Pharmacologic Chaperones as a Potential Treatment for X-Linked Nephrogenic Diabetes Insipidus
Virginie Bernier*,
Jean-Pierre Morello*,
Alexandro Zarruk,
Nicolas Debrand,
Ali Salahpour*,
Michèle Lonergan,
Marie-Françoise Arthus,
André Laperrière*,
Rémi Brouard,
Michel Bouvier* and
Daniel G. Bichet,
* Department of Biochemistry, Groupe de recherche universitaire sur le médicament, Departments of Medicine and Physiology, Université de Montréal, and Unité de recherche clinique, Centre de recherche et Service de néphrologie, Hôpital du Sacré-Coeur de Montréal, Québec, Canada; and Sanofi-Aventis, Paris, France
Address correspondence to: Dr. Daniel G. Bichet, Centre de recherche, Hôpital du Sacré-Coeur de Montréal, 5400 boulevard Gouin Ouest, Montréal, Québec, H4J 1C5 Canada. Phone: 514-338-2486; Fax: 514-338-2694; E-mail: daniel.bichet{at}umontreal.ca
Received for publication August 12, 2005.
Accepted for publication September 28, 2005.
In many mendelian diseases, some mutations result in the synthesisof misfolded proteins that cannot reach a transport-competentconformation. In X-linked nephrogenic diabetes insipidus, mostof the mutant vasopressin 2 (V2) receptors are trapped in theendoplasmic reticulum and degraded. They are unable to reachthe plasma membrane and promote water reabsorption through theprincipal cells of the collecting ducts. Herein is reportedtwo types of experiments: In vivo studies to assess clinicallya short-term treatment with a nonpeptide V1a receptor antagonist(SR49059) and in vitro studies in cultured cell systems. Inpatients, SR49059 decreased 24- h urine volume (11.9 ±2.3 to 8.2 ± 2.0 L; P = 0.005) and water intake (10.7± 1.9 to 7.2 ± 1.6 L; P < 0.05). Maximum increasein urine osmolality was observed on day 3 (98 ± 22 to170 ± 52 mOsm/kg; P = 0.05). Sodium, potassium, and creatinineexcretions and plasma sodium were constant throughout the study.In vitro studies indicate that the nonpeptide V1a receptor antagonistSR49059 and the V1a/V2 receptor antagonist YM087 (Conivaptan)rescued cell surface expression and function of mutant V2 receptors.Mutant V2 receptors with nonsense mutations were not affectedby the treatment. Misfolded V2 receptor mutants were rescuedin vitro and also in vivo by nonpeptide antagonists. This therapeuticapproach could be applied to the treatment of several hereditarydiseases that result from errors in protein folding and kinesis.
Although the activities of the protein synthesis quality controlsystems are generally advantageous to the cell, on occasionthis stringent monitoring process can lead to intracellularretention of salvageable proteins. In recent years, its hasbeen observed that a group of diseases stem from mutations thatpromote such retention and are collectively referred to as conformationalor protein-misfolding diseases (1,2). Nephrogenic diabetes insipidus(NDI) (3,4), which is characterized by a loss of arginine vasopressin(AVP)-mediated antidiuresis, is one of these diseases. In congenitalNDI that results from mutations in the AVPR2 gene that encodesthe V2 receptor, most missense mutations are misfolded, trappedin the endoplasmic reticulum, and unable to reach the basolateralcell surface to engage the circulating antidiuretic hormone,AVP (5–14).
