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전남의대학술지제 46 권제 3 호 Chonnam Medical Journal Vol. 46, No. 3, pp. 156 162 DOI: 10.4068/cmj.2010.46.3.156 시스플라틴유발신손상에서신장세뇨관세포자멸사및항산화제의신손상보호효과 전남대학교의과대학내과학교실, 1 생리학교실 배은희ㆍ이종은 1 ㆍ마성권ㆍ김수완 * Effects of Antioxidants in Cisplatin-Induced Renal Tubular Apoptosis Eun Hui Bae, JongUn Lee 1, Seong Kwon Ma and Soo Wan Kim* Departments of Internal Medicine and 1 Physiology, Chonnam National University Medical School, Gwangju, Korea The present study was designed to evaluate the possible renoprotective effects of alpha-lipoic acid (α- LA) in cisplatin-induced nephropathy and its role in inflammation and apoptosis in the kidney. Male Sprague-Dawley rats, weighing 180~200 g, were used. One group of rats was injected with cisplatin (6 mg/kg intraperitoneally). Another group of rats was injected with cisplatin and co-treated with α-la. Rats that received an injection of vehicle only served as controls. Four days later, the mrna expression of Bax and Bcl-2 was determined by real-time PCR. The protein expression of ED-1, cyclooxygenase-2 (COX2), heat shock protein 72 (HSP72), Bax, and Bcl-2 was determined in the kidney by immunoblotting. Apoptosis was determined by TUNEL staining. Cisplatin-treated rats had decreased creatinine clearance and increased plasma creatinine levels compared with controls. α-la treatment lowered plasma creatinine levels and increased creatinine clearance. In cisplatin-treated rats, the mrna expression of Bcl-2 was decreased, which was attenuated by α-la treatment. The protein expression of ED-1, COX 2, HSP72, cleaved caspase-3, and Bax in the kidney was increased compared with controls, whereas that of Bcl-2 was decreased, and these changes were counteracted by α-la treatment. TUNEL-positive cells were increased, in association with an increased protein expression of Bax and cleaved caspase-3 in the kidney of cisplatin-induced nephropathy, which was counteracted by α-la treatment. These findings suggest that α-la is effective in preventing the progression of cisplatin-induced nephropathy, the mechanism of which is associated with anti-inflammation and anti-apoptotic effects. Key Words: Oxidative stress; Cisplatin; Apoptosis; Inflammation 접수일 : 2010 년 11 월 7 일, 게재결정 : 2010 년 11 월 16 일 * 교신저자 : 김수완, 501-757, 전남대학교의과대학내과학교실, Phone: 062-220-6271, FAX: 062-225-8578, E-mail: skimw@chonnam.ac.kr 156

