热休克蛋白72抑制ATP耗竭时细胞色素C释放所诱导的肾小管上皮细胞凋亡

目的 研究热休克蛋白(HSP)72对ATP耗竭时细胞色素C释放所导致的肾小管上皮细胞凋亡的保护作用及其分子机制。方法 应用代谢抑制剂暂时性阻断细胞内ATP的生成,引起细胞凋亡。应用热处理细胞或编码HSP72 RNA的腺病毒感染细胞,诱导HSP72的表达。以Western印迹检测释放于胞浆内的细胞色素C。荧光肽法测定半胱氨酸天冬氨酸蛋白酶(caspase)3活性。Hoechst33342染色观察细胞凋亡的发生情况。结果 肾小管上皮细胞内ATP耗竭时,释放至胞浆内的细胞色素C的含量增多,caspase 3活性增强;细胞内ATP再恢复时,细胞色素C的释放和caspase 3活性进一步增加,细胞体积缩小,核浓染、固缩,形成凋亡小体。预先热处理后,各组细胞色素C的释放明显减少,caspase 3活性显著抑制(P<0.05,n=3)。高表达HSP72时,各时间点caspase 3活性的抑制程度与热处理组相似,细胞体积缩小,核浓染、固缩,凋亡小体的形成明显减少。结论 HSP72可抑制ATP耗竭时细胞色素C所导致的肾小管上皮细胞凋亡,其机制是抑制凋亡通路中细胞色素C的释放和caspase 3的激活。     

Smac/Diablo与X-连锁凋亡抑制蛋白在ATP耗竭再恢复诱导肾小管上皮细胞凋亡中的作用

目的 研究线粒体蛋白Smac/Diablo和细胞X-连锁凋亡抑制蛋白(XIAP)在ATP耗竭及再恢复导致肾小管上皮细胞凋亡中的作用和机制。 方法 应用代谢抑制剂暂时性阻断人肾小管上皮细胞(HK-2细胞)内ATP的生成,再换用含糖的培养液使细胞内ATP再恢复,诱导肾小管上皮细胞凋亡。应用Hoechst33342检测肾小管上皮细胞凋亡的发生。用间接免疫荧光检测Smac/Diablo在细胞内的分布。分别提取胞质蛋白和细胞总蛋白,以Western印迹检测胞质中Smac/Diablo、XIAP和活化半胱氨酸天冬氨酸蛋白酶3前体(pro-caspase-3)的蛋白水平。 结果 肾小管上皮细胞内ATP耗竭及再恢复时,Hoechst33342染色可见HK-2细胞核固缩和凋亡小体的形成;间接免疫荧光可见Smac/Diablo由线粒体释放至胞质;Western印迹可见胞质内Smac/Diablo的含量增多( P < 0.01);XIAP和pro-caspase 3的蛋白水平降低(P < 0.05)。 结论 肾小管上皮细胞内ATP耗竭及再恢复时,Smac/Diablo释放至胞质,XIAP蛋白水平降低,进而激活caspase 3,介导肾小管上皮细胞凋亡。     

半胱氨酸天冬氨酸3依赖的bcl-2的降解在ATP耗竭诱导肾小管上皮细胞凋亡中的作用

目的 研究ATP耗竭再恢复诱导的肾小管上皮细胞凋亡中,半胱氨酸天冬氨酸（caspase）3激活介导bcl-2降解的作用.方法 使用代谢抑制剂短暂阻断细胞内ATP的生成,诱导细胞凋亡.以Hoechst 33342染色观察凋亡细胞.应用编码人bcl-2 RNA的腺病毒感染细胞,使bcl-2在细胞内高表达.应用流式细胞仪,分析细胞凋亡和坏死的发生情况.以Western印迹检测caspase 3的活化以及bcl-2和bcl-2的降解产物.体外实验观察caspase 3和caspase 3特异性抑制剂DEVD对bcl-2降解产物的影响.结果 高表达bcl-2组与对照组比较,细胞ATP耗竭60min、90 min、120 min和再恢复60min后,可减少细胞凋亡50%,P＜0.05.肾小管上皮细胞ATP耗竭时,caspase 3被激活,bcl-2出现降解;细胞内ATP恢复时,bcl-2降解产物继续增多,并出现细胞凋亡.体外应用caspase 3可使bcl-2降解,而caspase3的特异性抑制剂DEVD能明显抑制caspase 3对bcl-2的降解.结论 肾小管上皮细胞ATP耗竭再恢复时,caspase 3激活及其介导的bcl-2降解在细胞凋亡中发挥重要作用.     

