Diabetes insipidus in mice with a mutation in aquaporin-2

Congenital nephrogenic diabetes insipidus (NDI) is a disease characterized by failure of the kidney to concentrate urine in response to vasopressin. Human kindreds with nephrogenic diabetes insipidus have been found to harbor mutations in the vasopressin receptor 2 (Avpr2) gene or the vasopressin-sensitive water channel aquaporin-2 (Aqp2) gene. Development of a treatment is rendered difficult due to the lack of a viable animal model. Through forward genetic screening of ethylnitrosourea-mutagenized mice, we report the identification and characterization of a mouse model of NDI, with an F204V mutation in the Aqp2 gene. Unlike previously attempted murine models of NDI, our mice survive to adulthood and more exactly recapitulate the human disorder. Previous in vitro experiments using renal cell lines suggest recessive Aqp2 mutations result in improper trafficking of the mutant water pore. Using these animals, we have directly proven this hypothesis of improper AQP2 translocation as the molecular defect in nephrogenic diabetes insipidus in the intact organism. Additionally, using a renal cell line we show that the mutated protein, AQP2-F204V, is retained in the endoplasmic reticulum and that this abnormal localization can be rescued by wild-type protein. This novel mouse model allows for further mechanistic studies as well as testing of pharmacological and gene therapies for NDI.     

Erythrocyte water permeability and renal function in double knockout mice lacking aquaporin-1 and aquaporin-3

Aquaporin (AQP) water channel AQP3 has been proposed to be the major glycerol and non-AQP1 water transporter in erythrocytes. AQP1 and AQP3 are also expressed in the kidney where their deletion in mice produces distinct forms of nephrogenic diabetes insipidus. Here AQP1/AQP3 double knockout mice were generated and analyzed to investigate the functional role of AQP3 in erythrocytes and kidneys. 53 double knockout mice were born out of 756 pups from breeding double heterozygous mice. The double knockout mice had reduced survival and impaired growth compared with the single knockout mice. Erythrocyte water permeability was 7-fold reduced by AQP1 deletion but not further reduced in AQP1/AQP3 null mice. AQP3 deletion did not affect erythrocyte glycerol permeability or its inhibition by phloretin. Daily urine output in AQP1/AQP3 double knockout mice (15 ml) was 9-fold greater than in wild-type mice, and urine osmolality (194 mosm) was 8.4-fold reduced. The mice remained polyuric after DDAVP administration or water deprivation. The renal medulla in most AQP1/AQP3 null mice by age 4 weeks was atrophic and fluid-filled due to the severe polyuria and hydronephrosis. Our data provide direct evidence that AQP3 is not functionally important in erythrocyte water or glycerol permeability. The renal function studies indicate independent roles of AQP1 and AQP3 in countercurrent exchange and collecting duct osmotic equilibration, respectively.     

Taurine in the osmoregulation of the Brattleboro rat

The function of taurine in mammalian osmoregulation was studied in the Brattleboro rat with hereditary hypothalamic diabetes insipidus (DI). DI rats are chronically dehydrated because of their inability to synthesize vasopressin. One day of water deprivation did not affect the water balance in rats with normal vasopressin synthesis, whereas DI rats were markedly dehydrated and lost considerably body weight. Taurine content and 3H-taurine accumulation by platelets were significantly higher in DI rats, with a further increase after one day of water deprivation. In DI rats, water deprivation also evoked a clear taurine increase in skeletal muscle and in the brain. These findings indicate that taurine has an osmoregulatory function in mammals.     

A Combined Outpatient and Inpatient Overnight Water Deprivation Test is Effective and Safe in Diagnosing Patients with Polyuria-Polydipsia Syndrome

