대한체질인류학회지제 30 권제 3 호 Korean J Phys Anthropol Vol. 30, No. 3 (2017) pp. 87~98 https://doi.org/10.11637/kjpa.2017.30.3.87 Original Article 저칼륨혈증흰쥐신장에서 Akt, p-akt, ERK 및 p-erk 단백발현의변화 배춘상, 조혜정, 안규윤 전남대학교의과대학해부학교실 (2017 년 6 월 15 일접수, 2017 년 8 월 10 일수정접수, 2017 년 8 월 10 일게재승인 ) 간추림 : 저칼륨혈증은신장의형태학적변화와대사성알칼리증을유발시키는것으로알려져있다. 이전결과들은저칼륨혈증의병태생리학적변화에 K + 평형조절이온채널, pump 유전자, NF-E2-related factor 2 (Nrf2) 전사유전자를포함한다양한유전자가관여할것이라는가능성을제시하였다. 이에본연구는칼륨제한식이기간에따른흰쥐신장내 Akt와 p-akt 및 ERK와 p-erk의발현및분포의변화를 Western 분석과면역조직화학방법으로관찰함으로써, 저칼륨혈증이신장에서 AKT/ERK 인산화에영향을미치는지를확인하였다. Western 분석소견에서 Akt 및 p-akt 단백질발현은칼륨제한식이가진행될수록증가하는양상을보였으며, ERK 및 p-erk 단백질발현은칼륨제한식이 2주군에서정상식이군에비해약간증가하는양상을보였다. 면역조직화학소견에서 Akt 단백의면역반응성은먼쪽곱슬세관, 겉질곧은부분및속질곧은부분에서중등도의발현을보였다. 칼륨제한식이군의 Akt의면역반응성은칼륨제한식이가진행될수록바깥속질집합관에서현저히증가하였다. 칼륨제한식이군의 p-akt의면역반응성은칼륨제한식이 2주군의먼쪽곱슬세관, 치밀반점과속질곧은부분에서증가하였고토리쪽곱슬세관에서는중등도의발현을보였다. 칼륨제한식이군의 ERK의면역반응성은칼륨제한식이 2주군과 3주군의바깥속질집합관에서현저히증가하였으며먼쪽곱슬세관, 겉질집합관에서는증등도의증가를보였다. 칼륨제한식이군의 p-erk의발현부위는정상식이군과비교해차이가없었으나면역반응성은칼륨제한식이 2주군의바깥속질집합관의핵에서현저히증가하였다. 이상의결과저칼륨혈증시 p-akt의발현은칼륨제한식이가길어질수록점진적으로증가하였지만, p-erk의발현은칼륨제한식이 2주군에서현저히증가되었다. 따라서저칼륨상태에서의 Akt 및 ERK 인산화의촉진은이온채널및이온수송체유전자조절뿐만아니라세포내신호전달에있어중요한역할에관여할것임을시사해주었다. 찾아보기낱말 : 저칼륨혈증, Akt, p-akt, ERK, p-erk, Western blot, 면역조직화학 서 론 K + 는세포내주요양이온으로세포막에걸쳐세포와 * 본논문은 2010~2011 학년도전남대학교병원의생명연구원학술연구비지원에의해이루어졌으며지원에감사드립니다. 저자 ( 들 ) 는 의학논문출판윤리가이드라인 을준수합니다. 저자 ( 들 ) 는이연구와관련하여이해관계가없음을밝힙니다. 교신저자 : 안규윤 ( 전남대학교의과대학해부학교실 ) 전자우편 : kyahn@jnu.ac.kr 전기적기울기의삼투평형을유지하는데매우중요하다 [1]. 또한 K + 는세포성장과분열, 산 -염기평형, 많은효소반응, 세포용적조절, 전기적인세포분극, 그리고근수축을포함한세포기능에중요한역할을담당하고있어모든세포의생리에필수적이다. 체내 K + 균형이정상인상태에서 K + 배설은섭취에따라조절되는데, 약 90% 는신장에의해배설되고, 주로소변이집합관을지나면서 K + 의분비가이루어지며이과정은알도스테론과 Na + 의이동에의해조절된다 [2]. K + 결핍은혈청 K + 농도가 3.0 meq/l 미만 c 2017 Korean Association of Physical Anthropologists This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. ISSN 2287-626X (Online) ISSN 1225-150X (Print)
88 배춘상, 조혜정, 안규윤 인경우를말하며전해질장애로암모니아대사증가, 산 - 염기평형장애, 대사성알칼리증을일으킨다 [3-5]. 이러한 암모니아대사는산분비및염기생성과밀접하게관련되어있으며산 -염기평형을조절하는주요기전으로작용한다 [6]. 만성적인 K + 결핍은신장세관의손상과사이질조직섬유화, 신장세관에서의 Osteopontin 발현증가및손상부위로의염증세포침윤을유발한다 [7]. 임등 [8] 은저칼륨혈증신장에서 H/K-ATPase, Na/K-ATPase, NHE-3 및 NBC-1에대한 inhibitor-sensitive 이온수송체의유전자발현이증가하며 Nrf2 mrna 단백질의발현이증가되어 K + 평형조절에이온채널이나 Nrf2 전사유전자를포함한다양한유전자가관여할것이라고하였다. Akt는 protein kinase B (PKB) 로알려져있는 serine/ threonine protein kinase로 phosphotidylinositol 3-kinase (PI3K)/Akt 신호전달경로이다. PI3K/Akt 신호전달경로는다양한성장인자및조절인자에의해활성화되어세포성장및생존, 역전사, 번역, 세포증식, 이동그리고세포주기를조절하는중요한역할을한다 [9-11]. PI3K 경로내에서신호가전달되면 Akt는세포질쪽에서세포막쪽으로이동하여구조적인변화를일으키고, 두개의아미노산이노출되어인산화되는데이는 Akt의활성화에필요하다. 