Mammalian aquaporins: diverse physiological roles and ... · Mammalian aquaporins: diverse physiological roles and potential clinical signiﬁcance A. S. Verkman Aquaporins have multiple

Mammalian aquaporins: diverse

physiological roles and potential

clinical significance

A. S. Verkman

Aquaporins have multiple distinct roles in mammalian physiology. Phenotypeanalysis of aquaporin-knockout mice has confirmed the predicted role ofaquaporins in osmotically driven transepithelial fluid transport, as occurs in theurinary concentrating mechanism and glandular fluid secretion. Aquaporinsalso facilitate water movement into and out of the brain in various pathologiessuch as stroke, tumour, infection and hydrocephalus. A major, unexpectedcellular role of aquaporins was revealed by analysis of knockout mice:aquaporins facilitate cell migration, as occurs in angiogenesis, tumourmetastasis, wound healing, and glial scar formation. Another unexpected roleof aquaporins is in neural function – in sensory signalling and seizure activity.The water-transporting function of aquaporins is likely responsible for theseroles. A subset of aquaporins that transport both water and glycerol, the‘aquaglyceroporins’, regulate glycerol content in epidermal, fat and othertissues. Mice lacking various aquaglyceroporins have several interestingphenotypes, including dry skin, resistance to skin carcinogenesis, impairedcell proliferation, and altered fat metabolism. The various roles of aquaporinsmight be exploited clinically by development of drugs to alter aquaporinexpression or function, which could serve as diuretics, and in the treatment ofbrain swelling, glaucoma, epilepsy, obesity and cancer.

The aquaporins (AQPs) are a family of small,hydrophobic, integral membrane proteins (~30kDa/monomer) that are expressed widely inthe animal and plant kingdoms, with 13members identified to date in mammals. AQPsare expressed in many epithelia and endotheliainvolved in fluid transport, such as kidneytubules, glandular epithelia and choroid plexus,

as well as in cell types that do not carry outsignificant fluid transport, such as skin and fatcells. In most cell types the AQPs resideconstitutively at the cell plasma membrane,with the notable exception of AQP2 in kidneycollecting duct, where vasopressin regulatesAQP2 trafficking between endosomes and thecell plasma membrane.

Departments of Medicine and Physiology, Cardiovascular Research Institute, 1246 Health SciencesEast Tower, University of California, San Francisco, CA 94143-0521, USA. Tel: +1 415 476 8530;Fax: +1 415-665-3847; E-mail: [email protected]

expert reviewshttp://www.expertreviews.org/ in molecular medicine

1Accession information: doi:10.1017/S1462399408000690; Vol. 10; e13; May 2008

&2008 Cambridge University Press

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High-resolution structures have been obtainedfor several AQPs, and show the assembly ofAQP monomers in tetramers, with individualmonomers containing six tilted a-helical domainsforming a barrel-like structure in which the firstthree and last three helices exhibit invertedsymmetry (Refs 1, 2). Molecular dynamicssimulations suggest tortuous, single-file passageof water through a narrow ,0.3 nm pore, inwhich steric and electrostatic factors preventtransport of protons and other small molecules(Ref. 3). AQPs 1, 2, 4, 5 and 8 are primarily waterselective, whereas AQPs 3, 7 and 9 (called‘aquaglyceroporins’) also transport glycerol andpossibly other small solutes. Water transportby some AQPs is inhibited by nonspecific,cysteine-sulphydral-reactive compounds such asmercuric chloride (HgCl2). There is considerableinterest, although little reported progress, inthe identification of nontoxic AQP-selectiveinhibitors, which could serve as valuableresearch tools and clinical therapies.

Tissue distribution and regulation studies haveprovided indirect evidence for the involvement ofAQPs in a variety of physiological processes.In the case of AQP2, nephrogenic diabetesinsipidus in subjects with AQP2 mutationsindicated the requirement of AQP2 for theformation of a concentrated urine (Ref. 4).Much of the knowledge of AQP functions inmammalian physiology has come fromphenotype analysis of mice lacking the variousmammalian AQPs. One paradigm that hasemerged is that tissue-specific AQP expressiondoes not mandate AQP involvement in aphysiologically important process, as wasfound for several AQPs in lung (Ref. 5) andintestine (Ref. 6), AQP4 in skeletal muscle(Ref. 7), AQP5 in sweat gland (Ref. 8), andAQP8 in multiple tissues (Ref. 9). Functionalanalysis of cells and tissues from knockout micehas also tested proposed roles of AQPs in gastransport and intracellular organellar function,as well as in AQP protein–protein interactions.Although there is evidence that some AQPsmay allow transport of CO2 and NH3 (Refs 10,11), physiological studies in mice and transportmeasurement in isolated tissues have providedevidence against a physiologically significantrole of AQPs in gas transport (Refs 12, 13, 14).Negative data were also found for the proposedinvolvement of AQPs in mitochondrial function(Ref. 15) and in a key AQP protein–protein

interaction in the central nervous system – aproposed interaction between AQP4 and theinwardly rectifying Kþ channel Kir4.1responsible for Kþ ion uptake by glial cellsduring neuroexcitation (Refs 16, 17).

Balancing the many negative phenotypestudies, data from knockout mice implicateimportant roles of AQPs in kidney, brain,eye, skin, fat and exocrine glands, suggestingtheir involvement in major organ functionsand disease processes, including urinaryconcentrating, brain swelling, epilepsy, glaucoma,cancer and obesity. This reviews focuses onthese AQP roles and their significance in normalorgan physiology and disease. Figure 1provides a schematic summary of the variousAQP roles in mammalian physiology, and isreferred to in the various sections below.

