Aquaporin-Mediated Fluid Regulation in the Inner Ear...The whole set-up of the middle and the inner ear from the general anatomy to terminally differentiated sensory hair cells is

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Cellular and Molecular Neurobiology [cemn] pp820-cemn-463429 April 22, 2003 22:44 Style file version Oct 23, 2000

Cellular and Molecular Neurobiology, Vol. 23, No. 3, June 2003 ( C© 2003)

Review

Aquaporin-Mediated Fluid Regulation in the Inner Ear

Eric Beitz,1,3 Hans-Peter Zenner,2 and Joachim E. Schultz1

Received September 30, 2002; accepted October 10, 2002

SUMMARY

1. The sensory functions of the inner ear (hearing and balance) critically depend onthe precise regulation of two fluid compartments of highly desparate ion composition, i.e.,the endolymph and the perilymph.

2. The parameters volume, ion composition, and pH need to be held at homeostasisirrespective of the hydration status of the total organism.

3. Specific cellular water channels, aquaporins, have been shown to be essential for thefluid regulation of several organs, e.g., kidney, lung, and brain.

4. Because of functional similarities of water regulation in the kidney and inner earthis review initially summarizes some aquaporin functions in the kidney and then focuseson 6 out of 11 mammalian aquaporins that are present in the inner ear (AQP1-6).

5. Their potential role in the inner ear fluid control will be discussed on the basis ofthe respective expression patterns and individual pore properties.

6. Further, a working model is presented of how the endolymphatic sac may contributeto inner ear fluid regulation.

KEY WORDS: inner ear; endolymph; fluid regulation; aquaporin; vasopressin.

INNER EAR FLUIDS, SOUND PERCEPTION, AND DISEASE

The inner ear is a tiny, yet anatomically and functionally highly sophisticated organfor sensing sound and gravity. The growth and development of the inner ear usuallyis completed at birth or shortly thereafter to enable young animals to communicatevocally with the mother animal and to stay and walk. Its size is principally delimitedby the length of the sound waves, which it can register and translate into a neuronalsignal. Therefore, much to the dismay of biochemists, its size does not vary very muchfrom the smallest to the largest animal species. For example, in man the length of

1 Department of Pharmaceutical Biochemistry, School of Pharmacy, University of Tubingen, 72076Tubingen, Germany.

2 Department of Otorhinolaryngology, University of Tubingen, 72076 Tubingen, Germany.3 To whom correspondence should be addressed at Department of Pharmaceutical Biochemistry, School

of Pharmacy, Morgenstelle 8, 72076 Tubingen, Germany; e-mail: [email protected].

315

0272-4340/03/0600-0315/0 C© 2003 Plenum Publishing Corporation

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316 Beitz, Zenner, and Schultz

the incapsuled cochlear spiral is about 3.5 cm. The sensory properties of the innerear are truly astounding: sustained sound perception in the kilo-Hertz range, reliablediscrimination of sound frequencies, which are only 0.2% apart, and perception ofa wide dynamic range of signals with amplitude differences of more than six ordersof magnitude. Without the inner ear’s degree of precision the analysis of complexauditory signals, such as speech, would be impossible. Further, irrespective of thephysical situation, the posture is constantly monitored and the respective musclesare constantly fine-tuned for balance and position control purposes.

The whole set-up of the middle and the inner ear from the general anatomy toterminally differentiated sensory hair cells is tailored to comply with these demands.First, an impedance matching of the sound signal by the ossicular chain takes placein the middle ear. The sound signals are then mechanically transmitted through vi-brations of the oval window membrane into the entirely fluid-filled inner ear. Thebasic elements for the conversion of mechanical signals into electrical signals areionic transduction channels, which are located in the stereocilia of sensory hair cells,allowing potassium ions to enter the cell. These channels are directly gated by the de-flection of the stereocilia possibly through so-called tip-links and thus open and closemechanically in response to the auditory signal. The resulting potassium influx givesrise to depolarization of the sensory hair cells, which are functionally linked at theirbasal sides to the afferent neurons of the cochlear nerve probably via glutaminergicsynapses (Dallos, 1996; Slepecky, 1996).

