The in vivo balance between B cell clonal expansion and elimination is regulated by CD95 both on B cells and in their micro-environment

In congenital nephrogenic diabetes insipidus, the renal collecting ducts are resistant to the antidiuretic action of arginine vasopressin or to its antidiuretic analog 1-deamino[8-D-arginine] vasopressin (dDAVP). This is a rare, but now well described entity secondary to either mutations in the AVPR2 gene that codes for the vasopressin antidiuretic (V2) receptor or to mutations in the AQP2 gene that codes for the vasopressin-dependent water channel. A majority (> 90%) of congenital nephrogenic diabetes insipidus patients have AVPR2 mutations: Of 115 families with congenital nephrogenic diabetes insipidus, 105 families had AVPR2 mutations, and 10 had AQP2 mutations. When studied in vitro, most AVPR2 mutations lead to receptors that are trapped intracellularly and are unable to reach the plasma membrane. A minority of the mutant receptors reach the cell surface but are unable to bind vasopressin or to trigger an intracellular adenosine 3:5-cyclic phosphate signal properly. Most of the reported mutations are secondary to a complete loss of function of the receptor, and only a few mutations have been associated with a mild phenotype. These advances provide diagnostic tools for physicians caring for these patients because, when the disease causing mutation has been identified, carrier and perinatal testing could be done by mutation analysis.     

Mutations in the vasopressin V2 receptor and aquaporin-2 genes in 12 families with congenital nephrogenic diabetes insipidus

Congenital nephrogenic diabetes insipidus (CNDI) is a rare inherited disorder characterized by renal tubular insensitivity to the antidiuretic effect of arginine vasopressin (AVP). In a large majority of the cases, nephrogenic diabetes insipidus is an X-linked recessive disorder caused by mutations in the AVP V2 receptor gene (AVPR2). In the remaining cases, the disease is autosomal recessive or dominant and, for these patients, mutations in the aquaporin 2 gene (AQP2) have been reported. Fourteen probands belonging to 12 families were analyzed by single-strand conformational polymorphism and direct sequencing of the AVPR2 and AQP2 genes. Ten mutations of the AVPR2 gene (six previously reported mutations and four novel mutations: G107E, W193X, L43P, and 15delC) were identified. Three mutations of the AQP2 gene were also identified in two patients: the first patient is homozygous for the R85X mutation and the second is a compound heterozygote for V168 M and S216P mutations. Extrarenal responses to infusion of the strong V2 agonist 1-desamino-8-D-arginine vasopressin allowed AVPR2- and AQP2-associated forms of CNDI to be distinguished in three patients. This test also identified an unexpectedly high urinary osmolality (614 mosmol/kg) in a patient with a P322S mutation of AVPR2 gene and a mild form of CNDI.     

AVPR2 variants and V2 vasopressin receptor function in nephrogenic diabetes insipidus

BACKGROUND: The AVPR2 gene encodes the type 2 vasopressin receptor, a member of the vasopressin/oxytocin receptor subfamily of G protein-coupled receptors. Disruption of AVPR2 causes X-linked congenital nephrogenic diabetes insipidus (NDI), yet the functional significance of most gene sequence variations found in association with NDI has not been proven. The large number of naturally occurring AVPR2 mutations constitutes a model system for studying the structure-function relationship of G protein-coupled receptors. This analysis can be aided by examining amino acid sequence variation and conservation among evolutionarily disparate members of the subfamily. METHODS: Twenty-five new NDI patients were evaluated by DNA sequencing for mutations in AVPR2. Receptors encoded by eighteen NDI alleles were tested for physiologic signaling activity in response to varying concentrations of arginine vasopressin (AVP) in a sensitive cell culture assay. Seventeen amino acid sequences from the vasopressin/oxytocin receptor subfamily were aligned and conserved residues were identified and correlated with the locations of NDI associated variations. RESULTS: Twenty-four variant alleles were found among the 25 new patients. Thirteen had no prior family history of expressed NDI. All 18 of the NDI-associated AVPR2 alleles tested for function demonstrated diminished response to stimulation with AVP. Twelve failed to respond at all, whereas six signaled only at high AVP concentrations. Evolutionarily conserved residues clustered in the transmembrane domains and in the first and second extracellular loops, and NDI-associated missense mutations appeared mostly in the conserved domains. CONCLUSIONS: Sporadic cases are frequent and they usually represent the X-linked rather than the autosomal form of NDI. Genetic and functional testing can confirm this in individual cases. Mutations in this study affecting ligand binding domains tend to retain partial signaling in vitro, whereas those that introduce a charged residue in a transmembrane domain are inactive. The minimal partial signaling observed in cultured cells is unlikely to correlate with clinically significant urine concentrating ability. Other AVPR2 mutations with milder effects on receptor function probably exist, but may not be expressed clinically as typical NDI.     

