Decreased sodium ion absorption across nasal epithelium of very premature infants with respiratory distress syndrome

Decreased sodium ion absorption across nasal epithelium of very premature infants with respiratory distress syndrome Pierre M, Barker, MBChB, C. W. Gowen, MD, Edward E, Lawson, MD, and Michael R. Knowles, MD From the Department of Pediatrics and Medicine, Universityof North Carolina at Chapel Hill, and the Department of Pediatrics,Universityof EasternVirginia and Hospital of the Kings Daughters, Norfolk, Virginia

Objective and study design: Successful adaptation to air breathing at birth depends on rapid absorption of fetal lung liquid that is mediated by activation of amiloride-sensitive sodium ion channels. To test the relationship between respiratory epithelial Na ÷ transport and development of respiratory distress syndrome (RDS), we measured nasal transepithelial potential difference (PD) in 31 very premature (-<30 weeks of gestation) newborn infants. Infants were retrospectively assigned to RDS (22 infants) and non-RDS (9 infants) groups on the basis of clinical and chest x-ray criteria. Results:Maximal nasal epithelial PD increased with birth weight (-1.2 mV/100 gm) and was lower in infants with RDS (-16.5 ± 0.6 mV) than in those without RDS (-22.0 ± 1.3 mV). Infants without RDS had PD values similar to normal fullterm infants. Amiloride inhibition of PD, an index of Na ÷ absorption,, was significantly lower, within the first 24 hours of life, in infants in whom RDS developed (3.8 ± 0.2 mV; 29.5% ± 0.8% inhibition) than in those without RDS (6. I ± 0.6 mV; 38.6% ± 0.5% inihibition). Maximal and amiloride-sensitive PD returned to normal during the recovery phase of RDS. Conclusions:We conclude that Na ÷ absorption across nasal epithelium increases wiith increasing birth weight and that impairment of Na ÷ absorption across the respiratory epithelia of very premature infants may contribute to the pathogenesis of RDS. (J Pediatr 1997;130:373-7)

During fetal life the lungs are expanded by liquid secretion that depends on active transport of the chloride ion across the pulmonary epithelium, t During labor and after delivery, liquid secretion diminishes and the airspaces are cleared by absorption of liquid out of the lung lumen. 2 Liquid absorption is driven by amiloride-blockable sodium ion transport 3, 4 and Supported by the Cystic Fibrosis Foundation. Submitted for publication April 17, 1996; accepted Aug. 19, 1996. Reprint requests: Pierre M. Barker, MBChB, Department of Pediatrics, University of North Carolina at Chapel Hill, 635 BurnettWomack Building, Manning Drive, Chapel Hill NC 27599-7220. Copyright © 1997 by Mosby-Year Book, Inc. 0022-3476/97/$5.00 + 0 9/21/77395

is initiated during labor and delivery, at least in part, by a sharp rise in circulating fetal epinephrine concentration. 2 Intraalveolar and interstitial pulmonary edema is a consistent histologic feature of respiratory distress syndrome, 5-7 See commentary, p. 342.

ENaC PD RDS

Epithelialsodium channel Potentialdifference [electrical] Respiratorydistress syndrome

but a primary etiologic role for ion transport abnormalities of the immature respiratory epithefium in this disease has not been shown. Because the capacity of the fetal lung to absorb 373

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Table. PD values for preterm infants with and without RDS and for healthy term infants RDS preterm

Mean PD (mY) Maximal PD (mV) Amilofide-sensitivePD (mY) Inhibition of PD by amiloride (%)

(<24 hr)

(72 hr)

(>7 days)

Non-RDS preterm (<24 hr)

Healthy term* (<24 hr)

-12.0 _+0.4 (13) -16.5 + 21)$ 3.8 _+0.2 (15)$ 29.5 _+0.8 (15)?

