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1 大韓獸醫學會誌 (2014) 第 54 卷第 4 號 Korean J Vet Res(2014) 54(4) : 225~231 < 원례보저 > Cisplatin 유도급성신부전에서 Klotho 단백질의발현 박소라 김태원 김영중 김현태 류시윤 정주영 * 충남대학교수의과대학 ( 접수 : 2014년 6월 14일, 수정 : 2014년 10월 24일, 게재승인 : 2014년 11월 5일 ) Localization of Klotho in cisplatin induced acute kidney failure So-Ra Park, Tae-Won Kim, Young-Jung Kim, Hyun-Tae Kim, Si-Yun Ryu, Ju-Young Jung* Department of Veterinary Medicine and Institute of Veterinary Science, Chungnam National University, Daejeon , Korea (Received: June 14, 2014; Revised: October 24, 2014; Accepted: November 5, 2014) Abstract : Klotho deficiency is an early event in acute kidney injury (AKI) that exacerbates acute kidney damage. The present study explored the expression of Klotho and inflammation related factors in cisplatin-induced AKI. Rats (n = 18) were treated with cisplatin intraperitoneal injection (5 mg/kg) or left untreated as controls (n = 6), then sacrificed at 5 (n = 6) and 10 days (n = 6) treatment. Five days after cisplatin injection, the serum kidney enzymes and kidney cell apoptosis were significantly increased. Moreover, the expression of Klotho was decreased when compared to the control group, especially in the cortex and outer medulla regions. In contrast, inflammation related signals including nuclear factor kappa B, tumor necrosis factor-α, and tumor necrosis factor-like weak inducer of apoptosis were enhanced. However, 10 days after cisplatin injection, Klotho expression was enhanced upon both IHC and Western blot analysis, with slightly recovered renal function and decreased apoptosis. Furthermore, inflammation related signals expression was decreased relative to the 5 days group. Overall, this study confirmed the opposite expression patterns between Klotho and inflammation related signals and their localization in cisplatin-induced AKI kidney. Keywords: acute kidney injury, inflammatory cytokine, Klotho, localization 서 론 급성신부전 (Acute Kidney Injury; AKI) 은콩팥혈류의감소, 사구체신염, 약물부작용등여러인자로인하여발생하며콩팥관형세포와세뇨관사이질염증의빠른악화로인한세뇨관손상및신기능의저하를보이며산화스트레스에의한콩팥세뇨관세포의사멸을특징으로한다 [16]. 시스플라틴 (cisplatin) 은여러종류의암에널리사용되는항암제중하나로뛰어난항암효과를가지고있지만과투여시보이는이독성 (ototoxicity) 및콩팥독성등으로인해그사용에주의를요구하고있다 [3]. 시스플라틴유래부작용중급성신부전은가장높은빈도를보이며, 사이질세포 (interstitial cell) 염증과유리기 (free radical) 로인한세뇨관손상을일으키고, tumor necrosis factor(tnf) 계열의인자와이들의생물학적활성인자인 nuclear factor-kappa B(NFκB) 가관여하여세뇨관세포의사멸을일으키는것으로알려졌다 [1, 13]. 