[Prevention of ischemia before cold ischemia by pharmcologic donor conditioning--experimental findings]. / Ischämieprotektion vor Kaltischämie durch pharmakologische Spenderkonditionierung--Experimentelle Befunde.

The loop-diuretic piretanide was used to study the influence of pharmacological donor pretreatment on immediate postischemic function in a pig model of kidney transplantation based on the results of a clinical pilot study [4]. Following laparotomy, both kidneys were flushed via a transaortal catheter with Eurocollins-solution and surgically removed. A cold ischemic period of 1 or 24 h was chosen. After that period, kidneys were reperfused with intraoperatively drawn heparinized blood for one hour. We used a special instrument for hemoperfusion of isolated organs which allows perfusion for several hours under steady-state conditions. Four groups were formed: Control and piretanide, 1 or 24 h cold ischemia. The perfusion of piretanide-treated organs resulted in a lower perfusion-resistance, calculated as pressure/flow-ratio or as pressure/glomerular filtrationratio. The flow in the pretreated group was higher, thus excluding a higher shunt-volume. In parallel, oxygen consumption as a parameter of postischemic function start and creatinine clearance were higher in the piretanide treated groups. The experiments demonstrate a superior postischemic function of pretreated kidneys in comparison to control organs after 1 and 24 hours of cold ischemia in this model.

Immunopathological observations after xenogeneic liver perfusions using donor pigs transgenic for human decay-accelerating factor.

BACKGROUND: Donor pigs transgenic for human decay-accelerating factor (hDAF) were used in a xenogeneic ex vivo liver perfusion model to study the effect of this modification on the development of hyperacute rejection. METHODS: Three transgenic pigs were hepatectomized after hypothermic portal and transaortal gravity perfusion. Livers from six nontransgenic pigs served as controls. All livers were perfused for 3 hr with human blood from two donors diluted to a hematocrit of 30%. Particular importance was placed on the use of an optimal perfusion technique incorporating the floating suspension of the organs in a waterbath and intermittent external pressurization. Biochemical, physiological, and immunological parameters were assessed. Tissue specimens taken before and after perfusion were analyzed using routine histology, electron microscopy, and immunohistology. RESULTS: Complement activation was more pronounced in the control group. AP50 and CH50 values fell to about 60% of the initial levels in control experiments, whereas they remained at 80% of the initial levels during perfusion of hDAF livers. After 180 min, pig tumor necrosis factor alpha levels were 7862+/-1645 pg/ml for unmodified livers and 2830+/-734 pg/ml in the hDAF group. Human tumor necrosis factor alpha levels were similar in both groups. Control livers showed marked morphological alterations and distinct deposition of complement factors, whereas livers expressing hDAF showed no signs of hepatocellular necrosis and almost no complement deposition beyond C3 activation. CONCLUSIONS: These results confirm that the transgenic expression of the human complement regulatory protein hDAF reduces complement activation and prevents hyperacute rejection in a xenogeneic liver perfusion model over the 3-hr evaluation period used in this study.

Morphology of hDAF (CD55) transgenic pig kidneys following ex-vivo hemoperfusion with human blood.

Discordant xenotransplantation of pig kidneys into man may be possible in the future using transgenic organs which regulate complement activity. It was the aim of this experimental study to characterize morphologic alterations of organs transgenic for human decay accelerating factor (hDAF/CD55) perfused with human blood since no data on function of these organs after exposure to human blood are available. An ex-vivo system was developed that allows computer driven pressure-controlled perfusion of kidneys including a separate cartridge oxygenator circuit. Following cold ischemia time of 1-4 hr, 8 kidneys from heterozygote transgenic animals (TG) and 9 control kidneys (C) were perfused with 500 ml freshly drawn heparinized human blood at physiological conditions. A histologic grading system from 0 to +4 was used to describe the histologic findings. Using a mouse antihuman DAF moAB, hDAF was stained on all TG kidneys both on glomerular capillary (4+) and vascular endothelium (2+), but there was no detectable hDAF-expression on controls. No difference in xenoantibody deposition on vascular endothelium was seen between both groups. There was comparable staining for complement fraction C4 in both groups, but significant reduction of C3 and C9 staining on glomerular and vascular endothelium in TG. P-selectin was expressed on a higher level in C (+4) compared with TG (+2). Neutrophil extravasation [NP-57 elastase] was higher in C (80.2 vs. 32.2 C vs. TG [values as n/high power field]). Tubular epithelial cell swelling and mild necrosis was paralleled by glomerular hemorrhage and platelet microthrombus formation in both groups as seen in transmission electron microscopy. The observed results allow the conclusion that hDAF expression on transgenic pig kidneys was sufficient to inhibit complement activation beyond C3 during xenoperfusion with human blood despite xenoantibody deposition.

