Original Paper

Received: August 6, 2009

Accepted after revision: December 22, 2009
Published online: $ $ $

Acta Haematol 957

DOI: 10.1159/000XXXXXX

Renal Dysfunction in Patients with

Beta-Thalassemia Major Receiving Iron
Chelation Therapy either with Deferoxamine
and Deferiprone or with Deferasirox
MarinaEconomou NikolettaPrintza AikateriniTeli VassilikiTzimouli
IoannaTsatra FotisPapachristou MirandaAthanassiou-Metaxa
First Department of Pediatrics, Aristotle University of Thessaloniki, Hippokration General Hospital,
Thessaloniki, Greece

Key Words
Cystatin C Iron chelation 2-Microglobulin
Renal function Thalassemia Urine markers

Abstract
There are limited studies on renal involvement in -thalassemia, mainly involving patients on deferoxamine, reporting
both glomerular and tubular dysfunction. The aim of the
present study was to investigate renal involvement in young
thalassemia patients, using both conventional and early
markers of renal dysfunction, and to correlate findings to
iron chelation therapy. Forty-two patients aged 423 years
were studied and, for analysis purposes, were divided into
two groups based on chelation therapy (group A receiving
deferasirox and group B receiving deferoxamine and deferiprone combination therapy). In addition to conventional renal biochemistries, creatinine clearance, estimated glomerular filtration rate, serum cystatin C (Cys C), fractional excretion of sodium, tubular phosphorus reabsorption and urine
calcium, protein, 2-microglobulin (2-MG) and glucose levels were measured. A considerable number of patients demonstrated impaired renal function with elevated Cys C levels
(36%), glomerular dysfunction with proteinuria (24%) and tubulopathy with hypercalciuria (35.5%) and elevated excre-

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tion of 2-MG (33.5%). Renal involvement seems to be present even in young patients with -thalassemia, therefore,
routine use of early markers of renal dysfunction is recommended. Further studies are needed in order to investigate
the role of new chelators in tubular function parameters.
Copyright 2010 S. Karger AG, Basel

Introduction

-Thalassemia major is caused by a genetic defect in

chain hemoglobin synthesis which results in chronic anemia. It is the most severe form in a diverse group of genetic disorders of hemoglobin synthesis, in which life can
only be sustained by regular blood transfusions [1]. Although transfusion therapy prolongs survival, the absence of a physiological iron excretion mechanism leads
to uneven accumulation of this metal in various body organs. Iron deposition principally affects the heart, liver
and endocrine glands and results in death, usually during
the second decade of life, should patients be left untreated
[2]. Therefore, lifelong chelation therapy is an essential
facet in the management of -thalassemia patients, extending their survival and dramatically improving their
quality of life. Deferoxamine (DFO), a parenterally adMarina Economou, MD, PhD
64 Th. Sofouli Street
GR546 55 Thessaloniki (Greece)
Tel./Fax +30 231 0857 111
E-Mail marina@med.auth.gr

09.02.2010 10:37:36

ministered iron chelator, has been in use for almost four

decades; during this period its benefits and limitations
have clearly been defined [3]. The orally active chelators
deferiprone and deferasirox (DFRA) have been widely
used as alternative efficacious chelation treatments during recent years [3, 4].
Although a large number of studies have been performed on different complications of thalassemia, little
information is available on renal involvement. Reports
investigating renal dysfunction in thalassemics have
been limited in number, mainly studying patients on
DFO. Additionally, they have not assessed novel, earlier
markers of renal and tubular function, such as serum cystatin C and urine 2-microglobulin (2-MG). The aim of
the present study was to investigate the presence of glomerular and/or tubular dysfunction in children and
young adults with -thalassemia major, using both common and advanced indexes, and to correlate possible
findings to iron chelation therapy.
Patients and Methods
The study included 42 children and young adults (22 males, 20
females) suffering from -thalassemia major. Patients were regularly transfused and were on a regular chelation program. Demographic data including age, sex, duration and volume of transfusion therapy as well as iron chelation regimen were recorded in all
patients. Patient age ranged from 4 to 23 years, with a mean of
13.66 8 5.11 years. Mean hemoglobin level was 9.35 8 0.45 mg/
dl and mean level of serum ferritin was 1,401.81 8 1,205.13 ng/
ml (range 1066,000 ng/ml). Iron chelation therapy consisted of
DFRA in 28 patients (group A) and combination of deferiprone
and DFO in 14 patients (group B). With regard to dosage, patients
on DFRA received orally 1535 mg/kg/day (mean 25.42 8 6.37
mg/kg/day) and patients on combination therapy received DFO
subcutaneously at 1148 mg/kg/day (mean 16.13 8 7.67 mg/kg/
day), 5 times weekly, and deferiprone orally at 6080 mg/kg/day
(mean 69.71 8 10.52). Fifteen healthy children of the same age
group served as controls.
Fasting blood was obtained for hematological, biochemical
and metabolic tests including hemoglobin, urea, serum creatinine
(Cr), potassium, sodium, magnesium, calcium, phosphate, parathyroid hormone, acid base-balance and total bicarbonates. A 24hour urine specimen was collected for the determination of Cr,
sodium, potassium, calcium, phosphorus and albumin. In addition to conventional renal biochemistries, serum cystatin C (Cys
C) and urinary 2-MG in a fresh, first morning urine sample were
measured, serving as early markers of renal and tubular dysfunction, respectively. Serum Cys C was examined by rate nephelometry (N Latex Cystatin C, N Protein Standard UY, Dade-Behring).
Assay sensitivity was 0.07 mg/l and normal values ranged from
0.53 to 0.95 mg/l. Urine 2-MG was assayed by rate nephelometry
(BN ProSpec, Dade-Behring). N Latex 2-microglobulin served
as reactant and N Protein Standard SL (Dade-Behring) was used
as antiserum. In urine samples with pH below 6.0 a dialysate of

