Hyperactive Vasopressin Receptors and Disturbed Water Homeostasis Nine V.A.M

PERSPECTIVE The Calling

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 impor- diated activation of adenylate cylase and the forma- tance for all terrestrial organisms. In most mam- tion of cyclic adenosine monophosphate (cAMP). mals, including humans, the maintenance of water cAMP, in turn, activates protein kinase A, which pro- balance is critically dependent on water intake, the motes the fusion of cytoplasmic vesicles containing sensation of thirst, and the regulation of water ex- aquaporin-2 water-channel proteins with the apical cretion in the kidney, which is under the control membrane. As a result, this normally water-tight of the antidiuretic hormone arginine vasopressin membrane becomes water-permeable. Driven by (AVP).1 In the past decade, our insight into the the osmotic gradient of sodium, water is then trans- AVP-mediated renal concentration mechanism has cellularly reabsorbed, entering the cells through substantially improved with the elucidation of the aquaporin-2 in the apical membrane and leaving roles of crucial molecular players in this process. the cells for the interstitium through aquaporin-3 The identification of disease-causing genes in he- and aquaporin-4, which reside in the basolateral reditary disorders of water balance has been ex- membrane. The withdrawal of AVP results in en- tremely helpful in identifying these pivotal mole- docytosis of the water channels, restoring the wa- cules. ter-impermeable state of the apical membrane. In normal physiology, AVP is secreted into the Proof of principle for the critical roles of both circulation by the posterior pituitary gland, in re- the V2 receptor and aquaporin-2 in orchestrating sponse to an increase in serum osmolality or a de- the AVP-mediated renal concentration mechanism crease in effective circulating volume (see diagram). came from the identification of mutations in the In the kidney, AVP binds to the V2 vasopressin genes encoding these proteins in patients with reeptor in the basolateral membranes of collect- congenital nephrogenic diabetes insipidus.2 This ing-duct cells in the last portion of the nephron rare genetic disorder is characterized by a failure to (see diagram). Occupancy of this receptor results, concentrate urine despite normal or elevated levels by means of signaling through a guanine nucleo- of AVP. Polyuria and polydipsia, usually associated tide–binding regulatory protein (G protein), in me- 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 re- ment of Human Genetics, Radboud University Nijmegen sult in severe neurologic deficits, growth retarda- Medical Centre, Nijmegen, the Netherlands. tion, or even death. Mutations in the gene encod-

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PERSPECTIVE Hyperactive Vasopressin Receptors and Disturbed Water Homeostasis

Increased plasma osmolality or decreased arterial circulating volume Thirst Increased fluid intake Decreased plasma osmolality or increased arterial circulating volume AVP Antidiuresis SIADH

Collecting Autosomal recessive and tubule dominant CNDI

H2O H2O

Aquaporin-4 Aquaporin-3 H2O H O H O channel H O channel 2 2 2 Aquaporin-2 H2O channel Stimulating G protein cAMP Vasopressin Protein type 2 receptor kinase A Arginine ATP vasopressin Adenylate cyclase Phosphoproteins Inactivating mutations: X-linked CNDI Activating mutations: NSIAD H2O

H2O H2O

Basolateral Apical

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.

