- Email: [email protected]
Genomic Organization and Developmental Expression of Aquaporin-5 in Lung
STUDIES I N I-IuMAN LUNG EPITHELIAL CELLS
Human alveolar carcinoma cells (A,549) were plated for 24 h prior to infection with MOls of 1 to .50 of AdMRCMV(X " AdMRCMVI3" and AdMRCMVI3-Gal. Cells infected with MOI s of 5 to 50 of AdMRCMV(X I demonstrated twofold to threefold increases in ouabaininhibitable SORb + upt ake 24 h after infection. No chan ges in Na +/K+-A'I'Pase function were noted following infection with AdMRCMVl31 or AdMRCMVI3-Gal. Nort hern blot analysis of total BNA using a rat-specific c DNA probe revealed the presence of (XI mRNA only in cells infected with AdMRCMV(XI ; no change in 131 message was not ed . Western blot analysis showed significantly increased (X l protein in cells infected with AdMRCMV(XI ' Th ese findings suggest that (Xl subunit may be the rate-limiting subunit in A,549 cells and that Na+/K+-ATPase function can be increased in thes e cells using gene transfer. The human (Xl isofonn is two to thr ee logs more sensitive to ouabai n than is the rat (X l isofonn. To dem onstrate transgene exp ression, ouabain sensitivity was determined in A549 cells by ascertaining the concentration of ouabain that produced a 50% reduction in HOBb + uptake (IC 50 ) . A549 cells were infected with AdMBCMV(XI at an MOl of 25, and 8°Bb + uptake was measured using ] 5 differ ent conce ntrations of ouabain (l X lO- lI to l XlO - 3 ) . IC 50 values were determined using a computerized nonline ar least squares regression function designed to test for the pr esenc e of one or two isozymes of a receptor with differ ent affinities for a ligand (ouabain). AdMRCMV(X I-infected cells demonstrated two distin ct IC,5o values (IC 50 (l ) =3..5 X 10- 7 , IC 50 (2)= 3.2X 10- 5 ) . Sham and AdMBCMVI3-Gal infected cells each demonstrated one IC50 that was not differ ent from the first (X l IC.5o. The identification of a second (Xl IC5 0 two logs greater th an that found in the sham or AdMRCMVI3Gal infect ed cells suggests that presenc e of two functional (XI isozymes: the endogenous ouabain-sensitive hum an (XI and the ouabain-resistant transgenic rat (XI ' These results demonstrate that adenoviruses can efficiently tra nsfe r genes to lung alveolar epithelial cells and augm ent Na+/K+-A'TPase abun danc e and function . The respons e to infection with individual subun its differs between cell types such that the 131 subunit could be rate-limiting for rat ATII cells and (Xl may be the ratelimiting subunit for hum an A549 cells in vit ro. Conceivably, gen e tran sfer to the alveolar epithelil~m may be app licable to the pr evention and treatm ent of pulmonary ede ma. R EFERENCES
1 Sznajder ]1. Olivera WG, Hidge KM, et al. Mechanisms of lung liquid clearan ce durin g hyperoxia in isolated rat lungs. Am J Hespir Crit Care Med 1995; 1.5 1:1519-25 2 Rutschman DB , Olivera W, Sznajder JI. Active transport and passive liquid movement in isolated perfused rat lungs. J Appl Physiol 1993; 75:1574-80 3 Goodman BE, Kim K, Crandall E D. Evidence for active sodium transport across alveolar epith elium of isolated rat luug. J Appl Physiol 1987; 62:2460-66 4 Matthay M, Wiener-Kronish J. Intact epithelial barrier lunction is critical for th e resolution of alveolar edema in hum ans.
Am Rev Hespir Dis 1990; 142:1250-57 5 Suzuki S, Zuege 0 , Berthiaum e Y. Sodium-indepe ndent modulation of?\a+-K+-ATPase activityby beta-adrenergic agonist in alveolar type II cells. Am J Physiol1995; 268:L983-L90 6 Olivera \V, Hidge K, Wood LD H, et al, Active sodium tran sport and alveolar epithelial Na-K-ATPase increase during subacute hyp eroxia in rats. Am J Physiol 1994; 266:L577L84 7 Schneeberger EE , McCarthy KM. Cytochemi cal localization of Na, K-ATPase in rat typ e II pneumocytes. J Appl Physiol 1986; 20:1.'584-89 8 O'Brodovich HM. The role of active Na + transport by lung epith elium in the clearance of airspace fluid. New Horizons 1995; 3:240-47 9 Ridge K, Ilekis J, Factor P, et al. Inhibition of the Na,K ATPase by transfect ion of alveolar type 2 cells with antisense RNA to the Na,K ATPase [abstract]. Am Rev Respir Dis 1992; 145:A832 10 McGrory WJ, Bautista OS, Graham FL. A Simple technique for the rescue of early region 1 mutations into infectious human adenovirus type 5. Virology 1988; 163:614-17
Genomic Organization and Developmental Expression of Aquaporin-5 in Lung* IH. Douglas Lee, uo. Landon S. King, MD ; S¢ren Nielsen, PhD; and Peter Agre, MD
(C HEST 1997; 11 1:111 5-11 3 5) iscovery of the aquaporin family of water transp~rters provided a molecular explanation for osmot ically driven water transport across cell membranes of mammalian and plant tissues, At present, ther e are six known mammalian aquaporins distributed in a wide variety of tissues and cell types . In general, the distribution of each aquapori n is uniq ue, tho ugh there are some scattered sites of overlap (reviewed by King and Agre l ) . Recent identification and characterization of the genes encoding these aquaporins have enabled investigators to begin establishing a role for aquaporins in the pat hogenesis of several disease states.s' Despite the wealth of evidence in recent years to explain the physiology of salt tran sport in the lung , the molecu lar mech anisms for transcellu lar movem ent of water in the lower respiratory tract remain poorly understood . Four aqua porins have been identified in the lung by Northern blot analysis or imrnunoblotting-" Cr able 1).
