TY - JOUR T1 - Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation JF - bioRxiv DO - 10.1101/2020.03.11.987586 SP - 2020.03.11.987586 AU - Yukiko Yasuoka AU - Takashi Fukuyama AU - Yuichiro Izumi AU - Yushi Nakayama AU - Hideki Inoue AU - Kengo Yanagita AU - Tomomi Oshima AU - Taiga Yamazaki AU - Takayuki Uematsu AU - Noritada Kobayashi AU - Yoshitaka Shimada AU - Yasushi Nagaba AU - Masashi Mukoyama AU - Tetsuro Yamashita AU - Yuichi Sato AU - Katsumasa Kawahara AU - Hiroshi Nonoguchi Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/03/11/2020.03.11.987586.abstract N2 - 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 detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except PEG-bound epoetin β pegol. The 22 kDa band from anemic patient urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo.Sever hypoxia (7% O2, 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 is the main and sole site of Epo production in control and severe hypoxia. Our method will completely change Epo doping and detection. ER -