대한내과학회지 : 제 78 권제 4 호 2010 원저 09-290 신혈관성고혈압흰쥐신장에서 Na, K-ATPase 단백발현감소 전남대학교의과대학 1 내과학교실, 2 생리학교실 마성권 1 오윤화 2 김인진 2 배은희 1 이종은 2 김수완 1 Decreased expression of Na,K-ATPase in the kidneys of rats with two-kidney, one-clip hypertension Seong Kwon Ma, M.D. 1, Yoon Wha Oh, Ph.D. 2, In Jin Kim, M.S. 2, Eun Hui Bae, M.D. 1, Jong Un Lee, M.D. 2, and Soo Wan Kim, M.D. 1 Departments of 1 Internal Medicine and 2 Physiology, Chonnam National University Medical School, Gwangju, Korea Background/Aims: This study investigated the role of Na,K-ATPase, the local renin-angiotensin-aldosterone system (RAAS), and atrial natriuretic peptide (ANP) system in the pathogenesis of renal tubular dysfunction and hypertension in rats with two-kidney, one-clip (2K1C) hypertension. Methods: Adult male Sprague-Dawley rats were made 2K1C hypertensive for 4 weeks. The renal expression of Na,K-ATPase was determined by immunoblotting. The mrna expression of renin, angiotensin-converting enzyme (ACE), aldosterone synthase (CYP11B2), mineralocorticoid receptor (MR), and the ANP system were determined in the kidney using real-time polymerase chain reaction. Results: The blood pressure was increased in the 2K1C rats, compared with controls. The plasma renin activity and serum aldosterone concentrations were increased, as were the urine output and fractional excretion of sodium. The expression of Na,K-ATPase protein was decreased in the clipped kidney, as compared with the control kidney, while it remained unchanged in the contralateral kidney. The mrna expression of renin, ACE1, CYP11B2, and MR was increased in the clipped kidney, but unchanged in the non-clipped kidney. The mrna expression of ACE2 did not differ between the groups. The expression of ANP mrna was increased in both clipped and non-clipped kidneys, as compared with control kidneys. Conclusions: The enhanced activity of the local RAAS may result in to ischemic tubular injury and the development of hypertension in 2K1C rats. The downregulation of Na,K-ATPase associated with tubular injury in the clipped kidney may account for the impaired tubular sodium reabsorption in 2K1C hypertension. (Korean J Med 78:477-484, 2010) Key Words: Renovascular hypertension; Na,K-ATPase; Aldosterone synthase; Atrial natriuretic peptide Received: 2009. 9. 17 Accepted: 2009. 11. 5 Correspondence to Soo Wan Kim, M.D., Department of Internal Medicine, Chonnam National University Medical School, 8 Hak-dong, Gwangju 501-757, Korea E-mail: skimw@chonnam.ac.kr * This work was supported by the grant from Korean Association of Internal Medicine (2006). - 477 -