Functional studies of twelve mutant V2 vasopressin receptors related to nephrogenic diabetes insipidus: molecular basis of a mild clinical phenotype

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.     

The molecular basis of nephrogenic diabetes insipidus

Nephrogenic diabetes insipidus (NDI) is characterized by resistance of the kidney to the action of arginine-vasopressin (AVP); it may be due to genetic or acquired causes. Recent advances in molecular genetics have allowed the identification of the genes involved in congenital NDI. While inactivating mutations of the vasopressin V2 receptor are responsible for X-linked NDI, autosomal recessive NDI is caused by inactivating mutations of the vasopressin-regulated water channel aquaporin-2 (AQP-2). About 70 different mutations of the V2 receptor have been reported, most of them missense mutations. The functionally characterized mutants show a loss of function due to defects in their synthesis, processing, intracellular transport, AVP binding, or interaction with the G protein/adenylyl cyclase system. Thirteen different mutations of the AQP-2 gene have been reported. Functional studies of three AQP-2 mutations reveal impaired cellular routing as the main defect. The great number of different mutations with various functional defects hinders the development of a specific therapy. Gene therapy may, however, eventually become applicable to the congenital forms of NDI. At present all gene-therapeutic approaches lack safety and efficiency, which is of particular relevance in a disease that is treatable by an adequate water intake. The progress with regard to the molecular basis of antidiuresis contributes to the understanding of acquired forms of NDI on a molecular level. Recent data show that lithium dramatically reduces the expression of AQP-2. Likewise, hypokalemia reduces the expression of this water channel. The exact mechanisms leading to this reduced expression of AQP-2 remain to be determined.     

Observations on the California mortality studies

Since the discovery of aquaporin water channels, insight into the molecular mechanism by which rapid osmotic water occurs across cell membranes has greatly improved. Aquaporin-2 is the vasopressin-responsive water channel in the collecting duct, and vasopressin control of water permeability in the collecting duct occurs in two ways: a short-term regulation and a long-term adaptation. In congenital nephrogenic diabetes insipidus, the kidney does not respond to vasopressin. Ninety percent of these patients carry a mutation in the gene coding for the vasopressin V2 receptor located on the X chromosome. Autosomal recessive and dominant forms of nephrogenic diabetes insipidus that are caused by mutations in the aquaporin-2 gene have now been described. This review focuses on recent insight in the molecular and cellular defect in autosomal nephrogenic diabetes insipidus.     

Bilateral deafness due to congenital CMV and not rubella reinfection

Two cases of autosomal recessive nephrogenic diabetes insipidus (NDI) were evaluated. Both cases were found to be compound heterozygote for missense mutations in the aquaporin-2 (AQP2) gene. To determine the structural-functional relationship, the mutated AQP2 proteins, T125M, G175R, A190T, and P262L, were expressed in Xenopus oocytes and examined by measurement of water permeability, immunoblot, and immunocytochemistry. Our results suggest that T125M and G175R are nonfunctional water channels, whereas the translocation to the plasma membrane is impaired in A190T and P262L.     

