Relief of Nocturnal Enuresis by Desmopressin Is Kidney and Vasopressin Type 2 Receptor Independent

JASN Express. Published on March 27, 2007 as doi: 10.1681/ASN.2006080907

Joris H. Robben,* Mozes Sze,* Nine V. Knoers,† Paul Eggert,‡ Peter Deen,* and Dominik Mu¨ller§ *Department of Physiology, Nijmegen Centre of Molecular Life Sciences, and †Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands; and ‡University Children’s Hospital, Kiel, and §Department of Pediatric Nephrology, Charite´Berlin, Berlin, Germany

Primary nocturnal enuresis (PNE) is a common problem in childhood and adolescence. Although various treatments are highly effective, a common underlying hypothesis on the pathogenesis is lacking. The success of desmopressin, a synthetic analogue of the antidiuretic hormone vasopressin, has been attributed to increased renal water reabsorption that is mediated by activation of the renal vasopressin V2 receptor (V2R). However, this effect does not explain other symptoms of PNE, such as the failure to arouse upon bladder distension. This study identified a family in which one child displayed PNE and coexisting nephrogenic diabetes insipidus, as a result of a novel nonsense mutation in the V2R gene (C358X). Cell-biologic investigations revealed that V2R-C358X is retained in the endoplasmic reticulum and is unstable, which explains his nephrogenic diabetes insipidus. Consistently, extrarenal V2R-mediated responses were absent in the patient who was treated with desmopressin. Administration of desmopressin, however, changed his PNE into nocturia, because he now still voided unchanged high urinary volumes at night but woke up and went to the bathroom. Withdrawal of desmopressin was accompanied by bedwetting, whereas reintroduction again relieved the symptoms. Therefore, these data indicate that neither a functioning renal concentration system nor a functional V2R is needed for the therapeutic benefit of desmopressin in PNE. Rather, it suggests that another vasopressin receptor and other organ(s) is the target for desmopressin to relieve PNE. J Am Soc Nephrol ●●: ●●●–●●●, ●●●●. doi: 10.1681/ASN.2006080907

he major role of the vasopressin type-2 receptor (V2R) Primary nocturnal enuresis (PNE) is defined as the persis- is the regulation of the body water homeostasis by tence of nightly bedwetting in children who are at least 5 yr of T determining the level of reabsorption of water from age and is one of the most frequent complaints in pediatric and pro-urine through aquaporin-2 (AQP2) water channels. In urologic practice. Although the frequency of enuretic episodes states of hypovolemia or hypernatremia, as well as during sleep in a child is not clearly defined, at least three episodes per week when there is functional antidiuresis, the release of the antidi- in a patient who has never been continuously dry for longer uretic hormone arginine-vasopressin (AVP) from the pituitary than 6 mo is generally accepted for the diagnosis of PNE (4,5). is increased. Upon binding of AVP to its V2R, it activates Despite a maturation rate of 15% per year, 0.5% of all cases adenylate cyclase via a stimulatory G protein. The subsequent remain in adulthood, with serious effects on self-esteem (6). increase of intracellular cAMP induces protein kinase A to Among various hypotheses on the pathophysiology of PNE, it phosphorylate, among other proteins, AQP2, which is then has been proposed that patients with PNE have insufficient redistributed from intracellular vesicles to the apical mem- nightly increase of AVP, leading to the production of large brane. Driven by the sodium and urea gradient, water is sub- amounts of dilute urine, thereby surpassing the bladder capac- sequently reabsorbed from the pro-urine through AQP2, result- ity (7). Successful application of a synthetic analogue of AVP, ing in concentrated urine. Removal of AVP reverses this desmopressin (dDAVP), a V2R agonist, seemed to confirm this process, restoring the water-impermeable state of the apical hypothesis. One of the major criticisms of this concept, how- membrane and occurrence of diluted urine. Mutations in the ever, is that patients with PNE do not wake up before, during, human V2R result in X-linked nephrogenic diabetes insipidus or after voiding, which is difficult to explain by an increased (NDI), a disorder in which patients are unable to concentrate urine production or by a small functional bladder capacity. their urine in response to AVP, resulting in the excretion of Here, we describe the identification of a family with two large volumes of diluted urine (1–3). brothers with NDI as a result of a mutation in the V2R gene (Xq28) leading to a premature stop codon (C358X) in the V2R Received August 26, 2006. Accepted February 11, 2007. protein. Besides NDI, one patient also experiences PNE. Ad- ministration of desmopressin and the use of alarm treatment Published online ahead of print. Publication date available at www.jasn.org. relieved PNE completely, whereas NDI remained unaffected. Address correspondence to: Dr. Dominik Mu¨ller, Charite´Department of Pediat- ric Nephrology, Augustenburger Platz 1, 13535 Berlin, Germany. Phone ϩ49-30- Cell biologic characterization of this novel mutation revealed 450-616147; Fax ϩ49-30-450-516912; E-mail: [email protected] instability and retention in the endoplasmic reticulum of the