최근에는, TNF 계열의사이토카인 (cytokine) 인 TNF-like weak inducer of apoptosis(tweak) 가수용체인 Fn14 와상호작용하여콩팥사이질에염증을유도하여급성신부전의진행에관여하는것으로보고되었다 [9]. Klotho 는노화방지와연관이있는단백질로수명, 불임증, 동맥경화증, 골다공증, 피부위축등의퇴행성질병과관련이있으며타장기보다콩팥에서주로발현되는것으로알려졌다 [10, 13]. 최근에는이러한 Klotho 의발현이콩팥손상시감소하는양상을나타내는것으로보고되었으며이러한경향은만성신부전동물모델에서도확인되었다 [4, 7, 8, 11, 19]. 또한노화에따른콩팥손상모델에서도활성산소의증가와함께 Klotho 의발현이감소하는것으로보고된바있다 [21]. 이처럼콩팥손상과 Klotho 의발현정도에대한연구는보고된바있지만콩팥손상시손상부위와관련하 *Corresponding author Tel: , Fax: jyjung@cnu.ac.kr The first two authors contributed equally to this work. 225

2 226 박소라 김태원 김영중 김현태 류시윤 정주영 여 Klotho 와사이토카인의콩팥내발현위치에따른연구는알려지지않았다. 따라서본실험을통해시스플라틴으로유발된급성신부전에서면역염색과 Western blot 을통해 Klotho 와급성신부전의진행에관여하는사이토카인인 TNFα, NF-κB, TWEAK 의발현위치및부위별발현양상에대해알아보고자한다. 재료및방법 실험동물본실험에서는 4 주령된 SPF Sprague-Dawley 수컷래트 ( 오리엔트바이오, 대한민국 ) 18 마리를 1 주간순화한후사용하였으며, 적절한온도, 습도 (22 ± 2 o C, 55 ± 5%, 12 시간광주기 ) 하에사료와식수는자유롭게제공되었으며모든동물은충남대학교실험동물센터에서사육되었다. 순화과정을거친래트는군당 6 마리씩세개의군으로나누었다. 첫번째군은대조군으로증류수를복강내투여한후 5 일뒤에부검을하였고, 두번째와세번째군은시스플라틴 (5 mg/ kg; Sigma, USA) 을복강내주입한각각 5 일과 10 일뒤에부검을하였다. 모든실험은충남대학교동물실험윤리위원회의규정에따라수행되었다. 콩팥기능측정래트의미정맥을통해채혈한후 4 o C 에서 30 분간거치한후 2,000 g 에서 10 분간원심분리하여상층액을분리했다. 수거한혈청을자동혈청분석기를이용하여혈청내의혈중요소질소수치와크레아티닌수치를분석하였다 (IDEXX Laboratories, USA). 조직보존부검에앞서래트의마취를위해 Tiletamine 과 Zolazepam 의합제 (30 mg/kg; Virbac, Carros, FRA) 를복강투여한후복대동맥을통한 PBS 관류로방혈을마친후 periodate lyine paraformaldehyde(plp) 로 5 분간관류하여콩팥을고정하였다. 적출된콩팥을 1~2 mm 의시상절편으로잘라 4 o C PLP 에서하루동안후고정뒤조직탈수과정을거쳐 polyester wax 로포매한후 microtome 을이용하여 5μm 로박절하여슬라이드절편을만들어면역염색과 H&E 염색에사용하였다. Terminal deoxynucleotidyl transferase dutp nick end labeling(tunel) 염색 ApoTag Peroxidase In situ Apoptosis Detection kit (Millipore, USA) 를이용하여 TUNEL 염색을하였다. 조직절편은자일렌을통해 polyester wax를제거하였고, 단계농도별알코올을이용하여함수시킨후 proteinase-k(20 μg/ ml; Millipore, USA) 를 15분간처리하여 PBS로 2회씻어낸후 equilibration buffer를 10초간, working-strength TdTenzyme을 37 o C에서 1시간동안처리하였다. 추후 stopwash buffer 를 10 분간처리하여반응을멈추고, PBS 로씻어낸뒤 anti-digoxigenin-peroxidase 로 30 분간처리후 3,3 - diaminobenzidin(dab) 을이용해발색하였다. 발현의분석은광학현미경 (Nikon eclipse 80i; Nikon, Japan) 을이용하여 100 배확대한후조직의겉질 (cortex), 바깥속질 (outer medullar), 속속질 (inner medullar) 에서각각염색된세포수대총세포수의비율을계산하여측정하였다. 면역조직화학염색조직절편을자일렌을이용하여 polyester wax를제거하고농도별알코올을이용하여함수시킨후 0.5% 트리톤 X-100 에 30분간처리하고 PBS를이용하여세척하였다. 비특이성결합부위는 PBS와 1:10으로희석된말혈청 (normal horse serum) 또는염소혈청 (normal goat serum) 을 1시간처리하여차단한후 Klotho(1 : 100; Abcam, UK), TNF-α(1 : 50; Abcam). TWEAK(1 : 50; Abcam), NF-κB(1 : 300; Abcam) 를 4 o C에서하루동안처리하였다. 추후 2차항체 (anti-mouse IgG, anti-rabbit IgG; Abcam) 처리과정을거친뒤 DAB로발색하고봉입과정을거쳐광학현미경을이용하여관찰하였다. Western blot 분석콩팥부위별분석을위하여적출한콩팥을겉질, 바깥속질, 속속질의부위별로나눈조직샘플을이용하여, 용해 buffer(50 mm Tris-HCl, ph 8.0, with 150 mm sodium chloride, 1% NP-40, 0.5% sodium deoxycholate 및 0.1% sodium dodecyl sulfate, with a protease inhibitor cocktail) 를통해만들어진단백질샘플은 Bradford assay로정량하여실험에사용하였다. 