线粒体介导细胞凋亡和心肌保护新途径

在细胞凋亡过程中,线粒体起着主开关作用,线粒体通透性转换孔开放,线粒体跨膜电位耗损,细胞色素C和凋亡诱导因子释放到胞质中,通过两条途径分别导致细胞凋亡,Bcl－2家族蛋白以线粒体为靶位调控凋亡。线粒体K_(ATP)通道开放可以预防线粒体跨膜电位耗损而减少细胞凋亡,从而揭示了线粒体K_(ATP)通道开放产生心肌保护的新途径。     

白蛋白超载通过线粒体经典途径诱导肾小管上皮细胞凋亡

目的研究线粒体经典途径在白蛋白诱导的肾小管上皮细胞凋亡中的作用。方法大鼠肾近曲小管细胞分别用0～40mg／ml低内毒素胎牛血清白蛋白处理24h,或用20rag／ml白蛋白处理0～24h,或在白蛋白处理同时加入蛋白水解酶抑制剂。处理后的细胞分别进行以下处理：固定后用Floechst 33342染色供计数凋亡细胞比例;以碘化丙啶染色观察坏死细胞;冰上裂解后供caspase-3活性测定;分离胞浆和线粒体组分供免疫印迹法分析Bax和细胞色素C的变化;固定后供免疫荧光染色。结果白蛋白呈剂量和时间依赖性诱导肾小管上皮细胞凋亡,并伴随有线粒体Bax活化、细胞色素C释放和蛋白水解酶激活,蛋白水解酶抑制剂可以抑制细胞凋亡。结论线粒体经典凋亡途径参与白蛋白诱导的肾小管上皮细胞凋亡。     

细胞色素C在缺血／再灌注损伤中的作用

细胞色素C为线粒体呼吸链复合体Ⅲ的成分，在复合体Ⅲ和Ⅳ之间传递电子。许多因素可刺激细胞色素C从线粒体释放，一旦其释放到胞浆中就可依赖于细胞内ATP水平诱导细胞凋亡或坏死。研究表明缺血／再灌注损伤与细胞色素c释放密切相关。现从细胞色素C的结构和特性、释放及其调控、缺血再灌注损伤对细胞色素C释放的影响以及其在缺血再灌注损伤中的作用等方面进行综述。     

细胞色素C在缺血/再灌注损伤中的作用

细胞色素C为线粒体呼吸链复合体Ⅲ的成分,在复合体Ⅲ和Ⅳ之间传递电子。许多因素可刺激细胞色素C从线粒体释放,一旦其释放到胞浆中就可依赖于细胞内ATP水平诱导细胞凋亡或坏死。研究表明缺血／再灌注损伤与细胞色素C释放密切相关。现从细胞色素C的结构和特性、释放及其调控、缺血再灌注损伤对细胞色素C释放的影响以及其在缺血再灌注损伤中的作用等方面进行综述。     

茶多酚作用于线粒体杀伤胰腺癌细胞的实验性研究

目的 探讨茶多酚特异性诱导胰腺癌细胞死亡的线粒体机制。方法 电子显微镜下观察茶多酚处理后胰腺癌细胞的形态学变化。三磷酸腺苷生物发光法检测茶多酚作用下胰腺癌细胞ATP含量的变化，MitoCapture法检测茶多酚作用下胰腺癌细胞的线粒体膜电位的变化。探讨细胞ATP含量与胰腺癌细胞凋亡与坏死之间的关系。Western blot技术检测茶多酚作用后胰腺癌细胞胞液中细胞色素C，caspase蛋白质含量的变化。结果 随着茶多酚浓度增高和不同浓度下作用时间延长，电镜下胰腺癌细胞线粒体肿胀，部分细胞呈坏死样改变，细胞ATP含量降低，MitoCapture阳性细胞百分数增加。ATP含量与BxPC-3细胞细胞MitoCapture阳性细胞呈负相关（r=-0．808，P〈0．01）。细胞色素C和procaspase-9释放到胞液中，胞液中可检测出活化的caspase-9，和caspase-3。结论茶多酚具有特异性诱导胰腺癌细胞死亡的功能，机制与细胞ATP含量降低，线粒体膜电位破坏，通透性增高，细胞色素C和procaspase-9释放，活化caspase-3启动胰腺癌细胞细胞凋亡过程，当ATP含量极度降低时部分胰腺癌细胞细胞出现坏死。     

