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Divisions of Neonatology and Cardiology, Hospital for Children and Adolescents, Helsinki University Central Hospital, Helsinki, Finland
ABSTRACT
Rationale: Fetal lung liquid secretion is coupled with chloride transport into the lung lumen. The postnatal clearance of lung liquid is dependent on osmotic force generated by active sodium absorption.
Objective: To study the interaction between airway epithelial sodium transport and postnatal lung function.
Methods: We determined lung compliance and nasal transepithelial potential difference as a measure of airway ion transport and epithelial sodium channel gene expression in 41 healthy newborn infants during the first 50 h after birth.
Measurements and Main Results: Lung compliance improved significantly during the study period, whereas nasal potential difference remained constant. There was a significant decrease in the expressions of and subunits of the epithelial sodium channel. A positive correlation existed between amiloride-sensitive nasal potential difference measured at 1–4 h of age and lung compliance at 21–27 h of age. We found no correlation between the molecular data and functional measurements.
Conclusions: An important part of pulmonary adaptation takes place during the first hour after birth. The improvement of lung compliance continues over the first postnatal days and coincides with down-regulation of epithelial sodium channel and subunit expression.
Key Words: ENaC epithelium ion transport lung compliance postnatal adaptation
The fetal pulmonary epithelium secretes liquid through active transport of Cl– into the luminal space (1–4). At birth, up-regulation of apical membrane sodium channels acting in concert with the basolateral Na/K/ATPase promotes the airway epithelium to switch from secretion to net absorption of liquid.
In rodents, clearance of lung fluid takes place over the first postnatal hours (5). This clearance has been considered to be connected to perinatal changes in circulating catecholamines and oxygen tension (6–10). Several experiments have underlined the role of Na+ channels in lung liquid absorption. Specifically, the amiloride-sensitive epithelial sodium channel (ENaC) may be crucial for transepithelial lung liquid movement. ENaC consists of three subunits: the , , and subunits. The subunit is a prerequisite for proper channel function (11). Application of the Na+-channel blocker amiloride into the airways of newborn guinea pigs results in delayed clearance of lung fluid, hypoxemia, and respiratory distress (12). In the newborn mouse, loss-of-function mutation of the ENaC gene results in early death due to respiratory distress (13).
The events leading to the switch from lung fluid secretion to absorption seem important for human pulmonary adaptation, and disturbances in this process may, along with the lack of surfactant, participate in the pathogenesis of respiratory distress of the newborn infant (2, 14–16). Infants who die of respiratory distress within the first postnatal hours have a significantly higher amount of lung fluid than affected infants who survive beyond 48 h (17). Human fetal pulmonary epithelial cells express all three ENaC subunits and actively transport Na+ and Cl– through apical pathways (18, 19). Airway epithelial ENaC expression differs significantly between preterm neonates with respiratory distress syndrome and healthy term neonates (20). Several studies have used transepithelial nasal potential difference (N-PD) as a method for measuring ion transport in the airway epithelia of newborn infants (21–23). In animals and humans, there have been few studies that attempt to connect the attributes of epithelial ion transport with measurements of pulmonary function in early postnatal adaptation.
We have evaluated the role of ion transport in airway epithelium in human postnatal pulmonary adaptation by comparing lung compliance during the first postnatal days, the activity of airway epithelial sodium transport measured in terms of N-PD, and the expression of ENaC subunits in newborn healthy term infants.
METHODS
Additional details on the methods for making these measurements are provided in an online supplement.
Subjects
The study subjects consisted of 41 healthy newborn infants. Key clinical data are shown in Table 1. The mothers were healthy, and all pregnancies were uneventful. Nineteen infants were delivered vaginally. The indications for an elective cesarean section were breech presentation or previous cesarean section. The infants were studied at 1–4, 21–27, and 45–50 h after birth. The study was approved by the institutional ethics committee, and informed consent was obtained from the parents. Seventeen of these infants were included in our previous study (14).