- The Korean Journal of Medicine: Vol. 78, No. 4, 2010 - 서론신혈관성고혈압은신장혈관의폐색에의하여발생하는고혈압으로, 2차성고혈압의주요한형태이며, 레닌- 안지오텐신-알도스테론계 (renin-angiotensin-aldosterone system, RAAS) 의활성증가가주된병인으로알려져있다. 즉, 신장으로의혈류감소로인하여 RAAS가활성화되고, 이로인하여혈중 angiotension II가증가되어, 혈관수축및말초저항성이증가되어고혈압이발생한다 1,2). 그러나 angiotension II는혈관수축을통한혈압상승작용외에도체액평형상태조절에중요한역할을하며, 신장혈관의폐색이발생한신장에서허혈성손상이발생하므로, 신혈관성고혈압에서신장세뇨관의기능장애가동반되었을것으로사료된다. 이전의보고들에의하면, 신혈관성고혈압에서체내의수분및나트륨평형상태의변조가발생하며소변량이증가함이보고되었으며 3,4), 혈관폐쇄가발생한신장의 aquaporin (AQP) 수분통로의발현감소가다뇨및요농축력장애의기전으로제시되었다 5). 신장세뇨관을통한체액의재흡수는세뇨관에분포하는나트륨수송체및 AQP 수분통로에의하여이루어지며, 고혈압및허혈성손상등의체액조절장애를수반하는여러질환들에서이의기능변조가보고되었다 6,7). 세뇨관에서의나트륨재흡수는두가지단계의기전에의해이루어지는데첫번째과정은세뇨관상피세포의기저외측막에서 Na,K- ATPase 작동에의한나트륨의능동적수송이며, 이어서내강측막의다른나트륨공동수송체를통한수동적이동이일어나게된다 8,9). 이러한과정을통하여나트륨이재흡수되면서삼투압경사가발생하고, 이로인하여수분이재흡수된다. 수분의재흡수는삼투압경사에의해열리는 AQP 수분통로단백을통하여이루어진다 10). 즉, Na,K-ATPase 가효율적으로작동하는것이체액조절에일차적으로중요한역할을하며, 이의조절에 angiotensin II, aldosterone 등의 RAAS 가관련되어있음이보고되었다 11-13). 신혈관성고혈압에서도 Na,K-ATPase 의효소활성도의변조가발생함이보고되었으나 14), 이의조절기전및구체적인양상에대하여는잘알려져있지않다. 신장의나트륨배설및혈압조절기능은여러전신및국소호르몬들에의하여조절되는데, 특히나트륨이뇨호르몬 (natriuretic peptide, NP) 계가중요한역할을한다. 신장은이러한 NP의중요한작용부위인동시에합성장소로알려져있으며, 신장에서심방나트륨이뇨호르몬 (atrial natriuretic peptide, ANP) 는사구체여과율을증가시키거나세뇨관의나트 륨재흡수를감소시켜나트륨이뇨와수분이뇨를일으키며, 고혈압의병인에도관련되어있다 15-17). 그러나신혈관성고혈압에서 ANP계의역할에대하여는정립되어있지않다 18-20). 과거전형적인내분비계로서의 RAAS의개념으로마지막작용물질인 angiotensin II와 aldosterone은여러조직에작용하여혈관수축및염분과수분항상성등에관여하는것으로알려져왔다. 또한, 최근에 angiotgensin-converting enzyme 2 (ACE2) 와 aldosterone synthase (CYP11B2) 의발견으로인하여 RAAS의새로운기능이알려지게되었다. ACE2 는 angiotensin I과 angiotensin II를분해하여 Ang-(1-7) 을생성시키고, 이는 angiotensin II의생리적작용과상반되는효과를나타낸다 21,22). CYP11B2는 aldosterone을합성하는효소로서주로부신에존재하는것으로알려져있으나, 최근에이효소가신사구체에서발현되며, 국소적으로 aldosterone을합성할수있음이보고되었다 23). 따라서신장에서도심장에서와비슷한기전으로사구체경화및이로인한고혈압의발생병인으로작용할가능성을시사한다. 또한고혈압에동반되는흔한신장손상인고혈압성사구체경화의병인으로작용할가능성이있다. 본연구는 two-kidney, one-clip (2K1C) 신혈관성고혈압을유발시킨흰쥐에서고혈압과더불어신장의 Na,K-ATPase 단백및 NP계의변화가동반되는지를조사하였다. 또한, 신장의국소 RAAS의변화여부를조사하였다. 대상및방법 1. 실험재료및신장기능측정실험재료는체중 200~250 g된 Sprague-Dawley 숫쥐를사용하였다. 전체실험과정은전남대학교의과대학실험동물사용윤리규정을준수하였다. 2K1C 고혈압을유발하기위하여 ketamine (50 mg/kg, i.p.) 마취후, 왼쪽신동맥에 0.25 mm 내경의 silver clip을끼웠다. 대조군은실험군과동일한방법으로처리하였으나, 클립을끼우지않았다. 4주일후에 tail-cuff 방법을이용하여수축기혈압을측정하였으며, 마취하지않은상태에서단두하여혈액을채취한후신속히신장을분리하고액체질소로얼려사용할때까지 -80 에서보관하였다. 동물들을대사상자에넣고유지하면서소변을채집하여요량, 크레아티닌, 나트륨을측정하였으며, 실험당일단두하여채취한혈액에서혈장크레아티닌, 나트륨을측정하였다. 또한, 혈장 renin 활성도및혈청 aldosterone을측정하였다. 혈장크레아티닌은 Jaffe method에의하여측정 - 478 -
- Seong Kwon Ma, et al. Renal expression of Na, K-ATPase in 2K1C hypertension - 하였으며 (AU5431, Olympus, Tokyo, Japan), 크레아티닌청소율 (creatinine clearance) 은다음과같은공식에의하여계산하였다. C Cr = U Cr UV/P Cr (C Cr, creatinine clearance; U Cr, urine creatinine concentration; UV, urine volume; P Cr, plasma creatinine concentration) 2. Western blot 분석신장조직을 250 mmol/l sucrose, 1 mmol/l EDTA, 0.1 mm phenylmethylsulfonyl fluoride (PMSF) 와 10 mm Tris-HCl buffer가함유된 ph 7.6 용액에넣고 3,000 rpm으로균질화하였다. 큰조직조각과핵질파편을 1,000 g, 15분저속회전으로제거하였다. 단백표본은 12.5% polyacrylamide resolving gel과 5% polyacrylamide stacking gel로구성된불연속계에서전기영동하여크기에따라분리하였다. 분리된단백은 40 V로 3시간동안전기영동법으로 nitrocellulose 막으로이동시켰다. 