Concluding remarks
I gratefully acknowledge the invaluable editorial assistance of my wife
Delphine Sayeed and the skillful help of Megan Flanagin in the preparation
of the figures.
The research in my laboratory was supported by National Institutes of
Health grants GM-53235 and GM-568501.
References
1. Arturson, G. Pathophysiology of the burn wound and pharmacological
treatment. Burns 22: 255–274, 1996.
2. Berton, G., S. R. Yan, L. Fumagalli, and C. A. Lowell. Neutrophil activation
by adhesion: mechanisms and pathophysiological implications. Int. J. Clin.
Lab. Res. 26: 160–177, 1996.
3. Bokoch, G. M. Chemoattractant signaling and leukocyte activation. Blood
86: 1649–1660, 1995.
4. Bone, R. C., R. A. Balk, F. B. Cerra, R. P. Dellinger, A. M. Fein, W. A.
Knaus, R. M. Schein, and W. J. Sibbald. Definitions for sepsis and organ
failure and guidelines for the use of innovative therapies in sepsis. The
ACCP/SCCM Consensus Conference Committee. American College of
Chest Physicians/Society of Critical Care Medicine. Chest 101:
1644–1655, 1992.
5. Downey, G. P., T. Fukushima, L. Fialkow, and T. K. Waddell. Intracellular
signaling in neutrophil priming and activation. Semin. Cell Biol. 6:
345–356, 1995.
6. Hallett, M. B., E. V. Davies, and A. K. Campbell. Oxidase activation in individual neutrophils is dependent on the onset and magnitude of the Ca2+
signal. Cell Calcium 11: 655–663, 1990.
7. Hallett, M. B., and D. Lloyds. Neutrophil priming: the cellular signals that
say “amber” but not “green.” Immunol. Today 16: 264–268, 1995.
8. Hinder, F., and D. L. Traber. Pathophysiology of the systemic inflammatory
response syndrome. In: Total Burn Care, edited by D. N. Herndon.
Philadelphia, PA: W. B. Saunders, 1996, p. 207–215.
9. Janeway, C. A., and P. Travers. Immunobiology. New York: Garland Publishing, 1996.
10. Morel, F., J. Doussiere, and P. V. Vignais. The superoxide-generating oxidase of phagocytic cells. Physiological, molecular, and pathological
aspects. Eur. J. Biochem. 201: 523–546, 1991.
11. Rane, M. J., S. L. Carrithers, J. M. Arthur, J. B. Klein, and K. R. McLeish.
Formyl peptide receptors are coupled to multiple mitogen-activated protein kinase cascades by distinct signal transduction pathways: role in activation of reduced nicotinamide adenine dinucleotide oxidase. J. Immunol.
159: 5070–5078, 1997.
12. Ryu, H., S. M. Jeong, C. D. Jun, J. H. Lee, J. D. Kim, B. S. Lee, and H. T.
Chung. Involvement of intracellular Ca2+ during growth hormone-induced
priming of human neutrophils. Brain Behav. Immun. 11: 39–40, 1997.
13. Sabeh, F., P. Hockberger, and M. M. Sayeed. Signaling mechanisms of elevated neutrophil O2– generation after burn injury (Part 2 of 2). Am. J. Physiol. Regulatory Integrative Comp. Physiol. 274: R476–R485, 1998.
14. Thelen, M., B. Dewald, and M. Baggiolini. Neutrophil signal transduction
and activation of the respiratory burst. Physiol. Rev. 73: 797–821, 1993.
15. Watson, F., and S. W. Edwards. Stimulation of primed neutrophils by soluble immune complexes: priming leads to enhanced intracellular Ca2+
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oxidase. Biochem. Biophys. Res. Commun. 247: 819–826, 1998.
Key Roles of Renal Aquaporins in Water Balance
and Water-Balance Disorders
Søren Nielsen, Tae-Hwan Kwon, Jørgen Frøkiær, and Mark A. Knepper
The discovery of aquaporins by Agre and co-workers provided an answer to the long-standing
biophysical question of how water can pass cell membranes. The identification and characterization
of several aquaporins expressed in the kidney has allowed detailed insight, at the molecular
level, into the fundamental physiology and pathophysiology of renal water metabolism.
R
enal regulation of body water balance involves reabsorption
of water in the proximal nephron and vasopressin-regulated
S. Nielsen, T.-H. Kwon, and J. Frøkiær are in the Department of Cell Biology,
Institute of Anatomy, University of Aarhus, DK-8000 Aarhus, Denmark, and
M. A. Knepper is in the Laboratory of Kidney and Electrolyte Metabolism,
National Heart, Lung, and Blood Institute, National Institutes of Health,
Bethesda, MD 20892.
