Abstract
Monocyte chemoattractant protein-1 (MCP-1) is a highly specific chemokine for monocytes and plays roles in pathogenesis of various renal diseases. The aim of this study is to investigate the effect of MCP1 2518 A/G polymorphism on the incidence and clinical course of focal segmental glomerulosclerosis (FSGS) in children. MCP1 2518 A/G genotype was identified by PCR-RFLP in 60 biopsy-proven FSGS patients, 76 steroid sensitive nephrotic syndrome (SSNS) patients, and 96 healthy children. MCP-1 levels in urine and serum were measured by ELISA in all patients and the correlations of genotype with MCP-1 levels and clinical outcome were evaluated. The genotype frequencies for MCP1 were similar in all groups. The percentage of patients who develop chronic renal failure was higher in patients with AA allele compared to GA or GG alleles (46% vs. 35% respectively, p < 0.01, Odds ratio: 1.59). Serum MCP-1 levels were similar in all groups, whereas urinary MCP-1 levels of the patients with FSGS (1680 pg/mg creatinine) were significantly higher than that of patients with SSNS (365 pg/mg creatinine, p < 0.05) and healthy controls (348 pg/mg creatinine; p < 0.05). Urinary MCP-1 levels were correlated with the degree of proteinuria in FSGS group (r = 0.529, p = 0.016). Our results suggest that the AA genotype might be a risk factor for the progression of renal disease in FSGS and MCP1 genotyping may help the physicians to predict prognosis in these patients.
Introduction
Focal segmental glomerulosclerosis (FSGS) is an important cause of end-stage renal disease (ESRD) in children and characterized with poor response to corticosteroids.Citation1 Over the past several years, the incidence of FSGS in adults and children appears to be increasing.Citation2 There is increasing evidence that the genetic background of the adults and children with FSGS affects the progression of the disease and the response to therapy.Citation2,Citation3
Although patients with FSGS may present with any degree of proteinuria, clinical concern is greatest for those who have nephrotic-range proteinuria because without treatment, they have an extremely poor prognosis and mostly progress to ESRD in 3–6 years.Citation4 It is widely recognized that the prognosis in nephrotic patients with primary FSGS is significantly improved if partial or complete remission of proteinuria is achieved.Citation5,Citation6
Monocyte chemoattractant protein-1 (CCL2/MCP-1) is a chemokine that mediates renal interstitial inflammation, tubular atrophy, and interstitial fibrosis by recruiting monocytes–macrophages into renal interstitium.Citation7 In a previous study, it has been shown that the degree of urinary albumin/creatinine ratio was correlated with urine MCP-1 levels and interstitial macrophage infiltration in patients with chronic kidney disease.Citation8 In vitro experiments have also demonstrated that tubular epithelial cells release MCP-1 when exposed to serum proteins in the apical side.Citation9 Urinary MCP-1 levels have been shown to be positively correlated with the degree of proteinuria in children with FSGS and IgA nephropathy.Citation10 There are several studies reporting the relationship between MCP1 2518 A/G polymorphism and prognosis of various renal diseases such as IgA nephropathy,Citation11,Citation12 diabetic nephropathyCitation13 and lupus nephritis,Citation14 however the association of this polymorphism with the clinical outcome in primary FSGS has not been determined yet. The aim of this study was to investigate the relationship between MCP1 2518 A/G polymorphism and urinary-serum MCP-1 levels with the incidence and clinical course of children with primary FSGS.
Methods
Patients
Sixty patients with biopsy-proven FSGS, 76 patients with steroid-sensitive nephrotic syndrome (SSNS), and 96 healthy children were included in the study. The study protocol was approved by the institutional ethical committee. Written informed consent for the genetic analysis was obtained from each family.
Clinical characteristics and laboratory findings including hematuria, hypertension, urinary protein/creatinine ratio, serum creatinine, albumin levels, and steroid sensitivity of the patients at the beginning and follow-up period were collected by standard clinical questionnaire forms. Serum and urine MCP-1 levels of the patients were measured during the active state of the disease (i.e. during nephrotic range proteinuria).
Inclusion criteria for FSGS group were to have biopsy-proven disease with age at disease onset between 1 and 16 years and the absence of any known cause for secondary FSGS. The inclusion criteria for SSNS group were to be responsive to initial standard 4 weeks prednisolone therapy with age at disease onset between 1 and 8 years and to have histopathologically minimal change disease for those patients who underwent a renal biopsy for any reason. Healthy controls had no personal or family history of renal disease and hypertension.
