Preclinical evaluation of resveratrol in sepsis-induced renal injury

描述（由申请人提供）：我提出在脓毒症期间，小管周围毛细血管灌注减少导致促氧化的小管周围微环境，有利于活性氧（ROS）和活性氮（RNS）的产生。我的初步数据显示，在小鼠脓毒症模型中，用天然抗氧化多酚白藜芦醇进行急性治疗，可以防止小管RNS的产生，并在盲肠结扎穿刺（CLP）诱导脓毒症后18h观察到小管周围毛细血管灌注的下降。此外，我已经产生了直接的证据，白藜芦醇可以清除RNS。基于这些发现，我假设白藜芦醇的RNS清除能力将保护小鼠肾小管免受clp诱导过程中产生的促氧化小管周围微环境的影响。目的1将通过小鼠肾小管上皮细胞的原代培养来确定白藜芦醇保护肾小管上皮免受RNS损伤的功效。目的2将研究白藜芦醇预防CLP引起的小管上皮细胞损伤的治疗潜力。将生成并分析白藜芦醇在导致肾损伤的时间过程中的剂量-反应曲线，以分析其对血清细胞因子谱、功能性小管周围毛细血管密度、体积流量、小管氧化剂生成（使用活体视频显微镜同时定量）和肾损伤（形态学和血清BUN/肌酐）的影响。Aim 3将在iNOS小鼠和过度表达超氧化物歧化酶（S0D1）以分别降低可用于RNS （ONOO）生成的NO和O2水平的小鼠中，建立RNS作为clp诱导小鼠肾损伤的关键介质。本研究的一个非常创新的方面是使用活体视频显微镜（IWM）实时研究CLP后毛细血管功能障碍发展和导致小管损伤的机制。这项研究计划将对我成为一名转化型临床科学家的整体训练有重要的贡献。在美国，每年有超过21万人死于败血症相关并发症。急性肾损伤发生在20%-50%的脓毒症患者中，这些脓毒症患者的死亡率极高（约75%），因为目前的治疗大多是支持性的，很大程度上无效。开发脓毒症肾损伤新治疗方法的主要障碍是缺乏关于脓毒症开始后可靶向的关键事件的知识。该建议研究新的治疗靶点，以治疗败血症，并有可能确定新的药物，以减少这种毁灭性的条件的死亡率。

DESCRIPTION (provided by applicant): I propose that during sepsis decreased peritubular capillary perfusion leads to a pro-oxidant peritubular microenvironment, which favors the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). My preliminary data show that acute treatment with the naturally occurring anti-oxidant polyphenol, resveratrol, prevented the generation of tubular RNS and the decline in peritubular capillary perfusion observed at 18h following induction of sepsis using the cecal ligation and puncture (CLP) in a murine model of sepsis. Moreover, I have generated direct evidence that resveratrol can scavenge RNS. Based on these findings, I hypothesize that the RNS scavenging ability of resveratrol will protect the mouse renal tubules from the pro-oxidant peritubular microenvironment produced during CLP-induced. Aim 1 will determine the efficacy of resveratrol to protect renal tubular epithelia from RNS using primary cultures of mouse renal tubular epithelial cells. Aim 2 will examine the therapeutic potential of resveratrol to prevent tubular epithelial cell injury caused by CLP. Dose-response curves for resveratrol over the time course leading to renal injury will be generated and analyzed for the effects on serum cytokine profile, functional peritubular capillary density, volumetric flow, tubular oxidant generation (each quantified simultaneously using intravital videomicroscopy), and renal injury (morphology and serum BUN/creatinine). Aim 3 will establish RNS as the key mediator of CLP-induced renal injury in the mouse using iNOS mice and in mice that over-express superoxide dismutase (S0D1) to reduce the levels of NO and O2" available for RNS (ONOO") generation, respectively. A very innovative aspect of this study is the use of intravital video microscopy (IWM) to study in real time the mechanisms by which capillary dysfunction develops and leads to tubular injury following CLP. This research proposal will contribute significant to my overall training to become a translational clinical scientist. Each year and more than 210,000 people die from sepsis-related complications in the United States. Acute kidney injury occurs in 20%-50% of septic patients and the mortality of these septic patients is extremely high (~75%) because current therapy is mostly supportive and largely ineffective. The major obstacle to the development of new therapeutic approaches for sepsis-induced kidney injury is the lack of knowledge regarding critical events that can be targeted after the initiation of sepsis. This proposal investigates new therapeutic targets to treat sepsis and has the potential to identify new agents to reduce mortality of this devastating condition.