Neonatal Mortality in an Aquaporin-2 Knock-in Mouse Model of Recessive Nephrogenic Diabetes Insipidus

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 diseasecausing AQP2 mutant.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 (1-4). AQP2 is the vasopressin (antidiuretic hormone)-regulated water channel (5, 6). Vasopressin induces the fusion of intracellular vesicles containing AQP2 with the apical plasma membrane resulting in increased water permeability (7-9). AQP2 is of considerable clinical importance in fluid and electrolyte balance. Mutations in human AQP2 cause hereditary non-X-linked nephrogenic diabetes insipidus (10 -13). Down-regulation of AQP2 expression occurs in several forms of acquired NDI (14), and AQP2 up-regulation is important in the pathophysiology of fluidretaining states such as congestive heart failure (15, 16). AQP3 and AQP4 are expressed constitutively at the basolateral membrane of collecting duct epithelial cells, with AQP3 mainly in cortical collecting duct and AQP4 in inner medullary collecting duct. Transgenic mice lacking AQP3 have low water permeability in cortical collecting duct and manifest NDI with marked polyuria and decreased urine osmolality (17). In contrast, mice lacking AQP4 show onl...

The Proteasome Is Involved in the Degradation of Different Aquaporin-2 Mutants Causing Nephrogenic Diabetes Insipidus

“…30 In our stable expression system, T126M AQP2 was synthesized as a high mannose-type 32-kd and a nonglycosylated 29-kd form, alike observed in kidney extracts of T126M AQP2 knock-in mice. 31 The two T126M AQP2 forms exhibited significant differences in their turnover rates. Although larger amounts of the nonglycosylated 29-kd form were initially synthesized, their average half-life was considerably shorter (t 1/2 ϭ 0.9 hours) than that of the glycosylated 32-kd form (t 1/2 ϭ 2.0 hours).…”

Functions of aquaporins in the eye

“…An anticipated phenotype of AQP deficiency is defective urinary concentrating function, which has been found in mice lacking AQPs 1-4 (Ma et al, 1997Yang et al, 2001) and in humans with mutations in AQP1 ) and AQP2 (Deen et al, 1994). The generation of a concentrated urine involves near-isosmolar fluid absorption by the proximal tubule, generation of a hypertonic extracellular space fluid in the renal medulla by countercurrent multiplication and exchange, and transepithelial osmotic equilibration across a highly water permeable collecting duct.…”

Vasopressin-independent mechanisms in controlling water homeostasis

“…To further clarify the role of AQP2 in concentrating urine, a number of mouse models have recently been developed. A mouse knockin model of AQP2-dependent nephrogenic diabetes insipidus (NDI) was generated; the mouse line was created by using a Cre-loxP strategy to insert a T126M mutation into the AQP2 gene, resulting in blocked delivery of mature AQP2 protein to the apical PM (Yang et al 2001). These knock-in mice generally died within 1 week of birth although they appeared outwardly normal.…”