단백질샘플은 6~15% 의 SDS-polyacrylamide gels을사용하였고, nitrocellulose membrane을이용하여전사하였다. 전사된막 (membrane) 은 5% skim milk (TBS-T) 로비특이성단백질을차단하였고각각의 1차항체 TNF-α(1 : 500), TWEAK(1 : 300), NF-κB(1 : 1000), Klotho (1 : 500) 를 4 o C에서하루동안처리하였다. TBS-T로세척을한막에 2차항체 (anti-mouse IgG, anti-rabbit IgG) 를 1시간처리한뒤, 항체와결합한막을 chemiluminescence system (Bio-Rad Laboratories, USA) 으로측정, Image Lab software (Bio-Rad Laboratories) 로분석하였다. 통계분석모든실험결과는 mean values ± standard error of the mean(sem) 으로나타내었으며통계분석은 Dunnet-test 를따르는 one-way ANOVA 를사용하였다. 통계적유의성은 p < 0.05 일경우유의한차이가있는것으로평가하였다. 결 콩팥기능평가각군의혈청혈중요소질소 (blood urea nitrogen) 와크레아 과

Cisplatin 유도급성신부전에서 Klotho 단백질의발현 227 Fig. 1. The levels of plasma blood urea nitrogen and Creatinine concentration after cisplatin injection (5 mg/kg).

3 Cisplatin 유도급성신부전에서 Klotho 단백질의발현 227 Fig. 1. The levels of plasma blood urea nitrogen and Creatinine concentration after cisplatin injection (5 mg/kg). The rats (n = 6/ group) were sacrificed 5 (5D) and 10 days (10D) after cisplatin (5 mg/kg) intraperitoneal injection. Values are expressed as mean ± SEM of 3~4 independent experiments, * p < 0.05, a significant difference in comparison with the control group. Fig. 2. Hematoxylin & Eosin stained kidney tissue from the cisplatin injected rat ( 400). The rats (n = 6/group) were sacrificed 5 (5D) and 10 days (10D) after cisplatin (5 mg/kg) intraperitoneal injection. The control group showed the normal morphology with well-preserved brush border membranes. In the 5D group, distinctive tubular epithelial desquamation (arrow) and intraluminal cast formation (*) were observed. In 10 days after cisplatin treated group, histopathological alterations were ameliorated than that of the 5 days group. Co: cortex, OM: outer medullar, IM: inner medullar. Scale bar = 50 μm. 티닌 (creatinine) 의농도는 Fig. 1 에서와같이나타났다. 시스플라틴투여후 5 일이경과한군의혈중요소질소와혈청크레아티닌의수치는 ± 26.1 mg/dl 과 7.78 ± 0.91 mg/dl, 10 일이경과한군에서는 ± mg/dl 과 3.46 ± 1.37 mg/dl 로대조군과비교했을때유의성있게증가했으나, 시스플라틴투여후 10 일째에는 5 일째와비교하여약 50% 의수치가감소해서대조군과비교하여유의성이있을뿐 5 일 째와는통계학적유의성이관찰되지않았다. 콩팥조직의병리조직학적형태의변화는 H&E 염색으로확인하였다 (Fig. 2). 대조군의콩팥조직은정상의조밀한세포질과함께정상의형태를보였으나, 시스플라틴투여후 5 일이지난콩팥에서겉질과바깥속질부위에서세뇨관의팽창, 표피의박리, 사멸세포등을관찰할수있으며, 속속질에서는내강의폐색이나타났다. 이러한형태적변화는시스

228 박소라 김태원 김영중 김현태 류시윤 정주영 Fig. 3. Terminal deoxynucleotidyl transferase dutp nick end labeling (TUNEL) stained tissue from the cisplatin injected rat ( 400).

