PERSPECTIVE
Fetal Lung Liquid Secretion
Insights Using the Tools of Inhibitors and Genetic Knock-out Experiments

Hugh O'Brodovich

Lung Biology Programme of the Hospital for Sick Children Research Institute, CIHR Group in Lung Development, and Departments of Paediatrics and Physiology, University of Toronto, Toronto, Ontario, Canada

At birth, the newborn infant's organ for gas exchange switches from the placenta to the lungs. Despite major advances in our understanding of the fundamental mechanisms involved in fetal lung development, respiratory disorders continue to be the major cause of perinatal morbidity and mortality. It is therefore not surprising that many investigators continue their research into the mechanisms responsible for the normal development of the fetal lung.

The initial key observation that led to our present understanding of fetal lung liquid production occurred in the 1940s when Jost and Policard (1) showed that the fluid within the fetal lung arises from the lung and did not, as it had been believed, represent aspirated amniotic fluid. At this time investigators speculated that the fluid was a transudate arising from the vasculature. However, experiments in the 1960s demonstrated that the fetal lung liquid has unusually high Cl ion concentrations (2) and that the movement of liquid into the developing fetal lung's lumen occurred as a result of the active transport of ions by the epithelium (3, 4).

What was the physiologic significance of this fluid secretion that distended the fetal lung and flowed out into the amniotic space? Clinical observations, including the recognition that oligohydramnios was associated with lung hypoplasia, suggested that the amount of fluid within the developing lung's lumen might be very important. A cause and effect relationship was demonstrated in chronically catheterized fetal lambs where increasing the amount of fluid within the lungs resulted in hyperplasia (5) associated with an increased DNA content per milligram of tissue (6); if the amount of fluid was decreased, the lungs were hypoplastic (5). These lamb studies are applicable to the human fetus since congenital laryngeal atresia is associated with hyperplastic lungs which are over-distended with fluid (7).

What are the cellular mechanisms that are responsible for the fetal lung liquid secretion? As in other ion transporting epithelia, the Na⁺/K⁺ ATPase is essential for normal fluid secretion. Studies in acutely isolated (8) and primary cultures (9) of fetal distal lung epithelia have outlined the ontogeny and functional importance of Na⁺/K⁺ ATPase as the "engine" for epithelial ion transport. However, the vectorial transport of ions by polarized lung epithelia can only take place if there are proteins on the basolateral (interstitial) side of the epithelia that permit ion entry into the cell and other proteins on the apical (lumenal) side that permit ion extrusion out of the cell. In vivo studies showing that fetal lung liquid production was reduced by the airspace instillation of bumetanide or furosemide (10) suggested a role for the Na⁺/K⁺/2Cl cotransporter in the Cl ion's entry into the cell during the secretory process. Electrophysiologic measurements combined with the use of pharmacologic inhibitors suggested that Cl exited through Cl permeant ion channels rather than moving across the membrane using other transporters such as a Na⁺/H⁺ antiport working in concert with a HCO₃/Cl anion exchanger (AE). The gene for cystic fibrosis transmembrane conductance regulator (CFTR) encodes a Cl permeant ion channel (11) and McCray and coworkers (12) showed that CFTR was present in the distal lung epithelium of the human lung. Although it was feasible that CFTR could be an (query the only) apical membrane exit pathway, experiments using a CFTR knockout mouse (13) demonstrated that there were Cl and fluid secretory pathways in the fetal murine lung that could operate independently from CFTR encoded Cl channel. These additional-alternative pathways are incompletely understood; however, they may involve the Ca⁺⁺ regulated Cl channel (14), the outwardly rectifying Cl channel (ORCC) (15), or a member(s) of the voltage gated ClC Cl ion channel family. Indeed, the ontogeny of ClC₂ (16) or ClC₃ (17) Cl channel expression is consistent with our understanding of fetal lung liquid secretion.

