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1 ORIGINAL ARTICLE Korean J Clin Lab Sci. 2016, 48(2): pissn eissn Korean J Clin Lab Sci. Vol. 48, No. 2, June Usefulness of the Neutrophil Gelatinase-Associated Lipocalin (NGAL) Kit for Acute Kidney Injury Patients at the Emergency Medical Center in Daegu Seung-Jin Lee 1 and Sangwook Park 2 1 Department of Laboratory Medicine, Daegu Catholic University Medical Center, Daegu 42472, Korea 2 Department of Biomedical Laboratory Science, College of Health, Kyungwoon University, Gumi 39160, Korea 대구지역응급의료센터에내원한급성콩팥손상환자의진단을위한호중구젤라티나제관련리포칼린키트의유용성 이승진 1, 박상욱 2 1 대구가톨릭대학교병원진단검사의학과, 2 경운대학교보건대학임상병리학과 Acute kidney injury (AKI) is a common syndrome resulting in kidney damage and malfunction within a few days or even a few hours. The diagnosis of AKI depends on routine biochemical tests, including serum creatinine, aspartate aminotransferase (AST), alanine aminotransaminase (ALT), blood urea nitrogen (BUN), and electrolytes. Plasma neutrophil gelatinase-associated lipocalin (NGAL) is a biomarker that shows correlation with the severity of acute infections and kidney injuries. The predictive value in other conventional assays for kidney functions has been reported to cause distraction for AKI syndrome. The aim of this study is to verify the predictive value of plasma NGAL in patients with established AKI. The NGAL kit for checkup demonstrates sensitivity of 300 (92.2%), 200 (95.6%), 100 (99.6%), specificity of 300 (95.1%), 200 (97.3%), 100 (99.4%), positive predictability of 300 (93.3%), 200 (93.4%), 100 (99.2%), and negative predictability of 300 (96.7%), 200 (97.7%), 100 (98.1%), respectively. The plasma NGAL compared with the enzyme-linked immunosorbent assay (ELISA) has been shown to be an early predictive biomarker of AKI. The NGAL kit, recently developed for point-of-care of plasma specimens, is thought to be a useful and reliable biomarker for the early diagnosis of decreased kidney functions. Keywords: Neutrophil gelatinase-associated lipocalin (NGAL), Acute kidney injury (AKI), Biomarker, Point of care testing Corresponding author: Sangwook Park Department of Biomedical Laboratory Science, College of Health, Kyungwoon University, 730, Gangdong-ro, Sandong-myeon, Gumi 39160, Korea Tel: Fax: spark367@ikw.ac.kr This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright 2016 The Korean Society for Clinical Laboratory Science. All rights reserved. Received: February 6, 2016 Revised 1 st : February 13, 2016 Revised 2 nd : March 1, 2016 Revised 3 rd : March 15, 2016 Revised 4 th : March 18, 2016 Accepted: March 21, 2016 서론 급성콩팥손상 (Acute Kidney Injury) 은콩팥기능이급격한저하로더이상체액, 전해질의평형을유지할수없고단백대사산물 의배설의지장을초래하는질환으로써, 입원환자의 5%, 중환자실환자의 30 50% 에서흔히발생하며, 그사망률이높게보고되고있다 [1]. 현재임상에서는혈청 Creatinine 수치와소변배출량 (Urine output) 이콩팥기능장애의진단적표지자로써가장많이

