Development of a Preclinical Model for An Erythropoietin Deficient Anemia

Abstract

The production of red blood cells is tightly controlled by the growth factor erythropoietin (EPO). Mice lacking a functional EPO gene die in-utero due to the complete absence of definitive red blood cells. Thus, studying the role of EPO in adult animals has been limited to the use of artificial anemia models such as phenylhydrazine (PHZ) treatment or phlebotomy. In addition, injections of rHuEPO have been shown to be a valuable tool to study erythropoiesis. However, these approaches have limited use as preclinical models since they do not recapitulate an anemia due to EPO deficiency as it is seen in patients with chronic kidney disease (CKD). These patients develop a chronic anemia due to the lack of a sufficient EPO gene expression response. In order to obtain a preclinical model for an EPO deficient anemia we developed a mouse model in which the EPO gene is flanked by two loxP sites (flox allele). In addition, these animals carry the Cre-ERT2 transgene which allows for a tamoxifen controlled activation of the Cre recombinase. Thus, these mice enable the inactivation of the EPO gene in adult animals by tamoxifen administration.After induction of Cre activity, mice heterozygous for the EPO allele (EPOko/flox) develop a moderate to severe anemia within 3 weeks. Although anemic, these mice display only low levels of EPO in their serum. However, within 3 months after induction of the knock-out the anemia resolves. This indicates that the Cre mediated excision of the EPO gene is insufficient and some EPO expressing cells remain. This mimics the clinical situation of patients with CKD.In order to study the response of EPO deficient mice to a severe anemia we treated animals with phenylhydrazine and observed the recovery of these animals. Interestingly, EPO deficient animals, although slightly delayed, fully recover from the induced anemia. Although the measured sEPO levels are significantly lower compared to control animals an increase in splenic erythropoiesis was observed in these animals. This indicates that the hypoxic response triggered by a sudden severe anemia is activating pathways other than EPO that promote erythropoiesis. Taken together, these results show that this novel mouse model will be useful tool as a preclinical model for the anemia associated with CKD. In addition, this model will allow the study of stress erythropoiesis under EPO limiting conditions. The Pfizer Institutional Animal Care and Use Committee reviewed and approved the animal use in these studies. The animal care and use program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International