Figure 1.

NGAL is increased in mice with ANCA-induced GN and is released by ANCA-activated murine and human neutrophils. (A) Circulating and urinary NGAL levels detected by ELISA were increased in mice with anti-MPO–induced NCGN (AAV) compared with control mice (Ctrl). (B) Representative immunoblots show strong NGAL protein expression in murine neutrophils (Neutro), but not in monocytes (Mono) that were isolated by cell sorting. Actin served as loading control. (C) NGAL protein by ELISA is increased in supernatants of murine neutrophils primed with TNFα and stimulated with 125 µg/ml anti-MPO IgG. Stimulation with murine anti-BSA IgG and 25 ng/ml PMA, respectively, served as controls. (D) Representative immunoblots show strong NGAL protein expression in human neutrophils, but not in monocytes that were purified by cell sorting. Actin served as loading control. (E) TNFα-primed human neutrophils were stimulated with three different human MPO-ANCA, PR3-ANCA, or control IgG preparations (125 µg/ml), respectively. Treatment with either 10⁻⁶ fMLP or buffer (Buf) served as controls. ANCA caused a strong release of neutrophil-specific NGAL 46-kDa homodimers into the supernatant by immunoblotting. A representative immunoblotting experiment is depicted together with the corresponding statistics of all experiments. (F) NGAL by ELISA is increased in supernatants of ANCA-stimulated human neutrophils treated as in (E). ***P>0.001.