L-Arginine/NO in Liver Transplant Preservation Injury

描述(由申请人提供)： 肝移植保存损伤是一种“冷”缺血/再灌注损伤。接受严重肝脏保存损伤的患者在ICU住院时间、感染、肾功能衰竭、胆道狭窄和同种异体移植排斥反应方面显著增加。由于受损的肝移植物释放精氨酸酶，移植后会出现严重的精氨酸缺乏状态。我们认为精氨酸在早期肝脏保护中起关键作用，部分是由NO合成介导的，而精氨酸缺乏进一步导致持续的肝移植损伤。补充L-精氨酸不会导致循环精氨酸水平升高，对肝脏损伤仅有轻微改善。因此，我们假设需要抑制精氨酸酶，以提高精氨酸的可用性，并将肝脏保存损伤所造成的损害降至最低。这是一种对抗器官损伤的全新方法；基本上对体内精氨酸酶阻断的影响一无所知。注射精氨酸酶抑制剂NU-羟基-L-精氨酸(NOR-NOHA)的初步数据显示，血清精氨酸酶活性受到抑制，循环精氨酸水平恢复。这导致了肝脏酶释放的显著减少和肝脏组织学的改善。在这个方案中，我们将追求两个目标来研究移植后精氨酸代谢的特征和确定精氨酸酶阻断对肝脏冷I/R损伤的影响：目的I：确定通过阻断精氨酸酶来提高精氨酸利用率是否能预防肝移植缺血/再灌注损伤。我们将确定移植后精氨酸代谢的特征，并确定在肝脏I/R损伤期间精氨酸转运是否发生改变。精氨酸酶阻断对血清和组织精氨酸水平的影响将被确定。将建立优化精氨酸供应以最大限度地保护器官的条件。精氨酸酶抑制对多胺/脯氨酸合成、尿素排泄、血压和同种异体免疫反应的潜在不良后果将被调查。目的II：探讨提高精氨酸利用率对移植肝损伤的保护作用机制。我们将确定精氨酸利用率增加的有益影响是否由NO合成介导。组织保护的二级机制将被确定，包括提高精氨酸浓度对中性粒细胞渗透、细胞因子释放、细胞凋亡、超微结构损伤、肝血流和HO-1表达的影响。所获得的信息将增加我们对肝移植损伤的分子病理生理学的了解，并将提供关于精氨酸酶阻断的体内效应的全新信息。我们希望这些发现具有广泛的意义，远远超出移植保存损伤，可能适用于任何形式的肝损伤，导致肝细胞精氨酸酶释放和精氨酸耗竭。

DESCRIPTION (provided by applicant): Liver transplant preservation injury represents a "cold" ischemia/reperfusion (I/R) injury. Patients receiving livers with severe preservation injury exhibit significant increases in the length of ICU stay, infection, renal failure, biliary strictures, and allograft rejection. A profound arginine-deficient state occurs post-transplant due to arginase release from the injured liver graft. We believe arginine is critical for early hepatic protection, mediated in part by NO synthesis, and that arginine deficiency further contributes to ongoing liver graft injury. L-arginine supplementation does not result in increased circulating arginine levels and achieves only modest improvement in liver injury. Therefore, we hypothesize that inhibition of arginase will be required to enhance arginine availability and minimize damage resulting from liver preservation injury. This is a completely novel approach to combat organ injury; essentially nothing is known about the effects of arginase blockade in vivo. Preliminary data with injection of the arginase inhibitor, Nu-hydroxy-nor-L-arginine (nor-NOHA), showed that serum arginase activity was inhibited and circulating arginine levels were restored. This resulted in a significant decrease in liver enzyirie release and improvement in liver histology. In this proposal, we will pursue two aims to characterize arginine metabolism post-transplant and determine the effects of arginase blockade on liver cold I/R injury: AIM I: TO DETERMINE WHETHER ENHANCING ARGININE AVAILABILITY THROUGH ARGINASE BLOCKADE PREVENTS LIVER TRANSPLANT ISCHEMIA/REPERFUSION INJURY. We will characterize arginine metabolism post-transplant and determine if arginine transport is modified during liver I/R injury. The effects of arginase blockade on serum and tissue arginine levels will be determined. The conditions that optimize arginine availability to maximize organ protection will be established. The potential adverse consequences of arginase inhibition on polyamine/proline synthesis, urea excretion, blood pressure, and alloimmurie response will be investigated. AIM II: TO IDENTIFY THE MECHANISMS OF PROTECTION MEDIATED BY IMPROVING ARGININE AVAILABILITY ON LIVER GRAFT INJURY. We will determine if the beneficial effects of enhanced arginine availability are mediated by NO synthesis. The secondary mechanisms of tissue protection will be identified including the effects of enhanced arginine concentration on neutrophil infiltration, cytokine release, apoptosis, ultrastructural damage, hepatic blood flow, and HO-1 expression. The information gained will increase our understanding of the molecular pathophysiology of liver graft injury, and will provide completely new information regarding the in vivo effects of arginase blockade. We expect these findings to have broad implications far beyond transplant preservation injury that may be applicable to any form of liver injury resulting in hepatocellular arginase release and arginine depletion.