AHA957.indd 2

Acta Haematol 957

1 N NaOH was added. Assay sensitivity was 0.07 mg/l and normal
values ranged from 0 to 0.2 mg/l. Creatinine clearance (CrC) was
evaluated in all children using the following formula: CrC (in milliliters per minute per 1.73 m2) = [(urine volume ! urine Cr)/
(serum Cr ! 1,440)] ! (1.73 m2 body surface). Estimated glomerular filtration rate (eGFR) was calculated using the Schwartz
formula for children under the age of 18 years: (height ! k)/Cr,
where height is calculated in centimeters, k = 0.44 for children
under 2 years and 0.55 for children over 2 years, and Cr is serum
creatinine [5, 6]. In young adults over the age of 18 years eGFR was
calculated according to the equation proposed by the Modification of Diet in Renal Disease Study Group [7]. Renal dysfunction
was defined as CrC or eGFR less than 90 ml/min/1.73 m2 and/or
abnormally elevated levels of serum Cys C. A renal ultrasound
was performed in 34/44 patients. Fractional excretion of sodium
(FENa) as well as calcium excretion and tubular reabsorption were
calculated by standard formulas. Hypercalciuria was defined as
urine Ca over 4 mg/kg/day. Proteinuria was defined as urinary
protein excretion over 100 mg/m2/day. Finally, the degree of iron
overload was calculated for each patient based on the mean serum
ferritin levels of a 1-year period prior to the study.
Statistics
Statistical analysis was performed using the Statistical Package for the Social Sciences for Windows (SPSS version 11.5); t test
and Mann-Whitney U test were used to compare differences between study groups for parameters with and without normal distribution, respectively. Pearson and Spearmans coefficient of correlation (r) and regression analysis model were used to determine
the correlations. A p value of !0.05 was considered to be statistically significant.

Results

All patients presented with normal blood biochemical

and metabolic parameters compared to controls (p 1
0.05), including serum Cr, urea, sodium, potassium, calcium, phosphate, magnesium, total proteins and albumin, acid-base balance and total bicarbonates.
With regard to glomerular function markers, mean serum Cr, CrC and eGFR were 0.7 8 0.16 mg/dl, 137 8
25.67 and 128 8 17.45 ml/min/1.73 m2, respectively, presenting no significant difference compared to controls
(p 1 0.05). Only 1 patient presented with a decreased
eGFR of 74 ml/min/1.73 m2.
Mean serum levels of Cys C were elevated in the patient cohort compared to controls (0.89 8 0.21 and 0.67
8 0.286 mg/l, respectively, p = 0.001). Specifically, 15/42
patients (36%) presented with abnormally elevated serum
Cys C. Children with elevated Cys C when compared to
those with normal levels demonstrated elevated 2-MG
levels (p = 0.017). A significant correlation between Cys
C and urine 2-MG levels was found (r = 0.325, p = 0.032).
However, there was no correlation between Cys C levels
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Table 1. Patient and control demographic and laboratory data

Age, years
Hemoglobin, mg/dl
Transfusions, ml/kg/month
Ferritin, ng/ml
Serum Cr, mg/dl
Urea, mg/dl
Cys C, mg/l
Urine protein, mg/m2/day
Urine calcium, mg/kg/day
Urine FENa, %
Urine P, reabsorption, %
2-MG, mg/l
CrC, ml/min/1.73 m2
eGFR, ml/min/1.73 m2
1

DFRA
(group A)

Deferiprone
DFO (group B)

Controls

p1

p2

p3

6.7583.01
9.480.48
16.3183.26
1,519.881,070.3
0.6580.21
35.5810.21
0.9280.39
250.568198.3
6.182.45
0.7480.59
9583.2
5.183.9
135.9829.2
127.98821.14

