Aquaporins in Health and Disease Landon S

Reviews MOLECULAR MEDICINE TODAY, FEBRUARY 2000 (VOL. 6)

Aquaporins in health and disease Landon S. King, Masato Yasui and Peter Agre

The molecular basis of membrane water-permeability remained elusive until the recent discovery of the aquaporin water-channel proteins. The fundamental importance of these proteins is suggested by their conservation from bacteria through plants to mammals. Ten mammalian aquaporins have thus far been identified, each with a distinct distribution. In the kidney, lung, eye and brain, multiple water-channel homologs are expressed, providing a network for water transport in those locations. It is increasingly clear that alterations in aquaporin expression or function can be rate-limiting for water transport across certain membranes. Aquaporins are likely to prove central to the pathophysiology of a variety of clinical conditions from diabetes insipidus to various forms of edema and, ultimately, they could be a target for therapy in diseases of altered water homeostasis.

APPROPRIATE regulation of membrane water-permeability is a acteristic of a channel protein. The size (28 kDa) and distribution fundamental requirement of all living organisms. In contrast to the (abundant in erythrocytes and the renal proximal tubule) of AQP1 were prevailing view of simple diffusion through a lipid bilayer, studies both similar to those of the physiologically defined water channel. performed over several decades have predicted the existence of a Finally, the number of copies of AQP1 in erythrocytes (~2ϫ105 per water-specific channel protein in certain membranes1. The molecular cell) was similar to that predicted for the water channel. Expression of identity of such a channel protein remained elusive until the early AQP1 in Xenopus laevis oocytes allowed definitive identification. 1990s. Investigation of the Rh protein of the erythrocyte membrane led When placed in hypotonic media, AQP1-expressing oocytes rapidly to the serendipitous identification of a novel 28 kDa protein2. As de- swell and rupture, but control oocytes exhibit little change in volume3. scribed below, this protein, now called Aquaporin-1 (AQP1), proved to Further studies in oocytes as well as in proteoliposomes reconstituted be the first molecular water channel3. Discovery of the aquaporin fam- with purified AQP1 protein demonstrated that the channel was specifi- ily of water-channel proteins has provided new insights into the mol- cally permeable to water, and was not permeated by other small molecular mechanisms of transcellular water movement. It is becoming ecules including protons, ions, urea and glycerol5. Recent data suggest apparent that aquaporin biology will prove relevant to the patho- that AQP1 is also permeable to CO2 (Ref. 6), but the magnitude of this physiology and perhaps even therapy of a wide array of conditions. permeability is much lower than that for water; the functional relevance of this observation is unclear. The archetype: function and structure of aquaporin-1 The structure of AQP1 is unique7. As noted, the AQP1 monomer con- Several features of AQP1 (initially called ‘CHIP 28’) suggested that it sists of six transmembrane domains, with N- and C-termini that are could be the long-sought water-channel protein4. Hydropathy analysis intracellular (Fig. 1). The first and second halves of the molecule have a of the AQP1 cDNA predicted six membrane-spanning domains char- high degree of internal homology, but are oriented to opposite sides of the lipid bilayer. A three amino acid motif of Asn-Pro-Ala (NPA) is found in both the N- and C-terminal halves of the molecule, a feature common Landon S. King MD* to all members of the aquaporin family. A cysteine in position 189, adja- Assistant Professor of Medicine cent to the carboxy-NPA motif, confers sensitivity to mercurial com- pounds, as described for the physiologically defined water channel8. Masato Yasui Mutation of residues surrounding the NPA motif in either half of the mol- Postdoctoral Fellow ecule reduces water permeability, strongly suggesting that these regions contribute to formation of the aqueous pore9. From the above observa- Peter Agre MD tions, an ‘hourglass’ model for the structure of the AQP1 monomer was Professor of Biological Chemistry and Medicine proposed. Loops B and E (Fig. 1) dip into the plane of the lipid bilayer, and the N- and C-terminal halves of AQP1 fold together to form the aque- Johns Hopkins University School of Medicine, 600 North Wolfe ous pore. Street, Blalock 910, Baltimore, MD 21287, USA. Reconstitution of purified AQP1 from erythrocytes into proteo- -Tel: ؉1 410 955 3467 liposomes has allowed analysis of their crystal structure. Electron dif -Fax: ؉1 410 955 0036 fraction of cryopreserved specimens at tilts of up to 60Њ (3–6 Å reso *e-mail: [email protected] lution) demonstrates six bilayer-spanning domains and an intrasubunit structure highly consistent with the proposed hourglass topology10,a

