顺铂治疗急性肾损伤的研究近况

顺铂肾毒性机制及其防护的研究进展作者：曾健劳山来源：《中国实用医药》2008年第10期顺铂(CDDP)是一种高效广谱的抗肿瘤药，容易引起胃肠道反应、骨髓抑制、耳毒性等不良反应。

尤其是它在肾脏中高聚集、高排泄、高代谢，其肾毒性作用尤为突出。

统计显示[1]，临床顺铂化疗肾损害发生率为25%~35%，如何降低其肾毒性，是目前急需解决的课题。

1 顺铂肾毒性机制顺铂在高氯环境下活性低，但当其进入低氯的细胞内液后活性增高，很快发生水合解离，生成带正电荷的水合配离子，受DNA静电引力的作用，向细胞核迁移，形成 cis-［Pt(NH3)2］2/DNA加合物。

加合作用改变了DNA正常复制模版的功能，引起DNA复制障碍，从而抑制肿瘤细胞的分裂[2]。

顺铂的肾毒性是否与这一机制有关尚未明确。

研究表明，氧化性损伤是顺铂肾毒性的机制之一。

顺铂可引起大鼠血浆和肾皮质巯基、谷胱甘肽过氧化物酶(GSH-Px)减少，丙二醛(MDA)的增高[3]。

顺铂在水合代谢过程中会产生大量的氧自由基，自由基引起肾损伤作用的表现在[4]:①膜脂质过氧化反应和膜蛋白功能的受损以及线粒体膜脂质过氧化导致线粒体功能抑制；②蛋白质功能抑制；③破坏核酸及染色体。

顺铂引起的肾小管上皮细胞内钙超载可能在其肾毒性过程中起了重要作用。

除了顺铂氧化应激造成的细胞膜和膜蛋白的损伤，可引发Ca2+内流的增加外，调节细胞内溶质钙的另一个重要因素是内质网(ER)的钙泵。

研究显示给予大鼠顺铂染毒后，可以引起肾脏ER钙泵的活性增加，这可能是顺铂引起的细胞毒的一个有用标记，还可能是顺铂引起肾毒的一个机制[5]。

钙超载引起的严重后果是它可使通过Ca2+介导的一些代谢过程活化，引起细胞的代谢混乱。

NO亦可能参与顺铂肾毒性的过程。

在体内NO是L-精氨酸(L-Arg)和分子氧在一氧化氮合成酶(NOS)催化下生成的。

NO在ARF中是一种具有双重作用的细胞因子，NO可以舒张血管改善微循环，从而保护肾功能。

顺铂致急性肾损伤的研究进展孟晓燕【摘要】顺铂为临床上广泛应用治疗多种实体肿瘤的化疗药物,但在正常组织器官中的不良反应,尤其是肾毒性限制了其临床应用.应用顺铂治疗后,大约1/3的患者可出现肾功能异常,甚至急性肾损伤.近年关于顺铂不良反应的研究聚焦于顺铂肾毒性的发生机制,尤其是在引起肾小管上皮细胞死亡、炎性反应的信号转导通路方面.最近自噬也被证实参与顺铂导致地细胞损伤,虽然已发现一些预防顺铂肾毒性的方法,但大多数保护方法均较局限,因此,更好地理解顺铂肾毒性作用机制,对于顺铂肾毒性防治将具有十分重要意义.该文就顺铂致急性肾损伤作用机制研究进展作一综述.【期刊名称】《医学综述》【年(卷),期】2014(020)021【总页数】3页(P3949-3951)【关键词】顺铂;肾毒性;急性肾损伤【作者】孟晓燕【作者单位】广西医科大学第四附属医院肾内科,广西柳州545005【正文语种】中文【中图分类】R969.320世纪70年代以来，顺铂已被临床广泛用于治疗多种恶性肿瘤，包括睾丸癌、卵巢癌、膀胱癌、宫颈癌、头颈部恶性肿瘤以及小细胞或非小细胞肺癌等，是治疗实体肿瘤最有效和常用的药物之一，但在正常组织中的严重不良反应常限制其临床应用，顺铂不良反应有耳毒性、胃肠道毒性、骨髓抑制、变态反应及肾毒性[1-2]，其中肾毒性最常见，顺铂治疗后，约1/3患者出现肾功能障碍导致急性肾衰竭[1,3-4]，其剂量相关的肾毒性大大限制了临床应用。

目前顺铂致肾损伤机制仍未完全明确，近年研究发现炎症介质、坏死、凋亡、氧化应激、自噬等均可能为顺铂致急性肾损伤原因，该文就顺铂致急性肾损伤作用机制的研究进展予以综述。

1 炎性介质Deng等[5]发现抗炎因子白细胞介素10可减轻顺铂导致的肾组织损伤及肾小管上皮细胞的死亡，由此提出在顺铂肾毒性中，有大量促炎性反应细胞因子及趋化因子参与。

