AJP - Lung AJP: Renal Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 290: L478-L484, 2006. First published October 7, 2005; doi:10.1152/ajplung.00124.2005
1040-0605/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/3/L478    most recent
00124.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Folkesson, H. G.
Right arrow Articles by Kitterman, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Folkesson, H. G.
Right arrow Articles by Kitterman, J. A.

Congenital diaphragmatic hernia prevents absorption of distal air space fluid in late-gestation rat fetuses

Hans G. Folkesson,1 Cheryl J. Chapin,2 LaMonta L. Beard,1 Robert Ertsey,2 Michael A. Matthay,2 and Joseph A. Kitterman2,3

1Department of Physiology and Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio; and 2Cardiovascular Research Institute and 3Department of Pediatrics, University of California, San Francisco, California

Submitted 21 March 2005 ; accepted in final form 4 October 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We hypothesized that congenital diaphragmatic hernia (CDH) may decrease distal air space fluid absorption due to immaturity of alveolar epithelial cells from a loss of the normal epithelial Na+ transport, as assessed by amiloride and epithelial Na+ channel (ENaC) and Na-K-ATPase expression, as well as failure to respond to endogenous epinephrine as assessed by propranolol. Timed-pregnant dams were gavage fed 100 mg of nitrofen at 9.5-day gestation to induce CDH in the fetuses, and distal air space fluid absorption experiments were carried out on 22-day gestation (term) fetuses. Controls were nitrofen-exposed fetuses without CDH. Absorption of distal air space fluid was measured from the increase in 131I-albumin concentration in an isosmolar, physiological solution instilled into the developing lungs. In controls, distal air space fluid absorption was rapid and mediated by beta-adrenoceptors as demonstrated by reversal to fluid secretion after propranolol. Normal lung fluid absorption was also partially inhibited by amiloride. In contrast, CDH fetuses continued to show lung fluid secretion, and this secretion was not affected by either propranolol or amiloride. CDH lungs showed a 67% reduction in {alpha}-ENaC and beta-ENaC expression, but no change in {alpha}1-Na-K-ATPase expression. These studies demonstrate: 1) CDH delays lung maturation with impaired distal air space fluid absorption secondary to inadequate Na+ uptake by the distal lung epithelium that results in fluid-filled lungs at birth with reduced capacity to establish postnatal breathing, and 2) the main stimulus to lung fluid absorption in near-term control fetuses, elevated endogenous epinephrine levels, is not functional in CDH fetuses.

amiloride sensitivity; distal air space epithelium; epinephrine; Na+ transport; pulmonary edema; neonatal respiratory distress syndrome

CONVERSION OF THE LUNG DISTAL air space epithelium from fluid secretion to absorption near term relies on endogenous epinephrine to stimulate Na+ and fluid absorption near term (30, 45), although some studies have suggested that other factors may also be involved (5, 6, 35). Apically localized amiloride-sensitive epithelial Na+ channels (ENaC) and basolaterally located Na-K-ATPases in the alveolar epithelial cells constitute the principal molecular mechanism for transepithelial Na+ transport (20, 23, 24, 31, 43).

Congenital diaphragmatic hernia (CDH) occurs once in 3,000 births (18) and causes pulmonary hypoplasia and hypertension leading to high mortality rates (41). Administration of the pesticide nitrofen to pregnant rats causes CDH with resultant pulmonary hypoplasia in 60% of the fetuses (2, 3), a condition that resembles CDH in human infants. Thus we decided to use this model to investigate the impact of CDH on absorption of fetal lung fluid at birth. A stimulus to this study was our clinical experience with two infants with severe pulmonary hypoplasia from CDH. The two infants were treated with extracorporeal membrane oxygenation (ECMO) because mechanical ventilation could not maintain adequate oxygenation and ventilation. Measurements showed that lung fluid absorption rates in these infants were far lower than expected based on previous studies in adult human lungs (see DISCUSSION for details).

The first objective of the present study was to investigate distal air space fluid absorption near term (22-day gestation) in rat fetuses with nitrofen-induced CDH. Because it has been shown that catecholamines may be an important stimulus for distal air space fluid absorption at birth (14, 45), our second aim was to investigate whether endogenous beta-adrenergic stimulation of absorption of fetal lung fluid was affected by the presence of CDH. Plasma samples were also obtained for measurements of endogenous epinephrine levels. Epithelial Na+ transport via amiloride-sensitive Na+ channels provides the driving force for distal air space fluid reabsorption in the lung (14, 23, 32). Therefore, the third aim was to determine whether the amiloride-sensitive fraction of distal air space fluid absorption in the fetal lungs was affected by CDH. A part of this third aim was to determine whether CDH affected lung ENaC and Na-K-ATPase expression.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Rats of the Sprague-Dawley strain (96 fetuses divided on 8 litters and 8 timed-pregnant rats; Charles River, Hollister, CA) were used in the study. Both male and female rat fetuses were used in the study. The timed-pregnant rats were housed in separate cages in temperature- and humidity-controlled units (20 ± 2°C and 55 ± 10% relative humidity). All studies were approved by the University of California San Francisco Committee on Animal Research.