From the above it is obvious that the inner ear fluids play important roles in theprocess of sound perception. Firstly, fluid is the basic medium in which the mechanicalsound signal travels hydrodynamically. This requires a tight volume control in orderto establish and maintain correct pressure and volume within the system. The delicatebalance between stiffness and mass that may be influenced by overpressure or volumeincrease has to be carefully regulated. Secondly, as a specialized fluid compartment,the endolymph serves as an extracellular reservoir and buffer of K+ ions that areneeded for signal transduction. In fact the inner ear as a labyrinthine tubular systemcontains two distinctly separated fluid compartments of defined, yet differing ioniccomposition. In the cochlea, the perilymph and the endolymph network, are dividedonly by the two cell layer thick Reissner’s membrane (Fig. 1(B)). The perilymphis considered to be a filtrate of the cerebrospinal fluid or blood and consequentlycontains a high Na+ concentration (about 145 mM), low K+ (about 5 mM), and 1–2 mM Ca2+. The endolymph in contrast is unique as an extracellular fluid in that itcontains only 1 mM Na+, yet 155 mM K+ and Ca2+ in the nanomolar range. Thus itvery much mirrors the cytosolic ion composition (Konishi et al., 1984; Salt et al., 1989;Sterkers et al., 1987). It is contained in a tube that runs throughout the gyrate cochlea(scala media), the vestibulum and which ends in a reservoir-like endolymphatic sac(Fig. 1). Between the cochlear endolymph and the endolymphatic sac, a rather staticion gradient exists. Actually, in the endolymphatic sac the fluid composition is moreakin to an extracellular fluid (about 140 mM Na+, 15 mM K+, and 0.5 mM Ca2+;Mori et al., 1987; Ninoyu and Meyer zum Gottesberge, 1986). For the production,absorption, and regulation of the endolymph volume and composition predominantlytwo anatomically distant sites are discussed. The stria vascularis in the scala media isthought to be the tissue, which extrudes K+ into the endolymph. Probably the stria

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Inner Ear Aquaporins 317

Fig. 1. Schematic drawings of the endolymph compartment of the innerear with indication of aquaporin expression sites. (A): The endolymphcompartment is shown as a solid black line running through the cochleaand the vestibulum ending up in the endolymphatic sac. (B): Shownhere is a section through one turn of the cochlea. The separation ofthe endolymph (scala media, SM) from the perilymph compartments(scala tympani and vestibuli, ST and SV) through the basiliar (BM)and Reissner’s membrane (RM) is clearly visible. The cell types, whichexpress aquaporins are labelled. The inset shows a microscopic pictureof a guinea pig cochlea from the same view. Abbreviations are BM –basiliar membrane, C – Claudius cells, D – Deiters cells, ESC – externalsulcus cells, H – Hensen’s cells, IHC – inner hair cells, IP – inner pillarcells, ISC – inner sulcus cells, OHC – outer hair cells, OP – outer pillarcells, RM – Reissner’s membrane, SL – spiral ligament, SM – scala media,SP – spiral prominence, ST – scala tympani, StV – stria vascularis, SV –scala vestibuli, TM – tectorial membrane.

vascularis also is involved in water transport. The function of the endolymphatic sac issomewhat more controversial. In the guinea pig, obliteration of the endolymphaticsac causes endolymphatic hydrops, i.e., endolymph volume increase, suggesting arole in endolymph transfer (Kimura and Schuknecht et al., 1965). Yet this would

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require a directional flow of fluid from the stria vascularis to the endolymphaticsac. So far, there was no flux detectable beyond a passive bidirectional diffusionof injected marker molecules into the scala media (Salt and Thalmann, 1988). Thesensory cells of the inner ear are exposed to the endolymph at their apical sideswhereas the basolateral parts are bathed in a perilymph environment (Fig. 1(B)).This contributes to a high electrochemical gradient of about 150 mV over the apicalend of the hair cells, which drives the K+ ions into the hair cells when the transductionchannels open. This empowers the cell to detect tiny signals with high precision.Furthermore, it allows the system to quickly reset because of a rapid K+ efflux viathe basolateral membrane. K+ ions are then discussed to be swiftly recycled into theendolymph compartment through the marginal cells of the stria vascularis involvinga gap junction network (Kikuchi et al., 2000). Hereditary mutations in gap junctionproteins (connexin 26) unavoidably result in deafness.

The close relationship between endolymph maintenance and sensory functionsis stressed by the finding that disturbances in the endolymph regulation causes bal-ance and hearing problems (Rybak, 1994). Such incidences may be precipitated byapplication of the loop diuretics furosemide or ethacrynic acid. The wanted thera-peutic effect of these drugs is due to the inhibition of the Na+-K+-2Cl− cotransporterlocalized in the ascending limb of the loop of Henle, which results in solute and waterdiuresis. In the inner ear, the presence of this very carrier as part of the potassiumrecycling machinery in the marginal cells of the stria vascularis is the basis for theototoxic effects of loop diuretics. An inhibition that may occur even after a sin-gle dose of furosemide, leads to an edema within the stria vascularis and probablyinterferes with the K+ and water transport into the endolymph compartment. Ob-viously, the inner ear shares a number of functional mechanisms for water and ionregulation with the kidney, which are the molecular basis for unwanted side effectsof diuretics (Humes, 1999). Notable are the oto- and nephrotoxic aminoglycosideantibiotics (gentamycin, kanamycin, etc.) and platinum-containing chemotherapeu-tics (cisplatin) although their effects on the fluid control in the stria vascularis aresecondary. As much as the water regulating system of the kidney and the hearingaparatus are concerned, this will be described in more detail later.

