Research Article: Diabetes Insipidus in Mice with a Mutation in Aquaporin-2

Date Published: August 19, 2005

Publisher: Public Library of Science

Author(s): David J Lloyd, Frank Wesley Hall, Lisa M Tarantino, Nicholas Gekakis, David Beier

Abstract: 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.

Partial Text: Nephrogenic diabetes insipidus (NDI) is a disease characterized by excessive urination and thirst, despite normal production of the antidiuretic hormone arginine vasopressin (AVP) [1]. The inherited forms are either X-linked as a consequence of mutation of the Avpr2 gene [2], or autosomal due to mutation of the Aqp2 gene [3]. Aquaporin-2 (AQP2) is a pore-forming protein belonging to a family of water channels [4], and it is expressed in collecting-duct principal cells in the kidney [5]. Generally these proteins permit the passage of water through the plasma membrane (PM) of cells, several of which carry out this role specifically in the process of water reabsorption from urine in the kidney. It has been established that aquaporins, although functional as a monomer, tetramerize before their insertion into the plasma membrane [4,6]. Furthermore these proteins can also be differentially targeted to distinct regions of the PM; for example, AQP2 is routed to the apical membrane of cells surrounding the collecting duct, whereas other aquaporins (AQP3 or 4) are inserted into the basolateral face. Unlike all other family members, AQP2 is not constitutively inserted into the plasma membrane. Under basal conditions, the protein resides in subapical intracellular vesicles; however, under conditions requiring water retention AQP2 translocates to the apical membrane, permitting water reabsorption [7,8]. For this process to occur, AVP binds its receptor, AVPR2, on the basolateral face of the collecting duct cells, leading to a rise in intracellular cAMP, ultimately resulting in phosphorylation of AQP2 at serine 256 by cAMP-dependent protein kinase [9] and its redistribution to the plasma membrane.

In a forward genetic screen that used ethylnitrosourea (ENU) to induce mutations in a founder animal whose offspring were then screened for abnormal whole body metabolism [19,20], we found a family of mice that urinated and drank excessively. Serum and urine analysis showed that plasma glucose levels were normal and there was no glucose in the urine (unpublished data). Hence, this was an example of diabetes insipidus.

Aqp2F204/F204V mice are viable and grow and reproduce normally. They are, however, severely defective in their ability to concentrate urine, leading to increased urine output and water intake, thus making them the first mouse model of NDI to survive to maturity. In humans, NDI is caused by mutations in Avpr2 or Aqp2. Knockout of the X-linked Avpr2 gene in mice [15] gave an NDI-like phenotype in male, hemizygous neonates, but the phenotype could not be assessed in adults as the mice died within 1 wk of birth. The adult heterozygous females showed a mild tendency toward increased urinary output and water intake and decreased urine osmolality. Knockout of the mouse Aqp2 gene has not been reported. A knock-in of a human disease-causing mutation (T126M), however, has been made [18]. These mice have a severe urine-concentrating defect resulting in dehydration and death within 1 wk of birth. Curiously, AQP2-T126M does localize properly in at least a subset of cells. The grossly abnormal collecting duct morphology makes it impossible to pinpoint the molecular defect in these knock-in mice.