Division of Nephrology and Endocrinology, University of Tokyo School of Medicine, Japan.
Lab Invest. 2011 Nov;91(11):1564-71. doi: 10.1038/labinvest.2011.114. Epub 2011 Aug 22.
Advanced chronic kidney disease (CKD) patients encounter anemia through insufficient erythropoietin (EPO) production by peritubular fibroblasts. Recent studies showed an increase in EPO production by pharmacological activation of hypoxia-inducible transcription factors (HIFs) in dialysis patients, suggesting that desensitization of the oxygen-sensing mechanism is responsible for the development of renal anemia. Our recent work demonstrated that indoxyl sulfate (IS), a uremic toxin, dysregulates oxygen metabolism in tubular cells. Here we provide evidence of an additional property that IS impairs oxygen sensing in EPO-producing cells. HepG2 cells were stimulated with cobalt chloride (CoCl(2)) or hypoxia under varying concentrations of IS. EPO mRNA was evaluated by quantitative PCR. Nuclear accumulation of HIF-α was evaluated by western blotting. Transcriptional activity of HIF was checked by hypoxia-responsive element (HRE)-luciferase reporter assay. The impact of IS was further evaluated in vivo by administering rats with indole, a metabolic precursor of IS, and subjecting them to CoCl(2) stimulation, in which renal EPO mRNA as well as plasma EPO levels were measured by quantitative PCR and enzyme-linked immunosorbent assay, respectively. Although IS induced cellular toxicity at relatively high concentrations (2.5 mM), EPO mRNA expression was significantly suppressed by IS at concentrations below cytotoxic ranges. In HepG2 cells, IS treatment decreased nuclear accumulation of HIF-α proteins and suppressed HRE-luciferase activity following hypoxia. Furthermore, administration of rats with indole suppressed renal EPO mRNA expression and plasma EPO levels, corroborating in vitro findings. Results of the present study provide a possible connection between a uremic toxin and the desensitization of the oxygen-sensing mechanism in EPO-producing cells, which may partly explain inadequate EPO production in hypoxic kidneys of CKD patients.
晚期慢性肾脏病 (CKD) 患者由于肾小管周围成纤维细胞产生的促红细胞生成素 (EPO) 不足而发生贫血。最近的研究表明，在透析患者中通过药理学激活低氧诱导转录因子 (HIF) 可增加 EPO 的产生，这表明氧感应机制的脱敏是导致肾性贫血发生的原因。我们最近的工作表明，尿毒症毒素吲哚硫酸酯 (IS) 可使肾小管细胞的氧代谢失调。在这里，我们提供了 IS 损害产生 EPO 细胞的氧感应能力的额外证据。用氯化钴 (CoCl(2)) 或缺氧刺激 HepG2 细胞，并在不同浓度的 IS 下进行实验。通过定量 PCR 评估 EPO mRNA。通过 Western blot 评估 HIF-α的核积累。通过缺氧反应元件 (HRE)-荧光素酶报告基因检测法检查 HIF 的转录活性。通过给予大鼠吲哚（IS 的代谢前体）并对其进行 CoCl(2) 刺激，进一步在体内评估 IS 的作用，通过定量 PCR 和酶联免疫吸附试验分别测量肾 EPO mRNA 以及血浆 EPO 水平。尽管 IS 在相对较高的浓度（≥2.5mM）下诱导细胞毒性，但 IS 在低于细胞毒性范围的浓度下可显著抑制 EPO mRNA 的表达。在 HepG2 细胞中，IS 处理可减少 HIF-α蛋白的核积累，并抑制缺氧后的 HRE-荧光素酶活性。此外，给大鼠施用吲哚可抑制肾 EPO mRNA 的表达和血浆 EPO 水平，与体外研究结果一致。本研究的结果提供了尿毒症毒素与产生 EPO 的细胞中氧感应机制脱敏之间的可能联系，这可能部分解释了 CKD 患者缺氧肾脏中 EPO 产生不足的原因。