线粒体介导细胞凋亡和心肌保护新途径

在细胞凋亡过程中，线粒体起着主开关作用，线粒体通透性转换孔开放，线粒体跨膜电位耗损，细胞色素C和凋亡诱导因子释放到胞质中，通过两条途径分别导致细胞凋亡，Bcl-2家族蛋白以线粒体为靶位调控凋亡。线粒体KATP通道开放可以预防线粒体跨膜电位耗损而减少细胞凋亡，从而揭示了线粒体KATP通道开放产生心肌保护的新途径。     

肾小管上皮细胞ATP耗竭再恢复时热休克蛋白72与Paxillin 的相互作用

目的 研究肾小管上皮细胞ATP耗竭再恢复时热休克蛋白(HSP)72与桩蛋白(Paxillin)的相互作用和意义。方法 应用代谢抑制剂暂时性阻断肾小管上皮细胞ATP的生成，引起细胞内的ATP耗竭； 换用含10 mmol/L葡萄糖的DMEM培养液，使细胞内ATP再恢复；以热处理细胞或编码HSP72 RNA的腺病毒感染细胞，诱导细胞高表达HSP72。Western印迹检测HSP72水平；间接免疫荧光和Western印迹检测Paxillin在细胞内的分布变化。免疫共沉淀观察HSP72与Paxillin的相互作用。结果 肾小管上皮细胞ATP耗竭再恢复时，对照组细胞内的Paxillin由局部黏附结构区域向细胞浆内弥散分布；HSP72由细胞浆转移至细胞膜。细胞高表达HSP72后，HSP72在细胞浆和细胞膜中的蛋白含量均增加(P < 0.05)；Paxillin由细胞浆向细胞膜的转移明显减少(P < 0.05)；Paxillin在细胞内的正常分布改善；HSP72和Paxillin之间的相互作用显著增加(P < 0.05)。 结论 肾小管上皮细胞ATP耗竭再恢复时，HSP72可保持Paxillin在细胞内的正常分布特点和区域，其机制可能是HSP72增加与Paxillin的相互作用，发挥分子伴侣的功能。     

Hexokinase regulates Bax-mediated mitochondrial membrane injury following ischemic stress

Hexokinase (HK), the rate-limiting enzyme in glycolysis, controls cell survival by promoting metabolism and/or inhibiting apoptosis. Since HK isoforms I and II have mitochondrial targeting sequences, we attempted to separate the protective effects of HK on cell metabolism from those on apoptosis. We exposed renal epithelial cells to metabolic stress causing ATP depletion in the absence of glucose and found that this activated glycogen synthase kinase 3β (GSK3β) and Bax caused mitochondrial membrane injury and apoptosis. ATP depletion led to a progressive HK II dissociation from mitochondria, released mitochondrial apoptosis inducing factor and cytochrome c into the cytosol, activated caspase-3, and reduced cell survival. Compared with control, adenoviral-mediated HK I or II overexpression improved cell survival following stress, but did not prevent GSK3β or Bax activation, improve ATP content, or reduce mitochondrial fragmentation. HK I or HK II overexpression increased mitochondria-associated isoform-specific HK content, and decreased mitochondrial membrane injury and apoptosis after stress. In vivo, HK II localized exclusively to the proximal tubule. Ischemia reduced total renal HK II content and dissociated HK II from proximal tubule mitochondria. In cells overexpressing HK II, Bax and HK II did not interact before or after stress. While the mechanism by which HK antagonizes Bax-mediated apoptosis is unresolved by these studies, one possible scenario is that the two proteins compete for a common binding site on the outer mitochondrial membrane.     

