Cell Biology of Vasopressin-induced Water Channels

Because of a lack of safe, effective and specific treatments for disorders of water balance, the ultimate goal of our work is to identify novel intracellular pathways by which plasma membrane accumulation of the aquaporin 2 water channel (AQP2) in kidney principal cells can be normalized in the absence of a properly functioning vasopressin receptor (V2R) signaling pathway. The overall objective of this proposal is to dissect newly- identified aquaporin 2 (AQP2) trafficking and regulatory pathways in order to provide actionable, basic information that can be translated into cell-specific clinical advances for the treatment of these conditions. Nephrogenic diabetes insipidus (NDI) is caused by renal insensitivity to VP, and results in excessive urine production, whereas water retention, often a result of inappropriate VP secretion, occurs in conditions such as congestive heart failure. Aim 1 addresses the hypothesis that AQP2 itself directs its intracellular trafficking itinerary as an “active” cargo protein rather than a passive bystander. We propose that AQP2 “catalyzes” compartment-specific actin remodeling via direct and indirect interactions with different cohorts of actin- regulatory proteins. These include actin itself, the Arp2/3 actin remodeling complex during exocytosis, and the actin binding protein ezrin during endocytosis. Aim 2 will identify the cellular crosstalk mechanism(s) by which a tug-of-war between the vasopressin receptor (VP/V2R - positive action) and epidermal growth factor receptor (EGF/EGFR - negative effect) regulate AQP2 trafficking and water balance. We will first explore how EGF and other EGFR ligands inhibit the antidiuretic effect of VP by receptor activation and downstream signaling. Then we will ask how inhibition of the EGFR pathway results in VP-independent AQP2 phosphorylation and membrane accumulation by activating a non-canonical kinase, P90 ribosomal S6 kinase (RSK). Techniques central to the proposed work include the molecular characterization of protein interactions, advanced super resolution microscopy and live cell imaging, enzyme activity assays, expression of modified proteins in cultured epithelial cells, and whole animal physiology. Understanding novel cellular mechanisms of AQP2 regulation and defining more specific participants in intracellular signaling will open unexplored avenues of research into the regulation of fluid and electrolyte homeostasis. We expect that our data will allow the development of more selective and cell specific pharmacological strategies to regulate AQP2 trafficking in water balance disorders.