The natural history of untreated X-linked NDI includes hypernatremia,hyperthermia, mental retardation, and repeated episodes of dehydrationin early infancy (15,16). In five new patients who were youngerthan 1 year and were from North America and in whom we providedmolecular testing over the past 12 mo, plasma sodium was inevery case >155 mEq/L at the time of diagnosis. We and othersinitially thought that close monitoring of infants whose AVPR2mutations were diagnosed pre- or perinatally not only wouldprevent episodes of dehydration but also would permit closeto normal growth and development. Although a low-sodium dietand distal tubule diuretics prescribed to these patients mayachieve a 20 to 30% decrease in urine output (17), the low-sodiumdiet is difficult to follow, and affected children continueto drink large amounts of water. As a result of a physiologicgastroesophageal reflux and to the large amount of water intheir stomach, these children often vomit, and, as a consequence,their nutritional intake is not optimal. There is a need, therefore,for a safe further reduction in urine output. We recently usedpharmacologic compounds to rescue misfolded mutant V2 receptorsby demonstrating in cultured cells that the nonpeptide V2-specificantagonists SR121463A and VPA-985 increased cell surface expressionand rescued the signaling activity of seven naturally occurringAVPR2 mutations (185_193del, L59P, L83Q, Y128S, S167L, A294P,and P322H) that are responsible for NDI by promoting their properfolding and maturation (18). These results that suggested thatsuch chaperoning of the receptor could represent a pharmacologictreatment for conformational diseases such as NDI have beenconfirmed by other investigators (19,20).
Here, we report a short-term trial that was conducted to testthe effect of a nonpeptide V1a receptor antagonist on decreasingurine output and increasing urine osmolality in patients withX-linked NDI. We used SR49059, a V1a receptor antagonist thatwas tested previously in normal volunteers and patients withputative vasopressin excess–related disorders (21–24).In addition, in vitro studies were done to rescue plasma membraneand signaling of a number of mutant V2 receptors with SR49059and YM087 (Conivaptan), a mixed V1-V2 antagonist (25–29).
Study Participants
We tested five adult male patients (weight 83.5 ± 3.9kg) who had X-linked NDI and bore the following AVPR2 mutationsthat were identified previously by sequencing: Three patients,who were 20, 42, and 41 yr of age and had R137H (30) (patients1, 2, and 3); one 21-yr-old with W164S (31) (patient 4); andone 20-yr-old with 185_193del (31) (patient 5). All had a documentedlifelong history of polyuria and polydipsia, and extensive previoustesting demonstrated a lack of urinary osmolality response toAVP or dDAVP. Specifically, maximal urine osmolalities (mOsm/kg)that were obtained during dDAVP infusions were, respectively,104, 145, and 248 (patients 1, 2, and 3); 85 (patient 4); and65 (patient 5) (32). Two patients were being treated with hydrochlorothiazide,which was discontinued for 1 wk before the study. None of thepatients described here were considered to have a mild phenotype(9). The following short-term trial conformed to the Declarationof Helsinki and was approved by the Ethics Committee at Hôpitaldu Sacré-Cur de Montréal, and all participantsapproved and signed a detailed informed consent.
SR49059 Administration for 2 D.
Patients were tested at the Clinical Research Unit of the Hôpitaldu Sacré-Coeur de Montréal and received a constantNa+, K+, osmotic and caloric diet for the 3 d of the study.A dietitian met the patients before the study and made a detailedevaluation of their usual diet during the previous month. Thisdiet was reproduced for the 3 d of the study. None of thesepatients followed a sodium-restricted diet. The dietitian metthe patients every day during testing and enforced the samediet throughout the study. Water intake was not restricted andwas recorded during the 3 d of the study. After 24-h controlmeasurements (day 1, no medication), SR49059 was administeredorally for the next 2 d; on day 2, the patients received 150mg at 8 a.m. and 300 mg at 1:00 p.m. and 6:00 p.m.; on day 3,the patients received 300 mg at 8:00 a.m., 1:00 p.m., and 6:00p.m. BP and pulse were measured every 30 min from 8:00 a.m.to 12:00 a.m. for the first two patients and every 2 h for thelast three patients. Urine volume was obtained by spontaneousvoiding every 30 min from 8:00 a.m. to 10:30 p.m. From 10:30p.m. to 8:00 a.m., depending on individual patients, all urineexcretion was measured at unspecific times; urine flow was calculated;and urine osmolality, Na+, K+, and creatinine were measuredon these samples. Plasma Na+ and plasma AVP were measured at7:30 a.m. and 1:30 p.m. each day. Urinary Na+, K+, creatinine,osmolality, and AVP were obtained on each urine sample.
SR49059 Administration for 7 Days.
