The glycoprotein Erythropoietin (Epo) is the main hormone that promotes erythropoiesis (formation of red blood cells). Recombinant forms of Epo (rHuEpo) and its derivatives (epoetins) have been available since the 1990s and Epo is used worldwide to treat anaemia resulting from chronic kidney disease or chemotherapy. A number of studies have addressed the effects of high levels of Epo in patients, with the goal of ensuring the maximal safety of the patients. However, the results are controversial and both positive and negative effects have been described. The use of Epo in human and veterinary medicine is still a matter of debate and there is a clear need for further investigations.
Studies of the effects of Epo treatment are currently performed by injecting Epo into a mouse model or by inducing specific pathological conditions in EPO-overexpressing mice. To our knowledge the present study is the first to combine the characteristics of the Tg(EPO) mouse with another mutation. To investigate the effects of oxygen-independent overexpression of Epo in the absence of Apolipoprotein E, we generated a new double-mutant mouse model Apoe-/-; Tg(EPO) by crossing the human Epo-overexpressing Tg(EPO) mouse with an apolipoprotein E knockout (Apoe-/-) mouse. For an effective breeding regime, all breeding animals were subjected to splenectomy. Physiological parameters such as life expectancy and body weight and blood data were compared to those of control groups. Furthermore, the concentration of sulfhydryl (SH) groups in whole blood and the levels of haemoglobin-nitric oxide (HbNO) complexes in erythrocyte pellets were investigated as indicators for oxidative stress.
The physiological and haematological characteristics of the new double-mutant mouse model are similar to those of the Tg(EPO) single mutant mouse. Mice overexpressing Epo (Tg(EPO) mice as well as double mutants) have an increased concentration of SH groups in the blood. Male Apoe knockout mice have significantly more HbNO complexes in erythrocyte pellets than male Apoe-/-; Tg(EPO) double-mutant mice. This suggests that mice with a high level of Epo are less susceptible to oxidative stress. Also striking is the difference in the number of HbNO complexes between male and female animals, independent of their genotype. Female mice tend to have more HbNO complexes than male animals.
In summary, the Tg(EPO) mouse can be used for crossbreeding to generate multiple mutant mouse models. Splenectomy is an effective method for lowering the haematocrit in Tg(EPO) mice and thus improving their fitness and usefulness for breeding. The procedure results in a lower number of breeding animals (reduction) and healthier animals (refinement) required for experiments to test the effects of high concentrations of Epo. The use of splenectomy should be considered when working with Epo-transgenic animals. The new double-mutant mouse provided interesting information on the effects of elevated levels of Epo on various physiological parameters. However, the model cannot be recommended for routine studies due to the difficulties in breeding and keeping the animals.
The results of the study indicate the necessity of further investigating the side-effects of an Epo therapy. Because Epo is used throughout the world, there is an urgent need for studies on the safety of its clinical use.