CDH induction. Nitrofen (2,4-dichloro-4"-nitrodiphenyl ether; Wako Chemicals, Osaka, Japan) was dissolved in 1 ml of olive oil by incubation at 50°C with rotation for 1 h. Timed-pregnant rats were at gestation day 9.5, anesthetized with ketamine (90 mg/kg body wt; Ketalar; Parke-Davis, Morris Plains, NJ) and xylazine (1.25 mg/kg body wt; Rompun; Bayer, Pittsburgh, PA), and gavage fed 100 mg of nitrofen. The timed-pregnant rats were then allowed to recover in their respective cages and were returned to where they were housed for the remainder of gestation.

Preparation of instillates. The 131I-labeled 5% albumin instillate was prepared by dissolving 50 mg/ml bovine serum albumin (Sigma, St. Louis, MO) in an isosmolar aqueous solution of 0.9% NaCl with 0.1 µCi 131I-labeled human serum albumin (Frosst Laboratories, Montreal, Canada). For studies of endogenous epinephrine dependence of distal air space fluid absorption, we added the general beta-adrenergic antagonist propranolol (10–4 M; Sigma) to the 131I-labeled 5% albumin instillate. For determination of fractional amiloride inhibition of distal air space fluid absorption, we added amiloride (10–3 M; ICN Biochemicals, Costa Mesa, CA) to the 131I-labeled 5% albumin instillate. We used 10–3 M amiloride because ~50% of amiloride becomes protein bound and another significant fraction escapes the air spaces, resulting in lower functional concentrations (32, 48).

Surgery. Timed-pregnant rats and their fetuses were anesthetized by maternal intramuscular injections of ketamine (180 mg/kg body wt) mixed with xylazine (2.5 mg/kg body wt). After all fetuses had been delivered, the female rats were euthanized with an overdose of intracardiac pentobarbital sodium (100 mg/kg body wt; Nembutal; Abbott Laboratories, Chicago, IL) followed by bilateral pneumothoraces.

The anesthetized timed-pregnant female rats were placed on a temperature-controlled pad heated to 38°C to maintain their body temperature, an abdominal hysterotomy was carried out by a midline laparotomy, and the fetuses were delivered one by one as described earlier (15). The success rate for the fetal surgery was 93 ± 9% (yielding 62 fetuses from 67 surgeries).

General experimental protocol. The 5% albumin instillate with the 131I-labeled albumin as a distal air space protein tracer was instilled into both lungs over 2–3 s through the tracheal cannula as described in detail in our earlier study (15). Lungs lacking significant radioactivity, i.e., when the radioactivity left in the lungs was <70% of instilled radioactivity, were discarded with their corresponding distal air space fluid samples because of uncertainty of instillate location in those fetuses. Immediately after the fluid collection, presence of clinical CDH was confirmed by autopsy of the thorax-abdomen. A diaphragm defect and herniation of abdominal viscera into the thorax were taken as evidence of fetal CDH. The fetuses were then grouped as CDH and nitrofen control animals and studied as such. Plasma was collected from littermate fetuses not used for the fluid absorption experiments at the same time as for the fluid absorption studies and measured by a commercially available ELISA (IBL, Hamburg, Germany). The inter-assay and intra-assay coefficients of variation for the ELISA were 6.2% and 15.2%, respectively.

Specific experimental protocols. All rat fetuses used for distal air space fluid absorption studies were studied for 30 min. All experimental groups contained fetuses from at least two different dams. The n values represent individual fetuses. Our previous study confirmed that the rate of alveolar fluid absorption did not differ between anesthetized rats and our in situ rat fetuses (15). Of 62 rat fetuses used for the distal air space fluid absorption studies, 45 fetuses (73% success rate) had >70% of the instilled radioactivity in the lungs and were divided into the experimental groups below.

Group 1: nitrofen control studies (22-day gestation: n = 6). Rat fetuses with no postmortem signs of CDH from two dams were instilled with 10 ml/kg body wt of the 131I-labeled 5% albumin instillate into the lungs.

Group 2: CDH studies (22-day gestation: n = 6). Rat fetuses with postmortem-confirmed CDH from two dams were instilled with 10 ml/kg body wt of the 131I-labeled 5% albumin instillate into the lungs.

Group 3: beta-adrenoceptor blockade studies (22-day gestation nitrofen control: n = 6, 22-day gestation CDH: n = 15). Rat fetuses from three dams were instilled with 10 ml/kg body wt of the 131I-labeled 5% albumin instillate containing 10–4 M propranolol into the lungs. Endogenous epinephrine levels were measured in plasma from littermates to rat fetuses used for distal air space fluid absorption studies in each group (n = 10 nitrofen control; n = 5 CDH; n = 12 dams).

Group 4: amiloride studies (22-day gestation nitrofen control: n = 6, 22-day gestation CDH: n = 6). Rat fetuses from two dams were instilled with 10 ml/kg body wt of the 131I-labeled 5% albumin instillate containing 10–3 M amiloride into the lungs.

Distal air space fluid absorption calculation. Distal air space fluid absorption or secretion was measured by the increase or decrease in distal air space protein concentration of the instillate over 30 min as previously reported (15). The increase in protein concentration due to removal of water from the air spaces is a direct reflection of distal air space fluid absorption, as demonstrated previously (7, 17, 25, 29). Data are presented as distal air space fluid absorption as percent of instilled volume. Net distal air space fluid absorption (DFA) was calculated from:

Formula
where VI is instilled volume (ml) corrected for preexisting lung fluid volume as described previously (15), and VF is final distal air space volume (ml) calculated from the 131I-labeled albumin concentrations in instilled and final distal air space fluids.

