陈东溟

内皮功能失调标志物Ang-1和Ang-2与危重病人急性肾损伤的相关研究

陈东溟

（沈阳急救中心沈河分中心，辽宁 沈阳 110000）

目的 探讨内皮功能失调标志物在危重病人合并急性肾损伤（AKI）中的作用。方法 对沈阳急救中心948例ICU患者的内皮功能标志物和炎症因子与AKI的相关性进行回顾研究，病人住院24小时内取血样，以血清肌酐定义AKI，在测定血管生成素1和2（angiopoietin，Ang-1，Ang-2）的同时也测定白介素6、17（IL-6、IL-17）和粒细胞集落刺激因子（G-CSF），以及各因子之间的相关性，共测量7天。结果 所有炎症因子和内皮功能失调标志物都与AKI相关，经过合并症与IL-6浓度的校正，每一个Ang-1浓度的标准差均与19%的AKI低风险相关（相对危险度（RR）=0.84，95%可信区间（CI）0.76～0.92，P＜0.01），而高浓度的Ang-2与AKI的高风险相关（RR=1.16，95%CI 1.12～1.21，P＜0.01）。结论 在危重病人中，Ang-1和Ang-2是除去炎症因子之外AKI的独立危险因素。

急性肾损伤；血管生成素；内皮功能失调；标志物；脓血症

急性肾损伤是（AKI）脓血症和全身炎症反应综合征（SIRS）的常见并发症，易增加危重患者的住院时间、透析需求和脓血症相关死亡率[1-7]。AKI是指48小时内肾功能突然衰退而至血肌酐升高，尿量减少，需要肾脏替代治疗或者以上均出现。然而，血肌酐作为早期AKI的指标，落后于肾小球滤过率[8-10]，以此很难预期患者的AKI风险和相关结局。因此对于评估严重AKI风险的化验检查方法，迫切需要新的敏感并且特异的生物标志物。

多年以来，中性粒细胞明胶酶蛋白（NGAL）被认为是最具希望的AKI标志物[11-13]，但是在危重病人范畴内的使用受到了限制，这是因为脓血症的炎症反应促使肝和肺中的NGAL合成增加，而与肾无关[10,14-16]。因此对于SIRS和多器官衰竭的病人，NGAL也许不是有效的AKI指标。

危重病人的AKI病因复杂，对其发病机制的深入研究可能发现新的生物标志物，先前认为，严重的全身性炎症提高了肿瘤坏死因子（TNF），白介素（IL）-6，粒细胞集落刺激因子（G-CSF）和其他炎症介质的表达，同时也对脓血症引起的器官功能障碍起了重要作用[17-20]。

然而，近来发现内皮细胞激活并导致血管屏障破坏成为器官功能障碍的关键病原机制[21-23]，此背景下的AKI的确切发病机制尚不清楚，有证据提示，微血管内皮细胞受损和功能失调对于缺血性AKI再灌注期的上皮细胞受损起到重要作用[9，24]。

血管生成素（Ang）是血管发育成熟和炎症反应的必要因子，Ang-1和拮抗剂Ang-2是分泌型内皮生长因子，与酪氨酸激酶受体（Tie2）的细胞外区域结合，后者主要在内皮细胞上表达，Ang-1具有稳定内皮的保护性，而Ang-2增加血管通透性，使脓血症结局恶化[25，28]。随着内皮激活，血管粘附分子上调，其中包括可溶性血管细胞粘附分子-1（sVCAM-1）[29]。内皮损伤和功能障碍的生物标志物与伴有全身性炎症的疾病的不良结局密切相关[30-32]，但其对AKI的临床意义有待进一步研究。

1 资料与方法

1.1 临床资料 纳入研究的患者年龄44～71岁，平均年龄（55.49±6.81）岁，其中36%为女性。在ICU住院24 h以上，同时具备SIRS诊断标准中的至少2项[33-34]。排除标准包括：主要脏器创伤，颅内出血，HIV，免疫抑制或者癌症。所有患者在进ICU后的第1天采血化验，随访至出院或死亡。本研究经过本中心伦理道德委员会批准。

