a mouse model for an erythropoietin-deficiency anemia · thus, patients with ckd display a chronic...

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INTRODUCTION The constant renewal of red blood cells (RBCs) via erythropoiesis is crucial to ensure proper tissue oxygenation (Jelkmann, 2007). In this respect, erythropoietin (EPO) has long been identified as a key factor in the regulation of erythropoietic output (Fang et al., 2008; Rankin et al., 2007; Sathyanarayana et al., 2008). During homeostasis, EPO is produced at low levels by the peritubular capillary endothelial cells in the kidney and is then released into the blood stream (Jelkmann, 2007; Koury et al., 1988). Following activation of the EPO-receptor (EPOR) complex, the expression of anti-apoptotic genes, e.g. Bcl2l1, is upregulated within erythroid progenitors, which in turn leads to their survival (Silva et al., 1996). Consequently, these cells are able to differentiate into mature reticulocytes. In times of low RBC counts, Epo expression is sharply upregulated and, in turn, erythropoietic output is dramatically increased to compensate for the loss of RBCs. This feedback system is tightly controlled by oxygen levels via hypoxia- inducible transcription factors (HIFs) (Rankin et al., 2007; Wang and Semenza, 1993). Chronic kidney disease (CKD) is characterized by the loss of kidney function and is initiated by diabetic nephropathy, hypertension and glomerulonephritis (Go et al., 2004; Shlipak et al., 2005). The leading cause of death in patients with CKD is cardiovascular disease, regardless of disease progression (Frank et al., 2004). Late-stage CKD patients exhibit a moderate-to-severe anemia from a reduction in renal EPO production (Means, 2003). Thus, patients with CKD display a chronic anemia in the presence of low serum EPO (sEPO) levels. Currently, recombinant human EPO (rHuEPO) is the approved treatment for patients with anemia due to CKD. Despite the success of rHuEPO as a therapy for anemia in CKD, various adverse effects have been reported (Frank et al., 2004; Goldberg et al., 1992). For example, patients can become EPO resistant and also hyporesponsive to the biologic (van der Putten et al., 2008). Furthermore, observations in a clinical trial with patients that suffer from anemia due to chemotherapy indicated that rHuEPO increases lethality in these patients (Bohlius et al., 2009). Results of another large clinical trial on patients with anemia and either diabetes or CKD determined that rHuEPO therapy did not reduce the risk of death due to either cardiovascular or renal complications in these patients (Pfeffer et al., 2009). The trial also indicated that rHuEPO therapy could lead to an increased risk of stroke in these patients. However, because rHuEPO rapidly raises the patient’s hemoglobin level, it is possible that the observed detrimental effects of rHuEPO therapy are attributable to the sharply increased levels of hemoglobin rather than being an effect of the drug itself (e.g. cross- reactivity to other targets). Nevertheless, on the basis of these observations it is now recommended that the patient’s hemoglobin level should not exceed 12 g/dl. Taken together, the development of erythropoiesis-stimulating agents (ESAs) that function without activating the EPOR complex would offer valuable alternative treatment options (Bunn, 2007; Wrighton et al., 1996). At present, few in vivo models exist that display a phenotype similar to the anemia of CKD: specifically, low RBC production in conjunction with depleted sEPO levels. Models involving chemical injections are used to directly destroy RBCs and lower RBC indices. For instance, the injection of phenylhydrazine (PHZ) results in the destruction of RBCs by binding to hemoglobin proteins within these cells (Augusto et al., 1982). The massive loss of RBCs causes the animal to undergo a stress-induced erythropoietic response but can also bring about deleterious secondary affects, e.g. liver damage (Jonen et al., 1982). Alternatively, oxygen levels can be reduced in hyperbaric chambers, resulting in low tissue oxygenation in the animal. However, regardless of the methods employed, these current techniques lead to a rapid increase in sEPO levels and are not representative of an EPO-deficiency anemia. To overcome this RESEARCH ARTICLE Disease Models & Mechanisms 763 Disease Models & Mechanisms 3, 763-772 (2010) doi:10.1242/dmm.004788 © 2010. Published by The Company of Biologists Ltd 1 Department of Inflammation, Pfizer Global Research and Development, 700 West Chesterfield Parkway, St Louis, MO 63017, USA 2 Genetically Modified Mice CoE, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, USA 3 Comparative Medicine, Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, USA *Present address: Pfizer Regenerative Medicine, 620 Memorial Drive, Cambridge, MA 02139, USA Author for correspondence (knut.niss@pfizer.com) SUMMARY In mammals, the production of red blood cells is tightly regulated by the growth factor erythropoietin (EPO). Mice lacking a functional Epo gene are embryonic lethal, and studying erythropoiesis in EPO-deficient adult animals has therefore been limited. In order to obtain a preclinical model for an EPO-deficient anemia, we developed a mouse in which Epo can be silenced by Cre recombinase. After induction of Cre activity, Epo KO/flox mice experience a significant reduction of serum EPO levels and consequently develop a chronic, normocytic and normochromic anemia. Furthermore, compared with wild-type mice, Epo expression in Epo KO/flox mice is dramatically reduced in the kidney, and expression of a well-known target gene of EPO signaling, Bcl2l1, is reduced in the bone marrow. These observations are similar to the clinical display of anemia in patients with chronic kidney disease. In addition, during stress-induced erythropoiesis these mice display the same recovery rate as their heterozygous counterparts. Taken together, these results demonstrate that this model can serve as a valuable preclinical model for the anemia of EPO deficiency, as well as a tool for the study of stress-induced erythropoiesis during limiting conditions of EPO. A mouse model for an erythropoietin-deficiency anemia Brandon M. Zeigler 1 , Janis Vajdos 2 , Wenning Qin 2 , Linda Loverro 3 and Knut Niss 1, * ,‡ Disease Models & Mechanisms DMM

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Page 1: A mouse model for an erythropoietin-deficiency anemia · Thus, patients with CKD display a chronic anemia in the presence of low serum EPO (sEPO) levels. Currently, recombinant human

INTRODUCTIONThe constant renewal of red blood cells (RBCs) via erythropoiesisis crucial to ensure proper tissue oxygenation (Jelkmann, 2007). Inthis respect, erythropoietin (EPO) has long been identified as a keyfactor in the regulation of erythropoietic output (Fang et al., 2008;Rankin et al., 2007; Sathyanarayana et al., 2008). Duringhomeostasis, EPO is produced at low levels by the peritubularcapillary endothelial cells in the kidney and is then released intothe blood stream (Jelkmann, 2007; Koury et al., 1988). Followingactivation of the EPO-receptor (EPOR) complex, the expression ofanti-apoptotic genes, e.g. Bcl2l1, is upregulated within erythroidprogenitors, which in turn leads to their survival (Silva et al., 1996).Consequently, these cells are able to differentiate into maturereticulocytes. In times of low RBC counts, Epo expression issharply upregulated and, in turn, erythropoietic output isdramatically increased to compensate for the loss of RBCs. Thisfeedback system is tightly controlled by oxygen levels via hypoxia-inducible transcription factors (HIFs) (Rankin et al., 2007; Wangand Semenza, 1993).

