Genomic damage in endothelial progenitor cells from uremic patients in hemodialysis.

Abstract

End stage renal disease (ESRD) is associated with a high incidence of cardiovascular disease and cancer. Patients undergoing hemodialysis show a reduced number and an impaired function of endothelial progenitor cells (EPCs), which in physiological conditions contribute to repair the vascular damage. In patients with ESRD, massive oxidative genome damage has been demonstrated but the role of HD in causing it is still a controversial issue. The aim of our study was to analyze the effects of a single HD session on the number of cells marked with CD34 (including sub-type cells known to be EPCs); we then evaluated the genomic damage in these cells using COMET assay.
We quantified CD34(+) cells in blood samples in 30 patients in hemodiafiltration treatment for 3.5 to 4 hours 3 times/week and in 30 healthy volunteers. In HD patients, blood samples were drawn at different time intervals: start of dialysis (T(0)), at the end of the treatment (T(end)) and 24 hours afterwards in the interdialytic day (T(inter)). Staining and analysis was performed using the ISHAGE (International Society of Hematotherapy and Graft Engineering) guidelines. EPCs count was conducted using a multiparameter flow cytometric lyse no-wash method. Genomic damage was evaluated by Comet assay.
The number of CD34(+) cells in the HD patients at the beginning of the dialysis session (T(0)) was significantly lower than in healthy controls. HD patients showed a significant increase in CD34 number at the end of the session (T(end)) with respect to T(0). In the interdialytic period (T(int)), the number of CD34(+) cells was significantly reduced with respect to T(end). COMET assay performed on CD34(+) cells showed a higher basal level of genomic damage in HD patients than in controls; it increased in a statistically significant manner after the hemodialysis session, while in the interdialytic period it came back to T(0) level.
Uremic status is characterized by lower levels of circulating EPCs, which increase after a single session of HD together with genomic damage to the CD34(+) cells.

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ORIGINAL ARTICLE
© 2010 Società Italiana di Nefrologia - ISSN 1121-8428
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AbstrAct
Introduction: End stage renal disease (ESRD) is asso-
ciated with a high incidence of cardiovascular disease
and cancer. Patients undergoing hemodialysis show a
reduced number and an impaired function of endothe-
lial progenitor cells (EPCs), which in physiological con-
ditions contribute to repair the vascular damage. In pa-
tients with ESRD, massive oxidative genome damage
has been demonstrated but the role of HD in causing it
is still a controversial issue. The aim of our study was to
analyze the effects of a single HD session on the num-
ber of cells marked with CD34 (including sub-type cells
known to be EPCs); we then evaluated the genomic
damage in these cells using COMET assay.
Patients and methods: We quantified CD34+ cells in
blood samples in 30 patients in hemodiafiltration treat-
ment for 3.5 to 4 hours 3 times/week and in 30 healthy
volunteers. In HD patients, blood samples were drawn
at different time intervals: start of dialysis (T0), at the
end of the treatment (Tend) and 24 hours afterwards in
the interdialytic day (Tinter). Staining and analysis was
performed using the ISHAGE (International Society
of Hematotherapy and Graft Engineering) guidelines.
EPCs count was conducted using a multiparameter
flow cytometric lyse no-wash method. Genomic dam-
age was evaluated by Comet assay.
Results: The number of CD34+ cells in the HD patients
at the beginning of the dialysis session (T0) was sig-
nificantly lower than in healthy controls. HD patients
showed a significant increase in CD34 number at the
end of the session (Tend) with respect to T0. In the inter-
dialytic period (Tint), the number of CD34+ cells was sig-
nificantly reduced with respect to Tend. COMET assay
performed on CD34+ cells showed a higher basal level
Genomic damage in endothelial progenitor
cells from uremic patients in hemodialysis
Michele Buemi 1, Chiara Costa 2, Fulvio Floccari 3,
Giuseppe Coppolino 1,4, Susanna Campo 1,
Davide Bolignano 1, Alessio Sturiale 1,
Antonio Lacquaniti 1, Antoine Buemi 1,
Saverio Loddo 5, Diana Teti 5
1 Chair of Nephrology, Department of Internal Medicine,
University of Messina, Messina - Italy
2 Department of Social Medicine and Community Health,
Occupational Health, University of Messina, Messina - Italy
3 Nephrology and Dialysis Unit, San Giovanni Evangelista
Hospital, Tivoli - Italy
4 Nephrology and Dialysis Unit, Pugliese-Ciaccio Hospital,
Catanzaro - Italy
5 Department of Experimental Pathology and Microbiology,
University of Messina, Messina - Italy
of genomic damage in HD patients than in controls;
it increased in a statistically significant manner af-
ter the hemodialysis session, while in the interdia-
lytic period it came back to T0 level.