structure also confirmed by others11,12. Atomic resolution of the structure and detailed analysis of the aqueous pore await further investigation. Crystallographic studies also confirm the tetrameric arrangement of (a) AQP1 in membranes as previously described. The requirement for Outside tetramerization is not yet understood, but presumably results from in- A C E stability of the asymmetric AQP1 monomer in a lipid environment. P N A C Aquaporins: the growing family N A P Aquaporins have now been identified in all levels of life as far back as Membrane prokaryotes. The first bacterial homolog, aqpZ, was identified in Escherichia coli, and is upregulated by, and confers a survival advantage B D Inside in, hypotonic conditions13. Saccharomyces contains two aquaporin genes, at least one of which forms a functional water channel14. As H2N might be predicted by their dependence on local environmental water, plants express numerous aquaporins15. Arabidopsis thaliana contains at least 23 aquaporin homologs in the expressed sequence tag library. Two functional groups of mammalian aquaporins are now being HOOC recognized16. The first, including AQP0, AQP1, AQP2, AQP4 and AQP5, are permeable only to water, as classically defined. A second group, including AQP3, AQP7 and AQP9, are highly permeable to C (b) water, but are also permeated by glycerol and other small molecules. A Outside Phylogenetically, this apparent dichotomy could have arisen from bac- E teria, which contain both a water channel gene (aqpZ in E. coli) and a P C N A glycerol transporter (glpF in E. coli). The structural explanation and A N physiological relevance of these differences is not known. Although P the sequence of AQP6 is similar to the water-selective group, the recent surprising observations of gated anion conductance indicates the func- Membrane 17 tional distinction is more complex than previously thought . The se- B Inside quence of AQP8 is intermediate between the water-selective and the D glycerol permeant groups, and functional definition is awaited18. H2N

Physiology and pathology of the aquaporins Ten mammalian aquaporins have been identified to date, each with a distinct tissue distribution. Multiple regulatory mechanisms are likely HOOC to be identified that will explain the specificity of ontogeny, distribu- Molecular Medicine Today tion and function of each aquaporin. With the exception of AQP2, these regulatory mechanisms are poorly understood. Below, we de- Figure 1. Hourglass model of aquaporin-1 (AQP1) topology. (a) The six trans- scribe aquaporin expression in several organs, with speculation about membrane domains of the AQP1 monomer are represented, as well as the their physiological roles. extracellular loops A, C, and E, and intracellular loops B and D. The two NPA motifs are located in loops B and E, which fold into the lipid bilayer. Cysteine Kidney 189 (C) is the residue that confers mercury-sensitivity to AQP1. (b) The NPA Of the 180 L of glomerular filtrate produced each day, 80–90% is reab- motifs in loops B and E come together to form the aqueous channel. sorbed in the proximal tubule and the descending thin limb of Henle’s loop – segments known to have constitutively high water permeability. The ascending thin and thick limbs of Henle’s loop, the distal convoluted inhibitable water permeability and the presence of AQP1 in endothelial tubule and the connecting tubule are known to be largely impermeable cells of the descending vasa recta, suggesting that AQP1 mediates to water. The remaining 10–20% of the glomerular filtrate is reabsorbed water transport in the vasa recta22. in a vasopressin-dependent fashion in the collecting duct. The water Discovery of the Colton blood-group antigen on AQP1 allowed the permeability of the individual nephron segments correlates closely with subsequent identification of rare Colton-null individuals who lack AQP1; aquaporin expression within each segment19 (Fig. 2). surprisingly, these individuals have no obvious clinical phenotype23. In AQP1 is abundant in the proximal tubule and the descending thin contrast, targeted gene disruption of the Aqp1 gene in mice revealed a limb of the nephron, where it constitutes approximately 4% of the marked urinary concentrating defect24. These studies add credence to brush-border protein20. AQP1 is present in reduced amounts on the speculation that rare AQP1-null humans have compensated water trans- basolateral membrane, and there is no intracellular pool. The density port defects that are subclinical under non-stressed conditions. and unit water conductance of AQP1 are sufficient to account for the AQP2 is located in the principal cells of the collecting duct. Under known water permeability of the proximal tubule and the descending resting conditions, AQP2 is found primarily in intracellular vesicles thin limb21. The renal medulla vascular supply, the vasa recta, is criti- beneath the apical membrane. In response to arginine vasopressin cal to the generation of an axial osmotic gradient in the medulla. (AVP) binding to the V2 receptor at the basolateral membrane, Ser256 Nielsen and colleagues recently demonstrated both mercury- of the AQP2 C-terminus is phosphorylated25. Vesicle-associated pro-

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where they probably provide an exit pathway for fluid reabsorbed in the collecting duct. Proximal tubule Intercalated cell Disruption of the Aqp4 gene in mice resulted (AQP1 and AQP6) in a mild urinary concentrating defect37. AQP3 transports glycerol as well as water; but the functional significance of this observation is unknown.