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News Physiol. Sci. • Volume 15 • June 2000
water reabsorption in the collecting duct. At least six aquaporins (AQP1, -2, -3, -4, -6, and -7) are presently known to be
expressed in the kidney (Table 1). AQP1 is highly abundant in
the proximal tubule and descending thin limb, and several studies have now underscored its important role in constitutive
water reabsorption in these segments. AQP1 is absent in other
tubule segments. At least three aquaporins are known to be
expressed in the kidney collecting duct, and they participate in
0886-1714/99 5.00 © 2000 Int. Union Physiol. Sci./Am.Physiol. Soc.
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Neutrophil intracellular signaling triggered by neutrophil
activating agents have been extensively studied in vitro. The
signaling pathways leading to neutrophil O2– generation
include both the classical PLC-mediated Ca2+/PKC sequence
as well as a variety of novel protein kinases not dependent on
the Ca2+ signal, namely PTK, MAPK, and PI3K. The recent burst
of knowledge about the roles of the novel kinases in modulating target proteins implicated in the O2– response generation
and the extensive presence of the Ca2+-independent pathways
has appropriately taken the limelight away from Ca2+ signaling.
The Ca2+-independent pathways do play critical roles not only
in actual generation of O2– by neutrophils but also in priming
neutrophils and enhancing their potential to produce O2– without an actual production. Nevertheless, the activation of
preprimed neutrophils could potentially occur via either the
Ca2+-independent or the Ca2+-dependent sequences as redundant pathways. Recent studies in neutrophils harvested from
inflammatory conditions prevailing after burn/trauma injuries
indicate that the activation of the primed neutrophils, in vivo,
may result from potentiating interactions between the Ca2+linked and the Ca2+-independent pathways.
vasopressin-regulated water reabsorption. AQP2 is the apical
water channel of collecting duct principal cells and is the chief
target for short-term regulation of collecting duct water permeability by vasopressin (9). In addition, collecting duct water
reabsorption is regulated on a long-term adaptational basis, and
several studies have provided strong support that long-term regulation of AQP2 expression is a key factor in this. Thus there is
a long-term regulation of the total abundance of AQP2 in collecting duct cells, which can then enter the short-term regulated
trafficking to regulated collecting duct water reabsorption.
AQP3 and AQP4 are both expressed in the basolateral plasma
membranes of collecting duct principal cells and represent
potential exit pathways for water reabsorbed via AQP2. Recent
studies have underscored the role of aquaporins, and especially
AQP2, in short-term and long-term regulation of body water
balance. Moreover, a series of studies has also implicated
important roles of AQP2 in several inherited and acquired water
balance disorders. AQP6 and AQP7 are also expressed in the
kidney, but their specific location is currently unknown.
Aquaporin structure
Aquaporins are water channels allowing passive flux of
water across the membrane. The prototypical aquaporin,
AQP1 or CHIP28, was discovered by Agre and colleagues
(for recent review, see Ref. 1). According to phylogenetic
properties and their specificity for water and other solutes,
aquaporins have been divided into two principal groups: the
“orthodox set” (AQPs), which selectively transport water, and
the “cocktail set” (aquaglyceroporins), which also carry other
small molecules such as glycerol (1). Although the structural
characteristics responsible for the transport specificity and
selectivity are poorly understood, the three-dimensional
structure and oligomeric organization of AQP1 is emerging.
Structurally, aquaporins have six membrane-spanning domains,
intracellular amino and carboxy terminals, and internal tandem repeats that are believed to be a consequence of an
ancient gene duplication (1). Of the five connecting loops in
AQP1, the B and E loops presumably dip into the lipid
bilayer and form “hemichannels” that connect between the
leaflets to form a single pathway within a symmetrical structure that resembles an hourglass. The three-dimensional
structure of AQP1 has recently been determined at 6-Å resolution by cryoelectron microscopy (15). AQP1 and possibly
several other aquaporins, such as AQP2 and AQP3, form
tetramers in the membrane. However, AQP4 in glial cells
and the basolateral plasma membrane of collecting duct
principal cells assembles rather uniquely in a multimeric
structure that, visualized by freeze-fracture, appears as
intramembrane particle square arrays.
Renal aquaporins
Absorption of water in the renal tubule depends on the
driving force for water reabsorption and osmotic equilibration of water across the tubular epithelium (9). The driving
force is established, in part, via active NaCl transport, and
generation of a hypertonic medullary interstitium results as a
consequence of countercurrent multiplication. This requires
active transport and low water permeability in some tubule
segments and high water permeability (constitutive or regulated) in other segments. It is now clear that osmotic water
transport across the tubule epithelium is chiefly dependent
on aquaporin water channels.
