Biology and Function of the Bladder Umbrella Cell Paracellular Barrier

描述（由申请人提供）：伞状细胞排列在膀胱、输尿管和肾盂的内表面，形成一个不可渗透的屏障，将尿空间与下面的肌肉层分开。这些细胞经历了深刻而可逆的形态变化，从空膀胱中大致倒置的伞形到膀胱充满时扁平鳞状的细胞，同时保持对细胞旁运输的紧密屏障。后一种功能依赖于紧密连接（TJ），这是一种位于上皮细胞外侧膜最上部的细胞间多蛋白复合物，可介导细胞粘附并调节管腔和下层组织之间的细胞旁运输。Claudins是一组四跨膜蛋白，是TJ的结构和功能成分。我们的初步结果表明，在实验填充过程中，伞细胞TJ环扩大，尿上皮细胞旁阻力明显下降。这些变化在排尿时是相反的。我们建议的中心假设是，在膀胱充盈和排尿过程中，基于clclin的孔的插入和移除分别伴随着TJ环的扩张和收缩，并且细胞旁运输的一个功能是向尿上皮下的细胞发出上皮拉伸程度的信号。第一个目的是探讨TJ组织和claudin重排如何促进膀胱充盈过程中细胞旁通透性的增加。基于尿上皮渗透途径的数学模型，我们将测量以下参数，以确定它们对填充期间细胞旁通透性降低的贡献：长度

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.