Static Lung Compliance
Static lung compliance during quiet non-REM sleep with regular respiration was measured by the double occlusion technique using a computerized pulmonary function testing device (Labmanager 4.52i; Erich Jaeger GmbH, Hoechberg, Germany) (24, 25). To ensure that the lung compliance measurements were performed at quiet non-REM sleep, 12 newborns were studied under polysomnographic follow-up. In the remaining subjects, quiet sleep was determined by direct observation of the absence of eye and body movements.
N-PD
Transepithelial N-PD measurement was used as a surrogate for distal airway epithelium as described previously (14). N-PD via both nostrils was performed on the floor of the nose, and the maximal stabile N-PD was measured for 10 s before infusions. The amiloride-sensitive sodium channel function was determined by perfusion with amiloride (10–4 M). Perfusion was continued for 2 min for each solution; during this time, a stable N-PD was achieved for 10–20 s. The N-PD measurement was discarded if the infant became restless and perfusion of amiloride could not be performed (Table 2).
Expression of ENaC in Nasal Epithelium
Sample collection, storage, and quantification of mRNA.
The samples were prepared and quantified as described previously (20). Total RNA quantification of the epithelial specimens was performed using a commercially available kit that included a standard RNA preparation and RiboGreen quantitation reagent (RiboGreen RNA Quantitation Kit; Molecular Probes, Eugene, OR). The emission at 520 nm of the adducts was measured after excitation at 480 nm using a spectrofluorometer (LS50B; Perkin Elmer, Shelton, CT), and the sample RNA content was deduced from the standard plot.
Reverse transcriptase–polymerase chain reaction.
Reverse transcription of RNA to cDNA was performed with TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions (26). Samples were analyzed by real-time polymerase chain reaction (PCR), which was performed with specific TaqMan predeveloped primers and probes (ENaC, ENaC, and ENaC; Applied Biosystems) using a Prism 7700 Sequence Detection System (Applied Biosystems). Primers and probes for cytokeratin 18 were designed with Primer Express software (Applied Biosystems). The PCR reactions were run as singleplex in duplicate wells. The ENaC expression of each sample was normalized against that of cytokeratin 18, which was used as an epithelial marker (ENaC: CK18, attomole per femtomole ). The repeated sampling of the nasal epithelium did not affect recovery of the turbinate tissue mRNA. The expression of the epithelial marker gene (CK-18) of the first sample was 0.27 ± 0.17 and remained at 0.24 ± 0.20 and 0.24 ± 0.17 fmol CK18/ng of total RNA at the following two sampling epochs (not significant). Tissue excised from a healthy turbinate during rhinoplasty served as a known standard after determination by quantitative competitive reverse transcriptase PCR as described previously (20, 27).
Demographic data are presented as mean ± SD, and the study data are presented as mean ± SEM. Comparisons were performed with the Friedman-Dunn test or the Mann-Whitney U test, which was corrected for multiple measurements. The Spearman test was used for correlations.
RESULTS
We found significant increases in lung compliance during the study period (Figure 1). The lung compliance increased from 1–4 to 45–50 h after birth by 6.4 ± 2.1 (p < 0.05) in the vaginally born infants and by 9.1 ± 2.6 ml/kPa/kg (p < 0.05) in the infants born by cesarean section. The comparison of the lung compliance values within each time epoch did not reveal differences dependent on the method of delivery.
All N-PD values remained constant (Table 2). N-PD at 1–4 h, 21–27 h, and 45–50 h after birth did not differ between infants born vaginally or by cesarean section. We found no correlation between the baseline N-PD and lung compliance. However, when the proportion of the amiloride-sensitive current, a surrogate for transepithelial Na+ transport, was tested against lung compliance, we found a significant correlation between amiloride-sensitive N-PD measured at 1–4 h and lung compliance measured at 21–27 h after birth (r = 0.478, p < 0.05, n = 18). No such correlation was found with earlier or later compliance measurements.