In the control group, TUNEL-positive cells were rarely expressed, whereas in the 5D group, TUNEL-positive cell was increased in all region including, Co, OM, and IM if compared to the control group.

4 228 박소라 김태원 김영중 김현태 류시윤 정주영 Fig. 3. Terminal deoxynucleotidyl transferase dutp nick end labeling (TUNEL) stained tissue from the cisplatin injected rat ( 400). The rats (n = 6/group) were sacrificed 5 (5D) and 10 days (10D) after cisplatin (5 mg/kg) intraperitoneal injection. In the control group, TUNEL-positive cells were rarely expressed, whereas in the 5D group, TUNEL-positive cell was increased in all region including, Co, OM, and IM if compared to the control group. In the 10D group, the expression of TUNEL positive cells were decreased in Co and OM region compared to 5D group without significant differences. Scale bar = 50 μm. 플라틴투여후 10 일이경과한조직에서는 5 일의조직에비해적게관찰되었다. 대조군의콩팥에서 TUNEL 양성세포, 즉자가사멸한세포는겉질, 바깥속질, 속속질의전부위에서거의찾아볼수없었다. 시스플라틴투여후 5 일째조직에서는겉질과바깥속질에서 TUNEL 양성세포수가크게증가했다. 반면, 시스플라틴투여후 10 일이지난콩팥에서는그양상이감소하였으며특히, 바깥속질부위에서현저한감소세를보였으나 5 일이지난군과통계학적유의성은없었다 (Fig. 3; p > 0.05). Klotho 와 NFκB, TNFα, TWEAK 의발현 Klotho 의발현을면역조직화학염색과 Western blot 을통하여발현부위별차이를알아보았다. 면역염색상에서정상콩팥의 Klotho 는주로겉질부위에서발현하며, 먼쪽곱슬세관 (distal convoluted tubule) 과연결세관 (connecting tubule) 에서발현하는것을확인하였고속속질부위의집합관세포 (collecting duct) 에서도발현됨을확인하였다 (Fig. 4). 이러한 Fig. 4. Immonohitochemical staining for the Klotho expression in the cisplatin-treated rat kidney ( 200). The rats (n = 6/group) were sacrificed 5 (5D) and 10 days (10D) after cisplatin (5 mg/kg) intraperitoneal injection. The positive stained cell was decreased in the 5D group at Co, OM, and IM region. In the 10D group, decreased expression was slightly recovered compared to the 5D group. OSOM: outer strip outer medulla, ISOM: inner strip outer medulla. Scale bar = 100 μm. 발현은시스플라틴투여후 5 일째에대조군과비교하면겉질부위의먼쪽곱슬세관과연결세관에서감소하며속질부위의집합관세포에서도감소하는것을확인하였다. 핵에집중되어있던염색상이옅어짐을관찰할수있었고시스플라틴투여 10 일후의조직에서는 5 일째의조직에서감소했던발현이증가한것을확인할수있었다. Western blot 에서도부위에따른차이는있었지만시스플라틴투여후에 Klotho 의발현이감소했음을확인할수있었다 (Fig. 5). 그러나겉질과바깥속질에서는 Klotho 의발현이감소하였지만속속질부위는시스플라틴투여후 5 일이지난콩팥에서그발현이감소하지않았다. NF-κB, TNF-α, TWEAK 는 Klotho 와는반대의발현양상을나타냈다. Klotho 의발현과는다르게대조군의먼쪽곱슬세관과연결세관에서발현이크게나타나지않았다. 면역염색에서 NF-κB 는시스플라틴투여후 5 일이된콩팥의토리쪽곱슬세관 (proximal convoluted tubule) 의핵에서그염색상을관찰할수있었으며 TNF-α 는대조군에서토리쪽곱슬세관의세포질의기저막 (basal membrane) 에서발현이나타났다. 또한시스플라틴투여 5 일째에서가장높은염색상을나타냈다. 10 일째에서는 5 일째보다발현이감소했다. TWEAK 은대조군의시스플라틴투여 5 일째와투여 10 일째

Cisplatin 유도급성신부전에서 Klotho 단백질의발현 229 Fig. 5. Western blot analysis for the Klotho, NF-κB, TNF-α and TWEAK expression in the cisplatin-treated rat kidney.