D
*From the Department s of Pulm onary and Critical Care Medicine (Drs. Lee and King), Medicin e and Biological Chemi stry (Drs. Lee , King, and Agre), Johns Hopkins University, Baltimore ; and Cell Biology, Inst itute of Anatomy (Dr. Nielsen ), University of Aarhus, Denmark. Supported in part bv ?\IB ROI grants EYI 1239, HL48268, and H L33991 (PA), NRSA F32 HL09420 (M.D .L.), and NRSA F32 H L09119-02 (L.K.); Dr. Lee was recipient of an Allen and Hanbu rys Pu lmon ary Fellowship Award. Reprint requests: Peter Agre, MD , Departmen t of Biological Chemistn], [ohns Hopkins University School of Medicine, 725 N W olfe St, Baltimore, MD 21205-2185; email: [email protected] CHEST / 111 / 6 / JUNE, 1997 SUPPLEMENT
111S
Table l-Aquaporins in Lung
Protein Aquaporin-I
Human Gene Location
Expression in Lung
7p14
Peribronchial endothelium, visceral pleura, rare alveolar pneu mocyies Aqu aporin-S 9p21 ---> 12 Basolateral surface of bronchial epithelium Aquaporin -t 18(111.2-12.1 Basolateral surface of bronchial and tracheal epithelium Aquaporin-f 12q13 Type I pneumocytes, trachea, bronchial submucosa
Aquapo rin-l (AQP l) is expressed in the lung in vascular endothelium, visceral pleura, and a subset of pneumocytes .' AQP4, the major brain aqu aporin," localiz es to th e basol at eral membrane of bronchial and trach eal epthe lium in lun g:' AQP3 is expressed in th e lun g exclusively on th e basolat eral sur face of trach eal epithelium ." The abs en ce of AQPs I , 3, and 4 in type I pn eumocytes or on th e apic al sur face of airway epithelial cells suggests th e presen ce of anothe r aqu aporin in tho se locations. Recently, th e complem entary DNA (cD NA) for th e fifth mammalian aquaporin (AQP5) was isolat ed from rat ; Northern blot analysis demonstrated AQP5 in rat salivary and lacrim al glands, co rnea, and lun g.6 Herein we discuss th e isolatio n and ch ar acterizatio n of th e human AQP5 cD NA and gen e, as well as the ontogeny and distribution of AQPS in rat lun g. Using th e rat AQP5 cDNA as a molecu lar probe, an AQP5 cDNA clon e was isolated from a human submaxillary gland library. Th e 1,348-base pairs (bp) insert contained a 79S-bp op en readin g frame encoding a 265 amino acid pro tein with 91 % homology to rat AQP5 .H Expression of human AQP5 in Xenopus laeois oocytes conferred mercurial-sen sitive osmotic water permeab ility equivalent to other aquaporins . A 7.4-kilobas e Sal I-Eco RI frag ment from a human genomic library was found to contain th e human AQP5 structural gen e . Nucl eotide seq ue ncing with pri mer s correspond ing to the human A QP5 cDNA identified four exons separat ed bv introns of 1.2, 0.05 , and 0.9 kiloh ases. Analvsis of the 5;-flanking region det e rmined AQP5 to be a TATA-Iess gene with a transcription initiation site .5 18 bp up stream of th e init iating methionin e. Genomic Southern blot analysis confirmed AQP5 to be a singl e copy gene th at localized to human chro moso me 12q13; this coincide s with th e chromosomal locations of th e homologous hum an ge ne s Ml.P? and AQP2 , 10 thus demonstrating the site of an aquaporin gen e cluster . Recently the ontogeny, distribution, and steroid-induc ed expression of AQPI in rat lung was described , implicating AQPI in the clearance of water in both the perinatal and adu lt rat lung .4 The absenc e of AQPI in type I or type Il pneumocytes suggested the existence of anoth er water channel in that location . To determine the ontogeny of AQP.5 in lung , membranes from feta l and postnatal rat lun gs were immunoblotted with affinitypuri fied anti-AQP5 antibody. In contrast to the pe rinatal 1128
