Biology and Function of the Bladder Umbrella Cell Paracellular Barrier

描述（由申请人提供）：伞细胞排列在膀胱、输尿管和肾盂的内表面，形成将泌尿空间与下面的肌肉层分开的不可渗透屏障。这些细胞经历了深刻的和可逆的形态学变化，从空膀胱中的大致倒置的伞状到膀胱充满时的扁平鳞状，同时保持对细胞旁转运的紧密屏障。后一种功能依赖于紧密连接（TJ），紧密连接是位于上皮细胞的侧膜的最上部的细胞间多蛋白复合物，其介导细胞粘附并调节管腔和下层组织之间的细胞旁转运。紧密连接蛋白是一组四跨膜蛋白，是TJ的结构和功能组分。我们的初步研究结果表明，在实验性充盈过程中，伞细胞TJ环扩张，尿路上皮的细胞旁阻力显着下降。这些变化在作废时会逆转。我们的建议的中心假设是，在膀胱充盈和排尿，插入和删除的密蛋白为基础的孔伴随扩张和收缩的TJ环，分别，和细胞旁运输的一个功能是信号的程度上皮拉伸的细胞下面的尿路上皮。第一个目的是探讨如何TJ组织和密蛋白重排促进膀胱充盈过程中细胞旁通透性增加。基于尿上皮渗透途径的数学模型，我们将对以下参数进行测量，以确定它们对充盈期间细胞旁渗透性降低的贡献：长度 单位面积上皮的连接数、侧间隙的阻力、TJ链数，以及 在实验性充盈过程中，连接阻力对claudin表达变化的响应。对于这些研究，我们将结合联合收割机原位腺病毒转导，以耗尽内源性封闭蛋白或过表达标记的封闭蛋白在伞细胞，生物化学，以评估封闭蛋白的表达在TJ的变化，和电生理学，以确定细胞旁通透性的变化。第二个目标将研究TJ如何适应膀胱充盈和排尿过程中细胞形状的变化。实验将检验这一假设，即TJ环扩张需要Rab13调节的胞吐，而环收缩涉及claudin内吞。为了确定在膀胱充盈和排尿过程中调节TJ环扩张和收缩的机制，我们将使用生物化学、原位腺病毒转导和形态学的组合。第三个目标是阐明充盈过程中细胞旁通透性增加的生理作用。在这里，我们将使用原位转导的孔形成或屏障样的claudins以及药理学演习，以改变上皮细胞的旁渗透性，然后评估如何增加或减少跨TJ的电导影响组织的功能subjobly上皮。

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.