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matic mutations that have been identified in certain Diseases Caused by Activating (Gain-of-Function) Mutations adenomas and malignant tumors. Such activating in G Protein–Coupled Receptors. mutations with germ-line transmission have been Disease G Protein–Coupled Receptor identified in only a few inherited disorders (see table). Testotoxicosis (familial male-limited pre- Luteinizing hormone receptor cocious puberty) (LHR) At present, more than 180 different mutations in AVPR2 have been described. These mutations all Autosomal dominant nonimmune hyper- Thyrotropin receptor (TSHR) thyroidism cause congenital nephrogenic diabetes insipidus and, consistently, are inactivating mutations, re- Congenital stationary blindness Rhodopsin sulting in receptor malfunction at different lev- Autosomal dominant hypocalcemia Calcium-sensing receptor (CaSR) els, such as reduced receptor expression at the cell Corticotropin-independent Cushing’s Melanocortin-2 receptor (MC2R) surface or disturbances in hormone binding and syndrome G protein coupling. Familial ovarian hyperstimulation Follitropin receptor (FSHR) In this issue of the Journal (pages 1884-1890), syndrome Feldman and coauthors describe two unrelated Jansen’s metaphyseal chondrodysplasia Parathyroid hormone/parathy- male infants with euvolemic hyponatremia and se- roid hormone–related protein (PTH/PTHrP) receptor rum hypo-osmolality, along with inappropriately elevated urine osmolality and urine sodium con- Nephrogenic syndrome of inappropriate Vasopressin type 2 receptor antidiuresis (NSIAD) (V2R) centrations. At first glance, the disorder in these boys resembles the syndrome of inappropriate antidiuretic hormone secretion (SIADH), a relatively common disorder characterized by insufficient ing the V2 receptor (AVPR2) cause the X-linked form suppression of AVP secretion in relation to the de- of congenital nephrogenic diabetes insipidus, and gree of hypo-osmolality, which leads to inappropri- mutations in the gene encoding aquaporin-2 are ate urine concentration. Since serum AVP levels responsible for both the autosomal recessive and were undetectably low in both boys, Feldman et al. autosomal dominant forms. ruled out SIADH and hypothesized that a hyperac- The V2 receptor is a typical member of the large tive V2 receptor could be the underlying cause of superfamily of G protein–coupled receptors, which the disorder. Sequencing of AVPR2 in the two pa- includes receptors for hormones, neurotransmit- tients revealed a hemizygous point mutation in ters, light, odorants, lipids, and ions. Once an ago- each. In an in vitro functional assay, both muta- nist binds to such a receptor, an intracellular signal tions were shown to lead to the production of a cascade is induced through the coupling and acti- constitutively active V2 receptor. The consequence vation of a G protein. G protein–coupled receptors was AVP-independent, and therefore inappropri- play key roles in almost all physiological functions, ate, activation of V2 receptor–mediated renal urine and mutations in the genes encoding these recep- concentration. Remarkably, and for reasons that tors have been identified in patients with a variety remain unexplained, neither patient showed any of inherited and acquired disorders, such as retini- clinical or biochemical sign of constitutive activa- tis pigmentosa, hypothyroidism and hyperthyroid- tion of extrarenal V2 receptors, which are known to ism, dwarfism, fertility disorders, obesity, and even mediate increases in circulating coagulation and fi- cancer.3 Most such mutations that are associated brinolytic factors and a decrease in diastolic blood with disease are inactivating mutations, resulting pressure after AVP stimulation. in reduced or total lack of response to the agonist. The two identified activating AVPR2 mutations These loss-of-function mutations are almost ex- result in substitutions of the same amino acid, lo- clusively germ-line mutations. cated in a part of the receptor protein that is highly Activating (gain-of-function) mutations in conserved among G protein–coupled receptors and G protein–coupled receptors, which induce consti- that is involved in G protein coupling. Elucidation tutive receptor activation in the absence of the ago- of the exact mechanism underlying the activation nist, occur less frequently and consist primarily of so- of these mutant V2 receptors will undoubtedly be

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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, Divi- luid homeostasis depends on proper water intake, governed by sions of Endocrinology (B.J.F., S.M.R., an intact thirst mechanism, and on urinary excretion of free water, mediated by E.A.H., R.H.L., W.L.M., S.E.G.) and Nephrol- f appropriate secretion of arginine vasopressin (AVP) (also known as antidiuretic ogy (M.M.-A., R.S.M., A.A.P.), and the De- 1 partment of Psychiatry (G.A.V.), University hormone). AVP exerts its antidiuretic action by binding to the V2 vasopressin receptor of California at San Francisco, San Francisco; (V2R), a G protein–coupled receptor, on the basolateral membrane of epithelial cells in and Quest Diagnostics Nichols Institute, San Juan Capistrano, Calif. (R.G.F.). Address the collecting duct of the kidney. Ligand binding activates the V2R, stimulating adenyl- reprint requests to Dr. Gitelman at the Uni- ate cyclase by means of Gs proteins. The resulting increase in intracellular cyclic AMP versity of California at San Francisco, Divi- (cAMP) promotes shuttling of intracellular vesicles containing the water channel aqua- sion of Pediatric Endocrinology, 513 Par- nassus Ave., Rm. S679, Box 0434, San porin-2 to the apical membrane of the collecting-duct cells, thereby increasing water Francisco, CA 94143, or at sgitelma@peds. permeability and inducing antidiuresis. ucsf.edu. Clinical disorders of water balance are common, and alterations in many steps of 1 Drs. Feldman and Rosenthal contributed this pathway have been described. Urinary concentrating defects associated with dia- equally to this article. betes 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 N Engl J Med 2005;352:1884-90. 2 Copyright © 2005 Massachusetts Medical Society. dominant lesions in aquaporin-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

