REGULATION OF TIGHT JUNCTIONAL PERMEABILITY IN RENAL EPITHELIAL CELLS

肾上皮细胞紧密连接通透性的调节

• 批准号: 6564384
• 负责人: JAMES M. ANDERSON
• 金额: $ 14.1万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6564384
• 关键词: MDCK cell; biological signal transduction; cell adhesion molecules; cell migration; cell motility; epithelium; freeze etching; immunoelectron microscopy; immunofluorescence technique; kidney cell; laboratory rat; membrane permeability; membrane transport proteins; protein structure function; renal ischemia /hypoxia; tight junctions; transfection

Description: (Abstract of the project) Tight junctions seal the paracellular space between renal epithelial cells allowing maintenance of electroosmotic gradients required for absorption and secretion. Transient renal ischemia results in breakdown of the tight junction paracellular barrier and loss of normal renal function. Occludin is the only known transmembrane protein of the tight junction and preliminary studies suggest it functions in creating the paracellular seal. The overall goals of this application are to determine the molecular basis by which occludin creates a paracellular seal, how the barrier properties are regulated by cytoplasmic components and signaling pathways and how occludin responds to reversible renal ischemia. We have recently shown occludin is an intercellular adhesion molecule and we will use full-length and mutated occludin expressed in both cultured fibroblasts and renal epithelial cell lines (MDCK and LLC-PK1) to determine the basis for adhesion. Peptides corresponding to portions of the two extracellular domains of occludin will be used to localize the regions most responsible for adhesion. Constructs lacking either the first or second loop will be expressed to determine which is most involved in adhesion. This information will be translated to cultured epithelial lines to determine if adhesion is the basis for creation of the paracellular seal as measured by transepithelial electrical resistance and the paracellular flux of radiolabeled tracer molecules. As a non-barrier function of occludin, we observe its expression in fibroblasts inhibits cell motility. A role in motility may be important in epithelial restitution following ischemic damage of the tubular epithelium. We will determine whether adhesion is the basis for this motility regulation. The role of cytoplasmic factors such as actin organization ATP levels and signaling pathways in conferring adhesion will be investigated in fibroblasts and cultured epithelial cell lines. Finally we will study the structural and biochemical alterations to occludin occurring during ATP depletion and recovery in cultured renal epithelial cell lines and in ischemia and recovery in an intact rat kidney model. Specifically we will determine changes in the phosphorylation and extractability of occludin and correlate these with changes in the cellular localization of occludin determined by immunofluorescence, immunogold TEM, and immunogold freeze fracture electron microscopy. Together these studies will advance our understanding of how the paracellular barrier is created and regulated in renal epithelia and how the barrier is disrupted and reforms following during transient ischemic injury.

描述：（项目摘要）紧密连接密封副细胞 肾上皮细胞之间的空间，允许维持电脑 吸收和分泌所需的梯度。瞬态肾脏缺血 导致紧密连接旁细胞屏障的崩溃和丧失 正常肾功能。 occludin是唯一已知的跨膜蛋白 紧密的结和初步研究表明，它在创建 细胞密封。该应用程序的总体目标是确定 分子基础，叶解蛋白会产生细胞细胞密封，屏障如何 性质受细胞质成分和信号通路的调节，以及 叶解素如何对可逆的肾脏缺血做出反应。我们最近显示了 occludin是一种细胞间粘附分子，我们将使用全长和 在培养的成纤维细胞和肾上皮中表达的突变咬合素 细胞系（MDCK和LLC-PK1）确定粘附的基础。肽 对应于胶囊的两个细胞外域的一部分将是 用于定位最负责粘附的区域。缺乏构造 第一个循环或第二个循环将被表达以确定哪个是最多的 参与粘附。这些信息将转化为文化 上皮线以确定粘附是否是创造的基础 副旁密封，通过跨越电阻和 放射性标记的示踪剂分子的细胞细胞通量。作为非级函数 在occludin中，我们观察到它在成纤维细胞中的表达抑制了细胞运动。一个 在缺血之后，在运动中的作用在上皮恢复中可能很重要 管状上皮的损害。我们将确定粘附是否是 这种运动调节的基础。细胞质因素的作用，例如 肌动蛋白组织ATP水平和信号途径赋予粘附 将在成纤维细胞和培养的上皮细胞系中进行研究。最后 我们将研究发生occludin的结构和生化改变 在ATP耗竭和培养的肾上皮细胞系的恢复期间， 在缺血和完整的大鼠肾脏模型中的恢复中。特别是我们会的 确定叶解蛋白和磷酸化的变化和可萃取性的变化 将这些与occludin的细胞定位变化相关 由免疫荧光，免疫胶质TEM和免疫冻结确定 断裂电子显微镜。这些研究将共同​​发展 了解如何在肾脏中创建和调节细胞细胞屏障 上皮以及如何破坏障碍以及在此期间进行改革 短暂性缺血性损伤。