Elevated urinary CD80 excretion in children with steroid-responsive nephrotic syndrome

Nakysa Hooman; Hasan Otukesh; Rozita Hosseini; Azar Nickavar; Farzaneh Dastan; Mahboubeh Jafari Sarouei; Parisa Honarpisheh

doi:10.4103/bbrj.bbrj_156_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 6 | Issue : 3 | Page : 367-371

Elevated urinary CD80 excretion in children with steroid-responsive nephrotic syndrome

Nakysa Hooman¹, Hasan Otukesh², Rozita Hosseini², Azar Nickavar², Farzaneh Dastan³, Mahboubeh Jafari Sarouei², Parisa Honarpisheh¹
¹ Department of Pediatric Nephrology, Ali-Asghar Clinical Research Development Center, School of Medicine, Ali-Asghar Children Hospital, Iran University of Medical Sciences, Tehran, Iran
² Department of Pediatric Nephrology, School of Medicine, Iran University of Medical Sciences, Ali-Asghar Children Hospital, Tehran, Iran
³ Chronic Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Date of Submission	01-Jun-2022
Date of Decision	21-Jul-2022
Date of Acceptance	22-Aug-2022
Date of Web Publication	17-Sep-2022

Correspondence Address:
Parisa Honarpisheh
Department of Pediatric Nephrology, Ali-Asghar Clinical Research Development Center, School of Medicine, Ali-Asghar Children Hospital, Iran University of Medical Sciences, Tehran
Iran

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_156_22

Abstract

Background: Idiopathic nephrotic syndrome (INS) is one of the most common glomerular diseases in children with different pathological types and different responses to corticosteroids. A definitive diagnosis is essential for planning the treatment and determining the prognosis of these patients and currently, kidney biopsy is the only method for definitive diagnosis. However, this is an invasive procedure. In addition, in some cases, the biopsy is contraindicated or tissue obtained on biopsy is insufficient and may not represent the underlying disease. According to the recent hypothesis about the role of circulating permeability factors in the pathogenesis of INS, urine protein analysis as a noninvasive method to determine the specific biomarkers of the disease is of great interest to nephrologists and can be useful. Methods: In this case − control study, we analyzed urinary CD80 (uCD80) levels in 51 patients with INS using a special CD80 enzyme-linked immunosorbent assay kit and were compared between different groups of patients. Results: The highest urine CD80/creatinine ratio was found in patients with active minimal change disease and steroid-responsive nephrotic syndrome in the relapse stage of the disease. Conclusion: A significant level of uCD80 is correlated with better renal function and a more favorable response to steroids in patients with INS. Therefore, it can be concluded that a high level of uCD80 is correlated with a good prognosis in these patients.

Keywords: Biomarker, CD80, idiopathic nephrotic syndrome

How to cite this article:
Hooman N, Otukesh H, Hosseini R, Nickavar A, Dastan F, Sarouei MJ, Honarpisheh P. Elevated urinary CD80 excretion in children with steroid-responsive nephrotic syndrome. Biomed Biotechnol Res J 2022;6:367-71

How to cite this URL:
Hooman N, Otukesh H, Hosseini R, Nickavar A, Dastan F, Sarouei MJ, Honarpisheh P. Elevated urinary CD80 excretion in children with steroid-responsive nephrotic syndrome. Biomed Biotechnol Res J [serial online] 2022 [cited 2023 Jun 10];6:367-71. Available from: https://www.bmbtrj.org/text.asp?2022/6/3/367/356149

