|Ahead of print publication
Urinary vascular cell adhesion molecule-1 as a marker of disease activity in lupus nephritis
Shivraj Padiyar1, Theophilus S Vijayakumar2, Samuel Hansdak3, John Mathew1
1 Department of Clinical Immunology and Rheumatology, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Nephrology, Christian Medical College, Vellore, Tamil Nadu, India
3 Department of Medicine, Christian Medical College, Vellore, Tamil Nadu, India
|Date of Submission||14-Oct-2020|
|Date of Acceptance||28-Dec-2020|
|Date of Web Publication||28-Oct-2021|
Department of Clinical Immunology and Rheumatology, Christian Medical College, Vellore, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Objectives: The objective of this study is to study the role of urinary vascular cell adhesion molecule-1 (uVCAM-1) as a marker for disease activity in lupus nephritis (LN).
Methodology: This was a diagnostic study where patients with active LN were taken as cases and those without LN were taken as disease controls. uVCAM-1 was correlated with the classes of LN and standard of care markers.
Results: There was a significant difference between the uVCAM-1 values in cases (59.69 [range: 0.07–13752.5]) pg/mg and controls (2.11 [range − 0.11 − 1138.5] pg/mg [P = 0.02]). Maximum levels of VCAM 1 were seen in Class 4 and Class 5 LN (P = 0.96). Although higher renal systemic lupus erythematosus disease activity index score had a higher median value of uVCAM 1, the values were not statistically significant (P = 0.2). There is a positive correlation between the uVCAM1 and anti-double-stranded DNA (anti-dsDNA) levels (r = 0.38) and a negative correlation between VCAM 1 levels and C3 (r = −0.19).The sensitivity of uVCAM1 for determining the disease activity was 65.2%, and the specificity was 75% at a cutoff value of more than 23.8 pg/mg.
Conclusions: uVCAM 1, although not in isolation, but along with the other standard of care markers may be useful in assessing the disease activity.
Keywords: Activity, biomarker, lupus nephritis, systemic lupus erythematosus disease activity index, urinary vascular cell adhesion molecule-11
| Introduction|| |
Systemic lupus erythematosus (SLE) is an autoimmune disorder with varied manifestations and can affect almost any system in the body. The reported prevalence of this disease worldwide is 20–150 cases/1 lakh population. Renal involvement is seen in about 60% cases of SLE. The course of lupus nephritis (LN) is variable with multiple remissions and relapses, and the prognosis depends upon the age of presentation, sex, class of nephritis, renal function at presentation, and optimal management. Five-year survival varies from 46% to 95%. Renal disease also leads to more cardiovascular-related deaths. Renal biopsy is the gold standard for the diagnosis and staging of LN, which is usually carried out after an abnormal urine analysis. However, renal disease precedes abnormalities in urine analysis, causing a considerable delay in the diagnosis. There are multiple contraindications for renal biopsy such as refractory hypertension, bleeding diathesis, solitary kidney, and technical feasibility in a critically ill patient among others. It is also associated with the risk of infection and bleeding, and obtaining a representative sample is a challenge.
The sensitivity and specificity of markers like anti-double-stranded DNA (antidsDNA) antibodies, complement levels, and anti-C1q antibodies are less than optimum. Complement deficiency (C4) can occur in the normal population, and hence, its use as a marker of disease activity is limited as false-positive rates are high. AntidsDNA Ab is a global marker of disease activity; however, it may be elevated in other associated conditions such as lupus vasculitis, cardiac or CNS lupus, hence decreasing its specificity. Renal SLE disease activity index (SLEDAI) is difficult to interpret in patients with urinary tract infections or in a catheterized sample. The determination of casts in microscopy requires expertise and timed evaluation of sample. Proteinuria cannot be solely relied upon, as it could also be a manifestation of sclerosed glomeruli.
Hence, the choice of these laboratory markers even on a background of the right clinical background has to be interpreted with caution. It is difficult to decide on tapering of immunosuppression based on these available serum biomarkers. Hence, there is an unmeet need of surrogate biomarkers that will correlate with the renal disease activity. A biomarker refers to a biological, genetic, or chemical molecules that can be measured and reproduced and represents the biological or pathological process or which can be used for assessing the treatment response. An ideal biomarker for SLE must have following characteristics: (1) Must be specific for lupus; (2) have a good correlation with kidney activity or damage; (3) be useful for serial monitoring; (4) be superior to routine available parameters; and (5) Cost-effective and easily available.
Vascular cell adhesion molecule-1 (VCAM-1) is an adhesion molecule belonging to the immunoglobulin super family. It plays an important role in the chemotactic process of immune cells – mainly transmigration. VCAM-1 has been found to be elevated in sera, urine of patients with LN, and it positively correlates with disease activity. Urinary biomarkers are preferred as it is noninvasive, can be easily collected, and direct representation of pathological process going on in the kidneys. This study is aimed to look at urinary VCAM (uVCAM-1) as a predictor of disease activity in LN.