막을 0.1% Tween-20을함유한 Tris-based saline buffer [TBST (Amresco, Solon, OH, USA), ph 7.4] 로세척한후비특이적결합을방지하기위해 5% 탈지분유 (NFM) 를포함한 TBST (NFM/TBST) 에서 1시간동안반응시켰다. Nitrocellulose 막을다시 0.2% NFM/TBST 용액에넣고 anti-mouse monoclonal Na,K-ATPase α1 subunit (1:1,000, provided by Dr. DM Fambrough, Johns Hopkins University Medical School) 24) 를첨가하여 2~3시간동안실온에서반응시켰다. 발광체를붙이기위해막을 2% NFM/TBST 용액에넣고 2차항체 [horseradish peroxidase-labeled goat anti-mouse IgG (1:1,000)] 를첨가하여 1시간동안반응시켰다. 고정된항체는 enhanced chemiluminescence [ECL (Amersham Pharmacia Biotech, Little Chalfont, UK)] 으로 photographic film (Hyperfilm ECL, Amersham Pharmacia Biotech, Little Chalfont, UK) 에서탐지하였다. 상대적단백량은자가방사법 (autoradiograms) 으로도출된신호를 transmitter scanning videodensitometer (Bioneer, Cheongwon, Korea) 로분석하여측정하였다. 3. Real-time polymerase chain reaction (PCR) 신장피질조직에 Trizol reagent (Invitrogen, Carlsbad, CA, USA) 를첨가하여균질화하였다. 여기에 chloroform을첨가하여 RNA를추출하고, isopropanol을첨가하여침전시켰다. RNA 침전물은 75% ethanol로세척한후증류수로희석하였다. 분리된 RNA의농도는 260 mm에서측정된 optical density 값을이용하여정량화하였다. cdna는총 RNA 5 µg을 oligo (dt) priming과 superscript reverse transcriptase II (Invitrogen, Carlsbad, CA, USA) 를이용하여역전사시켜서만들었으며, Smart Cycler II system (Cepheid, Sunnyvale, CA, USA) 을이용하여증폭시키고, SYBR Green 으로확인하였다. 각각의 PCR 반응은 10 μm forward primer, 10 μm reverse primer, 2 SYBR Green Premix Ex Taq (Takara Bio Inc., Shiga, Japan), 0.5 μl cdna와멸균증류수를포함하는최종 20 μl 의혼합물로시행하였으며, PCR Rotor-Gene TM 3000 Detecter System (Corbette Research, Mortlake, New South Wales, Australia) 를사용하였다. 실험에사용한 primer 들은표 1에기술하였다. PCR 반응후에는온도를 60 에서 95 로증가시켜얻어진해리곡선을확인하였다. 각각의 PCR 수행시에는 cdna가포함되지않은음성대조군도함께시행하였다. 2 - TC 을이용한상대정량법으로각각의 PCR 산물을비교하였으며, 결과는 GAPDH mrna 발현으로보정하여표시하였다 25). Table 1. The primers used in the polymerase chain reactions Primers Sequences GAPDH sense: ATCAAATGGGGTGATGCTGGTGCTG antisense: CAGGTTTCTCCAGGCGGCATGTCAG Renin sense: AGGCAGTGACCCTCAACACCAG antisense: CCAGTATGCAGGTCGTTCCT ACE1 sense: GCCTCCCCAACAAGACTGCCA antisense: CCACATGTCTCCCCAGCAGATG ACE2 sense: GGAGAATGCCCAAAAGATGA antisense: CGTCCAATCCTGGTTCAAGT CYP11B2 sense: TGAGACGTGGTGTGTTCTTGC antisense: GGCCTCCAAGAAGTCCCTTGC MR sense: TGGATGTGTCTATCATCGTT antisense: GGTCCTTCGTAGGCATAGA ANP sense: GGGGGTAGGATTGACAGGATT antisense: TCCGTGGTGCTGAAGTTTATT NPR A sense: AAGAGCCTGATAATCCTGAGTACT antisense: TTGCAGGCTGGGTCCTCATTGTCA NPR C sense: CGAGGTGCTTGTGCTATTGC antisense: GCGAGTACTCCGTGTCCTTG GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ACE1, angiotensin-converting enzyme 1; ACE2, angiotensin-converting enzyme 2; CYP11B2, aldosterone synthase; MR, mineralocorticoid receptor; ANP, atrial natriuretic peptide; NPR-A, natriuretic peptide receptor-a; NPR-C, natriuretic peptide receptor-c. - 479 -
