Neutrophil Extracellular Trap-Related Extracellular Histones Cause Vascular Necrosis in Severe GN

Abstract

Severe GN involves local neutrophil extracellular trap (NET) formation. We hypothesized a local cytotoxic effect of NET-related histone release in necrotizing GN. In vitro, histones from calf thymus or histones released by neutrophils undergoing NETosis killed glomerular endothelial cells, podocytes, and parietal epithelial cells in a dose-dependent manner. Histone-neutralizing agents such as antihistone IgG, activated protein C, or heparin prevented this effect. Histone toxicity on glomeruli ex vivo was Toll-like receptor 2/4 dependent, and lack of TLR2/4 attenuated histone-induced renal thrombotic microangiopathy and glomerular necrosis in mice. Anti-glomerular basement membrane GN involved NET formation and vascular necrosis, whereas blocking NET formation by peptidylarginine inhibition or preemptive anti-histone IgG injection significantly reduced all aspects of GN (i.e., vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment or activation of glomerular leukocytes, and glomerular crescent formation). To evaluate histones as a therapeutic target, mice with established GN were treated with three different histone-neutralizing agents. Anti-histone IgG, recombinant activated protein C, and heparin were equally effective in abrogating severe GN, whereas combination therapy had no additive effects. Together, these results indicate that NETrelated histone release during GN elicits cytotoxic and immunostimulatory effects. Furthermore, neutralizing extracellular histones is still therapeutic when initiated in established GN.

Necroinflammation in Kidney Disease

“…It is the release of histones that induces glomerular endothelial cell necrosis. 92 This became obvious because either the specific inhibition of NETosis or the neutralization of extracellular histones abrogated crescentic GN. 92 Similarly, NETs are also found in necrotic glomerular lesions in human renal vasculitis.…”

“…92 This became obvious because either the specific inhibition of NETosis or the neutralization of extracellular histones abrogated crescentic GN. 92 Similarly, NETs are also found in necrotic glomerular lesions in human renal vasculitis. 93 Complement-mediated cytotoxicity is another trigger for glomerular necroinflammation.…”

“…94 Glomerular cell necrosis induces DAMP release that further drives cytokine and chemokine release, leukocyte recruitment, and inflammation. 92 Infiltrating immune cells, in turn, further contribute to necroinflammation by NETosis (neutrophils), 92 cytokine-induced cell death (all proinflammatory leukocytes), 95 or direct T cell-related cytotoxicity. 96 Vascular wall rupture not only facilitates hematuria but also plasma leakage as a major driver of parietal epithelial cell hyperplasia and crescent formation.…”

“…101 Circulating neutrophils get increasingly involved and contribute to thrombus formation via NET formation, 102 which is associated with local histone release. Extracellular histones elicit three different impacts: they kill microvascular endothelial cells, 74,92,103 trigger platelet activation and further thrombosis, 104 and activate innate immunity via TLRs and the NLRP3 inflammasome. 103,105 As a proof of principle, histone injection into the renal artery induces widespread TMA and renal cell necrosis.…”

“…35 Along these lines, preventing NETosis with a PAD4 inhibitor or extracellular histone neutralization abrogates crescentic GN. 92 Autoacceleration of necroinflammation implies that the time point of intervention is of critical importance. Only preemptive blockade of necroinflammation can be maximally What is the role of apoptosis versus regulated necrosis inside the kidney?…”