induction of AQPI , AQP5 was expressed in rat lung I to 2 days after birth , with protein levels steadily increasing into adulthood.11 While AQPI expression was induced in both perinatal and adult rat lung by corticosteroids," AQP.5 was not indu ced in rat lung at any age. Immunoelectron microscopic localization demonstrated AQP5 in the apical membrane of type I pnem nocytes; AQP5 was not present in type II pneumocytes, airway epithe lium, or vascular structures. Th ese st udies have defin ed th e gene stru ctur e for A QP5 and localized the cell-sp ecific exp ression of AQP5 protein in alveolar epithe lium. Charact erization of th e AQP5 ge ne structure should aid future studies direct ed at de fining the promoter-specific ele me nts required for AQP5 gene regu lation during normal development and in clinical disorde rs of respiratory tissue. The abundance of AQP5 on th e apical surface of type I pn eumocytes suggests a role in alveolar transme mbrane wat er movement, though th e lack of a subapical or basolat eral location for AQP5 may indicate that oth er as yet undefin ed aquaporins may participate in th e movement of water acro ss the alveo lar epithelium , Additional studies aim ed at addressing this issue and determ ining th e pr eci se localization of AQP5 in upper airway epithelium ar e currently und erway.
R EFERENCES 1 King LS, Agre P. Pathophysiology of the aquaporin wate r channels. Annu Rev Physiol 1996; 58:619-48 2 Deen PMT , Verdijk MAJ, Knoers NVAM, et al. Requirement of human renal wate r channe l aq uapo rin-2 for vasop ressin-depe nde nt conc entration of urine. Scien ce 1994; 264:92-95 .3 Shiels A, Basnett S. Mutations in the fonnder of th e MIP gene family und erlie cataract develop ment in the mouse. Nat Genet 1996; 12:212-15 4 King LS, Nielsen S, Agre P. Aquaporin-I water chann el protein in lung: ontogeny, ste roid-induced expression, and distribution in rat. J Clin Invest 1996; 97:2183-91 5 Frigeri A, Gropper MA, Tur ck CW, et al. Immunolocalization of the mercurial-insensitive wate r channel and glycerol intrinsic pr otein in epithelial plasma membranes. Proc Natl Acad Sci USA 199.5; 92:4328-31 6 Raina S, Preston Glvl, Guggino \VB, et al. Molecular clonin g and characteri zation of an aquaporin cDNA from salivary, lacrimal, and resp iratory tissues. J Bioi Chern 199.5; 270: 1908-12 7 Jun g JS, Bhat BV, Preston GM, et al. Molecu lar characterization of an aquaporin cDNA from brain : candid ate osmoreceptor and regulator of water balance . Proc Natl Acad Sci USA 1994; 91:130.52-.56 8 Lee MD , Bhakta KY, Raina S, et al. The hum an aouaporin-S gene . J BioI Chern 1996; 271:8599-8604 9 Saito F, Sasaki S, Chepe linsky AB, et al. Hum an AQP2 and MIP genes, two memb ers of the MIl' family, map within chromosome band 12q13 on th e basis of two-color FISH . Cytoge net Cell Genet 1995; 68:4.5-48 10 Sasaki S, Fushimi K, Saito H, et al. Cloning, characterization, and chromosomal mappin g of human aquaporin of collecting duct. J Clin Invest 1994; 93:1250-56 11 King, LS, Nielsen , S, Agre, P. Aquaporin -.5is expressed in rat
Thomas L. Pett y 39t h Annual Aspen Lung Con fere nce: Genes and Gene Therap y
type I pneumocytes [abstract]. Am J Respir Crit Care Med 1996; 153:A508
Role of the Epithelial Sodium Channel in Lung Liquid Clearance* Edith Hununler, PhD; Pierre Barker, AID; Friedrich Beennann, PhD; John Gat zy , AID; Chan tal Verdulll o; Richard Boucher, MD ; and Bernard C. RossieI', l'vID
(CHE ST 1997; 111 :113 5) etal lung secretes into th e future airspaces fluid that arises from a con tinuous act ive secretion of CI - by th e lung 's epithelium . At birth, th e lungs rapid ly transform into a fluid (Na+)-absorbing organ to enable efficie nt gas exchange. Although involvem ent of an active transepithelial Na + tra nsport in th e lung during this transition to an air-filled organ has been demonstrated,1 2 the importance and the role of the amilor ide-sensitiv e epithelial sodium channel (E NaC ) in th ese processes has not been established . In preterrn rat " and human" lung, E NaC messen ger RNA transcripts are ba rely detectable , but expression levels increase considerably in the perinatal period and rem ain high th roughout adult life,3 suggesting a role for ENaC in maintaining a "dry" alveolar state postnatally. An amilorid e-sensitive electrogenic Na+ reabsorption has been documented in upp er and lower airways, but it was not established whether this sodium transport is mediated by EN aC in vivo . , The ENaC plays an important role in determining the final amount of sodium reabsorbed. ENaC is localized in the apica l membrane of tight epithelia lining th e distal part of the nephron , in the distal colon, in th e ducts of several exocrine glands , and in the alveolar cells of th e lung:5 ENaC is a hcteromultimeric protein mad e up of three homologous subunits (a , 13, and "1).6 Assemb ly and expression of functional active sodium cha nnels in vitro are strictly dep endent on the subunit aEN aC, the 13 and "I subunits being unable, by thems elves, to induce an amilotid e-sensitive sodium current. To elucidate the role of ENaC in viuo, we inactivated the mous e aENaC gene locus by gene targeting in mouse embryonic stem cells (E5).' At birt h, th e genotypic analysis of newborn animals from heterozygous mutant breeding pairs revea led that em b ryonic and fetal developmen t in aENaC( - 1- ) animals was not significa ntly impaired. \Vhereas most newborns developed a normal br eathing pattern afte r birt h, aENaC( - 1- ) neonates developed F
'From the Institut de Phannacologie et de Toxicologie de l'Universite, Lausanne, Switzerland (Drs. Hummler and Hossle r and Ms. Verdumo): University of North Carolina School of Medicine, Chapel Hill (Drs. Barker, Catzy, and Boucher); and the Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland (Dr. Beermann).
Reprint requests: Edith Huuun ler, PhD, l nstitui de Pharmacologie, C H-1005, Lausann e, Switzerland.
respi ratory distress and died within 40 h after birth. To evaluate the presence of aENaC-m ediated electrogenic Na+ transport in th e neonatal lung, pot ential difference (PD) was measured acro ss the epithe lia in cu ltured tracheal cysts derived fro m freshly excised lungs. Mean baselin e PD of aENaC-I- explants was lower than in cysts from +1+ or +1- airway epithe liu m, and in aENaC - I - animals, amiloride-sensitive elec tro gen ic Na + transport was compl etely abolished . Histologic ana lysis showed no differences in lung architecture or alveolar epithelial differentiation between aENaC - I - mice and controllittermates, but whole lobes were fllled with liquid and air was pr esent in a few subpleural alveoli of aENaC-I - mice . This was con finned by measuring whole lung wet -to -dry weigh t ratio (WID ), a parameter that reflects water content of th e lung . The ratios of fetal lun gs were similar in all three genotypes . Fifteen minutes after birth , the value s of wild -typ e and heterozygou s mutant mice were int ermediate , reaching values simila r to thos e of adu lt mous e lungs or 4 to 6 h afte r birth, indicating that the remainin g feta l lung liquid had been removed . Contrarily, in aENaC knockout mice , th e WI D values decreased slight ly from feta l values , with no further clearance by 4 to 6 h and even higher WID at 10 to 14 h, suggesting that liquid was reaccum ulating in those lun gs. In this study, we present evide nce of a direct re lationsh ip between the abs e nce of ENaC fun ction and neonatal death due to a d efective lung liquid clearance . We propose that active Na + transport across the newborn alveo lar epithe liu m, mediated by ENaC , is a critical factor in the suc ce ssfu l ad aptation of th e newborn to air b reat hing, and immaturity of this pathway may contribute to th e etiology of respiratory diseases associated with preteI'm delivery.
