Ischemia/Reperfusion Injury in Human Kidney Transplantation
1998; Elsevier BV; Volume: 153; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)65598-8
ISSN1525-2191
AutoresDicken D.H. Koo, Kenneth I. Welsh, Justin Roake, Peter J. Morris, Susan V. Fuggle,
Tópico(s)Transplantation: Methods and Outcomes
ResumoOrgans used for transplantation undergo varying degrees of cold ischemia and reperfusion injury after transplantation. In renal transplantation, prolonged cold ischemia is strongly associated with delayed graft function, an event that contributes to inferior graft survival. At present, the pathophysiological changes associated with ischemia/reperfusion injury in clinical renal transplantation are poorly understood. We have performed an immunohistochemical analysis of pre- and postreperfusion biopsies obtained from cadaver (n = 55) and living/related donor (LRD) (n = 11) renal allografts using antibodies to adhesion molecules and leukocyte markers to investigate the intragraft changes after cold preservation and reperfusion. Neutrophil infiltration and P-selectin expression were detected after reperfusion in 29 of 55 (53%) and 24 of 55 (44%) cadaver renal allografts, respectively. In marked contrast, neutrophil infiltration was not observed in LRD allografts, and only 1 of 11 (9%) had an increased level of P-selectin after reperfusion. Immunofluorescent double-staining demonstrated that P-selectin expression resulted from platelet deposition and not from endothelial activation. No statistically significant association was observed between neutrophil infiltration and P-selectin expression in the glomeruli or intertubular capillaries despite the large number of cadaver renal allografts with postreperfusion changes. Neutrophil infiltration into the glomeruli was significantly associated with long cold ischemia times and delayed graft function. Elevated serum creatinine levels at 3 and 6 months after transplantation were also associated with the presence of neutrophils and platelets after reperfusion. Our results suggest that graft function may be influenced by early inflammatory events after reperfusion, which can be targeted for future therapeutic intervention. Organs used for transplantation undergo varying degrees of cold ischemia and reperfusion injury after transplantation. In renal transplantation, prolonged cold ischemia is strongly associated with delayed graft function, an event that contributes to inferior graft survival. At present, the pathophysiological changes associated with ischemia/reperfusion injury in clinical renal transplantation are poorly understood. We have performed an immunohistochemical analysis of pre- and postreperfusion biopsies obtained from cadaver (n = 55) and living/related donor (LRD) (n = 11) renal allografts using antibodies to adhesion molecules and leukocyte markers to investigate the intragraft changes after cold preservation and reperfusion. Neutrophil infiltration and P-selectin expression were detected after reperfusion in 29 of 55 (53%) and 24 of 55 (44%) cadaver renal allografts, respectively. In marked contrast, neutrophil infiltration was not observed in LRD allografts, and only 1 of 11 (9%) had an increased level of P-selectin after reperfusion. Immunofluorescent double-staining demonstrated that P-selectin expression resulted from platelet deposition and not from endothelial activation. No statistically significant association was observed between neutrophil infiltration and P-selectin expression in the glomeruli or intertubular capillaries despite the large number of cadaver renal allografts with postreperfusion changes. Neutrophil infiltration into the glomeruli was significantly associated with long cold ischemia times and delayed graft function. Elevated serum creatinine levels at 3 and 6 months after transplantation were also associated with the presence of neutrophils and platelets after reperfusion. Our results suggest that graft function may be influenced by early inflammatory events after reperfusion, which can be targeted for future therapeutic intervention. Organs that are used for transplantation require effective ex vivo preservation from the moment the organ is retrieved to the time of transplantation. Hypothermic preservation solutions have been developed to maintain tissue viability by reducing metabolic activity and the accumulation of toxic substances during the cold ischemic period. Organs used for transplantation can undergo lengthy periods of cold ischemic storage after devascularization and cold perfusion, resulting in an increased susceptibility to damage upon reperfusion.In clinical renal transplantation, prolonged cold storage has been demonstrated in many studies to be strongly associated with delayed graft function (DGF).1Najarian JS Gillingham KJ Sutherland DE Reinsmoen NL Payne WD Matas AJ The impact of the quality of initial graft function on cadaver kidney transplants.Transplantation. 