CCR2 Polymorphism in Chronic Renal Failure Patients Requiring Long-Term Hemodialysis

Article (PDF Available)inInternal Medicine 50(21):2457-61 · January 2011with57 Reads
DOI: 10.2169/internalmedicine.50.5119 · Source: PubMed
Abstract
A number of chemokines and chemokine receptors are produced by intrinsic renal cells as well as by infiltrating cells during renal inflammation. The CCR2 chemokine receptor mediates leukocyte chemoattraction in the initiation and amplification phase of renal inflammation. The polymorphism, CCR2-V64I, changes valine 64 of CCR2 to isoleucine. We aimed to determine the frequency of CCR2-V64I polymorphism in patients with chronic renal failure requiring long-term hemodialysis. The PCR-based restriction fragment length polymorphism (PCR-RFLP) technique was used to assess the gene frequencies of CCR2-641 in CRF patients (n=210) and healthy controls (n=139) in the current study. The frequencies of the CCR2 genotype were 0.68 for V/V, 0.28 for V/I, and 0.4 for I/I in the CRF patients and 0.81 for V/V, 018 for V/I and 0.1 for I/I in healthy controls. The distribution of the CCR2-V64I mutant genotype was significantly different between subjects with CRF and healthy control subjects (X2=7.197 and p=0.027). We found that the CCR2-V64I polymorphism was significantly high in CRF patients. In addition to the contribution to disease pathogenesis, it was recently found that chemokines have therapeutic importance in chronic renal failure. The frequency of CCR2-V64I and other chemokine and chemokine receptor polymorphisms in renal pathologies must be further investigated in larger study populations and in different renal diseases.
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ORIGINAL ARTICLE
CCR2 Polymorphism in Chronic Renal Failure Patients
Requiring Long-Term Hemodialysis
Ilhan Sezgin
1
, Binnur Koksal
1
, Gokhan Bagci
1
,
Hande Kucuk Kurtulgan
1
and Ozturk Ozdemir
2
Abstract
Objective A number of chemokines and chemokine receptors are produced by intrinsic renal cells as well
as by infiltrating cells during renal inflammation. The CCR2 chemokine receptor mediates leukocyte
chemoattraction in the initiation and amplification phase of renal inflammation. The polymorphism, CCR2-
V64I, changes valine 64 of CCR2 to isoleucine. We aimed to determine the frequency of CCR2-V64I poly-
morphism in patients with chronic renal failure requiring long-term hemodialysis.
Methods and Patients The PCR-based restriction fragment length polymorphism (PCR-RFLP) technique
was used to assess the gene frequencies of CCR2-641 in CRF patients (n=210) and healthy controls (n=139)
in the current study.
Results The frequencies of the CCR2 genotype were 0.68 for V/V, 0.28 for V/I, and 0.4 for I/I in the CRF
patients and 0.81 for V/V, 018 for V/I and 0.1 for I/I in healthy controls. The distribution of the CCR2-V64I
mutant genotype was significantly different between subjects with CRF and healthy control subjects (X2=
7.197 and p=0.027).
Conclusion We found that the CCR2-V64I polymorphism was significantly high in CRF patients. In addi-
tion to the contribution to disease pathogenesis, it was recently found that chemokines have therapeutic im-
portance in chronic renal failure. The frequency of CCR2-V64I and other chemokine and chemokine receptor
polymorphisms in renal pathologies must be further investigated in larger study populations and in different
renal diseases.
Key words: CCR2 gene, chronic renal failure, chemokines
(Intern Med 50: 2457-2461, 2011)
(DOI: 10.2169/internalmedicine.50.5119)
Introduction
Chemokines are proinflammatory cytokines that function
in leukocyte chemoattraction (1). Chemokines can be di-
vided into four families based on their amino acid sequence
in relation to their cysteine moieties/differences in structure:
CC, CXC, CX
3
C and XC (2). CCR2 is a cognate receptor of
MCP1, a member of the CC family of chemokines (also
termed monocyte chemoactive and activating factor, or
CCL2), which is mainly expressed on monocytes (3).
