Pathophysiology of sepsis-induced renal injury

描述（由申请人提供）：尽管在该领域进行了数十年的深入研究，但革兰氏阴性脓毒症仍然是一个令人难以接受的高死亡率的可怕临床问题。脓毒症期间经常出现的多器官衰竭往往会带来迄今为止难以改变的严峻后果。脓毒症中急性肾损伤（AKI）的病理生理被认为是由免疫系统细胞上toll样受体（TLRs）刺激引发的炎症失控和促凝状态引起的。然而，干预全身炎症细胞因子、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收效甚微。本研究旨在探讨革兰氏阴性脓毒症中ARF和AKI的病理生理机制。该假设提出，全身性革兰氏阴性脓毒症的AKI部分是由于内毒素和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性败血症模型中小管-内毒素相互作用的大小和分布。利用活体动物的双光子显微镜，以及新型荧光探针，我们将测量由内毒素直接摄取引起的小管氧化应激和细胞凋亡。为了研究内毒素-小管相互作用在缺乏全身性细胞因子的情况下的病理生理学，我们将产生缺乏全身性TLR4的嵌合体小鼠。最后，我们将通过蛋白表达、共定位和抑制研究来探讨p53在介导细胞凋亡和氧化应激中的作用。在特定目标2中，我们将通过靶向活传递受体特异性siRNA的强大工具，确定TLR4、CD14和受体独立的液相内吞作用在小管内毒素摄取中的作用。TLR4或CD14的急性敲低对内毒素摄取、氧化应激和细胞凋亡的影响将通过活光子成像研究和传统的离体技术来确定。缺乏肾TLR4或CD14的嵌合体小鼠将提供一种额外的方法来研究这些分子在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、迄今尚未探索的败血症性肾损伤机制。通过揭示这样一个新的机制，我们的研究可以为未来的治疗提供基础，旨在干扰内毒素对肾细胞的直接有害影响。

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.