Hematology, Amgen Inc, Thousand Oaks, CA, USA.
Nephrol Dial Transplant. 2012 Jul;27(7):2733-45. doi: 10.1093/ndt/gfr698. Epub 2011 Dec 13.
Erythropoiesis-stimulating agents (ESAs) stimulate formation of red blood cells by binding to and activating Epo receptors (EpoR) on erythroid progenitor cells. Beyond successful treatment of anemia, ESAs have been reported to reduce damage following insult to organs, including the kidney, possibly via direct activation of EpoR. However, data on ESA effects outside hematopoietic functions are conflicting. Furthermore, limited use of appropriate EpoR-positive and EpoR-negative controls and lack of specific anti-EpoR antibodies make interpretation of data difficult. Recently positive and negative control cell types were validated and a sensitive and specific anti-EpoR antibody (A82) that detects low levels of EpoR protein was described.
A82 was used to measure EpoR protein levels in tissues, human renal cells and human cell lines by western blot analysis. Surface EpoR was examined on renal cells by measuring binding of [125I]-rHuEpo or antibodies. Renal cells and cell lines were treated with rHuEpo to see if phosphorylation of signaling proteins or proliferation/survival could be induced. Small inhibitory RNA (siRNA) were used to determine if EpoR knockdown affected cell viability.
Total EpoR protein was low to undetectable in tissues and renal cells with no detectable EpoR on cell surfaces. EpoR knockdown had no effect on viability of renal cell lines. RHuEpo had no detectable effect on intracellular signaling on renal cell lines with no growth-promoting or survival effect on primary human renal cells.
These results suggest that functional EpoR protein is absent on renal cells and that non-EpoR mechanisms should be explored to explain non-hematopoietic effects of ESAs.
促红细胞生成素刺激剂（ESAs）通过与红细胞祖细胞上的 Epo 受体（EpoR）结合并激活 EpoR，从而刺激红细胞的形成。除了成功治疗贫血外，ESA 还被报道可减少包括肾脏在内的器官损伤，这可能是通过 EpoR 的直接激活。然而，关于 ESA 在造血功能之外的作用的数据存在矛盾。此外，由于适当的 EpoR 阳性和 EpoR 阴性对照的使用有限以及缺乏特异性的抗 EpoR 抗体，使得数据的解释变得困难。最近，已经验证了阳性和阴性对照细胞类型，并描述了一种敏感和特异性的抗 EpoR 抗体（A82），该抗体可检测低水平的 EpoR 蛋白。
通过 Western blot 分析，使用 A82 测量组织、人肾细胞和人细胞系中的 EpoR 蛋白水平。通过测量[125I]-rHuEpo 或抗体的结合，检查肾细胞表面的 EpoR。用 rHuEpo 处理肾细胞和细胞系，观察是否可以诱导信号蛋白的磷酸化或增殖/存活。使用小干扰 RNA（siRNA）确定 EpoR 敲低是否影响细胞活力。
组织和肾细胞中的总 EpoR 蛋白水平低至无法检测，细胞表面也没有可检测到的 EpoR。EpoR 敲低对肾细胞系的活力没有影响。rHuEpo 对肾细胞系的细胞内信号没有可检测到的影响，对原代人肾细胞也没有促生长或生存作用。
这些结果表明，功能性 EpoR 蛋白不存在于肾细胞中，应该探索非 EpoR 机制来解释 ESA 的非造血作用。