Copyright © ●●●● by the American Society of Nephrology ISSN: 1046-6673/●●●●-0001 2 Journal of the American Society of Nephrology J Am Soc Nephrol ●●: ●●●–●●●, ●●●● mutant V2R protein, thereby explaining underlying NDI. These Ten micrograms of total RNA was run on a 1% Agarose/MOPS gel findings not only exclude a functioning urinary concentration with 7% formaldehyde and subsequently transferred to a nitrocellulose 32 system as a prerequisite for the beneficial action of desmopres- blot. V2R mRNA was detected using a [ P]-labeled cDNA probe of 0.65 sin in PNE but also reveal that this effect is V2R independent. kb corresponding to the PstI-HindIII fragment of the V2R coding sequence. Materials and Methods Immunoblotting and Immunocytochemistry Patient Analysis PAGE and immunoblotting were performed as described previously Serum and urinary electrolyte levels and osmolalities, vasopressin (12). Immunodetection was performed using 1:5000 diluted peroxidase- levels, and body weight and BP were measured by standard proce- conjugated mouse anti-FLAG antibodies (Sigma, St. Louis, MO). Im- dures. A thirst trial with additional infusion of desmopressin (0.3 munocytochemistry and confocal laser scanning microscopy were per- ␮g/kg body wt) at 7 h after the start of the thirst trial was done as formed as described previously (12). For immunocytochemistry under described previously (8). According to established standards, the test nonpermeabilized conditions, cells were incubated for1hat4°Cwith was stopped when clinical symptoms such as severe arterial hypoten- 1:100 diluted mouse anti-FLAG antibodies (Sigma) in PBS supple- sion and tachycardia occurred for more than three subsequent mea- mented with 1 mM MgCl and 0.1 mM CaCl . As secondary antibodies, surements or when body weight was reduced by Ͼ10% during the trial. 2 2 1:100-diluted goat anti-mouse coupled to Alexa-594 (Molecular Probes, The activities of von Willebrand factor and clotting factor VIII were Leiden, The Netherlands) was used. determined as described previously (8). When the indomethacin (3 mg/kg body wt per d) and hydrochlorothiazide (HCT; 1 mg/kg body wt per d) treatment was started, it Results reduced urinary volumes by approximately one third (daytime output Patient Analysis 5.1 ml/kg body wt; nighttime output 5.0 ml/kg body wt). Parent’s The oldest son of a white, nonconsanguineous family was informed consent was obtained for all studies, and all clinical experi- born in the 34th week of gestation (Figure 1). Polyhydramnios ments were performed in accordance to the Declaration of Helsinki. was noted during pregnancy. After birth, the child developed Isolation of genomic DNA, amplification of the V2R coding regions, poorly with vomiting and repeated episodes of hyperpyrexia. and sequence analyses were done as described previously (9,10). Age-related height and weight remained below the third per- ϩ centiles. High serum sodium (serum Na 158 mmol/L) and Expression Constructs polyuria (8.2 ml/kg body wt per h) pointed to a renal concen- The pEGFP-N1-wtV2R expression construct was a gift of Dr. Alex- tration defect. To establish whether our male proband had NDI, ander Oksche (FMP, Berlin, Germany). To allow N-terminal detection we performed a first thirst trial combined with desmopressin of the V2R, a FLAG-tag was fused to the N-terminus using the PCR infusion when he was 4 yr of age. This revealed unchanged with the sense primers 5Ј-ATGGACTACAAGGATGACGATGA- urinary osmolalities between the start (235 mosmol/kg H O) CAAGACCATGCTCATGGCGTCCACCAC-3Ј (FLAG-encoding se- 2 and end (229 mosmol/kg H O) of the treatment. Table 1 dis- quence in bold) and the antisense primer 5Ј-GCTGAGAAGGAGC- 2 GAGAAG-3Ј. In a second PCR reaction, using the same antisense plays the biochemical and physiologic data during this thirst primer and the sense primer 5Ј-GTCTACTCGAGCCACCATGGAC- trial. For confirmation of the diagnosis of NDI in the absence of TACAAG-3Ј, an XhoI site and the Kozak sequence (indicated in bold) mutation analysis, which was not available at that time, two were added to the 5Ј end of the first PCR product. The resulting 529-bp subsequent thirst-desmopressin trials were done at 4.5 and 5 yr PCR product was cut with XhoI and PstI and cloned into the XhoI/PstI of age. These revealed urinary osmolalities of 112 and 101 digested pEGFP-N1-V2R construct to yield pEGFP-N1-FLAG-V2R. mosmol/kg H2O (trial 1) and 150 and 138 mosmol/kg H2O Site-directed mutagenesis was performed on pEGFP-N1-wtV2R as de- (trial 2) before and after treatment, respectively. On the basis of Ј scribed previously (11) using the sense primer 5 -CAAGATGAGTCCT- these data, NDI was diagnosed. The child was started on HCT Ј GAACCACCGCCAGCTC-3 (C358X; mutated codon indicated in bold) and indomethacin after the last thirst trial was completed, and and its complementary antisense primer. After correct clones were the diagnosis of NDI was established. For further determina- digested with HindIII and BamHI (C358X), the mutation-containing fragment was isolated and cloned into the corresponding sites of the pEGFP-N1-FLAG-V2R. Sequence analysis of selected clones confirmed that only the desired mutations were introduced.