Introduction

Nephrotic syndrome (NS) is characterized by the triad of severe proteinuria (protein excretion ≥40 mg/m²/h or 1000 mg/m²/24 h or urine protein/creatinine (Cr) ratio ≥2 or >3+ proteinuria on dipstick), hypoalbuminemia (albumin <2·5 g/dL) and edema.^[1],[2] Minimal change disease (MCD) is the most common form of idiopathic NS (INS) in children and the second is focal segmental glomerulosclerosis (FSGS).^[3] Approximately 80%–90% of patients with INS achieve remission [Table 1]^{[18],[19],[20]} within 4 weeks of corticosteroid therapy and are considered to have steroid-sensitive NS (SSNS) [Table 1]. On biopsy, these patients probably have MCD. Ones with SSNS usually have a better outcome, with <5% progress to chronic kidney disease.^[1] The remaining 10%–20% of patients who have primary steroid-resistant NS (SRNS) [Table 1] are more likely to develop FSGS on biopsy, and 50% of them progress to end-stage renal disease within 5 years.^[4],[5] The etiology of INS and the exact mechanism of proteinuria in this disease are not fully understood. The proposed theories include immune dysfunction especially T-cell dysfunction, loss of normal podocyte structure, and genetic abnormalities.^[1] In recent years, circulating cytokines have been suggested in the pathogenesis of the disease and as a link between proteinuria and T-cell dysfunction.^[6] This is supported by the fact that patients with INS respond to corticosteroids and other immunosuppressive drugs.^[7] A definitive diagnosis is essential for planning the treatment of patients with NS and currently, kidney biopsy is the only method for definitive diagnosis. However, this is an invasive procedure and its results in the early stages of the disease are sometimes nondiagnostic and may be contraindicated in some patients.^[8] On the other hand, a biopsy can only give a small part of the kidney, so it may not be possible to accurately describe the disease without removing the affected area of the kidney. In other cases, the disease is so advanced that diagnostic features may be obscured. Therefore, it is necessary to find noninvasive biomarkers for the early detection of INS. Urinalysis can be used to screen for valuable biomarkers instead of a kidney biopsy. This is a simple, safe and accurate method that can be repeated to track the progression of the disease and monitor the response to treatment.^[9] Currently, urine protein analysis to determine specific biomarkers of the disease is of great interest to nephrologists, especially since this is a noninvasive method and was indicated to play a key role in the pathogenesis of INS.^[10] One of these noteworthy biomarkers is CD80. It is a protein that is located in the cell transmembrane and expressed in antigen-presenting cells (APCs). When CD80 binds to its ligand, CD28, on T-lymphocytes, it triggers a co-stimulation signal and promotes their activation.^[11] CD80 is not expressed in podocytes under normal conditions, but under certain conditions, podocytes can acquire the phenotype and/or function of APC and express CD80.^[12],[13] Several studies have demonstrated that the expression of CD80 in podocytes is correlated with functional changes in these cells that may cause proteinuria.^{[11],[14],[15]} These results have led clinical scientists to believe that altered expression of CD80 in podocytes may be involved in the etiology of MCD.^[13] Some podocyte antigens are secreted into the urine.^[16] This led us to hypothesize that CD80 could be detected in the urine of some patients with INS. Several studies have reported increased uCD80 levels in patients with MCD in the relapse stage [Table 1], but a slight increase in urinary excretion of CD80 in patients with MCD in remission or other glomerular disorders such as FSGS was reported.^[6],[17] Therefore, conducting a study to consider the urinary level of CD80 to predict the prognosis and type of NS is worthwhile. This study aimed to evaluate the uCD80 as a prognostic and diagnostic biomarker for differentiating MCD from FSGS NS and SSNS from SRNS in children.

Table 1: Definitions

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Methods

Ethical issue

All procedures performed in this study were according to the Ethical Standards of the Iran University of Medical Sciences Research Committee (Code: IR.IUMS.FMD.REC.1398.304) and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Both parents of participating children signed informed consent form before the study.

Study design

This study was conducted on 51 patients with INS who were referred to the Pediatric Nephrology Department of Ali-Asghar Children's Hospital (Tehran, Iran) from January 2019 to March 2020. Children with age ≥1 year and primary NS who had glomerular filtration rate (GFR) >60 cc/kg/min were included in the study. Children who met the following criteria were excluded from the study: (1) GFR <60cc/min/1.73 m²; (2) inherited NS (age <1 year); (3) secondary NS (e.g., low complement, malignancy, reflux nephropathy, positive serology for vasculitis).

Sampling

Morning urine samples of patients were collected in a sterile container and then centrifuged at 2000 rpm for 20 min. The supernatant was removed and stored at −20°C. The level of urinary CD80 was measured using an enzyme-linked immunosorbent assay (ELISA) kit (ELISA Genie, Human T-lymphocyte activation antigen CD80 ELISA kit, Ireland, code HUEB0376), and its value was reported as nanogram per gram (ng/g) Cr according to the manufacturer's instructions. The quantitative level of urine protein and Cr and serum Cr were measured. The estimated GFR (eGFR) was calculated using the Schwartz formula.