Respiratory tract Fluid requirements in the respiratory tract are complex. In the distal lung, removal of fluid in the perinatal period is critical in the transition from placental gas exchange to ex utero life. Descending thin limb Principal cell Throughout life, appropriate handling of water (AQP1) in the vascular, interstitial and airspace com- Collecting duct partments of the lung is essential for normal Aquaporins (AQP2, AQP3, gas exchange and lung defense. In the airways, AQP1 AQP4 AQP4 and AQP6) AQP2 AQP6 strict regulation of the airway surface liquid AQP3 layer is required for effective mucociliary Molecular Medicine Today transport. In both the airways and nasopharynx, inspired air must be humidified to prevent Figure 2. A single nephron showing aquaporin distribution. AQP1 is expressed in both the apical and baso- drying of the distal airways, and water must be lateral membrane of cells in the proximal tubule and descending thin limb, both highly water permeable. extracted from the expired air stream to mini- AQP6 is present in intracellular vesicles in proximal tubule epithelial cells, as well as in the acid secreting intercalated cells of the collecting duct. AQP2 is the collecting duct water-channel that translocates from mize breath-to-breath water loss. cytoplasmic vesicles to the apical membrane in the presence of vasopressin. AQP3 and AQP4 are present Four water channels have been identified in in the basolateral membrane of principal cells of the collecting duct. the respiratory tract and their distribution is complex38,39 (Fig. 3). In the rat, AQP1 is abundant in the apical and basolateral membrane of the microvasculature and visceral pleura. teins including vesicle-associated membrane protein-2 (VAMP-2) AQP5 is expressed in the apical membrane of both type I pneumocytes have been colocalized with AQP2 and might facilitate targeting of the and secretory cells in airway submucosal glands. AQP3 and AQP4 are AQP2-containing vesicles to the apical membrane26, where they could expressed in the basolateral membrane of different cells in airway and interact with the targeting receptor syntaxin-4 (Ref. 27). AQP2 re- nasopharyngeal epithelium. This non-overlapping distribution of aqua- distribution to the apical membrane has been closely correlated with a porins might provide a coordinated network for transcellular water dramatic increase in membrane water permeability. movement in the respiratory tract. The absence of known water chan- In contrast to genetically AQP1-deficient humans, AQP2 deficiency nels in the apical membrane of the airway epithelium and the basolat- produces a dramatic clinical phenotype. Nephrogenic diabetes insipidus eral membrane of type I pneumocytes is, however, provocative. (NDI) is a disease whose etiology is renal resistance to AVP, and whose Clearly, undiscovered aquaporins might exist in those locations. clinical hallmark is excretion of large volumes of dilute urine. Deen and Alternatively, transcellular water movement might not occur at every colleagues have now described multiple patients with autosomal recessive point across the respiratory epithelium, but instead take place only at NDI who have mutations in the AQP2 gene28, providing the first clear ex- select sites. ample that aquaporins can be rate-limiting for water transport. Acquired The phenomenon of perinatal lung water-clearance is well de- NDI is more common than the congenital form, and has a variety of scribed. In that context, the ontogeny of aquaporins in the lung is of causes. Nielsen and colleagues recently demonstrated that lithium29, bi- considerable interest. AQP1 is expressed in fetal rat lung late in ges- lateral ureteral obstruction30 and chronic hypokalemia31 – known causes tation, increases dramatically at birth, and is sustained at high levels of NDI – all produce marked reductions in AQP2 expression in animals, in adult animals39. Corticosteroids induce AQP1 expression in fetal with a concomitant decrease in urinary concentrating ability. At the other (and adult) rat lung, consistent with known acceleration of fetal lung end of the water imbalance spectrum, increased AQP2 expression has maturation by corticosteroids. AQP5 is expressed one to two days been demonstrated in conditions of fluid retention including congestive after birth in rat lung, with high levels of expression in adult ani- heart failure32,33, cirrhosis34 and pregnancy35. As we gain insight into mals40. In contrast to AQP1, AQP5 is not induced by corticosteroids. mechanisms regulating its function, AQP2 could prove to be a therapeutic AQP4 exhibits transient high level expression in distal lung two days target in numerous conditions of altered fluid balance. after birth. Although these observations predict participation of water Other aquaporins are also present in the nephron. AQP6 was re- channels in perinatal lung water-clearance, their precise roles in this cently identified as the first intracellular water channel. AQP6 is ex- process and in the pathophysiology of the premature lung remain to be pressed in vesicles in epithelial cells of the proximal tubule and in determined. intercalated cells of the collecting duct36. The unique characteristics of The distribution of aquaporins in the respiratory tract suggests in- AQP6 predict functional differences from other water channels, involvement in a variety of conditions. In the distal lung, altered expres- cluding regulation of acid–base balance17. AQP3 and AQP4 are ex- sion or function of AQP1 and AQP5 might play a role in the patho- pressed in the basolateral membrane of the collecting duct epithelium, genesis of pulmonary edema and pleural effusions. A functional role for