배은희외 3 인 : Effects of Antioxidant on Cisplatin Induced Nephropathy 157 서론시스플라틴은무기플래티넘 (inorganic platinum) 복합체로고형종양의치료에효과적인약제로광범위하게사용되어지고있다. 1-3 하지만시스플라틴은신장에서고농도로농축되어근위세뇨관세포의괴사를유발하고, 원위세관의세포자멸사를유발하는것으로알려져있다. 4 시스플라틴유발신독성은사구체여과율감소, 신장세뇨관괴사및세포자멸사를특징으로한다. 5 시스플라틴유발신독성의발생기전으로는약제자체의신세뇨관독성과신장혈관수축에의한혈역학적인변화에의한다고알려져있으며혈관수축의발생은산화질소와같은혈관확장인자와 endothelin 등의혈관수축인자의불균형에의할것으로생각된다. 6 그러므로이러한혈관수축 / 확장과관련된기전을차단하는물질의투여에의해시스플라틴유발신독성의발생을억제한다는동물실험을기초로하여임상적으로도시스플라틴유발신독성예방에사용하여연구되었으나실제로이러한예방효과가입증되지못하였다. 7 이러한 in vitro, in vivo 연구결과의불일치는시스플라틴유발신독성의병인기전이서로다른여러가지기전이복합되어작용하기때문으로생각되며이러한병인기전을완벽히규명하지못하였기때문이다. 신장세뇨관세포의염증반응과세포자멸사는시스플라틴유발신독성에서신장손상및이로인한사구체여과율감소의주요원인기전이다. 7 세포자멸사는크게내인성 ( 미토콘드리아매개성 ), 외인성 ( 수용체매개성 ) 및 caspase 비의존형기전으로나타난다. 그중내인성기전은미토콘드리아에서 cytochrome C가유리되는데이것은세포자멸사를촉진하는단백질인 Bax와억제하는단백질인 Bcl-2에의해서조절된다. 유리된 cytochrome C는 caspase 9을활성화하고이것이 caspase 3을활성화하여세포자멸사를초래한다. 8 Alpha-lipoic acid (α-la) 는 thiol 계항산화제화합물로서당뇨병으로인한혈관내피세포장애와신경내혈류를개선, 산화스트레스감소를통해당뇨병성말초신경병을개선시킨다고보고되고있다. 9 또한핵내전사인자 (NF-κB) 발현을억제하여경동맥손상흰쥐모델에서신생혈관증식을억제하였고, 10 패혈증흰쥐모델에서도내피세포에서 fractalkine 발현을억제하여염증반응을완화시키는효과가있는것으로보고되고있다. 11 기존의연구에의하면시스플라틴흰쥐에항산화제로잘알려진 N-acetyl cystein을투여한경우강력한신혈관수축물질로알려진 isoprostane 증가가감약되어신독성을예방함이보고되었다. 12 이처럼신독성모델에서항산화제치료가예방효 과의가능성이있음이제시되고있고, α-la가최근동물및임상에서다양한질환에효과가보고되고있으나시스플라틴유발신독성에서 α-la의치료효과및이의원인기전은확립되어있지않다. 본연구는시스플라틴신독성에서항산화제치료후신독성의회복여부와신장세뇨관세포자멸사가회복되는지를알아보고자하였다. 대상및방법 1. 실험동물실험동물은체중 180 200 g의 Sprgue-Dawley 숫쥐를사용하였다. 전체실험과정은전남대학교의과대학실험동물사용윤리규정에관한지침을준수하였다. 시스플라틴신독성을유발하기위하여복강내로시스플라틴 6 mg/kg를주사하였다. 항산화제 (α-la) 는 50 mg/kg로연속 4일간하루 1번복강내주사하였다. 정상대조군은생리식염수를복강내로주사하였다. 시스플라틴주입후 4일째에실험에이용하였다. 실험시작 3일전실험쥐들을대사상자 (metabolic cage) 에 3일간적응시킨후실험을시작하였다. 실험군 (n=7) 은 24 시간동안먹이섭취를박탈하였으나, 물의섭취는제한하지않았다. 대조군 (n=7) 은같은시간동안먹이와물을자유롭게공급한후실험에사용하였다. 2. 신기능측정동물을대사상자에넣고유지하면서매일소변을채집하였으며, 요량, 크레아티닌을측정하였다. 실험당일날 pentobarbital (50 mg/kg, i.p.) 마취하에개복후 5 ml 주사기를이용하여대정맥에서혈액을채취하여혈청크레아티닌을측정하였다. 크레아티닌농도는 Jaffe법 (Olympus 5431, Olympus Optical Co. Ltd., Tokyo, Japan) 으로측정하였다. 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

158 전남의대학술지제 46 권제 3 호 2010 II (Invitrogen, Carlsbad, CA, USA) 를이용하여역전사시켜서만들었으며, Smart Cycler II system (Cepheid, Sunnyvale, CA, USA) 을이용하여증폭시키고, SYBR Green으로확인하였다. 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 μl의혼합물로시행하였으며, PCR Rotor-Gene TM 3000 Detecter System (Corbette Research, Mortlake, New South Wales, Australia) 를사용하였다. 실험에사용한 primer 는 Bax (sense: AGGATGGCTGGGGAGACACCTG, antisense: CATGGGCTGGACACTGGACTTC) 와 Bcl-2 (sense: CGGGAGATCGTGATGAGGTACATC, antisense: AC AACATCGCTCTGTGGATGACTG) 이다. PCR 반응후에는얻어진결과는 Corbett Research Software을이용하여분석하였다. 