Podocyte-derived extracellular vesicles and urinary immune mediators are associated with lupus nephritis activity

Introduction: Lupus nephritis (LN) is a podocytopathy that affects 40-75% of patients with systemic lupus erythematosus (SLE). Since urinary extracellular vesicles (uEVs) can reflect early kidney damage, the detection of uEVs in SLE may suggest the onset of podocyte damage and renal inflammation. Thus, our aim is to evaluate uEVs as early biomarkers of kidney damage in patients with LN, in association with urinary levels of immune mediators. Methods: A cross-sectional study was performed with SLE patients and healthy donors (HD) (IRB#60747722.2.0000.5243). SLE disease activity was measured using the SLEDAI-2K (cutoff ≥5) and renal dysfunction was assessed by its renal domain, R-SLEDAI (cutoff ≥4). A first morning midstream urine was collected and immediately processed (2,000xg 5 min). uEVs were isolated by differential centrifugation (20,000xg 20 min 4ºC) and quantified by nanoscale flow cytometry (nFC, CytoFLEX S, Beckman Coulter) and nanoparticle tracking analysis (NTA, ZetaView). In nFC, polystyrene beads were used for size calibration (~100-900 nm) and positivity for Annexin V and podoplanin were considered for uEV identification. For negative controls during acquisition, solutions of 0.1% Triton-X, Milli Q water, Isotype, Annexin V buffer and antibody mixture were used. For NTA, the uEV suspension was diluted 1:50 in filtered PBS to a final volume of 1 mL. A multiplex assay was used to quantify urinary immune mediators. Importantly, the urinary levels of EVs and immune mediators were normalized by urinary creatinine. Results: We studied 70 SLE patients (42.5±13.9 years, 91.6% female), 28 patients (40%) with NL. NTA showed no differences in the number of particles/mL and medium diameter (nm) between groups. However, we observed higher counts of total and podocyte-derived uEVs (P=0.02; P=0.003) in patients with SLE when compared to HD using nFC. Interestingly, patients with active SLE and R-SLEDAI ≥4 presented higher podocyte uEVs counts (P=0.04). Similarly, active LN was also associated with presented higher podocyte uEVs (P=0.01) and these patients also presented higher urinary levels of IL-6 and CCL-2 (P=0.01; P=0.003, respectively). Lastly, we identified that urinary IL-5, -8, -9, -10, -12 and GM-CSF were significantly correlated with podocyte-derived uEVs in active LN. Conclusions: These findings suggest that podocyte-derived uEVs and urinary immune mediators are associated with SLE activity and LN, probably reflecting renal inflammation.