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Abstract
Hereditary non-X-linked nephrogenic diabetes insipidus (NDI) is caused by mutations in the aquaporin-2 (AQP2) water channel. In transfected cells, the human disease-causing mutant AQP2-T126M is retained at the endoplasmic reticulum (ER) where it is functional and targetable to the plasma membrane with chemical chaperones. A mouse knock-in model of NDI was generated by targeted gene replacement using a Cre-loxP strategy. Along with T126M, mutations H122S, N124S, and A125T were introduced to preserve the consensus sequence for N-linked glycosylation found in human AQP2. Breeding of heterozygous mice yielded the expected Mendelian distribution with 26 homozygous mutant offspring of 99 live births. The mutant mice appeared normal at 2-3 days after birth but failed to thrive and generally died by day 6 if not given supplemental fluid. Urine/serum analysis showed a urinary concentrating defect with serum hyperosmolality and low urine osmolality that was not increased by a V2 vasopressin agonist. Northern blot analysis showed up-regulated AQP2-T126M transcripts of identical size to wild-type AQP2. Immunoblots showed complex glycosylation of wild-type AQP2 but mainly endoglycosidase H-sensitive core glycosylation of AQP2-T126M indicating ER-retention. Biochemical analysis revealed that the AQP2-T126M protein was resistant to detergent solubilization. Kidneys from mutant mice showed collecting duct dilatation, papillary atrophy, and unexpectedly, some plasma membrane AQP2 staining. The severe phenotype of the AQP2 mutant mice compared with that of mice lacking kidney water channels AQP1, AQP3, and AQP4 indicates a critical role for AQP2 in neonatal renal function in mice. Our results establish a mouse model of human autosomal NDI and provide the first in vivo biochemical data on a disease-causing AQP2 mutant.
Highlights
The formation of concentrated urine by the kidney requires high osmotic water permeability across the collecting duct epithelium
In addition to the T126M mutation, mutations H122S, N124S, and A125T were engineered in exon 2 of the coding sequence to preserve the consensus site for N-linked glycosylation found in human AQP2
Two mutant mouse lines were generated: a CD1 line in which the Pol2neobpA cassette was retained in the genome and a hybrid CD1/FVB/N line in which the Pol2neobpA cassette was deleted by breeding with mice expressing cre-recombinase
Summary
The formation of concentrated urine by the kidney requires high osmotic water permeability across the collecting duct epithelium. Collecting duct epithelial cells express aquaporin water channels AQP2,1 AQP3, and AQP4 [1,2,3,4]. Treatment of transfected cells with chemical chaperones such as glycerol resulted in relocation of AQP2-T126M to the cell plasma membrane where it was able to function as a water channel and correct the defective cell phenotype [23, 24]. Therapy development requires demonstration that disease-causing AQP2 mutants are ER-retained in native kidney cells and that chaperone-modifying strategies are effective in vivo. The homozygous AQP2-T126M knock-in mice created here had NDI and expressed the mutant AQP2 protein in collecting duct epithelial cells, as engineered, but the mice had an unexpectedly severe phenotype with neonatal mortality.
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