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that SIADH is usually associated with measurably ports through a protocol approved by the institu- elevated serum levels of AVP. tional review board of the University of California at We describe two unrelated male infants whose San Francisco. clinical presentation was consistent with the presence of chronic SIADH but who had undetectable methods AVP levels. We postulated that novel activating mutations of the V2R might account for their unique mutation analysis presentation. Evaluation revealed novel activating Genomic DNA from the two patients and their mutations of the V2R leading to what we term mothers was isolated from whole blood with the use “nephrogenic syndrome of inappropriate antidi- of the Puregene Blood Kit (Gentra Systems). The uresis” (NSIAD). entire coding region of the V2R gene, AVPR2, was amplified as described previously.4 The resulting case reports amplicons were sequenced with multiple forward and reverse primers with the use of Big Dye (version Patient 1 presented at 3 months of age with irrita- 3.1) sequencing chemistry and an ABI Prism se- bility, and Patient 2 presented at 2.5 months of age quencer (model 3100, Applied Biosystems) accord- with two generalized seizures. Both children had ing to the manufacturer’s protocols. SeqScape soft- had unremarkable early neonatal courses. Both ware (Applied Biosystems) was used to assemble were exclusively bottle-fed formula (7 mmol of sodi- the sequence data and compare the results with um per liter). Both infants had mild systolic hyper- the AVPR2 reference sequence (GenBank accession tension with otherwise normal physical examina- number NT 025965). tions. Initial laboratory evaluations demonstrated hyponatremia with normal serum levels of potassi- construction of vasopressin expression um and bicarbonate (Table 1). Both children had constructs serum hypo-osmolality with inappropriately elevat- pCDNA3 (Invitrogen) was used as the control plas- ed urinary osmolality and urinary sodium levels. mid. Human wild-type V2R complementary DNA Both had low blood urea nitrogen and low or low- (cDNA),5 subcloned into pCDNA3, was used as a normal serum creatinine levels, low or suppressed normal control and template for mutagenesis. Site- plasma renin activity, and normal aldosterone lev- directed mutagenesis was performed with the use els, indicating euvolemia. Serum cortisol, thyroid- of a QuickChange II site-directed mutagenesis kit function tests, and coagulation studies were all (Stratagene). Mutations were confirmed by direct normal. Imaging studies of the head and chest sequencing. were unremarkable, with the exception of a small pars intermedia cyst in Patient 2. Despite clinical cell culture and transient transfection and laboratory presentations consistent with the For functional studies, COS-7 cells were cultured presence of SIADH, serum AVP levels were unde- in Dulbecco’s modified Eagle’s medium with 10 tectable in both patients (Quest Diagnostics Nichols percent fetal-calf serum and antibiotics. Cells were Institute). Both children were initially treated with plated in six-well plates (Falcon 3046, Becton Dick- fluid restriction, followed by the administration of inson) at approximately 95 percent confluence 24 an osmotic agent (urea), resulting in increased uri- hours before transfection with the use of Lipofecta- nary output and normalization of the serum sodi- mine 2000 (Invitrogen) according to the manufac- um level. turer’s protocol. Each well received 3 µg of plasmid Neither child had a family history of hyponatre- DNA and 500 ng of a cAMP-responsive luciferase mia or of an SIADH-like syndrome. Both patients reporter plasmid (pCREluc) containing 16 copies had no siblings. The mother of Patient 1, subse- of the consensus cAMP response element.6 To con- quently found to be heterozygous for an activat- trol for transfection efficiency, cells were cotrans- ing mutation of AVPR2 (see below), had normal fected with 50 ng of renilla luciferase reporter simultaneous serum sodium levels (140 mmol per plasmid (pRL-CMV, Promega) per well. Cells were liter) and serum and urine osmolality (293 and 795 incubated at 37°C in 5 percent carbon dioxide for mOsm per kilogram of water, respectively). 24 hours after transfection and were then lysed and The parents of the patients provided written in- assayed for luciferase activity with the use of the formed consent for the publication of the case re- Dual Luciferase Reporter Assay System (Promega),