Two patients who bore the R137H mutation (patients 1 and 3)were also treated 6 wk after the 2-d administration of SR49059for 7 d with the following dosages of SR49059: 750 mg on day1 (150 mg at 8:00 a.m., 300 mg at 1:00 p.m., and 300 mg at 6:00p.m.) and 900 mg (300 mg three times daily) for the following7 d. Urine and plasma measurements were obtained on days 1,6, and 9 (postdosing). On days 6 (on SR49059) and 9 (off SR49059),basal plasma samples were obtained for sodium at 8:00 a.m.,and urine was obtained every 30 min from 8:30 a.m. to 12:00p.m. for volume, osmolality, Na+, K+, creatinine, and AVP.
Cell Culture Studies V2 Receptor Mutant Expression.
Mammalian expression plasmids encoding the wild-type, 12 missensemutations (L59P, L83Q, Y128S, R113W, R137H, W164S, A165D, S167L,I209F, A294P, S315R, and P322H) two in-frame deletions (185_193deland V279del) or five nonsense mutations (W71X, S167X, Q180X,W284X, and R337X) were transiently or stably transfected inCOS-1 or HEK 293 cells, as described previously (18,33).
Immunofluorescence Microscopy and Flow Cytometry.
All immunofluorescence and flow cytometry studies were carriedout in COS-1 cells or HEK 293 as described previously (18,33).Briefly, V2 receptors were detected using antibodies directedagainst the myc- or Ha-epitope that was fused at the N-terminusof the constructs. For microscopy, immunoreactivity was assessedusing secondary Oregon-green–conjugated anti-mouse antibodies.For flow cytometry, the phycoerythrin-conjugated anti-mouseantibody was used.
cAMP Accumulation.
Total cAMP accumulation was measured in COS-1 cells or HEK 293by assessing the transformation of [3H]ATP into [3H]cAMP asdescribed previously (34).
Plasma AVP Measurements.
Plasma AVP was measured by RIA as described previously (35).
Statistical Analyses
Simple statistics were performed for the major variables ofinterest. Overall results were analyzed by two-way ANOVA. Comparisonbetween time points was performed using paired t test. A two-tailedP < 0.05 was considered statistically significant. Valuesare reported as mean ± SEM. All analyses were performedwith the statistical package SAS (SAS Institute Inc., Cary,NC).
Patient Studies Safety and Efficacy of SR49059 Administration in Adult Patients Who Bear the R137H, W164S, or 185_193del Mutations.
No significant changes in BP and pulse were encountered throughoutthe study, and no untoward clinical or biochemical abnormalitieswere observed. Intake and output; plasma sodium and potassiumand 24-h urine osmolar excretion; and Na+, K+, and creatinineexcretion are presented in Table 1 and Figure 1. SR49059 significantlydecreased 24-h urine volume and 24-h water intake from day 1to day 3. A maximal increase in urine osmolality was observedfrom 2:00 p.m. to 8:00 p.m. on day 3 (98 ± 22 to 170± 52 mOsm/kg; P = 0.05; Figure 1). Plasma Na+ was constant,indicating that the changes in urine volume and water intakewere secondary to the SR49059 administration and not to voluntarydecrease in water intake that would have led to increased plasmaNa+. Individual urine volume and urine osmolality responsesare presented for three patients, each bearing a different mutation(Figure 2, A, B, and C). The maximal urine osmolality duringtreatment was observed for patient 3 (Figure 2), who had a urineosmolality of 248 during a diagnostic dDAVP infusion (32). Tovarious extent, the treatment significantly decreased urinaryoutput and increased urine osmolality in all patients. PlasmaAVP levels were measured on day 1 (control), two times on day2 (8:00 a.m. and 2:00 p.m.), and four times on day 3 (8:00 a.m.,2:00 p.m., 8:00 p.m., and 12:00 a.m.). Plasma AVP values weresignificantly different among patients (from 1.97 to 6.24 pg/ml;ANOVA, P < 0.05), but no significant differences among daysor time effects were seen, and plasma values tended to decreaseduring the study (day 1, 5.39; day 2, 3.01, day 3, 3.34 pg/ml).
