Search

Familial neurohypophysial diabetes insipidus (FNDI) is a rare disease that is inherited in an autosomal dominant manner. In a previous study, we made a mouse model for FNDI, which showed progressive polyuria accompanied by inclusion bodies in the arginine vasopressin (AVP) neurons formed by aggregates in the endoplasmic reticulum. The present study was conducted to determine whether the activities of AVP neurons are related to the phenotype progression in the FNDI model. In the first experiment, female heterozygous mice were administered either desmopressin (dDAVP) or a vehicle (control) subcutaneously with osmotic minipumps for 30 days. The dDAVP treatment significantly decreased the urine volume, AVP mRNA expression, and inclusion bodies in the AVP neurons. Urine volume in the dDAVP group remained significantly less than the control for 14 days even after the minipumps were removed. In the second experiment, the males were fed either a 0.2% Na or 2.0% Na diet for 6 too. Urine AVP excretion was significantly increased in the 2.0% Na group compared with the 0.2% Na group for the first 2 mo but gradually decreased thereafter. Throughout the experiments, urine volume increased progressively in the 2.0% Na group but not in the 0.2% Na group. Immunohistochemical analyses revealed that inclusion bodies in the AVP cells had significantly increased in the 2.0% Na compared with the 0.2% Na group. These data demonstrated that activation of AVP neurons could accelerate the aggregate formation as well as the progression of the polyuria in the FNDI model mice. hereditary disease; aggregates doi: 10.1152/ajpregu.00529.2009

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 D 150E 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 ([P.sup.f] = 47.4 [+ or -] 12.2 x [10.sup.-4] cm/s) whereas D150E displays intermediate [P.sup.f] values ([P.sup.f] = 12.5 [+ or -] 3.0 x [10.sup.-4] cm/s) and G196D presents no specific water flux, similar to controls ([P.sub.f] = 2.1 [+ or -] 0.8 x [10.sup.-4] cm/s and 2.2 [+ or -] 0.7 x [10.sup.-4] cm/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. functional expression; Xenopus oocyte; water channel

Lithium (Li)-induced nephrogenic diabetes insipidus (NDI) has been attributed to the increased production of renal prostaglandin (PG)[E.sub.2]. Previously we reported that extracellular nucleotides (ATP/UTP), acting through [P.sub.2y2] receptor in rat medullary collecting duct (mCD), produce and release PG[E.sub.2]. Hence we hypothesized that increased production of PG[E.sub.2] in Li-induced NDI may be mediated by enhanced purinergic signaling in the mCD. Sprague-Dawley rats were fed either control or Li-added diet for 14 or 21 days. Li feeding resulted in marked polyuria and polydipsia associated with a decrease in aquaporin (AQP)2 protein abundance in inner medulla (~20% of controls) and a twofold increase in urinary PG[E.sub.2]. When acutely challenged ex vivo with adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma]S), UTP, or ADP, mCD of Li-fed rats showed significantly higher increases (50-130% over control diet-fed rats) in PGE2 production, indicating that more than one subtype of [P.sub.2y] receptor is involved. This was associated with a 3.4-fold increase in [P.sub.2y4], but not [P.sub.2y2], receptor mRNA expression in the inner medulla of Li-fed rats compared with control diet-fed rats. Confocal laser immunofluorescence microscopy revealed predominant localization of both [P.sub.2y2] and [P.sub.2y4] receptors in the mCD of control or Li diet-fed rats. Together, these data indicate that in Li-induced NDI 1) purinergic signaling in the mCD is sensitized with increased production of PG[E.sub.2] and 2) [P.sub.2y2] and/or [P.sub.2y4] receptors may be involved in the enhanced purinergic signaling. Our study also reveals the potential beneficial effects of [P.sub.2y] receptor antagonists in the treatment and/or prevention of Li-induced NDI. collecting duct; [P.sub.2] receptors; extracellular nucleotides; prostaglandin [E.sub.2]; cyclooxygenases; bipolar disorder; neurodegeneration; rat