The critical role of AQP1 in urinary concentration was
recently highlighted in studies using transgenic mice with
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FIGURE 1. Schematic representation of localization of different aquaporins in nephron and collecting duct system. AQP1 is present in proximal tubule and
descending thin limb. AQP2 is abundant in apical and subapical parts of collecting duct principal cells, whereas AQP3 and AQP4 are both present in basolateral
plasma membrane of collecting duct principal cells.
Table 1.
Aquaporins 1–9 in kidney and other organs
Number of
Amino Acids
Kidney
Localization
AQP1
Human
269
AQP2
Rat
271
AQP3
AQP4
AQP6
Rat
Rat
Rat
292
301
276
AQP7
AQP5
AQP8
Rat
Rat
Rat
269
265
263
Proximal tubules
descending thin limbs
Collecting duct principal
cells
Collecting duct
Medullary collecting duct
Collecting duct
intercalated cells, others
Cortex, medulla
AQP9
Human
295
Extrarenal
Localization
Subcellular
Distribution
Regulation
Multiple organs
APM/BLM
–
Testis
APM
VES
BLM
BLM
VES
Multiple organs
Brain and multiple organs
?
Testis, ?
Submandibular gland
Testis, pancreas, liver, colon,
heart, placenta
Liver, leuocytes, lung, spleen
APM ?
+++
+
–
+
?
?
?
?
AQP, aquaporin; APM, apical plasma membrane; BLM, basolateral plasma membrane; VES, intracellular vesicles; ?,
unknown; +++, significanty regulated; +, regulated; –, not regulated. Note that most of the renal AQPs have been cloned
from several species (human, rat, and mouse).
knockout of the AQP1 gene. These mice were polyuric and had
a reduced urinary concentrating capacity. Moreover, isolated
perfused proximal tubules and descending thin limbs had an
80 and 90% reduction in osmotic water permeability, respectively, illustrating an important role of AQP1 in water transport
across these tubule segments (14). Moreover, these studies also
emphasized the important role of transcellular rather than paracellular water transport in these tubule segments.
AQP2 (8) is expressed in principal cells of the cortical,
outer, and inner medullary collecting ducts (Fig. 1 and Table
1) and is abundant both in the apical plasma membrane and
subapical vesicles. AQP2 is the primary target for vasopressin
regulation of collecting duct water permeability (9). This conclusion was established in studies showing a direct correlation
between AQP2 expression and collecting duct water permeability in rats (6) and in studies demonstrating that humans
with mutations in the AQP2 gene (5) or rats with 95% reduction in AQP2 expression (10) have profound nephrogenic diabetes insipidus. As described below, body water balance is
regulated both by short-term and long-term mechanisms, and
it is now clear that AQP2 plays a fundamental role in both.
AQP3 and AQP4 are expressed in the collecting duct principal cell and are abundant in the basolateral plasma membranes, providing a potential exit pathway for water reabsorbed apically via AQP2. It was recently demonstrated that
there was a fourfold reduction in the osmotic water permeability of isolated perfused inner medullary collecting ducts
from mice lacking AQP4 (AQP4 gene knockout) (3). This indicates that AQP4 is responsible for a large fraction of water
transported out of the cell through the basolateral plasma
membrane in inner medullary collecting ducts. The lower
abundance of AQP4, together with the higher abundance of
AQP3 in cortical and outer medullary collecting ducts, raises
the possibility that AQP3 may play a more significant role in
these more proximal segments of the collecting duct.
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Two additional AQP cDNAs have been isolated from kidney: AQP6 and AQP7 (Table 1). AQP6 is abundant in intracellular vesicles in collecting duct intercalated cells, and
lower expression levels have also been noted in proximal
tubule and glomeruli. Preliminary data suggests that AQP7 is
present in the straight proximal tubule brush border (Table 1).
However, this remains to be established. Also the physiological roles of AQP6 and AQP7 remain to be identified.
Body water balance is regulated in part by short-term and
long-term regulation of the collecting duct water permeability. The acute vasopressin-induced increase in collecting duct
water reabsorption has been shown to involve vasopressinregulated trafficking of AQP2 between intracellular vesicles
and the apical plasma membrane. Long-term regulation of
AQP2 involves mechanisms that alter the total abundance of
AQP2 protein, thereby modulating the acute response by
changing the number of water channels in the cell that can
be recruited for vasopressin-regulated trafficking. The abundance of AQP3 also appears to be regulated by factors
related to water intake. Thus the short-term and long-term
mechanisms act together in a concerted fashion to regulate
body water balance (Fig. 2; see below).