2 50 Seung-Jin Lee and Sangwook Park. Usefulness of the NGAL Kit for Acute Kidney Injury Patients 활용하고있지만, 낮은민감도로인해조기진단표지자로활용하기에는한계점을가지고있다 [2]. 콩팥관련질환은여러만성질환처럼조기발견의중요성이강조되며 [3,4], 신장질환의선별검사에서성인의혈청 Creatinine 참고치는 1 mg/dl 내외이며혈청 Creatinine 수치의상승으로진단하는것이전통적인방법으로알려져있지만, 남녀성별차이와급성콩팥손상의초기에는그정도를제대로반영하지못한다는한계가있으며, 연령에따라서참고치분포가차이가남에도불구하고이에대한자료는부족하다 [5]. 최근급성콩팥손상의조기진단표지자 (Biomarker) 로써 NGAL (Neutrophil Gelatinase-Associated Lipocalin), KIM-1 (Kidney Injury Molecules-1), Interleukin-18, Cystatin C, 그리고 NAG (N-acetyl-beta-D-glucoseamidase) 등다양한생물학적표지자들이소개되고있으나그임상적의미가아직명확하게밝혀지지는않았다 [6]. 급성심질환에서는트로포닌 (Troponin) 과같은생물학적표지자가등장하면서진단과치료에많은개선이있었지만, 콩팥질환에서는많은치료약제개발이꾸준하게진전되어왔음에도불구하고실제사망률이나이환율은별로개선되지않았다. 이러한이유중에는급성콩팥부전을조기에찾아낼수있는생물학적표지자가없어서적절한치료시점을놓치기때문이었다. 다행히도, 최근에는기능적유전체학과단백질체학과같은첨단기술이개발되면서생물학적표지자로여러가지유전자산물이밝혀지고있다 [7]. 급성콩팥손상의원인들은다양하며콩팥의혈류량감소, 콩팥독성물질, 부신비대증등을포함한다. 이들진단은소변량감소와같은임상소견, BUN (Blood Urea Nitrogen)/Creatinine 비율의증가, 심한정도에따라대사성산증 (Metabolic acidosis), 고칼륨증 (Hyperpotassemia), 체액균형의변화및다른장기손상등을포함한합병증을유발하기도한다 [8]. NGAL은혈청 Creatinine 보다급성콩팥손상을조기진단하는생물학적표지자로써중성구나콩팥세뇨관의상피에서발현되는단백질인데여러가지원인으로급성콩팥손상이있게되면, 급속하게증가하는생물학적표지자로알려져있다. 급성콩팥손상시 Creatinine 의상승이전에소변에서지속적으로상승하는결과를보이므로, 급성콩팥손상을진단할수있는가장유용한표지자로보고되고있다. 현재는주로콩팥기능이상을조기에진단하고콩팥이식환자의예후를판단하는물질로활용되고있으며, 급성콩팥손상의발병시점이나지속기간을추정할수있다 [9]. 따라서 NGAL 검사키트의사용은 1차적으로응급처치를하는응급의료진에게객관적인진단적자료를제공할수가있고예방처치및응급검사에도움을줄수있으며 [10], 면역형광법 (Immunofluoresent assay) 을이용한 NGAL 정량검사는 NGAL의농도를신속하 게정량적으로측정할수있기때문에급성콩팥질환의평가와치료에좋은도구가될수있다 [11]. 본연구는 2013년 01월에서 2013년 12월사이에대구지역의응급의료센터를내원한콩팥질환환자들을대상으로 NGAL kit 검사를 ELISA (Enzyme-linked immunosorbent assay) 검사로측정한결과를비교하여 NGAL kit가급성콩팥질환환자의선별적인검사로써의가능성을알아봄으로써, 환자의조기진단과치료에도움을주고자본연구를실시하였다. 재료및방법 1. 연구대상 2013년 01월부터 2013년 12월까지대구광역시소재 C병원응급센터를내원하여치료를받은 20세이상의환자로진단명이급성콩팥손상이면서 NGAL 검사측정치가포함된 84명의혈액검체를대상으로하였다. NGAL kit의유용성평가연구를위해응급의료센터를내원한환자중 42명에게 NGAL검사를실시한후 ELISA 로측정한결과와비교분석하였다. 연구를진행하기전에개인정보수집과이용에관한제공동의서에자필서명한환자에대해서연구를실시하였고, 연구과정전반에걸쳐연구대상자에대한윤리적측면을고려하고, 개인식별자료는직접명시하지않았으며, 유추하여신상을파악할수있는어떠한자료도제시하지않았다. 본연구자는 Collaborative Institutional Training Initiative (CITI) Program Biomedical Research 과정을수료 (K ) 하였으며, 질병관리본부교육시스템에서생명윤리법관련온라인교육과정을수료 (15-A ) 하였다. 2. 검사방법 NGAL의정량검사는 NGAL kit (Alere Inc., San Diego, CA, USA) 를사용하였으며, EDTA 항응고전혈을이용하여 Triage Meter 면역형광분석기 (Biosite Inc., San Diego, CA, USA) 을통해 NGAL의농도를측정하였다. 검사장치 (device) 는형광나노입자가결합한 NGAL 특이단일클론항체를포함하는단일의플라스틱카트리지를사용한다. 검출한계는 ng/ml으로, 검사에소요되는시간은약 15분으로 Triage Meter 스크린에측정값이나타난다. ELISA를이용한 NGAL의측정은 3,500 rpm으로 10분간원심분리하여얻은혈청으로 Versa Max ELISA reader (R&D systems Inc., Minneapolis, MN, USA) 로측정하였다. 검사방법은혈청검체를 60분동안상온에서방치한후, 1X working buffer로희석하고 enzyme conjugate 를첨가하여검체를 30분동안상온에서방

