Genomic Organization and Developmental Expression of Aquaporin-5 in Lung

M. Douglas Lee, Landon S. King, Søren Nielsen and Peter Agre

Chest 1997;111;111S-113S DOI 10.1378/chest.111.6_Supplement.111S

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CHEST is the official journal of the American College of Chest Physicians. It has been published monthly since 1935. Copyright 1997 by the American College of Chest Physicians, 3300 Dundee Road, Northbrook, IL 60062. All rights reserved. No part of this article or PDF may be reproduced or distributed without the prior written permission of the copyright holder. (http://chestjournal.chestpubs.org/site/misc/reprints.xhtml) ISSN:0012-3692

Downloaded from chestjournal.chestpubs.org by guest on February 19, 2010 1997 by the American College of Chest Physicians Studies in Human Lung Epithelial Cells Am Rev Respir Dis 1990; 142:1250-57 5 Suzuki S, Zuege D, Berthiaume Y. Sodium-independent mod¬ Human alveolar carcinoma cells (A549) were plated ulation of Na+-K+-ATPase activity bybeta-adrenergic agonist in for 24 h prior to infection with MOIs of 1 to 50 of alveolar type II cells. Am J Physiol 1995; 268:L983-L90 AdMRCMVa!, AdMRCMVpi, and AdMRCMVp-Gal. 6 Olivera W, Ridge K, Wood LDH, et al. Active sodium Cells infected with MOIs of 5 to 50 of AdMRCMVc^ transport and alveolar epithelial Na-K-ATPase increase dur¬ demonstrated twofold to threefold increases in ouabain- ing subacute hyperoxia in rats. Am J Physiol 1994; 266:L577- inhibitable 86Rb+ uptake 24 h after infection. No changes L84 in Na+/K+-ATPase function were noted following infec¬ 7 Schneeberger EE, McCarthy KM. Cytochemical localization tion with AdMRCMVPi or AdMRCMVp-Gal. Northern of Na, K-ATPase in rat type II pneumocytes. J Appl Physiol blot of total RNA a cDNA 1986; 20:1584-89 analysis using rat-specific probe 8 O'Brodovich HM. The role of active revealed the of ax mRNA in cells infected Na+ transport by lung presence only in the clearance of fluid. New Horizons with AdMRCMVcq; no change in px message was noted. epithelium airspace Western blot showed increased 1995; 3:240-47 analysis significantly ax 9 K, Ilekis Factor P, et al. Inhibition of the in cells infected with These find¬ Ridge J, Na,K protein AdMRCMVcq. ATPase by transfection of alveolar type 2 cells with antisense ings suggest that ax subunit may be the rate-limiting RNA to the Na,K ATPase [abstract]. Am Rev Respir Dis subunit in A549 cells and that Na+/K+-ATPase function 1992; 145:A832 can be increased in these cells using gene transfer. 10 McGrory W7J, Bautista DS, Graham FL. A simple technique The human ax isoform is two to three logs more for the rescue of early region 1 mutations into infectious sensitive to ouabain than is the rat a2 isoform. To dem¬ human adenovirus type 5. Virology 1988; 163:614-17 onstrate transgene expression, ouabain sensitivity was determined in A549 cells by ascertaining the concentra¬ tion of ouabain that produced a 50% reduction in 86Rb+ uptake (IC50). A549 cells were infected with AdM- Genomic and RCMVcq at an MOI of 25, and 86Rb+ uptake was Organization measured using 15 different concentrations of ouabain Developmental Expression of (IX10-11 to 1X10~3). IC50 values were determined using Aquaporin-5 in Lung* a computerized nonlinear least squares regression func¬ tion to test for the of one or two designed presence M. Douglas Lee, MD; Landon S. King, MD; isozymes of a receptor with different affinities for a ligand S0ren Nielsen, PhD; and Peter Agre, MD (ouabain). AdMRCMVax-infected cells demonstrated two distinct IC50 values (IC50(1)=3.5X10~ IC50(2)- Sham and (CHEST 1997; 111:111S-113S) 3.2X10"5). AdMRCMVp-Gal infected cells "T\ of the of water transporters one was not iscovery aquaporin family each demonstrated IC50 that different from -"-^ a molecular for the