-14.3 +_0.6 (3) -19.5 + 0.9 (11) 4.6 _+0.4 (7) 33.1 _+ 1.7 (7)

-17.1 _+0.5 (7) -21.5 _+0.9 (12) 6.1 _+0.7 (8) 38.9 _+ 1.7 (8)

-16.0 -+ 0.4 (4) -22.0 + 1.3 (8) 6.1 _+0.6 (5) 38.6 -+ 0.5 (5)

-15.0 _+0.3 (40) -22.7 _+0.5 (40) -38.5 + 0.8 (25)

Number of newborninfants with measurementsavailableis shown in parentheses. *Data from Gowen CW, LawsonEE, BoucherRC, GatzyJT, KnowlesMR. J Pediatr 1988;113:121-7. $Significantlydifferentfrom non-RDS pretermgroup.

liquid and the expression or function of key components of the Na absorption mechanism (the amilofide-sensitive Na + channel, epithelial sodium channel, and Na+,K+-adenosineIriphospbatase) are dependent on fetal gestation, 2, s, 9 a "fetal" (i.e., liquid secretory) state may persist in the newborn lung of very premature infants. The critical dependence of neonatal respiratory adaptation on Na + absorption was shown recently when respiratory distress in c~-ENaCdeficient "knockout" mice resulted in death shortly after their birth. 4 It has been shown that hypoxia and respiratory difficulty develop in newborn guinea pigs when Na + absorption is blocked by addition of amilofide to the lung lumen. 10 Nasal potential difference reflects electrogenic transport of the dominant ions (Na + and C1-) across the respiratory epithelium and parallels ion transport patterns of lower respiratory epithelia.ll Nasal PD has been used extensively to diagnose cystic fibrosis, a condition characterized by amiloride-blockable Na + hyperabsorption in nasal and lower airway epithelia. 12,13 Abnormal nasal PD associated with cystic fibrosis and transient tachypnea of the newborn can be detected in the newborn period--even, in the case of cystic fibrosis, in preterm infants. 14 In normal term newborn infants, basal and amiloride-sensitive PD is similar to that in adults.15 We measured nasal PD in very premature newborn infants to test the relationship between maturity of Na ~-transport and the development of RDS. METHODS Our patient population consisted of 31 infants born at 30 weeks of gestation or less. We focused on infants born at that stage of gestation because the incidence of, and mortality rate from, RDS continues to be high in these infants. Infants were retrospectively assigned to an RDS group (22 infants) or a non-RDS group (9 infants). RDS was defined as respiratory distress associated with hypoxia, the need for mechanical ventilation for longer than 24 hours, and chest x-ray findings that were typical of this diagnosis. Eleven of the infants (seven with RDS, four without) were part of a study of sur-

factant replacement (the study was conducted before U.S. Food and Drug Administration approval for widespread surfactant use). Of these infants, 4 (57%) in the RDS group and 2 (50%) in the non-RDS group were given surfactant; the others were given placebo. We included in the analysis all infants who were born at 30 weeks of gestation or sooner and were tested. Nasal PD was measured within the first 24 hours in 29 of 31 infants (21 of 22 infants in the RDS group, 8 of 9 in the non-RDS group; mean time of study, 11 hours) and by 48 hours in all infants. In the RDS group, PD measurements were repeated in some infants at 36 hours (8 infants), 72 hours (10 infants), and after 7 days (12 infants). Measurements were repeated in some infants in the non-RDS group at 36 hours and 72 hours but not at later times. Because of technical difficulties (movement of the infant, electrical interference from nearby machinery, baseline drift), the mean PD and the amiloride-sensitive PD were not measured in all infants. PD was measured as described in detail previously.14, 15In brief, a recording bridge (Ringer filled polyethylene tubing) and a subcutaneous reference bridge (agar/Ringer-filled sterile 21-gauge needle) were linked by matched calomel electrodes to a high-impedance voltmeter. The recording bridge was perfused constantly with Ringer solution via an infusion pump (0.2 ml/min). We measured PD under the inferior turbinate at 0.25 cm intervals, moving from the anterior to the posterior site, as well as the mean PD (average of eight sites for each side) and the maximal PD (highest stable value). For amiloride superfusion studies, the region of the highest PD was revisited, and after a stable PD was recorded (_+ 10% PD drift for 30 seconds) amiloride HC1 (10-5 tool/L) was superfused over the recording area via a second catheter. The values shown represent the mean z SEM. Differences in mean values were tested by unpaired t tests or analysis of variance. Independent effects of baseline clinical parameters were determined by multivariate analysis. Differences with p values less than 0.05 were considered significant.

