Glutaraldehyde erythropoietin protects kidney in ischaemia/reperfusion injury without increasing red blood cell production

• Published in: British journal of pharmacology Vol. 168; no. 1; pp. 189 - 199
• Main Authors: Chattong, S, Tanamai, J, Kiatsomchai, P, Nakatsu, M, Sereemaspun, A, Pimpha, N, Praditpornsilpa, K, Rojanathanes, R, Sethpakadee, A, Tungsanga, K, Eiam‐Ong, S, Manotham, K
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01-01-2013
• Subjects: Anemia; Animals; Apoptosis; Apoptosis - drug effects; Bcl‐2; Blood; carbamylated; Cytoprotection; EPO; Erythrocyte Count; Erythropoiesis - drug effects; erythropoietin; Erythropoietin - chemistry; Erythropoietin - pharmacology; Female; glutaraldehyde erythropoietin; HEK293 Cells; Humans; ischaemia/reperfusion; JAK; Janus Kinase 2 - metabolism; Kidney - blood supply; Kidney - metabolism; Kidney - pathology; kidney injury; Mice; non‐erythropoietic; Protective Agents - pharmacology; Receptors, Erythropoietin - metabolism; Recombinant Proteins; Reperfusion Injury - blood; Reperfusion Injury - pathology; Reperfusion Injury - prevention & control; Research Papers
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1111/j.1476-5381.2012.02123.x

Background and Purpose
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.
Experimental Approach
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.
Key Results
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.
Conclusions and Implications
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.