Chronic kidney disease (CKD) is characterized by the loss ofkidney function and is initiated by diabetic nephropathy,hypertension and glomerulonephritis (Go et al., 2004; Shlipak etal., 2005). The leading cause of death in patients with CKD iscardiovascular disease, regardless of disease progression (Frank etal., 2004). Late-stage CKD patients exhibit a moderate-to-severeanemia from a reduction in renal EPO production (Means, 2003).Thus, patients with CKD display a chronic anemia in the presenceof low serum EPO (sEPO) levels.

Currently, recombinant human EPO (rHuEPO) is the approvedtreatment for patients with anemia due to CKD. Despite the successof rHuEPO as a therapy for anemia in CKD, various adverse effectshave been reported (Frank et al., 2004; Goldberg et al., 1992).For  example, patients can become EPO resistant and alsohyporesponsive to the biologic (van der Putten et al., 2008).Furthermore, observations in a clinical trial with patients that sufferfrom anemia due to chemotherapy indicated that rHuEPO increaseslethality in these patients (Bohlius et al., 2009). Results of anotherlarge clinical trial on patients with anemia and either diabetes orCKD determined that rHuEPO therapy did not reduce the risk ofdeath due to either cardiovascular or renal complications in thesepatients (Pfeffer et al., 2009). The trial also indicated that rHuEPOtherapy could lead to an increased risk of stroke in these patients.However, because rHuEPO rapidly raises the patient’s hemoglobinlevel, it is possible that the observed detrimental effects of rHuEPOtherapy are attributable to the sharply increased levels ofhemoglobin rather than being an effect of the drug itself (e.g. cross-reactivity to other targets). Nevertheless, on the basis of theseobservations it is now recommended that the patient’s hemoglobinlevel should not exceed 12 g/dl. Taken together, the developmentof erythropoiesis-stimulating agents (ESAs) that function withoutactivating the EPOR complex would offer valuable alternativetreatment options (Bunn, 2007; Wrighton et al., 1996).

At present, few in vivo models exist that display a phenotypesimilar to the anemia of CKD: specifically, low RBC production inconjunction with depleted sEPO levels. Models involving chemicalinjections are used to directly destroy RBCs and lower RBC indices.For instance, the injection of phenylhydrazine (PHZ) results in thedestruction of RBCs by binding to hemoglobin proteins within thesecells (Augusto et al., 1982). The massive loss of RBCs causes theanimal to undergo a stress-induced erythropoietic response but canalso bring about deleterious secondary affects, e.g. liver damage(Jonen et al., 1982). Alternatively, oxygen levels can be reduced inhyperbaric chambers, resulting in low tissue oxygenation in theanimal. However, regardless of the methods employed, thesecurrent techniques lead to a rapid increase in sEPO levels and arenot representative of an EPO-deficiency anemia. To overcome this

RESEARCH ARTICLE

Disease Models & Mechanisms 763

Disease Models & Mechanisms 3, 763-772 (2010) doi:10.1242/dmm.004788© 2010. Published by The Company of Biologists Ltd

1Department of Inflammation, Pfizer Global Research and Development, 700 WestChesterfield Parkway, St Louis, MO 63017, USA2Genetically Modified Mice CoE, Pfizer Global Research and Development, EasternPoint Road, Groton, CT 06340, USA3Comparative Medicine, Pfizer Global Research and Development, Eastern PointRoad, Groton, CT 06340, USA*Present address: Pfizer Regenerative Medicine, 620 Memorial Drive, Cambridge,MA 02139, USA‡Author for correspondence ([email protected])

SUMMARY

In mammals, the production of red blood cells is tightly regulated by the growth factor erythropoietin (EPO). Mice lacking a functional Epo geneare embryonic lethal, and studying erythropoiesis in EPO-deficient adult animals has therefore been limited. In order to obtain a preclinical modelfor an EPO-deficient anemia, we developed a mouse in which Epo can be silenced by Cre recombinase. After induction of Cre activity, EpoKO/flox miceexperience a significant reduction of serum EPO levels and consequently develop a chronic, normocytic and normochromic anemia. Furthermore,compared with wild-type mice, Epo expression in EpoKO/flox mice is dramatically reduced in the kidney, and expression of a well-known target geneof EPO signaling, Bcl2l1, is reduced in the bone marrow. These observations are similar to the clinical display of anemia in patients with chronickidney disease. In addition, during stress-induced erythropoiesis these mice display the same recovery rate as their heterozygous counterparts.Taken together, these results demonstrate that this model can serve as a valuable preclinical model for the anemia of EPO deficiency, as well as atool for the study of stress-induced erythropoiesis during limiting conditions of EPO.

A mouse model for an erythropoietin-deficiency anemia Brandon M. Zeigler1, Janis Vajdos2, Wenning Qin2, Linda Loverro3 and Knut Niss1,*,‡

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caveat, nephrectomy has been used to create an EPO-limitingenvironment in vivo. However, this model involves an invasiveprocedure and is therefore not appropriate for large-scaleexperiments such as screening of preclinical compounds. Inaddition, these animals experience a shortened lifespan, whichprevents the study of long-term effects of low EPO production.Finally, nephrectomized animals are still able to produce EPO insecondary tissues such as the liver (Tan et al., 1991).

The introduction of transgenic mice technology has also yieldedsome innovative mouse models for the study of EPO function;however, these are not without caveats. For example, Epo-knockoutmice are embryonic lethal, making it an ineffective model to studyanemia and erythropoiesis in the adult system (Wu et al., 1995).The authors determined that EPO was not necessary for erythroid-lineage commitment; rather, it facilitated the survival of erythroidprogenitors (CFU-E) that could then differentiate into RBCs. Inanother model, Maxwell and colleagues established a transgenicmouse in which the SV40-TAg sequence was inserted into the 5�sequence of the Epo gene. This gene modification leads to adramatic decrease in Epo expression and consequently the animalsdisplay a severe chronic anemia (Maxwell et al., 1993). However,the EPO-TAg mice also develop an immune response to EPO thatis probably due to a sensitizing effect of the expressed SV40 Tantigen (Rinsch et al., 2002).