顺铂触发的炎性反应可引起肾组织损伤及功能障碍，出现急性肾损伤[6]。

顺铂致大鼠急性肾损伤肾组织细胞凋亡及NGAL的相关性研究目的：研究顺铂（Cisplatin，CDDP）致大鼠急性肾损伤（AKI）模型中肾组织细胞凋亡及肾组织中性粒细胞明胶酶相关脂质运载蛋白（NGAL）变化及相关性。

方法：将36只健康雄性Wistar大鼠随机分为盐水对照组（CN组）与顺铂模型组（CP组），CP组为腹腔单次注射顺铂10 mg/kg，CN组腹腔注射同顺铂组等容量生理盐水，观察应用顺铂后12、24、48、96、144 h各组大鼠血清肌酐（Scr），肾组织细胞凋亡及NGAL变化。

结果：与CN组相比，CP组48 Scr 开始升高，144 h达峰值；CP组12 h肾组织凋亡细胞开始增多，48 h增加最明显；CP组12 h NGAL在肾小管上皮细胞中表达增强（P＜0.05），48 h表达最明显。

肾组织细胞凋亡同NGAL表达显著相关（r=0.925，P＜0.01）。

结论：细胞凋亡可能是CDDP致大鼠AKI早期致病机制之一，NGAL不仅是早期肾损伤的标记物，还可能参与AKI发病机制。

急性腎损伤（acute kidney injury，AKI）是常见的内科疾病，也是急危重症常见的合并症，与较高的死亡率和不良预后相关[1]。

关于我国AKI的一项横断面研究指出，2013年我国有（140～290）万AKI患者住院，AKI住院死亡率达12.4%，而71.6%的患者在诊断AKI时或之前有肾毒性药物应用[2]。

提示药物相关性AKI可能已经成为我国巨大的医疗负担。

顺铂（Cisplatin，CDDP）是一种广泛应用的抗癌药物，剂量相关的肾毒性大大限制了其临床应用，中性粒细胞明胶酶相关脂质运载蛋白（NGAL）是有前景的AKI早期损伤标记物，前期的工作也发现，CDDP致大鼠AKI早期即存在血、尿NGAL升高[3]，目前关于NGAL 研究主要集中于作为早期诊断，预后评估的生物学标记物及参与疾病发病两方面，在生理情况下或不同的疾病病程中，NGAL扮演着不同甚至截然相反的角色，国内不少研究仅关注于NGAL的早期诊断特性，忽略其生理特性及在疾病发病机制中的角色。

顺铂肾毒性机制及其防护的研究进展曾健劳山顺铂(CDDP)是一种高效广谱的抗肿瘤药,容易引起胃肠道反应、骨髓抑制、耳毒性等不良反应。

尤其是它在肾脏中高聚集、高排泄、高代谢,其肾毒性作用尤为突出。

统计显示[1],临床顺铂化疗肾损害发生率为25%~35%,如何降低其肾毒性,是目前急需解决的课题。

1顺铂肾毒性机制顺铂在高氯环境下活性低,但当其进入低氯的细胞内液后活性增高,很快发生水合解离,生成带正电荷的水合配离子,受DNA静电引力的作用,向细胞核迁移,形成c i s-[P t (NH3)2]2/DNA加合物。

加合作用改变了DNA正常复制模版的功能,引起DNA复制障碍,从而抑制肿瘤细胞的分裂[2]。

顺铂的肾毒性是否与这一机制有关尚未明确。

研究表明,氧化性损伤是顺铂肾毒性的机制之一。

顺铂可引起大鼠血浆和肾皮质巯基、谷胱甘肽过氧化物酶(G S H-Px)减少,丙二醛(M DA)的增高[3]。

顺铂在水合代谢过程中会产生大量的氧自由基,自由基引起肾损伤作用的表现在[4]:¹膜脂质过氧化反应和膜蛋白功能的受损以及线粒体膜脂质过氧化导致线粒体功能抑制;º蛋白质功能抑制;»破坏核酸及染色体。

