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1 Review Article J Korean Soc Transplant 2017;31: 급성신손상의동물모형 장혜련 성균관대학교의과대학삼성서울병원내과학교실 Animal Models for Acute Kidney Injury Hye Ryoun Jang, M.D. Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Acute kidney injury (AKI) is classified into three types according to its pathophysiology: prerenal, intrinsic renal, and post-renal AKI. Experimental models of AKI can be divided into two categories: in vivo and in vitro. Models can be further subdivided according to how AKI is simulated. The pathophysiology of intrinsic renal and post-renal AKI has been investigated using animal models. Most studies have been conducted in murine models using male mice or rats, while large mammals like sheep, pigs, and monkeys have been used in a limited number of studies. The intrinsic renal AKI model is divided into septic vs. aseptic AKI. Aseptic AKI is subdivided into ischemic vs. nephrotoxic AKI. Lipopolysaccharides (LPS) injection or cecal ligation and puncture (CLP) have been used to simulate the septic AKI model in rodents. Ischemic AKI is the most extensively investigated field to date because ischemic AKI is the most common and serious cause of AKI in both native kidneys and renal allografts. Ischemia-reperfusion injury (IRI) surgery has been used to induce ischemic AKI. There are two different methods of IRI surgery: laparotomy vs. flank approach. Warm temperature and male sex are critical to induction of sufficient grade of renal injury in this model. Many nephrotoxicants pertinent to human disease have been used to reproduce nephrotoxic AKI in rodent models. Cisplatin, a common chemotherapeutic agent, has many pathophysiologic features that overlap with IRI. Other nephrotoxicants such as gentamicin or glycerol were studied in the past, whereas much more attention has recently been devoted to environmental nephrotoxicants such as cadmium. However, variant susceptibility to different doses of nephrotoxicants is a big hurdle to set up a reproducible and consistent model of nephrotoxic AKI. Post-renal AKI is simulated with ureteral obstruction surgery, whereas the unilateral ureteral obstruction (UUO) model has frequently been used. Although some novel findings have been reported through numerous studies using murine AKI models, AKI still remains a challenging condition that lacks specific diagnostic or therapeutic tools because of species barriers or experimental settings. Animal AKI models using mammals genetically