Aims and objectives
- To study the role of uVCAM 1 levels in assessing disease activity in LN to compare uVCAM 1 levels with renal biopsy
- To compare uVCAM 1 levels with renal SLEDAI and standard of care markers (anti-dsDNA, C3 C4).
| Methodology|| |
This is a cross-sectional study conducted between June 2014 and May 2016 in the Christian Medical College, Vellore, India. SLE patients attending the rheumatology, nephrology, and medicine outpatient departments fulfilling the inclusion criteria were recruited after obtaining a written consent. The research proposal was approved by the Institutional Review Board [IRB Min. No.8907 dated 09.06.2014].
The study participants were divided into two groups:
- Disease control-SLE without active nephritis.
- SLE patients with renal SLEDAI of zero. These also included those individuals who were on treatment for LN but were in remission.
- SLE overlapping with other causes of renal disease
Cases- SLE with active nephritis.
- Renal SLEDAI more than zero those who are undergoing a biopsy was included in the study.
- SLE overlapping with other causes of renal disease
- Contraindications for a renal biopsy.
All the patients meeting the required criteria were enrolled into the study thus minimizing the chances of any selection bias. At enrollment, based on the investigation sent by the physician as a part of standard care of routine for the patients presenting with SLE, data were collected to calculate the renal SLEDAI. These patients were divided into two arms based on the renal SLEDAI score. Serum levels of anti-dsDNA and serum complements were done as a routine for these patients. Early morning spot urine sample was collected and was centrifuged at 3000G for 30 min to clear the sediments. Aliquots of the clear supernatant were frozen at −20° until the time of analysis. Care was taken to prevent repeated freeze thaw cycles. uVCAM 1 levels were measured using a commercial ELISA kit (Ray Biotech, USA). uVCAM 1 was corrected for urinary creatinine and was expressed in terms of pg/mg. Concentrations of respective molecules were ascertained from the standard curves constructed using manufacturer-supplied standards. Renal biopsy was done for the patients suspected to have active renal disease based on the renal SLEDAI, and the class of LN on biopsy was determined.
uVCAM-1 levels were correlated with routine markers (Anti-dsDNA, C3 C4) and renal SLEDAI. uVCAM-1 was also compared with activity in renal biopsy and class of LN. uVCAM-1 levels were also measured among a set of healthy patients to know the baseline normal value in our population. We recruited 15 healthy controls for the same purpose.
Clinical assessment, including a detailed history and physical examination, was done at the time of recruitment. All the hematological and biochemical tests and serological tests were done, and the parameters were noted in the data abstraction forms.
Sample size calculation
Sample size was calculated with a 10% precision and a sensitivity and specificity of 80 percent (obtained from the composite sensitivity and specificity of anti-double-stranded DNA and serum complements). This was done so that uVCAM-1 matches the sensitivity and specificity of the routinely used markers in clinical practice. In this study, 40 participants in each arm were taken to give a precision of 10–15.
The data entry was done in Excel. The results were analyzed using the SPSS software version 16 (SPSS version 16.0. Chicago, SPSS Inc.).
A 2 × 2 analysis for the diagnostic test was done. The validity and predictive value statistics were presented with 95% confidence interval. As VCAM1 provides levels, the best cutoff was identified using receiver operator characteristic (ROC) analysis.
| Results|| |
Eighty-three patients of SLE fulfilled the inclusion criteria, 9 of them were excluded, of which 5 of them refused consent, 2 of them had overlap with other glomerulonephritis, and biopsy was not feasible for the remaining 2. The remaining 74 patients were followed up for further analysis.
This study cohort comprised of 74 patients, with a mean (±standard deviation) age of 29.55 ± 9.62 years in controls, whereas 27.68 ± 9.25 years in cases. The baseline demographic and laboratory characteristics are tabulated in [Table 1].
Of the 44 cases, 31 (70%) of them had class 4 LN. Eleven percent of them had Class 3 and Class 5 LN.
The mean value of uVCAM-1/cr in healthy controls was 1.18 pg/mg. There was a significant difference between the uVCAM-1/cr values in cases and controls [Figure 1]. The median uVCAM-1 values in the cases were 59.69 (range: 0.07–13752.5) pg/mg, whereas in the controls were 2.11 (range: 0.11 − 1138.5) pg/mg (P = 0.02). The maximum levels of uVCAM-1 were seen in Class 4 and Class 5 LN (P = 0.96) [Figure 2]. However, this was not statistically significant. Furthermore, it was seen that the r2 was 0.023, which is a weak correlation. It was also noted that higher renal SLEDAI score had a higher median value of uVCAM-1 levels. However, the values were not statistically significant (P = 0.2) [Figure 3]. There was a small positive correlation between the uVCAM-1 levels and anti-dsDNA levels (r = 0.38) by Pearson correlation, but was not significant [Figure 4]. There was a nonsignificant negative correlation between VCAM 1 levels and C3 but was not statistically significant (r = −0.19) [Figure 5]. A ROC curve was plotted for determining the sensitivity and specificity, the area under the curve (AUC) was 0.65. The sensitivity of uVCAM-1 for determining the disease activity was 65.22%, and the specificity was 75% at a cutoff value of more than 23.8 pg/mg.