REFEHENCES J O'Brodovich II. Epithelial ion transport in the fetal and perinatal lung. Am J Physiol J991; 26J:C555-64 2 Olver RE, Ramsden CA, Strang LB, et al. The role of amiloride-hlockable sodium transport in adrenaline-induced lung liquid reabsorption in the fetal lamb. J Physiol 1986; 376:321-40 3 Tchepichev S, Ueda J, Canessa C, et al. Lung epithelial Na channel subunits are differentially regulated during development and by steroids. Am J Physiol J99.5; 269:C805-12 4 Voilley N, Lingueglia E, Champigny G, et al. The lung amiloride-scnsitlve J\'a+ channel: biophysical properties, pharmacology, ontogenesis, and molecular doni ng. Proc Natl Acad Sci USA 1994; 91:247-51 5 Due C, Farman N, Canessa CM, et al. Cell-specific expression of epithelial sodium channel a , 13 and "y subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry. J Cell BioI 1994; 127:1907-21 6 Canessa CM, Schild L, Buell G, et al. Amiloride-sensitive epithelial Na + channel is made of three homologous subunits. Nature 1994; 367:463-67 7 Hummler E, Barker P, Gatzy J, et al. Early death due to defective neonatal lung liquid clearance in aENaC- deHcient mice. Nat Genet 1996; 12:325-28 CHEST / 111/ 6/ JUNE, 1997 SUPPLEMENT
1135
Human alveolar carcinoma cells (A,549) were plated for 24 h prior to infection with MOls of 1 to .50 of AdMRCMV(X " AdMRCMVI3" and AdMRCMVI3-Gal. Cells infected with MOI s of 5 to 50 of AdMRCMV(X I demonstrated twofold to threefold increases in ouabaininhibitable SORb + upt ake 24 h after infection. No chan ges in Na +/K+-A'I'Pase function were noted following infection with AdMRCMVl31 or AdMRCMVI3-Gal. Nort hern blot analysis of total BNA using a rat-specific c DNA probe revealed the presence of (XI mRNA only in cells infected with AdMRCMV(XI ; no change in 131 message was not ed . Western blot analysis showed significantly increased (X l protein in cells infected with AdMRCMV(XI ' Th ese findings suggest that (Xl subunit may be the rate-limiting subunit in A,549 cells and that Na+/K+-ATPase function can be increased in thes e cells using gene transfer. The human (Xl isofonn is two to thr ee logs more sensitive to ouabai n than is the rat (X l isofonn. To dem onstrate transgene exp ression, ouabain sensitivity was determined in A549 cells by ascertaining the concentration of ouabain that produced a 50% reduction in HOBb + uptake (IC 50 ) . A549 cells were infected with AdMBCMV(XI at an MOl of 25, and 8°Bb + uptake was measured using ] 5 differ ent conce ntrations of ouabain (l X lO- lI to l XlO - 3 ) . IC 50 values were determined using a computerized nonline ar least squares regression function designed to test for the pr esenc e of one or two isozymes of a receptor with differ ent affinities for a ligand (ouabain). AdMRCMV(X I-infected cells demonstrated two distin ct IC,5o values (IC 50 (l ) =3..5 X 10- 7 , IC 50 (2)= 3.2X 10- 5 ) . Sham and AdMBCMVI3-Gal infected cells each demonstrated one IC50 that was not differ ent from the first (X l IC.5o. The identification of a second (Xl IC5 0 two logs greater th an that found in the sham or AdMRCMVI3Gal infect ed cells suggests that presenc e of two functional (XI isozymes: the endogenous ouabain-sensitive hum an (XI and the ouabain-resistant transgenic rat (XI ' These results demonstrate that adenoviruses can efficiently tra nsfe r genes to lung alveolar epithelial cells and augm ent Na+/K+-A'TPase abun danc e and function . The respons e to infection with individual subun its differs between cell types such that the 131 subunit could be rate-limiting for rat ATII cells and (Xl may be the ratelimiting subunit for hum an A549 cells in vit ro. Conceivably, gen e tran sfer to the alveolar epithelil~m may be app licable to the pr evention and treatm ent of pulmonary ede ma. R EFERENCES
1 Sznajder ]1. Olivera WG, Hidge KM, et al. Mechanisms of lung liquid clearan ce durin g hyperoxia in isolated rat lungs. Am J Hespir Crit Care Med 1995; 1.5 1:1519-25 2 Rutschman DB , Olivera W, Sznajder JI. Active transport and passive liquid movement in isolated perfused rat lungs. J Appl Physiol 1993; 75:1574-80 3 Goodman BE, Kim K, Crandall E D. Evidence for active sodium transport across alveolar epith elium of isolated rat luug. J Appl Physiol 1987; 62:2460-66 4 Matthay M, Wiener-Kronish J. Intact epithelial barrier lunction is critical for th e resolution of alveolar edema in hum ans.
Am Rev Hespir Dis 1990; 142:1250-57 5 Suzuki S, Zuege 0 , Berthiaum e Y. Sodium-indepe ndent modulation of?\a+-K+-ATPase activityby beta-adrenergic agonist in alveolar type II cells. Am J Physiol1995; 268:L983-L90 6 Olivera \V, Hidge K, Wood LD H, et al, Active sodium tran sport and alveolar epithelial Na-K-ATPase increase during subacute hyp eroxia in rats. Am J Physiol 1994; 266:L577L84 7 Schneeberger EE , McCarthy KM. Cytochemi cal localization of Na, K-ATPase in rat typ e II pneumocytes. J Appl Physiol 1986; 20:1.'584-89 8 O'Brodovich HM. The role of active Na + transport by lung epith elium in the clearance of airspace fluid. New Horizons 1995; 3:240-47 9 Ridge K, Ilekis J, Factor P, et al. Inhibition of the Na,K ATPase by transfect ion of alveolar type 2 cells with antisense RNA to the Na,K ATPase [abstract]. Am Rev Respir Dis 1992; 145:A832 10 McGrory WJ, Bautista OS, Graham FL. A Simple technique for the rescue of early region 1 mutations into infectious human adenovirus type 5. Virology 1988; 163:614-17
Genomic Organization and Developmental Expression of Aquaporin-5 in Lung* IH. Douglas Lee, uo. Landon S. King, MD ; S¢ren Nielsen, PhD; and Peter Agre, MD
(C HEST 1997; 11 1:111 5-11 3 5) iscovery of the aquaporin family of water transp~rters provided a molecular explanation for osmot ically driven water transport across cell membranes of mammalian and plant tissues, At present, ther e are six known mammalian aquaporins distributed in a wide variety of tissues and cell types . In general, the distribution of each aquapori n is uniq ue, tho ugh there are some scattered sites of overlap (reviewed by King and Agre l ) . Recent identification and characterization of the genes encoding these aquaporins have enabled investigators to begin establishing a role for aquaporins in the pat hogenesis of several disease states.s' Despite the wealth of evidence in recent years to explain the physiology of salt tran sport in the lung , the molecu lar mech anisms for transcellu lar movem ent of water in the lower respiratory tract remain poorly understood . Four aqua porins have been identified in the lung by Northern blot analysis or imrnunoblotting-" Cr able 1).