closer to human like monkeys would be valuable to simulate human AKI more appropriately. Key Words: Acute kidney injury, Animal model 중심단어 : 급성신손상, 동물모형 Received September 15, 2017 Accepted September 17, 2017 Corresponding author: Hye Ryoun Jang Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul 06351, Korea Tel: , Fax: hyeryoun.jang@samsung.com 급성신손상과동물모형개요 그동안연구되어온여러가지신질환에대한동물모형중에서급성신손상 (acute kidney injury, AKI) 은임상적중요성과만성신질환동물모형에비해신손상유도후관찰기간이상대적으로짧은장점등으로인해가장 Copyright c the Korean Society for Transplantation, 2017

2 J Korean Soc Transplant ㆍ September 2017 ㆍ Volume 31 ㆍ Issue 3 활발히연구된분야이다 (1,2). 급성신손상의전통적인분류기준은병태생리를바탕으로하며, 크게신전성 (pre-renal), 신성 (intrinsic renal), 신후성 (post-renal) 급성신손상으로나뉜다 (Fig. 1). 급성신손상의실험모형 (experimental model) 은크게두가지로구분할수있는데, 신세관세포 (renal tubular cell) 배양실험을주축으로하는 in vitro 모형과동물에서여러가지방법으로급성신손상을직접유도하는 in vivo 모형이다. In vivo 모형은다시 septic AKI 모형과 aseptic AKI 모형으로나뉘어진다 (3). Aseptic AKI 모형에는허혈성급성신손상모형과신독성급성신손상모형이속한다 (Fig. 2). 허혈성급성신손상동물모형 (ischemic AKI animal model) 허혈성급성신손상은다양한포유동물에서실험이진행되었는데, 생쥐 (mouse) 나쥐 (rat) 를이용하는설치류실 Fig. 1. Classification of acute kidney injury. Abbreviations: ATN, acute tubular necrosis; AGN, acute glomerulonephritis; RPGN, rapidly progressive glomerulonephritis. 험이가장많았고, 그밖에돼지나토끼, 양, 원숭이등의포유동물에서도실험이이루어지고있다. 허혈성급성신손상은정상신장 (native kidney) 와이식신장 (renal allograft) 모두에서신손상의주요원인을차지하고, 이식신장에서는특히이식신기능지연 (delayed graft function, DGF) 의원인이므로최근 30여년간매우활발히연구된분야이다. 대부분의주요연구결과가설치류실험으로밝혀졌고, 허혈방법에따라 warm ischemia와 cold ischemia 로구분된다. Warm ischemia 는실험동물의체온을유지하는상태에서수술로신장에허혈을유도하였다가재관류시키면서신손상을유도하는것인데, 신문부 (renal pedicle) 를결찰 (clamping) 하기위해노출하는수술방법의차이에따라개복술 (laparotomy) 과 flank approach로나뉜다. 설치류에서개복술은마취후배면을면도하고정중앙절개 (midline incision) 로개복한후에복막을절개하고장을젖힌후양측또는편측신문부를결찰하였다가일정한허혈유도시간이지난후신문부의 microvascular clip을제거하여허혈신장을재관류시킨다. 설치류에서 flank approach 수술은동물을마취한후에엎드린자세 (prone position) 로두고면도한후신장을노출시켜신문부를결찰한다. 두방법모두수컷설치류에서체온이 37 내외로잘유지되도록하면서수술하는것이중요하다. 개복술이좀더침습적이기때문에같은허혈시간을두었을때더심한신손상을유발하는경향이있다. 양측신문부를결찰하는양측허혈-재관류신손상 (bilateral renal ischemia-reperfusion injury, IRI) 수술은허혈성급성신손상의초기신손상기 (injury phase) 의신손상기전을연구하는데주로쓰이고, 왼쪽신문부만을결찰하는편측허혈-재관륜신손상 (unilateral 또는 left renal IRI) 수술은회복기 (recovery phase) 의신세관재생 (renal tubular regeneration) 과간질섬유화 Fig. 2. Acute kidney injury (AKI) animal models. Adapted from Fig. 1 of reference [3]. 112