두아미노산중하나는 Akt1의 kinase domain에위치하는 308번 threonine으로서 phosphoinositide-dependent kinase 1 (PDK1) 에의해인산화되어활성고리 (activation loop) 의안정화에관여하고, 다른하나는 Akt1의 C-terminal domain에위치하는 473번 serine으로서 PDK2에의해인산화되어 Akt의활성화에관여한다 [12]. 이러한 Akt 활성화는 insulin 수용체를통해 PI3K/Akt 경로를자극하고신장의사구체비대및세포사멸을초래하여당뇨병성신장에서 Akt의인산화를증가시키고 [13], AQP-2 수분통로단백조절을위한또다른신호전달경로로제안되어신장세포의 1차배양에서 AQP-2 발현증가를통해 PI3K/Akt 경로를자극한다고한다 [14]. Mitogen-activated protein kinase (MAPK) 는 extracellular regulated protein kinase (ERK), c-jun N-terminalkinase (JNK) /stress-activated protein kinase (SAPK), serine/ threonine protein kinase인 p38 MAPK로분류되며세포의분화, 발생, 세포신호전달등의다양한세포기능을담당하는것으로알려져있다 [15]. 이중 ERK는성장인자, mitogen 등에의해서활성화되며세포유사분열및생존, 세포증식, 분화에관여한다 [16]. p38 MAPK는일반적으로세포스트레스와 proinflammatory cytokine에의해활성화되어염증반응에중요한역할을하며 apoptosis 등에관여한다 [17]. Balbi 등 [18] 은생쥐신장의발생과정에관여하 는안지오텐신 II 수용체의발현과 MAPK 발현의연관성을관찰함으로써안지오텐신 II의작용이 MAPK 경로에의해서조절된다고하였다. 또한신장의산화적스트레스는 ERK와 JNK 경로를자극하여신장세포의대사를조절하며 [19], MAPK 경로중에하나혹은여러개가저해되면신장세포암의성장이억제된다고한다 [20]. 이와같이 MAPK는신장조직에서다양한세포작용에관여하며, 흰쥐의토리쪽세관세포에서저농도 ouabain는 ERK 의존성경로를통해 Akt의인산화를자극하고세포증식을유도한다고한다 [21]. 또한저칼륨혈증흰쥐신장에서는 Nrf2 단백질의발현이증가되며 [22], Nrf2의인산화활성조절요인으로 MAPK, protein kinase C, PI3K 등이있으며, 활성산소종을생성하는여러약제나항생제들에유도된산화적스트레스는 MAPK 신호전달계의활성화를초래한다고한다. 이상의보고를감안해볼때 Akt와 p-akt 및 ERK 와 p-erk의단백발현변화는저칼륨혈증시신장의이온수송체유전자조절뿐만아니라세포내신호전달에있어중요한역할을할것으로생각된다. 이에본연구는칼륨제한식이기간에따른흰쥐신장내 Akt와 p-akt 및 ERK와 p-erk의발현및분포의변화를 Western 분석및면역조직화학적방법으로관찰하고자하였다. 재료및방법 1. 실험동물본실험에사용된동물은체중 230 g 내외의성숙 Sprague-Dawley계숫컷흰쥐 30마리로다섯군으로구분하였다. 제1군은정상식이 (150 meq K + /kg, TD88082, Harlan Teklad, U.S.A.) 를, 제2군은칼륨제한식이 (potassium-free diet, TD88081, Harlan Teklad, U.S.A.) 3일, 제3군은칼륨제한식이 1주, 제4군은칼륨제한식이 2주, 제5군은칼륨제한식이 3주로식이적응을시켰다. 칼륨제한식이군은먹이섭취가변화될것으로여겨져날마다적은양을먹은군의양으로먹이를조절하였다. 2. 단백분리와 Western 분석칼륨제한식이기간에따른신장조직을적출하여액체질소에담가급속동결시킨후, 조직의일부를 Nonidet P- 40 buffer (150 mm NaCl, 1 mm benzamidine, 50 mm Tris ph 8.0, Trypsin inhibitor 1 μg/ml, 5 mm EDTA ph 8.0, 1 mm PMSF, NP-40 1%) 에넣고 homogenizer로분쇄하
저칼륨혈증 Akt, p-akt, ERK 및 p-erk 단백발현 89 여균질액을만들어얼음속에 30분간방치하였다. 균질액은 3,000 rpm으로 15분간원심시켜상층액을분리한다음다시 15,000 rpm으로 30분간원심하고단백농도는 spectrophotometer로정량화하였다. 