To facilitate the creation of a new model for the anemia of EPOdeficiency, we developed a conditional Epo-knockout mouse byinserting loxP sites into the Epo gene (Claxton et al., 2008; Li et al.,2006). By crossing this floxed Epo allele to an inducible Cretransgene (Rosa26-CreERT2) Epo was postnatally ablated (Seibleret al., 2003). After induction of Cre activity, Epo expression andsEPO levels were substantially reduced. Accordingly, theseconditional-knockout mice experienced a significant reduction inRBCs, although mean corpuscular hemoglobin (MCH) and meancorpuscular volume (MCV) remained normal. In addition, ananalysis of the bone marrow progenitors of Epo-knockout animalsshowed no effect on the erythroid progenitors BFU-E and CFU-E.Therefore, these mice develop a chronic, normocytic andnormochromic anemia in conjunction with low sEPO levels thatcorrelate to the clinical display of patients with anemia due to CKD.However, in contrast to patients with CKD, these animals do notdevelop inflammation or uremia and therefore will allow the studyof erythropoiesis in an EPO-limiting environment without the biasof possible secondary complications. Taken together, these resultsdemonstrate that this mouse model can serve as a preclinical toolto study chronic anemia due to EPO deficiency.

RESULTSConditional deletion of Epo leads to a reduction in Epo mRNA andsEPO levelsWe analyzed the impact of Epo deletion in adult mice by employinga conditional-knockout strategy (Claxton et al., 2008; Seibler et al.,2003). To induce Epo deletion, mice in which exon 2 through 4 wasflanked by loxP sites were generated (Fig. 1A,B). These exonsencode for the amino acid sequences necessary for EPOR binding.After obtaining germline transmission, EpoWT/flox mice werecrossed with each other to obtain homozygous Epoflox/flox mice.These mice seemed phenotypically normal to their wild-typecounterparts (data not shown). Epoflox/flox animals were bred to the

EIIa-Cre mice, which ubiquitously express Cre, to generate Epo/

embryos. These embryos died at 12.5 days post-coitum (dpc) withsevere defects in erythropoiesis (Fig. 1C). An absence of definitiveRBCs in the Epo/ embryos was observed in the fetal liver;however, erythroid precursors were present in the yolk sac of theseanimals, as verified by CD71+/Ter119+ staining (data not shown).Thus, the phenotype of Epo/ embryos is similar to previouslydescribed Epo-knockout mice (Suzuki et al., 2002; Wu et al., 1995).

To investigate the effects of Epo deletion in the adult, Epoflox/flox

and EpoWT/flox mice were bred to mice that contained a tamoxifen-inducible Cre allele, Rosa26WT/CreERT2. The resulting animals(Epoflox/flox) displayed normal hematocrit (HCT) levels. Whentransgenic mice reached 8 weeks of age, tamoxifen wasadministered through subcutaneous pellet implantation, activatingthe Cre. Tamoxifen was released at 1 mg/day for 25 days, ensuringproper Cre activation and resulting in the Epoflox allele becomingEpo. At 30 days after pellet implantation, Epo deletion was verifiedby quantitative reverse transcriptase PCR (qRT-PCR) (Fig. 1D).Under steady-state conditions, we observed a reduction of Epokidney expression in Epo/ mice compared with EpoWT/

littermates. However, owing to low baseline expression levels ofEpo, an estimation of knockout efficiency by qRT-PCR wasambiguous. To overcome this limitation, we analyzed Epoexpression levels during an acute hypoxic stimulus, induced by PHZadministration. At 2 days after PHZ treatment, Epo expression levelsin the kidney of Epo/ animals were approximately 95% reducedcompared with EpoWT/ animals. Also, sEPO levels were monitoredduring the acute hypoxic event. In this respect, it is worth notingthat homeostatic levels of sEPO are undetectable with currenttechnologies owing to the low protein concentration in normalmouse serum. Therefore, sEPO levels were also monitored in miceundergoing a stress-induced erythropoietic response due to PHZtreatment. In agreement with the Epo mRNA data, sEPO levels inEpo/ mice were approximately 10% of those of EpoWT/ mice afterPHZ treatment (data not shown and see below). These resultsindicate that induction of Cre activity leads to a severe ablation ofEpo mRNA as well as a reduction in sEPO levels. qRT-PCR analysiswas next performed on a gene known to be directly regulated byEPO signaling, Bcl2l1. In agreement with the Epo mRNA and sEPOdata, we observed a significant reduction of Bcl2l1 expression inthe bone marrow (Fig. 1E). Taken together, these data demonstratethat the induced deletion of the Epo gene results in a decrease insEPO levels, which in turn leads to decreased EPO signaling in thebone marrow.

rHuEPO rescues the EpoWT/ and Epo/ mice phenotypeWe next investigated whether the erythroid precursors of Epo/

mice are hypersensitive to EPO and whether a smallerconcentration of EPO would be able to illicit the same effect asbasal levels of the protein in transgenic mice. To evaluate this,rHuEPO was injected into Epo/ and EpoWT/flox animals at twodifferent concentrations. A low dose (120 U/kg), representing theapproximate levels observed in the Epo/ mice, and a highconcentration (1200 U/kg) were selected to evaluate theerythropoietic responses. At 4 days after rHuEPO administration,whole blood was analyzed and reticulocytes measured (Table 1 andFig. 2A). In the placebo as well as the 120 U/kg rHuEPO group,Epo/ mice displayed a comparable low percentage of reticulocytes.

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This suggests that the Epo/ mice are not hypo-responsive to EPO.By contrast, all transgenic animals in the 1200 U/kg rHuEPO groupshowed a significant increase in reticulocyte and erythroid indicesin the blood. These results suggest that erythroid precursors ofEpo/ were not hyper-responsive to EPO because, under high EPOconcentrations, reticulocyte counts in the Epo/ mice were thesame as in EpoWT/flox animals.

