| Abstract|| |
There is no single biomarker to detect lupus nephritis (LN) activity. Renal biopsy is still the gold standard method but it is invasive and mainly used in the initial assessment of the patients. Urinary tumor necrosis factor-like weak inducer of apoptosis (uTWEAK) and urinary monocyte chemo-attractant protein-1 (uMCP-1) can be secreted in the urine of active LN. The aim of the study is to assess the potential role of uTWEAK and uMCP-1 in lupus patients and to determine their correlation with disease activity. This is a case-control study conducted on a total of 114 subjects; 92 systemic lupus erythematosus (SLE) patients and 22 healthy volunteers. The patients were recruited from the rheumatology unit at the internal medicine department, Tanta University Hospital, Tanta, Egypt. The patients and controls were subjected to full history taking, complete clinical examination, routine laboratory tests, uTWEAK and uMCP-1 measurement, assessment of the disease activity using SLE Disease Activity Index (SLEDAI), and renal SLEDAI (rSLEDAI) scores. uTWEAK and uMCP-1 levels were higher in SLE with active nephritis group than those of other SLE groups and controls. There was a significant positive correlation between uTWEAK and uMCP-1 levels in lupus patients with proteinuria, anti-dsDNA, SLEDAI and r-SLEDAI and a negative correlation with C3 and C4. TWEAK showed a sensitivity of 80.43% and 100% and specificity of 50% and 100% in detecting lupus activity and LN activity, respectively. Furthermore, uMCP-1 showed a sensitivity of 82.6% and 100% and specificity of 50% and 100% in detecting lupus activity and LN activity, respectively. uTWEAK and uMCP-1 are new, easily obtained, accurate markers with high sensitivity and specificity in the detection of LN activity.
|How to cite this article:|
Elsaid DS, Abdel Noor RA, Shalaby KA, Haroun RA. Urinary Tumor Necrosis Factor-Like Weak Inducer of Apoptosis (uTWEAK) and Urinary Monocyte Chemo-attractant Protein-1 (uMCP-1): Promising Biomarkers of Lupus Nephritis Activity?. Saudi J Kidney Dis Transpl 2021;32:19-29
|How to cite this URL:|
Elsaid DS, Abdel Noor RA, Shalaby KA, Haroun RA. Urinary Tumor Necrosis Factor-Like Weak Inducer of Apoptosis (uTWEAK) and Urinary Monocyte Chemo-attractant Protein-1 (uMCP-1): Promising Biomarkers of Lupus Nephritis Activity?. Saudi J Kidney Dis Transpl [serial online] 2021 [cited 2021 Sep 25];32:19-29. Available from: https://www.sjkdt.org/text.asp?2021/32/1/19/318522
| Introduction|| |
Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder affecting multiple organs and characterized by a variety of autoantibodies, immune complex deposition in tissues and possible subsequent development of glomerulonephritis. Lupus nephritis (LN), remains a frequent and severe manifestation of SLE. Renal involvement in SLE is still one of the strongest predictors for morbidity and mortality. Although over the last decades, the therapeutic options for LN have increased leading to better results, the survival rate among patients with LN is still about 75% after 15 years.
Measurement of disease activity in SLE is essential to evaluate outcomes, differences among SLE patient groups, responses to a new drug proposed, and also for assessing disease longitudinally for observational and clinical trials. A lot of scoring systems are used to evaluate SLE activity but no single biomarker can be used to predict lupus flares, especially LN.
Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional pro-inflammatory cytokine that belongs to the TNF-ligand superfamily. it leads to activation of several intracellular signal transduction cascades, including the nuclear factor kappa B and mitogen-activated protein kinase pathways, thus controlling many cellular activities including proliferation, migration, differentiation, apoptosis, angiogenesis, and inflammation., A lot of studies supported a role for TWEAK in the pathogenesis of LN and provided strong evidence for urinary TWEAK (uTWEAK) as a clinical biomarker for LN.,,
Monocyte chemo-attractant protein-1 (MCP-1/CCL2) is one of the key chemokines that regulate the migration and infiltration of monocytes/macrophages. It is a leukocyte chemotactic factor that is involved in mediating inflammation and injury in LN. In murine models of LN, genetic depletion or blockade of MCP-1 ameliorates glomerular and interstitial inflammation and hence renal damage. In human LN, increased expression of MCP-1 on endothelial cells, renal epithelial cells, and infiltrating mononuclear cells in the tubulo-interstitial regions can be demonstrated by immunohistochemical staining and in situ hybridization., Hence, the aim of this study is to assess the potential role of uTWEAK, and urinary MCP-1 (uMCP-1) in lupus patients and to determine their correlation with disease activity.
