The Calling
PERSPECTIVE
school, coming to the profession for timeless rea- rate life events from their meaning for those who
sons — because of a physician they admire, or be- live them. In literature, the two are united. That is
cause they want to serve, or because they have suf- reason enough to keep reading. And writing.
fered or witnessed suffering. Perhaps some lucky
ones even today have been called to medicine 1. Maugham WS. Of human bondage. New York: Bantam Books,
1991.
through the medium of a book. If they have a love 2. Fraser A, ed. The pleasure of reading. London: Bloomsbury,
of literature, reading may well help them to discov- 1992:74-8.
er a way to understand and identify with the ambi- 3. Trautmann J. The wonders of literature in medical education.
Mobius 1982;2(3):23-31.
tions, sorrows, and joys of the people whose lives 4. Allison D. Skin: talking about sex, class & literature. Ithaca,
are put in their hands. In medicine, we often sepa- N.Y.: Firebrand Books, 1994.
Hyperactive Vasopressin Receptors and Disturbed Water
Homeostasis
Nine V.A.M. Knoers, M.D., Ph.D.
Related article, page 1884
Regulation of water homeostasis is of vital importance for all terrestrial organisms. In most mammals, including humans, the maintenance of water
balance is critically dependent on water intake, the
sensation of thirst, and the regulation of water excretion in the kidney, which is under the control
of the antidiuretic hormone arginine vasopressin
(AVP).1 In the past decade, our insight into the
AVP-mediated renal concentration mechanism has
substantially improved with the elucidation of the
roles of crucial molecular players in this process.
The identification of disease-causing genes in hereditary disorders of water balance has been extremely helpful in identifying these pivotal molecules.
In normal physiology, AVP is secreted into the
circulation by the posterior pituitary gland, in response to an increase in serum osmolality or a decrease in effective circulating volume (see diagram).
In the kidney, AVP binds to the V2 vasopressin
reeptor in the basolateral membranes of collecting-duct cells in the last portion of the nephron
(see diagram). Occupancy of this receptor results,
by means of signaling through a guanine nucleotide–binding regulatory protein (G protein), in me-
diated activation of adenylate cylase and the formation of cyclic adenosine monophosphate (cAMP).
cAMP, in turn, activates protein kinase A, which promotes the fusion of cytoplasmic vesicles containing
aquaporin-2 water-channel proteins with the apical
membrane. As a result, this normally water-tight
membrane becomes water-permeable. Driven by
the osmotic gradient of sodium, water is then transcellularly reabsorbed, entering the cells through
aquaporin-2 in the apical membrane and leaving
the cells for the interstitium through aquaporin-3
and aquaporin-4, which reside in the basolateral
membrane. The withdrawal of AVP results in endocytosis of the water channels, restoring the water-impermeable state of the apical membrane.
Proof of principle for the critical roles of both
the V2 receptor and aquaporin-2 in orchestrating
the AVP-mediated renal concentration mechanism
came from the identification of mutations in the
genes encoding these proteins in patients with
congenital nephrogenic diabetes insipidus.2 This
rare genetic disorder is characterized by a failure to
concentrate urine despite normal or elevated levels
of AVP. Polyuria and polydipsia, usually associated
with hypernatremia, are typical features of this disorder. The disease is particularly serious in infants,
Dr. Knoers is a professor of clinical genetics at the Depart- in whom recurrent episodes of dehydration may rement of Human Genetics, Radboud University Nijmegen sult in severe neurologic deficits, growth retardaMedical Centre, Nijmegen, the Netherlands.
tion, or even death. Mutations in the gene encod-
n engl j med 352;18
www.nejm.org
may 5, 2005
1847
The New England Journal of Medicine
Downloaded from www.nejm.org at CTR HOSPITAL UNIVERSITAIRE VAUDOIS on November 6, 2010. For personal use only. No other uses without permission.
Copyright © 2005 Massachusetts Medical Society. All rights reserved.
Hyperactive Vasopressin Receptors and Disturbed Water Homeostasis
PERSPECTIVE
A
Increased plasma osmolality or
decreased arterial circulating volume
Thirst
Increased fluid intake
Decreased plasma
osmolality or
increased arterial
circulating volume
AVP
Antidiuresis
SIADH
B
Collecting
tubule
Autosomal recessive and
dominant CNDI
H2O
Aquaporin-4
H2O channel
H2O
Stimulating
G protein
cAMP
Vasopressin
type 2 receptor
Arginine
vasopressin
Adenylate
cyclase
H2O
Aquaporin-3
H2O channel
H2O
Protein
kinase A
ATP
Phosphoproteins
Inactivating mutations: X-linked CNDI
Activating mutations: NSIAD
H2O
Aquaporin-2
H2O channel
H2O
H2O
Apical
Basolateral
Physiology of Water Homeostasis in Humans (Panel A) and Pathway of AVP Signaling in Renal Collecting-Duct Cells Involved in Regulating
Water Excretion (Panel B).
The cells of the collecting duct are polarized into an apical surface that faces the lumen of the collecting duct, through which the tubular fluid
flows, and a basolateral surface that faces the circulating blood. The relevant disorders of water balance include the syndrome of inappropriate antidiuretic hormone secretion (SIADH), congenital nephrogenic diabetes insipidus (CNDI), and nephrogenic syndrome of inappropriate
antidiuresis (NSIAD). The aquaporin-2 water channel is regulated by vasopressin, and the aquaporin-3 and aquaporin-4 water channels are
constitutively expressed. AVP denotes arginine vasopressin, and cAMP cyclic adenosine monophosphate.
