In Vitro Model of Sepsis-Induced Renal Epithelial Reactive Nitrogen Species Generation
In Vitro Model of Sepsis-Induced Renal Epithelial Reactive Nitrogen Species Generation
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DOI：
10.1093/toxsci/kfq058
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发表时间：
2010-06-01
影响因子：
3.8
通讯作者：
Mayeux, Philip R.
中科院分区：
文献类型：
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作者：
Pathak, Elina;Mayeux, Philip R.
脓毒症诱导的急性肾损伤（阿基）是一种复杂的疾病，其特征在于肾小管上皮细胞产生诱导型一氧化氮合酶（iNOS）衍生的活性氮（RNS）。虽然大多数体外脓毒症模型使用脂多糖和细胞因子的组合来模拟暴露于被认为在脓毒症中起作用的炎症介质，但这些模型的相关性有限。为了解决对更接近地模拟脓毒症期间的肾小管微环境的模型的需求，我们开发了一种体外模型，其中在盲肠结扎和穿刺（CLP）或假手术（无脓毒症）后4小时用含有从小鼠收集的5%血清的培养基处理mIMCD-3（鼠肾小管上皮）细胞。暴露于CLP血清后，诱导的iNOS信使RNA发生和NO生成显着增加相比，假手术。这种增加伴随着RNS增加，如通过5-（和-6）-羧基-2，7 '-二氯二氢荧光素二乙酸酯（羧基-H2 DCF-DA）和2-（3，6-二氨基-9H-咕吨-9-基）-苯甲酸甲酯（二氢罗丹明123）的氧化所测量的，以及在用CLP血清处理的细胞中的中度细胞毒性，与在经受CLP的小鼠中观察到的相似。由于iNOS已被证明在脓毒症诱导的阿基中起重要作用，因此在该体外模型中测试了iNOS抑制剂L-N-6-（1-亚氨基乙基）-赖氨酸（L-NIL）。L-NIL完全阻断NO生成，RNS生成和细胞毒性，类似于其在体内的作用。因此，这种新的体外模型表现出许多在体内观察到的特征，这表明它是一个相关的模型，用于研究脓毒症诱导的肾上皮RNS的产生和损伤的机制。
Sepsis-induced acute kidney injury (AKI) is a complex disease characterized by generation of inducible nitric oxide synthase (iNOS)-derived reactive nitrogen species (RNS) by the renal tubular epithelium. While most in vitro models of sepsis use combinations of lipopolysaccharide and cytokines to simulate exposure to inflammatory mediators thought to play a role in sepsis, the relevance of these models is limited. To address the need for a model that more closely mimics the tubular microenvironment during sepsis, we developed an in vitro model where mIMCD-3 (murine tubular epithelial) cells are treated with media containing 5% serum collected from mice at 4 h after cecal ligation and puncture (CLP) or sham surgery (no sepsis). After exposure to CLP serum, induction of iNOS messenger RNA occurred and NO generation was significantly increased compared to sham. This increase was accompanied by increased RNS as measured by oxidation of 5-(and-6)-carboxy-2,7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCF-DA) and 2-(3,6-diamino-9H-xanthen-9-yl)-benzoic acid, methyl ester (dihydrorhodamine 123) and moderate cytotoxicity in cells treated with CLP serum, similar to what is observed in mice subjected to CLP. Since iNOS has been shown to play an important role in sepsis-induced AKI, the iNOS inhibitor L-N-6-(1-iminoethyl)-lysine (L-NIL) was tested in this in vitro model. L-NIL completely blocked NO generation, RNS generation, and cytotoxicity, similar to its effects in vivo. Therefore, this new in vitro model exhibits many of the characteristics observed in vivo, suggesting that it is a relevant model for studying the mechanism of sepsis-induced renal epithelial RNS generation and injury.