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Aquaporin‐2 (AQP2) is a homotetrameric water channel responsible for the final water reuptake in the kidney. Disease‐causing AQP2 mutations induce nephrogenic diabetes insipidus (NDI), a condition that challenges the bodily water balance by producing large urinary volumes. In this study, we characterize three new AQP2 mutations identified in our lab from NDI patients (A120D, A130V, T179N) along the previously reported A47V variant. Using Xenopus oocytes, we compared the key functional and biochemical features of these mutations against classical recessive (R187C) and dominant (R254Q) forms, and once again found clear functional recovery features (increased protein stability and function) for all mutations under study. This behaviour, attributed to heteromerization to wt‐AQP2, challenge the classical model to NDI which often depicts recessive mutations as ill‐structured proteins unable to oligomerize. Consequently, we propose a revised model to the cell pathophysiology of AQP2‐related NDI which accounts for the functional recovery of recessive AQP2 mutations., Mutations to AQP2 induce nephrogenic diabetes insipidus (NDI), where low/inactive water channels cause large urinary waste of possible fatal outcome. Our past studies have shown that several AQP2 mutations could be functionally recovered by associating to wild‐type AQP2 (wt‐AQP2) into the protein complex (tetramer). This study presents the characterization of three new AQP2 mutations from actual case‐studies in regard to recovery capacities, and conclude by proposing a revised pathophysiological model to NDI, one where AQP2 mutations are now presented as mostly recoverable through wt‐AQP2 association into functional water channels.

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the GLA gene encoding the &alpha, galactosidase A enzyme. This enzyme cleaves the last sugar unit of glycosphingolipids, including globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), and galabiosylceramide (Ga2). Enzyme impairment leads to substrate accumulation in different organs, vascular endothelia, and biological fluids. Enzyme replacement therapy (ERT) is a commonly used treatment. Urinary analysis of Gb3 isoforms (different fatty acid moieties), as well as lyso-Gb3 and its analogues, is a reliable way to monitor treatment. These analogues correspond to lyso-Gb3 with chemical modifications on the sphingosine moiety (&minus, C2H4, &minus, C2H4+O, &minus, H2, &minus, H2+O, +O, +H2O2, and +H2O3). The effects of sample collection time on urinary biomarker levels between ERT cycles were not previously documented. The main objective of this project was to analyze the aforementioned biomarkers in urine samples from seven Fabry disease patients (three treated males, three treated females, and one ERT-na&iuml, ve male) collected twice a day (morning and evening) for 42 days (three ERT cycles). Except for one participant, our results show that the biomarker levels were generally more elevated in the evening. However, there was less variability in samples collected in the morning. No cyclic variations in biomarker levels were observed between ERT infusions.

Objectives Orphan medicinal products (OMPs) often receive market authorization under conditions imposed by regulators for ongoing postauthorization surveillance (PAS) to answer questions that remain at the time of market entry. This surveillance may be provided through industry-funded registries (IFRs). Nevertheless, data in these registries may not be of sufficient quality to answer these questions and may not always be accessible for regulatory review. We propose that a mandatory independent registry is an efficient and cost-effective tool for PAS for OMPs. Methods Using data from the Canadian Fabry Disease Initiative, we reviewed costs per unique patient from sites participating in both the independent national registry and IFRs for Fabry disease and compared data completeness from the Canadian Fabry Disease Initiative to that in published documents from IFRs. Results The costs of data collection through the independent registry were 17% to 36% (depending on site) lower than costs to collect data in the IFRs, and completeness of data collected through the independent registry was higher than that through the IFRs. Data from the independent registry were reviewed annually to guide indications for publicly funded Fabry disease therapy. Even when enrollment ceased to be a requirement to receive therapy, 77% of patients continued to enroll in the registry, suggesting the structure was acceptable to patients. Conclusions Independent registries are cost-effective and efficient tools and should be mandated by regulatory agencies as the preferred tool for PAS for OMPs. Countries with publicly funded health systems should consider investment in registry infrastructure for OMPs.

Fabry disease is an X-linked lysosomal storage disorder with marked variability in the phenotype and genotype. Glycosphingolipids such as globotriaosylceramide (Gb3) isoforms/analogs, globotriaosylsphingosine (lyso-Gb3) and analogs, and galabiosylceramide (Ga2) isoforms/analogs may accumulate in biological fluids and different organs. The aims of this study were to: 1) develop/validate a novel UHPLC-MS/MS method for relative quantitation of Gb3 in leukocytes (unfractionated white blood cells), B lymphocytes and monocytes; 2) evaluate these biomarkers in a cohort of Fabry patients and healthy controls; and 3) assess correlations between these biomarkers, treatment and genotype. Whole blood, plasma and urine samples from 21 Fabry patients and 20 healthy controls were analyzed. Samples were purified by liquid-liquid extraction and analyzed by UHPLC-MS/MS in positive electrospray ionization. Methylated Gb3 isoforms were detected, showing that a methylation process occurs at the cellular level. Our results show that there were no significant differences in the distribution of the different Gb3 isoforms/analogs in blood cells between Fabry patients and healthy controls. In leukocyte, Gb3[(d18:1)(C14:0)], Gb3[(d18:1)(C16:0)], Gb3 [(d18:1)(C16:0)]Me, Gb3 [(d18:1)(C16:1)], Gb3 [(d18:1)(C18:0)], Gb3 [(d18:1)(C18:1)], Gb3 [(d18:1)(C20:1)], Gb3 [(d18:1)(C24:2)], Gb3 [(d18:1)(C26:1)] and total Gb3 allowed good discrimination between male Fabry patients and male controls, patients having higher biomarker levels than controls. Regarding B lymphocytes and monocytes, the same tendency was observed without reaching statistical significance. A positive concordance between mutation types and biomarker levels in white blood cells was established. Our results might provide a deeper mechanistic comprehension of the underlying biochemical processes of Gb3 biomarkers in white blood cells of Fabry patients.

