Pediatr Nephrol (2012) 27:941948

DOI 10.1007/s00467-011-2094-4

Plasma and urinary levels of cytokines in patients

with idiopathic hypercalciuria
Augusto C. S. Santos Jr & Eleonora M. Lima &
Maria Goretti M. G. Penido & Katia D. Silveira &
Mauro M. Teixeira & Eduardo A. Oliveira &
Ana Cristina Simes e Silva

Received: 2 April 2011 / Revised: 2 December 2011 / Accepted: 5 December 2011 / Published online: 7 January 2012
# IPNA 2012

Abstract
Background Recent studies suggest that cytokines modulate
bone turnover. Idiopathic hypercalciuria (IH) seems to be
associated with bone mineral loss. Therefore, the aim of this
study was to assess cytokines involved in bone turnover in
patients with IH.
Methods Plasma and spot-urine levels of interleukin (IL)1, IL-6, IL-8, tumor necrosis factor alpha (TNF-), transforming growth factor 1 (TGF-1), and monocyte chemoattractant protein (MCP-1) were measured in 70 children
and adolescents with IH and in 37 healthy controls. Patients
with IH were subdivided according to their calciuria at the
time of sample collection: 4 mg/kg/day (persistent IH,
n027) and below 4 mg/kg/day (controlled IH, n043).
Cytokines were determined by enzyme-linked immunoassay.
Results Plasma and urinary concentrations of IL-1, IL-6,
IL-8, and TNF- were undetectable in all groups. No differences were found between controlled and persistent hypercalciuria for plasma and urinary levels of MCP-1 and TGF1. On the other hand, MCP-1 levels were significantly
higher in both subgroups of IH in comparison to healthy
controls. Furthermore, urinary MCP-1 levels of IH patients
correlated positively with bone mineral content (p00.013).

A. C. S. Santos Jr : E. M. Lima : M. G. M. G. Penido :

E. A. Oliveira : A. C. Simes e Silva (*)
Pediatric Nephrology Unit, Department of Pediatrics,
Faculty of Medicine,
Belo Horizonte, MG, Brazil
e-mail: acssilva@hotmail.com
K. D. Silveira : M. M. Teixeira
Laboratory of Immunopharmacology, Department of Biochemistry
and Immunology, Institute of Biological Sciences,
Federal University of Minas Gerais (UFMG),
Belo Horizonte, MG, Brazil

Conclusion Although cytokine measurements did not allow

the differentiation between persistent and controlled IH, our
findings suggest that MCP-1 might play a role in patients
with IH.
Key words Biomarkers . Cytokines . MCP-1 . Idiopathic
hypercalciuria . Bone mineral loss

Introduction
Idiopathic hypercalciuria (IH) was first described by
Albright et al. [1], who defined it as an excessive urinary
calcium loss accompanied by normal serum calcium levels.
IH is the most common metabolic abnormality in patients
with nephrolithiasis, accounting for 3050% of calciumoxalate stone formers [24].
The pathogenesis of IH is not yet fully understood.
However, it is generally considered that IH is caused by an
alteration in calcium homeostasis at sites where large
amounts of calcium must be precisely controlled [5].
Several studies have shown decreased bone mineral density
(BMD) in patients with IH [618]. This progressive decrease in bone mineral content suggests that osteoclasts
and osteoblasts might play a key role in the chain of events
leading to hypercalciuria. The function of osteoblasts and
osteoclasts and the resulting balance between bone formation and resorption are regulated by multiple mediators with
the participation of cytokines [1925]. In IH, cytokines may
be responsible for triggering specific alterations to bone
metabolism, which in turn contribute to the development
of excessive bone remodeling, with the possible predominance of bone mass resorption over formation [1925].
In the setting of IH, we hypothesized that the measurement of cytokines as non-invasive biomarkers could

942

improve the diagnostic capability or help determine the risk

of persistent hypercalciuria and of bone mineral loss.
Therefore, we measured plasma and spot-urine levels of
interleukin 1 beta (IL-1), interleukin 6 (IL-6), interleukin
8 (IL-8), tumor necrosis factor (TNF-), transforming
growth factor 1 (TGF-1) and monocyte chemoattractant
protein (MCP-1) in children and adolescents with IH and in
sex- and age-matched healthy subjects.