13 4. 단백분리및 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 xg, 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 1 시간동안반응시켰다. Nitrocellulose 막을다시 0.2% NFM/TBST 용액에넣고 1차항체 (ED-1& HSP 72): Santa Cruz Ciotechmology, Santa Cruz, CA, USA; COX2: Cayman Chemical Company, MI, USA, Bax and Bcl-2: Cell Signaling Techmology, Beverly, MA, USA) 를첨가하여밤동안 4 o C에서반응시켰다. 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)] 반응시켜 Image Reader (LAS-3000 Imaging System, Fuji Photo Film, Tokyo, Japan) 로분석하였다. 5. TUNEL stain 백서를실험당일에 pentobarbital (50 mg/kg, i.p.) 마취하에복부를중앙절개한뒤대동맥과하대정맥을노출시킨다. 신동맥상하대동맥을 clamp하고하대정맥을박리하고 phosphate buffered saline (PBS) 을대동맥을통하여역행성으로관류함으로써 5마리씩각군백서를고정한다. 신장을제거하고 formalin에두시간고정하고 paraffin에 embed한다. 조직을 xylene으로 5분씩 3번씻어내고 absolute ethanol로 5분간 2번 95% ethanol로 3분간 1번, 70% ethanol로 3분간 1번 PBS로 5분간 1번세척하였다. Proteinase K (proteinase K, Dako) 로실온에서 15분간전처치후에 3% hydrogen peroxide in PBS에 5분간씻어냈다. Equilibration buffer를슬라이드에투입하고 terminal deoxynucleotidyl transferase와함께 37 o C 에서 1시간배양하였다. 이후실온에서 anti-digoxigenin peroxide conjugate에 30분간처리한뒤 peroxidase substrate 를충분히슬라이드에공급하고 haematoxylin에 10분간대조염색을실시하였다. Apoptosis는 Apoptag plus peroxidase in situ apoptosis detection kit (Chemicon International, Temecula, USA) 를이용하여측정하였다. 6. 통계처리실험결과는평균 ± 표준오차로표시하였으며, 실험군간차이에대한통계적유의성분석은 ANOVA 또는비쌍체 t-검정법을사용하였다. 결과 1. 신장의기능시스플라틴단독투여군에서혈청크레아티닌의상승과사구체여과율의감소를보였으며, 이는 α-la 투여로호전되었다 (Fig. 1). 2. 염증계의단백발현변화세포염증반응시증가한다고알려져있는 ED-1, COX2, HSP72의단백발현이시스플라틴단독투여군에서현저히증가되었으며이는 α-la 투여로호전되었다 (Fig. 2). 3. 세포자멸사계의 real-time PCR발현변화세포자멸사를조장한다고알려진 Bax의 mrna의발현

배은희외 3 인 : Effects of Antioxidant on Cisplatin Induced Nephropathy 159 Fig. 1. Effects of alpha lipoic acid (α-la) on serum creatinine level and creatinine clearance. *p<0.05 compared with control. p<0.05 compared with cisplatin-treated rats. Fig. 2. Effects of alpha lipoic acid (α-la) on the protein expression of ED-1, COX2 and HSP70 in the kidney. *p<0.05 compared with control. p<0.05 compared with cisplatin-treated rats. 이시스플라틴단독투여군에서증가, α-la 투여로호전되는경향을보였으나통계학적의의는없었다. 그러나 Bcl-2의 mrna의발현은시스플라틴단독투여군에서감소, α-la 투여로호전되었다 (Fig. 3). 4. 세포자멸사계의단백발현변화 Bax와 cleaved caspase 3 단백발현이시스플라틴단독투여군에서증가, α-la 투여로호전되었다. 세포자멸사를 억제한다고알려진 Bcl-2의단백발현은시스플라틴단독투여군에서감소하는경향을보였으나통계적으로의의는없었고, α-la 투여로호전되었다 (Fig. 4). 5. TUNEL 염색시스플라틴단독투여군의신피질에서 TUNEL 양성세포가증가된것을관찰할수있었으며, α-la 투여는이러한 TUNEL 염색반응정도를감약시켰다 (Fig. 5).