CCR2 protein has 374 amino acids and the polymor-
phism, CCR2-V64I (also named CCR2G190A) is a transi-
tion mutation that changes valine 64 of CCR2 to isoleucine.
Studies indicate that CCR2 V64I mutation does not affect
the CCR2 expression level (4). The amino-terminal domain
of CCR2 is necessary for binding of MCP1. CCR2 muta-
tions have been associated with insulin-dependent diabetes
mellitus (5), a reduced risk for severe coronary artery dis-
ease (6), and a delay in the progression to AIDS in human
immunodeficiency virus (HIV)-infected individuals (7).
Locally secreted chemokines mediate leukocyte recruit-
ment during the initiation and amplification phase of renal
inflammation. In the initiation phase, renal cells release
chemokines and induce leukocyte infiltration to the place of
injury. In the amplification phase the infiltrating leukocytes
Department of Medical Genetics, Faculty of Medicine, Cumhuriyet University, Turkey and
Department of Medical Genetics Faculty of Medi-
cine, Canakkale Onsekiz Mart University, Turkey
Received for publication January 7, 2011; Accepted for publication July 8, 2011
Correspondence to Dr. Ilhan Sezgin, isezgin@tnn.net
Intern Med 50: 2457-2461, 2011 DOI: 10.2169/internalmedicine.50.5119
2458
contribute to renal damage by releasing inflammatory and
profibrotic factors (8). Termination of the chemokine signal
is critical for the resolution of the inflammatory process (9).
In the case of chronic disease process, even if the initial in-
jury to the kidney subsides, chemokine-mediated leukocyte
recruitment can be maintained or exacerbated by other
mechanisms such as infection, activation of rennin-
angiotensin system, hypoxia, and proteinuria (10).
Chemokines also have been implicated in acute cardiac
and renal allograft rejection. It was hypothesized that for
acute allograft rejection monocytes and T-effector cells are
directed into the transplant and produce a characteristic tu-
bular or vascular infiltrate. The complex process of ex-
travasation and influx of leukocyte subsets into the site of
tissue injury appears to be mediated by specifically expres-
sion of the CC-chemokine MCP-1 together with the corre-
sponding chemokine receptor CCR2 can be detected in
mononuclear cells infiltrating the kidney graft (11, 12). The
association of human chemokine receptor genetic variants,
CCR5-Delta32, CCR5-59029-A/G, CCR2-V64I, CX3CR1-
V249I, and CX3CR1-T280M, with outcome of renal trans-
plant recipients was examined by Abdi et al and significant
reductions were found in the risk of acute renal transplant
rejection in recipients who possessed the CCR2-64I al-
lele (13). Kang et al found a significant increase for the risk
of late acute rejection in recipients who were homozygous
for the MCP-1-2518G polymorphism and no difference in
the incidence of rejection among recipients stratified by the
CCR2-V64I genotype (14).
We aimed to determine the frequency and possible role of
inherited CCR2 gene mutation in patients with chronic renal
failure.
Materials and Methods
Study population
Two hundred and ten Turkish patients (average age of
57.4±14.3) who suffered from chronic renal impairment and
required hemodialysis for approximately 5.0±4.4 years in
the four different dialysis centres from Sivas city were en-
rolled in the current study. Mean hemodialysis duration was
5.4±4.9 hours per week. One hundred thirty-nine age-
matched healthy individuals with no history of Type II dia-
betes, hypertension, renal, cardiac, or family history of renal
disease were selected as healthy controls.
CCR2 V64I genotyping
Peripheral blood samples obtained from both the subjects
in the CRF and control groups were collected in tubes con-
taining 1 mL of EDTA and stored at -20. Total genomic
DNA was obtained from a 100 μL peripheral blood sample
with an Invitek kit extraction technique (Invisorb spin blood;
Invitek, Berlin, Germany). Genotype was determined by po-
lymerase chain reaction/restriction fragment length polymor-
phism (PCR-RFLP) analysis.