A well-documented clinical manifestation of endolymph dysregulation isMeniere’s disease (Hamann and Arnold, 1999). Here, either the underlying over-production or reduced absorption of the endolymph may have severe pathophys-iological consequences, such as endolymphatic hydrops or a permeability increaseof tight junctions resulting in vertigo attacks, hearing loss, tinnitus, and a sensationof fullness in the inner ear (Zenner et al., 1994). The mechanisms that lead to en-dolymphatic hydrops are not known. The fact that quite often only one ear is affectedand the presence of macrophages in the endolymphatic sac led to the hypothesis ofan allergic autoimmune reaction. Other speculations are a neural or viral etiology.Our own finding that systemically applied vasopressin, the regulator of water perme-ability of the kidney collecting duct, evokes endolymphatic hydrops in guinea pigs(Kumagami et al., 1998) will be discussed later in conjunction with a model for thefluid regulation of the endolymphatic sac. Taken together, water regulation of theinner ear fluid compartments is of utmost importance for functional stability of thehearing and balancing system.

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CELLULAR WATER CHANNELS—AQUAPORINS

The presence of pore-like molecules dedicated to transmenbraneous waterfluxes was suggested by the ingenius regulation of the water permeability in thecollecting duct, by the unexpectedly low Arrhenius activation energy observed forthe rapid water flux across certain cell membranes (<5 kcal/mol vs. >10 kcal/molin a plain lipid bilayer), and the inhibitory effect of Hg2+ ions on water fluxes. Thediscovery of cellular water channels, the proteins were named aquaporins (AQP),provided the molecular basis and consequently dramatically enhanced the field (Agreet al., 2001). Aquaporins are integral membrane proteins with a high specificity forthe transport of water or small uncharged solutes, such as glycerol or urea acrosscell membranes. Importantly, the permeation strictly follows the existing osmotic orchemical gradient as the driving force. Structurally, all aquaporins share the sameoverall topology with six membrane spanning helices and two short pore heliceswhich protrude into the membrane and form a pseudo-seventh transmembrane do-main (Fig. 2; Kozono et al., 2002). The latter guides passing molecules by hydrogenbonds through the pore-forming center of the protein. Independent of the monomericoperation, the water channels form a tetrameric unit in the membrane. In humans,11 isoforms (AQP0 to AQP10) exist. They have marked differences in their waterand solute permeabilities and in the mode of regulation as well as in their tissuedistribution and subcellular localization.

So far, the best examined mammalian water channels are those with a pro-nounced function in kidney physiology, i.e., aquaporins AQP1 to AQP4 (Nielsen

Fig. 2. Structure of an aquaporin monomer as seenfrom the intracellular space. The membrane spanninghelices are labeled consecutively with numerals (1–6)and the two short pore helices are marked with aster-isks. The site of the actual water pore is indicated by theblack circle in the center of the monomer. The diamondlabels the position of the fourfold axis of the tetramericaquaporin.

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et al., 2002). They significantly contribute to the regulation of urine volume. AQP1 islocated in the descending part of the loop of Henle, and facilitates the reabsorptionof 60–80% of the primary urine volume. This is a hormone-independent process,which is driven solely by the hypotonicity of the primary urine with respect to thehigh osmolarity in the surrounding tissue (up to 1200 mOsm/kg). The reabsorptionof another 10–20% in the collecting duct is fine-tuned by the antidiuretic hormonevasopressin (ADH). This involves the activation of the vasopressin V2-receptor, anincrease in intracellular cAMP and, consequently, the phosphorylation of AQP2 atSer256, which is stored in cytosolic vesicles. The phosphorylation event triggers byan as yet unknown mechanism the directional translocation of these vesicles to theapical plasma membrane. There, AQP2 is integrated into the plasmalemma and in-creases the water permeability. The entering water leave the cell basolaterally viaAQP3 and AQP4 into the surrounding tissue. This upregulation of the apical waterpermeability is fully reversible, i.e., dephosphorylation of membrane-bound AQP2triggers its removal via targeted endocytosis and probably replenishes the cytosolicvesicle pool for another round of shuttling. Disturbances in this network (lack ofvasopressin, mutations in the V2-receptor or in the AQP2 protein) can cause dia-betes insipidus. Even temporary challenges, such as the downregulation of AQP2expression during a lithium treatment may lead to polyuria.

The physiological role of aquaporins in the kidney and other fluid controllingtissues seems rather straight forward. However, puzzling novel observations indicateadditional functions for aquaporins apart from fluid regulation. For example, AQP0may also be important for the structural maintenance of cell to cell interactions offiber cells in the eye lens (Fotiadis et al., 2000). Further, in acid secreting intercalatedcells of the kidney collecting duct AQP6 represents an intracellular pore protein,which may operate as an ion channel (Yasui et al., 1999). A number of observationshave always indicated that distinct similarities in the physiological processes exist,which are responsible for the fluid regulation in the kidney and in the inner ear. Theadvance in biomolecular technologies in recent decades has enabled us to investigatethe molecular basis for these obvious similarities, as outlined later.

