Department of Surgery, The Methodist Hospital and Research Institute, Houston, TX 77030, USA.
Shock. 2012 Aug;38(3):320-7. doi: 10.1097/SHK.0b013e31826359d0.
Kidney ischemia-reperfusion injury (IRI) activates cellular and soluble mediators that drive lung inflammatory cascades, tumor necrosis factor receptor 1 (TNFR1)-mediated programmed cell death, and microvascular barrier dysfunction, leading to acute lung injury. We hypothesized that lung microvascular endothelial cells (ECs), with their integral role in maintaining the lung-semipermeable barrier, were key cellular targets of TNFR1-mediated apoptosis during ischemic AKI. Male C57/BL6 mice and Sprague-Dawley rats underwent 60 min of bilateral renal pedicle occlusion (IRI) or sham laparotomy (sham) and were killed at 4 or 24 h. Colocalization with TUNEL, DAPI, and CD34 was performed to identify EC-specific apoptosis. Mouse ECs (CD45/CD31) isolated with novel tissue digestion techniques and magnetic microbead sorting underwent quantitative real-time polymerase chain reaction SuperArray analysis with 84 apoptosis-related genes. In parallel, rat lung microvascular ECs grown to confluence were treated with serum from rats obtained following sham or kidney IRI. Rat lung microvascular ECs treated +/- etanercept, a TNF-α/TNFR1 signaling inhibitor, underwent custom real-time polymerase chain reaction analysis for proapoptotic and TNF superfamily transcriptional events, and apoptosis was identified with caspase 3 and poly(ADP-ribose) polymerase activity assays. In vivo, TUNEL-positive cells colocalized with CD34 in whole-lung tissue and isolated lung ECs demonstrated a proapoptotic transcriptome during ischemic AKI. In vitro, ischemic AKI incited proapoptotic (FasL, Dapk1, Bcl10) and TNF superfamily (TNFR1, TNFR2, TNF-α) gene activation and increased caspase 3 and poly(ADP-ribose) polymerase activity at 24 h versus sham. Compared with vehicle, treatment of rat lung microvascular ECs with etanercept inhibited proinflammatory gene activation (E-selectin, intercellular adhesion molecule 1, interleukin 6, RhoB) and apoptosis during ischemic AKI. Ischemic AKI drives distinct proinflammatory and proapoptotic changes in the pulmonary EC transcriptome with TNFR1-dependent caspase activation and programmed cell death. Further investigation of potential EC mechanisms of kidney-lung crosstalk during AKI may identify potential therapeutic targets for this deadly disease.
肾缺血再灌注损伤 (IRI) 激活细胞和可溶性介质，驱动肺炎症级联反应、肿瘤坏死因子受体 1 (TNFR1) 介导的程序性细胞死亡和微血管屏障功能障碍，导致急性肺损伤。我们假设，肺微血管内皮细胞 (EC) 在维持肺半通透性屏障方面具有重要作用，是缺血性 AKI 期间 TNFR1 介导的细胞凋亡的关键细胞靶点。雄性 C57/BL6 小鼠和 Sprague-Dawley 大鼠接受双侧肾蒂夹闭 (IRI) 或假手术 (sham) 60 分钟，然后在 4 或 24 小时处死。通过 TUNEL、DAPI 和 CD34 的共定位来鉴定 EC 特异性凋亡。使用新型组织消化技术和磁性微珠分选分离小鼠 EC（CD45/CD31），进行 84 个与凋亡相关基因的定量实时聚合酶链反应 SuperArray 分析。同时，将培养至汇合的大鼠肺微血管内皮细胞用 sham 或肾 IRI 后大鼠血清处理。用 TNF-α/TNFR1 信号抑制剂 etanercept 处理的大鼠肺微血管内皮细胞进行定制实时聚合酶链反应分析，检测促凋亡和 TNF 超家族转录事件，并通过 caspase 3 和聚（ADP-核糖）聚合酶活性测定鉴定凋亡。在体内，TUNEL 阳性细胞与全肺组织中的 CD34 共定位，分离的肺 EC 在缺血性 AKI 期间表现出促凋亡的转录组。在体外，与 sham 相比，缺血性 AKI 可引发促凋亡（FasL、Dapk1、Bcl10）和 TNF 超家族（TNFR1、TNFR2、TNF-α）基因激活，并在 24 小时增加 caspase 3 和聚（ADP-核糖）聚合酶活性。与载体相比，用 etanercept 处理大鼠肺微血管内皮细胞可抑制缺血性 AKI 期间的促炎基因激活（E-选择素、细胞间黏附分子 1、白细胞介素 6、RhoB）和凋亡。缺血性 AKI 导致肺 EC 转录组中出现独特的促炎和促凋亡变化，与 TNFR1 依赖性半胱天冬酶激活和程序性细胞死亡有关。进一步研究 AKI 期间肾脏-肺部相互作用中潜在的 EC 机制可能为这种致命疾病确定潜在的治疗靶点。
