Aquaporins– the Waterways of Nature
Ofra Gohar, Ph.D., Co-Director, Antibody Group
The importance of the Aquaporin water channels was underscored by awarding the
2003 Nobel Prize in Chemistry to Peter Agre “for the discovery of water channels”. Many
scientific articles have been published describing the Aquaporins’ structure, function
and tissue distribution. Among the many methods that were used and contributed to
the enormous progress in the field, antibodies played an important role. This article
describes the use of Alomone Labs antibodies serving as a powerful tool in the ongoing
research.
Introduction
Water is a major component of the cell, forming
between 70 and 95% of its mass. It can cross
lipid bilayers of all biological membranes by
simple diffusion. However, diffusion is a slow,
non-regulated process which cannot account for
selective transmembrane water permeation as
occurs in many fluid transporting tissues during
normal physiological processes. This suggested
the existence of additional pathways for water
flux. The recent discovery of water channels,
Aquaporins (AQPs), provides a molecular
explanation for the rapid and regulated transport
of water through lipid bilayers1.
The Aquaporin family consists of 13, small and
hydrophobic proteins named AQP0 to AQP121,24.
The water channels share six transmembrane
domains with an intracellular N- and C-termini
structure but differ in their tissue distribution
as well as in their regulatory mechanism. The
functional channel is a tetramer comprised of
four functionally independent pores. They are
expressed in a subset of epithelia that have 10 to
100-fold higher capacities for water permeation
such as kidney tubules and red blood cells1.
These channels facilitate osmotically driven water
transport and are divided into two groups based
on their permeability characteristics: AQP1, AQP2,
4
AQP4, AQP5 and AQP8 are considered primarily as
water selective channels. AQP3, AQP7, AQP9 and
AQP10 transport water as well as glycerol and are
also called Aquaglyceroporins24.
Aquaporins in the Kidney
Water re-absorption occurs in different regions
of the kidney and involves several members of
the Aquaporin family: AQP1, AQP2, AQP3, AQP4,
AQP6 and AQP7.
association with decreased expression of AQP1,
AQP2 and AQP3 as was demonstrated using
Anti-Aquaporin 1 (#AQP-001), Anti-Aquaporin
2 (#AQP-002) and Anti-Aquaporin 3 (#AQP-003)
antibodies8 (Figure 1). Co-treatment of rats with
cisplatin and α-Lipoic acid prevented kidney
dysfunction and restored AQP1-AQP3 expression
as was demonstrated using all three Alomone
Labs respective antibodies8,4,7.
Aquaporin 2 and Aquaporin 3
Aquaporin 2 is mainly expressed in the principal
cells of renal collecting ducts. It is primarily
abundant in intracellular vesicles and apical
membranes1. Regulation of AQP2 trafficking from
intracellular vesicles to the cell surface appears
to be a complex process that is regulated by
vasopressin1,10,22.
An important role in maintaining water
homeostasis was first attributed to Aquaporins.
This discovery subsequently led to countless of
studies directed to explore their role in kidney
failure, injury or transplantation.
Aquaporin 1
Aquaporin 1 is selectively permeated by water
driven by osmotic gradients. AQP1 is expressed
in the apical and basolateral membranes of renal
proximal tubules as well as in the descending
thin limbs of Henle’s loop and the descending
vasa recta endothelia1,24. Studies with mice
lacking AQP1 show that the channel has a critical
role in urine concentration13. Induction of renal
dysfunction by injection of cisplatin in rats
demonstrated urine concentration defects in
Aquaporin 3 is expressed at the basolateral
membrane of principal cells in the collecting
ducts. Although it is a member of the Aquaporin
family, its biophysical function is slightly different
since it is also permeable to glycerol, and is
therefore considered to be an Aquaglyceroporin
channel1.
Anti-Aquaporin 2 antibody was used in many
works to demonstrate alteration in AQP2
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expression following different treatments or
in different pathological statuses. Analysis
of kidneys from deoxycorticosterone acetate
(Doca)-salt hypertensive rats demonstrated
a significantly enhanced expression of AQP2
in the cortex and outer medulla compared to
control groups as well as enhanced shuttling
as was demonstrated by western blot analysis
of cortex, outer medulla and inner medulla
of DOCA-salt kidneys using Anti-Aquaporin 2
antibody10. Increased expression of medullary
AQP1, AQP2 and AQP3 was also demonstrated
in spontaneously hypertensive rats using AntiAquaporin 2 and Anti-Aquaporin 3 antibodies11.