Epithelial fluid transportActive fluid secretion and absorptionAQPs are expressed in many epithelia, such askidney tubules, glands, choroid plexus, ciliarybody and alveoli, where they increasetransepithelial osmotic water permeability. Oneconsequence of increased transepithelial waterpermeability is active (also referred to as‘facilitated’ or ‘near-isosmolar’) fluid secretionand absorption. Active fluid transport involvesthe creation of an osmotic gradient (generallyquite small) across an epithelium by activeion/solute transport, which drives watertransport through highly water-permeableepithelial cell membranes (Fig. 1a). Reducedepithelial cell osmotic water permeability canconsequently impair active fluid transport andosmotic water equilibration, resulting in thesecretion (or absorption) of a reduced volumeof inappropriately hypertonic fluid. Thisprediction has been confirmed in AQP5-knockout mice in salivary gland (Refs 18, 19)and airway submucosal gland (Ref. 20).Defective fluid secretion has also been foundin AQP1-knockout mice in choroid plexus(Ref. 21), which produces cerebrospinal fluid,and in ciliary epithelium (Ref. 22), whichproduces ocular aqueous fluid. In each of thesesystems the rate of transepithelial fluidsecretion, normalised to epithelial surface area,is very high, such that the reduced but non-zero water permeability in AQP deficiencyimpacts on transepithelial osmotic equilibration.Of note, because of the substantial intrinsic water




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permeability of lipid bilayers, AQP deficiency isgenerally not associated with more than a five- totenfold reduction in transepithelial osmoticwater permeability. Very rapid, activetransepithelial fluid absorption occurs in kidneyproximal tubule, where the majority of fluidfiltered by the glomerulus is reabsorbed.Proximal-tubule fluid absorption is impaired inmice lacking the proximal-tubule water channel

AQP1 (Ref. 23), resulting in inappropriatelyhypertonic absorbed fluid (Ref. 24).

By contrast to these examples of AQP-dependent transepithelial fluid secretion andabsorption, there are examples where AQPdeletion does not affect active fluid secretionor absorption. In lung alveolus, although deletionof AQP1 or AQP5 each reduces airspace-capillarywater permeability by about tenfold, active fluid

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absorption is not impaired under normalphysiological conditions (Refs 25, 26), includingduring rapid airspace fluid absorption in theneonatal lung and during stresses such as lunginjury (Ref. 27). Similarly, deletion of AQPsdoes not impair active fluid absorption in theairways (Ref. 28), peritoneal cavity (Ref. 29) andpleural cavity (Ref. 30), or fluid secretion by thesweat gland (Ref. 8). The common feature ofthese examples is that the area-normalised ratesof transepithelial fluid transport aresubstantially lower than those where AQPdeletion impairs fluid transport. Thus, aspredicted from the considerations in Fig. 1a,whether AQPs are required to facilitatetransepithelial fluid transport depends on therate of fluid transport.

Osmotic equilibration across kidneytubules and the urinary concentratingmechanismAnother anticipated role of AQPs is in watertransport across kidney tubules andmicrovessels (vasa recta), which is required forthe formation of a concentrated urine. Themajor AQPs expressed in the kidney are AQPs1, 2, 3, 4 and 7 (Fig. 2a). Deletion of the genesfor AQP1 and/or AQP3 in mice results inmarked polyuria, as seen in 24 h urinecollections (Fig. 2b) (Refs 31, 32). Measurementof urine osmolalities in mice before and after36 h water deprivation (Fig. 2c) shows thaturinary osmolality in AQP1-null mice is lowand does not increase with water deprivation,

resulting in severe dehydration. AQP3-null miceare able to generate a partially concentratedurine in response to water deprivation, whereasAQP4-null mice manifest only a mild defect inmaximum urinary concentrating ability (Ref. 33).

AQP1 deletion produces polyuria andunresponsiveness to water deprivation by twodistinct mechanisms: impaired near-isosmolarwater reabsorption in the proximal tubule, asdescribed above, and reduced medullaryhypertonicity resulting from impairedcountercurrent multiplication and exchange(a consequence of low water permeabilityin the thin descending limb of Henle andouter medullary descending vasa recta).Transepithelial osmotic water permeabilityin the isolated microperfused S2 segment ofproximal tubule was reduced by about fivefoldin AQP1-knockout mice (Ref. 23), indicatingthat most water transport in the proximaltubule is transcellular and AQP1 dependent.AQP1 also provides the major route fortransepithelial water permeability in the thindescending limb of Henle and outer medullarydescending vasa recta (Refs 34, 35). Theseresults support the conclusions that AQP1 is theprincipal water channel in these segments,and that AQP1 plays a key role in theantidiuretic kidney in the generation of thehypertonicity of the medullary interstitium bycountercurrent multiplication and exchange.The aquaglyceroporin AQP7 is expressed in asmall distal segment (S3 segment) of theproximal tubule; however, its deletion in mice is

Figure 1. Aquaporin functions in mammalian physiology. (Legend; see previous page for figure.) Water-transporting functions of aquaporins (AQPs) are shown in the top half of the figure; glycerol-transportingfunctions are shown in the lower half of the figure. (a) AQP facilitates rapid, near-isomolar transepithelial fluidsecretion: AQP deficiency in an epithelium such as salivary gland slows osmotic water transport into theacinar lumen, resulting in the secretion of a reduced volume of a hypertonic fluid. Numbers representhypothetical fluid osmolalities. (b) Expression of AQPs 2, 3 and 4 in the kidney collecting duct facilitates theproduction of a concentrated urine: AQP deficiency reduces transepithelial water permeability, preventingosmotic equilibration of lumenal fluid and impairing urinary concentrating ability. Numbers representhypothetical fluid osmolalities. (c) Proposed mechanism of AQP-facilitated cell migration, showing waterentry into protruding lamellipodia in migrating cells. (d) AQP4-dependent neuroexcitation, showing AQP4-facilitated water transport in glial cells, which communicate with neurons through changes in extracellularspace volume and Kþ concentration. (e) AQP3 facilitates glycerol entry in the epidermis, allowing waterretention (glycerol functions as an osmolyte) and biosynthesis: in AQP deficiency the steady-state glycerolcontent in epidermis and stratum corneum in skin is reduced, accounting for reduced skin hydration.(f) Proposed mechanism of AQP3-facilitated cell proliferation involving increased cellular glycerol andconsequent increased ATP energy and biosynthesis. (g) AQP7 facilitates glycerol escape from adipocytes:adipocyte hypertrophy is seen in AQP7 deficiency, possibly as a result of impaired AQP7-dependentglycerol escape from adipocytes, resulting in cellular glycerol accumulation and increased triglyceridecontent. See text for further explanations. Abbreviations: G3P, glycerol-3-phosphate; TG, triglyceride.




