Endothelial Cell Ferroptosis Promotes Renal Damage in ANCA-Induced Glomerulonephritis

Background: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are systemic autoimmune diseases characterized by inflammation of small blood vessels and organ damage, including necrotizing and crescentic glomerulonephritis (NCGN). ANCA are circulating immunoglobulin G (IgG) autoantibodies binding to and activating neutrophil granulocytes and monocytes. The persistent inflammation leads to renal cell necrosis. Ferroptosis is a form of programmed cell death characterized by the production of ROS-and iron-dependent lipid peroxidation leading to membrane rupture. The Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) enzyme regulates the generation of lipid peroxides. We tested the hypothesis that endothelial cell ferroptosis contributes to ANCA-associated NCGN. Methods: To analyse the biological significance of ferroptosis in endothelial cells in vivo, we generated MPO -/- ACSL4 EC mice, which lack ACSL4 specifically in endothelial cells. MPO -/- ACSL4 EC mice were immunized with murine MPO, irradiated and subsequently transplanted with hematopoietic cells from C/57BL6J (WT) mice. Mice were sacrificed and analyzed 6-8 weeks following transplantation. Ferroptosis was investigated in vitro using human umbilical vein endothelial cells and ANCA-stimulated neutrophils. Cell death and lipid peroxidation were detected by flow cytometry. Ferrostatin-1 (Fer-1) and siRNA against ACSL4 were used to inhibit ferroptosis. Results: We found increased lipid peroxidation (4-HNE staining) in kidney section of mice with AAV. MPO -/- ACSL4 EC chimeric mice showed reduced renal damage as indicated by less necrotic and crescentic glomeruli supporting the notion that endothelial cell ferroptosis is important for the development of MPO-ANCA-induced NCGN. In vitro experiments revealed that ANCA-activated neutrophils induced endothelial cell death and this effect was prevented by ferroptosis inhibition with Fer-1 and siRNA against ACSL4. In contrast, inhibition of either necroptosis or apoptosis did not prevent endothelial cell death. Finally, ferroptosis inhibition alleviated the accumulation of lipid peroxides and endothelial dysfunction induced by ANCA-activated neutrophils. Conclusions: ANCA-activated neutrophils induce ferroptosis in endothelial cells in vitro and endothelial cell ferroptosis contributes to ANCA-associated NCGN in a murine AAV model.

Background: Lupus nephritis (LN) is a prevalent and severe complication of systemic lupus erythematosus (SLE).Non-invasive diagnostics are limited and current therapies have inadequate response rates.This raises a great need for understanding the cellular and molecular mechanisms for LN development to identify novel diagnostics and therapeutic targets.We previously reported the TNIP1 variant rs4958881 as risks for LN.TNIP1 encodes the protein ABIN1, which is a polyubiquitin binding protein that negatively regulates the immune regulatory transcription factor NF-κB. NF-κB activation leads to the expression of inflammatory cytokines and chemokines.We hypothesize that patients with the risk variant for TNIP1 have increased serum levels of NF-κB-regulated cytokines and chemokines.
Methods: To test our hypothesis, we acquire serum from 30 LN patients in an active flare (UPCR > 500 mg/g) and 7 healthy control individuals.Of the 30 LN patients, 17 patients had the TNIP1 rs4958881 variant.These serum samples were analyzed with a bio-plex array containing antibodies for 48 human cytokine/chemokine regulated by NF-κB.
Conclusions: Our findings indicate that LN patients have an increased serum levels of a panel of specific inflammatory cytokines and chemokines and the presence of TNIP1 risk allele leads to a differential response.These data provide a list of candidate diagnostic markers and potential therapeutic targets and important insight into novel mediators of immune-phenotypes in LN.
Funding: NIDDK Support

FR-PO578 Poster Friday
Glomerular Diseases: Lupus and Vasculitis

Enhanced Neutrophil Activity in Lupus Nephritis Patients With TNIP1 Variant rs4958881
David W. Powell, Makayla Brady, Shweta Tandon, Madhavi J. Rane, Michelle T. Barati, Dawn J. Caster.University of Louisville School of Medicine, Louisville, KY.
Background: There is need for understanding cellular and molecular mechanisms of lupus nephrits (LN) to define novel diagnostics and therapeutic.We previously reported the TNIP1 variant rs4958881 as risks for LN.TNIP1 encodes ABIN1, which negatively regulates the immune regulatory NF-κB.Glomerular neutrophil accumulation has been shown in LN patients and we have shown that transgenic mice with loss of ABIN1 function also develop glomerular influx of neutrophils.Release of neutrophil extracellular traps (NETs) is implicated in loss of immune tolerance in SLE and enhanced serums levels of NETs and antibodies against NET components have been reported in LN patients.Thus, we hypothesize that LN patients with the TNIP1 rs4958881 risk variant have increased NET production/release and recruitment of neutrophils due to NF-κB over activation.
Methods: We tested our hypothesis by performing in vitro transwell migration (chemotaxis) and supernatant DNA measurements (NET release) assays with neutrophils isolated from 6 LN patients with and 6 LN patients without the TNIP1 variant rs4958881 and 6 healthy control subjects.Neutrophil functions were measured in response to known positive activators (FMLF for chemotaxis and PMA for NET production).Interferon (IFN)-γ is prominent activator of neutrophils that was recently implicated in LN and we found that serum levels of IFN-γ are enhanced in LN patients with the rs4958881 variant.Thus, stimulation with IFN-γ was also assessed.

Enhanced
Serum Levels of NF-κB-Regulated Immune Modulators in Lupus Nephritis Patients With TNIP1 Variant rs4958881 David W. Powell, Makayla Brady, Shweta Tandon, Dawn J. Caster.University of Louisville School of Medicine, Louisville, KY.