顺铂引起的肾小管上皮细胞内钙超载可能在其肾毒性过程中起了重要作用。

除了顺铂氧化应激造成的细胞膜和膜蛋白的损伤,可引发Ca2+内流的增加外,调节细胞内溶质钙的另一个重要因素是内质网(ER)的钙泵。

研究显示给予大鼠顺铂染毒后,可以引起肾脏ER钙泵的活性增加,这可能是顺铂引起的细胞毒的一个有用标记,还可能是顺铂引起肾毒的一个机制[5]。

钙超载引起的严重后果是它可使通过Ca2+介导的一些代谢过程活化,引起细胞的代谢混乱。

NO亦可能参与顺铂肾毒性的过程。

在体内NO是L-精氨酸(L-A rg)和分子氧在一氧化氮合成酶(NO S)催化下生成的。

NO在AR F中是一种具有双重作用的细胞因子,NO可以舒张血管改善微循环,从而保护肾功能。

REVIEW ARTICLECisplatin-induced nephrotoxicity and targets of nephroprotection:an updateNeife Aparecida Guinaim dos Santos •Maria Augusta Carvalho Rodrigues •Nadia Maria Martins •Antonio Cardozo dos SantosReceived:26January 2012/Accepted:14February 2012/Published online:1March 2012ÓSpringer-Verlag 2012Abstract Cisplatin is a highly effective antitumor agent whose clinical application is limited by the inherent nephrotoxicity.The current measures of nephroprotection used in patients receiving cisplatin are not satisfactory,and studies have focused on the investigation of new possible protective strategies.Many pathways involved in cisplatin nephrotoxicity have been delineated and proposed as tar-gets for nephroprotection,and many new potentially pro-tective agents have been reported.The multiple pathways which lead to renal damage and renal cell death have points of convergence and share some common modulators.The most frequent event among all the described pathways is the oxidative stress that acts as both a trigger and a result.The most exploited pathways,the proposed protective strategies,the achievements obtained so far as well as conflicting data are summarized and discussed in this review,providing a general view of the knowledge accu-mulated with past and recent research on this subject.Keywords Cisplatin ÁNephrotoxicity ÁNephroprotection ÁOxidative stress ÁApoptosis ÁMolecular mechanisms ÁMitochondriaCisplatinCisplatin (cisplatinum or cis-diamminedichloroplatinum (II),CDDP)is a highly effective chemotherapeutic drugwhose anticancer activity was accidentally discovered by the physicist–biologist Barnett Rosenberg,during his studies addressing the effect of a platinum electrode-gen-erated electric field on the division processes of Esche-richia coli .He observed that the cellular division was inhibited and a filamentous growth was induced by elec-trolysis products that were afterward identified as platinum compounds.Based on this observation,he and his col-leagues investigated the antitumor activity of platinum compounds in leukemia L1210-and Sarcoma 180-bearing mice.The antitumor efficacy of cisplatin was then dis-covered (Rosenberg et al.1965,1967,1969).The clinical use of cisplatin was approved by the FDA in December 1978(FDA database).Since then,the application of cisplatin has been broadened to several types of cancer and it has been used both alone or com-bined with other drugs:as first-line treatment,as adjuvant,or even as neoadjuvant therapy of other procedures such as surgery or radiotherapy.Currently,the use of cisplatin is approved to treat bladder cancer,cervical cancer,malig-nant mesothelioma,non-small cell lung cancer,ovarian cancer,squamous cell carcinoma of the head and neck,and testicular cancer (National Cancer Institute database).Additionally,cisplatin has been used to treat other types of cancer when the first-line treatment has failed or yet in specific situations that preclude the standard treatment (Candelaria et al.2006;Helm and States 2009;Goffin et al.2010;Campbell and Kindler 2011;Ismaili et al.2011a ,b ).Cisplatin chemotherapy is limited by tumor cells resis-tance and severe side effects such as nephrotoxicity,neurotoxicity,ototoxicity,and emetogenicity (Wang and Lippard 2005;Pabla and Dong 2008).Among these factors,nephrotoxicity has been reported as the major limiter in cisplatin therapy (Arany and Safirstein 2003).N.A.G.dos Santos ÁM.A.Carvalho Rodrigues ÁN.M.Martins ÁA.C.dos Santos (&)Department of Clinical,Toxicological Analyses and FoodSciences of School of Pharmaceutical Sciences of Ribeira˜o Preto,University of Sa˜o Paulo,Ribeira ˜o Preto,SP,Brazil e-mail:acsantos@p.brArch Toxicol (2012)86:1233–1250DOI 10.1007/s00204-012-0821-7The susceptibility of kidneys to cisplatin toxicity Kidneys are particularly affected by cisplatin,and this has been attributed mainly to(a)high concentration of cisplatin in the kidneys and(b)the renal transport systems.Cisplatin is eliminated predominantly by the kidneys;the biliary and the intestinal excretion of this drug are minimal.During the excretion process,the drug is concentrated and even non-toxic blood levels of cisplatin might reach toxic levels in kidneys.In fact,it has been reported that the concentration of cisplatin in epithelial tubular cells isfivefold higher than in blood(Rosenberg1985;Bajorin et al.1986;Gordon and Gattone1986;Kuhlmann et al.1997;Schenellmann2001). The nephrotoxicity induced