D
*From the Department s of Pulm onary and Critical Care Medicine (Drs. Lee and King), Medicin e and Biological Chemi stry (Drs. Lee , King, and Agre), Johns Hopkins University, Baltimore ; and Cell Biology, Inst itute of Anatomy (Dr. Nielsen ), University of Aarhus, Denmark. Supported in part bv ?\IB ROI grants EYI 1239, HL48268, and H L33991 (PA), NRSA F32 HL09420 (M.D .L.), and NRSA F32 H L09119-02 (L.K.); Dr. Lee was recipient of an Allen and Hanbu rys Pu lmon ary Fellowship Award. Reprint requests: Peter Agre, MD , Departmen t of Biological Chemistn], [ohns Hopkins University School of Medicine, 725 N W olfe St, Baltimore, MD 21205-2185; email: [email protected] CHEST / 111 / 6 / JUNE, 1997 SUPPLEMENT
111S
Table l-Aquaporins in Lung
Protein Aquaporin-I
Human Gene Location
Expression in Lung
7p14
Peribronchial endothelium, visceral pleura, rare alveolar pneu mocyies Aqu aporin-S 9p21 ---> 12 Basolateral surface of bronchial epithelium Aquaporin -t 18(111.2-12.1 Basolateral surface of bronchial and tracheal epithelium Aquaporin-f 12q13 Type I pneumocytes, trachea, bronchial submucosa
Aquapo rin-l (AQP l) is expressed in the lung in vascular endothelium, visceral pleura, and a subset of pneumocytes .' AQP4, the major brain aqu aporin," localiz es to th e basol at eral membrane of bronchial and trach eal epthe lium in lun g:' AQP3 is expressed in th e lun g exclusively on th e basolat eral sur face of trach eal epithelium ." The abs en ce of AQPs I , 3, and 4 in type I pn eumocytes or on th e apic al sur face of airway epithelial cells suggests th e presen ce of anothe r aqu aporin in tho se locations. Recently, th e complem entary DNA (cD NA) for th e fifth mammalian aquaporin (AQP5) was isolat ed from rat ; Northern blot analysis demonstrated AQP5 in rat salivary and lacrim al glands, co rnea, and lun g.6 Herein we discuss th e isolatio n and ch ar acterizatio n of th e human AQP5 cD NA and gen e, as well as the ontogeny and distribution of AQPS in rat lun g. Using th e rat AQP5 cDNA as a molecu lar probe, an AQP5 cDNA clon e was isolated from a human submaxillary gland library. Th e 1,348-base pairs (bp) insert contained a 79S-bp op en readin g frame encoding a 265 amino acid pro tein with 91 % homology to rat AQP5 .H Expression of human AQP5 in Xenopus laeois oocytes conferred mercurial-sen sitive osmotic water permeab ility equivalent to other aquaporins . A 7.4-kilobas e Sal I-Eco RI frag ment from a human genomic library was found to contain th e human AQP5 structural gen e . Nucl eotide seq ue ncing with pri mer s correspond ing to the human A QP5 cDNA identified four exons separat ed bv introns of 1.2, 0.05 , and 0.9 kiloh ases. Analvsis of the 5;-flanking region det e rmined AQP5 to be a TATA-Iess gene with a transcription initiation site .5 18 bp up stream of th e init iating methionin e. Genomic Southern blot analysis confirmed AQP5 to be a singl e copy gene th at localized to human chro moso me 12q13; this coincide s with th e chromosomal locations of th e homologous hum an ge ne s Ml.P? and AQP2 , 10 thus demonstrating the site of an aquaporin gen e cluster . Recently the ontogeny, distribution, and steroid-induc ed expression of AQPI in rat lung was described , implicating AQPI in the clearance of water in both the perinatal and adu lt rat lung .4 The absenc e of AQPI in type I or type Il pneumocytes suggested the existence of anoth er water channel in that location . To determine the ontogeny of AQP.5 in lung , membranes from feta l and postnatal rat lun gs were immunoblotted with affinitypuri fied anti-AQP5 antibody. In contrast to the pe rinatal 1128
induction of AQPI , AQP5 was expressed in rat lung I to 2 days after birth , with protein levels steadily increasing into adulthood.11 While AQPI expression was induced in both perinatal and adult rat lung by corticosteroids," AQP.5 was not indu ced in rat lung at any age. Immunoelectron microscopic localization demonstrated AQP5 in the apical membrane of type I pnem nocytes; AQP5 was not present in type II pneumocytes, airway epithe lium, or vascular structures. Th ese st udies have defin ed th e gene stru ctur e for A QP5 and localized the cell-sp ecific exp ression of AQP5 protein in alveolar epithe lium. Charact erization of th e AQP5 ge ne structure should aid future studies direct ed at de fining the promoter-specific ele me nts required for AQP5 gene regu lation during normal development and in clinical disorde rs of respiratory tissue. The abundance of AQP5 on th e apical surface of type I pn eumocytes suggests a role in alveolar transme mbrane wat er movement, though th e lack of a subapical or basolat eral location for AQP5 may indicate that oth er as yet undefin ed aquaporins may participate in th e movement of water acro ss the alveo lar epithelium , Additional studies aim ed at addressing this issue and determ ining th e pr eci se localization of AQP5 in upper airway epithelium ar e currently und erway.