Endoplasmic reticulum stress-associated caspase 12 mediates cisplatin-induced LLC-PK1 cell apoptosis

Reactive oxygen metabolites are important mediators in cisplatin-induced apoptosis in renal tubular epithelial cells (LLC-PK1). Mitochondria have been implicated to play a principal role in cisplatin-induced apoptosis. Caspase 12, an endoplasmic reticulum (ER)-specific caspase, participates in apoptosis under ER stress. Cytochrome P450 system is crucial to the generation of reactive oxygen metabolites and is present at high concentration in the ER. The direct role of caspase 12 in any model of renal injury has not previously been described. In this study, cleavage of procaspase 12 preceded that of caspases 3 and 9 after cisplatin treatment of LLC-PK1 cells. The active form of caspase 8 was not detected throughout the course of study. Preincubation of the LLC-PK1 cells with the caspase 9 inhibitor did not attenuate caspase 3 activation and provided no significant protection. Caspase 3 inhibitor provided only modest protection against cisplatin-induced apoptosis. LLC-PK1 cells that were transfected with anti-caspase 12 antibody significantly attenuated cisplatin-induced apoptosis. Taken together, these data indicate that caspase 12 plays a pivotal role in cisplatin-induced apoptosis. It is proposed that the oxidative stress that results from the interaction of cisplatin with the ER cytochrome P450 leads to activation of procaspase 12, resulting in apoptosis.     

制大黄-川芎药对对造影剂肾病大鼠肾小管上皮细胞凋亡的影响

目的：探讨制大黄-川芎药对对造影剂肾病（CIN）大鼠肾小管上皮细胞凋亡的影响及机制。方法：将32只雄性SD大鼠分为正常组（A组）、模型组（B组）、药对高剂量组（C组）、药对低剂量组（D组）。C、D组于造模前7天每日灌胃药对水煎液,灌胃量分别为成人标准体重（60kg）常规用量50、20倍。造模后24h处死动物,测定血清肌酐、尿素氮,HE染色观察肾脏病理改变,TUNEL染色检测肾小管上皮细胞凋亡,western印迹检测肾组织Caspase-3表达。结果：建模后24h与A组相比,B组血清肌酐、尿素氮均明显升高（P〈0.01）;病理形态学检测提示模型大鼠发生明显肾间质水肿、肾小管上皮细胞胞浆空泡样变、肾小管上皮细胞凋亡（P〈0.01）,Caspase-3蛋白表达显著增多（P〈0.01）。与B组相比,C、D组血清肌酐、尿素氮明显回落（P分别〈0.01or〈0.05）,肾脏病理改变显著为轻,肾小管上皮细胞凋亡指数显著减少（P〈0.01）,Caspase-3蛋白表达明显降低（P〈0.01）。结论：caspase3依赖的肾小管上皮细胞凋亡参与了大鼠造影剂肾病的发生,制大黄-川芎药对能通过抑制caspase3抑制肾小管上皮细胞凋亡,并进而保护CIN大鼠肾功能。     

川芎嗪与N-乙酰半胱氨酸抑制对比剂肾病大鼠肾小管上皮细胞凋亡的比较

目的评价川芎嗪抑制对比剂。肾病大鼠。肾小管上皮细胞凋亡的效应。方法建立对比剂肾病大鼠模型,分正常组（A）、模型组（B）、N-乙酰半胱氨酸组（C）、川芎嗪组（D）。C、D组在造模前3d每日分别腹腔注射150mg／kgN-乙酰半胱氨酸、80mg／kg川芎嗪。测定肾功能,观察。肾脏病理改变,TUNEL染色检测肾小管上皮细胞凋亡,Western印迹检测肾组织Caspase3表达。结果与A组相比,B组血肌酐、尿素氮、血清胱抑素C明显升高,肾间质水肿、肾小管上皮细胞空泡样变及细胞凋亡（P〈O．01）、Caspase-3蛋白表达增多（P〈0．05）。与B组相比,C、D组血肌酐、尿素氮、血清胱抑素C明显回落,病理改变显著减轻,肾小管上皮细胞凋亡指数显著减少（P〈0．01）,Caspase3蛋白表达降低（P〈O．05）,C、D组在上述检测中差异不显著（P〉0．05）。结论Caspase3依赖的肾小管上皮细胞凋亡参与了对比剂肾病的发生,川芎嗪通过抑制Caspase3抑制。肾小管上皮细胞凋亡并保护大鼠肾功能,效果与N_乙酰半胱氨酸组相近。     

Prior heat stress inhibits apoptosis in adenosine triphosphate-depleted renal tubular cells.

