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Our research examines cellular mechanisms and physiological signals responsible for regulating the expression of physiologically important transporting proteins in plasma membranes of kidney epithelial cells. We are especially interested in the intracellular trafficking pathways followed by the vasopressin sensitive water channel, aquaporin 2 (AQP2), in normal function and in water balance disorders such as hereditary and acquired nephrogenic diabetes insipidus (NDI). Principal cells in the collecting duct (CD) respond to vasopressin by increasing the water permeability of their apical plasma membranes. This process involves AQP2 phosphorylation events that control the insertion (exocytosis) and removal (endocytosis) of AQP2 to reversibly shift its location from intracellular vesicles to the plasma membrane. These events are essential for urine concentration to occur. We use both in vivo studies and in vitro transfected epithelial cells in which water channel shuttling has been reconstituted by transfection with AQP2 cDNA. A major current project is to use high throughput screening of chemical libraries to discover new drugs that mimic vasopressin action to correct NDI. Another major interest is acid base transport by CD intercalated cells (IC), which are responsible for distal acid secretion. IC use a similar but distinct vesicle shuttling mechanism to control the number of proton pumps (a vacuolar H+ATPase) in their plasma membrane. This regulates acid secretion and allows IC to help kidneys eliminate an acid load. In a recent study, we performed a protemic analysis to produce a detailed map of the V-ATPase “interactome” and we identified several new regulatory proteins that associate with the V-ATPase to regulate its function. These are now being dissected using convergent cell, molecular and physiological techniques. Our methods include high resolution immunogold electron microscopy and confocal fluorescence imaging; biochemical assays of acidification and water transport in cells and isolated vesicles; enzyme activity assays; molecular biology approaches such as siRNA knockdown; whole animal water and electrolyte balance studies using wild type and genetically engineered mice. While our data are specifically relevant to renal function, the work is also of significance to the biology of epithelial cells in general.
Dennis Brown, PhD
Professor of MedicineDirector, MGH Program in Membrane BiologyAssociate Director, MGH Center for Systems Biology
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