Patients and methods

Study design
This cross-sectional study included patients with confirmed
diagnosis of IH and a group of sex- and age-matched
healthy subjects as controls.
Patients with IH
This group included a sample of children and adolescents
with well-established IH, followed up at the Pediatric
Nephrology Unit of our institution from 2009 to 2011,
whose parents gave their consent to participate in the study
protocol. Hypercalciuria was defined by serum calcium
within normal limits and 24-h urinary excretion of calcium
equal to or higher than 4 mg/kg per day for both genders in
two nonconsecutive samples, on an unrestricted diet [26,
27]. In order to define the diagnosis of IH, all patients were
submitted to a systematic protocol to investigate the possibility of hypercalciuria secondary to diseases and conditions
that might affect urinary calcium excretion, as described
elsewhere [26, 28]. Briefly, the protocol included: blood
gas analysis, serum electrolytes, erithrogram, urea, creatinine, uric acid, PTH, TSH, T4, spot urine, and 24-h urinary
concentrations of calcium, citrate, uric acid, oxalate, cystine,
and creatinine [26, 28]. Patients with known diseases or use
of medication that could affect calcium excretion, bone
remodeling or monocyte function were excluded [6].
Therefore, a total of 81 patients with confirmed IH were
invited to participate in the study. From this group, 11 patients
refused to participate. The remaining 70 patients were then
divided in two subgroups according to their urinary calcium
excretion at the time of urine and blood sample collection.
Patients with calcium excretion equal or superior to
4 mg/kg/day were allocated to the persistent IH group
(n027). Patients with calcium excretion below 4 mg/kg/day
were allocated to the controlled IH group (n043) [26].
Controls
The control group consisted of sex- and age-matched
healthy subjects from our Pediatric Primary Care Center.

Pediatr Nephrol (2012) 27:941948

Healthy status was determined through the subjects medical

history and either a parental report or self-report to rule out
the presence of chronic or acute diseases.
Study protocol
All participants were interviewed and underwent physical
examination at the time of blood and urine collection. Blood
and urine samples were obtained simultaneously in patients
and controls. Age, gender, race, weight, height, body mass
index, systolic and diastolic blood pressure, serum creatinine, calciuria, citraturia, phosphaturia, magnesuria, BMD,
family history of nephrolithiasis, presence of calculus, past
history of extracorporeal shock lithotripsy, and symptoms
were analyzed in all IH patients. The study protocol did not
interfere with medical prescriptions for IH. Treatment basically included the long-term administration of potassium
citrate (0.5 to 1 mEq/kg/day) associated with hydrochlorothiazide (0.5 to 1 mg/kg/day) for 3 to 6 months to control
high urinary calcium excretion. Medical prescriptions were
also recorded for analysis.
Regarding the control group, clinical variables (age, gender, race, weight, height, body mass index, and systolic and
diastolic blood pressure) were also obtained. In order to exclude
from the control group all subjects with impaired renal function
and with increased urinary calcium excretion, plasma creatinine
and spot-urine levels of creatinine and of calcium were assayed
in the same sample collected for cytokine measurements.
Therefore, all controls included in the study exhibited plasma
creatinine between reference values and spot-urine calcium:
creatinine ratios lower than 0.20.
Bone mineral density was assessed by dual energy X-ray
absorptiometry at the lumbar spine (L1L4) using a Lunar
Prodigy Primo DXA System (GE Healthcare Lunar,
Madison, WI, USA) in 46 patients with IH. Bone density
was expressed in g/cm2 and was also stratified as Z-score >1
SD and 1 SD according to previous studies [12, 17, 18,
2931]. Z scores were calculated in relation to a population of
individuals of the same gender, age range, and ethnicity [12,
17, 18, 2931].
Blood sampling
After informed consent, all subjects were subjected to blood
collection. Blood sampling occurred on only one occasion, at
the same time as other routine examinations. The samples
were collected into sterile citrate tubes, which were immediately immersed in ice, and processed within 30 min of collection. Cells were sedimented by centrifugation at 700 g for
10 min at 4C. Then the supernatant was collected and re-spun
for another 20 min at 1,300 g to sediment platelets.
Cell-free plasma was aliquoted into 0.5-mL samples and
stored at 80C until measurement.