Regulation of AQP2 trafficking by vasopressin
As illustrated in Fig. 3, a marked redistribution of AQP2
from intracellular vesicles to the apical plasma membrane
occurs in response to vasopressin stimulation. This was
demonstrated in isolated perfused inner medullary collecting
ducts that were fixed for immunoelectron microscopy. Conversely, removal of vasopressin induced a decrease in AQP2
in the apical plasma membrane in association with the reappearance of AQP2 in intracellular vesicles. These changes in
the subcellular distribution of AQP2 were paralleled by changes
in water permeability of the same tubules. This provided direct
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Species
evidence that vasopressin-regulated trafficking of AQP2 represents the cellular mechanism underlying the acute regulation of body water balance by vasopressin, thereby providing
support for the original “shuttle hypothesis” proposed by
Wade and colleagues (reviewed in Ref. 9) based on studies in
toad urinary bladders. Consistent with this, vasopressin treatment of rats in vivo was found to be associated with redistribution of AQP2 to the apical plasma membrane of collecting
duct cells, whereas treatment with vasopressin V2 receptor
antagonist produced an internalization of AQP2 from the apical plasma membrane to intracellular vesicles and multivesicular bodies (reviewed in Refs. 9 and 12).
Several laboratories have been successful in reconstituting elements of vasopressin regulation of AQP2 in cultured cell systems (2). Through transfection of AQP2 or
AQP2-c-myc into MDCK or LLCPK1 cells, increased AQP2
plasma membrane labeling and transcellular water flow
have been seen in response to vasopressin or forskolin
treatment, whereas AQP2 plasma membrane levels and
water transport were reduced in response to removal of
vasopressin or forskolin. The studies also indicated that
AQP2 may be subjected to recycling during repeated challenges to forskolin or vasopressin.
AQP2 contains a consensus site for PKA phosphorylation
in the cytoplasmic carboxy terminus (Ser256). Using AQP2transfected LLCPK1 cells, it was shown that PKA-mediated
phosphorylation of Ser256 is critical for vasopressin-induced
trafficking of AQP2 from intracellular vesicles to the plasma
membrane (2). Consistent with this, it has been demonstrated
that AQP2 is phosphorylated in the PKA consensus site in
response to vasopressin treatment of kidney tissue slices (13).
Antibodies have recently been developed that selectively recognize AQP2 phosphorylated at Ser256. These antibodies
labeled both the apical plasma membrane and vesicles, and
it was demonstrated that phosphorylation of this serine was
regulated via V2 receptors (4). Thus it appears likely that
PKA phosphorylation/dephosphorylation of AQP2 may be
involved in the regulated trafficking of AQP2 to and from the
plasma membrane (Fig. 2). It remains unknown whether
phosphorylation of other serines or threonines (by other
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FIGURE 2. Regulation of AQP2 trafficking and expression in collecting duct principal cells. Vasopressin (AVP) acts on V2 receptors in basolateral plasma membrane.
Through GTP-binding protein GS, adenylyl cyclase (AC) is activated, which accelerates production of cAMP from ATP. Then cAMP binds to the regulatory subunit of
protein kinase A (PKA), which activates the catalytic subunit of PKA. PKA phosphorylates AQP2 in intracellular vesicles and possibly other cytosolic or membrane proteins. Molecular apparatus involved in trafficking of AQP2 is shown in enlarged square (bottom). Microtubule motor protein dynein and dynein-associated protein
dynactin is associated with AQP2-bearing vesicles, as is myosin-1. At apical plasma membrane, vesicle-targeting receptors VAMP-2 and syntaxin-4 may participate
along with soluble vesicle-targeting receptors (among them NSF: n-ethylmaleimide-sensitive fusion protein) in docking and fusion of AQP2-bearing vesicles with apical plasma membrane. As shown in intact cell, cAMP may also participate in long-term regulation of AQP2 expression by increasing levels of catalytic subunit of PKA
in nuclei. This is thought to phosphorylate transcription factors such as cAMP responsive element binding protein (CREB) and c-jun/c-fos. Binding of these factors is
then in turn thought to increase gene transcription of AQP2, resulting in synthesis of AQP2 protein, which can then enter regulated trafficking system.
kinases) may be involved in the regulated exocytic or endocytic events as well.
Vesicle-targeting receptors, the so-called SNARE proteins, are
believed to play a key role in synaptic vesicle targeting, docking, and fusion. VAMP2, which is a vesicle SNARE, has been
found associated with AQP2-bearing vesicles, and, recently, target SNAREs such as syntaxin-4 and SNAP23 have been identified in collecting duct principal cells, using RT-PCR and
immunocytochemistry, where they are localized in the apical
plasma membrane of collecting duct principal cells. This supports the view that SNARE vesicle-targeting receptors may play
a role in vasopressin regulation of AQP2 trafficking (Fig. 2).
However, functional data to support this view is awaited.