3 Korean J Clin Lab Sci. Vol. 48, No. 2, June 치한다. 다시 working buffer로희석하고 TMB (Tetramethylbenzidine) 기질액을첨가하여 working substrate solution 을만든다. 20분동안방치한후, 정지액 (Stop solution) 을넣어반응을정지시킨다음, 450 nm에서흡광도를측정하였다. NGAL kit 검사결과와 ELISA 검사결과를비교하여민감도, 특이도, 양성예측도, 그리고음성예측도를구하였다. 3. 통계분석자료의분석은윈도우용 SPSS version 18.0 (SPSS Institute, Chicago, IL, USA) 를이용하여분석하였다. 변수는 t-test를시행하였고, 결과값은평균과표준편차로표시하였다. 범주형자료의비교에는 x 2 을사용하였고, 기대값이작은경우에는 Fisher의직접확률계산법을이용하였다. 모든통계분석을위한유의수준 (p) 은 p<0.05 로설정하였다. 결과 1. 연구대상자의특성및생화학적혈액검사총 84명의연구대상자중남자가 50명 (59.5%) 이었고, 여자가 34명 (40.5%) 이었고, 연령대는 60세미만이 21명 (25.0%) 이었으며, 60세이상이 63명 (75.0%) 이었다 (Table 1). 일반적으로시행되는응급 7종생화학적혈액검사에서 AST (Aspartate aminotransferase) 112.3±35.6 U/L, ALT (Alanine aminotransferase) 95.7±29.5 U/L, BUN (Blood Urea Nitrogen) 84.6±35.6 mg/dl, Creatinine 5.3±2.4 mg/dl, Sodium 138.2±3.2 meq/l, Potassium 4.2±1.2 meq/l, Chlorine 98.5±3.9 meq/l, NGAL 625.3±125.8 ng/ml이었다 (Table 2). 2. 혈액검사와 NGAL 과의상관관계응급 7종생화학적혈액검사와 NGAL과의상관관계에서 NGAL 이증가할수록, Creatinine 이유의하게증가하였다 (p-value=0.018). AST와 ALT 의상관계수는각각 0.168, 로통계적으로유의하지않았고, BUN, Sodium, Potassium, Chlorine 또한통계적으로유의하지않았다 (Table 3). 3. NGAL kit와 ELISA 민감도특이도응급의료센터를내원한환자의 NAGL kit와 ELISA의민감도는 300 (92.2%), 200 (95.6%), 100 (99.6%), 특이도는 300 (95.1%), 200 (97.3%), 100 (99.4%) 으로조사되었으며, 이에따른양성예측도는 300 (93.3%), 200 (93.4%), 100 (99.2%), 음성예측도는 300 (96.7%), 200 (97.7%), 100 (98.1%) 이였다 (Table 4). 고찰급성콩팥손상 (Acute kidney injury, AKI) 은입원환자의입원기간은물론사망률을증가시키고장기적으로는만성신장병의발 Table 1. General characteristics in patient Variable N % Gender Male Female Total Age < Table 2. Clinical blood test indicators Variable Mean SD AST (U/L) ALT (U/L) BUN (mg/dl) Creatinine (mg/dl) Na (meq/l) K (meq/l) Cl (meq/l) NGAL (ng/ml) Abbreviation: SD, standard deviation; AST, aspartate aminotransferase; ALT, alanine aminotransaminase; BUN, blood urea nitrogen; NGAL, neutrophil gelatinase-associated lipocalin. Table 3. Correlation coefficients between blood tests and NGAL Variable NGAL p-value AST (U/L) ALT (U/L) BUN (mg/dl) Creatinine (mg/dl) Na (meq/l) K (meq/l) Cl (meq/l) Table 4. Characteristics of NGAL and ELISA at various cut-off values Variable Cut-off point (ng/ml) Sensitivity (%) Specificity (%) Positive predictive value (%) Negative predictive value (%) Abbreviation: ELISA, enzyme-linked immunosorbent assay.

