Extracellular vesicles reflect the immune-complex glomerulonephritis in pristane-induced lupus - A Pilot Study

Introduction: Pristane-induced lupus is an environmental model, which develops serum autoantibodies and immune-complex glomerulonephritis. Extracellular vesicles (EVs) are increased in proinflammatory states and can be isolated from urine (uEVs) or directly from the kidney (kEVs). Thus, we aimed to evaluate uEVs and kEVs in Pristane-induced Lupus nephritis.

METHODS: BALB/c female mice received a single intraperitoneal injection of 0.5 mL of pristane (n=7) or saline (n=5). After 7 months, renal tissue and urine samples were collected. We investigated the presence of IgG deposit by indirect immunofluorescence assay and kEVs were obtained after the tissue incubation in a dissociation solution (Collagen D, DNAse I) for 30 minutes at 37C°. EVs isolation, from both urine and tissue, was performed using differential centrifugation; 500xg, 5min and 20000xg, 20 min at 4ºC. Also, we have used transmission electron microscopy, nanoparticle tracking analysis (NTA), and nanoscale flow cytometry (nFC, CytoFLEX S) for EV detection. In nFC, EVs were identified by size (Gigamix beads, 100-900nm) and Annexin V and Anti-podoplanin positivity. kEVs were normalized to kidney weight and uEVs to urinary creatinine. This study was approved by the Universidade Federal Fluminense’s animal ethics committee (CEAU-UFF: 4714311022).

RESULTS: IgG deposition was detected only in the Pristane group. Analysis of uEVs showed no differences between groups either by nFC (p=0.2) or NTA (p=0.3). As for kEVs, we observed an increase in mean particle diameter by NTA, (Pristane 180nm ± 6.5nm vs. Saline 120nm ± 2.9nm, p=0.03). In addition, total kEVs and podocyte-derived kEVs were elevated in Pristane when analyzed by nFC (p=0.02 and p=0.05, respectively).

CONCLUSION: Our study suggests that kEVs are related to the presence of IgG and nephritis, which was not associated with urinary levels of EVs.