However, similar work with DOCA-salt
hypertensive rats done by Bae et al. resulted with
opposite results, demonstrating a decrease in
AQP1-3 expression4.The discrepancies between
the two experiments, might have been a result
of methodological differences, Na+ and water
balance differences and the different sensitivity to
Na+ and water intake.
Figure 1. Expression of AQP1, AQP2 and AQP3 in the Outer Medulla from Control and
Cisplatin-Treated Rats.
The decrease in AQP expression is associated in
many cases with the obstruction of kidneys or
kidney failure. Immunohistochemical staining
of obstructive kidneys compared to controls
demonstrated a decrease in expression of all
three AQPs using Anti-Aquaporin 1, 2 and 3
antibodies9. Expression of AQP1-3 was decreased
in kidneys with left obstructed ureter compared to
its contralateral kidney that was left untouched.
Alomone Labs antibodies directed against AQP13 demonstrated diminished expression of these
channels in the ureteral-obstructed kidney by
immunohistochemical staining as well as by
western blot analysis27.
Induced denervation in rats by painting the renal
vessels with 10% phenol through a midline
abdominal incision, led to a decrease in AQP2
expression, demonstrated by western blot
analysis and by immunohistochemical staining
using Alomone Labs respective antibody12 (Figure
2).
Following renal transplantation, patients often
develop tubular disorders, particularly due to
(subclinical) acute rejection. Following kidney
transplantation in rats, it was demonstrated
that the following exhibited a decrease in
expression and function: Na+/H+ Exchanger Type
3 (NHE-3) in the proximal tubule, Epithelial Na+
Channel (ENaC), and AQP2 (detected with AntiAquaporin 2 antibody) in the cortical collecting
ducts. These data strongly suggest that shortly
after transplantation, Na+ and water imbalances
occur18.
Immunohistochemical staining of AQP1, AQP2, and AQP3 in the outer medulla from control and cisplatin-treated rats using
Anti-Aquaporin 1 (#AQP-001), Anti-Aquaporin 2 (#AQP-002) and Anti-Aquaporin 3 (#AQP-003) antibodies. A) Immunoreactivity
for AQP1 was most prominent in the apical membrane of proximal tubules. B) The AQP1 labeling decreased markedly with
cisplatin treatment. C) The abundance of AQP2 labeling was shown exclusively in the collecting duct principal cells, both in
the apical region and throughout the cytoplasm. D) The AQP2 labeling decreased by cisplatin. E) AQP3 was localized to the
basolateral membrane of the collecting duct principal cells. F) The AQP3 labeling decreased in the outer medullary collecting
duct in response to cisplatin. *, S3 segment of the proximal tubule; •, collecting duct. Magnification, X350.
Adapted from reference 8 with permission of the American Society of Nephrology.
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Figure 2. Expression of AQP2 in Control, Denervated and Contralateral Kidneys.
AQP2
Control
Denervated
Contralateral
CCD
OMCD
IMCDi
Aquaporin 6
Aquaporin 6 is predominantly expressed in
intracellular vesicles in α-intercalated cells in
the collecting duct of the kidney (intercalated
cells, make up a third of the cells in the cortical
collecting duct and mediate acid-base transport).
AQP6 was found to allow permeation of
anions following activation with acidic pH or Hg2+
ions.
AQP6 was shown to bind calmodulin (a Ca2+binding protein). AQP6-expressing CHO-K1 cell
lysates were mixed with calmodulin beads and
AQP6 was pulled down in the presence of Ca2+
or EGTA. Immunoblot using Anti-Aquaporin 6
antibody (#AQP-006) confirmed the presence
of AQP6 in the sample, thereby confirming the
binding of calmodulin to the water channel17. The
binding of AQP6 to calmodulin may be important
in deciphering the physiological role of AQP6 in
the kidney.
Aquaporins in the Brain
IMCDm
IMCDt
Immunohistochemical staining of AQP2 in control, denervated and contralateral kidneys using Anti-Aquaporin 2 antibody
(#AQP-002) demonstrated marked decrease in AQP2 expression in the inner medullary collecting ducts, and a slight
decrease in the expression of AQP2 in the cortical and outer medullary collecting ducts. In addition, AQP2 immunoreactivity
was diffusely dispersed throughout the cytoplasm with decreased apical labeling in the denervated kidney, being
Several AQPs were found to be expressed in the
brain: AQP1, AQP4, AQP5 and AQP9. AQP1 was
detected in epithelial cells in the choroid plexus
whereas AQP4, AQP5 and AQP9 were localized in
astrocytes and ependymal cells (ependymal cells
are involved in the production of cerebrospinal
fluid, and are the thin epithelial membrane cells
lining the ventricular system of the brain and
the spinal cord and are one of the four types
of neuroglia in the central nervous system)3.