Table 1. Water, Na+, and K+ intake; 24-h urine osmolar excretion; Na+, K+, and creatinine excretion; and plasma sodium during day 1 (control) or SR49059 administration (days 2 and 3)
Figure 1. (A) Urine volume, urine osmolality, and water intake on day 1 and after SR49059 administration (day 3) in five adult male patients with X-linked nephrogenic diabetes insipidus (NDI). (B) The same values are described for the afternoon period (2:00 p.m. to 8:00 p.m.) when the effect of SR49059 was suspected to be maximal. Mean values (± SEM) are presented. *P < 0.05, paired t test.
Figures 2. Urine volume and osmolality before (day 1) and after (days 2 and 3) SR49059 administration to individual patients who bore the R137H (A), W164S (B), and 185_193del (C) mutations. Note that the distances observed between the two lines on days 2 and 3 represent the mirror images of urine volume and osmolality. Urine volume and osmolalities that were obtained during the control, second, and third nights are indicated by round circles. These data were obtained from 9:30 p.m. to 8:00 a.m. for patient 3; 11:00 p.m. to 8:00 a.m. for patient 2, and 11:30 p.m. to 8:00 a.m. for patient 5. Note that the magnitude of the volume and osmolalities observed were different among these three patients who bore different mutations but that SR49059 induces a consistent effect.
Longer treatment was tested for two patients who bore the R137Hmutations. Reduced urine volumes and increased urine osmolalitieswere maintained for the 7 d of the treatment with SR49059 (Figure 3).
Figure 3. Urine volume and osmolality on day 6 of a 7-d treatment with SR49059 and on day 9, 2 d after treatment. Patient 5 (see Figure 2A) had a urine osmolality of approximately 400 mOsm/kg after SR49059 (300 mg) administration. Urine osmolality then decreased to 200 during the remaining of the morning observation. On day 9, 2 d after cessation of SR49059, urine osmolality values were similar to the control values obtained on day 1 (see Figure 2A).
Cell Culture Studies SR49059.
The effect of the nonpeptide V1a antagonist SR49059 was assessedon cell surface expression and function of two missense (R137Hand W164S), one in-frame deletion (185_193del), and one nonsense(W284X) mutation. Immunofluorescence microscopy showed thatunder basal conditions, all V2 mutants were poorly expressedat the cell surface of transfected COS-1 cells (Figure 4A).A 16-h treatment with SR49059 increased the plasma membranetargeting of three of the V2 receptor mutants but not the nonsensemutant receptor. Figure 4B shows the quantitative assessmentof the increase in cell surface expression for the 185_193delV2 receptor mutant using flow cytometry.
Figure 4. SR49059 treatment on cell surface expression of four V2 receptor mutants. (A) Immunofluorescence microscopy of nonpermeabilized COS-1 cells that transiently expressed wild-type, R137H, W164S, 185_193del, or W284X mutations and were incubated or not for 16 h with 10–5 M SR49059. (B) Cell surface receptor expression was measured by flow cytometry analysis of cells that stably expressed the 185_193del mutant V2 receptor incubated in the absence or presence of 10–5 M SR49059.
The increased cell surface expression of R137H, W164S, and 185_193delV2 receptor mutants after treatment with SR49059 resulted ina significant potentiation of the AVP-mediated cAMP production,suggesting that SR49059 restored function by acting as a pharmacologicchaperone. In contrast, the SR49059 treatment had no effecton AVP-stimulated cAMP accumulation in cells that expressedW284X V2 receptors (Figure 5A).
Figure 5. Signaling activity of COS-1 and HEK293 cells after treatment with SR49059. (A) Potentiation of arginine vasopressin (AVP)-stimulated cAMP accumulation was measured in COS-1 cells that transiently expressed wild-type, R137H, W164S, 185_193del, or W284X V2 mutant receptors after a 16-h pretreatment of 10–5 M SR49059. Cells that were not treated with SR49059 () are compared with treated cells (). The fold increases are given above the solid bars. The value of one obtained for the wild-type V2 receptors indicates that SR49059 does not alter its maximal efficacy. (B) Duration of the effect of SR49059 pretreatment on AVP-stimulated cAMP accumulation. HEK 293 cells that stably expressed the 185_193del V2 mutant receptor were treated with 10–5 M SR49059 for 16 h. At the end of the treatment, the antagonist was removed by successive washing, and the AVP-stimulated cAMP accumulation was determined at indicated times after the washing procedures.
