Aquaporin Water Channels in Kidney
Authors: PETER AGRE
Journal: Journal of the American Society of Nephrology (2000)

As a first year medical student, I attended nearly 200 lectures, but I have clear recollections of only two. On the first day that autumn, Albert Lehninger canceled his planned lecture and spoke to us of a recent paper that he said would revolutionize biomedical research—the discovery of reverse transcriptase. The following spring, James L. Gamble, Jr. lectured to us about renal physiology and the important contributions of a man named Homer Smith who had conducted research in a shed on Mount Desert Island in Maine. At the time, the idea of a researcher at an idyllic site studying marine and tidal organisms struck me as particularly appealing. Later that summer, I set out on a bicycle tour of Nova Scotia by way of Bar Harbor where Dr. Gamble and his family had warmly invited me to stop at their summer home. In addition to some delicious meals and a visit to multiple well-known sights in the area, Dr. Gamble drove me past the site of Homer Smith's laboratory where 20 years later I was to spend time as a visiting scientist. In many ways, the words of Smith were to have particular meaning for me. The history of renal physiology has erred, more often than not, by attempts at oversimplification. The problems of water and salt excretion appear to be extremely complex, and especially liable to this danger. Homer W. Smith, 1937 The Physiology of the Kidney (1) Water is the major component of all living cells, so the ability to absorb and release water must be considered a fundamental property of life. As recognized by Homer Smith, the kidney is the champion organ with regard to fluid transport. The epithelia lining the tubules in kidney are covered by plasma membranes, which control the solute composition of the enclosed compartments by regulating the entry of ions, small uncharged solutes, and water into and out of cells. Movement of these fluids from lumen to interstitium or in the reverse direction is clearly related to the high degree of cellular polarity in epithelial tissues whose cells have distinct apical and basolateral membranes. The identification and characterization of the molecular entities responsible for the function of biologic membranes has been a major goal of physiologists during the past two decades. Using biochemical purification and functional reconstitution techniques, multiple transporters have been identified and characterized. In addition, expression cloning has led to the discovery of other transporters. Despite this, the molecular identities of water transporters remained unknown until a series of fortuitous events occurred in our laboratory several years ago. The plasma membranes of all cells are known to exhibit finite water permeability. Nevertheless, simple diffusion does not account for all water movement through biologic membranes, and specialized water channels were predicted to explain the high water permeability of certain tissues (reviewed in reference (2). In particular, the water permeability of red cell membranes is much higher than that observed for other cell types and artificial lipid bilayers. The activation energy of this process (Ea < 5 kcal/mol) is equivalent to the diffusion of water in solution (reviewed in reference (3), indicating that water moves through aqueous pathways across these membranes. The observation of that HgCl2 and certain organomercurials reversibly inhibit the water permeability predicted that water channels are proteins with accessible sulfhydryl groups (reviewed in reference (4). Water permeability has been characterized in each segment of the nephron (reviewed in reference (5). Renal proximal tubules and descending thin limbs of Henle's loop are known to have constitutively high water permeability allowing for the reabsorption of the majority of the water in glomerular filtrate. In contrast, the ascending thin limbs and thick limbs are relatively impermeable to water but are highly permeable to ions and other solutes. Renal collecting ducts are extremely important in clinical water balance, since collecting duct water permeability is regulated by vasopressin (antidiuretic hormone). Early studies of toad urinary bladder provided a model of vasopressin-regulated water permeability; stimulation of the basolateral membrane of this epithelium with antidiuretic hormone induces a redistribution of intracellular particles to the apical membrane and increases the cellular water permeability (6, 7). Nevertheless, attempts to identify the membrane water channels were unsuccessful due to the ubiquity of water, the presence of free sulfhydryls in many proteins, and relatively high diffusional permeability of most membrane bilayers (reviewed in reference (8). Aquaporin-1 A serendipitous observation accompanied by the wisdom of a former mentor led to the recognition of the first molecular water channel. During