Western blot protocols: general protocol. Lung tissue from eight CDH and six nitrofen control fetuses was pooled by group and homogenized in T-Per reagent (Pierce, Rockford, IL) containing the protease inhibitors aprotinin (30 µg/ml, Sigma) and leupeptin (1 µg/ml, Sigma) with a Tissue Tearor homogenizer on ice. The tissue homogenate was centrifuged at 13,000 g for 5 min at +4°C. The supernatant (membrane and cytosol fraction) was collected and aliquoted in multiple vials for each sample and snap-frozen in liquid nitrogen. One vial was designated to be used for determining total protein concentration of the sample to ensure equal loading of the electrophoresis gel. Aliquots were stored at –80°C until analysis.

Polyacrylamide gel electrophoresis and transfer onto nitrocellulose membrane (Pierce) were carried out using standard protocols. After electrophoresis and transfer, the nitrocellulose membrane was placed in blocking buffer [SuperBlock Dry Blend blocking buffer in Tris-buffered saline (TBS), Pierce] for 1 h. Primary antibody incubations were carried out overnight at +4°C on an orbital shaker.

ENaC. The anti-ENaC antibodies used were a gift from Dr. James D. Stockand at University of Texas Health Science Center and were directed against residues 137–161 of Xenopus laevis {alpha}-ENaC and residues 624–647 of beta-ENaC (42). These antibodies specifically recognize membrane proteins of appropriate sizes (85–90 kDa for {alpha}-ENaC and 90–95 kDa for beta-ENaC) in rats. After blocking, the membranes were incubated with the primary antibody [anti-{alpha}-ENaC (rabbit) and anti-beta-ENaC (rabbit), respectively, diluted 1:1,000]. After incubation, the membranes were washed 5x with wash buffer (pH 7.5, TBS with 0.1% Tween 20). After the washing process, the membranes were incubated with the enzyme-conjugated secondary antibody (goat anti-rabbit IgG, diluted 1:1,000) for 1 h at room temperature. After incubation, the membranes were washed again. Then, the substrate solution (SuperSignal West Femto substrate solution, Pierce) was added to the blot and incubated for 5 min. The luminescence signal was detected using a Kodak image analyzer and analyzed densitometrically using TotalLab software (Nonlinear Dynamics, Newcastle upon Tyne, United Kingdom).

Na-K-ATPase. The anti-Na-K-ATPase antibody was obtained from Upstate Cell Signaling Solutions (Waltham, MA) and was directed against residues 338–518 of the {alpha}1-subunit of the Na-K-ATPase. The antibody specifically recognizes a membrane protein of appropriate size (~95 kDa for the {alpha}1-subunit of the Na-K-ATPase) in rats. A rat heart microsomal protein preparation was run on the gel as a positive control. After blocking, the membrane was incubated with the primary polyclonal antibody [anti-{alpha}1-Na+-K+-ATPase (rabbit), diluted 1:1,000]. After incubation, the membrane was washed 5 x 10 min in wash buffer. Then, the membrane was incubated with horseradish-conjugated secondary antibody (goat anti-rabbit IgG, diluted 1:1,000) for 1 h at room temperature. After being incubated and washed, the substrate solution (SuperSignal West Femto) was added to the blot and incubated for 5 min. The luminescence signal was detected and densitometrically analyzed as above.

Statistics. All data are presented as means ± SD. Data were analyzed with one-way ANOVA with Tukey's test as post hoc. Differences were considered significant when a P value of <0.05 was reached. The n for the different groups represents individual fetuses.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Incidence of CDH after nitrofen exposure. In the current study, the incidence of CDH after the nitrofen exposure was 48 ± 21% based on 96 fetuses and 8 litters. This CDH incidence was similar to previously published data (4).

Distal air space fluid absorption in CDH and nitrofen control fetal lungs. Distal air space fluid absorption in nitrofen-exposed control rat fetuses was normal (Fig. 1) and not different from the previously published distal air space fluid absorption of 18 ± 8% in normal 22-day gestation rat fetuses (15). Distal air space fluid absorption in our earlier study (15) was not different from that in the adult rat when extrapolated to 1 h (16 ± 2%) in the 40-h-old rats (data from Ref. 15) and compared with that in adult rats (17 ± 2%; data from Ref. 16). In CDH fetuses, there was no net distal air space fluid absorption; in fact, the lungs remained in the fluid secretory state with a net distal air space fluid secretion (Fig. 1).


Figure 1
View larger version (15K):
[in this window]
[in a new window]
  		Fig. 1. Distal air space epithelial fluid absorption over 30 min in nitrofen-exposed rat fetuses with and without postmortem-confirmed congenital diaphragmatic hernia (CDH) after instillation of the 5% albumin solution. Values are means ± SD; n is given in Specific experimental protocols. *P < 0.05 compared with 22-day gestation nitrofen control rat fetuses (ANOVA with Tukey's test as post hoc).

 
Sensitivity to endogenous beta-adrenergic stimulation. Circulating epinephrine levels were elevated in 22-day gestation rat fetuses [normal range in adult rats 10–100 pg/ml (10, 36, 37)] and unaffected by the presence of CDH (Fig. 2). The plasma from the dams displayed equally high circulating plasma epinephrine levels as the rat fetuses.


Figure 2
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 2. Plasma epinephrine levels in nitrofen-exposed rat fetuses with and without postmortem-confirmed CDH and in 2 dams.