DISTRIBUTION OF AQUAPORIN ISOFORMS IN THE INNER EAR

After the first aquaporin proteins had been characterized there was little doubtthey would also be involved in the fluid regulation of the inner ear. This has beeninvestigated by several groups using the reverse transcriptase-polymerase chain re-action (RT-PCR) and immunological techniques. Despite the immense difficultiesin the dissection of clearly defined tissue samples and sections from the inner earbecause of the tiny size and a highly complex anatomy enclosed in a hard boney shellconsiderable progress has been made. Below we review what is known about theexpression and localization of various aquaporin isoforms in anatomically and func-tionally defined areas of the inner ear (Table I). Additionally, we will summarize dataon signal transduction systems which may directly contribute to regulatory inputs. InFig. 1 the standard anatomical outlay of the inner ear is depicted and the areas areindicated, which were prepared by microdissection (endolymphatic sac, vestibulum,

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Tabl

eI.

Aqu

apor

inE

xpre

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nP

atte

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+/+

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tibu

lum

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epit

helia

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ls,

Stan

kovi

cet

al.,

1995

,Tak

umi

(RT-

PC

R,I

F)

linin

gce

llsof

the

spir

allig

amen

t,et

al.,

1998

,Bei

tzet

al.,

1999

,en

doly

mph

atic

sac

fibro

cyte

sL

iand

Ver

kman

,200

1.A

QP

2+/+

End

olym

phat

icsa

cal

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2-re

cept

or,v

esic

le(R

T-P

CR

,IF

)tr

ansp

ortp

rote

ins.

Kum

agam

iet

al.,

1998

,Bei

tzet

al.,

1999

,M

erve

set

al.,

2000

,M

hatr

eet

al.,

2002

.A

QP

3+/−

End

olym

phat

icsa

cB

eitz

etal

.,19

99(R

T-P

CR

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QP

4+/+

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tibu

lum

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ngce

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the

M1

alm

osta

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M1,

M23

(RT-

PC

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tu,

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lym

phat

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cco

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1:M

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cells

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RN

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rM

1in

inne

rsu

lcus

cells

endo

lym

phat

icsa

c.Ta

kum

iet

al.,

1998

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tzet

al.,

1999

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ndV

erkm

an,2

001.

AQ

P5

+/−

Coc

hlea

Ext

erna

lsul

cus

cells

,ap

ical

turn

son

ly.

(RT-

PC

R,I

F)

spir

alpr

omin

ence

Bei

tzet

al.,

1999

,M

hatr

eet

al.,

1999

.A

QP

6+/−

End

olym

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cB

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,unp

ublis

hed.

(RT-

PC

R)

Not

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heda

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cted

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mer

ase

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acti

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ce–

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este

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otor

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itu

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idiz

atio

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and

the

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icul

arce

llty

pes

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sion

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show

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osp

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ants

(M1

and

M23

);th

era

tios

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don

Wes

tern

blot

ting

are

give

n.

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organ of Corti, stria vascularis, Reissner’s membrane) or sections through the cochleaand vestibulum and investigated by RT-PCR or immunofluorescence.

AQP1 was present in most areas of the inner ear at the mRNA and protein level(Beitz et al., 1999; Huang et al., 2002; Li and Verkman, 2001; Stankovic et al., 1995;Takumi et al., 1998). Immunofluorescence was predominantly detected in fibrocytesin close proximity to the bone capsule, in the spiral ligament, in cells below thebasilar membrane and in mesothelial cells lining the scala tympani in the cochlea.Notably, AQP1 was not found in epithelial cells nor in cells involved in the endolymphformation. Thus, a decisive involvement of AQP1 water permeability in endolymphor perilymph regulation is rather unlikely. It may have an as yet undefined role inthe maintenance of the bone and the basilar membrane.

In the 4-day-old rat, AQP2 mRNA was present exclusively in the endolymphaticsac (Beitz et al., 1999). In addition, AQP3 and AQP4 mRNAs were detected in thistissue. Immunofluorescence confirmed the expression of AQP2 in neonatal rats atDay 18 after gestation (Merves et al., 2000). Recent evidence indicates that AQP2may also be expressed in the mammalian cochlea in particular structures borderingthe endolymph (Mhatre et al., 2002). The findings for the endolymphatic sac wereparticularly striking because the coexpression of the three AQP isoforms AQP2,AQP3, and AQP4 so far was only known to occur in the principal cells, which arethe main cell type lining the collecting duct in the kidney. There, AQP2 trafficking isregulated by vasopressin-dependent vesicle shuttling as mentioned earlier. Not sur-prisingly then, the presence of mRNAs for the vasopressin receptor and the majorvesicle transport proteins VAMP2, syntaxin-4, and rab3a in the endolymphatic sacof rats was demonstrated by RT-PCR (Beitz et al., 1999; Kumagami et al., 1998). Thelining of the collecting duct of the kidney is interspersed with acid secreting cells,so-called intercalated cells, which express the intracellular AQP6 (type A interca-lated cells; Yasui et al., 1999). In the inner ear, protons need to be actively secretedinto the endolymph in the cochlea in order to maintain a pH of 7.4 because of thestrongly positve potential of the endolymph (+80 mV) a constant passive efflux ofprotons occurs from this compartment. This problem is somewhat less pressing inthe endolymphatic sac, where the pH of the endolymph is around 6.6 (Karet et al.,1999). Recently, by RT-PCR with mRNA from the endolymphatic sac of 9-day-oldrats we obtained a weak signal, which turned out to be the expected AQP6 product(unpublished data). How the intracellular AQP6 may be involved in pH regulationof the endolymph certainly is a pressing question. The presence of AQP2 and 6 inthe endolymphatic sac extends the similarities with the kidney collecting duct frommorphology to the molecular level.

