HETEROGENEITY CELL CALCIUM DISTRIBUTION RENAL HYPOXIA

异质性细胞钙分布肾缺氧

• 批准号: 3236752
• 负责人: EDORIS ANN LEFURGEY
• 金额: $ 14.51万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-15 至 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3236752
• 关键词: acidity /alkalinity; adenine nucleotides; calcium metabolism; cell death; homeostasis; hypoxia; kidney circulation; laboratory rabbit; mannitol; mitochondria; organelles; renal ischemia /hypoxia; renal tubular transport

Our long range goal is to define the role which calcium plays in early events leading to renal ischemic cell injury. To do so, it is vital to identify the sites of release, uptake and binding of calcium within the cells. Electron probe x-ray microanalysis (EPXMA) offers and unexcelled means of measuring elemental contents within individual cells and subcellular organelles, so that changes in the amounts of localized calcium can be quantitated at chosen intervals during the time course leading to irreversible cell injury. Preliminary experiments using EPXMA in proximal tubules have demonstrated a large degree of heterogeneity in the cellular and intracellular distribution of calcium. Such heterogeneity brings into question numerous previous studies which have assumed that there is a more homogeneous distribution. The proximal tubule suspension facilitates experimental maneuvers which permit examination of the earliest events leading to irreversible cell damage and eliminates secondary effects that, in an intact animal model, arise from compromising the function of the renovascular system in toto. We shall perform, in parallel, experiments which will measure biochemical, physiological, ultrastructural and microchemical variables on the same suspension. Experimental conditions will also be examined that preserve renal function during anoxia, such as the presence of adenine nucleotides, mannitol, and low pH in the bathing medium. Experiments will be performed to measure cytosolic free calcium using methyl ester dyes (fura-2) under identical conditions. EPXMA will be used to validate these measurement by determining the compartmentation of the dyes. This powerful technique (EPXMA) judiciously applied to a well-characterized renal preparation, in combination with the established array of physiological, biochemical, and ultrastructural measurements, is expected to provide unique direct information on the role of calcium in hypoxic cell injury in the kidney.

我们的长期目标是确定钙在 导致肾缺血性细胞损伤的早期事件。 要做到这一点，就是 对于确定释放、吸收和结合的位点至关重要 细胞内的钙。 电子探针X射线微量分析 (EPXMA) 提供无与伦比的元素测量方法 单个细胞和亚细胞器内的内容物，以便 局部钙含量的变化可以定量 在导致不可逆细胞的时间过程中选择的间隔 受伤。 在近曲小管中使用 EPXMA 的初步实验 已证明细胞内存在很大程度的异质性 以及钙的细胞内分布。 这样的异质性 使之前的许多研究受到质疑 假设存在更均匀的分布。 近端小管悬液有利于实验 允许检查最早事件的演习 导致不可逆的细胞损伤并消除继发性 在完整的动物模型中，由于妥协而产生的影响 肾血管系统的整体功能。 我们将 并行进行测量生化的实验， 生理、超微结构和微量化学变量 相同的悬浮液。 还将检查实验条件 在缺氧期间保护肾功能，例如存在 腺嘌呤核苷酸、甘露醇和沐浴时的低 pH 值 中等的。 将进行实验来测量细胞质 在相同条件下使用甲酯染料 (fura-2) 测定游离钙 状况。 EPXMA 将用于验证这些测量 通过确定染料的区室。 这个强大的 技术（EPXMA）明智地应用于特征良好的 肾脏制剂，结合已建立的阵列 生理、生化和超微结构测量，是 预计将提供有关作用的独特直接信息 钙在肾脏缺氧细胞损伤中的作用。