Pathophysiology of sepsis-induced renal injury

描述(由申请人提供)：革兰氏阴性败血症仍然是一个可怕的临床问题，尽管在该领域进行了数十年的密集研究，但死亡率仍高得令人无法接受。脓毒症期间经常出现的多器官衰竭往往会带来严重的后果，到目前为止还很难改变。脓毒症急性肾损伤(AKI)的病理生理机制被认为是由于免疫系统细胞上Toll样受体(TLRs)的刺激引起的失控炎症和促凝状态所致。然而，全身炎症细胞因子的干预、抗凝治疗和强有力的血流动力学支持在预防AKI和提高总死亡率方面都收效甚微。本研究旨在探讨革兰氏阴性脓毒症ARF和AKI的病理生理学新假说。该假说认为，系统性革兰氏阴性脓毒症的AKI部分原因是内毒素与肾小管细胞之间的直接相互作用。在具体目标1中，我们将测量小鼠和大鼠全身脓毒症模型中小管-内毒素相互作用的大小和分布。在活体动物中使用双光子显微镜，结合新的荧光探针，我们将测量直接摄取肾小管内毒素所导致的肾小管氧化应激和细胞凋亡。为了在缺乏系统性细胞因子的情况下检测内毒素-小管相互作用的病理生理学，我们将产生缺乏系统性TLR4的嵌合体小鼠。最后，将通过蛋白表达、共定位和抑制研究来研究P53在介导细胞凋亡和氧化应激中的作用。在特定的目标2中，我们将通过靶向实时递送受体特异性siRNA的强大工具来确定TLR4、CD14和受体非依赖性的液相内吞作用在肾小管内毒素摄取中的作用。急性TLR4或CD14基因敲除对内毒素摄取、氧化应激和细胞凋亡的影响将通过实时双光子成像研究和传统的体外技术来确定。缺乏肾脏TLR4或CD14的嵌合体小鼠将提供另一种方法来研究这些分子在肾小管内毒素摄取中的作用。这些特定的目标将决定内毒素与肾小管细胞相互作用的大小、影响和机制。我们相信，他们将建立一种新的、迄今尚未探索的脓毒症所致肾损伤的机制。通过揭示这样一种新的机制，我们的研究可以为未来旨在干扰内毒素对肾细胞的直接有害影响的治疗奠定基础。

DESCRIPTION (provided by applicant): Gram negative sepsis remains a formidable clinical problem with an unacceptably elevated mortality despite decades of intensive research in the field. The multi organ failure that frequently sets in during sepsis often ushers a grim outcome that so far has been difficult to alter. The pathophysiology of acute kidney injury (AKI) in sepsis is thought to result from uncontrolled inflammation and a procoagulant state triggered by stimulation of Toll-like receptors (TLRs) on cells of the immune system. Nevertheless, interference with systemic inflammatory cytokines, anticoagulation therapy and vigorous hemodynamic support all met with little success in preventing AKI and improving overall mortality. It is the purpose of this proposal to explore a novel hypothesis regarding the pathophysiology of ARF and AKI in gram negative sepsis. The hypothesis proposes that AKI in systemic gram negative sepsis results in part from a direct interaction between endotoxin and renal tubular cells. In specific aim 1, we will measure the magnitude and distribution of tubule- endotoxin interactions in mice and rat models of systemic sepsis. Using 2 photon microscopy in live animals, along with novel fluorescent probes, we will measure tubular oxidative stress and apoptosis that result from direct tubular endotoxin uptake. To examine the pathophysiology of endotoxin-tubule interactions in the absence of systemic cytokines, we will generate chimera mice lacking systemic TLR4. Finally, the role of p53 in mediating apoptosis and oxidative stress will be examined through protein expression, co localization and inhibitory studies. In specific aim 2, we will determine the roles of TLR4, CD14 and receptor-independent fluid-phase endocytosis in tubular endotoxin uptake with the powerful tool of targeted live delivery of receptor-specific siRNA. The impact of acute TLR4 or CD14 knockdown on endotoxin uptake, oxidative stress and apoptosis will be determined with live 2 photon imaging studies as well as traditional ex vivo techniques. Chimera mice lacking renal TLR4 or CD14 will provide an additional approach to examine the role of these molecules in tubular endotoxin uptake. These specific aims will determine the magnitude, impact and mechanism of the interaction between endotoxin and tubular cells. We believe they will establish a novel and so far unexplored mechanism underlying sepsis-induced renal injury. By exposing such a new mechanism, our studies could be at the basis of future therapies that aim at interfering with the direct detrimental effects of endotoxin on renal cells.