1Medizinische Klinik IV, Universitätsklinikum Hamburg Eppendorf, 2Abteilung für Nephrologie und Klinische Immunologie, Universitätsklinikum Aachen and 3Institut für Pathologie, Universitätsklinikum Hamburg Eppendorf, Germany
Correspondence and offprint requests to: Oliver M. Steinmetz, MD, Zentrum für Innere Medizin, Medizinische Klinik IV, University of Hamburg, Martinistraße 52, D-20246 Hamburg, Germany. Email: o.steinmetz{at}uke.uni-hamburg.de
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Abstract |
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Methods. Blood samples from 207 patients with biopsy proven IgA nephropathy and 140 ethnically, age and sex-matched healthy controls were collected and genomic DNA was extracted. MCP-1 2518 genotype was assessed by PCR, followed by restriction fragment length polymorphism analysis. Genotype distribution between the two groups was compared by 2 test. Cumulative renal survival was assessed by KaplanMeier plot and log-rank analysis.
Results. 111 (53.6%) patients had the MCP-1 2518 wild-type A/A, 83 (40.1%) were heterozygous for the G allele and 13 (6.3%) patients showed homozygosity. The allelic distribution was not significantly different in the control group of 140 healthy blood donors (P = 0.71). Renal survival analysis of patients did not reveal statistically significant differences in cumulative survival (P = 0.32), median survival time and 5 year survival rate between the wild-type group and carriers of the G allele. Furthermore, the number of infiltrating CD68-positive monocytes/macrophages into the kidneys of patients with IgA nephropathy was not statistically different between the groups.
Conclusion. Our data indicate that no association exists between the 2518 A/G polymorphism and susceptibility to IgA nephropathy or its clinical course.
Keywords: CCL2; chemokine; IgA nephropathy; inflammation; MCP-1
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Introduction |
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Interestingly, a correlation between the presence of mononuclear cells (monocytes and lymphocytes) in the renal tissue and disease progression of IgA nephropathy was recently described [46]. In general, the directional recruitment of leucocytes is regulated by chemokines and their counteracting chemokine receptors [7]. In human and experimental models of glomerulonephritis it was shown that monocyte chemoattractant protein-1 (MCP-1), a ligand of the C-C chemokine receptor 2 (CCR2) plays a pivotal role in the attraction of monocytes into the renal tissue [811]. Therefore, genetic polymorphisms in the regulatory region of the MCP-1 gene that exert an impact on its expression could play a role in the susceptibility and progression of IgA nephropathy.
Rovin et al. [12] described a biallelic A/G polymorphism at position 2518 of the MCP-1 gene. The polymorphism proved functionally important, as peripheral blood mononuclear cells of individuals with G/G and A/G genotype produced significantly more MCP-1 after stimulation with IL-1ß than those with Caucasian wild-type A/A [12,13]. An association of the presence of G at position 2518 with the presence of cutaneous vasculitis could be shown in patients with systemic lupus erythematodes [14] as well as an association with development of coronary artery aneurysms after acute Kawasaki disease [13]. Furthermore, the G/G genotype was identified as a genetic risk factor for severe coronary artery disease [15] and a correlation between the incidence and severity of asthma and the G allele at position 2518 has been shown [16]. These findings suggest an important role for MCP-1 and the A/G polymorphism in its regulatory region in inflammatory processes.
The aim of our study was to evaluate whether the A/G polymorphism at position 2518 in the regulatory region of the MCP-1 gene is associated with the incidence and progression of IgA nephropathy in Caucasian patients.
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Materials and methods |
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Approval for the study was given by the local ethics committee on February 1, 2000 and has been conducted according to the Declaration of Helsinki principles.