1848
n engl j med 352;18
www.nejm.org
may 5, 2005
The New England Journal of Medicine
Downloaded from www.nejm.org at CTR HOSPITAL UNIVERSITAIRE VAUDOIS on November 6, 2010. For personal use only. No other uses without permission.
Copyright © 2005 Massachusetts Medical Society. All rights reserved.
Hyperactive Vasopressin Receptors and Disturbed Water Homeostasis
PERSPECTIVE
Diseases Caused by Activating (Gain-of-Function) Mutations
in G Protein–Coupled Receptors.
Disease
G Protein–Coupled Receptor
Testotoxicosis (familial male-limited precocious puberty)
Luteinizing hormone receptor
(LHR)
Autosomal dominant nonimmune hyperthyroidism
Thyrotropin receptor (TSHR)
Congenital stationary blindness
Rhodopsin
Autosomal dominant hypocalcemia
Calcium-sensing receptor (CaSR)
Corticotropin-independent Cushing’s
syndrome
Melanocortin-2 receptor (MC2R)
Familial ovarian hyperstimulation
syndrome
Follitropin receptor (FSHR)
Jansen’s metaphyseal chondrodysplasia
Parathyroid hormone/parathyroid hormone–related protein (PTH/PTHrP) receptor
Nephrogenic syndrome of inappropriate
antidiuresis (NSIAD)
Vasopressin type 2 receptor
(V2R)
ing the V2 receptor (AVPR2) cause the X-linked form
of congenital nephrogenic diabetes insipidus, and
mutations in the gene encoding aquaporin-2 are
responsible for both the autosomal recessive and
autosomal dominant forms.
The V2 receptor is a typical member of the large
superfamily of G protein–coupled receptors, which
includes receptors for hormones, neurotransmitters, light, odorants, lipids, and ions. Once an agonist binds to such a receptor, an intracellular signal
cascade is induced through the coupling and activation of a G protein. G protein–coupled receptors
play key roles in almost all physiological functions,
and mutations in the genes encoding these receptors have been identified in patients with a variety
of inherited and acquired disorders, such as retinitis pigmentosa, hypothyroidism and hyperthyroidism, dwarfism, fertility disorders, obesity, and even
cancer.3 Most such mutations that are associated
with disease are inactivating mutations, resulting
in reduced or total lack of response to the agonist.
These loss-of-function mutations are almost exclusively germ-line mutations.
Activating (gain-of-function) mutations in
G protein–coupled receptors, which induce constitutive receptor activation in the absence of the agonist, occur less frequently and consist primarily of so-
n engl j med 352;18
www.nejm.org
may 5, 2005
matic mutations that have been identified in certain
adenomas and malignant tumors. Such activating
mutations with germ-line transmission have been
identified in only a few inherited disorders (see
table).
At present, more than 180 different mutations
in AVPR2 have been described. These mutations all
cause congenital nephrogenic diabetes insipidus
and, consistently, are inactivating mutations, resulting in receptor malfunction at different levels, such as reduced receptor expression at the cell
surface or disturbances in hormone binding and
G protein coupling.
In this issue of the Journal (pages 1884-1890),
Feldman and coauthors describe two unrelated
male infants with euvolemic hyponatremia and serum hypo-osmolality, along with inappropriately
elevated urine osmolality and urine sodium concentrations. At first glance, the disorder in these
boys resembles the syndrome of inappropriate antidiuretic hormone secretion (SIADH), a relatively
common disorder characterized by insufficient
suppression of AVP secretion in relation to the degree of hypo-osmolality, which leads to inappropriate urine concentration. Since serum AVP levels
were undetectably low in both boys, Feldman et al.
ruled out SIADH and hypothesized that a hyperactive V2 receptor could be the underlying cause of
the disorder. Sequencing of AVPR2 in the two patients revealed a hemizygous point mutation in
each. In an in vitro functional assay, both mutations were shown to lead to the production of a
constitutively active V2 receptor. The consequence
was AVP-independent, and therefore inappropriate, activation of V2 receptor–mediated renal urine
concentration. Remarkably, and for reasons that
remain unexplained, neither patient showed any
clinical or biochemical sign of constitutive activation of extrarenal V2 receptors, which are known to
mediate increases in circulating coagulation and fibrinolytic factors and a decrease in diastolic blood
pressure after AVP stimulation.
The two identified activating AVPR2 mutations
result in substitutions of the same amino acid, located in a part of the receptor protein that is highly
conserved among G protein–coupled receptors and
that is involved in G protein coupling. Elucidation
of the exact mechanism underlying the activation
of these mutant V2 receptors will undoubtedly be
1849
The New England Journal of Medicine
Downloaded from www.nejm.org at CTR HOSPITAL UNIVERSITAIRE VAUDOIS on November 6, 2010. For personal use only. No other uses without permission.
Copyright © 2005 Massachusetts Medical Society. All rights reserved.
