| Abstract|| |
Idiopathic nephrotic syndrome (NS) is one of the most common kidney diseases of childhood. In this study, we assessed urine Vitamin-D binding protein (VDBP) and neutrophil gelatinase-associated lipocalin (NGAL) levels as a predictor of steroid responsiveness in idiopathic NS. This cross-sectional study included children with steroid-resistant NS (SRNS) (n = 28), steroid-sensitive NS (SSNS) (n = 28), and healthy controls (n = 28). Urine levels of VDBP and NGAL were measured using a commercially available ELISA kit and normalized to urine creatinine (Cr). Urine microalbumin (MALB) was measured using nephelometer, and MALB/Cr was calculated. Urine Vitamin-D binding protein (uVDBP) and urine neutrophil gelatinase-associated lipocalin (uNGAL) levels were statistically significantly higher (P < 0.001) in patients with SRNS (701.12 ± 371.64 ng/mL and 28.42 ± 15.40 ng/mL, respectively) than in patients with SSNS (252.87 ± 66.34 ng/mL and 8.86 ± 5.54 ng/mL, respectively) and normal controls (34.74 ± 14.10 ng/mL and 6.79 ± 1.32 ng/mL, respectively). Estimated glomerular filtration rate shows a significant negative correlation with MALB/Cr, uVDBP, and uNGAL. However, uVDBP and uNGAL showed a much higher discriminatory ability for differentiating SRNS from MALB/Cr. uVDBP and uNGAL at the cutoff value of 303.81 and 13.1 ng/mL, respectively, yielded the optimal sensitivity (82% and 86%) and specificity (78% and 89%) to distinguish SRNS from SSNS. Urine levels of VDBP and NGAL can predict steroid responsiveness in patients with idiopathic NS.
|How to cite this article:|
Choudhary A, Mohanraj P S, Krishnamurthy S, Rajappa M. Association of Urinary Vitamin D Binding Protein and Neutrophil Gelatinase-Associated Lipocalin with Steroid Responsiveness in Idiopathic Nephrotic Syndrome of Childhood. Saudi J Kidney Dis Transpl 2020;31:946-56
|How to cite this URL:|
Choudhary A, Mohanraj P S, Krishnamurthy S, Rajappa M. Association of Urinary Vitamin D Binding Protein and Neutrophil Gelatinase-Associated Lipocalin with Steroid Responsiveness in Idiopathic Nephrotic Syndrome of Childhood. Saudi J Kidney Dis Transpl [serial online] 2020 [cited 2021 Aug 1];31:946-56. Available from: https://www.sjkdt.org/text.asp?2020/31/5/946/301201
| Introduction|| |
Idiopathic nephrotic syndrome (NS) is one of the most common diseases affecting children, characterized by edema, proteinuria, and hypo-albuminemia. The usual histopathological variety in childhood NS is minimal change disease (MCD), but focal segmental glomeru-losclerosis (FSGS) is also known, especially in steroid-resistant NS (SRNS). The prognosis of NS depends on the histopathological changes underlying the disease. Biopsy-proven FSGS is steroid resistant in 70% of cases and is associated with poor outcome, whereas MCD is usually steroid sensitive and has a better prognosis.,, Recently, the incidence of SRNS has been reported to have increased, possibly due to increasing FSGS in children, even though the total incidence of patients diagnosed with NS remains same. FSGS is associated with high recurrence rate as well as leads to end-stage renal disease (ESRD).
In adults, an invasive renal biopsy is the gold standard for the diagnosis of NS. However, in children, renal biopsy is not done at the time of presentation because MCD is the most common histopathological variety, which is usually steroid sensitive. Moreover, multiple biopsies are needed to accurately diagnose FSGS, due to the focal nature of the disease and their effectiveness in predicting the outcome is less., Hence, currently, there is no diagnostic test that could predict steroid responsiveness and long-term prognosis in children with NS to differentiate SRNS from steroid-sensitive NS (SSNS). All children diagnosed with NS were started initially on high-dose corticosteroids daily for a long period and monitored for the response to steroid to serve both diagnostic and therapeutic purposes. A noninvasive diagnostic test that could be performed easily with minimal risk and provide information on the steroid responsiveness and long-term prognosis of children with NS could go a long way in designing better therapeutic strategy and avoiding unnecessary potential exposure to immuno-suppressants for a prolonged period. Therefore, identification of a marker in urine that differentiates SRNS from SSNS gains significance.
