J Am Soc Nephrol 11: 764 –777, 2000
HOMER W. SMITH AWARD LECTURE
Aquaporin Water Channels in Kidney
PETER AGRE
Departments of Biological Chemistry and Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland.
As a first year medical student, I attended nearly 200 lectures,
but I have clear recollections of only two. On the first day that
autumn, Albert Lehninger canceled his planned lecture and
spoke to us of a recent paper that he said would revolutionize
biomedical research—the discovery of reverse transcriptase.
The following spring, James L. Gamble, Jr. lectured to us about
renal physiology and the important contributions of a man
named Homer Smith who had conducted research in a shed on
Mount Desert Island in Maine. At the time, the idea of a
researcher at an idyllic site studying marine and tidal organ-
isms struck me as particularly appealing. Later that summer, I
set out on a bicycle tour of Nova Scotia by way of Bar Harbor
where Dr. Gamble and his family had warmly invited me to
stop at their summer home. In addition to some delicious meals
and a visit to multiple well-known sights in the area, Dr.
Gamble drove me past the site of Homer Smith’s laboratory
where 20 years later I was to spend time as a visiting scientist.
In many ways, the words of Smith were to have particular
meaning for me.
The history of renal physiology has erred, more often
than not, by attempts at oversimplification. The prob-
lems of water and salt excretion appear to be ex-
tremely complex, and especially liable to this danger.
Homer W. Smith, 1937
The Physiology of the Kidney (1)
Water is the major component of all living cells, so the
ability to absorb and release water must be considered a fun-
damental property of life. As recognized by Homer Smith, the
kidney is the champion organ with regard to fluid transport.
The epithelia lining the tubules in kidney are covered by
plasma membranes, which control the solute composition of
the enclosed compartments by regulating the entry of ions,
small uncharged solutes, and water into and out of cells.
Movement of these fluids from lumen to interstitium or in the
reverse direction is clearly related to the high degree of cellular
polarity in epithelial tissues whose cells have distinct apical
and basolateral membranes. The identification and character-
Received November 29, 1999. Accepted December 10, 1999.
This lecture was updated and modified from an earlier review with permission
(119).
Correspondence to Dr. Peter Agre, Department of Biological Chemistry, Johns
Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-
2185. Phone: 410-955-7049; Fax: 410-955-3149; E-mail: pagre@jhmi.edu
1046-6673/1104-0764
Journal of the American Society of Nephrology
Copyright © 2000 by the American Society of Nephrology
ization of the molecular entities responsible for the function of
biologic membranes has been a major goal of physiologists
during the past two decades. Using biochemical purification
and functional reconstitution techniques, multiple transporters
have been identified and characterized. In addition, expression
cloning has led to the discovery of other transporters. Despite
this, the molecular identities of water transporters remained
unknown until a series of fortuitous events occurred in our
laboratory several years ago.
The plasma membranes of all cells are known to exhibit
finite water permeability. Nevertheless, simple diffusion does
not account for all water movement through biologic mem-
branes, and specialized water channels were predicted to ex-
plain the high water permeability of certain tissues (reviewed
in reference (2). In particular, the water permeability of red cell
membranes is much higher than that observed for other cell
types and artificial lipid bilayers. The activation energy of this
process (Ea ⬍ 5 kcal/mol) is equivalent to the diffusion of
water in solution (reviewed in reference (3), indicating that
water moves through aqueous pathways across these mem-
branes. The observation of that HgCl2 and certain organomer-
curials reversibly inhibit the water permeability predicted that
water channels are proteins with accessible sulfhydryl groups
(reviewed in reference (4).
Water permeability has been characterized in each segment
of the nephron (reviewed in reference (5). Renal proximal
tubules and descending thin limbs of Henle’s loop are known
to have constitutively high water permeability allowing for the
reabsorption of the majority of the water in glomerular filtrate.
In contrast, the ascending thin limbs and thick limbs are
relatively impermeable to water but are highly permeable to
ions and other solutes. Renal collecting ducts are extremely
important in clinical water balance, since collecting duct water
permeability is regulated by vasopressin (antidiuretic hor-
mone). Early studies of toad urinary bladder provided a model
of vasopressin-regulated water permeability; stimulation of the
basolateral membrane of this epithelium with antidiuretic hor-
mone induces a redistribution of intracellular particles to the
apical membrane and increases the cellular water permeability
(6,7). Nevertheless, attempts to identify the membrane water
channels were unsuccessful due to the ubiquity of water, the
presence of free sulfhydryls in many proteins, and relatively
high diffusional permeability of most membrane bilayers (re-
viewed in reference (8).