Studies from the 1970s have made it clear that the
cytoskeleton is involved in the regulation of osmotic water
permeability by antidiuretic hormone. Recently, dynein, a
microtubule-based motor protein, was shown to be associated with AQP2-bearing vesicles (reviewed in Ref. 12). Dynactin was also found to be associated with AQP2-bearing
vesicles (reviewed in Ref. 12). Since dynactin is believed to
link vesicles via dynein to microtubules, this further supports
the view that microtubule-based motor proteins, and associated proteins, may be involved in vasopressin-regulated trafficking of AQP2 (Fig. 2). Actin, together with myosin-1, has
also been hypothesized to be involved in AQP2 trafficking.
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Regulation of AQP2 protein abundance
The ability of the kidney to increase or decrease the urinary
concentrating capacity in response to changes in hydration status is dependent on regulation of solute and water transport in
different tubule segments. It has been demonstrated that in the
collecting duct there is an adaptational regulation of the
osmotic water permeability (9), and several studies have established that this response is associated with changes in the total
number of AQP2 water channels per cell (9, 12). Water restriction or chronic vasopressin treatment induces an increase in
AQP2 levels that is paralleled by an increase in collecting duct
water permeability (9). Conversely, water loading or treatment
with V2 receptor antagonists decreases the overall abundance
of AQP2. The adaptational changes in AQP2 abundance in
turn change the levels of AQP2 available for short-term regulation of trafficking to/from the apical plasma membrane to regulate body water balance. Both vasopressin-dependent and
vasopressin-independent regulation are involved in controlling
AQP2 expression (Fig. 2). This long-term increase in AQP2
abundance is, at least in part, ascribed to regulation of AQP2
gene transcription, possibly involving a cAMP response element in the 5’-flanking region of the AQP2 gene. Mice that
have inherently high levels of cAMP-phosphodiesterase activity and hence low cytosolic levels of cAMP in the collecting
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FIGURE 3. Immunoelectron microscopical demonstration of AQP2 trafficking in isolated perfused collecting ducts perfused in absence (top left) or presence (bottom left) of vasopressin. Before AVP administration (PRE-AVP), AQP2 is primarily located in intracellular vesicles with very little labeling of apical plasma membrane. Forty minutes after AVP administration (AVP) AQP2 labeling is mainly confined to apical plasma membrane with little labeling of intracellular vesicles. At
right, AQP2 labeling density in apical plasma membrane (top) and osmotic water permeability of same collecting ducts (bottom) are shown. In response to vasopressin, there was a significant increase in AQP2 labeling density of apical plasma membrane, whereas 40 min after removal of vasopressin labeling was markedly
reduced. Osmotic water permeability of same tubules changed in parallel. Adapted from Ref. 12.
Table 2. Water balance disorders associated with aquaporin
dysregulation
Acquired neprogenic diabetes insipidus
Lithium treatment
Hypokalemia
Hypercalcemia
Postobstructive nephropathy
Bilateral ureteral obstruction
Unilateral ureteral obstruction
Diseases/conditions with water retention
Congestive heart failure
Hepatic cirrhosis
Nephrotic syndrome
Pregnancy
Other diseases/conditions
Syndrome of inappropriate antidiuretic hormone
secretion/vasopressin escape
Primary polydipsia
Chronic renal failure
Acute renal failure
Low protein diet
Age-induced reduction in urinary concentrating capacity
duct have very low expression levels of AQP2 and severe
polyuria (12). This supports the view that cAMP plays a role in
regulating AQP2 expression levels (Fig. 2).
Roles of AQP2 in diseases or conditions with altered
water balance
The first demonstration that AQP2 was essential for urinary concentration came from a study by Deen et al. (5).
They found mutated and nonfunctional AQP2 in patients
with very severe nephrogenic diabetes insipidus (non-Xlinked NDI). Subsequently, it was demonstrated that Brattleboro rats, which are vasopressin deficient and have extreme
polyuria and therefore have central diabetes insipidus, have
reduced expression of AQP2 and very low levels in the apical plasma membrane.
In contrast to the rare inherited forms of diabetes insipidus
(central and nephrogenic), acquired forms of nephrogenic
diabetes insipidus are much more common. A series of studies has been aimed at testing whether reduced expression
and apical targeting of AQP2 might play a role in these
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Genetic defects
Central diabetes insipidus
Hereditary nephrogenic diabetes insipidus
X-linked:
Mutations in vasopressin V2 receptor gene
Non-X-linked:
Mutations in AQP2 gene
Diabetes insipidus +/+ severe mice (increased
cAMP-phosphodieserase levels)
polyuric conditions. For this purpose, several classic experimental protocols were used. Prolonged lithium administration
to rats causes a 95% downregulation of AQP2 expression and
a similar reduction in the apical plasma membrane levels of
AQP2. This was associated with the development of extreme
polyuria (10), strongly supporting the view that dysregulation
of AQP2 plays a fundamental role in the development of
polyuria in acquired NDI (10). It was subsequently demonstrated that hypokalemia and hypercalcemia, which are relatively common electrolyte disorders and well-known causes
of acquired neprogenic diabetes insipidus, were also associated with downregulation of AQP2 expression and targeting.
