Department of Physiology, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara 252-0374, Kanagawa, Japan.
Department of Nephrology, Kumamoto University Graduate School of Medical Sciences, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Kumamoto, Japan.
Molecules. 2022 Feb 8;27(3):1119. doi: 10.3390/molecules27031119.
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.
贫血是慢性肾衰竭的主要并发症。为了治疗这种贫血，已经使用了脯氨酰羟化酶结构域酶（PHD）抑制剂和促红细胞生成素刺激剂（ESA）。虽然 PHD 抑制剂可迅速刺激促红细胞生成素（Epo）的产生，但这些药物给药后 Epo 产生的确切部位尚未确定。我们开发了一种新的 Epo 蛋白检测方法，该方法采用去糖基化结合 Western blot。通过蛋白质去糖基化，可以定量检测组织中极其广泛范围的 Epo 含量。使用这种方法，我们研究了 PHD 抑制剂 Roxadustat（ROX）和严重缺氧对大鼠各种组织中 Epo 产生的影响。我们观察到 ROX 增加了肾脏和肝脏中的 Epo mRNA 表达。然而，在肾脏中检测到 Epo 蛋白，但在肝脏中未检测到。Epo 蛋白也在唾液腺、脾脏、附睾和卵巢中检测到。然而，两种 PHD 抑制剂（ROX）和严重缺氧仅在肾脏中增加了 Epo 蛋白的丰度。这些数据表明，虽然 Epo 在许多组织中产生，但 PHD 抑制剂和严重缺氧仅在肾脏中调节 Epo 的产生。