Aquaporin-1
A serendipitous observation accompanied by the wisdom of
a former mentor led to the recognition of the first molecular
J Am Soc Nephrol 11: 764 –777, 2000
water channel. During the biochemical purification of the
32-kD core polypeptide of the red cell Rh blood group antigen,
a 28-kD polypeptide was noted and assumed to represent a
proteolytic fragment of Rh (9). Antiserum raised in rabbits
reacted exclusively with the 28-kD band, indicating that it is
unrelated to Rh (10). The 28-kD protein contained hydropho-
bic amino acids and exists in two forms: a nonglycosylated
28-kD polypeptide and a 40- to 60-kD N-glycosylated polypep-
tide. The protein proved easy to purify due to its limited
solubility in the detergent N-lauroylsarcosine. Biochemical
studies showed that the protein is a tetramer with intracellular
N- and C-termini and yielded the N-terminal amino acid se-
quence (11), which permitted cDNA cloning from an erythroid
library (12). The deduced amino acid sequence of the 28-kD
polypeptide was found to be related to a functionally undefined
family of putative membrane channel proteins including major
intrinsic protein of lens (13). At the same time, radiation
inactivation studies of water permeability by renal vesicles
yielded a target size of 30 kD (14). Abundance of the 28-kD
polypeptide in highly water-permeable tissues, red cells, renal
proximal tubules, and descending thin limbs (10) led the late
John C. Parker (1935–1993) at the University of North Caro-
lina at Chapel Hill to predict that it may be the sought-after
water channel now known as aquaporin-1 (AQP1). Soon to
follow was the identification of multiple related proteins that
comprise the aquaporin family of proteins and explain mem-
brane water permeability in diverse tissues and organisms
(8,15).
Measurement of Membrane Water Permeability
The measurement of water transport across cell membranes
posed an experimental challenge. First proposed by Eric
Windhager of Cornell Medical School, the Xenopus oocyte
expression system has proven to be a valuable method for
functional analysis of candidate water channel RNAs, since the
oocytes normally exhibit low membrane water permeability
(16,17). Fortunately, our colleague Bill Guggino at Johns Hop-
kins was very experienced in oocyte expression. When injected
with cRNA corresponding to the 28-kD polypeptide (18), the
oocytes became remarkably permeable to water (Pf approxi-
mately 200 ⫻ 10⫺4 cm/s) and exploded in hypotonic buffer.
Water-injected control oocytes swelled minimally (Figure 1).
Moreover, the AQP1 oocytes behaved like cells containing
native water channels: low activation energy, reversible inhi-
bition by HgCl2, and no measurable increase in ion conduc-
tance (18). The direction of water flow through AQP1 is
determined by the orientation of the osmotic gradient (19).
The ability to isolate milligrams of AQP1 from human red
cells permitted direct biophysical studies of the protein. Highly
purified AQP1 protein from human red cells reconstituted with
pure phospholipid yielded proteoliposomes of approximately
100 nm in diameter (20). The change in volume of AQP1
proteoliposomes was compared to liposomes containing no
reconstituted protein by measuring quenching of internal car-
boxyfluorescein. These measurements yielded the unit water
permeability (Pf approximately 3 ⫻ 109 water molecules/
subunit/s). The proteoliposomes exhibited water permeability
Homer W. Smith Award Lecture 765
Figure 1. Water permeability of aquaporin-1 (AQP1) expressed in
Xenopus oocytes. When transferred to hypo-osmolar buffer for 2 min,
control water-injected oocytes exhibit negligible swelling (left). Un-
der the same conditions, oocytes previously injected with AQP1
cRNA rapidly swell and explode (right). Reproduced and modified
with permission from Annu Rev Biochem (119).
similar to water channels in native membranes: reversible
inhibition by HgCl2 and low activation energy. Permeation by
other small solutes or even protons showed AQP1 to be water-
selective (21). Thus, AQP1 is both necessary and sufficient to
explain the well-recognized membrane water permeability of
the red cell. AQP1 functions as a constitutively active, water-
selective pore. Although forskolin was once reported to induce
a cation current in AQP1-expressing oocytes (22), multiple
other scientific groups have failed to reproduce this effect (23).
Recently, the permeation by CO2 has been evaluated, and rates
of pH change are higher in oocytes expressing AQP1 (24).
Because permeation of lipid bilayers by CO2, NO, and O2 may
be high, the potential physiologic relevance of AQP1 perme-
ation by gases deserves further study (25), and the possibility
of yet undiscovered transport functions cannot be excluded.
