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Urinary Monocyte Chemoattractant Protein-1 as A Diagnostic Marker of Lupus Nephritis

Background: Lupus nephritis accounts for significant morbidity and mortality in patients with systemic lupus erythematosus (SLE). Many biomarkers for renal involvement in SLE have been suggested in Egyptian patients; one of them is Monocyte chemoattractant protein-1 (MCP-1) which is one of the key chemokines that has chemotactic effect for monocytes and macrophages to sites of inflammation and may share in the pathogenesis of lupus nephritis (LN). Aim: The study aimed at assessing the role of MCP-1 in the early diagnosis of lupus nephritis and to explore any correlation its levels with disease activity and renal status. Subjects and Methods: The study was done as a case control study where 60 SLE patients with lupus nephritis (30 patients with active LN and 30 patients with inactive LN) in addition to 30 healthy volunteers as control group were enrolled in the study and MCP-1 levels was determined using ELISA technique. Results: Urinary MCP-1 levels in SLE studied patients’ groups was significantly higher than its level in control group (p = 0.0001) and it was significantly higher in active LN than in non-active LN subgroups (P value =0.0001). Conclusion: Urinary MCP-1 can be used as a marker for LN activity.


Introduction
Systemic lupus erythematosus (SLE) is an autoimmune disease that may affect multiple organ systems. One of the most serious complications of SLE is renal damage. It occurs in 40-70% of all patients (1) . Lupus nephritis (LN) accounts for significant morbidity and mortality in patients with systemic lupus erythematosus (SLE) (2,3) . Many biomarkers for renal involvement in SLE have been suggested in Egyptian patients (4)(5)(6) . Monocyte chemoattractant protein-1 (MCP1) is one of the key chemokines that has potent chemotactic action for monocytes and macrophages to sites of inflammation (7) . The MCP-1 was significantly increased in Egyptian SLE patients especially those with an increased intima media thickness (8) . MCP-1 is produced by many cell types, including mesangial, podocyte, and monocyte cells in response to various proinflammatory stimuli such as tumor necrosis factor alpha (TNF-α). These cells subsequently mediate tissue injury and contribute to the development of renal dysfunction (7) . The Level of MCP-1 in urine was found to be significantly greater in patients with a renal flare than in patients with stable renal (9,10) . The aim of the study was to assess the role of urinary monocyte chemoattractant protein-1 (MCP-1) in the early diagnosis of lupus nephritis (LN) and to explore any correlation of its levels with disease activity and renal status.

Patient and Methods
The study was done as a case control study aiming at assessing the role of urinary MCP-1 in the early diagnosis of lupus nephritis and to correlate the levels with disease activity and renal status. 60 SLE patients with lupus nephritis attending the Suez Canal University hospitals outpatient's clinic were enrolled in the study (30 patients with active LN and 30 patients with inactive LN) in addition to 30 healthy volunteers as control group. The studied population was subjected to detailed medical history , clinical examination and laboratory investigation where Systemic Lupus Erythematosus disease activity score (SLEDAI) assessment was used to define lupus activity in addition to basic laboratory investigation ; serological markers of activity (e.g.: C3 , C4 , ESR ≥ 100 ), evidence of renal flare (e.g.; urinary sediments and 24 hour urinary protein excretion rate) which were done by an automated analyzer (COBAS INTE-GRA 400 Automated Chemistry Analyzer) and urinary MCP-1 assay was done using the Quantikine® Human MCP-1 Immunoassay which is a 3. 5-4. 5-hour solid phase ELISA used for MCP-1 assay according to the manufacturer instruction. All laboratory work was done in SCU clinical pathology laboratory. Collected data was coded, entered, and analyzed using Statistical Package for the Social Sciences (SPSS) version 24 software for analysis. Differences between frequencies such as age, gender, clinical pattern, and severity, was compared using Chisquare or Fisher exact tests. Differences between means were compared by ttest. Pearson correlation coefficient test was used to evaluate the inter-correlations between the studied variables. Logistic regression analysis of the dependent variable and other studied variables was performed. Statistical significance was considered at P-value <0.05 and highly significant at P-value <0.01. The study was conducted after the Suez Canal university, faculty of medicine ethical committee approval.

