title: Glutaraldehyde erythropoietin protects kidney in ischaemia/reperfusion injury without increasing red blood cell production.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  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
  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.
authors.affiliation: Renal Unit, Department of Medicine, Lerdsin General Hospital, Bangkok, Thailand.
authors.forename: S
authors.initials: S
authors.lastname: Chattong
authors.forename: J
authors.initials: J
authors.lastname: Tanamai
authors.forename: P
authors.initials: P
authors.lastname: Kiatsomchai
authors.forename: M
authors.initials: M
authors.lastname: Nakatsu
authors.forename: A
authors.initials: A
authors.lastname: Sereemaspun
authors.forename: N
authors.initials: N
authors.lastname: Pimpha
authors.forename: K
authors.initials: K
authors.lastname: Praditpornsilpa
authors.forename: R
authors.initials: R
authors.lastname: Rojanathanes
authors.forename: A
authors.initials: A
authors.lastname: Sethpakadee
authors.forename: K
authors.initials: K
authors.lastname: Tungsanga
authors.forename: S
authors.initials: S
authors.lastname: Eiam-Ong
authors.forename: K
authors.initials: K
authors.lastname: Manotham
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D019610:Cytoprotection; D004906:Erythrocyte Count; D004920:Erythropoiesis / Q000187:drug effects*; D004921:Erythropoietin / Q000737:chemistry / Q000494:pharmacology*; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D053614:Janus Kinase 2 / Q000378:metabolism; D007668:Kidney / Q000098:blood supply* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D020011:Protective Agents / Q000494:pharmacology; D017467:Receptors, Erythropoietin / Q000378:metabolism; D011994:Recombinant Proteins; D015427:Reperfusion Injury / Q000097:blood* / Q000473:pathology / Q000517:prevention & control*
chemical_list: D020011:Protective Agents; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin; D053614:Janus Kinase 2