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Table 1. Characteristics of the Two Patients.*

Characteristic Patient 1 Patient 2 Age-Matched Controls

Age at presentation (mo) 3.0 2.5 — Clinically significant findings Irritability Generalized seizures — Blood pressure (mm Hg) Systolic 80–128 93–118 108† Diastolic 34–92 37–57 66† Serum and plasma studies Sodium (mmol/liter) 123 118 134–143 Potassium (mmol/liter) 4.6 4.7 3.4–4.9 Chloride (mmol/liter) 91 86 98–107 Bicarbonate (mmol/liter) 28 21 23–32 Creatinine (mg/dl) <0.3 0.3 0.3–0.7 Urea nitrogen (mg/dl) <5 3 8–23 Aldosterone (ng/dl) 10 24‡ 6–68 Plasma renin activity (ng of angiotensin I/ml/hr) 3.8 <1‡ <15 Osmolality (mOsm/kg) 252 247 285–293 AVP (pg/ml) <1 <1‡ 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§ 4–20 60 Min after intravenous synthetic corticotropin 36.5 — >20 (15µg/kg) Coagulation studies von Willebrand factor antigen (%) 104 100 46–155 Ristocetin cofactor (%) 108 65 56–155 Factor VIII activity (%) 153 123 50–150 Fibrinogen (mg/dl) 198 360 170–435 d-Dimer fragment Negative Negative Negative Prothrombin time (sec) 11.7 10.9 9.2–11.9 Partial-thromboplastin time (sec) 24.3 32.5 21.3–34.8 Urine studies Osmolality (mOsm/kg) 284 390‡ 300–900 Sodium (mmol/liter) 35 75‡ — 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.

