Thrombotic Microangiopathic Glomerulopathy in Human Decay Accelerating Factor–Transgenic Swine-to-Baboon Kidney Xenografts

Abstract

Models of pig-to-baboon xenografting were examined to identify the mechanisms and pathologic characteristics of acute humoral xenograft rejection (AHXR). Thymus and kidney (composite thymokidney) from human decay accelerating factortransgenic swine were transplanted into baboons (n ‫؍‬ 16) that were treated with an immunosuppressive regimen that included extracorporeal immunoadsorption of anti-␣Gal antibody and inhibition of complement activation. Morphologic and immunohistochemical studies were performed on protocol biopsies and graftectomy samples. All renal xenografts avoided hyperacute rejection. However, graft rejection coincided with the increase of anti-␣Gal antibody in the recipient's circulation. The 16 xenografts studied were divided into two groups dependent on the rapid return (group 1) or gradual return (group 2) of anti-␣Gal antibody after immunoadsorption. In group 1 (n ‫؍‬ 6), grafts were rejected to day 27 with development of typical AHXR, characterized by marked interstitial hemorrhage and thrombotic microangiopathy in the renal vasculature. In group 2 (n ‫؍‬ 10), grafts also developed thrombotic microangiopathy affecting mainly the glomeruli by day 30 but also showed minimal evidence of interstitial injury and hemorrhage. In the injured glomeruli, IgM and C4d deposition, subsequent endothelial cell death and activation with upregulation of von Willebrand factor and tissue factor, and a decrease of CD39 expression developed with the formation of fibrin-platelet multiple microthrombi. In this model, the kidney xenografts, from human decay accelerating factor-transgenic swine, in baboons undergo AHXR. In slowly evolving AHXR, graft loss is associated with the development of thrombotic microangiopathic glomerulopathy. Also, anti-␣Gal IgM deposition and subsequent complement activation play an important role in the mechanism of glomerular endothelial injury and activation and the formation of multiple microthrombi.

Thrombotic Microangiopathy Associated with Humoral Rejection of Cardiac Xenografts from α1,3-Galactosyltransferase Gene-Knockout Pigs in Baboons

“…19,22,28,[35][36][37][38] Endothelial cell death can cause the loss of vascular integrity, exposure of thrombogenic matrix, and loss of endothelial function, such as the inhibition of coagulation and inflammation. 28,35 Endothelial cell activation, with an increase in vWF and TF expression, and the linked decrease in CD39 expression, can also contribute to graft injury, because this is associated with a shift from an anticoagulant into a procoagulant state.…”

“…Frozen tissue sections were stained by the direct immunofluorescent technique, using fluorescein isothiocyanate (FITC)-conjugated rabbit polyclonal antibody to human IgG, IgM, C3, and fibrinogen (all from DAKO, Carpinteria, CA); and the indirect immunofluorescent technique, using anti-human C4d mAb (Quidel, San Diego, CA), polyclonal rabbit anti-human C4d antibody (American Research Products, Inc., Belmont, MA) and anti-human C5b-9 mAb (DAKO). The following primary antibodies were stained by the standard avidin-biotin-peroxidase complex (ABC) technique 19 : 1) anti-swine CD31 (PCAM1) mAb (Serotec, Raleigh, NC) and anti-MHC class II mAb (ISCR3) 20 that detect capillary endothelium in swine grafts; 2) polyclonal rabbit anti-tissue factor (TF) antibody (the cross-reactive anti-porcine TF antibody was kindly provided by Prof. Yale Nemerson, Mount Sinai School of Medicine, New York, NY), 21,22 which detects TF on porcine activated endothelial cells; 3) anti-pig CD39 mAb, 23 which detects NTP diphosphohydrolase on porcine endothelial cells; 4) anti-human CD41 mAb (5B12, DAKO), which detects baboon platelets; 5) polyclonal rabbit anti-human von Willebrand factor (vWF, DAKO), which detects vWF in endothelial cells and thrombi; and 6) anti-proliferating cell nuclear antigen (PCNA) mAb (PC10, DAKO), which detects proliferating cells. To detect platelet-fibrin thrombi in xenografts, two-color immunohistochemistry for CD41 (Texas Red) and fibrinogen (FITC) was performed.…”

Thrombotic Microangiopathy

“…In fact, CRF-transgenic pig renal grafts succumb to DIC and microangiopathy when transplanted into nonimmunosuppressed baboons, suggesting that it is an early and major host response to vascularized xenografts (7,10). This was confirmed by the work of Shimizu et al (8), where abnormal thromboregulation preceded any cellular response to the graft. This is in contradistinction to prior animal studies that predicted that an innate, immune-mediated accumulation of natural killer (NK) cells and macrophages would be the predominant immune response if HAR could be averted (11).…”

“…However, a proper understanding of the pathogenesis of AHXR has been difficult because of a lack of appropriate animal models and difficulty in completely absorbing anti-␣Gal antibody from the host or removing the ␣Gal epitope from the donor pig. The article by Shimizu et al in this issue of the JASN has clarified many of these complex issues, especially as it relates to renal xenotransplantation (8). In their study, HAR was averted by using kidney grafts from human decay accelerating factor (hDAF) transgenic pigs, and treating the donor with cobra venom factor.…”

“…The study by Shimizu et al identifies several predisposing factors for thrombotic microangiopathy (8). First and foremost, anti␣Gal antibody has long been suspected as a significant factor in this process, and its rapid reappearance correlated strongly with more severe renal injury.…”