Pediatr Nephrol (2012) 27:941948

Urine sampling
A single urine sample was obtained from all patients on the
same day as blood collection from 7.30 a.m. to 9.00 a.m.
After homogenization, 10 mL of the collected urine were
centrifuged at 4C for 20 min at 1,300 g. Cell-free urine was
aliquoted into 0.5-mL tubes and stored at 80C until
measurement.
Cytokines measurement
Plasma and urinary levels of IL-1, IL-6, IL-8, TNF-,
TGF-1, and MCP-1 were measured by specific enzymelinked immunoassay (ELISA) kits (R&D Systems,
Minneapolis, MN, USA), following the manufacturers
instructions, as described elsewhere [32]. Urine cytokine
levels were expressed as absolute concentrations (pg/mL)
as well as concentrations standardized for urine creatinine
measured in the same urine spot (pg/mg cr). All samples
were assayed in duplicate in two separate assays with interassay variation below 5%. Our intra-assay variation for the
ELISA measurements was below 3%. Specifically for the
measurement of TGF-1, we used a Quantikine kit (R&D
Systems). Samples were activated before the TGF-1 assay.
Sample activation basically comprised biochemical steps
(acidification followed by neutralization of the pH) in order
to activate latent TGF-1 to immunoreactive TGF-1 detectable by the Quantikine TGF-1 immunoassay, as recommended by the manufacturer and previously described
[32]. The detection limits were 0.1 g/mL (IL-1),
0.039 pg/mL (IL-6), 6 pg/mL (IL-8), 0.106 pg/mL (TNF-),
6 pg/mL (TGF-1), and 8 pg/mL (MCP-1).
Statistical analysis
Values are expressed as medians and interquartile range
(25th percentile, 75th percentile) or means and standard
deviation (SD), when appropriate. The MannWhitney and
the KruskalWallis tests were used to compare nonparametric continuous variables. Means were compared using nonpaired Students t test. Dichotomous variables were compared using the two-sided Fisher's exact test. Correlation
among plasma cytokines, urinary cytokines, and BMD was
performed using a nonparametric test (Spearman rank correlation test). The level of significance was set at p<0.05.
Ethical aspects
The Ethics Committee of the Federal University of Minas
Gerais approved the study. Informed consent was obtained
from all parents and, when appropriate, also from the included patients and healthy controls. The research protocol
did not interfere with any medical recommendations or

943

prescriptions. Subject follow-up was guaranteed even in

cases of refusal to participate in the study.

Results
General clinical characteristics
A total of 70 patients with IH and 37 healthy controls were
included in the analysis. Clinical and laboratory characteristics were obtained at the same time as the cytokine measurements and summarized in Table 1. No differences were
observed in general clinical characteristics among patients
with persistent IH, controlled IH, and healthy controls
(Table 1). Both subgroups of patients with IH and healthy
controls were normotensive and had normal serum creatinine levels at the time of sample collections (Table 1).
Except for the increased prescription of hydrochlorothiazide
in patients with persistent IH (p<0.05), there were no other
differences in clinical and laboratory variables between
patients with persistent and those with controlled IH. Signs
and symptoms at initial presentation of IH were also very
similar in both subgroups of IH patients (Table 1).
Bone mineral density measurements
A total of 46 patients underwent BMD measurements during
the study period: 20 patients with persistent IH and 26
patients with controlled IH. No differences were detected
in the number of patients with a Z-score 1 SD and with a
Z-score 2 SD between these subgroups of patients with
IH (Table 2).
Association of plasma and urinary cytokine concentrations
with urinary calcium excretion
In IH subgroups (persistent and controlled) and in healthy
controls, plasma and urinary concentrations of IL-1, IL-6,
IL-8, and TNF- were below the detection limits of the
ELISA kits. Plasma and spot-urine concentrations of
MCP-1 and TGF-1 were detectable in both subgroups of
patients with IH. However, the median value for plasma and
spot-urine concentrations of TGF-1 was zero in both IH
subgroups (data not shown). In the control group, TGF-1
levels were undetectable in the majority of samples, while
MCP-1 concentrations were measurable in plasma and spoturine. As shown in Table 3, no significant differences were
verified between controlled and persistent IH patients for
plasma and spot-urine (absolute and standardized for creatinine) levels of MCP-1. On the other hand, plasma and
urinary levels of MCP-1 were significantly higher in both
groups of IH patients in comparison to healthy controls
(Table 3).