The PCR mixture in a 25-μL final volume consisted of
12.5 μL PCR master mix (Fermentas, St. Leon-Rot, Ger-
many), 9.5 μL ddH2O, 1 μL of each primer, and 1 μL
DNA. The G to A mutation at position 190 of CCR2 gene
was determined by PCR-RFLP. The following primers were
used for amplification: forward 5´-CAT TGC AAT CCC
AAA GAC CCA CTC-3´ and reverse 5´-TTG GTT TTG
TGG GCA ACA TGA TGG-3´. Initial denaturation at 94
for 5 minutes was followed by 33 cycles of denaturation at
94 for 30 seconds, annealing at 56 for 30 seconds and
extension at 72 for 30 seconds. Final extension step was
at 72 for 5 minutes.
PCR product (5 μl) was digested for 2 hours at 65 with
2.5 U of BsaBI restriction endonuclease (Fermentas). Diges-
tion products were analyzed by electrophoresis on 3% aga-
rose gel in TBE buffer and visualized using ethidium bro-
mide staining. Samples with a single 173 bp band were
identified as having GG genotype, samples with two bands,
149 bp and 24 bp as AA genotype and those with three
bands, 173 bp, 149 bp and 24 bp as GA heterozygotes.
Statistical analysis
Statistical analysis was performed using SPSS software
package, version 15.0. Differences in the distribution of
chemokine polymorphisms between cases and controls were
tested using chi square test. Results were considered signifi-
cant when the p value was less than 0.05.
Results
Hypertension, Type II Diabetes, atherosclerosis and hae-
matological diseases were found in 52.38%, 27.62%,
11.42% and 2.9% of the patients respectively. 11.2% of pa-
tients have parental consanguinity and 2.5% of patients have
FMF history in their families. There was no renal disease in
82.6% of patients before chronic renal failure (Table 1).
For the entire group of patients with CRF, the frequencies
of the CCR2 V64I polymorphisms as GG, GA and AA
genotypes were 68.1% (n=143), 28% (n=58), and 4.3%(n=9)
respectively. Distribution of CCR2 V64I polymorphisms
(GG, GA, AA) in healthy controls were 80.6% (n=112),
18% (n=25), and 1.4% (n=2), respectively (Table 2). The
distribution of the CCR2-V64I mutant genotype was signifi-
cantly different between subjects with CRF and healthy con-
trol subjects (X2= X2=7.197 and p=0.027). Distribution of
CCR2 genotypes in three different disease sub-groups of
CRF patients were also evaluated and no significant associa-
tion was found in these diseases and CCR2 genotypes (Ta-
ble 3).
Discussion
Clarifying the chemokine network that functions in leuko-
cyte recruitment may represent a promising therapeutic op-
tion for progressive renal disorders (8). The importance of
chemokine and chemokine receptors in renal diseases was
Intern Med 50: 2457-2461, 2011 DOI: 10.2169/internalmedicine.50.5119
2459
Table
1.
Demographic Findings of
Study and Control Group
HT: Hypertension, DM: Diabetes Mellitus,
FMF: Familial Mediterranean Fever
Patients Controls
Demographic
findings
% (n) %(n)
male 51.90 (109) 50
female 48.10 (101) 95
Average age 57.4±14.3 57.24±9.71
Parental
consanguinity
9.52 (20) unknown
HT 52.38 (110) 0
DM 27.62 (58) 0
Atherosclerosis 11.42 (24) 0
Table
2.
