Role of peroxynitrite anion in renal hypothermic preservation injury.

BACKGROUND: Peroxynitrite anions may play a role in normothermic renal ischemia and reperfusion. The purpose of this study was to determine if endogenous peroxynitrite anion is involved in renal preservation injury. METHODS: Experiments were conducted in isolated canine renal tubules and in a canine autotransplant model of hypothermic preservation injury. RESULTS: Isolated renal tubules demonstrated progressive loss of membrane transport function after reperfusion with increasing cold storage times in UW solution as assessed by tetraethylammonium cation transport (TEA). This transport defect was not altered by reperfusion in the presence of WW85, a peroxynitrite decomposition catalyst. Likewise, tubule LDH release was not altered by WW85. Renal tubules did not demonstrate any evidence of peroxynitrite formation after cold storage (0-120 h) or after subsequent reperfusion in vitro as measured by nitrotyrosine adduct formation. Addition of exogenous peroxynitrite (1 mM) directly to freshly isolated renal tubules produced strong nitrotyrosine signals but failed to alter membrane function (TEA uptake). Conversely, SIN-1, a peroxynitrite generator molecule, failed to produce a nitrotyrosine signal in extracted renal tubule proteins but significantly impaired transport function. Finally, function of cold stored canine autografts was not affected by the scavenging of peroxynitrite anions (WW85) before kidney harvest and immediately at reperfusion. Tissue biopsies from cold stored kidney autografts also failed to show evidence of peroxynitrite synthesis either after cold storage (72 h) or after kidney transplantation (60 min reperfusion). CONCLUSIONS: This study concludes that peroxynitrite anions are not formed and are not involved in renal preservation injury.

Prolonged hypothermia causes primary nonfunction in preserved canine renal allografts due to humoral rejection.

Canine kidney preservation models have historically used autotransplants to avoid the complications of rejection, although clinically all transplants are allografts. This study investigated the effects of preservation time and method on early kidney function in a canine allograft vs. autograft model. Kidneys were harvested from beagles, preserved by cold storage (CS) in UW solution for 0, 24 or 72 h, or by machine perfusion (MP) with Belzer MPS for 72 h. In some experiments 45 min of warm ischemia (WI) was performed in situ before harvest. Allograft recipients received steroid immunosuppression. Kidney function was assessed by serum creatinine and survival for 7 days. Allografts preserved for 0 and 24 h performed as well as autografts. Allografts preserved for 72 h by either CS or MP had a higher incidence of primary nonfunction (PNF) compared with autografts, as determined by survival (50% vs. 100%, p < 0.003). Primary nonfunction kidneys had thrombotic microangiopathy, vascular and peritubular capillary binding of IgM and complement C4d, and evidence of circulating donor-specific antibodies; all consistent with humoral rejection. These responses were dependent on hypothermia time and were not attributable to ischemia, immunosuppression, preservation solution, or cellular rejection. In conclusion, prolonged hypothermia can cause PNF in allografts owing to acute humoral rejection.

Primitive endothelial cell lines from the porcine embryonic yolk sac.

Endothelial cell lines have been established from cells that were isolated from porcine yolk sacs from day 18 and day 22 embryos and propagated in vitro under various growth conditions. After expansion in vitro, the general properties of the cells proved similar for the different media used. The endothelial cells expressed cell surface receptors for acetylated low-density lipoprotein and also expressed cell surface-associated angiotensin-converting enzyme. The cells showed a characteristically high level of binding for Bandeiraea simplicifolia lectin I and Dolichos biflorus agglutinin but did not bind significant amounts of Ulex europaeus lectin I. The cells expressed low but serologically detectable levels of Class I major histocompatibility complex (MHC) antigens but failed to bind antibodies directed against Class II MHC antigens. Alpha5beta1 integrins were weakly expressed, whereas vascular cell adhesion molecule-1 (CD106) and alphavbeta3 integrins were not detected. Three-dimensional tube formation was readily observed in cultures grown on Matrigel and occurred even in uncoated plastic dishes in the absence of Matrigel. In contrast to most of the adult porcine endothelial cells, yolk sac-derived endothelial cells did not possess serologically detectable receptors for porcine growth hormone (GH), an observation consistent with the finding that GH did not increase the proliferative rate of these cells. Electron microscopic examination demonstrated the presence of Weibel-Palade bodies, tight endothelial cell junctions, and typical rough endoplasmic reticulum. Exposure of the cells to either concanavalin-A-stimulated porcine splenocyte culture supernatants or to human tumor necrosis factor alpha did not cause upregulation of VCAM-1 or Class II MHC antigens. Addition of porcine interferon-gamma led to an increase in the level of expression of Class I MHC. Yolk sac endothelial cells from day 22 embryos showed a low but detectable level of expression of Class II MHC antigens, whereas the endothelial cells from day 18 embryos showed no expression of Class II antigens after interferon-gamma stimulation. The cells maintained competence to develop vascular structures in vitro and could do so after coinjection with murine tumor cells into adult, immunocompromised mice.