first The identification of a second two provided explanation osmotically ax IC50. a1 IC50 driven water transport across cell membranes of mamma¬ in or logs greater than that found the sham AdMRCMVP- lian and tissues. At there are six known Gal infected cells that of two functional plant present, suggests presence mammalian aquaporins distributed in a wide variety of ax isozymes: the endogenous ouabain-sensitive human ax tissues and cell In the distribution of each and the ouabain-resistant rat types. general, transgenic aY. aquaporin is unique, though there are some scattered sites These results demonstrate that adenoviruses can effi¬ of (reviewed and Recent identifi¬ transfer to alveolar cells and overlap by King Agre1). ciently genes lung epithelial cation and characterization of the genes encoding these augment Na+/K+-ATPase abundance and function. The have enabled to establish¬ to infection with individual subunits differs aquaporins investigators begin response ing a role for aquaporins in the pathogenesis of several between cell types such that the Px subunit could be disease states.23 the wealth of evidence in recent for rat ATII cells and be the rate- Despite rate-limiting a! may years to explain the physiology of salt transport in the lung, limiting subunit for human A549 cells in vitro. Conceiv¬ the molecular mechanisms for transcellular movement of ably, gene transfer to the alveolar epithelium may be water in the lower respiratory tract remain poorly under¬ applicable to the prevention and treatment of pulmonary stood. edema. Four aquaporins have been identified in the lung by Northern blot or References analysis immunoblotting4'6 (Table 1). 1 Sznajder JI, Olivera WG, Ridge KM, et al. Mechanisms of *From the Departments of Pulmonary and Critical Care Medi¬ lung liquid clearance during hyperoxia in isolated rat lungs. cine (Drs. Lee and King), Medicine and Biological Chemistry Am J Respir Crit Care Med 1995; 151:1519-25 (Drs. Lee, King, and Agre), Johns Hopkins University, Balti¬ 2 Rutschman DH, Olivera W, Sznajder JI. Active transport and more; and Cell Biology, Institute of Anatomy (Dr. Nielsen), passive liquid movement in isolated perfused rat lungs. J Appl University of Aarhus, Denmark. 75:1574-80 Supported in part by NIH ROI grants EY11239, HL48268, and Physiol 1993; HL33991 NRSA F32 HL09420 and NRSA 3 Goodman BE, Kim K, Crandall ED. Evidence for active (P.A.), (M.D.L.), F32 HL09119-02 (L.K.); Dr. Lee was recipient of an Allen and sodium transport across alveolar epithelium of isolated rat Hanburys Pulmonary Fellowship Award. lung. J Appl Physiol 1987; 62:2460-66 Reprint requests: Peter Agre, MD, Department ofBiological Chem¬ 4 Matthay M, Wiener-Kronish J. Intact epithelial barrier func¬ istry, Johns Hopkins University School ofMedicine, 725 N Wolfe St, tion is critical for the resolution of alveolar edema in humans. Baltimore, MD 21205-2185; email: [email protected]

CHEST/ 111 /6/JUNE, 1997 SUPPLEMENT 111S Downloaded from chestjournal.chestpubs.org by guest on February 19, 2010 1997 by the American College of Chest Physicians Table 1.Aquaporins in Lung induction of AQP1, AQP5 was expressed in rat lung 1 to 2 after with levels into Human days birth, protein steadily increasing Gene adulthood.11 While AQP1 expression was induced in both Protein Location Expression in Lung perinatal and adult rat lung by corticosteroids,4 AQP5 was not induced in rat lung at any age. Immunoelectron Aquaporin-1 7pl4 Peribronchial endothelium, visceral localization demonstrated in the rare alveolar microscopic AQP5 apical pleura, pneumocytes membrane of I was not Aquaporin-3 9p21 -» 12 Basolateral surface of bronchial type pneumocytes; AQP5 present epithelium in type II pneumocytes, airway epithelium, or vascular Aquaporin-4 18qll.2-12.1 Basolateral surface of bronchial