3 Hye Ryoun Jang: Animal Models for Acute Kidney Injury (interstitial fibrosis) 과정을연구하는데주로쓰인다. 허혈성급성신손상의설치류모형실험을통해신손상기와회복기에중요한역할을하는여러가지면역학적요소가자세히보고되었다 (3-5). 또한, 허혈성급성신손상에서발생하는타장기 ( 뇌, 심장, 폐, 간, 장, 골수등 ) 의기능이상도주로설치류실험을통해밝혀졌다 (6-9). 과거에는주로급성신손상의초기신손상기에관해연구가집중적으로이루어졌지만, 최근 10여년동안급성신손상의회복기및급성신손상으로부터만성신질환으로진행하는과정에대한관심이높아지면서 (10,11) 이에관한연구도설치류편측허혈-재관류신손상 (unilateral renal ischemia-reperfusion injury) 모형을이용하여진행되었다 (12). Warm ischemia 로유도하는설치류허혈성급성신손상모형의주요단점은수컷설치류만을사용한다는성별제한과신손상정도가실험온도에큰영향을받는다는점이다. Cold ischemia는 4 o C의장기보관용액 (University of Wisconsin solution) 안에서실험동물신장의신문부를결찰하는방법으로연구되는데, 주로설치류가실험에사용된다. 독성급성신손상동물모형 (nephrotoxic AKI animal model) 여러가지약제나독소가독성급성신손상을유발하는데, 동물모형에많이사용되는약제또는독소는시스플라틴 (cisplatin), 젠타마이신 (gentamicin), 글리세롤 (glycerol), 카드뮴 (cadmium), 조영제 (contrast dye), 엽산 (folic acid), 광방기 (aristolochic acid), 와파린 (warfarin) 등이다. 이중에서시스플라틴은광범위하게쓰이고있는주요항암제로서독성급성신손상의유발약제로많은연구에사용되었다 (13,14). 고용량의시스플라틴을 1 2회주사하거나, 저용량의시스플라틴을반복적으로주사하여급성신손상을유도하는데실험의목적과평가지표에따라투여용량과일정이다르며, 시스플라틴을피하주사하거나복강내주사한다. 글리세롤은횡문근융해증에의한급성신손상을동물모형에서구현하는데사용되었으며, 동물의다리근육에글리세롤을주사하고 24시간동안수분섭취를막는방법으로급성신손상을유도하였다 (15). 다른방법으로는글리세롤투여전에과당 (sucrose) 을주사하거나출혈을유발하는방법이있다. 와파린으로유도한급성신손상모형에서는흔히혈뇨가동반되며, 조직학적으로적혈구원주 (erythrocytes casts) 에의한신세관의폐색과유리된헤모글로빈으로부터산화스트레스 (oxidative stress) 가증가되어신손상이유발되는특징을보인다. 이모형은 5/6 신절제술을시행한쥐에서만구현가능한데 (1), 이는만성신질환환자들에서와파린에의한급성신손상의발생위험성이증가하는임상적특징에잘부합한다. 조영제신독성도설치류에서조영제투여가신장에끼치는조직학적및기능적변화에대해연구되었는데 (16), 고령의수컷고혈압쥐 (spontaneously hypertensive rats, SHR) 에조영제를투여하여심신증후군 (cardiorenal syndrome) 환자에서발생한조영제신독성 (contrast-induced nephropathy, CIN) 을구현한연구도있다 (17). 독성급성신손상은임상적으로연구필요성이높고비교적비침습적으로유도가능하며실험방법을숙지하는데걸리는시간이상대적으로짧다는장점이있다. 반면, 실험에사용되는약제가환자들에게투여되는투약일정과다르고, 실제임상적상황을구현하기어려우며, 약제또는독소에대한실험동물의감수성차이로인해개체간급성신손상의중증도차이가상대적으로크게발생하는재현성문제가있다. 패혈증에의한급성신손상동물모형 (septic AKI animal model) 패혈증과급성신손상은병태생리학적상호작용을보이는데, 패혈증으로인해신장조직에서염증과산화스트레스증가및세포자멸사등이초래되면서급성신손상이발생하고악화된다 (18). 패혈증에의한급성신손상을설치류에서유도하는방법은 lipopolysaccharide (LPS) 를투여하거나광범위한복막염을일으키는수술적방법이다 (19). LPS는 E.coli, K. pneumoniae, P. aeruginosa, Salmonella enterica, Salmonella typhosa, Serratia marcescens 등의세균으로부터유래된것을주로사용하며, mg/kg 용량으로 1회복강내투여하는데, 수컷설치류를사용하며 LPS의종류와동물의종류에따라 LPS의투여용량을결정한다. 복막염을유발하는수술적방법에는맹장 (cecum) 을결찰하였다가천공시키는방법 (cecal ligation and puncture, CLP) 과상행결장을천공시키는 colon ascendens stent peritonitis (CASP) 방법이있다. CLP 방법은염증반응과혈역학적불안정성을모두구현한다는장점이있지만, 더복잡하고재현성이낮은문제가있다. 이러한설치류모형의단점은실험동물의신장구조및기능적측면과면역체계가모두인간과다르다는종간의차이이다. 설치류뿐만아니라, 돼지와같은큰포유동물에서도패혈증에의한급성신손상에관한연구가진행되고있다 (20). 113