1.2 方法 将所有血样稀释到合适范围，在同一天以电化学发光免疫测定法测试IL-6，IL-8，G-CSF，TNFR-1，Ang-1，Ang-2，和sVCAM-1，界定各个因子的范围如下，IL-6，IL-8和TNFR-1：0.08～2 500 pg/mL；G-CSF：0.12～5 000 pg/mL；Ang-1：3～100 000 pg/mL；Ang-2：0.5～10 000 pg/mL；sVCAM-1：0.05～1 000 pg/mL。超出范围的只取界值，部分测量值缺失归因为样品量不足（～5%）。

1.3 诊断标准 从入ICU至住院7 d，每天测量血肌酐，以KDIGO标准定义[35]AKI，即48 h内血肌酐升高0.3 mg/dL，或7 d内升高1.5倍。严重的AKI是指KDIGO的2或者3期。因为本研究不以AKI为主要终点，所以未测量每小时尿量与肾脏替代治疗的数据。

1.4 协变量 从住院的电子病历中调取病人的自然情况，诊疗过程和并发症等信息。急性生理学及慢性健康状况评分系统Ⅲ（Acute Physiology，Age，Chronic EvaluationⅢ，APACHEⅢ）和序贯器官衰竭估计评分（Sequential Organ Failure Assessment，SOFA）计算同上[36-37]。入院时血肌酐为基础血肌酐，慢性肾脏病（CKD）定义为CKD-EPI[38]公式计算的肾小球滤过率低于60 mL/min/1.73 m2。

1.5 统计学方法 以SPSS19.0统计分析数据，连续变量以“x±s”或中位数表示，组间比较用方差分析；分类变量用频数和百分数表示，组间比较用χ2检验或Fisher精确检验；生存分析用Kaplan-Meier生存分析曲线，Log-Rank检验；死亡危险因素分析用Cox回归分析；以P＜0.05为差异有统计学意义。

2 结果

2.1 根据KDIGO标准，506（53%）例出现AKI。正如预期的那样，患者出现越严重的AKI则具有越高的APACHE III评分和SOFA评分，也越有可能以内科原因入ICU治疗。见表1。

2.2 所有测得的血清标志物浓度与AKI各阶段相关（P＜0.0025）。较高阶段AKI病人具有更高中位数浓度的炎症标志物：IL-6，IL-8，IL-17，G-CSF和TNFR-1，同时具有更高中位数浓度的内皮标志物Ang-2，Ang-2/Ang-1和sVCAM-1，而Ang-1中位数浓度较低。见表2。

2.3 内皮因子与炎症因子之间及各自内部亦有中度相关，其中，最明显的是Ang-2和TNFR-1（P=0.60）以及Ang-2和IL-6（P=0.49）。

表1 AKI患者基本资料（x±s，n=948）Table 1 Baseline characteristics by AKIstage(x±s,n=948)

2.4 在经过人口特征、生活方式和合并症的校正之后，所有标志物的高表达与患者住院期间各个阶段的AKI风险相关。特别是标准差高表达的Ang-1伴随着预计降低19%的AKI校正风险（P＜0.01）而标准差高表达的Ang-2伴随着预计升高22%的AKI校正风险（P＜0.01）。见表3。

2.5 与之相类似的是，所有高浓度的内皮因子和炎症因子均与住院期间2～3阶段的AKI相关，而且内皮因子与住院期间2～3阶段的AKI相关性需要经过APACHEⅢ评分和循环IL-6的校正。

3 讨论

本研究报道了总体的ICU患者人群中内皮标志物与AKI的相关性，结果与先前的研究一致，即炎症标志物与多种危重疾病人群的AKI相关[40]。近来有几个相对小规模的研究探索了内皮标志物与AKI的相关性，其中，心外科病人的病例对照研究发现，在术后发展成为AKI的病人中，血清Ang-2水平比对照组有很大程度的增加[41-42]。在一个ICU危重病人初始肾脏替代治疗的横向研究中发现，循环Ang-2与AKI发展和28天的死亡风险有关[43]。