AQP1 Structure
The late 1990s have proven to be a time of great progress in
the elucidation of membrane protein structures. Hydropathy
analysis of the deduced amino acid sequence of AQP1 suggests
that the protein spans the lipid bilayer (12). Similar to the
homolog major intrinsic protein from lens (now referred to as
AQP0), the deduced amino acid sequence of AQP1 is com-
prised of two internal repeats (Figure 2), and each half contains
the signature motif Asn-Pro-Ala (NPA) (26). Hydropathy anal-
ysis predicts six bilayer-spanning domains, although connect-
ing loops B and E are also somewhat hydrophobic. Location of
loop C between the two halves of the molecule is therefore
critical to the topology. In different recombinants, BamHI
restriction sites were introduced into the cDNA for insertion of
a DNA encoding a 29-residue epitope. Expression in oocytes
confirmed that several of the recombinants remained func-
tional, and using antibody binding and vectorial proteolysis,
loop C was shown to reside at the extracellular surface of the
oocytes. Thus the N- and C-terminal halves of the molecule
exhibit a uniquely obverse symmetry with the two halves
766 Journal of the American Society of Nephrology
Figure 2. Membrane topology of AQP1 subunit. The Asn-Pro-Ala
signature motifs are indicated (NPA). Polyhedrons enclose sequence-
related repeats in obverse orientation. Sites of N-linked glycan and
surface polymorphism are represented. Reproduced and modified with
permission from Annu Rev Biochem (119).
oriented at 180 degrees to each other (27) (Figure 2). Other
studies showed that Cys-189 is the site of mercurial inhibition
(28), and loops B and E were found to be functionally essential
for water permeability. Together, these studies led to the “hour-
glass” model (29), in which loops B and E overlap midway
between the leaflets of the bilayer, creating a constitutively
open, narrow aqueous pathway (Figure 3). Although not un-
derstood in detail, multiple studies indicate that the AQP1 is a
tetramer comprised of functionally independent aqueous pores
(29,30).
AQP1 protein from human red cell membranes has provided
increasingly detailed molecular insight into the protein’s struc-
ture. Shadowed freeze-fracture electron microscopic analyses
of reconstituted AQP1 confirmed the tetrameric assembly of
AQP1 (21,31). Attenuated total reflection-FTIR (Fourier trans-
form infrared spectroscopy) of highly purified red cell AQP1
reconstituted into membrane crystals has been directly com-
pared to proteins with known structures, bacteriorhodopsin,
and the porin OmpF. These studies demonstrated that AQP1
lacks beta structure and contains multiple alpha helices tilted at
21 to 27 degrees (32). Using fluorescently labeled antibody
specific for the extracellular domain of loop A (Co antigen),
the lateral mobility of AQP1 in red cell membranes was shown
to be regulated by passive steric hindrance from the membrane
skeleton (33).
The structure of the AQP1 protein is being solved at increas-
ing levels of resolution. Reconstitution of highly purified red
cell AQP1 into membranes under controlled conditions yields
membrane crystals with highly uniform lattices with p4221
symmetry. These resealed vesicles retain full water permeabil-
ity, providing the opportunity to define the structure of AQP1
J Am Soc Nephrol 11: 764 –777, 2000
Figure 3. Hourglass model for AQP1 membrane topology. (Top) Each
AQP1 subunit contains six bilayer-spanning domains comprised of
two obversely symmetrical structures (TM1–3 ⫽ hemipore-1 and
TM4 – 6 ⫽ hemipore-2). (Bottom) When NPA motifs in loops B and
E are juxtaposed, they form a single aqueous channel spanning the
bilayer (the “hourglass”) flanked by the mercury-sensitive residue
(C189). Reproduced and modified with permission from J Biol Chem
(29).
in a biologically active state (34). Microscopic examination of
negatively stained membranes showed that one side of the
tetramer contains subunits widely separated around a central
stain-filled depression (the extracellular surface) or closely
surrounding a central area (cytoplasmic surface) (35). Cryo-
electron microscopic studies have revealed the presence of
multiple bilayer-spanning domains (36 –38), and atomic force
microscopy further defined the orientation of the right-handed
helix bundle and extramembrane dimensions of AQP1 (39).
Electron crystallography of cryopreserved specimens using a
liquid helium-cooled stage and tilts of up to 60 degrees re-
vealed the presence of AQP1 tetramers with individual sub-
units containing six bilayer-spanning alpha helices viewed at a
resolution of 6 Å (40 – 42). These studies have revealed that the
AQP1 subunit has a structure similar to the proposed hourglass
(Figure 4 and cover photo). The next challenge is to merge
structural understandings derived from molecules with unam-
biguous landmarks.
Tissue Distribution of AQP1
In the initial report, AQP1 was noted to be abundant in the
apical and basolateral membranes of renal proximal tubules
J Am Soc Nephrol 11: 764 –777, 2000
Figure 4. Stereoscopic view of AQP1 at 6 Å resolution established by
cryoelectron microscopy. Six tilted, bilayer-spanning alpha helices
(numbered) in a single subunit surround the internal hourglass formed
from loop B (green) and loop E (orange). Reproduced and modified
with permission from Nature (40).
and descending thin limbs of Henle’s loop (10). This was
subsequently confirmed with polyclonal rabbit antiserum (43)
and with affinity-purified Ig specific for the N- and C-terminal
domains of AQP1 (44,45) (Figures 5 and 6). In all studies,
AQP1 is constitutively present in the apical plasma membranes
(including the brush borders) and in basolateral membranes as
well. A similar pattern of expression is found in the descending
thin limbs of Henle’s loop (Figure 7). AQP1 at the lumenal and
ablumenal surfaces of the tubule provides the transcellular
pathway for water following small osmotic gradients at the
apical and basolateral surfaces of the cell. Quantification of
AQP1 at these sites by dilutional immunoblotting and by
enzyme-linked immunosorbent assay verified that AQP1 alone
could serve as the conduit for the huge volumes of water
reabsorbed in the proximal nephron (44,46). Immunohisto-
chemical studies have also demonstrated AQP1 in renal vasa
recta (47).