Background Inherited nephrogenic diabetes insipidus (NDI) is a rare disorder characterized by impaired urinary concentrating ability. Little clinical data on long-term outcome exists. Method This was a single-centre retrospective medical record review of patients with a diagnosis of NDI followed between 1985 and 2017. We collected available data on growth, weight, school performance, complications and comorbidities. Results We identified 36 patients with available data and a clinical diagnosis of NDI, which was genetically confirmed in 33 of them. Patients presented at a median age of 0.6 years and median length of follow-up was 9.5 years. Chief symptoms at presentation were faltering growth, vomiting/feeding concerns, polyuria/polydipsia, febrile illness and hypernatraemic dehydration. Median weight standard deviation scores (SDS) improved from −2.1 at presentation to 0.2 at last follow-up. In contrast, height SDS remained essentially unchanged at −1.1 at presentation and −0.9 at last follow-up. Most patients were treated with prostaglandin synthesis inhibitors and thiazides, yet weaned off during school age without an obvious change in urine output. Median estimated glomerular filtration rate at last follow-up was 81 mL/min/1.73 m2. Urological complications were noted in 15 patients, constipation in 11 and learning difficulties in 5. Median age at resolution of nocturnal enuresis was 11 years. Estimated median daily fluid intake at median age of 13 years was 3800 mL/m2. Conclusion The overall prognosis in inherited NDI is favourable with regular treatment. As expected, most complications were related to polyuria. There is an apparent loss of efficacy of medications during school age. Our data inform the prognosis and management of patients with NDI.

High plasma copeptin, a marker of vasopressin (VP) secretion, has been shown to be associated with the metabolic syndrome and development of type 2 diabetes in humans. The present study was designed to determine the long-term influence of plasma VP concentration in a rodent model prone to metabolic dysfunction. Obese Zucker rats and their lean counterparts were submitted for 4 weeks to one of three protocols inducing different levels of VP. Circulating VP was either reduced by increasing the daily water intake (low-VP), or increased by a chronic i.p. infusion of VP (high-VP). The control rats had normal VP levels that depended on their own regulation of water intake and VP secretion. Compared with controls with normal VP, lean rats with high-VP had a higher fasting glycaemia after 4 weeks. In obese rats, high-VP promoted hyperinsulinaemia, glucose intolerance, assessed by glucose and insulin tolerance tests, and an impaired response to a pyruvate challenge. Conversely, treatment with a selective arginine vasopressin receptor 1A (V1aR) antagonist reduced glucose intolerance. Low-VP obese rats had unchanged glucose tolerance but exhibited a drastic decrease in liver steatosis compared with control obese rats, associated with low hepatic triacylglycerol and cholesterol content, and reduced expression of hepatic lipogenic genes. These effects were independent of changes in body adiposity, and plasma sodium and osmolality did not differ among groups. These findings show a causal relationship between the VP–hydration axis and the metabolic risk. Therapeutic perspectives include diet recommendations regarding hydration, but also potential pharmacological interventions targeting the VP V1aR.

Fabry disease is an X-linked lysosomal storage disorder with marked variability in the phenotype and genotype. Glycosphingolipids such as globotriaosylceramide (Gb

Aquaporin-2 (AQP2) is a water channel responsible for the final water reabsorption in renal collecting ducts. Alterations in AQP2 function induce nephrogenic diabetes insipidus (NDI), a condition characterized by severe polyuria and polydipsia. Three patients affected with severe NDI, who were compound heterozygous for the AQP2 mutations D150E and G196D, are presented here along with a mildly affected D150E homozygous patient from another family. Using Xenopus oocytes as an expression system, these two mutations (G196D and D150E) were compared with the wild-type protein (AQP2-wt) for functional activity (water flux analysis), protein maturation, and plasma membrane targeting. AQP2-wt induces a major increase in water permeability ( Pf= 47.4 ± 12.2 × 10−4cm/s) whereas D150E displays intermediate Pfvalues ( Pf= 12.5 ± 3.0 × 10−4cm/s) and G196D presents no specific water flux, similar to controls ( Pf= 2.1 ± 0.8 × 10−4cm/s and 2.2 ± 0.7 × 10−4cm/s, respectively). Western blot and immunocytochemical evaluations show protein targeting that parallels activity levels with AQP2-wt adequately targeted to the plasma membrane, partial targeting for D150E, and complete sequestration of G196D within intracellular compartments. When coinjecting AQP2-wt with mutants, no (AQP2-wt + D150E) or partial (AQP2-wt + G196D) reduction of water flux were observed compared with AQP2-wt alone, whereas complete loss of function was found when both mutants were coinjected. These results essentially recapitulate the clinical profiles of the family members, showing a typical dominant negative effect when G196D is coinjected with either AQP2-wt or D150E but not between AQP2-wt and D150E mutant.

