|Year : 2015 | Volume
| Issue : 3 | Page : 507-515
|Urinary monocyte chemoattractant protein-1 as a biomarker of lupus nephritis activity in children
Emad E Ghobrial1, Azza A El Hamshary1, Ashraf G Mohamed2, Yomna A Abd El Raheim2, Ahmed A Talaat2
1 Department of Pediatrics, Faculty of Medicine, Cairo University, Cairo, Egypt
2 Department of Pediatrics, National Research Center, Cairo, Egypt
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|Date of Web Publication||20-May-2015|
| Abstract|| |
Systemic lupus erythematosus (SLE) is a life-long, life-limiting and multi-systemic autoimmune disease. Glomerulonephritis is one of the most serious manifestations of SLE. Younger children have an increased incidence, severity and morbidity of lupus nephritis (LN) compared with adult-onset disease. Monocyte chemoattractant protein-1 (MCP-1) enhances leukocyte adhesiveness and endothelial permeability in the kidneys of murine and human LN models. Our study aimed to assess the role of urinary MCP-1 in the early diagnosis of LN activity. Sixty children, of whom 45 children aged from six to 12 years old and of both sexes (15 SLE patients without nephritis, 15 active LN and 15 inactive LN) fulfilling the American College of Rheumatology Classification Criteria for SLE were studied in comparison with 15 healthy subjects. We investigated the serum and urinary MCP-1 in all groups using the enzyme-linked immunosorbent assay test. Urinary MCP-1 was significantly higher in active LN in comparison with inactive LN and controls, and also significantly higher in inactive LN in comparison with SLE without nephritis and controls. There was also a significant difference between SLE without nephritis and controls. Serum MCP-1 was significantly higher in the group with active LN in comparison with the inactive group and SLE without nephritis and controls, but there was no significant difference between SLE and controls. The urinary MCP-1 level correlated well with SLE disease activity as measured by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Urinary MCP-1 correlates positively with proteinuria, blood urea nitrogen level and creatinine and negatively with hemoglobin and creatinine clearance. We concluded that measurement of MCP-1 in urine may be useful for monitoring the severity of renal involvement in SLE. We recommend measuring urinary MCP-1 in pediatric SLE for the early diagnosis of LN and for the evaluation of the severity of renal involvement.
|How to cite this article:|
Ghobrial EE, El Hamshary AA, Mohamed AG, Abd El Raheim YA, Talaat AA. Urinary monocyte chemoattractant protein-1 as a biomarker of lupus nephritis activity in children. Saudi J Kidney Dis Transpl 2015;26:507-15
|How to cite this URL:|
Ghobrial EE, El Hamshary AA, Mohamed AG, Abd El Raheim YA, Talaat AA. Urinary monocyte chemoattractant protein-1 as a biomarker of lupus nephritis activity in children. Saudi J Kidney Dis Transpl [serial online] 2015 [cited 2021 Jan 20];26:507-15. Available from: https://www.sjkdt.org/text.asp?2015/26/3/507/157350
| Introduction|| |
Glomerulonephritis is one of the most serious manifestations of systemic lupus erythematosus (SLE). In spite of the improvement in the medical care of SLE in the past two decades, the prognosis of lupus nephritis (LN) still remains poor. 
Approximately half of lupus patients develop LN and approximately 10% overall will progress to dialysis or transplantation.  Renal involvement in SLE is a common manifestation and a strong predictor of poor outcome. 
LN usually arises within five years of diagnosis; however, renal failure rarely occurs before fulfilling the American College of Rheumatology classification criteria. 
The current most widely used biomarkers for the early detection of chronic kidney disease or acute kidney injury are proteinuria, serum creatinine and blood urea nitrogen (BUN). All of these are less than optimal and tend to denote later stages of involvement when therapies may be less effective. 
Monocyte chemoattractant protein-1 (MCP-1) is produced by renal mesangial cells, endothelial cells, tubular epithelial cells and smooth muscle cells. This molecule has been described to play a main role in progression of inflammatory processes in kidney disease linked to SLE. 
