Novel Oxygen Carriers Based on Hexacoordinate Globins to Prevent Renal Toxicity

项目摘要/摘要 输血是治疗严重贫血或失血的最常见的疗法，并提供不可否认的生命- 在各种临床情况下节省福利。尽管如此，献血的长期短缺和持续 术后感染和免疫并发症的风险(大大降低，但不能通过筛查消除) 输血促使人们寻找全血的替代品。纯化的血红蛋白，而这些血红蛋白- 从它衍生出来的碱性氧气载体(HBOC)是一种潜在的血液替代品 输血。然而，在临床研究中，HBOCs一直显示出不良反应，包括 严重高血压、肾损伤和衰竭等终末器官损害。渗出与一氧化氮(NO) 这种蛋白质的清除作用被认为是这些效应的基础。我们实验室已经研制出重组突变体 基于快速生成的六配位珠蛋白脑红蛋白(RNgb)和细胞球蛋白(RCgb)的蛋白质 NO通过还原亚硝酸盐，潜在地抵消任何NO清除作用。因此，我们提出了这样的假设 RNgb和rCgb的特定突变可用于设计新一代氧载体 这会降低高血压和肾脏毒性。为了验证这一假设，我们将使用UV-Vis 光谱学鉴定众多rNgb和rCgb突变体的功能，并用 理想的氧气传输特性，不会产生。我们还将把这些蛋白质封装在 人造类脂膜，产生不能渗出的含有蛋白质的脂质体。第二，我们 将评估在体内降低高铁血红蛋白的细胞色素b5作为一种潜在的还原系统。 突变蛋白，作为持续的蛋白质氧化，阻止氧结合和亚硝酸盐还原为NO。最后，我们 将产生脂质体(如目标1)，其中包含一种有希望的蛋白质，用于氧气输送和还原 系统在AIM 2中进行评价，并通过静脉给药给药。输液后，我们 将测量血压、肾功能和肾脏组织病理学，以确定是否有任何不良反应 现在时。NRSA奖将为我提供一个独特的机会来利用现有的研究和 我的导师Mark Gladwin博士的开发基础设施，以获得动物模型的高级培训 疾病、蛋白质工程和血红素生物化学。我的导师Dr的资源和经验 Gladwin是一位一氧化氮和珠蛋白生物化学以及来自 从实验室到临床，再加上加州大学提供的大量资源 匹兹堡，将使我能够发展新的翻译研究技能，特别是发展 新的治疗策略。NRSA还将支持我完成#年博士学位的要求 生物工程和论文答辩，以及我回到医学院，在那里我将开始发展 我的临床技能为我最终在学术医学领域的职业生涯做准备。

PROJECT SUMMARY/ABSTRACT Blood transfusions are the most common therapy for severe anemia or blood loss, and offer undeniable life- saving benefits in a variety of clinical situations. Nevertheless, chronic shortages of donor blood and continued risks of infectious and immune complications (greatly reduced but not eliminated by screening) post- transfusion have spurred a search for alternatives to whole blood. Purified hemoglobin, and the hemoglobin- based oxygen carriers (HBOCs) that have been derived from it, are one potential substitute for blood transfusions. In clinical studies, however, HBOCs have consistently demonstrated adverse effects, including severe hypertension, renal injury and failure, and other end-organ damage. Extravasation and nitric oxide (NO) scavenging by the protein is thought to underlie these effects. Our lab has developed recombinant mutant proteins based on the hexacoordinate globins neuroglobin (rNgb) and cytoglobin (rCgb) that rapidly generate NO via reduction of nitrite, potentially offsetting any NO scavenging. We, therefore, propose the hypothesis that specific mutations of rNgb and rCgb can be used to engineer a new generation of oxygen carriers that cause reduced hypertension and renal toxicity. To test the hypothesis, we will use UV-Vis spectroscopy to characterize the function of numerous rNgb and rCgb mutants, and identify those mutants with ideal characteristics for oxygen transport and NO generation. We will also encapsulate these proteins within artificial lipid membranes, creating protein-containing liposomes that will be unable to extravasate. Second, we will evaluate cytochrome b5, which reduces methemoglobin in vivo, as a potential reducing system for these mutant proteins, as persistent protein oxidation prevents oxygen binding and nitrite reduction to NO. Finally, we will create liposomes (as in aim 1) containing a promising protein for oxygen delivery as well as the reducing system evaluated in aim 2, and administer these liposomes to mice via venous infusion. Following infusion, we will measure blood pressure, kidney function, and kidney histopathology to determine if any adverse effects are present. The NRSA award will provide me a unique opportunity to leverage the existing research and development infrastructure of my mentor, Dr. Mark Gladwin, to gain advanced training in animal models of disease, protein engineering, and heme biochemistry. The resources and experience of my mentor Dr Gladwin, an expert in NO and globin biochemistry, as well as the translational development of drugs from the laboratory to the clinic, combined with the expansive array of resources available at the University of Pittsburgh, will allow me to develop new translational research skills with a specific focus on development of novel therapeutic strategies. The NRSA will also support my completion of the requirements for a PhD in Bioengineering and defense of my thesis, as well as my return to medical school, where I will begin to develop my clinical skillset in preparation for an eventual career in academic medicine.