BACKGROUND: This study tested the following hypotheses: (a) renal tubular epithelial cells subjected to transient adenosine triphosphate (ATP) depletion undergo apoptosis, and (b) induction of heat stress proteins (HSPs) inhibits cell death following ATP depletion, possibly by interacting with anti-apoptotic signal proteins. METHODS: To simulate ischemia in vivo, cells derived from opossum kidney proximal tubule (OK) were subjected to ATP depletion (5 mM cyanide, 5 mM 2-deoxy-D-glucose, and 0 mM glucose) for 1 to 1. 5 hours, followed by recovery (10 mM glucose without cyanide). The presence of apoptosis was assessed by morphological and biochemical criteria. The effect of prior heat stress or caspase inhibition on apoptosis and cell survival were assessed. RESULTS: In the ATP-depleted cell, both Hoechst dye and electron microscopy revealed morphological features that are typical of apoptosis. On an agarose gel, a "ladder pattern" typical of endonucleosomal DNA degradation was observed. Prior heat stress reduced the number of apoptotic-appearing cells, significantly decreased DNA fragmentation, and improved cell survival compared with controls (73.0 +/- 1% vs. 53.0 +/- 1.5%; P < 0.05). Two different caspase inhibitors also improved survival, suggesting that apoptosis is a cause of cell death in this model. Compared with ATP-depleted controls, prior heat stress inhibited the pro-apoptotic changes in the ratio of Bcl2 to BAX, proteins known to regulate the apoptotic set point in renal cells. HSP 72, a known cytoprotectant, co-immunoprecipitated with Bcl2, an anti-apoptotic protein. Prior heat stress markedly increased the interaction between HSP 72 and Bcl2. CONCLUSIONS: Transient ATP depletion causes apoptosis in tubular epithelial cells. Prior HS inhibits apoptosis and improves survival in these cells. Novel interactions between HSP 72 and Bcl2 may be responsible, at least in part, for the protection afforded by prior heat stress against ATP depletion injury.     

DNA fragmentation reduced by antioxidants following ischaemia-reperfusion in the isolated perfused rat kidney

SUMMARY: The role of oxygen-derived free radicals (OFR) in modifying structure and function after ischaemia-reperfusion (IR) injury was studied in isolated perfused rat kidneys (IPRK). Control kidneys were studied after 20 min of ischaemia followed by 15 or 60 min of reperfusion. the xanthine oxidase inhibitor allopurinol and the hydroxyl radical scavenger dimethylthiourea (DMTU) were used to prevent OFR-related damage. Morphological injury was assessed in cortex, inner and outer medulla and compared with indices of global renal function (inulin clearance, fractional sodium excretion and renal vascular resistance). Apoptosis was assessed using both morphological criteria and in situ end-labelling (ISEL) to identify DNA fragmentation. Tubular damage, as evidenced by cellular blebbing, tubular cast formation, epithelial necrosis, and occasional apoptosis, was greatest in the straight proximal tubule and thick ascending limb (TAL) in the outer zone of the outer medulla. Pretreatment with allopurinol or DMTU did not significantly improve renal function. However, structural damage and luminal debris were diminished in allopurinol- and DMTU-treated kidneys. These changes may lead to functional improvement after more prolonged reperfusion. In situ end-labelling was more frequent in distal tubular epithelial cells after IR than either morphological evidence of apoptosis or necrosis. Decreased ISEL was observed after pretreatment with both allopurinol and DMTU. the data demonstrate that OFR produce DNA damage after IR, increasing ISEL. This probably represents reversible DNA damage rather than incipient apoptosis. Thus, antioxidants reduce or prevent DNA and cellular injury after IR and may reduce functional impairment after prolonged reperfusion.