Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation

Abstract 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 d...

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Main Authors: Yukiko Yasuoka, Takashi Fukuyama, Yuichiro Izumi, Yushi Nakayama, Hideki Inoue, Kengo Yanagita, Tomomi Oshima, Taiga Yamazaki, Takayuki Uematsu, Noritada Kobayashi, Yoshitaka Shimada, Yasushi Nagaba, Masashi Mukoyama, Tetsuro Yamashita, Yuichi Sato, Jeff M. Sands, Katsumasa Kawahara, Hiroshi Nonoguchi
Format: Article
Language:English
Published: Wiley 2020-06-01
Series:Physiological Reports
Subjects:
Online Access:https://doi.org/10.14814/phy2.14485
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spelling doaj-5ec73d617102476aa3f117aab7606d782020-11-25T03:33:52ZengWileyPhysiological Reports2051-817X2020-06-01812n/an/a10.14814/phy2.14485Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylationYukiko Yasuoka0Takashi Fukuyama1Yuichiro Izumi2Yushi Nakayama3Hideki Inoue4Kengo Yanagita5Tomomi Oshima6Taiga Yamazaki7Takayuki Uematsu8Noritada Kobayashi9Yoshitaka Shimada10Yasushi Nagaba11Masashi Mukoyama12Tetsuro Yamashita13Yuichi Sato14Jeff M. Sands15Katsumasa Kawahara16Hiroshi Nonoguchi17Department of Physiology Kitasato University School of Medicine Sagamihara JapanDivision of Biomedical Research Kitasato University Medical Center Kitamoto JapanDepartment of Nephrology Kumamoto University Graduate School of Medicine Kumamoto JapanDepartment of Nephrology Kumamoto University Graduate School of Medicine Kumamoto JapanDepartment of Nephrology Kumamoto University Graduate School of Medicine Kumamoto JapanDepartment of Molecular Diagnostics Kitasato University School of Allied Health Sciences Sagamihara JapanDepartment of Physiology Kitasato University School of Medicine Sagamihara JapanDivision of Biomedical Research Kitasato University Medical Center Kitamoto JapanDivision of Biomedical Research Kitasato University Medical Center Kitamoto JapanDivision of Biomedical Research Kitasato University Medical Center Kitamoto JapanDivision of Internal Medicine Kitasato University Medical Center Kitamoto JapanDivision of Internal Medicine Kitasato University Medical Center Kitamoto JapanDepartment of Nephrology Kumamoto University Graduate School of Medicine Kumamoto JapanDepartment of Biological Chemistry and Food Sciences Faculty of Agriculture Iwate University Morioka JapanDepartment of Molecular Diagnostics Kitasato University School of Allied Health Sciences Sagamihara JapanRenal Division Department of Medicine Emory University School of Medicine Atlanta GA USADepartment of Physiology Kitasato University School of Medicine Sagamihara JapanDivision of Internal Medicine Kitasato University Medical Center Kitamoto JapanAbstract 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 β 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% 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 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.https://doi.org/10.14814/phy2.14485anemiadeglycosylationerythropoiesis‐stimulating agentserythropoietinhypoxia
collection DOAJ
language English
format Article
sources DOAJ
author Yukiko Yasuoka
Takashi Fukuyama
Yuichiro Izumi
Yushi Nakayama
Hideki Inoue
Kengo Yanagita
Tomomi Oshima
Taiga Yamazaki
Takayuki Uematsu
Noritada Kobayashi
Yoshitaka Shimada
Yasushi Nagaba
Masashi Mukoyama
Tetsuro Yamashita
Yuichi Sato
Jeff M. Sands
Katsumasa Kawahara
Hiroshi Nonoguchi
spellingShingle Yukiko Yasuoka
Takashi Fukuyama
Yuichiro Izumi
Yushi Nakayama
Hideki Inoue
Kengo Yanagita
Tomomi Oshima
Taiga Yamazaki
Takayuki Uematsu
Noritada Kobayashi
Yoshitaka Shimada
Yasushi Nagaba
Masashi Mukoyama
Tetsuro Yamashita
Yuichi Sato
Jeff M. Sands
Katsumasa Kawahara
Hiroshi Nonoguchi
Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation
Physiological Reports
anemia
deglycosylation
erythropoiesis‐stimulating agents
erythropoietin
hypoxia
author_facet Yukiko Yasuoka
Takashi Fukuyama
Yuichiro Izumi
Yushi Nakayama
Hideki Inoue
Kengo Yanagita
Tomomi Oshima
Taiga Yamazaki
Takayuki Uematsu
Noritada Kobayashi
Yoshitaka Shimada
Yasushi Nagaba
Masashi Mukoyama
Tetsuro Yamashita
Yuichi Sato
Jeff M. Sands
Katsumasa Kawahara
Hiroshi Nonoguchi
author_sort Yukiko Yasuoka
title Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation
title_short Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation
title_full Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation
title_fullStr Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation
title_full_unstemmed Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation
title_sort erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by western blotting with deglycosylation
publisher Wiley
series Physiological Reports
issn 2051-817X
publishDate 2020-06-01
description Abstract 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 β 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% 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 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.
topic anemia
deglycosylation
erythropoiesis‐stimulating agents
erythropoietin
hypoxia
url https://doi.org/10.14814/phy2.14485
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