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not associated with significant impairment inurinary concentrating ability, but rather with animpairment of glycerol clearance, whosesignificance remains unclear (Ref. 36).

AQP3 and AQP4 are expressed at the basolateralmembrane of collecting duct epithelium, withrelatively greater expression of AQP3 in corticaland outer medullary collecting duct, and AQP4in inner medullary collecting duct. In contrast toAQP1 deficiency, countercurrent multiplication

and exchange mechanisms in AQP3/AQP4-nullmice are basically intact. The polyuria inAQP3-null mice results from reduced osmoticwater permeability of cortical collecting ductbasolateral membrane (Ref. 32). Reducedtransepithelial osmosis across the collecting ductepithelium interferes with osmotic waterextraction from fluid flowing through the tubulelumen (as shown in Fig. 1b), resulting in theexcretion of an inappropriately large volume of

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Figure 2. Impaired urinary concentrating function in aquaporin deficiency.Deletion of aquaporins (AQPs) inkidney results in increased urinary output and reduced urinary osmolality. (a) Sites of AQP expression in kidney,showing AQP1 expression in the proximal tubule, thin descending limb of Henle (TDLH) and outer medulllarydescending vasa recta, AQP2 expression at the lumen membrane and endosomes in collecting duct, andAQP3 and AQP4 at the basolateral membrane in collecting duct. (b) 24 h urine collections showing polyuriain mice lacking AQP1 and AQP3, individually and together. (c) Urine osmolalities before and after 36 h waterdeprivation (standard error shown). Data summarised from Refs 31, 32 and 33. (The nomenclature style Aqpis used to denote mouse aquaporin genes here, but the style AQP is used throughout the main text of thisarticle to denote mammalian AQP proteins in general.)




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dilute urine. AQP4-null mice manifest only a mildimpairment in maximal urinary concentratingability (Ref. 33), despite a fourfold reduced waterpermeability in microperfused inner medullarycollecting duct (Ref. 37), in part because of therelatively greater quantity of fluid absorbed bycortical versus inner medullary segments of thecollecting duct. Several mouse models of AQP2gene deletion and mutation (reviewed in Ref. 38)support the conclusion from humans withnephrogenic diabetes insipidus that AQP2 is themajor vasopressin-regulated water channelwhose apical membrane targeting in collectingduct during antidiuresis is crucial for theformation of a concentrated urine. Becausetransepithelial water transport in collecting ductis transcellular, the impairment in urinaryconcentration resulting from reduced waterpermeability (Fig. 1b) can result from reducedwater permeability of the serial apical (AQP2-containing) or basolateral (AQP3/AQP4-containing) membrane barriers.

Brain swellingAnother major AQP role related to its watertransport function is in brain water balance.AQP4 is expressed in glial cells (astrocytes)throughout the brain and spinal cord, particularlyat sites of fluid transport at blood–brain andbrain–cerebrospinal-fluid (CSF) interfaces. AQP4expression is polarised to glial cell foot processesin contact with blood vessels, and in the denseglial cell processes that form the glia limitanslining the CSF-bathed pial and ependymalsurfaces in the subarachnoid space and theventricles. AQP4 provides the major route forwater transport across glial cell membranes.Osmotic water permeability in glial cells culturedfrom AQP4-null mice was sevenfold lower thanthat from wild-type mice (Ref. 39). Also, greatlyslowed accumulation of brain water was found inAQP4-null mice in response to serum hypo-osmolality, as monitored by a noninvasive near-infrared optical method (Ref. 40), or by brainwet-to-dry weight ratios (Ref. 41).

Classification of brain oedemaAccording to the Klatzo classification (Ref. 42),brain oedema can be classified as cytotoxic (cellswelling) oedema or vasogenic (leaky vessel)oedema (Fig. 3a). In cytotoxic oedema, excesswater moves from the vasculature into the brainparenchyma through an intact blood–brain

barrier. The forces driving water flow to formcytotoxic oedema are osmotic, generated inwater intoxication by reduced plasmaosmolality, and in ischaemia and otherpathologies by impaired Naþ/Kþ-ATPasepump function with consequent Naþ and wateraccumulation in brain cells. When the blood–brain barrier becomes disrupted (as in braintumour or abscess), water is driven byhydrostatic forces from the vasculature into theextracellular space of the brain in an AQP4-independent manner to form vasogenicoedema. Excess brain water is eliminatedprimarily through the glia limiting membraneinto the CSF, and to a lesser extent backthrough the blood–brain barrier into the blood.The blood–brain barrier may become animportant route for water elimination inobstructive hydrocephalus when other routes orwater exit are impaired.