944

Pediatr Nephrol (2012) 27:941948

Table 1 Clinical and biochemical characteristics in healthy subjects

(control group) and in patients with idiopathic hypercalciuria (IH)
divided according to the level of urinary calcium excretion into

persistent IH (4 mg/kg/day) and controlled IH (<4 mg/kg/day) at

the time of blood and urine sampling

Characteristics

Control group (n037)

Persistent IH (n027)

Controlled IH (n043)

Age (years)

15.434.82

16.995.24

14.634.42

0.28

Follow-up (years)

10.926.36

8.605.24

0.15
1.00

Gender (%)
Male

21 (56.76)

15 (55.56)

25 (58.13)

16 (43.24)

12 (44.44)

18 (41.87)

22 (59.46)
15 (40.54)

16 (59.26)
11 (40.74)

28 (65.11)
15 (34.89)

0.80

Percentile of weight

6318

6625

6221

0.18

Percentile of height
Percentile of BMI

6715
5510

7014
579

6517
5613

0.22
0.58

Percentile of SBP

5813

6318

6017

0.32

Percentile of DBP
Serum creatinine (mg/dl)

6418
0.670.15

6721
0.730.17

6519
0.650.18

045
0.08

Calciuria (mg/24 h)
Calciuria (mg/kg/day)
Citraturia mg/24 h

259.2488.01
5.401.53
598.10295.53

107.1447.33
2.340.84
482.34192.73

<0.0001
<0.0001
0.09

Phosphaturia mg/24 h
Magnesuria mg/24 h
Prescription of potassium citrate (%)

686.70241.76
97.5837.47
23 (85.18)

706.90196.14
97.7374.54
32 (74.41)

0.83
0.99
0.38

Prescription of hydrochlorothiazide (%)

Family history of nephrolithiasis (%)
Presence of calculus oin ultrasound (%)

14 (51.85)
21 (77.78)
8 (29.63)

6 (13.95)
31 (72.09)
20 (46.51)

0.001
0.77
0.21

History of lithotripsy (%)

Recurrent abdominal pain (%)a
Macroscopic hematuria (%)a

4 (14.81)
12 (44.44)
6 (22.22)

8 (18.60)
20 (46.51)
13 (30.23)

0.76
1.0
0.58

Microscopic hematuria (%)a

5 (18.52)
3 (11.11)
1 (3.70)

5 (11.63)
5 (11.63)
0 (0)

0.49
1.0
0.39

Female (%)
Race
White (%)
Non-white (%)

Urinary tract infection (%)a

Nephrolithiasis (%)a

Values are expressed as meansstandard deviation for continuous variables. Number of individuals and percentages refers to categorical variables.
Analysis of variance followed by StudentNewmanKeuls test compared continuous variables and Fisher's exact test was used for percentage
comparisons
BMI 0 body mass index; SBP 0 systolic blood pressure; DBP 0 diastolic blood pressure
a

Signs and symptoms at initial presentation

There was a trend toward a positive correlation

between plasma and spot-urine levels of MCP-1

standardized to creatinine in patients with IH (r00.24,

p00.08).

Table 2 Bone mineral density (BMD) Z-score in 46 patients with idiopathic hypercalciuria (IH) divided according to the level of urinary calcium
excretion into persistent IH (4 mg/kg/day) and controlled IH (<4 mg/kg/day)
Characteristics

Persistent IH (n020)

Controlled IH (n026)

BMD L1L4 >1 (%)

BMD L1L4 1 and >2 (%)
BMD L1L4 2 (%)

10 (50.00)
9 (45.00)
1 (5.00)

14 (53.84)
9 (34.62)
3 (11.54)

1.00
0.55
0.62

Fishers exact test for dichotomous variables

Pediatr Nephrol (2012) 27:941948

945

Table 3 Median and interquartile range (25th percentile, p25, and

75th percentile, p75) of plasma and urinary (absolute and standardized
to creatinine) levels of monocyte chemoattractant protein (MCP-1) in

healthy subjects (control group) and in patients with persistent and

controlled idiopathic hypercalciuria (IH group)