- 대한내과학회지 : 제 78 권제 4 호통권제 596 호 2010 - Table 2. Changes in blood pressure and renal functional parameters Control (n=8) 2K1C (n=10) Body weight (g) 347.5±3.1 325.0±7.8 * Right kidney (g) 1.25±0.02 1.80±0.08 * Left kidney (g) 1.24±0.03 0.90±0.05 * Systolic blood pressure (mmhg) 121.8±3.9 173.6±7.6 * Plasma renin activity (ng/ml/h) 35.4±5.0 92.3±8.1 * Serum aldosterone (ng/dl) 7.3±2.3 81.7±29.0 * Creatinine clearance (ml/min) 1.63±0.13 1.55±0.08 Urine output (ml/day) 13.6±1.2 19.0±2.0 * FE Na (%) 0.32±0.08 0.61±0.10 * Values are the mean±sem. * p<0.05 vs. control. 2K1C, two-kidney, one-clip; FE Na, fractional sodium excretion. 4. 통계분석 실험결과는평균 ± 표준오차로표시하였으며, 실험군간차이에대한통계적유의성분석은 ANOVA 또는비쌍체 t- 검정법을사용하였다. 결 1. 혈압및신장기능의변화 표 2는혈압및신장의기능적지표들을요약하였다. 실험군에서수축기혈압이유의하게증가하였으며, 혈장레닌활성도및혈청알도스테론농도가증가하였다. 또한, 실험군의클립을끼운좌측신장은대조군의좌측신장에비하여 과 무게가감소하였으며, 실험군의클립을끼우지않은우측신장의무게는대조군의우측신장에비하여증가하였다. 사구체여과율은양군간차이가없었으나, 실험군에서소변량및나트륨분획배설이유의하게증가하였다. 2. Na,K-ATPase 단백발현 Na,K-ATPase α1 subunit 의단백발현을신장의피질및외수질에서조사하였다. 클립신장의단백발현이대조군에비하여유의하게감소하였다. 클립을끼우지않은실험군의반대쪽신장에서는클립신장에비하여단백발현이증가하였고, 대조군과는유의한차이가없었다 ( 그림 1). 3. 레닌-안지오텐신-알도스테론계의 mrna 발현신장조직의 renin 및 ACE1의 mrna 발현은실험군의클립신장에서대조군에비하여증가하였고, 클립을끼우지않은반대쪽신장에서는클립신장에비하여감소하였다. 반대쪽신장과대조군사이에는발현의차이가없었다. ACE2 mrna 발현은대조군, 클립신장및반대쪽신장조직에서모두유의한차이가없었다 ( 그림 2). CYP11B2 및 mineralocorticoid receptor (MR) 의 mrna 발현역시클립신장에서대조군에비하여유의하게증가하였으며, 반대쪽신장과대조군사이에는발현의차이가없었다 ( 그림 3). 4. ANP 및수용체 mrna 발현클립신장및반대쪽신장의 ANP mrna 발현이대조군에비하여유의하게증가하였다. 하지만 natriuretic peptide receptor (NPR)-A 및 NPR-C mrna 발현은세군간에유의 Figure 1. The protein expression of Na,K-ATPase α1 subunit in the cortex and outer stripe of the outer medulla (Cortex/OSOM) and inner stripe of the outer medulla (ISOM) of the kidney. Each column represents mean±sem of rats (control=8, clipped=10, non-clipped=10). * p<0.05 vs. control, # p<0.05 vs. clipped kidney. - 480 -
- 마성권외 5 인. 2K1C 고혈압에서나트륨수송계변화 - Figure 2. The mrna expression of renin, angiotensin-converting enzyme 1 (ACE1), and angiotensin-converting enzyme 2 (ACE2) in the cortex of the kidney. Legends as in Fig. 1. Figure 3. The mrna expression of aldosterone synthase (CYP11B) and mineralocorticoid receptor (MR) in the cortex of the kidney. Legends as in Fig. 1. Figure 4. The mrna expression of atrial natriuretic peptide (ANP), natriuretic peptide receptor-a (NPR-A), and natriuretic peptide receptor-c (NPR-C) in the cortex of the kidney. Legends as in Fig. 1. 한차이가없었다 ( 그림 4). 고찰본연구에서일측신동맥클립에의하여전신의 RAAS의항진과함께고혈압이발생하였다. 이는신혈관성고혈압이유발되었음을나타낸다. 2K1C 고혈압에서클립신장으로혈류량이감소하게되면, 신장의저관류상태를회복시키기위하여 RAAS의활성이증가하며, 혈중 angiotensin II에의한전신혈관수축으로인하여고혈압이발생한다 1,2). 그러나 RAAS는신장의국소호르몬으로써신장세뇨관에서의수분및염분재흡수를조절한다. 특히, 근위세뇨관에높은농 도로존재하며, type 3 Na + /H + exchanger의작용을직접적으로자극하여, 나트륨재흡수를증가시킴이보고되었다 26). 또한, aldosterone 합성을증가시켜서원위세뇨관에서의나트륨재흡수를증가시키는것으로알려져있다 11-13). 즉, angiotensin II의세뇨관에대한작용은수분및나트륨의재흡수를증가시켜서, 체액량을증가시키는작용을한다. 그러나 angiotensin II-infusion에의하여유발된고혈압에서는혈압증가에대한적응기전으로써오히려수분및나트륨배설이증가하며신장의 AQP2 및 Na-K-2Cl 수송체발현이감소된다 27). 또한, 신장혈관폐색상태가지속되면, angiotensin II 에의한혈관내피세포기능저하, 염증유발 cytokines 증가, 산화스트레스등에의하여신장조직의섬유화가발생하 - 481 -
