defective respiratory amiloride-sensitive sodium transport predisposes to pulmonary oedema and...

J Physiol 560.3 (2004) pp 857–865 857

Defective respiratory amiloride-sensitive sodium transportpredisposes to pulmonary oedema and delaysits resolution in mice

Marc Egli1,2, Herve Duplain1,2, Mattia Lepori1,2, Stephane Cook1,2, Pascal Nicod1, Edith Hummler3,Claudio Sartori1,2 and Urs Scherrer1,2

1Department of Internal Medicine and 2the Botnar Center for Clinical Research, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland3Institute of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland

Pulmonary oedema results from an imbalance between the forces driving fluid into theairspace and the biological mechanisms for its removal. In mice lacking theα-subunit of the amiloride-sensitive sodium channel (αENaC(−/−)), impairedsodium transport-mediated lung liquid clearance at birth results in neonatal death.Transgenic expression of αENaC driven by a cytomegalovirus (CMV) promoter(αENaC(−/−)Tg+) rescues the lethal pulmonary phenotype, but only partiallyrestores respiratory sodium transport in vitro. To test whether this may also be truein vivo, and to assess the functional consequences of this defect on experimentalpulmonary oedema, we measured respiratory transepithelial potential difference (PD)and alveolar fluid clearance (AFC), and quantified pulmonary oedema during experimentalacute lung injury in these mice. Both respiratory PD and AFC were roughly 50% lower(P < 0.01) in αENaC(−/−)Tg+ than in control mice. This impairment was associated witha significantly larger increase of the wet/dry lung weight ratio in αENaC(−/−)Tg+ than incontrol mice, both after exposure to hyperoxia and thiourea. Moreover, the rate of resolutionof thiourea-induced pulmonary oedema was more than three times slower (P < 0.001) inαENaC(−/−)Tg+ mice.αENaC(−/−)Tg+ mice represent the first model of a constitutivelyimpaired respiratory transepithelial sodium transport, and provide direct evidence thatthis impairment facilitates pulmonary oedema in conscious freely moving animals. Thesedata in mice strengthen indirect evidence provided by clinical studies, suggesting thatdefective respiratory transepithelial sodium transport may also facilitate pulmonary oedemain humans.

(Received 19 April 2004; accepted after revision 9 August 2004; first published online 12 August 2004)Corresponding author U. Scherrer: Department of Internal Medicine, BH 10.642, Centre Hospitalier UniversitaireVaudois, CH-1011 Lausanne, Switzerland. Email: [email protected]

Pulmonary oedema is a life-threatening condition,resulting from a persistent imbalance between theforces driving fluid into the airspace and the biologicalmechanisms for its removal (Staub, 1974). Activevectorial transepithelial sodium transport, mediatedmainly by the apical amiloride-sensitive sodium channel(ENaC) and basolateral ouabain-sensitive sodium pump(Na+–K+-ATPase) (Matalon & O’Brodovich, 1999), isa major driving force of alveolar fluid clearance (AFC)(Matthay et al. 1998; Sartori et al. 2001). Mice withdeletion of the gene encoding for the α-subunit ofthe amiloride-sensitive sodium channel (αENaC(−/−))(Canessa et al. 1994) die within the first hours of life

C. Sartori and U. Scherrer contributed equally to this work

from failure to clear the fetal lung fluid (Hummler et al.1996). However it is difficult to extrapolate observationsmade during the first hours of life to the adult period,as the perinatal transition is characterized by uniquephysiological changes in the lung, as reflected by AFC ratesin newborn guinea pigs that are nearly two times fasterthan after maximal pharmacological stimulation in adultlife (Finley et al. 1998).