Cytotoxic brain oedemaPhenotype analysis of AQP4-null mice hasprovided compelling evidence for AQP4-facilitated brain water accumulation in cytotoxicoedema and for AQP4-facilitated brain waterelimination in vasogenic oedema. Waterintoxication, produced experimentally in miceby intraperitoneal water injection, is an exampleof pure cytotoxic oedema in which water isdriven osmotically into the brain through anintact blood–brain barrier. AQP4-null micehave remarkably improved survival followingwater intoxication compared with wild-typemice (Fig. 3b), with reduced brain wateraccumulation and glial cell foot-processswelling (Ref. 43). Reduced brain swelling andimproved clinical outcome was also found inAQP4-null mice in a model of ischaemic strokeproduced by transient middle cerebral arteryocclusion (Ref. 43) and in a model of bacterialmeningitis produced by intracisternalstreptococcus injection (Ref. 41). Reduced brainswelling in water intoxication was also reportedin a-syntrophin-null mice, which secondarilymanifest disrupted brain AQP4 expression(Ref. 44). AQP4 inhibition may thus provide anew approach to reduce brain swelling incytotoxic oedema, which would complementthe currently available therapies, includingdecompressive craniectomy and intravenousmannitol administration – techniques that havechanged little over the last century. Recently,




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Aquaporin 4 deficiency slows brain water accumulation in cytotoxic oedema, and brain water elimination in vasogenic oedemaExpert Reviews in Molecular Medicine 2008 Published by Cambridge University Press

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Figure 3. Aquaporin 4 deficiency slows brain water accumulation in cytotoxic oedema, and brain waterelimination in vasogenic oedema. (a) Routes of water movement in brain during oedema formation andelimination. Oedema formation involves water movement through the blood–brain barrier, which is intact incytotoxic oedema and disrupted in vasogenic oedema. Oedema elimination involves water movementacross the glia limitans, ependyma and blood–brain barrier. Aquaporin 4 (AQP4) expression in glial cells isshown as blue circles. Oedema elimination schematics adapted from Ref. 46 (& 2004 FASEB), withpermission. (b) Water intoxication model of cytotoxic oedema. AQP4-null mice show improved survival afteracute water intoxication produced by intraperitoneal water injection. Adapted from Ref. 43. (c) Increasedelevation in intracranial pressure in AQP4-null mice during continuous intraparenchymal infusion of artificialcerebrospinal fluid (0.5 ml/min). The recordings show intracranial pressure with fluid infusion begun at thearrows. Adapted from Ref. 46 (& 2004 FASEB), with permission. (d) Accelerated progression ofhydrocephalus in AQP4 deficiency, as shown by the larger size of lateral ventricles in AQP4-null miceat 5 days after kaolin injection. Adapted from Ref. 48.




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greatly improved outcome with neuronalpreservation was found in AQP4-null mice in amodel of spinal cord compression injury(Ref. 45), which is likely a consequence ofreduced water entry into the spinal cord inAQP4 deficiency by a cytotoxic-like mechanism.

Vasogenic brain oedema andhydrocephalusBy contrast to these examples of cytotoxicoedema, AQP4 deletion in mice increases brainwater accumulation and worsens outcome invasogenic brain oedema and hydrocephalus. Ina model of pure vasogenic oedema producedby continuous intraparenchymal fluid infusion,there was increased brain water accumulationwith greater elevation in intracranial pressure inAQP4 deficiency (Ref. 46) (Fig. 3c). Similarfindings were obtained in other examples ofvasogenic oedema, including brain tumour,brain abscess and focal cortical freeze injury(Refs 46, 47), supporting the conclusion that invasogenic oedema fluid is eliminated primarilyby an AQP4-dependent route. Finally, in akaolin-injection model of obstructivehydrocephalus, producing what has been called‘interstitial oedema’, AQP4-null mice developmarked ventricular enlargement (Fig. 3d),probably due to reduced transependymal waterclearance (Ref. 48).

AQP4 is thus a major determinant of fluidmovement into and out of the brain. Manybrain pathologies, such as impact injury andtoxic encephalopathies, produce brain oedemaby a combination of cytotoxic and vasogenicmechanisms, each with a different time courseand severity, so it is difficult a priori to predictwhether and when AQP4 inhibition would bebeneficial or detrimental.

Swelling of ocular tissuesAs in the brain, AQPs in the eye are likely to beimportant in fluid balance and pathology insome ocular tissues. The eye expresses severalAQPs at putative sites of fluid transport. Theexpression of MIP (major intrinsic protein, alsoreferred to as AQP0) in lens fibre has beenknown for many years. Mutations in AQP0 inhumans are associated with congenital cataracts(Ref. 49), and recent data suggest theinvolvement of AQP0 in lens fibre cell adhesion(Ref. 1). AQP1 is expressed in cornealendothelium, and at sites of aqueous fluid

production (ciliary epithelium) and outflow(trabecular meshwork). AQP3 is expressed inthe conjunctival epithelium. AQP4 is expressedin Muller cells in retina, and is coexpressedwith AQP1 in nonpigmented ciliary epithelium.AQP5 is expressed in corneal epithelia. Thisexpression pattern provides indirect evidencefor AQP involvement in intraocular pressureregulation (AQP1 and AQP4) (Ref. 22), cornealand lens transparency (AQP0, AQP1 andAQP5) (Refs 50, 51), visual signal transduction(AQP4) (Ref. 52), tear film homeostasis (AQP3and AQP5) (Ref. 53), and conjunctival barrierfunction (AQP3) (Ref. 53). These possibilitieshave been examined systematically byphenotype analysis of AQP-knockout mice. Wefocus here on AQP involvement in ocular tissueswelling.

