBD-bound albumin instilled in the air space was not detected in the perfusate or bath compartments in any of the experimental groups. However, the movement of FITC-albumin from the pulmonary vascular compartment into the air space increased slightly at the fifth hour (Fig. 3). The difference between the EBD-albumin and FITC-albumin movement measurements probably represents a higher sensitivity of the FITC detection, which moves from a large (90-ml) space into a much smaller compartment (5 ml), whereas EBD-albumin is moving from a small compartment to an 18-fold-larger compartment, probably falling below the level of detection of our spectrophotometric approach. The [Na+] (~140 mM) was similar in the air-space instillate, pleural bath, and pulmonary circulation and did not change significantly over the 5-h experiments.


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  		Fig. 3.   Movement of FITC-albumin from pulmonary circulation into alveolar space increased slightly at 5th hour in isolated perfused, fluid-filled rat lung model. Bars, means ± SE. ** P < 0.05 compared with 1st and 2nd hours.

Terbutaline (10-5 M) perfused through the pulmonary circulation increased lung liquid clearance by ~132% compared with the basal period (Fig. 4), which is consistent with previously reported stimulation of edema clearance by beta -adrenergic agonists (3, 11, 14, 19-21, 23). Lung liquid clearance decreased by ~75% in rats perfused with the Na+-K+-ATPase-antagonist ouabain (5 × 10-4 M) through the pulmonary circulation, similar to other communications using the same model (1, 8, 20) (Fig. 5). Neither terbutaline nor ouabain changed the passive flux of small and large solutes (Table 1). Stable perfusion pressures were maintained, and flow rates did not change significantly in any experimental group.


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  		Fig. 4.   Terbutaline perfused through pulmonary circulation increased lung liquid clearance in isolated perfused rat lungs. Bars, means ± SE; CT, control (1st hour); Terb, 10-5 M terbutaline (2nd hour). * P < 0.01 compared with CT.


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  		Fig. 5.   Ouabain perfused through pulmonary circulation decreased lung liquid clearance in isolated perfused rat lungs. Ouab, 5 × 10-4 M ouabain (2nd hour); bars, means ± SE. * P < 0.01 compared with CT.

To assess whether lung tissue integrity was preserved over the 5-h experiments, total RNA extracted at the end of isolated perfused rat lung experiments and analyzed by agarose gel electrophoresis showed that the 18S and 28S ribosomal RNAs were intact compared with ribosomal RNAs extracted from fresh lung tissue (Fig. 6).


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  		Fig. 6.   Lung tissue structure is preserved after 5-h experiments in isolated perfused rat lung model. IL, isolated perfused, fluid-filled rat lung experiments over 5 h. Agarose gel electrophoresis shows that 18S and 28S ribosomal RNAs are intact compared with CT fresh lung tissue (n = 3/group).

Also, the Na+-K+-ATPase activity measured in BLM of AT2 cells isolated at the end of 5-h experiments was comparable to the Na+-K+-ATPase function determined in freshly isolated AT2 cells from control rat lungs (Fig. 7).


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  		Fig. 7.   Na+-K+-ATPase activity in alveolar type II cells isolated at end of 5-h IL experiments did not change significantly compared with CT rat lungs (n = 3/group). Bars, means ± SE.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have developed a physiological model to measure the active and passive movement of liquid and solutes across the rat lung epithelium, whereby this preparation can be utilized for up to 5 h without significant alteration in the epithelial function. Therefore, we reason that the same lung preparation could be used to examine the stimulatory or inhibitory effects of different agents added to the pulmonary circulation after a control period. The lung liquid clearance and passive movement of small solutes did not change significantly over 5 h; meanwhile, there was only a slight increase in albumin movement during the fifth hour compared with the 60-min studies (see Fig. 3). This minimal increment in the permeability for albumin may indicate the initial deterioration of the isolated lung preparation, but it still allows us to accurately assess lung liquid clearance and active ion transport, as we have previously reported in rats exposed to hyperoxic lung injury where the albumin flux was significantly above control levels (15, 16, 21, 25).

The isolated perfused, liquid-filled lung model has been used to investigate the alveolar epithelium transport properties over the last decade (1, 3, 4, 8, 12, 15, 16, 18, 21-23, 25). The physiological ex vivo model has provided valuable information about lung edema clearance regulation in normal and pathological conditions. However, this model has some limitations that must be considered. For example, it is not possible to determine exactly which cell type is responsible for the active ion transport and what the relative contribution is of small-airway epithelium to overall pulmonary edema resolution. Also, the rate of active ion transport is lower than that of passive bidirectional flux, and thus small changes in lung liquid clearance may not be easily demonstrated (18). Nevertheless, in this model the hydrostatic and colloid-osmotic pressure gradients across the alveolar-capillary barrier, pH, temperature, and composition of the solution added into the air space and pulmonary circulation can be tightly controlled. In agreement with previous short-term studies using the same preparation (1, 3, 4, 8, 12, 15, 18, 21-23, 25), we have demonstrated that the isolated perfused rat lung model is stable at least for 5 h, and with the experimental conditions tightly controlled, the results are highly reproducible. Thus this new approach allows us to improve the ability to study temporal changes in lung edema clearance because we are using the same preparation as a control, thus avoiding interindividual variability.

To evaluate the functional stability of the long-term preparation, we measured the total RNA in lung tissue and the Na+-K+-ATPase activity in BLM of AT2 cells isolated after the 5-h isolated perfused lung experiments. We have shown that total RNA and Na+-K+-ATPase function in alveolar epithelial cells are preserved in the isolated lung preparation after 5 h compared with control rat lungs (see Figs. 6 and 7).

beta -Adrenergic agonists stimulate active Na+ transport and lung edema clearance in vivo, in isolated perfused lungs, and in cultured AT2 cells (3, 9, 11, 17, 20, 22). In agreement with these previous studies, we have shown that terbutaline increases active Na+ transport and lung liquid clearance in isolated perfused rat lungs (see Fig. 4). The stimulatory effect of beta -adrenergic agonists could be demonstrated by using the same preparation as a control; the increase in active Na+ transport is of a magnitude similar to that in previous reports (3, 11). In the same model, ouabain significantly inhibited active Na+ transport and lung edema clearance without affecting the passive movement of small and large solutes across the alveolar-capillary membrane. The inhibitory effect of ouabain on the Na+-K+-ATPase function reported by our study agrees with previous communications using a similar model (1, 8).

In summary, our data demonstrate for the first time that the isolated perfused, fluid-filled rat lung model is stable for at least 5 h and can be utilized to evaluate the effects of different interventions by using the same preparation. This could improve the accuracy of our physiological studies evaluating lung edema clearance and active Na+ transport pathways.

    ACKNOWLEDGEMENTS

This research was supported in part by National Heart, Lung, and Blood Institute Grant HL-48129 and grants from the American Heart Association (96012890), the Research and Education Foundation of the Michael Reese Medical Staff, and Pontificia Universidad Católica de Chile. K. Ridge is the recipient of the National Research Service Award.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: J. I. Sznajder, Dept. of Medicine, Michael Reese Hospital and Medical Center, 2929 S. Ellis Ave., Baum 101, Chicago, IL 60616.

Received 1 April 1998; accepted in final form 5 June 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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J APPL PHYSIOL 85(4):1572-1577
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