Urinary MCP-1 was shown to be a sensitive and specific biomarker of renal SLE flare and its severity in adults with SLE, even in those who were on immunosuppressive agents. However, there is little data on biomarkers in childhood SLE. 
| Objective|| |
The purpose of this study was to assess the relationship of urinary and serum MCP-1 with disease activity in pediatric patients with LN as a non-invasive biomarker.
| Patients and Methods|| |
This cross-sectional study was performed in the Rheumatology clinic at the New Children Hospital, Faculty of Medicine, Cairo University, including 45 patients regularly following in the clinic and 15 children as the control group.
The 45 patients' ages ranged from six to 12 years, and included both sexes. Consent from the parents of all patients and controls were taken and approval was taken from the medical ethics committee of the National Research Centre and Cairo University.
The 45 patients were divided into three groups.
- Group I included 15 patients with SLE without clinically apparent renal disease.
- Group II included 15 patients with SLE with clinically apparent renal disease and in activity.
- Group III included 15 patients with SLE with clinically apparent renal disease and in inactive form.
The control group included 15 children and age and sex-matched healthy subjects as a control group.
Age ranged from six to 12 years old. All the patients fulfilled at least four of 11 revised American Colleges of Rheumatology (ACR) classification criteria for SLE. Patients were deemed to have renal involvement with LN based on clinical and histopathological findings and confirmation of LN. SLE patients without LN (non-nephritis) fulfilled the ACR classification criteria for SLE in the absence of renal involvement at the time of analysis. Children of the control group were age and sex matched, had no known medical illnesses or active infection and were not taking any drugs at the time of study.
Patients with primary and secondary hypertension (except renal hypertension), coronary heart disease, congestive heart failure, chronic respiratory diseases, chronic liver diseases or diabetes mellitus were excluded from the study.
The medical records were reviewed to screen for any pre-existing renal disease among patients and to obtain pediatric SLE-specific information. Review of system information and the results of routine laboratory testing at the time of the study visits were recorded. Relevant demographic data of all participants were obtained.
All patients and controls were subjected to full history taking, with special emphasis on disease activity in SLE patients measured by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)  and laying stress on age, duration of the disease, urinary symptoms, SLE manifestations [e.g., joint pains, rash, cutaneous photosensitivity, central nervous system (CNS) symptoms, including seizures], symptoms of hypertension and type of therapy received by the patient. Thorough clinical examination was performed for all patients, including vital signs and anthropometric measurements (weight and height), skin rash distribution, joint affectation, chest examination, heart examination, abdominal examination and CNS examination. All available data about LN were recorded.
Laboratory testing (for patients only) were carried out in the form of serum creatinine and BUN (using a multi-channel auto-analyzer; Hitachi, Japan), complete blood count [using a hematology counter (Cell Dyne 3700, Abbott laboratories, North Chicago, USA)], urine analysis (urine was centrifuged and the supernatant was examined by direct microscopy), urinary protein to creatinine ratio (using a coulter Beckman auto-analyzer, Synchron CX9, Beckman, Houston, Texas, USA), erythrocyte sedimentation rate (ESR) by the Westergen method, anti-DNA by manually employing the indirect immunofluorescent test and complement 3 and 4 levels manually by radial immunodiffusion.
MCP-1 in serum and urine was determined using an enzyme-linked immunosorbent assay (ELISA) kit (15), which was supplied by R&D Systems Europe, Ltd, Abingdon, Oxfordshire, UK.
Assessment of disease activity of SLE was carried out by the SLEDAI. Percutaneous renal biopsies had been taken from all patients with renal involvement. Renal histopathology was classified according to the World Health Organization criteria for LN.
Sample collection and storage
The serum sample of about 3 mL of fasting (6-8 h) venous blood samples was taken from each child participating in the study. A serum separator tube (SST) was used and was allowed to clot for 30 min, followed by centrifugation for 15 min at 1000× g. The serum was then removed and the sample stored at ≤-20°C.
The urine sample was aseptically collected using the first urine of the day (mid-stream), voided directly into a sterile container, centrifuged and then stored at ≤-20°C.