Data collection

Required information includes demographic data, immunosuppressive drugs received by each patient at the time of the study, histopathological results of biopsies, quantitative level of uCD80/Cr (ng/g) and calculated eGFR (cc/min/1.73 m²) of the patients recorded in a prepared checklist.

Data analysis

Mann–Whitney U-test was used to compare the median of the data between the two groups. Quantitative data are presented as the median (interquartile range [IQR]) and a P < 0.05 was considered significant using SPSS version 21 21 (IBM Corp., Armonk, N.Y., USA) software.

Results

In this study, 21 out of 51 patients (41.2%) had biopsy-proven MCD and 12 patients (23.5%) were biopsy-proven FSGS and 18 patients (35.3%) were not biopsied. Based on the clinical response to corticosteroids, 18 out of 51 patients were steroid-sensitive (9 patients in relapse and 9 patients in remission), and 33 patients were steroid-resistant (24 patients in relapse and 9 patients in remission) [Table 2].

Table 2: Distribution of patient's baseline data in the study population

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Fifty-one patients with mean ± standard deviation age of 8.45 ± 3.83 years and a weight of 30.57 ± 14.80 kg were included in the study and 49% of them were male.

In patients with SSNS, eGFR (cc/min/1.73 m²) was shown to be significantly higher than in the steroid-resistant group (P < 0.001). Furthermore, eGFR was revealed to be significantly higher in patients with MCD compared to the patients with FSGS (P = 0.001) [Table 3].

Table 3: Comparison of estimated glomerular filtration rate between different groups of patients with nephrotic syndrome using Mann-Whitney U-test

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uCD80/Cr ratio (ng/g) also differed by sensitivity to steroid, disease activity, and disease type. A significant increase was found in uCD80/Cr excretion in patients with MCD-relapse compared to patients with MCD-remission (median IQR: 206 [222] ng/g vs. 17 (12) ng/g; P = 0.003]. In contrast, no significant difference was found in uCD80 excretion in FSGS patients in the relapse and remission stages [22.50 (8.50) ng/g vs. 14 (10.50) ng/g; P = 0.105; [Figure 1]. No significant difference was found in urinary CD80 levels between MCD and FSGS patients in remission (P = 0.334) but uCD80 was significantly higher in patients with MCD-relapse compared to FSGS-relapse status (median [IQR) was 206.00 [222.00] for MCD-relapse and 22.50 [8.50] for FSGS-relapse; P = 0.003) [Table 4].

Table 4: Comparison of urinary CD80 level between different groups of patients with nephrotic syndrome using Mann-Whitney U-test

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Figure 1: Comparison of the urinary CD80/creatinine ratio (nanogram per gram creatinine) between MCD and FSGS patients in relapse and remission. MCD: Minimal change disease, FSGS: focal segmental glomerulosclerosis

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Figure 2: Comparison of the urinary CD80/creatinine ratio (Nanogram per gram creatinine) between steroid-responsive and steroid-nonresponsive patients in relapse and remission

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Analysis of the uCD80 concentration in steroid-responsive and steroid-resistant groups indicated that uCD80/Cr ratio was significantly higher in the relapse stage of SSNS than those in the remission stage of SSNS and relapse stage of SRNS (median [IQR]: 402 [332] ng/g versus 17 [8] ng/g, P < 0.001) and (median [IQR]: 402 [332] ng/g vs. 124 [221] ng/g, P = 0.001), respectively. In contrast, there was no significant difference in the urinary CD80 concentration between SSNS and SRNS in the remission stage (median [IQR: 17 [8] ng/g vs. 18 [7] ng/g, P = 0.757) [Table 4].