We evaluated a baby boy with severe combined immunodeficiency (SCID) and X-linked nephrogenic diabetes insipidus (NDI). This patient had less than 10% CD3+ T cells, almost all of which were positive for CD4 and CD45RO. Genetic studies demonstrated a 34.4 kb deletion at Xq28 which included AVPR2, the gene responsible for NDI; ARHGAP4, a hematopoietic specific gene encoding a GTPase-activating protein; and a highly conserved segment of DNA between ARHGAP4 and ARD1A, a gene involved in the response to hypoxia. Other patients with NDI, but without immunodeficiency, have had deletions that remove all ARHGAP4 except exon 1; however, no other patients have had deletions of the highly conserved intragenic region between ARHGAP4 and ARD1A. X chromosome inactivation studies, done on sorted cells from the mother and grandmother of the patient, carriers of the deletion, demonstrated exclusive use of the non-mutant X chromosome as the active X in CD4 and CD8 T cells. Surprisingly, NK cells, monocytes and neutrophils from these women demonstrated preferential use of the mutant X chromosome as the active X. These results are consistent with an X-linked form of SCID, due to the loss of regulatory elements that control the response to hypoxia in hematopoietic cells.

Background. An increase in urinary albumin excretion (UAE) represents an early predictor of glomerular damage in diabetes mellitus (DM) and a risk factor for cardiovascular complications in hypertension. Vasopressin is elevated in DM and in some forms of hypertension. Previous studies in rats suggested that this hormone could play a role in the albuminuria observed in chronic renal failure or diabetic nephropathy, but no information is available concerning the mechanism of these effects and the possible influence of vasopressin on UAE in the healthy kidney. The present study was thus designed to evaluate whether vasopressin influences UAE in normal rats and humans, whether this effect is V 2 -receptor-dependent, and whether it is mediated by the renin-angiotensin system. Methods. UAE was measured in normal Wistar rats and healthy humans, or in subjects with various forms of diabetes insipidus (DI), before and after acute or chronic infusion of the vasopressin V 2 receptor agonist dDAVP®. Chronic dDAVP administration was also performed in normal Wistar rats previously submitted to either chronic angiotensin-converting enzyme inhibition (ACEI) or chronic blockade of ATI receptors (ARB). Results. In rats, acute or chronic dDAVP infusion increased UAE significantly and reversibly (4-fold and 6-fold, respectively). In healthy subjects, acute infusion of dDAVP tripled UAE (P

Mutations in the Aquaporin-2 gene, which encodes a renal water channel, have been shown to cause autosomal nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. Most AQP2 missense mutants in recessive NDI are retained in the endoplasmic reticulum (ER), but AQP2-T125M and AQP2-G175R were reported to be nonfunctional channels unimpaired in their routing to the plasma membrane. In five families, seven novel AQP2 gene mutations were identified and their cell-biologic basis for causing recessive NDI was analyzed. The patients in four families were homozygous for mutations, encoding AQP2-L28P, AQP2-A47V, AQP2-V71M, or AQP2-P185A. Expression in oocytes revealed that all these mutants, and also AQP2-T125M and AQP2-G175R, conferred a reduced water permeability compared with wt-AQP2, which was due to ER retardation. The patient in the fifth family had a G>A nucleotide substitution in the splice donor site of one allele that results in an out-of-frame protein. The other allele has a nucleotide deletion (c652delC) and a missense mutation (V194I). The routing and function of AQP2-V194I in oocytes was not different from wt-AQP2; it was therefore concluded that c652delC, which leads to an out-of-frame protein, is the NDI-causing mutation of the second allele. This study indicates that misfolding and ER retention is the main, and possibly only, cell-biologic basis for recessive NDI caused by missense AQP2 proteins. In addition, the reduced single channel water permeability of AQP2-A47V (40%) and AQP2-T125M (25%) might become of therapeutic value when chemical chaperones can be found that restore their routing to the plasma membrane.

Mutations in the aquaporin-2 gene (AQP2), encoding the vasopressin-regulated water channel of the renal collecting duct, are responsible for the autosomal recessive or dominant forms of congenital nephrogenic diabetes insipidus. We describe two new families with normal hypotensive and coagulation responses following the administration of desamino-8-D-arginine AVP, a clinical suggestion of normal vasopressin-2 receptors. The patients were compound heterozygotes for point mutations at nucleotide position 170 (CAG to CCG; Q57P) and at position 299 (GGA to GTA; G100V) in exon 1 of the AQP2 gene. Expression of the G57P and G100V AQP2 proteins in Xenopus oocytes showed only 1.3-fold and 1.2-fold increase, respectively, in the water permeability in contrast to 8.0-fold increase in oocytes injected with wild-type cRNA. Immunoblots of oocyte lysate revealed the intensities of the 29-kDa bands were comparable among oocytes injected with wild-type and mutant cRNAs. Immunocytochemistry showed the plasma membrane was not stained in oocytes injected with cRNA of Q57P and of G100V. These results provide evidence that the Q57P and G100V mutations in congenital nephrogenic diabetes insipidus are attributable to the misrouting of AQP2.

