Effects of Roxadustat on Erythropoietin Production in the Rat Body.

Yukiko Yasuoka,Tetsuro Yamashita,Haruki Omiya,Katsumasa Kawahara,Susan M Wall,Taiga Yamazaki,Hideki Inoue,Masashi Mukoyama,Takayuki Uematsu,Hiroshi Nonoguchi,Noriko Takahashi,Yuichi Sato,Takashi Fukuyama,Yasushi Nagaba,Truyen D Pham,Noritada Kobayashi,Jeff M Sands,Tomomi Oshima,Izumi Yokoyama ,Yuji Shimada

doi:10.3390/molecules27031119

Abstract

Anemia is a major complication of chronic renal failure. To treat this anemia, prolylhydroxylase domain enzyme (PHD) inhibitors as well as erythropoiesis-stimulating agents (ESAs) have been used. Although PHD inhibitors rapidly stimulate erythropoietin (Epo) production, the precise sites of Epo production following the administration of these drugs have not been identified. We developed a novel method for the detection of the Epo protein that employs deglycosylation-coupled Western blotting. With protein deglycosylation, tissue Epo contents can be quantified over an extremely wide range. Using this method, we examined the effects of the PHD inhibitor, Roxadustat (ROX), and severe hypoxia on Epo production in various tissues in rats. We observed that ROX increased Epo mRNA expression in both the kidneys and liver. However, Epo protein was detected in the kidneys but not in the liver. Epo protein was also detected in the salivary glands, spleen, epididymis and ovaries. However, both PHD inhibitors (ROX) and severe hypoxia increased the Epo protein abundance only in the kidneys. These data show that, while Epo is produced in many tissues, PHD inhibitors as well as severe hypoxia regulate Epo production only in the kidneys.

Highlights

Anemia is common world-wide [1] and is a major complication of chronic renal failure [2]
Treatment of the anemia of chronic kidney failure was revolutionized with the development of human recombinant erythropoietin (Epo) and erythropoiesis stimulating agents (ESAs) [4]
We observed that Epo deglycosylation with PNGase increases Epo assay sensitivity ~10 fold. Using this Epo assay, we investigated which tissues increase Epo production in response to the Prolyl Hydroxylase Domain (PHD) inhibitor, ROX or in response to severe hypoxia

Summary

Introduction

Anemia is common world-wide [1] and is a major complication of chronic renal failure [2] Hypoxia and anemia both stimulate Epo production in the kidneys [3,4,5] by inhibiting the Prolyl Hydroxylase Domain (PHD), which stimulates hypoxia-inducible factor (HIF)1α/2α [6]. PHD inhibitors stimulate HIF production in these other tissues, which has benefits beyond the treatment of anemia, including a reduction in myocardial infarction, atherosclerosis and peripheral artery disease [19]. This HIF1α/2α stimulation promotes cancer progression as well as other cardiac and metabolic diseases [20,21,22,23,24,25]

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