Labile Heme Aggravates Renal Inflammation and Complement Activation After Ischemia Reperfusion Injury

Abstract

Background: Ischemia reperfusion injury (IRI) plays a major role in solid organ transplantation. The length of warm ischemia time is critical for the extent of tissue damage in renal IRI. In this experimental study we hypothesized that local release of labile heme in renal tissue is triggered by the duration of warm ischemia (15 vs. 45 min IRI) and mediates complement activation, cytokine release, and inflammation. Methods: To induce IRI, renal pedicle clamping was performed in male C57BL/6 mice for short (15 min) or prolonged (45 min) time periods. Two and 24 h after experimental ischemia tissue injury labile heme levels in the kidney were determined with an apo-horseradish peroxidase assay. Moreover, renal injury, cytokines, and C5a and C3a receptor (C5aR, C3aR) expression were determined by histology, immunohistochemistry and qPCR, respectively. In addition, in vitro studies stimulating bone marrow-derived macrophages with LPS and the combination of LPS and heme were performed and cytokine expression was measured. Results: Inflammation and local tissue injury correlated with the duration of warm ischemia time. Labile heme concentrations in renal tissue were significantly higher after prolonged (45 min) as compared to short (15 min) IRI. Notably, expression of the inducible heme-degrading enzyme heme oxygenase-1 (HO-1) was up-regulated in kidneys after prolonged, but not after short IRI. C5aR, the pro-inflammatory cytokines IL-6 and TNF-α as well as pERK were up-regulated after prolonged, but not after short ischemia times. Consecutively, neutrophil infiltration and up-regulation of pro-fibrotic cytokines such as CTGF and PAI were more pronounced in prolonged IRI in comparison to short IRI. In vitro stimulation of macrophages with LPS revealed that IL-6 expression was enhanced in the presence of heme. Finally, administration of the heme scavenger human serum albumin (HSA) reduced the expression of pro-inflammatory cytokines, C3a receptor and improved tubular function indicated by enhanced alpha 1 microglobulin (A1M) absorption after IRI. Conclusions: Our data show that prolonged duration of warm ischemia time increased labile heme levels in the kidney, which correlates with IRI-dependent inflammation and up-regulation of anaphylatoxin receptor expression.

Interplay of Heme with Macrophages in Homeostasis and Inflammation

“…Accordingly, studies from Nath and colleagues have shown a detrimental role of heme released from hemoproteins in kidney injury [98][99][100]. Furthermore, in an experimental model of renal ischemia-reperfusion injury (IRI), we recently detected high levels of local labile heme in kidneys, which was dependent on the IRI-time and correlated with the IRI-mediated inflammation [101]. The source of heme in this setting is currently not clear but indicates that extracellular labile heme might aggravate an inflammatory response [101].…”

“…Furthermore, in an experimental model of renal ischemia-reperfusion injury (IRI), we recently detected high levels of local labile heme in kidneys, which was dependent on the IRI-time and correlated with the IRI-mediated inflammation [101]. The source of heme in this setting is currently not clear but indicates that extracellular labile heme might aggravate an inflammatory response [101]. Alternatively, labile heme levels in the intravascular space might mediate inflammatory effects on macrophages via secondary factors such as complement activation [102,103].…”

TLR4 Signaling by Heme and the Role of Heme-Binding Blood Proteins

“…Heme toxicity and its pro-inflammatory effects have been demonstrated in experimental disease models like sickle cell disease (SCD), malaria, sepsis, atypical hemolytic uremic syndrome, arteriosclerosis, or ischemia-reperfusion injury ( 27 , 41 , 49 – 52 ). The damaging effects of free heme can be blocked by intracellular factors like heme oxygenases and ferritin, and extracellular factors such as various plasma proteins, respectively ( Figure 1 ).…”

Heme-Oxygenase and Kidney Transplantation: A Potential for Target Therapy?

“…The period of warm ischemia has a high influence on the induction of IRI-related cellular stress. In a recent study by Wang et al [ 56 ], the cellular responses to two different periods of warm renal ischemia (15 vs. 45 min) were studied. Labile heme concentrations in renal tissue were significantly higher after prolonged warm ischemia (45 min) as compared to short warm ischemia (15 min).…”