Frequent, Low-Dose Erythropoietin A Mechanistic Approach to Mitigate Adverse Cardiovascular Effects of Erythropoietin

Erythropoietin (EPO) is the most widely prescribed cytokine, yet the benefits and potential
side effects of different dosing regimens are poorly understood. It is now recognized that
erythropoietin administered at high doses to patients with chronic kidney disease, results in
an increased risk of morbidity and mortality from heart disease and stroke. However, the
mechanisms that mediate this increased risk of cardiovascular disease is not known. There are
two receptors for erythropoietin the homodimeric EPO receptor (EPOR) and the heterodimeric
beta common receptor ( CR)/EPOR. The investigators have demonstrated that activation of the
heterodimeric CR/EPOR only occurs with high doses of EPO. Our exciting, published,
preliminary data also demonstrates that the CR is in a complex with vascular endothelial
growth factor receptor-2 (VEGFR-2) and that high doses of EPO activate VEGFR-2 through the
CR, resulting in the deleterious effects of VEGFR-2 activation on the cardiovascular system.
This is particularly important in patients with kidney disease since they are already at a
high risk of cardiovascular disease. Moreover in advanced kidney disease cyanate derived from
the high urea levels can non-enzymatically form an amide bond with EPO. This carbamylated EPO
(cEPO) has no effect on hemoglobin, but still activates the heterodimeric CR/EPOR. To date
there have been no studies that have directly measured levels of cEPO or activation of the
CR/EPOR in patients with kidney disease. Our hypothesis is that the administration of
low-doses of EPO more frequently will result in lower levels of total and carbamylated
erythropoietin decreased activation of VEGFR-2 via the heterodimeric CR/EPOR and consequently
decreased inflammation and atherosclerosis. The investigators will directly test this
hypothesis by randomly allocating 120 patients with chronic kidney disease to either low-
dose EPO given thrice weekly or the same cumulative dose, a high-dose, administered once
every 2 weeks. Our hypothesis predicts that low-dose EPO will be as effective at correcting
anemia, but will demonstrate less progression of carotid artery plaque, as assessed by
non-contrast magnetic resonance imaging, as compared to the high-dose, EPO given every 2
weeks. To delineate how EPO affects blood vessels, the investigators will isolate endothelial
cells from blood vessels in 20 patients who are assigned to low-dose EPO and 20 allocated to
high-dose EPO. Within these cells the investigators will investigate the signaling pathways
that are triggered by activation of CR/EPOR. In a substudy of 20 subjects with kidney disease
randomized to low-dose EPO or to high-dose EPO, as well as 20 healthy controls receiving a
single dose of high- or low-dose EPO, the investigators will determine how kidney function
and dosing affects levels of total and carbamylated erythropoietin. The investigators' study
will not only provide us with a thorough understanding of the mechanism by which EPO mediates
the increased risk of atherosclerosis, but a clinical strategy to avoid the side effects of
EPO therapy and a tool to quantify the cardiovascular risk of EPO and newer erythropoiesis
stimulating agents by assessing activation of the heterodimeric CR/EPOR.

Inclusion Criteria:

The investigators will enroll Veterans who fulfill the following criteria:

- stage 3, 4, or 5 CKD (estimated glomerular filtration rate of less than 60 ml/min/1.73
m2) on at least two separate occasions greater than 3 months apart; and

- candidates for EPO therapy as per the National Kidney Foundation's Kidney Disease
Outcomes Quality Initiative guidelines (hemoglobin < 10 gm/dL and anemia of CKD).

Exclusion Criteria:

The investigators will exclude any Veteran who meets any of the following criteria:

1. pregnant, planning to become pregnant in the next year, or breast feeding;

2. uncontrolled hypertension (blood pressure > 180/100 mm Hg despite optimal
antihypertensive medications);

3. active gastrointestinal bleeding (visible blood or positive tests for stool occult
blood accompanied by a decrease in hemoglobin);

4. likely to have EPO resistance;

5. an adverse cardiovascular event in the prior three months;

6. active or recent (within the last 3 months) severe, systemic infection;

7. active inflammatory disease such as lupus, rheumatoid arthritis, or vasculitis
requiring immunosuppressive or immunomodulatory medications;

8. history of solid organ transplantation;

9. expected off-dialysis survival of less than one year (as determined by the estimated
glomerular filtration slope and the treating physician;

10. active cancer (undergoing chemotherapy or radiation within the last 3 months) or
primary bone marrow disease such as myelofibrosis; or

11. a contraindication for an MRI or individuals who cannot comply with the study
protocol. The investigators will exclude healthy subjects that meet a, b, f, g, h, or
j.