Biology and Function of the Bladder Umbrella Cell Paracellular Barrier

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DESCRIPTION (provided by applicant): Umbrella cells line the inner surface of the urinary bladder, ureters, and renal pelvis, forming an impermeable barrier that separates the urinary space from the underlying muscle layer. These cells experience profound and reversible morphologic changes from a roughly inverted umbrella shape in empty bladders to one that is flat and squamous as the bladder fills, all the while maintaining a tight barrier to paracellular transport. The latter function is dependent on the tight junction (TJ), an intercellular multi-protin complex located at the upper-most portion of the lateral membrane of epithelial cells that mediates cell adhesion and modulates paracellular transport between the lumen and the underlying tissue. Claudins, a group of tetraspan membrane proteins, are structural and functional component of the TJ. Our preliminary results indicate that during experimental filling the umbrella cell TJ ring expands and the paracellular resistance of the uroepithelium drops significantly. These changes are reversed upon voiding. The central hypothesis of our proposal is that during bladder filling and voiding, insertion and removal of claudin-based pores accompany expansion and contraction of the TJ ring, respectively, and that one function of paracellular transport is to signal the degree of epithelial stretch to the cells underlying the uroepithelium. The first aim explores how TJ organization and claudin rearrangements promote increased paracellular permeability during bladder filling. Based on a mathematical model of the uroepithelium permeation pathways we will conduct measurements of the following parameters to determine their contribution to the decrease in paracellular permeability during filling: length of junction per unit area of the epithelium, resistance of the lateral space, TJ strand number, and junctional resistance during experimental filling in response to changes in claudin expression. For these studies we will combine in situ adenoviral transduction to deplete endogenous claudins or overexpress tagged-claudins in umbrella cells, biochemistry to assess changes in the expression of claudins at the TJs, and electrophysiology to determine changes in paracellular permeability. The second aim will investigate how the TJ accommodates cell shape changes during bladder filling and voiding. Experiments will examine the hypothesis that TJ ring expansion requires Rab13-regulated exocytosis, while ring contraction involves claudin endocytosis. To define the machinery that regulates TJ ring expansion and contraction during bladder filling and voiding we will use a combination of biochemistry, in situ adenoviral transduction, and morphology. The goal of the third aim is to elucidate the physiologic role of increased paracellular permeability during filling. Here, we will use in situ transduction of pore-forming or barrier-like claudins as well as pharmacological maneuvers to alter the paracellular permeability of the epithelium, and then assess how increased or reduced conductance across the TJs affects the function of tissues subjacent to the epithelium.