The principle of the assay, storage and calculation of results were followed as per the manufacturer's instructions.
| Statistical Analysis|| |
All patient information was tabulated and processed using Statistical Package for the Social Sciences (SPSS) version 19 and Microsoft Excel software program, and the results were represented graphically. For quantitative variables, means and medians (as a measure of central tendency), standard deviation, range and minimum and maximum (as measures of variability) were used. Frequency and percentage are presented for qualitative variables. The one-way analysis of variance was used to test the differences between groups. The Duncan multiple comparison test was used to test the significant differences between each of two groups. The chi square test was used to compare the distributions between groups. The Pearson correlation coefficient test was used to test the significant correlations between the quantitative parameters within each group. A P-value less than 0.05 was considered significant.
| Results|| |
The study included 45 patients and 15 controls.
Group I included 15 patients with SLE without clinically apparent renal disease. There were six (40%) males and nine (60%) females. Their ages ranged from six to 12 years old and the mean age was 9.80 ± 1.424 years.
Group II included 15 patients with SLE with clinically apparent renal disease and in activity. There were six (40%) males and nine (60%) females. Their ages ranged from eight to 12 years old and the mean age was 10.8 ± 1.207 years.
Group III included 15 patients with SLE with clinically apparent renal disease and in inactive form. There were six (40%) males and nine (60%) females. Their ages ranged from six to 12 years old and the mean age was 9.27 ± 1.792 years.
All patients of the three groups fulfilled at least four of the ACR preliminary criteria for diagnosis of SLE.
The control group included 15 children who were age and sex matched healthy subjects.
There were four (26.6%) males and 11 (73.3%) females. Their ages ranged from six to 12 years old and the mean age was 9.87 ± 1.959 years.
There was no significant difference regarding age and sex between all four groups.
[Table 1] shows the demographic and laboratory data of the three patient groups and the control group. There were statistically significant differences between the four groups as regards the age of onset of SLE and LN duration, being higher in Group III. [Figure 1] shows comparison of the sex distribution among the four groups.
|Table 1: Demographic and laboratory data of the patients and controls (mean ± SD).|
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As regards the total SLEDAI score in Groups I, II and III, in Group I, 10 patients (66.7%) were inactive (0-4) and five patients (33.3%) were mild (>4-8) with mean ± SD of 5.0 ± 1.9. In Group II, four patients (26.7%) were mild, nine patients (60%) were moderate (8-12) and two patients (13.3%) were severe (>12) with mean ± SD of 9.6 ± 1.4. In Group III, all the 15 patients (100%) were inactive with mean ± SD of 2.6 ± 1.1.
Patients in the active LN group (Group II) had a significantly lower hemoglobin level (P = 0.009) and complement C3 and C4 (P = 0.000), glomerular filtration rate (GFR) (P = 0.000) and serum albumin (P = 0.000) than inactive LN, SLE without nephritis and control groups. In addition, Group II had a significantly higher level of BUN and creatinine levels than other groups (P = 0.000), as shown in [Table 1].
Urinary MCP-1 correlates positively with SLEDAI (P = 0.05), BUN level (P = 0.037) and creatinine (P = 0.057) and negatively with hemoglobin (P = 0.04) and creatinine clearance (P = 0.002), as shown in [Table 2].
|Table 2: Correlation between serum and urine MCP-1 and some laboratory parameters in lupus nephritis patients (Groups II and III).|
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We measured the correlation between serum and urinary MCP-1 and the levels of proteinuria, GFR, hemoglobin, BUN and creatinine at the time of measurement of serum and urinary MCP-1. There was a positive significant correlation between urinary MCP-1 and SLEDAI proteinuria and BUN and a negative significant correlation between urine MCP-1 and creatinine clearance and hemoglobin. No significant correlation was found between serum MCP-1 and these parameters [Table 2].
The mean value of serum MCP-1 in Group II (352.36 ± 22.02 pg/mL) was significantly higher (P = 0.000) than Group III (311.98 ± 32.26 pg/mL), and both groups were significantly higher than the control group and Group I. There was also no significant difference between Group I SLE and the control group, as shown in [Table 3].