Conclusions: Uremic status is characterized by
lower levels of circulating EPCs, which increase af-
ter a single session of HD together with genomic
damage to the CD34+ cells.
Key words: Cardiovascular risk, COMET assay, En-
dothelial progenitor cells, Genomic damage, Hemo-
dialysis
IntroductIon
During hemodialysis treatment (HD), the endothelium is a
main target of damage caused by mechanical stimuli, such
as blood pressure and shear stress, and by immunological
stimuli (1). Damaged endothelial cells (ECs) may be repaired
by the migration and co-option of pre-existing vascular wall
ECs and/or by the recruitment of stem cells, mobilized from
bone marrow. These cells become endothelial progenitor
cells (EPCs) and circulate in blood to the site of injured vas-
cular tissue. EPCs express CD34, a surface marker which
is also expressed by a small number of hematopoietic stem
cells, including clonogenic progenitors like CFU-GM (colo-
nies containing both neutrophils and monocytes), BFU-E
(erythroblastic precursors) and BFU-MK (megakaryocyte
progenitors) (2). Although the molecular mechanisms that
promote the homing and recruitment of bone marrow-de-
rived progenitor cells to remodeling tissues remain unclear,
the evidence that these cells promote tissue repair is strong

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(3). However, the numbers of EPCs in chronic kidney disease
patients are significantly lower than in control populations
and this decrease is related to glomerular filtration rate de-
cline (4, 5). Patients undergoing HD show a further reduction
in the number and impaired function of EPCs (6). Although
the causes of this phenomenon are still unknown, several
hypotheses can be put forward: it may be due to a delayed
differentiation into EPCs; to reduced stem-cell mobilization
from bone marrow; or to an increased consumption of EPCs
to repair the injured vascular tissue (7).
One explanation could be that apoptosis is increased in
progenitor cells. CD34+ cells show a marked tendency to
undergo apoptosis (8), as do various cell lines in HD pa-
tients (9).This behavior has been attributed to an increase
in oxidative stress following an alteration in the balance
between the production of oxidating substances and the
activity of antioxidating systems. Up to now there have
been no scientific data concerning the genomic damage
caused by oxidative stress in CD34+ stem cells in vivo in
HD. In this study we aimed to analyze the effects of a sin-
gle acetate-free hemodiafiltration session on the number of
circulating CD34+ cells, using COMET assay technique to
evaluate the extent of genomic damage in these cells.
MAterIAls And Methods
Patients
Between September 2006 and March 2007 we enrolled
30 patients (14 women, 16 men, mean age 54.8±10.5 yrs,
mean dialysis age 2.70±1.60 years, residual glomerular
filtration rate 2.3±0.6 mL/min), receiving dialytic treat-
ment in 4-hour sessions, 3 times a week. Patients’ clinical
condition was stable. No racial differences were present.
All the patients’ dry weight had been stable for at least
three months, they had achieved a normotensive edema-
free state and had an adequate dialysis delivery (Kt/V >=
1.3). Throughout the study period, each patient complied
with fluid and food restrictions, a constant ultrafiltra-
tion volume being maintained. Patients were on dialysis
for primary interstitial nephritis (n=10), polycystic kidney
disease (PKD; n=6), glomerulonephritis (n=8) or chronic
pyelonephritis (n=6). Subjects were excluded if they had a
condition leading to a catabolic state, such as malignan-
cy, infection requiring antibiotics within two months prior
to enrolment, or corticosteroid treatment. Other criteria
for exclusion were: diabetes mellitus, smoking, secon-
dary hypertension, statin therapy. All HD patients, except
those with PKD (n=4), were usually given subcutaneous
erythropoietin 2 times a week (mean dosage 65±7 UI/Kg
body weight). Patients underwent treatment with ACE
inhibitors, calcium antagonists, angiotensin receptor an-
tagonists, nitrate agents, diuretics, vitamin D analogue
and calcium-containing phosphate binders. None of the
subjects enrolled had undergone surgery in the six mon-
ths prior to the study.
