Preclinical evaluation of resveratrol in sepsis-induced renal injury

描述(由申请人提供)：我认为在脓毒症期间，肾小管周围毛细血管灌注量减少会导致促氧化的肾小管周围微环境，这有利于活性氧(ROS)和活性氮(RNS)的产生。我的初步数据显示，在使用盲肠结扎和穿刺术(CLP)诱导脓毒症小鼠模型18小时后，使用自然产生的抗氧化剂多酚白藜芦醇的急性治疗可以防止管状RNS的生成和管周毛细血管灌注量的下降。此外，我还提供了白藜芦醇可以清除RNS的直接证据。基于这些发现，我推测白藜芦醇清除RNS的能力将保护小鼠肾小管免受CLP诱导的促氧化小管周围微环境的影响。目的1利用原代培养的小鼠肾小管上皮细胞，研究白藜芦醇对肾小管上皮细胞的保护作用。目的2研究白藜芦醇对CLP所致肾小管上皮细胞损伤的治疗作用。将绘制白藜芦醇在导致肾损伤的一段时间内的剂量-反应曲线，并分析其对血清细胞因子谱、功能性肾小管周围毛细血管密度、体积流量、管状氧化剂生成(每个都使用活体视频显微镜同时定量)和肾损伤(形态和血清尿素氮/肌酐)的影响。目的3将利用iNOS小鼠和过度表达超氧化物歧化酶(S0D1)的小鼠分别建立RNS作为CLP诱导的小鼠肾损伤的关键介质，以降低可用于RNS(ONOO)生成的NO和O2“的水平。这项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肾小管损伤的机制。这项研究建议将对我成为一名翻译型临床科学家的整体培训做出重大贡献。在美国，每年有超过21万人死于败血症相关的并发症。脓毒症患者中有20%-50%发生急性肾损伤，这些患者的死亡率极高(~75%)，因为目前的治疗大多是支持性的，大多无效。脓毒症所致肾损伤的新治疗方法发展的主要障碍是缺乏对脓毒症发生后可针对的危重事件的了解。这项建议研究了治疗脓毒症的新的治疗靶点，并有可能找到新的药物来降低这种毁灭性疾病的死亡率。

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.