MCP1 Genotyping
Genomic DNA was extracted from peripheral leukocytes by using standard procedures. The MCP1 genotypes were identified by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) assay using PvuII restriction enzyme (Life Technologies, Carlsbad, CA). A 930-bp fragment including the distal regulatory segment of the MCP1 5′-flanking region (nucleotides −2746 to −1817) was analyzed. Primers used were 5′-CCG AGA TGT TCC CAG CAC AG-3′ (forward) and 5′-CTG CTT TGC TTG TGC CTC TT-3′ (reverse). PCR products were digested by PvuII and the products were separated by gel-electrophoresis. Samples with a single 930-bp band were identified as AA, samples with three bands of 930, 708, and 222 bp were typed as AG, and those with two bands of 708 and 222 bp were typed as GG.
MCP-1 Measurements
Serum and urinary MCP-1 levels were measured by solid-phase enzyme linked immunosorbent assay (ELISA) in all patients (Quantikine MCP-1 ELISA; R&D Systems, Minneapolis, MN). All assays were done in duplicate. The mean variation in each sample was <5%. The minimum detectable MCP-1 level with this kit is 5 pg/mL. There is no cross-reactivity with the closely related chemokines MIP-1α, MIP-1β, and MCP-2,-3, and -4. Urine MCP-1 levels were normalized according to urine creatinine levels.
Statistical analysis
The results are presented as mean ± standard deviation (SD) unless otherwise specified. Mann–Whitney U test for paired samples was used for comparison between groups. Correlations were calculated with Pearson product moment correlation coefficient. p < 0.05 was considered statistically significant.
Results
The characteristics of children with FSGS and SSNS are demonstrated in . Control group was age (72.3 ± 39.9 months) and sex (40 males, 36 females) matched with the FSGS group. Mean age at the diagnosis was higher in FSGS group than that in SSNS group (p < 0.01, ). Five patients in FSGS group had hypertension at initial presentation whereas none in SSNS group. Serum albumin and creatinine levels were similar in both groups. Microscopic hematuria was a more common finding in the FSGS group (p < 0.01, ). The urinary protein/creatinine ratio in SSNS group was higher than that in FSGS group (p < 0.01, ). In both groups, mean follow-up duration was about 5.5 years. During the follow-up period, renal functions of all patients with SSNS remained stable whereas 25 patients (41.6%) in FSGS group developed chronic renal failure ().
Table 1. Clinical and laboratory features of the patients with FSGS and SSNS at initial presentation and the disease course.
The MCP1 genotypes of all groups are presented in . Genotype and allel frequencies were comparable in all groups (p > 0.05). The MCP1 genotype had no effect on age at diagnosis, hypertension, degree of proteinuria, and steroid sensitivity (data not shown). The FSGS patients who progressed to chronic renal failure or who had stable renal functions during the follow-up were comparable in terms of age at diagnosis, sex, prevalence of hypertension, and serum creatinine level at presentation (data not shown). The percentage of patients who develop chronic renal failure was significantly higher in AA genotype compared to GA and GG genotype (46% vs. 35% respectively, p < 0.01, Odds ratio: 1.59, ).
Table 2. MCP1 2518 genotypic and allele frequencies of patients with FSGS, SSNS and healthy controls.
Table 3. The genotypic frequencies in patients with and without chronic renal failure in FSGS group.
There was no correlation between urinary and serum MCP-1 level in all groups, but a positive correlation was found between urinary MCP-1 level and urinary protein/creatinine ratio in FSGS group (r = 0.529, p = 0.016). There was no association between serum MCP-1 level and degree of proteinuria. Serum MCP-1 levels were similar in all groups, however urinary MCP-1 levels of FSGS group were higher than SSNS group (p < 0.05, ) and healthy controls (p < 0.05, ). There was no correlation between MCP1 genotype and urine or serum MCP-1 level.
Table 4. Urinary CCL2/MCP-1 levels in patients with focal segmental glomerulosclerosis and SSNS, and control group.
Discussion
Clinical course of FSGS is highly variable and correlated with severity of interstitial inflammation, which plays an important role in progression of the disease. Recruitment of monocytes/macrophages by MCP-1 and other chemokines to interstitium is a key process, which leads to chronic inflammation and eventually glomerular sclerosis.Citation15,Citation16 Previous studies showed the relationship between MCP1 2518A/G polymorphism and severity of various renal diseases.Citation12–14 In this study, we investigated for the first time the relationship between MCP1 2518A/G polymorphism and disease course in a group of FSGS patients.