AQP4 and AQP9 appear to be implicated in brain
homeostasis and in central plasma osmolarity
regulation.
Aquaporin 1
contrasted by prominent apical labeling in the control. CCD, cortical collecting duct; OMCD, outer medullary collecting duct;
IMCDi, IMCDm, IMCDt, initial, middle, terminal parts of inner medullary collecting ducts respectively. Scale bars, 20 μm.
Adapted from reference 12 with permission of S. Karger AG.
Figure 3. Expression of AQP1 in Brain Tumors.
A
B
Immunohistochemical staining of primary glioblastoma multiforme (GBM) tumors (A) and normal adult brain
temporal cortex (B) specimens using Anti-Aquaporin 1 antibody (#AQP-001). AQP1 immunostaining in GBM
revealed a combination of cytoplasmic and cell membrane staining, whereas normal brain was negative.
Adapted from reference 15 with permission of the American Physiological Society.
6
Aquaporin 1 was proposed to be the major watertransporting protein in the choroid plexus. It
appears to be expressed in capillary endothelial
cells in the systemic circulation but not in
capillaries of the rat cerebrovascular system.
AQP1 immunolabeling using Anti-Aquaporin 1
antibody, was observed in various tumor cell
lines of primary glioblastoma multiforme (GBM)15
(Figure 3).
Aquaporin 4
Aquaporin 4 is the predominant AQP expressed in
the brain, in the perivascular margin of astroglial
cells, in the blood-brain barrier (BBB) and in
ependyma and pial surfaces in contact with
cerebrospinal fluid2,23. The distribution of AQP4
suggests that it provides an exit for excess brain
water in sever pathophysiologic conditions.
However, in brain edema, water enters the brain
through the intact blood-brain barrier, following
osmotic force. Therefore, in AQP4 -/- mice, after
experiencing water intoxication and focal cerebral
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ischemia have reduced edema formation and
survive better than wild-type mice in a model of
brain edema caused by acute water intoxication14.
Neuromyelitis optica (NMO), is an autoimmune
inflammatory disorder in which a person's
own immune system attacks the optic nerves
and spinal cord. The targeted protein, in some
patients with NMO has been identified as AQP4.
Immunohistochemical staining of primate
cerebellum from healthy and NMO patients,
using Anti-Aquaporin 4 antibody (#AQP-004),
demonstrated loss of AQP4 immunoreactivity
in NMO cerebellum compared to healthy
subject and also demonstrated that NMO-IgG
immunoreactivity completely overlaps with AQP4
immunoreactivity25.
numerous cell types and is involved in a wide
range of disease processes such as lung
inflammation and edema6,16. MLE-12 cells were
treated with NOC-18 (a nitric oxide donor) for 2
hours and stained with Anti-Aquaporin 5 antibody
(#AQP-005)16, (Figure 4). While most of the AQP5
signal was associated with the plasma membrane
(Figure 4A), staining on cell membrane was
markedly decreased, and increased intracellular
staining was observed compared to control
cells, following treatment with NOC-1816 (Figure
4B). This signal was abolished with treatment of
methyl-β-cyclodextrin, an endocytosis inhibitor
(Figure 4C). The decrease in AQP5 expression
along with the increase in NO metabolites, eNOS,
and iNOS was also described in acute lung injury
(ALI) induced by bleomycin inhalation6.
Aquaporins in the Lungs
Aquaporins in other Tissues
Four AQPs have been identified in the respiratory
tract: AQP1, AQP3, AQP4 and AQP5.
Taste Buds
Aquaporin 1
Aquaporin 1 is expressed in the apical and
basolateral membrane of the microvascular
endothelium, as well as in the visceral pleura.
Aquaporin 5
Aquaporin 5 is selectively expressed in the apical
plasma membrane of various secretory glands
such as the airway submucosal glands, and
alveolar type I epithelial cells in the lungs16,23.
It was demonstrated that nitric oxide decreases
cell surface expression of AQP516. Nitric oxide
(NO) is a ubiquitous molecule produced by
Immunohistochemical staining of AQP1 and
AQP2 using Anti-Aquaporin 1 and Aquaporin
2 antibodies revealed expression of both
channels in rat taste buds. AQP1 and AQP2 were
immunolabeled predominantly in the basolateral
membrane. Double labeling demonstrated
overlapping between AQP1 and AQP2 in many but
not all taste cells26 (Figure 5).