Discussion
Lupus nephritis is a frequent and serious complication of Systemic Lupus Erythematosus and is associated with considerable morbidity and mortality (3) . Early detection of renal involvement during the course of SLE is important for improving the outcome (11) . The ordinary serological markers used in clinical practice such as serum C3, C4 and anti-ds-DNA antibodies may be unreliable indicators of LN as they lack both sensitivity and specificity for prediction of active or relapsing LN. Moreover, serum creatinine is also an unreliable marker as significant kidney injury may occur before it rises (12) . Other urinary parameters like proteinuria and urinary sediments are also non-specific markers (13) and therefore the need for finding such a marker is under research. So, our study aimed to assess the role of urinary MCP-1 as noninvasive biomarker predicting Lupus activity and determine its correlations with standard laboratory markers and disease activity indices. The present study showed that the mean levels of urinary MCP-1 in active SLE/ LN was significantly higher than the nonactive SLE / LN. This finding was the same in an earlier study by Tucci which examined the role of a functional MCP-1 polymorphism in SLE and LN. They showed that, U MCP-1 values were significantly higher in patients with LN (14) . In another study investigating the urinary biomarker in SLE patients, they found that mean Urinary MCP-1 levels in SLE renal flares, were significantly higher than its levels in non-renal flares (15) . The current study also provided evidence that urinary MCP-1is significantly higher in SLE/ LN patients whether with active or non-active lesions when compared to healthy controls. patients with rheumatoid arthritis, 11 non-SLE patients with renal impairment, and 40 healthy volunteers. The expression of MCP-1 was significantly higher in active LN groups than in all other groups, and there was a close correlation between MCP-1 expression and the overall SLE disease activity index score and the SLE disease activity index renal score (16) . In Egypt, one earlier study showed urinary MCP-1 levels were significantly elevated in patients with active LN compared to those with inactive disease and control (17) . The associations between urinary MCP-1 excretion and serological lupus activity markers remain controversial. In the current study both C3 and C4 was significantly associated with urinary MCP-1 levels; the same finding was reported by after a 2 months follow up of LN patients with renal flare (18)  reported that urinary MCP-1 levels and anti-dsDNA positivity were highly associated (10,19) , whereas in 2014 Watson et al. in their longitudinal study involving 64 pediatric SLE patients reported an association between urinary MCP-1 and serum C3 (20) .
The present study showed that the increase of urinary ACR was met by increase of urinary MCP-1 and both were significantly associated with each other. In the same context, In 2004 the study by Tucci et al showed that urinary MCP-1 and 24-hr urinary protein excretion were positively correlated (14) . Furthermore, studying the glomerular expression of MCP-1 in SLE pediatric population revealed a correlation between increased glomerular MCP-1 expression and albuminuria (21) . In another study, a positive correlation between urinary excretion of MCP-1 and proteinuria was observed (22) . Nevertheless, in contrast to the previous results; the early work of Dai et al in 2001 could not detect any correlation between urinary MCP-1 and urinary protein excretion in LN patients and the same finding was reported in the later work in 2012 by Mirfeizi and his colleagues (23,24) . The ROC curve in the present study showed  (25) . But other studies suggest a higher cut-off point of 82pg/ml, Urinary MCP-1 with sensitivity of 88.5% and a specificity of 46.3% for identifying LN (24) . Amore investigation using renal pathology tissue showed that urinary MCP-1 had a sensitivity of 97% and a specificity of 100% in detecting active lupus nephritis and Glomerular MCP1 had a sensitivity of 64% and specificity of 95% while tubular MCP1 had a sensitivity of 4% and specificity of 20% in detecting active LN; Therefore, urinary and glomerular MCP-1 are more sensitive and specific for detection of activity of lupus nephritis (17) .

Conclusion
Urinary MCP-1 can be used as a predictor or an adjunctive marker for LN activity and may identify early relapse of LN leading to earlier treatment and better outcome.

Limitation of the study
Certain limitations were encountered in this study which need to be addressed in the future. 1) The small sample size which may limit generalization of the study results hence a large population observational trial may be needed. 2) Using longitudinal study designs correlating multiple urinary MCP-1with multiple histological checkpoints using kidney biopsies and serological disease activity markers. And 3) Clinical trials investigating the ability of urinary MCP-1 in risk stratification of lupus nephritis is needed.