Kinetic analysis of the effect of SR49059 on the function ofone of the V2 mutant receptors (185_193del) in HEK 293 cellsis shown in Figure 5B. The potentiation effect peaked at 2 hafter treatment but was maintained for at least 12 h after theantagonist. The relatively slow onset of the effect is consistentwith the notion that the drug acts by favoring folding and cellsurface trafficking of newly synthesized receptors (36). Themodest increase in AVP-stimulated cAMP production that was observedin cells during the course of the experiment most likely reflectsaccumulation of a small number of 185_193del V2 mutant receptorsat the cell surface.
YM087.
Because the clinical development of SR49059 was interrupted(vide infra), we also tested the ability of YM087, a dual V1aand V2 vasopressin receptor antagonist (27), to rescue cellsurface expression and function of 19 naturally occurring V2Rmutants in COS-1 cells. As shown in Figure 6, YM087 promotedcell surface expression and potentiated AVP-mediated cAMP productionfor 10 naturally occurring mutations tested. As was the casefor SR49059, YM087 had no effect on the nonsense mutations selectedas negative controls: W284X (Figure 6), W71X, S167X, Q180X,and R337X (data not shown).
Figure 6. YM087 treatment on cell surface expression and AVP-stimulated cAMP accumulation of nine missense (L59P, L83Q, Y128S, R137H, W164S, A165D, S167L, A294P, P322H), one in-frame deletion (185_193del), and one nonsense (W284X) V2 receptors in COS-1 cells. cAMP units are the same as used in Figure 5.
To date, no specific treatment that is aimed at restoring thefunction of the mutant V2 receptor is available to treat patientswith X-linked NDI. Volume contraction and thiazide diuretics,amiloride, and indomethacin are acting only indirectly by decreasingthe amount of tubular fluid presented to the distal tubule (37,38).These indirect forms of treatment are most effective in patientswho have mild to moderate forms of X-linked NDI and bear incompleteloss-of-function mutations. These patients with mild to moderatedisease are rare, and most patients are completely unresponsiveto AVP or dDAVP (32). Here, we present evidence that nonpeptidevasopressin antagonists are potential specific treatments ofthis disease. It has been argued that the diversity of mutationsin NDI may complicate the search for a universal therapeuticstrategy for these patients (39). However, because approximately50% of all NDI mutations are missense, a pharmacologic chaperone-basedtherapy could represent a potential general treatment of thisprotein-misfolding disease.
Manning et al. (40) designed in the 1970s numerous vasopressinand oxytocin receptor agonists and antagonists. Their clinicaluse in humans, however, was deceptive because antagonists inrats were found to be agonists in humans. This was found laterto be due to different molecular structures of the receptorsthat are responsible for different affinities for agonists andantagonists in human and rat species (41). The random screeningof chemical compounds resulted in the development of oral nonpeptidevasopressin receptor antagonists now called "Vaptans," Vap forvasopressin, tan for antagonists (42). The structure of thesecompounds imitates the structure of the native hormone AVP,and these antagonists interfere with the binding pocket of AVP(43). During the past few years, various selective, orally activeAVP V1a (OPC-21268, SR49059 [Relcovaptan]), V2 (OPC-31260, OPC-41061[Tolvaptan], VPA-985 [Lixivaptan], SR121463A and B, VP-343,and FR-161282), and mixed V1a/V2 (YM-087 [Conivaptan], JTV-605,and CL-385004) receptor antagonists have been studied intensivelyin various animal models and have reached phase III clinicaltrials for some of them (44).
We gave SR49059, a potent and selective, orally active, nonpeptideV1a receptor antagonist to five patients with V2 receptor defects.Previous in vitro binding experiments of human V1a receptorsobtained from platelets, adrenals, aortic smooth muscles, andnonpregnant myometrium demonstrated that SR49059 was a selectiveV1a antagonist with inhibition constants (Ki) ranging from 1.5to 6.5 nM (21). SR49059 displayed competitive nanomolar affinityfor V1a receptors but weak affinities for human and nonhumanV2, V1b, and oxytocin receptors with Ki ranging from 220 to1080 nM (21).