Hemodialysis techniques
All patients, regularly treated with acetate free hemo-
diafiltration (HD), were studied on a midweek day. Each
HD patient was treated with a dialysis bath (SAFEBAG
93G, Hospal Spa, Italy) containing NaCl (284.31 g/L),
KCl (5.22 g/L), CaCl (10.29 g/L), MgCl2 (2.63 g/L), and
glucose (35 g/L). The dialysis prescription was: duration
(4 hours), blood flow (300 mL/min), bicarbonate infusion
(2000 mL/h with BIOSOL sacks AFB145- Hospal), dialysis
membrane (AN69), and mean weight loss, set at 2.5 kg. All
the HD sessions were conducted using Integra® monitors
(Hospal, Bologna).
Control group
The control group consisted of 30 subjects (15 women, 15
men; mean age 59.8±12.5 years) without evidence in their
clinical history or clinical examination of atherosclerotic
disease, hypertension, diabetes, or hypercholesterolemia.
Moreover, none of the control subjects were taking any
drugs. Data from HD patients and control group are sum-
marized in Table I.
Study design
Peripheral venous blood was taken from HD patients and
control subjects via a 20-gauge butterfly inserted into a fore-
arm vein. Blood samples were obtained from HD patients at
different time intervals: start of dialysis (T0), at the end of the
treatment (Tend), and 24 hours after treatment (Tinter). To mini-
mize circadian variation in the control group, all blood samples
were taken at the same time of the day corresponding to T0 in
HD patients. All samples were processed within 2 hours after
collection. The study was approved by the local Ethics Com-
mittee and informed consent was obtained from all subjects.
EPC characterization and quantification
Staining and analysis were performed following the ISHAGE
(International Society of Hematotherapy and Graft Engineer-
ing) guidelines (10, 11). All peripheral blood specimens were
collected into 0.34 M K3EDTA anticoagulant and stored at a
constant temperature of 4°C and all processing was com-

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Buemi et al: Genomic damage in hemodialysis
pleted within 2 hours. The circulating CD34+ count was con-
ducted using PROCOUNTTM (Becton-Dickinson Biosciences,
San Jose, CA, USA), which is a multiparameter flow cytomet-
ric lyse no-wash method, performed in a TRUCOUNTTM tube
(BD Biosciences) with a known number of fluorescent beads
as the internal control, and differentiating nucleated cells from
debris as a threshold reagent. Using reverse pipetting, a 50
µL sample was incubated for 15 minutes at room tempera-
ture with 10 µL of PROCOUNTTM CD34 reagent in a TRU-
COUNTTM tube, followed by a 30-minute incubation with 450
µL of FACS lysing solution (BD Biosciences). 7-AAD (7-Ami-
no-Actinomycin D- VIAPROBETM BD Pharmigen) was added
to determine viable cells. Using a FACSort (BD Biosciences)
flow cytometer, and based on the SSC/forward scatter (FSC)
profile, a minimum of 60,000 events were acquired for each
sample. For the analysis of data by LYSIS II software (ver-
sion 1.0), three dot plots were created: SSC versus FL1-H;
FL1-H versus FL2-H; SSC versus FL3-H. For every sample, a
sequential seven-gate strategy was followed and manual ad-
justments were made to identify the different regions (CD34+
cells, nucleated cells, CD45+ cells, and beads). The total ab-
solute number of CD34+ cells was calculated as:
TABLE I
CLINICAL CHARACTERISTICS OF HD PATIENTS AND CONTROL GROUP
Variables
HD patients
N=30
Healthy subjects
N=30
Age (years)54.8±10.559.8±12.5
Sex (M/F)14/1615/15
Duration of dialysis, years2.70±1.60-
Body mass index, Kg/m2
24.6±4.8029.3±8.62
Hemoglobin, g/dL 11.4±1.44*14.3±1.12
Hematocrit36.3±4.94*44.3±4.12
HD dose Kt/V 1.30±0.28-
Parathyroid hormone, pg/mL368±234*27±10
Cholesterol mg/dL 203±60.2* 114±30.5
HDL cholesterol mg/dL45.7±17.2 60.1±9.12
LDL cholesterol mg/dL143.6±34.4 90.7±6.45
Triglycerides mg/dL125±76.4 120±29.12
CD34+/µL 2.65±0.31* 4.31±0.49
COMET126.10±15.57*** 67.67±9.57
* p<0.05 vs. controls; *** p<0.001 vs. controls; non-significant differences are not indicated.