1994; 57: 812-816Crossref PubMed Scopus (118) Google Scholar, 2Peters TG Shaver TR Ames JE Santiago-Delpin EA Jones KW Blanton JW Cold ischemia and outcome in 17,937 cadaveric kidney transplants.Transplantation. 1995; 59: 191-196Crossref PubMed Scopus (93) Google Scholar, 3Troppmann C Gillingham KJ Benedetti E Almond PS Gruessner RW Najarian JS Matas AJ Delayed graft function, acute rejection, and outcome after cadaver renal transplantation: the multivariate analysis.Transplantation. 1995; 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63: 968-974Crossref PubMed Scopus (827) Google Scholar DGF is broadly defined as the requirement for dialysis within the first week after transplantation and results in complications in the immunosuppressive management of the transplant patient, prolonged hospitalization, and potentially detrimental effects to subsequent graft function and survival.9Rosenthal JT Danovitch GM Wilkinson A Ettenger RB The high cost of delayed graft function in cadaveric renal transplantation.Transplantation. 1991; 51: 1115-1118Crossref PubMed Scopus (138) Google Scholar, 10Yokoyama I Uchida K Kobayashi T Tominaga Y Orihara A Takagi H Effect of prolonged delayed graft function on long-term graft outcome in cadaveric kidney transplantation.Clin Transplant. 1994; 8: 101-106PubMed Google Scholar Some studies have suggested that DGF has little or no effect on graft survival, especially when the compounding effects of acute rejection are taken into account.4Troppmann C Gillingham KJ Gruessner RWG Dunn DL Payne WD Najarian JS Matas AJ Delayed graft function in the absence of rejection has no long-term impact.Transplantation. 1996; 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63: 968-974Crossref PubMed Scopus (827) Google Scholar This effect has recently been highlighted in a multivariate analysis of 37,216 primary cadaver renal allografts from the U.S. Renal Data System, in which DGF was shown to be an independent factor in determining poor short- and long-term graft survival, regardless of both the incidence of early rejection episodes and the degree of human leukocyte antigen (HLA) matching.8Ojo AO Wolfe RA Held PJ Port FK Schmouder RL Delayed graft function: risk factors and implications for renal allograft survival.Transplantation. 1997; 63: 968-974Crossref PubMed Scopus (827) Google ScholarAlthough it is widely accepted that prolonged cold storage has a detrimental effect upon graft function, the precise mechanisms by which this occurs are not completely understood. During cold ischemic storage of organs before transplantation, biochemical events occur within the tissue leading to free radical-mediated damage upon reperfusion of the vascularized graft (Reviewed in 14Korthuis RJ Granger DN Reactive oxygen metabolites, neutrophils, and the pathogenesis of ischemic-tissue/reperfusion.Clin Cardiol. 1993; 16: I19-I26Crossref PubMed Scopus (65) Google Scholar, 15Granger DN Kvietys PR Perry MA Leukocyte—endothelial cell adhesion induced by ischemia and reperfusion.Can J Physiol Pharmacol. 1993; 71: 67-75Crossref PubMed Scopus (104) Google Scholar, 16Grace PA Ischaemia-reperfusion injury.Br J Surg. 1994; 81: 637-647Crossref PubMed Scopus (628) Google Scholar, 17Land W Messmer K The impact of ischaemia/reperfusion injury on specific and non-specific, early and late chronic events after organ transplantation.Transplant Rev. 1996; 10: 108-127Abstract Full Text PDF Scopus (149) Google Scholar). Free radicals appear to mediate tissue injury through lipid peroxidation and the activation of endothelial cells, resulting in functional and structural cell damage.In vitro experiments on human umbilical vein endothelial cells have shown that reactive oxygen species induce adhesion molecule expression, resulting in activation and increased binding of neutrophils.18Patel KD Zimmerman GA Prescott SM McEver RP McIntyre TM Oxygen radicals induce human endothelial cells to express GMP-140 and bind neutrophils.J Cell Biol. 1991; 112: 749-759Crossref PubMed Scopus (524) Google Scholar, 19Sellak H Franzini E Hakim J Pasquier C Reactive oxygen species rapidly increase endothelial ICAM-1 ability to bind neutrophils without detectable upregulation.Blood. 