| Patients and Methods|| |
This is a case-control study conducted on a total of 114 subjects; 92 SLE patients who were recruited from the internal medicine department, rheumatology unit (inpatient wards and outpatient clinics), Tanta University hospital, Tanta, Egypt and 22 age- and sex-matched healthy volunteers were allocated as a control group. The included SLE patients met systemic lupus international collaborating clinics criteria for SLE classification 2012. We excluded patients with: diabetes mellitus, overlap syndrome, urinary tract infections, and end-stage renal disease.
This study is in agreement with the ethical guidelines of the Declaration of Helsinki and it follows the ethical standards of the Tanta faculty of medicine. Informed consents from all patients were obtained in accordance with the local ethical committee. Privacy of all patients’ data was granted as there was a code number for every patient file that includes all investigations.
All patients and controls were subjected to: full history taking, complete clinical examination, routine laboratory tests as complete blood count, blood urea, serum creatinine, erythrocyte sedimentation rate (ESR), C-reactive protein, liver function tests, urine analysis, and protein quantification in 24 h urine. Serological tests such as anti-double-stranded deoxyribonucleic acid (anti-ds DNA) and antinuclear antibody (ANA) were assessed by enzyme-linked immunosorbent assay (ELISA) and the complement factors (C3 and C4 levels) by radial immunodiffusion. uTWEAK levels and uMCP-1 levels were measured by human TWEAK ELISA kits and human MCP-1 ELISA kits from Thermo Fisher Scientific, USA. The fresh urine samples were centrifuged to remove sediments and supernatants were frozen in small aliquots without further manipulation at –80°C for later analysis. We measured absorbance A values by Microplate reader at 450 nm wavelength and then calculated uTWEAK concentrations and uMCP-1 concentrations according to the standard curve.
Assessment of the disease activity using SLE disease activity index 2000 update (SLEDAI-2k) which consists of 24 variables with weighted score for each variable. The maximum possible score is 105; patients with active disease have 8 or more points. LN activity was assessed by renal SLEDAI (r-SLEDAI). r-SLEDAI (SLEDAI-2K renal scores) consists of the four kidney-related items of the SLEDAI -2K, including hematuria (>5 red blood cells/high-power field), pyuria (>5 white blood cells/high-power field), proteinuria (>0.5 g/24 h or urine protein/creatinine ratio >0.5) and urinary casts (heme, granular, or red blood cell). The presence of each of the parameters is scored as 4 points; the renal activity score can therefore range from 0 to 16. For our inclusion criteria, any r-SLEDAI score ≥8 was taken as an indicator of active LN.
According to the 2000 SLEDAI scores, our patients were divided into four groups: patients with active LN (n = 24); patients with non-active LN (n = 23); patients with active SLE without renal involvement (n = 22); and patients with non-active SLE without renal involvement (n = 23) in addition to 22 as a healthy control group.
| Statistical Analysis|| |
The data were analyzed using the IBM SPSS Statistics version 23.0 (IBM Corp., Armonk, NY, USA). Descriptive statistics were expressed as mean and standard deviation for numerical variables with normal distribution, and median and interquartile range) for numerical variables with the abnormal distribution. The Kolmogorov–Smirnov test was used to analyze the normal distribution of the variables. Kruskal–Wallis test, for abnormally distributed quantitative variables, to compare between more than two studied groups, and Post Hoc Test (Dunn’s for multiple comparisons test) for pairwise comparisons. ANOVA test, for normally distributed quantitative variables, to compare between more than two groups, and post hoc test (LSD) for pairwise comparisons.
