title: Effect of TNF-alpha blockade on coagulopathy and endothelial cell activation in xenoperfused porcine kidneys.
abstract: BACKGROUND
  Following pig-to-primate kidney transplantation, endothelial cell activation and xenogenic activation of the recipient's coagulation eventually leading to organ dysfunction and microthrombosis can be observed. In this study, we examined the effect of a TNF-receptor fusion protein (TNF-RFP) on endothelial cell activation and coagulopathy utilizing an appropriate ex vivo perfusion system.
  
  
  METHODS
  Using an ex vivo perfusion circuit based on C1-Inhibitor (C1-Inh) and low-dose heparin administration, we have analyzed consumptive coagulopathy following contact of human blood with porcine endothelium. Porcine kidneys were recovered following in situ cold perfusion with Histidine-tryptophan-ketoglutarate (HTK) organ preservation solution and were immediately connected to a perfusion circuit utilizing freshly drawn pooled porcine or human AB blood. The experiments were performed in three individual groups: autologous perfusion (n = 5), xenogenic perfusion without any further pharmacological intervention (n = 10), or with addition of TNF-RFP (n = 5). After perfusion, tissue samples were obtained for real-time PCR and immunohistological analyses. Endothelial cell activation was assessed by measuring the expression levels of E-selectin, ICAM-1, and VCAM-1.
  
  
  RESULTS
  Kidney survival during organ perfusion with human blood, C1-Inh, and heparin, but without any further pharmacological intervention was 126 ± 78 min. Coagulopathy was observed with significantly elevated concentrations of D-dimer and thrombin-antithrombin complex (TAT), resulting in the formation of multiple microthrombi. Endothelial cell activation was pronounced, as shown by increased expression of E-selectin and VCAM-1. In contrast, pharmacological intervention with TNF-RFP prolonged organ survival to 240 ± 0 min (max. perfusion time; no difference to autologous control). Formation of microthrombi was slightly reduced, although not significantly, if compared to the xenogenic control. D-dimer and TAT were elevated at similar levels to the xenogenic control experiments. In contrast, endothelial cell activation, as shown by real-time PCR, was significantly reduced in the TNF-RFP group.
  
  
  CONCLUSIONS
  We conclude that although coagulopathy was not affected, TNF-RFP is able to suppress inflammation occurring after xenoperfusion in this ex vivo perfusion model.
authors.affiliation: Klinik für Allgemein-, Viszeral- und Transplantationschirurgie, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Wolf
authors.initials: W
authors.lastname: Ramackers
authors.affiliation: Klinik für Allgemein-, Viszeral- und Transplantationschirurgie, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Klose
authors.affiliation: Klinik für Haematologie, Haemostaseologie, Onkologie und Stammzelltransplantation, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Tiede
authors.affiliation: Klinik für Haematologie, Haemostaseologie, Onkologie und Stammzelltransplantation, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Werwitzke
authors.affiliation: Klinik für Haematologie, Haemostaseologie, Onkologie und Stammzelltransplantation, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Dennis
authors.initials: D
authors.lastname: Rataj
authors.affiliation: Klinik für Anaesthesiologie und Intensivmedizin, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Friedrich
authors.affiliation: Klinik für Anaesthesiologie und Intensivmedizin, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Kai
authors.initials: K
authors.lastname: Johanning
authors.affiliation: Klinik für Allgemein-, Viszeral- und Transplantationschirurgie, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Florian W R
authors.initials: FW
authors.lastname: Vondran
authors.affiliation: Klinik für Allgemein-, Viszeral- und Transplantationschirurgie, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Bergmann
authors.affiliation: Klinik für Allgemein-, Viszeral- und Transplantationschirurgie, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Schuettler
authors.affiliation: Institut für Pathologie, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Clemens Luitpold
authors.initials: CL
authors.lastname: Bockmeyer
authors.affiliation: Institut für Pathologie, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Jan Ulrich
authors.initials: JU
authors.lastname: Becker
authors.affiliation: Klinik für Allgemein-, Viszeral- und Transplantationschirurgie, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Klempnauer
authors.affiliation: Klinik für Allgemein-, Viszeral- und Transplantationschirurgie, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Winkler
mesh_terms: D000818:Animals; D001778:Blood Coagulation Disorders / Q000517:prevention & control*; D042783:Endothelial Cells / Q000276:immunology; D000068800:Etanercept / Q000008:administration & dosage; D005260:Female; D005947:Glucose; D006085:Graft Survival; D006801:Humans; D007166:Immunosuppressive Agents / Q000008:administration & dosage; D066298:In Vitro Techniques; D007249:Inflammation / Q000517:prevention & control; D007668:Kidney / Q000276:immunology / Q000473:pathology; D016030:Kidney Transplantation / Q000009:adverse effects* / Q000379:methods*; D008353:Mannitol; D019188:Organ Preservation Solutions; D010477:Perfusion / Q000009:adverse effects / Q000379:methods; D011189:Potassium Chloride; D011343:Procaine; D013552:Swine; D013927:Thrombosis / Q000209:etiology / Q000517:prevention & control; D014183:Transplantation, Heterologous / Q000009:adverse effects* / Q000379:methods; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors*
chemical_list: C039984:Bretschneider cardioplegic solution; D007166:Immunosuppressive Agents; D019188:Organ Preservation Solutions; D014409:Tumor Necrosis Factor-alpha; D008353:Mannitol; D011343:Procaine; D011189:Potassium Chloride; D005947:Glucose; D000068800:Etanercept
keywords: TNF-alpha; coagulation; porcine endothelium; xenoperfusion; xenotransplantation