1994; 83: 2669-2677Crossref PubMed Google Scholar, 20Arnould T Michiels C Remacle J Hypoxic human umbilical vein endothelial cells induce activation of adherent polymorphonuclear leukocytes.Blood. 1994; 83: 3705-3716Crossref PubMed Google Scholar, 21Rainger GE Fisher A Shearman C Nash GB Adhesion of flowing neutrophils to cultured endothelial cells after hypoxia and reoxygenation in vitro.Am J Physiol. 1995; 269: H1398-H1406PubMed Google Scholar, 22Pinsky DJ Naka Y Liao H Oz MC Wagner DD Mayadas TN Johnson RC Hynes RO Heath M Lawson CA Stern DM Hypoxia-induced exocytosis of endothelial cell Weibel-Palade bodies: a mechanism for rapid neutrophil recruitment after cardiac preservation.J Clin Invest. 1996; 97: 493-500Crossref PubMed Scopus (246) Google Scholar, 23Ichikawa H Flores S Kvietys PR Wolf RE Yoshikawa T Granger DN Aw TY Molecular mechanisms of anoxia/reoxygenation-induced neutrophil adherence to cultured endothelial cells.Circ Res. 1997; 81: 922-931Crossref PubMed Scopus (171) Google Scholar Furthermore, in experimental animal models, in situ cold ischemia followed by reperfusion of the kidney has been shown to lead to increased expression of adhesion molecules and neutrophil infiltration within hours of reperfusion, followed by mononuclear cell infiltration and up-regulation of major histocompatibility complex (MHC) class II expression several days later.24Takada M Nadeau KC Shaw GD Marquette KA Tilney NL The cytokine-adhesion molecule cascade in ischemia/reperfusion injury of the rat kidney: inhibition by a soluble P-selectin ligand.J Clin Invest. 1997; 99: 2682-2690Crossref PubMed Scopus (475) Google Scholar, 25Takada M Chandraker A Nadeau KC Sayegh MH Tilney NL The role of the B7 costimulatory pathway in experimental cold ischemia/reperfusion injury.J Clin Invest. 1997; 100: 1199-1203Crossref PubMed Scopus (199) Google Scholar This increase in immunogenicity resulting from the early nonspecific inflammatory events may intensify subsequent alloimmune responses and play a major role in determining the quality of graft function in the long term.26Shoskes DA Partrey NA Halloran PF Increased major histocompatibility complex antigen expression in unilateral ischemic acute tubular necrosis in the mouse.Transplantation. 1990; 49: 201-207Crossref PubMed Scopus (237) Google Scholar, 27Goes N Urmson J Ramassar V Halloran PF Ischemic acute tubular necrosis induces an extensive local cytokine response.Transplantation. 1995; 59: 565-572Crossref PubMed Scopus (243) Google Scholar, 28Tullius SG Tilney NL Both alloantigen-dependent and -independent factors influence chronic allograft rejection.Transplantation. 1995; 59: 313-318Crossref PubMed Scopus (381) Google ScholarStudies investigating the effects of ischemia/reperfusion injury in clinical renal transplantation are limited and have relied mainly on the measurement of a marker of lipid peroxidation, malondialdehyde.29Rabl H Khoschsorur G Colombo T Tatzber F Esterbauer H Human plasma lipid peroxide levels show a strong transient increase after successful revascularization operations.Free Radical Biol Med. 1992; 13: 281-288Crossref PubMed Scopus (57) Google Scholar, 30Pincemail J Defraigne JO Franssen C Bonnet P Deby-Dupont G Pirenne J Deby C Lamy M Limet M Meurisse M Evidence for free radical formation during human kidney transplantation.Free Radical Biol Med. 1993; 15: 343-348Crossref PubMed Scopus (48) Google Scholar, 31Davenport A Hopton M Bolton C Measurement of malondialdehyde as a marker of oxygen free radical production during renal allograft transplantation and the effect on early graft function.Clin Transplant. 1995; 9: 171-175PubMed Google Scholar Although these studies showed elevated levels of malondialdehyde in plasma after reperfusion of the graft, potential correlations with subsequent graft function were not examined. A more informative method of investigating reperfusion injury of renal allografts would be to analyze biopsies obtained immediately after transplantation.During the early era of transplantation, biopsies were frequently obtained an hour after revascularization, when hyperacute rejection was suspected. A neutrophil infiltration in the glomeruli was an indicator of hyperacute rejection of the allograft,32Kincaid-Smith P Morris PJ Saker BM Ting A Marshall VC Immediate renal-graft biopsy and subsequent rejection.Lancet. 