Due to the lack of an adult in vivo model of defectiverespiratory sodium transport, the study of the specificcontribution of this transport to the pathogenesis ofpulmonary oedema was limited to measurements ofexogenous fluid clearance in anaesthetized and ventilatedanimals, and in ex vivo animal preparations over shortperiods of time, during experimental interventionsintended to stimulate or inhibit transepithelial sodium

C© The Physiological Society 2004 DOI: 10.1113/jphysiol.2004.066704

858 M. Egli and others J Physiol 560.3

transport (Matthay et al. 1982; Pittet et al. 1994; Garatet al. 1997, 1998; Finley et al. 1998; Campbell et al. 1999;Charron et al. 1999; Folkesson et al. 2000; Hardimanet al. 2001). Introduction of a rat αENaC transgeneunder a heterologous CMV promoter into the αENaCknockout background (αENaC(−/−)Tg+) restores ENaCresponsiveness to physiological and pharmacologicalstimuli in respiratory cells in vitro (Olivier et al. 2002),and rescues the lethal pulmonary phenotype (Hummleret al. 1997). αENaC(−/−)Tg+ mice develop normally.However, compared with the endogene, mRNA expressionof the transgene is lower in the kidney, colon, andlung (Hummler et al. 1997; Olivier et al. 2002), andamiloride-sensitive sodium transport is impaired intracheal explants in vitro (Olivier et al. 2002). We hypo-thesized that αENaC(−/−)Tg+ mice may represent an invivo model of defective transepithelial respiratory sodiumtransport, and allow us to study its role in the pathogenesisof pulmonary oedema in adult life.

We therefore measured nasal and trachealtransepithelial potential difference (PD), an indexof the electrogenic transport of Na+ and Cl− ions acrossthe distal respiratory epithelium (Boucher et al. 1980;Knowles et al. 1982; Grubb et al. 1994; Kelley et al. 1997),and AFC in αENaC(−/−)Tg+ and control mice in vivo.We found that both PD and AFC were defective. Toexamine the functional consequences of this defect, wecompared the severity and time course of thiourea- andhyperoxia-induced pulmonary oedema (Cunningham &Hurley, 1972; Mais & Bosin, 1984; Zuege et al. 1996; Songet al. 2000) in αENaC(−/−)Tg+ and control mice.

Methods

General experimental protocol

The total number of mice used in this study(αENaC(−/−)Tg+ mice and αENaC(+/+)Tg- controllittermates) was 172. Mice were matched for age(12–16 weeks), sex and weight in all the experimentalgroups. Generation of the transgenic mice on a NMRIgenetic background and breeding were as previouslydescribed (Hummler et al. 1997). Animals were housedin standard cages and light conditions, and fed standardrodent chow and water ad libitum. The experiments wereapproved by the institutional reviewing board on animalexperimentation.

Measurement of nasal and tracheal transepithelial PD.For the measurement of nasal PD, a modification ofpreviously described techniques was used (Boucheret al. 1980; Grubb et al. 1994; Ghosal et al. 1996).Briefly, mice were anaesthetized (ketamine, 0.1 mg (g bodyweight)−1 and xylazine, 0.01 mg (g body weight)−1; i.p.,supplemented with additional doses throughout the

experiment if necessary, to maintain an adequate depthof anaesthesia, i.e lack of motor and heart rate responseto tail and/or paw pinch), and placed on a heatingtable to keep their body temperature between 37◦C and38◦C. A stretched PE-10 tubing filled with pre-warmed(37◦C) Ringer solution was inserted into the nostrilof the spontaneously breathing mouse. The intranasalrecording bridge and a subcutaneous reference bridge (anagar/Ringer solution-filled sterile 21-gauge needle), werelinked by matched electrodes (Dri-Ref 5, World PrecisionInstruments Inc. Sarasota, FL, USA) to a high impedancevoltmeter (ISOMIL, World Precision Instruments Inc.).The recording site was located at a depth of 2 mm fromthe nares. Once in place, the recording bridge wasstabilized, and a stable plateau value was obtained for atleast 30 s. The recording site in each nostril was revisitedtwice, and nasal PD was expressed as the average of thefour measurements obtained for each animal.

Tracheal PD was measured by placing the recordingbridge into the proximal part of the trachea in miceundergoing mechanical ventilation for the measurementof AFC (see below).

Measurement of in vivo AFC. AFC was measured bystandard gravimetric method in anaesthetized (as above),paralysed (pancuronium, 0.01 mg (g body weight)−1,i.p), tracheotomised, mechanically ventilated (MouseVentilator Model 687, Harvard Apparatus, Inc.) mice.After 15 min of a stable baseline period, mice were instilledwith Ringer solution containing 5% bovine serum albumin(BSA; Sigma) supplemented with concentrated salinesolution to make it isosmolar (320 mosm l−1) with mouseplasma (Ma et al. 1998). Pre-warmed (37.5◦C) solution(8 µl g−1) was instilled via the endotracheal cannula intoboth lungs. An alveolar fluid sample was collected byaspiration 15 min after instillation. AFC was expressedas percentage of the instilled fluid volume absorbed after15 min, calculated from the final-to-instilled total albuminconcentration ratio (Garat et al. 1998; Charron et al. 1999;Fukuda et al. 2000; Folkesson et al. 2000).