the biochemical purification of the 32-kD core polypeptide of the red cell Rh blood group antigen, a 28-kD polypeptide was noted and assumed to represent a proteolytic fragment of Rh (9). Antiserum raised in rabbits reacted exclusively with the 28-kD band, indicating that it is unrelated to Rh (10). The 28-kD protein contained hydrophobic amino acids and exists in two forms: a nonglycosylated 28-kD polypeptide and a 40- to 60-kD N-glycosylated polypeptide. The protein proved easy to purify due to its limited solubility in the detergent N-lauroylsarcosine. Biochemical studies showed that the protein is a tetramer with intracellular N- and C-termini and yielded the N-terminal amino acid sequence (11), which permitted cDNA cloning from an erythroid library (12). The deduced amino acid sequence of the 28-kD polypeptide was found to be related to a functionally undefined family of putative membrane channel proteins including major intrinsic protein of lens (13). At the same time, radiation inactivation studies of water permeability by renal vesicles yielded a target size of 30 kD (14). Abundance of the 28-kD polypeptide in highly water-permeable tissues, red cells, renal proximal tubules, and descending thin limbs (10) led the late John C. Parker (1935-1993) at the University of North Carolina at Chapel Hill to predict that it may be the sought-after water channel now known as aquaporin-1 (AQP1). Soon to follow was the identification of multiple related proteins that comprise the aquaporin family of proteins and explain membrane water permeability in diverse tissues and organisms (8, 15). Measurement of Membrane Water Permeability The measurement of water transport across cell membranes posed an experimental challenge. First proposed by Eric Windhager of Cornell Medical School, the Xenopus oocyte expression system has proven to be a valuable method for functional analysis of candidate water channel RNAs, since the oocytes normally exhibit low membrane water permeability (16, 17). Fortunately, our colleague Bill Guggino at Johns Hopkins was very experienced in oocyte expression. When injected with cRNA corresponding to the 28-kD polypeptide (18), the oocytes became remarkably permeable to water (Pf approximately 200 × 10-4 cm/s) and exploded in hypotonic buffer. Water-injected control oocytes swelled minimally (Figure 1). Moreover, the AQP1 oocytes behaved like cells containing native water channels: low activation energy, reversible inhibition by HgCl2, and no measurable increase in ion conductance (18). The direction of water flow through AQP1 is determined by the orientation of the osmotic gradient (19).Figure 1.: Water permeability of aquaporin-1 (AQP1) expressed in Xenopus oocytes. When transferred to hypo-osmolar buffer for 2 min, control water-injected oocytes exhibit negligible swelling (left). Under the same conditions, oocytes previously injected with AQP1 cRNA rapidly swell and explode (right). Reproduced and modified with permission from Annu Rev Biochem (119).The ability to isolate milligrams of AQP1 from human red cells permitted direct biophysical studies of the protein. Highly purified AQP1 protein from human red cells reconstituted with pure phospholipid yielded proteoliposomes of approximately 100 nm in diameter (20). The change in volume of AQP1 proteoliposomes was compared to liposomes containing no reconstituted protein by measuring quenching of internal carboxyfluorescein. These measurements yielded the unit water permeability (Pf approximately 3 × 109 water molecules/subunit/s). The proteoliposomes exhibited water permeability similar to water channels in native membranes: reversible inhibition by HgCl2 and low activation energy. Permeation by other small solutes or even protons showed AQP1 to be water-selective (21). Thus, AQP1 is both necessary and sufficient to explain the well-recognized membrane water permeability of the red cell. AQP1 functions as a constitutively active, water-selective pore. Although forskolin was once reported to induce a cation current in AQP1-expressing oocytes (22), multiple other scientific groups have failed to reproduce this effect (23). Recently, the permeation by CO2 has been evaluated, and rates of pH change are higher in oocytes expressing AQP1 (24). Because permeation of lipid bilayers by CO2, NO, and O2 may be high, the potential physiologic relevance of AQP1 permeation by gases deserves further study (25), and the possibility of yet undiscovered transport functions cannot be excluded. AQP1 Structure The late 1990s have proven to be a time of great progress in the elucidation of membrane protein structures. Hydropathy analysis of the deduced amino acid sequence of AQP1 suggests that the protein spans the lipid bilayer (12). Similar to the homolog major intrinsic protein from lens (now referred to as AQP0), the deduced amino acid sequence of AQP1 is comprised of two internal repeats (Figure 2), and each half contains the