Apoptotic Death of Renal Tubular Cells in Experimental Sepsis
Apoptotic Death of Renal Tubular Cells in Experimental Sepsis
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DOI：
10.1089/sur.2006.018
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发表时间：
2008-06-01
影响因子：
2
通讯作者：
Bramis, John
中科院分区：
文献类型：
--
作者：
Messaris, Evangelos;Memos, Nikolaos;Bramis, John
背景和目的：脓毒症引起的肾功能障碍长期以来被认为是血流动力学不稳定和随后的局部缺血的结果。最近的数据表明细胞凋亡也可能与此有关。本研究的目的是评估脓毒症大鼠肾实质细胞凋亡和bax、bcl-2、caspase-8和细胞色素c蛋白表达的作用。方法：采用盲肠结扎穿刺（CLP）法诱导62只雄性Wistar大鼠脓毒症，并在6、12、24、36、48或60小时后安乐死。十只经过假处理的动物作为对照组。另一组 50 只动物接受 CLP，然后监督 60 小时。使用光学和透射电子显微镜、原位缺口末端标记 (TUNEL) 和使用 7-氨基放线菌素 D (7-AAD) 的流式细胞术评估肾细胞凋亡。使用 M30 抗体评估 Caspase 介导的细胞凋亡。免疫组织化学检测凋亡调节蛋白 B 细胞淋巴瘤 2 (bcl-2)、bcl-2 相关 x 蛋白 (bax)、caspase-8 和细胞色素 c 的表达。 结果：脓毒症增加肾实质炎症浸润 (p < 0.001) 和坏死 (p < 0.001)。细胞凋亡明显比对照动物的肾脏更常见（p = 0.02）。 TUNEL 技术染色的细胞核主要存在于非幸存者的肾小管细胞中 (p = 0.05)。脓毒症诱导后6 h，各类细胞死亡的时间分布显着增加，随后下降。脓毒症动物肾脏中 Caspase 生成的细胞角蛋白 18 (CK18) 新表位 (M30) 明显比对照大鼠更丰富，峰值出现在术后 6 小时和 60 小时 (p < 0.001)。此外，CLP 后 6 小时启动凋亡的细胞明显比 48 小时更常见 (p = 0.014)。 Caspase-8 蛋白免疫检测遵循与细胞死亡相同的时间模式，最早在 CLP 后 6 小时增加，此后减少 (p = 0.013)。 Bax 蛋白表达在脓毒症早期显着升高 (p = 0.037)，而线粒体依赖性途径的其他成员保持不变。死于脓毒症的动物中，bax- (p = 0.037) 和 caspase-8- (p = 0.031) 免疫反应性肾细胞的患病率显着更高。结论：脓毒症动物的肾组织细胞凋亡明显高于对照组。细胞死亡标记物的时间分布显示出一致的模式，使得早期脓毒症可能是细胞凋亡事件的引发者。
Background and Purpose: Renal dysfunction attributable to sepsis was long considered a result of hemodynamic instability and subsequent local ischemia. Recent data show that apoptosis may be implicated also. The purpose of this study was to evaluate the role of apoptosis and the expression of the bax, bcl-2, caspase-8, and cytochrome c proteins in the renal parenchymal cells of rats with sepsis.Methods: Sepsis was induced using cecal ligation and puncture (CLP) in 62 male Wistar rats, which were euthanized 6, 12, 24, 36, 48, or 60 h later. Ten sham-treated animals served as a control group. Another group of 50 animals were subjected to CLP and then supervised for 60 h. Renal apoptosis was evaluated using light and transmission electron microscopy, in situ nick-end labeling (TUNEL), and flow cytometry using 7-amino-actinomycin D (7-AAD). Caspase-mediated apoptosis was assessed using M30 antibody. The expression of the apoptosis-regulator proteins B-cell lymphoma 2 (bcl-2), bcl-2-associated x protein (bax), caspase-8, and cytochrome c was detected immunohistochemically.Results: Sepsis increased inflammatory infiltration (p < 0.001) and necrosis (p < 0.001) in renal parenchyma. Apoptosis was significantly more common than in the kidneys of control animals (p = 0.02). Nuclei stained by the TUNEL technique were predominant in the tubular cells of non-survivors (p = 0.05). The time distribution of all types of cell death was increased significantly 6 h after the induction of sepsis, and declined subsequently. Caspase-generated cytokeratin 18 (CK18) new epitope (M30) was significantly more abundant in the kidneys of animals with sepsis than in control rats, with peaks at 6 h and 60 h post-procedure (p < 0.001). In addition, cells initiating apoptosis were significantly more common at 6 h than at 48 h post-CLP (p = 0.014). Caspase-8 protein immunodetection followed the same time pattern as cell death, increasing as early as 6 h post-CLP and decreasing thereafter (p = 0.013). Bax protein expression was elevated significantly early in the course of sepsis (p = 0.037), whereas the other members of the mitochondrial-dependent pathway remained constant. Animals dying from sepsis had a significantly greater prevalence of bax- (p = 0.037) and caspase-8- (p = 0.031) immunoreactive renal cells.Conclusion: Apoptosis in renal tissue was significantly more common in animals with sepsis than in controls. The time distribution of cell death markers showed a consistent pattern, making early sepsis the likely initiator of the apoptotic events.