Measurement of lung barrier permeability. To estimatethe leak of a vascular tracer protein (125I-albumin,injected intravenously 1 h before the end of theexperiments) into the extravascular compartments ofthe lung (lung interstitium and air spaces), the totalextravascular [125I]albumin accumulation in alveolarliquid recovered from the air spaces and the lunghomogenate was measured, and expressed as extravascularplasma equivalents (Rezaiguia et al. 1997).

Measurement of pulmonary oedema. As an index oflung oedema, the amount of extravascular lung waterwas calculated according to established techniques (Pittet

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J Physiol 560.3 Defective respiratory sodium transport and pulmonary oedema 859

et al. 1994, 2001; Zuege et al. 1996). Briefly, animalswere anaesthetized, as described above, and killed byexsanguination, the lungs were removed and the wetweight was recorded. The lungs were then placed inan incubator at 80◦C for 24 h, and the dry weight wasrecorded. For each animal, the wet/dry weight ratio wascalculated. Lung homogenate supernatant haemoglobincontent was measured to calculate bloodless lung wet/dryweight ratios.

Classical wet/dry weight ratios were used to studythe time course of the resolution of thiourea-inducedpulmonary oedema. Bloodless wet/dry lung weight ratioswere used to compare pulmonary oedema in animalswith hyperoxic or thiourea-induced lung injury in orderto exclude confounding effects of alveolar haemorrhageduring hyperoxia.

Lung histology. Mice were killed as described above. Thetrachea was cannulated and connected to a syringe beforethoracotomy, in order to prevent lung collapse and tore-adjust the lung volume to the thorax volume beforefixation. Lungs were fixed by sequential immersion inisosmolar 1.5% glutaraldehyde, osmium tetroxide anduranyl acetate. Blocks (1 mm3) were excised from theright middle lobe, embedded in Epon resin, cut in 1-µmthin sections and stained with toluidine blue for lightmicroscopic examination (Bachofen et al. 1993).

Specific experimental protocols

Nasal and tracheal PD. Baseline measurements (n = 6mice for each group) were performed before and afteradministration of drug-free water as vehicle aerosol(in order to control for non-specific effects of theaerosolization on nasal PD). The values after vehicleaerosol were used as baseline values. The aerosol wasgenerated by a nebulizing system (Respirgard-II, MarquestInc., Englewood, CO, USA) run at 8 l min−1 for 2 min,resulting in aerosolization of 1 ml min−1 of aerosolsolution. Anaesthetized mice were breathing the aerosolthrough a custom-built open-flow face mask. Vehicleaerosol did not alter nasal PD. In the same animals wealso measured the amiloride-sensitive fraction of nasal PDafter a 2-min aerosolization of amiloride (10−3 m dissolvedin water) (Ghosal et al. 1996; Tomlinson et al. 1999). Micewere allowed at least 48 h of recovery between the twomeasurements. Amiloride and vehicle were administeredin random order.

The effects of mechanical ventilation on nasal PDand the relationship between nasal and tracheal PDwere studied in mechanically ventilated mice prepared asdescribed in the section on AFC, above (n = 5 mice foreach group).

AFC under normal conditions. Baseline AFC wasquantified in αENaC(−/−)Tg+ and control mice (n = 6

for each group) by instillation of the 5% BSA solutionprepared as described above.

To measure the amiloride-sensitive fraction of AFCin αENaC(−/−)Tg+ and control mice (n = 6 for eachgroup), Amiloride (10−3 m) was added to the 5% BSAsolution.