signature motif Asn-Pro-Ala (NPA) (26). Hydropathy analysis predicts six bilayer-spanning domains, although connecting loops B and E are also somewhat hydrophobic. Location of loop C between the two halves of the molecule is therefore critical to the topology. In different recombinants, BamHI restriction sites were introduced into the cDNA for insertion of a DNA encoding a 29-residue epitope. Expression in oocytes confirmed that several of the recombinants remained functional, and using antibody binding and vectorial proteolysis, loop C was shown to reside at the extracellular surface of the oocytes. Thus the N- and C-terminal halves of the molecule exhibit a uniquely obverse symmetry with the two halves oriented at 180 degrees to each other (27) (Figure 2). Other studies showed that Cys-189 is the site of mercurial inhibition (28), and loops B and E were found to be functionally essential for water permeability. Together, these studies led to the “hour-glass” model (29), in which loops B and E overlap midway between the leaflets of the bilayer, creating a constitutively open, narrow aqueous pathway (Figure 3). Although not understood in detail, multiple studies indicate that the AQP1 is a tetramer comprised of functionally independent aqueous pores (29, 30).Figure 2.: Membrane topology of AQP1 subunit. The Asn-Pro-Ala signature motifs are indicated (NPA). Polyhedrons enclose sequence-related repeats in obverse orientation. Sites of N-linked glycan and surface polymorphism are represented. Reproduced and modified with permission from Annu Rev Biochem (119).Figure 3.: Hourglass model for AQP1 membrane topology. (Top) Each AQP1 subunit contains six bilayer-spanning domains comprised of two obversely symmetrical structures (TM1-3 = hemipore-1 and TM4-6 = hemipore-2). (Bottom) When NPA motifs in loops B and E are juxtaposed, they form a single aqueous channel spanning the bilayer (the “hourglass”) flanked by the mercury-sensitive residue (C189). Reproduced and modified with permission from J Biol Chem (29).AQP1 protein from human red cell membranes has provided increasingly detailed molecular insight into the protein's structure. Shadowed freeze-fracture electron microscopic analyses of reconstituted AQP1 confirmed the tetrameric assembly of AQP1 (21, 31). Attenuated total reflection-FTIR (Fourier transform infrared spectroscopy) of highly purified red cell AQP1 reconstituted into membrane crystals has been directly compared to proteins with known structures, bacteriorhodopsin, and the porin OmpF. These studies demonstrated that AQP1 lacks beta structure and contains multiple alpha helices tilted at 21 to 27 degrees (32). Using fluorescently labeled antibody specific for the extracellular domain of loop A (Co antigen), the lateral mobility of AQP1 in red cell membranes was shown to be regulated by passive steric hindrance from the membrane skeleton (33). The structure of the AQP1 protein is being solved at increasing levels of resolution. Reconstitution of highly purified red cell AQP1 into membranes under controlled conditions yields membrane crystals with highly uniform lattices with p4221 symmetry. These resealed vesicles retain full water permeability, providing the opportunity to define the structure of AQP1 in a biologically active state (34). Microscopic examination of negatively stained membranes showed that one side of the tetramer contains subunits widely separated around a central stain-filled depression (the extracellular surface) or closely surrounding a central area (cytoplasmic surface) (35). Cryoelectron microscopic studies have revealed the presence of multiple bilayer-spanning domains (36,37,38), and atomic force microscopy further defined the orientation of the right-handed helix bundle and extramembrane dimensions of AQP1 (39). Electron crystallography of cryopreserved specimens using a liquid helium-cooled stage and tilts of up to 60 degrees revealed the presence of AQP1 tetramers with individual subunits containing six bilayer-spanning alpha helices viewed at a resolution of 6 Å (40,41,42). These studies have revealed that the AQP1 subunit has a structure similar to the proposed hourglass (Figure 4 and cover photo). The next challenge is to merge structural understandings derived from molecules with unambiguous landmarks.Figure 4.: Stereoscopic view of AQP1 at 6 Å resolution established by cryoelectron microscopy. Six tilted, bilayer-spanning alpha helices (numbered) in a single subunit surround the internal hourglass formed from loop B (green) and loop E (orange). Reproduced and modified with permission from Nature (40).Tissue Distribution of AQP1 In the initial report, AQP1 was noted to be abundant in the apical and basolateral membranes of renal proximal tubules and descending thin limbs of Henle's loop (10). This was subsequently confirmed with polyclonal rabbit antiserum (43) and with affinity-purified Ig specific for the N- and C-terminal domains of AQP1 (44, 45) (Figures 5 and 6). In all studies, AQP1 is constitutively present in the apical plasma membranes (including the brush borders) and in basolateral membranes as well. A similar pattern of expression is found in the descending thin limbs of Henle's loop (Figure 7). AQP1 at the lumenal and ablumenal surfaces of the tubule provides the transcellular pathway for water following small osmotic gradients at the apical and basolateral surfaces of the cell. Quantification of AQP1 at these sites by dilutional immunoblotting and by enzyme-linked immunosorbent assay verified that AQP1 alone could serve as the conduit for the huge volumes of water reabsorbed in the proximal nephron (44, 46). Immunohistochemical studies have also demonstrated AQP1 in renal vasa recta (47).Figure 5.: Light and electron microscopic studies of AQP1 in rat proximal tubules. (a) Immunohistochemical analysis of S3 segment stained with Meier reagent exhibits strong staining over apical brush border and basolateral membranes (arrows). (b) Tangentially sectioned S3 segment stained with peroxidase-conjugated goat-anti-rabbit IgG shows no reaction over collecting duct (C). Bars, 20 μm. (c) Immunogold electron microscopy of S3 segments in longitudinal section. (d) Cross section. Magnification, ×30,000. Reproduced and modified with permission from J Cell Biol (44).Figure 6.: Diagram representing transcellular water permeation of proximal tubule cell via AQP1.Figure 7.: Light and electron microscopic studies of AQP1 in rat descending thin limbs of Henle's loop. (a) Immunohistochemical analysis of outer medulla reveals labeling of apical and basolateral membranes of descending thin limbs (D) but not of ascending thin limbs (T) or collecting ducts (C). (b) Higher magnification. Bars, 20 μm. (c) Immunogold electron microscopy of descending thin limb from short-looped nephrons shows staining of cytoplasmic leaflets of apical membranes including microvilli (arrow) and basolateral membranes (BM). (d) Long-looped nephron. Magnification, ×45,000. Reproduced and modified with permission from J Cell Biol (44).AQP1 is also abundant in capillary endothelia in other organs. In lung, AQP1 is present in peribronchiolar capillary endothelium, where expression is induced by glucocorticoids (48,49,50) acting through the classic glucocorticoid response elements in the proximal gene promoter (51). In brain, AQP1 has been defined in the apical membrane of choroid plexus epithelia, the site of cerebrospinal fluid secretion. AQP1 is also present at multiple locations in the eye: nonpigmented ciliary epithelium, lens epithelium, and corneal endothelium (48, 52). Although not present in intestinal mucosa, AQP1 is rich in cholangiocytes, where it appears to be regulated by secretin (53). Developmental expression of AQP1 has been found to be complex in the rat model, and at least three patterns are recognized: (1) transient expression before birth (periosteum, cardiac endothelium); (2) permanent expression beginning at the time of birth (cornea, lung, kidney); (3) constitutive life-long expression (choroid plexus) (50, 54, 55). AQP1 Deficiency AQP1 protein is not essential for survival in humans or mice. Humans have been identified who totally lack the AQP1 protein (56). Due to their linkage to human chromosome 7p14, it was recognized that the Colton blood group antigen (Co) may reside on the AQP1 protein. Co was shown to be an Ala/Val polymorphism at the 45th residue located on the first extracellular loop of red cell AQP1 (57) (Figure 2). The Co null blood phenotype is exceedingly rare, and our laboratory has studied Co null probands from three unrelated kindreds. Each was born in a remote rural area and is believed to have been the offspring of parental consanguinity. All three were found to be homozygous for naturally occurring disruptions in the AQP1 gene. Nonsense mutations were identified in two probands: deletion of exon 1 and frameshift in exon 1. The third proband is homozygous for a missense mutation: replacement of proline by a leucine at the end of the first transmembrane domain that produces an unstable polypeptide (56). These Co null individuals are all women who developed anti-Co during pregnancy. Although the exceedingly rare blood group phenotype makes them impossible to match for blood transfusion, it was surprising that none of them exhibited any other obvious clinical phenotype, and it is uncertain whether this is explained by naturally occurring backup systems or the existence of compensating mutations. Evaluation of these Co null humans has been undertaken, and preliminary analyses revealed that they manifest a major concentration defect in the proximal tubules but are not polyuric. When fluid-deprived for up to 24 h, the Co null individuals concentrate their urine to less than half the normal level, and administration of exogenous vasopressin produced no additional effect (L. King and P. Agre, unpublished observations). Mice