12.1085.31
9.280.35
17.6681.79
1,147.48838
0.8280.29
34.28813.85
0.8480.31
210.58159.27
4.181.98
1.1480.73
9682
0.2580.11
131823.89
122.31819.01

12.6684.2
13.281.82

38.5813.18
0.7580.13
37.18815.67
0.6780.286
79.34829.1
1.880.91
1.2180.57
96.582.8
0.280.08
139.9835.43
131.56825.11

0.007
NS
NS
0.033
0.011
NS
NS
NS
0.045
0.04
NS
0.001
NS
NS

0.006
0.0001

0.0001
0.045
NS
0.0001
0.007
0.0001
0.03
NS
0.0001
NS
NS

NS
0.0001

0.0001
NS
NS
0.001
0.011
0.0001
NS
NS
0.009
NS
NS

Group A versus group B; 2 Group A versus controls; 3 Group B versus controls.

and age, iron chelation therapy, ferritin, CrC, eGFR, proteinuria or urine calcium levels (p 1 0.05).
With regard to urine findings, none of the patients
presented with phosphaturia, abnormal FENa or glycosuria. Urinary calcium excretion was found to be elevated
in patients compared to controls (5.3 8 3.54 and 1.8 8
0.91 mg/kg/day, respectively, p ! 0.0001). Hypercalciuria
was detected in 15/42 patients (35.5%). Patients with hypercalciuria compared with those with normal urine calcium excretion presented increased proteinuria (p =
0.007) and a significant correlation between urine calcium and proteinuria was found (r = 0.552, p = 0.001). Performing linear regression analysis, proteinuria was found
to be the best predictor of urine calcium levels (B = 2.096,
= 2.273 and p = 0.025), while there was no relationship
to the rest of the variables examined (p 1 0.05). Serum
parathyroid hormone was normal in all patients. In 2/42
patients there was a positive history of nephrolithiasis,
1 patient receiving DFRA and 1 receiving combination
therapy. Six patients (14.5%) had a positive family history
of nephrolithiasis, including the 2 patients mentioned
above.
Mean urinary 2-MG was elevated in patients compared to controls (4.35 8 9.4 and 0.2 8 0.08 mg/l, respectively, p ! 0.0001). Abnormal excretion of 2-MG was
found in 14/42 patients (33.5%) and in none of the controls. Children with elevated 2-MG presented additionally with increased Cys C compared to those with normal
2-MG (p = 0.002).

Finally, urine protein, a glomerular function marker,

was found elevated in patients compared to controls
(223.28 8 125.84 and 79.34 8 29.1 mg/m2/day, respectively, p ! 0.0001). Proteinuria was found in 10/42 patients (24%) and none of the controls. Proteinuria was
correlated with calciuria (as mentioned above), no other
correlation found with regard to age, hemoglobin, iron
chelation therapy, ferritin, CrC, eGFR or 2-MG (p 1
0.05).
Regarding iron chelation therapy, mean levels of blood
and urine parameters as well as comparisons between the
two groups studied are shown in table1. Group A and
group B were on comparative erythrocyte volume transfusions (p = 0.948). Patients in group A were found to be
younger compared to patients in group B (p = 0.007), presenting with higher serum ferritin levels (p = 0.033), elevated urinary calcium and 2-MG levels (p = 0.045 and
p = 0.001, respectively), decreased FENa (p = 0.04) and decreased serum Cr (p = 0.011).

Renal Dysfunction in -Thalassemia

with Iron Chelation Therapy

Acta Haematol 957

AHA957.indd 3

Discussion

Renal involvement in thalassemic patients has not

been studied to the same extent as other complications of
the disease involving organs like the heart or the endocrine glands. Additionally, reports have been limited to
patients receiving DFO and have used common markers
unable to demonstrate early renal changes. These studies
3

