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

Abstract

Introduction: Nephrotic syndrome (NS) is a characterized by massive proteinuria, edema, hypoalbuminemia, and dyslipidemia. Glucocorticoids (GCs), the primary therapy for >60 years, are ineffective in approximately 50% of adults and approximately 20% of children. Unfortunately, there are no validated biomarkers able to predict steroid-resistant NS (SRNS) or to define the pathways regulating SRNS. Methods: We performed proteomic analyses on paired pediatric NS patient plasma samples obtained both at disease presentation before glucocorticoid initiation and after approximately 7 weeks of GC therapy to identify candidate biomarkers able to either predict steroid resistance before treatment or define critical molecular pathways/targets regulating steroid resistance. Results: Proteomic analyses of 15 paired NS patient samples identified 215 prevalent proteins, including 13 candidate biomarkers that predicted SRNS before GC treatment, and 66 candidate biomarkers that mechanistically differentiated steroid-sensitive NS (SSNS) from SRNS. Ingenuity Pathway Analyses and protein networking pathways approaches further identified proteins and pathways associated with SRNS. Validation using 37 NS patient samples (24 SSNS/13 SRNS) confirmed vitamin D binding protein (VDB) and APOL1 as strong predictive candidate biomarkers for SRNS, and VDB, hemopexin (HPX), adiponectin (ADIPOQ), sex hormone-binding globulin (SHBG), and APOL1 as strong candidate biomarkers to mechanistically distinguish SRNS from SSNS. Logistic regression analysis identified a candidate biomarker panel (VDB, ADIPOQ, and matrix metalloproteinase 2 [MMP-2]) with significant ability to predict SRNS at disease presentation (P ¼ 0.003; area under the receiver operating characteristic curve ¼ 0.78). Conclusion: Plasma proteomic analyses and immunoblotting of serial samples in childhood NS identified a candidate biomarker panel able to predict SRNS at disease presentation, as well as candidate molecular targets/pathways associated with clinical steroid resistance.

Plasma Cytokine Profiling to Predict Steroid Resistance in Pediatric Nephrotic Syndrome

“…As has been reported previously, children with SRNS presented at a later age than from those with SSNS (9.5 vs. 5.7 years; P = 0.004). 14 , 17 , 24 As expected, this difference in age was also reflected in significant differences in height and weight between the 2 cohorts ( P < 0.001, see Table 2 ). Moreover, although the SSNS cohort comprised 23% African Americans and 42% Caucasians, the SRNS cohort comprised 43% of each, thus reflecting a significantly greater incidence of SRNS in African American children ( P = 0.001).…”

“…Although we utilized statistical analyses of the cytokine profiles generated from plasma samples of children with SSNS and SRNS to answer these questions in the present study, we and others have also made other efforts toward the same goal using proteomic, metabolomic, and transcriptomic approaches to identify additional interesting candidates that are either predictive of SRNS or molecularly associated with SRNS. 14 , 15 , 16 , 17 , 18 Over the past few years, serum concentrations of a few cytokines have been associated with relapse in NS. For example, elevated serum levels of IL-8 have been associated with NS relapse, and anti-IL8 antibodies have been shown to neutralize the ability of peripheral blood mononuclear cell culture supernatant to induce albuminuria in rats.…”

“…To try to better identify those patients highly unlikely to respond to GC, and thus avoid its unnecessary toxicity, a few studies have tried to identify urinary, plasma, and salivary biomarkers for SRNS in children using cytokine profiling and omics approaches such as proteomics, metabolomics, and transcriptomics. 8 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 As a result, recently T lymphocytes expressing inflammatory cytokines and macrophage migration inhibitory factor in plasma, and MCP-1 in urine have been implicated in persistent proteinuria and SRNS in childhood NS. 8 , 19 , 20 However, despite these efforts, we still lack understanding of the molecular mechanisms regulating GC resistance in NS, and there remain no plasma or urine biomarkers that have been validated to reliably predict GC resistance in NS prior to initiation of GC therapy.…”

Nephrotic syndrome‐associated hypercoagulopathy is alleviated by both pioglitazone and glucocorticoid which target two different nuclear receptors

“…Genetics are known to play a role in steroid-responsiveness (Adeyemo et al, 2018;Asharam et al, 2018;Govender et al, 2019;Gribouval et al, 2018Gribouval et al, , 2019. However, while genetic and other mechanisms underlying steroid-responsiveness in this small cohort were not evaluated in the present study, they have been the subject of previously reported biomarker studies (Agrawal et al, 2020;Gooding et al, 2020). Importantly, none of the children in this small cohort were diagnosed with venous thromboembolism, which occurs in ~3% of childhood NS cases (Kerlin et al, 2012a).…”

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

“…This enabled us to use multiple approaches (see Figure 1) to analyze and interpret the dataset, in terms of predicting, mechanistically defining, and differentiating biomarkers in children with SSNS versus SRNS. Notably, this exact same group of patient samples also has undergone plasma proteomics analyses, 44 and we are now initiating studies to identify additional relevant molecular pathways and biomarkers of steroid resistance in NS using approaches to integrate the proteomics and metabolomics datasets.…”