AQP4, the aquaporin with the highest water permeability, is also expressed in thesensory parts of the inner ear (Beitz et al., 1999; Huang et al., 2002; Li and Verkman,2001; Takumi et al., 1998). It is not identified in the hair cells themselves, i.e., inner,outer, and vestibular hair cells, but in supporting cells within the cochlea (Hensen’scells, Claudius cells, inner sulcus cells) and the vestibular end organs (Fig. 1). Inter-estingly, there is a significant difference in the ratio of two splice variants of AQP4(M1 and M23) which are present in the vestibulum and the cochlea (Takumi et al.,1998). The M23 splice variant lacks the first 22 amino acids and appears 2–3 kDasmaller on Western blots than the full length M1 AQP4 isoform. In the cochlea the

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AQP4 M23 isoform is by far predominant whereas in the vestibulum both variantsare expressed in reasonable amounts (M1:M23 = 1:3; Takumi et al., 1998). Poten-tial functional consequences of these differences in the expression of AQP4 splicevariants are enigmatic at present and have to await the elucidation of functionaldifferences between M1 and M23 AQP4 splice variants.

A highly regionalized expression pattern is also observed for AQP5 (Beitz et al.,1999). RT-PCR established the presence of AQP5 mRNA in the organ of Corti and inReissner’s membrane. By immunofluorescence AQP5 was localized in external sulcuscells at the spiral prominence of the scala media. Curiously, only the upper turns ofthe cochlea appear to express AQP5 (Mhatre et al., 1999). Because of the spatialresolution of an auditory stimulus along the length of the cochlea low frequenciesstimulate hair cells in the apical turns whereas high frequencies stimulate close tothe base. Although highly speculative one may envision that AQP5 may play an asyet undefined role in low frequency perception which is considered the “primitivetrait” in vertebrate hearing.

AQUAPORIN KNOCKOUTS AND HEARING IMPAIRMENT

Mouse aquaporin knockout models are available for AQP1, AQP3, AQP4, andAQP5 (Verkman, 2000). Of those AQP4 turned out to the only water pore, which isessential for normal for hearing (Li and Verkman, 2001). The fact that the deafness ofAQP4 knockout mice was overlooked for 4 years indicates that only very subtle be-havioral differences exist in rodents with impaired hearing. A morphological analysisof AQP4 knockout mice did not reveal any abnormalities of the inner ear. In fact, thisanimal has only a mild urinary concentration defect due to impaired water reabsorp-tion in the kidney collecting duct (Li and Verkman, 2001). This raises the questionabout the basic function of AQP4. The AQP4 expression pattern and localizationin neuronal tissues may provide a hint. Usually, AQP4 is localized to the basolat-eral membrane of various types of supporting cells in the brain (astroglial cells), theeye (Muller cells) and, as already mentioned earlier, the inner ear (Hensen’s andClaudius cells). These cells accompany neurons, bipolar cells or sensory hair cells,respectively, which carry considerable K+-ion fluxes during excitation. The highlypermeable AQP4 water channel would functionally fit into such a scenario when onetakes the transient osmotic imbalance in the surrounding interstitium into accountwhich may occur locally because of the charge movements. A concurrent water fluxthrough the AQP4 pore of the supporting cells may at least partially offset local swellsin K+ ion concentrations. Currently, this is considered as a general paradigm in thephysiology of excitable tissues (Li and Verkman, 2001). The deaf AQP4 knockoutmice strikingly demonstrate that the AQP4 water permeability of the supportingcells is functionally almost as important for excitation as is the ion flux through K+

channels itself.AQP1, AQP3, and AQP5 knockout mice appear to have normal hearing as

deduced from auditory brain stem response measurements questioning the proposedinvolvement of AQP5 in low frequency hearing (Li and Verkman, 2001; Mhatre et al.,1999). But the immuno localization of AQP5 was done in rat cochleae leaving thepossibility of interspecies differences in this case. AQP6 knockout mice are not yet

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available. In addition to AQP1 knockout mice there are rare human null mutants.These individuals exhibit a surprisingly mild phenotype (Preston et al., 1994). Onlya slight urinary concentration defect was noted after water deprivation (King et al.,2001); hearing deficits were not reported.

The lack of AQP2 knockout mice is due to a simple reason: it is lethal and anAQP2 knock-in mouse model of autosomal recessive nephrogenic diabetes insipidusdoes not survive beyond the first week after birth (Verkman, 2000). Clearly, this modelis not suitable for hearing analysis because of the later onset of hearing in mice, whichis around postnatal Day 12. On the other hand, there are humans with mutations inthe AQP2 gene, which lead to nephrogenic diabetes insipidus by misfolding or anintracellular routing failure of the water channel (Deen et al., 2000). A single reportdescribes two related patients with nephrogenic diabetes insipidus who also sufferfrom Meniere’s disease (Comacchio et al., 1992). To conclude from this single casethat a nonfunctional AQP2 leads to hearing problems would certainly be premature.Screening for AQP2 mutations in individuals with Meniere’s disease has revealedno sequence alterations (Mhatre et al., 2002). Below, we propose a model for theregulation of the endolymph, which is congruent in many respects with the regulatoryfeatures established for the kidney collecting duct.