The
new england journal
of
medicine
brief report
Nephrogenic Syndrome
of Inappropriate Antidiuresis
Brian J. Feldman, M.D., Ph.D., Stephen M. Rosenthal, M.D.,
Gabriel A. Vargas, M.D., Ph.D., Raymond G. Fenwick, Ph.D., Eric A. Huang, M.D.,
Mina Matsuda-Abedini, M.D., Robert H. Lustig, M.D., Robert S. Mathias, M.D.,
Anthony A. Portale, M.D., Walter L. Miller, M.D., and Stephen E. Gitelman, M.D.
summary
The syndrome of inappropriate antidiuretic hormone secretion (SIADH) is a common
cause of hyponatremia. We describe two infants whose clinical and laboratory evaluations were consistent with the presence of SIADH, yet who had undetectable arginine
vasopressin (AVP) levels. We hypothesized that they had gain-of-function mutations in
the V2 vasopressin receptor (V2R). DNA sequencing of each patient’s V2R gene (AVPR2)
identified missense mutations in both, with resultant changes in codon 137 from arginine to cysteine or leucine. These novel mutations cause constitutive activation of the
receptor and are the likely cause of the patients’ SIADH-like clinical picture, which we
have termed “nephrogenic syndrome of inappropriate antidiuresis.”
From the Department of Pediatrics, Divisions of Endocrinology (B.J.F., S.M.R.,
E.A.H., R.H.L., W.L.M., S.E.G.) and Nephrology (M.M.-A., R.S.M., A.A.P.), and the Department of Psychiatry (G.A.V.), University
of California at San Francisco, San Francisco;
and Quest Diagnostics Nichols Institute,
San Juan Capistrano, Calif. (R.G.F.). Address
reprint requests to Dr. Gitelman at the University of California at San Francisco, Division of Pediatric Endocrinology, 513 Parnassus Ave., Rm. S679, Box 0434, San
Francisco, CA 94143, or at sgitelma@peds.
ucsf.edu.
Drs. Feldman and Rosenthal contributed
equally to this article.
N Engl J Med 2005;352:1884-90.
Copyright © 2005 Massachusetts Medical Society.
1884
f
luid homeostasis depends on proper water intake, governed by
an intact thirst mechanism, and on urinary excretion of free water, mediated by
appropriate secretion of arginine vasopressin (AVP) (also known as antidiuretic
hormone).1 AVP exerts its antidiuretic action by binding to the V2 vasopressin receptor
(V2R), a G protein–coupled receptor, on the basolateral membrane of epithelial cells in
the collecting duct of the kidney. Ligand binding activates the V2R, stimulating adenylate cyclase by means of Gs proteins. The resulting increase in intracellular cyclic AMP
(cAMP) promotes shuttling of intracellular vesicles containing the water channel aquaporin-2 to the apical membrane of the collecting-duct cells, thereby increasing water
permeability and inducing antidiuresis.
Clinical disorders of water balance are common, and alterations in many steps of
this pathway have been described.1 Urinary concentrating defects associated with diabetes insipidus may result from a deficiency of AVP or from nephrogenic causes, such
as X-linked, inactivating mutations in the V2R or autosomal recessive or autosomal
dominant lesions in aquaporin-2.2 Conversely, the syndrome of inappropriate antidiuretic hormone secretion (SIADH) manifests as an inability to excrete a free water
load, with inappropriately concentrated urine and resultant hyponatremia, hypo-osmolality, and natriuresis. SIADH occurs in the setting of euvolemia, without evidence of
renal disease or thyroxine or cortisol deficiency. Though usually transient, SIADH may
be chronic; it is often associated with drug use or a lesion in the central nervous system
or lung. When the cardinal features of SIADH were defined by Bartter and Schwartz,3
AVP levels could not be measured. Subsequently, radioimmunoassays have revealed
n engl j med 352;18
www.nejm.org
may 5, 2005
The New England Journal of Medicine
Downloaded from www.nejm.org at CTR HOSPITAL UNIVERSITAIRE VAUDOIS on November 6, 2010. For personal use only. No other uses without permission.
Copyright © 2005 Massachusetts Medical Society. All rights reserved.
brief report
that SIADH is usually associated with measurably
elevated serum levels of AVP.
We describe two unrelated male infants whose
clinical presentation was consistent with the presence of chronic SIADH but who had undetectable
AVP levels. We postulated that novel activating mutations of the V2R might account for their unique
presentation. Evaluation revealed novel activating
mutations of the V2R leading to what we term
“nephrogenic syndrome of inappropriate antidiuresis” (NSIAD).
case reports
Patient 1 presented at 3 months of age with irritability, and Patient 2 presented at 2.5 months of age
with two generalized seizures. Both children had
had unremarkable early neonatal courses. Both
were exclusively bottle-fed formula (7 mmol of sodium per liter). Both infants had mild systolic hypertension with otherwise normal physical examinations. Initial laboratory evaluations demonstrated
hyponatremia with normal serum levels of potassium and bicarbonate (Table 1). Both children had
serum hypo-osmolality with inappropriately elevated urinary osmolality and urinary sodium levels.
Both had low blood urea nitrogen and low or lownormal serum creatinine levels, low or suppressed
plasma renin activity, and normal aldosterone levels, indicating euvolemia. Serum cortisol, thyroidfunction tests, and coagulation studies were all
normal. Imaging studies of the head and chest
were unremarkable, with the exception of a small
pars intermedia cyst in Patient 2. Despite clinical
and laboratory presentations consistent with the
presence of SIADH, serum AVP levels were undetectable in both patients (Quest Diagnostics Nichols
Institute). Both children were initially treated with
fluid restriction, followed by the administration of
an osmotic agent (urea), resulting in increased urinary output and normalization of the serum sodium level.
Neither child had a family history of hyponatremia or of an SIADH-like syndrome. Both patients
had no siblings. The mother of Patient 1, subsequently found to be heterozygous for an activating mutation of AVPR2 (see below), had normal
simultaneous serum sodium levels (140 mmol per
liter) and serum and urine osmolality (293 and 795
mOsm per kilogram of water, respectively).
