Division of Kidney and Dialysis, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan.
Department of Physiology, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan.
Biochem Biophys Res Commun. 2014 Jun 27;449(2):222-8. doi: 10.1016/j.bbrc.2014.05.014. Epub 2014 May 14.
Erythropoietin production has been reported to occur in the peritubular interstitial fibroblasts in the kidney. Since the erythropoietin production in the nephron is controversial, we reevaluated the erythropoietin production in the kidney. We examined mRNA expressions of erythropoietin and HIF PHD2 using high-sensitive in situ hybridization system (ISH) and protein expression of HIF PHD2 using immunohistochemistry in the kidney. We further investigated the mechanism of erythropoietin production by hypoxia in vitro using human liver hepatocell (HepG2) and rat intercalated cell line (IN-IC cells). ISH in mice showed mRNA expression of erythropoietin in proximal convoluted tubules (PCTs), distal convoluted tubules (DCTs) and cortical collecting ducts (CCDs) but not in the peritubular cells under normal conditions. Hypoxia induced mRNA expression of erythropoietin largely in peritubular cells and slightly in PCTs, DCTs, and CCDs. Double staining with AQP3 or AE1 indicated that erythropoietin mRNA expresses mainly in β-intercalated or non α/non β-intercalated cells of the collecting ducts. Immunohistochemistry in rat showed the expression of HIF PHD2 in the collecting ducts and peritubular cells and its increase by anemia in peritubular cells. In IN-IC cells, hypoxia increased mRNA expression of erythropoietin, erythropoietin concentration in the medium and protein expression of HIF PHD2. These data suggest that erythropoietin is produced by the cortical nephrons mainly in the intercalated cells, but not in the peritubular cells, in normal hematopoietic condition and by mainly peritubular cells in hypoxia, suggesting the different regulation mechanism between the nephrons and peritubular cells.
促红细胞生成素的产生已被报道发生在肾脏的肾小管周间质成纤维细胞中。由于肾单位内的促红细胞生成素产生存在争议，我们重新评估了肾脏中的促红细胞生成素产生。我们使用高灵敏度原位杂交系统（ISH）检查了促红细胞生成素和 HIF PHD2 的 mRNA 表达，并用免疫组织化学检查了 HIF PHD2 的蛋白表达。我们进一步通过体外低氧条件下使用人肝肝细胞（HepG2）和大鼠闰细胞系（IN-IC 细胞）研究了促红细胞生成素产生的机制。在正常条件下，ISH 显示小鼠近端曲管（PCT）、远端曲管（DCT）和皮质集合管（CCD）中有促红细胞生成素的 mRNA 表达，但无管周细胞。缺氧诱导促红细胞生成素的 mRNA 表达主要在管周细胞中，在 PCT、DCT 和 CCD 中略有表达。与 AQP3 或 AE1 的双重染色表明，促红细胞生成素的 mRNA 主要表达在集合管的β闰细胞或非α/非β闰细胞中。在大鼠中免疫组化显示 HIF PHD2 在集合管和管周细胞中的表达及其在管周细胞中的贫血增加。在 IN-IC 细胞中，缺氧增加了促红细胞生成素的 mRNA 表达、培养基中促红细胞生成素的浓度和 HIF PHD2 的蛋白表达。这些数据表明，在正常造血条件下，皮质肾单位主要在闰细胞中产生促红细胞生成素，而不是在管周细胞中，在缺氧条件下主要由管周细胞产生，提示肾单位和管周细胞之间存在不同的调节机制。