추출한총단백 100 μg을 8% polyacrylamide gel에전기영동한다음 nitrocellulose membrane에 4 에서 20 ma로 12시간전이하였다. Membrane은 TBS-T buffer (20 mm Tris, 150 mm NaCl, 1 M HCl, ph 7.6, 0.1% Tween 20) 에 5% skim milk가첨가된 blocking buffer에실온에서 2시간동안처리한후 1 차항체 Akt (Santa Cruz Biothchnology, California, USA; sc-1619, 1 : 3,000), p-akt (Santa Cruz Biothchnology, California, USA; sc-16646r, 1 : 3,000), ERK (Cell Signaling Technology, Danvers, MA, USA; 9102, 1 : 3,000), p-erk (Cell Signaling Technology, Danvers, MA, USA; 9101, 1 : 3,000) 에 4 에서 14시간반응시켰다. 다시 TBS-T로 10 분씩 3번수세하고 peroxidase conjugated goat anti-rabbit IgG (Santa Cruz, USA; sc-2004, 1 : 4,000) 로부치하였다. TBS-T로 3번수세하고발색제인 ECL용액 (Amersham, USA) 에 5분간반응시킨후시간별로현상하였다. 3. 면역조직화학염색동물을 pentobarbital sodium (50 mg/kg, ip) 으로마취하여복강을노출시킨후대동맥을통해 phosphate buffered saline (PBS; 137 mm NaCl, 2.7 mm KCl, 4.3 mm Na 2HPO 4, 1.4 mm KH 2PO 4) 과 paraformaldehyde-lysine-periodate (PLP) 고정액으로관류고정시켰다. 상기고정액에 16시간동안 4 고정후 PBS로 3회세척하고 ethanol로탈수과정을거친후 wax (polyethylene glycol 400 distearate, Polysciens Inc. Warrington, PA, USA) 포매하였다. 포매된조직은회전식절편기로 6 μm 절편을얻어면역조직화학실험에이용하였다. 면역조직화학반응에사용된일차항체는 Akt (Santa Cruz Biothchnology, California, USA; sc-1619, 1 : 800), p-akt (Santa Cruz Biothchnology, California, USA; sc-16646r, 1 : 400), ERK (Cell Signaling Technology, Danvers, MA, USA; 9102, 1 : 200), p-erk (Cell Signaling Technology, Danvers, MA, USA; 9101S, 1 : 400) 였다. 면역조직화학염색은 Vector ABC Kit (Vector Laboratories, California, USA) 를이용하였다. 즉, 탈왁스과정을거친절편은내재성 peroxidase 활성을제거하기위해 60% methanol에녹인 3% H 2O 2 에 30분부치시킨다음 PBS로세척하였다. 5% normal horse serum, 5% normal goat serum, 10% normal goat serum으로 30분간반응시켜주변부의비특이적반응을억제하고일차항체에 4 에서 14~16시간반응시켰다. 반응시킨조직은 PBS로 5분씩 3 번세척하고 biotinylated anti-goat IgG과 biotinylated antirabbit IgG에 30분간부치시킨다음 avidin-biotin conjugate 에 30분간반응시켰다. 반응이끝난절편은 PBS로세척후 3,3-diaminobenzidine tetrahydrochloride (DAB) 으로 7 분간발색시킨다음탈수과정을거친후 permount로봉입하여광학현미경으로관찰하였다. 결과 1. Western 분석 Akt와 p-akt 단백은 60 kda 정도이고칼륨제한식이가진행될수록증가하는양상을보였고, 칼륨제한식이 1주군에서정상식이군에비해증가하기시작하여칼륨제한식이 3주군에최고의발현을보였다. ERK 단백은 42,44 kda 정도이고칼륨제한식이 2주군에서정상식이군에비해약간증가하였지만나머지칼륨제한식이군에서는정상식이군과유사한발현을보였다. p-erk 단백은 42,44 kda 정도이고칼륨제한식이 2주군에서최고의발현을보였고칼륨제한식이 3주군에서는약간감소하였으나칼륨제한식이 1주군이나정상식이군보다는증가하였다 (Fig. 1). 2. 면역조직화학소견 Akt와 p-akt 및 ERK와 p-erk에대한신장내발현분포를알아보기위해서면역조직화학실험을실시하였다. 일차항체를사용하지않은대조염색에서는대조군과실험군모두에서면역반응성을관찰할수없었다. 정상식이군 Akt 단백의면역반응성은먼쪽곱슬세관, 겉질곧은부분및속질곧은부분에서중등도의발현을보였고, 겉질및바깥속질집합관에서는낮게발현되었다. 칼륨제한식이 Akt의발현부위는정상식이군과비교해차이가없었으나면역반응성은칼륨제한식이 1주군의바깥속질집합관에서증가하기시작하여칼륨제한식이 2주군에서현저히증가하였다. 그외먼쪽곱슬세관, 겉질및속질곧은부분에서는중등도의증가를보였다 (Fig. 2). 정상식이군 p-akt 단백의면역반응성은토리쪽곱슬세관과속질곧은부분에서중등도의발현을보였고, 먼쪽곱슬세관과치밀반점에서는낮게발현되었다. 칼륨제한식이군의 p-akt 의발현부위는정상식이군과비교해차이가없었으나면역반응성은칼륨제한식이 1, 2주군의먼쪽곱슬세관, 치밀반점과속질곧은부분에서높게발현되었고토리쪽곱슬세관에서는중등도의증가를보였다 (Fig. 3).