1968; 2: 748-749Abstract PubMed Google Scholar, 33Williams GM Hume DM Hudson RP Morris PJ Kano K Milgrom F "Hyperacute" renal-homograft rejection in man.N Engl J Med. 1968; 279: 611-618Crossref PubMed Scopus (330) Google Scholar but subsequent reports did not show a direct correlation between neutrophil infiltration and either hyperacute rejection or acute rejection episodes.34Perloff LJ Goodloe SJ Jenis EH Light JA Spees EK Value of one-hour renal-allograft biopsy.Lancet. 1973; 2: 1294-1295Abstract PubMed Scopus (10) Google Scholar, 35McDicken IW Hawking KM Lameyer LD Blok APR Westbroek DL Prognostic value for immediate function of one-hour renal allograft biopsy.Br Med J. 1975; 4: 559Crossref PubMed Google Scholar, 36Valenzuela R Hamway SA Deodhar SD Braun WE Banowsky LH Magnusson MO Osborne DG Histologic, ultrastructural, and immunomicroscopic findings in 96 one hour human renal allograft biopsy specimens.Hum Pathol. 1980; 11: 187-195Abstract Full Text PDF PubMed Scopus (10) Google Scholar In the modern era of transplantation, hyperacute rejection has been virtually eliminated because of improvements in antibody screening, crossmatching, and immunosuppression, and thus there have been few studies of postreperfusion biopsies. In one recent study of postreperfusion biopsies, polymorphonuclear leukocytes were detected in biopsies from cadaver renal allografts, and this was found to be associated with long cold-storage times.37Gaber LW Gaber AO Tolley EA Hathaway DK Prediction by postrevascularization biopsies of cadaveric kidney allografts of rejection, graft loss, and preservation nephropathy.Transplantation. 1992; 53: 1219-1225Crossref PubMed Scopus (36) Google Scholar Interpretation of these results is complicated by the presence of hyperacute rejection in 4 of 57 allografts studied and by the fact that prereperfusion biopsies were not available for comparison; thus, it is not clear whether these cells entered upon reperfusion or were already present within the donor kidney.To investigate the potential effects of cold ischemic damage and reperfusion injury in renal transplantation, we have performed an immunohistochemical study on renal allograft biopsies obtained immediately after transplantation and, for comparison, on biopsies from the same kidney before transplantation. This has enabled us to analyze changes resulting from reperfusion of the allograft, while excluding pre-existing factors associated with the donor kidneys. Furthermore, biopsies from living/related donor (LRD) renal allografts with minimal cold ischemia times have been obtained for comparison. The results from the analysis have been related to relevant donor parameters and factors relating to graft function and rejection.Materials and MethodsPatients and Biopsy MaterialBiopsy material was obtained from transplants of cadaveric (n = 55) and living/related (n = 11) kidney allografts performed at the Oxford Transplant Centre. Wedge biopsies were obtained from all transplanted kidneys at two time points: 1) prereperfusion, after nephrectomy, flushing with ice-cold hypertonic citrate (Marshall's solution) and storage, but before implantation, and 2) postreperfusion, approximately 20 to 40 minutes after reperfusion of the kidney, immediately before wound closure. In five of the cadaver kidneys, additional biopsies were obtained at the time of nephrectomy, immediately after flushing, and before the period of cold storage. All biopsies were snap frozen in liquid nitrogen and stored at −80°C.After transplantation, all patients received standard triple-therapy immunosuppression (cyclosporine, azathioprine, and steroids).38Jones RM Murie JA Allen RD Ting A Morris PJ Triple therapy in cadaver renal transplantation.Br J Surg. 1988; 75: 4-8Crossref PubMed Scopus (36) Google Scholar Details relating to important clinical parameters and outcome indicators are given in Table 1. There were no significant differences observed between cadaver and LRD renal allografts in donor age; HLA-A, -B, and -DR mismatches; number of retransplants; anastomosis time; and recipient sex.Table 1Clinical Factors and Graft Outcome Indicators after TransplantationClinical DetailsCadaver (n = 55)LRD (n = 11)Donor age (±SD)41 ± 14.841 ± 11.3Cold ischemia time (hours)± SD*Significant difference between cadaver and LRD groups;P < 0.01.24.7 ± 91.8 ± 0.5Positive crossmatch†Positive crossmatch resulting from non-HLA, autoreactive IgM antibodies.7 of 550 of 11DGF‡DGF is defined as the requirement for dialysis in the first week after transplantation.8 of 551 of 11Recipient age (±SD)*Significant difference between cadaver and LRD groups;P < 0.01.47.8 ± 1229.4 ± 12Serum creatinine, μmol/L (3 months)± SD154.7 ± 52142.3 ± 37Serum creatinine, μmol/L (6 months)± SD154.2 ± 51143.7 ± 31No. of rejection episodes (0:1:2:3)29:16:5:54:5:2:0* Significant difference between cadaver and LRD groups;P < 0.01.