Impact of heme and heme degradation products on acute kidney injury associated with Shiga toxin 2-induced hemolytic-uremic syndrome

溶血性尿毒症综合征（HUS）是肠出血性大肠杆菌（EHEC）感染的一种严重的肠外并发症。细菌性志贺毒素（Stx），特别是Stx2，在溶血性尿毒综合征发病机制中发挥重要作用，引发血栓性微血管病变，随后引发溶血性贫血、血小板减少症和急性肾损伤的典型三联征。最近，细胞外“游离”血红素作为危及生命感染的持续因素的核心作用，即使是非常中度的溶血，也已得到承认。然而，溶血、“游离”血红素和血红素降解产物（HHDPs）以及血红蛋白和血红素中和蛋白和血红素分解代谢酶（血红素加氧酶1，HO1）对溶血性尿毒综合征急性肾损伤程度的影响，作为伴有炎症的溶血性贫血的范例，需要进一步研究。与其他形式的溶血性贫血形成鲜明对比的是，溶血性尿毒综合征患者血清中促红细胞生成素（EPO）水平平行下降。这可能是由于炎症通过抑制EPO基因表达或溶血性尿毒综合征的特征直接损害肾脏并伴有肾性贫血。众所周知，多效性激素EPO具有器官保护特性，这是独立于其完善的造血作用。越来越多的证据表明，EPO的组织保护作用是由促红细胞生成素受体和b共同受体（bcR）的异络合物介导的，后者在药理学上不同于促红细胞生成素受体同二聚体。因此，溶血/血红素依赖性损伤和EPO/ bcr依赖性保护之间的严重失衡可能加重溶血性尿毒综合征的肾功能障碍。了解这种不平衡可能带来新的治疗策略。在拟议的子项目中，我们旨在确定溶血/细胞外血红素和EPO/bcR信号轴在胡斯综合征相关急性肾损伤发展中的作用。在翻译方法中，我们将专门研究(i)溶血和HHDPs形成的作用（ii）血红蛋白和血红素清除蛋白的影响（iii） HO1的影响，以及（iv） EPO的影响以及EPO在肾功能障碍和肾损伤中的bcr介导的潜在组织保护作用。我们将在stx2介导的实验性溶血性尿毒症模型中系统地解决这些问题，方法是使用缺乏触珠蛋白、血凝素、HO1或bcR的小鼠。这些机制的小鼠研究将通过定量分析溶血性尿毒综合征患者血清样本中的HHDPs和EPO，并将结果与这些患者肾功能不全的程度/结果相关联来加以补充。拟议的研究将使我们能够表征溶血/血红素和EPO/bcR信号轴在Stx2传播的肾功能障碍和损伤的发病机制中的影响，这是终末期肾脏疾病的一个重要危险因素。

The hemolytic-uremic syndrome (HUS) is a serious extra-intestinal complication of infections with enterohemorrhagic Escherichia coli (EHEC). Bacterial Shiga toxins (Stx), in particular Stx2, play an essential role in HUS pathogenesis by triggering thrombotic microangiopathy and subsequently the typical triad of symptoms of hemolytic anemia, thrombocytopenia and acute kidney injury. Recently, the central role of extracellular "free" heme as a perpetuating factor in lifethreatening infections, even with a very moderate degree of hemolysis, has been acknowledged. However, the impact of hemolysis, "free" heme and heme degradation products (HHDPs) as well as hemoglobin- and heme-neutralizing proteins and heme-catabolizing enzymes (heme oxygenase 1, HO1) on the degree of acute kidney injury in HUS, as a paradigm for hemolytic anemia with concomitant inflammation, requires investigation. In striking contrast to other forms of hemolytic anemia, a parallel decrease in erythropoietin (EPO) serum levels has been described in HUS patients. This might be due to either inflammation via inhibition of EPO gene expression or a HUS characteristic direct damage to the kidneys with concomitant renal anemia. The pleiotropic hormone EPO is known to have organ protective properties, which are independent of its well-established hematopoietic effects. There is increasing evidence that the tissue-protective effects of EPO are mediated by a heterocomplex of the erythropoietin receptor and the b-common receptor (bcR) that is pharmacologically distinct from the erythropoietin receptor homodimer conveying erythropoiesis. Thus, a critical imbalance between hemolysis/heme-dependent injury and EPO/bcR-dependent protection might aggravate renal dysfunction in HUS. Understanding this imbalance carries the potential for novel therapeutic strategies. In the proposed subproject we aim to establish the role of hemolysis/extracellular heme and the EPO/bcR signaling axis in the development of HUS-associated acute kidney injury. In a translational approach we will specifically investigate (i) the role of hemolysis and formation of HHDPs (ii) the impact of hemoglobin- and heme-scavenging proteins (iii) the impact of HO1, and (iv) the impact of EPO and the potential bcR-mediated tissue protective effects of EPO on renal dysfunction and injury in HUS. We will address these objectives systematically in a model of Stx2-mediated experimental HUS by employing mice deficient for haptoglobin, hemopexin, HO1 or bcR. These mechanistic murine studies will be complemented by quantification of HHDPs and EPO in serum samples from HUS patients and by correlating the results to the degree/outcome of renal dysfunction in these patients. The proposed studies will enable us to characterize the impact of hemolysis/heme and the EPO/bcR signaling axis in the pathogenesis of Stx2 propagated renal dysfunction and injury, a significant risk factor for end-stage kidney disease.