Comparison of CCR2 G190A Genotype and Allele Frequency
between Study Group and Healthy Controls
Genotype Case (n=210) % Control (n=139)% p
GG (143) 68.1 (112) 80.6
GA (58) 27.6 (25) 18
X2=7.107
AA (9) 4.3 (2) 1.4
p=0.027
lellA
G (344) 81.90 (249) 89.57
A (76) 18.10 (29) 10.43
investigated by blocking these ligands and receptors with
neutralizing antibodies and chemokine receptor antagonists
and targeted disruption of genes encoding chemokine recep-
tor genes in animal models (8). The strategy of blocking
MCP-1/CCR2 interaction found to be effective in preventing
macrophage-induced tissue damage. Neutralizing antibodies
against CCL2/MCP-1 blocked glomerular infiltration of
macrophages in the rat anti-thymocyte antibody-induced
glomerulonephritis model (15). Treatment anti-CCL2/MCP-1
antibodies reduced proteinuria and glomerular macrophage
influx in rat nephrotoxic serum nephritis (16). Consistently,
a preliminary report demonstrated that blockade of CCR2
with a series of antagonists ameliorated disease, including
glomerular injury, in the rat model (17). Kitagawa et al
found that the therapeutic strategy of blocking CCR2 may
prove beneficial for progressive fibrosis via the decrease in
infiltration and activation of macrophages in the diseased
kidneys (3). Chemokines and their receptors have many
other functions in addition to leukocyte migration to sites of
tissue injury. These includes homeostatic functions in leuko-
cyte development, cell trafficking during immune surveil-
lance, hematopoiesis and, angiogenesis (10). In a knockout
mice study, mice lacking CCR2 for CCL2/MCP-1, the pro-
teinuria and glomerular pathology of nephrotic serum ne-
phritis, were worse despite reduced glomerular macrophage
infiltration. This study indicates that the absence of CCR2
may influence other immune mechanisms in addition to lo-
cal cell infiltration (18). In a preliminary study Bıyıklı et al
(2006), found no significant difference in CCR2 and CCR5
polymorphisms between 25 children with biopsy proven fo-
cal segmental glomerulosclerosis (FSGS) and 40 healthy
age-matched controls (19). In the current study we find that
the frequency of CCR2-V64I mutant genotype was signifi-
cantly higher than in healthy controls. This higher mutation
frequency may be related to the heaviness of the CRF and
especially in the CRF with chronic disease. CCR2 is only
one of the chemokine receptors that is expressed by inflam-
matory cells after renal injury. Chararacteristic chemokines
and chemokine receptors participate in particular pathologic
changes at specific time points. Chemokine cascades in
ischemia-reperfusion injury pathologic change of tubular ne-
crosis after ischemic acute kidney injury are mainly medi-
ated by the action of CCR2 on macrophages. Regeneration
of tubular epithelial cells in the late phase of the injury was
mediated by IP-10, a CXC class chemokine, producing
macrophages. Chronic interstitial fibrosis was mediated by
the action of CX3CR1 on macrophages and platelets (20).
Monocyte/macrophage infiltration has been detected in the
glomeruli of rats with streptozotocin-induced diabetes and in
renal biopsy specimens from patients with diabetic nephro-
pathy, suggesting that the secretion of chemokines is en-
hanced in diabetes and that monocyte recruitment to renal
tissues and differentiation to macrophages may be associated
with the development or progression of diabetic nephropa-
thy (21-23). Although Prasad et al and Nakajima et al found
an association between CCR5 polymorphisms and diabetic
Intern Med 50: 2457-2461, 2011 DOI: 10.2169/internalmedicine.50.5119
2460
Table
3.