 
Because endogenous plasma epinephrine levels in near-term rat fetuses (22-day gestation) were high and alveolar fluid absorption in adult rats can be stimulated by exogenous administration of beta-adrenergic agonists (21), we studied whether distal air space fluid absorption would be sensitive to inhibition by the beta-adrenergic antagonist propranolol and if such a sensitivity would vary with the presence of CDH. Addition of propranolol significantly inhibited distal air space fluid absorption in 22-day gestation nitrofen control rat fetuses but had no effect in rat fetuses with postmortem-confirmed CDH (Fig. 1).

Involvement of amiloride-sensitive Na+ channels. We used amiloride to determine the contribution of the amiloride-sensitive Na+ channels for distal air space fluid absorption in 22-day gestation nitrofen control and CDH-positive rat fetuses. Addition of amiloride to the 131I-labeled 5% albumin instillate decreased distal air space fluid absorption in the 22-day gestation nitrofen control rat fetuses but had no effect in rat fetuses with postmortem-confirmed CDH (Fig. 1).

Expression of {alpha}-ENaC, beta-ENaC, and the {alpha}1-Na-K-ATPase subunit. ENaC and Na-K-ATPase expression were investigated in nitrofen-exposed control and CDH rat fetal lungs by Western blot. Both the {alpha}-ENaC and beta-ENaC subunits were significantly decreased in expression in the CDH-positive fetal lungs (Fig. 3). The optical density (OD) values were normalized to the nitrofen control OD values. In fact, as seen in the figure, it is difficult to see a positive band in either of the CDH-positive fetal lungs on the displayed representative blot.


Figure 3
View larger version (26K):
[in this window]
[in a new window]
  		Fig. 3. Expression of {alpha}-epithelial Na+ channel ({alpha}-ENaC) and beta-ENaC subunits in lung tissue from CDH-positive fetal lungs and nitrofen-exposed fetal control (NC) lungs. Left: representative Western blots of {alpha}-ENaC (top) and beta-ENaC (bottom). Right: summary graphs of the optical density (OD) analyses of the Western blots. The OD graphs were normalized to the OD of the nitrofen control fetal lungs. Values are means ± SD, n = 6 nitrofen control rat fetuses, n = 8 CDH rat fetuses for each marker. *P < 0.05 compared with 22-day gestation nitrofen control rat fetuses (ANOVA with Tukey's test as post hoc). MW, molecular weight.

 
In contrast, the expression of the {alpha}1-Na-K-ATPase subunit was unchanged between the nitrofen-exposed control rat fetal lungs and the CDH-positive lungs (Fig. 4). The OD values were normalized to the nitrofen control OD values.


Figure 4
View larger version (23K):
[in this window]
[in a new window]
  		Fig. 4. Expression of the {alpha}1-Na-K-ATPase subunit in lung tissue from CDH-positive fetal lungs and nitrofen-exposed fetal control lungs. Left: representative Western blot of the {alpha}1-Na-K-ATPase. Right: summary graph of the OD analysis of the Western blot. The OD graph was normalized to the OD of the nitrofen control fetal lungs. Values are means ± SD, n = 6 nitrofen control rat fetuses, n = 8 CDH rat fetuses for each marker. H, positive control sample for the {alpha}1-Na-K-ATPase subunit from heart cell membrane microsomes.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The majority of infants make the transition from intrauterine life to postnatal life without complications, but only hours before birth, the lungs are filled with an essentially protein-free isosmolar solution that has been actively secreted by the pulmonary epithelium. Complications such as CDH may have a serious impact on the ability and rate of fluid removal from the distal air spaces at birth and drastically affect the ability to oxygenate the newborn. We therefore determined the rate of distal air space fluid absorption in developing rat fetuses with and without nitrofen-induced confirmed CDH. Normally, fetal rat lungs of 22-day gestation have the ability to rapidly clear the distal air spaces from the fetal lung fluid, a fluid absorption that depends on plasma epinephrine and beta-adrenoceptor stimulation and lung epithelial Na+ absorption (15). The nitrofen-exposed control rats in the current study displayed alveolar fluid absorption rates indistinguishable from those previously published in normal healthy rat fetuses of the same gestational age (15). Moreover, the responses to propranolol and amiloride were identical in the nitrofen-exposed control rat fetuses and normal healthy rat fetuses (15). Importantly, rat fetuses with confirmed postmortem CDH completely lacked the ability to clear the fetal distal air spaces from fluid. These lungs remained in the fetal fluid secretory stage, in sharp contrast to their nitrofen-exposed control littermates, in which the distal air spaces cleared the fetal distal air space fluid at a normal rate. Moreover, the CDH fetal lungs were nonresponsive to beta-adrenoceptor stimulation by the elevated fetal plasma epinephrine levels, plasma levels that were no different than in nitrofen-exposed control fetal lungs. Also, amiloride had no effect on the fluid movement across the distal air space epithelia in the CDH-positive fetal lungs.