Several markers have been identified in urine that is excreted at different levels in SRNS and SSNS. Neutrophil gelatinase-associated lipocalin (NGAL) and Vitamin D-binding protein (VDBP) shows more promise as urinary markers for predicting steroid responsiveness in children with NS. NGAL is a protein found mainly in neutrophils and epithelial tissues, such as distal portion of the nephron in the kidney. NGAL is upregulated in the case of renal injury and acts as a biomarker for acute kidney injury. A recent study has found that higher urine and plasma NGAL levels are associated with the progress of disease severity in chronic kidney disease (CKD). As SRNS is associated with rapid progression of disease severity and poor prognosis than that of SSNS, urinary NGAL levels may help to predict steroid responsiveness in NS. Another pilot study has shown higher urinary levels of NGAL in SRNS than that of SSNS.
VDBP is a circulating plasma protein in the alpha globulin region mainly produced in the liver. Its production is relatively stable in children and adults, though some physiological changes such as pregnancy can increase its levels. The main function of the protein is its ability to bind 25-hydroxyvitamin D. Other biological roles of VDBP to include fatty acid transport, extracellular actin binding, and enhanced chemotaxis action of complement factor C5a by preventing its degradation. Vitamin D deficiency is seen most commonly in both types of NS, which is mainly due to the excretion of a high amount of VDBP in urine. VDBP has similar molecular weight and isoelectric point to that of albumin and excreted in patients with NS. Previous studies have found that the serum levels of VDBP are low in patients with NS and negatively correlate with the urinary levels of VDBP. Urine VDBP also found to correlate positively with urine albumin levels. They have also shown that urine VDBP failed to differentiate SRNS from SSNS. In contrast, some of the studies have found that Vitamin D deficiency is more pronounced in SRNS than SSNS and VDBP can distinguish SRNS from SSNS.
Hence, urine NGAL and VDBP act as a proven marker for prognosis in patients with renal disorders. However, the interrelationship between NGAL and VDBP excretion with steroid responsiveness is not studied to date. There is a lacuna in the literature regarding the ability of urinary NGAL and VDBP in distinguishing SRNS from SSNS. Hence, we undertook to study the urinary levels of NGAL and VDBP as predictors of steroid responsiveness in idiopathic NS.
| Methods|| |
This was a cross-sectional study, conducted in the Department of Biochemistry, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, in collaboration with the Department of Pediatrics from February 2017 to March 2018. The study was approved by the Institute Ethics Committee (Human Studies), JIPMER, Puducherry, India (Approved as project number JIP/IEC/2016/ 1016), and written informed consent was obtained from the parents/legal guardians of all the study participants, prior to their enrollment in the study. The study protocol conformed to the ICMR Biomedical Guidelines for Research in Human Participants.
The study involved two groups – cases and controls. Cases were further subdivided into patients with idiopathic NS who are steroid sensitive (SSNS) and patients with the idio-pathic NS who are steroid resistant (SRNS). Consecutive idiopathic nephrotic syndrome patients attending JIPMER pediatric nephrology clinic fulfilling the inclusion and exclusion criteria were recruited as cases under SRNS and SSNS (28 participants in each group) and 28 healthy patients attending general pediatric outpatient department were recruited as controls. Patients aged (1–12 years) diagnosed with idiopathic NS attending JIPMER pediatric nephrology clinic were recruited as cases. Healthy individuals without any history or evidence of renal disease attending general pediatric outpatient depart-ment were recruited as controls. Exclusion criteria for cases and controls included a history of gross hematuria, inflammation, infection or acute kidney injury, active or recurrent urinary tract infection, and NS secondary to systemic disease such as IgA nephropathy and lupus nephritis.
Sample size calculation
The sample size was calculated based on a prior study done by Bennett et al on urine N-GAL levels in patients with SRNS, those with SSNS and healthy controls. The mean values were 17.3, 6.3, and 6.5 in the three groups. The mean sum of squares within the groups was found to be 573.425. Using this, the calculated effect size (f) comes to 0.56. With type I error as 5% and power as 90%, the calculated sample size was 28 in each group.
