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）紧密连接密封细胞旁 肾上皮细胞之间的空间允许维持电渗 吸收和分泌所需的梯度。短暂性肾缺血 导致紧密连接的细胞旁屏障的破坏和 肾功能正常封闭蛋白是唯一已知的跨膜蛋白， 紧密连接和初步研究表明，它的功能，在创造 细胞旁密封此应用程序的总体目标是确定 闭合蛋白的分子基础，它创造了一个细胞旁密封，屏障是如何 性质由细胞质组分和信号传导途径调节， occludin对可逆性肾缺血的反应我们最近的研究表明 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细胞中表达突变的occludin 细胞系（MDCK和LLC-PK 1），以确定粘附的基础。肽 对应于闭合蛋白的两个细胞外结构域的部分将被 用于定位最负责粘附的区域。缺乏结构 第一或第二循环将被表示以确定哪一个最 参与粘附。这些信息将被翻译成文化 上皮细胞系，以确定粘附是否是产生 通过跨上皮电阻测量的细胞旁密封， 放射性标记的示踪分子的细胞旁通量。作为一个非屏障功能 occludin，我们观察到它在成纤维细胞中的表达抑制细胞运动。一 运动的作用可能是重要的上皮修复缺血后 肾小管上皮损伤。我们将确定附着力是否是 这是运动调节的基础。细胞质因子的作用， 肌动蛋白组织ATP水平和信号通路在赋予粘附 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occludin发生的结构和生化变化 在培养的肾上皮细胞系中ATP消耗和恢复期间， 在完整大鼠肾脏模型中的缺血和恢复。具体来说，我们将 确定闭合蛋白的磷酸化和可提取性的变化， 将这些与occludin的细胞定位变化相关联， 通过免疫荧光、免疫金TEM和免疫金冷冻测定 断口电子显微镜这些研究将共同推动我们的 了解细胞旁屏障是如何在肾脏中产生和调节的 上皮细胞以及屏障如何被破坏，以及在 短暂性缺血性损伤