Cell Culture and Transfection Maintenance of MDCK type I cells and generation of stable clones were performed as described previously (8). COS-M6 cells were main- tained and transfected with 1 ␮g of expression construct (unless indicated otherwise) as described previously (11). Before analysis, COS cells that transiently expressed FLAG-wtV2R-GFP or FLAG-V2R- C358X were seeded in six multiwell plates (Costar, Cambridge, MA), and grown for 2 d. Figure 1. Pedigree of the family. f, Patients with nephrogenic RNA Isolation and Northern Blotting diabetes insipidus (NDI). PNE, primary nocturnal enuresis; wt, Transiently transfected COS cells were trypsinized and subsequently wild-type. The vasopressin V2 receptor (V2R) gene is located counted. Total RNA of 5 million cells was isolated using the RNeasy kit on the X-chromosome; therefore, male family members have (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. only one copy of the gene. J Am Soc Nephrol ●●: ●●●–●●●, ●●●● Relief of Nocturnal Enuresis by Desmopressin 3

Table 1. Thirst trial: Given are the biochemical and clinical data of the patient during the first thirst triala

7h Parameter Before 1 h 2 h 3 h 4 h 5 h 6 h (Desmo- 8h 9h pressin) (End)

Body weight (kg) 12.2 12.0 11.8 11.5 11.0 10.9 10.5 10.4 10.3 10.1 Blood pressure (mmHg) 73/55 83/56 95/67 84/56 79/51 65/49 59/45 51/43 48/34 45/37 Heart rate (bpm) 102 103 97 112 114 111 123 122 131 133

S-Osmol (mosmol/kg H2O) 297 302 314 321 325 326 329 330 335 339 U-Osmol (mosmol/kg H2O) 235 238 225 205 204 210 223 222 229 229 U-Vol (ml/kg per h) 10.5 10.3 10.2 12.5 9.5 8.7 8.2 8.3 8.8 9.1 ϩ U-Ca2 (mmol/kg per 24 h) 0.04 Vasopressin (ng/L) 84 75 94 Platelet count (per nl) 420 396 432 434 444 431 444 471 495 491

ϩ aS-Osmol, serum osmolality; U-Osmol, urinary osmolality; U-Vol, urinary volume; U-Ca2 , urinary calcium.