MODEL OF THE FLUID REGULATION IN THE ENDOLYMPHATIC SAC

Four experimental observations form the basis for our water regulation model ofthe endolymphatic sac (Fig. 3). (A) The endolymph is hypertonic to the perilymph andthe serum (305 mOsm/kg vs. 285 mOsm/kg, Konishi et al., 1984). (B) AQP2 is presentin the endolymphatic sac and key regulators of AQP2 are identical to the kidneycollecting duct (Beitz et al., 1999; Kumagami et al., 1998). (C) The endolymphaticsac shows a very high endocytotic activity in the basal state and vasopressin inhibitsendocytosis in vitro (Kumagami et al., 1998). (D) Vasopressin application leads toendolymphatic hydrops in vivo (Kumagami et al., 1998; Takeda et al. 2000).

The sole driving force for water movement over epithelia is osmosis. This means,that the hypertonicity of the endolymph facilitates a unidirectional water flow intothe endolymph compartment. Because of the rather small differences in osmolalitybetween the endolymph and perilymph (10–20 mOsm/kg) the rate of the nonfacili-tated water diffusion is very slow unless augmented by the presence of aquaporins.So far, AQP2 is the only established water pore, which has been unequivocally local-ized to the endolymph lining epithelium. Nevertheless, the subcellular localization inthe endolymphatic sac is to be determined. The presence of the major componentsof the kidney AQP2 regulatory equipment strongly suggests that a similar AQP2shuttling process will also operate in the endolymphatic sac. Vasopressin stimulationat the basolateral site should therefore increase the number of AQP2 water channelsin the apical membrane via a cAMP-triggered phosphorylation thus greatly enhanc-ing the water influx into the endolymph compartment (see Fig. 3(A) for a model).The physiological function of the vasopressin effect might be an effective short-termprotection of the endolymph volume during periods of hypovolemia. However, it isknown when vasopressin levels are pathologically increased over a prolonged period

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Fig. 3. Model of the vasopressin-dependent water regula-tion in the endolymphatic sac. Shown is a cell under basaland stimulated conditions. (A): In the presence of vasopressinAQP2 water channels are inserted into the apical membraneallowing osmotic water flux into the endolymph compartment.(B): Without vasopressin pinocytosis of endolymph fluid dom-inates whereas AQP2 water channels are stored intracellularlyleading to a reduction of the endolymph volume. See text forfurther explanations.

of time, this will ultimately result in endolymphatic hydrops in agreement with theabove proposal (Kumagami et al., 1998). In diabetes insipidus patients the vaso-pressin regulatory system is inoperative (because of defects in vasopressin biosyn-thesis or release, the vasopressin receptor or the AQP2 protein). According to theproposed model those patients cannot increase the water influx into the endolymphcompartment and, thus, are usually essentially protected against the development ofa hydrops.

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Fig. 3. (Continued.)

How can the endolymph volume be reduced? The only possibility to transportwater against an osmotic gradient out of the endolymph compartment is by pinocyto-sis of the cell layer lining the fluid compartment. Two ways are possible by which thecells could dispose of the internalized hyperosmolar fluid load: by transpinocytosisor generation of localized, transbasolateral water and ion efflux. Pinocytosis wouldinvolve a directional transport of the ingested vesicles from the apical to the baso-lateral site and subsequent disposal into the microvasculature. The alternative is theion secretion over the basolateral membrane and a concurrent water flux (throughAQP3 and AQP4). Which of the two possibilities actually has been realized is unclearat present and must wait until appropriately designed experiments are feasible—anarduous task if one considers the known difficulties in handling the endolymphaticsac or primary cultures of it. The excess fluid in the interstitium can finally enterthe network of surrounding fenestrated capillaries. It has been demonstrated that in

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the basal state, i.e., without hormonal stimulation, endocytosis is rapid. Vasopressininhibits pinocytosis from the endolymphatic sac and this process may potentiallycontribute passively to an increase of the endolymph volume in addition to thevasopressin-stimulated water transport through AQP2 (Kumagami et al., 1998). Theunderlying mechanism for the inhibition of endocytosis in the endolymphatic sac byvasopressin is unknown so far.

In summary, this “leaky boat” model predicts a local endolymph turn-over byregulated osmotic water influx into the endolymph through AQP2 (the leaks in theboat) and the simultaneous removal of endolymph fluid by isosmotic pinocytosis(scooping out the water bucket by bucket). The overall process is hormonally bal-anced via vasopressin with respect to the systemic hydration status. Additional localregulatory mechanisms may exist. So far, mainly two further observations point in thisdirection. First, it was reported that the endolymphatic sac secretes a hormone, “sac-cin,” with diuretic activity (Qvortrup et al., 1996). Second, the seven transmembrane,calcium sensing receptor (CaSR), is expressed in the endolymphatic sac indicating thepotential of a cellular responsiveness towards changing Ca2+ concentrations (Beitzet al., 1999). This receptor has rather prominent roles in the parathyroid and thekidney in the calcium homeostasis by triggering parathyroid hormone secretion andrenal calcium reabsorbation (Hendy et al., 2000).