structures. and tracheal epithelium These studies have defined the gene structure for Aquaporin-5 12ql3 Type I pneumocytes, trachea, AQPS and localized the cell-specific expression of bronchial submucosa AQPS protein in alveolar epithelium. Characterization of the AQPS gene structure should aid future studies directed at defining the promoter-specific elements is in the in vascular required for AQPS gene regulation during normal Aquaporin-1 (AQP1) expressed lung and in clinical disorders of endothelium, visceral pleura, and a subset of pneumo¬ development respiratory cytes.4 AQP4, the major brain aquaporin,7 localizes to tissue. The abundance of AQP5 on the apical surface of the basolateral membrane of bronchial and tracheal type I pneumocytes suggests a role in alveolar trans- epthelium in lung.5 AQP3 is expressed in the lung membrane water movement, though the lack of a exclusively on the basolateral surface of tracheal epithe¬ subapical or basolateral location for AQP5 may indicate lium.5 The absence of AQPs 1, 3, and 4 in type I that other as yet undefined aquaporins may participate pneumocytes or on the apical surface of airway epithe¬ in the movement of water across the alveolar epithe¬ lial cells suggests the presence of another aquaporin in lium. Additional studies aimed at addressing this issue those locations. Recently, the complementary DNA and determining the precise localization of AQP5 in (cDNA) for the fifth mammalian aquaporin (AQP5) was upper airway epithelium are currently underway. isolated from rat; Northern blot analysis demonstrated AQP5 in rat salivary and lacrimal glands, cornea, and lung.6 Herein we discuss the isolation and characteriza¬ References tion of the human cDNA and as well as the AQP5 gene, 1 P. of the water and distribution of in rat King LS, Agre Pathophysiology aquaporin ontogeny AQP5 lung. channels. Annu Rev 1996; 58:619-48 the rat cDNA as a molecular an Physiol Using AQP5 probe, 2 Deen PMT, Knoers NVAM, et al. cDNA clone was isolated from a human submax- Verdijk MAJ, Require¬ AQPS ment of human renal water channel aquaporin-2 for illary gland library. The 1,348-base pairs (bp) insert vasopressin-dependent concentration of urine. Science contained a 795-bp open reading frame encoding a 265 1994; 264:92-95 amino acid protein with 91% homology to rat AQP5.8 3 Shiels A, Basnett S. Mutations in the founder of the MIP gene in Expression of human AQP5 Xenopus laevis oocytes family underlie cataract development in the mouse. Nat conferred mercurial-sensitive osmotic water permeabil¬ Genet 1996; 12:212-15 ity equivalent to other aquaporins. A 7.4-kilobase Sal 4 King LS, Nielsen S, Agre P. Aquaporin-1 water channel I-EcoRI fragment from a human genomic library was protein in lung: ontogeny, steroid-induced expression, and found to contain the human AQPS structural gene. distribution in rat. J Clin Invest 1996; 97:2183-91 Nucleotide sequencing with primers corresponding to 5 Frigeri A, Gropper MA, Turck CW, et al. Immunolocalization the human AQPS cDNA identified four exons separated of the mercurial-insensitive water channel and glycerol intrin¬ introns of 1.2, 0.5, and 0.9 kilobases. of the sic protein in epithelial plasma membranes. Proc Natl Acad by Analysis Sci USA 92:4328-31 determined to be a TATA-less 1995; 5'-flanking region AQPS 6 Raina S, Preston GM, W7B, et al. Molecular with a initiation site 518 Guggino cloning gene transcription bp upstream and characterization of an cDNA from of the methionine. Genomic Southern blot aquaporin salivary', initiating lacrimal, and respiratory tissues. J Biol Chem 1995; 270: analysis confirmed AQPS to be a single copy gene that 1908-12 localized to human chromosome 12ql3; this coincides 7 Jung JS, Bhat RV, Preston GM, et al. Molecular character¬ with the chromosomal locations of the