2020, Vol. 49文章信息
- 郑悦, 郭金杰, 黄晓金, 侯玲, 杜悦
- ZHENG Yue, GUO Jinjie, HUANG Xiaojin, HOU Ling, DU Yue
- KIM-1和NGAL对儿童紫癜性肾炎后急性肾损伤的诊断价值
- Significance of kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin in the diagnosis of children with acute kidney injury caused by Henoch-Schönlein purpura nephritis
- 中国医科大学学报, 2020, 49(2): 156-160
- Journal of China Medical University, 2020, 49(2): 156-160
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文章历史
- 收稿日期：2019-04-01
- 网络出版时间：2019-12-23 11:51
引用本文 |
2. 辽宁省健康产业集团抚矿总医院儿科, 辽宁 抚顺 113000;
3. 大连医科大学附属第二医院儿科, 辽宁 大连 116000
2. Department of Pediatrics, Liaoning Health Industry Group General Hospital of Fushun Mining Bureau, Fushun 113000, China;
3. Department of Pediatric, The Second Hospital of Dalian Medical University, Dalian 116000, China
紫癜性肾炎(Henoch-Schönlein purpura nephritis, HSPN)是过敏性紫癜引起的肾脏损害, 是儿童常见的继发性肾小球疾病之一。HSPN后急性肾损伤(acute kidney injury on HSPN, A-on-HSPN)是儿童急性肾损伤(acute kiney injury, AKI)的重要原因之一, 严重威胁患儿生命。因此, A-on-HSPN的早期诊断至关重要。血清肌酐、尿素氮等作为传统肾脏损伤指标, 易受年龄、性别、肌容积等因素的影响, 敏感度低, 具有滞后性, 当它们出现显著变化时往往错过了最佳的治疗时机, 因此, 发现更敏感、更特异的指标对A-on-HSPN的早期诊治、降低病死率具有重要意义。近年来, 越来越多的学者开始关注肾损伤分子-1(kidney injury molecule-1, KIM-1)和中性粒细胞明胶酶相关脂质运载蛋白(neutrophil gelatinase-associated lipocalin, NGAL)。既往研究[1-5]表明, KIM-1及NGAL在脓毒症相关、药源性、体外循环心脏术后及心脏骤停后的AKI具有早期诊断的意义。但是, 关于A-on-HSPN中KIM-1、NGAL的研究较少。本研究拟分析血、尿及肾脏组织KIM-1、NGAL水平及尿KIM-1、NGAL与肾小球滤过率(glomerular filtration rate, GFR)和尿蛋白定量的相关性, 探讨其对A-on-HSPN早期诊断的价值, 以期获得A-on-HSPN的早期诊断生物标志物。
1 材料与方法 1.1 研究对象本研究为单中心前瞻性临床对照研究, 选择2018年1月至2018年10月在中国医科大学附属盛京医院小儿肾脏风湿免疫科收治的14岁以下HSPN患儿25例作为研究对象。其中, HSPN肾病综合征型患儿(HSPN组) 16例, 男7例, 女9例, 平均年龄(10.22±2.91)岁; A-on-HSPN患儿(A-on-HSPN组) 9例, 男3例, 女6例, 平均年龄(7.56±1.42)岁。
所有研究对象符合2009年中华医学会儿科学分会肾脏学组制定的《儿童常见肾脏疾病诊治循证指南(二) :紫癜性肾炎的诊治循证指南(试行)》 [6]HSPN的诊断标准。HSPN组24 h尿蛋白定量>50 mg/ (kg·24 h), 血清白蛋白≤25 g/L; A-on-HSPN组AKI诊断符合2012年改善全球肾脏病预后组织的诊断标准[7]。所有患儿均无慢性肾脏病、其他自身免疫性疾病、肿瘤性疾病、血液系统疾病, 且近期无泌尿系统感染、脓毒症病史。所有患儿的监护人均签署了自愿参加本研究的知情同意书。
1.2 研究方法收集患儿清晨6:00的静脉血和清晨6:00至7:00的尿液各5 mL, 3 000 r/min离心15 min, 取上清1 mL, -80 ℃冻存。采用酶联免疫吸附法(试剂盒购自美国Santa Cruz Biotechnology公司)检测血、尿中NGAL、KIM-1含量。