Thiourea-induced pulmonary oedema. Thiourea causesacute lung oedema by increasing vascular permeability(Cunningham & Hurley, 1972). In normal mice, itseffect peaks around 4 h after injection, and the timefor resolution of the oedema is ∼12 h (Mais & Bosin,1984). Preliminary experiments revealed a dose–responserelationship with a maximal effect at a dose of 40 mg kg−1

i.v. This dose was subsequently used.Lungs were excised 4 h after intravenous injection of

thiourea or saline for lung histology or measurement ofthe bloodless wet/dry lung weight ratio. To study the timecourse of lung oedema, classical wet/dry weight ratioswere measured 3, 4, 5, 6 and 7 h (n = 4–6 mice pergroup at each time point) after thiourea injection. Therate of resolution of pulmonary oedema was expressedas percentage decrease per hour of the peak increaseof the wet/dry lung weight ratio over baseline values.To quantify AFC during thiourea-mediated lung injury,αENaC(−/−)Tg+ and control mice (n = 5 for eachgroup) were instilled with 5% BSA 4 h after thioureaadministration (40 mg kg−1 i.v). In order to accountfor the initial dilution of the instillate by the presenceof pulmonary oedema, we collected an additional alveolarfluid sample 1 min after instillation, and calculatedAFC from the albumin concentration changes over thefollowing 15 min in these groups (Hardiman et al.2001).

Hyperoxia-induced pulmonary oedema. Hyperoxic lunginjury was induced by exposing the mice to an inspiredO2 fraction (F i,O2 ) > 98% in a sealed Plexiglas chamber.Lung histology, bloodless wet/dry lung weight ratio andAFC were measured after 72 h of hyperoxia, using thetechniques described in the previous sections.

All measurements and calculations were carried out byan investigator who was unaware of mouse genotype.

Statistical analysis

Data were analysed with the JMP software package (SASInstitute Inc.). Statistical analysis was performed withtwo-way ANOVA for between-group comparisons as afunction of time, and with two-tailed paired or unpairedt tests for single comparisons. Relations between variableswere analysed by calculating Pearson’s product-momentcorrelation coefficient. Unless otherwise indicated, dataare given as means ± s.d. A P-value below 0.05 wasconsidered to indicate statistical significance.



Figure 1. Nasal PD in αENaC(−/−)Tg+ and control miceNasal PD (a marker of transepithelial sodium transport) inanaesthetized, ventilated, transgenic αENaC(−/−)Tg+ and controlmice after nebulization with drug-free water as vehicle (BL) or 10−3 M

amiloride (AM). Data are mean ± S.E.M. for six mice under eachcondition (each animal served as its own control). #P < 0.01 versuscontrol littermates; ∗P < 0.01 versus corresponding vehicle inhalation.

Results

Nasal and tracheal PD

Baseline nasal PD was almost 40% lower inαENaC(−/−)Tg+ than in control mice (12.2 ± 1.4 mVversus 20.0 ± 3.2 mV, P < 0.001, Fig. 1). Similarly, trachealPD was significantly lower in αENaC(−/−)Tg+ thanin control mice (8.3 ± 1.8 mV versus 13.1 ± 2.8 mV,P < 0.05). Amiloride induced a significant decrease ofthe nasal PD in both wild-type and transgenic mice.However, this decrease was more than five times smallerin the transgenic than in the control mice (1.8 ± 2.1versus 10.1 ± 4.2 mV, P < 0.001). Residual nasal PDsafter amiloride were comparable in the two groups(10.4 ± 1.6 mV versus 9.9 ± 1.4 mV, Fig. 1). Nasal PD wascomparable in spontaneously breathing and mechanicallyventilated mice, and in ventilated mice, nasal and trachealPDs were closely correlated (r = 0.95, P < 0.001, Fig. 2).

Figure 2. Correlation between tracheal and nasal PDCorrelation between tracheal and nasal PD in anaesthetized,ventilated, transgenic αENaC(−/−)Tg+ (�) and control mice (�).r = 0.95; P < 0.001.

Figure 3. AFC in αENaC(−/−)Tg+ and control miceAFC in anaesthetized, ventilated, transgenic αENaC(−/−)Tg+ andcontrol mice under baseline conditions (BL), and after inhibition ofsodium transport by amiloride (AM, 10−3 M). Data are mean ± S.E.M.of six experiments for each group, and are expressed as percentage ofinstilled albumin solution absorbed within 15 min. #P < 0.001 versuscontrol mice; ∗P < 0.001 versus corresponding baseline.