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7,8 as described previously. Data are presented as discussion mean (±SE) luciferase activity expressed in arbitrary units and adjusted for renilla luciferase activity in G protein–coupled receptors constitute the largest three experiments, each performed in triplicate. gene family of receptors involved in signal transduc- All five types of samples (pCDNA3 vector alone and tion and are responsible for regulating many phys- vector with wild-type V2R, the R137C mutant, the iological processes.10 Many diseases are caused by R137L mutant, or the R137H mutant) were run si- mutations in G protein–coupled receptors.11,12 multaneously under the same conditions during For some G protein–coupled receptors, a particular each of the three experiments. disease state has been ascribed to inactivating mutations that render the receptors unresponsive to li- results gand, whereas a converse condition has been linked to gain-of-function mutations, resulting in consti- Genomic DNA was isolated from both boys and tutive activation. their mothers, and the V2R gene, AVPR2, was se- Although many different inactivating mutations quenced directly. AVPR2 is X-linked, and therefore, of the X-linked V2R have been described that cause paternal DNA is not informative. Each patient car- nephrogenic diabetes insipidus,2 to our knowledge, ried a mutation in codon 137 of AVPR2. In Patient 1, no naturally occurring activating mutations of the nucleotide 770 was mutated from cytosine to thym- V2R have been reported previously. The two cases ine, changing arginine to cysteine at codon 137 described here are characterized by chronic SIADH (R137C); in Patient 2, nucleotide 771 was mutated but with undetectable AVP levels, constituting a from guanine to thymine, changing arginine to leu- novel example of gain-of-function mutations caused cine at codon 137 (R137L) (Fig. 1A). Arginine 137 by a hemizygous V2R mutation. In the light of these maps to the predicted second cytoplasmic loop, findings, we suggest referring to all SIADH-like near the cytoplasmic boundary of the third trans- conditions as syndromes of inappropriate anti- membrane domain; this same amino acid is mutat- diuresis (SIAD) and that these two case reports con- ed to histidine (R137H) in a form of familial neph- stitute a subtype, NSIAD. To our knowledge, these rogenic diabetes insipidus (Fig. 1B). The mother of cases are the only reported examples in which mu- Patient 1 was heterozygous for the R137C muta- tations affecting the same amino acid cause two tion, whereas the mother of Patient 2 was homozy- different genetic diseases: R137H causes nephro- gous for wild-type AVPR2, suggesting that Patient 2 genic diabetes insipidus, and R137L and R137C had a spontaneous mutation. cause NSIAD (Fig. 1B). To evaluate the effect of these novel mutations The mechanism by which these missense muta- on V2R function, we developed a functional assay tions constitutively activate the V2R requires further for V2R. Production of cAMP has previously been investigation. V2R, a class 1b G protein–coupled re- used to assess many G protein–coupled receptors, ceptor,12,13 exists in the plasma membrane in equi- including V2R9; we adopted our previous pro- librium with inactive and active conformations.10 cedures8 to use with V2R. COS-7 cells transiently The binding of ligand shifts the equilibrium to the transfected with the vector alone, wild-type V2R, or active state, permitting coupling with intracellular the R137H nephrogenic diabetes insipidus mutant G proteins and activation of intracellular effectors. induced low levels of cAMP (Fig. 2). However, bas- With activation, V2R is desensitized through phos- al levels of cAMP production in cells expressing phorylation by specific G protein–coupled recep- V2R with the R137C mutation were four times the tor kinases. Subsequent recruitment of b arrestin levels in cells expressing wild-type V2R (P=0.01), to the phosphorylated receptor terminates the and cells expressing the R137L mutant had 7.5 signal by blocking further interaction with G pro- times the level of activity of cells expressing wild- teins and also initiates receptor internalization type V2R (P<0.004) (Fig. 2). These results indicate through its ability to bind clathrin and other en- that these novel mutations create a constitutively docytic adapters.14 active V2R and provide an explanation for the hy- By comparison with other class 1 G protein– ponatremia with increased urinary osmolality in our coupled receptors, the highly conserved motif of patients. The condition in both patients is clinically aspartic acid, arginine, and tyrosine or histidine similar to SIADH, despite the fact that AVP levels (DRY/H) in V2R at the junction of the third trans- were undetectable. membrane domain and second intracellular loop

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A Wild Type Patient 1 Patient 2

R137 R137C R137L

T T S A M P S L M –NH2 S A P H L G V S L P N S P S S Q R F E D R R T G P A P A E P W L P L F G K D C L D D R A A S G G E A A R T W R P P W G E A L T D L R C V L C V E A D T Y W E Q R R N V A G Extracellular L P A T A A L V A Q W W P L A V G K F I Q L F V S F Y I A L L V L F L L L Q F M F L M I A V V L Q F V F L V V A M P V A P L A A L G M L A P W S Cell A D A A Y S L L T C N L L V L S L L G V L S membrane G S C S S F I V Y C T L L H M Y A A V N A N I G I W A C I V P W V F V L A A V V Q V I A L H M L L T L Y L I T L V I F M A S Intracellular A P D P R R S F C C R E S L A R A H R I V S R H V L R R W N T A A S S G G H R137 W K S E L R R G I S R R H V S A C L T A H A V R V A P A P G P P G S M S G C S E D Q P G L L G E A P T Y R H G P S S G T E T A S R R S S L P G R A K G R R R G D T S S –COOH M T C L NSIAD D L R H H: Congenital R nephrogenic diabetes A 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.

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appears to be critical for receptor function. The ar- 9 ginine residue in the DRY/H motif appears to be in- P<0.004 variant, though the aspartic acid and tyrosine may 8 be replaced by other amino acids without altering the function (e.g., from aspartic acid to glutamic 7 15 acid). The arginine residue in the DRY/H motif 6 corresponds to R137, the site of the inactivating 5 R137H mutation in nephrogenic diabetes insipidus P=0.01 and the activating R137C and R137L mutations in 4

