Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation
Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation
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DOI：
10.14814/phy2.14485
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发表时间：
2020-06-01
影响因子：
2.5
通讯作者：
Nonoguchi, Hiroshi
中科院分区：
文献类型：
--
作者：
Yasuoka, Yukiko;Fukuyama, Takashi;Nonoguchi, Hiroshi
用Western blotting检测促红细胞生成素(EPO)蛋白需要对样品进行预纯化。我们建立了一种新的蛋白印迹方法来检测血浆和尿液中的EPO。用免疫印迹法直接检测尿液和组织中的EPO及尿液中的促红细胞生成素(ESAs)。血浆EPO和ESA不是直接应用检测到的，而是在去糖基化后用我们的Western blotting检测到的。除聚乙二醇化的Epoetinβ-pegol外，EPO和ESA的宽带通过脱糖作用移动到22 kDa。一例贫血患者尿液中的22 kDa条带经LC/MS证实含有人促红细胞生成素。严重缺氧(7%O-2，O-4小时)引起大鼠肾脏脱糖EPO表达增加400倍，这与EPO基因表达和血浆EPO浓度的增加是一致的。免疫组织化学显示EPO在对照组大鼠肾组织中有表达，但在间质细胞中无表达；低氧可增加间质细胞中EPO的表达，但不能增加肾小管中EPO的表达。这些数据表明，无论是直接在尿液中还是在血液中脱糖后，都可以通过Western印迹检测到内源性EPO和所有ESA，并且在对照和严重缺氧时，EPO产生的主要部位是肾脏，而不是肝脏。我们的方法将使EPO的兴奋剂检测和检测变得容易。
The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG-bound epoetin beta pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Severe hypoxia (7% O-2,O- 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney but not the liver is the main site of Epo production in control and severe hypoxia. Our method will make the tests for Epo doping and detection easy.