There was no significant change in the expression of ENaC (Figure 2) in the serial samples. On the contrary, the expression of ENaC decreased from 12.0 ± 1.4 amol/fmol CK18 (n = 34) at 1–4 h to 4.0 ± 0.8 amol/fmol CK18 (n = 27, p < 0.05 vs. 1–4 h) at 21–27 h and 2.6 ± 0.7 amol/fmol CK18 (n = 26, p < 0.001 vs. 1–4 h) at 45–50 h after birth. ENaC expression was 19.1 ± 5.9 amol/fmol CK18 (n = 35) at 1–4 h, 8.7 ± 4.5 amol/fmol CK18 (n = 27, p < 0.05 vs. 1–4 h) at 21–27 h, and 2.2 ± 0.7 amol/fmol CK18 (n = 24, p < 0.01 vs. 1–4 h) at 45–50 h after birth. No correlation existed between the expression of ENaC subunit genes with baseline and amiloride-sensitive N-PD.
Because the infants delivered by cesarean section showed a tendency to have a greater increase in lung compliance (Figure 1; albeit not statistically significant), we quantified the difference in ENaC subunit expression between the two groups. At 21–27 h after birth, there was a statistically significant difference in the expression of ENaC between infants born vaginally (5.0 ± 1.6 amol/fmol CK18, n = 12) and infants born by cesarian section (9.7 ± 1.6 amol/fmol CK18, n = 17, p < 0.05; Figure 3). At 21–27 h after birth, the expression of ENaC was lower in infants born vaginally (0.5 ± 0.2 amol/fmol CK18, n = 11) than in infants born by cesarian section (14.3 ± 7.4 amol/fmol CK18, n = 16, p < 0.05).
DISCUSSION
In the present study on healthy infants, we observed a significant increase in lung compliance during the first days of life. The lung compliance values obtained were slightly higher than previous reference data (25). However, the present measurements were performed with special attention to sleep-stage analysis, and this could partly explain our higher lung compliance readings. Previous reports have described that alveolar ventilation, functional residual capacity (16), and lung compliance (14) improve over the first postnatal days, but there are limited data available on the changes over the first postnatal hours. We did not observe a significant correlation between the initial compliance and gestational age. The initial compliance was not dependent on the timing of the first measurement. It is therefore possible that an essential part of postnatal lung liquid clearance takes place already within the first postnatal hours (28, 29).
We have previously reported amiloride-sensitive N-PD to correlate with the change in lung compliance (14), but in this study we set a more strict time line for the present indices of postnatal adaptation that is closer to delivery. The baseline N-PD representing the net transepithelial movement of anionic and cationic compounds remained constant during the study period. In addition, the amiloride-sensitive proportion of the N-PD remained at 40.0 ± 2.2%, which is somewhat less than reported previously (21, 23). We explain this slight discordance by methodologic differences that have previously shown slightly conflicting readings in amiloride sensitivity between infants born vaginally and via cesarean section (21, 23). However, in line with the present findings, a previous study reported only minute changes or no significant changes in the proportional amiloride-sensitive N-PD of healthy newborn infants depending on whether delivery was preceded by labor (21). As previously described (23), we detected no significant difference in amiloride-sensitive proportions of the baseline N-PD between infants delivered by the two methods. Because the electrophysiologic and lung functional data were similar in our study subjects, it is possible that the two groups of newborn infants have been in a comparable clinical state (e.g., in terms of the catecholamine levels) (30). However, our measurements performed at the early time point demonstrated a novel finding that the baseline and amiloride-sensitive N-PD had stabilized within a couple of hours after birth. This suggests that if a physiologic surge in perinatal airway ENaC happened, it must, at least in humans, take place in an acute fashion. Alternatively, it is possible that amiloride-insensitive Na+-absorptive mechanisms, as described in other models of excess lung fluid (31, 32), are equally important for perinatal liquid clearance and that these pathways are activated simultaneously with ENaC.
The role of the ENaC subunit has been considered crucial for channel function especially in the lung, whereas the pulmonary and subunits may possess facilitatory properties (13). There is no knowledge available on antenatal ENaC-subunit expression in humans. Postnatally, we found no statistically significant change over time in the expression of ENaC. There could be several explanations for the finding. First, postnatal ENaC expression may have peaked before our first sampling. Second, there may be a brief surge in ENaC mRNA levels (33). Third, because the ENaC is the prerequisite pore-forming subunit for maximal Na-channel activity and because the subunit expressions are differentially regulated, it is possible that antenatal factors are important for bringing ENaC expression to its constitutional level (34).