160 전남의대학술지제 46 권제 3 호 2010 Fig. 3. Effects of alpha lipoic acid (α-la) on mrna expression of Bax and Bcl-2 in the kidney. *p<0.05 compared with control. p<0.05 compared with cisplatin-treated rats. Fig. 4. Effects of alpha lipoic acid (α-la) on the protein expression of Bax, Bcl-2 and cleaved caspase 3 in the kidney. *p<0.05 compared with control. p<0.05 compared with cisplatin-treated rats. 고찰시스플라틴유발신독성에서신기능장애및사구체여과율감소의병인으로산화스트레스의증가, 14 염증반응활성화 15 및이로인한신장세뇨관세포손상및세포자멸사가 16 중요한역할을함이알려져있다. 시스플라틴유발신독성에서는다양한염증유발성싸이토카인및케모카인들이유도된다. 이러한염증반응은신조직손상및신기능저하를 일으키는것으로알려져있는데, 시스플라틴유발신독성에서는특히 tumor necrosis factor (TNF)-α가염증반응을일으키는주요매개물로연구되고있다. 17 대부분의연구들에서 TNF-α의활성화를억제시키는경우시스플라틴유발신독성이감소하는것으로나타났다. 18,19 그러나시스플라틴유발신독성에서 TNF-α가유도되는기전및이에따른염증반응의증가에의해신독성이일어나는기저기전에대해서는아직까지연구가진행되고있다. 또한 cisplatin

배은희외 3 인 : Effects of Antioxidant on Cisplatin Induced Nephropathy 161 Fig. 5. Apoptosis evaluated by TUNEL staining in the kidney sections from different groups of rats. The dark brown dots represent TUNELpositive nuclei. G: glomerulus. 유발신독성에서 TNF-α 외에다른염증관련싸이토카인들의역할은아직규명되지않았다. 최근들어시스플라틴유발신독성의진행에다른면역성신증의경우와마찬가지로대식세포의침윤및이로인한염증반응이중요한병리기전인것으로보고되고있다. 20 신조직내로유입된대식세포는 TNF-α와같은염증성 chemokine과 cytokine의합성을촉진하고, 단백분해효소를분비하여세뇨관세포들을활성화시킨다. 활성화된세포들이다시염증성매개체를분비하여그결과신손상이더욱악화된다. 본연구에서도대식세포의표지자인 ED-1이 cisplatin에서증가, α-la 투여로호전되었다. 또한염증표지자인 COX2, HSP72의단백발현이시스플라틴단독투여군에서현저히증가되었으며, 이는 α-la 투여로호전되었다. 신손상염증반응에중요한역할을한다고잘알려진 COX2 뿐만아니라, HSP도여러스트레스한상황에서유도되며고혈압을포함한다양한질환의병태생리에관여하는것으로알려져있다. 21 HSP는 HSP25, HSP32, HSP 72에대한연구가많이진행되고있는데, HSP70은다양한스트레스상황에서방어역할은하는것으로알려져있다. 또한, HSP70을과발현시킨신세포에시스플라틴처리했을때신세포손상이억제된다는기존보고 22 가있어본연구에서시스플라틴군에서증가한 HSP72은신손상을보호하기위해증가했을것으로생각된다. 이러한사실은 α-la가시스플라틴에의해유발된신장세뇨관염증반응을완화시키고이로인한신장손상을차단할수있음을시사한다. 세포자멸사는염증반응과같은병적인원인에의해발생하기도하고정상적인세포의성장과정에서일어날수도있다. 세포자멸사가발생하면세포질의크기가축소되고핵의염색질의응축이일어나 apoptic body를생성하게된다. 세 포자멸사는 DNA fragmentation이나 TUNEL 염색으로확인할수있는데, 많은연구자들에서세포자멸사가시스플라틴신독성의원인이된다는것이밝혀졌다. 5,7 본연구에서시스플라틴유발신독성흰쥐신장세뇨관에서 TUNEL 양성세포가유의하게증가하였으며, α-la 치료는이러한 TUNEL 염색반응정도를감약시켰다. 따라서시스플라틴유발신독성병인에신장세뇨관세포자멸사가관여하며, α-la에의한신기능개선효과는세포자멸사감소에기인할것으로보인다. Bcl-2 계열의유전자들은여러가지자극에의해세포자멸사를조절하는단백질을만드는데, 이단백질중 Bcl-2와 Bcl-xl은세포자멸사를억제하고 Bax는세포자멸사를조장하는것으로알려져있다. 23 따라서이런단백질의발현여부에따라신장세뇨관세포들이자멸하거나살아남게된다. Bcl-2는세포내의여러기관에존재하는 26 kda의막단백질로서여러종류의유해자극으로부터세포의손상을방지하는역할을한다고알려져있다. 24,25 비록정확한기전은모르지만 Bcl-2는 endoplasmic reticulum (ER) 에서비정상적인칼슘유리를방해하여세포자멸사를억제하는것으로알려져있다. 26 Bax는 21 kda의막단백질로서 ER에서칼슘을배출시켜 ER의칼슘을고갈시키고미토콘드리아의칼슘섭취를조장시켜세포자멸사를조장한다. 27 본연구에서시스플라틴유발신독성에서 Bax 단백발현은유의하게증가하였으며, Bcl-2 발현은감소하였다. 시스플라틴유발신손상이발생하면 cytochrome-c가유리되고이것이 apoptosis activating factor (apaf-1) 와 procaspase 9과결합하여 apoptosome을형성한다. 이것이 caspase 9을활성화시키고활성화된 caspase 9가 caspase 3를활성화시킨다. 7 본연구에서또한 α-la 치료는시스플라틴유발신독성에서 Bax