Genotypic Distribution of Etiologic Reasons Causing Renal Damage
Patients Patients Patients
Genotypes
With
HT (%)
Without
HT(%)
p
With
DM(%)
Without
DM(%)
p
With
Atherosclerosis
Without
Atherosclerosis
p
GG
75(68.18) 68(68) 41(70.69) 102(67.1) 14(58.33) 129(69.35)
GA
32(29.09) 26(26) X
2
=1.491 14(24.14) 44(28.95) X
2
=0.578 10(41.67) 48( 25.81) X2=3.347
AA
3(2.72) 6(6)
p=0.4
3(5.17) 6(3.95)
p=0.749
0(0) 9(4.84)
p=0.176
TOTAL
110(100) 100(100) 58(100) 152(100) 24(100) 186(100)
Allel
A
182 (%82.73) 162 (%81) 96(%82.76) 248(81.58) 38 (79.17) 306 (82.26)
G
38 (%17.27) 38 (%19) 20(%17.24) 56(18.42) 10(20.83) 66 (17.24)
nephropaty they did not find any association between CCR2
genotypes and diabetic nephropathy (23, 24). Muntinghe et
al have investigated the interaction between CCR5 genotype
and levels of high-sensitivity C-reactive protein (hsCRP) and
found that the CCR5Δ32 polymorphism attenuates the ad-
verse effects of inflammation on overall and cardiovascular
mortality in ESRD (25). Joo et al have genotyped single nu-
cleotide polymorphism (SNPs) in the MCP-1G-2518A,
CCR2G46295A, RANTES C-28G and G-403A in 177 dia-
betic end-stage renal disease (ESRD) patients and 184 pa-
tients without renal involvement (controls) in order to inves-
tigate the effects of these SNPs on DN in Korean patients
with type 2 DM. They found no associations of MCP-1,
CCR2 and RANTES promoter SNPs with diabetic ESRD in
the Korean population (26). We have evaluated the relation-
ships between CCR2 genotypes and disease sub-groups
which cause CRF in patients. No significant associations
were found between CCR2 genotypes and DM, HT and
atherosclerosis (Table 3). These results have shown that in
the current study group the presence of DM, HT and athero-
sclerosis did not affect the results. In order to reveal the role
of chemokines in renal pathogenesis further investigations
should be carried out using other chemokines and
chemokine receptors in different renal diseases.
The authors state that they have no Conflict of Interest (COI).
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2011 The Japanese Society of Internal Medicine
http://www.naika.or.jp/imindex.html
    • "The studies investigating the role of polymorphisms and mutations in genes encoding chemokines and chemokine receptors in renal diseases have increased steadily. In our previous studies, we demonstrated that the polymorphisms in MCP-1 and CCR2 genes have increased the risk of CRF development [21, 22]. CX3CR1 is present on both interstitial and glomerular infiltrating leukocytes [10]. "
    [Show abstract] [Hide abstract] ABSTRACT: Purpose We aimed to investigate the associations of fractalkine receptor (CX3CR1) V249I, T280M and CCR5- 59029 A/G gene polymorphisms in chronic renal failure (CRF) subjects undergoing hemodialysis and to evaluate possible associations of these polymorphisms with hypertension (HT), diabetes mellitus (DM) and atherosclerosis (AS). Methods A total of 225 CRF subjects undergoing hemodialysis and 201 healthy controls were enrolled in the study. CRF subjects were divided into three major subgroups according to comorbidities including HT (n = 127), DM (n = 65) and AS (n = 33). Genotyping was done using polymerase chain reaction–restriction fragment length polymorphism method. Results The II genotype and I allele frequencies of CX3CR1 V249I polymorphism were found significantly more frequent in CRF subjects, CRF subjects with DM and CRF subjects with AS compared with controls (p < 0.05 for all comparisons). G allele frequency of CCR5 polymorphism was found significantly more prevalent in CRF subjects with DM than that of controls. Further, GG genotype and G allele frequencies of CCR5 polymorphism were significantly more prevalent in CRF subjects with AS compared with controls (p < 0.05). We also explored these polymorphisms among CRF subjects with and without following comorbidities: HT, DM, AS. We found significant association between CRF subjects with HT and without HT in terms of genotype and allele frequencies of V249I polymorphism (p < 0.05). CX3CR1 T280M polymorphism was not found significantly different in none of the comparisons. Conclusion These data demonstrate possible associations between CX3CR1 V249I and CCR5-59029 A/G polymorphisms and/or HT, DM and AS in CRF subjects.
