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: 6517952
• 负责人: ANDREW K GELASCO
• 金额: $ 13.35万
• 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/6517952
• 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） 该提案提供了一种为候选人奠定基础的方法 定量生物医学研究的职业生涯。他的正式博士学位。培训是 主要研究无机化学，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 和生物医学研究，他可以从那里研究这些系统 分子水平到细胞水平。在此获奖期间，博士。 Gelasco 希望培养细胞生物学和生物化学方面的技能 需要通过教学和实验室经验。然后，使用这些新的 他将结合化学和光谱学背景的技能 开发独立的研究项目，调查酶的作用和 信号事件，可能通过生物金属中心介导， 肾衰竭病理学。他应该非常适合“分子基础” 针对细胞生物学途径，特别是信号的“疾病”类型方法 转导，因为化学、光谱和 生化技术应提供有效的武器库以用于 解决复杂的生物医学问题。为了实现这些目标，一项研究计划 已做好准备，这应该提供充足的机会来发展新技能和 获得所需的细胞生物学和生物医学的理论背景 为了 Gelasco 博士的长期目标。 该研究计划的总体目标是调查以下因素的影响： 硫酸吲哚酚对细胞氧化还原激活途径的影响，特别是涉及的途径 参与 ERK 的激活，因为 ERK 是主要的贡献信号 介导肾小球细胞增殖和肾肥大。这 待检验的主要假设是尿毒症毒素激活 ERK 硫酸吲哚酚很可能是细胞氧化还原改变的结果 通过活性氧 (ROS) 的过量产生，并且 ROS 生成的信号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、生物化学、 细胞生物学和微物理测量技术应用于研究 硫酸吲哚酚在这些系统中的作用。初步工作将涉及 扩展了最近对硫酸吲哚酚激活 ERK 的观察 氧化还原微物理测量与电子顺磁共振 (EPR) 的结合 光谱法测量 ROS 参与这一过程的程度。氧化还原 微物理测量将利用以下方法探测活细胞的氧化还原状态 通过新技术的电化学电位测量方法， 在任何其他设置中不可用。造成这种情况的另一个因素 特别独特的提案是提供新的布鲁克 EPR 我们实验室的光谱仪，我们将在并行实验中使用它来测量 硫酸吲哚酚处理的系膜和血管平滑肌中ROS的产生 肌肉细胞。这种生物物理技术的开发将提供一种手段 不仅是为了确定硫酸吲哚酚是否会急剧改变 ROS 水平 在培养细胞中，但要确定产生哪些自由基ROS并 精确测量产生的 ROS 水平 这些方法应该是 广泛适用于研究肾细胞中兴奋剂诱导的信号传导事件 途径。