AFC under normal conditions

Consistent with the findings for nasal PD, baseline AFCwas 55% lower in αENaC(−/−)Tg+ than in controlmice (6.0 ± 1.9 versus 13.4 ± 1.9%, P < 0.001, Fig. 3).The amiloride-induced decrease of AFC in control mice(−69%, P < 0.001 versus baseline) was nearly three timeslarger than in αENaC(−/−)Tg+ mice (−24%, P = 0.36versus baseline). The amiloride-insensitive fractions ofAFC were comparable in the two groups (Fig. 3).

There was a significant correlation between AFC andnasal PD (r = 0.81, P < 0.01, Fig. 4)

Thiourea-induced lung oedema

At baseline, the macroscopic appearance of the lungswas normal in transgenic and control mice. The blood-less wet/dry lung weight ratios were similar in bothgroups (4.18 ± 0.33 versus 4.26 ± 0.56, P = 0.73, Fig. 5),and no alveolar fluid was detectable on histologicalexamination (Fig. 7A and B), indicating that under

Figure 4. Correlation between nasal PD and AFCCorrelation between nasal PD and AFC in anaesthetized, ventilated,transgenic αENaC(−/−)Tg+ (�) and control mice (�). r = 0.81;P < 0.01.



Figure 5. Bloodless wet/dry lung weight ratio inαENaC(−/−)Tg+ and control miceBloodless wet/dry lung weight ratio in control (�) and transgenicαENaC(−/−)Tg+ mice (�) under baseline conditions, 4 h afterintraperitoneal administration of thiourea, and after 72 h of hyperoxia(F i,O2 > 98%). Data are mean ± S.E.M. for eight mice per group andcondition. #P < 0.001 versus control littermates; ∗P < 0.05 versuscorresponding baseline.

normal conditions, defective AFC in αENaC(−/−)Tg+mice was not associated with fluid accumulation inthe lung. In contrast, after thiourea administration, thepeak increase of the bloodless wet/dry weight ratio wassignificantly larger in the transgenic than in the controlmice (5.87 ± 0.38 versus 4.82 ± 0.25, P < 0.01; Fig. 5),and the rate of resolution of pulmonary oedema wasmore than three times slower (6.9 versus 22.2% decreaseof the wet/dry weight ratio per hour, P < 0.001, Fig. 6).Three hours after the peak increase, the wet/dry lungweight ratio had returned to near-baseline values inwild-type animals, whereas 80% of the excess waterwas still present in the lungs of the transgenic mice.Consistent with these findings, histological examinationof the lung, harvested 4 h after thiourea administration,revealed alveolar oedema that was more marked inαENaC(−/−)Tg+ than in control mice (Fig. 7C and D).

Figure 6. Time course of thiourea-induced pulmonary oedemain αENaC(−/−)Tg+ and control miceTime course of thiourea-induced pulmonary oedema as reflected bythe classical wet/dry lung weight ratio. Resolution of pulmonaryoedema was significantly slower (P < 0.001) in transgenicαENaC(−/−)Tg+ (�) than in control mice (�). Data are mean ± S.E.M.for n = 4–6 mice per group and time point.

Hyperoxia-induced lung oedema

After hyperoxia, the differences between the two groupswere even more pronounced than after thiourea.The increase in bloodless wet/dry weight ratio wasnearly six times larger in αENaC(−/−)Tg+ than incontrol mice (from 4.3 ± 0.6 to 7.2 ± 1.2 versus from4.2 ± 0.3 to 4.6 ± 0.6, respectively, P < 0.001; Fig. 5).αENaC(−/−)Tg+ mice showed more severe symptoms(lethargy and respiratory distress) of lung oedema; atthe opening of the chest, their lungs appeared dark redand stiffened, whereas the lungs of control mice wereonly slightly discoloured. Histological examination ofthe lung revealed marked alveolar oedema with entire

Figure 7. Photomicrographs of lung oedema inαENaC(−/−)Tg+ and control micePhotomicrographs of representative lung sections of transgenic (rightpanels) and control mice (left panels) under baseline conditions (A andB), 4 h after thiourea administration (C and D), and after 72 h ofexposure to hyperoxia (E and F). Arrows indicate the presence ofalveolar oedema. Experimental pulmonary oedema is more severe intransgenic αENaC(−/−)Tg+ than in control mice. The interstitialcomponent of oedema appears comparable in the two groups (l00×magnification; scale bar, 50 µm).



acini being fluid-filled and collapsed in αENaC(−/−)Tg+mice, whereas alveolar oedema was barely detectable incontrol mice (Fig. 7E and F). Interstitial oedema appearedcomparable in the two groups.