Cornea and lens swellingIn cornea, endothelial cell AQP1 and epithelialcell AQP5 are involved in corneal stromal waterbalance, and thus maintenance of cornealtransparency. Corneal thickness is reducedcompared with normal in AQP1-null mice andincreased in AQP5-null mice (Ref. 51). In anexperimental model of corneal swellingproduced by exposure of the ocular surface tohypo-osmolar fluid, the recovery of cornealtransparency and thickness after hypotonicswelling was greatly delayed in AQP1-nullmice. AQP1 is also expressed in the epithelialcell layer surrounding the lens, where it plays arole in lens water balance (Ref. 50). AlthoughAQP1 deletion did not alter baseline lensmorphology or transparency, loss of lenstransparency was greatly increased in an invitro model of cataractogenesis produced byincubation of lenses in high-glucose solutions.Cataract formation was also greatly acceleratedin AQP1-null mice in an in vivo model ofcataractogenesis produced by acetaminophentoxicity. Notwithstanding lack of a clear-cutmechanism for these observations, the resultssuggest the interesting possibility of reducingcorneal and lens oedema by AQP1 upregulation.

Retinal swellingThere is also evidence implicating AQP4 in retinalswelling. AQP4 is expressed in Muller cells inretina, where it is involved in light signaltransduction (see section ‘Neural signaltransduction’ below). Based on the protection




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against cytotoxic brain oedema conferred byAQP4 gene deletion, the possibility was testedthat AQP4 deletion protects the retina in atransient ischaemia–reperfusion modelproduced by 45–60 min elevation in intraocularpressure to 120 mmHg (Ref. 54). Retinalstructure and cell number were remarkablypreserved in AQP4-null mice, particularly inthe inner nuclear and plexiform layers of retinawhere Muller cells are concentrated. Retinalfunction and cell survival were also improvedin AQP4-null mice, with electroretinographicevidence of significant attenuation of thereduction in b-wave amplitudes. Whether theneuroprotective effects of AQP4 deletion inretina can be exploited in the therapy of humanocular disease remains to be explored.

Cell migrationImpaired angiogenesis in AQP1 deficiencyPhenotype analysis of AQP1-null mice led to thediscovery of AQP involvement in cell migration.Given the expression of AQP1 in tumourmicrovessels (Ref. 55), the involvement of AQP1in tumour angiogenesis was tested (Ref. 56).AQP1 deletion in mice reduced tumour growthfollowing subcutaneous injection of melanomacells (Fig. 4a), which was associated withincreased tumour necrosis and reduced blood-

vessel formation within the tumour bed. Inexperiments to elucidate the mechanism ofdefective tumour angiogenesis in AQP1deficiency, it was found that cultured aorticendothelial cells from AQP1-null mice migratedseveral-fold slower towards a chemotacticstimulus than AQP1-expressing endothelialcells. Other processes involved in angiogenesis,including endothelial cell proliferation andadhesion, were not impaired in AQP1deficiency. Transfection of AQP1 or other AQPsinto cells that do not express AQPs increasedtheir migration, suggesting the involvementof AQP-facilitated cell membrane waterpermeability in cell migration. In the migratingcells, AQP1 becomes polarised to the front endof cells (Fig. 4b), and is associated withincreased turnover of cell membraneprotrusions (lamellipodia), suggesting thatAQPs at the leading edge of migrating cellsfacilitate their migration.

Reduced tumour spread, glial scarring andwound healing in AQP deficiencyFollow-up experiments showed that AQPsfacilitate cell migration independent of AQPand cell type. AQP4 facilitates astrocyte cellmigration (Refs 57, 58), AQP3 facilitatesmigration of corneal epithelial cells (Ref. 59)

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Figure 4. Impairment in tumourgrowthandendothelial cellmigration in aquaporin 1deficiency.Aquaporin1 (AQP1)deletion impairs tumourangiogenesisbecause, inpart, of reducedmigrationof endothelial cells. (a, left)Reduced tumour size in AQP1-null mouse, two weeks after subcutaneous injection of one million B16F10melanoma cells. (a, right) Tumour growth data (ten mice per group). (b) AQP1 protein (green) polarisation tolamellipodia (arrows) in a migrating CHO cell. Graph in part a and image in part b reprinted from Ref. 56.




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and epidermal cells (Ref. 60), and AQP1 facilitatesthe migration of cultured renal proximal tubulecells (Ref. 61), B16F10 melanoma and 4T1 breastcancer cells (Ref. 62). These studies alsodemonstrated that AQP-facilitated cellmigration participates not only in angiogenesisbut also in other processes including tumourcell spread, glial scar formation, and woundhealing. AQP1 expression in tumour cellsincreases their migration across endothelialbarriers, local invasiveness and metastaticpotential (Ref. 62). AQP4 deletion in glial cellsreduces their migration toward a stab wound invivo (Ref. 58) and the rate of glial scarformation (Ref. 57). AQP3 deletion impairsclosure of cutaneous wounds (Ref. 60) andcorneal wounds (Ref. 59). Although not yettested, AQPs may also be involved in organregeneration and immune cell chemotaxis.

Mechanisms of AQP-facilitated cellmigrationThe enhanced cell migration found for multiplestructurally different AQPs, independent oftheir modulation method (transfection,knockout, RNA inhibition), suggests that AQP-facilitated water transport is the responsiblemechanism. AQPs might accelerate cellmigration by facilitating rapid changes in cellvolume that accompany changes in cell shapeas cells squeeze through the narrowextracellular space. Water flow across the cellmembrane may also allow migrating cells togenerate hydrostatic forces to push apartadjacent stationary cells. This mechanism,however, does not account for the polarisationof AQPs to the front end of migrating cells orfor AQP enhancement of lamellipodialdynamics, which support a role for watermovement across the leading edge of migratingcells, as was proposed previously (Ref. 63).According to this hypothesis, actindepolymerisation and ion influx increasecytoplasmic osmolality at the front end ofthe migrating cell, driving water influx acrossthe plasma membrane (Fig. 1c). Water influxwould thus expand the adjacent plasmamembrane by increased local hydrostaticpressure, followed by actin repolymerisation tostabilise the cell membrane protrusion. There isevidence that regional hydrostatic pressurechanges within cells do not equilibratethroughout the cytoplasm on scales of 10 mm

and 10 s (Ref. 64), and could thus contribute tothe formation of localised cell membraneprotrusions. Further studies, including directmeasurements of water flow across the leadingedge of migrating cells, are needed to validatethese ideas.