by cisplatin is dose-dependent and therefore limits the increase of doses,compromising the efficacy of the therapy(Hanigan and Devarajan2003). The toxic effects occur primarily in the renal proximal tubules,particularly in the epithelial tubular cells of S-3 segment(Werner et al.1995).Glomeruli and distal tubules are also affected afterward.Impairment of the renal func-tion is found in approximately25–35%of patients treated with a single dose of cisplatin(Han et al.2009).Decrease of20–40%of glomerularfiltration,increased BUN(blood urea nitrogen),and increased serum creatinine concentra-tions as well as reduced serum magnesium and potassium levels are frequent in patients treated with cisplatin(Ries and Klastersky1986;Kintzel2001;Han et al.2009).The high concentration of cisplatin in kidneys favors its cellular uptake by passive diffusion(Gale et al.1973; Gately and Howell1993),and this was once considered the main process through which cisplatin entered and accu-mulated in cells.More recently,active transport systems have gained importance and have been associated with tumor cells resistance as well as the toxicity of cisplatin (Ishida et al.2002;Pabla et al.2009;Burger et al.2011). The facilitated transport systems which have been associ-ated with cisplatin nephrotoxicity are those mediated by the organic cation transporter OCT2and more recently,the copper transporter Ctr1.In2002,Ishida and colleagues proposed that cisplatin uptake was mediated by the copper transporter Ctr1in yeast and mammals(Ishida et al.2002). Although Ctr1is highly expressed in kidney(Sharp2003), it wasfirst associated with cisplatin uptake by non-renal cells and only recently a study associated Ctr1with cis-platin uptake in renal cells and therefore nephrotoxicity (Pabla et al.2009).OCT2is highly expressed in the basolateral membrane of proximal tubules and has been reported to participate in the renal accumulation of cisplatin(Ludwig et al.2004;Ciarimboli et al.2005; Yonezawa et al.2005).It has been reported that OCT1/2double-knockout mice treated with cisplatin presented only a mild nephrotoxicity as well as reduced renal platinum accumulation when compared to wild-type mice(Ciarimboli et al.2005). Additionally,it was reported that the concomitant admin-istration of imatinib,a cationic anticancer agent,with cis-platin prevented cisplatin-induced nephrotoxicity by inhibiting the OCT2-mediated renal accumulation of cis-platin(Tanihara et al.2009).In vivo and in vitro studies have shown that cimetidine inhibits cisplatin renal damage without affecting its antitumor activity(Katsuda et al. 2010).However,in another study with cimetidine in vivo, only a partial protection against cisplatin-induced nephro-toxicity was observed.The nephroprotective action of cimetidine has been attributed to(i)a competitive inhibi-tion of cisplatin transport by OCT2,since cimetidine is an organic cation and therefore an OCT substrate(Ciarimboli et al.2005);and(ii)inhibition of cytochrome P450with blockade of iron release and consequently inhibition of hydroxyl radicals generation(Baliga et al.1998).The protective effect of cimetidine has also been shown in a clinical trial with nine patients treated with cisplatin, verapamil,and cimetidine(Sleijfer et al.1987).Another strategy to blockade cisplatin uptake in renal cells is the inhibition of Ctr1.In fact,it has been reported that CTR1-deficient cells accumulate less platinum in their DNA and are more resistant to the cytotoxic effect of cisplatin than the CTR1-replete cells(Lin et al.2002).The antitumor mechanism versus the nephrotoxic mechanismThe molecule of cisplatin is formed by a central platinum ion linked to2chloride ions and2ammonia molecules. Neither the antitumor activity nor the nephrotoxicity of cisplatin results from the heavy metal platinum itself,since both effects are stereospecific to the cis isomer,not occurring with the trans isomer(Goldstein and Mayor 1983).Instead,the cytotoxicity of cisplatin is related to highly reactive aquated metabolites,whose formation is determined by the concentration of chloride ions.As the intracellular concentration of chloride(20mM)is lower than the blood concentration(100mM),cisplatin remains unaltered in the bloodstream,but undergoes hydrolysis in the intracellular environment,originating positively charged molecules in which one or two chloride ions have been replaced by water.These aquated forms easily react with the nuclear DNA,forming covalent bonds with purine bases,primarily at the N7position,resulting in1,2-intra-strand crosslinks,which are the main responsible for the genotoxic effects of cisplatin.These crosslinks between DNA and cisplatin lead to the impairment of replication and transcription,resulting in cell cycle arrest and