4 52 Seung-Jin Lee and Sangwook Park. Usefulness of the NGAL Kit for Acute Kidney Injury Patients 생과도연관이있으며, 많은연구에서혈청 Creatinine은사구체여과율이현저히감소된이후에변화를보여, 정확한예후를판정하고조기에예방적치료를시행하는근거로는부적합하다는의견이지배적이다 [12]. 임상적으로급성콩팥기능부전을판단할수있는혈청 Creatinine 의상승이나소변량의감소등은콩팥손상이진행된이후에나타나며 [13], 신장이식후소변량의변동이천천히감소하는경우, 급성거부반응과감별진단이임상적으로진단하기는매우어려워초기콩팥손상을알려주는예민한표지자의필요성이제기되어왔다 [14]. 사구체여과율 (Glomerular filtration rate, GFR) 은콩팥이일정시간동안특정물질을완전히제거할수있는혈장량으로정의되며, 콩팥기능평가의중요한표지자로이용되었다 [15]. 사구체여과율측정의표준방법은 Inulin, Iohexol, 51 Cr-EDTA, 99mTC-labeled diethylenetriamine pentaacetic acid (DTPA), 125 I-labeled iothalamate 등외부표지자의제거율을측정하는것이다. 하지만, 환자에게표지약제를직접주입해야하고시간이많이걸려상용검사로는시행하기어렵기때문에 Creatinine, BUN, 2-microglobulin 등을이용하여사구체여과율을추정하는방법이사용되고있으며, 그중혈청 Creatinine이임상적으로가장많이사용되고있다 [16]. NGAL은활성화된호중구에서초기에발견된 lipocalin superfamily 에속하는 25 kda의분자량이작은단백질이며, 콩팥의허혈성손상, 감염, 악성종양, 신세뇨관손상등이일어났을때, 콩팥의헨넬고리 (Henle s loop) 나원위세뇨관에서발현된다. 사구체에서여과된후, 근위세뇨관에서도재흡수되어발현될수있으며혈장및소변에서도발견할수있다 [17]. NGAL 정량검사는소변및혈액 ( 혈장또는혈청 ) 에서급성콩팥손상시생성되는 NGAL을측정함으로써, 급성콩팥손상의조기진단및손상의정도를평가하는검사이다. 이러한 NGAL의발현은콩팥독성손상또는허혈에의한콩팥손상에서의미있게증가하며, 급성콩팥손상의생체표지자로콩팥기능이상의조기진단과콩팥이식환자의예후를판단하는중요한물질로알려져있다 [18]. 최근새로운급성콩팥손상의표지자를발견하려는노력이있었음에도불구하고여전히혈청 Creatinine이유일한지표로사용되고있는것이현실이며급성콩팥손상의표지자는조기발견과진단, 그리고정확한예후를판정하는지표로사용되어야하지만, 급성콩팥손상의발생이단순히신장만의문제로국한되는것이아니라다양한원인질환및다른전신장기와연관하여나타나므로이러한임상적요구를충족하는새로운표지자를찾기란어려운일이다. Yilmaz A 등 [19] 의연구에서는콩팥질환환자에서 NGAL 농도 가상승하는것을확인하였고급성콩팥질환의초기진단및중증도추정에 NGAL을이용할수있다고하였다. 또한응급실을기반으로한연구에서는 NGAL 의측정값은콩팥관련질환의좋은지표이며콩팥관련질환에서 NGAL을이용할수있다고하였다 [20]. 본연구에서는대구지역응급의료센터에내원한급성콩팥손상환자의일반적인특성을조사하고검사실결과와의상관관계를분석하였다. 응급 7종생화학적혈액검사와 NGAL과의상관관계에서 NGAL이증가할수록 Creatinine 이유의하게증가하였다. 하지만 AST, ALT, BUN, Sodium, Potassium 그리고, Chlorine 의상관관계는통계적으로유의하지않았다. NAGL kit와 ELISA의민감도는 300 (92.2%), 200 (95.6%), 100 (99.6%), 특이도는 300 (95.1%), 200 (97.3%), 100 (99.4%) 로조사되었으며, 양성예측도는 300 (93.3%), 200 (93.4%), 100 (99.2%), 음성예측도는 300 (96.7%), 200 (97.7%), 100 (98.1%) 로조사되어, 선행연구 [19,20] 에서밝혀진바와같이 NGAL 정량검사는혈액검체에서급성콩팥손상의정도를평가하는방법으로조기진단및예후를예측함에있어서신속하고유용하게이용할수있을것으로생각한다. 본연구의한계점으로는 NGAL 수치는체액상태, 약물등의영향을받을수있으며, 이에대한기준이나평가가현재까지는명확하지않다는점이다 [21]. 또한 NGAL 측정상한치가 1300 ng/ml이기때문에 1300 ng/ml 이상인환자의평가가제한적이고, 조사지역이단일지역이라는점은보완할필요가있다. 따라서통계적유의성을나타내려면충분한피험자의수치와추가적인검사요인뿐만아니라전국지역을대상으로하는연구가필요할것으로생각되고, 앞으로이러한제한점들을보완하는전향적인다기관연구가필요할것으로사료된다. 이러한한계점에도불구하고, 응급의료센터의콩팥질환환자들에게 NGAL kit검사는간단하고신속하게결과를확인할수있는현장검사 (Point of care testing, POCT) 로서, 급성콩팥손상과관련된콩팥기능에대한평가와치료에도움이될수있을것으로사료되고, 이러한현장검사의단점으로는훈련을받지않은비전문가에의해검사가행해지거나, 정도관리 (Quality control) 가이루어지지않은상황에서검사가시행되고결과보고가체계적으로이루어지지않을수도있다는점이다. 따라서현장검사는그유용성이확실하게입증된검사에한해서실시되고, 정도관리시스템이구축되어지속적인관리가이루어질수있는경우에한해도입되고운영되어야할것이다. 마지막으로 NGAL kit의사용은급성콩팥손상환자에게 1차적으로접하고응급처치를하는응급의료진에게정확하고객관적인진단적자료를제공함으로써예방처치및응급검사에많은도움을

