Early Cellular Rejection of Pig Kidney Perfused With Manipulated Human Blood to Avoid Hyperacute Rejection A. Vega, A. Ramos, F. Val, M. Lo ´ pez-Hoyos, J.C. Ruiz, A.L.M. de Francisco, J. Castillo, M.G. Fleitas, and M. Arias T HE HUMORAL and cellular factors that initiate and support xenograft hyperacute rejection (HAR) are still not well understood. 1 To achieve a better understand- ing of xenograft rejection mechanisms, we have developed an ex vivo perfusion model of pig-to-human kidney xeno- transplantation. 2 The purpose of this study was to evaluate human antiporcine HAR in an ex vivo perfusion circuit, where human blood was manipulated to get some immu- nosuppression effect. MATERIALS AND METHODS Pig kidneys were connected to an ex vivo perfusion circuit with perfusion pressure, flow rate, and diuresis control. Recirculating perfusate was made by 450 cc of either autologous pig (1) or human blood (2 to 6), diluted by Ringer’s lactate solution to a total starting volume of 600 mL. Each of the five human blood groups was specifically dealt with: (1) unmanipulated, (2) complement-de- pleted by heat serum inactivation at 56°C for 30 min, (3) platelets depleted by selective centrifugation, (4) leukocytes depleted by passing through a selective deleukocyte filter, and (5) xenoantibod- ies depleted by staphylococcus protein A column immunoadsorp- tion. The number of experiments was five per group. The experi- ments took place for 180 min or until HAR was evident. Blood samples were collected from the circuit at baseline and 5, 15, 30, 45, 60, 90, 120, and 180 min after starting hemoperfusion. Biopsies were taken from all hemoperfused kidneys at the end of each experiment and fixed in formalin or frozen. RESULTS Graft function declined and HAR appeared after 15 to 90 minutes in porcine kidneys perfused with unmanipulated human blood. Light microscopy studies of these xenoper- fused kidneys indicated HAR with diffuse haemorrhage, thrombosis, and glomerular injury. We did not observe HAR in the 3 hours of the experiment when any type of manipulated human blood was used as perfusate, and some of these experiments did not show rejection. However, some biopsies of pig kidneys perfused with manipulated human blood demonstrated absence of HAR and presence of acute cellular rejection with different degrees of intersti- tial infiltrates of mononuclear cells. DISCUSSION The most striking observation from our data was the demonstration of an acute cellular rejection in less than 3 hours in this ex vivo model of xenotransplantation. This model accurately depicts the pathological entity of human antiporcine HAR. 3 These studies further corroborate that HAR is a complex immune response involving antibody, coagulation system, complement cascade, and inflammatory cells. 4 So, it may be valuable in the isolated evaluation of any of these effector arms simulating in vivo conditions. 5 Reduction in circulation of any of these effector elements (xenoantibodies, complement, platelets, or leukocytes) sub- stantially prolonged the survival of kidney xenograft avoid- ing HAR 6 and developing a fast acute cellular rejection, which may be an important tool to evaluate xenograft rejection because in other experimental models the rise of cellular rejection takes longer. REFERENCES 1. Lawson JH, Platt JL: Transplantation 62:303, 1996 2. Bremier ME, Svalander CT, Haraldson B, et al: Scand J Urol Nephrol 30:213, 1996 3. Fiane AE, Videm V, Scholz T, et al: Transplant Proc 27:3560, 1995 4. Daniels LJ, Platt JL: Kidney Int 58:S28, 1997 5. Kroshus TJ, Bolman RM, Dalmasso AP: Transplantation 62:5, 1996 6. Baldwin WM, Sanfilippo F: Curr Opin Organ Transplantation 2:121, 1997 From the Nephrology (A.R., J.C.R., A.L.M.dF., M.A.), Surgery (A.V., J.C., M.G.F.), Pathology (F.V.), and Immunology Units, (M.L-H.) H.U. “Marque ´ s de Valdecilla”, Santander, Spain. Address reprint requests to Dr Ramos, Hospital Marques de Valdecilla, Servicio de Nefrologia, Avda de Valdecilla, s/n 39008, Santander, Spain. © 1999 by Elsevier Science Inc. 0041-1345/99/$–see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0041-1345(99)00483-2 Transplantation Proceedings, 31, 2643–2644 (1999) 2643