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RESULTS Infants in the RDS and non-RDS groups were matched for gestational age (27.9-+ 0.4 weeks, non-RDS group; 27.2 _+ 0.3 weeks, RDS group), but the mean birth weight of the non-RDS group (110t -+ 52 gm) was significantly higher than that of the RDS group (935 _+ 44 gm; p <0.05). There was a tendency for Apgar scores to be lower in the RDS group (1-minute score: non-RDS group, 4.5 + 0.8; RDS group, 3.6 + 0.4; 5-minute score: non-RDS group, 7.4+-0.5; RDS group, 6 . 3 + 0 . 3 ; p >0.05 at 1 and 5 minutes). In accordance with the known influence of gender in RDS, boys were strongly overrepresented in the RDS group (77%) and underrepresented in the non-RDS group (20%). Clinical outcome. In the non-RDS group, five of the nine infants underwent intubation (mean duration of ventilation, 17 + 5 hours; range, 4 to 24 hours) and all required some supplemental oxygen (mean duration, 47 -+ 12 hours; range, 24 hours to 7 days). All infants in the RDS group were supported with mechanical ventilation for 7 days or longer. Four infants in the RDS group (18%) died of respiratory failure during the first month of fife. Of the remaining 18 infants with RDS, the mean duration of ventilator treatment was 26 + 5 days;. All except one infant with RDS required 02 for more than 30 days, but only nine required 02 after 36 weeks' postconceptional age (mean duration of 02 therapy was 63 + 11 days, with the exclusion of one infant who required 02 for 33 months). Five of the surviving infants with

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Fig. 2. Inhibition of nasal PD by amiloride in 15 infants with RDS and five healthy infants. Individual values are shown. The mean (_+SEM) inhibition of PD (38.5% -+ 0.6%) by amiloride in term infants is shown by the dashed line. (Mean data from Gowen CW, Lawson EE, Boucher RC, Gatzy JT, Knowles MR. J Pediatr 1988;113:121-7.) RDS had lower respiratory problems after the first year of fife. PD measurements. Infants in the RDS group had significantly lower maximal PD values (-16.5 -+ 0.6 mV) measured within the first 24 hours, in comparison with measurements in infants i n t h e non-RDS group (-22.2 + 1.3 mV; Table). Maximal PD measured in the first 24 hours increased with gestational age at birth (-1.5 mV/wk; p <0.05) and with birth weight (-1.2 mV/100 gm; p <0.01; Fig. 1) and was lower in the RDS group than in the non-RDS group (p <0.001; Table). Inhibition of PD by amiloride was significantly less in the RDS group than in the non-RDS group (p <0.001; Fig. 2). These differences were evident when data were expressed as the absolute change in PD after amiloride (APD, 3.8 _+ +0.2 mV in the RDS group; APD, 6.1 +- 0.6 mV in the non-RDS group) or as the percentage of inhibition of basal PD after amiloride (29.5% _+ 0.8% inhibition in the RDS group; 38.6% _+ 0.5% inhibition in the non-RDS group; Table). Differences between the RDS and non-RDS groups in maximal and amiloride-sensitive PD were independent of differences in birth weight between the groups. The decreased PD seen in the RDS group was clearly linked to a smaller amiloride-sensitive PD (Fig. 3). In the RDS group, maximal and amiloride-sensitive PD increased significantly during the first 72 hours of life, and after 7 days all bioelectric parameters were very similar to those of preterm and normal term infants without RDS who were studied in the first 24 hours (Table).