The vasopressin-regulated water channel aquaporin-2 (AQP2) is known to tetramerize in the apical membrane of the renal tubular cells and contributes to urine concentration. We identified three novel mutations, each in a single allele of exon 4 of the AQP2 gene, in three families showing autosomal dominant nephrogenic diabetes insipidus (NDI). These mutations were found in the C-terminus of AQP2: a deletion of G at nucleotide 721 (721 delG), a deletion of 10 nucleotides starting at nucleotide 763 (763–772del), and a deletion of 7 nucleotides starting at nucleotide 812 (812–818del). The wild-type AQP2 is predicted to be a 271–amino acid protein, whereas these mutant genes are predicted to encode proteins that are 330–333 amino acids in length, because of the frameshift mutations. Interestingly, these three mutant AQP2s shared the same C-terminal tail of 61 amino acids. In Xenopus oocytes injected with mutant AQP2 cRNAs, the osmotic water permeability (Pf) was much smaller than that of oocytes with the AQP2 wild-type (14%–17%). Immunoblot analysis of the lysates of the oocytes expressing the mutant AQP2s detected a band at 34 kD, whereas the immunoblot of the plasma-membrane fractions of the oocytes and immunocytochemistry failed to show a significant surface expression, suggesting a defect in trafficking of these mutant proteins. Furthermore, coinjection of wild-type cRNAs with mutant cRNAs markedly decreased the oocyte Pf in parallel with the surface expression of the wild-type AQP2. Immunoprecipitation with antibodies against wild-type and mutant AQP2 indicated the formation of mixed oligomers composed of wild-type and mutant AQP2 monomers. Our results suggest that the trafficking of mutant AQP2 is impaired because of elongation of the C-terminal tail, and the dominant-negative effect is attributed to oligomerization of the wild-type and mutant AQP2s. Segregation of the mutations in the C-terminus of AQP2 with dominant-type NDI underlies the importance of this domain in the intracellular trafficking of AQP2.

Over 155 mutations within the V2 vasopressin receptor (AVPR2) gene are responsible for nephrogenic diabetes insipidus (NDI). The expression and subcellular distribution of four of these was investigated in transfected cells. These include a point mutation in the seventh transmembrane domain (S315R), a frameshift mutation in the third intracellular loop (804delG), and two nonsense mutations that code for AVPR2 truncated within the first cytoplasmic loop (W71X) and in the proximal portion of the carboxyl tail (R337X). RT-PCR revealed that mRNA was produced for all mutant receptor constructs. However, no receptor protein, as assessed by Western blot analysis, was detected for 804delG. The S315R was properly processed through the Golgi and targeted to the plasma membrane but lacked any detectable AVP binding or signaling. Thus, this mutation induces a conformational change that is compatible with endoplasmic reticulum (ER) export but dramatically affects hormone recognition. In contrast, the W71X and R337X AVPR2 were retained inside the cell as determined by immunofluorescence. Confocal microscopy revealed that they were both retained in the ER. To determine if calnexin could be involved, its interaction with the AVPR2 was assessed. Sequential coimmunoprecipitation demonstrated that calnexin associated with the precursor forms of both wild-type (WT) and mutant receptors in agreement with its general role in protein folding. Moreover, its association with the ER-retained R337X mutant was found to be longer than with the WT receptor suggesting that this molecular chaperone also plays a role in quality control and ER retention of misfolded G protein-coupled receptors.

It is clinically useful to distinguish between two types of hereditary nephrogenic diabetes insipidus (NDI): a ‘pure’ type characterized by loss of water only and a complex type characterized by loss of water and ions. Patients with congenital NDI bearing mutations in the vasopressin 2 receptor gene, AVPR2, or in the aquaporin-2 gene, AQP2, have a pure NDI phenotype with loss of water but normal conservation of sodium, potassium, chloride and calcium. Patients with hereditary hypokalemic salt-losing tubulopathies have a complex phenotype with loss of water and ions. They have polyhydramnios, hypercalciuria and hypo- or isosthenuria and were found to bear KCNJ1 (ROMK) and SLC12A1 (NKCC2) mutations. Patients with polyhydramnios, profound polyuria, hyponatremia, hypochloremia, metabolic alkalosis and sensorineural deafness were found to bear BSND mutations. These clinical phenotypes demonstrate the critical importance of the proteins ROMK, NKCC2 and Barttin to transfer NaCl in the medullary interstitium and thereby to generate, together with urea, a hypertonic milieu. This editorial describes two new developments: (i) the genomic information provided by the sequencing of the AQP2 gene is key to the routine care of these patients, and, as in other genetic diseases, reduces health costs and provides psychological benefits to patients and families and (ii) the expression of AQP2 mutants in Xenopus oocytes and in polarized renal tubular cells recapitulates the clinical phenotypes and reveals a continuum from severe loss of function with urinary osmolalities 200 mOsm/kg H2O.

Aquaporin-2 (AQP2), located at the luminal side of the collecting duct principal cells, is a water channel responsible for the final concentration of urine. Lack of function, often occurring through mistargeting of mutated proteins, induces nephrogenic diabetes insipidus (NDI), a condition characterized by large urinary volumes. In the present study, two new mutations (K228E and V24A) identified in NDI-affected individuals from distinct families along with the already reported R187C were analysed in comparison to the wild-type protein (AQP2-wt) using Xenopus laevis oocytes and a mouse collecting duct cell-line (mIMCD-3). Initial data in oocytes showed that all mutations were adequately expressed at reduced levels when compared to AQP2-wt. K228E and V24A were found to be properly targeted at the plasma membrane and exhibited adequate functionality similar to AQP2-wt, as opposed to R187C which was retained in internal stores and was thus inactive. In coexpression studies using oocytes, R187C impeded the functionality of all other AQP2 variants while combinations with K228E, V24A and AQP2-wt only showed additive functionalities. When expressed in mIMCD-3 cells, forskolin treatment efficiently promoted the targeting of AQP2-wt at the plasma membrane (90%) while K228E only weakly responded to the same treatment (approximately 20%) and both V24A and R187C remained completely insensitive to the treatment. We concluded that both V24A and K228E are intrinsically functional water channels that lack a proper response to vasopressin, which leads to NDI as found in both compound mutations studied (K228E + R187C and V24A + R187C). The discrepancies in plasma membrane targeting response found in both expression systems stress the need to evaluate such data using mammalian cell systems.