4 J Korean Soc Transplant ㆍ September 2017 ㆍ Volume 31 ㆍ Issue 3 폐쇄성급성신손상동물모형 (post-renal or obstructive AKI model) 편측요관만결찰하는 unilateral ureteral obstruction (UUO) 모형에서는요관결찰후신세관내압력이상승하면서신세관허혈이이차적으로유도되어신원손상이유발된다 (1). UUO 모형에서는신세관압력상승에의한 mechanical stress와염증세포침윤및섬유화가진행되면서신손상이진행한다. 편측요관만결찰하므로반대쪽신장기능이유지되어혈청크레아티닌은상승하지않지만, UUO로손상된신장에서는간질염증 (interstitial inflammation) 과신세관세포손상 (tubular cell injury) 및섬유화 (fibrosis) 와같은만성신질환의특징이모두나타난다. 따라서, UUO 모형은요로폐쇄자체의효과보다는급성신손상에서만성신질환으로이행하는병태생리기전을연구하는데더많이쓰이고있다. 급성신손상동물모형에서평가척도 급성신손상의정도를평가하는척도는크게기능적변화를비교하는방법과신장조직의구조적변화를분석하는방법으로나뉜다. 급성신손상의동물모형에서기능적평가는혈중요소질소 (blood urea nitrogen, BUN), 혈청또는혈장크레아티닌 (creatinine) 과시스타틴 C (cystatin C), 혈장또는요 neutrophil gelatinase-associated lipocalin (NGAL) 및 kidney injury molecule-1 (KIM-1) 등을측정하는것이다. 설치류급성신손상모형에서는동물이살아있는상태에서얻을수있는혈액검체량이적으므로, 어떠한기능 적평가항목을측정할것인지를미리결정하여소량의검체를효율적으로사용하여야한다. 신장조직의구조적변화는조직을여러가지방법으로염색하여평가하는데, H&E 염색이나 PAS 염색으로신세관과사구체의손상정도를평가하고, Masson s trichrome 염색으로섬유화정도를분석한다. 신장조직내염증세포의침윤이나특정표지인자의발현은면역조직화학염색 (immunohistochemistry) 이나면역형광염색 (immunofluorescence staining) 으로평가한다. 그밖에도신장조직내에침윤한염증세포를유세포분석기법 (flow cytometry analysis of kidney mononuclear cells) 으로분석할수있으며 (21), 신장조직내여러가지사이토카인이나케모카인을 multiplex assay 또는 real-time PCR 기법으로분석할수있다. 급성신손상동물모형의특징과한계점 급성신손상동물모형은설치류실험에서가장많이구현되었는데, 설치류급성신손상모형에는주로수컷설치류가사용된다. 암컷설치류에서는같은실험방법으로급성신손상이잘유도되지않기때문인데, 반면전신성홍반성낭창에의한신염 (lupus nephritis) 모형은암컷설치류를사용한다. 이처럼신장질환의종류에따라특정성별의동물을사용해야하는점은동물모형의특징인동시에한계점이기도하다. 환자대상연구에서 aminoglycoside 의신독성에의한독성급성신손상이여성에서더호발한다는보고가다수있었지만, 메타분석에의하면여성성별이독립적인위험인자는아닌것으로보고되었다 (22). Table 1. Difference in renal anatomy and function: rodent vs. human Rodent Human Anatomy Papilla One Multiple Nephron per kidney 14,000 in mice 200,000 to 1.8 (up to 2.7) million 22,000 25,000 in rats Glomerulus diameter 70 m in mice 200 m PTEC Vacuolated in male mice Distal tubule Brush border (+) in mice Brush border ( ) Outer stripe of OM Much more developed Loop of Henle Higher ratio of long loop of Henle (1/3) Lower ratio of long loop of Henle (1/7) Function Concentration Higher: 4,000 mosm/kg 1200 mosm/kg Proteinuria mg/day in mice 2 15 mg/day in rats only in males age dependent increase <150 mg/day: mainly uromodulin (Tamm-Horsfall protein) Abbreviations: PTEC, proximal tubular epithelial cells; OM, outer medulla. 114

5 Hye Ryoun Jang: Animal Models for Acute Kidney Injury 설치류급성신손상동물모형실험에는주로 8 12주령정도의수컷을사용하는데, 실제임상에서는고령의환자들에서급성신손상의발생률이증가하므로이점역시동물실험의결과를일반화하는데걸림돌로작용한다. 특정면역학적요소를결손시키거나과발현시키는방향으로유전학적인조작이가해진동물 ( 대부분설치류 ) 실험을통해, 급성신손상연구분야 ( 특히허혈성급성신손상의병태생리분야 ) 에서큰발전이이루어졌다. 그러나, 특정면역학적요소의유전학적조작을통해신손상을악화시키거나완화시키는병태생리학적인역할이알려진면역학적요소를실제임상에적용하였을때기대한결과를얻지못한경우가대부분인데 (23), 이는동물실험에서손상된신장조직내부에서일어나는변화의유기적인관계를전체적으로파악하지못하고특정요소에만지나치게집중하였기때문일수있다. 또한, 동물과인간사이에신장의해부학적및기능적차이가있기때문일가능성도높다. 