The parents of the patients provided written informed consent for the publication of the case re-
n engl j med 352;18
ports through a protocol approved by the institutional review board of the University of California at
San Francisco.
methods
mutation analysis
Genomic DNA from the two patients and their
mothers was isolated from whole blood with the use
of the Puregene Blood Kit (Gentra Systems). The
entire coding region of the V2R gene, AVPR2, was
amplified as described previously.4 The resulting
amplicons were sequenced with multiple forward
and reverse primers with the use of Big Dye (version
3.1) sequencing chemistry and an ABI Prism sequencer (model 3100, Applied Biosystems) according to the manufacturer’s protocols. SeqScape software (Applied Biosystems) was used to assemble
the sequence data and compare the results with
the AVPR2 reference sequence (GenBank accession
number NT 025965).
construction of vasopressin expression
constructs
pCDNA3 (Invitrogen) was used as the control plasmid. Human wild-type V2R complementary DNA
(cDNA),5 subcloned into pCDNA3, was used as a
normal control and template for mutagenesis. Sitedirected mutagenesis was performed with the use
of a QuickChange II site-directed mutagenesis kit
(Stratagene). Mutations were confirmed by direct
sequencing.
cell culture and transient transfection
For functional studies, COS-7 cells were cultured
in Dulbecco’s modified Eagle’s medium with 10
percent fetal-calf serum and antibiotics. Cells were
plated in six-well plates (Falcon 3046, Becton Dickinson) at approximately 95 percent confluence 24
hours before transfection with the use of Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Each well received 3 µg of plasmid
DNA and 500 ng of a cAMP-responsive luciferase
reporter plasmid (pCREluc) containing 16 copies
of the consensus cAMP response element.6 To control for transfection efficiency, cells were cotransfected with 50 ng of renilla luciferase reporter
plasmid (pRL-CMV, Promega) per well. Cells were
incubated at 37°C in 5 percent carbon dioxide for
24 hours after transfection and were then lysed and
assayed for luciferase activity with the use of the
Dual Luciferase Reporter Assay System (Promega),
www.nejm.org
may 5, 2005
1885
The New England Journal of Medicine
Downloaded from www.nejm.org at CTR HOSPITAL UNIVERSITAIRE VAUDOIS on November 6, 2010. For personal use only. No other uses without permission.
Copyright © 2005 Massachusetts Medical Society. All rights reserved.
The
new england journal
of
medicine
Table 1. Characteristics of the Two Patients.*
Characteristic
Patient 1
Patient 2
Age-Matched Controls
3.0
2.5
—
Irritability
Generalized seizures
—
Systolic
80–128
93–118
108†
Diastolic
34–92
37–57
66†
123
118
Age at presentation (mo)
Clinically significant findings
Blood pressure (mm Hg)
Serum and plasma studies
Sodium (mmol/liter)
Potassium (mmol/liter)
4.6
4.7
134–143
3.4–4.9
Chloride (mmol/liter)
91
86
98–107
Bicarbonate (mmol/liter)
28
21
23–32
Creatinine (mg/dl)
<0.3
Urea nitrogen (mg/dl)
<5
3
8–23
Aldosterone (ng/dl)
10
24‡
6–68
Plasma renin activity (ng of angiotensin I/ml/hr)
Osmolality (mOsm/kg)
3.8
252
AVP (pg/ml)
<1
0.3
<1‡
247
<1‡
0.3–0.7
<15
285–293
1.0–13.3
Thyrotropin (mIU/liter)
0.77
4.58
1.7–9.1
Free thyroxine (ng/dl)
1.09
0.97
0.8–1.8
Cortisol (µg/dl)
Baseline
13.3
10.3, 43§
60 Min after intravenous synthetic corticotropin
(15µg/kg)
36.5
—
4–20
>20
Coagulation studies
von Willebrand factor antigen (%)
104
Ristocetin cofactor (%)
Factor VIII activity (%)
Fibrinogen (mg/dl)
100
46–155
108
65
56–155
153
123
50–150
198
Negative
d-Dimer fragment
360
Negative
170–435
Negative
Prothrombin time (sec)
11.7
10.9
9.2–11.9
Partial-thromboplastin time (sec)
24.3
32.5
21.3–34.8
284
390‡
300–900
35
75‡
—
Urine studies
Osmolality (mOsm/kg)
Sodium (mmol/liter)
Magnetic resonance image of the head
Normal
Pars intermedia cyst,
otherwise normal
—
Chest radiograph
Normal
Normal
—
* To convert values for creatinine to micromoles per liter, multiply by 88.4. To convert values for urea nitrogen to millimoles
per liter, multiply by 0.357. To convert values for aldosterone to nanomoles per liter, multiply by 0.0277. To convert values
for AVP to picomoles per liter, multiply by 0.923. To convert values for free thyroxine to picomoles per liter, multiply by
12.87. To convert values for cortisol to nanomoles per liter, multiply by 27.59. To convert values for fibrinogen to micromoles per liter, multiply by 0.0294.
† Measurement is given for the 95th percentile.
‡ The value was obtained during an episode of hyponatremia after initial presentation.
§ Two baseline levels were obtained on separate occasions.
1886
n engl j med 352;18
www.nejm.org
may 5 , 2005
The New England Journal of Medicine
Downloaded from www.nejm.org at CTR HOSPITAL UNIVERSITAIRE VAUDOIS on November 6, 2010. For personal use only. No other uses without permission.