AFC and lung barrier permeability duringexperimental pulmonary oedema

AFC. The difference of AFC between αENaC(−/−)Tg+ and control mice was maintained duringexperimental pulmonary oedema (thiourea: αENaC(−/−)Tg+, 5.7 ± 2.6; control, 15.7 ± 4.1%, P < 0.01;hyperoxia: αENaC(−/−)Tg+, 5.8 ± 2.6; control, 10.0 ±0.9%, P < 0.01).

Lung barrier permeability (125I-albumin studies). Baselinealbumin flux out of the vascular space was comparablein αENaC(−/−)Tg+ and control mice (47.7 ± 6.0 and41.8 ± 7.6 µl g−1 of lung tissue, respectively). Thioureaand hyperoxia significantly and comparably increasedthe lung vascular albumin leak in wild-type andtransgenic mice (thiourea: 85.8 ± 2.8 and 98.7 ±9.0 µl g−1 lung tissue, respectively; hyperoxia: 63.1 ±11.5 and 77.1 ± 14.5 µl g−1 lung tissue, respectively),suggesting that differences in lung barrierpermeability did not contribute to the augmentedsusceptibility to pulmonary oedema in the transgenicmice.

Discussion

There is abundant evidence that active transepithelialsodium transport is an important driving force forthe removal of liquid from the airspace of the lungunder normal (Matthay et al. 1982) and pathologicalconditions (Pittet et al. 1994; Yue & Matalon, 1997; Garatet al. 1997; Campbell et al. 1999; Charron et al. 1999).However its quantitative importance in the pathogenesis ofpulmonary oedema has been difficult to establish becauseof the lack of a suitable model of impaired respiratorysodium transport in vivo. Here we provide such a model,by demonstrating that αENaC(−/−)Tg+ mice had aroughly 50% lower respiratory transepithelial PD and AFCcompared to control mice. This constitutive impairmentof the respiratory sodium transport had importantpathophysiological consequences, as it augmented theseverity and delayed the resolution of experimentalpulmonary oedema in conscious, freely moving mice.

Transepithelial PD has been used extensively as anindirect bioelectric marker of in vivo respiratory sodiumtransport in mice (Grubb et al. 1994; Kelley et al. 1997)and humans (Boucher et al. 1980; Knowles et al. 1982;Kerem et al. 1999; Sartori et al. 2002). Here we showthat nasal and tracheal PD were almost 50% smallerin αENaC(−/−)Tg+ mice than in wild-type littermates.Consistent with earlier data (Boucher et al. 1980; Knowles

et al. 1982), the values measured in the upper and lowerairways were closely correlated. The defect of therespiratory ion transport in αENaC(−/−)Tg+ micewas almost entirely related to its amiloride-sensitivecomponent, as evidenced by residual PDs that werecomparable after amiloride treatment in the twogroups. The significantly smaller respiratory PD inαENaC(−/−)Tg+ mice was mirrored by a quantitativelysimilar impairment of AFC. The amiloride-sensitivefraction of the AFC almost entirely accounted for thisimpairment. Moreover, there was a close relationshipbetween respiratory PD and AFC measurements. The latterobservation represents the first direct demonstration thatthe respiratory PD is a marker of AFC in the more distalairways.

Taken together, these data indicate that respiratorytransepithelial sodium transport and sodiumtransport-driven AFC are significantly impaired inαENaC(−/−)Tg+ mice, and that the amiloride-sensitive,ENaC-mediated component accounts for the main partof this impairment. These findings are in accordance withdata showing decreased pulmonary mRNA levels of thetransgene in αENaC(−/−)Tg+ mouse lungs (Hummleret al. 1997), a markedly lower amiloride-sensitive fractionof the transepithelial short-circuit current in primarycultures of tracheal cells from αENaC(−/−)Tg+ micein vitro (Hummler et al. 1997), and an almost abolishedamiloride-sensitive rectal PD in adult transgenic micein vivo (Hummler et al. 1997). The insensitivity toamiloride in vivo, in the present and these earlier studies,is probably related to a low expression of the transgenerather than to defective regulation of ENaC, becausetracheal explants from ENaC transgenic mice respondnormally to pharmacological and environmental stimuli(Olivier et al. 2002).

