Department of Nephrology, Hannover Medical School, Hanover, Germany.
Institute for Transfusion Medicine, Hannover Medical School, Hanover, Germany.
Front Immunol. 2019 Dec 20;10:2975. doi: 10.3389/fimmu.2019.02975. eCollection 2019.
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. 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, studies stimulating bone marrow-derived macrophages with LPS and the combination of LPS and heme were performed and cytokine expression was measured. 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. 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. 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.
缺血再灌注损伤(IRI)在实体器官移植中起着重要作用。热缺血时间的长短对肾 IRI 组织损伤的程度至关重要。在这项实验研究中,我们假设肾脏组织中不稳定血红素的释放是由热缺血时间(15 分钟与 45 分钟 IRI)触发的,并介导补体激活、细胞因子释放和炎症。为了诱导 IRI,雄性 C57BL/6 小鼠的肾蒂夹闭时间分别为短时间(15 分钟)和长时间(45 分钟)。实验性缺血后 2 小时和 24 小时,用脱辅基辣根过氧化物酶测定法测定肾脏中的不稳定血红素水平。此外,通过组织学、免疫组织化学和 qPCR 分别测定肾损伤、细胞因子以及 C5a 和 C3a 受体(C5aR、C3aR)的表达。此外,还进行了用 LPS 和 LPS 与血红素组合刺激骨髓来源的巨噬细胞的研究,并测量了细胞因子的表达。炎症和局部组织损伤与热缺血时间的长短相关。与短时间(15 分钟)IRI 相比,长时间(45 分钟)IRI 后肾脏组织中的不稳定血红素浓度显著升高。值得注意的是,在长时间 IRI 后,诱导血红素降解酶血红素加氧酶-1(HO-1)的表达上调,但在短时间 IRI 后则没有上调。C5aR、促炎细胞因子 IL-6 和 TNF-α以及 pERK 在长时间但不在短时间缺血后上调。随后,与短时间 IRI 相比,在长时间 IRI 中,中性粒细胞浸润和促纤维化细胞因子如 CTGF 和 PAI 的上调更为明显。用 LPS 刺激巨噬细胞后发现,血红素存在时 IL-6 的表达增强。最后,血红素清除剂人血清白蛋白(HSA)的给药降低了促炎细胞因子、C3a 受体的表达,并改善了 IRI 后的肾小管功能,表现为增强的α 1 微球蛋白(A1M)吸收。我们的数据表明,长时间的热缺血时间增加了肾脏中的不稳定血红素水平,这与 IRI 依赖性炎症和过敏毒素受体表达上调有关。