Familial neurohypophysial diabetes insipidus (FNDI), an autosomal dominant disorder, is mostly caused by mutations in the gene of neurophysin II (NPII), the carrier protein of arginine vasopressin (AVP). Previous studies suggest that loss of AVP neurons might be the cause of polyuria in FNDI. Here we analyzed knockin mice expressing mutant NPII that causes FNDI in humans. The heterozygous mice manifested progressive polyuria as do patients with FNDI. Immunohistochemical analyses revealed that inclusion bodies that were not immunostained with antibodies for mutant NPII, normal NPII, or AVP were present in the AVP cells in the supraoptic nucleus (SON), and that the size of inclusion bodies gradually increased in parallel with the increases in urine volume. Electron microscopic analyses showed that aggregates existed in the endoplasmic reticulum (ER) as well as in the nucleus of AVP neurons in 1-mo-old heterozygous mice. At 12 too, dilated ER filled with aggregates occupied the cytoplasm of AVP cells, while few aggregates were found in the nucleus. Analyses with in situ hybridization revealed that expression of AVP mRNA was significantly decreased in the SON in the heterozygous mice compared with that in wild-type mice. Counting cells expressing AVP mRNA in the SON indicated that polyuria had progressed substantially in the absence of neuronal loss. These data suggest that cell death is not the primary cause of polyuria in FNDI, and that the aggregates accumulated in the ER might be involved in the dysfunction of AVP neurons that lead to the progressive polyuria. hereditary disease; arginine vasopressin; endoplasmic reticulum; inclusion bodies

Prostaglandin E2 may antagonize vasopressin-stimulated salt absorption in the thick ascending limb and water absorption in the collecting duct. Blockade of prostaglandin E2 synthesis by nonsteroidal anti-inflammatory drugs (NSAIDs) enhances urinary concentration, and these agents have antidiuretic effects in patients with nephrogenic diabetes insipidus (NDI) of different etiologies. Because renal prostaglandins are derived largely from cyclooxygenase-2 (COX-2), we hypothesized that treatment of NDI with a COX-2 inhibitor may relieve polyuria through increased expression of Na-K-2C1 cotransporter type 2 (NKCC2) in the thick ascending limb and aquaporin-2 (AQP2) in the collecting duct. To test this hypothesis, semiquantitative immunoblotting and immunohistochemistry were carried out from the kidneys of lithium-induced NDI rats with and without COX-2 inhibition. After male Sprague-Dawley rats were fed an LiCl-containing rat diet for 3 wk, the rats were randomly divided into control and experimental groups. The COX-2 inhibitor DFU (40 mg*[kg.sup.-]*[day.sup.-1]) was orally administered to the experimental rats for an additional week. Treatment with the COX-2 inhibitor significantly relieved polyuria and raised urine osmolality. Semiquantitative immunoblotting using whole-kidney homogenates revealed that COX-2 inhibition caused significant increases in the abundance of AQP2 and NKCC2. Immunohistochemistry for AQP2 and NKCC2 confirmed the effects of COX-2 inhibition in lithium-induced NDI rats. The upregulation of AQP2 and NKCC2 in response to the COX-2 inhibitor may underlie the therapeutic mechanisms by which NSAIDs enhance antidiuresis in patients with NDI. cyclooxygenase-2; lithium; aquaporin-2; Na-K-2C1 cotransporter; DFU

In mammals, the hormonal regulation of water homeostasis is mediated by the aquaporin-2 water channel (Aqp2) of the collecting duct (CD). Vasopressin induces redistribution of Aqp2 from intracellular vesicles to the apical membrane of CD principal cells, accompanied by increased water permeability. Mutations of AQP2 gene in humans cause both recessive and dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. In this study, we generated a line of mice with the distal COOH-terminal tail of the Aqp2 deleted (Aqp[2.sup.[DELTA]230]), including the protein kinase A phosphorylation site (S256), but still retaining the putative apical localization signal (221-229) at the COOH-terminal. Mice heterozygous for the truncation appear normal. Homozygotes are viable to adulthood, with reduced urine concentrating capacity, increased urine output, decreased urine osmolality, and increased daily water consumption. Desmopressin increased urine osmolaiity in wild-type mice but had no effect on Aqp[2.sup.[DELTA]230/[DELTA]230] mice. Kidneys from affected mice showed CD and pelvis dilatation and papillary atrophy. By immunohistochemical and immunoblot analyses using antibody against the N[H.sub.2]-terminal region of the protein Aqp[2.sup.[DELTA]230/[DELTA]230] mice had a markedly reduced protein abundance. Expression of the truncated protein in MDCK cells was consistent with a small amount of functional expression but no stimulation. Thus we have generated a mouse model of NDI that may be useful in studying the physiology and potential therapy of this disease. mouse; aquaporin-2; diabetes insipidus; protein trafficking; protein phosphorylation doi:10.1152/ajprenal.00308.2006