09.02.2010 10:37:52

have mainly demonstrated the presence of proximal tubular dysfunction in thalassemic patients, being attributed to chronic anemia, iron overload and/or dose-dependent DFO toxicity [811].
In the present study 42 young patients with -thalassemia major, well maintained on regular blood transfusions and receiving iron chelation therapy at conventional dosage, were studied. In addition to common renal indexes, earlier markers such as Cys C and 2-MG were
assessed. The results of the study show impaired renal
function at both glomerular and tubular level. Specifically, results demonstrate impaired renal function with
elevated Cys C (36%), significant tubulopathy with hypercalciuria (35.5%) and elevated urine excretion of 2MG (33.5%), as well as glomerular dysfunction with proteinuria (24%). Although more than half of the patients
studied (54.5%) had ferritin levels higher than 1,000 ng/
ml, ferritin was not found to be an independent predictor
neither for glomerular nor for tubular dysfunction. However, it is well understood that ferritin levels do not sufficiently reflect the degree of hemosiderosis and that iron
deposition in the renal tubules may not be removed sufficiently by iron chelators [12].
In accordance with previous reports, common indexes
such as electrolytes, urea and Cr were normal in the patients participating in the present study [12]. CrC and
eGFR also presented no significant difference compared
to controls (p 1 0.05). Koren et al. [11] reported that subcutaneous administration of DFO was associated with a
significant decrease in GFR in 40% of the thalassemia
major patients studied and with a mild decrease in another 40%. However, DFO nephrotoxicity has been shown
to be dose-dependant. In the present study only 1 patient
presented with a decreased eGFR of 74 ml/min/1.73 m2.
The patient had a low ferritin level (458 ng/ml) and was
on combination therapy with conventional DFO dosage.
Serum Cr is known to be an unreliable indicator of
changes in kidney function as it is affected by factors unrelated to renal function, such as muscle mass, protein
intake, inflammatory illness and hepatic disease. Additionally, Cr is partially secreted by renal tubules and frequently overestimates GFR. An extensive search is being
conducted to find a serum marker able to detect renal
function impairment, especially at an early stage [13].
Previous studies have demonstrated the superiority of serum Cys C compared to Cr in the evaluation of GFR, particularly when there is a minor reduction in GFR [14, 15].
Cys C is an age- and gender-independent proteinase inhibitor, with low molecular weight that is produced in all
nucleated cells. Its appearance rate in blood is constant.
4

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Acta Haematol 957

It is freely filtered in the renal glomeruli and totally reabsorbed and metabolized in the proximal tubule. Thus, the
serum concentration of Cys C is mainly determined by
GFR [16]. The present study is the first to assess Cys C
elevation as an early marker of renal dysfunction in patients with -thalassemia. Both findings of elevated mean
serum Cys C levels in the patient cohort and of increased
Cys C presence in a large percentage of patients, with
concurrent normal CrC and eGFR, are indicative of the
advantages of Cys C measurement in evaluating early
changes of renal function in thalassemic patients.
In the patients studied, eGFR and Cys C presented no
significant difference with regard to iron chelation regimen. The comparatively lower values of serum Cr in patients on DFRA may not reflect a real difference in renal
function, but could be attributed to the younger age and
smaller weight of these patients, given that serum Cr level is affected by muscle mass.
In agreement with previous reports, hypercalciuria
was found in a large number of patients studied [10, 12].
Hypercalciuria has been formerly attributed to hypoparathyroidism and related treatment with calcium and
vitamin D [12]. However, in the patient group studied it
reflects proximal tubular damage, as no patient suffered
from hypoparathyroidism or received relative supplements. In accordance is the finding of elevated 2-MG
excretion, a low molecular weight protein which, under
normal circumstances, is freely filtered at the glomerulus
but almost totally reabsorbed by renal tubules [17]. Elevation of 2-MG in urine is a sensitive and reliable early
marker of tubular dysfunction [18, 19].
The cause of renal tubular dysfunction in thalassemic
patients is not clear. It seems to be multifactorial, attributed mainly to chronic hypoxia resulting from continuing anemia and to hemosiderosis, excess free iron being
a catalyst of lipid peroxidation and leading to cell damage
[8, 20, 21]. The role of DFO has been studied with conflicting results, while the possible effect of the new chelating agents remains to be seen [11]. In the present study,
patients on DFRA, although younger, presented with increased hypercalciuria and elevated 2-MG levels compared with patients on combination therapy. Given that
these patients had also comparatively higher ferritin levels, it is difficult to assess the specific role of the chelator
versus the effect of iron overload in the tubular dysfunction noted.
With regard to urine FENa, increased values were
found in patients belonging to group B who received
combination therapy. Although DFO was given in much
lower doses and in a subcutaneous mode compared to
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09.02.2010 10:37:53

patients from older reports who received DFO intravenously as monotherapy, increase in FENa could, in a similar way, be attributed to DFO therapy. The effect has
been known to reverse when discontinuing the drug [11].
Finally, in the present study, serum Cys C levels were
correlated to urine 2-MG excretion and calciuria was
correlated to proteinuria. This is an interesting finding as
serum Cys C and urine 2-MG are both early markers of
renal dysfunction (tubular and glomerular, respectively)
while calciuria and proteinuria indicate both marked and

chronic damage. The presence of proteinuria and microalbuminuria has been attributed by the literature to
chronic anemia and iron overload [10, 21].
In conclusion, renal disorders are present even in
young patients with -thalassemia. As renal dysfunction
may not be detected by routine tests, use of early markers
is recommended in this patient cohort. Further studies
are needed in order to investigate the role of new chelators
in tubular function parameters, such as urine calcium
and 2-MG excretion.

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