5 Korean J Clin Lab Sci. Vol. 48, No. 2, June 줄수있을것으로생각한다. 요약 급성심질환환자는트로포닌 (troponin) 과같은생물학적표지자 (biomarker) 가등장하면서진단과치료에많은개선이있었다. 콩팥질환에서는많은치료약제개발이꾸준하게진전해왔음에도불구하고실제사망률이나이환률은별로개선되지않았다. 급성콩팥손상환자를대상으로응급의료센터에서측정한 NGAL의측정치는 NGAL이증가할수록 Creatinine 이유의하게증가하였다. 그러나, AST, ALT, BUN, Na, K, Cl 과의상관관계는통계적으로유의하지않았다. NAGL kit와 ELISA의민감도는 300 (92.2%), 200 (95.6%), 100 (99.6%), 특이도는 300 (95.1%), 200 (97.3%), 100 (99.4%) 로조사되었으며양성예측도는 300 (93.3%), 200 (93.4%), 100 (99.2%), 음성예측도는 300 (96.7%), 200 (97.7%), 100 (98.1%) 로조사되었으며, 급성콩팥손상의정도를평가하는검사로써조기진단및예후를예측함에있어서환자의치료의결정에유용하게이용될수있다. 향후응급의료센터에서콩팥질환환자의중증도를평가하고치료방향을결정하며, 예후를예측하는데있어서 NGAL kit 검사가도움이될수있다고생각한다. Acknowledgements: None Funding: None Conflict of interest: None References 1. Lisowska MB. Serum and urinary biomarkers of acute kidney injury. Blood Purification. 2010;29: Haase FA, Bellomo R, Devarajan P, Story D, Matalanis G, Dragun D, et al. Novel and conventional serum biomarkers predicting acute kidney injury in adult cardiac surgery a prospective cohort study. Critical Care Medicine. 2009;37: Striker GE. Report on a workshop to develop management recommendations for the prevention of progression in chronic renal disease. Nephrol Dial Transplant. 1995;10: Hostetter TH. Prevention of the development nd progression of renal disease. J Am Soc Nephrol. 2003;14(Suppl):S Kwon SY, Na YA. Concentration of serum and urine creatinine in children and adolescents. Korean J Clin Lab Sci. 2014; 46: Han JS. Urinary diagnostic indices in acute kidney injury. The Korean Journal of Nephrology. 2009;28: Haase M, Bellomo R, Devarajan P, Schlattmann P, Haase F. Accuracy of NGAL in diagnosis and prognosis in AKI: a systematic review and meta-analysis. Am J Kidney Dis. 2009;54: Yang HN, Boo CS, Kim MG, Jo SK, Cho WY, Kim HK. Urine nutrophil glatinase-asociated lipocalin an independent predictor of adverse outcomes in acute kidney injury. American Journal of Nephrology. 2010;31: Vaidya VS, Ferguson MA, Bonventre JV. Biomarkers of acute kidney injury. Annual Review of Pharmacology and Toxicology. 