62 MOLECULAR MEDICINE TODAY, FEBRUARY 2000 (VOL. 6) Reviews

Figure 3. Aquaporin expression in the respiratory tract. (a) AQP1 is expressed in the apical and basolateral membrane of endothelial cells. AQP5 is present in the apical membrane of type I pneumocytes. (b) In tracheal epithelium, AQP3 is present in the (a) Alveolar airspace basal cells, and AQP4 is present in the basolateral membrane of the ciliated cells. No aquaporins have been identified in the apical membrane or in the goblet cells. AQP1 Type II is expressed in endothelial cells of the vascular plexus beneath the airway. (c) In se- pneumocyte cretory glands of the nasopharyngeal epithelium, AQP5 is present in the apical membrane of secretory cells, whereas AQP3 and AQP4 are expressed in the basolateral Type I membrane of the same cells. H2O pneumocyte

AQP1 in the distal lung is supported by recent investigation in Aqp1 knockout mice41. When compared with control animals, Aqp1-null mice demonstrated a tenfold reduction in osmotic water permeability, Basement and a twofold decrease in hydrostatic permeability. Additionally, perfusion membrane of isolated distal airways has demonstrated water-channel-mediated transport across the distal airway epithelium42. Water channel expression Capillary in the epithelium, subepithelial vasculature and subepithelial glands of the airways and nasopharynx (Fig. 3) predicts participation in airway hu- (b) Respiratory airspace midification, as well as generation and regulation of the airway surface liquid38. Alterations in the airway surface liquid layer are central to the pulmonary manifestations of cystic fibrosis, and might play a role Goblet cell in some forms of asthma. As with the kidney, it is increasingly likely that aquaporins will participate in multiple aspects of lung water Ciliated homeostasis. cell Brain The physical constraints of the bony cranium demand tight regulation of intracranial fluid. Cerebrospinal fluid is made by the choroid plexus, a specialized structure located in the walls of the lateral, third and fourth ventricles consisting of a vascular core covered by a secretory epithelium. Immunolocalization studies demonstrate that AQP1 is abundant in apical membrane microvilli of the choroid epithelium; it is expressed there from early gestation in the fetal rat. Colocalization of AQP1 with a Basal Na–K ATPase in the apical membrane strongly suggests a role for the cells water channel in cerebrospinal fluid production1. AQP4 was cloned from both lung43 and brain44 cDNA libraries, although the brain appears to be its predominant distribution. AQP4 is ex- Basement pressed in the foot processes of astroglial cells, specifically in the perivas- membrane cular membrane, suggesting that it plays a role in the regulation of H2O extravascular brain water45. Additionally, AQP4 is abundant in the lamel- Fibroblasts lae adjacent to magnocellular cells in the supraoptic and paraventricular nuclei, the site of arginine vasopressin production. From there, AVP is Capillary then transported along axons to the posterior pituitary, where it can sub- (c) Airway gland sequently be released. These AQP4-containing lamellar structures might participate in the sensation and/or the transduction of osmotic signals to the magnocellular cells. Recent studies have demonstrated a marked reduction in AQP4-mediated water permeability by phorbol diesters46. Phosphorylation-mediated gating or trafficking of water channels would allow rapid regulation of intracerebral membrane water permeability.