MCP-1 promoter genotyping
Genomic DNA was isolated from peripheral blood leucocytes using a commercial kit (QIAamp DNA Blood Mini Kit). The identification of the polymorphism was carried out using PCR, followed by a restriction fragment length polymorphism (RFLP) assay, using a PvuII site, which is introduced by the presence of the G nucleotide. The regulatory region of the MCP-1 gene (1817 to 2746) was amplified by PCR, resulting in a 930 bp fragment. Primers used were 5'-CCGAGATGTTCCCAGCACAG-3' (forward) and 5'-CTGCTTTGCTTGTGCCTCTT-3' (reverse). Five microlitres of genomic DNA (50 ng) were added to 20 µl of amplification buffer containing 12.7 µl of H2O, 2.5 µl of 10x PCR buffer, 1.5 µl of MgCl2 (50 mM), 1 µl of dNTPs (10 mM each), 0.3 µl of Taq DNA polymerase (5 U/µl), 1.5 µl of forward and 1.5 µl of reverse primer (10 pmol/µl each). PCR was run for 40 cycles using the following temperature profile: denaturation at 94°C for 60 s, annealing at 55°C for 60 s, extension at 72°C for 1 min 30 s, followed by a single final extension step at 72°C for 10 min. Eight microlitres of the PCR products were digested with 10 U of PvuII in 10x buffer and H2O up to a final volume of 20 µl at 37°C for 2 h 30 min.
The resulting products were separated by gel-electrophoresis in 1.5% agarose gels, containing ethidium bromide in a final concentration of 0.5 µg/ml. Samples showing only a 930 bp band were assigned as A/A, samples showing two bands of 708 and 222 bp were considered G/G and samples showing three bands at 930, 708 and 222 bp were typed A/G [12]. Of 42 randomly selected samples from the IgA collective (20%), 21 were amplified and digested twice and 21 were analysed by automatic DNA sequencing after PCR amplification as a control for possible mismatches introduced by the Taq polymerase. All 42 samples were in agreement (data not shown).
Statistical methods and design
Allelic and genotypic frequencies were obtained by direct counting. Statistical comparison of genotypic frequencies between patients with IgA nephropathy and healthy controls was carried out by 2 test. For survival studies, due to the small number of individuals with the G/G genotype, these patients were included in the same group as patients with the A/G genotype and compared with the group with the Caucasian wild-type genotype A/A. Differences of nominal variables (proteinuria, hypertension and sex) between the two groups were compared by
2 test. Differences of continuous variables (creatinine, age and time of follow-up) were calculated by MannWhitney U-test. The study was designed to have a power of 80% to detect a difference in hazard ratio between the two groups of 2.5 or more with a two-sided significance level of P = 0.05 referring to the log-rank test. Cumulative renal survival was analysed by KaplanMeier plot and log-rank test. Analysis of covariables was carried out according to the Cox proportional hazard model. The number of infiltrating CD68-positive cells into renal biopsies of patients with IgA nephropathy and A/A, A/G or G/G genotype was compared by KruskalWallis test. SPSS 10.1.3 was used for statistical analyses.
Immunohistochemical staining of CD68 in biopsies from patients with IgA nephropathy
Paraffin tissue sections (34 µm thick) were protease treated for antigen retrieval and subsequently incubated with anti-CD68 (KP1 Zymed, 1:2) for 30 min. Sections were then incubated with rabbit anti-mouse-IgG (Z 259 DAKO, 1:135) for 15 min, followed by application of APAAP complex (Progen, 1:300) for 15 min. The last two steps were carried out twice, followed by staining with neufuchsin for 30 min.
The infiltration of CD68-positive cells into the tubulointerstitium of kidneys from patients with IgA nephropathy was quantified by counting all positive cells in 30 high-power fields (x1000) for each biopsy section. The number of CD68-positive cells in glomeruli was quantified by counting all positive infiltrating cells in all glomerular cross sections of each biopsy section and subsequent calculation of the mean value per glomerular cross section.
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Results |
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Genotyping of MCP-1 promoter polymorphism
The MCP-1 2518 genotype was identified by PCR, followed by RFLP analysis (Figure 1). The genotypic frequency of the 207 IgA nephropathy patients was as follows: 111 (53.6%) patients had the A/A genotype, which can be regarded as wild-type for a Caucasian population; 83 (40.1%) patients had the A/G genotype; 13 (6.3%) patients had the G/G genotype; the frequency of the G allele was 26.3% (109/414). The distribution in the group of healthy controls was not statistically different: 81 (57.9%) patients showed the A/A, 50 (35.7%) patients the A/G and nine (6.4%) patients the G/G genotype (2 test: P = 0.71). The frequency of the G allele was 24.29% (68/280). Thus, the distribution of the genotypes in the two collectives was according to the HardyWeinberg equilibrium and no association of the A/G polymorphism and susceptibility to IgA nephropathy was detectable.