Deletion of Epo in the adult causes a chronic anemia as seen inCKD patientsInduced EpoWT/ and Epo/ mice were monitored for more than300 days in order to evaluate the long-term effects of EPOdeficiency in the adult. Over the initial induction period, a 40%decrease in HCT levels was observed in Epo/ animals (Fig. 2B).Complete blood count (CBC) indices partially rebounded andstabilized after the first 30 days, although their HCT levels were

still 25% below pre-induction levels. This observation suggestedthat tamoxifen might have a deleterious effect on erythropoiesis.To evaluate whether tamoxifen caused adverse effects, tamoxifen

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Preclinical model for anemia of CKD RESEARCH ARTICLE

Table 1. rHuEPO injection into transgenic mice

Genotype Treatment HCT (%) RBCs (103/µµl) Hb (g/dl)

Epo / PBS

rHuEPO (120 U/kg)rHuEPO (1200 U/kg)

32.7±2.2

32.1±2.537.7±2.3

7.4±0.2

7.1±0.48.3±0.3

10.6±0.4

10.8±0.812.6±0.5

Epoflox/WT PBS

rHuEPO (120 U/kg)

rHuEPO (1200 U/kg)

42.6±2.1

42.3±2.7

48.7±2.0

9.5±0.3

9.6±0.2

10.3±0.3

14.1±0.4

14.4±0.5

15.4±0.5

120 U/kg and 1200 U/kg of rHuEPO were injected subcutaneously into transgenic mice

(post pellet). PBS was subcutaneously injected into the mice and served as a negative

control. Mice were bled 4 days after injections via mandibular bleed, and whole blood of

transgenic and wild-type mice were analyzed using a Cell-Dyne 3700. Three mice were

used for each data set.

Fig. 1. Generation and molecular evaluationof the Epo conditional-knockout mouse.(A)Schematic of the Epo, Epoflox and Epo alleles.The 5� loxP site (white triangle on locus) wasinserted into intron 1 of the Epo gene, 94 bpupstream from exon (green boxes) 2. The 3� loxPsite was inserted into intron 4 of the Epo gene,86 bp downstream of exon 4. Thephosphoglycerate kinase promoter (PGK)/NEOcassette (white box) flanked by the 5� and 3� frtsites were inserted upstream from the 3� loxPsite. The expected sizes of the DraI restrictionfragments that hybridize with the 5� probe(black bar) and the XbaI fragments that hybridizewith the 3� probe (black bar) are indicated aboveeach allele, before and after homologousrecombination. D, DraI; Xb, XbaI. (B)Molecularconfirmation of homologous recombination.Southern blot showing four embryonic stem cellclones with correct targeting after homologousrecombination (prior to Epo excision) at both the5� and 3� ends. Top graph shows DraI digest;bottom, XbaI digest. Clone BB198 was used togenerate the Epoflox mouse. (C)Embryos weredissected at 12 dpc. Red arrow points to fetalliver with defective erythropoiesis in the Epo/

mouse, whereas the white arrow points to anEpoWT/ embryo in which erythropoiesis isoccurring. (D)qRT-PCR data analysis of Epoexpression levels in the kidney and heart. Geneexpression data are presented as the foldchange relative to EpoWT/WT kidney tissue frommice treated with PHZ (normalized to a value of1) and represent averages from threeindependent experiments. P-values werecalculated using a two-tailed Student’s t-test. Foreach sample, expression values were normalizedto cyclophilin mRNA. (E)qRT-PCR data analysis ofBcl2l1 expression levels in the bone marrow.Gene expression data are presented as the foldchange relative to EpoWT/WT bone marrow tissue(normalized to a value of 1) and representaverages from three independent experiments.For each sample, expression values werenormalized to cyclophilin mRNA.

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pellets were implanted into EpoWT/flox and Epoflox/flox animalslacking the Rosa26CreERT2 allele. Therefore, these animals experiencetamoxifen without the activation of Cre recombinase. After

tamoxifen administration, both Epoflox/flox Cre-negative (CreNeg) andEpoWT/flox CreNeg animals showed no reduction in the levels of HCT,RBCs and hemoglobin (Hb), indicating that it did not have adeleterious effect on erythropoiesis (Fig. 2B and Table 2). However,another explanation for the observed mild rebound could be thatthe activated CRE-ERT2 protein produces some toxic effects. It isknown that the CRE protein is not well tolerated by cells and highlevels of CRE can lead to cell death. In this respect it is curious tonote that the HCT values slightly rebounded after removal of theinducer. Taken together, it seems that the induction of CRE-ERT2by tamoxifen affects erythropoiesis. For this reason, all subsequentstudies were performed with animals that were at least 50 days post-induction.

Between days 40 and 250 of the observation period, Epo/ miceshowed a 25% reduction in HCT values compared with EpoWT/flox

CreNeg animals. At 50 days after pellet implant, a reduction in RBC,Hb and HCT levels was observed in Epo/ mice compared withEpoWT/ mice (Table 2). The MCH, MCV and MHCV [the amountof hemoglobin relative to the size of the cell (hemoglobinconcentration) per RBC] values remained normal in Epo/ animalsand no changes in other hematopoietic lineages were observed, suchas in the number of megakaryocytic progenitors (data not shown).These findings are in corroboration with previously describedobservations in Epo-knockout mice (Jelkmann, 2007; Wang andSemenza, 1993). Taken together, these data show that the Epoconditional deletion led to the development of a chronic,normocytic and normochromic anemia.

Loss of Epo leads to a decrease in mature erythroid cells in thebone marrowEpo/, EpoWT/ and EpoWT/WT bone marrow and spleen cells weretested for their potential to differentiate into committed erythroidprogenitors. The number of progenitor colonies in bone marrowand spleen were statistically similar in Epo/, EpoWT/ andEpoWT/WT mice (Fig. 3). This observation is comparable to thepreviously described Epo-knockout mice (Wu et al., 1995). Also,no differences in either size nor morphology were detected withinthe BFU-E and CFU-E colonies of Epo/, EpoWT/ or EpoWT/WT

cells. To further evaluate the erythron, bone marrow and spleenerythroid precursors were analyzed using CD71 and Ter119markers (Zhang et al., 2003). Epo/ mice experienced no reductionin the CD71+/Ter119Neg cell population, which represents an early

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Fig. 2. Cellular analysis of Epo transgenic mice. (A)rHuEPO rescue intransgenic Epo mice. rHuEPO was injected at either 120 U/kg (low dose) and1200 U/kg (high dose) into EpoWT/WT and Epo/ mice. At 4 days after injection,whole blood was measured for reticulocytes by thiazole orange staining.Experiments were performed in triplicate. (B)Hematocrit readings oftransgenic mouse after pellet implantation. Epo/ and EpoWT/, with orwithout the EIIa-Cre allele, were analyzed over a 140-day time course. n6Epo/; n5 EpoWT/; n11 EpoWT/flox CreNeg. P-values on graph are a statisticalcomparison between Epo/ and EpoWT/.