Absolute No. CD cells/μL =
Alkaline Comet Assay
Immediately after treatment, blood aliquots were col-
lected to be processed by the comet assay. The assay
was performed essentially according to Kim BS et al with
minor modifications (12). Briefly, the blood cells were sus-
pended in 0.7% (w/v) low melting point agarose (Bio-Rad
Lab., Hercules, CA, USA) and wicked between a lower
layer of 1% (w/v) normal melting point agarose (Bio-Rad)
and a upper layer of 0.7% (w/v) low melting point agarose
on microscope slides (Carlo Erba, Milan, Italy). The slides
were then immersed in a lysing solution (2.5 MNaCl, 100
mMNa2 EDTA, 10 mMTris, pH 10) containing 10% DMSO
(Carlo Erba) and 1% Triton X 100 (Sigma Chemical Co.,
St. Louis, MO, USA), overnight at 4°C. At lysis comple-
Bead count per test
sample volume (μL)
No. of CD34 cells x
No. of beads collected

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tion, the slides were placed in a horizontal gel electropho-
resis tank with fresh alkaline electrophoresis buffer (300
mM NaOH, 1 mM Na2 EDTA, pH 13) and left in the solu-
tion for 25 minutes at 4°C to allow the DNA to unwind and
the alkali-labile sites to express. Electrophoresis was car-
ried out at 4°C for 25 minutes, 30 V (1 V/cm), and 300 mA,
using a Bio-Rad 300 power supply. After electrophoresis,
the slides were fixed basically according to the protocol
proposed by Singh and Lai (1998). Slides were immersed
in 0.3 M sodium acetate in ethanol for 30 minutes. Micro-
gels were then dehydrated in absolute ethanol for 2 hours
and immersed for 5 minutes in 70% ethanol. Slides were
air dried at room temperature. Immediately before scor-
ing, slides were stained with 12 mg/mL ethidium bromide
(Sigma) and examined at 200 magnification with an Olym-
pus fluorescent microscope (BX51, Olympus America,
Inc., Melville, NY, USA). Slides were analyzed by a com-
puterized image analysis system (Delta Sistemi, Rome,
Italy). One hundred and fifty cells were randomly selected
in each slide and scored from 0 to 4 on the basis of Comet
tail length: undamaged cells (score 0) looked like an in-
tact nucleus without tail, while damaged cells appeared
as comets. Final score was calculated by the formula:
Comet score = ( n cells scored 1) + (2 x n cells scored 2)
+ (3 x n cells scored 3) + (4 x n cells scored 4). To evalu-
ate the amount of DNA damage, computer-generated tail
moment values (tail length x fraction of total DNA in the
tail) were used.
Statistical analysis
The mean and SDs of all variables were calculated. Circulat-
ing EPC count was calculated as absolute number of cells
per micro liter (µL). A statistical analysis of variance of groups
was performed by a 1-way ANOVA followed by a Fisher Test
for comparison between variance values in the single obser-
vation times. P<0.05 was considered significant. The comet
assay scores were evaluated by the Mann–Whitney and t-
test where appropriate. Statistical significance is present
if p<0.05 . Results were given as (mean ±SD ). The SPSS
11.0 statistical package and Microsoft Excel were used for
tabulation and analysis. Some graphs were drawn by us-
ing Prism Statistical software (version 4.00; Graphpad, San
Diego, CA, USA).
results
The number of CD34+ cells in the HD patients at the begin-
ning of the dialysis session (T0) was significantly lower than
in healthy controls (2.65±0.31 vs. 4.31±0.49; p<0.05; Fig.