R EFERENCES 1 King LS, Agre P. Pathophysiology of the aquaporin wate r channels. Annu Rev Physiol 1996; 58:619-48 2 Deen PMT , Verdijk MAJ, Knoers NVAM, et al. Requirement of human renal wate r channe l aq uapo rin-2 for vasop ressin-depe nde nt conc entration of urine. Scien ce 1994; 264:92-95 .3 Shiels A, Basnett S. Mutations in the fonnder of th e MIP gene family und erlie cataract develop ment in the mouse. Nat Genet 1996; 12:212-15 4 King LS, Nielsen S, Agre P. Aquaporin-I water chann el protein in lung: ontogeny, ste roid-induced expression, and distribution in rat. J Clin Invest 1996; 97:2183-91 5 Frigeri A, Gropper MA, Tur ck CW, et al. Immunolocalization of the mercurial-insensitive wate r channel and glycerol intrinsic pr otein in epithelial plasma membranes. Proc Natl Acad Sci USA 199.5; 92:4328-31 6 Raina S, Preston Glvl, Guggino \VB, et al. Molecular clonin g and characteri zation of an aquaporin cDNA from salivary, lacrimal, and resp iratory tissues. J Bioi Chern 199.5; 270: 1908-12 7 Jun g JS, Bhat BV, Preston GM, et al. Molecu lar characterization of an aquaporin cDNA from brain : candid ate osmoreceptor and regulator of water balance . Proc Natl Acad Sci USA 1994; 91:130.52-.56 8 Lee MD , Bhakta KY, Raina S, et al. The hum an aouaporin-S gene . J BioI Chern 1996; 271:8599-8604 9 Saito F, Sasaki S, Chepe linsky AB, et al. Hum an AQP2 and MIP genes, two memb ers of the MIl' family, map within chromosome band 12q13 on th e basis of two-color FISH . Cytoge net Cell Genet 1995; 68:4.5-48 10 Sasaki S, Fushimi K, Saito H, et al. Cloning, characterization, and chromosomal mappin g of human aquaporin of collecting duct. J Clin Invest 1994; 93:1250-56 11 King, LS, Nielsen , S, Agre, P. Aquaporin -.5is expressed in rat
Thomas L. Pett y 39t h Annual Aspen Lung Con fere nce: Genes and Gene Therap y
type I pneumocytes [abstract]. Am J Respir Crit Care Med 1996; 153:A508
Role of the Epithelial Sodium Channel in Lung Liquid Clearance* Edith Hununler, PhD; Pierre Barker, AID; Friedrich Beennann, PhD; John Gat zy , AID; Chan tal Verdulll o; Richard Boucher, MD ; and Bernard C. RossieI', l'vID
(CHE ST 1997; 111 :113 5) etal lung secretes into th e future airspaces fluid that arises from a con tinuous act ive secretion of CI - by th e lung 's epithelium . At birth, th e lungs rapid ly transform into a fluid (Na+)-absorbing organ to enable efficie nt gas exchange. Although involvem ent of an active transepithelial Na + tra nsport in th e lung during this transition to an air-filled organ has been demonstrated,1 2 the importance and the role of the amilor ide-sensitiv e epithelial sodium channel (E NaC ) in th ese processes has not been established . In preterrn rat " and human" lung, E NaC messen ger RNA transcripts are ba rely detectable , but expression levels increase considerably in the perinatal period and rem ain high th roughout adult life,3 suggesting a role for ENaC in maintaining a "dry" alveolar state postnatally. An amilorid e-sensitive electrogenic Na+ reabsorption has been documented in upp er and lower airways, but it was not established whether this sodium transport is mediated by EN aC in vivo . , The ENaC plays an important role in determining the final amount of sodium reabsorbed. ENaC is localized in the apica l membrane of tight epithelia lining th e distal part of the nephron , in the distal colon, in th e ducts of several exocrine glands , and in the alveolar cells of th e lung:5 ENaC is a hcteromultimeric protein mad e up of three homologous subunits (a , 13, and "1).6 Assemb ly and expression of functional active sodium cha nnels in vitro are strictly dep endent on the subunit aEN aC, the 13 and "I subunits being unable, by thems elves, to induce an amilotid e-sensitive sodium current. To elucidate the role of ENaC in viuo, we inactivated the mous e aENaC gene locus by gene targeting in mouse embryonic stem cells (E5).' At birt h, th e genotypic analysis of newborn animals from heterozygous mutant breeding pairs revea led that em b ryonic and fetal developmen t in aENaC( - 1- ) animals was not significa ntly impaired. \Vhereas most newborns developed a normal br eathing pattern afte r birt h, aENaC( - 1- ) neonates developed F