Copyright © 2005 Massachusetts Medical Society. All rights reserved.
brief report
as described previously.7,8 Data are presented as
mean (±SE) luciferase activity expressed in arbitrary
units and adjusted for renilla luciferase activity in
three experiments, each performed in triplicate.
All five types of samples (pCDNA3 vector alone and
vector with wild-type V2R, the R137C mutant, the
R137L mutant, or the R137H mutant) were run simultaneously under the same conditions during
each of the three experiments.
results
Genomic DNA was isolated from both boys and
their mothers, and the V2R gene, AVPR2, was sequenced directly. AVPR2 is X-linked, and therefore,
paternal DNA is not informative. Each patient carried a mutation in codon 137 of AVPR2. In Patient 1,
nucleotide 770 was mutated from cytosine to thymine, changing arginine to cysteine at codon 137
(R137C); in Patient 2, nucleotide 771 was mutated
from guanine to thymine, changing arginine to leucine at codon 137 (R137L) (Fig. 1A). Arginine 137
maps to the predicted second cytoplasmic loop,
near the cytoplasmic boundary of the third transmembrane domain; this same amino acid is mutated to histidine (R137H) in a form of familial nephrogenic diabetes insipidus (Fig. 1B). The mother of
Patient 1 was heterozygous for the R137C mutation, whereas the mother of Patient 2 was homozygous for wild-type AVPR2, suggesting that Patient 2
had a spontaneous mutation.
To evaluate the effect of these novel mutations
on V2R function, we developed a functional assay
for V2R. Production of cAMP has previously been
used to assess many G protein–coupled receptors,
including V2R9; we adopted our previous procedures8 to use with V2R. COS-7 cells transiently
transfected with the vector alone, wild-type V2R, or
the R137H nephrogenic diabetes insipidus mutant
induced low levels of cAMP (Fig. 2). However, basal levels of cAMP production in cells expressing
V2R with the R137C mutation were four times the
levels in cells expressing wild-type V2R (P=0.01),
and cells expressing the R137L mutant had 7.5
times the level of activity of cells expressing wildtype V2R (P<0.004) (Fig. 2). These results indicate
that these novel mutations create a constitutively
active V2R and provide an explanation for the hyponatremia with increased urinary osmolality in our
patients. The condition in both patients is clinically
similar to SIADH, despite the fact that AVP levels
were undetectable.
n engl j med 352;18
discussion
G protein–coupled receptors constitute the largest
gene family of receptors involved in signal transduction and are responsible for regulating many physiological processes.10 Many diseases are caused by
mutations in G protein–coupled receptors.11,12
For some G protein–coupled receptors, a particular
disease state has been ascribed to inactivating mutations that render the receptors unresponsive to ligand, whereas a converse condition has been linked
to gain-of-function mutations, resulting in constitutive activation.
Although many different inactivating mutations
of the X-linked V2R have been described that cause
nephrogenic diabetes insipidus,2 to our knowledge,
no naturally occurring activating mutations of the
V2R have been reported previously. The two cases
described here are characterized by chronic SIADH
but with undetectable AVP levels, constituting a
novel example of gain-of-function mutations caused
by a hemizygous V2R mutation. In the light of these
findings, we suggest referring to all SIADH-like
conditions as syndromes of inappropriate antidiuresis (SIAD) and that these two case reports constitute a subtype, NSIAD. To our knowledge, these
cases are the only reported examples in which mutations affecting the same amino acid cause two
different genetic diseases: R137H causes nephrogenic diabetes insipidus, and R137L and R137C
cause NSIAD (Fig. 1B).
The mechanism by which these missense mutations constitutively activate the V2R requires further
investigation. V2R, a class 1b G protein–coupled receptor,12,13 exists in the plasma membrane in equilibrium with inactive and active conformations.10
The binding of ligand shifts the equilibrium to the
active state, permitting coupling with intracellular
G proteins and activation of intracellular effectors.
With activation, V2R is desensitized through phosphorylation by specific G protein–coupled receptor kinases. Subsequent recruitment of b arrestin
to the phosphorylated receptor terminates the
signal by blocking further interaction with G proteins and also initiates receptor internalization
through its ability to bind clathrin and other endocytic adapters.14
By comparison with other class 1 G protein–
coupled receptors, the highly conserved motif of
aspartic acid, arginine, and tyrosine or histidine
(DRY/H) in V2R at the junction of the third transmembrane domain and second intracellular loop
www.nejm.org
may 5, 2005
1887
The New England Journal of Medicine
Downloaded from www.nejm.org at CTR HOSPITAL UNIVERSITAIRE VAUDOIS on November 6, 2010. For personal use only. No other uses without permission.
Copyright © 2005 Massachusetts Medical Society. All rights reserved.