The impairment of the respiratory sodium transportand AFC in αENaC(−/−)Tg+ mice had no patho-physiological consequences under normal conditions,as evidenced by the normal wet/dry lung weightratio and lung histology in both groups. However,in the presence of augmented alveolar fluid floodingafter exposure to hyperoxia or thiourea administration,αENaC(−/−)Tg+ mice showed an exaggerated increaseof the wet/dry lung weight ratio and more severepulmonary oedema on histological examination. Thisdifference does not appear to be related to differencesin blood pressure, sympathetic tone or propensity todevelop heart failure between the two groups (data notshown). The more severe pulmonary oedema was relatedspecifically to defective sodium transport-dependent fluidclearance, as the difference of AFC between the twogroups persisted during experimental pulmonary oedema,whereas the alveolo-capillary barrier permeability inαENaC(−/−)Tg+ and control mice was comparableunder all experimental conditions.



Earlier in vivo studies on the role of transepithelialsodium transport in the pathogenesis of pulmonaryoedema were limited to the measurement of the clearanceof exogenous liquid over short time periods, duringexperimental interventions intended to alter the trans-epithelial sodium transport in anaesthetized, paralysedand ventilated animals (Matthay et al. 1982; Pittetet al. 1994; Garat et al. 1997, 1998; Finley et al. 1998;Campbell et al. 1999; Charron et al. 1999; Folkesson et al.2000; Hardiman et al. 2001). Here, αENaC(−/−)Tg+mice allowed us, for the first time, to directly assessthe functional consequences of an impaired respiratorytransepithelial sodium transport on the time course ofexperimental pulmonary oedema over an extended timeperiod of up to 72 h in conscious, freely moving micein vivo.

A few studies in humans have addressed thepotential importance of respiratory transepithelial

Figure 8. Effects of reduced or absent pulmonary expression of αENaC on pulmonary fluid homeostasisSchematic diagram showing the effects of reduced or absent pulmonary expression of αENaC on pulmonary fluidhomeostasis under normal conditions, and under pathological conditions associated with augmented alveolarfluid flooding. Absence of pulmonary αENaC expression in αENaC(−/−) mice results in neonatal death becauseof failure to clear the fluid from the fetal lung. Decreased pulmonary αENaC expression in αENaC(−/−)Tg+mice impairs transepithelial sodium and water transport. Under normal conditions, this impairment of AFC has nopathophysiological consequences. However in the presence of augmented alveolar fluid flooding after experimentallung injury, this impairment augments the severity and delays the resolution of pulmonary oedema.

sodium transport in fluid homeostasis of the lung. Inpremature infants, pulmonary oedema in respiratorydistress syndrome is associated with a transient decreaseof the nasal PD (Barker et al. 1997). In patients susceptibleto high-altitude pulmonary oedema, nasal PD islower than in mountaineers resistant to this condition(Sartori et al. 2004), and prophylactic stimulation oftransepithelial sodium transport with a β-adrenergicagonist prevented pulmonary oedema duringhigh-altitude exposure in susceptible subjects (Sartoriet al. 2002). The present demonstration of a closerelationship between respiratory PD, AFC andsusceptibility to experimental pulmonary oedemain mice, strengthens the indirect evidence provided bythese clinical studies, and is consistent with the novelconcept that defective respiratory transepithelial sodiumtransport may facilitate pulmonary oedema in humans(Fig. 8).



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Acknowledgements

The assistance of Reynald Olivier and Caroline Mathieu isgratefully acknowledged. Special thanks go to Camille Angladafor his invaluable help in setting up the measurement equipmentand to Professor Hans Bachofen and Mrs Ursula Gerber for theirexpert help in processing and histological analysis of lung tissue.This work was supported by grants from the Swiss NationalScience Foundation (32.46797.96 and 3238–051157.97), theProfessor Dr Max Cloetta Foundation and the Placide NicodFoundation.


defective respiratory amiloride-sensitive sodium transport predisposes to pulmonary oedema and...

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