Neural signal transductionImpaired neural signal transduction inAQP4 deficiencyAQP4 appears to play an unexpected role inneural function. AQP4 is expressed insupportive cells adjacent to electrically excitablecells, as in glia versus neurons in brainand spinal cord, Muller versus bipolar cellsin retina, and supportive versus hair cells in theinner ear. Electrophysiological measurementsindicated impaired auditory and visual signaltranduction in AQP4-null mice, seen asincreased auditory brainstem responsethresholds (Refs 65, 66) and reducedelectroretinographic potentials (Ref. 52). Inbrain, seizure susceptibility in response to theconvulsant pentylenetetrazol was reduced inAQP4-null mice (Ref. 67). In freely movingmice, electrically induced seizures followinghippocampal stimulation, as measured byelectroencephalography, showed greaterthreshold and remarkably longer duration inAQP4-null mice (Ref. 68). In agreementwith these findings, a-syntrophin-deficientmice developed more-severe behaviouralseizures than wild-type mice followinghyperthermia (Ref. 69). Recently, defectiveolfaction was found in AQP4-null mice asdemonstrated in behavioural studies andodorant-induced electro-olfactogram responses(Ref. 70).

Possible mechanisms of impairedneuroexcitation in AQP4 deficiencyThe mechanisms for altered neuroexcitation inAQP4 deficiency are unclear at present(Fig. 1d). Delayed Kþ uptake from brainextracellular space (ECS) in AQP4 deficiencyhas been suggested, which may account for theprolonged seizure phenotype. Measurements of[Kþ] in brain cortex in living mice using Kþ-sensitive microelectrodes showed significantslowing of Kþ clearance following electricalstimulation (Ref. 68). Using a Kþ-sensitivefluorescent dye applied directly to the brain inliving mice following craniectomy, altered Kþ




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wave dynamics were found in a cortical spreadingdepression model of neuroexcitation, again withdelayed Kþ clearance (Ref. 71). How delayed Kþ

reuptake from the ECS is related to AQP4deficiency is not known. It has been proposedthat AQP4 associates in a functionallysignificant manner with Kir4.1, such that thatreduced Kþ-channel function in AQP4deficiency might account for the delay in Kþ

clearance. However, recent patch-clamp studiesin astroglia (Ref. 16) and Muller cells (Ref. 17)provide evidence against this mechanism.Another possible mechanism involves ECSexpansion in AQP4 deficiency, which mayaccount in part for reduced seizuresusceptibility and prolonged seizure duration inAQP4-null mice. An expanded ECS wouldprovide a larger aqueous volume to dilute Kþ

released into the ECS during neuroexcitation,thereby slowing changes in ECS Kþ

concentration. There is evidence for anexpanded ECS in AQP4 deficiency from corticalsurface photobleaching (Ref. 72) andmicrofibreoptic photobleaching (Ref. 73)measurements of the diffusion of fluorescentlylabelled macromolecules in mouse brain. Itremains unclear, however, whether ECSexpansion in AQP4 deficiency could accountfully for the altered ECS Kþ dynamics.Perhaps reduced water permeability inAQP4 deficiency may be responsible fordefective neuroexcitation function by amechanism involving impaired cell volumeresponses. Alternative possible mechanismsinclude AQP4 interaction with key ionchannels, perhaps through PDZ-domaininteractions, and maladaptive regulation inAQP4 deficiency of other transporters involvedin neuroexcitation.

Glycerol transport by theaquaglyceroporins

For many years the physiological significance ofglycerol transport by the aquaglyceroporinswas unclear. Phenotype studies of mice lackingaquaglyceroporins have produced a numberof remarkable findings for the involvementof AQP3 in epidermal biology and cellproliferation, and of AQP7 in adipocytemetabolism. A recent report on AQP9-null miceshowed a subtle phenotype suggestive ofimpaired hepatic glycerol uptake (Ref. 74),although the mechanism remains to be

established as does its proposed significance todiabetes.

AQP3 and skin functionThe stratum corneum (SC) is the most superficiallayer of skin, consisting of a lamellar lipid layerand terminally differentiated keratinocytesthat originate from actively proliferatingkeratinocytes in lower epidermis. SC hydrationis an important determinant of skin appearanceand physical properties, and depends onseveral factors including the external humidity,and SC structure, lipid/protein composition,barrier properties, and concentration of water-retaining osmolytes.

AQP3 is expressed strongly in the basal layer ofkeratinocytes (Fig. 5a). SC hydration is reduced inAQP3-null mice as measured by high-frequencyskin conductance (Ref. 75) (Fig. 5b), which is alinear index of SC water content. Exposure ofmice to high humidity or occlusion increasedSC hydration in wild-type, but not AQP3-nullmice, indicating that water transport throughAQP3 is not a rate-limiting factor intransepidermal water loss. If reduced SChydration is related to a balance betweenevaporative water loss from the SC and waterreplacement through AQP3-containing basalkeratinocytes, then preventing water loss byhigh humidity or occlusion should havecorrected the defect in SC hydration in AQP3-null mice, which it did not. Skin phenotypeanalysis also indicated delayed barrier recoveryafter SC removal by tape-stripping in AQP3-null mice, as well as decreased skin elasticityand delayed wound healing (Ref. 75).