AQP1 is also abundant in capillary endothelia in other
organs. In lung, AQP1 is present in peribronchiolar capillary
endothelium, where expression is induced by glucocorticoids
(48 –50) acting through the classic glucocorticoid response
elements in the proximal gene promoter (51). In brain, AQP1
has been defined in the apical membrane of choroid plexus
epithelia, the site of cerebrospinal fluid secretion. AQP1 is also
present at multiple locations in the eye: nonpigmented ciliary
epithelium, lens epithelium, and corneal endothelium (48,52).
Although not present in intestinal mucosa, AQP1 is rich in
cholangiocytes, where it appears to be regulated by secretin
(53). Developmental expression of AQP1 has been found to be
complex in the rat model, and at least three patterns are
recognized: (1) transient expression before birth (periosteum,
cardiac endothelium); (2) permanent expression beginning at
the time of birth (cornea, lung, kidney); (3) constitutive life-
long expression (choroid plexus) (50,54,55).
AQP1 Deficiency
AQP1 protein is not essential for survival in humans or
mice. Humans have been identified who totally lack the AQP1
Homer W. Smith Award Lecture 767
protein (56). Due to their linkage to human chromosome 7p14,
it was recognized that the Colton blood group antigen (Co)
may reside on the AQP1 protein. Co was shown to be an
Ala/Val polymorphism at the 45th residue located on the first
extracellular loop of red cell AQP1 (57) (Figure 2). The Co
null blood phenotype is exceedingly rare, and our laboratory
has studied Co null probands from three unrelated kindreds.
Each was born in a remote rural area and is believed to have
been the offspring of parental consanguinity. All three were
found to be homozygous for naturally occurring disruptions in
the AQP1 gene. Nonsense mutations were identified in two
probands: deletion of exon 1 and frameshift in exon 1. The
third proband is homozygous for a missense mutation: replace-
ment of proline by a leucine at the end of the first transmem-
brane domain that produces an unstable polypeptide (56).
These Co null individuals are all women who developed an-
ti-Co during pregnancy. Although the exceedingly rare blood
group phenotype makes them impossible to match for blood
transfusion, it was surprising that none of them exhibited any
other obvious clinical phenotype, and it is uncertain whether
this is explained by naturally occurring backup systems or the
existence of compensating mutations. Evaluation of these Co
null humans has been undertaken, and preliminary analyses
revealed that they manifest a major concentration defect in the
proximal tubules but are not polyuric. When fluid-deprived for
up to 24 h, the Co null individuals concentrate their urine to
less than half the normal level, and administration of exoge-
nous vasopressin produced no additional effect (L. King and P.
Agre, unpublished observations).
Mice homozygous for targeted AQP1 gene disruption have
been raised (58). The recent development of an Aqp1 gene
knockout has revealed that renal water reabsorption in mice is
very dependent on AQP1 protein, since the Aqp1 null animals
became hyperosmolar after fluid restriction. Detailed classical
physiologic evaluation of the isolated proximal tubules from
the Aqp1 null mice were undertaken to establish that trans-
membrane water permeability was reduced by 80%, and led to
the observation of a compensatory reduction in glomerular
filtration (59,60).
Other Renal Aquaporins
The first functional definition of one member of a protein
family often predicts the functions of the other members, and this
has certainly been the case for the aquaporins. Homology cloning
has been undertaken by multiple laboratories, and 10 mammalian
homologs have been reported, while preliminary evidence for
additional homologs is emerging. Homology cloning of aquapor-
ins is most frequently undertaken using polymerase chain ampli-
fication with degenerate oligonucleotide primers corresponding to
the most highly conserved domains, the NPA sequences within
loops B and E (61). All members of the family are believed to be
water channels, because all contain structural motifs similar to
AQP1, but each has features apparently needed for functional and
regulatory differences. The complete sequencing of the human
genome will almost certainly identify new aquaporin homologs.
Although an oversimplification, the 10 known mammalian aqua-
porins have tentatively been broken into two subgroups: water-
768 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 764 –777, 2000

Figure 5. Light and electron microscopic studies of AQP1 in rat proximal tubules. (a) Immunohistochemical analysis of S3 segment stained
with Meier reagent exhibits strong staining over apical brush border and basolateral membranes (arrows). (b) Tangentially sectioned S3 segment
stained with peroxidase-conjugated goat-anti-rabbit IgG shows no reaction over collecting duct (C). Bars, 20 ␮m. (c) Immunogold electron
microscopy of S3 segments in longitudinal section. (d) Cross section. Magnification, ⫻30,000. Reproduced and modified with permission from
J Cell Biol (44).