even-tually apoptosis(Jamieson and Lippard1999;Wong and Giandomenico1999;Cohen and Lippard2001;Wang andLippard2005).The apoptosis triggered by DNA damage is mediated by the tumor suppressor gene p53that activates pro-apoptotic genes and repress anti-apoptotic genes(Jiang et al.2004;Norbury and Zhivotovsky2004;Jiang and Dong2008).The dividing tumor cells are particularly susceptible to DNA damage,and the anticancer activity of cisplatin has been mainly attributed to DNA adducts for-mation(Eastman1999;Hanigan and Devarajan2003). However,some studies have suggested that nuclear DNA adducts formation may not be the only determinant of cisplatin pharmacological effect and that mitochondrial DNA(mtDNA)might be a more common target of cis-platin binding,due to its weaker repair(Olivero et al.1997; Gonzalez et al.2001;Yang et al.2006;Cullen et al.2007).In adult humans,proximal tubular cells are non-divid-ing;therefore,the formation of adducts with DNA might not play a key role in cisplatin nephrotoxicity(Wainford et al.2008).Besides nuclear and mitochondrial DNA, cisplatin targets other cellular components such as RNA, proteins,and phospholipids and distinct mechanisms have been associated with the toxic effects of cisplatin on healthy renal cells.Oxidative damage and inflammatory events might explain the effects on other cellular constit-uents and have been associated with cisplatin-induced nephrotoxicity(Cvitkovic1998;Ali and Al Moundhri 2006;Yao et al.2007;Pabla and Dong2008).Several lines of evidence indicate that cisplatin nephrotoxicity is mainly associated with mitochondria-generated oxygen reactive species(ROS)(Matsushima et al.1998;Somani et al. 2000;Chang et al.2002;Wang and Lippard2005;Santos et al.2007;Santos et al.2008).Alterations in renal hemodynamic modulators have also been associated with the toxic effects of cisplatin on kidneys(Hye Khan et al. 2007).It has been suggested that cisplatin is conjugated with reduced glutathione(GSH)in the liver and reaches the kidney as a cisplatin–GSH conjugate,which is cleaved to a nephrotoxic metabolite mainly by the action of gamma-glutamyl transpeptidase(GGT),an enzyme primarily located in the brush border of the proximal convoluted tubule of the kidney.The metabolite formed is a highly reactive thiol/platinum compound that interacts with mac-romolecules leading eventually to renal cell death(Ward 1975;Wainford et al.2008).The interference in this bio-transformation pathway has been proposed as an approach to prevent the formation of the nephrotoxic metabolite and therefore,minimizing cisplatin nephrotoxicity.It has been demonstrated that GGT-deficient mice are resistant to the nephrotoxic effects of cisplatin(Hanigan et al.2001). Additionally,studies have demonstrated that inhibition of GGT with acivicin,both in mice and in rats,protected against the nephrotoxicity of cisplatin(Hanigan et al.1994; Townsend and Hanigan2002).The participation of other enzymes such as aminopeptidase N(AP-N),renal dipepti-dase(RDP),and cysteine-S-conjugate beta-lyase(C–S lyase)in this toxificant pathway has been reported.The following sequence has been proposed:after cisplatin–GSH conjugates are secreted into the proximal tubule lumen and cleaved by GGT,a cysteine–glycine conjugate is formed and then cleaved by the cell surface aminopeptidases, AP-N,or RDP,to a cysteine conjugate,which is then reabsorbed into proximal tubular cells andfinally metabo-lized by C–S lyase to toxic reactive thiols resulting in nephrotoxicity(Hanigan et al.1994;Townsend and Hani-gan2002;Townsend et al.2003;Zhang and Hanigan2003). The inhibition of C–S lyase with amino oxyacetic acid was protective in mice treated with15mg/kg cisplatin(Town-send and Hanigan2002);however,opposing data have been reported.According to a more recent study,AP-N,RDP, and CS-lyase inhibition were non-protective against neph-rotoxicity in mice treated with10mg/kg cisplatin and/or in rats treated with6mg/kg cisplatin(Wainford et al.2008).A second-generation platinum-protecting disulfide drug named BNP7787(disodium2,2-dithio-bis-ethane sulfo-nate,dimesna,Tavocept TM)was developed to specifically inactivate the toxic platinum species found in normal organs in order to reduce or prevent common toxicities of platinum chemotherapeutic drugs(Hausheer et al.1998). BNP7787is selectively taken up by the kidneys where it is converted into mesna(Ormstad and Uehara1982). BNP7787may accumulate in renal tubular cells,where it can exert its protective effects against cisplatin-induced nephrotoxicity by direct covalent conjugation of mesna with cisplatin(Hausheer et al.2011a).Besides the forma-tion of this inactive adduct with cisplatin,other mecha-nisms might be involved in the protection:(a)inhibition of GGT,(b)inhibition of