Podocyte extracellular vesicles and immune mediators as urinary biomarkers in active lupus nephritis

Abstract

Urinary extracellular vesicles (uEVs) and immune mediators have emerged as potential minimally invasive renal biomarkers. Even though active lupus nephritis (LN) is associated with immune complex deposition, tissue inflammation, and podocyte damage, it remains unclear how these parameters are simultaneously altered in systemic lupus erythematosus (SLE). Thus, we aimed to evaluate uEVs as biomarkers in LN, in association with urinary immune mediators. In this cross-sectional study, uEVs were isolated from SLE patients and healthy donors by differential centrifugation and characterized and/or quantified by electron microscopy, nanoscale flow cytometry, and nanoparticle tracking analysis (NTA). Urinary immune mediators were assessed by a multiplex assay. We included 82 patients (42.6 ± 11.3 years-old, 91.4% female), of whom 56.1% (n = 46) had LN, and 18 healthy donors (37.5 ± 8.2 years-old, 83.3% female). No differences were found for particle size/concentration by NTA, but higher counts of total (P = 0.03) and podocyte-derived (P = 0.01) uEVs were observed in SLE patients, especially in active LN (P = 0.02; P = 0.03). We also identified higher urinary levels of cytokines such as IL-6, IL-8, and CCL-2 according to SLE activity and LN (P < 0.05). Significant correlations were observed between uEVs, immune mediators, R-SLEDAI-2K, proteinuria, and albuminuria in active LN. Lastly, the combinatory analysis of podocyte uEVs, IL-6, IFN-γ, IL-8, uCCL-2 and CCL-3 showed a good predictive power to detect active LN (AUC = 0.88, P = 0.0009). Our results suggest that urinary podocyte-derived uEVs and cytokines are associated with LN activity, which may reflect podocyte injury mediated by inflammation. Thus, the combined application of these biomarkers could help to identify patients with podocyte damage and renal inflammation.

Keywords: Extracellular vesicles; Immune mediators; Lupus nephritis; Podocyte; Urine.

Fig. 1

Characterization of urinary extracellular vesicles in patients with systemic lupus erythematosus (SLE) and healthy donors (HD). Electron micrograph of the uEV isolate (scale bars 250 nm) (A). Comparison of particle’s mean diameter and counts by NTA between SLE and HD (B), according do SLE activity (SLEDAI-2K) (C) and inactive/active LN (D). HD: healthy donors; LN: lupus nephritis; SLE: systemic lupus erythematosus.

Fig. 2

Analysis of uEVs in SLE patients by nanoscale flow cytometry. (A) Representative dot plot flow cytometry charts. (B) Comparison of uEVs in SLE patients and healthy donors (C) and according to disease activity (SLEDAI-2 K). (D) Comparison renal involvement (E) and number of immunosuppressive drugs administered to SLE patients. P-values were assessed using Manny-Whitney test. HD: healthy donors; LN: lupus nephritis; SLE: systemic lupus erythematosus; SLEDAI-2K: Systemic Lupus Erythematosus Disease Activity Index; uCr: urinary creatinine; uEVs: urinary extracellular vesicles.

Fig. 3

Analysis of urinary immune mediators in SLE patients according to disease activity based on SLEDAI-2 K (A) and renal involvement/lupus nephritis (B). P-values were assessed using Kruskal Wallis with Dunn post-test. CCL: Chemokine ligand; IFN: interferon; IL: interleukin; LN: lupus nephritis; SLEDAI-2K: Systemic Lupus Erythematosus Disease Activity Index; u: urinary.