- The Korean Journal of Medicine: Vol. 78, No. 4, 2010 - 며, 신장수질의저산소증을유발하여허혈성손상을일으킨다 28,29). 이전의보고에의하면, 신장의허혈성손상시 Na,K-ATPase 등의나트륨수송체발현이감소되고, 나트륨이뇨가발생함이보고되었다 7). 이처럼 angiotensin II의신장세뇨관에대한작용은전신혈압및신장혈류상태에따라다르게나타난다. 본연구에서는실험군에서소변량의증가와나트륨분획배설이증가하였으며, 이와함께클립신장의 Na,K-ATPase 단백발현이감소하였다. 따라서클립신장의 Na,K-ATPase 발현감소가 2K1C 고혈압에서발생하는다뇨및나트륨이뇨의발생에기여하였을것으로생각되며, 이는신장세뇨관의허혈성손상과관련되었을것으로생각된다. 본연구에서클립신장의 renin, ACE1, CYP11B2, MR의 mrna 발현이모두증가되었으며, 이는신장조직의국소 RAAS의활성증가가고혈압및세뇨관손상에중요한역할을하고있음을시사한다. RAAS는혈압및체액조절, 심혈관계및신장질환의병태생리에관련되어있으며, 주로 angiotensin 및 aldosterone이중요한역할을하는것으로알려져있다. 그러나수많은연구에도불구하고 RAAS은아직까지도고혈압및장기손상의발생기전을명확히설명하는데있어서많은한계를가지고있다 30). 최근에 RAAS의구성요소들인 renin-like enzyme, angiotensinogen, ACE1, ACE2, angiotensin II receptor, CYP11B2, MR 등이혈관벽의평활근세포와내피세포, 심장, 뇌및신장에존재함이알려지게되었다 31-33). 즉, RAAS의국소호르몬으로서의작용이심혈관계및신장질환의병태생리에중요한역할을함이알려지게되었다. 특히최근에발견된 ACE2는 angiotensin I을 angiotensin (1-9) 와 (1-7) 로변환시키며 angiotensin (1-7) 은혈관확장, 이뇨, 항증식, 산화질소분비자극등의작용을하여 angiotensin II type 1 receptor (AT1R) 에결합한 angiotensin II와는반대되는작용을나타낸다. 따라서고혈압의발생및유지에 ACE1-angiotensin II-AT1R 축과 ACE2-angiotensin-(1-7)-Mas receptor 축이모두중요하게작용한다 31). 실제로고혈압의병인에서신장에서 ACE1/ACE2 발현의상대적비율이 RAAS 의활성을보여주는표지로이용될수있으며, 본태성고혈압환자에서 ACE1/ACE2 비율이증가되어있다 33). 본연구에서 2K1C 고혈압흰쥐의클립신장에서 ACE1 mrna 발현은증가하였으나 ACE2 발현은유의한변화를보이지않았다. 이러한결과는 ACE2에비교하여상대적으로활성화된 ACE1에의한 angiotensin II 합성증가및이로인한혈관수축및고혈압발생에기여할것으로생각된다. 신장조직의 CYP11B2는 RAAS의새로이알려진구성요소로써이효소에의한 aldosterone 합성증가는신사구체에서국소적으로세포비대, 기질합성증가및이로인한사구체경화를증가시킴이제시되었다 34). 본연구에서신혈관성고혈압의클립신장에서 CYP11B2 mrna 발현이유의하게증가되었다. 이러한결과는부신이외의신장조직에서자체적으로 aldosterone을합성함을증명하는결과이며, 국소적으로 aldosterone 합성증가가사구체경화및고혈압의병인으로작용할것임을나타내는결과이다. 또한 aldosterone이작용하는 MR는신장집합관뿐만아니라, 메산지움세포에존재하여메산지움세포에서국소적으로합성되는 aldosterone 이결합하는수용체로사구체경화의병인으로작용함이제시되었다 35). 따라서 2K1C 고혈압에서클립신장에서나타나는 CYP11B2 및 MR 발현증가는메산지움세포의증식및사구체경화를유발하여신기능악화및고혈압발생의병인으로작용할것으로보인다. ANP계는 RAAS와는길항적관계로혈압조절에깊이관여하고있다. 최근연구에서신장은이뇨호르몬의중요한작용부위이지만또한신장자신이이뇨호르몬의중요한합성장소임을밝히고있다. Immunoreactive ANP, brain natriuretic peptide (BNP) 및 C-type natriuretic peptide (CNP) 가사람신장에서검출되었고, 사람및흰쥐에서 ANP, BNP 및 CNP 유전자발현이확인되었으며, 신장 ANP계는심장 ANP 계와독립적으로조절된다 36,37). 실제로 DOCA-salt 고혈압및당뇨쥐에서혈장 renin 활성감소와더불어신장 ANP mrna 합성과요중 ANP 배설증가를보였다 38,39). 이러한소견은신장내 ANP 합성이체액용적증가및고혈압에반응하여증가하며, 체액변화및혈압조절에혈장 ANP보다더욱중요하게작용할것을시사한다. 본연구에서클립신장의 ANP mrna 발현이대조군신장에비하여증가하였다. 이는 Na,K-ATPase 발현감소와함께나트륨이뇨의기전으로생각된다. 그러나 ANP의수용체인 NPR-A 및 NPR-C의발현은유의한차이가없었다. 이전의보고에의하면, angiotensin II 가 ANP에의하여자극되는 cgmp 합성을감소시켜서 NPR 을하향조절한다고하였다 40). 따라서 2K1C 고혈압에서의 RAAS 활성도증가가 NPR 발현에영향을주었을것으로생각된다. 이에대하여는신장조직의 guanylyl cyclase 활성도측정등의추후의연구를요한다. 또한, 실험군의클립을끼우지않은신장에서도 ANP 발현이증가되었다. 본실험에서클립신장이위축되고, 반대쪽신장이비후되었으므로, 반대쪽신장의상대적인사구체여과율증가및혈압의증가에 - 482 -