In these two conditions, the downregulation of AQP2 was
much more modest, as was the polyuria, further supporting a
role of AQP2 downregulation. Again, it should be emphasized that it is very likely that these conditions are also associated with other defects, e.g., in solute transport in other
segments, that may also contribute to the polyuria.
A relatively common condition associated with impaired
urinary concentrating is obstruction of the urinary tract.
Experimental bilateral obstruction of the ureters for 1 day was
found to be associated with significant downregulation of
AQP2 in rats. This downregulation persisted after release of
obstruction and was associated with development of prolonged polyuria. Likewise, after 1 day of unilateral ureteral
obstruction, a condition in which there are no overall changes
in urine production and solute excretion rates, both AQP2
mRNA and AQP2 protein levels were downregulated in the
obstructed kidney. In addition, AQP2 levels were moderately
reduced in the unobstructed kidney, suggesting that AQP2
downregulation may also be important for the compensatory
increase in urine output from this kidney (excreting about
twice as much urine to compensate for the obstructed kidney). Solute free water clearance changed in parallel in both
the obstructed and unobstructed kidneys in a pattern that
matched the reduction in AQP2 expression closely. This further supports the view that AQP2 downregulation, together
with other defects, e.g., in solute transport, plays a role in the
development of polyuria in postobstructive nephropathy.
In two recent studies it was demonstrated that rats with
congestive heart failure and renal water retention have
increased levels of AQP2 expression and a marked redistribution of AQP2 water channels with increased targeting to the
apical plasma membrane. It is important that this was only
seen in rats with severe congestive heart failure and not in
rats with compensated heart failure (i.e., in rats with
increased left-ventricular end-diastolic filling pressure but no
hyponatremia). This supports the view that increased AQP2
expression and targeting, in conjunction with the altered
renal handling of sodium, may participate in the retention of
water and development of hyponatremia in severe heart failure. Both the increased expression and targeting of AQP2
may be ascribed to increased baroreceptor-mediated vasopressin release. It was recently demonstrated that AQP2
expression levels were also increased in pregnant rats, a condition that is associated with water retention. This raises the
possibility that increased AQP2 expression may play a role in
the development of water retention in pregnancy as well.
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and AQP3 are severely downregulated, providing a potential
mechanism contributing to this collecting defect. Also, ischemiainduced acute renal failure is known to be associated with
polyuria of unknown mechanisms. Although clearly severe
defects have been disclosed in proximal tubules and thick
ascending limbs, the collecting duct also appears to be
involved. Two models are generally used: unilateral or bilateral
clamping (of the renal pedicle or selectively of the renal artery)
for 30, 45, or 60 min followed by 1–5 days of recovery. Aquaporin expression, including expression of AQP2 and AQP3, is
significantly reduced after unilateral and bilateral clamping,
and this was associated with significant polyuria. These data
provide direct evidence that there is a collecting duct defect
and implicate a role of aquaporin downregulation in the development of the polyuria. It should be emphasized that recent
studies of experimental nephrotic syndrome, chronic renal failure, and acute renal failure have demonstrated that, in addition
to the downregulation of aquaporins, there is also dysregulation
of a number of solute transporters. This reinforces the important
issue that urinary concentration and dilution depends critically
on both active transport processes (which are also involved in
establishing the driving force for water reabsorption) and on
aquaporins for osmotic equilibration. Continued studies of
aquaporins, as well as of solute transporters, are likely to provide details to further elucidate the molecular basis for regulation and dysregulation of body water balance.
References
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F328–F331, 1998.
3. Chou, C. L., T. Ma, B. Yang, M. A. Knepper, and A. S. Verkman. Fourfold
reduction of water permeability in inner medullary collecting duct of aquaporin-4 knockout mice. Am. J. Physiol. Cell Physiol. 274: C549–C554, 1998.
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H. van-Os, and B. A. van-Oost. Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine. Science 264: 92–95, 1994.
6. DiGiovanni, S. R., S. Nielsen, E. I. Christensen, and M. A. Knepper. Regulation of collecting duct water channel expression by vasopressin in Brattleboro rat. Proc. Natl. Acad. Sci. USA 91: 8984–8988, 1994.
7. Ecelbarger, C. A., S. Nielsen, B. R. Olson, T. Murase, E. A. Baker, M. A.
Knepper, and J. G. Verbalis. Role of renal aquaporins in escape from vasopressin-induced antidiuresis in rat. J. Clin. Invest. 99: 1852–1863, 1997.