The mean value of urinary MCP-1 in Group II (556.14 ± 71.84 pg/mL) was significantly higher (P = 0.000) than Group III (464.95 ± 76.2 pg/mL) and also significantly higher than both Group IV (202.42 ± 45.479 pg/mL) and Group I (225.107 ± 49.806 pg/mL). There was also a significant difference between Group I (SLE) and the control group, as shown in [Figure 2].
|Figure 2: Comparison of MCP-1 distribution in urine in the different groups.|
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There was a direct significant correlation between urinary MCP-1 and disease activity index (P = 0.004), although there is no significant difference between serum MCP-1 and disease activity index (P = 0.6) as shown in [Table 4].
|Table 4: Correlation between MCP-1 in serum and urine according to activity.|
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There was a positive significant correlation between urine MCP-1 in moderate and severe activity and mild and moderate activity while there was no significance in the serum.
| Discussion|| |
SLE occurring in children typically has both a more active disease course and a higher incidence of renal involvement compared with adults. In spite of its diagnostic strengths, renal biopsy, the gold standard in confirming the diagnosis and class of LN, is an invasive procedure with potential complications.  Clinical and laboratory biomarkers currently available fail to reliably predict the severity of the underlying disease process. In addition, conventional clinical parameters are not sensitive or specific enough for detecting ongoing disease activity in the SLE.
Cytokines, chemokines and the immunoregulatory networks in which they participate play a principal role in the pathogenesis of this chronic disease.  Measurement of cytokines in urine is therefore an encouraging approach with increasing clinical applications in SLE.  Identifying biomarkers that identify severe and active LN may provide a reliable alternative to the invasive biopsy. MCP-1 is a cytokine expressed in response to pro-inflammatory stimuli. Its presence within the glomerulus correlates with a poor renal prognosis in childhood LN. Urinary concentrations also correlate with histological diagnosis. 
Concerning the activity of disease, for Group I, 66.7% of patients were lupus inactive, 33.3% had mild disease activity and none had moderate to severe disease activity with a mean ± SD SLEDAI of 5 ± 1.9. For Group II, none were inactive, 26.7% had mild disease activity, 60% had moderate disease activity and 13.3% had severe disease activity with mean ± SD SLEDAI of 9.6 ± 1.404. For Group III, 100% were inactive LN with a mean ± SD of 2.6 ± 1.1.
In this study, we showed that urinary MCP-1 was significantly higher in patients with active LN when compared with patients with inactive LN and control patients (P = 0.000). Also, the inactive LN patients showed a significantly higher urinary MCP-1 than lupus non-nephritis patients and controls (P = 0.000).
We also found that serum MCP-1 was significantly higher in active LN patients when compared with patients with inactive renal disease, lupus non-nephritis and controls (P = 0.000), but there were no significant difference in serum MCP-1 between the lupus non-nephritis patients and controls.
In accordance with our study, Marks et al in 2010  proved that there were significantly increased urinary MCP-1 levels in the LN patients compared with the healthy controls whose values were significantly higher than the lupus non-nephritis children. There were no differences in plasma MCP-1 levels between SLE patient groups and controls. These results provide evidence of increased urinary - but not plasma - MCP-1 levels in children with LN, which correlates well with SLE disease activity. The lack of significant increase of circulating levels of MCP-1 in the serum of patients with nephritis may be due to the possibility that locally produced MCP-1 is excreted into urine rather than circulated in the blood, and to the extremely short half-life of MCP-1 in serum.