selective channels (Orthodox aquaporins) and channels permeated
by water, glycerol, and other small molecules (Aquaglyceropor-
ins) (Figure 8). The Human Genome Organization has established
an Aquaporin Nomenclature System for designation of novel
family members (15), and updates are available on the Internet
(http://www.gene.ucl.ac.uk/nomenclature).
AQP2, the Vasopressin-Regulated Water Channel
Water permeability of renal collecting ducts is regulated by
vasopressin. The failure to demonstrate AQP1 in collecting
ducts by immunohistochemistry (10) led to the prediction that
other members of the aquaporin family must exist at this site
(18). Using the homology cloning approach, a cDNA was
reported to encode a renal collecting duct water channel, AQP2
(62). The combined efforts of multiple groups of investigators
have established major clinical significance for AQP2 (re-
viewed in reference (63). AQP2 is expressed predominantly in
the principal cells of the renal collecting duct (62,64,65).
Regulation of AQP2 appears to be complex. The intracellular
trafficking of AQP2 is regulated by vasopressin through short-
term exocytosis to the plasma membrane, whereas long-term
biosynthetic mechanisms are activated in response to chronic
thirsting.
Vasopressin has been known for more than two decades to
J Am Soc Nephrol 11: 764 –777, 2000
Figure 6. Diagram representing transcellular water permeation of
proximal tubule cell via AQP1.
cause the redistribution of intracellular vesicles to the apical
surface of principal cells, a process referred to as the “mem-
brane shuttle mechanism” (66). Immunolocalization of AQP2
in sections of rat kidney after treatment with vasopressin sup-
ports the shuttle hypothesis (65,67,68). A much-cited study of
isolated rat renal collecting ducts showed that in the absence of
vasopressin, water permeability was low and AQP2 resided
predominantly in intracellular vesicles (69). Administration of
vasopressin caused the water permeability to rise fivefold and
was accompanied by a redistribution of AQP2 to the apical
membrane. After removal of vasopressin, the water permeabil-
ity declined and surface AQP2 was reinternalized (Figure 9).
The mechanism of vasopressin action is through a surface V2
receptor coupled to activation of adenylyl cyclase, inducing
phosphorylation of the C terminus of the AQP2 protein (Figure
10). Cytoplasmic vesicles containing AQP2 are then targeted
to the cell surface via the vesicle-targeting proteins syntaxin-4
and synaptobrevin-2. The molecular details of this pathway are
being evaluated and appear to be complex (70). The stoichi-
ometry of subunit phosphorylation and the possible presence of
other transporters in the vesicle are being established (reviewed
in reference (63). The involvement of a heterotrimeric member
of the Gi protein family is known to occur during membrane
trafficking in renal epithelial cells (71). The long-term mech-
anisms for regulation of AQP2 biosynthesis and removal are
also being evaluated. Identification of a cAMP regulatory
element in the 5⬘ flanking DNA of AQP2 suggests that tran-
scriptional regulation may be occurring (72,73). Reduced ex-
Homer W. Smith Award Lecture 769
pression of AQP2 protein has been found during the postan-
tidiuretic state known as “escape from vasopressin” (74).
Much clinical significance has been ascribed to AQP2, and
this protein may be involved in most, if not all, imbalances of
water metabolism. For example, impaired renal water reab-
sorption is associated with reduced levels of AQP2. Nephro-
genic diabetes insipidus (NDI) is a disorder that occurs when
vasopressin levels are elevated, but the kidney fails to respond
to the hormone. Mutations in genes encoding renal vasopressin
V2 receptors have been demonstrated in many patients with the
X-linked disorder (reviewed in reference (75). A family with
recessively inherited NDI and normal V2 receptors was found
to have mutations in the AQP2 gene causing loss of function
(76). Multiple other AQP2 mutations in aqueous pore domains
have been found in other patients with recessively inherited
NDI (77,78). One family with dominantly inherited NDI was
recently identified with a mutation in the C terminus of AQP2
(79). The products of the mutant and the normal AQP2 allele
have been shown to oligomerize, thereby restricting membrane
trafficking of both polypeptides. Individuals with NDI result-
ing from the dominant mutations experience a less severe form
of polyuria (Daniel Bichet, personal communication).
Secondary decreases in AQP2 expression probably occur
with relative frequency in medical clinics. Lithium is widely
prescribed for treatment of bipolar disorder, but polyuria is a
potentially problematic side effect. While lithium-induced
polyuria may be managed by increasing fluid intake, this can
cause difficulties in elderly individuals or in patients deprived
of oral intake, such as in preparation for surgery. Lithium has
been found to cause a striking reduction (90%) in AQP2
expression in rats (80). Other forms of polyuria have been also
evaluated, and reduced AQP2 expression was also documented
after release of urinary obstruction, such as after prostate
surgery (81), in chronic hypokalemia-induced polyuria (82),
and is suspected in nocturnal enuresis (83).
