RENAL ISCHEMIA--CELLULAR DISRUPTION AND RESTITUTION

肾缺血——细胞破坏和恢复

基本信息

批准号：
6229083
负责人：
NORMAN J SIEGEL
金额：
$ 8.18万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-07-01 至 2001-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6229083
关键词：
adenosine triphosphate ankyrins cellular pathology confocal scanning microscopy cytoskeleton genetic transcription immunocytochemistry intermolecular interaction laboratory rat nuclear magnetic resonance spectroscopy nucleotide metabolism renal ischemia /hypoxia sodium potassium exchanging ATPase spectrin stress proteins ubiquitin western blottings

项目摘要

The overall objectives of the proposed studies are to define the cellular and molecular mechanisms which are responsible for the loss of cellular organization and disassembly of the cytoskeleton following renal ATP depletion and to delineate those adaptive and reparative processes which result in restitution of cellular structure during early recovery from an ischemic insult. Three specific aims will be investigated: 1) The pattern and sequence of disassociation and restitution of the cytoskeleton in response to ATP depletion will be studied using an integrated approach in which cellular ATP is determined by 31P NMR spectroscopy in vivo and the molecular processing of cytoskeletal proteins and integral membrane proteins is evaluated at graded levels of ATP depletion. Detached or unstable proteins will be detected by detergent solubility and Western immunoblotting. The disruption and reconstitution of the cytoskeleton will be confirmed and the intracellular localization of proteins will be defined by confocal and immunocytochemical electron microscopy. To assess the molecular processing involved in the restoration of cellular structure, the message (mRNA) and the specific transcription rate for each of the cytoskeletal proteins and for Na/K ATPase (alpha and beta subunits) will be determined at specific ATP levels and reflow intervals. 2) The mechanism of disassembly of the cytoskeleton-integral membrane protein complex will be determined by an assessment of the binding of ankyrin and fodrin to Na/K ATPase. An ankyrin binding assay will be used to asses the susceptibility of specific binding sites to graded reductions In cellular ATP. 3) The molecular process which contributes to the restitution of cellular structure and integrity during recovery from energy depletion will be determined by an assessment of stress protein responses, particularly heat shock protein 70-72 and ubiquitin. The principal focus of this group of studies will be the hypothesis that stress proteins participate in the rescue and/or disposal of disassembled cytoskeletal proteins and/or Na/K ATPase during recovery from an ischemic insult. Co- precipitation and colocalization studies will be utilized to assess the structural and functional interaction of the stress proteins with specific cytoskeletal proteins during the restitution of cellular integrity. These studies will provide important new information concerning the basic biological mechanisms by which renal epithelial cells are injured and through which recovery of sublethally injured cells is attained.

拟议研究的总体目标是定义细胞以及导致细胞丧失的分子机制肾 ATP 后细胞骨架的组织和分解耗尽并描绘那些适应性和修复过程导致细胞结构在早期恢复过程中恢复缺血性损伤。将研究三个具体目标：1）模式以及细胞骨架解离和恢复的顺序将使用综合方法研究对 ATP 消耗的反应细胞 ATP 是通过体内 31P NMR 光谱测定的细胞骨架蛋白和整合膜的分子加工蛋白质按 ATP 消耗的分级水平进行评估。独立或不稳定的蛋白质将通过洗涤剂溶解度和Western检测来检测免疫印迹。细胞骨架的破坏和重建将得到证实，并且蛋白质的细胞内定位将被确定通过共聚焦和免疫细胞化学电子显微镜定义。评估参与细胞修复的分子加工结构、信息（mRNA）和每个的具体转录率细胞骨架蛋白和 Na/K ATP 酶（α 和 β 亚基）将根据特定的 ATP 水平和回流间隔来确定。 2) 的细胞骨架整合膜蛋白的分解机制复合物将通过评估锚蛋白和的结合来确定胞质蛋白 (fodrin) 转化为 Na/K ATP 酶。锚蛋白结合测定将用于评估细胞中特定结合位点对分级减少的敏感性 ATP。 3) 有助于恢复的分子过程从能量耗尽恢复期间的细胞结构和完整性将通过对应激蛋白反应的评估来确定，特别是热休克蛋白 70-72 和泛素。主要关注点这组研究的假设是应激蛋白参与拆卸的细胞骨架的救援和/或处置缺血性损伤恢复期间的蛋白质和/或 Na/K ATP 酶。共同将利用降水和共定位研究来评估应激蛋白与特定的结构和功能相互作用细胞完整性恢复期间的细胞骨架蛋白。这些研究将提供有关基本原理的重要新信息肾上皮细胞损伤的生物学机制通过它实现亚致死损伤的细胞的恢复。