8. Fushimi, K., S. Uchida, Y. Hara, Y. Hirata, F. Marumo, and S. Sasaki.
Cloning and expression of apical membrane water channel of rat kidney
collecting tubule. Nature 361: 549–552, 1993.
9. Knepper, M. A. Molecular physiology of urinary concentrating mechanism: regulation of aquaporin water channels by vasopressin. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 272: F3–F12, 1997.
10. Marples, D., S. Christensen, E. I. Christensen, P. D. Ottosen, and S.
Nielsen. Lithium-induced downregulation of aquaporin-2 water channel
expression in rat kidney medulla. J. Clin. Invest. 95: 1838–1845, 1995.
11. Nielsen, S., C. L. Chou, D. Marples, E. I. Christensen, B. K. Kishore, and
M. A. Knepper. Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to
plasma membrane. Proc. Natl. Acad. Sci. USA 92: 1013–1017, 1995.
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Hepatic cirrhosis is another serious chronic condition
associated with water retention. Hepatic cirrhosis can be
experimentally induced by chronic administration of carbon
tetrachloride. In studies using intraperitoneal administration,
cirrhosis was found to be associated with a moderately
increased AQP2 protein and mRNA expression. Cirrhosis can
also be experimentally induced by ligation of the common
bile duct, which can produce a compensated state of cirrhosis with respect to salt and water handling. These rats display
an impaired vasopressin-regulated water reabsorption as
determined by the reduced effect of V2 receptor antagonists
in inducing water excretion. Consistent with this, AQP2 levels were significantly decreased in the rats with compensated
cirrhosis. Thus dysregulation of AQP2 may be involved in the
dynamic changes in water handling in hepatic cirrhosis.
“Vasopressin escape” describes the condition (physiological or pathophysiological) in which the normal hydroosmotic
effect of vasopressin is relieved or reduced. Several experimental studies have been undertaken to elucidate the role of
aquaporins in this setting. Rats were chronically infused with
1-desamino-[8-D-arginine]vasopressin (dDAVP) in osmotic
minipumps and were divided into two groups. One group of
rats was water loaded, and the other group was allowed free
access to water. Despite the continued administration of
dDAVP, water-loaded rats exhibited a marked downregulation
of AQP2 and developed polyuria compared with the antidiuretic control rats (7). Thus the rats escape from the action of
vasopressin. This downregulation of AQP2 is likely to represent a physiologically appropriate way to reduce the capacity
to reabsorb water and thereby prevent hyponatremia and
water intoxication. The signaling transduction pathways
involved in this are not well understood, but vasopressin-independent regulation is clearly involved. Thus the existence and
potential importance of a vasopressin-independent regulation
of AQP2 expression has gained considerable support and is
likely to play a physiological and pathophysiological role.
Disturbed renal water handling is a main characteristic of
complex renal diseases such as nephrotic syndrome, chronic
renal failure, and acute renal failure. A series of different classic
experimental models have been used to examine the potential
role of aquaporins in these water balance disorders. With
respect to nephrotic syndrome, there are defects in the mechanisms responsible both for urinary dilution and urinary concentration. The reasons for these disturbances are incompletely
understood. The reduced urinary diluting ability is likely a result
of nonosmotic elevation in plasma vasopressin levels, which
may then increase free water reabsorption. In rats with
puromycin aminonucleoside (PAN)-induced nephrotic syndrome or with adriamycin-induced nephrotic syndrome, an
extensive reduction in AQP2 and AQP3 expression was seen,
suggesting that the impaired urinary concentrating capacity in
nephrotic syndrome could in part be ascribed to this. It was
speculated that this response seems to be physiologically
appropriate to reduce a further extracellular fluid volume
expansion (9, 12). Chronic renal failure is also characterized by
a defect in urinary concentration, and several studies using isolated perfused tubules have disclosed a defect in the collecting
duct water handling. Recently, it was demonstrated that AQP2
12. Nielsen, S., T.-H. Kwon, B. Mønster Christensen, D. Promeneur, J. Frøkiær,
and D. Marples. Physiology and pathophysiology of renal aquaporins.
J. Am. Soc. Nephrol. 10: 647–663, 1999.
13. Nishimoto, G., M. Zelenina, D. Li, M. Yasui, A. Aperia, S. Nielsen, and A.
C. Nairn. Arginine vasopressin stimulates phosphorylation of aquaporin-2
in rat renal tissue. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 276:
F254–F259, 1999.
14. Schnermann, J., C. L. Chou, T. Ma, T. Traynor, M. A. Knepper, and A.
S. Verkman. Defective proximal tubular fluid reabsorption in transgenic aquaporin-1 null mice. Proc. Natl. Acad. Sci. USA 95:
9660–9664, 1998.
