Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
Brigham and Women's Hospital, Harvard Medical School, Boston, MA, United States of America.
PLoS One. 2019 Dec 27;14(12):e0220483. doi: 10.1371/journal.pone.0220483. eCollection 2019.
E. coli associated Hemolytic Uremic Syndrome (epidemic hemolytic uremic syndrome, eHUS) caused by Shiga toxin-producing bacteria is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney injury that cause acute renal failure in up to 65% of affected patients. We hypothesized that the mannose-binding lectin (MBL) pathway of complement activation plays an important role in human eHUS, as we previously demonstrated that injection of Shiga Toxin-2 (Stx-2) led to fibrin deposition in mouse glomeruli that was blocked by co-injection of the anti-MBL-2 antibody 3F8. However, the markers of platelet thrombosis in affected mouse glomeruli were not delineated. To investigate the effect of 3F8 on markers of platelet thrombosis, we used kidney sections from our mouse model (MBL-2+/+ Mbl-A/C-/-; MBL2 KI mouse). Mice in the control group received PBS, while mice in a second group received Stx-2, and those in a third group received 3F8 and Stx-2. Using double immunofluorescence (IF) followed by digital image analysis, kidney sections were stained for fibrin(ogen) and CD41 (marker for platelets), von-Willebrand factor (marker for endothelial cells and platelets), and podocin (marker for podocytes). Electron microscopy (EM) was performed on ultrathin sections from mice and human with HUS. Injection of Stx-2 resulted in an increase of both fibrin and platelets in glomeruli, while administration of 3F8 with Stx-2 reduced both platelet and fibrin to control levels. EM studies confirmed that CD41-positive objects observed by IF were platelets. The increases in platelet number and fibrin levels by injection of Stx-2 are consistent with the generation of platelet-fibrin thrombi that were prevented by 3F8.
产志贺毒素的大肠杆菌相关溶血尿毒症综合征（流行型溶血尿毒综合征，eHUS）的特征是血小板减少症、微血管病性溶血性贫血和急性肾损伤，高达 65%的受影响患者会导致急性肾衰竭。我们假设补体激活的甘露糖结合凝集素（MBL）途径在人类 eHUS 中发挥重要作用，因为我们之前的研究表明，注射志贺毒素-2（Stx-2）会导致小鼠肾小球中的纤维蛋白沉积，而同时注射抗 MBL-2 抗体 3F8 则可以阻止这种沉积。然而，受影响的小鼠肾小球中血小板血栓形成的标志物尚未确定。为了研究 3F8 对血小板血栓形成标志物的影响，我们使用了我们的小鼠模型（MBL-2+/+ Mbl-A/C-/-；MBL2 KI 小鼠）的肾切片。对照组小鼠接受 PBS 注射，第二组接受 Stx-2 注射，第三组接受 3F8 和 Stx-2 注射。使用双重免疫荧光（IF）后进行数字图像分析，对肾切片进行纤维蛋白（原）和 CD41（血小板标志物）、血管性血友病因子（血管内皮细胞和血小板标志物）和 podocin（足细胞标志物）的染色。对来自患有 HUS 的小鼠和人类的超薄切片进行电子显微镜（EM）检查。注射 Stx-2 会导致肾小球中纤维蛋白和血小板数量增加，而同时注射 3F8 和 Stx-2 会将血小板和纤维蛋白减少到对照水平。EM 研究证实，IF 观察到的 CD41 阳性物为血小板。注射 Stx-2 引起的血小板数量和纤维蛋白水平增加与血小板纤维蛋白血栓的形成一致，而 3F8 可防止这种血栓形成。