We had hypothesized that lung fluid absorption was decreased in CDH based on our previous experience with two infants with CDH in 1996 and 1998. Because they had severe pulmonary hypoplasia due to CDH (2, 18, 26, 40, 41), mechanical ventilation was not sufficient for adequate oxygenation and ventilation; thus both infants were treated with ECMO. Based on studies in experimental animals (1, 47), we attempted to stimulate lung growth in these infants by filling their lungs and airways with fluid at a constant positive pressure of 12 cmH2O. This was accomplished by attaching a tube to the endotracheal tube and instilling a protein-free, isosmolar fluid into the lungs via the tube. The pressure was maintained for 48 h by adding fluid as needed to keep the meniscus of the fluid at a level of 12 cm above the infants' midthorax. We recorded the amount of fluid needed to maintain the pressure. We assumed that the rate at which we added fluid represented the rate of fluid absorption by the lungs. Based on fluid absorption studies from adult lungs, we expected a lung fluid absorption rate between 2 and 9 ml/h, representing 11–55%/h (11, 24, 38, 46). Similar approaches have been subsequently reported using perfluorocarbon filling of the developing air spaces to promote lung growth ex utero (13, 19, 44). To our surprise, fluid was absorbed at an initial rate of only 0.8 ml/h, representing 5%/h, which later slowed down to ~0.3 ml/h, representing 2%/h (Fig. 5), thus suggesting a significant abnormality in the normal perinatal processes of conversion of the fetal lungs from being a fluid-secreting organ to becoming one of fluid absorption. Some of the initial fluid absorption rate could have been caused by an initial distention of the lung air spaces as the fluid is instilled. In addition, it is possible that some fluid leaked from the air spaces of the damaged lungs. In either case, it would result in an overestimation of lung fluid absorption, thus further strengthening our observation of the subnormal distal lung fluid absorption rate in these lungs made hypoplastic by the CDH. This very low rate of fluid absorption may contribute to the respiratory insufficiency seen clinically in infants with CDH.


Figure 5
View larger version (21K):
[in this window]
[in a new window]
  		Fig. 5. Fluid absorption in 2 CDH-positive human infants after fluid filling of their lungs with an isosmolar solution to 12 cmH2O pressure. The regression line represents the mean of the 2 infants' data. Initially, the lungs were filled with 14 ml of the isosmolar solution, which generated the desired pressure of 12 cmH2O. Fluid absorption was measured and expressed as volume added needed to maintain a constant pressure of 12 cmH2O over 46 h. The left inset represents a regression analysis of the initial 15 h of the study, and the right inset represents a regression analysis of the last 30 h of the study.

 
What may regulate distal air space fluid absorption at birth? Endogenous epinephrine can stimulate reabsorption of fetal lung fluid near term (8, 9, 12, 14, 45), levels that also have been shown to increase during labor and delivery (9). We demonstrated in our previous study that plasma epinephrine levels near term stimulated the rate of fluid absorption in fetal rats (15). Therefore, the involvement of endogenous epinephrine in regulating distal air space fluid absorption in CDH fetuses near term was determined in two ways. First, endogenous epinephrine plasma levels were measured in the nitrofen control and CDH rat fetuses and correlated with the ability to clear excess distal air space fluid in nitrofen-exposed control and CDH rat fetuses. Second, functional inhibition of beta-adrenoceptor stimulation by addition of propranolol to the instilled fluid determined the extent of receptor stimulation. Endogenous epinephrine levels were significantly elevated in the 22-day gestation nitrofen control and CDH rat fetuses (Figs. 1 and 2) at the time when distal air space fluid absorption normally was at its greatest (15). However, plasma epinephrine failed to stimulate distal air space fluid absorption in CDH-positive fetal lungs. As a confirmation of the importance of epinephrine and beta-adrenergic receptors for absorption of fetal lung fluid at birth, distal air space fluid absorption in 22-day gestation nitrofen control rat lungs was highly sensitive to propranolol inhibition, but CDH-positive lungs were insensitive to propranolol inhibition (Fig. 1). All of the results suggest that the defect in beta-adrenoceptor stimulation of distal air space fluid absorption in the CDH-positive fetal lungs resides either at the beta-adrenoceptor level, at the beta-adrenoceptor signaling level, and/or at the Na+ transport protein level. Although not investigated in the current study, beta-adrenoceptor downregulation has been demonstrated to be important in impairing distal lung fluid absorption in adult rats and mice (27, 39), and deficiencies in beta-adrenoceptor signaling may also be involved (28). Another possible explanation may reside within the mesenchyme. Pinter and colleagues (34) demonstrated that when, as in fetuses from diabetic mothers, there was more mesenchymal cells present, the expression of the {alpha}1-Na-K-ATPase decreased. However, for this to be solely the mechanism, one would expect that the expression of both transporters studied to change in a similar way, i.e., decrease. This was not the case in our studies, where ENaC expression decreased and {alpha}1-Na-K-ATPase expression was unchanged after CDH induction. Our results do not exclude any of the above mechanisms and thus do not permit us to make a definite conclusion, but the amiloride data and the ENaC protein expression data suggest that the principal mechanism for the attenuated distal air space fluid absorption in the CDH rat fetuses resides at the level of the apical Na+ uptake transport proteins.