Fig. 1 - A) Differences in CD34+ number between HD patients
and control subjects; * p<0.05. B) Variation in CD34+ number
at the beginning of hemodialysis session (T0), at the end of
the session (Tend) and in the interdialytic period (Tint); * p<0.05
vs. T0 ; ** p<0.05 vs. Tend.
Fig. 2 - A) Differences in COMET score between hemodialysis
patients and control subjects; * p<0.05. B) Variation in COM-
ET score at the beginning of Hemodialysis session (T0), at the
end of the session (Tend) and in the interdialytic period (Tint);
* p<0.05 vs. T0 ; *** p<0.001 T0vs Tend.

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Buemi et al: Genomic damage in hemodialysis
1A). Moreover, HD patients showed a statistically significant
increase in CD34 number at the end of the session (Tend) with
respect to T0 (3.27±0.780 vs. 2.65±0.310; p<0.05; Fig. 1B). In
the interdialytic period (Tint), the number of CD34+ cells was
significantly reduced with respect to Tend, coming back to
values similar to starting levels (2.64±0.257 vs. 3.27±0.780;
p<0.05; Fig. 1B). The COMET assay performed on CD34+
cells at T0 showed a higher basal level of genomic damage in
HD patients than in controls (126.10±15.57 vs. 67.67±9.57;
p<0.001; Fig. 2A). Genomic damage increased in a statisti-
cally significant manner after HD session (Tend) (164.0±24.12
vs. 126.10±15.57; p<0.05; Fig. 2B), while in the interdialytic
period it came back to T0 level, significantly reduced with re-
spect to Tend as previously observed for the CD34+ number
(119.23±19.63 vs. 164.0±24.12; p<0.05; Fig. 2B; Tab. II).
dIscussIon
The results of our study confirm the reduced number of
circulating EPCs in patients undergoing HD, previously de-
scribed by Eizawa et al (8) and by Choi et al (13). Moreover,
these cells in HD patients appear characterized by intensive
genomic damage, even before starting the HD session. This
datum confirms the observed direct correlation between
renal impairment severity and genomic damage extension
in CRF patients (14). After HD the number of circulating
CD34+ cells significantly increased. In HD patients, CD34+
cells also show a higher level of genomic damage quantified
by COMET assay at the end of the hemodialysis session,
notwithstanding the fact that we used highly biocompatible
dialysis membranes in our study. This finding confirms that
hemodialysis per se can determine genomic damage, as we
reported in a previous work (15, 16).
A key role in the induction of genomic damage is prob-
ably played by oxidative stress, which characterizes end
TABLE II
VARIATION IN CD34+ NUMBER AND GENOMIC DAMAGE
CD34+
COMET
T0
Tend
Tint
2.65±0.310
3.27±0.780*
2.64±0.257**
126.10±15.57
164.0±24.12*
119.23±19.63**
Evaluated by COMET assay at the beginning of hemodialysis
session (T0), at the end of the session (Tend) and in the interdia-
lytic period (Tint).
* p<0.05 vs T0 ; ** p<0.05 vs Tend.
stage renal disease and is amplified by hemodialytic treat-
ment (17). It seems that a single hemodialysis session can
strongly stimulate the mobilization of CD34+ cells in our pa-
tients (6) and, on the other hand, it can cause accelerated
deterioration of these cells because of the HD-induced
genomic damage. Defining the study design, we chose to
minimize the potential effects of erythropoietin administra-
tion, including in the study only patients who received EPO
every fifteen days and performing the analysis on the four-
teenth or fifteenth day, immediately before the following
administration. The treatment with EPO, which represents a
powerful stimulus for EPC mobilization, may have led, how-
ever, to an underestimation of the difference between the
numbers of CD34 in HD patients versus healthy controls (7,
18). Several hypotheses can be put forward to explain the
reduced number of CD34+ population in HD patients. One
explanation could be that apoptosis is increased in pro-
genitor cells. Various cell lines show a marked tendency to
undergo apoptosis in HD patients. This behavior has been
attributed to an increase in oxidative stress following an
alteration in the balance between the production of oxidat-
ing substances and the activity of antioxidating systems.
