Exploring regulated membrane protein trafficking of the aquaporin-2 water channel in renal epithelial cells

Aquaporin-2 (AQP2) is a kidney water channel that plays a critical role in urine concentration and body water homeostasis by shuttling to and from the plasma membrane of collecting duct principal cells in response to plasma levels of the hormone, vasopressin (VP). Inappropriate water retention as well as excessive diuresis have both been linked to defects in the AQP2 trafficking system. The aim of my doctoral research was to dissect the mechanisms by which VP coordinates exocytosis and endocytosis of AQP2, as well as to uncover new insights into how other physiological signals may affect AQP2 trafficking. The development and characterization of a novel fluorimetry-based ssYFP assay that quantifies exocytosis is described, and using the assay we reveal that phosphorylation of AQP2 at S256 may not be as important in the AQP2 exocytosis arm of the cycle as previously imagined. Using high-resolution microscopic techniques, an increase in AQP2 cell-surface expression due to exposure to a hyperosmotic environment is reported, as well as previously undiscovered disturbances in vesicular movement and induction of autophagy in the acute response to hypertonic stress. A series of smaller studies employing microscopic and fluorescence-based techniques explore non-VP mediated pathways and show that the hormone calcitonin also increases AQP2 cell-surface expression through both exocytosis and endocytosis, whereas a nitric oxide donor and statin may affect only endocytosis. Additionally, the role of actin and a putative actin-modulating AQP2 binding partner, arhgap8, are explored with drugs, overexpression and siRNA methodologies. Finally, the adaptation of the ssYFP exocytosis assay to 384-well format is described and ∼50 out of 4,000 molecules screened are identified as putative enhancers or suppressors of exocytosis in renal epithelial cells, providing novel candidates for future research in this exciting field.