Hypoxia-induced apoptosis in cultured glomerular endothelial cells: Involvement of mitochondrial pathways
Hypoxia-induced apoptosis in cultured glomerular endothelial cells: Involvement of mitochondrial pathways
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DOI：
10.1046/j.1523-1755.2003.00301.x
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发表时间：
2003-12-01
影响因子：
19.6
通讯作者：
Nangaku, M
中科院分区：
文献类型：
--
作者：
Tanaka, T;Miyata, T;Nangaku, M
背景肾小球内皮细胞（GENs）在损伤后肾小球毛细血管网的保存和重建中起着关键作用，从而维持组织的氧合。越来越多的证据表明，不能维持微循环导致不可逆的肾小球损伤和肾小球硬化。在这方面，在缺氧环境中的内皮细胞的行为是感兴趣的。我们将培养的GENs暴露于缺氧，并通过膜联蛋白V测定观察凋亡。我们通过实时聚合酶链反应（PCR）、免疫细胞化学和免疫沉淀检测了线粒体信号传导，重点是Bcl 2和Bax。此外，我们还研究了Bcl 2过表达模型对缺氧的反应。低氧处理在24小时诱导12.8%+/-1.1%GENs凋亡，在24小时诱导19.8%+/-0.9%GENs凋亡，随后再给氧8小时。Bcl 2 mRNA表达在24小时下降至0.45 +/-0.15倍，而Bax mRNA表达在复氧后1小时增加至7.3 +/-1.2倍，并伴有从胞浆到线粒体的易位。这些变化与线粒体膜电位的降低和caspase-9活性的增加有关。Bcl 2的过表达和Bax的抑制均能保护GENs免受缺氧损伤。我们认为Bcl 2和Bax的数量和定位的变化有助于缺氧介导的GENs在体外的凋亡。需要进一步研究缺氧环境中的肾小球内皮细胞损伤和细胞内信号传导，以更好地了解并最终预防慢性肾病的进展。
Background. Glomerular endothelial cells (GENs) play a key role in the preservation and reconstruction of the glomerular capillary network following injury, thus maintaining the tissue oxygenation. Accumulating evidence has shown that failure to maintain the microcirculation leads to irreversible glomerular injury and glomerular sclerosis. In this regard, the behavior of endothelial cells in a hypoxic milieu is of interest.Methods. We exposed cultured GENs to hypoxia and observed apoptosis by annexin V assay. We examined mitochondrial signaling, focusing on Bcl2 and Bax by real-time polymerase chain reaction (PCR), immunocytochemistry, and immunoprecipitaion. Furthermore, we examined the response to hypoxia in an overexpression model of Bcl2.Results. Hypoxic treatment induced apoptosis in 12.8%+/-1.1% of GENs at 24 hours, and in 19.8%+/-0.9% at 24 hours followed by 8 hours of reoxygenation. The expression of Bcl2 mRNA decreased to 0.45-+/-0.15-fold at 24 hours, whereas that of Bax increased to 7.3-+/-1.2-fold 1 hour after reoxygenation, accompanied by translocation from the cytosol to mitochondria. These changes were associated with a decrease in mitochondrial membrane potentials and an increase in caspase-9 activity. Both overexpression of Bcl2 and inhibition of Bax protected GENs from hypoxic injury.Conclusion. We conclude that changes of quantity and localization of Bcl2 and Bax contribute to hypoxia-mediated apoptosis of GENs in vitro. Further investigation into glomerular endothelial cell injury and intracellular signaling in a hypoxic milieu is required to better understand and ultimately prevent progression of chronic kidney disease.