A systematic analysis of SC and epidermalultrastructure and composition revealedreduced glycerol content in SC and epidermis(Fig. 5c), with normal glycerol in dermis andserum, suggesting reduced glycerol transportfrom blood into the epidermis in AQP3deficiency through the basal keratinocytes(Fig. 1e). No significant differences in wild-typeversus AQP3-null mice were found in SCstructure, cell turnover, lipid profile, proteincontent, and the concentrations of amino acids,ions and other small solutes (Ref. 76). Theseobservations suggest that reduced epidermaland SC glycerol content is responsible for theabnormal skin phenotype in AQP3-null mice.Because glycerol is a water-retaining osmolyte,or ‘natural moisturising factor’, reduced SC




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glycerol reduces SC hydration and skin elasticity;furthermore, because of its biosynthetic role in theepidermis (see next section), reduced epidermalglycerol is predicted to delay barrier recoveryfunction and wound healing. In support of thishypothesis, glycerol replacement by topical orsystemic routes corrected the phenotypeabnormalities in AQP3-null mice, and SCglycerol content correlated well with SC watercontent (Ref. 77). These findings indicate an

important role for AQP3 and glycerol inepidermal function, providing a rationalscientific basis for the long-standing practice ofincluding glycerol in cosmetic and skinmedicinal preparations.

AQP3 and cell proliferationRecent data support the unexpected involvementof AQP3 in cell proliferation in certain cell types.Remarkably, mice lacking AQP3 failed to produce

Aquaporin 3 deficiency reduces skin hydration and prevents skin tumour formationExpert Reviews in Molecular Medicine 2008 Published by Cambridge University Press

90%

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Figure 5. Aquaporin 3 deficiency reduces skin hydration and prevents skin tumour formation.Aquaporin 3(AQP3) is expressed in the basal layer of keratinocytes in normal skin, and its deletion in mice produces dry skinbecause of reduced skin glycerol content, and resistance to tumourigenesis. (a) Immunofluorescence showingAQP3 expression (yellow/green) in basal layer of epidermis in mice. Abbreviations: D, dermis; E, epidermis; SC,stratumcorneum. Image reproduced fromRef. 75 (&2008TheAmericanSociety forBiochemistryandMolecularBiology), with permission. (b) Reduced statum corneum water content in AQP3-null mice, measured by high-frequency skin surface conductance (five mice per group, *P , 0.01). Skin conductance was measured after24 h exposure to relative humidity of 10, 40 or 90%; ‘occluded’ indicates a plastic occlusion dressing thatprevents evaporative water loss. Reprinted from Ref. 75 (& 2008 The American Society for Biochemistry andMolecular Biology), with permission. (c) Reduced glycerol content in stratum corneum and epidermis ofAQP3-null mice (*P , 0.01). Adapted from Ref. 76 (& 2008 The American Society for Biochemistry andMolecular Biology), with permission. (d, left) Absence of cutaneous papillomas in AQP3-null mice treatedwith an initiator (once) and twice-weekly applications of a promoter for 20 weeks. Arrows point topapillomas. (d, right) Percentage of mice with papillomas after initiator treatment. Part d adapted fromRef. 78 (& 2008 American Society for Microbiology), with permission.




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cutaneous papillomas in an inducer–promotermodel of skin cancer, whereas wild-type miceproduced multiple tumours (Fig. 5d) (Ref. 78).The motivation for studying AQP3 and skintumours was the strong expression of AQP3 inbasal cells in human skin squamous cellcarcinomas, and data showing AQP3-facilitatedcell proliferation in several cell types. Woundhealing and corneal epithelial cell proliferationare impaired in AQP3 deficiency (Ref. 59), as isthe healing of cutaneous wounds (Ref. 60) andregeneration of the colonic epithelium inexperimental colitis (Ref. 79).

Experiments to establish the cellularmechanisms responsible for the impairedtumourigenesis phenotype showed impairedpromoter-induced cell proliferation in AQP3-null or -knockdown keratinocyte cell cultures.AQP3-deficient keratinocytes had reducedcontent of glycerol, its metabolite glycerol-3-phosphate, and ATP, without impairmentof mitochondrial function. Glycerolsupplementation or AQP3 adenoviral infection(but not AQP1 adenoviral infection) correctedthe defects in keratinocyte proliferation andincreased ATP. Further studies revealedcorrelations between cell proliferation, and ATPand glycerol content. It was proposed thatAQP3-facilitated glycerol transport is animportant determinant of epidermal cellproliferation and tumourigenesis by amechanism in which glycerol is a key regulatorof cellular ATP energy (Fig. 1f). The mechanismalso shows glycerol biosynthetic incorporationinto lipids, and positive feedback in which cellproliferation increases AQP3 expression. Thesefindings have potential implications in theprevention and therapy of skin and othercancers, and raise concerns in the use ofcosmetics containing ingredients that increaseepidermal AQP3 expression whose goal is toimprove skin moisture and appearance (Ref. 80).

AQP7 and fat metabolismAQP7 is expressed in the plasma membrane ofadipocytes. Although wild-type and AQP7-nullmice grow at similar rates as assessed by mouseweight, over time AQP7-null mice developsignificantly greater fat mass compared withwild-type mice (Ref. 81). Adipocytes from adultAQP3-null mice are several-fold larger thanthose from wild-type mice, suggesting thatthe greater fat mass in the AQP7-null mice is

a consequence of adipocyte hypertrophy.Concentrations of glycerol and triglycerides inserum were unaffected by AQP7 deletion, butadipocyte glycerol and triglycerideconcentrations were significantly elevated inAQP7-null mice, suggesting a mechanism forthe progressive adipocyte hypertrophy in AQP7deficiency. Plasma membrane glycerolpermeability was reduced significantly inadipocytes of AQP7-null mice, as was glycerolrelease. However, lipolysis, as measured by freefatty acid release from isolated adipocytes, wassimilar in wild-type and AQP7-deficient mice,as was lipogenesis, as assayed from theincorporation of [14C]glucose into triglycerides.From these results, we proposed a simplemechanism for progressive triglycerideaccumulation in AQP7-deficient adipocytes(Fig. 1g), in which reduced plasma membraneglycerol permeability in AQP7 deficiencyproduces an increased glycerol concentration inadipocyte cytoplasm, resulting in increasedglycerol-3-phosphate and triglyceride biosynthesis.Similar conclusions about fat metabolism inAQP7 deficiency were reported independently(Ref. 82), although with some relatively minordifferences in phenotype findings comparedwith our results. It was speculated that AQP7plays an important role in the pathogenesisof human obesity (reviewed in Ref. 83),although whether this is the case remains to bedetermined.