    Full-text · Article · Jul 2016
    • "The following primers were used for CCR2 V64I polymorphism: forward 5'-CAT TGC AAT CCC AAA GAC CCA CTC-3' and reverse 5'-TTG GTT TTG TGG GCA ACA TGA TGG-3'. PCR was performed with an initial denaturation step at 94°C for 5 minutes followed by 33 cycles of denaturation step at 94°C for 30 seconds, annealing step at 56°C for 30 seconds, extension step at 72°C for 30 seconds, and final extension step at 72°C for 5 minutes (Sezgin et al., 2011). PCR product (5µl) was digested overnight at 37°C using 2.5U of PvuII restriction endonuclease (Fermentas, Germany) for MCP-1 -2518 A>G polymorphism, and 2 hour sat 65 °C using 2.5U of BsaBI restriction endonuclease (Fermentas, Germany) for CCR2 V64I polymorphism. "
    [Show abstract] [Hide abstract] ABSTRACT: Article History In the current study, we aimed to investigate the possible role of MCP-1-2518 A>G and CC chemokine receptor 2 (CCR2) V64I polymorphisms in patients with lung cancer. Sixty-five patients with lung cancer (57 with NSCLC and 8 with SCLC) and 57 healthy controls were enrolled. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used. Genotype distribution of monocyte chemoattractant protein 1 (MCP-1)-2518 A>G polymorphism in lung cancer patients was as follows: 30 for AA, 30 for AG and 5 for GG genotype. Frequency of minor G allele in patients and controls were found as 30.8% and 23.7%, respectively. In patients, genotype distribution of CCR2 V64I polymorphism was as follows: 47 for GG, 16 for GA, and 2 for AA. Frequency of minor A allele was found in patients and controls as 15.4% and 19.2%, respectively. Genotype distribution and allele frequencies of MCP-1 and CCR2 polymorphism were not statistically different between patients and controls (p values >0.05 for both polymorphisms). In lung cancer patients, there was no significant association between smoking status and MCP-1 and CCR2 polymorphisms. Similarly, no significant association was found between distant organ metastasis and both gene polymorphisms. Our findings suggest that MCP-1-2518 A>G and CCR2 V64I polymorphisms are not associated with genetic susceptibility to lung cancer and lung cancer metastasis.
    Full-text · Article · Jun 2016
    • "Genotyping of CCR5-D32 (rs333) was determined by conventional PCR, using specific primers according to Aoki et al. [22], and CCR2-V64I (rs1799864) was assessed by PCR followed by restriction fragment length polymorphism (RFLP-PCR) using specific primers according to Sezgin et al. [23]. Samples were amplified using genomic DNA (100 ng) and the buffer kit plus 1.25 units of Taq polymerase (Invitrogen TM , Carlsbad, CA, USA) at 25 lL of final volume, in Mastercycler Gradient thermal cycler (Eppendorf, Hamburg, Germany). "
    [Show abstract] [Hide abstract] ABSTRACT: Many tumor cells express chemokines and chemokine receptors, and these molecules can affect both tumor progression and anti-tumor immune response. Genetic polymorphisms of some chemokine receptors were found to be closely related to malignant tumors, especially in metastasis process, including breast cancer (BC). Considering this, it was investigated a possible role for CCR2-V64I (C-C chemokine receptor 2) and CCR5-Δ32 (C-C chemokine receptor 5) genetic variants in BC context. Patients were divided into subgroups according to immunohistochemical profile of estrogen (ER) and progesterone (PR) receptors and the human epidermal growth factor receptor 2 (HER2) overexpression. No significant associations were found in relation to susceptibility (CCR2-V64I: OR 1.32; 95 % CI 0.57-3.06; CCR5-∆32: OR 1.04; 95 % CI 0.60-1.81), clinical outcome (tumor size, lymph nodes commitment and/or distant metastasis, TNM staging and nuclear grade) or therapeutic response (recurrence and survival). However, it was found a significant correlation between CCR2-V64I allelic variant and HER2 immunohistochemical positive samples (p = 0.026). All in all, we demonstrate, for the first time, a positive correlation between CCR2 receptor gene polymorphism and a subgroup of BC related to poor prognosis, which deserves further investigation in larger samples for validation.
    Full-text · Article · Feb 2015
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