2000 Global Researcher Conference Proceeding

March 10 - 12, 2000

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Conference: 2000 Global Researcher Conference
Title: Phenotype of aquaporin knockout mice, and AQP2 misprocessing in NDI
Authors: Verkman, Alan S.; Manley, Geoffery T.; Ma, Tonghui; Song, M.D., Yuanlin; Tamarappoo, B.K.; Umenishi, Dr. F.; Yang, Baoxue
Institutions: University of California, S.F., Oregon Health & Science University, University of California, San Francisco, University of California, University of Colorado Health Sciences Center
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Verkman

New insights into water channel physiology have come from phenotype analysis of aquaporin knockout mice. To date we have characterized transgenic mice deficient in water channels AQP1, AQP3, AQP4 and AQP5, as well as double knockout mice deficient in pairs of aquaporins. In kidney, AQP1 or AQP3 deletion produced NDI. AQP1 deletion results in defective proximal tubule fluid reabsorption and defective countercurrent multiplication, resulting in a hypotonic medullary interstitium because of low water permeability in thin descending limb of Henle and outer medullary descending vasa recta. AQP3 deletion results in decreased water permeability in the basolateral membrane of cortical collecting duct. AQP4 deletion produces only a mild NDI despite a 4-fold decrease in water permeability in inner medullary collecting duct. Many interesting extrarenal phenotypes in AQP null mice have been found, such as defective saliva secretion in AQP5 null mice, protection from injury-induced brain edema in AQP4 null mice, and low lung water permeabilities in AQP1 and AQP5 null mice. The general paradigm supported by our studies is that aquaporins facilitate rapid near-isosmolar transepithelial fluid absorption/secretion, as well as rapid vectorial water movement driven by osmotic gradients. However the tissue-specific expression of an aquaporin does not indicate physiological significance. For example, despite AQP4 expression in skeletal muscle plasmalemma and gastric parietal cells, the AQP4 null mice have normal muscular function and gastric acid secretion. The data in AQP null mice suggest that aquaporins are suitable targets for drug discovery; we are thus carrying out high-throughput screening of combinatorial libraries to discover pharmacologically-useful aquaporin blockers.

We have been interested in cellular mechanisms by which mutant AQP2 proteins cause NDI and the development of novel therapeutic strategies. Some AQP2 mutants that cause autosomal recessive human NDI (eg. T126M) result in retention of AQP2 at the endoplasmic reticulum of mammalian cells. Interestingly the ER-retained AQP2 appears to be fully functional and able, with appropriate treatment, to target to the plasma membrane. Chemical chaperones such as glycerol and TMAO correct the defective phenotype in cell culture models of NDI. Detergent extraction and limited proteolysis experiments suggest that ER-retained mutant AQP2s are mildly misfolded and that the folding defect is corrected by chemical chaperone treatment. In addition, ER-retained mutant AQP2s undergo accelerated degradation by an apparently novel ER-dependent mechanism. A mouse knock-in model of human NDI is being generated to test the efficacy of chemical chaperones and other agents in vivo. Another area of investigation is the mobility and trafficking of aquaporins utilizing green fluorescent protein (GFP)-aquaporin chimeras and biophysical methods including photobleaching recovery and fluorescence correlation spectroscopy. An interesting result is that translational diffusion of GFP-AQP2 is remarkably decreased after cAMP stimulation, and that the decreased diffusion is blocked by cytoskeletal inhibitors and mutating the consensus serine for phosphorylation at the C-terminus. ER-remained AQP2 mutants diffuse rapidly through the ER membrane, but are slowed selectively by maneuvers which upregulate molecular chaperones. Basic knowledge of the cell biology of AQP2 trafficking should be helpful in the engineering of new therapies for human NDI.

Verkman, et al., studied mice engineered to lack specific aquaporins (AQPs) in order to gain insight into the functions they perform in the body. Mice lacking either AQP1 or AQP3 would both have NDI, but the physiology of the NDI would differ depending on whether the mice were lacking AQP1 or AQP3. Mice lacking AQP4 experienced a milder form of NDI despite having a four-fold decrease in their ability to transport water through the inner medullary section of the kidney collecting duct. The researchers’ studies support the consensus that AQPs help fluid flow through specific cell membranes.

Each AQP type locates itself in specific tissues of the body. However, the research of Verkman, et al., indicates the AQPs’ presence in their specific tissue is not always necessary for those tissues to carry out their primary functions. For example, mice lacking AQP4 have normal muscular function and gastric acid secretion even though AQP4 normally are located in muscle plasma membranes and gastric cells.

In other research, Verkman, et al., have investigated how certain chemical chaperones such as glycerol and TMAO can restore function to certain mutant AQP2s in laboratory cell cultures. These mutants under study are slightly misfolded (i.e. they do not have a normal AQP2 shape) and their research suggests that the chaperones are able to correct this.

The researchers are developing a mouse model of human NDI to test the efficacy of chemical chaperones in living subjects. They are also further investigating how and where AQP2 moves and is directed to specific locations within the cell. Results from these investigations should help develop new therapies for NDI.