Na-K/2Cl transporter inhibition for reduction of postischemic kidney failure tested in autologous reperfusion.

Postischemic kidney function may be influenced by donor conditioning. The sulfamoyl-benzoate "piretanide" (P) is a diuretic agent with an inhibitory effect on the luminal Na-K-2CL-transporter system in the ascending part of the loop of Henle. A clinical pilot study demonstrated a lower rate of organ dysfunction following transplantation in humans when the donor organs were pretreated with piretanide. In an experimental ex vivo model the effect of piretanide on immediate organ function following long or short cold ischemia was studied. Porcine kidneys (n = 36) were removed after in situ transaortal hypothermic flushing with 21 Eurocollins solution. Following short storage (1 h, n = 18) or long storage (24 h, n = 18) the kidneys were reperfused with intraoperatively drawn heparinized autologous blood diluted with Ringer's lactate to a hematocrit of 25%. Urine flow was higher in the piretanide-pretreated group (p), especially after long storage. The electrolyte loss was comparable in both groups. Postischemic endogenous creatinine clearance was significantly elevated in the treatment group (4.45 +/- 0.6 ml/min per 100 mg in P vs 1.91 +/- 0.4 ml/min per 100 mg, in control, P < 0.05 Mann-Whitney test). Renal hemodynamics were improved by piretanide, resulting in significantly lower resistance and allowing higher flow during pressure-controlled perfusion. O2 consumption, representing general metabolic activity, was higher after long storage, indicating an earlier recovery from cold ischemia. In this ex vivo model, autologous reperfusion of porcine kidneys could be improved by piretanide pretreatment. Autoregulation of kidney vasculature was maintained as well as functional parameters such as creatinine clearance or gluconeogenesis. Therefore, piretanide may be used in larger clinical trials to further improve organ quality in times of donor shortage.

Production of proinflammatory cytokines and adhesion molecules in ex-vivo xenogeneic kidney perfusion.

Xenogeneic transplantation of solid organs is limited due to hyperacute rejection. In concordant systems, the mechanisms of rejection can be studied due to cross-reactivity of mediators with anti-human monoclonal antibodies. The aim of this study was to obtain information about the kinetics of proinflammatory cytokines and production of soluble adhesion molecules in the acute phase of reperfusion, eight kidneys from rhesus monkeys were perfused ex-vivo with human blood (group B/0) for 1 hour in a closed system. Blood levels of IL-1b, IL-6, TNFalpha, soluble ICAM, and E-selectin were measured using an ELISA technique under steady-state conditions. Cytokine levels rose significantly within the 60-min interval (IL-1b, 6.1 +/- 2.6-161.1 +/- 98.5 pg/ml; IL-6, 30.2 +/- 7.7-274.2 +/- 75.8 pg/ml; TNFalpha, 544.2 +/- 363.6-1651.0 +/- 25.7 pg/ml; P < 0.05). Immediately after the beginning of reperfusion, soluble ICAM-1 and selectin levels were abnormally high and rose constantly throughout the observation period, reaching significance at 60 min. High levels of proinflammatory cytokines may lead to an induction of adhesion molecules, thus, upregulating the leukocyte-endothelial interaction in a complement-independent mechanism. Specific pretreatment with monoclonal antibodies against ICAM-1, LFA-1, or other soluble mediators may be useful in down-regulating hyperacute rejection in trans-species transplantation.