Known anti-apoptotic stimuli, such as bcl-2, are reduced in
patients on HD (19). In patients on long-term HD, Herbrig
found that the migratory activity of EPCs was markedly al-
tered, as was their adhesion to the proteic matrix and to
endothelial cells with respect to a healthy population: this
observation may partly explain the low CD34+ levels de-
tected in our HD population. A further possible explanation
for this phenomenon could be a reduced nitric oxide pro-
duction in patients on HD due to an increase in the endoge-
nous inhibitors of nitric oxide synthase, such as ADMA (20,
21). The mobilization and differentiation of EPCs is in fact
affected by nitric oxide and by bone marrow-expressed en-
dothelial NO synthase (eNOS) (18). Our hypothesis is that
in uremic patients, bone marrow responds by a continuous
release of stem cells into the circulation as an immediate re-
sponse to endothelial damage. In recent times we showed
that revascularization performed on HD patients affected
by critical peripheral artery disease stimulated EPC release
from bone marrow (22). After their differentiation, EPCs can
contribute to damage repair, but they would be ‘consumed’
in proportion to the damage repaired. This, together with
reduced migratory activity, a marked apoptotic tendency
and reduced nitric oxide production, would explain the re-
duced number of EPCs found before dialysis.
But what about their increase after the session? The increase
in the stem cell population about to undergo endothelial mat-
uration as a response to pathological conditions has already
been demonstrated by other authors. Although the molecu-

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lar mechanisms that promote the homing and recruitment
of bone marrow-derived progenitor cells to remodeling tis-
sues remain unclear, the evidence that these cells promote
tissue repair is strong (3). Wojakowski et al and Massa et
al have shown that EPCs are mobilized within a few hours
after acute myocardial infarction, the level of these cells in
peripheral blood remaining high for up to several days or
weeks in patients with acute myocardial infarction, as com-
pared with patients with stable angina, and healthy control
subjects (23, 24). Furthermore, in patients with vascular
surgical trauma, Gill et al demonstrated that the number of
EPCs increased six hours after the trauma and that this in-
crease persisted for 72 hours after the event (25). The find-
ings reported in the literature show that hemodiafiltration
can induce hemodynamic stress of limited severity and ef-
ficacy and could influence the severity and the rhythm of
the physiopathological response of stem cells to an acute
traumatic event. This may explain why the increase in pro-
genitor cells disappeared within 24 hours in our patients.
These findings may indicate a development of CD34+ cells
into elements directed to transform into endothelium and to
repair lesions. It is then likely that repeated cardiovascular
injury, together with the underlying uremia in the HD patient,
lead to a depletion of the bone marrow reserve of progenitor
cells. In agreement with our findings, Chan et al demonstrat-
ed that nocturnal dialysis, which prolongs the duration of
the dialysis session from 8 to 10 hours during sleep for 5 to
6 nights a week, thereby increasing urea clearance and re-
ducing hemodynamic stress on the cardiovascular system,
is associated with a restored number and migratory function
of EPCs compared to traditional dialysis (26). In their recent
study Laufs et al showed that moderate exercise in healthy
subjects increased the levels of EPCs; we believe this find-
ing demonstrates that limited hemodynamic stress can be
an effective stimulus to mobilize endothelial precursors (27).
Our group recently demonstrated that EPC levels increased
rapidly after sudden hemodynamic stress caused by dipping
the patients’ hands in icy water (cold presser test) (28).
conclusIons
In conclusion: 1) in this paper we confirm that a uremic sta-
tus is characterized by lower levels of circulating EPCs; 2)
we demonstrate that these levels increase in response to a
single session of HD together with an increase in genomic
damage to CD34+ cells; 3) we assume that the observed
reduction in CD34 may be correlated with defective neo-
vascularization, and endothelial dysfunction typical of ESRD
patients as reported by Herbrig et al (7).
Financial support: No financial support has been obtained from
any institution or company except for logistic support from the
authors’ affiliated departments.
Conflict of interest statement: None declared.
Address for correspondence:
Prof. Michele Buemi
Via Salita Villa Contino, 30
Pal. Bianca
98100 Messina, Italy
buemim@unime.it
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Received: November 18, 2008
Revised: July 12, 2009
Accepted: August 05, 2009