Biology and Function of the Bladder Umbrella Cell Paracellular Barrier

描述(申请人提供)：伞状细胞排列在膀胱、输尿管和肾盆的内表面，形成一个不透水的屏障，将尿路与下面的肌肉层隔开。这些细胞经历了深刻和可逆的形态变化，从空膀胱中大致倒置的伞形到随着膀胱充盈而扁平和扁平的形状，同时保持着对细胞旁运输的紧密屏障。后一种功能依赖于紧密连接(TJ)，这是一种细胞间多蛋白复合体，位于上皮细胞侧膜的最上面部分，介导细胞黏附并调节管腔和下层组织之间的细胞旁运输。克劳丁(Claudins)是TJ的结构和功能成分，是一组四聚糖膜蛋白。我们的初步结果表明，在实验充盈过程中，伞状细胞TJ环扩张，尿路上皮细胞旁阻力显着下降。这些变化在排尿时被逆转。我们的建议的中心假设是，在膀胱充盈和排尿过程中，基于克劳丁的毛孔的插入和移除分别伴随着TJ环的扩张和收缩，细胞旁运输的一个功能是向尿路上皮下的细胞发出上皮伸展程度的信号。第一个目的是探索TJ组织和Claudin重排如何在膀胱充盈过程中促进细胞旁通透性的增加。基于尿路上皮渗透途径的数学模型，我们将对以下参数进行测量，以确定它们在充盈过程中对细胞旁通透性降低的贡献：长度 单位面积上皮的连接、侧间隙的阻力、TJ链数和 实验填充过程中对claudin表达变化的反应中的连接阻力。对于这些研究，我们将结合原位腺病毒转导来耗尽伞状细胞中内源性Claudins或过表达标记Claudins，结合生物化学来评估TJ处Claudins表达的变化，并结合电生理学来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。实验将检验这样的假设，即TJ环的扩张需要Rab13调节的胞吐作用，而环的收缩涉及克拉丁的内吞作用。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，我们将结合生物化学、原位腺病毒转导和形态学。第三个目标是阐明充填过程中细胞旁通透性增加的生理作用。在这里，我们将使用形成孔洞或屏障样克拉丁蛋白的原位转导以及药物操作来改变上皮细胞旁的通透性，然后评估跨TJ的电导增加或减少如何影响上皮细胞下组织的功能。

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.