Increased Urine Excretion of Neutrophil Granule Cargo in Active Proliferative Lupus Nephritis

Abstract

Lupus nephritis (LN) occurs in over half of patients with systemic lupus erythematosus (SLE), but the cellular and molecular events that contribute to LN are not clearly defined. We reported previously that neutrophil degranulation participates in glomerular injury in mouse models of acute LN. The current study tests the hypothesis that glomerular recruitment and subsequent activation of neutrophils results in urine excretion of neutrophil granule constituents that are predictive of glomerular inflammation in proliferative LN. Urine and serum levels of 11 neutrophil granule proteins were measured by antibody-based array in proliferative LN patients and healthy donors (HD), and results were confirmed by ELISA. Glomerular neutrophil accumulation was assessed in LN patient biopsies who contributed urine for granule cargo quantitation and normal kidney tissue by microscopy. Degranulation was measured by flow cytometry in neutrophils isolated from LN patients and HD controls by cell surface granule markers CD63 (azurophilic), CC66b (specific) and CD35 (secretory). Nonparametric statistical analyses were performed and corrected for multiple comparisons. Eight granule proteins (myeloperoxidase, neutrophil elastase, azurocidin, olfactomedin-4, lactoferrin, alpha-1-acid glycoprotein 1 (α-1AG), MMP-9, and cathelicidin) were significantly elevated in urine from patients with active proliferative LN by array and/or ELISA, while only neutrophil elastase was increased in LN serum. Urine excretion of α-1AG declined in patients who achieved remission. The majority of LN glomeruli contained ≥ 3 neutrophils. Basal levels of specific granule markers were increased in neutrophils from LN patients, compared to HD controls. Serum from patients with active LN stimulated specific and secretory, but not azurophilic granule release by HD neutrophils. Circulating neutrophils in patients with LN are primed for enhanced degranulation. Glomerular recruitment of those primed neutrophils leads to release and urine excretion of neutrophil granule cargo that serves as a urine marker of active glomerular inflammation in proliferative LN.