'From the Institut de Phannacologie et de Toxicologie de l'Universite, Lausanne, Switzerland (Drs. Hummler and Hossle r and Ms. Verdumo): University of North Carolina School of Medicine, Chapel Hill (Drs. Barker, Catzy, and Boucher); and the Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland (Dr. Beermann).
Reprint requests: Edith Huuun ler, PhD, l nstitui de Pharmacologie, C H-1005, Lausann e, Switzerland.
respi ratory distress and died within 40 h after birth. To evaluate the presence of aENaC-m ediated electrogenic Na+ transport in th e neonatal lung, pot ential difference (PD) was measured acro ss the epithe lia in cu ltured tracheal cysts derived fro m freshly excised lungs. Mean baselin e PD of aENaC-I- explants was lower than in cysts from +1+ or +1- airway epithe liu m, and in aENaC - I - animals, amiloride-sensitive elec tro gen ic Na + transport was compl etely abolished . Histologic ana lysis showed no differences in lung architecture or alveolar epithelial differentiation between aENaC - I - mice and controllittermates, but whole lobes were fllled with liquid and air was pr esent in a few subpleural alveoli of aENaC-I - mice . This was con finned by measuring whole lung wet -to -dry weigh t ratio (WID ), a parameter that reflects water content of th e lung . The ratios of fetal lun gs were similar in all three genotypes . Fifteen minutes after birth , the value s of wild -typ e and heterozygou s mutant mice were int ermediate , reaching values simila r to thos e of adu lt mous e lungs or 4 to 6 h afte r birth, indicating that the remainin g feta l lung liquid had been removed . Contrarily, in aENaC knockout mice , th e WI D values decreased slight ly from feta l values , with no further clearance by 4 to 6 h and even higher WID at 10 to 14 h, suggesting that liquid was reaccum ulating in those lun gs. In this study, we present evide nce of a direct re lationsh ip between the abs e nce of ENaC fun ction and neonatal death due to a d efective lung liquid clearance . We propose that active Na + transport across the newborn alveo lar epithe liu m, mediated by ENaC , is a critical factor in the suc ce ssfu l ad aptation of th e newborn to air b reat hing, and immaturity of this pathway may contribute to th e etiology of respiratory diseases associated with preteI'm delivery.
REFEHENCES J O'Brodovich II. Epithelial ion transport in the fetal and perinatal lung. Am J Physiol J991; 26J:C555-64 2 Olver RE, Ramsden CA, Strang LB, et al. The role of amiloride-hlockable sodium transport in adrenaline-induced lung liquid reabsorption in the fetal lamb. J Physiol 1986; 376:321-40 3 Tchepichev S, Ueda J, Canessa C, et al. Lung epithelial Na channel subunits are differentially regulated during development and by steroids. Am J Physiol J99.5; 269:C805-12 4 Voilley N, Lingueglia E, Champigny G, et al. The lung amiloride-scnsitlve J\'a+ channel: biophysical properties, pharmacology, ontogenesis, and molecular doni ng. Proc Natl Acad Sci USA 1994; 91:247-51 5 Due C, Farman N, Canessa CM, et al. Cell-specific expression of epithelial sodium channel a , 13 and "y subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry. J Cell BioI 1994; 127:1907-21 6 Canessa CM, Schild L, Buell G, et al. Amiloride-sensitive epithelial Na + channel is made of three homologous subunits. Nature 1994; 367:463-67 7 Hummler E, Barker P, Gatzy J, et al. Early death due to defective neonatal lung liquid clearance in aENaC- deHcient mice. Nat Genet 1996; 12:325-28 CHEST / 111/ 6/ JUNE, 1997 SUPPLEMENT
1135