In adult lungs, active Na+ uptake through apical amiloride-sensitive ENaC channels and basolateral Na-K-ATPases is a key mechanism for generating the driving force for alveolar epithelial water reabsorption (for review see Refs. 22 and 24). Amiloride inhibits ENaC and impairs reabsorption of fetal lung fluid in several species (14, 32, 33). Therefore, we investigated whether inhibition of distal air space fluid absorption by amiloride differed with and without nitrofen-induced CDH. Interestingly, the results showed that CDH-positive lungs completely lacked amiloride sensitivity, whereas normal amiloride sensitivity was observed in nitrofen-exposed control lungs (Fig. 1). We also investigated potential variations in {alpha}-ENaC and beta-ENaC subunit expression in fetal lungs with and without confirmed CDH and found a significant decrease in lung expression of both ENaC subunits in CDH-positive lungs compared with nitrofen-exposed control fetal lungs. These data suggest that the principal impairment in CDH-positive lungs may reside at the level of ENaC expression and at the apical epithelial cell membrane. To further investigate the involvement of the Na+ transport machinery, we studied the expression of the basolaterally expressed Na-K-ATPase, specifically the {alpha}1-Na-K-ATPase subunit. Interestingly, the expression of the {alpha}1-Na-K-ATPase subunit was unchanged between CDH-positive lungs and nitrofen-exposed control lungs, further suggesting that the defect of distal fluid absorption seen in CDH critically depends on ENaC expression and not on Na-K-ATPase expression. However, since we did not study Na-K-ATPase activity, we cannot exclude that the actual activity of the basolateral Na-K-ATPase was altered by CDH.

In conclusion, our data demonstrate that the presence of anatomically confirmed CDH attenuates normally developing Na+ uptake and absorption of fetal lung fluid in preparation for air-breathing postnatal life. Our data also suggest that the principal defect in the mechanism responsible for distal lung fluid absorption, i.e., the transepithelial Na+ transport, resides in the apical cell membrane and the ENaC channel. This conclusion is also functionally supported by the distal lung fluid absorption studies with amiloride showing no inhibition in the CDH-positive lungs. Our findings suggest that, in addition to the well-recognized problems of pulmonary hypoplasia and pulmonary hypertension, human infants with CDH may have decreased development of the normal fluid-absorptive function of the lungs that may contribute to a potentially fatal respiratory insufficiency after birth.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute (NHLBI) Grants RO1-HL-51854 and NHLBI Program Project Grant HL-24075.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. G. Folkesson, Dept. of Physiology and Pharmacology, Northeastern Ohio Universities College of Medicine, 4209 State Route 44, PO Box 95, Rootstown, OH 44272-0095 (e-mail: hgfolkes{at}neoucom.edu)