162 전남의대학술지제 46 권제 3 호 2010 발현증가및 Bcl-2 발현감소를회복시켰으며, caspase 3발현증가역시회복시켰다. 이러한효과는시스플라틴유발신독성에서보이는세포자멸사회복의중요한원인기전으로보인다. 이상의결과시스플라틴유발신손상은항산화제인 α- LA에의해회복되었으며그원인기전에는항염증반응및세포자멸사억제효과에기인하는것으로보인다. 알 이논문은전남대학교병원임상의학연구소학술연구비 (CRI08055-1) 에의하여연구되었음. 림 References 1. Einhorn LH, Williams SD. The role of cis-platinum in solid-tumor therapy. N Engl J Med 1979;300:289-91. 2. Einhorn LH, Donohue J. Cis-diamminedichloroplatinum, vinblastine, and bleomycin combination chemotherapy in disseminated testicular cancer. Ann Intern Med 1977;87:293-8. 3. Rozencweig M, von Hoff DD, Slavik M, Muggia FM. Cisdiamminedichloroplatinum (II). A new anticancer drug. Ann Intern Med 1977;86:803-12. 4. Arany I, Safirstein RL. Cisplatin nephrotoxicity. Semin Nephrol 2003; 23:460-4. 5. Kim SW, Lee JU, Nah MY, Kang DG, Ahn KY, Lee HS, et al. Cisplatin decreases the abundance of aquaporin water channels in rat kidney. J Am Soc Nephrol 2001;12:875-82. 6. Li Q, Bowmer CJ, Yates MS. Effect of arginine on cisplatin-induced acute renal failure in the rat. Biochem Pharmacol 1994;47:2298-301. 7. Pabla N, Dong Z. Cisplatin nephrotoxicity: mechanisms and renoprotective strategies. Kidney Int 2008;73:994-1007. 8. Pabla N, Dong Z. Cisplatin nephrotoxicity: mechanisms and renoprotective strategies. Kidney Int 2008;73:994-1007. 9. El Midaoui A, de Champlain J. Prevention of hypertension, insulin resistance, and oxidative stress by alpha-lipoic acid. Hypertension 2002;39:303-7. 10. Lee KM, Park KG, Kim YD, Lee HJ, Kim HT, Cho WH, et al. Alpha-lipoic acid inhibits fractalkine expression and prevents neointimal hyperplasia after balloon injury in rat carotid artery. Atherosclerosis 2006;189:106-14. 11. Sung MJ, Kim W, Ahn SY, Cho CH, Koh GY, Moon SO, et al. Protective effect of alpha-lipoic acid in lipopolysaccharide-induced endothelial fractalkine expression. Circ Res 2005;97:880-90. 12. Salahudeen A, Poovala V, Parry W, Pande R, Kanji V, Ansari N, et al. Cisplatin induces N-acetyl cysteine suppressible F2-isoprostane production and injury in renal tubular epithelial cells. J Am Soc Nephrol 1998;9:1448-55. 13. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402-8. 14. Davis CA, Nick HS, Agarwal A. Manganese superoxide dismutase attenuates Cisplatin-induced renal injury: importance of superoxide. J Am Soc Nephrol 2001;12:2683-90. 