Renal biomarkers in juvenile-onset systemic lupus erythematosus

Background: Juvenile-onset Systemic Lupus Erythematosus (JSLE) is a rare, but severe, autoimmune condition with lupus nephritis (LN) occurring in up to 80% of patients and predisposing to an increased mortality. Aims/Objectives: As part of the UK JSLE Cohort Study this study aimed to identify whether standard and/or novel biomarkers are useful for monitoring LN and to assess whether novel urine biomarkers are released from the podocyte (kidney epithelial cell). Method/Results: A prospective translational study encompassing a clinical cross sectional biomarker analysis, a longitudinal observational biomarker study and scientific laboratory investigation. The study had full ethical approval. Cross sectional investigation identified whether (a) urine biomarkers (monocyte chemoattractant protein-1, MCP1; alpha-1-acid glycoprotein, AGP; interferon producing protein-10, IP10; neutrophil gelatinase associated lipocalin, NGAL) or (b) plasma biomarkers (interferon-gamma, IFN-γ; IP10; tumour necrosis factor alpha, TNF-α; interleukin 6, IL6; IL1β; MCP1; IL13; vascular endothelial growth factor, VEGF) are increased in JSLE patients with active renal disease. JSLE patients (n=60) and healthy controls (n=23) were recruited. Of JSLE patients, 45 were female (75%) and aged 14.7 (12.2-16.1) years. All had ≥ 4 ACR SLE classification criteria (median 5, range 4-9). Urinary MCP1 concentration increased in patients with JSLE active renal disease compared to JSLE non-active renal disease (582 (251-2100) pg/mgCr; 207 (121-280) pg/mgCr; p=0.018) and healthy controls (117 (68-193) pg/mgCr; p=0.005), AGP concentration also increased (JSLE active renal disease: 1517 (753-2389) ng/mgCr; JSLE non-active renal disease: 485 (212-967) ng/mgCr; p=0.027; healthy controls 313 (108-729) ng/mgCr; p=0.013). On cross sectional testing IP10 and NGAL showed no significant difference. Plasma analysis demonstrated increased IL-13 and a trend toward increased IFN-γ, TNF-α, IL-6 and VEGF in patients with JSLE active renal disease (n=12). For prospective longitudinal analysis, JSLE samples were collected at clinical reviews and standard/novel biomarkers assessed against disease activity. The longitudinal JSLE cohort (n=64), seen at 3 (IQR: 2-5) clinical reviews over 364 (182-532) days were aged 14.1 (11.8-15.8) years and 80% female. Active renal episodes had increased concentration of; MCP-1, NGAL, ESR, anti-double stranded DNA, urine albumin:creatinine ratio (UACR), creatinine, and reduced complement 3 (C3), C4 and lymphocytes. On multivariate analysis, MCP-1 and C3 were independent variables (p<0.001) of active renal disease. MCP-1 was an excellent predictor of improved renal disease (AUC: 0.81; p=0.013; specificity 71%, sensitivity 70%); NGAL was a good predictor of worsened activity (AUC 0.76; p=0.04; specificity 60%, sensitivity 61%). An in vitro model was developed using a human podocyte cell line. Podocytes were cultured in macrophage media (activated with IFN-γ or inactive), and subsequently TNF-α, and protein/gene biomarker, TNF-α receptor (TNFR) podocyte expression quantified. Podocyte MCP-1 gene expression increased when exposed to active macrophage media (8.87x10-3+1.3x10-3) compared to inactive media (1.29x10-3+0.9x10-3; p<0.01), as did protein analysis. Podocyte NGAL expression was not directly related to macrophage exposure. The podocyte adopted a pro-inflammatory TNFR2 predominance when exposed to active macrophages. Conclusion Novel biomarkers perform well for monitoring and predicting JSLE associated renal disease. The podocyte up regulates MCP-1 and adopts a pro-inflammatory TNFR2 pathway; the podocyte may be responsible for urinary MCP-1. Further studies are required to determine NGAL signalling. Impact: Increased local, national and international awareness of lupus nephritis. Advancing biomarkers from planning/discovery to prospective validation. Understanding biomarker regulation using in vitro techniques may explain their renal specificity. Biomarker commercialization would truly improve the renal management of JSLE patients.