2002 Global Researcher Conference Proceeding
April 26 - 28, 2002
|Conference:||2002 Global Researcher Conference|
|Title:||New insights in water channel physiology revealed by transgenic mouse models|
|Authors:||Verkman, Alan S.; Song, M.D., Yuanlin; Ma, Tonghui; Manley, Geoffery T.; Hara, M.; Solenov, E.; Thiagarajah, J.; Zhang, D.; Vetrivel, L; Oshio, K.; Yang, Baoxue|
|Institutions:||University of California, S.F., University of California, University of California, San Francisco, U.C. S.F., UCSF|
Our laboratory has utilized transgenic null or mutant mice to systematically evaluate the physiology of mammalian aquaporins 1-5. In kidney, deletion of AQP1 produces moderate polyuria with an inability to generate concentrated urine even after dehydration because of defective proximal tubule fluid absorption and countercurrent multiplication/exchange. Deletion of AQP3 results in marked polyuria below of low collecting duct water permeability, with partial response to vasopressin, whereas deletion of AQP4 produces only a mild defect in maximal urinary concentrating ability. Urinary diluting function is normal in aquaporin null mice. Many interesting extra-renal phenotypes of aquaporin null mice were found. Mice lacking AQP1 have reduced pain sensation, reduced intraocular pressure, defective corneal fluid extrusion, and impaired fat processing. Mice lacking AQP3 manifest very dry stratum corneum in skin. Mice lacking AQP4 have impaired hearing and vision, reduced brain swelling after injury, and increased seizure threshold. Mice lacking AQP5 have reduced agonist-stimulated fluid secretion by salivary and airway submucosal glands. However, many phenotype studies were negative, such as normal airway/lung and skeletal muscle function despite expression of multiple AQPs, and normal secretion of sweat despite AQP5 expression, indicating that tissue-specific aquaporin expression does not indicate physiological significance. A general paradigm from studies on transgenic mouse models of AQP deletion is that AQPs facilitate rapid near-isosmolar transepithelial fluid absorption / secretion, as well as rapid vectorial water movement driven by osmotic gradients. Another paradigm is the involvement of AQP4 in neural signal transduction, which may result from coupled water and potassium recycling in supportive cells adjacent to electrically excitable cells (astrocyte-neuron in brain, Muller cell-bipolar cell in eye, supportive cell-hair cell in cochlea). However for some phenotypes, such as reduced pain perception and dry skin, the mechanistic basis of the defect in aquaporin null mice is not yet clear. The transgenic mouse studies suggest that aquaporin inhibitors may have clinical indications as diuretics and in the treatment of cerebral edema, elevated intraocular pressure, and other conditions of abnormal fluid homeostasis.
We have also continued to study the pathophysiology of NDI caused by AQP2 mutation. Over the past two years an AQP2-T126M knock-in mouse model of human NDI was generated by targeted gene replacement. The mice were very polyuric and died of renal insufficiency in the first week of life - generation of conditional knock-in mice is needed. Photobleaching was used to study the diffusional mobility of green fluorescent protein-labeled AQP2-T126M at the endoplasmic reticulum (ER). Interestingly, the ER retention of mutant AQP2 is not caused by reduced mobility or significant aggregation or interaction with slowly-moving membrane proteins. Another program is high-throughput screening of diverse compound collections to identify AQP inhibitors, a strategy that might be useful to identify small molecule that correct AQP2 or V2R misprocessing - such a project is in progress to identify compounds that correct DF508-CFTR misprocessing in cystic fibrosis.
Verkman, et al., developed transgenic mice lines designed to be missing specific aquaporins (AQPs) in order to study the roles the AQPs play in mouse physiology. Mice lacking AQP1 have a reduced ability to concentrate urine, reduced pain sensation and eye pressure, defective corneal fluid extrusion and abnormal fat processing. Mice lacking AQP3 also have reduced urine concentration ability and dry skin. Mice missing AQP4 have mildly diminished urine concentrating ability, impaired hearing and vision, reduced brain swelling after injury and increased seizure threshold.
Verkman’s studies indicate that just because certain AQPs appear in specific bodily locations does not mean they have a direct function at that location. However, the AQPs are most important for rapid secretion or absorption of fluid across cell walls. The research team’s ongoing study of mutant AQP2 in relation to NDI has eliminated several reasons why some mutants of AQP2 are retained by the cell’s quality control machinery in the endoplasmic reticulum. It is not caused by the AQP2 having reduced mobility or dense clustering or interacting with slow moving membrane proteins. The researchers are also working with a screening method to identify AQP inhibitors that might also be useful to develop new therapies for NDI.