Secondary increases in AQP2 expression may also be com-
mon in clinical medicine and may cause problems with exces-
sive renal water retention. Patients with congestive heart fail-
ure often succumb to refractory pulmonary edema, and
increased expression of AQP2 has been documented in rat
models of incompletely compensated congestive heart failure
(84,85). Increased expression of AQP2 may also explain fluid
retention known to complicate pregnancy (86), cirrhosis, and
syndrome of inappropriate antidiuretic hormone (87). AQP2
may be an effective target for pharmacologic intervention in
some of these disorders.
Multifunctional AQP3
Using the homology cloning approach, three scientific
groups isolated cDNAs encoding AQP3, a member of the
aquaporin family with distinctive biophysical functions (88 –
90). Previously cloned aquaporins were only permeable to
water, but AQP3 was noted to be genetically closer to the
known Escherichia coli glycerol transport protein GlpF (Fig-
ure 8), and AQP3 is permeated by glycerol. AQP3 was also
reported to be slightly permeable to urea, but the biologic
significance of this is uncertain, since unrelated proteins spe-
770 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 764 –777, 2000

Figure 7. Light and electron microscopic studies of AQP1 in rat descending thin limbs of Henle’s loop. (a) Immunohistochemical analysis of
outer medulla reveals labeling of apical and basolateral membranes of descending thin limbs (D) but not of ascending thin limbs (T) or
collecting ducts (C). (b) Higher magnification. Bars, 20 ␮m. (c) Immunogold electron microscopy of descending thin limb from short-looped
nephrons shows staining of cytoplasmic leaflets of apical membranes including microvilli (arrow) and basolateral membranes (BM). (d)
Long-looped nephron. Magnification, ⫻45,000. Reproduced and modified with permission from J Cell Biol (44).

cialized for urea transport have been identified (91). The struc-
tural explanation for how AQP3 may permit transport of water
and glycerol remains uncertain, and primary sequence differ-
ences in aquaglyceroporins include a motif (GLYY) in loop C
and an aspartate residue following the second NPA motif
(NPARD) rather than the motif (NPARS) found in orthodox
aquaporins. The possibility that the AQP3 has separate water-
and glycerol-transporting domains has been proposed by some
investigators (92), however, the existence of a single pore that
permits the flow of water or glycerol has been reported by
others (93). Although the significance is unclear, AQP3 ap-
pears to be gated shut at acid pH (94).
No humans have yet been identified with AQP3 deficiency,
and the clinical importance of this molecule remains to be
established. Because AQP3 is expressed at the basolateral
membranes of principal cells in the collecting duct (Figure 10),
J Am Soc Nephrol 11: 764 –777, 2000
Figure 8. Phylogenetic comparisons of aquaporins. Mammalian aqua-
porins and Escherichia coli homologs. Water-selective “orthodox
aquaporins” and AqpZ (gray) and “aquaglyceroporins” and GlpF
(white). Reproduced and modified with permission from Annu Rev
Biochem (119).
a role in renal water reabsorption is expected (95–97). AQP3
appears to be regulated at a biosynthetic level similar to AQP2.
In addition to kidney, AQP3 is also expressed in lower levels
at multiple other sites, including airways (95). AQP3 is strik-
ingly abundant in nasopharyngeal epithelium, where it is sus-
pected to play a role in generation of mucosal secretions and
allergic rhinitis (50,98).
Brain AQP4
Although primarily expressed in brain, AQP4 is also present
in the basolateral membranes of collecting duct principal cells
in the inner medulla (99). The protein presumably provides the
exit pore from these cells during vasopressin-dependent water
reabsorption. Disruption of the mouse Aqp4 gene has been
reported to result in a defect in renal concentration (100).
AQP4 is the predominant aquaporin in brain (101,102).
Residing at the perivascular margin of astroglial cells, the
protein may provide an exit for excess brain water in severe
clinical problems such as cerebral edema (103). AQP4 is
abundant in glial lamellae surrounding vasopressin secretory
neurons (103) and in ependymal cells lining the cerebrospinal
fluid-filled cavities (95). In addition, AQP4 is expressed in
retina and optic nerve, where the protein resides in Müller cell
endfeet adjacent to the vitreous body and vascular endothelium
(104).
AQP4 is present in fast twitch skeletal muscle in rat (105).
Fast twitch fibers accumulate high concentrations of lactate, so
Homer W. Smith Award Lecture 771
Figure 9. Immunogold electron microscopy of AQP2 in rat collecting
duct principal cells before (top) or after (bottom) exposure to vaso-
pressin. Magnification, ⫻44,000. Reproduced and modified with per-
mission from Proc Natl Acad Sci USA (69).
