cDNA and Genomic Cloning of Mouse Aquaporin-2: Functional Analysis of an Orthologous Mutant Causing Nephrogenic Diabetes Insipidus

Title: cDNA and Genomic Cloning of Mouse Aquaporin-2: Functional Analysis of an Orthologous Mutant Causing Nephrogenic Diabetes Insipidus
Authors: Yang, Baoxue; Ma, Tonghui; Xu, Zhidong; Verkman, Alan S.
Publisher: Genomics
Date Published: April 01, 1999
Reference Number: 482
As the first step in generating a transgenic mouse model of nephrogenic diabetes insipidus (NDI), we have analyzed the mouse aquaporin-2 (Aqp2) cDNA and 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-acid 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 only in kidney (medulla > cortex); transcript expression was increased approximately 20-fold in 48-h water-deprived mice. Immunoblot analysis revealed a 29-kDa glycoprotein in mouse kidney. Sequence comparison of the Aqp2 cDNA with 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, but was retained at the endoplasmic reticulum (ER) in transfected mammalian cells. The chemical chaperone glycerol produced a redistribution of Aqp2-T126M from ER to plasma membrane/endosomes. These results establish a basis for an Aqp2-T126M transgenic knock-in model of NDI. © 1999 Academic Press.

This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

Several different mutations in the aquaporin-2 gene have been identified as causes of non-X-linked hereditary nephrogenic diabetes insipidus (NDI). The aquaporin-2 (AQP2) water channel proteins that these mutated aquaporin-2 genes help synthesize are able to channel water (their primary function), but they are unable to travel to the cell membrane. This is where the AQP2s must be if they are to function as water channels (i.e. if they are to be inserted into the cell membrane so water can channel through them). Instead, the mutant AQP2s are retained in a part of the cell called the endoplasmic reticulum (ER). Experiments with laboratory cell cultures suggest that the use of chemical chaperones such as glycerol or trimethylamine oxide (TMAO) may help mutant AQP2s that are retained in the ER to leave the ER and travel to their job site, the cell membrane. Chemical chaperones help misformed AQP2s conform to normal shape, thus letting them pass through the ER¹s quality control mechanism. In order to test the effectiveness of these chemical chaperones, researchers must first develop a line of test animals (in this case, mice) that have a mutant form of the AQP2 gene that will help produce AQP2s that can function as water channels, but that are misshapen so they are retained in the ER.

Yang, et al., report on their analysis of a mouse aquaporin (Aqp2) gene that could fulfill this role. The researchers analyzed the Aqp2 cDNA and gene and generated a mutated Aqp2 that was similar to an NDI-causing human AQP2 called AQP2-T126M. The mouse form of this gene, Aqp2-T126M, has similar molecular, functional and physiological characteristics as the AQP2-T126M. It can act as a channel for water, but it is retained in the ER, so it cannot travel to the cell membrane to act as a channel for water. Further, in laboratory cell cultures, the chemical chaperone glycerol produced a redistribution of Aqp2-T126M from the cell ER to the cell membrane. This establishes the Aqp2-T126M gene as a candidate gene to develop a mouse line that bears the Aqp2-T126M gene. Mice from this line would than inherit the non-X-linked hereditary form of NDI. They could serve as a live model on which to test the effectiveness of chemical chaperones on this form of NDI.