Eye Five aquaporins have been identified in non-overlapping domains in the 47 eye (Fig. 4). MIP (AQP0) comprises approximately half of the lens- Basement fiber-cell protein, and has recently been shown to function as a low ca- membrane Aquaporins pacity water channel. Two different mutations of the Mip gene in mice H2O AQP1 AQP4 lead to congenital cataracts48. AQP1 in the corneal endothelium and in AQP3 AQP5 the lens anterior epithelium, as well as AQP5 in the corneal epithelium, might participate in reducing the water content of those tissues, an im- Molecular Medicine Today portant feature for maintaining transparency of the cornea and the lens.

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Glossary The outstanding questions Intercalated cells – Renal collecting duct epithelial cells that par- • What structural features of the aquaporin molecules dictate ticipate in acid secretion (in contrast to collecting duct principal cells, the specificity of their permeability? which reabsorb water). • Are aquaporins always open and permeable when inserted in the membrane, or can they be gated? Proteoliposomes – Vesicles made in vitro by mixing different • How is aquaporin expression regulated and, in organs with combinations of lipid and proteins. more than one aquaporin, is this regulation coordinated? • Can aquaporin expression or function be altered to affect Targeting receptor – A membrane protein that interacts with pathophysiologic processes? vesicle-associated proteins to direct trafficking of vesicles to the • What role do aquaporins play in epithelia or endothelia that plasma membrane. are thought to have high paracellular permeability?

Vesicle-associated proteins – One of a small group of integral membrane-proteins found in intracellular vesicles, which facilitate specificity of docking and fusion with the plasma membrane. dence of only low-grade hemolysis. A combination of washout of the medullary interstitial gradient and low-level expression of AQP3 in erythrocytes51 could partially explain the surprisingly normal erythrocyte survival in AQP1-null humans. Expression of AQP1 in the anterior ciliary epithelium and canals of Schlemm suggests a role in secretion and uptake of aqueous humor. Salivary and lacrimal glands AQP1 is also present in the iris, where high water permeability is AQP5 was cloned from a salivary gland library, and is similar to the thought to facilitate the rapid shape changes that occur with pupillary other aquaporins in water transport capacity52. AQP5 is abundantly ex- constriction. The presence of AQP4 in the end-feet of Müller cells in the pressed in the apical membrane of secretory cells in the salivary and retina suggests that it plays a role in the light-dependent hydration of the lacrimal glands, but is not present in the basolateral membrane or in space around photoreceptors49. Finally, AQP3 in the bulbar conjunctiva duct cells38. AQP5 has a protein kinase A consensus in a cytoplasmic might play a role in the hydration of the protective covering of the eye. loop, similar to that of AQP2. Phosphorylation of the protein is an ap- pealing, though as yet unproven, explanation for the rapid onset of sali- Erythrocytes vation and lacrimation in response to the appropriate stimuli. Erythrocytes were the source for the initial identification and purifi- Adenoviral-mediated transfer of the AQP5 gene is being evaluated as cation of AQP1 (Ref. 50). Although water channels are thought to a potential therapy for damaged salivary glands53. facilitate erythrocyte survival during the transit through the hypertonic Curiously, the distribution of AQP5 coincides almost exactly with renal medulla, AQP1-deficient humans are not anemic and have evi- the organ involvement of Sjögren’s disease, an immunologically mediated process causing dry eyes, dry mouth and desiccation of the tra- cheobronchial secretions. The antigen(s) driving immune destruction of the involved organs is unknown, but it is compelling to consider that either primary or secondary dysfunction of AQP5 could be involved. AQP5 Lacrimal gland Corneal epithelium Concluding remarks Discovery of the aquaporin family of membrane proteins has provided AQP1 new insights into the molecular mechanisms of membrane water-per- Lens epithelium Corneal endothelium meability. The conservation of aquaporins from prokaryotes to mam- Nonpigmented epithelium mals is consistent with the central role that water plays in all forms of (ciliary and iris) life. Historically, the focus of discussions on membrane permeability Trabecular meshwork has been solute transport. However, it is increasingly clear that, under some circumstances, membrane water permeability might be regulated MIP (AQP0) independently from solute transport, and that aquaporin expression or Lens fiber cells function can be rate-limiting for water movement. Further investigation AQP3 AQP4 of the structure, regulation and function of aquaporins should greatly Conjunctiva Retinal glia enhance our ability to both understand and manipulate membrane (Müller cells) water-permeability.

Molecular Medicine Today Acknowledgments. This work was supported by the National Institutes of Health (L.K. and Figure 4. Aquaporin expression in the eye. Five aquaporins have been identi- P.A.), the Cystic Fibrosis Foundation (L.K. and P.A.) and the Human Frontier Science fied in the eye, with non-overlapping distribution in different parts of the eye as Program (M.Y.). shown above. References

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