tion of whether daytime and nighttime urine volumes differed molecular cause of NDI. with the patient under adequate NDI treatment, 12-h diurnal and nocturnal urine volumes were measured on repetitive mea- Patient Responses to Desmopressin surements (four samplings) over a 12-mo period. However, the Ever since birth and despite the indomethacin/HCT treat- median diurnal (8.5 ml/kg body wt; range 6.9 to 11.2) and ment started at age 5, the patient wet his bed at night without nocturnal (8.8 ml/kg body wt; range 5.2 to 11.4) urine volumes waking up. At the age of 10 and during an observation period did not differ. Note that measurements were used only when of 3 mo, he wet his bed on average at least three times per week bedwetting had not occurred during the night. (as assessed by a chart completed by the parents; Table 2), X-linked NDI is due to mutations in the V2R gene, whereas consistent with the diagnosis of PNE. Then, desmopressin ad- mutations in the AQP2 water channel gene cause autosomal ministration (20 ␮g intranasally before going to bed) was initi- NDI. Besides their ability to induce urine concentration, AVP ated 1 mo after indomethacin and HCT administration was and desmopressin are known to increase levels and therewith stopped. After 3 d, the child did not wet his bed anymore but the activities of von Willebrand factor and blood clotting factor woke up and went to the toilet instead. Withdrawal of the VIII in plasma. Because this occurs through activation of the medication after 3 mo of dryness resulted in recurrence of PNE, V2R, this response is absent in patients with inactivating mu- whereas reintroduction after 4 wk again led to disappearance of tations of the V2R gene but is unaffected in patients with NDI PNE. The treatment left the urinary volume unchanged (before and AQP2 gene mutations (8). To discriminate between these 9.1 ml/kg body wt per h [9.0 daytime and 9.2 nighttime] and two, we therefore these factors measured after infusion of des- 9.3 ml/kg body wt per h during treatment [9.2 daytime and 9.4 mopressin (0.3 ␮g/kg body wt). However, no changes in the nighttime]). Reintroduction of indomethacin and HCT treat- activity of von Willebrand factor and clotting factor VIII were ment, together with desmopressin, reduced urinary volume found (Figure 2), which indicated that a V2R defect is the

Table 2. Frequency of enuretic episodes for periods of various medical treatmentsa

Enuretic Observation Events/Wk Treatment Period (Median (mo) ͓Range͔)

HCT and Indomethacin 3 6 (4 to 7) Off medication 1 5 (4 to 7) Desmopressin 3 0 (0 to 1) Off medication 1 5 (4 to 7) Desmopressin 3 0 (0 to 1) Desmopressin, HCT, 3 0 (0 to 1) Figure 2. Activity of von Willebrand factor and clotting factor VIII (%) after dDAVP infusion (0.3 ␮g/kg) in the patient with indomethacin ␮ PNE-NDI. At time 0 min, desmopressin (0.3 g/kg body wt) aDosages were 1 and 3 mg/kg body wt per d for was infused, followed by measurement of von Willebrand fac- hydrochlorothiazide (HCT) and indomethacin and 20 ␮g tor and clotting factor VIII responses at the indicated time nasal spray/d for desmopressin. Note that the thirst trial was points. Methods after Bichet et al. (8). performed during daytime. 4 Journal of the American Society of Nephrology J Am Soc Nephrol ●●: ●●●–●●●, ●●●● again by approximately one third. The number of enuretic events during the various periods is given in Table 2.

V2R Gene Mutation and Sibling Responses For identification of the V2R gene mutation, sequence analysis of the V2R gene was done. Genomic DNA analysis of the patient revealed a mutation at nucleotide position 1074 (NM_000054), substituting a cytosine for an adenine (NM_000054). This substitution leads to the introduction of a premature stop codon (C358X) in the extreme C-terminus of the V2R. His brother, who also has NDI, harbors the same C358X mutation, and neither showed increased activity of blood clotting factors upon desmopressin administration (data not shown). In contrast to the proband, he did not wet his bed after 4 yr of age but went to the toilet three to five times every night Figure 3. V2R-C358X is not expressed in the plasma membrane of COS cells. Nonpermeabilized or permeabilized (indicated) for voiding. The sister of the proband also experienced PNE but COS cells that transiently expressed FLAG-V2R-C358X or had no signs of impaired renal concentration ability in a con- FLAG-V2R-GFP were subjected to immunocytochemistry using centration test, and sequence analysis did not reveal a mutation anti-FLAG antibodies, followed by confocal laser scanning mi- in her V2R gene (data not shown). Initiation of desmopressin croscopy. administration (20 ␮g intranasally) resolved her PNE.