AP-N,and(c)inhibition of C–S lyase(Hausheer et al.2010,2011b).Additionally,it was reported that BNP7787does not interfere in the antitumor activity of cisplatin in human ovarian cancer cell lines in vitro or in nude mice bearing human ovarian cancer xenografts(Boven et al.2002).The drug is currently undergoing global Phase III studies(Hausheer et al.2011a). Mechanisms of cell death in cisplatin-induced nephrotoxicityCisplatin induces two models of cell death:apoptosis and necrosis.Initially,only necrosis was associated with the renal damage induced by cisplatin(Goldstein and Mayor 1983);afterward,the induction of apoptosis was also demonstrated.A study published in1996demonstrated that high concentrations of cisplatin(800l M)induced necrosis in primary cultures of mouse proximal tubular cells,while lower concentrations(8l M)led to apoptosis(Lieberthalet al.1996).More recently,several studies have demon-strated that both the mechanisms of cell death are induced by cisplatin in vivo (Baek et al.2003;Tsuruya et al.2003;Wang and Lippard 2005).The relative contribution of both types of cell death,apoptosis,and necrosis,to cisplatin nephrotoxicity has not been established yet (Bonegio and Lieberthal 2002;Faubel et al.2004).However,apoptosis has been in the spotlight in the last years.Necrosis has been mainly associated with high doses of cisplatin,severe mitochondrial damage,and ATP depletion,whereas apoptosis is a process dependent on ATP energy and therefore associated with the milder mitochondrial altera-tions resulting from therapeutic doses (Lieberthal et al.1998;Ueda et al.2000;Hanigan and Devarajan 2003;Wang and Lippard 2005).Different apoptotic pathways are triggered by cisplatin in renal tubular epithelial cells (RTEC).The main reported pathways are (a)the intrinsic pathway,which is triggered by mitochondria and (b)the extrinsic pathway,which is mediated by TNF (tumor necrosis factor)receptor/ligand and Fas (APO -1or CD95)/Fas ligand systems (Ramesh and Reeves 2002).Additionally,the endoplasmic reticulum stress (ER stress)pathway has also been demonstrated in cisplatin-induced apoptosis in RTEC (Liu and Baliga 2005).The mechanisms of nephrotoxicity induced bycisplatin are summarized in Fig.1,and the potential cytoprotectors which interfere in these pathways are sum-marized in Table 1.Intrinsic or mitochondrial apoptotic pathwayMitochondrial injury in RTEC leads to the release of apoptogenic factors,including cytochrome c ,Smac/DIA-BLO,Omi/HtrA2,and apoptosis-inducing factor or AIF (Daugas et al.2000a ;Servais et al.2008).The migration of cytochrome c to cytosol is a key event in caspases acti-vation,and the following sequence of events has been described:formation of Apaf-1/cytochrome c apoptosome,caspase-9activation,and ultimately the activation of the executioner caspase-3(Lee et al.2001;Park et al.2002;Cullen et al.2007).Smac/DIABLO and Omi/HtrA2inhibit the suppressors of apoptosis,IAPs (inhibitor of apoptosis proteins),which interfere in the cytochrome c /Apaf-1/caspase-9activating pathway.Omi/HtrA2can also pro-mote apoptosis through its serine protease activity,a mechanism independent of caspases (Du et al.2000;Cil-enti et al.2005).AIF is a protein that translocates to the nucleus and promotes apoptosis without the activation of caspases (Daugas et al.2000a).Fig.1Multiple pathways involved in cisplatin-induced nephrotoxicityTable1Nephroprotective agents,targeted pathways,molecular mechanisms,and experimental models used in studies Target/class Agents Mechanisms of actionand experimental modelReferencesCisplatin transport and accumulation Imatinib Reduced the toxicity and platinumconcentration in OCT2-expressing HEK293cells and ratkidneysTanihara et al.(2009)Cimetidine*Competitive inhibition of cisplatintransport at OCT2in vitro and inratsKatsuda et al.(2010)Acivicin Inhibition of GGT in mice and rats Hanigan et al.(1994),Townsendand Hanigan(2002)Amino oxyacetic acid Inhibition of C–S lyase in mice Townsend and Hanigan(2002)BNP7787*Formation of inactive adducts,inhibition of GGT,inhibition ofAP-N,inhibition of C–S lyase invitro and in miceHausheer et al.(2010),Hausheer et al.(2011a,b)Saline,saline plus mannitol,and saline plus furosemideHydration/diuresis,increases therate of cisplatin elimination inhumansGonzales-Vitale et al.(1977),Hayes et al.(1977),Frick et al.(1979),Santoso et al.(2003)Oxidative stress (antioxidants)Vitamin C ROS scavenging and/or ironchelating,decreasing oxidativestress,and avoiding activation ofapoptosis in vitro and rodentmodelsTarladacalisir et al.(2008)Vitamin E Ajith et al.(2009)Vitamin A Dillioglugil et al.(2005) Resveratrol Do Amaral et al.(2008)Quercetin Francescato et al.(2004)Caffeic acid phenethyl ester(CAPE)Ozen et al.(2004)Naringenin Badary et al.(2005)Lycopene Atessahin et al.