Cornea and Retina
In the cornea, AQP1 is found in the endothelial
cells and has been shown to function in osmotic
water transport in mice.
The effect of ozonated solution on the cornea
morphology was studied, where AQP1 and ZO-1
(tight junction-associated protein) expression
was tested along with the protective effect of
ascorbic acid (AA). Immunohistochemical staining
using Anti-Aquaporin 1 antibody, showed that the
expression of AQP1 in the AA group was similar to
that in the control21.
In the retina, AQP4 is expressed in Müller cells
(associated with bipolar cells). AQP4 expression
in Müller cells from AQP4+/+ and AQP4-/- mice was
compared using Anti-Aquaporin 4 antibody19.
Depletion of Dp71, a cytoskeleton protein
associated to the membrane, leads to
physiological alterations in Müller cells which are
similar to those observed in injured or diseased
retinas. This involves a mislocation of K+ and
water channels (Kir4.1 and AQP4) demonstrated
by immunohistochemical staining and western
blot analyses using Anti-Aquaporin 4 antibody
and Anti-Kir4.1 antibody (#APC-035). AQP4
mislocation resulted in dysregulation of water
transport through Müller cells20.
New Aquaporin Antibodies
Aquaporin 7
Aquaporin 7 is permeable to water, urea and
glycerol. It is expressed in a number of tissues;
ovary, testis, kidney, adipose tissue and islet
cells. The effect of urea and glycerol on rat
pancreatic β-cell membrane potential, cell
volume and insulin secretion was investigated.
Immunohistochemical staining and western
blot analyses assessed AQP7 expression and
localization in these cells5 (Figure 6). It was
concluded that glycerol and urea can activate
β-cells via their rapid uptake across the β-cell
plasma membrane, possibly via AQP7 resulting
Figure 4. The Effect of NO-Donors on the Subcellular Localization of AQP5.
MLE-12 cells were incubated with NOC-18,
and stained with Anti-Aquaporin 5 antibody
(#AQP-005), (green). In control cells, most of
AQP5 signals are associated with the plasma
membrane (A). After 2 hours of treatment with
NOC-18, AQP5 staining on the membrane is
markedly decreased, and intracellular staining
is increased (B). This translocation of AQP5
staining was abolished by the use of methyl-βcyclodextrin (C).
Adapted from reference 16 with permission of
Elsevier.
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7
in cell swelling, VRAC (volume-regulated anion
channel) activation, electrical activity leading
to depolarization, and insulin release. Glycerol
appears to exert an additional effect, possibly
related to its intracellular metabolism.
Figure 5. Expression of AQP1 and AQP2 in Rat Taste Buds.
Aquaporin 8
Aquaporin 8 is expressed in the liver, pancreas,
intestine, salivary gland, testes and heart and is
primarily water selective. Immunohistochemical
staining of rat pancreas and lung demonstrated
the expression of AQP8 in both tissues, using
Anti-Aquaporin 8 antibody (#AQP-008), (Figure 7).
Aquaporin 9
Aquaporin 9 is expressed in the liver, white blood
cells, testis and brain. It is permeable to water and
small solutes. Immunohistochemical staining of
rat liver and testis demonstrated the expression
of AQP9 in both tissues, using Anti-Aquaporin 9
antibody (#AQP-009), (Figure 8).
Immunohistochemical staining of rat taste buds double labeled with Anti-Aquaporin 1
Alomone Labs is following with great interest
after the developing research of Aquaporins and
keeps updating and offering new antibodies,
intracellular as well as extracellular, to meet
research needs.
(#AQP-001) and Anti-Aquaporin 2 (#AQP-002) antibodies. DIC image of a vallate taste
bud (A) and the same taste bud labeled with Anti-Aquaporin 1 antibody (green, B) and
Anti-Aquaporin 2 (red, C). D) Merged images, highlighting the lack of apical labeling in
these taste buds (arrowhead). Scale bars, 10 μm.
Adapted from reference 26 with permission of Oxford University Press.
Figure 6. Expression of AQP7 in Rat Pancreatic Islet Cells.
A
B
C
D
Immunohistochemical staining of rat pancreatic β-cells, double
labeled with Anti-Aquaporin 7 antibody (#AQP-007), insulin and
glucagon. A) AQP7 labeling (green) overlaps with that of insulin
(red), but not with that of glucagon (red, B).
C) AQP7 labeling (green) overlaps with that of somatostatin (in
red, indicated by arrows). D) Negative control performed in the
absence of primary antibodies. Scale bar, 100 μm.
Adapted from reference 5 with permission of S. Karger AG.