Discussion

In this study, patients were subdivided into different types based on disease activity (relapse vs. remission), type of NS (MCD vs. FSGS), and response to steroids (SSNS vs. SRNS) and uCD80 levels. We found a significant increase in uCD80 values in patients with MCD-relapse compared to FSGS-relapse. On the other hand, while there was no significant difference in the uCD80 between FSGS patients in the relapse and remission status, patients with MCD-relapse had higher uCD80 values than those with MCD-remission. These results indicate uCD80/Cr may differentiate MCD from FSGS in the active stage of the disease and a high uCD80/Cr ratio can favor MCD and avoid repeat biopsy in steroid-resistant MCD patients. Consist to our study, several research reported a significant increase in the urinary level of CD80 in cases with MCD who were in relapse compared to those in remission or those with FSGS and concluded that urinary excretion of CD80 can be a valuable biomarker to distinguish between MCD and FSGS and between MCD in relapse and remission.^{[21],[22],[23]} Their results showed urine-soluble CD80 is increased in relapsed MCD, which may be related to the pathogenesis of the disease and CD80 expression in the podocytes of patients with MCD in the relapse stage of the disease. However, Minamikawa et al.^[24] did not find a significant difference in uCD80 levels between patients with MCD and other glomerular diseases including FSGS. They suggested uCD80 level correlated with the severity of proteinuria and podocyte injury rather than the type of NS. In another study, Ahmed et al.^[25] showed that urinary CD80 levels in children with MCD were significantly higher than those in the control and non-MCD groups. However, contrary to our results, they found no significant difference between MCD patients in remission and relapse states. This discrepancy can be a consequence of differences in the ELISA kit and standard levels.

In our study, urinary excretion of CD80 in patients with SSNS was significantly higher than in SRNS in the relapse stage. In addition, in cases with SSNS, the uCD80 level was significantly different between relapse and remission stages but there was no significant difference between patients with SRNS in relapse and remission stages. These data indicated that an increase in urinary CD80 is closely correlated with relapse in patients with SSNS and can be a valuable biomarker for steroid responsiveness in children presenting with INS. Cara-Fuentes et al.^[26] evaluated the importance of urinary CD80 as a biomarker of eligible response to steroids in MCD patients and suggested that increased levels of uCD80 may be a sensitive biomarker for steroid responsiveness and that their presence is also correlated with long-term preservation of kidney function. Zhao et al.^[27] evaluated urinary CD80 levels in adult patients with biopsy-proven MCD. They showed in MCD patients, urinary CD80 levels were higher in steroid-sensitive cases than in steroid-resistant cases. They concluded that glucocorticoid therapy leads to complete remission only in MCD patients with high urinary CD80 levels. Our patients also showed significantly higher uCD80 in the relapse stage than in the remission stage. Therefore, increased uCD80 can reflect disease activity in patients with NS. Consist to these findings, Liao et al.,^[28] showed the predictable role of CD80 in the relapse of patients with NS. Ling et al.^[29] also showed that urinary CD80 levels reflect progression and remission in children with NS.

In our study, patients with MCD and SRNS have higher eGFR and better renal function than patients with FSGS and SRNS, respectively. Considering that patients with higher eGFR also had higher uCD80/Cr levels, it may be concluded that the high level of uCD80/Cr ratio has correlated with better renal function and favorable prognosis in these patients.

These data indicate that the measurement of uCD80 can be a useful and noninvasive tool to discriminate between MCD and FSGS patients and prediction of response to steroids and therefore prognosis of the disease in children with INS.

Conclusion

The results of our study showed that an increased level of urinary CD80 is associated with a favorable response to steroid treatment and better prognosis in children presenting with INS and may be a valuable noninvasive biomarker in these patients. Therefore, the use of urinary CD80 facilitates the early detection of high-risk patients and may lead to better treatment choices.

Limitations of study

This study like other studies has some limitations including the small sample size, due to the limited study time frame, and fewer referred patients to our hospital secondary to the COVID-19 pandemic. Studies with a larger sample size could propose a cutoff level of uCD80/Cr to discriminate MCD from FSGS and prediction of sensitivity to steroid therapy in pediatrics with NS.

Acknowledgment

The authors would like to thank Vice-Chancellor for the research of the Iran University of medical sciences for support of this research project. We are grateful to the staff of Gholhak clinical laboratory for cooperation in conducting laboratory tests and special thanks to parents who allowed enrolling their children in this study.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors declare that none of the authors have any competing interests.

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