AQP4 may facilitate rapid water flux needed to restore osmotic
equilibrium. Of particular interest is the unexplained observa-
tion that AQP4 is reduced in fast twitch fibers in the MDX
mouse model of Duchennes muscular dystrophy (105).
Based on their absence in tissues from Aqp4 null mice, it has
been proposed that AQP4 is the molecular basis of square
arrays within astroglial membranes (106). This has recently
been confirmed. Direct anti-AQP4 immunolabeling of freeze-
fracture replicas from brain and spinal cord directly demon-
strated AQP4 in the majority of square arrays (107). It has also
recently been reported that protein kinase C activated by phor-
bol diesters produces a 90% reduction in water permeability by
AQP4 (108). Although this suggests that AQP4 may be ac-
tively regulated, important control experiments are still needed.
Nevertheless, AQP4 may reversibly open and close the blood-
brain barrier to the movement of water, a process that may be
of great significance in various clinical problems of brain
ischemia.
AQP6, an Intracellular Ion Channel Permeated by
Anions
Another sequence-related protein initially referred to as
WCH-3 (now designated AQP6) was identified in rat kidney
by homology cloning several years ago (109), but the protein
was not functionally defined. A highly related protein initially
772 Journal of the American Society of Nephrology
Figure 10. Diagram representing vasopressin-regulated transcellular
water permeation of apical membrane of collecting duct principal cell
via AQP2 and constitutive water permeation of basolateral membrane
via AQP3.
referred to as hKID was identified in human kidney, and the
chromosomal locus at human 12q13 coincided with that of
several other aquaporins. AQP6 was also reported to have
extremely low inherent water permeability and was further
inhibited by HgCl2 (110). The apparent lack of functional
information reduced the scientific interest in this protein.
Recently, there has been renewed interest in AQP6. This
aquaporin has been shown to reside in intracellular vesicles at
three sites in rat kidney: within foot processes of glomerular
podocytes, in subapical vesicles of proximal tubules, and in
alpha-intercalated cells of collecting duct (111). Exploring the
hypothesis that AQP6 may have a low basal activity which
may be activated, functional studies of AQP6 were recently
undertaken in Xenopus laevis oocytes using protein kinase A
and protein kinase C to phosphorylate the protein; however, no
increase in water permeability was noted. Using the known
aquaporin inhibitor HgCl2, AQP6 unexpectedly became acti-
vated and was accompanied by a large electrical current (112).
AQP6 was observed to colocalize with H⫹-ATPase in alpha-
intercalated cells of the collecting duct (Figure 11), suggesting
that low pH may be the natural activator of the protein. Expo-
sure to low pH produced slight increases in water permeability,
and the membrane currents were notably activated below pH
5.5 but were rapidly reduced after return to pH 7.5 (Figure 12).
J Am Soc Nephrol 11: 764 –777, 2000
Figure 11. Immunogold electron microscopy of AQP6 and H⫹-
ATPase in alpha-intercalated cell of rat collecting duct. Anti-AQP6
(small particles, arrowheads) is found over intracellular vesicles,
whereas anti-H⫹-ATPase (large particles, arrows) is also found at the
apical plasma membrane. Magnification, ⫻83,000. Reproduced and
modified with permission from Nature (112).
Ion replacement techniques showed that the AQP6 currents
were anion-selective. Moreover, AQP6 contains a unique pos-
itively charged residue lysine that is predicted to reside at the
cytoplasmic mouth of the pore (Figure 13). Replacement of
this residue with a negative charge K72E produced a loss of ion
selectivity.
Together these studies have documented highly unusual
behavior for an aquaporin and suggest that some members of
this protein family may participate in acid secretion or other
processes in addition to simple transmembrane water move-
ments. These studies of AQP6 suggest that the functional
repertoire of aquaporins may be much broader than previously
thought. The intracellular vesicles in alpha-intercalated cells
are believed to permit renal acid secretion by generating HCl.
Much work has firmly established the role of the H⫹-ATPase
(reviewed in references (113) and (114). To maintain electro-
neutrality, a pathway for anion secretion must exist, and the
ClC-5 has been located at this site (115). However, this protein
is known to become inactive at low pH (116). Thus, the role of
AQP6 in alpha-intercalated cells may be to function as a
chloride channel during later stages of acid secretion.
Perspectives
Organs including kidney, lung, eye, and brain have tissues
with complex expression patterns. Some of these tissues have
multiple different aquaporins expressed even within individual
cells. Precise subcellular locations are being elucidated by light
J Am Soc Nephrol 11: 764 –777, 2000 Homer W. Smith Award Lecture 773

Figure 12. Electrophysiologic analyses of Xenopus laevis oocytes expressing AQP6 and water-injected control oocytes. (a) Osmotic water
permeability at pH 4 and 7.5. (b) Representative currents of AQP6 or water-injected oocytes at indicated pH values. (c) Representative
current-voltage plots of AQP6 oocytes at pH 7.5 (E), after 1 min at pH 4.0 (F), or 1 min after return to pH 7.5 (Œ). (Inset) Continuous current
measurement. (d) Conductances measured at indicated pH values. Reproduced and modified with permission from Nature (112).