90 배춘상, 조혜정, 안규윤 Normal LK3D LK1W LK2W LK3W AKT p-akt ERK p-erk β-actin 60 kda 60 kda 44 kda 42 kda 44 kda 42 kda 43 kda A 1.2 B 1.2 1 1 AKT Relative optical density 0.8 0.6 0.4 0.2 p-akt Relative optical density 0.8 0.6 0.4 0.2 0 Normal LK3D LK1W LK2W LK3W 0 Normal LK3D LK1W LK2W LK3W C 1.2 D 1.2 1 1 ERK Relative optical density 0.8 0.6 0.4 0.2 p-erk Relative optical density 0.8 0.6 0.4 0.2 0 Normal LK3D LK1W LK2W LK3W 0 Normal LK3D LK1W LK2W LK3W Fig. 1. Western blotting showed the protein level of Akt, p-akt, ERK, p-erk in Low K + (LK) diet rat kidney. (A) The protein level of Akt was gradually increased in LK3D, LK 1W, 2W, and 3W diet rat kidney in comparison with the normal diet rat kidney. (B) The protein level of p-akt was gradually increased in LK 1W, 2W, and 3W diet rat kidney compared to the normal diet rat kidney. (C) The protein level of ERK was increased in LK 1W, 2W, 3W diet rat kidney compared to the normal diet rat kidney. (D) The protein level of p-erk was markedly increased in LK 2W diet rat kidney compared to the normal diet rat kidney. Data are expressed as mean±s.e.m, and each experiment conducted 3 repeats per conditions. β-actin was used as control. Differences were considered significant at *p<0.05, **p<0.001 (ANOVA one way statistical analysis compared to the normal group) (Normal: normal diet rat kidney, LK3D: Low K + diet rat kidney for 3 days, LK1W: Low K + diet rat kidney for 1 week, LK2W: Low K + diet rat kidney for 2 weeks, LK3W: Low K + diet rat kidney for 3 weeks). 정상식이군 ERK 단백의면역반응성은먼쪽곱슬세관, 바깥속질집합관에서중등도의발현을보였으며, 겉질집합관, 겉질곧은부분및속질곧은부분은낮게발현되었다. 칼륨제한식이군의 ERK의발현부위는정상식이군과비교해차이가없었으나면역반응성은칼륨제한식이 2주군과 3주군의바깥속질집합관에서현저히증가하였다. 그외먼쪽곱슬세관, 겉질집합관및속질곧은부분에서는증등도의 증가를보였다 (Fig. 4). 정상식이군 p-erk 단백의면역반응성은겉질및바깥속질집합관의핵에서중등도의발현을보였고, 먼쪽곱슬세관, 속속질집합관에서는낮게발현되었다. 칼륨제한식이군의 p-erk의발현부위는정상식이군과비교해차이가없었으나면역반응성은칼륨제한식이 1주군은정상식이군과유사하였으나칼륨제한식이 2주군의바깥속질집합관의핵에서현저히증가하였고그
저칼륨혈증 Akt, p-akt, ERK 및 p-erk 단백발현 91 CTX AKT OM A D Normal B E LK1W C F LK2W Fig. 2. Images showed the expression of Akt in Low K + (LK) diet rat kidney. We checked the expression of Akt in kidney using 3,3ʹ-Diaminobenzidine (DAB) staining. (A~C) Renal cortex, (D~F) outer medulla. In normal group, immunoreactivity of Akt was moderately detected in the distal convoluted tubule (DCT) and cortical thick ascending limb (CTAL) and outer medullary thick ascending limb (MTAL), and weakly in cortical collecting duct (CCD) and outer medullary