Comprehensive evaluation of long-term trends in occupational exposure: Part 1. Description of the database

The regulation of water excretion by the kidney is one of the few physiologic processes that are prominent in everyday life. This process predominantly occurs in renal collecting duct cells, where transcellular water reabsorption is induced after binding of the pituitary hormone arginine-vasopressin to its vasopressin type-2 receptor and the subsequent insertion of aquaporin-2 (AQP2) water channels in the apical membrane of these cells. Removal of the hormone triggers endocytosis of AQP2 and restores the water-impermeable state of the collecting duct cells. Nephrogenic diabetes insipidus is characterized by the inability of the kidney to concentrate urine in response to vasopressin; the vasopressin type-2 receptor and the AQP2 water channel have both been shown to be involved in this disease. This article focuses on mutations in the vasopressin V2 receptor and aquaporin-2 water channel identified in nephrogenic diabetes insipidus patients, and on the effects of these mutations on the transport and function of these proteins upon expression in cell systems.     

Molecular biological studies on patients with nephrogenic diabetes insipidus]

Nephrogenic diabetes insipidus is a rare, mostly X-linked recessive disorder characterised by renal tubular resistance to the antidiuretic effect of arginine vasopressin. The gene responsible for the X-linked nephrogenic diabetes insipidus, the G-protein-coupled vasopressin V2-receptor, has been localised on the Xq28 region. In this study four patients were investigated with molecular genetic methods. Diagnosis was based on clinical symptoms and lack of increase of urinary osmolality after administration of the arginine vasopressin, or the synthetic vasopressin analogue DDAVP. Three different mutations (C112R, N317K, W323S) were found in three patients, while no mutation was detected in the fourth patient. Since earlier histiocytosis X has been diagnosed in this patient, this patient has probably central diabetes insipidus. Although the main symptoms of the disease can be found in all patients, there are significant differences in the seriousness of the symptoms as well as in some other symptoms. The explanations might be the different mutations in the V2-receptor gene and the various other genetic and environmental factors; these findings provide further evidence that X-linked nephrogen diabetes insipidus results from defects in the V2-receptor gene.     

Autosomal recessive nephrogenic diabetes insipidus caused by an aquaporin-2 mutation

Vasopressin V2 receptors, expressed from an x-chromosomal gene, are involved in antidiuresis, but also in release of coagulation factor VIII and von Willebrand factor (vWF). The present study describes autosomal recessive nephrogenic diabetes insipidus (NDI) in a large cluster of patients in Israel's Lower-Galilee. Evidence for an intact V2 receptor was concluded by their normal increase in factor VIII and vWF after desmopressin infusion. Thus, in these patients a defect in the pathway beyond the V2 receptor was suspected. The recent cloning of the human Aquaporin-2 gene enabled us to test this gene as a candidate for such a postreceptor defect. Direct sequencing of the Aquaporin-2 gene revealed a G298T substitution causing a Gly100Stop nonsense mutation in the third transmembrane region. Because this putative disease-causing mutation was identified in index patients of different families, we suggest that all patients are descendants of a common ancestor. Thus, this new entity is characterized by an autosomal recessive NDI. The differential response of clotting factors and urine osmolality to desmopressin may provide a simple tool for clinical diagnosis of a V2-postreceptor defect. The early stop-codon of Aquaporin-2 results in complete resistance to vasopressin antidiuretic effect.     

Novel mutations in aquaporin-2 gene in female siblings with nephrogenic diabetes insipidus: evidence of disrupted water channel function

Novel mutations of the aquaporin-2 (AQP2) gene have been detected in Japanese female siblings with autosomal-recessive nephrogenic diabetes insipidus. The patients were compound heterozygote for point mutations at nucleotide position 374 (C374T) and at position 523 (G523A) in exon 2 of the AQP2 gene, resulting in substitution of methionine for threonine at codon 125 (T125M) and arginine for glycine at codon 175 (G175R). The water permeability (Pf) of oocytes injected with wild-type complementary RNA increased 9.0-fold compared with the Pf of water-injected oocytes, whereas the increases in the Pf of oocytes injected with T125M and G175R complementary RNA were only 1.7-fold and 1.5-fold, respectively. Immunoblot and immunocytochemistry indicated that the plasma membrane expressions of T125M and G175R AQP2 proteins were comparable to that of the wild-type, suggesting that although neither the T125M nor G175R mutation had a significant effect on plasma membrane expression, they both distorted the structure and function of the aqueous pore of AQP2. These results provide evidence that the nephrogenic diabetes insipidus in patients with T125M and G175R mutations is attributable not to the misrouting of AQP2, but to the disrupted water channel function.     

