Extracellular histones cause vascular necrosis in severe glomerulonephritis

Crescentic glomerulonephritis is characterized by glomerular
necrosis. Dying cells release intracellular proteins that act as
danger-associated molecular patterns to activate the innate immune
system. Previously, we have demonstrated that dying tubular cells
release histones, which can kill endothelial cells and activate the
toll-like receptor 2/4 (TLR2/4). This drives tubulointerstitial
inflammation in septic or post-ischemic acute kidney injury (AKI).
Furthermore, other groups have also reported that extracellular
histones cause organ damage during acute lung injury, stroke,
peritonitis and retinal dysfunction, and that blocking
extracellular histones represents a beneficial approach during the
disease progression. In this thesis, we investigated whether
extracellular histones can elicit similar pathogenic effects during
necrotizing glomerulonephritis. To do so, we used an animal model
based on the necrotizing type of severe glomerulonephritis.
Necrotic glomerulonephritis was induced in mice by a single
intravenous injection of 100µl sheep anti-GBM antiserum. The impact
of histone neutralization was studied by using an antibody isolated
from the BWA-3 clone, which had the capacity to neutralize released
extracellular histones in-vivo and in-vitro. After 7 days, mice
were sacrificed and kidneys were collected for further data
analysis. Proteinuria was assessed in spot urine samples. Anti-GBM
treated mice showed increased proteinuria (albumin/creatinine
ratio), plasma creatinine and BUN levels. This was associated with
a reduced number of podocytes, increased crescentic glomeruli and
the infiltration of neutrophils and macrophages into the kidney.
Interestingly, neutralization of extracellular histones
significantly reduced proteinuria leading to less podocyte damage.
This was linked to an improved renal function defined by lower
plasma creatinine and BUN levels, and with a decrease in neutrophil
and macrophage infiltration and activation in kidney. Histone
blockade also significantly reduced renal mRNA expression of TNF-α
and fibrinogen in the glomerular capillaries, which was associated
with less glomerulosclerosis, crescents and tubular atrophy.
In-vitro studies demonstrated that extracellular histones and
NETs-related histones kill glomerular endothelial cells, podocytes
and parietal epithelial cells in a dose-dependent manner.
Histone-neutralizing agents such as anti-histone IgG, activated
protein C or heparin prevented this cytotoxic effect. Stimulation
of BMDCs with histones upregulated the expression of the activation
marker including MHC-II, CD48, CD80 and CD86 significantly as well
as increased the production of TNF-α and IL-6. It has been
previously reported by others including us that in biopsies from
patients with ANCA-associated vasculitis showed an over-expression
of the TLR2/4 receptor compared to the healthy glomeruli. Histone
toxicity on glomeruli ex-vivo was also dependent on the TLR2/4
receptor axis given that the lack of TLR2/4 attenuated
histone-induced renal thrombotic microangiopathy and glomerular
necrosis in mice. Anti-GBM glomerulonephritis involved NET
formation and vascular necrosis, while blocking NET formation via
PAD inhibitor or pre-emptive anti-histone IgG injection
significantly reduced all parameters of glomerulonephritis
including vascular necrosis, podocyte loss, albuminuria, cytokine
induction, recruitment and activation of glomerular leukocytes, and
glomerular crescent formation. Finally, to evaluate histones as a
therapeutic target, mice with established glomerulonephritis were
treated with three different histone-neutralizing agents such as
anti-histone IgG, recombinant activated protein C and/or heparin.
Interestingly, all agents were equally effective in abrogating
severe glomerulonephritis, while combination therapy had no
additive effect. In summary, the results of this thesis indicate
that NET-related histones released during glomerulonephritis elicit
cytotoxic and immunostimulatory effects and that neutralizing
extracellular histones, therefore, represents a potential
therapeutic approach when applied during already established
glomerulonephritis.