Assessment of clinical parameters
Patients attending JIPMER pediatric nephrology clinic, diagnosed with idiopathic NS classified as SRNS and SSNS based on their urinary protein level after treatment with oral prednisolone, were recruited as cases and healthy controls without any history or evidence of renal disease attending general pediatric outpatient department were recruited as controls in the study. Diagnostic criteria for NS include the following: heavy proteinuria: urine protein 3+ or 4+ by dipstick test or urine spot protein:creatinine (Cr) ratio >2 in a morning urine specimen, hypoalbuminemia (serum albumin <2.5 g/dL) and serum cholesterol >200 mg/dL. SSNS is defined by the presence of remission based on their urine protein level, with the use of oral prednisolone within four weeks after initial diagnosis. SRNS is defined as the absence of such remission despite oral prednisolone treatment at a dose of 2 mg/kg for >4 weeks.
The clinical characteristics were recorded in a predetermined proforma. Gender, age, blood pressure, duration of symptoms, body mass index, and intake of concomitant medications were recorded in all the study participants.
Assessment of biochemical parameters
Urine was collected as part of a routine clinic visit, centrifuged for 5 min at 5000 ×g, aliquoted, and stored at −80°C. No more than two freeze-thaw cycles were permitted for use in the study. Demographic and clinical data (urinalysis, most recent serum Cr, steroid-response history, current remission/relapse status, pathology, and immunosuppressant/drug intake) were obtained at the time of enrollment. Urine Cr was assayed using fully automated clinical chemistry autoanalyzer. Microalbumin was assayed using nephelometer to calculate the MALB/Cr levels. Urine NGAL and VDBP were assayed using commercially available ELISA kits. Estimated glomerular filtration rate (eGFR) was calculated from serum Cr using the new Schwartz formula and classified to CKD stage.
| Statistical Analysis|| |
Statistical analysis was performed using IBM SPSS Statistics version 20.0 (IBM Corp., Armonk, NY, USA), and GraphPad Prism. Categorical data were described using percentages and frequencies were compared by using the Chi-square test. The continuous data were expressed as mean with standard deviation. The normality of continuous data was assessed by the Kolmogorov–Smirnov test. Comparison of the various parameters between cases and controls was done by one-way ANOVA and Tukey honestly significant difference test. Pearson's correlation was performed to study the association between the levels of eGFR, MALB/Cr, urine VDBP, and urine NGAL. Receiver operator characteristic (ROC) curve analysis was carried out for biochemical parameters such asurine microalbumin, MALB/Cr, urine VDBP, and urine NGAL to identify the predictors of steroid response in idiopathic NS. Sensitivity and specificity of a combination of parameters were computed and compared with their likelihood ratio obtained when the parameters were assessed alone. The analysis was carried out at 5% level of significance, and P <0.05 was considered statistically significant.
| Results|| |
Fifty-six children with idiopathic NS and 28 normal healthy controls were included in the study. Among the 56 cases with NS, 28 were grouped as SSNS and the remaining were grouped as SRNS. The clinical and biochemical characters of the study participants in SSNS and SRNS group are depicted in [Table 1]. The baseline characters were comparable between the three groups of SRNS, SSNS, and controls, as shown in [Table 2].
|Table 1: Clinical and biochemical characteristics of children with SSNS and SRNS.|
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The biochemical parameters were compared between cases (SSNS, SRNS) and controls. There was a significant decrease in the level of eGFR as calculated by Schwartz formula in SRNS (66.43 ± 18.0 mL/min, P <0.001) group as compared to SSNS (94.76 ± 20.52 mL/min) even though it was above the critical level of 60 mL/min in both the groups. The level of urine microalbumin was significantly higher (P < 0.001) in SRNS group as compared to both SSNS and controls. Similarly, the albumin-Cr ratio (MALB/Cr) was also found to be higher in SRNS (2826.77 ± 2465.28 mg/g) as compared to both groups. Urine Vitamin-D binding protein (uVDBP) and urine neutrophil gelatinase-associated lipocalin (uNGAL) levels were significantly higher (P < 0.001) in patients with SRNS (701.12 ± 371.64 ng/mL and 28.42 ± 15.40 ng/mL, respectively) than in patients with SSNS (252.87 ± 66.34 ng/mL and 8.86 ± 5.54 ng/mL, respectively) and normal controls (34.74 ± 14.10 ng/mL and 6.79 ± 1.32 ng/mL, respectively). There was a significant difference in the levels of uVDBP and uNGAL between SSNS and SRNS as well [Table 2] and [Figure 1]. eGFR shows a significant negative correlation with MALB/Cr, uVDBP, and uNGAL [Figure 2],[Figure 3],[Figure 4]. We also found that MALB/Cr showed a significant positive correlation with uVDBP, and uNGAL. Similarly, uVDBP and uNGAL also showed a strong positive correlation between each other [Table 3].
|Figure 1: Comparison of the study parameters.|
*P-value significant compared to control, #P-value significant compared to steroid-sensitive nephrotic syndrome.
