A Novel Biomarker Panel to Identify Steroid Resistance in Childhood Idiopathic Nephrotic Syndrome

Abstract

Idiopathic nephrotic syndrome (NS) is the most common glomerular disorder of childhood. Response to initial treatment with corticosteroids is an indicator of prognosis, as resistant patients often present more progressive disease. In this cross-sectional pilot study, we set out to discover a panel of noninvasive biomarkers that could distinguish steroid-resistant nephrotic syndrome (SRNS) from steroid-sensitive nephrotic syndrome (SSNS). Information gleaned from such a panel could yield more individualized treatment plans and prevent unnecessary steroid exposure in patients unlikely to respond. Urine was collected from 50 pediatric patients diagnosed with idiopathic NS at Cincinnati Children’s Hospital Medical Center. Isobaric tags for relative and absolute quantitation (iTRAQ) was used to discover 13 proteins that were differentially expressed in SSNS vs SRNS in a small 5 × 5 discovery cohort. Suitable assays were found for 9 of the 13 markers identified by iTRAQ and were used in a 25 SRNS × 25 SSNS validation cohort. Vitamin D–binding protein (VDBP), alpha-1 acid glycoprotein 1 (AGP1), alpha-1 acid glycoprotein 2 (AGP2), alpha-1-B glycoprotein (A1BG), fetuin-A, prealbumin, thyroxine-binding globulin and hemopexin, and alpha-2 macroglobulin were measured and combined with urine neutrophil gelatinase–associated lipocalin (NGAL), which had been previously shown to distinguish patients with SRNS. Urinary VDBP, prealbumin, NGAL, fetuin-A, and AGP2 were found to be significantly elevated in SRNS using univariate analysis, with area under the receiver operating characteristic curves (AUCs) ranging from 0.65 to 0.81. Multivariate analysis revealed a panel of all 10 markers that yielded an AUC of 0.92 for identification of SRNS. A subset of 5 markers (including VDBP, NGAL, fetuin-A, prealbumin, and AGP2) showed significant associations with SRNS and yielded an AUC of 0.85.

Predicting and Defining Steroid Resistance in Pediatric Nephrotic Syndrome Using Plasma Proteomics

“…podocyte actin cytoskeleton, 29 and to distinguish SRNS via a urine proteomics approach, although it was not identified as a candidate on validation by enzymelinked immunosorbent assay (ELISA). 16 Our studies showed that HPX could discriminate patients with SSNS versus SRNS pretreatment, and that steroid treatment significantly increased plasma HPX levels in SSNS (but not SRNS) patients.…”

“…17,[21][22][23][24][25][26] More recently, proteomic profiling of patients with NS based on their histology and clinical phenotype has yielded a panel of proteins able to distinguish between these different groups. 16,17 These studies had important differences from the current study. First, both of these studies analyzed urine samples.…”

“…Second, neither of these studies included analyses of pretreatment samples that could enable identification of truly predictive biomarkers before initiation of any therapy. Third, the NS classification of Choi et al 17 was based on histology (minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy) and included only adult patients, whereas the classification of Bennett et al 16 was similarly based on SRNS versus SSNS in children. From the discovery set, a few proteins, such as VDB, antithrombin-III (SERPINC1), and HPX, were common between our predictive markers of SRNS versus SSNS, and the markers of Choi et al, 17 which were able to distinguish among minimal change disease, focal segmental glomerulosclerosis, and membranous nephropathy cases.…”

“…From the discovery set, a few proteins, such as VDB, antithrombin-III (SERPINC1), and HPX, were common between our predictive markers of SRNS versus SSNS, and the markers of Choi et al, 17 which were able to distinguish among minimal change disease, focal segmental glomerulosclerosis, and membranous nephropathy cases. However, none of these markers were validated in the study by Choi et al 17 In the Bennett et al 16 study (which had more commonalties in study design), of their 13 identified proteins, VDB, HPX, APOA1, TBG (thyroxine-binding globulin or SERPINA7), a closely related protein to Fetuin-B (Fetuin-A) and zinc-alpha 2 glycoprotein (AZGP1) were commonly identified in our discovery sets of either predictive or mechanistic biomarkers (Tables 2 and 3). Of these, VDB, HPX, APOA1, Fetuin-B, and AZGP1 were also identified in our predictive set, of which VDB was validated in both studies.…”

“…[9][10][11][12][13][14][15] Recently, a few studies have approached urinary biomarker identification using proteomics in patients with NS either classified by histology or by clinical response. 16,17 The present studies were designed to test the primary hypothesis that proteomic analyses with subsequent validation of paired plasma samples from children with SSNS and SRNS can be used to identify biomarkers predictive of steroid resistance at the time of disease presentation before therapy ( Figure 1a). The secondary aim of the present study was to identify mechanistic molecular pathways or targets associated with clinical steroid resistance in NS (Figure 1b).…”