In most cases, nephrogenic diabetes insipidus results from mutations in the V2 vasopressin receptor (V2R) gene that cause intracellular retention of improperly folded receptors. We previously reported that cell permeable V2R antagonists act as pharmacological chaperones that rescue folding, trafficking, and function of several V2R mutants. More recently, the vasopressin antagonist, SR49059, was found to be therapeutically active in nephrogenic diabetes insipidus patients. Three of the patients with positive responses harbored the mutation R137H, previously reported to lead to constitutive endocytosis. This raises the possibility that, instead of acting as a pharmacological chaperone by favoring proper maturation of the receptors, SR49059 could mediate its action on R137H V2R by preventing its endocytosis. Here we report that the β-arrestin-mediated constitutive endocytosis of R137H V2R is not affected by SR49059, indicating that the functional rescue observed does not result from a stabilization of the receptor at the cell surface. Moreover, metabolic labeling revealed that R137H V2R is also poorly processed to the mature form. SR49059 treatment significantly improved its maturation and cell surface targeting, indicating that the functional rescue of R137H V2Rs results from the pharmacological chaperone action of the antagonist.

Contains fulltext : 57229.pdf (Publisher’s version ) (Closed access) Vasopressin regulates water homeostasis through insertion of homotetrameric aquaporin-2 (AQP2) water channels in the apical plasma membrane of renal cells. AQP2 mutations cause recessive and dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. Until now, all AQP2 mutants in recessive NDI were shown to be misfolded, retained in the endoplasmic reticulum (ER) and unable to interact with wild-type (wt)-AQP2, whereas AQP2 mutants in dominant NDI are properly folded and interact with wt-AQP2, but, due to the mutation, cause missorting of the wt-AQP2/mutant complex. Here, patients of two families with recessive NDI appeared compound heterozygotes for AQP2-A190T or AQP2-R187C mutants, together with AQP2-P262L. As mutations in the AQP2 C-tail, where P262 resides, usually cause dominant NDI, the underlying cell-biological mechanism was investigated. Upon expression in oocytes, AQP2-P262L was a properly folded and functional aquaporin in contrast to the classical mutants, AQP2-R187C and AQP2-A190T. Expressed in polarized cells, AQP2-P262L was retained in intracellular vesicles and did not localize to the ER. Upon co-expression, however, AQP2-P262L interacted with wt-AQP2, but not with AQP2-R187C, resulting in a rescued apical membrane expression of AQP2-P262L. In conclusion, our study reveals a novel cellular phenotype in recessive NDI in that AQP2-P262L acts as a mutant in dominant NDI, except for that its missorting is overruled by apical sorting of wt-AQP2. Also, it demonstrates for the first time that the recessive inheritance of a disease involving a channel can be due to two cell-biological mechanisms.

Item does not contain fulltext Vasopressin regulates body water conservation by redistributing aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical surface of renal collecting ducts, resulting in water reabsorption from urine. Mutations in AQP2 cause autosomal nephrogenic diabetes insipidus (NDI), a disease characterized by the inability to concentrate urine. Here, we report a frame-shift mutation in AQP2 causing dominant NDI. This AQP2 mutant is a functional water channel when expressed in Xenopus oocytes. However, expressed in polarized renal cells, it is misrouted to the basolateral instead of apical plasma membrane. Additionally, this mutant forms heterotetramers with wild-type AQP2 and redirects this complex to the basolateral surface. The frame shift induces a change in the COOH terminus of AQP2, creating both a leucine- and a tyrosine-based motif, which cause the reversed sorting of AQP2. Our data reveal a novel cellular phenotype in dominant NDI and show that dominance of basolateral sorting motifs in a mutant subunit can be the molecular basis for disease.

Item does not contain fulltext Autosomal nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin, is caused by mutations in the Aquaporin-2 (AQP2) gene. Analysis of a new family with dominant NDI revealed a single nucleotide deletion (727deltaG) in one AQP2 allele, which encoded an AQP2 mutant with an altered and extended C-terminal tail. When expressed in oocytes, the tetrameric AQP2-727deltaG was retained within the cell. When co-expressed, AQP2-727deltaG, but not a mutant in recessive NDI (AQP2-R187C), formed hetero-oligomers with wild-type (wt) AQP2 and reduced the water permeability of these oocytes, because of a reduced plasma membrane expression of wt-AQP2. Expressed in renal epithelial cells, AQP2-727deltaG predominantly localized to the basolateral membrane and late endosomes/lysosomes, whereas wt-AQP2 was expressed in the apical membrane. Upon co-expressing in these cells, wt-AQP2 and AQP2-727deltaG mainly co-localized to late endosomes/lysosomes. In conclusion, hetero-oligomerization of AQP2-727deltaG with wt-AQP2 and consequent mistargeting of this complex to late endosomes/lysosomes results in absence of AQP2 in the apical membrane, which can explain dominant NDI in this family. Together with other mutants in dominant NDI, our data reveal that a misrouting, instead of a lack of function, is a general mechanism for the 'loss of function' phenotype in dominant NDI and visualizes for the first time a mislocalization of a wild-type protein to late endosomes/lysosomes in polarized cells after oligomerization with a mutant protein.