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|Figure 2: Correlation analysis of microalbumin/creatinine with estimated glomerular filtration rate (R < -0.501, P < 0.001).|
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|Figure 3: Correlation analysis of urine Vitamin-D binding protein with estimated glomerular filtration rate (R < -0.263, P < 0.05).|
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|Figure 4: Correlation analysis of urine neutrophil gelatinase-associated lipocalin with estimated glomerular filtration (R < -0.325, P < 0.01).|
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ROC curve analysis was used to assess the ability of MALB, MALB/Cr, uVDBP, and uNGAL as a predictor of steroid response in idiopathic NS. It was observed that MALB/Cr, uVDBP, and uNGAL showed increased sensitivity, specificity and likelihood ratio [Figure 5].
|Figure 5: ROC showing comparison of all the study parameters.|
ROC: Receiver operating characteristic.
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uVDBP and uNGAL showed a much higher discriminatory ability for differentiating SRNS than MALB/Cr. uVDBP and uNGAL at cutoff values of 303.81 and 13.1 ng/mL, respectively, yielded the optimal sensitivity (82% and 86%) and specificity (78% and 89%) to distinguish SRNS from SSNS [Table 4]. Among the three, uNGAL showed the highest sensitivity, specificity, and likelihood ratio for predicting steroid response in patients with NS. The cutoff values of 819.9, 303.81, and 13.1 for MALB/Cr, uVDBP, and uNGAL, respectively, showed high sensitivity, specificity, and likelihood ratio for the population under study [Table 5].
| Discussion|| |
SRNS is usually associated with poor prognosis and can result in progression to ESRD. Currently, there are no diagnostic markers for differentiating SRNS from SSNS. The diagnosis is usually made by observing the response after treatment with corticosteroids. Alternatively, diagnosis can be made by invasive renal biopsy to study the histopathological type of NS to predict steroid responsiveness and long-term prognosis, but this is usually avoided in SSNS of childhood. In our study, we evaluated the efficacy of uVDBP and uNGAL in predicting steroid responsiveness in patients with NS.
SRNS group has lower level of eGFR as compared to SSNS group in our study. eGFR is a marker of renal function widely used to estimate the severity of kidney disease. Lower eGFR in SRNS indicates that the SRNS group has a significant decrease in the renal function as compared to the SSNS group. This is consistent with the established fact that SRNS is more severe form of NS and has a poor prognosis as compared SSNS which usually has better prognosis and remission on treatment with steroids. Our results show that the levels of MALB/Cr, uVDBP, and uNGAL are increased in SRNS as compared to both SSNS and healthy controls. One of the possible explanations for increased excretion of uVDBP in NS is that being low-molecular-weight proteins, it can be filtered freely through the glomerulus, but the reabsorption of uVDBP depends on the structural and functional integrity of megalin and cubulin receptors in renal tubules. Chronic tubular injury to the kidney, which is common due to rapid progression of disease in SRNS, could potentially lead to increased excretion of uVDBP. An earlier study has found increased excretion of uVDBP could be a potential biomarker for fibrosis and interstitial damage by using adriamycin-induced nephrosis in a rat model. In humans, uVDBP was found to increase with microalbuminuria and also in patients with overt proteinuria associated with CKD. This suggests that uVDBP could be a potential marker for tubular damage in patients with NS.
uNGAL is recognized as a marker of ischemic renal injury and also a prognostic marker for CKD., A recent study has shown increased levels of uNGAL in SRNS as compared to SSNS and healthy controls, but did not compare levels between SRNS and SSNS and also suggested to use it as a marker for prognosis in these patients. An earlier study has also shown that uNGAL is increased in the case of acute kidney injury and can act as a marker for renal tubular injury. A previous study also found that uNGAL levels are associated with severity of disease in CKD. This is in line with our hypothesis that because SRNS patients with greater risk of progression to ESRD will have more tubular damage than those of SSNS, which has a good prognosis.