Table 2. CBC measurements before and after induction of CRE activity

Genotype HCT (%) Hb (g/dl) RBCs (103/µµl) PLT (103/µl) WBCs (%)

CBC measurements before pellet implantation

EpoWT/flox CreNeg 46.3±2.2 16.4±0.9 10.4±0.5 866±137 7.0±1.0

Epo /flox CreNeg 37.7±1.1 15.3±0.4 8.5±0.2 1088±193 6.5±1.3

EpoWT/flox 47.2±3.3 16.4±1.2 10.6±0.7 850±192 7.9±2.3

Epoflox/flox 41.4±2.4 13.8±0.8 8.8±0.7 990±128 8.1±2.3

CBC measurements 50 days after pellet implantation

EpoWT/flox CreNeg 44.4±1.3 15.6±0.5 9.7±0.4 848±137 6.8±1.2

Epo /flox CreNeg 38.9±1.4 14.4±0.5 8.8±0.2 1194±140 5.1±0.9

EpoWT/flox 44.3±1.5 14.9±0.6 9.3±0.4 1004±380 6.5±2.5

Epoflox/flox 34.7±1.4 11.7±0.7 7.4±0.3 982±318 5.8±1.5

CBC counts of EPO transgenic mice before and 50 days after pellet implantation. PLT, platelet count; WBC, white blood cell count (% of total blood). n=11 EPO / ; n=9 EPOWT/ ; n=15

Epoflox/flox CreNeg; n=17 EpoWT/flox CreNeg. P-values before pellet implantation: HCT EpoWT/flox CreNeg to EpoWT/ P=0.003 and Epo / to Epoflox/flox CreNeg P=0.05. After implantation: HCT EpoWT/flox

CreNeg to EpoWT/ P=0.03 and Epo / to Epoflox/flox CreNeg P=0.01.

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erythroid stage (Fig. 4). In addition, no significant increase in bonemarrow or spleen CD71+/Ter119+ erythroblast populations wasobserved. By contrast, Epo/ mice displayed a decrease in theCD71Neg/Ter119+ population, which represent a more matureerythroid stage in the bone marrow. Epo/ mice displayed a 2.5-fold decrease in the CD71Neg/Ter119+ population compared withEpoWT/WT mice. Taken together, these results demonstrate that,whereas EPO reduction in Epo/ mice did not have an effect onthe percentages of early-stage progenitors, these mice had lowernumbers of intermediate progenitors in their hematopoieticcompartments.

Epo/ mice recover normally from an acute hypoxic eventNext, we investigated whether Epo/ mice could respond to anacute erythropoietic crisis. Although HCT levels were significantlylower in the Epo/ animals under steady conditions, sEPO levelswere similar (68 pg/ml vs 76 pg/ml, respectively; Fig. 5A). Micewere treated with PHZ to induce an acute hypoxic response andwere monitored for 14 days. EpoWT/ and Epo/ animals showedno differences in the rate of recovery from the acute anemia, asmonitored by their rise in HCT levels (Fig. 5A). As expected,EpoWT/ mice showed a substantial increase in sEPO levelscompared with Epo/ mice. At 2 days after PHZ injection, sEPOlevels were more than tenfold higher in EPOWT/ mice comparedwith the Epo/ animals (1000-10,000 pg/ml vs 100-800 pg/ml,respectively). Furthermore, no difference in spleen size wasobserved between EpoWT/ and Epo/ mice, indicating thatextramedullary erythropoiesis was normal in these animals. FACSanalysis revealed no significant differences in erythroid progenitorpopulations in either bone marrow or spleen during recovery (Fig.5B). These data suggest that, although EPO production is severelydiminished in Epo/ mice, the erythropoietic system is fullycapable to respond to an acute hypoxic event.

Epo/ mice display low EPO levels in correlation with low HCTlevels, similar to CKD patientsFinally, HCT values were compared with sEPO levels in Epo/,EpoWT/ and EpoWT/WT transgenic mice (Fig. 6). The inverserelationship between sEPO levels and HCT values has beenobserved and is characterized by low HCT values in correlationwith significantly increased sEPO levels (Artunc and Risler, 2007).As shown in Fig. 6, this relationship was observed in EpoWT/WT

mice, whereas heterozygous animals (EpoWT/) displayed lowersEPO level compared with HCT levels. In addition, Epo/ miceshowed a dramatic reduction of sEPO in relation to low HCT levels.For example, Epo/ mice with an HCT of 20% displayed an sEPOlevel of 100-300 pg/ml. In sharp contrast to this, EpoWT/WT animalswith an HCT of 30% were able to produce sEPO levels well above10,000 pg/ml.

DISCUSSIONIn this study, we describe the generation and characterization ofa mouse model for the anemia of CKD. To create this model, aninducible Cre-loxP system was used that allowed for the deletionof Epo in the adult animal. After induction of Cre, the level ofsEPO significantly decreased and the animals developed amoderate anemia with normal MCV and MCH indices. However,the reduction in sEPO levels did not affect early erythroid

progenitors in the bone marrow, as demonstrated by CFU-Cassays and FACS analysis. These results are in agreement withpreviously published data of EPO-deficient animals (Wu et al.,1995).

Some variations in the model characterized here versuspreviously generated Epo-knockout mice were observed. Forinstance, animals heterozygous for the Epo gene (EpoWT/ andEpo/flox) consistently displayed a reduced HCT level compared withEpoWT/WT animals. Furthermore, the amount of late-stage erythroidprecursors in the bone marrow (CD71Neg/Ter119+ cells) wasreduced in a dose-dependant manner. This decrease could be theresult of an early egress of reticulocytes. It has been shown thatunder hypoxic conditions sinusoid pores in the bone marrow widenand release reticulocytes (Stohlman et al., 1954). In agreement withthis, a minor increase in peripheral blood reticulocytes of Epo/

animals was observed. These differences could be the result of theEpo locus design. The model described here was created byinserting loxP sites into the Epo allele. Although this insertionshould not affect the expression of the Epo gene, it is possible thatthe integration influenced expression levels. We did not, however,find any known enhancer sequences within the region of loxPintegration. Nevertheless, the significantly lower HCT in theEpo/flox CreNeg mice compared with EpoWT/ animals indicates thatthe insertion of the loxP sites decreased Epo expression levels.Finally, a potential deleterious effect of the Cre-recombinaseprotein on erythropoiesis can be ruled out because EpoWT/flox mice

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Fig. 3. Erythroid progenitor capacity of Epo transgenic mice. Tissue wascollected, processed through a 40-m strainer and placed into MethoCult3434. (A)Spleen cells were plated at a concentration of 1�105 cells perMethoCult dish and counted 2 days later for CFU-E colonies. (B)Bone marrowcells were plated at 1�104 cells per dish and counted 2 days later for CFU-Ecolonies. (C)Spleen cells were plated at a concentration of 1x105 cells perMethoCult dish and counted 4 days later for BFU-E colonies. (D)Bone marrowcells were plated at a 1�104 cells per dish and counted 4 days later for BFU-Ecolonies.