Antidiuretic hormone (arginine vasopressin) binds to and activates V2 receptors in renal collecting tubule cells. Subsequent stimulation of the Gs/adenylyl cyclase system promotes insertion of water pores into the luminal membrane and thereby reabsorption of fluid. In congenital nephrogenic diabetes insipidus (CNDI), an X-linked recessive disorder, the kidney fails to respond to arginine vasopressin. Here we report that an affected male of a family with CNDI has a deletion in the open reading frame of the V2 receptor gene, causing a frame shift and premature termination of translation in the third intracellular loop of the receptor protein. A normal receptor gene was found in the patient's brother. Both the normal and the mutant allele were detected in his mother. A different mutation, causing a codon change in the third transmembrane domain of the V2 receptor, was found in the open reading frame of an affected male but not in the unaffected brother belonging to another family suffering from CNDI.

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease caused by mutations in the arginine vasopressin receptor 2 gene (AVPR2). Thirty-three novel AVPR2 mutations were identified in 62 families that were not included in our previous studies. This study describes the diversity of mutations observed in a total of 117 families, the number of affected people at the time of diagnosis, skewed X chromosome inactivation in severely affected females, the inferred parental origin of de novo mutations, and it provides estimates of incidence. Among 117 families, there were 82 different putative disease-causing mutations. Based on haplotype analysis, it can be inferred that when the same AVPR2 mutation is identified in different families that were not known to be related, the mutations most likely arose independently. More than half of the families had only one affected male; two families presented with a severely affected female and no family history of NDI. A de novo mutation arose during oogenesis in the mother in 20% of isolated cases. The estimate of about 8.8 per million male live births of the incidence of X-linked NDI in the province of Quebec, Canada may be representative of the general population except in Nova Scotia and New Brunswick, where the incidence is more than six times higher. Documentation of the diversity of mutations will assist in revealing the full spectrum of clinical variation. Discussion of genetic and population genetic aspects of X-linked NDI may contribute to early diagnosis and treatment.

Over 150 mutations within the coding sequence of the V2 vasopressin receptor (V2R) gene are known to cause nephrogenic diabetes insipidus (NDI). A large number of these mutant receptors fail to fold properly and therefore are not routed to the cell surface. Here we show that selective, nonpeptidic V2R antagonists dramatically increase cell-surface expression and rescue the function of 8 mutant NDI-V2Rs by promoting their proper folding and maturation. A cell-impermeant V2R antagonist could not mimic these effects and was unable to block the rescue mediated by a permeant agent, indicating that the nonpeptidic antagonists act intracellularly, presumably by binding to and stabilizing partially folded mutants. In addition to opening new therapeutic avenues for NDI patients, these data demonstrate that by binding to newly synthesized mutant receptors, small ligands can act as pharmacological chaperones, promoting the proper folding and maturation of receptors and their targeting to the cell surface.

Mutations in the aquaporin-2 (AQP2) water channel gene cause autosomal recessive nephrogenic diabetes insipidus (NDI). Here we report the first patient with an autosomal dominant form of NDI, which is caused by a G866A transition in the AQP2 gene of one allele, resulting in a E258K substitution in the C-tail of AQP2. To define the molecular cause of NDI in this patient, AQP2-E258K was studied in Xenopus oocytes. In contrast to wild-type AQP2, AQP2-E258K conferred a small increase in water permeability, caused by a reduced expression at the plasma membrane. Coexpression of wild-type AQP2 with AQP2-E258K, but not with an AQP2 mutant in recessive NDI (AQP2-R187C), revealed a dominant-negative effect on the water permeability conferred by wild-type AQP2. The physiologically important phosphorylation of S256 by protein kinase A was not affected by the E258K mutation. Immunoblot and microscopic analyses revealed that AQP2-E258K was, in contrast to AQP2 mutants in recessive NDI, not retarded in the endoplasmic reticulum, but retained in the Golgi compartment. Since AQPs are thought to tetramerize, the retention of AQP2-E258K together with wild-type AQP2 in mixed tetramers in the Golgi compartment is a likely explanation for the dominant inheritance of NDI in this patient.

In X-linked nephrogenic diabetes insipidus (NDI) the urine of male patients is not concentrated after the administration of the antidiuretic hormone arginine-vasopressin. This disease is due to mutations in the V2 receptor gene that maps to chromosome region Xq28. In 1969, Bode and Crawford suggested that most NDI patients in North America shared common ancestors of Ulster Scot immigrants who arrived in Halifax in 1761 on the ship Hopewell. A link between this family and a large Utah kindred was also suggested. DNA was obtained from 17 affected male patients from the "Hopewell" kindred and from four additional families from Nova Scotia and New Brunswick who shared the same Xq28 NDI haplotype. The Utah kindred and two families (Q2, Q3) from Quebec were also studied. The "Hopewell" mutation, W71X, is a single base substitution (G-->A) that changes codon 71 from TGG (tryptophan) to TGA (stop). The W71X mutation was found in affected members of the Hopewell and of the four satellite families. The W71X mutation is the cause of X-linked NDI for the largest number of related male patients living in North America. Other families (Utah, Q2 and Q3) that are historically and ethnically unrelated bear other mutations in the V2 receptor gene.

The mechanisms of the stimulation of coagulation factor release and of the vasodilating properties of l-desamino[8-D-arginine]vasopressin (dDAVP) have similarities and our laboratory (Bichet et al., 1988, 1989, 1991) and others (Mannucci et al., 1975; Liard, 1988a, 1988b; Hirsch et al., 1989) have pioneered the concept of extrarenal V2 receptors.