在B超引导下行经皮肾穿刺活检术取合格肾组织, 采用免疫组化法检测患儿肾小管中NGAL、KIM-1的表达(试剂盒购自美国R & D公司)。
1.3 统计学分析采用SPSS 17.0软件进行统计分析。计量资料以x±s表示, 组间比较采用t检验。计数资料采用χ2检验比较。P < 0.05为差异有统计学意义。采用Pearson相关分析对KIM-1、NGAL与GRF、24 h尿蛋白定量的相关性进行分析。
2 结果 2.1 临床资料HSPN组和A-on-HSPN组分别有10例和6例患儿行肾活检并做免疫组化染色。2组性别、年龄、体质量、尿蛋白水平均无统计学差异(P > 0.05)。而A-on-HSPN组血CystatinC、血清肌酐(serum creatinine, SCr)以及血、尿β2微球蛋白(β2 microglobulin, β2-MG)、NGAL、KIM-1水平均显著高于HSPN组, 差异有统计学意义(P < 0.05)。A-on-HSPN组少数患儿SCr正常, 但血和尿NGAL、KIM-1水平均明显升高。见表 1。
| Item | HSPN group (n = 16) | A-on-HSPN group (n = 9) | t | P |
| Gender (male/female) | 7/9 | 3/6 | - | 0.691 |
| Age (year) | 10.22±2.91 | 7.56±1.42 | 2.286 | 0.052 |
| Body weight (kg) | 34.62±13.52 | 28.72±9.28 | 1.160 | 0.258 |
| CysC (mg/L) | 0.83±0.16 | 2.53±0.86 | 6.076 | < 0.001 |
| SCr (μmol/L) | 52.22±10.83 | 91.22±16.34 | 7.020 | < 0.001 |
| Urine protein (g/d) | 2.83±0.91 | 3.18±1.50 | 0.563 | 0.589 |
| GFR (mL·min-1·1.73 m-2) | 130.53±5.46 | 68.60±11.79 | 17.297 | < 0.001 |
| Urine β2-MG (mg·L-1) | 0.61±0.31 | 14.01±18.92 | 2.880 | 0.008 |
| Serum β2-MG (mg·L-1) | 1.57±0.37 | 5.79±3.22 | 3.903 | 0.005 |
| Urine KIM-1 (pg·mL-1) | 16.69±8.94 | 329.20±157.36 | 5.974 | < 0.001 |
| Serum KIM-1 (pg·mL-1) | 8.96±0.92 | 147.91±68.15 | 6.161 | < 0.001 |
| Urine NGAL (ng·mL-1) | 422.89±45.60 | 1 627.62±470.39 | 8.094 | < 0.001 |
| Serum NGAL (ng·mL-1) | 219.95±44.80 | 312.82±33.17 | 8.445 | < 0.001 |
| CysC, cystatinC; SCr, serum creatinine; GFR, glomerular filtration rate; β2-MG, β2 microglobulin; KIM-1, kidney injury molecule-1;NGAL, neutrophil gelatinase-associated lipocalin. | ||||
2.2 NGAL和KIM-1在肾小管中的表达
肾组织活检KIM-1、NGAL免疫组化结果显示, HSPN组未见棕黄色阳性颗粒, 染色浅, A-on-HSPN组棕黄色阳性颗粒多, 染色深, 且主要位于肾小管上皮细胞, A-on-HSPN组KIM-1、NGAL表达水平明显高于HSPN组, 差异有统计学意义(P < 0.05)。见图 1和表 2。
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| A, NGAL expression in A-on-HSPN group; B, NGAL expression in HSPN group; C, KIM-1 expression in A-on-HSPN group; D, KIM-1 expression in HSPN group. 图 1 NGAL及KIM-1在肾组织的表达免疫组织化学染色×200 Fig.1 Expression of NGAL and KIM-1 in renal tissue Immunohistochemical staining×200 |
| Parameter | A-on-HSPN group (n = 6) | HSPN patients (n = 10) | t | P |