15. Faubel S, Lewis EC, Reznikov L, Ljubanovic D, Hoke TS, Somerset H, et al. Cisplatin-induced acute renal failure is associated with an increase in the cytokines interleukin (IL)-1beta, IL-18, IL-6, and neutrophil infiltration in the kidney. J Pharmacol Exp Ther 2007;322:8-15. 16. Kaushal GP, Kaushal V, Hong X, Shah SV. Role and regulation of activation of caspases in cisplatin-induced injury to renal tubular epithelial cells. Kidney Int 2001;60:1726-36. 17. Wang J, Wei Q, Wang CY, Hill WD, Hess DC, Dong Z. Minocycline up-regulates Bcl-2 and protects against cell death in mitochondria. J Biol Chem 2004;279:19948-54. 18. Bhatt K, Feng L, Pabla N, Liu K, Smith S, Dong Z. Effects of targeted Bcl-2 expression in mitochondria or endoplasmic reticulum on renal tubular cell apoptosis. Am J Physiol Renal Physiol 2008;294: F499-507. 19. He H, Lam M, McCormick TS, Distelhorst CW. Maintenance of calcium homeostasis in the endoplasmic reticulum by Bcl-2. J Cell Biol 1997;138:1219-28. 20. Lithgow T, van Driel R, Bertram JF, Strasser A. The protein product of the oncogene bcl-2 is a component of the nuclear envelope, the endoplasmic reticulum, and the outer mitochondrial membrane. Cell Growth Differ 1994;5:411-7. 21. Hamet P, Malo D, Tremblay J. Increased transcription of a major stress gene in spontaneously hypertensive mice. Hypertension 1990;15:904-8. 22. Komatsuda A, Wakui H, Oyama Y, Imai H, Miura AB, Itoh H, et al. Overexpression of the human 72 kda heat shock protein in renal tubular cells confers resistance against oxidative injury and cisplatin toxicity. Nephrol Dial Transplant 1999;14:1385-90. 23. Ramesh G, Reeves WB. TNF-alpha mediates chemokine and cytokine expression and renal injury in cisplatin nephrotoxicity. J Clin Invest 2002;110:835-42. 24. Ramesh G, Brian Reeves W. Cisplatin increases TNF-alpha mrna stability in kidney proximal tubule cells. Ren Fail 2006;28:583-92. 25. Zhang B, Ramesh G, Norbury CC, Reeves WB. Cisplatin-induced nephrotoxicity is mediated by tumor necrosis factor-alpha produced by renal parenchymal cells. Kidney Int 2007;72:37-44. 26. Yamate J, Tatsumi M, Nakatsuji S, Kuwamura M, Kotani T, Sakuma S. Immunohistochemical observations on the kinetics of macrophages and myofibroblasts in rat renal interstitial fibrosis induced by cisdiamminedichloroplatinum. J Comp Pathol 1995;112:27-39. 27. Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science 1998;281:1322-6.