The antidiuretic (V2) agonist, desmopressin (dDAVP), stimulates release of the clotting factors von Willebrand factor (vWF) and factor VIIIc (FVIIIc) in humans. dDAVP (3.0 μg/kg) stimulated release of vWF and FVIIIc, and was associated with hypotension and tachycardia in the pentobarbital-anesthetized rhesus monkeys (Macaca mulatta). The V2 receptor antagonist, SK&F 105494 (30 μg/kg), had no effect on clotting factor release, blood pressure or heart rate, but prevented dDAVP stimulation of clotting factor release, hypotension, and tachycardia. The results support the hypothesis that dDAVP stimulation of clotting factor release and vasodilation are mediated by low-affinity, V2-like receptor mechanisms.

Platelet vasopressin receptors in patients with congenital nephrogenic diabetes insipidus. Arginine-vasopressin(AVP), interacts with at least two types of receptors: V 1 receptors which mediate the aggregating effects of AVP on human blood platelets and other AVP actions on vascular smooth muscle and hepatocytes; and V 2 receptors which mediate the antidiuretic effects on renal tubules. Congenital nephrogenic diabetes insipidus (CNDI) is a rare X-linked disorder in humans with abnormal renal and extrarenal V 2 -receptor responses. However, the V 1 receptor responses are apparently normal, since in these patients blood pressure increases in response to AVP. To assess V 1 receptor responses, binding studies ( 3 H-AVP) were done on intact platelets obtained from 6 male patients with CNDI, 10 normal subjects and 4 patients with autosomal dominant central diabetes insipidus (ADCDI). The affinity constant (0.68 ± 0.04 vs. 0.59 ± 0.06 nm) and the number of specific binding sites per platelet (101 ± 6 vs. 86 ± 12) were similar in the normal subjects and the patients with CNDI. However, the number of binding sites per platelet was increased in the patients with ADCDI (189 ± 12). Platelet aggregation induced by AVP was equivalent in the three groups Platelet-fraction AVP was elevated in patients with CNDI and undetectable in patients with ADCDI. These results suggest that the structure and the function of V 1 platelet receptor-effector pathway are normal in patients with CNDI.

Objective: We describe the effects of withdrawal of enzyme replacement therapy (ERT) in FD. Methods: Patients were followed up prospectively after ERT withdrawal that occurred due to lack of government reimbursement of this licensed therapy. Results: Seventeen patients (13 males, 4 females) aged 22 to 72 years (mean age, 41.6 years) received ERT intravenously every 2 weeks for a mean of 32.8 months (range, 4-63 months). Three received agalsidase beta (Fabrazyme ® 1 mg/kg); 10 received agalsidase alfa (Replagal ® 0.2 mg/kg); and 4 received both in turn. One patient was lost to follow-up. Mean estimated glomerular filtration rate (eGFR) (adjusted modification of diet in renal disease formula) was 95.9 mL/min/1.73 m 2 pre-ERT, 88.9 mL/min/l.73 m 2 with ERT, and 83.3 mL/min/1.73 m 2 after a mean of 14.8 months off ERT. Class 3 to 5 chronic kidney disease (eGFR

We measured glycine release from ([2-3H]glycine)-labelled GSH and glucose formation from maltose incubated with rat kidney whole cortex homogenate, thin cortex slices or collagenase-treated tubule fragments. Liberation of glycine was inhibited (74-83%) by serine borate (20 mM), indicating a gamma-glutamyltransferase-dependent hydrolysis of GSH. In whole cortex homogenate, the GSH cleavage activity was 17.4 +/- 0.6 nmol GSH degraded/mg protein per min (mean +/- S.D.); cleavage activity by intact slices was 3.5 +/- 0.7 (P less than 0.001 relative to whole cortex homogenate) and in tubule fragments 9.4 +/- 0.8 (P less than 0.001). Homogenizing the tissue preparation increased cleavage rate in slices about 4-fold (12.4 +/- 2.9; P less than 0.005 relative to intact slice) but did not change the rate in tubule fragments (9.8 +/- 0.5). Maltose cleavage activity in whole cortex homogenate was 512 +/- 22 nmol glucose formed/mg protein per min, in slices 162 +/- 12, and in tubules 884 +/- 48. These findings imply that substrate in the incubation medium has a limited access to the luminal membrane of cortex slices but not of tubule fragments. They further imply that basolateral membrane is preferentially exposed in the slice preparation.

Arginine-vasopressin (AVP) immunoreactivity (Ir) has been found to be elevated in platelet-rich plasma. PlatAVP was defined as platelet-rich plasma Ir minus platelet-poor plasma Ir (Pavp). PlatAVP, Pavp, and synthetic AVP were found to have identical retention time on high performance liquid chromatography analysis and similar mobility on thin-layer chromatography. During a standard osmotic suppression-stimulation test, Pavp increased with plasma osmolality (Posm, mosmol/kg H2O); Pavp (pg/ml) = 0.98 (Posm -274.4), r = 0.57, P less than 0.001, n = 65; but PlatAVP was not significantly correlated with Posm and remained at 5 pg/ml. This PlatAVP concentration was estimated to represent a true intraplatelet AVP concentration of 0.4 to 3.7 X 10(-9) M. Binding studies on intact human platelets demonstrated specific binding sites for [3H]AVP (n = 16; BMax = 98 +/- 30 binding sites/platelet; Kd = 0.72 +/- 0.24 nM). This in vitro affinity association constant (Kd) was close to the estimated in vivo intraplatelet AVP concentration. Measurement of PlatAVP could estimate vasopressin bound to a specific platelet receptor.