NSIAD. The R137H mutant behaves as a constitu- Reporter Units tively desensitized receptor, since it is phosphory- 3 lated, binds to b arrestin (thereby blocking its ability 2 to activate G proteins), and is sequestered in intracellular vesicles.16 Relative No. of cAMP-Inducible Luciferase 1 Constitutively activated mutant G protein–cou- 0 Control Wild-Type R137H R137C R137L pled receptors have also been created by altering this V2R Mutant Mutant Mutant 17 DRY/H motif — for example, through in vitro mu- V2R V2R V2R tation of aspartate to alanine at codon 136 in the V2R.18 The NSIAD mutations found in this domain Figure 2. Basal Levels of cAMP Production in Cells Expressing Wild-Type and Mutant V2R. may stabilize the receptor in an active conforma- The ability of the mutant V2R from each patient to induce cAMP, as measured tion, activating G proteins and downstream signal- by a cAMP-inducible luciferase reporter, was evaluated. Each bar represents ing events in the absence of ligand. In theory, such cells transfected with the renilla luciferase expression vector, the cAMP-induc- a gain of function could also affect many other as- ible luciferase reporter plasmid, and pCDNA3 either alone (control), reflect- pects of V2R biology, including phosphorylation, ing basal cAMP levels in the cells, or with the following inserts subcloned into internalization, down-regulation, and recycling to the pCDNA3 plasmid: wild-type V2R; R137H mutant V2R, found in some patients with nephrogenic diabetes insipidus; R137C mutant V2R, found in Pa- the cell membrane. tient 1; or R137L mutant V2R, found in Patient 2. Each value represents the The effects of the constitutive activation of V2R mean (±SE) of three independent experiments with all five samples run simul- may not be limited to the kidney. V2R is also ex- taneously, each performed in triplicate. pressed in endothelial cells, where it appears to mediate vasodilation after the administration of the vasopressin analogue desmopressin19,20 and to Patients with NSIAD would be expected to have mediate the rise in circulating levels of von Wille- low AVP levels. However, since the AVP assay is not brand factor and tissue plasminogen activator.21 optimized to identify low values, we recommend We thought that these responses, which are absent sequencing the V2R gene to identify specific mu- in patients with nephrogenic diabetes insipidus,22 tations before the diagnosis of NSIAD is final- might be constitutively activated in our patients ized. Other patients may be identified who have with NSIAD. However, we found no clinical or lab- the NSIAD phenotype but without V2R mutations, oratory evidence of coagulopathy in either patient suggesting the presence of additional defects in (Table 1). this signaling cascade. One such possibility would The frequency of NSIAD is not known, but it be an activating mutation in aquaporin-2. may not be rare. Previous studies of patients with Treatment of NSIAD poses a challenge. Water SIADH have noted variable patterns of AVP secre- restriction improved serum sodium levels and os- tion, particularly in response to water loading and molality in both infants but limited calorie intake water restriction.23 As many as 10 to 20 percent of in these formula-fed infants. Agents that act down- affected patients have AVP levels at or below the stream from the V2R, such as demeclocycline or limits of detection by radioimmunoassay. Thus, lithium, might antagonize the constitutively acti- some of these patients may in fact have NSIAD due vated receptors, but they have potentially limiting to V2R-activating mutations. Such patients would adverse effects. AVP antagonists are under clinical probably have clinical presentations similar to those development but would probably be ineffective, giv- in our patients, although the severity and clinical en the ligand-independent nature of the lesion.24 course may depend on the nature of the mutation. Ideally, one might be able to use an inverse agonist