The
new england journal
of
medicine
A
Wild Type
Patient 1
Patient 2
R137L
R137C
R137
B
S
P
S
P
L
N
S
S
Q
E
L
P
L
D
D
R T
A
R
A
E
L
L
A
S
L
F
I
V
V
A
A
G L S V
A L V N
A
L
L
Intracellular
A
Extracellular
R
R
G
V
A
D
L
F
R R G
G
V
A
T T S A
M L
M –NH2
P
R F R
G
D
T
A
R
L P
H
L
S P
S
P
A E P W
F
G
D
C
R
A
A
A
S G G
P
P E
R
G
E
W
L
L
D
T
V
C
V
E
C
W
Y
D T
R
V
G
A
R N
T W
A
A P
A
W
A Q
V K
L
F V
I
Q
A L
Y
L L
I F
L V
M
F
L Q
F
M L
V
L
F
Q
F
V
M
P
L A
P
V A
G M
Cell
L
S L
W A
P
C
S
L
T L
A
Y
N S
L
L
membrane
S S
G I
C T
V Y V
S L A F
Y
A A
M
V
N
V
P W
I L
C Q
V I
A W V
I
A
V L
M T
L
Y A
I F
S
V L
L
M T
F
R
C C
D
P
R
E
R
S S
A
L
I
H
S
R
V
H
R
R
V
L
N
T
S
A
G
S
A
S E L R
W
R137
K
S
R
I
H
A
V S
L
T
C
V
H
A
A
V
P
R
A
A
G
P
P
P
S
G
S
M
G
C S E D Q P G L
L
E
G
T
P
A
Y R H
G P S
T
S
G
T
A
E
S
R
R
S S
L A K
P G
R
D T S S
G R R R G
–COOH
K
W
A
L
Q
L P G
L F V
L A A
C L
H
I G
H V
I
P
A
W
H
M
C
NSIAD
L
H
R
A
T
R
L
D
H: Congenital
nephrogenic
diabetes
insipidus
I
Figure 1. Nucleotide Sequence of the Wild-Type and Two Mutant AVPR2 Genes in the Affected Region (Panel A) and Diagram of V2R (Panel B).
In Panel A, the normal CGC sequence encoding R137 is changed to TGC (R137C) in Patient 1 and to CTC (R137L) in Patient 2. In Panel B,
R137 is indicated. The inset highlights changes in R137 that result in either congenital nephrogenic diabetes insipidus or NSIAD.
1888
n engl j med 352;18
www.nejm.org
may 5 , 2005
The New England Journal of Medicine
Downloaded from www.nejm.org at CTR HOSPITAL UNIVERSITAIRE VAUDOIS on November 6, 2010. For personal use only. No other uses without permission.
Copyright © 2005 Massachusetts Medical Society. All rights reserved.
brief report
n engl j med 352;18
9
Relative No. of cAMP-Inducible Luciferase
Reporter Units
appears to be critical for receptor function. The arginine residue in the DRY/H motif appears to be invariant, though the aspartic acid and tyrosine may
be replaced by other amino acids without altering
the function (e.g., from aspartic acid to glutamic
acid).15 The arginine residue in the DRY/H motif
corresponds to R137, the site of the inactivating
R137H mutation in nephrogenic diabetes insipidus
and the activating R137C and R137L mutations in
NSIAD. The R137H mutant behaves as a constitutively desensitized receptor, since it is phosphorylated, binds to b arrestin (thereby blocking its ability
to activate G proteins), and is sequestered in intracellular vesicles.16
Constitutively activated mutant G protein–coupled receptors have also been created by altering this
DRY/H motif 17 — for example, through in vitro mutation of aspartate to alanine at codon 136 in the
V2R.18 The NSIAD mutations found in this domain
may stabilize the receptor in an active conformation, activating G proteins and downstream signaling events in the absence of ligand. In theory, such
a gain of function could also affect many other aspects of V2R biology, including phosphorylation,
internalization, down-regulation, and recycling to
the cell membrane.
The effects of the constitutive activation of V2R
may not be limited to the kidney. V2R is also expressed in endothelial cells, where it appears to mediate vasodilation after the administration of the
vasopressin analogue desmopressin19,20 and to
mediate the rise in circulating levels of von Willebrand factor and tissue plasminogen activator.21
We thought that these responses, which are absent
in patients with nephrogenic diabetes insipidus,22
might be constitutively activated in our patients
with NSIAD. However, we found no clinical or laboratory evidence of coagulopathy in either patient
(Table 1).
The frequency of NSIAD is not known, but it
may not be rare. Previous studies of patients with
SIADH have noted variable patterns of AVP secretion, particularly in response to water loading and
water restriction.23 As many as 10 to 20 percent of
affected patients have AVP levels at or below the
limits of detection by radioimmunoassay. Thus,
some of these patients may in fact have NSIAD due
to V2R-activating mutations. Such patients would
probably have clinical presentations similar to those
in our patients, although the severity and clinical
course may depend on the nature of the mutation.
P<0.004
8
7
6
5
P=0.01
4
3
2
1
0
Control
Wild-Type
V2R
R137H
Mutant
V2R
R137C
Mutant
V2R
R137L
Mutant
V2R
Figure 2. Basal Levels of cAMP Production in Cells Expressing Wild-Type
and Mutant V2R.
The ability of the mutant V2R from each patient to induce cAMP, as measured
by a cAMP-inducible luciferase reporter, was evaluated. Each bar represents
cells transfected with the renilla luciferase expression vector, the cAMP-inducible luciferase reporter plasmid, and pCDNA3 either alone (control), reflecting basal cAMP levels in the cells, or with the following inserts subcloned into
the pCDNA3 plasmid: wild-type V2R; R137H mutant V2R, found in some patients with nephrogenic diabetes insipidus; R137C mutant V2R, found in Patient 1; or R137L mutant V2R, found in Patient 2. Each value represents the
mean (±SE) of three independent experiments with all five samples run simultaneously, each performed in triplicate.