실제로급성신손상연구에가장많이쓰인동물인설치류와인간은신장의구조적측면과기능적측면모두에서여러가지차이를보인다. Table 1에서정리된바와같이설치류신장의유두 (papilla) 는하나이지만, 인간신장에는여러개의유두가존재한다. 신원의수와사구체의크기는당연히큰차이를보이며, 설치류신장에는길이가긴헨레고리 (loop of Henle) 가상대적으로더많다. 따라서, 설치류의요농축능력이 4,000 mosm/kg로인간의 1,200 mosm/kg보다훨씬더높다. 급성신손상의기타모형 급성신손상에대한연구가활발하게이루어지면서원숭이와같은영장류에서도급성신손상연구가진행되고있지만, 고가의실험비용과사육및관리문제, 윤리적인문제등으로인해큰제약이있다. Zebrafish 는포유류처럼발달된신장을가지지못했지만재생능력이뛰어난 mesonephros를가지고있어서, 신원의재생 (neonephrogenesis) 을연구하는분야에서유용하게쓰이고있다 (24). 그밖에도조직공학적으로신질환모형을연구하거나 (25), 줄기세포를활용하는연구도진행되고있다. 결론 급성신손상의동물모형실험을통해급성신손상의병태생리분야에서큰발전이이루어졌는데, 최근수십년간가장많이연구된분야인허혈성급성신손상에서는 유전적으로조작된생쥐를적절히활용하여병태생리의많은부분이알려지게되었다. 설치류실험의한계를극복하고자토끼나돼지, 양, 원숭이등대형포유동물도실험에이용되고있지만, 동물관리와재원및윤리적인문제등으로인해연구에제한이있다. 급성신손상은조기진단및치료가아직어려운분야이므로, 동물의성별과연령을고려하고적절한실험방법을미리잘계획하여체계적인실험을진행함으로써학문적및의료기술적으로이분야를더욱발전시켜야할것이다. REFERENCES 1) Ortiz A, Sanchez-Nino MD, Izquierdo MC, Martin-Cleary C, Garcia-Bermejo L, Moreno JA, et al. Translational value of animal models of kidney failure. Eur J Pharmacol 2015;759: ) Skrypnyk NI, Siskind LJ, Faubel S, de Caestecker MP. Bridging translation for acute kidney injury with better preclinical modeling of human disease. Am J Physiol Renal Physiol 2016;310:F ) Jang HR, Rabb H. Immune cells in experimental acute kidney injury. Nat Rev Nephrol 2015;11: ) Jang HR, Rabb H. The innate immune response in ischemic acute kidney injury. Clin Immunol 2009;130: ) Jang HR, Ko GJ, Wasowska BA, Rabb H. The interaction between ischemia-reperfusion and immune responses in the kidney. J Mol Med (Berl) 2009;87: ) Scheel PJ, Liu M, Rabb H. Uremic lung: new insights into a forgotten condition. Kidney Int 2008;74: ) Faubel S, Edelstein CL. Mechanisms and mediators of lung injury after acute kidney injury. Nat Rev Nephrol 2016;12: ) Liu M, Liang Y, Chigurupati S, Lathia JD, Pletnikov M, Sun Z, et al. Acute kidney injury leads to inflammation and functional changes in the brain. J Am Soc Nephrol 2008;19: ) Grams ME, Rabb H. The distant organ effects of acute kidney injury. Kidney Int 2012;81: ) Zager RA. Progression from acute kidney injury to chronic kidney disease: clinical and experimental insights and queries. Nephron Clin Pract 2014;127: ) Chawla LS, Eggers PW, Star RA, Kimmel PL. Acute kidney injury and chronic kidney disease as interconnected syndromes. N Engl J Med 2014;371: ) Jang HR, Gandolfo MT, Ko GJ, Satpute SR, Racusen L, Rabb H. B cells limit repair after ischemic acute kidney injury. J Am Soc Nephrol 2010;21: ) Liu M, Chien CC, Burne-Taney M, Molls RR, Racusen LC, 115