In our study, we observed significant negative correlation between eGFR and various urinary parameters such as uVDBP, uNGAL, and MALB/Cr. A previous study has reported that eGFR below 60mL/min/1.73 m2 is positively correlated with albumin excretion in urine, but it did not comment on the correlation of albumin excretion and eGFR ≥60 mL/min/1.73 m2. Another study has reported that elevated ACR is associated with a faster decline in eGFR in diabetic patients.
These studies imply that in healthy controls with normal GFR, there is no significant albumin excretion, but when GFR starts to decline due to loss of nephrons owing to the progression of the disease, there is a compensatory hyperfiltration in the remaining nephrons, causing increased albumin excretion in urine. The positive correlation between albumin excretion and eGFR has been reported in literature only when the eGFR is significantly lower than normal limits. In our study, the eGFR of the participants is well above the critical limits of ≥60 mL/min/1.73 m2. Further decrease in eGFR below the critical levels may decrease the filtration and may decrease the albumin excretion. Hence, there may be an inverse relation between albumin excretion and eGFR especially when eGFR is ≥60 mL/min/1.73 m2.
There is a significant positive correlation of uVDBP and uNGAL with MALB/Cr, suggesting that high levels of albumin levels in urine are associated with increased levels of uVDBP and uNGAL. SRNS compared to SSNS, being a progressive form of NS with poor prognosis, is expected to have increased tubular damage, also resulting in the excretion of LMW proteins such as uVDBP and uNGAL in urine. A previous study has also found a significant positive correlation of uVDBP and uNGAL levels with proteinuria in patients of CKD.
The results from the ROC curve analysis evaluate the ability of different urine markers to predict steroid responsiveness in children with idiopathic NS. We observed that uNGAL showed the highest sensitivity, specificity, and likelihood ratio than uVDBP and MALB/Cr. MALB/Cr, uVDBP, and uNGAL showed an AUC of 0.876, 0.897, and 0.901, respectively, indicating that all the three parameters have significant predicting power. AUC of 1.0 is a perfect predictor and AUC of 0.5 is considered to have no discrimination power. uNGAL with AUC of 0.901 is considered a better predictor of steroid responsiveness in NS. Further, we found that cutoff value of 819.9, 303.8, and 13.1 for MALB/Cr, uVDBP, and uNGAL, respectively, showed the highest likelihood ratio of 3.2, 3.8, and 8.0, respectively. This study shows that MALB/Cr, uVDBP, and uNGAL can be used to predict the steroid responsiveness accurately in NS.
Thus, we conclude that uVDBP and uNGAL can differentiate SRNS from SSNS. Although these biomarkers cannot replace biopsy for diagnosis of steroid response in NS, it can be used to gain valuable information to clinicians for personalized treatment to individuals with NS.
Our study is not without limitations. This was a single-center cross-sectional pilot study with small sample size done on patients who were already on treatment before the start of the study. A large multicenter prospective study to determine urine VDBP and NGAL levels at baseline before the start of treatment and following up of the uVDBP and uNGAL levels with the progress of disease would be required to confirm our preliminary findings. Histopathological diagnosis is also required to confirm whether uVDBP and uNGAL can predict the steroid response. Because steroid-sensitive patients do not undergo biopsy, it is very difficult to confirm the hypothesis with certainty.
To determine the different markers for differentiating steroid-resistant idiopathic NS, larger cohort studies with longer duration of follow-up are required to assess the effect of treatment on renal dysfunction markers in idiopathic NS. Future studies on the molecular basis of renal dysfunction markers (involving genotyping, gene expression and microRNA studies) in the pathogenesis of renal dysfunction in idiopathic NS are required to validate our findings further.
| Acknowledgment|| |
The present study was funded by the JIPMER Intramural Research Grant. Funding from JIPMER, Puducherry, India in the name of the corresponding author is gratefully acknowledged.
Mohanraj P. S. is currently working as Assistant Professor at All India Institute of Medical Sciences, Gorakhpur. Amit Choudhary and Mohanraj P. S. contributed equally for this research and hence are co-first authors for this study.
Conflict of interest: None declared.
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Department of Biochemistry, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry-605006
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]