Hepcidin kinetics in hemodialysis patients.

Anemia is common in patients with renal insufficiency and erythropoietin
(EPO)-deficiency is by far the major cause. Since 1998 patients with CKD are
widely treated with erythropoietin agents (ESA). About 10% of patients are
hypo- or unresponsive to ESA. This is problematic: several recent studies
indicate that ESA-resistance is associated with increased cardiovascular
morbidity and mortality. The underlying mechanism remains to be clarified, but
the adverse cardiovascular effects may be due to some off-target effect of ESA.
Iron deficiency contributes to ESA-resistance and the majority of HD patients
receive IV iron aiming to maintain ferritin levels between 200-600 ng/ml.
However, traditional markers of iron status are inaccurate in HD and iron is a
potential toxin. Hepcidin is a key regulator of iron metabolism. Recently
hepcidin over-expression in mice was associated with resistance to ESA.
Moreover, antibody treatment neutralized hepcidin in vivo and facilitated
anemia treatment in these mice. Hepcidin could thus become an important tool to
predict ESA responsiveness, and to guide treatment with ESA and IV iron.
Hepcidin could even become a potential target of treatment in patients with
CKD.

Serum hepcidin levels are elevated in hemodialysis patients and may contribute
to functional iron impairment and thus to ESA-resistance. Kinetics of hepcidin
in hemodialysis patients has not been studied in detail. This study is designed
to provide an answer to the following questions:

1. Do the type of dialysis membrane, the dialysis mode, and/or the dialysis
duration influence hepcidin clearance?
2. Does administration of IV EPO/iron influence serum hepcidin levels in
patients requiring hemodialysis?
3. Are changes in hepcidin levels after dialyses and/or therapeutic
interventions quickly counter-regulated in dialysis patients?

Results will be used for the design and execution of large multicenter
pan-European study on hepcidin as a tool to predict ESA-responsiveness, to
guide ESA and iron treatment in CKD.

1. Do the type of dialysis membrane (e.g. low vs high-flux, polysulphon vs
polyamide vs polyacrylonitrile), the dialysis mode
(hemodiafiltration/hemodialysis), and the dialysis duration influence hepcidin
clearance?
In order to evaluate the in vivo effect of the dialysis technique on serum
hepcidin kinetics, serum hepcidin levels will be measured before and 30 minutes
after the end of the hemodialysis session. In addition samples will be drawn
from the arterial (blood flowing to the dialyzer) and the venous (blood flowing
to the patient) line to assess the arterio-venous differences at the beginning
and at the end of the dialysis session. Dialysate samples will be collected at
the same time points in order to calculate clearance.

Various conditions will be compared:
• Several different dialysis membranes (low-flux vs high-flux, polyamide vs
polysulphonvs polyacrylonitrile).
• Hemodialysis versus hemofiltration

8-10 patients will be studied during each experiment in a crossover design.
Patients will be studied using at least 2 and at most 5 different dialysis
techniques. During each session 50 ml blood will be collected. Thus, maximal
250 ml blood will be drawn in total. Patients will not receive IV iron or EPO
during experimental dialysis sessions.

2. Does administration of IV EPO/iron influence serum hepcidin levels in
patients requiring hemodialysis?
To evaluate the short-term effects of IV iron and EPO serum hepcidin levels
will be measured before administration of IV iron or IV EPO and 1,2,4 and 8
hours afterwards. Samples will be collected from the arterial line. Studies
will be conducted in patients who are regularly treated with IV iron/EPO
(n=8-10 in each group, total 16-20) before and after withholding IV iron and
EPO for at least 2 weeks. Total number of samples amounts to 5 per session ( 50
ml blood). A patient will be studied during maximal 4 different conditions.

3. Are changes in hepcidin levels after dialyses and therapeutic interventions
quickly counter regulated in dialysis patients?
After interpretation of data obtained by the above experiments, regulation of
hepcidin will be studied more extensively in those patients/conditions where
serum hepcidin levels were most notably altered (see point 1 and 2). In these
patients, similar experiments will be performed resulting in considerably
decreased hepcidin levels. Serum hepcidin levels will be then monitored at
regular intervals (1,2,4, 8 and 24 hours) after the nadir of hepcidin. In total
50-80 ml blood will be drawn per patient.

We hypothesize that the kinetics of serum hepcidin may be partly dependent on
iron status and inflammatory responses. Therefore, in all studies blood samples
will be drawn for assessment of baseline ferritin, sTfR, iron, transferrin, %
hypochromic erythrocytes, hs-CRP, IL-6, Hb, Ht, MCV, MCH, ret Hb , and
reticulocytes.

Hepcidin-25 and its two smaller isoforms hepcidin-22 and 20 will be quantitated
by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF-MS), preceded by hepcidin enrichment by weak cation exchange
(Peters, NDT 2009; Swinkels, PLoS One 2008).

Studies will be performed in a time period of 6-9 months. Theoretically,
patients can participate in experiments described under 1,2, and 3.The total
volume of blood may be a burden to patients. Therefore, we will arrange the
experiments in such a manner that the total amount of blood will be limited to
400 ml during 9 months.

Nature and extent of the burden and risks are considered to be minimal.