Endothelial but not systemic ferroptosis inhibition protects from antineutrophil cytoplasmic antibody-induced crescentic glomerulonephritis.

Abstract

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are systemic autoimmune diseases featuring small blood vessel inflammation and organ damage, including necrotizing crescentic glomerulonephritis (NCGN). Persistent vascular inflammation leads to endothelial and kidney cell necrosis. Ferroptosis is a regulated cell death pathway executed by reactive oxygen species and iron-dependent lipid peroxidation culminating in cell membrane rupture. Here we show that ANCA-activated neutrophils induced endothelial cell (EC) death in vitro that was prevented by ferroptosis inhibition with Ferrostatin-1, Liproxstatin-1 and small inhibiting RNA against the enzyme AcylCoA Synthetase Long Chain Family Member 4 (ACSL4). In contrast, neither necroptosis nor apoptosis inhibition affected EC death. Moreover, both ferroptosis inhibitors alleviated lipid peroxide accumulation in EC. Increased lipid peroxidation was detected in kidney sections of AAV mice by immunohistochemistry. We generated MPO^-/- ACSL4^flox Tie2-Cre⁺ mice lacking ACSL4 specifically in EC (ACSL4^ΔEC) to study the significance of endothelial ferroptosis in vivo. ACSL4^ΔEC chimeric mice, but not control mice (ACSL4^WT), were protected from NCGN in a MPO-AAV bone-marrow transplantation model. These data establish that EC ferroptosis contributes to ANCA-induced glomerulonephritis. However, systemic pharmacological ferroptosis inhibition with Ferrostatin-1 or Liproxstatin-1 did not protect from NCGN in a murine AAV model. Ferrostatin-1 treatment both directly activated T cell proliferation and indirectly myeloid-mediated T cell proliferation and polarization in vitro. Conceivably, both effects may cancel the beneficial effect of endothelial ferroptosis inhibition. Mechanistically, we describe the importance of EC ferroptosis for the development of AAV. However, the lack of protection with systemic pharmacological ferroptosis inhibition should discourage clinicians from evaluating this treatment strategy in clinical AAV studies.