6 J Korean Soc Transplant ㆍ September 2017 ㆍ Volume 31 ㆍ Issue 3 Colvin RB, et al. A pathophysiologic role for T lymphocytes in murine acute cisplatin nephrotoxicity. J Am Soc Nephrol 2006;17: ) Park JH, Jang HR, Kim DH, Kwon GY, Lee JE, Huh W, et al. Early, but not late treatment with human umbilical cord blood-derived mesenchymal stem cells attenuates cisplatin nephrotoxicity through immunomodulation. Am J Physiol Renal Physiol [in press 2017 Mar 29]. 15) Zurovsky Y. Models of glycerol-induced acute renal failure in rats. J Basic Clin Physiol Pharmacol 1993;4: ) Kiss N, Hamar P. Histopathological evaluation of contrast-induced acute kidney injury rodent models. Biomed Res Int 2016;2016: ) Zhang J, Fallahzadeh MK, McCullough PA. Aging male spontaneously hypertensive rat as an animal model for the evaluation of the interplay between contrast-induced acute kidney injury and cardiorenal syndrome in humans. Cardiorenal Med 2016;7: ) Alobaidi R, Basu RK, Goldstein SL, Bagshaw SM. Sepsis-associated acute kidney injury. Semin Nephrol 2015;35: ) Buras JA, Holzmann B, Sitkovsky M. Animal models of sepsis: setting the stage. Nat Rev Drug Discov 2005; 4: ) Chvojka J, Sykora R, Krouzecky A, Radej J, Varnerova V, Karvunidis T, et al. Renal haemodynamic, microcirculatory, metabolic and histopathological responses to peritonitis-induced septic shock in pigs. Crit Care 2008;12:R ) Ascon DB, Lopez-Briones S, Liu M, Ascon M, Savransky V, Colvin RB, et al. Phenotypic and functional characterization of kidney-infiltrating lymphocytes in renal ischemia reperfusion injury. J Immunol 2006;177: ) Neugarten J, Golestaneh L. The effect of gender on aminoglycoside-associated nephrotoxicity. Clin Nephrol 2016; 86: ) Salmela K, Wramner L, Ekberg H, Hauser I, Bentdal O, Lins LE, et al. A randomized multicenter trial of the anti-icam-1 monoclonal antibody (enlimomab) for the prevention of acute rejection and delayed onset of graft function in cadaveric renal transplantation: a report of the European Anti-ICAM-1 Renal Transplant Study Group. Transplantation 1999;67: ) McKee RA, Wingert RA. Zebrafish renal pathology: emerging models of acute kidney injury. Curr Pathobiol Rep 2015;3: ) Desrochers TM, Palma E, Kaplan DL. Tissue-engineered kidney disease models. Adv Drug Deliv Rev 2014;69-70:

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( )Kju269.hwp 만성세균성전립선염모델흰쥐에서 의항염효과 Anti-inflammatory Effect of Lycopene on Chronic Bacterial Prostatitis Rat Model Cho Hwan Yang, Dong Wan Sohn, Yong-Hyun Cho From the Department of Urology, The Catholic University