| KIM-1 | 0.64±0.14 | 0.16±0.32 | 2.957 | 0.032 |
| NGAL | 0.47±0.11 | 0.12±0.23 | 2.903 | 0.034 |
2.3 尿KIM-1、NGAL含量与GFR及24 h尿蛋白定量的相关性分析
Pearson相关分析结果显示, 本研究所有患儿尿KIM-1、NGAL含量均与GFR呈负相关, 而与24 h尿蛋白定量无明显相关。见图 2。
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| 图 2 尿KIM-1、NGAL与GFR及24 h尿蛋白定量的相关性 Fig.2 Correlation between urinary KIM-1, NGAL and GFR, 24 h urine protein |
3 讨论
A-on-HSPN是临床常见的危重症之一, 目前A-on-HSPN的诊断主要依靠检测尿量、尿肌酐、尿素氮等, 但是, 当尿肌酐、尿素氮水平明显升高时, 患者的肾功能损伤往往已较为严重。因此, 临床上需要能早期发现肾功能损伤的更敏感的生物标志物, 以便早期诊断、治疗肾功能损伤, 从而降低病死率, 改善预后。
KIM-1是一种分子量为38.7×103的Ⅰ型跨膜糖蛋白[8], 在正常的肾脏组织中表达量很低, 但当发生缺血性或药物性肾损伤时, KIM-1在肾小管上皮细胞内的表达量急剧上升[9-11]。啮齿类动物和人类KIM-1的表达上调主要发生在近端小管, 特别是S3段[12-13]。研究[11, 14]表明, KIM-1同时参与肾脏的损伤和修复过程, 是肾小管增殖和再生的重要标志物。发生肾脏损伤时, KIM-1的细胞外区域在基质金属蛋白酶的作用下可裂解成可溶片段, 排入尿中, 并在体外中性环境中稳定存在, 因此, 通过检测尿中KIM-1水平可以间接评价肾损伤的情况[15]。
NGAL是一种分子量为25×103的脂质运载蛋白。NGAL在成人子宫、骨髓、胃、前列腺、气管、结肠和正常肾脏远端小管上皮、髓质集合管中均有稳定的低水平表达[16]。尽管NGAL在人体多种器官中有稳定的表达, 但正常情况下, 肾小球滤过后, 由于大部分NGAL通过近端小管被重吸收, 尿中浓度较低, 因此, 在正常情况下, 尿液中不能被检出[17]。NGAL在免疫防御[18-21]、肿瘤发生发展[22]的过程中均起重要作用。同时, NGAL还可通过减轻肾小管上皮细胞凋亡、促进细胞增殖、上皮形成、诱导受损细胞再生等机制对肾脏发挥保护作用[23-24]。当脓毒症、药物中毒、急性缺血再灌注损伤导致AKI时, 血、尿中NGAL水平明显上升[25]。其机制为发生AKI时, 肺、肝脏等远处器官和免疫细胞NGAL表达上调, 血清NGAL合成增加, 从而导致血浆中NGAL水平升高[26-27]。同时, 肾小球滤过膜通透性增加, 引起NGAL漏出增加, 近端小管对NGAL重吸收作用减弱, 最终导致尿NGAL水平升高。
本研究通过检测A-on-HSPN组和HSPN组患儿血、尿KIM-1及NGAL水平发现, A-on-HSPN组的血、尿中KIM-1、NGAL水平明显高于HSPN组, A-on-HSPN组患儿血及尿中KIM-1、NGAL的改变可能早于SCr, 少数患儿SCr正常时血、尿KIM-1及NGAL已高于正常, 说明血及尿中KIM-1、NGAL的敏感性明显优于SCr, 可以在SCr升高之前预测AKI的发生。
本研究中, 10例HSPN组及6例A-on-HSPN组患儿做了肾活检, 通过免疫组化方法检测肾组织内NGAL、KIM-1的表达情况, 结果发现, AKI-on-HSPN组较HSPN组棕黄色阳性颗粒表达多, 染色深, 即KIM-1和NGAL表达水平明显高于HSPN组。提示肾组织NGAL及KIM-1检测在儿童A-on-HSPN的诊断中具有一定的价值。
本研究中, 尿KIM-1、NGAL与GFR、24 h尿蛋白定量的相关分析结果显示, 尿KIM、NGAL水平均与GFR水平呈负相关, 而与24 h尿蛋白定量无明显相关。由此推测, HSPN患儿只有在发生肾损伤时, 尿KIM、NGAL才会明显升高, 而即使大量蛋白尿时, KIM、NGAL也不一定明显上升。再次证明KIM、NGAL是A-on-HSPN的有价值的生物标志物。
本研究存在的不足之处如下:纳入患者均来自同一家医院, 样本量较少, 未对肾脏病理和AKI进行分期; 未留取不同时段的血、尿标本, 以进一步观察血、尿KIM-1及NGAL在整个病程中的动态变化。
综上所述, KIM-1和NGAL在A-on-HSPN的诊断中具有重要作用。血、尿标本留取简单, 且不受年龄、性别、肌肉容量、肾前因素等的影响, 是一种快速、敏感、且较特异的A-on-HSPN早期诊断的生物学标志物[28]。
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