collecting duct (OMCD). The immunoreactivity is prominently increased in outer medullary collecting duct especially in K + -depleted diet 2 weeks (Akt: Akt, CTX: renal cortex, OM: outer medulla, Normal: normal diet rat kidney, LK1W: Low K + diet rat kidney for 1 week, LK2W: Low K + diet rat kidney for 2 weeks. Scale bar: 30 μm, CTAL: cortical thick ascending limb (arrow), MTAL: outer medullary thick ascending limb (asterisk)). 외겉질집합관, 먼쪽곱슬세관에서는중등도의증가를보였다 (Fig. 5). 고찰 PI3K/Akt 및 MARK/ERK 신호전달경로는다양한성장 인자및조절인자에의해활성화되며, 이로인해세포성장및생존, 역전사, 번역, 세포증식, 이동및세포주기를조절하는중요한역할을한다 [9-11]. 또한최근 PI3K/Akt 및 MAPK/ERK 신호전달경로의활성화는악성세포에서세포사멸신호에대한중추적인메카니즘으로생각되었고 [23] 세포의분화, 발생, apoptosis와같은다양한세포반응을조절하는 serine/threonine kinase로알려져있으며이러한
92 배춘상, 조혜정, 안규윤 CTX p-akt OM A D Normal B E LK1W C F LK2W Fig. 3. Images showed the expression of p-akt in Low K + (LK) diet rat kidney. We checked the expression of p-akt in kidney using 3,3ʹ-Diaminobenzidine (DAB) staining. (A~C) Renal cortex, (D~F) outer medulla. In normal group, p-akt is moderately detected in the proximal convolute tubule (PCT) and outer medullary thick ascending limb, and weakly in distal convoluted tubule and macula densa (MD). The expressed localization of p-akt proteins in K + -depleted groups was not different from normal group, but the immunoreactivity is significantly increased in distal convoluted tubule, macula densa and outer medullary thick ascending limb in K + -depleted diet 1 and 2 weeks groups (p-akt: phosphorylation of Akt, CTX: renal cortex, OM: outer medulla, Normal: normal diet rat kidney, LK1W: Low K + diet rat kidney for 1 week, LK2W: Low K + diet rat kidney for 2 weeks. Scale bar: 30 μm, MD: macula densa (arrow), MTAL: outer medullary thick ascending limb (asterisk)). kinase는신장조직에서다양한세포작용과연관성이있는것으로보고되었다. 신장세포의신호전달과정에미치는영향에대한연구는미약하며, Akt와 p-akt 및 ERK와 p-erk 단백발현이저칼륨혈증신장에서신호전달에미치는영향을관찰하고자하였다. 이에본연구는정상및칼륨제한식이기간에따른흰쥐신장에서 Akt와 p-akt 및 ERK와 p-erk 단백발현및분포의변화를알아보고자 Western 분석과면역조직화학적방법을관찰한것이다. Western 분석결과 Akt와 p-akt 단백은 60 kda 정도이고칼륨제한식이가진행될수록증가하는양상을보였고, 칼륨제한식이 1주군에서정상식이군에비해증가하기시작하여칼륨제한식이 3주군에최고의발현을보였다. 면