774 Journal of the American Society of Nephrology
Figure 13. Hourglass model for AQP6 showing conserved Asn-Pro-
Ala motifs (NPA), functionally important cysteines (C), and selectiv-
ity-determining lysine (K). Reproduced with permission from Nature
(112).
and electron microscopy, and generally it is found that indi-
vidual aquaporin homologs are expressed at unique intracellu-
lar sites. Because of its major importance in transport, this
review has focused on aquaporins in the mammalian kidney,
since this organ has been studied most extensively and multiple
aquaporins have been documented. It is generally believed that
renal aquaporins function together to provide transcellular wa-
ter flow. In principal cells of collecting duct, AQP2 traffics to
the apical membrane where water enters from the lumen (Fig-
ure 9), while AQP3 and AQP4 reside at the basolateral mem-
branes in the outer medulla and in the inner medulla, respec-
tively, where they appear to provide the outflow pathways to
the interstitium. Because the precise roles of AQP6 and other
uncharacterized aquaporins are still unclear, it is fair to say that
we still do not fully understand the importance of aquaporins in
kidney or other organs.
The list of 10 recognized mammalian aquaporins is still
growing, and aquaporins are being identified in invertebrates,
microbes, and plants. In particular, plants are likely to use
aquaporins for numerous physiologic processes involving hy-
draulics. Accordingly, the list of recognized plant aquaporins is
already three times the length of the mammalian list. Sequenc-
ing the entire genomes from microbes led to recognition of
microbial aquaporin homologs, and the much-awaited se-
quence of the human genome will certainly contain genes for
still unrecognized members of the aquaporin family. It will be
a major challenge to define the ontogeny and the sites of
expression. At the same time, the recent functional surprises
found with AQP3 and AQP6 indicate that aquaporins may not
simply serve as water transporters. It is also likely that cotrans-
port proteins that are genetically unrelated to aquaporins may
also transport water along with the specific solutes (117). The
already recognized primary and secondary roles of aquaporins
in human pathophysiology suggest that other clinical problems
such as cataracts, glaucoma, brain edema, or body temperature
J Am Soc Nephrol 11: 764 –777, 2000
regulation may involve members of this protein family. Plant
geneticists are already utilizing aquaporin genes in attempts to
develop pest- or drought-resistant organisms. Thus, the cellular
and molecular biology of the aquaporins is far from under-
stood.
In summary, there are no small problems. Problems
that appear small are large problems that are not
understood. Nature is a harmonious mechanism
where all parts, including those appearing to play a
secondary role, cooperate in the functional whole.
No one can predict their importance in the future. All
natural arrangements, however capricious they may
seem, have a function.
Santiago Ramón y Cajal, 1897
Advice for a Young Investigator (118)
Acknowledgments
This work was supported by grants from National Institutes of
Health, the Cystic Fibrosis Foundation, and the Human Frontier
Science Program. Credit for our studies should be shared with our
colleagues and the members of their groups: William B. Guggino,
Søren Nielsen, Mark Knepper, Landon King, Andreas Engel, Yoshi-
nori Fujiyoshi, Ole Petter Ottersen, Mark Zeidel, Carel van Os, Peter
Deen, Bruce Baum, Nick LaRusso, Erhard Bremer, Suresh Ambud-
kar, Erik Ilsø Christensen, John Rash, Carolyn Bondy, David Anstee,
John Moulds, Bill Bishai, Peter Maloney, Narla Mohandas, Tom
Pallone, and Thomas Zeuthen. Several junior scientists have been
particularly helpful: Thomas Walz, Mahmood Amiry-Moghaddam,
Erlend Nagelhus, Tae-Hwan Kwon, Akihiro Hazama, Kaoru Mit-
suoka, Teruhisa Hirai, Miriam Echevarria, and Bernard Heymann.
Former members of my own laboratory contributed much to these
studies: Andy Asimos, Brad Denker, Barb Smith, Greg Preston, Jin
Sup Jung, Surabhi Raina, Giuseppe Calamita, Mingqi Lu, Mélanie
Bonhivers, Matt Hall, Doug Lee, John Mathai, Ruben Baumgarten,
Sabine Mulders, Chulso Moon, Christy Turtzo, Kushal Bhakta, and
Zev Waldman. Our present laboratory group is continuing the aqua-
porin story: John Neely, Mario Borgnia, Jennifer Carbrey, Masato
Yasui, Jason Hoffert, Virginia Leitch, and David Kozono. Generous
long-term support and guidance have been provided by John C.
Parker, Anita Aperia, Arvid Maunsbach, Joseph Handler, and Dan
Lane.
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