On the contrary, Kaneko et al (1999)  found an increase of serum MCP-1 with the progression of disease activity in SLE patients compared with healthy controls. Moreover, Vega et al (2010)  found that SLE patients, even in the absence of symptoms, have higher MCP-1 levels in plasma than healthy controls. In agreement, Watson et al (2012)  have confirmed that MCP-1 distinguishes patients with active LN when compared with patients with inactive renal disease using a disease activity tool, regardless of previous renal involvement. In 2012, Rosa et al  found that in the active LN group, the concentration of urinary MCP-1 was significantly higher, a finding similar to our study. In contrast to our study, in the study by Wada et al (1996),  it was demonstrated that the serum MCP-1 level was significantly higher in patients with LN as compared with the control group. In 1995, Noris et al  found that patients with active LN had higher levels of urinary MCP-1 compared with the inactive group and control group. Also, the serum MCP-1 level in active LN patients was higher than that of the control group.
Similar to our study, Rovin et al in 2004  found that urinary MCP-1 of patients with renal involvement was significantly higher than that of healthy control subjects and higher than SLE patients with non-renal flare. Also, they found that urinary MCP-1 in patients with proliferative forms of SLE glomerulonephritis (WHO classes II, III and IV) was higher than that of non-proliferative forms (WHO class V).
In our study, the urinary MCP-1 level correlated well with SLE disease activity as measured by SLEDAI (P = 0.004). There were no statistically significant differences (P = 0.6) in the serum MCP-1 levels between those patients with score ≥8 (moderate and severe activity) and others with score <8 (mild and inactive), although the urinary levels were significantly higher for urinary MCP-1 (P = 0.004). In addition, urinary MCP-1 correlates positively with proteinuria (P = 0.011), BUN level (P = 0.037) and creatinine (P = 0.057) and negatively with hemoglobin (P = 0.04) and creatinine clearance (P = 0.002), such that the urinary MCP-1 correlates with severity of nephritis, while no correlation was found with serum MCP-1 and total disease activity index.
There was a positive significant correlation between urinary MCP-1 and SLEDAI (P = 0.05), proteinuria (P = 0.01) and BUN (P = 0.037) and a negative significant correlation between urine MCP-1 and creatinine clearance (P = 0.002) and hemoglobin (P = 0.04). No significant correlation was found between serum MCP-1 and these parameters. Therefore, urinary MCP-1 correlates with the severity of nephritis.
The mean value of serum MCP-1 in Group II (active LN) was significantly higher than Group III (inactive LN), and both groups were significantly higher than the control group (IV) and SLE without nephritis (Group I). There was also no significant difference between Group I (SLE) and Group IV (control group).
In accordance with our study, Alzawawy et al in 2009  found that serum MCP-1 was significantly higher in SLE patients with nephritis than in the control group, while no significant difference was found between SLE patients without nephritis and the control group. Urinary MCP-1 in patients with active LN was significantly higher than that in patients with inactive LN and the control group. Also, urinary MCP-1 in SLE patients with nephritis was significantly higher than both lupus without nephritis and control groups, and it correlated positively with proteinuria and negatively with creatinine clearance and hemoglobin; thus, urinary MCP-1 correlates with the severity of nephritis.
The sample size was the only limitation of the study. The small number of patients was due to the fact that many patients reached the adolescent and adult age groups; therefore, they were referred to the adult rheumatology clinic of Cairo University to be followed there. Therefore, the number of patients in the pediatric rheumatology clinic became less.
| Conclusion|| |
Measurement of MCP-1 in urine seems to be a useful method for monitoring the severity of renal involvement in SLE using a non-invasive urinary biomarker and the SLEDAI as a disease activity tool. Urinary but not serum MCP-1 may be a useful technique for the assessment of renal involvement in patients with LN and with activity of LN as it shows a good correlation with some clinical and laboratory parameters of disease relapse.
Measurement of urinary MCP-1 in pediatric SLE patients can be used for the evaluation of disease activity and evaluation of renal involvement. Further studies are recommended to clarify the role of MCP-1 in the follow-up of LN.
| Acknowledgments|| |
The authors thank all the patients who participated in the study and their parents.
Conflicts of Interest: None declared.
This study was approved by the ethical scientific committee in the Cairo University hospital and was conducted in accordance with the University for human research. All caretakers have given their informed consent.
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Dr. Emad E Ghobrial
Department of Pediatrics, Faculty of Medicine, Cairo University, Cairo
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]
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