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that carry no Cre allele show the same decrease in HCT as theEpoWT/flox mice after induction of Cre activity, thus becomingEpoWT/.

An interesting finding in this study is that the severe reductionof sEPO in the adult leads to a moderate rather than a severeanemia. In this respect it is important to recognize that someresidual Epo mRNA and sEPO remained in the Epo/ mice evenafter 20 days of continuous induction. Cre expression in these micewas driven by the ubiquitously expressed Rosa26 locus; however,expression of this locus is variable between tissues (Zambrowiczet al., 1997). Therefore, it is possible that a number of peritubularcells within the kidney escape Cre activity, leading to the residualEPO production. Alternatively, EPO production in secondarytissues such as liver or lung could contribute to the observedresidual EPO. However, several previous observations argue againstthis view. First, EPO from secondary tissues only accounts for asmall percentage of the total circulating EPO (Fried et al., 1969).Second, liver and lung tissues have been shown to express thehighest levels of the CRE-ERT2 transgene and therefore excisionof the Epo allele in these tissues is likely to be of a high degree(Jullien et al., 2008). Finally, in preliminary experiments weattempted to quantify hepatic Epo mRNA levels in Epo/ mice aswell as control animals but were not able to detect significant levels(data not shown). Because the renal Epo mRNA expression andsEPO levels measured in the whole blood serum are in tightagreement, it seems unlikely that EPO derived from different Cre-negative tissues is responsible for the prevention of a severeanemia. Furthermore, Epo/ mice have a tenfold reduction of Bcl2l,a well-characterized downstream mediator of EPO activity, in thebone marrow (Dolznig et al., 2002). Taken together, these data showthat the induction of Cre activity leads to a significant but

incomplete loss of Epo expression throughout the adult animal inthis model.

The commonly used preclinical anemia models are not suitedto study the anemia of EPO deficiency, owing to the normal stateof the Epo response in these animals. However, several transgenicapproaches have been undertaken to overcome this hurdle. In anattempt to characterize Epo-expressing cells in vivo, Maxwell andcolleagues established a transgenic mouse model in which theSV40-TAg sequence was inserted into the 5� end of the Epo gene(EPO-TAg) (Maxwell et al., 1993). Mice homozygous for this alleledevelop a severe chronic anemia with sEPO levels below thedetection limit. By contrast, the Epo/ mice described heredisplayed a mild-to-moderate chronic anemia with measurablesEPO levels, albeit much lower than expected for the HCTobserved. In this respect it is important to note that a majordifference between the EPO-TAg mice and the animals describedhere is the nature of the Epo gene modification. Whereas theEpo/ mice are obtained via induced Cre-mediated excision, theEPO-TAg mice are derived from breeding carriers of the modifiedallele so that all cells in the animal carry the modified Epo allele.By contrast, the less severe loss of sEPO levels in the Epo/ miceis probably the result of incomplete deletion of the Epo allele.Thus, the Epo/ mice exhibit a phenotype that is more closelyrelated to the clinical display of anemia in CKD. Furthermore,EPO-TAg mice develop an immune response to EPO, probablyowing to a sensitizing effect of the expressed SV40 T antigen(Rinsch et al., 2002). Thus, the EPO-TAg mice represent a criticalEPO-deficient anemia, whereas the anemia observed in Epo/

mice seems to be more closely related to the clinical presentationof anemia in CKD patients. In another model, Gruber andcolleagues reported that the inducible deletion of the Hif2

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Fig. 4. Representative flow cytometricanalysis of bone marrow, spleen andwhole blood from EpoWT/WT, Epo/ andEpoWT/ mice. Tissue was collected,processed through a 40-m strainer, andstained for anti-CD71 and -Ter119antibodies for 45 minutes. nexperiments4.Whole blood was diluted and stained forthiazole orange for 30 minutes, thenanalyzed on BD FACS Caliber.nexperiments4.

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transcription factor in the adult animal results in an anemia thatis similar to the one described here for the Epo/ model (Gruberet al., 2007). However, the observed sEPO levels in these animalsare significantly higher than those observed in Epo/ mice andtherefore are not representative of an EPO-deficiency anemia.Because HIF transcription factors are known to regulate severalgenes, it can be suspected that the anemia observed in theseanimals is multifactorial and not exclusively based on EPOdeficiency (Mastrogiannaki et al., 2009; Rankin et al., 2007;Sathyanarayana et al., 2008).

A surprising discovery of this study is that Epo/ mice recoverat a normal rate from an induced acute hypoxic stimulus. Whereas,overall, Epo/ mice display lower HCT and sEPO levels, therecovery rate of these animals is similar to their wild-typecounterparts. The low levels of sEPO measured in Epo/ micecannot account for the normal recovery to the hypoxic stressbecause injections of a low EPO dose had no effect onerythropoietic output. Further studies are needed to determinewhether, under severe hypoxic conditions, erythroid precursorsdislodge from the bone marrow in the Epo/ mice and populatethe spleen or whether expansion of splenic precursors alone issufficient to account for the observed normal response. Anotherpossible explanation for the observed normal recovery rate is thaterythroblasts in the Epo/ mice become hypersensitive to EPO(Perry et al., 2007). However, injecting low concentrations ofrHuEPO, comparable to the sEPO levels observed in Epo/ miceunder anemic conditions, are in contrast with this hypothesisbecause we did not observe an increase in erythropoiesis.