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CD68 immunohistochemistry
To analyse whether the MCP-1 2518 genotype affects the recruitment of monocytes/macrophages into the renal tissue, immunohistochemical examination of renal biopsies from 23 patients with IgA nephropathy was performed. The results revealed no significant differences in interstitial and glomerular infiltration of CD68-positive monocytes/macrophages between the three groups [cells/glomerular cross section: A/A (n = 9) 9.2±5.1; A/G (n = 9) 10.7±4.9; G/G (n = 5) 8.4±3.7; P = 0.71; cells/30 high power fields: A/A 380±127; A/G 447±184; G/G 403±104; P = 0.71] (Figures 3 and 4). Patients in the three groups did not differ significantly in clinical basis data (age at biopsy, creatinine at biopsy, proteinuria, hypertension and gender) (analysis by KruskalWallis resp. 2 test, data not shown).
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Discussion |
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The present study is, to the best of our knowledge, the first to investigate a potential influence of the MCP-1 A/G promoter polymorphism at position 2518 on the incidence and clinical course of IgA nephropathy. The presence of G at position 2518 has previously been shown to be associated with a higher production of MCP-1 by peripheral blood mononuclear cells after stimulation with IL-1ß [12,13]. The G allele was found in 22% of African Americans, 29% of Caucasians and 47% of Mexicans and Asians, suggesting ethnic variation. Knowing that the progression of IgA nephropathy correlates with the presence of mononuclear cells in the kidney [46], an enhanced production of the chemokine MCP-1, which is known to be a major chemoattractant for monocytes and T lymphocytes [811,18], might therefore exert an effect on the clinical course. Mice lacking MCP-1 presented with a strongly impaired recruitment of monocytes and an ameliorated disease severity in experimental models of glomerulonephritis [19,20], stressing the unique importance of MCP-1, despite the existence of other chemokines that bind to CCR2 and/or attract monocytes in vitro [18]. Furthermore, other studies have reported a significant impact of this polymorphism on other inflammatory processes. An association of the presence of G at position 2518 with cutaneous vasculitis among patients with systemic lupus erythematodes [14], severity of asthma in children [16] and coronary artery aneurysm after acute Kawasaki disease [13] has been shown.
We studied the MCP-1 2518 A/G polymorphism in 207 patients with biopsy proven primary IgA nephritis and a group of 140 matched controls. The distribution of genotypic frequencies was neither statistically different between these two groups nor between three subgroups of patients with a slower progression rate of IgA nephropathy and the total collective of patients with IgA nephropathy. These findings strongly argue against a role of the MCP-1 2518 A/G polymorphism for susceptibility to IgA nephropathy and against an influence on the progression rate of this disease.
In addition, immunohistochemical staining of biopsies for CD68 from the different genotypes showed no significant differences in monocyte/macrophage infiltration into glomeruli and tubulointerstitium. The results provide evidence against a major pathophysiological role of the MCP-1 2518 A/G polymorphism in renal monocyte/macrophage recruitment.
Due to collection in dialysis centres, our study group contains a high number of patients who reached ESRD. In this collective, no association between the renal survival and the MCP-1 2518 genotype could be detected.
Certain aspects, however, remain unclear and require further studies. The group of patients homozygous for the G allele included in this study was too small for separate statistical analysis and was therefore included in the group of patients with the A/G genotype. This approach leaves unanswered whether a possible effect of homozygosity on renal survival exists. This question could be addressed by investigation of a population with IgA nephropathy from an ethnic group with a high incidence of the G allele at position 2518, e.g. Asians. Several previous studies, however, have revealed a very similar 10 year renal survival of adult patients with IgA nephropathy in many populations with a low frequency of the G allele, e.g. Germany and on the other hand Japan, where the G allele and thus, homozygotes, are much more frequent [3].
As it has previously been shown that urinary MCP-1 levels correlate with disease severity of IgA nephropathy [21], the effect of the 2518 promotor polymorphism on intrarenal expression of MCP-1 would be another interesting subject to study.
In summary, our data suggest that the MCP-1 2518 A/G gene polymorphism is not associated with the incidence and progression of IgA nephropathy in a Caucasian population.
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Acknowledgments |
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Conflict of interest statement. None declared.
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Notes |
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References |
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