The antidiuretic hormone arginine vasopressin interacts with two types of receptors: V1, which mediates the effects of vasopressin on vascular smooth muscle, and V2, which mediates the antidiuretic effects on renal tubules. Resistance of the renal tubules to arginine vasopressin and to the antidiuretic V2-specific agonist 1-desamino[8-D-arginine] vasopressin (dDAVP) occurs in congenital nephrogenic diabetes insipidus, a rare X-linked disease, although the V1-receptor responses remain intact. The extrarenal actions of dDAVP in normal persons are a decrease in blood pressure, an increase in plasma renin activity, and stimulation of the release of factor VIIIc and von Willebrand factor. We measured the response of mean arterial pressure, pulse rate, plasma renin activity, factor VIIIc, and von Willebrand factor to an infusion of dDAVP (0.3 microgram per kilogram of body weight) in seven male patients with congenital nephrogenic diabetes insipidus, six obligatory carriers of the gene for nephrogenic diabetes insipidus, five patients with central diabetes insipidus, and four normal subjects. In the normal subjects and the patients with central diabetes insipidus, dDAVP decreased mean arterial pressure (by 10 to 15 percent) and increased pulse rate (by 20 to 25 percent), renin activity (by 65 percent), and the release of coagulation factors (twofold to threefold) (all changes were significant, P less than 0.01). None of these changes were observed in the patients with congenital nephrogenic diabetes insipidus, and minimal responses were observed in the obligatory carriers. These results confirm the existence of extrarenal vasopressin V2-like receptors, which may be defective in patients with congenital nephrogenic diabetes insipidus.

Epinephrine and dDAVP administration in patients with congenital nephrogenic diabetes insipidus. Evidence for a pre-cyclic AMP V 2 receptor defective mechanism. We recently showed that the administration of the antidiuretic V 2 specific agonist, l-desamino[8-D-arginine]vasopressin (dDAVP), to seven male patients with congenital nephrogenic diabetes insipidus (CNDI) did not cause a decrease in blood pressure nor an increase in plasma renin activity or factor VIIIc or von Willebrand factor release. In normal subjects, plasma renin activity, coagulation factors and plasma cyclic AMP are stimulated not only by dDAVP but also by the administration of epinephrine. In the present study, we measured tissue plasminogen activator (activity and antigenicity), von Willebrand factor multimers, plasma and urinary cyclic AMP concentrations following dDAVP or epinephrine administration. We infused epinephrine into three male patients with CNDI. Factor VIIIc and tissue plasminogen activator augmented by 75 to 100% and von Willebrand Factor multimers were increased; plasma renin activity and plasma cyclic AMP concentration increased by 200%. None of these values changed when the same subjects as well as eleven other male patients with CNDI received dDAVP. Furthermore, dDAVP administration increased plasma cyclic AMP concentrations in normal subjects, but not in 14 male patients with CNDI. These results demonstrate the specificity of the extrarenal V 2 receptor defect expressed in our patients. The lack of a plasma cyclic AMP response to the administration of dDAVP would suggest an altered pre-cyclic AMP stimulation mechanism.

We investigated the process of neonatal hyperprolinuria in dog and rat. Plasma proline varied only 2-fold in the puppy whereas prolinuria increased up to 12-fold from birth to the 10th day declining thereafter to reach adult values (

Publisher Summary Ontogeny represents intensification of specific transport systems. It is applied to postnatal changes in the renal handling of proline and glycine. Increasing single-nephron filtration accounts for increasing iminoglycinuria in the early postnatal period. This chapter presents that backflux from immature distal tubule epithelium is not a source of neonatal iminoglycinuria. Mature pars recta accumulates amino acids avidly and, if this phenomenon occurs in the immature nephron also, it is possible that backflux of intracellular amino acids from this segment contributes to neonatal iminoglycinuria. The specific schedule of intensification characterizes the maturation of proline and glycine reabsorption. The chapter reviews that a mosaic of events contributes to the ontogeny of prolinuria. The proliferation of the brush-border membrane and lengthening of nephron segments change the general parameters of reabsorption, while specific intensification of selective carriers, changes in permeability, and oscillations of intracellular metabolism each contribute to the specific schedule of ontogeny.

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease with defective renal and extrarenal arginine vasopressin V2 receptor responses due to mutations in the AVPR2 gene in Xq28. To study the cause of loss of function of mutant V2 receptors, we expressed 12 mutations (N55H, L59P, L83Q, V88M, 497CC-->GG, deltaR202, I209F, 700delC, 908insT, A294P, P322H, P322S) in COS-7 cells. Eleven of these, including P322H, were characterized by a complete loss of function, but the mutation P322S demonstrated a mild clinical and in vitro phenotype. This was characterized by a late diagnosis without any growth or developmental delay and a significant increase in urine osmolality after intravenous 1-deamino[D-Arg8]AVP administration. In vitro, the P322S mutant was able to partially activate the Gs/adenylyl cyclase system in contrast to the other V2R mutants including P322H, which were completely inactive in this regard. This showed not only that Pro 322 is important for proper V2R coupling, but also that the degree of impairment is strongly dependent on the identity of the substituting amino acid. Three-dimensional modeling of the P322H and P322S mutant receptors suggested that the complete loss of function of the P322H receptor could be due, in part, to hydrogen bond formation between the His 322 side chain and the carboxyl group of Asp 85, which does not occur in the P322S receptor.