† Positive crossmatch resulting from non-HLA, autoreactive IgM antibodies.‡ DGF is defined as the requirement for dialysis in the first week after transplantation. Open table in a new tab ImmunohistochemistryCryostat tissue sections (7 μm) from wedge biopsies were stained with monoclonal antibodies (mAbs) using an indirect immunoperoxide technique as previously described.39Fuggle SV McWhinnie DL Chapman JR Taylor HM Morris PJ Sequential analysis of HLA-class II antigen expression in human renal allografts: induction of tubular class II antigens and correlation with clinical parameters.Transplantation. 1986; 42: 144-150Crossref PubMed Scopus (110) Google Scholar The sections were stained with the following mAbs: 5D11 (anti-E-selectin (CD62E)), 4B2 (anti-vascular cellular adhesion molecule-1 (VCAM-1) (CD106)), and 14C11 (anti-intercellular adhesion molecule-1 (ICAM-1) (CD54)), all obtained from British Biotechnology Ltd (Oxford, UK); F10.89.4 (anti-CD45 leukocyte common marker40Dalchau R Kirkley J Fabre JW Monoclonal antibody to a human leukocyte-specific membrane glycoprotein probably homologous to the leukocyte-common (L-C) antigen of the rat.Eur J Immunol. 1980; 10: 737-744Crossref PubMed Scopus (176) Google Scholar); UCHT-1 (anti-CD3 T cell marker41Beverley PC Callard RE Distinctive functional characteristics of human "T" lymphocytes defined by E rosetting or a monoclonal anti-T cell antibody.Eur J Immunol. 1981; 11: 329-334Crossref PubMed Scopus (329) Google Scholar); UCHM-1 (anti-CD14 macrophage/monocyte marker42Hogg N MacDonald S Slusarenko M Beverley PC Monoclonal antibodies specific for human monocytes, granulocytes and endothelium.Immunology. 1984; 53: 753-767PubMed Google Scholar); EBM/11 (anti-CD68 macrophage/monocyte marker43Kelly PM Bliss E Morton JA Burns J McGee JO Monoclonal antibody EBM/11: high cellular specificity for human macrophages.J Clin Pathol. 1988; 41: 510-515Crossref PubMed Scopus (140) Google Scholar); G1 (anti-P-selectin (CD62P)44McEver RP Martin MN A monoclonal antibody to a membrane glycoprotein binds only to activated platelets.J Biol Chem. 1984; 259: 9799-9804Abstract Full Text PDF PubMed Google Scholar); 1G10 (anti-CD15s neutrophil marker45Bernstein ID Andrews RG Cohen SF McMaster BE Normal and malignant human myelocytic and monocytic cells identified by monoclonal antibodies.J Immunol. 1982; 128: 876-881PubMed Google Scholar); 5B12 (anti-CD41 platelet-specific marker) and anti-neutrophil elastase, (DAKO Ltd., High Wycombe, Bucks, UK). An anti-dog Thy-1 (F3.20.746McKenzie JL Fabre JW Studies with a monoclonal antibody on the distribution of Thy-1 in the lymphoid and extracellular connective tissues of the dog.Transplantation. 1981; 31: 275-282Crossref PubMed Scopus (99) Google Scholar) mAb was used as a negative control.Briefly, mAb bound to the sections was detected using a peroxidase-conjugated rabbit anti-mouse immunoglobulin (Ig) (DAKO Ltd.) preincubated with human AB serum to prevent nonspecific binding. The reaction was developed using 3,3′-diaminobenzidine tetrachloride (Sigma Ltd., Poole, Dorset, UK) and H2O2, counterstained with Harris' Haematoxylin (Merck Ltd., Atherstone, UK), dehydrated, and mounted in dextropropoxyphene mountant (Merck Ltd.). The signal for E-selectin and P-selectin was enhanced by incubating with a further antibody, a peroxidase-conjugated swine anti-rabbit Ig (DAKO Ltd.) preincubated with human AB serum.To determine the origin of leukocyte infiltration into the postreperfusion biopsies, pre- and postreperfusion biopsies from transplants mismatched for HLA-A2 or B17 were stained with an anti-HLA-A2/B17 antibody (MA2.147McMichael AJ Parham P Rust N Brodsky F A monoclonal antibody that recognizes an antigenic determinant shared by HLA-A2 and B17.Hum Immunol. 