New Mutations in the AQP2 Gene in Nephrogenic Diabetes Insipidus Resulting in Functional but Misrouted Water Channels

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Title: New Mutations in the AQP2 Gene in Nephrogenic Diabetes Insipidus Resulting in Functional but Misrouted Water Channels
Authors: Mulders, Sabine M.; Knoers, Nine; van Lieburg, Angenita; Monnens, Leo A.H.; Leumann, MD, Ernst; Wuhl, MD, Elke; Schober, MD, Edith; Rijss, Johan P.L.; van Os, Carel; Deen, Peter M.T.
Publisher: Journal of the American Society of Nephrology
Date Published: February 01, 1997
Reference Number: 1
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Nephrogenic diabetes insipidus (NDI) is characterized by the inability of the kidney to concentrate urine in response to vasopressin. The autosomal recessive form of NDI is caused by mutations in the AQP2 gene, encoding the vasopressin-regulated water channel of the kidney collecting duct. This report presents three new mutations in the AQP2 gene that cause NDI, resulting in A147T-, T126M-, or N68S-substituted AQP2 proteins. Expression of the A147T and T126M mutant AQP2 proteins in Xenopus oocytes revealed a relatively small, but significant increase in water permeability, whereas the water permeability of N68S expressing oocytes was not increased. cRNA encoding missense and wild-type AQP2 were equally stable in oocytes. Immunoblots of oocyte lysates showed that only the A147T mutant protein was less stable than wild-type AQP2. The mutant AQP2 proteins showed, in addition to the wild-type 29-kd band, an endoplasmic reticulum-retarded form of AQP2 of approximately 32 kd. Immunoblotting and immunocytochemistry demonstrated only intense labeling of the plasma membranes of oocytes expressing wild-type AQP2. In summary, two mutant AQP2 proteins encoded in NDI are functional water channels. Therefore, the major cause underlying autosomal recessive NDI is the misrouting of AQP2 mutant proteins.
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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)

Aquaporins (AQP) are water-transporting proteins that serve as a means to increase the flow of water through the membranes of the cells in which they reside. Four different types of AQPs -- AQP1, AQP2, AQP3 and AQP4 -- reside in different parts of the kidney. AQP2, the focus of this research project, lies in the principal cells of kidney-collecting ducts and is regulated by the antidiuretic hormone, vasopressin. AQP2 lives beneath the top of the collecting cells and when stimulated by a process initiated by vasopressin it is shuttled in little sacs to the top of the cell membrane where it makes that section of the membrane become much more water permeable. After the vasopressin leaves, AQP2 is shuttled out of the membranes and back into the cell's interior.

Mutations of the AQP2 gene have been shown to be the cause of one form of nephrogenic diabetes insipidus (NDI), a disorder characterized by the inability to concentrate urine in response to vasopressin. Researchers did not have enough information to decide if the mutations of the AQP2 gene resulted in a nonfunctional AQP2 or a functional AQP2 that could not get to the cell membrane where it is needed to do its job. Mulders, et al., report on three unrelated patients with NDI caused by AQP2 gene mutations.

Mulders, et al., examined AQP2 gene samples from the three patients and found they all had different forms of the same type of mutations -- a missense mutation which changes a part of the gene's sequence so that the gene creates a different amino acid than it is supposed to. In patient #1's AQP2 gene, the amino acid alanine was located where threonine should have been; in patient #2, methionine was where threonine should have been; in patient #3, aspargine was where a serine should have been.

Testing showed patient #3's AQP2 did not increase the cell\'s water permeability, whereas the AQP2 of patient #1 and #2 did, though it did not increase it to near normal standards. The mutant AQP2s low or absent ability to make the cell membrane water permeable could have been due to:

  1. low stability of CRNA in the cells where the AQP2s were being tested,
  2. low stability of the AQP2s themselves,
  3. an impairment in the cell's ability to get AQP2 to the cell membrane where it is supposed to do its work,
  4. the mutations rendering the AQP2s nonfunctional.

Further testing eliminated options one, two, and four for patients #1 and #2, leading the researchers to believe these particular mutations resulted in the inability of the AQP2s to reach their work site. That is, the AQP2s were present in the cell, and they were functional, but they couldn't get to the membrane at the top of the cell where they are needed to increase the membrane's water permeability. Apparently their AQP2s get stuck in a canal-like part of the cell called the endoplasmic reticulum (ER) and are unable to reach the cell membrane. In the ER, newly synthesized proteins (such as AQP2s) undergo further modifications, and if they do not pass the ER's quality control (say, the protein is improperly folded), then the ER will not let them leave and will, in fact, degrade them.

Patient #3's AQP2 mutation also impaired its ability to travel to its work site. But this mutation also rendered the aquaporin non-functional. Other research had shown correlations between location of the mutation within the aquaporin gene and AQP2's ability to function. Patient #3's mutation was a N68S mutation, which is located in the B loop of the AQP2 protein. All AQP2s with mutations in either the B loop or the E loop are nonfunctional - they wouldn\'t work even if they got to the work site. Patient #2's mutation occurred in the C loop of the AQP2 protein and patient #l's near the D loop. (Please see diagram of a normal AQP2.) Mutations in these parts of the AQP2 leave them functional, but unable to travel to their work site. Future studies will try to understand the mechanisms that cause the AQP2 to get stuck in the ER. This could lead to treatment of AQP2-related NDI.