- Seong Kwon Ma, et al. Renal expression of Na, K-ATPase in 2K1C hypertension - 대한보상기전으로작용할것으로생각된다. 이상의결과로신혈관성고혈압유발흰쥐에서전신및신장의국소 RAAS의활성증가가고혈압및세뇨관손상에기여할것으로사료되며, 신장 ANP계의활성증가는고혈압에대한보상기전으로작용할것으로보인다. 또한, 세뇨관손상과관련된클립신장의 Na,K-ATPase 발현감소가다뇨및나트륨재흡수장애에관여할것으로보인다. 요약목적 : 본연구는 two kidney, one clip (2K1C) 신혈관성고혈압을유발한흰쥐에서신장의 Na,K-ATPase, 레닌- 안지오텐신-알도스테론계 (renin-angiotensin-aldosterone system, RAAS) 및심방나트륨이뇨호르몬 (atrial natriuretic peptide, ANP) 계가고혈압및신장손상의병태생리에관련되어있는지를조사하고자하였다. 방법 : 실험재료는 Sprague-Dawley 숫쥐를사용하였다. 2K1C 고혈압을유발하기위하여 ketamine (50 mg/kg, i.p.) 마취후왼쪽신동맥에 0.25 mm 내경의 silver clip을끼웠다. 4 주일후에 tail-cuff 방법을이용하여수축기혈압을측정하였으며, 마취하지않은상태에서단두하여신장을적출하였다. 신장조직에서 Na,K-ATPase α1 subunit 단백발현을 Western blot 분석법에의하여조사하였다. 또한, renin, angiotensinconverting enzyme (ACE), aldosterone synthase (CYP11B2), mineralocorticoid receptor (MR) 및 ANP계의 mrna 발현을 real-time polymerase chain reaction으로조사하였다. 결과 : 실험군에서수축기혈압이유의하게증가하였으며, 혈장레닌활성도및혈청알도스테론농도가증가하였다. 또한, 소변량및나트륨분획배설이유의하게증가하였다. Na,K-ATPase α1 subunit의단백발현은클립신장에서대조군에비하여유의하게감소하였고, 클립을끼우지않은실험군의반대쪽신장의단백발현이클립신장에비하여증가하였다. 신장조직의 renin, ACE1, CYP11B2 및 MR의 mrna 발현은실험군의클립신장에서대조군에비하여증가하였고, 클립을끼우지않은반대쪽신장에서는클립신장에비하여유의하게감소하였다. ACE2 mrna 발현은대조군, 실험군의클립신장및반대쪽신장모두에서유의한차이가없었다. 또한, 클립신장및반대쪽신장의 ANP mrna 발현이대조군에비하여유의하게증가하였다. 결론 : 신혈관성고혈압유발흰쥐에서전신및신장의국소 RAAS의활성증가가고혈압및세뇨관손상에기여할 것으로사료되며, 신장 ANP계의활성증가는고혈압에대한보상기전으로작용할것으로보인다. 또한, 세뇨관손상과관련된클립신장의 Na,K-ATPase 발현감소가다뇨및나트륨재흡수장애에관여할것으로생각된다. 중심단어 : 신혈관성고혈압 ; 나트륨수송체 ; 알도스테론 ; 나트륨이뇨호르몬 REFERENCES 1) Garovic V, Textor SC. Renovascular hypertension: current concepts. Semin Nephrol 25:261-271, 2005 2) Garovic VD, Textor SC. Renovascular hypertension and ischemic nephropathy. Circulation 112:1362-1374, 2005 3) Machida J, Ueda S, Yoshida M, Soejima H, Ikegami K. Role of sodium and renal prostaglandin E2 in the maintenance of hypertension in the chronic phase of two-kidney one-clip renovascular hypertension in rabbits. Nephron 49:74-80, 1988 4) Ploth DW, Roy RN, Huang WC, Navar LG. Impaired renal blood flow and cortical pressure autoregulation in contralateral kidneys of Goldblatt hypertensive rats. Hypertension 3:67-74, 1981 5) Lee JU, Oh YW, Kim SW. Altered renal expression of aquaporin-2 water channels in rats with experimental two-kidney, one clip hypertension. J Korean Med Sci 16:462-466, 2001 6) Kim SW, Wang W, Kwon TH, Knepper MA, Frokiaer J, Nielsen S. Increased expression of ENaC subunits and increased apical targeting of AQP2 in the kidneys of spontaneously hypertensive rats. Am J Physiol Renal Physiol 289:F957-F968, 2005 7) Kwon TH, Frokiaer J, Han JS, Knepper MA, Nielsen S. Decreased abundance of major Na+transporters in kidneys of rats with ischemia-induced acute renal failure. Am J Physiol Renal Physiol 278:F925-F939, 2000 8) Katz AI, Doucet A, Morel F. Na-K-ATPase activity along the rabbit, rat, and mouse nephron. Am J Physiol 237:F114-F120, 1979 9) Kashgarian M, Biemesderfer D, Caplan M, Forbush B 3rd. Monoclonal antibody to Na,K-ATPase: immunocytochemical localization along nephron segments. Kidney