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DISCUSSION The principal cause of RDS is thought to be deficient surfactant production and secretion in the lungs of preterm infants. Widespread use of surfactant has decreased the morbidity and mortality rates associated with this disease. 16 However, RDS continues to be a significant cause of death in very premature infants, and the incidence of bronchopulmonary dysplasia has not decreased since the introduction of surfactant replacement therapy. 16 These observations suggest that surfactant deficiency is not the sole cause of these two disease entities. Our data indicate that RDS in very premature infants is associated with an abnormality of ion transport, specifically, an incomplete switch of lung liquid movement from the "fetal" (i.e., liquid [C1-] secretory) state to the "postnatal" (i.e., liquid [Na ÷] absorptive) state. Ion and liquid flow in immature human (and other species) fetal lung is dominated by C1- (and liquid) secretion, with little inhibition achieved by the Na + channel blocker amiloride.2' 17 By contrast, the postnatal alveolar epithelia show no evidence of the secretion of C1- but have a high fraction of amiloride-sensitive PD. In the present study, basal and amiloride-sensitive PD increased with advancing gestational age and birth weight, suggesting a gestation-dependent maturation of Na + transport. The residual, amiloride-insensitive PD was similar in the RDS group (8.8 -+ 0.5 mV) and the non-RDS group (9.9 + 1.2 mV). This residual PD may reflect the ongoing,

active secretion of C1- (liquid secretion), amiloride-insensifive Na + absorption (liquid absorption), or both. It is possible that amiloride-insensitive Na + channels may participate in liquid clearance in newborn lung, but previous studies of mice 4 and guinea pig 1° suggest that the dominant path for Na + absorption from the lung lumen is through an amiloride-sensitive channel. The strongest supportive evidence for the critical role of o~-ENaC in perinatal liquid absorption, which links defective perinatal liquid absorption to RDS, comes from the recent study of ~-ENaC "knockout" newborn mice. 4 The respiratory distress and early death observed in these mice were associated with high ("fetal" -like) water content of the lungs, which showed no other pathologic changes. Likewise, the lungs of preterm monkeys with early RDS 8 and of low birth weight infants who died of this disease5 showed waterlogged alveoli and distal airways. These latter studies suggest that the intraalveolar edema found in RDS comes both from inadequate perinatal fiquid clearance and from secondary influx of liquid through damaged epithelium. Failure to activate fully the liquid absorptive mechanism in labor has been proposed as a mechanism for the apparent waterlogging of lungs in transient tachypnea of the newborn, which is often associated with elective cesarean delivery in term infants. In an earlier study by Gowen et al., 15 infants with transient tachypnea of the newborn and infants born by elective cesarean delivery had a smaller fraction of amiloride-

The Journal ~,f Pediatrics Volume 130, Number 3

sensitive PD (30.9% and 31.8%, respectively) than did healthy infants (38.5%). These data are consistent with submaximal activation of existing Na + channels in the mature lung epithelium. We propose that, in RDS, waterlogging of the lungs results from deficiency of the Na + channels in the immature lung. Infants in whom RDS did not develop had values of mean, maximal, and amiloride-sensitivePD similar to those which we reported for term infants.13 In contrast, infants in whom RDS developed had low PD values and low amiloridesensitive PD values within the first 24 hours. Inhibition of PD by amiloride was the best discriminator between the non-RDS and the RDS preterm groups, with no overlap between the two groups in measurements made within the first 24 hours (Fig. 2). Some "normalization" of PD measurements was evident within the first 72 hours, and after 7 days the PD values were in the range reported for healthy term infants13 (Fig. 3; Table). This trend toward"normalization" of PD suggests that ion transport changes precede the onset of, and the recovery from, RDS and are not secondary to the disease process. The differences in PD measurements between the RDS and the non-RDS groups fit well with the known hormone and gestation dependence of Na + absorption and ENaC expression in fietal lung], 17These maturational events coincide with a brisk rise in endogenous triiodothyronine and cortisol in human cord blood that is evident after 30 weeks of gestation. 18 It is likely, then, that ENaC expression and consequently liquid absorptive capacity may be deficient in the lungs of some very premature newborn infants. Because nasal PD parallels ion transport patterns in more distal regions of the human airway, l° our results suggest that deficient amiloride-sensitive Na + absorption plays a pathogenic role in the development of RDS. If further study confirms an association among "fetal" patterns of ion transport across the nasal epithelium, perinatal hormone deficiency, and the onset of RDS and bronchopulmonary dysplasia, then prenatal or early neonatal therapy that targets ion transport immaturity 'may improve the outcome of RDS in very premature infants. REFERENCES

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