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. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alcorn D, Adamson TM, Lambert TF, Maloney JE, Ritchie BC, and Robinson PM. Morphological effects of chronic tracheal ligation and drainage in the fetal lamb lung. J Anat 123: 649–660, 1977.[Web of Science][Medline]
  2. Alfonso LF, Arnaiz A, Alvarez FJ, Qi B, Diez-Pardo JA, Vallis-i-Soler A, and Tovar JA. Lung hypoplasia and surfactant system immaturity induced in the fetal rat by prenatal exposure to nitrofen. Biol Neonate 69: 94–100, 1996.[Web of Science][Medline]
  3. Allan DW and Greer JJ. Pathogenesis of nitrofen-induced congenital diaphragmatic hernia in fetal rats. J Appl Physiol 83: 338–347, 1997.[Abstract/Free Full Text]
  4. Babiuk RP, Thébaud B, and Greer JJ. Reductions in the incidence of nitrofen-induced diaphragmatic hernia by vitamin A and retinoic acid. Am J Physiol Lung Cell Mol Physiol 286: L970–L973, 2004.[Abstract/Free Full Text]
  5. Baines DL, Folkesson HG, Norlin A, Bingle CD, Yuan H, and Olver RE. The influence of mode of delivery, hormonal status and postnatal O2 environment on epithelial sodium channel (ENaC) expression in perinatal guinea-pig lung. J Physiol 522: 147–157, 2000.[Abstract/Free Full Text]
  6. Barker PM, Walters DV, Markiewicz M, and Strang LB. Development of the lung liquid reabsorptive mechanism in fetal sheep: synergism of triiodothyronine and hydrocortisone. J Physiol 433: 435–449, 1991.[Abstract/Free Full Text]
  7. Berthiaume Y, Broaddus VC, Gropper MA, Tanita T, and Matthay MA. Alveolar liquid and protein clearance from normal dog lungs. J Appl Physiol 65: 585–593, 1988.[Abstract/Free Full Text]
  8. Bland RD and Nielson DW. Developmental changes in lung epithelial ion transport and liquid movement. Annu Rev Physiol 54: 373–394, 1992.[CrossRef][Web of Science][Medline]
  9. Brown MJ, Olver RE, Ramsden CA, Strang LB, and Walters DV. Effects of adrenaline and of spontaneous labour on the secretion and absorption of lung liquid in the fetal lamb. J Physiol 344: 137–152, 1983.[Abstract/Free Full Text]
  10. Charron PD, Fawley JP, and Maron MB. Effect of epinephrine on alveolar liquid clearance in the rat. J Appl Physiol 87: 611–618, 1999.[Abstract/Free Full Text]
  11. Chesnutt MS, Nuckton TJ, Golden J, Folkesson HG, and Matthay MA. Rapid alveolar epithelial fluid clearance following lung lavage in pulmonary alveolar proteinosis. Chest 120: 271–274, 2001.[Abstract/Free Full Text]
  12. Enhorning G, Chamberlain D, Contreras C, Burgoyne R, and Robertson B. Isoxsuprine-induced release of pulmonary surfactant in the rabbit fetus. Am J Obstet Gynecol 129: 197–202, 1977.[Web of Science][Medline]
  13. Fauza DO, Hirschl RB, and Wilson JM. Continuous intrapulmonary distension with perfluorocarbon accelerates lung growth in infants with congenital diaphragmatic hernia: initial experience. J Pediatr Surg 36: 1237–1240, 2001.[CrossRef][Web of Science][Medline]
  14. Finley N, Norlin A, Baines DL, and Folkesson HG. Alveolar epithelial fluid clearance is mediated by endogenous catecholamines at birth in guinea pigs. J Clin Invest 101: 972–981, 1998.[Web of Science][Medline]
  15. Folkesson HG, Matthay MA, Chapin CJ, Porta NFM, and Kitterman JA. Pre- and postnatal lung development, maturation, and plasticity: distal air space epithelial fluid clearance in near-term rat fetuses is fast and requires endogenous catecholamines. Am J Physiol Lung Cell Mol Physiol 282: L508–L515, 2002.[Abstract/Free Full Text]
  16. Folkesson HG, Norlin A, Wang Y, Abedinpour P, and Matthay MA. Dexamethasone and thyroid hormone pretreatment upregulate alveolar epithelial fluid clearance in adult rats. J Appl Physiol 88: 416–424, 2000.[Abstract/Free Full Text]
  17. Folkesson HG, Pittet JF, Nitenberg G, and Matthay MA. Transforming growth factor-{alpha} increases alveolar liquid clearance in anesthetized ventilated rats. Am J Physiol Lung Cell Mol Physiol 271: L236–L244, 1996.[Abstract/Free Full Text]
  18. Harrison MR, Adzick NS, Estes JM, and Howell LJ. A prospective study of the outcome for fetuses with diaphragmatic hernia. JAMA 271: 382–384, 1994.[Abstract/Free Full Text]
  19. Hirschl RB, Philip WF, Glick L, Greenspan J, Smith K, Thompson A, Wilson J, and Adzick NS. A prospective, randomized pilot trial of perfluorocarbon-induced lung growth in newborns with congenital diaphragmatic hernia. J Pediatr Surg 38: 283–289; discussion 283–289, 2003.[CrossRef][Medline]
  20. Hummler E, Barker P, Gatzy J, Beermann F, Verdumo C, Schmidt A, Boucher R, and Rossier BC. Early death due to defective neonatal lung liquid clearance in {alpha}ENaC-deficient mice. Nat Genet 12: 325–328, 1996.[CrossRef][Web of Science][Medline]
  21. Jayr C, Garat C, Meignan M, Pittet JF, Zelter M, and Matthay MA. Alveolar liquid and protein clearance in anesthetized ventilated rats. J Appl Physiol 76: 2636–2642, 1994.[Abstract/Free Full Text]
  22. Matalon S, Benos DJ, and Jackson RM. Biophysical and molecular properties of amiloride-inhibitable Na+ channels in alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 271: L1–L22, 1996.[Abstract/Free Full Text]
  23. Matalon S, Bridges RJ, and Benos DJ. Amiloride-inhibitable Na+ conductive pathways in alveolar type II pneumocytes. Am J Physiol Lung Cell Mol Physiol 260: L90–L96, 1991.[Abstract/Free Full Text]
  24. Matthay MA, Folkesson HG, and Clerici C. Lung epithelial fluid transport and the resolution of pulmonary edema. Physiol Rev 82: 569–600, 2002.[Abstract/Free Full Text]
  25. Matthay MA, Landolt CC, and Staub NC. Differential liquid and protein clearance from the alveoli of anesthetized sheep. J Appl Physiol 53: 96–104, 1982.[Abstract/Free Full Text]
  26. Miyazaki E, Ohshiro K, Taira Y, and Puri P. Altered insulin-like growth factor I mRNA expression in human hypoplastic lung in congenital diaphragmatic hernia. J Pediatr Surg 33: 1476–1479, 1998.[CrossRef][Web of Science][Medline]