Patients with NSIAD would be expected to have
low AVP levels. However, since the AVP assay is not
optimized to identify low values, we recommend
sequencing the V2R gene to identify specific mutations before the diagnosis of NSIAD is finalized. Other patients may be identified who have
the NSIAD phenotype but without V2R mutations,
suggesting the presence of additional defects in
this signaling cascade. One such possibility would
be an activating mutation in aquaporin-2.
Treatment of NSIAD poses a challenge. Water
restriction improved serum sodium levels and osmolality in both infants but limited calorie intake
in these formula-fed infants. Agents that act downstream from the V2R, such as demeclocycline or
lithium, might antagonize the constitutively activated receptors, but they have potentially limiting
adverse effects. AVP antagonists are under clinical
development but would probably be ineffective, given the ligand-independent nature of the lesion.24
Ideally, one might be able to use an inverse agonist
www.nejm.org
may 5, 2005
1889
The New England Journal of Medicine
Downloaded from www.nejm.org at CTR HOSPITAL UNIVERSITAIRE VAUDOIS on November 6, 2010. For personal use only. No other uses without permission.
Copyright © 2005 Massachusetts Medical Society. All rights reserved.
brief report
that would suppress receptor activity in the absence
of agonist; two potential nonpeptide V2R inverse
agonists have been studied in vitro.18 In the absence
of a definitive therapy, we have successfully treated
our patients with urea to induce an osmotic diuresis.25 This approach has occasionally been used for
the treatment of chronic SIADH in adults.26
Study of the V2R has been important for understanding the physiology of water balance and has
served as a prototype for G protein–coupled receptor biology. Further characterization of NSIAD may
offer additional insights into fluid homeostasis
and clinical disease, as well as expand our understanding of G protein–coupled receptor signaling.
Supported in part by a training grant (T32DK07161, to Drs. Feldman and Huang) and a grant (M01RR01271, to the Pediatric Clinical Research Center) from the National Institutes of Health.
Dr. Rosenthal reports having received grant support and speaking honoraria from Pfizer and having served on an advisory board
for Tercica and Pfizer. Dr. Vargas reports having received grant support from Wyeth and Janssen. Dr. Lustig reports having received lecture fees from Novo Nordisk and grant support from Novartis. Dr.
Mathias reports having equity interest in AstraZeneca, Pfizer, Merck,
and SIRNA Therapeutics; receiving grant support from Satellite
Healthcare and Genentech; and receiving speaking honoraria from
Shire. Dr. Fenwick is employed by Quest Diagnostics, owns stock in
the company, and developed the clinical assay used for sequencing
AVPR2 in this study.
We are indebted to Ian Ocrant and Kamer Tezcan for referring the
patients; to Mariel Birnbaumer (National Institute of Environmental
Health Sciences, Division of Intramural Research) for sharing the
V2R cDNA; to Christian Vaisse (University of California at San Francisco) for the cAMP-responsive luciferase reporter plasmid; to Mark
von Zastrow (University of California at San Francisco), Jon Nakamoto (Quest Diagnostics), and Hillel Gitelman for thoughtful discussions and review of the manuscript; and to Izabella Damm (University of California at San Francisco) for technical support.
refer enc es
1. Robertson GL. Antidiuretic hormone:
normal and disordered function. Endocrinol
Metab Clin North Am 2001;30:671-94.
2. Morello JP, Bichet DG. Nephrogenic diabetes insipidus. Annu Rev Physiol 2001;63:
607-30.
3. Bartter FC, Schwartz WB. The syndrome
of inappropriate secretion of antidiuretic
hormone. Am J Med 1967;42:790-806.
4. Bichet DG, Arthus MF, Lonergan M, et
al. X-linked nephrogenic diabetes insipidus mutations in North America and the
Hopewell hypothesis. J Clin Invest 1993;92:
1262-8.
5. Birnbaumer M, Gilbert S, Rosenthal W.
An extracellular congenital nephrogenic diabetes insipidus mutation of the vasopressin
receptor reduces cell surface expression,
affinity for ligand, and coupling to the Gs/
adenylyl cyclase system. Mol Endocrinol
1994;8:886-94.
6. Vaisse C, Clement K, Durand E, Hercberg S, Guy-Grand B, Froguel P. Melanocortin-4 receptor mutations are a frequent and
heterogeneous cause of morbid obesity. J Clin
Invest 2000;106:253-62.
7. Stables J, Scott S, Brown S, et al. Development of a dual glow-signal firefly and
Renilla luciferase assay reagent for the analysis of G-protein coupled receptor signalling.
J Recept Signal Transduct Res 1999;19:395410.
8. Fluck CE, Martens JW, Conte FA, Miller
WL. Clinical, genetic, and functional characterization of adrenocorticotropin receptor
mutations using a novel receptor assay.
J Clin Endocrinol Metab 2002;87:4318-23.
9. Rosenthal W, Antaramian A, Gilbert S,
Birnbaumer M. Nephrogenic diabetes insipidus: a V2 vasopressin receptor unable to
1890
stimulate adenylyl cyclase. J Biol Chem 1993;
268:13030-3.
10. Pierce KL, Premont RT, Lefkowitz RJ.
Seven-transmembrane receptors. Nat Rev
Mol Cell Biol 2002;3:639-50.
11. Spiegel AM, Weinstein LS. Inherited
diseases involving G proteins and G proteincoupled receptors. Annu Rev Med 2004;55:
27-39.
12. Seifert R, Wenzel-Seifert K. Constitutive
activity of G-protein-coupled receptors: cause
of disease and common property of wildtype receptors. Naunyn Schmiedebergs Arch
Pharmacol 2002;366:381-416.