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brief report

that would suppress receptor activity in the absence Dr. Rosenthal reports having received grant support and speak- of agonist; two potential nonpeptide V2R inverse ing honoraria from Pfizer and having served on an advisory board 18 for Tercica and Pfizer. Dr. Vargas reports having received grant sup- agonists have been studied in vitro. In the absence port from Wyeth and Janssen. Dr. Lustig reports having received lec- of a definitive therapy, we have successfully treated ture fees from Novo Nordisk and grant support from Novartis. Dr. our patients with urea to induce an osmotic diure- Mathias reports having equity interest in AstraZeneca, Pfizer, Merck, 25 and SIRNA Therapeutics; receiving grant support from Satellite sis. This approach has occasionally been used for Healthcare and Genentech; and receiving speaking honoraria from the treatment of chronic SIADH in adults.26 Shire. Dr. Fenwick is employed by Quest Diagnostics, owns stock in Study of the V2R has been important for under- the company, and developed the clinical assay used for sequencing AVPR2 in this study. standing the physiology of water balance and has We are indebted to Ian Ocrant and Kamer Tezcan for referring the served as a prototype for G protein–coupled recep- patients; to Mariel Birnbaumer (National Institute of Environmental tor biology. Further characterization of NSIAD may Health Sciences, Division of Intramural Research) for sharing the V2R cDNA; to Christian Vaisse (University of California at San Fran- offer additional insights into fluid homeostasis cisco) for the cAMP-responsive luciferase reporter plasmid; to Mark and clinical disease, as well as expand our under- von Zastrow (University of California at San Francisco), Jon Naka- standing of G protein–coupled receptor signaling. moto (Quest Diagnostics), and Hillel Gitelman for thoughtful dis- cussions and review of the manuscript; and to Izabella Damm (Uni- Supported in part by a training grant (T32DK07161, to Drs. Feld- versity of California at San Francisco) for technical support. man and Huang) and a grant (M01RR01271, to the Pediatric Clini- cal Research Center) from the National Institutes of Health.

references 1. Robertson GL. Antidiuretic hormone: stimulate adenylyl cyclase. J Biol Chem 1993; nists with partial agonist and inverse ago- normal and disordered function. Endocrinol 268:13030-3. nist activities. FEBS Lett 1998;441:470-5. Metab Clin North Am 2001;30:671-94. 10. Pierce KL, Premont RT, Lefkowitz RJ. 19. Hirsch AT, Dzau VJ, Majzoub JA, Crea- 2. Morello JP, Bichet DG. Nephrogenic dia- Seven-transmembrane receptors. Nat Rev ger MA. Vasopressin-mediated forearm vaso- betes insipidus. Annu Rev Physiol 2001;63: Mol Cell Biol 2002;3:639-50. dilation in normal humans: evidence for a 607-30. 11. Spiegel AM, Weinstein LS. Inherited vascular vasopressin V2 receptor. J Clin 3. Bartter FC, Schwartz WB. The syndrome diseases involving G proteins and G protein- Invest 1989;84:418-26. of inappropriate secretion of antidiuretic coupled receptors. Annu Rev Med 2004;55: 20. Kaufmann JE, Iezzi M, Vischer UM. Des- hormone. Am J Med 1967;42:790-806. 27-39. mopressin (DDAVP) induces NO production 4. Bichet DG, Arthus MF, Lonergan M, et 12. Seifert R, Wenzel-Seifert K. Constitutive in human endothelial cells via V2 receptor- al. X-linked nephrogenic diabetes insipi- activity of G-protein-coupled receptors: cause and cAMP-mediated signaling. J Thromb dus mutations in North America and the of disease and common property of wild- Haemost 2003;1:821-8. Hopewell hypothesis. J Clin Invest 1993;92: type receptors. Naunyn Schmiedebergs Arch 21. Mannucci PM, Ruggeri ZM, Pareti FI, 1262-8. Pharmacol 2002;366:381-416. Capitanio A. 1-Deamino-8-d-arginine vaso- 5. Birnbaumer M, Gilbert S, Rosenthal W. 13. Bockaert J, Pin JP. Molecular tinkering pressin: a new pharmacological approach to An extracellular congenital nephrogenic dia- of G protein-coupled receptors: an evolu- the management of haemophilia and von betes insipidus mutation of the vasopressin tionary success. EMBO J 1999;18:1723-9. Willebrand’s diseases. Lancet 1977;1:869- receptor reduces cell surface expression, 14. Luttrell LM, Lefkowitz RJ. The role of 72. affinity for ligand, and coupling to the Gs/ beta-arrestins in the termination and trans- 22. Bichet DG, Razi M, Lonergan M, et al. adenylyl cyclase system. Mol Endocrinol duction of G-protein-coupled receptor sig- Hemodynamic and coagulation responses 1994;8:886-94. nals. J Cell Sci 2002;115:455-65. to 1-desamino[8-D-arginine] vasopressin in 6. Vaisse C, Clement K, Durand E, Herc- 15. Probst WC, Snyder LA, Schuster DI, patients with congenital nephrogenic diabe- berg S, Guy-Grand B, Froguel P. Melanocor- Brosius J, Sealfon SC. Sequence alignment tes insipidus. N Engl J Med 1988;318:881-7. tin-4 receptor mutations are a frequent and of the G-protein coupled receptor super- 23. Zerbe R, Stropes L, Robertson G. Vaso- heterogeneous cause of morbid obesity. J Clin family. DNA Cell Biol 1992;11:1-20. pressin function in the syndrome of inap- Invest 2000;106:253-62. 16. Barak LS, Oakley RH, Laporte SA, Caron propriate antidiuresis. Annu Rev Med 1980; 7. Stables J, Scott S, Brown S, et al. Devel- MG. Constitutive arrestin-mediated desen- 31:315-27. opment of a dual glow-signal firefly and sitization of a human vasopressin receptor 24. Verbalis JG. Vasopressin V2 receptor Renilla luciferase assay reagent for the analy- mutant associated with nephrogenic diabe- antagonists. J Mol Endocrinol 2002;29:1-9. sis of G-protein coupled receptor signalling. tes insipidus. Proc Natl Acad Sci U S A 2001; 25. Huang EA, Geller DH, Gitelman SE. The J Recept Signal Transduct Res 1999;19:395- 98:93-8. use of oral urea in the treatment of chronic 410. 17. Parnot C, Miserey-Lenkei S, Bardin S, syndrome of inappropriate antidiuretic hor- 8. Fluck CE, Martens JW, Conte FA, Miller Corvol P, Clauser E. Lessons from constitu- mone secretion (SIADH) in children. Pediatr WL. Clinical, genetic, and functional char- tively active mutants of G protein-coupled Res 2004;55:161A. abstract. acterization of adrenocorticotropin receptor receptors. Trends Endocrinol Metab 2002; 26. Decaux G, Prospert F, Penninckx R, mutations using a novel receptor assay. 13:336-43. Namias B, Soupart A. 5-Year treatment of J Clin Endocrinol Metab 2002;87:4318-23. 18. Morin D, Cotte N, Balestre MN, et al. the chronic syndrome of inappropriate secre- 9. Rosenthal W, Antaramian A, Gilbert S, The D136A mutation of the V2 vasopressin tion of ADH with oral urea. Nephron 1993; Birnbaumer M. Nephrogenic diabetes insip- receptor induces a constitutive activity which 63:468-70. idus: a V2 vasopressin receptor unable to permits discrimination between antago- Copyright © 2005 Massachusetts Medical Society.