Int 28:899-913, 1985 10) Knepper MA, Wade JB, Terris J, Ecelbarger CA, Marples D, Mandon B, Chou CL, Kishore BK, Nielsen S. Renal aquaporins. Kidney Int 49:1712-1717, 1996 11) Feraille E, Doucet A. Sodium-potassium-adenosinetriphosphatase-dependent sodium transport in the kidney: hormonal control. Physiol Rev 81:345-418, 2001 12) Verrey F, Schaerer E, Zoerkler P, Paccolat MP, Geering K, Kraehenbuhl JP, Rossier BC. Regulation by aldosterone of Na+,K+-ATPase mrnas, protein synthesis, and sodium transport in cultured kidney cells. J Cell Biol 104:1231-1237, 1987 13) El Mernissi G, Doucet A. Short-term effect of aldosterone on renal sodium transport and tubular Na-K-ATPase in the rat. - 483 -
- 대한내과학회지 : 제 78 권제 4 호통권제 596 호 2010 - Pflugers Arch 399:139-146, 1983 14) Akabane S, Natsume T, Matsushima Y, Deguchi F, Kuramochi M, Ito K. Alterations in renal Na+K+ATPase activity and [3H]ouabain binding in Goldblatt hypertensive rabbits. J Hypertens 3:469-474, 1985 15) Huang CL, Lewicki J, Johnson LK, Cogan MG. Renal mechanism of action of rat atrial natriuretic factor. J Clin Invest 75:769-773, 1985 16) Nonoguchi H, Sands JM, Knepper MA. ANF inhibits NaCl and fluid absorption in cortical collecting duct of rat kidney. Am J Physiol 256:F179-F186, 1989 17) Ogawa T, Linz W, Scholkens BA, de Bold AJ. Variable renal atrial natriuretic factor gene expression in hypertension. Hypertension 33:1342-1347, 1999 18) Huang WC, Wu JN. Blunted renal responses to atrial natriuretic peptide and its reversal by unclipping in one-kidney, one clip Goldblatt hypertensive rats. J Hypertens 15:181-189, 1997 19) Fenoy FJ, Salazar FJ, Hernandez I, Pinilla JM, Quesada T. Effect of a chronic infusion of atrial natriuretic peptide on sodium balance in normotensive and two-kidney, one-clip hypertensive rats. Clin Physiol Biochem 8:256-260, 1990 20) Huang WC, Wu JN, Chao DH. Differential renal responses to atrial natriuretic peptide in two-kidney, one clip goldblatt hypertensive rats. J Cardiovasc Pharmacol 13:7-15, 1989 21) Iyer SN, Chappell MC, Averill DB, Diz DI, Ferrario CM. Vasodepressor actions of angiotensin-(1-7) unmasked during combined treatment with lisinopril and losartan. Hypertension 31:699-705, 1998 22) Ferrario CM, Chappell MC, Tallant EA, Bronsnihan KB, Diz DI. Counterregulatory actions of angiotensin-(1-7). Hypertension 30:535-541, 1997 23) Xue C, Siragy HM. Local renal aldosterone system and its regulation by salt, diabetes, and angiotensin II type 1 receptor. Hypertension 46:584-590, 2005 24) Kim SW, Wang W, Sassen MC, Choi KC, Han JS, Knepper MA, Jonassen TE, Frøkiær J, Nielsen S. Biphasic changes of epithelial sodium channel abundance and trafficking in common bile duct ligation-induced liver cirrhosis. Kidney Int 69:89-98, 2006 25) Bae EH, Oh YW, Park JW, Ma SK, Choi KC, Lee J, Kim SH, Kim SW. Gentamicin