5 Cisplatin 유도급성신부전에서 Klotho 단백질의발현 229 Fig. 5. Western blot analysis for the Klotho, NF-κB, TNF-α and TWEAK expression in the cisplatin-treated rat kidney. The rats (n = 6/group) were sacrificed 5 (5D) and 10 days (10D) after cisplatin (5 mg/kg) intraperitoneal injection. Values are expressed as mean ± SEM for triplicate experiments, * p < 0.05, a significant difference in comparison with the control group, # p < 0.05, a significant difference in comparison with the 5D group. 의발현에서차이는보이지않았으나, 토리쪽곱슬세관의핵에서대조군보다는발현이증가했으며 (Fig. 6), Western blot 결과에서도사이토카인의발현은시스플라틴투여후 5 일째에증가하며 10 일째에는감소하는양상을보였다. 세부부위별로살펴보면 NF-κB 는시스플라틴투여 5 일째의전부위에서많이증가했고, TNF-α 는시스플라틴투여 5 일째군에서겉질보다바깥속질과속속질에서증가하였고 10 일째에비교적감소하는것으로나타났다. 하지만, TWEAK 의발현은시스플라틴투여 5 일째에전부위에서증가하였으며, 증가한발현양상은시스플라틴을투여한뒤 5 일과 10 일이지난콩팥에서유의성이관찰되지않았다 (Fig. 5). 고 과량의시스플라틴은세뇨관세포에직접손상을입혀산화스트레스를유발하고활성산소를증가시켜세포사멸을일으키며연쇄적인염증반응에인한콩팥손상으로신부전을발생시키는것으로알려졌다 [1, 12]. 본실험에서는혈중요소질소과크레아티닌의농도를측정하여콩팥기능의저하여부를확인하였다. 혈중요소질소와크레아티닌은콩팥의상태를나타내는지표로, 혈중요소질소는 7~21 mg/dl, 크레아티닌은 0.7 ± 0.13 mg/dl 의정상범위를가진다. 콩팥손상으로인하여사구체여과율과콩팥혈류가감소하면혈중요소질소와혈청크레아티닌의수치는증가하게된다. 본실험에서시스플라틴투여후 5 일이경과한군의혈중요소질소과 찰 Fig. 6. Immonohitochemical staining for the NF-κB, TNF-α and TNF-like weak inducer of apoptosis (TWEAK) expression in the cisplatin-treated rat kidney ( 200). The rats (n = 6/ group) were sacrificed 5 (5D) and 10 days (10D) after cisplatin (5 mg/kg) intraperitoneal injection. Positive stained cell (arrows) was shown in the proximal tubules (star) from the S3 segments of proximal tubules of OSOM. Scale bar = 100 μm. 크레아티닌의수치는대조군과비교했을때각각 7 배가량증가하였다. 또한시스플라틴투여후 5 일째의조직에서병리조직학적인변화와함께자가사멸한세포를 H&E 와 TUNEL 염색을통해서확인하였다. 위와같은손상은시스플라틴투여후 10 일이지난조직을통해그손상이감소하였음을확인하였다. 최근연구에따르면 Klotho 의발현은허혈로인한콩팥손상시급격하게감소하였다가손상이회복됨에따라복구되는것으로밝혀졌다 [5]. 이렇듯 Klotho 가콩팥손상과회복시빠르게반응하여발현이감소했다가증가하는특성으로말미암아콩팥손상의지표로활용하는방법도연구중이다 [6]. 또한허혈모델에서 Klotho 가콩팥세포의자가사멸을줄여주는것으로밝혀졌으며 Klotho 를제거한마우스에서콩팥에손상을주었을경우그손상의정도가 wildtype 에비해크게나타나는것으로 Klotho 의손상에따른보호효과적인측면또한연구되고있다 [14, 20]. Klotho 는정상적으로콩팥의세뇨관세포에서발현된다고알려졌으며정상콩팥의먼쪽곱슬세관에서높게발현하고, 토리쪽곱슬세관에서는상대적으로낮게발현하는것으로보고된바있다 [6]. 본실험에서면역염색실험결과, Klotho 의발현은겉질의연결세관, 먼쪽곱슬세관및토리일부에서도나타남을확인하였으며, 바깥속질과속속질의집합관세포에서도발현이나타났다. 하지만 Western blot 의결과상속속질부위의 Klotho 의발현은시스플라틴투여후에도감소하지않았다.