Previous investigations in experimental animals suggest that humoral and environmental factors influence expression, composition, and function of ENaC subunits and that these factors may have differential effects on the ENaC subunits (8, 19, 20, 26, 33–36). Of these factors, the changes in glucocorticoid hormone or physiochemical factors may be important (34), but these variables could not be included in the present study. The present study is in line with these earlier observations and suggests that the subunits' expressions are differentially regulated. First, the gene expression of ENaC and ENaC, but not that of ENaC, differed slightly but significantly depending on whether the method of delivery was vaginal or cesarean section. This finding is in line with some recent observations from animals (31). Second, one of the key findings of the present study was the significant decrease in the expression of ENaC and ENaC over the first days of life. Recent reports suggest a regulatory role for ENaC within the lung (35, 36). Mice overexpressing ENaC demonstrate similar symptoms as in cystic fibrosis, including mucus obstruction, goblet cell metaplasia, neutrophilic inflammation, and poor bacterial clearance (35). In the light of these previous experimental data, it is interesting that we observed the highest human postnatal ENaC subunit expression preceding the significant improvement in lung compliance. Accordingly, we conjecture that ENaC may have a regulatory role in the adaptation to air breathing. Alternatively, the rapid decrease in ENaC may be a reflection of mRNA stability or postnatal translational efficiency, both of which may differ between the subunits (37). We also found a significant decrease in the ENaC over the follow-up period. The implications of this latter observation are unclear.
We recognize some limitations to our study. The time of our first measurement (1–4 h after birth) was chosen because of difficulties in reaching all patients within an hour after birth. In light of our results, an early adaptation could have been missed by this chronology of sampling. Also, it can be argued whether the nasal epithelium can be used as a surrogate for measuring changes in the pulmonary epithelium. We favor the view that humoral factors during postnatal adaptation have an effect throughout the airway epithelium, and therefore changes in nasal epithelium should reflect changes taking place throughout the respiratory tract. Although the expression of all ENaC subunits in preterm infants with respiratory distress syndrome was recently reported to be significantly lower than the levels at term (14), the question of whether measurements of gene expression can be used to draw conclusions on functional proteins remains relevant. This is underscored by the fact that in the present study of human postnatal adaptation, we found no significant connection of ENaC expression between lung compliance and transepithelial ion transport. This observation suggests that translational and post-translational processes are pivotal in the regulation of the efficacy of ion transport in airway epithelium. Finally, we did not observe changes in the amiloride-sensitive proportion of the N-PD during the postnatal follow-up period. We cannot completely exclude the possibility of trauma perturbing the transepithelial ion movement affecting our measurements despite the fact that the baseline N-PD and epithelial RNA recovery in the biopsy specimen remained constant.
In summary, in healthy term infants we demonstrate an increase in lung compliance during the first 2 d of life. There was a significant correlation between the earliest amiloride-specific N-PD and lung compliance at approximately 24 h of postnatal age. A high perinatal airway expression of - and ENaC at birth followed by a significant decrease after the first hours of extrauterine life suggests that ENaC is under regulation during the immediate postnatal period. Finally, the present findings suggest that after successful postnatal transformation to air breathing, the maintenance of low amounts of the airway epithelial lining fluid may be shared by ion transport mechanisms other than ENaC.
Acknowledgments
The authors thank the personnel of the Neonatal Unit of the Hospital for Children and Adolescents for their kind cooperation, Marita Suni and Marjatta Vallas for their excellent technical assistance, and the Pediatric Graduate School of the University of Helsinki for support. The authors thank Professor Markku Heikinheimo, M.D., for his constructive criticism during preparation of the manuscript, and Professor of Biometrics Seppo Sarna, Ph.D., for his help with statistical analyses.
FOOTNOTES
Supported by the Foundation for Pediatric Research, the National Graduate School of Clinical Investigation, the Finnish Special Governmental Subsidy for Health Sciences, Finska Lkaresllskapet, and the Sigrid Juselius Foundation.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200501-052OC on November 4, 2005
Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject mater of this manuscript.
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