Intracellular retention of a functional vasopressin V2 receptor (V2R) is a major cause of congenital nephrogenic diabetes insipidus (NDI) and rescue of V2R mutants by nonpeptide antagonists may restore their basolateral membrane (BM) localization and function. However, the criteria for efficient functional rescue of G protein-coupled receptor (GPCR) mutants at clinically feasible antagonist concentrations are unknown. We found that the four nonpeptide antagonists SR49059, OPC31260, OPC41061, and SR121463B induced maturation and rescued the BM expression of eight of nine different V2R mutants, stably expressed in physiologically relevant polarized cells. The extent of maturation and rescued BM expression correlated with the antagonists' concentration and affinity for the V2R. Displacement of the antagonists by AVP and subsequent cAMP generation inversely correlated with the antagonists' affinities for the V2R but is partially influenced by antagonist-specific aspects. Despite limited increases in maturation and cellsurface expression of V2R mutants, the low-affinity SR49059 optimally induced functional rescue at high concentrations, due to its easy displacement by vasopressin. At clinically feasible antagonist concentrations, however, only the high-affinity antagonists OPC31260 and OPC41061 induced functional rescue, as at these concentrations the extent of BM expression became limited. In conclusion, functional rescue of mutant V2Rs at clinically feasible concentrations is most effective with high-affinity antagonists. As OPC31260 and OPC41061 are clinically safe, they are promising candidates to relieve NDI. Moreover, as numerous other diseases are caused by endoplasmic reticulum-retained GPCRs for which cell-permeable antagonists become available, our finding that high-affinity antagonists are superior is anticipated to be important for pharmacotherapy development of these diseases. Madin-Darby canine kidney cells; misfolding; antagonist; water transport

Transgenic mouse models of defective urinary concentrating ability produced by deletion of various membrane transport or receptor proteins, including aquaporin-2 (AQP2), are associated with neonatal mortality from polyuria. Here, we report an inducible mouse model of AQP2 gene deletion with severe polyuria in adult mice. LoxP sequences were inserted into introns 1 and 2 in the mouse AQP2 gene by homologous recombination in embryonic stem cells. Mating of germ-line AQP2-loxP mice with tamoxifen-inducible Cre-expressing mice produced offspring with inducible homozygous Cre-AQP2-loxP, which had a normal phenotype. Tamoxifen injections over 10 days resulted in AQP2 gene excision, with undetectable full-length AQP2 transcript in kidney and a >95% reduction in immunoreactive AQP2 protein. Urine osmolality decreased from ~2,000 to water transport; water channel; transgenic mouse; NDI; polyuria doi:10.1152/ajprenal.00494.2005

In the renal collecting duct, water reabsorption is regulated by the antidiuretic hormone vasopressin (AVP). Binding of this hormone to the vasopressin V2 receptor (V2R) leads to insertion of aquaporin-2 (AQP2) water channels in the apical membrane, thereby allowing water reabsorption from the pro-urine to the interstitium. The disorder nephrogenic diabetes insipidus (NDI) is characterized by the kidney's inability to concentrate pro-urine in response to AVP, which is mostly acquired due to electrolyte disturbances or lithium therapy. Alternatively, NDI is inherited in an X-linked or autosomal fashion due to mutations in the genes encoding V2R or AQP2, respectively. This review describes the current knowledge of the cell biological causes of NDI and how these defects may explain the patients' phenotypes. Also, the increased understanding of these cellular defects in NDI has opened exciting initiatives in the development of novel therapies for NDI, which are extensively discussed in this review. osmoregulation; protein trafficking; genetic disorder; collecting duct; lithium; pharmacological chaperones doi:10.1152/ajprenal.00491.2005

