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 NET-related
histone release during GN elicits cytotoxic and immunostimulatory effects. Furthermore, neutralizing extracellular
histones is still therapeutic when initiated in established GN.