Cytokine

Control group (n037)

Persistent IH (n027)

Controlled IH (n043)

Plasma MCP-1 (pg/mL)

12.37 (0.00, 32.75)

125.38 (86.90, 157.64)*

129.52 (103.95, 168.48)**

Urinary MCP-1 (pg/mL)

21.82 (3.36, 47.55)

209.68 (95.26, 239.16)*

213.28 (109.87, 278.29)**

Urinary MCP-1 (pg/mg cr)

0.47 (0.00, 0.86)

2.83 (1.38, 4.80)*

4.08 (1.85, 8.66)**

*p<0.05 for the comparison between the persistent IH group and the control group
**p<0.05 for the comparison between controlled IH vs the control group. No differences were detected in the comparison of persistent and
controlled IH

Association of plasma and urinary cytokine concentrations

with bone mineral density
Patients were stratified according to their BMD Z-score into
two groups: >1 SD, n028; 1SD; n018, as shown in
Table 4. The comparison between these groups did not
reveal differences in general clinical findings or in 24h urinary calcium excretion.
The comparison of plasma and urinary concentrations of
MCP-1 and TGF-1 in patients with BMD Z-score>1 SD
and1 SD did not reveal significant differences (Table 5).
However, there was a positive correlation between urinary
levels of MCP-1 and bone mineral content (r00.379,
p00.013).

in the comparison between plasma and spot-urine levels of

MCP-1 (absolute and standardized for creatinine) in these
age groups among healthy subjects (data not shown).
Association of plasma and urinary cytokine concentrations
with hydrochlorothiazide prescription
In order to verify if the use of hydrochlorothiazide influenced MCP-1 levels, IH patients were also divided according to the prescription (n020) or not (n050) of this
medication at the time of blood and urine sampling. As
shown in Table 7, no significant differences were detected
in plasma and spot-urine (absolute and standardized to creatinine) levels of MCP-1 in relation to the prescription or not
of hydrochlorothiazide.

Association of plasma and urinary cytokine concentrations

with age groups
Discussion
In order to detect possible changes in cytokine levels related
to age, the patients with IH were stratified into the following
age groups: school (age12 years, n018) and adolescent
(age >12 years, n052). The absolute levels of MCP-1 (pg/
mL) were significantly higher in adolescents than in school
age children (p00.02). However, this difference was not
observed when values were standardized to creatinine
(p00.61, Table 6). Healthy controls were also stratified into
the same age groups: school (age 12 years, n010) and
adolescent (age >12 years, n027). There were no differences
Table 4 Patients characteristics
according to bone mineral density (BMD) IH idiopathic
hypercalciuria Z-score

Students t test compared continuous variables and Fisher's

exact test for percentage
comparisons

To our knowledge, this is the first study that simultaneously

measure bone turnover cytokines in plasma and spot-urine
in pediatric patients with IH. Our results showed that single
measurements of bone turnover cytokines seem not to be
useful in distinguishing patients with persistent hypercalciuria or reduced BMD Z-score. Indeed, many cytokines were
below the detection limits for ELISA kits in patients with IH
and in healthy controls. On the other hand, the chemokine
MCP-1 is significantly higher in plasma and spot-urine

Characteristics

BMD Z-score >1 SD (n028)

BMD Z-score 1 SD (n018)

BMD Z-score
Age (years)
Gender
Male (%)
Female (%)
Persistent IH (%)
BMI (kg/m2)
Calciuria (mg/24 h)

0.20.68
13.384.96

1.650.58
15.154.29

<0.001
0.20

15 (53.57)
12 (42.86)
9 (32.14)
18.653.95
161.78112.04

10 (55.56)
9 (50.00)
9 (50.00)
19.193.74
175.7793.75

1.00
0.23
0.66
0.65

946

Pediatr Nephrol (2012) 27:941948

Table 5 Median and interquartile range (25th percentile, p25, and

75th percentile, p75) of plasma and urinary (absolute and standardized
to creatinine) levels of monocyte chemoattractant protein (MCP-1) and
TGF-1 in patients according to bone mineral density (BMD) Z-score
Cytokines