In this study we demonstrate that the anemia of Epo/ mice issimilar to the clinical presentation of anemia in CKD patients

(Artunc and Risler, 2007). These patients suffer from a loss of kidneyfunction and develop a severe anemia from the lack of EPOproduction (van der Putten et al., 2008). Thus, these patients displaya low HCT in conjunction with low sEPO levels, similar to theobservation in Epo/ mice (Artunc and Risler, 2007). In addition,CKD patients are able to maintain low sEPO levels and aretherefore not completely deprived of EPO, similar to the modeldescribed here. It should be noted that patients with CKD displaynormal hepatic EPO production, in contrast to the model describedhere. In Epo/ mice, the Epo gene is excised ubiquitously and

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Fig. 5. Recovery of Epo/ transgenicmice from an acute hypoxic response.(A)HCT levels after PHZ injectionmonitored in Epo/ and EpoWT/ mice (Ai);sEPO levels after PHZ injection of Epo/

and EpoWT/ mice (Aii); spleen:body weightratio after PHZ injection of Epo/ andEpoWT/ mice (Aiii). Each experiment wascarried out in triplicate. (B)Representativeflow cytometric analysis of spleen andbone marrow 2 days post PHZ injection,from Epo/ and EpoWT/ mice stained forCD71 and Ter119. nexperiments3.

Fig. 6. Epo/ transgenic mice display a similar HCT:sEPO ratio as CKDpatients. CBC data compiled from all studies (includes data from PHZ mouseand Cre induction initiating Epo gene knockout). n29 Epo/ (square); n27EpoWT/ (circle); n12 Epo/flox CreNeg (triangle).

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therefore hepatic EPO is unlikely to be a major source of circulatingEPO. Also, MCH and MCV values are unaffected in both Epo/

mice and CKD patients. However, Epo/ mice do not show signsof abnormal kidney function, inflammation or uremia, in contrastto patients with CKD. Thus, these animals will allow the study oferythropoiesis under EPO-limiting conditions free of secondarycomplications. The control of erythropoiesis by EPO signalingseems to be comparable between CKD patients and the modeldescribed here. However, patients with CKD develop a more severeanemia in late stages of the disease despite low levels of sEPO, incontrast to the Epo/ mice. This suggests that other factors arecontributing to the anemia in these patients. Because CKD patientsexperience a significant amount of inflammation, it is possible thatfactors that oppose erythropoiesis are increased in these patients.In this respect it is of interest that it has been shown that the ironregulator hepcidin can suppress CFU-E development and levels ofhepcidin are increased in CKD patients (Ashby et al., 2009;Tomosugi et al., 2006). Therefore, the anemia of CKD seems to bemultifactorial rather than an anemia of EPO deficiency alone. TheEpo/ mice described here will offer a preclinical model to studythe biology of the anemia of CKD. For example, treating theseanimals with hepcidin might help to dissect the role of thisregulator in the anemia of CKD.

In summary, our data show that the conditional deletion of Epoin the adult animal results in an anemia of EPO deficiency. We didnot observe any changes in the behavior, body weight or activitylevel of the animal during the time observed (>50 weeks postinduction), and therefore the development of a chronic moderateanemia seems to be well tolerated by the animals. Also, thesetransgenic animals have the ability to survive for long term studies,unlike previously used models such as those using nephrectomy.Thus, Epo/ mice will be a valuable tool for the development ofnew therapeutic approaches for the treatment of the anemia ofCKD. In addition, this model will allow the study of erythropoiesisunder EPO-limiting conditions as well as studies analyzing thecontribution of extra-renal EPO production to erythropoiesis.Furthermore, analyzing the stress-induced erythropoietic responsein these animals has the potential to discover new erythropoiesis-regulating mechanisms that are masked by EPO in the currentlyused models. Elucidating these mechanisms has the potential toidentify new target opportunities for the treatment of anemia.Finally, these animals will allow the study of the effects of EPOoutside the erythropoietic system. For example, EPO has beenshown to ameliorate overall cardioprotective activity and theEpo/ mice could provide a valuable model to study these effectsin detail.

METHODSGeneration of Epo conditional-knockout miceA 10-kb targeting vector encompassing all five Epo exons andflanking genomic regions was created using recombinogenicengineering technology (Liu et al., 2003). A loxP site was insertedinto intron 1 followed by a neomycin-resistance cassette and asecond loxP site inserted into intron 4. The targeting vector waslinearized by NotI restriction-enzyme digestion and electroporatedinto the Bruce4 embryonic stem cell line (Lakso et al., 1996). Clonescorrectly targeted at both homology arms were identified bySouthern blot and injected into blastocysts to generate chimeras.

The Epo allele with loxP sites flanking exons 2-4 is termed Epoflox

throughout this manuscript. Mice were genotyped for Cre-excisionusing primers complementary to exon 4: reverse: 5�-GCCATA -GAAGTTTGGCAAGG-3�, forward: 5�-ACCCGAAG CAGTG -AAGTGAG-3�. Epoflox/flox mice (C57BL/6) were bred to EIIa-Cremice [FVB/N-Tg(EIIa-cre)C5379Lmgd/J; Jackson Laboratories, BarHarbor, ME], which ubiquitously express Cre early in embryonicdevelopment, to delete exons 2-4 of Epo. Deletion was confirmedby qRT-PCR and sEPO ELISA. An Epo allele with exons 2-4 deletedis described as Epo throughout this paper.

Epoflox/flox mice were bred to mice carrying an inducible Creallele, Rosa26WT/CreERT2 [B6.129S4-Gt(Rosa)26Sortm1Sor/J], fromJackson Laboratories to postnatally ablate Epo upon tamoxifenexposure (Seibler et al., 2003). Tamoxifen was administered viasubcutaneous pellet (from Innovated Research of America,Sarasota, FL) implant and released at a rate of 1 mg/day for 20 days(Claxton et al., 2008). To induce an acute stress response, PHZ(Sigma-Aldrich, St Louis, MO) was intraperitonally injected at aconcentration of 40 mg/kg (Agosti et al., 2009; Nakano et al., 2005).To rescue transgenic mice, rHuEPO (Amgen, Seattle, WA) wasdiluted in PBS and injected at 120 U/kg (for low-dose experiments)and 1200 U/kg (for high-dose experiments) in 500 l per mousesubcutaneously (Albertengo et al., 1999). 4 days later, mouse wholeblood was collected via heart stick and analyzed by CBC and flowcytometric analysis. The animal care and use program is fullyaccredited by the Association for Assessment and Accreditationof Laboratory Animal Care, International.