1980; 1: 121-129Crossref PubMed Scopus (224) Google Scholar and a monomorphic anti-HLA-A, -B, and -C antibody (PA2.648Brodsky FM Parham P Barnstable CJ Crumpton MJ Bodmer WF Monoclonal antibodies for analysis of the HLA system.Immunol Rev. 1979; 47: 3-62Crossref PubMed Scopus (559) Google Scholar), as a control for the presence of HLA class I antigens.Double-Immunofluorescent StainingDouble-immunofluorescent staining was performed to clarify the origin of the increased expression of P-selectin detected in the postreperfusion biopsies. All incubations were performed for 30 minutes at room temperature in the dark. Acetone-fixed cryosections of pre- and postreperfusion biopsies were first incubated with antibodies to either P-selectin (IgG1) or ICAM-1 (IgG2a), and after washing, bound antibody was detected with the appropriate Texas Red-conjugated isotype-specific goat anti-mouse Ig antibodies (Southern Biotechnology Associates, Birmingham, AL) preincubated with human AB serum. Sections were then incubated with a fluorescein isothiocyanate (FITC)-conjugated anti-CD41 (platelet-specific) antibody (5B12, DAKO Ltd). After a final washing, slides were mounted in Vectashield (Vector Laboratories, Peterborough, UK) and analyzed by fluorescent microscopy. The specificity of the staining procedure was confirmed by including isotype control antibodies to ensure that there was no nonspecific binding of the secondary antibodies. All antibodies were used in isolation to check that the binding was in no way altered by the double-staining protocol.Assessment of StainingStaining of endothelial and leukocyte markers was scored by two independent observers (SVF and DDHK) without knowledge of the clinical status of the patients. Minor differences in the scoring were resolved by conference. The semiquantitative grades given for E-selectin, P-selectin, and CD41 detected on endothelium were scored as follows: 0, negative; 1, predominantly negative, with an isolated positive vessel; 2, focus of positive vessels/occasional positive vessels; and 3, multiple foci/positive vessels throughout biopsy. A significant increase in the level of expression of adhesion molecules after reperfusion was considered as an increase in grades of ≥1. Changes between grades 0 and 1 were not considered significant.Leukocytes were quantified and expressed as 1) mean number of positive cells/glomerulus per section, with a minimum of three glomeruli required for inclusion in the analysis, and 2) mean number of positive cells in the intertubular areas per field of view (×10 objective). A significant increase in glomerular infiltration after reperfusion was taken as an increase in mean glomerular count of ≥1.5, and an increase in intertubular infiltration was scored as an increase of ≥10 positive cells.Statistical AnalysesStatistical analyses of the immunohistochemical results and the clinical data were performed using the Student's t-test, Fisher's exact, and χ2Peters TG Shaver TR Ames JE Santiago-Delpin EA Jones KW Blanton JW Cold ischemia and outcome in 17,937 cadaveric kidney transplants.Transplantation. 1995; 59: 191-196Crossref PubMed Scopus (93) Google Scholar tests.ResultsImmunohistochemical Changes after ReperfusionBiopsies obtained from donor kidneys before and after reperfusion were stained with mAbs to leukocyte subpopulations and endothelial adhesion molecules, to provide information about the changes that may occur immediately after reperfusion in cadaveric and LRD transplants. In addition, five biopsies were obtained at the time of donor nephrectomy and compared with prereperfusion biopsies from the same kidney to determine changes arising from cold storage.Leukocyte SubpopulationsThe major leukocyte population in the prereperfusion biopsies comprised CD14/CD68+ macrophages localized in the interstitial areas, but occasional CD3+ T lymphocytes were detected. There were no quantifiable differences in either of these cell populations immediately after reperfusion. In contrast, an increase in neutrophil infiltration, as determined by staining with antibodies to CD15s and neutrophil elastase, was observed in 29 of 55 (53%) cadaver renal allografts after reperfusion (Figure 1). In the 29 cadaver allografts in which a neutrophil infiltration was detected, the infiltration was localized in the glomeruli of 16 of 29 (55%) (Figure 2, a and b) and in the intertubular regions of 25 of 29 (86%) cadaver allografts (Table 2). The increase in neutrophil infiltration after reperfusion was restricted to cadaver allografts; there was no neutrophil infiltration detected in the postreperfusion biopsies of LRD renal allografts (Table 2). Furthermore, no changes occurred during the period of cold storage as determined by comparison between the five biopsies obtained at donor nephrectomy and the corresponding prereperfusion biopsies taken after cold storage.Figure 2Immunohistological changes observed after reperfusion
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