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

Abstract

31The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification 32 of the sample. We developed a new Western blot method to detect plasma and urinary Epo using 33 deglycosylation. Epo in urine and tissue and erythropoiesis-stimulating agents (ESAs) in urine 34 were directly detected by our Western blotting. Plasma Epo and ESAs were detected by our 35 Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 36 kDa by deglycosylation except PEG-bound epoetin  pegol. The 22 kDa band from anemic 37 patient urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain 38 human Epo. 39 Sever hypoxia (7% O 2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat 40 kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo 41 concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial 42 cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not 43 in tubules. 44 These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly 45 in urine or after deglycosylation in blood, and that the kidney is the main and sole site of Epo 46 production in control and severe hypoxia. Our method will completely change Epo doping and 47 detection. 4 48 49

Differentiation of endogenous Erythropoietin and exogenous ESAs by Western blotting with deglycosylation

“…We cut the 22 kDa band of deglycosylated Epo isolated from an anemic patient's urine and analyzed it by LC/MS. We found that the band contained human Epo, proving the correctness of our method 13 . Using this method, we compared endogenous Epo and exogenous ESAs.…”

“…LC/MS analysis detected endogenous rhEpo and exogenous ESAs both in glycosylated and deglycosylated forms as deglycosylated Epo of 22 kDa (Suppl Table 1). We have shown that endogenous Epo is detected as a 22 kDa band in our previous report 13 . Our data clearly show that LC/MS analysis cannot differentiate endogenous rhEpo and exogenous ESAs since LC/MS analysis needs trypsin digestion of samples 14 .…”

“…Pre-puri cation of Epo or loading a large amount of samples are required to observe Epo by Western blotting or SDS-PAGE with Coomassie brilliant blue staining [9][10][11] . We have developed a new method for the detection of Epo in urine, plasma/serum and tissue by Western blotting 12,13 . Pre-puri cation of Epo is not required in our method.…”

“…They had to load a huge amount of the samples. Epo protein expression by Western blot has been reported but prepuri cation of Epo from samples were required, which caused di culty for quantitative analysis 12,13 . Prepuri cation of Epo from sample is not required in our method.…”