The article by Gillie and colleagues (18) in this issue represents a significant advance in our understanding of the mechanisms involved in fetal lung liquid secretion. These investigators used a combination of genetic, cell biologic, and electrophysiologic approaches to provide an important new insight into Cl secretion in the developing murine lung. They studied lungs from both wild type and Na⁺/K⁺/2Cl type 1 (NKCC1) knock-out fetal mice. Assuming that there is no undetected leakage in their knock-out mouse, their results prove that the NKCC1 is involved in the secretory process and that it is not the only pathway for the basolateral entry of Cl ions. They also show that the undetermined additional-alternative pathway(s) is (are) up-regulated in the NKCC1 knock-out fetal mouse. Since NKCC1 mice survive without obvious respiratory impairment (19), it is likely that these additional-alternative Cl entry pathways permit adequate lung liquid production for normal murine fetal lung development. Their experiments with pharmacologic inhibitors of ion transporting pathways provided data supporting their speculation that Na⁺/H⁺ antiport and the AE work in concert to provide a route for Cl entry across the basolateral membrane. Both the Na⁺/H⁺ antiport and the AE have been previously identified in distal lung epithelia (20) where they participate in the regulation of cell volume and intracellular pH. Although Gillie and coworkers provide some data to the contrary (18), it is likely premature to exclude other potential Cl entry pathways such as the Na⁺/HCO₃ symport (23). Further experiments will be required to determine which pathway(s) is (are) physiologically important in vivo.

There are some important caveats to the study by Gillie and colleagues, some of which arise from technical difficulties when working with murine fetuses or limitations to the albeit state-of-the-art techniques that they used. Fetal lung liquid secretion starts at a very early stage of lung development; however, these investigators were only able to study 17 to 19 d fetuses (term = 19 d). Since many ion transporting pathways are developmentally regulated, it is unknown whether their results can be extrapolated to earlier time points of murine fetal lung development. It should also be noted that water contents were determined from acutely isolated fetal lungs, yet their bioelectric studies and cyst volume experiments were conducted up to several days later, after the explants were exposed to ambient gas tensions that are not similar to those seen by the fetus in vivo. The switch from a fetal to a postnatal pO₂ alters the cyst formation of late gestation fetal lung explants (24) and the Na⁺ transporting characteristics of primary cultures of fetal distal lung epithelium (25), so it is unknown whether nonfetal ambient gas tensions affected the physiologic response of the explants that they studied. Pharmacologic inhibitors have their limitations and as acknowledged by the authors, many of the agents they utilized are not specific. For example, 0.1 mM amiloride not only blocks amiloride sensitive Na⁺ channels but also inhibits the Na⁺/H⁺ exchanger (26). There also may be cation or anion transport pathways that are not blocked by the agents that were used. This is relevant since their data show that the NKCC1 knock out mice had decreased lung water content relative to wild type mice yet had similar rates of lateral cyst wall expansion. Although the authors speculate that there were changes in secretion rates, one must also consider the possibility of changes in amiloride insensitive absorptive pathways.

The work by Gillie and colleagues highlights some of the strengths and limitations of using mouse genetic experiments to understand human physiology and disease processes. The principal strength is that the investigator can isolate one factor, in this case the presence or absence of the NKCC1 cotransporter, and not have concerns over such issues as effectiveness or specificity of pharmacologic inhibitors. The authors (18) have now provided conclusive proof of the importance of the NKCC1 cotransporter in murine fetal lung liquid production. The major limitations relate to the up-regulation of alternative cellular functions and our desire to understand and prevent or treat human disease. Physicians must understand the fundamental mechanisms responsible for the development of fetal lung hypoplasia and since some cases of human lung hypoplasia likely result from defective fetal lung liquid secretion, studies such as those by Gillie and coworkers are very important. However, it is unknown whether the human and murine fetal lung utilizes identical ion transport pathways. Indeed, their measurements of the murine lung liquid's pH, HCO₃, and Cl concentrations suggest some of the pathways may differ between the two species. Although the murine lung liquid Cl ion concentration is similar to values obtained from primates, the HCO₃ ion concentration is markedly different (Table 1). The reason for the difference in the marked HCO₃ concentrations of the fetal lung liquid from fetal lambs, guinea pigs, and mice versus dogs and primates is unknown (Table 1). The relevance of such observations is highlighted by previous genetic experiments in mice where the phenotype of "knock-outs" in the mouse and human are profoundly different for CFTR (27, 28) and the epithelial Na⁺ channel (30).

	Footnotes

Address correspondence to: Hugh O'Brodovich, M.D., FRCP(C), Paediatrician-in-Chief, Hospital for Sick Children, Professor and Chair of Paediatrics, University of Toronto, R. S. McLaughlin Foundation Chair in Paediatrics at The Hospital for Sick Children, Hospital for Sick Children, 555 University Ave., Toronto, ON, Canada M5G-1X8. E-mail: hugh.obrodovich{at}sickkids.on.ca

(Received in original form May 4, 2001).

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