저칼륨혈증 Akt, p-akt, ERK 및 p-erk 단백발현 93 CTX ERK OM A D Normal B E LK2W C F LK3W Fig. 4. Images showed the expression of ERK in Low K + (LK) diet rat kidney. We checked the expression of ERK in kidney using 3,3ʹ-Diaminobenzidine (DAB) staining. (A~C) Renal cortex, (D~F) outer medulla. In normal group, ERK is moderately detected in the distal convoluted tubule and outer medullary collecting duct, weakly in cortical collecting duct, cortical thick ascending limb and outer medullary thick ascending limb. The immunoreactivity is prominently increased in outer medullary collecting duct especially in K + -depleted diet 2 and 3 weeks (ERK: ERK, CTX: renal cortex, OM: outer medulla, Normal: normal diet rat kidney, LK2W: Low K + diet rat kidney for 2 week, LK3W: Low K + diet rat kidney for 3 weeks. Scale bar: 30 μm. CTAL: cortical thick ascending limb (arrow)). 역조직화학소견에서 Akt 단백의면역반응성은먼쪽곱슬세관, 겉질곧은부분및속질곧은부분에서중등도의발현을보였고, 겉질및바깥속질집합관에서는낮게발현되었다. 칼륨제한식이 Akt의면역반응성은칼륨제한식이 1주군의바깥속질집합관에서증가하기시작하여칼륨제한식이 2주군에서현저히증가하였다. 그외먼쪽곱슬세관, 겉질및속질곧은부분에서는중등도의증가를보였다. 이와같 은소견은저칼륨혈증에서여러이온수송체의유전자가이들부위에서발현하고칼륨제한식이가진행될수록단백발현이점진적으로증가한다는소견과도일치하였다 [24-26]. 정상식이군 p-akt 단백의면역반응성은토리쪽곱슬세관과속질곧은부분에서중등도의발현을보였고, 먼쪽곱슬세관과치밀반점에서는낮게발현되었다. 칼륨제한식이군의 p-akt의면역반응성은칼륨제한식이 1, 2주군
94 배춘상, 조혜정, 안규윤 CTX p-erk OM A D Normal B E LK1W C F LK2W Fig. 5. Images showed the expression of p-erk in Low K + (LK) diet rat kidney. We checked the expression of p-erk in kidney using 3,3ʹ-Diaminobenzidine (DAB) staining. (A~C) Renal cortex, (D~F) outer medulla. In normal group, p-erk is moderately detected in nucleus of cortical and outer medullary collecting duct and weakly in distal convoluted tubule and inner medullary collecting duct. The expressed localization of p-erk proteins in K + -depleted groups was not different from normal group, but the immunoreactivity is prominently increased in the nucleus of outer medullary collecting duct especially in K + -depleted diet 2 weeks (p-erk: phosphorylation of ERK, CTX: renal cortex, OM: outer medulla, Normal: normal diet rat kidney, LK1W: Low K + diet rat kidney for 1 week, LK2W: Low K + diet rat kidney for 2 weeks. Scale bar: 30 μm. DCT: distal convoluted tubule (arrow)). 의먼쪽곱슬세관, 치밀반점과속질곧은부분에서높게발현되었고토리쪽곱슬세관에서는중등도의증가를보였다. 이와같은소견은일측성요관폐쇄후생쥐신장의면역조직화학적소견에서 p-akt 단백이겉질의토리쪽세관, 먼쪽곱슬세관에서발현되었다는결과와도일치하였고 [27], in vivo cryotechnique (IVCT) 기법을이용한저산소증생쥐신장에서 p-akt 단백이토리쪽세관세포질에서발현되 었다는보고 [28] 와도유사하였다. 또한저산소증신장에서 phospho-(ser/thr) PKA/C substrate(p-pk-s), Na/HCO 3 cotransporter-1 (NBC-1) 등의유전자가이들부위에서발현하였다는보고를감안하면 Protein kinase (PKs) 가신장의요세관에작용하여신호전달활성영역에서단백질인산화에관여하였을것으로생각되었다. 칼륨제한식이군의 p-akt 단백발현이칼륨제한식이가길어질수록점진