Objective: The water deprivation test (WDT) is widely used for the differential diagnosis of the polyuria-polydipsia syndrome (PPS). However, it is inconvenient and may not always be precise in differentiating partial forms of diabetes insipidus (DI) from primary polydipsia (PP). The aim of this study was to evaluate the results of a combined outpatient and inpatient overnight WDT protocol that included an overnight unsupervised period concerning its feasibility and safety.Methods: We performed a retrospective analysis of clinical data and laboratory results of 52 patients with PPS undergoing WDT at a single center.Results: PP was the most frequent diagnosis, followed by complete central DI (cCDI), partial central DI (pCDI), and nephrogenic DI (NDI). Over 90% of the patients showed an expected increase in serum osmolality at the end of the dehydration period. There were no reports of complications during the overnight deprivation period. Post-dehydration urine osmolality and urine-to-serum osmolality ratio significantly differentiated all the groups (P<.05), except for cCDI and NDI, which could be differentiated by basal and post-dehydration vasopressin (AVP) levels (P<.05 for both). Although these measurements were useful for differentiating patients according to their allocation groups, results from WDT and direct AVP levels may often require a comprehensive diagnostic approach, particularly in the challenging groups of PP and pCDI.Conclusion: A combined outpatient and inpatient overnight WDT protocol is safe and feasible when the test is performed with special care at experienced centers. Newer diagnostic tools are expected to improve the accuracy of PPS diagnosis.Abbreviations: AQP2 = aquaporin-2; AVP = vasopressin; CDI = central diabetes insipidus; cCDI = complete central diabetes insipidus; DDAVP = desmopressin; DI = diabetes insipidus; IQR = interquartile range; MRI = magnetic resonance imaging; Na⁺ = sodium; NDI = nephrogenic diabetes insipidus; pCDI = partial central diabetes insipidus; PP = primary polydipsia; PPS = polyuria-polydipsia syndrome; S_osm = serum osmolality; U_osm = urine osmolality; WDT = water deprivation test     

Effects of adrenalectomy and hypercorticism on the ACTH content of the anterior and posterior pituitary in rats with inherited diabetes insipidus (Brattleboro strain).

The effects of adrenalectomy (Adx) and hypercorticism on the ACTH content in the anterior (AH) and the neurointermediate lobe (NIL) of the pituitary in Long Evans (+/+), heterozygous (+/DI) and homozygous (DI/DI) Brattleboro rats were determined using dispersed adrenal cells bioassay. Adx decreased the NIL-ACTH content in +/DI and DI/DI rats and left it unchanged in the +/+ rats. Adx increased the AH-ACTH content in the three groups. Hypercorticism had a delayed decreasing effect both in the AH and in the NIL in all rats, with one exception for the NIL in DI/DI rats. Conversely to what appeared in Wistar rats, in Long Evans and Brattleboro rats the corticosterone administered in drinking water was unable to reduce the increase in AH-ACTH activity. These data suggest that Brattleboro, and, to a lesser extent, Long Evans rats from which the former are derived present some particularities in the regulation of their corticotropic function at the AH and the NIL level. We also observed that NaCl (0.9%) added to drinking water and hypercorticism are two factors able to increase diabetes insipidus in homozygous rats without modifying the water intake in Long Evans and heterozygous rats.     

Reversed polarized delivery of an aquaporin-2 mutant causes dominant nephrogenic diabetes insipidus

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.     

Glycosylation is important for cell surface expression of the water channel aquaporin-2 but is not essential for tetramerization in the endoplasmic reticulum

Aquaporin-2 (AQP2) is a pore-forming protein that is required for regulated reabsorption of water from urine. Mutations in AQP2 lead to nephrogenic diabetes insipidus, a disorder in which functional AQP2 is not expressed on the apical cell surface of kidney collecting duct principal cells. The mechanisms and pathways directing AQP2 from the endoplasmic reticulum to the Golgi complex and beyond have not been defined. We found that approximately 25% of newly synthesized AQP2 is glycosylated. Nonglycosylated and complex-glycosylated wild-type AQP2 are stable proteins with a half-life of 6-12 h and are both detectable on the cell surface. We show that AQP2 forms tetramers in the endoplasmic reticulum during or very early after synthesis and reaches the Golgi complex in 1-1.5 h. We also report that glycosylation is neither essential for tetramerization nor for transport from the endoplasmic reticulum to the Golgi complex. Instead, the N-linked glycan is important for exit from the Golgi complex and sorting of AQP2 to the plasma membrane. These results are important for understanding the molecular mechanisms responsible for the intracellular retention of AQP2 in nephrogenic diabetes insipidus.     

Defective protein phosphorylation in renal medulla of vasopressin-resistant mice

Protein phosphorylation was compared in renal medulla of normal mice and a strain with hereditary vasopressin-resistant diabetes insipidus (DI mice). The phosphorylation of a specific pair of proteins (M_r 185,000 and 175,000) in the particulate fraction was found to be impaired in DI mice in a fashion that correlated with the severity of the concentrating defect. Defective protein phosphorylation in DI mice may be related to the pathogenesis of cellular unresponsiveness to vasopressin.     