Area under the curve (AUC) calculations of nonparametric receiver operating characteristic (ROC) curves were used to compare the ability of uTWEAK and uMCP-1 to distinguish between specific groups of patients. In addition, sensitivity and specificity characteristics were derived from the ROC curves and were used to identify cutoff point values for defining high and low uTWEAK and uMCP-1 levels.
| Results|| |
The clinical and laboratory data of healthy subjects and SLE patients are summarized in [Table 1] and [Table 2]. As the study was designed to match the age and gender between cases and controls, no statistically significant difference was observed for these parameters (P >0.05).
|Table 2: The clinicopathologic characteristics of the SLE patients (cont.)|
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Statistically significant increase in the blood urea, ESR 1st h, ANA, anti-ds DNA and proteinuria were observed, and significant decrease in the glomerular filtration rate (GFR), red blood cells, hemoglobin, platelets, serum albumin, serum protein, C3, C4 among the SLE patients as compared to controls.
Also uTWEAK level and uMCP-1 level of SLE patients group were significantly higher than those of the control group and after post hoc tests there was also a significant difference between all groups with the highest level among active LN then inactive LN, active lupus without nephritis, and inactive lupus without nephritis, respectively [Table 3].
A significant positive correlation was present in SLE patients between uTWEAK and uMCP-1 levels and blood urea, serum creatinine, eGFR, proteinuria, anti-ds DNA, SLEDAI, and r-SLEDAI and negatively with, C3 and C4 as shown in [Table 4].
|Table 4: The correlations of uTWEAK and uMCP-1 and different parameters among SLE patients.|
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ROC curves analysis of uTWEAK in detecting SLE disease, SLE activity, LN and LN activity revealed an AUC of 1.00, 0.76, 1.00, and 1.00, respectively, as well as a sensitivity of 100%, 80.43%, 100%, and 100% and specificity of 100%, 50%,100%, and 100%, respectively; while for uMCP-1 it revealed an AUC of 0.99, 0.77, 0.99, and 1.00, respectively, as well as a sensitivity of 90.91%, 82.61%, 95.74%, and 100% and specificity of 96.74%, 50.00%, 93.33%, and 100% respectively [Table 5] and [Figure 1], [Figure 2].
|Figure 1: (a-d) Receiver operating characteristic curves analysis of uTWEAK in detecting SLE disease, LN, SLE activity and LN activity.|
uTWEAK: Urinary tumor necrosis factor-like weak inducer of apoptosis, SLE: Systemic lupus erythematosus, LN: Lupus nephritis.
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|Figure 2: (a-d) Receiver operating characteristic curves analysis of urinary MCP-1 in detecting SLE disease, LN, SLE activity and LN activity.|
uMCP-1: Urinary monocyte chemo-attractant protein-1, SLE: Systemic lupus erythematosus, LN: Lupus nephritis.
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|Table 5: Receiver operating characteristic curves analysis of uTWEAK and uMCP-1 in the diagnosis of SLE, SLE activity and lupus nephritis among the studied lupus patients.|
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| Discussion|| |
Early detection of LN activity is a very important tool which will help to decrease morbidity and mortality in LN. However, renal biopsy still remains the gold standard method to assess activity in renal tissues. Its main use is in the initial assessment of the patient to decide the degree of glomerulonephritis and the most suitable line of treatment and also for relapses as well as for assessing refractory disease. However, it is an invasive and costly tool which also carries some risks., Re-biopsy can be difficult in most of patients due to its invasive nature and complications, many physicians are still using proteinuria, hypo-complementemia and high titer of anti-ds DNA to diagnose renal flares but sometimes these parameters have limitations because proteinuria may occur due to permanent glomerular damage not actually due to recent activity and also the low C3, C4 and anti-ds DNA have low sensitivity and specificity., Hence, there is a need for a novel, accurate, specific and easily obtained biomarker that can be used for early detection of lupus flares, especially LN. TWEAK is a 249-amino acid cytokine that is a member of the TNF-ligand superfamily. The binding of TWEAK to its sole receptor, Fn14, mediates multiple biologic effects as upregulation of pro-inflammatory mediators known to be essential in LN pathogenesis, including MCP-1 an induction of cell death and apoptosis., In this case–control study we tried to assess the role of uTWEAK and MCP-1 as new biomarkers of LN, so we conducted our study on 114 persons; 22 healthy control, 24 active LN patients; 23 with non-active LN; 22 patients with active SLE without renal involvement; and 23 patients with non-active SLE without renal involvement. We measured SLEDAI, anti-dsDNA, C3, C4, proteinuria and the level of uTWEAK and MCP-1 in all groups and compared to the results. We found that uTWEAK and MCP1 levels higher in SLE with active nephritis group than those of other SLE groups and controls, also there was a significant positive correlation between uTWEAK and uMCP-1 levels in lupus patients and urea, serum creatinine, proteinuria, ESR 1st h, Anti-ds DNA and r-SLEDAI and negatively with C3 and C4.