Role of aquaporin water channels in kidney and lung

Several aquaporin-type water channels are expressed in mammalian kidney and lung: AQP1 in lung microvessels and kidney proximal tubule, thin descending limb of Henle, and vasa recta; AQP2 in apical membrane of collecting duct epithelium; AQP3 and AQP4 in basolateral membranes of airway and collecting duct epithelium; and AQP5 in alveolar epithelium. Novel quantitative fluorescence methods demonstrated very high water permeabilities of the alveolar epithelial and endothelial barriers, and moderately high water permeability across distal airways. In the kidney, water permeability is high in proximal tubule and thin descending limb of Henle, and regulated by vasopressin in collecting duct. The author's laboratory has studied the role of aquaporins in organ physiology using transgenic knockout mice lacking specific aquaporins. AQP1 null mice are mildly growth-retarded, manifest a severe urinary concentrating defect, and have reduced water permeability between airspace and capillary compartments. AQP4 null mice appear normal grossly except for a mild defect in maximum urinary concentrating ability. AQP2-deficient humans have hereditary non-X-linked nephrogenic diabetes insipidus (NDI). In transfected mammalian cells, many NDI-causing AQP2 mutants are retained in the endoplasmic reticulum. The author's laboratory has found that "chemical chaperones," that is, small compounds that promote protein folding in vitro, are able to correct defective AQP2 trafficking in cell culture models. The transgenic mouse and mammalian cell models are thus beginning to provide clues about the role of aquaporins in normal physiology and disease.     

Defective aquaporin-2 trafficking in nephrogenic diabetes insipidus and correction by chemical chaperones

Five single-point aquaporin-2 (AQP2) mutations that cause non-X-linked nephrogenic diabetes insipidus (NDI) were characterized to establish the cellular defect and to develop therapeutic strategies. In Xenopus oocytes expressing AQP2 cRNAs, single-channel water permeabilities of mutants L22V, T126M, and A147T were similar to that of wild-type AQP2, whereas R187C and C181W were nonfunctional. In [35S]methionine pulse-chase experiments in transiently transfected CHO cells, half-times for AQP2 degradation were approximately 4 h for wild-type AQP2 and L22V, and mildly decreased for T126M (2.7 h), C181W (2.4 h), R187C (2.0 h), and A147T (1.8 h). Immunofluorescence showed three distinct AQP2-staining patterns: plasma membrane and endosomal staining (wild-type, L22V), endoplasmic reticulum (ER) staining (T126M > A147T approximately R187C), or a mixed pattern of reticular and perinuclear vesicular staining. Immunoblot of fractionated vesicles confirmed primary ER localization of T126M, R187C, and A147T. To determine if the AQP2-trafficking defect is correctable, cells were incubated with the "chemical chaperone" glycerol for 48 h. Immunoblot showed that glycerol produced a nearly complete redistribution of AQP2 (T126M, A147T, and R187C) from ER to membrane/endosome fractions. Immunofluorescence confirmed the cellular redistribution. Redistribution of AQP2 mutants was also demonstrated in transfected MDCK cells, and using the chaperones TMAO and DMSO in place of glycerol in CHO cells. Water permeability measurements indicated that functional correction was achieved. These results indicate defective mammalian cell processing of mutant AQP2 water channels in NDI, and provide evidence for pharmacological correction of the processing defect by chemical chaperones.     

Congenital nephrogenic diabetes insipidus: a difficult diagnosis?

In five patients (a boy aged 10 years, a boy aged 3 months, his brother aged 1 week, the brother of the mother of the last-mentioned two boys who had died at the age of one, and a girl of kindergarten age) congenital nephrogenic diabetes insipidus was diagnosed. This rare syndrome (prevalence 1:500,000) is caused by renal insensitivity to the antidiuretic hormone arginine vasopressin. In infancy the symptoms of this disorder are aspecific, and the main symptoms of the disease, polyuria and polydipsia, often remain unnoticed at this young age. A simple anamnesis and a few laboratory tests should suggest the diagnosis. Early diagnosis and genetic counselling are possible as the molecular effects involved have been elucidated.     