homologous ization of an aquaporin cDNA from brain: candidate osmo- human genes MIP9 and AQP2,10 thus demonstrating the receptor and regulator of water balance. Proc Natl Acad Sci site of an aquaporin gene cluster. USA 1994; 91:13052-56 the distribution, and steroid-in¬ 8 Lee MD, Bhakta KY, Raina S, et al. The human aquaporin-5 Recently ontogeny, Biol Chem 271:8599-8604 duced expression of AQP1 in rat lung was described, gene. J 1996; in the clearance of water in both the 9 Saito F, Sasaki S, Chepelinsky AB, et al. Human AQP2 and implicating AQP1 MIP two members of the MIP within and adult rat The absence of AQP1 in genes, family, map perinatal lung.4 type chromosome band 12ql3 on the basis of two-color FISH. I or type II pneumocytes suggested the existence of Cell Genet 1995; 68:45-48 another water channel in that To determine the Cytogenet location. 10 Sasaki S, Fushimi K, Saito H, et al. Cloning, characterization, ontogeny of AQP5 in lung, membranes from fetal and and chromosomal mapping of human aquaporin of collecting postnatal rat lungs were immunoblotted with affinity- duct. J Clin Invest 1994; 93:1250-56 purified anti-AQP5 antibody. In contrast to the perinatal 11 King, LS, Nielsen, S, Agre, P. Aquaporin-5 is expressed in rat

112S Thomas L. Petty 39th Annual Aspen Lung Conference: Genes and Gene Therapy Downloaded from chestjournal.chestpubs.org by guest on February 19, 2010 1997 by the American College of Chest Physicians type I pneumocytes [abstract]. Am J Respir Crit Care Med respiratory distress and died within 40 h after birth. To 1996; 153:A508 evaluate the presence of aENaC-mediated electrogenic Na+ transport in the neonatal lung, potential difference (PD) was measured across the epithelia in cultured tra¬ cheal cysts derived from freshly excised lungs. Mean Role of the Epithelial Sodium baseline PD of aENaC./. explants was lower than in cysts from 4-/+ or +/. airway epithelium, and in Channel in Lung Liquid aENaC./. animals, amiloride-sensitive electrogenic Clearance* Na+ transport was completely abolished. Histologic analysis showed no differences in lung archi¬ tecture or alveolar epithelial differentiation between Edith Hummler, PhD; Pierre Barker, MD; aENaC./. mice and control but whole lobes Friedrich Chantal littermates, Beermann, PhD; John Gatzy, MD; Verdumo; were filled with and air was in a few Richard Boucher, MD; and Bernard C. Bossier, MD liquid present subpleural alveoli of aENaC./. mice. This was con¬ firmed by measuring whole lung wet-to-diy weight ratio (CHEST 1997; 11L113S) a that reflects water content ofthe "C1 etal secretes into the future fluid that (W/D), parameter lung. lung airspaces The ratios of fetal were similar in all three .*- arises from a continuous active secretion of CI. lungs geno¬ by Fifteen minutes after the values of the At birth, the transform types. birth, wild-type lung's epithelium. lungs rapidly and mutant mice were intermediate, reach¬ into a fluid organ to enable efficient gas heterozygous (Na+)-absorbing ing values similar to those of adult mouse lungs or 4 to 6 h exchange. Although involvement of an active transepithe- after birth, that the fetal lial Na+ in the this transition to an indicating remaining lung liquid transport lung during had been removed. in aENaC knockout mice, air-filled organ has been demonstrated,12 the importance Contrarily, and the role of the amiloride-sensitive sodium the W/D values decreased slightly from fetal values, with epithelial no further clearance 4 to 6 h and even W/D at channel (ENaC) in these processes has not been estab¬ by higher 10 to 14 h, suggesting that liquid was reaccumulating in lished. In preterm rat3 and human4 lung, ENaC messen¬ those ger RNA are detectable, but lungs. transcripts barely expression In this study, we present evidence of a direct rela¬ levels increase considerably in the perinatal period and between the absence of ENaC function and remain adult life,3 a role for tionship high throughout suggesting neonatal death due to a defective clearance. ENaC in maintaining a "dry" alveolar state postnatally. An lung liquid amiloride-sensitive Na+ has We propose that active Na+ transport across the new¬ electrogenic reabsorption born alveolar mediated ENaC, is a been documented in and lower but it was epithelium, by upper airways, critical factor in the successful of the new¬ not established whether this sodium is mediated adaptation transport born to air and of this ENaC in vivo. breathing, immaturity pathway by may contribute to the etiology of respiratory diseases The ENaC plays an important role in determining the associated with final amount of sodium reabsorbed. ENaC is localized in preterm delivery. the apical membrane oftight epithelia lining the distal part of the in the distal in the ducts of several nephron, colon, References exocrine glands, and in the alveolar cells of the lung.5 ENaC is a heteromultimeric protein made up of three 1 O'Brodovich H. Epithelial ion transport in the fetal and subunits (a, (3, and 7).6 and expres¬ perinatal lung. Am J Physiol 1991; 26LC555-64 homologous Assembly 2 et sion of functional active sodium channels in vitro are Olver RE, Ramsden CA, Strang LB, al. The role of amiloride-blockable sodium in adrenaline-induced on the subunit aENaC, the and transport strictly dependent (3 7 lung liquid reabsorption in the fetal lamb. J Physiol 1986; subunits being unable, by themselves, to induce an amilo¬ 376:321-40 ride-sensitive sodium current. 3 Tchepichev S, Ueda J, Canessa C, et al. Lung epithelial Na To elucidate the role of ENaC in we inactivated vivo, channel subunits are differentially regulated during develop¬ the mouse aENaC gene locus by gene targeting in mouse ment and by steroids. Am J Physiol 1995; 269:C805-12 embryonic stem cells (ES).7 At birth, the genotypic anal¬ 4 Voilley N, Lingueglia E, Champigny G, et al. The lung ysis of newborn animals from heterozygous mutant breed¬ amiloride-sensitive Na+ channel: biophysical properties, phar¬ ing pairs revealed that embryonic and fetal development macology, ontogenesis, and molecular cloning. Proc Natl Acad in aENaC(./.) animals was not Sci USA 1994; 91:247-51 significantly impaired. et Whereas most newborns a normal 5 Due C, Farman N, Canessa CM, al. Cell-specific expression developed breathing of sodium channel and subunits in aldoste- after birth, aENaC(./.) neonates epithelial a, p 7 pattern developed rone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry. J Cell Biol 1994; *From the Institut de Pharmacologie et de Toxicologie de 127:1907-21 I'Universite, Lausanne, Switzerland (Drs. Hummler and Rossier 6 Canessa Schild Buell et al. Amiloride-sensitive and Ms. Verdumo); University' of North Carolina School of CM, L, G, Medicine, Chapel Hill (Drs. Barker, Gatzy, and Boucher); and epithelial Na+ channel is made of three homologous subunits. the Swiss Institute for Experimental Cancer Research, Epalin- Nature 1994; 367:463-67 ges, Switzerland (Dr. Beermann). 7 Hummler E, Barker P, Gatzy J, et al. Early death due to Reprint requests: Edith Hummler, PhD, Institut de Pharmacolo¬ defective neonatal lung liquid clearance in aENaC-deficient gie, CH-1005, Lausanne, Switzerland. mice. Nat Genet 1996; 12:325-28

CHEST/111/6/JUNE, 1997 SUPPLEMENT 113S Downloaded from chestjournal.chestpubs.org by guest on February 19, 2010 1997 by the American College of Chest Physicians Genomic Organization and Developmental Expression of Aquaporin-5 in Lung M. Douglas Lee, Landon S. King, Søren Nielsen and Peter Agre Chest 1997;111; 111S-113S DOI 10.1378/chest.111.6_Supplement.111S

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