Clinical implications/applicationsNotwithstanding differences in mouse versushuman physiology, the involvement of AQPs inmajor physiological processes in mouse modelslikely has a number of clinical implications. Therequirement of AQPs for the formation of aconcentrated urine suggests that AQPinhibitors, or ‘AQP-aquaretics’, would act asunique diuretics with potential utility indiuretic-refractory oedematous states such assevere congestive heart failure. Inhibitors ofvarious AQPs are also predicted to havepotential efficacy in reducing water entry intothe brain in cytotoxic oedema, in improvingthe outcome following spinal cord injury,in reducing aqueous fluid production inglaucoma, in inhibiting glial scar formation,in reducing angiogenesis and tumour spread, inreducing cell proliferation in certain cancers,and in increasing seizure threshold in epilepsy.




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Drugs that increase AQP function, acting forexample by increasing AQP expression, arepredicted to have potential efficacy in reducingfat mass in obesity, in accelerating brain waterclearance in vasogenic oedema, and inpromoting wound healing and tissueregeneration following injury. Validation ofthese predictions will require the developmentof appropriate AQP-specific modulators.Another AQP-related clinical application is indisease diagnosis, as demonstrated for serumAQP4 autoantibodies in diagnosing theoptic–spinal form of multiple sclerosis(Ref. 84), and for urinary AQP2 protein indistinguishing among various aetiologies ofnephrogenic diabetes insipidus (Ref. 85).Other AQP-related disease markers are likely tobe identified. The possibility of AQPpolymorphisms contributing to humandisease is largely unexplored. It would beworthwhile, for example, to investigatepolymorphisms in AQP4 in brain diseases suchas hydrocephalus, in AQP3 in skin diseases,and in various AQPs in cancer. Last, themodulation of AQP expression in disease statesmay be clinically important. In some casesaltered AQP expression appears to be amaladaptive response, as in the case of reducedrenal AQP2 expression in various forms ofpolyuria, which further impairs urinaryconcentrating ability (Ref. 86), and AQP4upregulation in various aetiologies of brainswelling, which may exacerbate brainwater accumulation. There is an expandingliterature on altered AQP expression in humandiseases, with evidence for altered AQP3expression in skin diseases (Ref. 87), AQP4expression in epilepsy (Ref. 88), andAQP7 expression in obesity and metabolicdiseases (Ref. 89). In most cases, however, itwill likely be difficult to establish the cellularmechanisms and clinical significance of suchobservations.

Research in progress and outstandingresearch questions

There remain many unanswered questions aboutthe roles of AQPs in mammalian physiology, aswell as exciting opportunities for clinicalapplications. Although water transport has beenstudied for many decades and AQP proteinswere identified in the early 1990s, many of thenew AQP cellular functions were recognised

only in the past few years, so it is likely thatadditional new AQP functions will bediscovered. Much work remains in the preciseelucidation of cellular mechanisms responsiblefor AQP involvement in cell migration, neuralsignal transduction and in vasogenic brainoedema, and in the precise role of theaquaglyceroporins in cellular metabolism andproliferation. Finally, as mentioned in theprevious section, identification of chemical AQP-selective modulators is a high priority inongoing research, as small-molecule AQPinhibitors and upregulators have the potential toserve as new tools to study AQP function and aspotential therapies for major human diseases.

Acknowledgements and fundingThe contributions of many collaborators andtrainees who shaped the ideas presented hereare greatly acknowledged: Drs D. Binder,M. Hara-Chikuma, T. Ma, G. Manley,M. Papadopoulos, S. Saadoun, Y. Song,J. Thiagarajah, B. Yang and many others. TheAQP mechanism and mouse phenotype studieswere supported primarily by the NationalInstitutes of Health, through awards R37DK35124, R37 EB00415, R01 EY13574, R01HL59198, R01 HL73856, and P30 DK72517.

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3640-3645




Mam

malianaq

uaporins

:diverse

phy

siological

roles

andpotentialc

linical

significa

nce

Further reading, resources and contacts

General information on aquaporins:

http://www.ucsf.edu/verklabhttp://www.ks.uiuc.edu/Research/aquaporins/http://en.wikipedia.org/wiki/Aquaporin

Aquaporin meetings:

http://www.congre.co.jp/aqp2007/http://www.scanbalt.org/sw14185.asp

Nephrogenic Diabetes Insipidus Foundation:

http://www.ndif.org

Features associated with this article

FiguresFigure 1. Aquaporin functions in mammalian physiology.Figure 2. Impaired urinary concentrating function in aquaporin deficiency.Figure 3. Aquaporin 4 deficiency slows brain water accumulation in cytotoxic oedema, and brain water

elimination in vasogenic oedema.Figure 4. Impairment in tumour growth and endothelial cell migration in aquaporin 1 deficiency.Figure 5. Aquaporin 3 deficiency reduces skin hydration and prevents skin tumour formation.

Citation details for this article

A. S. Verkman (2008) Mammalian aquaporins: diverse physiological roles and potential clinical significance.Expert Rev. Mol. Med. Vol. 10, e13, May 2008, doi:10.1017/S1462399408000690




Mam

malianaq

uaporins

:diverse

phy

siological

roles

andpotentialc

linical

significa

nce

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Mammalian aquaporins: diverse physiological roles and ... · Mammalian aquaporins: diverse physiological roles and potential clinical signiﬁcance A. S. Verkman Aquaporins have multiple