Vasopressin processing defects in the Brattleboro rat: implications for hereditary central diabetes insipidus in humans?

The arginine vasopressin (AVP) precursor gene of mammals contains three exons encoding the principal domains of the polyprotein precursor, including vasopressin (exon A), neurophysin (exon B), and glycopeptide (exon C). The AVP precursor (preprohormone) is processed and transported through the endoplasmic reticulum (ER), Golgi apparatus, and secretory vesicles, and finally, mature AVP is secreted from the posterior pituitary into the circulation. The exact steps of these processes during AVP translation and posttranslation events are not yet well elucidated. Defects in peptide processing are associated with several genetic disorders, including central diabetes insipidus (CDI). In the Brattleboro rat with CDI, the mRNA and protein of AVP are present in the hypothalamus, but no circulating AVP is detectable, thus suggesting a processing defect, transport defect, or both. The mutated AVP gene precursor of Brattleboro rat has a deletion of a single base, guanine, in the neurophysin coding region that leads to a frameshift resulting in the loss of the normal stop codon. It has been reported that the mutated precursor is trapped in the ER and does not reach the Golgi apparatus. Recent studies examined AVP secretion in cultured COS cells transfected with various constructs from wild-type and mutated Brattleboro AVP gene precursors. The wild-type in vitro studies demonstrated that intact neurophysin, but not the glycoprotein coding region, is necessary for normal AVP processing and secretion. Next, the results demonstrated that the guanine defect in the neurophysin coding region and the prolonged C-terminus accounted for the processing defect in the Brattleboro rat with CDI. These defects no doubt impair the folding and configuration necessary for normal processing of the AVP gene precursor in the ER. In hereditary CDI in humans, the majority of the mutations have also been shown to occur in the neurophysin coding region. However, in contrast to the recessive defect in the Brattleboro rat, in human CDI, neurotoxicity and denigration of the magnocellular neurons have been observed, and dominant inheritance occurs. Moreover, all mutations are missense, nonsense, or deletions in human CDI rather than the shift in reading frame and preserved neurons that is observed with the Brattleboro rat. Thus, the results from studies in the Brattleboro rat may only be partially applicable to hereditary CDI in humans.     

Opioid dependence in 15 patients after a work injury

A 50-year-old Japanese man had been suffering from polydipsia and polyuria for 2 months without any other specific symptoms. His daily urinary output reached 5 liters. On admission, no abnormalities of the kidneys, heart, thyroid, adrenals, pituitary or hypothalamus were detected by laboratory tests and MRI of the head. Pure psychogenic polydipsia was ruled out because his urine volume did not decrease sufficiently with 18 h of water deprivation and the subsequent injection of aqueous vasopressin. Plasma arginine vasopressin (AVP) levels against plasma osmolality remained within the normal range during the test. These results indicated that diabetes insipidus in this case was caused by renal insensitivity to AVP. The symptoms disappeared spontaneously, and marked improvement was observed in a second water deprivation test 1 month later, although the maximum urine concentration was still subnormal. The combination of both latent insufficiency of AVP secretion and impairment of the renal countercurrent system induced by psychogenic polydipsia was speculated as a possible mechanism for the transient nephrogenic diabetes insipidus in this case.     

Diabetes insipidus

Diabetes insipidus, characterized by the excretion of copious volumes of unconcentrated urine, results from a deficiency in the action of the antidiuretic hormone arginine vasopressin and can be caused by any of four fundamentally different defects, including impaired secretion (neurohypophyseal diabetes insipidus), impaired renal response (nephrogenic diabetes insipidus), excessive fluid intake (primary polydipsia), or increased metabolism of the hormone (gestational diabetes insipidus). Differentiation between their causes, pathophysiology, and treatment methods is essential for effective management and is best achieved by a combination of hormonal, clinical, and neuroradiologic observations. Understanding of the genetic forms has advanced greatly and may soon lead to improved methods of prevention, diagnosis, and treatment.     

