Pathways of caspase-mediated apoptosis in autosomal-dominant polycystic kidney disease (ADPKD).
Pathways of caspase-mediated apoptosis in autosomal-dominant polycystic kidney disease (ADPKD).
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DOI：
10.1111/j.1523-1755.2005.00155.x
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发表时间：
2005-03
影响因子：
19.6
通讯作者：
Y. Tao;Jun Kim;M. Stanley;Zhibin He;S. Faubel;R. Schrier;C. Edelstein
中科院分区：
文献类型：
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作者：
Y. Tao;Jun Kim;M. Stanley;Zhibin He;S. Faubel;R. Schrier;C. Edelstein
背景：我们最近发现常染色体显性遗传性多囊肾病(ADPKD)的HAN：SPRD大鼠肾脏细胞凋亡增加。Caspase-3和caspase-7是细胞凋亡的主要介导者。Caspase-3和caspase-7介导的细胞凋亡有两条途径：(1)涉及死亡受体Fas、Fas配体(FasL)和caspase-8的“外在”途径；(2)涉及Bcl2蛋白、caspase-2、细胞色素c释放和caspase-9的“线粒体”或“内在”途径。本研究旨在探讨3周龄HAN：SPRD大鼠ADPKD的细胞凋亡途径。方法采用荧光底物法检测caspase活性。用核糖核酸酶保护法和免疫印迹法分别检测mRNA和蛋白质的表达。观察半胱氨酸天冬氨酸氨基转移酶抑制剂对多囊肾组织半胱氨酸氨基转移酶活性的影响。结果Cy/Cy纯合子(Cy/Cy)组Caspase-3和Caspase-7活性高于杂合子(Cy/+)和正常对照(+/+)。核糖核酸酶保护实验显示caspase-3基因表达无差异。免疫印迹结果显示，Cy/Cy组较+/+组和Cy/+组caspase-3、caspase-7蛋白表达增加。Cy/Cy组caspase-8和caspase-9活性较Cy/+和+/+肾高出100%以上。免疫印迹显示Cy/Cy肾组织caspase-8和caspase-9蛋白表达均增加。Cy/Cy肾细胞色素c释放增加，caspase-2蛋白和活性增加。在核糖核酸酶保护实验中，+/+、Cy/+和Cy/Cy肾的FasL基因表达差异无统计学意义。Cy/Cy大鼠短期应用半胱氨酸氨基转移酶抑制剂IDN-8050可抑制肾脏中caspase-3和caspase-7的活性。结论ADPKD患者Cy/Cy肾组织caspase-9蛋白表达增加，细胞色素c释放增加，caspase-2蛋白和活性增加，提示caspase-2参与了内源性途径。Caspase-8蛋白表达增加，表明参与了外在途径。未发现FasL mRNA的差异，提示该外源性途径不依赖于死亡受体配体FasL。
BACKGROUND We have recently demonstrated an increase in apoptosis in Han:SPRD rat kidneys with autosomal-dominant polycystic kidney disease (ADPKD). Caspase-3 and caspase-7 are major mediators of apoptosis. There are two pathways of caspase-3 and caspase-7-mediated apoptosis: (1) the "extrinsic" pathway involving the death receptor Fas, Fas ligand (FasL), and caspase-8 and (2) the "mitochondrial" or "intrinsic" pathway involving Bcl-2 proteins, caspase-2, cytochrome c release, and caspase-9. The aim of the present study was to investigate the pathways of apoptosis in 3-week-old Han:SPRD rats with ADPKD. METHODS Fluorescent substrates were used to measure caspase activity. mRNA and protein was determined by ribonuclease protection assays and immunoblotting, respectively. The effect of caspase inhibitors on caspase activity in polycystic kidneys was determined. RESULTS Caspase-3 and caspase-7 activity was more than 100% increased in homozygous (Cy/Cy) compared to heterozygous (Cy/+) and normal littermate control (+/+) kidneys. Ribonuclease protection assays demonstrated no difference in caspase-3 mRNA. On immunoblotting, there was an increase in the proform of caspase-3 and caspase-7 in Cy/Cy compared to +/+ and Cy/+ kidneys. Caspase-8 and caspase-9 activity was more than 100% increased in Cy/Cy compared to Cy/+ and +/+ kidneys. On immunoblotting, there was an increase of the proform of both caspase-8 and caspase-9 in Cy/Cy kidneys. There was also an increase in cytochrome c release into the cytosol and an increase in caspase-2 protein and activity in Cy/Cy kidneys. On ribonuclease protection assay there was no difference in FasL mRNA between +/+, Cy/+, and Cy/Cy kidneys. Short-term treatment of Cy/Cy rats with the caspase inhibitor IDN-8050 resulted in inhibition of caspase-3 and caspase-7 activity in the kidney. CONCLUSION In Cy/Cy kidneys with ADPKD, there was an increase of the proform of caspase-9, an increase in cytochrome c release into the cytosol, and an increase in caspase-2 protein and activity demonstrating involvement of the intrinsic pathway. There was an increase in the proform of caspase-8 demonstrating involvement of the extrinsic pathway. No differences in FasL mRNA were seen suggesting that the extrinsic pathway is independent of the death receptor ligand, FasL.