(2005)DMTU Santos et al.(2008)DMSO Jones et al.(1991)Carvedilol Rodrigues et al.(2010,2011) Captopril El-Sayed el et al.(2008)Allopurinol plus ebselem Lynch et al.(2005)Edaravone Satoh et al.(2003),Iguchi et al.(2004) Desferrioxamine(DFO)Kadikoylu et al.(2004)Oxidative stress (antioxidants–thiols)Diethyldithiocarbamate(DDTC),GSH,D-methionine,sodium thiosulphate(STS)Restoration of thiol enzymesfunction,free radical scavenging,formation of non-toxic adducts,reduction of cisplatin uptake byrenal cells in vitroCvitkovic(1998),Wu et al.(2005),Bae et al.(2009)N-acetylcysteine(NAC)Blockade of intrinsic and extrinsicapoptotic pathways induced bycisplatin in SCLC,SKOV3,andU87MG human tumor cell linesWu et al.(2005)Alpha-lipoic acid Maintenance of activities of NOand ET systems and inhibition ofthe development of apoptosis inratsBae et al.(2009)Amifostine(WR2721)**Scavenger of ROS tested in vitro,in rodents and humansCvitkovic(1998)Cisplatin can trigger the mitochondrial apoptotic path-way through different stimuli such as increased ROS generation and the activation of pro-apoptotic proteins (Hanigan and Devarajan2003),which permeabilize the outer mitochondrial membrane and induce the release of cytochrome c(Lee et al.2001;Park et al.2002),AIF(Seth et al.2005)and Omi/HtrA2(Cilenti et al.2005).Mitochondrial dysfunction is considered a key event in cisplatin-induced renal damage.Decline in membrane electrochemical potential,disturbance in calcium homeo-stasis,reduced ATP synthesis,and impaired mitochondrial respiration have been demonstrated in kidneys of rats treated with cisplatin(Santos et al.2007;Rodrigues et al. 2010).It is known that cisplatin can damage complexes I,II, III,and IV of the mitochondrial respiratory chain, increasing the generation of superoxide anions at com-plexes I,II,and III.Superoxide anions might originateTable1continuedTarget/class Agents Mechanisms of actionand experimental modelReferencesApoptosis TNFR1-deficient mice Avoid TNF activation of apoptosiswhen ROS production isincreased in mice models Tsuruya et al.(2003)TNF-a-deficient mice Ramesh and Reeves(2002) TNFR2-deficient mice Ramesh and Reeves(2003) Trichostatin A(TSA)Inhibition of p53activation inRTPC cell lineDong et al.(2009)Suberoylanilide hydroxamic acid(SAHA)Inhibition of p53activation andreduction of Bax translocationand cytochrome c release inRTPC and HCT116colon cancercellsDong et al.(2009)Pituitary adenylate cyclase-activating polypeptide (PACAP38)Inhibition of p53expression,inhibition of caspase-7cleavagein HK-2cells and miceLi et al.(2010,2011)Erythropoietin(EPO)Up-regulation of anti-apoptoticproteins expression,down-regulation of pro-apoptoticproteins and reduction ofcaspase-3activity in ratsRjiba-Touati et al.(2012)Rottlerin Inhibition of PKC delta in mice Pabla et al.(2011)Inflammation Salicylate Anti-inflammatory action in rats Li et al.(2002)GM6001and pentoxifylline Inhibition of TNF-a withantagonists and blunted the up-regulation of cytokines such asTGF-b,RANTES,MIP-2,MCP-1,and IL-1b in miceRamesh and Reeves(2002)Pituitary adenylate cyclase-activating polypeptide (PACAP38)Reduction of TNF-a levels in HK-2cells and miceLi et al.(2010,2011)Quercetin*Inhibition of TNF-a and NOÁproduction through attenuationof NF-kB activity in ratsSanchez-Gonzalez et al.(2011a,b) Celecoxib Inhibition of COX-2Jia et al.(2011)Abnormal hemodynamics Captopril Inhibition of renin-angiotensinsystem,prostaglandins,andendothelin-1in ratsEl-Sayed el et al.(2008),Saleh et al.(2009)Losartan Blockage of angiotensin IIreceptor in ratsSaleh et al.(2009)Aminophylline Competitive antagonist ofadenosine in ratsHeidemann et al.(1989)BN-52063Platelet-activating factor(PAF)antagonist in ratsdos Santos et al.(1991),Dos Santos et al.(1991)*Does not change cisplatin-antitumor action in the experimental model **Approved to be used in humanshydroxyl radicals by partial reduction catalyzed by transi-tion metals,mainly iron(Fenton reaction)(Kruidering et al. 1994,1997;Turrens2003;Yao et al.2007).Hydroxyl radicals are very strong oxidants,and their induction has been demonstrated in kidneys of rats treated with cisplatin (Matsushima et al.1998;Santos et al.2008).The oxidative damage induced by cisplatin has been associated with depletion of the non-enzymatic(GSH and NADPH)and the enzymatic antioxidant defense system(superoxide dismu-tase,catalase,glutathione peroxidase,glutathione trans-ferase,and glutathione reductase)in rat kidneys (Hannemann et al.1991;Sadzuka et al.1992;Antunes et al.2000;Kadikoylu et al.2004).Lipoperoxidation, oxidation of cardiolipin,oxidation of sulfhydryl protein, increased carbonylated proteins levels,decreased activity of aconitase,cytochrome c release,increased activity of caspase-9,and caspase-3have also been