15. Walz, T., T. Hirai, K. Murata, J. B. Heymann, K. Mitsuoka, Y. Fujiyoshi, B.
L. Smith, P. Agre, and A. Engel. The three-dimensional structure of aquaporin-1. Nature 387: 624–627, 1997.
Atrial Natriuretic Peptide: Regulator of Chronic
Arterial Blood Pressure
Luis Gabriel Melo, Stephen C. Pang, and Uwe Ackermann
A
trial natriuretic peptide (ANP) is the most abundant of a
family of at least three structurally and functionally related
peptide hormones that exert widespread effects on cardiovascular and renal function. Under normal hemodynamic conditions, ANP is predominantly synthesized, stored, and secreted
in a regulated fashion by modified myocytes of the cardiac
atria. However, in pathophysiological conditions of hemodynamic overload, such as in congestive heart failure, ventricular
synthesis of the peptide, which is negligible under normal conditions, is reactivated and contributes significantly to the circulating pool of the peptide. ANP is also synthesized in lesser
amounts in some peripheral tissues, in the vasculature, and in
central nervous structures, where the peptide may exert
autocrine and paracrine modulatory effects on autonomic nervous function and neurohormone release. The biologically
active 28-amino acid peptide is cleaved from the carboxy end
of a prohormone and released in response to stretch of the
secretory myocytes, consequent to an increase in central
venous pressure. On release, ANP exerts its biological effects
by interacting with a membrane-bound guanylate cyclaselinked receptor (NPR-A) and subsequently stimulating intracellular cGMP synthesis. A second receptor subtype (NPR-C) is
the preponderant ANP binding site in most tissues and is primarily involved in clearance of the peptide from the circulation (for a comprehensive review, see Ref. 2).
When administered acutely, ANP elicits potent and shortlasting natriuresis and diuresis and systemic hypotension in a
wide variety of mammalian and nonmammalian species
(2).The renal excretory effects of the hormone are due, in part,
to direct inhibition of tubular sodium reabsorption in the inner
L. G. Melo and U. Ackermann are in the Department of Physiology of the
University of Toronto, 1 King’s College Circle, Toronto, ON M5S 1A8,
Canada. S. C. Pang is in the Department of Anatomy and Cell Biology, Faculty of Health Sciences, Queen’s University, Kingston, ON K7L 3N6, Canada.
0886-1714/99 5.00 © 2000 Int. Union Physiol. Sci./Am.Physiol. Soc.
medullary collecting duct and to inhibition of salt- and waterconserving mechanisms, such as the sympathetic nervous system, the renin-angiotensin (ANG)-aldosterone hormonal axis,
and antidiuretic hormone. The acute hypotensive effect of
ANP, on the other hand, is mediated primarily by a renal-independent reduction in cardiac output, consequent to a
decrease in intravascular volume. The hypotension is further
compounded by attenuation of autonomic reflex compensatory increases in heart rate and vascular resistance. (2).
Despite the extensive characterization of acute cardiovascular/renal actions of ANP, progress in elucidating a role for
this hormone in chronic regulation of blood pressure and fluid
and electrolyte balance was hampered by the lack of suitable
experimental models of ANP-induced disease or selective
pharmacological receptor antagonists. These difficulties have
been overcome, in part, with the introduction of genetic
mouse models expressing life-long alterations in ANP bioactivity. Recent work in these murine models provides evidence
that ANP contributes to long-term maintenance of blood pressure constancy and may play an essential role in mediating
the cardiovascular and renal adaptations to chronically elevated
dietary salt intake. This review summarizes and evaluates the
current evidence for a role of ANP in chronic regulation of
arterial pressure and fluid-electrolyte balance.
ANP and chronic regulation of blood pressure
The earliest evidence that ANP may participate in chronic
regulation of arterial blood pressure (ABP) originated with the
observation that prolonged infusion (3–7 days) of ANP into
conscious animals, resulting in plasma levels of the hormone
in the high physiological-to-pathophysiological range, causes
a sustained reduction in ABP of ~15–20 mmHg (4). Furthermore, the hypotension occurs in the absence of detectable
changes in cardiac output, intravascular volume, or absolute
News Physiol. Sci. • Volume 15 • June 2000
143
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Recent findings in atrial natriuretic peptide (ANP) transgenic and gene knockout mouse models
uncovered a tonic vasodilatory effect of this hormone that contributes to chronic blood pressure
homeostasis. With elevated salt intake, ANP-mediated antagonism of the renin-angiotensin
system is essential for blood pressure constancy, suggesting that a deficiency in ANP activity
may underlie the etiology of sodium-retaining disorders.