MOLECULAR MECHANISMS OF DELAYED KIDNEY GRAFT FUNCTION

J. Ma¹, H. Wu¹, P. Wang¹, K.R. Wyburn¹, S. Chadban^2
1Transplantation Research Group, Central Clinical School, University of Sydney, Sydney/NSW/AUSTRALIA, ²Transplantation Lab, Central Clinical School, University of Sydney, Sydney/NSW/AUSTRALIA

Body: HMGB1, a nuclear factor that is released extracellularly as an inflammatory cytokine, is also an endogenous ligand for Toll like receptor 4 (TLR4). We have reported that TLR4 signaling mediates kidney ischemia reperfusion injury (IRI). Here we hypothesise that endogenous HMGB1 mediates cell injury and inflammation in kidney IRI via TLR4 signalling. We aimed to determine 1) whether endogenous HMGB1 contributes to kidney IRI; 2) whether this is via a direct HMGB1-TLR4 interaction; 3). therapeutic potential of neutralizing antibody to HMGB1 in kidney IRI. Methods. Wild-type (WT) and TLR4-/- mice were randomized to receive anti-HMGB1 neutralizing Ab (300 ug per animal), rHMGB1, or appropriate controls by ip injection 1 hour before ischemia or immediately after reperfusion. Kidney ischemia was induced for 22 min followed by reperfusion. Samples were collected day 1 and 5 after reperfusion. Primary TEC cultures were stimulated in vitro with rHMGB1, LPS or medium only. Results. Both mRNA and protein levels of HMGB1 were significantly increased in IRI kidney. Immunofluorescent staining demonstrated expression of HMGB1 by tubular epithelial cells. Mice pretreated with neutralizing anti HMGB1 antibody were protected against kidney IRI, with lower serum creatinine and less tubular damage versus control mice (p<0.05-0.001). Tubulo-interstitial neutrophil (day 1, p<0.001) and macrophage (day 5, p <0.001) infiltration were markedly less in anti-HMGB1 antibody treated mice versus controls. IL6 & TNF-a mRNA levels were significantly reduced in anti-HMGB1 antibody treated IRI kidneys (p<0.05-0.001). Conversely, administration of rHMGB1 post reperfusion d kidney IRI in WT mice. TLR4^-/- mice were protected against kidney IRI, a finding that was unchanged by administration of either anti-HMGB1 Ab or rHMGB1. In vitro, rHMGB1 induced marked up-regulation of TNFa expression through NFkB signaling pathway in WT TECs , compared to negative control, however this was not observed in TLR4^-/- TECs. As with pretreatment, treatment of neutralizing antibody to HMGB1 after reperfusion also protected against kidney IRI. Conclusion. Endogenous HMGB1 is a mediator of kidney damage following IRI, which may operate through the TLR4 pathway. Administration of a neutralizing antibody to HMGB1 either prior to or soon after IRI afforded significant protection, indicating clinical potential.

Disclosure: All authors have declared no conflicts of interest.