associated with the renal damage induced by cisplatin(Kaushal et al.2001; Park et al.2002;Santos et al.2007).Cytochrome c is attached to the inner mitochondrial membrane(IMM),and its release occurs due to the loss of the mitochondrial membrane integrity.The mitochondrial membrane is a target of the oxidative species that attack proteins and lipids,particularly the anionic phospholipid cardiolipin, located in IMM.As cardiolipin holds cytochrome c attached to IMM,its oxidation contributes to cytochrome c release to cytosol(Petrosillo et al.2003).Cardiolipin is also a target of caspase-2and Bid,a pro-apoptotic protein from the Bcl2family,which promotes a link between the extrinsic and intrinsic apoptotic pathways,since it is acti-vated by caspase-8(extrinsic pathway)and acts on mito-chondria promoting the apoptotic intrinsic pathway (Enoksson et al.2004;Campbell et al.2008;El Sabbahy and Vaidya2011).Besides increasing mitochondrial ROS generation,cisplatin activates the pro-apoptotic proteins Bax and Bak,upstream mitochondrial injury.These pro-teins induce the permeabilization of the outer mitochon-drial membrane and therefore,cytochrome c release and caspases activation(Lee et al.2001;Park et al.2002; Cullen et al.2007).The nephrotoxicity induced by cisplatin is attenuated in Bax/Bak-knockout cells and in Bax-defi-cient mice(Jiang et al.2006;Wei et al.2007a).Erythro-poietin(EPO),a renal cytokine which regulates hematopoiesis,has been shown to reduce apoptosis during cisplatin nephrotoxicity by the up-regulation of anti-apop-totic proteins expression,down-regulation of pro-apoptotic protein levels,and reduction of caspase-3activity(Rjiba-Touati et al.2012).Besides the apoptosis dependent of caspases activation, cisplatin can also trigger a mitochondrial mediated and caspase-independent apoptotic pathway through the apop-tosis-inducing factor(AIF),a protein located in the mito-chondrial intermembrane space and present in renal epithelium.When the outer mitochondrial membrane is damaged,AIF translocates to the nucleus inducing chro-matin condensation and large-scale DNA fragmentation. The anti-apoptotic Bcl-2protein preserves the mitochon-drial membrane integrity,preventing both the release of cytochrome c and translocation of AIF to the nucleus (Daugas et al.2000b;Adams and Cory2001).The release of AIF has been reported to be dependent on caspase-2, which is activated by PIDD,a p53-induced protein with death domain.Caspase-2permeabilizes the outer mito-chondrial membrane and damages anionic phospholipids, causing release of pro-apoptotic factors such as cyto-chrome c and AIF.Inhibition of caspase-2and inhibition of AIF have been reported as protective against cisplatin-induced renal damage(Daugas et al.2000b;Enoksson et al. 2004;Seth et al.2005;Jiang and Dong2008;Servais et al. 2008).The transcriptional factor p53activates pro-apoptotic genes encoding Bax,Bak,PUMA-a,PIDD,and the ER-iPLA2(Ca2?-independent phospholipase A2)and down-regulates the anti-apoptotic proteins Bcl-2and Bcl-xL, leading to the mitochondrial apoptotic pathway(Seth et al. 2005;Jiang et al.2006;Jiang and Dong2008;Servais et al. 2008).The involvement of ROS,particularly hydroxyl radicals,in p53activation during cisplatin nephrotoxicity has been suggested(Jiang et al.2007),and the crucial role of hydroxyl radicals in cisplatin nephrotoxicity has been demonstrated(Santos et al.2008).Due to the importance of ROS and oxidative stress in the induction of apoptotic cell death,particularly of the intrinsic pathway,one of the most studied approaches to protect against cisplatin nephrotoxicity is the use of natural and synthetic antioxidants.Experimental studies have reported the protective effects of natural compounds such as vitamins C(Tarladacalisir et al.2008),E(Ajith et al. 2009),and A(Dillioglugil et al.2005);resveratrol(Do Amaral et al.2008),quercetin(Francescato et al.2004), and caffeic acid phenethyl ester(Ozen et al.2004); naringenin(Badary et al.2005)and lycopene(Atessahin et al.2005),as well as synthetic compounds such as DMTU(Santos et al.2008),DMSO(Jones et al.1991), carvedilol(Rodrigues et al.2010),allopurinol plus ebselem (Lynch et al.2005),edaravone(Satoh et al.2003;Iguchi et al.2004),desferrioxamine(DFO)(Kadikoylu et al. 2004),and many others.Antioxidants protect kidneys from cisplatin damage mainly by free radical scavenging or iron chelation(Koyner et al.2008).As ROS plays a role in the inflammatory pathway,antioxidants may also interfere positively in the inflammatory process.The nephroprotec-tive effect of quercetin,for example,seems to be related with its antioxidant activity as well as with its capacity to inhibit renal inflammation and tubular cell apoptosis. Quercetin has been shown to inhibit lipopolysaccharide-。