The proposed model could account for a further important feature in endolymphregulation: it would remove “used” fluid and replace it with AQP2-“filtered” liquidand thus clean the endolymph from accumulating metabolites, cellular debris, andother unwanted material, which may accumulate therein.

CONCLUSION

Aquaporins have proven to be absolutely essential for the sensory functions ofthe inner ear (AQP4) or are very good candidates for an involvement in the regulationof the endolymph volume (AQP2) and pH control (AQP6). The observation thata sustained stimulation of the vasopressin-AQP2 system leads to endolymphatichydrops in guinea pigs is intriguing and might be of use for a rational therapeuticapproach of Meniere’s disease.

REFERENCES

Agre, P., Borgnia, M. J., Yasui, M., Neely, J. D., Carbrey, J., Kozono, D., Beitz, E., Hoffert, J., Leitch, V.,and King, L. S. (2001). Discovery of the aquaporins and their impact on basic and clinical physiology.Curr. Topics Membranes 51:1–38.

Beitz, E., Kumagami, H., Krippeit-Drews, P., Ruppersberg, J. P., and Schultz J. E. (1999). Expressionpattern of aquaporin water channels in the inner ear of the rat. The molecular basis for a waterregulation system in the endolymphatic sac. Hear. Res. 132:76–84.

Comacchio, F., Boggian, O., Poletto, E., Beghi, A., Martini, A., and Rampazzo, A. (1992). Meniere’sdisease in congenital nephrogenic diabetes insipidus: Report of two twins. Am. J. Otol. 13:477–481.

Dallos, P. (1996). Overview: Cochlear neurobiology. In Dallos, P., Popper, A. N., and Fay, R. R. (eds.), TheCochlea, Springer, New York, pp. 1–43.

Deen, P. M., van Balkom B. W., and Kamsteeg, E. J. (2000). Routing of the aquaporin-2 water channel inhealth and disease. Eur. J. Cell Biol. 79:523–530.

Fotiadis, D., Hasler, L., Muller, D. J., Stahlberg, H., Kistler, J., and Engel, A. (2000). Surface tongue-and-groove contours on lens MIP facilitate cell-to-cell adherence. J. Mol. Biol. 300:779–789.

P1: FLT



Hamann, K.-F., and Arnold, W. (1999). Meniere’s disease. Adv. Otorhinolaryngol. 55:137–168.Hendy, G. N., D’Souza-Li, L., Yang, B., Canaff, L., and Cole, D. E. (2000). Mutations of the calcium-sensing

receptor (CASR) in familial hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, andautosomal dominant hypocalcemia. Hum. Mutat. 16:281–296.

Huang, D., Chen, P., Chen, S., Nagura, M., Lim, D. J., and Lin, X. (2002). Expression patterns of aquaporinsin the inner ear: Evidence for concerted actions of multiple types of aquaporins to facilitate watertransport in the cochlea. Hear. Res. 165:85–95.

Humes, H. D. (1999). Insights into ototoxicity. Analogies to nephrotoxicity. Ann. N.Y. Acad. Sci. 884:15–18.Karet, F. E., Finberg, K. E., Nelson, R. D., Nayir, A., Mocan, H., Sanjad, S. A., Rodriguez-Soriano, J.,

Santos, F., Cremers, C. W., Di Pietro, A., Hoffbrand, B. I., Winiarski, J., Bakkaloglu, A., Ozen, S.,Dusunsel, R., Goodyer, P., Hulton, S. A., Wu, D. K., Skvorak, A. B., Morton, C. C., Cunningham,M. J., Jha, V., and Lifton, R. P. (1999). Mutations in the gene encoding B1 subunit of H+-ATPasecause renal tubular acidosis with sensorineural deafness. Nat. Genet. 21:84–90.

Kikuchi, T., Adams, J. C., Miyabe, Y., So, E., and Kobayashi, T. (2000). Potassium ion recycling pathwayvia gap junction systems in the mammalian cochlea and its interruption in hereditary nonsyndromicdeafness. Med. Electron Microsc. 33:51–56.

Kimura R. S., and Schuknecht H. F. (1965). Membranous hydrops in the inner ear of the guinea pig afterobliteration of the endolymphatic sac. Pract. Otorhinolaryngol. 27:343–354.

King, L. S., Choi, M., Fernandez, P. C., Cartron, J.-P., and Agre, P. (2001). Defective urinary concentratingability due to a complete deficiency of aquaporin-1. N. Engl. J. Med. 345:175–179.

Konishi, T., Hamrick, P. E., and Mori, H. (1984). Water permeability of the endolymph-perilymph barrierin the guinea pig cochlea. Hear. Res. 15:51–58.