저칼륨혈증 Akt, p-akt, ERK 및 p-erk 단백발현 95 적으로증가함을보였주었는데, 이는 Western 분석소견과일치하였으며이러한 Akt 활성화는인슐린수용체를통해 PI3K/Akt 경로를자극하고 streptozotocin 당뇨유발흰쥐신장의 p-akt 단백발현을증가시켰다는소견 [29], 신세포암에서활성화된 Akt는세포사멸을억제하고인산화를통해세포증식을촉진하여 p-akt 단백발현이증가한다는소견과도일치하였다 [30]. 이와같이 PI3K/Akt 신호전달경로가신장조직에서다양한성장인자및조절인자에의해활성화되며, 이로인해세포성장및생존, 역전사, 번역, 세포증식, 이동및세포주기에중요한역할을한다는이전의연구를감안하면 [9,10], 저칼륨혈증신장에서 Akt와 p-akt 단백발현이높게나타나활성화되어세포내신호전달조절인자로관여하였을가능성이추측되었다. Western 분석결과 ERK 단백은 42,44 kda 정도이고칼륨제한식이 2주군에서정상식이군에비해약간증가하였지만나머지칼륨제한식이군에서는정상식이군과유사한발현을보였다. p-erk 단백은 42,44 kda 정도이고칼륨제한식이 2주군에서최고의발현을보였고칼륨제한식이 3주군에서는약간감소하였다. 면역조직화학소견에서 ERK 단백의면역반응성은먼쪽곱슬세관, 바깥속질집합관에서중등도의발현을보였으며, 겉질집합관, 겉질곧은부분및속질곧은부분은낮게발현되었다. 칼륨제한식이군의 ERK의면역반응성은칼륨제한식이 2주군과 3주군의바깥속질집합관에서현저히증가하였다. 그외먼쪽곱슬세관, 겉질집합관및속질곧은부분에서는증등도의증가를보였다. 이와같은소견은당뇨유발흰쥐신장에서 ERK 단백이먼쪽곱슬세관과집합관에서발현되었다는보고 [31] 와일측성요관폐쇄후흰쥐신장의면역조직화학소견에서 ERK 단백이속질곧은부분과바깥속질집한관에서발현되었다는소견 [32] 과일치하였다. 정상식이군 p- ERK 단백의면역반응성은겉질및바깥속질집합관의핵에서중등도의발현을보였고, 먼쪽곱슬세관, 속속질집합관에서는낮게발현되었다. 칼륨제한식이군의 p-erk의면역반응성은칼륨제한식이 1주군은정상식이군과유사하였으나칼륨제한식이 2주군의바깥속질집합관의핵에서현저히증가하였고그외겉질집합관, 먼쪽곱슬세관에서는중등도의증가를보였고속속질집합관핵에서도높게발현되었다. 이는일측성요관폐쇄후 p-erk 단백이바깥수집집합관의핵에서발현되었다는보고 [32], 머리부터발끝까지작용하는높은중력가속도에노출된흰쥐신장집합관의핵에서증가되었다는소견 [33] 과도일치하였다. 칼륨제한식이군의 ERK와 p-erk 단백발현이칼륨제한식이기간이길어질수록증가함을보여주었는데, 이는일측성요관폐쇄로유발된신장의섬유화가 ERK 단백을 활성화시켜산화스트레스에영향을주어 p-erk 단백의겉질집합관과바깥수질집합관에서증가되었다는소견과도일치하였다 [34]. 이는 MAPK가세포내신호전달등의다양한세포기능을담당하며저칼륨혈증시 ERK 단백발현이이온수송체유전자조절뿐만아니라세포내신호전달에있어중요한역할에관여할것임을암시해주었다. 또한 MAPK가 Nrf2를핵내로이동시켜여러 DNA 단백질과결합하도록인산화및안정화에중요한역할을하고 [35] Puerarin이생쥐신장에서 ERK/Nrf2/ARE 경로를조절함으로써 CCl 4 에의해유도된염증을완화시켰다는보고를감안하면 [36] 세포내신호전달에있어중요한역학을하는 MARK (EKR, p38 MARK, c-jun, JNK) 에의해산화스트레스나염증반응조건하에 Nrf2는핵내로이동하여 ARE의단백질과결합하여표적유전자의발현을조절할것으로생각되어진다. 이상의소견은저칼륨혈증에서신장 Akt와 p-akt 및 ERK와 p-erk의단백발현양상은상호간에차이가있었으며칼륨제한식이가길어질수록증가함을보여주었고, 이온수송체단백의발현부위와유사하여이들유전자의조절뿐만아니라세포내신호전달에있어중요한역할을할것이라는가능성을시사해주었다. 그러나본연구는신장의 Akt와 p-akt 및 ERK와 p-erk의단백발현과분포의변화를알아보는데국한되었고, 세포내신호전달에있어중요한역학을하는 MARK (EKR, p38 MARK, c-jun, JNK) 가 Nrf2의세포핵내로이동하여 ARE의단백질과결합하여표적유전자의발현을조절하는기전에대한연구는시행하지못했다. 또한저칼륨혈증에서 Akt와 p-akt 및 ERK와 p-erk 유전자발현변화가세포내신호전달에직접관여했는지는추후여러가지생물학적인방법을이용하여연구해야할과제라고생각된다. REFERENCES 1. Lin SH, Yang SS, Chau T. A Practical Approach to Genetic Hypokalemia. Electro Electrolyte Blood Press. 2010; 8:38-50. 2. Giebisch G. Renal potassium transport: mechanisms and regulation. Am J Physiol 1998; 274:F817-33. 3. Jones JW, Sebatian A, Hulter HN, Scharmbelan M, Sutton JM, Biglieri EG. Systemic and renal acid-base effedts of chronic dietary potassium depletion in humans. Kidney Int. 1982; 21:402-10. 4. Hernadez RE, Schambelan M, Cogan MG, Colman J, Morris RC Jr, Sebastain A. Diratary NaCl dertermines se-
