cDNA and genomic cloning of mouse aquaporin-2: Functional analysis of an orthologous mutant causing nephrogenic diabetes insipidus

As the first step in generating a transgenic mouse model of nephrogenic dia betes insipidus (NDI), we have analyzed the mouse aquaporin-2 (Aqp2) cDNA a nd gene and generated a mutated Aqp2 orthologous to NDI-causing human AQP2- T126M, Aqp2 cDNA was isolated from mouse kidney and encoded a 271-amino-aci d protein with 90.4% identity to human AQP2, Expression in Xenopus oocytes indicated that Aqp2 encoded a mercurial-sensitive, water-selective channel. Northern blot analysis showed a single 1.7-kb Aqp2 transcript expressed on ly in kidney (medulla > cortex); transcript expression was increased simila r to 20-fold in 48-h water-deprived mice. Immunoblot analysis revealed a 29 -kDa glycoprotein in mouse kidney. Sequence comparison of the Aqp2 cDNA wit h a 5.5-kb mouse genomic DNA indicated three introns (lengths 2.4, 0.9, and 0.6 kb) separating four exons with boundaries at amino acids 120, 175, and 202, Genomic Southern blot analysis revealed a single-copy Aqp2 gene. The mutant Aqp2-T126M was water permeable when expressed in Xenopus oocytes, bu t was retained at the endoplasmic reticulum (ER) in transfected mammalian c ells, The chemical chaperone glycerol produced a redistribution of Aqp2-T12 6M from ER to plasma membrane/endosomes. These results establish a basis fo r an Aqp2-T126M transgenic knock-in model of NDI. (C) 1999 Academic Press.