2008;48: Lee SJ, Lim SO, Jeong JY, Park MJ, Park JE. The clinical pathology characteristics and tetanus quick stick evaluation for tetanus patients in daegu emergency medical center. Korean J Clin Lab Sci. 2014;46: Dent CL, Ma Q, Dastrala S, Bennett M, Mitsnefes MM, Barasch J, et al. Plasma neutrophil gelatinase-associated lipocalin predicts acute kidney injury morbidity and mortality after pediatric cardiac surgery: a prospective uncontrolled cohort study. Critical Care. 2007;11:R127. DOI: /cc Al Ismaili Z, Palijan A, Zappitelli M. Biomarkers of acute kidney injury in children: discovery, evaluation, and clinical application. Pediatr Nephrol. 2011;26: Choi MJ, Park SH, Kim CD, Kim YL, Kwon TH, Kim IS, et al. Identification of TGF- -induced gene product, ig-h3 in ischemic acute renal failure. The Korean Journal of Nephrology. 2007;26: Lee SR, Kwon OJ, Koh BH, Kwak JY. Serial changes of serum creatinine and resistive index to predict acute rejection after renal transplantation. J Korean Soc Transplant. 1999; 13: Ha JS, Ryoo NH, Kim JR, Jun DS, Kim HC, Kim YJ. Cystatin C as a marker for early renal impairment. Korean J Lab Med. 2004;24: Laterza OF, Price CP, Scott MG. Cystatin C an improved estimator of glomerular filtration rate. Clinical Chemistry. 2002; 48: Mishra J, Ma Q, Prada A. Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol. 2003;14: Grande AD, Giuffrida C, Carpinteri G, Narbone G, Pirrone G, Mauro AD, et al. A novel biomarker for the early diagnosis of acute kidney injury in the emergency department. European Review for Medical and Pharmacological Sciences. 2009; 13: Yilmaz A, Sevketoglu E, Gedikbasi A, Karyagar S, Kiyak A, Mulazimoglu M, et al. Early prediction of urinary tract infection with urinary neutrophil gelatinase-associated lipocalin. Pediatr Nephrol. 2009;24: Nickolas TL, O'Rourke MJ, Yang J, Sise ME, Canetta PA, Barasch N, et al. Sensitivity and specificity of a single emergency department measurement of urinary neutrophil gelainase-associated lipocalin for diagnosing acute kidney injury. Ann Intern Med. 2008;148: Lee JY, Kim JY, Park SO, Lee KR, Baek KJ, Hong DY. Plasma neutrophil gelatinase-associated lipocalin is an early marker of acute kidney injury. J Korean Soc Emerg Med. 2013;24:

012임수진

012임수진 Received : 2012. 11. 27 Reviewed : 2012. 12. 10 Accepted : 2012. 12. 12 A Clinical Study on Effect of Electro-acupuncture Treatment for Low Back Pain and Radicular Pain in Patients Diagnosed with Lumbar