越来越多的证据提示全身炎症和内皮激活构成AKI的基础[41，43-46]。TNF是一种由活化的巨噬细胞、单核细胞和中性粒细胞释放的炎症标志物，已经显露出对脓血症和感染性AKI的重要作用[20，47-49]。肾脏内皮细胞被TNF激活，进而保持促炎症反应状态，并且潜在地使肾组织对后续的损害敏感[47-48]。对动物模型的研究成果论证了Ang-1可能提高了鼠科动物AKI时内皮增值细胞的保护能力[50-51]。急性的内皮细胞病变可能导致了血管反应性、通透性、白细胞粘附，凝血和微血管收缩自动调节的改变，从而使AKI持续进展。

表2 AKI各阶段的血清标志物浓度Table 2 Plasma biomarkers concentrations according to AKIstage

表3 血清标志物浓度与AKI各阶段相关Table 3 Associations of biomarkers with acute kidney injury in any stage

在这个大的ICU病人队列研究中，我们发现循环中的炎症和内皮标志物浓度与AKI的高风险显著相关。

这些生物标志物可能揭示了危重患者肾脏损伤的途径，同时也可能被用来预测个体的AKI风险。我们的研究结果支持如下的假说：在明显的肾脏细胞损害发生之前，微循环和组织氧化的变化使病人倾向于发生肾脏损害[52]。这个概念特别适用于炎症细胞和内皮细胞被明显激活的SIRS[16]。微血管系统和内皮细胞共同调节组织血流灌注、凝血、炎症和血管通透性。AKI严重损害了肾脏的内皮系统，导致了微血管功能失调，进行性的缺血和进一步的病变[53-54]。

虽然我们找到了在危重患者中内皮标志物与AKI的独立相关性，但是我们的研究仍有局限性。其中最重要的是，我们的观察研究的准确性有潜在地不确定因素，这是因为诸如疾病的严重性等特征很可能与内皮功能失调和AKI的风险都有关。虽然我们努力对潜在的混杂因素进行了校正，但是通过我们的研究仍然不能建立内皮功能失调和AKI发生发展之间的因果关系。第二，本研究不是把AKI作为主要终点，没有收集足够详细的尿量数据；AKI仅仅通过肌酐的改变进行回顾性评价[34]。没有获得肾脏替代治疗的数据，该数据原本可以提供附加的AKI严重性分级。此外，脓血症降低了肌酐的生成，也就限制了肌酐作为AKI标志物的作用[55]。一小部分病人没有足够的血样用来同时测量生物标志物。这些问题可能导致相关性的减弱。第三，我们未能排除反转因果关系的可能性，其中，在肌酐升高之前，受损的肾脏自身可能释放内皮标志物，从而参与了Ang-2的升高和Ang-1的降低[56]。最后，本研究的目标人群来自同一家医院，这可能使得研究结果有狭义。

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Relationship between markers ofendothelial dysregulation(Ang1,Ang2)and acute kidney injury in criticalpatients

Chen Dong-ming
(Shenhe Sub Centre,Shenyang Emergency Centre,Shenyang,Liaoning,110000,China)

Objective To search the role of endothelialdysregulation with acute kidney injury(AKI)in criticalpatients.Methods We retrospectively assessed the associations of AKIwith biomarkers of endothelialfunction and inflammation among 948 subjects admitted to the intensive care unit(ICU)atShenyang emergency centre.From plasma obtained within 24 h ofenrollment,we measured angiopoietin(Ang)-1 and Ang-2 alongside biomarkers of inflammation,including interleukin(IL)-6,IL-17 and granulocyte colony-stimulating factor.We tested for associations between standardized concentrations of biomarkers and AKI,defined by serum creatinine,from ICU admission to up to 7 days later.Results Allbiomarkers of inflammation and endothelialdysfunction were associated with AKI.After adjustmentfor comorbidities,and IL-6 concentration,every standard deviation of Ang-1 concentration was associated with a 19%lower risk of AKI(relative risk(RR)=0.84,95%confidence interval(CI)0.76-0.92,P＜0.01).Conversely,higher Ang-2 concentration was associated with higher risk of AKI(RR per standard deviation=1.16,95%CI 1.12-1.21,P＜0.01).Conclusion In critically patients,plasma concentration of the Ang-1 and Ang-2 are associated with AKI,independently of inflammation.

Acute kidney injury;Angiopoietin;Endothelialdysfunction;Marker;Sepsis

10.3969/j.issn.1009-4393.2017.22.024