When comparing our results with other studies we found that some researchers tested both uTWEAK and MCP-1 in lupus patients. Schwartz et al tested the uTWEAK many times and concluded that uTWEAK is a reliable novel biomarker of LN activity and it correlates with SLEDAI and urinary MCP-1 but not with proteinuria. They emphasized that uTWEAK is not less than serum TWEAK in active LN.,, Xuejing et al also concluded that uTWEAK levels were correlated with all active indexes of LN including MCP-1. Xu et al also tested their levels in 31 lupus patients and 10 controls and they found that uTWEAK is increased in active LN with positive correlation with activity index, urinary proteins, anti-ds DNA, MCP-1 and El-Shehaby et al who studied 73 lupus patients and 23 control and they found that uTWEAK and uMCP-1 were significantly elevated in LN patient with high sensitivity and specificity. On the other hand, studies done only on uTWEAK as that of Reyes-Martínez et al, who tested 44 lupus patients and found that uTWEAK can adequately detect renal lupus activity, but cannot predict the degree of histological activity in active lupus nephropathy. In a meta-analysis on eight studies Lee and Song concluded that serum and urinary TWEAK are significantly higher in patients with active LN than in those with inactive LN and that urinary TWEAK levels were positively correlated with renal disease activity.
As regard urinary MCP-1 our study was in agreement with several studies as that of Noris et al, Mirfeizi et al, Watson et al, and Alharazy et al. who found that uMCP-1 was elevated in the urine of LN patients than lupus patients without renal involvements. Interestingly, Rovin et al measured uMCP-1 in lupus patients with follow-up every two months and found that its level is markedly elevated in LN than non-nephritis group or control groups. Furthermore, the level of uMCP-1 was high in urine (2–4) months before lupus flares. In another study, Marks et al. who proved that uMCP-1 increased in active LN and he used BILAG index to measure the disease activity. In our study by ROC curves analysis of uTWEAK and uMCP-1 in detecting SLE disease, SLE activity, LN and LN activity the uTWEAK showed a sensitivity of 100%, 80.43%,100% and 100% and specificity of 100%, 50%, 100% and 100% respectively; while for uMCP-1 it revealed a sensitivity of 90.91%, 82.61%, 95.74% and 100% and specificity of 96.74%, 50.00%, 93.33% and 100% respectively. Some studies reported high sensitivity and specificity of uTWEAK and uMCP-1 like Selim et al also concluded that uTWEAK has sensitivity of 77.3% and specificity of 90% in detecting lupus activity Reyes-Martínez et al who reported a sensitivity of 81% and specificity of 75% for uTWEAK in the diagnosis of active LN, for uMCP-1 in the detection of lupus activity, Taha et al found that its sensitivity was 97% and specificity was 100%. and Mirfeizi et al reported sensitivity of 88.5% and a specificity of 46.3% for uMCP-1 in identifying LN.
Although our study has some limitations as we did not follow-up the patients by measurement of these urinary cytokines after receiving the treatments or attacks of new lupus flares. However, we compared their levels not only in active and non-active LN patients but also in active and non-active lupus without renal involvement and this design enabled us to be sure that their elevation was specifically due to LN activity not due to lupus activity as a whole. Hence, we can conclude from the present findings that both uTWEAK and uMCP-1 are new, easily obtained, accurate markers with high sensitivity and specificity in the detection of LN activity and further prospective studies should be performed to prove their beneficial effect in the early detection of lupus flares.
| Acknowledgment|| |
All authors would like to acknowledge the patients for participation in this work.
Conflicts of interest: None declared.
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Dina Samir Elsaid
Department Biochemistry, Faculty of Science, Ain Shams University, Cairo
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]