An adapted passive model of anti-MPO dependent crescentic glomerulonephritis reveals matrix dysregulation and is amenable to modulation by CXCR4 inhibition (1)

Anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitides (AAV) are severe inflammatory disorders involving necrotizing inflammation of small blood vessels, often leading to irreversible tis- sue damage and organ failure. The involvement of pathogenic autoantibodies (targeting myeloperoxidase, MPO, and proteinase 3, PR3), which can lead to the activation of neutrophils and their adhesion to glomerular capillary walls, resulting in focal necrosis, extravasation of plasma proteins (e. g. fibrinogen), hemorrhage, and thrombosis.

Mouse model To induce AAV, female C57Bl6J mice (bred in house) were administered 100 ul sheep anti-rat glomerular basement membrane (aGBM) serum via tail vein (i.v.) at day 0. At day 5, mice were administered 0.75 mg of murine monoclonal anti-MPO (aMPO) antibody clones 6D1 and 100 ul LPS (0.5 mg/g) one hour later. Mouse monoclonal antibodies 6D1 (IgG2b) and 6G4 (IgG2c) directed against murine myeloperoxidase (MPO) were generated as described for anti-MPO antibody clone 8F4.

In order to prime glomeruli for MPO-dependent injury, we administer a sub-proteinuric dose of anti-GBM serum, shown to be essential for injury in a murine model caused by autoimmunity to MPO. Following this treatment regimen, mice developed severe injury as demonstrated by assessment of macrohematuria, dipstick hematuria (3+) and proteinuria (> 2+), and approximately 20% loss of initial body weight. On day 12, renal injury was confirmed by histology in 70% of glomeruli, with 10% showing fresh necrosis and 60% crescents. In contrast, robust injury was not observed after single administration of either LPS (0.5 mg/g) + IgG2a/IgG2b isotype mAbs or anti-GBM serum alone (50 ul or 100 ul). Injury was also not induced by a combination of anti-GBM serum anti-MPO mAbs, anti-GBM + IgG2a/IgG2b isotype mAbs with LPS, or anti-MPO mAbs + LPS. Significant induction of Vascular Cell Adhesion Molecule 1 (Vcam-1), Intercellular Adhesion Molecule 1 (Icam-1) and a trend towards higher E-selectin mRNA transcripts, altogether supporting endothelial cell damage in AAV model mice. The up-regulation of ICAM-1 and VCAM-1 were confirmed at the protein level.

Supplemental data.

Dipstick measurements showed that severe hematuria was sustained at high levels (3+) for approximately 10 days before a slight decrease, and continued to be strong (2+) until the end of observation at day 29. Moreover, proteinuria remained at high levels (3+) until day 29. Mice did not recover body weight loss within 29d of observation. Sirius red stain revealed substantial glomerular and tubulointerstitial scarring, attaining highest levels at 29d post injury.

A modified murine AAV model demonstrates robust anti-MPO antibody-dependent, immune-mediated injury in form of fibrinoid necrosis and glomerular cres- cents after 12d, and extensive scarring of glomeruli and tubulointerstitium after 29d with persistent genera- tion of acute lesions. The model reflects pertinent clinical features of AAV, notably severe hematuria, proteinuria and weight loss. Anti-GBM serum recruits leukocytes (especially neutrophils) to the glomerulus, where a deposition of MPO occurs, murine monoclonal anti-MPO antibodies then target persistent inflammation to the glomerular basement membrane. LPS is a common trigger of immunological responses, for instance to bacterial infections, which are often observed prior to onset of AAV in humans.

1. Abou Faycal, Oszwald, Feilen, Contreras, An adapted passive model of anti-MPO dependent crescentic glomerulonephritis reveals matrix dysregulation and is amenable to modulation by CXCR4 …. Matrix Biol. (2022) (available at https://www.sciencedirect.com/science/article/pii/S0945053X22000014).