  27. Morgan EE, Hodnichak CM, Stader SM, Maender KC, Boja JW, Folkesson HG, and Maron MB. Alveolar epithelial ion and fluid transport: prolonged isoproterenol infusion impairs the ability of beta2-agonists to increase alveolar liquid clearance. Am J Physiol Lung Cell Mol Physiol 282: L666–L674, 2002.[Abstract/Free Full Text]
  28. Morgan EE, Stader SM, Hodnichak CM, Mavrich KE, Folkesson HG, and Maron MB. Postreceptor defects in alveolar epithelial beta-adrenergic signaling after prolonged isoproterenol infusion. Am J Physiol Lung Cell Mol Physiol 285: L578–L583, 2003.[Abstract/Free Full Text]
  29. Norlin A, Finley N, Abedinpour P, and Folkesson HG. Alveolar liquid clearance in the anesthetized ventilated guinea pig. Am J Physiol Lung Cell Mol Physiol 274: L235–L243, 1998.[Abstract/Free Full Text]
  30. Norlin A and Folkesson HG. Alveolar fluid clearance in late gestational guinea pigs after induction of labor: mechanisms and regulation. Am J Physiol Lung Cell Mol Physiol 280: L606–L616, 2001.[Abstract/Free Full Text]
  31. O'Brodovich H, Canessa C, Ueda J, Rafii B, Rossier BC, and Edelson J. Expression of the epithelial Na+ channel in the developing rat lung. Am J Physiol Cell Physiol 264: C491–C496, 1993.[Abstract/Free Full Text]
  32. O'Brodovich H, Hannam V, Seear M, and Mullen JB. Amiloride impairs lung water clearance in newborn guinea pigs. J Appl Physiol 68: 1758–1762, 1990.[Abstract/Free Full Text]
  33. Olver RE, Ramsden CA, Strang LB, and Walters DV. The role of amiloride-blockable sodium transport in adrenaline-induced lung liquid reabsorption in the fetal lamb. J Physiol 376: 321–340, 1986.[Abstract/Free Full Text]
  34. Pinter E, Peyman JA, Snow K, Jamieson JD, and Warshaw JB. Effects of maternal diabetes on fetal rat lung ion transport. Contribution of alveolar and bronchiolar epithelial cells to Na+,K+-ATPase expression. J Clin Invest 87: 821–830, 1991.[Medline]
  35. Pitkänen O, Tanswell AK, Downey G, and O'Brodovich H. Increased PO2 alters the bioelectric properties of fetal distal lung epithelium. Am J Physiol Lung Cell Mol Physiol 270: L1060–L1066, 1996.[Abstract/Free Full Text]
  36. Pittet JF, Brenner TJ, Modelska K, and Matthay MA. Alveolar liquid clearance is increased by endogenous catecholamines in hemorrhagic shock in rats. J Appl Physiol 81: 830–837, 1996.[Abstract/Free Full Text]
  37. Pittet JF, Wiener-Kronish JP, McElroy MC, Folkesson HG, and Matthay MA. Stimulation of lung epithelial liquid clearance by endogenous release of catecholamines in septic shock in anesthetized rats. J Clin Invest 94: 663–671, 1994.[Web of Science][Medline]
  38. Sakuma T, Folkesson HG, Suzuki S, Okaniwa G, Fujimura S, and Matthay MA. beta-Adrenergic agonist stimulated alveolar fluid clearance in ex vivo human and rat lungs. Am J Respir Crit Care Med 155: 506–512, 1997.[Abstract]
  39. Sartori C, Fang X, McGraw DW, Koch P, Snider ME, Folkesson HG, and Matthay MA. Selected contribution: long-term effects of beta2-adrenergic receptor stimulation on alveolar fluid clearance in mice. J Appl Physiol 93: 1875–1880, 2002.[Abstract/Free Full Text]
  40. Smith NP, Jesudason EC, and Losty PD. Congenital diaphragmatic hernia. Paediatr Respir Rev 3: 339–348, 2002.[CrossRef][Medline]
  41. Stege G, Fenton A, and Jaffray B. Nihilism in the 1990s: the true mortality of congenital diaphragmatic hernia. Pediatrics 112: 532–535, 2003.[Abstract/Free Full Text]
  42. Stockand JD, Bao HF, Schenck J, Malik B, Middleton P, Schlanger LE, and Eaton DC. Differential effects of protein kinase C on the levels of epithelial Na+ channel subunit proteins. J Biol Chem 275: 25760–25765, 2000.[Abstract/Free Full Text]
  43. Voilley N, Lingueglia E, Champigny G, Mattéi MG, Waldmann R, Lazdunski M, and Barbry P. The lung amiloride-sensitive Na+ channel: biophysical properties, pharmacology, ontogenesis, and molecular cloning. Proc Natl Acad Sci USA 91: 247–251, 1994.[Abstract/Free Full Text]
  44. Walker GM, Kasem KF, O'Toole SJ, Watt A, Skeoch CH, and Davis CF. Early perfluorodecalin lung distension in infants with congenital diaphragmatic hernia. J Pediatr Surg 38: 17–20; discussion 17–20, 2003.[CrossRef][Medline]
  45. Walters DV and Olver RE. The role of catecholamines in lung liquid absorption at birth. Pediatr Res 12: 239–242, 1978.[Web of Science][Medline]
  46. Ware LB, Golden JA, Finkbeiner WE, and Matthay MA. Alveolar epithelial fluid transport capacity in reperfusion lung injury after lung transplantation. Am J Respir Crit Care Med 159: 980–988, 1999.[Abstract/Free Full Text]
  47. Wilson JM, Difiore JW, and Peters CA. Experimental tracheal ligation prevents the pulmonary hypoplasia associated with fetal nephrectomy: possible application for congenital diaphragmatic hernia. J Pediatr Surg 28: 1433–1440, 1993.[CrossRef][Medline]
  48. Yue G and Matalon S. Mechanisms and sequelae of increased alveolar fluid clearance in hyperoxic rats. Am J Physiol Lung Cell Mol Physiol 272: L407–L412, 1997.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
P.-H. Rolland, P. de Lagausie, E. Stathopoulos, O. Lepretre, G. Viudes, G. Gorincour, G. Hery, C. de Magnee, O. Paut, and J.-M. Guys
Phasic hemodynamics and reverse blood flows in the aortic isthmus and pulmonary arteries of preterm lambs with pulmonary vascular dysfunction
Am J Physiol Heart Circ Physiol, December 1, 2008; 295(6): H2231 - H2241.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
T. Li, S. Koshy, and H. G. Folkesson
Involvement of {alpha}ENaC and Nedd4-2 in the conversion from lung fluid secretion to fluid absorption at birth in the rat as assayed by RNA interference analysis
Am J Physiol Lung Cell Mol Physiol, October 1, 2007; 293(4): L1069 - L1078.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/3/L478    most recent
00124.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Folkesson, H. G.
Right arrow Articles by Kitterman, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Folkesson, H. G.
Right arrow Articles by Kitterman, J. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.