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of great help in clarifying the relationships be- teraction of the renal epithelial sodium channel tween the structure and function of the V2 receptor with an important regulatory protein called Nedd4; and possibly other G protein–coupled receptors; it failure of this interaction prevents the removal of may even guide the design of rational drugs for V2 the renal epithelial sodium channel from the cell receptor–associated disease. surface, leading to constitutive expression at the This report adds a new example to the currently cell membrane and increased sodium reabsorp- short list of gain-of-function mutations as causes tion. This mechanism accounts for the develop- of genetic disorders of tubular transport. The iden- ment of hypertension. An increased awareness of tification and functional analysis of such disease- the possibility of gain-of-function mutations, trig- associated gain-of-function mutations, however, gered by the report of Feldman et al., may well lead, have proved important to our understanding of the in the near future, to the elucidation of other unex- physiology of renal tubular transport. For instance, plained genetic disorders of tubular transport. functional analysis of activating mutations in the 1. Verbalis JG. Disorders of body water homeostasis. Best Pract renal epithelial sodium channel identified in Lid- Res Clin Endocrinol Metab 2003;17:471-503. dle’s syndrome, an autosomal dominant form of 2. Morello JP, Bichet DG. Nephrogenic diabetes insipidus. salt-sensitive hypertension, provided further in- Annu Rev Physiol 2001;63:607-30. 3. Spiegel AM, Weinstein LS. Inherited diseases involving sight into normal regulation of this channel. The G proteins and G protein-coupled receptors. Annu Rev Med activating mutations were shown to disrupt the in- 2004;55:27-39.

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