6 230 박소라 김태원 김영중 김현태 류시윤 정주영 이는정상적으로요가농축되는속속질부위의특성상정상상태에서도삼투로인한지속적인손상을받고있어 Klotho 의발현이다른부위와는다르게발현이된것으로추측된다. 급성신부전에서시스플라틴은 TNF-α 와같은사이토카인을유도하며 [15, 17], 세포의분화, 증식을조절하는 TWEAK 및 NF-κB 와세뇨관세포에작용하여콩팥손상을일으킨다고알려졌다 [4, 9, 17, 18]. 또한, 염증으로인한콩팥의손상시 TWEAK 와 TNF-α 가 NF-κB 를통해콩팥의 Klotho 를감소시킨다는연구가보고된바있다 [13]. 본실험을통해서면역염색과 Western blot 을통한단백질발현양상을관찰한결과, Klotho 발현은사이토카인의발현양상과반대의경향을나타낸다는이전의연구와일치하는것을확인하였다. 또한면역염색상에서시스플라틴의특징적인손상부위인바깥속질바깥줄무늬의토리쪽곱슬세관토리쪽 3 분절부위 (s3 segment of proximal tubule in the outer stripe of outer medulla) 에서염증성사이토카인의발현이가장활발함을확인하였으며, 염증성사이토카인은 Klotho 의발현이감소한 5 일째에증가하고손상이수복된것으로추정되는 10 일째에는그발현이감소하였다. TWEAK 는 TNF 와기능적인측면은유사하지만 TNF 에비해상대적으로더넓게발현되는것으로알려졌다 [2]. 본실험에서 5 일째에증가한 TWEAK 의발현은 10 일째군에서도그발현이지속되었다. 본연구를통해, 시스플라틴에의한콩팥손상시발생하는염증반응과 Klotho 발현양상은관련이있으며, 손상이생긴후수복됨에따라손상부위에서의 Klotho 발현이부위별로복구되는것을확인하였다. 추후 Klotho 의발현기전에대한추가연구를통해콩팥손상에따른방어기전으로서 Klotho 의새로운시각을제공할수있을것이다. 감사의글 본연구는충남대학교학술연구비지원에의해이루어졌습니다. References 1. Arjumand W, Seth A, Sultana S. Rutin attenuates cisplatin induced renal inflammation and apoptosis by reducing NFκB, TNF-α and caspase-3 expression in wistar rats. Food Chem Toxicol 2011, 49, Chicheportiche Y, Bourdon PR, Xu H, Hsu YM, Scott H, Hession C, Garcia I, Browning JL. TWEAK, a new secreted ligand in the tumor necrosis factor family that weakly induces apoptosis. J Biol Chem 1997, 272, Fuertes MA, Castilla J, Alonso C, Pérez JM. Cisplatin biochemical mechanism of action: from cytotoxicity to induction of cell death through interconnections between apoptotic and necrotic pathways. Curr Med Chem 2003, 10, Gao HX, Campbell SR, Burkly LC, Jakubowski A, Jarchum I, Banas B, Saleem MA, Mathieson PW, Berman JW, Michaelson JS, Putterman C. TNF-like weak inducer of apoptosis (TWEAK) induces inflammatory and proliferative effects in human kidney cells. Cytokine 2009, 46, Hu MC, Kuro-o M, Moe OW. Klotho and chronic kidney disease. Contrib Nephrol 2013, 180, Hu MC, Moe OW. Klotho as a potential biomarker and therapy for acute kidney injury. Nat Rev Nephrol 2012, 5, Izquierdo MC, Perez-Gomez MV, Sanchez-Niño MD, Sanz AB, Ruiz-Andres O, Poveda J, Moreno JA, Egido J, Ortiz A. Klotho, phosphate and inflammation/ageing in chronic kidney disease. Nephrol Dial Transplant 2012, 27 (Suppl 4), iv Izquierdo MC, Sanz AB, Sánchez-Niño MD, Pérez-Gómez MV, Ruiz-Ortega M, Poveda J, Ruiz-Andrés O, Ramos AM, Moreno JA, Egido J, Ortiz A. Acute kidney injury transcriptomics unveils a relationship between inflammation and ageing. Nefrologia 2012, 32, Justo P, Sanz AB, Sanchez-Niño MD, Winkles JA, Lorz C, Egido J, Ortiz A. Cytokine cooperation in renal tubular cell injury: the role of TWEAK. Kidney Int 2006, 70, Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y, Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, Shiraki-Iida T, Nishikawa S, Nagai R, Nabeshima Y. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 1997, 390, Mezzano S, Aros C, Droguett A, Burgos ME, Ardiles L, Flores C, Schneider H, Ruiz-Ortega M, Egido J. NF-κB activation and overexpression of regulated genes in human diabetic nephropathy. Nephrol Dial Transplant 2004, 19, Miller RP, Tadagavadi RK, Ramesh G, Reeves WB. Mechanism of cisplatin nephrotoxicity. Toxins (Basel) 2010, 2, Moreno JA, Izquierdo MC, Sanchez-Niño MD, Suárez- Alvarez B, Lopez-Larrea C, Jakubowski A, Blanco J, Ramirez R, Selgas R, Ruiz-Ortega M, Egido J, Ortiz A, Sanz AB. The inflammatory cytokines TWEAK and TNFα reduce renal Klotho expression through NFκB. J Am Soc Nephrol 2011, 22, Panesso MC, Shi M, Cho HJ, Paek J, Ye J, Moe OW, Hu MC. Klotho has dual protective effects on cisplatin-induced acute kidney injury. Kidney Int 2014, 85, Ramesh G, Reeves WB. TNFR 2 -mediated apoptosis and necrosis in cisplatin-induced acute renal failure. Am J Physiol Renal Physiol 2003, 285, F Remuzzi G, Ruggenenti P, Benigni A. Understanding the nature of renal disease progression. Kidney Int 1997, 51, Sanz AB, Justo P, Sanchez-Niño MD, Blanco-Colio LM, Winkles JA, Kreztler M, Jakubowski A, Blanco J, Egido J, Ruiz-Ortega M, Ortiz A. The cytokine TWEAK modulates renal tubulointerstitial inflammation. J Am Soc Nephrol 2008, 19, Sanz AB, Sanchez-Niño MD, Izquierdo MC, Jakubowski A, Justo P, Blanco-Colio LM, Ruiz-Ortega M, Selgas R, Egido J, Ortiz A. TWEAK activates the non-canonical NFκB pathway in murine renal tubular cells: modulation of CCL21. PLoS One 2010, 5, e Schmitt R, Cantley LG. The impact of aging on kidney repair. Am J Physiol Renal Physiol 2008, 294, F

7 Cisplatin 유도급성신부전에서 Klotho 단백질의발현 Sugiura H, Yoshida T, Tsuchiya K, Mitobe M, Nishimura S, Shirota S, Akiba T, Nihei H. Klotho reduces apoptosis in experinental ischaemic acute renal failure. Nephrol Dial Transplant 2005, 20, Zuo Z, Lei H, Wang X, Wang Y, Sonntag W, Sun Z. Agingrelated kidney damage is associated with a decrease in klotho expression and an increase in superoxide production. Age 2011, 33,

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α α α α α α α α 太陰調胃湯加減方 dbdb 마우스 肝에 대한 아디포사이토카인 및 발현에 미치는 영향 SREBPs 와 섞어서 하고 마커 또한 하여 전기 영동을 하였다 전기영동을 한 후에 에 를 쪼여서 각 를 확인하였다 이 를 프로그램을 이용해 수치화하 여 분석하였다 肝 조직 동결 절편 분리한 조직은 로 시간 동안 고정 시킨 후 에 세척한 후 물기를