homozygous for targeted AQP1 gene disruption have been raised (58). The recent development of an Aqp1 gene knockout has revealed that renal water reabsorption in mice is very dependent on AQP1 protein, since the Aqp1 null animals became hyperosmolar after fluid restriction. Detailed classical physiologic evaluation of the isolated proximal tubules from the Aqp1 null mice were undertaken to establish that transmembrane water permeability was reduced by 80%, and led to the observation of a compensatory reduction in glomerular filtration (59, 60). Other Renal Aquaporins The first functional definition of one member of a protein family often predicts the functions of the other members, and this has certainly been the case for the aquaporins. Homology cloning has been undertaken by multiple laboratories, and 10 mammalian homologs have been reported, while preliminary evidence for additional homologs is emerging. Homology cloning of aquaporins is most frequently undertaken using polymerase chain amplification with degenerate oligonucleotide primers corresponding to the most highly conserved domains, the NPA sequences within loops B and E (61). All members of the family are believed to be water channels, because all contain structural motifs similar to AQP1, but each has features apparently needed for functional and regulatory differences. The complete sequencing of the human genome will almost certainly identify new aquaporin homologs. Although an oversimplification, the 10 known mammalian aquaporins have tentatively been broken into two subgroups: water-selective channels (Orthodox aquaporins) and channels permeated by water, glycerol, and other small molecules (Aquaglyceroporins) (Figure 8). The Human Genome Organization has established an Aquaporin Nomenclature System for designation of novel family members (15), and updates are available on the Internet (http://www.gene.ucl.ac.uk/nomenclature).Figure 8.: Phylogenetic comparisons of aquaporins. Mammalian aquaporins and Escherichia coli homologs. Water-selective “orthodox aquaporins” and AqpZ (gray) and “aquaglyceroporins” and GlpF (white). Reproduced and modified with permission from Annu Rev Biochem (119).AQP2, the Vasopressin-Regulated Water Channel Water permeability of renal collecting ducts is regulated by vasopressin. The failure to demonstrate AQP1 in collecting ducts by immunohistochemistry (10) led to the prediction that other members of the aquaporin family must exist at this site (18). Using the homology cloning approach, a cDNA was reported to encode a renal collecting duct water channel, AQP2 (62). The combined efforts of multiple groups of investigators have established major clinical significance for AQP2 (reviewed in reference (63). AQP2 is expressed predominantly in the principal cells of the renal collecting duct (62, 64, 65). Regulation of AQP2 appears to be complex. The intracellular trafficking of AQP2 is regulated by vasopressin through shortterm exocytosis to the plasma membrane, whereas long-term biosynthetic mechanisms are activated in response to chronic thirsting. Vasopressin has been known for more than two decades to cause the redistribution of intracellular vesicles to the apical surface of principal cells, a process referred to as the “membrane shuttle mechanism” (66). Immunolocalization of AQP2 in sections of rat kidney after treatment with vasopressin supports the shuttle hypothesis (65, 67, 68). A much-cited study of isolated rat renal collecting ducts showed that in the absence of vasopressin, water permeability was low and AQP2 resided predominantly in intracellular vesicles (69). Administration of vasopressin caused the water permeability to rise fivefold and was accompanied by a redistribution of AQP2 to the apical membrane. After removal of vasopressin, the water permeability declined and surface AQP2 was reinternalized (Figure 9). The mechanism of vasopressin action is through a surface V2 receptor coupled to activation of adenylyl cyclase, inducing phosphorylation of the C terminus of the AQP2 protein (Figure 10). Cytoplasmic vesicles containing AQP2 are then targeted to the cell surface via the vesicle-targeting proteins syntaxin-4 and synaptobrevin-2. The molecular details of this pathway are being evaluated and appear to be complex (70). The stoichiometry of subunit phosphorylation and the possible presence of other transporters in the vesicle are being established (reviewed in reference (63). The involvement of a heterotrimeric member of the Gi protein family is known to occur during membrane trafficking in renal epithelial cells (71). The long-term mechanisms for regulation of AQP2 biosynthesis and removal are also being evaluated. Identification of a cAMP regulatory element in the 5′ flanking DNA of AQP2 suggests that transcriptional regulation may be occurring (72, 73). Reduced expression of AQP2 protein has been found during the postantidiuretic state known as “escape from