The absolute bioavailability of the nonmicronized formulationF1 used in this study was low and variable (5.3 ± 4.7%)with a Tmax of approximately 3 h and a terminal half-life ofapproximately 23 h. A 300-mg dose of the F1 formulation wasreported in the Clinical Investigator Brochure to increase plasmaconcentration to 18.5 ng (33.5 micromolar, MW of SR49059 is620.5) 3 h after the administration of SR49059 to normal volunteers.In our in vitro studies, a 16-h pretreatment with 10 micromolar(10–5 M) concentration of SR49059 or YM087 (MW 535.04)rescued cell surface expression and cAMP production.
Because in our clinical studies the maximal urine osmolalitywas observed, in general, 2 to 3 h after the oral administrationof 300 mg of SR49059, these in vivo and in vitro results agreewith these pharmacokinetics data. We demonstrated that SR49059,a V1a receptor antagonist that shows moderate affinity for theV2 receptor (275 nM) (21), rescued plasma membranes and signalingof the R137H, W164S, and 185_193del mutants, a confirmationof previous results obtained with the selective V2 receptorantagonists SR121463A, SR121463B, and VPA-985 (18,33) and othermutant V2 receptors (19,20). It is hypothesized that these compoundswill enter the cell and the endoplasmic reticulum compartmentand may stabilize the misfolded mutant receptor to a conformationthat will permit further maturation through the endoplasmicreticulum and Golgi compartments. These nonpeptide vasopressinantagonists, with different affinities for V1 or V2 receptors,may be seen as a mold on which the unstable mutant receptorwill wrap itself, perhaps hiding its hydrophobic residues anddecreasing free energy (2). In recent in vitro results (45),we demonstrated that in vitro treatment with SR121463 for 16h led to an important decrease in the polyubiquitination immunoreactivesignal, indicating that the increased receptor maturation isaccompanied by a decrease in the proportion of receptor beingtargeted to the polyubiquitination-dependent degradation pathway.
In five patients who had X-linked NDI and harbored three differentAVPR2 mutations, SR49059 had beneficial effects on urine volumeand osmolality starting a few hours after administration. Thislag in efficacy is compatible with our previous in vitro observations(18), demonstrating that the pharmacologic chaperones need topermeate the cell and favor folding and trafficking of functionalmutant receptors to the cell surface to be active. That nonsensemutations could not be rescued by the antagonist treatment isconsistent with such a proposed mode of action. In a recentstudy, we demonstrated that the -arrestin–mediated constitutiveendocytosis of the V2 receptor (46) is not affected by SR49059(33). The functional rescue observed in vitro and in our clinicalstudy thus is unlikely to result from a stabilization of theV2 receptor at the cell surface.
Urine osmolality increased by 50% on day 3 from 2:00 p.m. to8:00 p.m., and a maximum urine osmolality of 430 mOsm/kg (Figure 2A)was documented in patient 3, who was able to increase hisurine osmolality only to 248 mOsm/kg during a previous dDAVPinfusion (32). The urine osmolality changes were not secondaryto increased endogenous plasma AVP concentrations. The effectson urine concentration occurred with no change in BP or pulse,an observation consistent with the lack of hemodynamic effectobserved in hypertensive patients after the administration of300 mg of SR49059 (22). The excretion of tonomoles being constant(Table 1), doubling urine osmolality will half the urine output.In patients with a mean urinary output of 12 L/d, a theoreticaldecrease of urine volume to 6 L/d could be obtained providedthat a sustained drug effect could be reached through optimizationof the drug regimen. A nonsignificant decrease in sodium excretionwas observed, possibly indicating that these patients were notstrictly in Na+ balance and/or pointing to the possible restorationof an AVP antinatriuretic effect (47). The highest increasein urine osmolality and consequent decrease in urine volumewas observed with patient 3 with a maximal increase in urineosmolality after dDAVP of 248 mOsm/kg. Further clinical studieswill needed to test whether the ability to rescue will dependon basal receptor function.