Groupsa

Median (p25, p75)

Plasma MCP-1
(pg/mL)

>1

135.61 (87.15, 185.89)

0.88

145.96 (104.80, 165.58)

>1

0 (0, 4.36)

8.95 (0, 27.20)

>1

5.14 (1.79, 8.58)

Plasma TGF-1
(pg/mL)
Urinary MCP-1
(pg/mg cr)
Urinary MCP-1
(pg/mL)
Urinary TGF-1
(pg/mg cr)
Urinary TGF-1
(pg/mL)

1
>1

3.68 (2.67, 8.56)

209.68 (121.51, 237.42)

0.67

1
>1

225.16 (131.79, 296.24)

0 (0, 0.24)

0.36

0 (0, 0.21)

>1

0 (0, 5.976)

0 (0, 8.87)

0.48

Groups

Median (p25, p75)

Plasma MCP-1
(pg/mL)

School

134.39 (99.36, 187.23)

0.82

Adolescent
School

129.15 (103.95, 163.12)

0 (0, 21.78)

0.69

Adolescent
School

0 (0, 11.15)
6.08 (1.31, 8.58)

0.61

Adolescent
School

3.18 (1.65, 7.29)

121.51 (72.34, 203.74)

0.02

Adolescent
School

220.95 (137.86, 296.37)

0 (0, 0.20)

0.74

Adolescent

0 (0, 0.21)

School

0 (0, 5.15)

Adolescent

0 (0, 11.01)

Median comparisons were made by MannWhitney test

Plasma MCP-1
(pg/mL)

Use of hydrochlorothiazide

124.77 (103.46,
163.12)
127.33 (92.75,
158.13)
2.71 (1.65, 6.97)

0.77

No use of hydrochlorothiazide
Use of hydrochlorothiazide
No use of hydrochlorothiazide

3.39 (1.26, 7.29)

Use of hydrochlorothiazide

189.25 (131.29,
289.93)
209.68 (83.49,
237.42)

Use of hydrochlorothiazide

0.93

0.63

Median comparisons were made using the MannWhitney test

Cytokines

Urinary TGF-1
(pg/mL)

Urinary MCP-1
(pg/mL)

Table 6 Median and interquartile range (25th percentile, p25, and

75th percentile, p75) of plasma and urinary (absolute and standardized
to creatinine) levels of monocyte chemoattractant protein (MCP-1) and
TGF-1 in patients equal to or under 12 years old (school, n018) and
above 18 years old (adolescent, n052)

Urinary TGF-1
(pg/mg cr)

Median (p25, p75)

0.81

samples of patients with IH compared with healthy controls.

In addition, there was a positive correlation between urinary
MCP-1 levels and bone mineral content.

Urinary MCP-1
(pg/mL)

IH

Urinary MCP-1
(pg/Ll cr)

BMD Z-score SD. Median comparisons were made using the Mann
Whitney test

Urinary MCP-1
(pg/mg cr)

Cytokines

0.06

Plasma TGF-1
(pg/mL)

Table 7 Median and interquartile range (25th percentile, p25, and

75th percentile, p75) of plasma and urinary (absolute and standardized
to creatinine) levels of monocyte chemoattractant protein (MCP-1) in
patients with idiopathic hypercalciuria (IH) in the use of hydrochrorothiazide (n020) and those not under this treatment (n050)