The mice used in the study were generated by Pfizer’s GeneticallyModified Mice department and are available to the scientificcommunity. To obtain these mice please contact the senior authorof this manuscript.

qRT-PCRKidney and heart tissues were extracted then flash frozen andground down to a powder with a mortar and pestle. Total RNAwas extracted using RNeasy Mini Kit from Qiagen (Valencia, CA).First-strand cDNA was generated using reverse transcriptaseSuperscriptIII and oligo (dT)20 primers. Real-time PCR wasperformed in duplicate on an Applied Biosystems (Carlsbad, CA)HT-7900 7900 Real-Time PCR.

The following primers were used for SYBR-Green detection:Epo forward: 5�-AGGAGGCAGAAAATGTCACG-3�, Epo reverse:5�-GGCCTTGCCAAACTTCTATG-3�; Cyclophilin forward:5�-AGAGAAATTTGAGGATGAGAACTTCA-3�, Cyclophilinreverse: 5�-TTGTGTTTGGTCCAGCATTTG-3. Absolutequantification of each gene was calculated by the standard curvemethod using ten-fold dilutions of a positive control (PHZ treated,EpoWT/WT kidney cDNA). Bcl2l1 primers were acquired fromQiagen, Unigene number, Mm.238213. Expression of individualgenes was normalized to cyclophilin expression.

Cellular analysisFor flow cytometric analysis, bone marrow and spleen samples werepassed through a 40-m nylon mesh filter to create a single-cellsuspension. 2�106 cells/ml were treated with 1 L Fc blockingantibody (2.4G2) for 20 minutes at 4°C then incubated with anti-CD71-FITC (C2) and anti-Ter-119-APC (BD/Pharmingen, San Jose,CA) for 45 minutes at 4°C in the dark. Samples were washed with

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PBS and suspended in 200 l FACS buffer. Isotype controls wereAPC-Rat IgG2b and FITC-Rat IgG1 for Ter119 and CD71,respectively, from BD/Pharmingen. Cells were processed on a BDCaliber flow cytometer and the raw data analyzed using FlowJosoftware (Tree Star, Portland, OR).

Mouse whole blood was analyzed using a CELL-DYN 3700(Abbott Laboratories). The remaining blood was spun and serumcollected for ELISA analysis. sEPO levels were detected by mouseELISA hypoxia kit (Meso-Scale Discovery, Gaithersburg, MD) andevaluated using a MSD Sector Imager 6000. For whole bloodreticulocytes measurement, cells were stained with thiazole orangefrom Sigma-Aldrich for 30 minutes at room temperature in thedark and the fluorescence intensity was measured (Maltby et al.,2009).

For colony-forming assay (StemCell Technologies, Vancouver,BC) were performed as previously described (Zeigler et al., 2006).Bone marrow cells were plated at a density of 1�104 cells in 4 mlof MethoCult per 35-mm dish and spleen cells were plated 1�105

cells per dish. The number of CFU-Es was determined 2 days afterculture and BFU-Es were counted on day 4.

ACKNOWLEDGEMENTSThe authors thank the Comparative Medicine group for the upkeep of animalsused in this study. The authors also thank Monica Hultman and Eva Nagiec forexpertise in qRT-PCR and MSD ELISA Technology. Thanks to William L. Blake, MaryK. Bauchmann and Marsha L. Roach for their assistance in electroporation, andDiane M. Nadeau for her contribution in microinjection. Finally, thanks to DonWojchowski and his lab for helpful advice on experimental design.

COMPETING INTERESTSAt the time of this study all authors were employed and funded by Pfizer Inc.

AUTHOR CONTRIBUTIONSB.Z. and K.N. performed experiments, analyzed the results, made the figures andwrote the manuscript. K.N. designed the mouse model and research project. J.V.and W.Q. created the transgenic mouse used for experiments. L.L. designed thebreeding strategy and maintained the colony.

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Artunc, F. and Risler, T. (2007). Serum erythropoietin concentrations and responses toanaemia in patients with or without chronic kidney disease. Nephrol. Dial. Transplant.22, 2900-2908.

Ashby, D. R., Gale, D. P., Busbridge, M., Murphy, K. G., Duncan, N. D., Cairns, T. D.,Taube, D. H., Bloom, S. R., Tam, F. W., Chapman, R. S. et al. (2009). Plasmahepcidin levels are elevated but responsive to erythropoietin therapy in renaldisease. Kidney Int. 75, 976-981.

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TRANSLATIONAL IMPACT

Clinical issueAnemia characterized by low erythrocyte counts and reduced serumerythropoietin (sEPO) is commonly observed in patients with chronic kidneydisease (CKD). Anemia decreases the capacity of the blood to efficientlyoxygenate the tissues of the body and therefore decreases the patient’s qualityof life. Although recombinant human EPO is an approved treatment, thisbiologic is not effective in certain subsets of CKD patients and also haspotential adverse effects. Therefore, the development of erythropoiesis-stimulating agents would offer valuable alternative treatment options.However, there has been a lack of experimental models that accuratelyrepresent the EPO-deficiency anemia seen in CKD patients and in which newtherapies can be tested.

ResultsDeletion of the Epo gene in mice causes embryonic lethality; therefore, tocreate a new model of EPO-deficient anemia, the authors of this study developan Epo conditional-knockout mouse by inserting loxP sites into the Epo gene.By crossing mice carrying this floxed Epo allele with mice carrying an inducibleCre transgene, Epo is postnatally ablated. When Cre activity is induced in thenew strain, Epo expression is substantially reduced. These mice develop achronic, normocytic and normochromic anemia, and display low sEPO levels, aphenotype that resembles the clinical symptoms of patients that suffer fromanemia caused by CKD. Surprisingly, although Epo conditional-knockout micedisplay lower hematocrit and reduced sEPO levels compared with wild-typemice, their rate of recovery following an induced acute hypoxic stimulus issimilar. This suggests that there are other pathways that can compensate forthe loss of EPO during hypoxic stress.

Implications and future directionsThe Epo conditional-knockout mouse model will provide a tool for preclinicalstudies of EPO-deficiency anemia caused by CKD. In addition, because theseanimals do not develop the inflammation or uremia observed in CKD patients,this model will allow the study of erythropoiesis in an EPO-limitingenvironment in the absence of secondary complications. Furthermore,analyzing responses of these mice to hypoxic stress has the potential touncover new erythropoiesis-regulating mechanisms that are masked by EPO incurrently used models of anemia. Finally, this model will allow investigation ofpotential roles of EPO outside the erythropoietic system.

doi:10.1242/dmm.006718

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