decreasese guanylyl cyclase activity in rat glomerulus. Kidney Blood Press Res 30:81-87, 2007 26) Geibel J, Giebisch G, Boron WF. Angiotensin II stimulates both Na+-H+ exchange and Na+/HCO3- cotransport in the rabbit proximal tubule. Proc Natl Acad Sci U S A 87:7917-7920, 1990 27) Klein JD, Murrell BP, Tucker S, Kim YH, Sands JM. Urea transporter UT-A1 and aquaporin-2 proteins decrease in response to angiotensin II or norepinephrine-induced acute hypertension. Am J Physiol Renal Physiol 291:F952-F959, 2006 28) Lerman LO, Nath KA, Rodriguez-Porcel M, Krier JD, Schwartz RS, Napoli C, Romero JC. Increased oxidative stress in experimental renovascular hypertension. Hypertension 37:541-546, 2001 29) Lerman L, Textor SC. Pathophysiology of ischemic nephropathy. Urol Clin North Am 28:793-803, 2001 30) Lakkis J, Lu WX, Weir MR. RAAS escape: a real clinical entity that may be important in the progression of cardiovascular and renal disease. Curr Hypertens Rep 5:408-417, 2003 31) Re RN. Mechanisms of disease: local renin-angiotensin-aldosterone systems and the pathogenesis and treatment of cardiovascular disease. Nat Clin Pract Cardiovasc Med 1:42-47, 2004 32) Dostal DE, Baker KM. The cardiac renin-angiotensin system: conceptual, or regulator of cardiac function? Circ Res 85:643-650, 1999 33) Kurdi M, De Mello WC, Booz GW. Working outside the system: an update on the unconventional behavior of the renin-angiotensin system components. Int J Biochem Cell Biol 37:1357-1367, 2005 34) Xue C, Siragy HM. Local renal aldosterone system and its regulation by salt, diabetes, and angiotensin II type 1 receptor. Hypertension 46:584-590, 2005 35) Nishiyama A, Yao L, Fan Y, Kyaw M, Kataoka N, Hashimoto K, Nagai Y, Nakamura E, Yoshizumi M, Shokoji T, Kimura S, Kiyomoto H, Tsujioka K, Kohno M, Tamaki T, Kajiya F, Abe Y. Involvement of aldosterone and mineralocorticoid receptors in rat mesangial cell proliferation and deformability. Hypertension 45:710-716, 2005 36) Totsune K, Takahashi K, Murakami O, Satoh F, Sone M, Saito T, Mouri T, Abe K. Natriuretic peptides in the human kidney. Hypertension 24:758-762, 1994 37) Kim SW, Lee J, Park JW, Hong JH, Kook H, Choi C, Choi KC. Increased expression of atrial natriuretic peptide in the kidney of rats with bilateral ureteral obstruction. Kidney Int 59:1274-1282, 2001 38) Lee YJ, Shin SJ, Tan MS, Hsieh TJ, Tsai JH. Increased renal atrial natriuretic peptide synthesis in rats with deoxycorticosterone acetate salt treatment. Am J Physiol 271:F779-F789, 1996 39) Shin SJ, Lee YJ, Tan MS, Hsieh TJ, Tsai JH. Increased atrial natriuretic peptide mrna expression in the kidney of diabetic rats. Kidney Int 51:1100-1105, 1997 40) Smith JB, Lincoln TM. Angiotensin decreases cyclic GMP accumulation produced by atrial natriuretic factor. Am J Physiol 253:C147-C150, 1987-484 -