MCP1 2518 A/G polymorphism affects a region that regulates the transcription of this gene.Citation17 This polymorphism was originally described to affect MCP-1 production by peripheral mononuclear cells in response to an inflammatory stimulus.Citation18 The effect of the presence of A or G alleles at this position on MCP-1 production is controversial. Rovin et al. reported that G at 2518 position causes increased in vitro MCP-1 production by mononuclear cells in response to IL-1β.Citation18 However, Kim et al. reported that A at 2518 position was associated upregulation of MCP1 and results in severe proteinuria in patients with lupus nephritis.Citation19 We demonstrated that the MCP1 genotype frequencies for AA, GA and GG were similar in FSGS, SSNS, and control groups, which suggest that MCP1 2518 genotype is not a risk factor for the development of FSGS. However, the percentage of patients who progressed to chronic renal failure was significantly higher in those carrying AA genotype when compared to those who had GA or GG genotypes. This observation may suggest that MCP1 2518 A/G polymorphism might decrease the risk of progression to chronic renal failure in FSGS patients. However, this polymorphism seems to have no effect on the clinical parameters at the presentation and severity of proteinuria. Similarly, Steinmetz et al.Citation11 reported that 2518 A/G polymorphism had no effect on the incidence and progression of IgA nephropathy in a group of Caucasian patients with a mean follow-up period of 45.5 months. However, although not statistically significant, they also noted that the incidence of end-stage renal disease in patients with the GG genotype was lower than that in patients with the AA or AG genotype. Mori et al.Citation12 performed a similar study in Japanese patients with IgA nephropathy. They found that the AA genotype was an independent risk factor for the progression of the IgA nephropathy, and was closely associated with renal survival. This discrepancy may be explained by ethnicity and also the duration of follow-up, which was longer (average 135.9 months) in the latter study. MCP1 2518 A/G polymorphism affects the promoter region of the gene and the effect of this polymorphism on transcription in FSGS patients is yet to be determined.
Increased urinary MCP-1 levels have been associated with disease activity and poor prognosis in various renal diseases.Citation8 In our study, serum MCP-1 levels of FSGS group were comparable with the healthy controls and there was no correlation between urinary and serum MCP-1 levels in all study groups. Our findings suggest that increased urinary excretion of MCP-1 in the patients with FSGS is most likely due to enhanced production of MCP-1 in kidney, presumably induced by excessive exposure to plasma proteins filtered from the damaged glomeruli. We also showed that urinary MCP-1 levels correlated with degree of proteinuria in FSGS patients. Similarly, previous studies reported the increased urinary MCP-1 levels and the correlation between degree of proteinuria and urinary MCP-1 levels in patients with FSGS and IgA nephropathy.Citation10 A number of studies have demonstrated a correlation between degree of proteinuria and progression rate of chronic kidney disease, which have led to the hypothesis that proteinuria might be an independent player for disease progression rather than simply being a marker of glomerular dysfunction.Citation20–22 It has also been demonstrated that high protein load in renal tubular cells up-regulates MCP-1 expression and thereby MCP-1 production.Citation9,Citation23 Interestingly, despite more severe proteinuria in SSNS group, the patients with FSGS had higher urinary MCP-1 levels. This finding suggests that the role of MCP-1 in pathogenesis of FSGS is not a direct result of proteinuria. Proteinuria itself may be a result of immunological insult to glomeruli, in which MCP-1 plays a critical role in this scenario. It has been shown that podocytes can be detected in urine specimens of FSGS patients but not in healthy controls or minimal-change nephrotic syndrome patients.Citation24 Inflammatory cytokines such as TGF-β is overexpressed in kidney biopsy specimens of FSGS patientsCitation25 and cultured podocytes express MCP-1 in presence of TGF-β.Citation26,Citation27 Thus, another explanation for the increased urinary MCP-1 levels in FSGS patients may be the presence of MCP-1 expressing podocytes in urine of these patients but not in SSNS or control groups. The major limitation of the current study is limited patient number. Further studies investigating the role of MCP-1 genotype in a larger group of FSGS patients and functional studies investigating the effect of MCP-1 2518 A/G polymorphism on proteinuria and MCP-1 production by podocytes and macrophages may help to clarify the role of MCP-1 in this disease.
In conclusion, AA allele might be a risk factor for disease progression in FSGS and MCP-1 genotyping may be useful in these patients to predict the prognosis. This approach might also guide the physicians to determine the patients who need more aggressive treatment.
Declaration on interest
The authors report no conflict of interest. This study was supported by The Scientific and Technological Research Council of Turkey [Grant number: SBAG2685(103S024)].
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