Cellular Effects of Indoxyl Sulfate-A Uremic Toxin

硫酸吲哚酚-尿毒症毒素的细胞作用

• 批准号: 6604074
• 负责人: ANDREW K GELASCO
• 金额: $ 13.62万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6604074
• 关键词: biological signal transduction; cellular respiration; electron spin resonance spectroscopy; enzyme activity; free radical oxygen; glycolysis; glycosides; kidney cell; membrane potentials; mitogen activated protein kinase; oxidation reduction reaction; renal toxin; tissue /cell culture; vascular smooth muscle

DESCRIPTION (provided by applicant) This proposal provides a means of laying the groundwork for the candidate's career in quantitative biomedical research. His formal Ph.D. training was primarily in inorganic chemistry with an emphasis on metal-oxygen interactions as models for biological systems. He has developed an interest in biochemical and biomedical research from where he could study these systems from the molecular to the cellular level. In the course of this award period, Dr. Gelasco expects to develop skills in cell biology and biochemistry that are needed, through both didactic and laboratory experience. Then, using these new skills in combination with a chemistry and spectroscopy background he will develop independent research projects investigating the role of enzymatic and signaling events, potentially mediated through biological metal centers, in renal failure pathology. He should be very well suited for a "molecular basis of disease"-type approach toward cell biology pathways and in particular signal transduction, because the combination of chemical, spectroscopic and biochemical techniques should provide an effective arsenal to use toward solving complex biomedical problems. Toward these ends, a research plan has been prepared which should provide ample opportunity to develop new skills and to obtain the theoretical background in cell biology and biomedicine required for Dr. Gelasco's long-term goals. The overall goal of the research proposal is to investigate the effects of indoxyl sulfate on cellular redox-activated pathways, especially those involved in the activation of ERK, because ERK is a major contributing signal intermediary in proliferation of glomerular cells and renal hypertrophy. The major hypothesis to be tested is that the activation of ERK by the uremic toxin indoxyl sulfate is the result of alteration of cellular redox, most likely through the overproduction of reactive oxygen species (ROS), and that the ROS generated are specific for different signaling events in this activation cascade. This project will involve a combination of biophysical, biochemical, cell biological and microphysiometric techniques applied to the investigation of the effects of indoxyl sulfate in these systems. Initial work will involve expanding upon recent observations of ERK activation by indoxyl sulfate with a combination of redox microphysiometry and electron paramagnetic resonance (EPR) spectroscopy to measure the extent of ROS involvement in this process. Redox microphysiometric measurements will probe the redox state of living cells using an electrochemical potentiometric method through a novel technique, which is not available in any other setting. An additional factor that makes this proposal particularly unique is the availability of a new Bruker EPR spectrometer in our lab, which we will use in parallel experiments to measure the production of ROS in indoxyl sulfate-treated mesangial and vascular smooth muscle cells. This biophysical technique will be developed to provide a means not only to determine whether indoxyl sulfate acutely alters the levels of ROS in cultured cells, but to determine which free radical ROS are produced and to precisely measure the levels of ROS produced These methods should then be broadly applicable to studying stimulant-induced signaling events in renal cell pathways.

描述（由申请人提供） 该建议提供了为候选人奠定基础的一种方法 定量生物医学研究的职业。他的正式博士训练是 主要是无机化学，重点是金属氧相互作用 作为生物系统的模型。他对生化产生了兴趣 以及他可以从那里研究这些系统的生物医学研究 分子至细胞水平。在这个奖项期间，博士 Gelasco希望发展细胞生物学和生物化学技能 需要通过教学和实验室经验。然后，使用这些新 与化学和光谱背景结合的技能他将 开发独立研究项目，调查酶促和 信号事件，可能通过生物金属中心介导的信号事件 肾衰竭病理学。他应该非常适合“分子基础 疾病“型”细胞生物学途径，特别是信号的方法 转导，因为化学，光谱和 生化技术应提供有效的武器库 解决复杂的生物医学问题。在这些目的方面，研究计划已经 已准备好，应该提供足够的机会来发展新技能和 为了获得细胞生物学和生物医学的理论背景 为Gelasco博士的长期目标。 研究建议的总体目标是研究 硫酸硫酸盐在细胞氧化还原的途径上，尤其是涉及的途径 在ERK的激活中，因为ERK是主要的贡献信号 肾小球细胞和肾脏肥大的增殖中介。这 要测试的主要假设是尿毒症毒素激活ERK 吲哚基硫酸盐是细胞氧化还原改变的结果，很可能 通过反应性氧（ROS）的过量生产，ROS 生成的特定于此激活中的不同信号事件 级联。该项目将涉及生物物理，生化， 细胞生物学和微生物测量技术应用于研究 这些系统中硫酸硫酸盐的作用。最初的工作将涉及 扩大了硫酸硫酸硫酸盐最近观察到的ERK激活 氧化还原微生物测量和电子顺磁共振（EPR）的组合 测量该过程中ROS参与程度的光谱。氧化还原 微观生理测量结果将使用 通过一种新技术的电化学计量方法，即 在任何其他设置中都不可用。使这个的另一个因素 提案特别独特的是新的Bruker EPR的可用性 我们实验室中的光谱仪，我们将在并行实验中使用该光谱仪来测量 硫酸硫酸盐处理的肾小球和血管平滑中ROS的产生 肌肉细胞。将开发这种生物物理技术来提供一种手段 不仅要确定硫酸硫酸盐是否急性改变ROS的水平 在培养的细胞中，但要确定产生哪种自由基ROS并至 精确测量产生的ROS的水平，应该是 广泛适用于研究肾细胞中刺激性诱导的信号传导事件 途径。