Aldose reductase-deficient mice develop nephrogenic diabetes insipidus

Aldose reductase (ALR2) is thought to be involved in the pathogenesis of various diseases associated with diabetes mellitus, such as cataract, retinopathy, neuropathy, and nephropathy. However, its physiological functions are not well understood. We developed mice deficient in this enzyme and found that they had no apparent developmental or reproductive abnormality except that they drank and urinated significantly more than their wild-type littermates. These ALR2-deficient mice exhibited a partially defective urine-concentrating ability, having a phenotype resembling that of nephrogenic diabetes insipidus.     

Detection of aquaporin-2 in the plasma membranes of oocytes: a novel isolation method with improved yield and purity

Aquaporin-2 (AQP2) water channel mutations cause autosomal recessive and dominant nephrogenic diabetes insipidus (NDI). Expressed in oocytes, a mutant in dominant (AQP2-E258K), but not in recessive (AQP2-R187C), NDI conferred a specific dominant-negative effect on wild-type (wt) AQP2 water permeability (Pf) only at low expression levels. Since at these levels, the yield of conventional-isolated plasma membranes was too low, an improved technique to semiquantify AQP2 in the plasma membrane was needed. Antibodies against the C-loop of AQP2 were not applicable since they were unspecific and introduction of a tag into this loop caused misfolding and ER retardation. Membrane-impermeable biotin analogues turned out to label intracellular AQP2 proteins. Therefore, a method has been developed which generates a high yield of nearly pure plasma membranes, which enables semiquantification of plasma membrane proteins expressed at low levels in oocytes. Our new method allows for phenotype-genotype correlation studies in a wide range of channelopathies.     

Diabetes Insipidus in Pregnancy: Etiology,Evaluation, and Management

ObjectiveTo review the approach to a patient with diabetes insipidus during pregnancy.MethodsThis review examines the normal physiology of water homeostasis, the related changes that occur during pregnancy, and the pathophysiology of diabetes insipidus in pregnancy. Associated complications, evaluation, and management are discussed.ResultsDiabetes insipidus can complicate up to 1 in 30000 pregnancies. Diabetes insipidus during pregnancy has a variety of causes, some that predate the pregnancy and others that begin during gestation. Polyuria and polydipsia can occur or be exacerbated in women with overt or subclinical central or nephrogenic diabetes insipidus. These women have either decreased central secretory reserve or impaired renal responsiveness to vasopressin. In addition, women can experience diabetes insipidus de novo in pregnancy through the actions of placental vasopressinase, which causes accelerated degradation of vasopressin. This form of diabetes insipidus may be associated with increased complications of pregnancy, including preeclampsia. Management of central diabetes insipidus and transient diabetes insipidus of pregnancy can be achieved with 1-deamino-8-D-arginine vasopressin (desmopressin acetate) (DDAVP), a vasopressin analogue. Nephrogenic diabetes insipidus is typically resistant to both DDAVP and vasopressin and underlying causes should be addressed.ConclusionsIncreased awareness of diabetes insipidus in pregnancy may lead to early diagnosis and appropriate treatment that will reduce the risks of maternal and fetal morbidity. Overall, growing experience with DDAVP has shown that it is a safe and effective treatment for diabetes insipidus caused by a variety of factors. (Endocr Pract. 2009;15:377-382)     

Phenotypic expression of the mutant gene diabetes insipidus in rats and criteria of genotyping by phenotype

The autosomal semidominant mutant gene di (diabetes insipidus) is manifested in homozygotes in the form of diabetes insipidus with water consumption from 25 to 100% of body weight per day. The heterozygotes di/+ drink water at a rate higher than 5% but lower than 25%. The level of water consumption in rats with +/+ genotype does not exceed 5% of body weight per day. Segregation analysis of F1 animals yielded by various crosses showed that genotyping of di/di homozygotes is absolutely reliable at 30% and higher level of the water consumption per day.     

