The Cytoskeletal System in Preservation Injury

描述（由申请人提供）：肾移植是终末期肾衰竭患者唯一可行的治疗方法。然而，严重的器官短缺使美国成千上万的人在等待名单上受苦和死亡。扩大供体池，包括高风险供体，如果这些移植物能得到妥善保存，可能会解决这一短缺问题。然而，近20年来，人们对器官摘取后的器官保存损伤知之甚少，在这方面也没有取得重大进展。因此，本提案的目的是确定涉及细胞骨架系统的肾保存损伤的新分子机制，并利用这一知识指导未来的治疗方法。初步结果表明，细胞骨架蛋白ezrin在肾低温贮藏过程中发生失能，ezrin表达的增加可显著保护肾小管上皮细胞免受低温贮藏损伤。相反，ezrin knock-down的作用正好相反，这表明是因果关系。本研究将通过分子和遗传方法改变ezrin的功能，测试ezrin在细胞和肾脏冷库损伤模型中诱导细胞保护的可能机制。这些研究将确定ezrin在其适当的配置下是否在冷缺血和再灌注过程中保护细胞膜结构和保持细胞线粒体功能。将使用肾上皮细胞模型，其中细胞表达不同数量的野生型和突变型ezrin蛋白。高阶保存损伤模型也将使用表达突变ezrin基因的转基因ezrin敲入小鼠的离体肾小管和全肾。在这些研究结束时，我们将收集到ezrin如何参与冷藏肾保存损伤以及这种参与是否具有临床相关性的有用分子信息。这些见解与先前确定的机制一起，可能提出改善肾脏保存的可能治疗靶点，这将允许从高风险供体中收集不太健壮的肾脏，以帮助缓解供体短缺。

DESCRIPTION (provided by applicant): Kidney transplantation is the only viable treatment for patients with end stage renal failure. However, the severe organ shortage leaves thousands in the US to suffer and die on waiting lists. Expanding the donor pool to include high risk donors may solve this shortage if those grafts could be preserved properly. However, organ preservation injury that is suffered after organ harvest is not well understood and little major advancement have been made in this area in the last 20 years. Therefore, the objective of this proposal is to identify novel molecular mechanisms of renal preservation injury involving the cytoskeleton system and to use this knowledge to direct future therapeutic approaches. Preliminary data show that the cytoskeleton protein ezrin fails during renal cold storage and that augmenting expression of ezrin significantly protects renal tubule epithelial cells from preservation injury. Conversely, ezrin knock-down does the opposite, which suggests causation. This proposal will test likely mechanisms of ezrin-induced cytoprotection in cell and kidney cold storage injury models by using molecular and genetic approaches to alter ezrin functionality. These studies will determine if ezrin, in its proper configuration, protects cell membrane structure and preserves cell mitochondrial function during cold ischemia and reperfusion. Renal epithelial cell models will be used where the cells express different amounts of wild type and mutant ezrin protein. Higher order models of preservation injury using isolated renal tubules and whole kidneys derived from transgenic ezrin knock-in mice expressing mutant ezrin genes will also be used. At the end of these studies, we will have gathered useful molecular information of how ezrin is involved in cold storage renal preservation injury and if this involvement is clinically relevant. These insights, together with previously identified mechanisms, may suggest likely therapeutic targets to improve renal preservation, which will allow harvesting of less robust kidneys from high risk donors to help alleviate the donor shortage. PUBLIC HEALTH RELEVANCE: Kidney transplantation is the treatment of choice for patients in end stage renal failure but there are not enough usable donor kidneys available. This need to expand the donor pool has increased the need to understand how to better preserve organs for transplantation, which in turn requires a more complete understanding of the mechanisms of organ preservation injury. This proposal is relevant to human health because it will bridge some of the key knowledge gaps in our understanding of the basic molecular mechanisms of how kidneys are injured after they are removed from the donor. This will reveal novel therapeutic targets to focus treatments and help alleviate the donor shortage.