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Figure 7. Expression of AQP8 in Rat Lung.
Expression of AQP4 in Rat Kidney.
Immunohistochemical staining of AQP8 in
rat lung using Anti-Aquaporin 8 antibody
(#AQP-008). AQP8 labeling (brown) is
specific for the bronchiolar epithelium.
Hematoxilin is used as the counterstain.
Experimental procedure and figure
Immunohistochemical staining of rat kidney paraffin
processed at Alomone Labs Ltd.
embedded sections using Anti-Aquaporin 4 antibody (#AQP004), (1:100). Staining is present in the thin segments of the
Loop of Henle in the outer medulla (arrows). Hematoxilin is
used as the counterstain.
Experimental procedure and figure processed at Alomone
Labs Ltd.
Figure 8. Expression of AQP9 in Rat Testis.
Immunohistochemical staining of AQP9 in
rat testes using Anti-Aquaporin 9 antibody
(#AQP-009). AQP9 labeling (brown)
Western blot analysis of rat brain membranes:
appears in the columnar epithelium of the
1. Anti-Aquaporin 4 antibody (#AQP-004), (1:1000).
epididymus (arrows). Hematoxilin is used
2. Anti-Aquaporin 4 antibody, preincubated with
as the counterstain.
the control fusion protein.
Experimental procedure and figure
Experimental procedure and figure processed at
processed at Alomone Labs Ltd.
Alomone Labs Ltd.
Related Products
References
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1. Agre, P. et al. (2002) J.Physiol. 542.1, 3.
2. Agre, P. (2000) J. Am. Soc. Nephrol. 11, 764.
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4. Bae, E.H. et al. (2009) Nephrol. Dial. Transplant. 24, 2692.
5. Best, L. et al. (2009) Cell. Physiol. Biochem. 23, 255.
6. Jung, A.S. et al. (2004) Intensive Care Med. 30, 489.
7. Kang, D.G. et al. (2004) Biol. Pharm. Bull. 27, 366.
8. Kim, S.W. et al. (2001) J. Am. Soc. Nephrol. 12, 875.
9. Kim, S.W. et al. (2001) J. Am. Soc. Nephrol. 12, 2019.
10. Lee, J. et al. (2000) Clin. Exp. Hyper. 22, 531.
11. Lee, J. et al. (2006) Kidney Blood Press. Res. 29, 18.
12. Lee, J. et al. (2006) Nephron Physiol. 103, 170.
13. Ma, T. et al. (1998) J. Biol. Chem. 273, 4296.
15. Markert, J.M. et al. (2001) Physiol. Genomics 5, 21.
16. Nagai, K. et al. (2007) Biochem. Biophys. Res. Commun. 354, 579.
17. Rabaud, N, E. et al. (2009) Biochem. Biophys. Res. Commun. 383,
54.
18. Reuter, S. et al. (2008) Eur. J. Physiol. 456, 1075.
19. Ruiz-Ederra, J. et al. (2007) J. Biol. Chem. 282, 21866.
20. Sene, A. et al. (2009) PLoS One 4, e7329.
21. Suzuki, H. et al. (2009)] Jpn. J. Ophthalmol. 53, 151.
22. Valenti, G. et al. (2005) Endocrinology 146, 5063.
23. Verkman, A.S. (2005) J. Cell Sci. 118, 3225.
24. Verkman, A.S. (2009) J.Exp. Biol. 212, 1707.
25. Vincent, T. et al. (2008) J. Immunol. 181, 5730.
Compound
Cat. #
Aquaporin Channel Antibodies
Anti-Aquaporin 1_______________________________
Anti-Aquaporin 2_______________________________
Anti-Aquaporin 2-ATTO-550______________________
Anti-Aquaporin 3_______________________________
Anti-Aquaporin 3-ATTO-594______________________
Anti-Aquaporin 4_______________________________
Anti-Aquaporin 5_______________________________
Anti-Aquaporin 6_______________________________
Anti-Aquaporin 7_______________________________
Anti-Aquaporin 8_______________________________
Anti-Aquaporin 9_______________________________
AQP-001
AQP-002
AQP-002-AO
AQP-003
AQP-003-AR
AQP-004
AQP-005
AQP-006
AQP-007
AQP-008
AQP-009
26. Watson, K.J. et al. (2007) Chem. Senses 32, 411.
Inward Rectifier K+ Channel Antibodies
27. Yeum, C.H. et al. (2003) Scand. J. Urol. Nephrol. 37, 99.
Anti-Kir4.1_ ____________________________________ APC-035
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