Fig 1.
Paired ischemic porcine kidney perfusion models.
Porcine kidneys are retrieved from the slaughterhouse and flushed 25min after sacrifice (warm ischemia time). Kidneys are then put on cold storage for 3h until the first period of cold machine perfusion at 4°C. Subsequently, kidneys are flushed one more time before being assigned to their group. Model 1 (green) includes 2 groups; 4h of cold perfusion vs 4h of warm perfusion. Regular perfusate sampling is done during this final perfusion period and at the end, biopsies are taken for further analysis. Model 2 (gray) includes 2 groups of warm perfusion; one kidney from a pair received non-anticoagulant heparin (0.31 mg/mL or 54 U/mL), whilst the other did not receive treatment. A total of 7 kidney pairs were used, from which 3 were perfused up to 4h and the other 4 pairs were perfused up to 8h. Regular perfusate sampling was done and tissue was collected at the end of perfusion.
Fig 2.
Release of extracellular histone H3 is dependent on the amount of ischemic injury.
Extracellular histone H3 was determined semi-quantitatively using Western blotting. Perfusate levels of total histone H3 increased over time in the warm ischemic injury group, whilst remaining constant in the cold ischemia group. Data are presented as median and whiskers include interquartile range.*Indicates significant differences between groups (p<0.05).
Fig 3.
EM images of anti-histone H3 immunolabelling after 4 hours of warm ischemia in moderately damaged renal cells.
Histone H3 (few gold particles indicated with black arrows) was scarcely detected by immuno-EM using antibody labelling with 10 nm gold particles. Top row: representative images are shown for histone H3 localization in a proximal tubule with histone localization in an ovoid cytoplasmic structure (left low magnification, right high magnification). Bottom row: extracellular histone H3 is present in ovoid and spheroid in glomerular endothelial cytoplasmic structures (left low magnification, right high magnification). Scale bar represents 2 μm or 500 nm as indicated.
Fig 4.
EM images of anti-histone H3 immunolabelling after 4 hours of warm ischemia in severely damaged glomerular cells.
Histone H3 was detected by immuno-EM using antibody labelling with 10 nm gold particles (visible as small black dots). Top row: representative images are shown for histone H3 localization surrounding a podocyte with foot process effacement (asterisk, left image), with histones in the cytoplasm (thick arrow and in extracellular debris (thin arrows) in a “bug shot pattern”, right image). Bottom row: extracellular histone H3 is released from an endothelial cell with loss of endothelial fenestrations and subendothelial accumulation of electronlucent material (left image enlarged area in right image) with histone localisation in extracellular debris (thin arrows, right image). Scale bar represents 1 μm or 200 nm or 500 nm as indicated.
Fig 5.
Temperature-dependent cytotoxicity of extracellular histone H3.
Endothelial cells (EA.hy926) were incubated with 10 μg/mL histone H3 in serum-free DMEM for 1 hour at 4°C, 22°C or 37°C. Left panel: representative images from histone treated cells. Nuclei were stained using Hoechst 33342 (in blue) and necrotic cells were stained using PI (in green). Right panel: The number of PI positive cells were quantified in 6 random image fields using ImageJ software. Data is expressed as the mean ± SD of the average percentage of PI positive cells per condition (n = 5).
Fig 6.
Time-dependent release of histone H3 in heparin treated and untreated kidneys with warm ischemic injury.
Extracellular histone H3 was determined in kidneys with or without circulating nonanticoagulant heparin. Kidneys treated with heparin showed a similar release in total histone H3 (A) but a more rapid and pronounced increase in levels of a lower molecular weight histone H3 (LMW-H3) variant (B) compared to untreated counter kidneys. (C) A representative Western blot image of full-length histone H3 (17 kDa apparent MW) and LMW-H3 (15 kDa apparent MW) in perfusate of untreated SNMP kidney at t = 5, 6, 7 and 8 hours of perfusion. *Indicates significant differences between groups (p<0.05).