Cell Biologic Analysis transiently transfected COS cells with different amounts of For determination of the molecular cause of NDI, MDCK F-V2R-GFP or F-V2R-C358X expression constructs. After 2 d, cells, which are an appropriate model for renal principal cells cells were lysed for immunoblotting or total RNA was isolated (13), were stably transfected with constructs that encode wild- and subjected to Northern blotting. As shown in Figure 4, for type V2R or V2R-C358X. Both were N-terminally fused to a similar or somewhat higher levels of F-V2R-C358X mRNA com- FLAG (F) epitope tag to allow detection (from the extracellular pared with that of F-V2R-GFP (Figure 4, top), the expression side). Under conditions that would allow detection throughout levels of F-V2R-C358X (35 kD) were clearly lower than those of the cells, F-V2R expression was easily detected in the basolat- V2R-GFP (60 and 75 kD; Figure 4 bottom). In fact, the expres- eral membrane of many stably transfected clones, whereas sion of F-V2R-GFP stayed relatively constant with decreasing F-V2R-C358X expression was not observed (data not shown). amounts of mRNA, whereas the F-V2R-C358X levels rapidly This suggested that V2R-C358X is unstable. For further inves- tigation of this, COS cells were transiently transfected with expression constructs that encode F-V2R-GFP and F-V2R- C358X. Here, the green fluorescence protein (GFP) cDNA was cloned in frame at the C-terminus of F-V2R and F-V2R-C358X, which, as a result of the stop codon in F-V2R-C358X, would yield expression of green F-V2R only. Two days after transfection, the cells were incubated with anti-FLAG antibodies in ice-cold PBS for1htolabel only V2R at the cell surface and fixed. Parallel-transfected cells were fixed, permeabilized, and incubated with anti-FLAG antibodies to stain V2R at any loca- tion. Subsequently, all cells were incubated with secondary antibodies and subjected to immunocytochemistry. Confocal laser scanning microscopy analysis revealed that F-V2R-C358X was not expressed at the cell surface, whereas the F-V2R Figure 4. FLAG-V2R-C358X is an unstable protein. COS cells showed clear membrane localization (Figure 3, left). Analysis of were transiently transfected with decreasing amounts of ex- permeabilized cells, however, showed expression of F-V2R and pression constructs for FLAG-V2R-GFP or FLAG-V2R-C358X F-V2R-C358X, indicating that V2R-C358X was retained in the (indicated). Subsequently, mRNA was isolated and subjected to cell and that the lack of plasma membrane expression of V2R- electrophoresis followed by Northern blotting. V2R mRNA was detected using a radioactively labeled V2R probe (top). Of cells C358X was not due to the absence of expression (Figure 3, transfected in parallel, lysates were generated and analyzed by right). This explains the NDI phenotype in the patients, because PAGE followed by Western blotting. FLAG-V2R-GFP and intracellularly retained receptors are not accessible to AVP and, FLAG-V2R-C358X were detected using anti-FLAG antibodies. thus, also not to desmopressin. Approximate masses (in kD) of the observed bands are indi- To determine semiquantitatively whether V2R-C358X is un- cated on the left. M, mock (transfected). Note the aspecific band stable, we analyzed the expression of F-V2R-GFP and F-V2R- of 70 kD. As found by others, the 120-kD band likely represents C358X, normalized for their amounts of mRNA. For this, we a dimer of V2R. J Am Soc Nephrol ●●: ●●●–●●●, ●●●● Relief of Nocturnal Enuresis by Desmopressin 5 decreased, which underscores the instability of V2R-C358X nism, reducing the urine volume does not seem to be essential compared with wild-type V2R. for the therapeutic effect of desmopressin. However, because activation of the renal V2R also stimulates sodium and urea Discussion uptake (23,24), a remaining role for renal V2R could not be Although PNE is common and extensively studied, the cause completely ruled out. of its inherited or sporadic forms remains unknown (14). Three In this study, we have identified a (novel) V2R mutation as entirely different therapeutic options (alarm treatment, tricyclic the underlying cause of NDI in a family, because dehydration antidepressants, and desmopressin) have been demonstrated as combined with desmopressin administration did not reduce the being highly efficient in prospective controlled trials, but, re- urine volume or increase the blood levels of clotting factors in gardless of dosage, a group of nonresponders exists for all the patients with NDI. Average urinary osmolalities were as treatments. Therefore, for obtaining more insight into this com- expected for congenital NDI, and a significant renal concentrat- plex disorder, attempts have been made to subgroup PNE. ing ability could be excluded. The molecular characterization of One classification has been made on the basis of nocturnal the mutant V2R demonstrated that the encoded protein is un- urinary volume or functional bladder capacity and their re- stable and does not reach the plasma membrane, where it is sponse upon desmopressin treatment (15). This drug has been normally bound by AVP or desmopressin to induce the intra- used successfully since the 1970s for the treatment of