New insights into the dynamic regulation of water and acid-base balance by renal epithelial cells

Maintaining tight control over body fluid and acid-base homeostasis is essential for human health and is a major function of the kidney. The collecting duct is a mosaic of two cell populations that are highly specialized to perform these two distinct processes. The antidiuretic hormone vasopressin (VP) and its receptor, the V2R, play a central role in regulating the urinary concentrating mechanism by stimulating accumulation of the aquaporin 2 (AQP2) water channel in the apical membrane of collecting duct principal cells. This increases epithelial water permeability and allows osmotic water reabsorption to occur. An understanding of the basic cell biology/physiology of AQP2 regulation and trafficking has informed the development of new potential treatments for diseases such as nephrogenic diabetes insipidus, in which the VP/V2R/AQP2 signaling axis is defective. Tubule acidification due to the activation of intercalated cells is also critical to organ function, and defects lead to several pathological conditions in humans. Therefore, it is important to understand how these "professional" proton-secreting cells respond to environmental and cellular cues. Using epididymal proton-secreting cells as a model system, we identified the soluble adenylate cyclase (sAC) as a sensor that detects luminal bicarbonate and activates the vacuolar proton-pumping ATPase (V-ATPase) via cAMP to regulate tubular pH. Renal intercalated cells also express sAC and respond to cAMP by increasing proton secretion, supporting the hypothesis that sAC could function as a luminal sensor in renal tubules to regulate acid-base balance. This review summarizes recent advances in our understanding of these fundamental processes.     

Characterization of Three Vasopressin Receptor 2 Variants: An Apparent Polymorphism (V266A) and Two Loss-of-Function Mutations (R181C and M311V)

Arginine vasopressin (AVP) is released from the posterior pituitary and controls water homeostasis. AVP binding to vasopressin V2 receptors (V2Rs) located on kidney collecting duct epithelial cells triggers activation of Gs proteins, leading to increased cAMP levels, trafficking of aquaporin-2 water channels, and consequent increased water permeability and antidiuresis. Typically, loss-of-function V2R mutations cause nephrogenic diabetes insipidus (NDI), whereas gain-of-function mutations cause nephrogenic syndrome of inappropriate antidiuresis (NSIAD). Here we provide further characterization of two mutant V2Rs, R181C and M311V, reported to cause complete and partial NDI respectively, together with a V266A variant, in a patient diagnosed with NSIAD. Our data in HEK293FT cells revealed that for cAMP accumulation, AVP was about 500- or 30-fold less potent at the R181C and M311V mutants than at the wild-type receptor respectively (and about 4000- and 60-fold in COS7 cells respectively). However, in contrast to wild type V2R, the R181C mutant failed to increase inositol phosphate production, while with the M311V mutant, AVP exhibited only partial agonism in addition to a 37-fold potency decrease. Similar responses were detected in a BRET assay for β-arrestin recruitment, with the R181C receptor unresponsive to AVP, and partial agonism with a 23-fold decrease in potency observed with M311V in both HEK293FT and COS7 cells. Notably, the V266A V2R appeared functionally identical to the wild-type receptor in all assays tested, including cAMP and inositol phosphate accumulation, β-arrestin interaction, and in a BRET assay of receptor ubiquitination. Each receptor was expressed at comparable levels. Hence, the M311V V2R retains greater activity than the R181C mutant, consistent with the milder phenotype of NDI associated with this mutant. Notably, the R181C mutant appears to be a Gs protein-biased receptor incapable of signaling to inositol phosphate or recruiting β-arrestin. The etiology of NSIAD in the patient with V266A V2R remains unknown.     

The Clinical Physiology of Water Metabolism: Part II: Renal Mechanisms for Urinary Concentration; Diabetes Insipidus

The renal reabsorption of water independent of solute is the result of the coordinated function of the collecting duct and the ascending limb of the loop of Henle. The unique juxtaposition of the ascending and descending portions of the loop of Henle and of the vasa recta permits the function of a counter-current multiplier system in which water is removed from the tubular lumen and reabsorbed into the circulation. The driving force for reabsorption is the osmotic gradient in the renal medulla which is dependent, in part, on chloride (followed by sodium) pumping from the thick ascending loop of Henle. Urea trapping is also thought to play an important role in the generation of a hypertonic medullary interstitium. Arginine vasopressin (AVP) acts by binding to receptors on the cell membrane and activating adenylate cyclase. This, inturn, results in the intracellular accumulation of cyclic adenosine monophosphate (AMP) which in some fashion abruptly increases the water permeability of the luminal membrane of cells in the collecting duct. As a consequence, water flows along an osmotic gradient out of the tubular lumen into the medullary interstitium.Diabetes insipidus is the clinical condition associated with either a deficiency of or a resistance to AVP. Central diabetes insipidus is due to diminished release of AVP following damage to either the neurosecretory nuclei or the pituitary stalk. Possible causes include idiopathic, familial, trauma, tumor, infection or vascular lesions. Patients present with polyuria, usually beginning over a period of a few days. The diagnosis is made by showing that urinary concentration is impaired after water restriction but that there is a good response to exogenous vasopressin therapy. Nephrogenic diabetes insipidus can be identified by a patient''s lack of response to AVP. Nephrogenic diabetes insipidus is caused by a familial defect, although milder forms can be acquired as a result of various forms of renal disease. Central diabetes insipidus is eminently responsive to replacement therapy, particularly with dDAVP, a long lasting analogue of AVP. Nephrogenic diabetes insipidus is best treated with a combination of thiazide diuretics as well as a diet low in sodium and protein.     