Kozono, D., Yasui, M., King, L. S., and Agre, P. (2002). Aquaporin water channels: Atomic structuremolecular dynamics meet clinical medicine. J. Clin. Invest. 109:1395–1399.

Kumagami, H., Loewenheim, H., Beitz, E., Wild, K., Schwartz, H., Yamashita, K., Schultz, J. E., Paysan, J.,Zenner, H. P., and Ruppersberg, J. P. (1998). The effect of anti-diuretic hormone on the endolymphaticsac of the inner ear. Pflugers Arch. 436:970–975.

Li, J., and Verkman, A. S. (2001). Impaired hearing in mice lacking aquaporin-4 water channels. J. Biol.Chem. 276:31233–31237.

Merves, M., Bobbitt, B., Parker, K., Kishore, B. K., and Choo, D. (2000). Developmental expression ofaquaporin 2 in the mouse inner ear. Laryngoscope 110:1925–1930.

Mhatre, A. N., Steinbach, S., Hribar, K., Hoque, A. T., and Lalwani, A. K. (1999). Identification of aqua-porin 5 (AQP5) within the cochlea: cDNA cloning and in situ localization. Biochem. Biophys. Res.Commun. 264:157–162.

Mhatre, A., Jero, J., Chiappini, I., Bolasco, G., Barbara, M., and Lalwani, A. (2002). Aquaporin-2 ex-pression in the mammalian cochlea and investigation of its role in Meniere’s disease. Hear. Res.170:59–69.

Mori, N., Ninoyu, O., and Morgenstern, C. (1987). Cation transport in the ampulla of the semicircularcanal and in the endolymphatic sac. Arch. Otorhinolaryngol. 244:61–65.

Nielsen, S., Frokiaer, J., Marples, D., Kwon, T. H., Agre, P., and Knepper, M. A. (2002). Aquaporins in thekidney: From molecules to medicine. Physiol. Rev. 82:205–244.

Ninoyu, O., and Meyer zum Gottesberge, A. M. (1986). Ca++ activity in the endolymphatic space. Arch.Otorhinolaryngol. 243:141–142.

Preston, G. M., Smith, B. L., Zeidel, M. L., Moulds, J. J., and Agre, P. (1994). Mutations in aquaporin-1 inphenotypically normal humans without functional CHIP water channels. Science 265:1585–1587.

Qvortrup, K., Rostgaard, J., and Holstein-Rathlou, N. H. (1996). The inner ear produces a natriuretichormone. Am. J. Physiol. 270:F1073–F1077.

Rybak, L. P. (1994). Ototoxicity of loop diuretics. Otolaryngol. Clin. North Am. 26:829–844.Salt, A. N., Inamura, N., Thalmann, R., and Vora, A. (1989). Calcium gradients in inner ear endolymph.

Am. J. Otolaryngol. 10:371–375.Salt, A. N., and Thalmann, R. (1988). Interpretation of endolymph flow results: A comment on ‘Lon-

gitudinal flow of endolymph measured by distribution of tetraethylammonium and choline in scalamedia.’ Hear. Res. 33:279–284.

Slepecky, N. B. (1996). Structure of the mammalian cochlea. In Dallos, P., Popper, A. N., and Fay, R. R.(eds.), The Cochlea, Springer, New York, pp. 44–129.

Stankovic, K. M., Adams, J. C., and Brown, D. (1995). Immunolocalization of aquaporin CHIP in theguinea pig inner ear. Am. J. Physiol. 269:C1450–C1456.

Sterkers, O., Ferrary, E., Saumon, G., and Amiel, C. (1987). Na and nonelectrolyte entry into inner earfluids of the rat. Am. J. Physiol. 253:F50–F58.

Takeda, T., Takeda, S., Kitano, H., Okada, T., and Kakigi, A. (2000). Endoloymphatic hydrops inducedby chronic administration of vasopressin. Hear. Res. 140:1–6.

P1: FLT



Takumi, Y., Nagelhus, E. A., Eidet, J., Matsubara, A., Usami, S., Shinkawa, H., Nielsen, S., and Ottersen, O.P. (1998). Select types of supporting cell in the inner ear express aquaporin-4 water channel protein.Eur. J. Neurosci. 10:3584–3595.

Verkman, A. S. (2000). Physiological importance of aquaporins: Lessons from knockout mice. Curr. Opin.Nephrol. Hypertens. 9:517–522.

Yasui, M., Hazama, A., Kwon, T. H., Nielsen, S., Guggino, W. B., and Agre, P. (1999). Rapid gating andanion permeability of an intracellular aquaporin. Nature 402:184–187.

Zenner, H. P., Reuter, G., Zimmermann, U., Gitter, A. H., Fermin, C., and LePage, E. L. (1994). Transitoryendolymph leakage induced hearing loss and tinnitus: Depolarization, biphasic shortening and lossof electromotility of outer hair cells. Eur. Arch. Otorhinolaryngol. 251:143–153.

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Aquaporin-Mediated Fluid Regulation in the Inner Ear...The whole set-up of the middle and the inner ear from the general anatomy to terminally differentiated sensory hair cells is