PNE with cellular cAMP signaling cascade. the rationale of increased distal tubular water reabsorption and Of further importance, one of the NDI-affected boys also a consequently reduced nightly urinary volume. This concept experienced PNE. Considering the nonfunctional V2R and the has later been fueled by a study that demonstrated an insuffi- repetitive replacement of PNE by nocturia after initiation of cient nightly increase in endogenous AVP in some patients desmopressin treatment, our data clearly indicate that the V2R with PNE, leading subsequently to high urinary volume of is not essential for the action of desmopressin in the treatment poorly concentrated urine (7). Consequently, a low functional of PNE. It has been noted that a placebo-drug effect in PNE is bladder capacity would be rapidly surpassed during sleep, not negligible. Although we cannot rule out such an effect here, leading to enuresis, which is reduced by desmopressin. How- the rapid recurrence of PNE upon withdrawal and its disap- ever, several investigators were not able to divide patients with pearance again by reintroduction of desmopressin indicate that PNE into such subgroups or to find positive correlations for the relief of PNE in our patient with PNE-NDI depends on drug urinary volume and vasopressin levels as predictors for the application. Moreover, PNE in our patient seems unrelated to desmopressin response in PNE (16). In addition, the largest hypercalciuria, because urinary calcium levels in our patient study on PNE and desmopressin treatment showed that only were low (Table 1). dosage, low voiding frequency, and older age are favorable Although desmopressin will reduce the nightly urine volume factors for dryness upon treatment (17). Therefore, the Stan- in patients without NDI-PNE and our data are derived from dardization Committee of the International Children’s Conti- one patient only, our results indicate that the success of des- nence Society has recently questioned the clinical usefulness of mopressin in PNE involves neither the renal concentrating such subgrouping approaches (18). mechanism nor the V2R. It is unclear which receptor/tissue is Another hypothesis to explain the cause of some PNE is involved in this effect. Possibly, desmopressin acts on a protein detrusor-sphincter dyssynergies and bladder instability, be- in the spinal cord or the neural network of the bladder and the cause some patients with PNE benefit from treatment with urethra, because these tissues have been suggested to be in- anticholinergics drugs, such as oxybutynin. However, a con- volved in nocturnal enuresis (25). More likely, however, des- founding factor here is that these patients also have daytime mopressin increases arousability by acting on a non-V2R in the symptoms and are therefore not monosymptomatic. Also, sev- central nervous system, because the alarm treatment and tricy- eral studies have suggested a role for idiopathic hypercalciuria clic antidepressants act on the central nervous system, and in PNE (19,20). This hypothesis, however, is also highly dis- increasing evidence is obtained for a delayed central nervous puted, because others came to opposite conclusions (21). For all cortical and brainstem maturational function in children and of these hypotheses, however, it is difficult to rationalize how adolescents with PNE (26–29). If this holds true, then one of the desmopressin, the alarm, and tricyclic antidepressants would best candidate proteins for mediating the action of desmopres- relieve nocturnal enuresis if nocturnal enuresis would have a sin in nocturnal enuresis is the human V1b receptor, because common cause. this protein has a similar affinity for desmopressin as the hu- This study and another recent study of ours provide data that man V2R and is localized in the brain (30,31). Determination of desmopressin, the alarm, and tricyclic antidepressants may act an involvement of the V1b receptor in PNE has to await Food on the same tissue/organ. We showed that two patients with and Drug Administration–approved specific agonists for this PNE and co-segregating NDI as a result of a mutation in the receptor. AQP2 gene responded to the treatment with desmopressin (22). Although these patients still had large nocturnal urinary vol- Acknowledgments umes, they aroused on the sensation of a full bladder and went This project is supported by a grant from the Dutch Kidney Foun- to the bathroom with desmopressin treatment. In this way, dation (PC104) to P.D. and N.V.K. desmopressin has converted PNE into nocturia. Because these J.R. set up the techniques to investigate V2R, set out design of cloning patients have an impaired renal urine concentrating mecha- and analysis, and interpreted the in vitro data; M.S. performed cloning 6 Journal of the American Society of Nephrology J Am Soc Nephrol ●●: ●●●–●●●, ●●●● of DNA and transfection of cells; N.V.K. participated in writing the Length of treatment, vasopressin secretion, and response. report; P.E. was the treating physician involved in making the diagno- Arch Dis Child 67: 184–188, 1992 sis; P.D. supervised the cell biological work and wrote the report; and 17. 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