Vasopressin administration in the first month of life: effects of growth and water metabolism in hypothalamic diabetes insipidus rats

Rats homozygous for the mutant gene for diabetes insipidus (Brattleboro strain) are stunted in growth compared to rats heterozygous for the mutant gene and normal rats without the mutant gene. The hypothesis was tested that normal growth depends upon the presence of vasopressin. It was expected that replacement therapy of vasopressin rats homozygous for diabetes insipidus would make possible a normal growth rate similar to that of rats heterozygous for diabetes insipidus. Rats heterozygous and homozygous for diabetes insipidus were treated with 0.25 U (Days 0-9) and 0.5 U (Days 10-29) of vasopressin during the first month of life. During the treatment period, vasopressin significantly increased the urine osmolatities of the homozygous rats demonstrating the renal effectiveness of the vasopressin. The results showed that remedial vasopressin administration could not produce normal growth rates in homozygous rats and may be detrimental. Six weeks following vasopressin treatment, homozygous, diabetes insipidus rats which had received vasopressin had increased 24 hr water intakes and decreased urine osmolalities compared to control, homozygous rats, Heterozygous rats also had decreased urine osmolalities resulting from vasopressin six weeks after the cessation of vasopressin treatment.     

Surfactant homeostasis in corticotropin-releasing hormone deficiency in adult mice

Familial neurohypophyseal diabetes insipidus (FNDI) is an autosomal dominant disease caused by deficiency in the antidiuretic hormone arginine vasopressin (AVP) encoded by the AVP-neurophysin II (AVP-NPII) gene on chromosome 20p13. In this study, we analyzed two families with FNDI using direct automated fluorescent, solid phase, single-stranded DNA sequencing of PCR-amplified AVP-NPII DNA. In one of the families, affected individuals presented a novel nonsense mutation in exon 3 of the gene, consisting in a G to T transition at nucleotide 2101, which produces a stop signal in codon 82 (Glu) of NPII. The premature termination eliminates part of the C-terminal domain of NPII, including a cysteine residue in position 85, which could be involved in the correct folding of the prohormone. In the second family, a G279A substitution at position -1 of the signal peptide was observed in all affected individuals. This missense mutation, which replaces Ala with Thr, is frequent among FNDI patients and is thought to reduce the efficiency of cleavage by signal peptidases.     

Decreased aquaporin-2 expression and apical plasma membrane delivery in kidney collecting ducts of polyuric hypercalcemic rats

Hypercalcemia is frequently associated with a urinary concentrating defect and overt polyuria. The molecular mechanisms underlying this defect are poorly understood. Dysregulation of aquaporin-2 (AQP2), the predominant vasopressin-regulated water channel, is known to be associated with a range of congenital and acquired water balance disorders including nephrogenic diabetes insipidus and states of water retention. This study examines the effect of hypercalcemia on the expression of AQP2 in rat kidney. Rats were treated orally for 7 d with dihydrotachysterol, which produced significant hypercalcemia with a 15 +/- 2% increase in plasma calcium concentration. Immunoblotting and densitometry of membrane fractions revealed a significant decrease in AQP2 expression in kidney inner medulla of hypercalcemic rats to 45.7 +/- 6.8% (n = 11) of control levels (100 +/- 12%, n = 9). A similar reduction in AQP2 expression was seen in cortex (36.9 +/- 4.2% of control levels, n = 6). Urine production increased in parallel, from 11.3 +/- 1.4 to a maximum of 25.3 +/- 1.9 ml/d (P < 0.01), whereas urine osmolality decreased from 2007 +/- 186 mosmol/kg x H2O to 925 +/- 103 mosmol/kg x H2O (P < 0.01). Immunocytochemistry confirmed a decrease in total AQP2 labeling of collecting duct principal cells from kidneys of hypercalcemic rats, and reduced apical labeling. Immunoelectron microscopy demonstrated a significant reduction in AQP2 labeling of the apical plasma membrane, consistent with the development of polyuria. In summary, the results strongly suggest that AQP2 downregulation and reduced apical plasma membrane delivery of AQP2 play important roles in the development of polyuria in association with hypercalcemia.