vasopressin” (74).Figure 9.: Immunogold electron microscopy of AQP2 in rat collecting duct principal cells before (top) or after (bottom) exposure to vasopressin. Magnification, ×44,000. Reproduced and modified with permission from Proc Natl Acad Sci USA (69).Figure 10.: Diagram representing vasopressin-regulated transcellular water permeation of apical membrane of collecting duct principal cell via AQP2 and constitutive water permeation of basolateral membrane via AQP3.Much clinical significance has been ascribed to AQP2, and this protein may be involved in most, if not all, imbalances of water metabolism. For example, impaired renal water reabsorption is associated with reduced levels of AQP2. Nephrogenic diabetes insipidus (NDI) is a disorder that occurs when vasopressin levels are elevated, but the kidney fails to respond to the hormone. Mutations in genes encoding renal vasopressin V2 receptors have been demonstrated in many patients with the X-linked disorder (reviewed in reference (75). A family with recessively inherited NDI and normal V2 receptors was found to have mutations in the AQP2 gene causing loss of function (76). Multiple other AQP2 mutations in aqueous pore domains have been found in other patients with recessively inherited NDI (77, 78). One family with dominantly inherited NDI was recently identified with a mutation in the C terminus of AQP2 (79). The products of the mutant and the normal AQP2 allele have been shown to oligomerize, thereby restricting membrane trafficking of both polypeptides. Individuals with NDI resulting from the dominant mutations experience a less severe form of polyuria (Daniel Bichet, personal communication). Secondary decreases in AQP2 expression probably occur with relative frequency in medical clinics. Lithium is widely prescribed for treatment of bipolar disorder, but polyuria is a potentially problematic side effect. While lithium-induced polyuria may be managed by increasing fluid intake, this can cause difficulties in elderly individuals or in patients deprived of oral intake, such as in preparation for surgery. Lithium has been found to cause a striking reduction (90%) in AQP2 expression in rats (80). Other forms of polyuria have been also evaluated, and reduced AQP2 expression was also documented after release of urinary obstruction, such as after prostate surgery (81), in chronic hypokalemia-induced polyuria (82), and is suspected in nocturnal enuresis (83). Secondary increases in AQP2 expression may also be common in clinical medicine and may cause problems with excessive renal water retention. Patients with congestive heart failure often succumb to refractory pulmonary edema, and increased expression of AQP2 has been documented in rat models of incompletely compensated congestive heart failure (84, 85). Increased expression of AQP2 may also explain fluid retention known to complicate pregnancy (86), cirrhosis, and syndrome of inappropriate antidiuretic hormone (87). AQP2 may be an effective target for pharmacologic intervention in some of these disorders. Multifunctional AQP3 Using the homology cloning approach, three scientific groups isolated cDNAs encoding AQP3, a member of the aquaporin family with distinctive biophysical functions (88,89,90). Previously cloned aquaporins were only permeable to water, but AQP3 was noted to be genetically closer to the known Escherichia coli glycerol transport protein GlpF (Figure 8), and AQP3 is permeated by glycerol. AQP3 was also reported to be slightly permeable to urea, but the biologic significance of this is uncertain, since unrelated proteins specialized for urea transport have been identified (91). The structural explanation for how AQP3 may permit transport of water and glycerol remains uncertain, and primary sequence differences in aquaglyceroporins include a motif (GLYY) in loop C and an aspartate residue following the second NPA motif (NPARD) rather than the motif (NPARS) found in orthodox aquaporins. The possibility that the AQP3 has separate water-and glycerol-transporting domains has been proposed by some investigators (92), however, the existence of a single pore that permits the flow of water or glycerol has been reported by others (93). Although the significance is unclear, AQP3 appears to be gated shut at acid pH (94). No humans have yet been identified with AQP3 deficiency, and the clinical importance of this molecule remains to be established. Because AQP3 is expressed at the basolateral membranes of principal cells in the collecting duct (Figure 10), a role in renal water reabsorption is expected (95,96,97). AQP3 appears to be regulated at a biosynthetic level similar to AQP2. In addition to kidney, AQP3 is also expressed in lower levels at multiple other sites, including airways (95). AQP3 is strikingly abundant in nasopharyngeal epithelium, where it is suspected to play a role in generation of mucosal secretions and allergic rhinitis (50, 98). Brain AQP4 Alth