Vasopressin increases urine concentration by stimulating plasma membrane accumulation of aquaporin-2 (AQP2) in collecting duct principal cells, allowing bulk water flow across the collecting duct from lumen to interstitium down an osmotic gradient. Mutations in the vasopressin type 2 receptor (V2R) cause hereditary X-linked nephrogenic diabetes insipidus (NDI), a disease characterized by excessive urination and dehydration. Recently, we showed that inhibition of endocytosis by the cholesterol-depleting drug methyl-[beta]-cyclodextrin (m[beta]CD) induces plasma membrane accumulation of AQP2 in transfected renal epithelial cells overexpressing epitope-tagged AQP2. Here, we asked whether m[beta]CD could induce membrane accumulation of AQP2 in situ using the isolated, perfused kidney (IPK). By immunofluorescence and electron microscopy, we show that AQP2 was shifted from a predominantly intracellular localization to the apical membrane of principal cells following 1-h perfusion of Sprague-Dawley rat kidneys with 5 mM m[beta]CD. Quantification of staining revealed that the intensity of AQP2 was increased from 647 [+ or -] 114 (control) to 1,968 [+ or -] 299 units (m[beta]CD; P < 0.001), an effect similar to that seen alter perfusion with 4 nM dDAVP (1,860 [+ or -] 298, P < 0.001). Similar changes were observed following m[beta]CD perfusion of kidneys from vasopressin-deficient Brattleboro rats. No effect of m[beta]CD treatment on the basolateral distribution of AQP3 and AQP4 was detected. These data indicate that AQP2 constitutively recycles between the apical membrane and intracellular vesicles in principal cells in situ and that inducing apical AQP2 accumulation by inhibiting AQP2 endocytosis is a feasible goal for bypassing the defective V2R signaling pathway in X-linked NDI. diabetes insipidus; collecting duct; endocytosis; immunocytochemistry; aquaporin recycling

To investigate the underlying causes for aldose reductase deficiency-induced diabetes insipidus, we carried out studies with three genotypic groups of mice. These included wild-type mice, knockout mice, and a newly created bitransgenie line that was homozygous for both the aldose reductase null mutation and an aldose reductase knockin transgene driven by the kidney-specific cadherin promoter to direct transgene expression in the collecting tubule epithelial cells. We found that from early renal developmental stages onward, urine osmolality did not exceed 1,000 mosmol/kg[H.sub.2]O in aldose reductase-deficient mice. The functional defects were correlated with significant renal cellular and structural abnormalities that included cell shrinkage, apoptosis, disorganized tubular and vascular structures, and segmental atrophy. In contrast, the transgenic aldose reductase expression in the bitransgenic mice largely but incompletely rescued urine concentrating capacity and significantly improved renal cell survival, cellular morphology, and renal structures. Together, these results suggest that aldose reductase not only plays important roles in osmoregulation and medullary cell survival but may also be essential for the full maturation of the urine concentrating mechanism. urine concentration; diabetes insipidus

Lithium (Li) treatment for 4 wk has previously been shown to increase the fraction of intercalated ceils in parallel with a decrease in the fraction of principal cells in the kidney collecting duct (Christensen BM, Marples D, Kim YH, Wang W, Frokiaer J, and Nielsen S. Ant J Phvsiol Cell Physiol 286: C952-C964, 2004; Kim YH, Kwon TH, Christensen BM, Nielsen J, Wall SM, Madsen KM, Frckiaer J, and Nielsen S. Am J Physiol Renal Physiol 285: F1244-F1257, 2003). To study how early this fractional change starts, the origin of the cells and the possible mechanism behind the changes, we did time course studies in rats subjected to different durations of Li treatment (i.e., for 4, 10, and 15 days). Increased urine output was already observed at day 4 of Li treatment with decreased AQP2 levels although not statistically significant. At days 10 and 15, both a significant polyuria and down-regulation in AQP2 expression were observed. At day 10, the density of [H.sup.+]-ATPase-positive cells was increased in the IMCD of Li-treated rats and this was further pronounced at day 15. Some of the [H.sup.+]-ATPase-positive cells did not costain with [Cl.sup.-]/HC[O.sup.-.sub.3]. exchanger AE1, indicating that they were not fully differentiated to type AIC. By double labeling for either [H.sup.4]-ATPase and proliferating-cell nuclear antigen (PCNA) or for AQP4 and PCNA, we found that proliferation mainly occulted in proximal IMCD cells at day 4 and it increased toward the middle part of the IMCD in response to prolonged Li treatment. Most cells expressing PCNA were stained with AQP4 but not with [H.sup.+]-ATPase. Triple-labeling for [H.sup.+]-ATPase, AQP4, and PCNA showed a subset of cells negative for all three proteins or only positive for PCNA. In contrast, a 4-wk recovery period after 4 wk of Li treatment reversed the enhanced proliferative rate to the control levels. In conclusion, the Li-induced increase in the density of intercalated cells is associated with a high proliferative rate of principal cells in the IM-1 and IM-2 rather than a selective proliferation of intercalated cells as expected. This is likely to contribute to the remodeling of the collecting duct after Li treatment. cell proliferation; collecting duct principal cell; collecting duct intercalated cells; lithium-induced nephrogenic diabetes insipidus