|Figure 1: Scatter plot showing urinary VCAM-1 values in cases and controls. VCAM: Vascular cell adhesion molecule|
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|Figure 2: Scatter plot showing the correlation of VCAM 1 levels with class in lupus nephritis. VCAM: Vascular cell adhesion molecule|
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|Figure 3: Scatter plot showing correlation of VCAM 1 levels with renal systemic lupus erythematosus disease activity index in patients with active nephritis. VCAM: Vascular cell adhesion molecule|
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|Figure 4: Scatter plot showing the correlation of vascular cell adhesion molecule 1 levels with anti-dsDNA. dsDNA: Double-stranded DNA|
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|Figure 5: Scatter plot showing the correlation of urinary vascular cell adhesion molecule-1 levels with C3. VCAM: Vascular cell adhesion molecule|
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| Discussion|| |
This study was aimed at assessing the correlation between this biomarker and disease activity and fill in the lacunae of nonavailability of data on Indian patients.
In our study, majority (70%; n = 31) of the patients who had renal biopsy were found to have Class 4 nephritis, which is similar to existing literature.
The uVCAM 1 levels were significantly higher in those with active renal disease than in controls (P = 0.02). The mean uVCAM 1 in the controls was found to be 1.18 (±1.4) pg/mg of creatinine. Among the different classes of LN, Class 4 and Class 5 had a higher VCAM level than the other classes and are consistent with earlier findings with similar study population, but the value was not statistically significant (P = 0.96).
Higher uVCAM1 levels were seen in higher renal SLEDAI; however, this was not statistically significant. This is in contrast with the positive correlation between renal SLEDAI and uVCAM 1 levels (r = 0.2) of statistical significance (P = 0.011) observed in another study.
The ROC curve showed an AUC to be 0.65, which is not a very good correlation. A similar study done by Mok et al. showed a slightly higher ROC value (AUC 0.73). The sensitivity of this test was found to be 65.28% and specificity was 75% in our study [Figure 6]. This test had a positive predictive value of 55% and negative predictive value of 81% at a value of 23.8 pg/mg. A prior study done by Nazri et al. showed a sensitivity of 71% and specificity of 65% for uVCAM-1.
|Figure 6: Receiver operator characteristic curve showing sensitivity and specificity of urinary vascular cell adhesion molecule-1 levels in determining disease activity|
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Based on the findings of this study, it appears that uVCAM 1 can be used as a marker for active renal disease. Since the sensitivity and specificity is not very high, the predictive values may not be satisfactory on its own, but in combination with other standard of care markers, it will be of use, especially to rule out ambiguities. A recently done study in the pediatric population showed that the addition of uVCAM-1 to other urinary biomarkers increased the AUC to 0.952 to detect active LN. Leukocytic infiltration is a hallmark of severe renal disease and one of the morphological features contributing to the elevated disease activity in LN. Since nephritis is involved in the acute phase of inflammation when leukocytic infiltration is ongoing and since VCAM-1 levels are likely to recede with reduced activity and when chronicity sets in, tracking VCAM-1 levels longitudinally may help monitor disease activity over time.
- Although this study showed that uVCAM 1 is elevated in active renal disease, it did not have statistically significant correlation with biopsy findings. This could be explained possibly by the fact that many people (about 50%) were already on either steroids or second line immunosuppressant. Being a tertiary referral center, this bias was difficult to remove and could have been the major confounding factor. Whether prior doses of steroids significantly reduce the levels of uVCAM 1 is not known. Second, the ideal way to look at the activity in biopsy would be to do NIH activity and chronicity indices, which was not done here due to feasibility issues
- We have assessed the activity at one point in time and not looked at the longitudinal follow up. Hence, by this study, we cannot conclude on the usefulness of this molecule for serial monitoring. However, recently, a published study showed that uVCAM 1 levels drops when the disease goes into remission, highlighting its role in monitoring the disease
- The uVCAM levels were lower than expected. Since most data in literature are from Caucasian participants from the West, whether the levels are truly lower in Asians/Indians are not clear and need further investigation with a larger sample size
- Despite the advantage of ease of using urine as the biomarkers, there are few pitfalls here. Timing of collection – early morning or day time collection, changes in urinary pH due to physiological or pathological conditions, presence of bacteriuria can alter the results.
Even though this study has drawbacks, it still is relevant in the current scenario. This is a first study done in Indian patients. Unlike the previous studies, this study has correlated the values of uVCAM 1 with Class of LN in renal biopsy. Although not statistically significant uVCAM 1 values were found to be higher in Class 4 and Class 5 LN, further studies are needed to establish the role of uVCAM 1 in monitoring the disease and prediction of flares of LN through a longitudinal study.
| Conclusions|| |
uVCAM-1 levels, although not in isolation, but in association with other standard disease activity markers can help better in predicting the renal disease activity.
We are thankful to Dr Suceena Alexander, Associate Professor, Department of Nephrology for helping in this study.
Financial support and sponsorship
A FLUID Research grant (Institutional grant) was approved for the purpose of this study. The funds were used for the purchasing the VCAM 1 ELISA kits and for detecting urine creatinine for all the individuals.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]