0.56

There is very little information concerning the role of

MCP-1 in bone metabolism. The principal function of
MCP-1 is the recruitment of monocytes [33, 34]. In vitro
and in vivo studies indicate that MCP-1 induces the recruitment of monocytes to bone, which, in turn, is associated
with an increase in osteoblast number [35, 36]. MCP-1 is
typically not expressed in normal bone or by normal osteoblasts. Upon stimulation by inflammatory mediators and
growth factors [3739], the expression of MCP-1 and the
recruitment of monocytes are increased in both osseous
inflammation and during bone remodeling. Indeed, monocytes seem to have different functional roles in areas of bone
formation and resorption [33, 35]. The recruitment of monocytes in areas of bone formation is associated with a decrease
in the number of osteoclasts, while in bone-resorbing areas,
recruitment of cells of the monocytic lineage is associated
with formation of osteoclasts [33, 35]. In this context, the
increased levels of MCP-1 in IH patients compared with
healthy subjects and the positive correlation between urinary
MCP-1 and BMD may indicate that this chemokine might
play a role in bone remodeling of patients with IH.
Alternatively, no differences in MCP-1 levels were found
when patients were stratified according to the persistence of
high urinary calcium excretion and according to the prescription of hydrochlorothiazide.
In addition, the frequency of reduced BMD Z-scores was
similar in both subgroups of IH patients, also suggesting that
the persistence of hypercalciuria might not be directly linked
to high bone resorption in our group of patients. We believe
that the process of bone remodeling in IH is very complex
and different mechanisms might be activated in spite of the
levels of urinary calcium excretion. Therefore, MCP-1 could
be locally produced by osteoblasts and signaling toward
bone formation or bone resorption depending on the area
of expression and on the interactions with other mediators.

Pediatr Nephrol (2012) 27:941948

In our study, plasma and urinary levels of IL-1, IL-6,

IL-8, and TNF- were below detectable limits. Other
authors, by using different methodologies, were able to
evaluate these cytokines in patients with IH [18, 20, 22,
23, 39]. Freundlich et al. showed that the mRNA expression
of IL-1 in peripheral blood mononuclear cells of children
with IH did not differ from that of healthy controls [23].
Pacifici et al. described an association between IL-1 activity and bone resorption [39]. Weisinger et al. used unstimulated blood monocytes to show increased expression of IL1, IL-6, and TNF- mRNA in patients with IH [18]. These
authors also described a correlation between basal production of IL-1, but not IL-1, and decreased trabecular bone
[20]. Indeed, these more refined methodologies have
allowed the evaluation of cytokine expression in bone tissue
or in peripheral mononuclear cells [18, 20, 22, 23, 39].
However, we opted to use conventional plasma and spot
urine samples in order to ease collection and to evaluate the
utility of these tests for patients with IH in clinical practice.
We are aware of the limitations of our study. The main
possible weakness is the cross-sectional design. Once
patients with IH probably decrease their calcium bone mineral content progressively, serial densitometries might be
necessary to accurately evaluate bone mineral loss.
Another weakness was the fact that our patients were not
on standard diets during sample collections. Diets rich in
protein and salt can significantly affect calciuria by different
mechanisms from those involved in bone remodeling in IH
[40]. Parathyroid hormone and vitamin D are also important
variables not concomitantly measured with cytokines in our
research protocol [41, 42]. In this study, age was addressed
as a possible confounder. Bone remodeling regulatory
mechanisms may vary according to age, once children and
adolescents experience different stages in skeletal development [43]. In spite of that, no significant difference in
cytokine measurements was found in the comparison between school age and adolescents in the control group and in
patients with IH.
Nevertheless, some aspects of the study may increase the
strength of our findings, such as the sample size, strict
inclusion criteria, well-established protocols for cytokine
measurements, and the homogeneity among groups. Our
sample size was considerably larger than those of previous
studies on cytokines in IH [20, 22, 39, 44]. Except for
calciuria and the prescription of hydrochlorothiazide, there
were no differences between the controlled and persistent IH
groups. Indeed, the increased prescription of hydrochlorothiazide in the persistent group seemed not to influence
cytokine measurements.
In conclusion, single bone turnover cytokine measurements were not useful in differentiating persistent and controlled IH. However, we found that MCP-1 levels were
significantly higher in IH compared with healthy subjects

947

and that spot-urine MCP-1 concentrations and BMC correlated positively. Future studies are necessary to evaluate
whether this chemokine plays a role in bone remodeling in
children with IH.
Acknowledgements This study was partially supported by CNPq
(Brazilian National Research Council) and FAPEMIG (Foundation of
Research Support of Minas Gerais). Dr. A.C. Simes e Silva, Dr. E.A.
Oliveira, Dr E.M. Lima, and Dr. M.M. Teixeira had a scientific productivity grant from the CNPq. Dr. A.C. Simes e Silva and Dr. E.A.
Oliveira also received the Grant INCT-MM (FAPEMIG: CBB-APQ00075-09 / CNPq 573646/2008-2).
Conflicts of interest None.

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