The proof-of-principle results obtained in this study indicatethat pharmacologic chaperone-based therapy could be appliedto other missense mutations or in-frame deletions or insertionsthat are responsible for X-linked NDI. Among the 207 familieswho had X-linked NDI and were referred to our laboratory (48;unpublished data), 66 of the 155 different putative disease–causingmutations are missense mutations that potentially are amenableto rescue by these pharmacologic chaperones.
Unfortunately, the clinical development of SR49059 has beeninterrupted during the course of these studies as a result ofpossible interference with the cytochrome P450 metabolic pathway.We would have liked to administer SR49059 to other patientswith congenital NDI caused by a noncorrectable defect such asnonsense V2 receptor mutants or aquaporin mutants, but thiswas not possible. We also wanted to test whether other vasopressinantagonists, which could be clinically developed, may also actas pharmacologic chaperones for NDI-causing mutations. Anothernonpeptide vasopressin receptor antagonist that is in advancedclinical testing phase and has an excellent safety profile foranother application (27), YM087, was found to rescue cell surfaceexpression and function of nine missense V2 mutant receptors.This provides supporting data that will allow us and othersto test this compound and additional vasopressin ligands, includingnonpeptide vasopressin agonists in patients with X-linked NDI,when they become available for such trials.
In addition to be a promising avenue for the treatment of X-linkedNDI, stabilization of protein conformation, using small cell–permeableligands, may represent a generally applicable rescue strategyfor different diseases resulting from improper protein foldingand targeting. These diseases include cystic fibrosis, osteogenesisimperfecta, 1-antitrypsin deficiency, NDI caused by mutationsin AQP2, Gitelman syndrome, Fabry disease, and many others (49–58).
Acknowledgments
This study was supported by grants MOP 8126 from the CanadianInstitutes of Health Research (D.G.B.) and the Kidney Foundationof Canada (D.G.B. and M.B.) and la Fondation J. Rodolphe-LaHaye (D.G.B.). J.P.M. was supported by a studentship from theMRC/PMAC Health Program. V.B. was supported by a studentshipfrom the Quebec Society of Hypertension, the Heart and StrokeFoundation of Canada, and the Fonds de Recherche en Santédu Québec. A.S. was supported by a studentship from theCanadian Institute for Health Research. M.B. holds a CanadaResearch Chair in Signal Transduction and Pharmacology, andD.G.B. holds a Canada Research Chair of Kidney Disease.
We thank C. Serradeil-LeGal (Sanofi-Aventis) and R.E. Desjardins(Yamanouchi Pharma America, Inc., Paramus, NJ) for the generousgift of SR49059 and YM087, respectively; L. Cournoyer and S.Sénéchal for technical assistance; M. Moreau forassistance with the figures; T.M. Fujiwara for critical readingof the manuscript; and D. Binette for graphical and secretarialexpertise. We also thank Dr. M. Caron for providing the R137Hconstruction and Dr. F. Madore for statistical advice.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org
V.B. and J.-P.M. contributed equally to this work.
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Top
Abstract
Introduction
ur de Montréal, and all participants approved and signed a detailed informed consent. 
-arrestin–mediated constitutive endocytosis of the V2 receptor (46) is not affected by SR49059 (33). The functional rescue observed in vitro and in our clinical study thus is unlikely to result from a stabilization of the V2 receptor at the cell surface. 
1-antitrypsin deficiency, NDI caused by mutations in AQP2, Gitelman syndrome, Fabry disease, and many others (49–58). 
) are compared with treated cells (
). The fold increases are given above the solid bars. The value of one obtained for the wild-type V2 receptors indicates that SR49059 does not alter its maximal efficacy. (B) Duration of the effect of SR49059 pretreatment on AVP-stimulated cAMP accumulation. HEK 293 cells that stably expressed the 185_193del V2 mutant receptor were treated with 10–5 M SR49059 for 16 h. At the end of the treatment, the antagonist was removed by successive washing, and the AVP-stimulated cAMP accumulation was determined at indicated times after the washing procedures.