13. Bockaert J, Pin JP. Molecular tinkering
of G protein-coupled receptors: an evolutionary success. EMBO J 1999;18:1723-9.
14. Luttrell LM, Lefkowitz RJ. The role of
beta-arrestins in the termination and transduction of G-protein-coupled receptor signals. J Cell Sci 2002;115:455-65.
15. Probst WC, Snyder LA, Schuster DI,
Brosius J, Sealfon SC. Sequence alignment
of the G-protein coupled receptor superfamily. DNA Cell Biol 1992;11:1-20.
16. Barak LS, Oakley RH, Laporte SA, Caron
MG. Constitutive arrestin-mediated desensitization of a human vasopressin receptor
mutant associated with nephrogenic diabetes insipidus. Proc Natl Acad Sci U S A 2001;
98:93-8.
17. Parnot C, Miserey-Lenkei S, Bardin S,
Corvol P, Clauser E. Lessons from constitutively active mutants of G protein-coupled
receptors. Trends Endocrinol Metab 2002;
13:336-43.
18. Morin D, Cotte N, Balestre MN, et al.
The D136A mutation of the V2 vasopressin
receptor induces a constitutive activity which
permits discrimination between antago-
n engl j med 352;18
www.nejm.org
nists with partial agonist and inverse agonist activities. FEBS Lett 1998;441:470-5.
19. Hirsch AT, Dzau VJ, Majzoub JA, Creager MA. Vasopressin-mediated forearm vasodilation in normal humans: evidence for a
vascular vasopressin V2 receptor. J Clin
Invest 1989;84:418-26.
20. Kaufmann JE, Iezzi M, Vischer UM. Desmopressin (DDAVP) induces NO production
in human endothelial cells via V2 receptorand cAMP-mediated signaling. J Thromb
Haemost 2003;1:821-8.
21. Mannucci PM, Ruggeri ZM, Pareti FI,
Capitanio A. 1-Deamino-8-d-arginine vasopressin: a new pharmacological approach to
the management of haemophilia and von
Willebrand’s diseases. Lancet 1977;1:86972.
22. Bichet DG, Razi M, Lonergan M, et al.
Hemodynamic and coagulation responses
to 1-desamino[8-D-arginine] vasopressin in
patients with congenital nephrogenic diabetes insipidus. N Engl J Med 1988;318:881-7.
23. Zerbe R, Stropes L, Robertson G. Vasopressin function in the syndrome of inappropriate antidiuresis. Annu Rev Med 1980;
31:315-27.
24. Verbalis JG. Vasopressin V2 receptor
antagonists. J Mol Endocrinol 2002;29:1-9.
25. Huang EA, Geller DH, Gitelman SE. The
use of oral urea in the treatment of chronic
syndrome of inappropriate antidiuretic hormone secretion (SIADH) in children. Pediatr
Res 2004;55:161A. abstract.
26. Decaux G, Prospert F, Penninckx R,
Namias B, Soupart A. 5-Year treatment of
the chronic syndrome of inappropriate secretion of ADH with oral urea. Nephron 1993;
63:468-70.
Copyright © 2005 Massachusetts Medical Society.
may 5 , 2005
The New England Journal of Medicine
Downloaded from www.nejm.org at CTR HOSPITAL UNIVERSITAIRE VAUDOIS on November 6, 2010. For personal use only. No other uses without permission.
Copyright © 2005 Massachusetts Medical Society. All rights reserved.
PERSPECTIVE
Hyperactive Vasopressin Receptors and Disturbed Water Homeostasis
of great help in clarifying the relationships between the structure and function of the V2 receptor
and possibly other G protein–coupled receptors; it
may even guide the design of rational drugs for V2
receptor–associated disease.
This report adds a new example to the currently
short list of gain-of-function mutations as causes
of genetic disorders of tubular transport. The identification and functional analysis of such diseaseassociated gain-of-function mutations, however,
have proved important to our understanding of the
physiology of renal tubular transport. For instance,
functional analysis of activating mutations in the
renal epithelial sodium channel identified in Liddle’s syndrome, an autosomal dominant form of
salt-sensitive hypertension, provided further insight into normal regulation of this channel. The
activating mutations were shown to disrupt the in-
1850
teraction of the renal epithelial sodium channel
with an important regulatory protein called Nedd4;
failure of this interaction prevents the removal of
the renal epithelial sodium channel from the cell
surface, leading to constitutive expression at the
cell membrane and increased sodium reabsorption. This mechanism accounts for the development of hypertension. An increased awareness of
the possibility of gain-of-function mutations, triggered by the report of Feldman et al., may well lead,
in the near future, to the elucidation of other unexplained genetic disorders of tubular transport.
1. Verbalis JG. Disorders of body water homeostasis. Best Pract
Res Clin Endocrinol Metab 2003;17:471-503.
2. Morello JP, Bichet DG. Nephrogenic diabetes insipidus.
Annu Rev Physiol 2001;63:607-30.
3. Spiegel AM, Weinstein LS. Inherited diseases involving
G proteins and G protein-coupled receptors. Annu Rev Med
2004;55:27-39.
n engl j med 352;18
www.nejm.org
may 5, 2005
The New England Journal of Medicine
Downloaded from www.nejm.org at CTR HOSPITAL UNIVERSITAIRE VAUDOIS on November 6, 2010. For personal use only. No other uses without permission.
Copyright © 2005 Massachusetts Medical Society. All rights reserved.