The development of renal scar is multifactorial in
children with vesicou-reteral reflux (VUR) [1]. Recent studies have
implicated tissue macrophage accumulation in the development of renal
scar [2]. The secreted phosphorylated protein osteopontin (OPN) is
expressed in a variety of cells including proximal renal tubular cells,
and is likely involved in the macrophage infiltration in various models
of tubulointerstital disease [3-5].
There are a limited number of clinical studies
examining the role of OPN in renal diseases. In these studies, the
effects of T/T genotype and T allele frequency on the development of the
disease and severity of the disease have generally been studied [6-10].
The aim of this study was to investigate whether OPN gene C/T
polymorphism plays a role in the development of VUR, the degree of VUR,
the development of renal scar and the severity of renal scarring.
Methods
A total of 78 patients (53 girls) with VUR treated in
Ege University Medical School were enrolled. The mean age (SD) at
diagnosis was 4.1 (3.5) years (range 1 month to 18 years) and the mean
(SD) follow-up period was 4.1 (3.0) years. Patients with VUR secondary
to neurogenic bladder, lower urinary tract obstruction, duplicated
collecting system, ectopic kidney, and additional urinary tract
malformation were excluded. The frequency of urinary tract infections
(UTI) and the rate of scarring during the follow-up period, as well as
the family history of VUR were also recorded.
Voiding cystourethrogram was obtained as soon as the
child has completed the course of antibiotic therapy of the UTI.
Patients with VUR were divided into two groups; Group 1: low-grade
(grades I, II and III), and Group 2: high-grade (grades IV and V). DMSA
scintigraphy was performed 4-6 months after acute infection in order to
allow acute reversible lesions to resolve. Scar grading was made as type
I no more than two scarred areas, type II more than two scarred areas,
type III generalized damage, type IV kidney with little or no uptake
[13].
In order to determine the OPN gene
polymorphism heterogeneity, the control group consisted of 61 healthy
children (33 boys; mean age: 5.4 ± 3.9 yr) without a history of
urological problems or family history of VUR. All of the patients and
controls were recruited from the same racial, ethnic, and the same
geographical and environment stratification. This study was approved by
our Institutional Review Board and informed consent was obtained from
parents and control subjects.
Genomic DNA preparation and Genotyping:
Genomic DNA from patients and controls was extracted from peripheral
blood using a QIAamp Blood kit (Qiagen, Hilden, Germany). DNA
concentration was determined by the PicoGreen dsDNA quantitation kit
(Molecular Probes Inc., Eugene,OR) and diluted as 100 ng/µL. OPN
(SPP1) gene polymorphism was genotyped as described previously [10].
Amplification was carried out an a GeneAmp PCR System
9700 (PE Applied Biosystems, Foster City, CA,USA). The PCR product was
incubated for 16 hours with 0.5 U AciI enzyme (New England
Biolabs, Beverly, MA) [10]. Additionally, 20% randomly selected samples
for each of the three possible genotypes had formerly been confirmed by
sequencing and served as standards.
SPSS version 10.0 for Windows was used for
statistical analyses. Differences in categorical variables between
groups were tested with Fisher exact and chi-square tests. Differences
in continuous variables between groups were tested by the non-parametric
Mann-Whitney U test. The compatibility of all the variables to normal
distribution was assessed by one-example Kolmogorov-Smirnov test. In the
multivariate analysis, results were expressed by the odds ratio (OR) and
the 95% confidence interval; P <0.05 was considered significant.
Results
Forty seven patients showed unilateral and 31
patients showed bilateral VUR. Of these, 57 patients (Group 1) had
low-grade reflux (grade I in 4, grade II in 19, grade III in 34) and 21
patients (Group 2) had high-grade reflux (grade IV in 16, grade V in 5).
Forty patients had no renal scar and 38 showed a renal scar. Renal scar
was seen in 21 out of 57 patients (36.8%) in Group 1, whereas 17 out of
21 patients (80.9%) developed renal scar in Group 2. Renal scar type 1
was seen in 15 (19.2%), type II in 10 (12.7%), type III in 7 (9.2%) and
type IV in 6 (7.6%) patients. On comparing the OPN gene C/T
polymorphism between the patient and control groups, T allele frequency
was found to be higher in control as compared to the patient group (0.38
vs 0.28) (P>0.05). Similarly, T allele frequency was not
statistically different between patients with and without renal scar
(0.34 vs 0.23) and patients with low- and high-grade reflux (0.25
vs 0.40).
In one-way analysis, reflux grade was found to be the
only risk factor for the development of renal scar (P<0.01, OR
7.2, 95% CI 2.16 , 24.55). Other risk factors such as age, sex,
bilateral reflux, family history, frequency of UTI and T allele
frequency had no effect on renal scarring. On multivariate analysis,
reflux grade was also the only factor renal scarring (P <0.01, OR
4.83, 95% CI 2.14 - 10.91).The T allele frequency correlated
positively with scar grading (0.20 in type I, 0.25 in type II, 0.50 in
type III, 0.67 in type IV) (P <0.05). The T allele frequency was
0.22 in type I and type II (n=25) and 0.58 in type III and type
IV type IV renal scar (n=13). This difference was statistically
significant (P <0.05). T allele frequency was not statistically
different between patients with no renal scars and type I plus type II
group (0.23 vs 0.22) (P >0.05).
T allele frequency and reflux grade increased the
development of scarring 4-fold and 8.5-fold, respectively. Age, sex,
bilateral reflux, family history, and frequency of UTI were found to be
insignificant risk factors on scar severity score in VUR cases (P
> 0.05). On multivariate analysis, T allele frequency was the only
factor statistically significantly associated with the susceptibility to
scar severity (P <0.01, OR 26.4, 95% CI 1.71, 40.8), whereas
other factors were insignificant (P >0.05).
Discussion
In the present study, we investigated OPN gene
C/T polymorphism (on exon 7, position 9250) in children with VUR.
Although statistically insignificant, T allele frequency was found to be
higher in control group as compared to patient group, suggesting that T
allele frequency plays no significant role in the development of VUR.
Similarly, no statistical difference was observed between low- and
high-grade VUR cases, indicating that the presence of TT genotype or the
high T allele frequency does not play a role in the degree of reflux. To
our knowledge, this is the first study focusing on the T allele
frequency and VUR in children from same ethnicity.
Although the T allele frequency tended to be
higher in patients with renal scar, this did not reach statistical
significance. Additionally, univariate and multivariate analyses also
showed that the T allele frequency had no effect on the development of
renal scars. Increased T allele frequency was progressively associated
with the increased scar grading. Besides this, the T allele frequency
increased the risk of the development of renal scar 4-fold and
26.4-fold, respectively.
The mechanism by which the molecular change induces
renal fibrosis is not clearly known [12]. While the present study, does
not prove that OPN gene polymorphism is the sole factor associated
either with development of scars or their severity, further studies are
necessary.
Contributes: All authors contributed to study
design, conduct of study and writing the manuscript.
Funding: None; Competing interests: None
stated.
References
What This Study Adds?
• Increased osteopontin gene T allele
frequency might correlate with the increased severity of renal
scars.
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