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98 배춘상, 조혜정, 안규윤 Alteration of Akt, p-akt, ERK, and p-erk Proteins Expression in the Kidney of Hypokalemic Rat Choon Sang Bae, Hye Jung Cho, Kyu Yoon Ahn Department of Anatomy, College of Medicine, Jeonnam University Abstract : Hypokalemia causes metabolic alkalosis and morphological changes of the kidney. K + balance is regulated not only by ion channels or pump gene, but also by various genes including NF-E2-related factor 2 (Nrf2). Previous study suggested the possibility that Akt and ERK kinase may be involved in Nrf2 transcriptional gene activation. In present study, we investigate the alterations of Akt, p-akt, ERK, p-erk protein in both normal kidney and K + -deficient diet kidney using Western blot analysis, and immunohistochemisrty. Our western blot data showed that the expression of Akt and p-akt was increased gradually in K + -depleted diet (from 1W-3W) compared to normal group. The expression of ERK and p-erk was markedly increased in K + -depleted diet 2W in comparison with normal group. Based on our immunostaining results, Akt protein immunoreactivity was prominently increased in outer medullary collecting duct, especially in K + -depleted diet 2 weeks. The localization of p-akt proteins in K + -depleted groups was not different from normal group, but the immunoreactivity was significantly increased in distal convoluted tubule, macula densa and outer medullary thick ascending limb in K + -depleted diet 1 and 2 weeks groups. ERK protein immunoreactivity was prominently increased in outer medullary collecting duct, especially in K + -depleted diet 2 and 3 weeks. The localization of p-erk proteins in K + -depleted groups was not different from normal group, but the immunoreactivity was prominently increased in the nucleus of outer medullary collecting duct especially in K + -depleted diet 2 weeks. Taken together, we suggest that the expression of p-akt was gradually increased in K + -depleted groups of kidney, but the expression of p-erk was markedly increased in K + -depleted diet 2 week group. Hence, the promotion of AKT and ERK phosphorylation in hypokalemic condition may be involved in the regulation of ion channels, ion transporters and subsequent intracellular signal transduction. Keywords : Hypokalemia, Metabolic alkalosis, Akt, p-akt, ERK, p-erk, Immunohistochemisty Correspondence to : Kyu Yoon Ahn (Department of Anatomy, College of Medicine, Jeonnam University) E-mail : kyahn@jnu.ac.kr