Renal Unit, Department of Medicine, Lerdsin General Hospital, Bangkok, Thailand.
Br J Pharmacol. 2013 Jan;168(1):189-99. doi: 10.1111/j.1476-5381.2012.02123.x.
Recombinant human erythropoietin (rHuEPO) is currently the mainstay of renal anaemia treatment. Recently, rHuEPO has been shown to provide pleiotrophic tissue protection in various pathological conditions. However, the benefits of rHuEPO beyond anaemia treatment are limited because it increases red blood cell mass. Carbamylated erythropoietin (CEPO) is the first rHuEPO derivative that lacks erythropoietic activity but retains tissue protection properties. Since carbamylation targets lysine residues on rHuEPo, we hypothesized that targeted lysine modifications of rHuEPO may result in a novel non-erythropoietic erythropoietin.
rHuEPO was subjected to various targeted lysine modifications. In vitro cytoprotection and apoptosis were evaluated using P19 and HEK293 cells. In vivo erythropoiesis was performed by administering the derivatives to animals for 2 weeks. Renoprotection was tested on an ischaemia/reperfusion (I/R) model.
We synthesized a novel derivative, a glutaraldehyde erythropoietin (GEPO). This construct abolished in vivo erythropoiesis. Biochemical characterization showed that GEPO was more electrostatically negative than rHuEPO. Immunoprecipitation experiments revealed that GEPO bound to the IL3RB/EPOR heterotrimeric receptor and ameliorated cellular apoptosis via the activation of Bcl-2. Notably, Bcl-2 activation was suppressed by the JAK2 inhibitor, tyrphostin AG490. In vivo experiments showed that GEPO also ameliorated kidney damage due to I/R injury both functionally and histologically.
Herein, we describe a novel lysine-modified rHuEPO, glutaradehyde-EPO (GEPO), obtained from a simple reaction. This derivative has no erythropoietic properties but retains cell-protective characteristics both in vitro and in vivo, with promise for future use as an adjunctive treatment of kidney disease.
重组人促红细胞生成素（rHuEPO）目前是治疗肾性贫血的主要药物。最近，rHuEPO 已被证明在各种病理条件下具有多效性的组织保护作用。然而，rHuEPO 除了治疗贫血之外的益处是有限的，因为它会增加红细胞量。氨甲酰化促红细胞生成素（CEPO）是第一种缺乏促红细胞生成活性但保留组织保护特性的 rHuEPO 衍生物。由于氨甲酰化作用于 rHuEPo 上的赖氨酸残基，我们假设 rHuEPO 的靶向赖氨酸修饰可能会产生一种新型的非促红细胞生成素促红细胞生成素。
对 rHuEPO 进行了各种靶向赖氨酸修饰。使用 P19 和 HEK293 细胞评估体外细胞保护和细胞凋亡。通过对动物进行 2 周的衍生物给药来进行体内红细胞生成。在缺血/再灌注（I/R）模型上进行了肾保护作用的测试。
我们合成了一种新型衍生物，戊二醛促红细胞生成素（GEPO）。这种构建物消除了体内的红细胞生成。生化特性表明，GEPO 的静电电势比 rHuEPO 更负。免疫沉淀实验表明，GEPO 与 IL3RB/EPOR 异源三聚体受体结合，并通过激活 Bcl-2 来减轻细胞凋亡。值得注意的是，Bcl-2 的激活被 JAK2 抑制剂 tyrphostin AG490 抑制。体内实验表明，GEPO 还可以改善 I/R 损伤引起的肾脏损伤的功能和组织学表现。
本文描述了一种新型的赖氨酸修饰的 rHuEPO，戊二醛促红细胞生成素（GEPO），通过简单的反应获得。该衍生物没有促红细胞生成作用，但保留了体外和体内的细胞保护特性，有望成为治疗肾脏疾病的辅助治疗方法。
