A novel mechanism in recessive nephrogenic diabetes insipidus: wild-type aquaporin-2 rescues the apical membrane expression of intracellularly retained AQP2-P262L.

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.

Cell-biologic and functional analyses of five new Aquaporin-2 missense mutations that cause recessive nephrogenic diabetes insipidus.

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.

Heteroligomerization of an Aquaporin-2 mutant with wild-type Aquaporin-2 and their misrouting to late endosomes/lysosomes explains dominant nephrogenic diabetes insipidus.

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.

Desmopressin for nocturnal enuresis in nephrogenic diabetes insipidus.

We have investigated two unrelated families, in which two children had inherited primary nocturnal enuresis, and nephrogenic diabetes insipidus caused by new mutations in the aquaporin-2 gene (AQP2). The mutant AQP2 proteins were inactive, suggesting that administration of desmopressin could not concentrate the urine in these patients. However, treatment with desmopressin resolved primary nocturnal enuresis completely. This observation questions the notion that desmopressin resolves primary nocturnal enuresis through pharmacological manipulation of renal concentrating ability only. Desmopressin might also act on extrarenal targets such as the central nervous system.