Electron microscopic studies on the pars intermedia in normal and in mice with hereditary nephrogenic diabetes insipidus

Summary The ultrastructure of the pars intermedia (PI) of the normal VII +/+ and hereditary nephrogenic diabetes inspidus DI Os/+ mice has been studied with particular reference to the morphology of the glandular cells and their innervation. Four types of cells were observed in both the genotypes of mice, 1) the light glandular cell, 2) the dark cell, 3) a type of cell similar to ependymal cells and 4) a small percentage of typical ACTH cells, observed mostly on the PI border of the cleft and rarely in the centre of PI. The predominant light glandular cells contain mainly two types of membrane bound granules: 1) electron dense core granules, which measure 1500–2500 Å and 2) electron lucent vesicles, which measure 3000–4000 Å in diameter. Granules of intermediate size with various density are also present in both types of mice. The electron dense core granules are predominant in DI Os/+ mice, whereas, electron lucent vesicles are predominant in the normal VII +/+ mice. Similar uniform size membrane bound electron dense granules have been observed in ACTH cells of PI and pars distalis. From earlier experimental evidences and the present observations, it is concluded that the dense core granules in PI may be synthesizing ACTH or ACTH-like substance. It is also discussed that these dense core granules may further mature and give rise to MSH in the form of electron lucent vesicles. If it is so, PI light glandular cells may have dual functions, of producing MSH and ACTH. One of the functions of ependymal-like cells, may be the transport of PI secretion.Three types of nerve endings are observed throughout the PI, making synaptic contact with the predominant cell type. The innervation is more in DI Os/+ mice than in normal mice. The classification of these nerves is according to Bargmann and co-workers 1) peptidergic neurosecretory fibers, contain mainly membrane bound dense core granules, measuring 1200 to 1800 Å, and are the classic neurosecretory granules; 2) adrenergic fibers, measuring 700–900 Å; 3) cholinergic fibers, measuring 300–400 Å. Adrenergic and cholinergic fibers are more towards the hypophysial cleft. The increased innervation, the synaptic contact, the extremely hypertrophied PI and the greater activity of its light glandular cells in the DI Os/+ mice show the PI is under the influence of the nervous system.This study was supported by MRC of Canada Grant No. MA-3759.     

Heterogeneous AVPR2 gene mutations in congenital nephrogenic diabetes insipidus.

Mutations in the AVPR2 gene encoding the receptor for arginine vasopressin in the kidney (V2 ADHR) have been reported in patients with congenital nephrogenic diabetes insipidus, a predominantly X-linked disorder of water homeostasis. We have used restriction-enzyme analysis and direct DNA sequencing of genomic PCR product to evaluate the AVPR2 gene in 11 unrelated affected males. Each patient has a different DNA sequence variation, and only one matches a previously reported mutation. Cosegregation of the variations with nephrogenic diabetes insipidus was demonstrated for two families, and a de novo mutation was documented in two additional cases. Carrier detection was accomplished in one family. All the variations predict frameshifts, truncations, or nonconservative amino acid substitutions in evolutionarily conserved positions in the V2 ADHR and related receptors. Of interest, a 28-bp deletion is found in one patient, while another, unrelated patient has a tandem duplication of the same 28-bp segment, suggesting that both resulted from the same unusual unequal crossing-over mechanism facilitated by 9-mer direct sequence repeats. Since the V2 ADHR is a member of the seven-transmembrane-domain, G-protein-coupled receptor superfamily, the loss-of-function mutations from this study and others provide important clues to the structure-function relationship of this and related receptors.     

Functional rescue of the constitutively internalized V2 vasopressin receptor mutant R137H by the pharmacological chaperone action of SR49059

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 beta-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.