Dicken D.H. Koo

The role of mitochondria in ischemia/reperfusion injury

In organ transplantation, ischemia/reperfusion injury is a multifactorial process that leads to organ damage and primary graft dysfunction. Injury to the organ is mediated by a complex chain of events that involves depletion of energy substrates, alteration of ionic homeostasis, production of reactive oxygen species, and cell death by apoptosis and necrosis. There is increasing evidence that mitochondria play a role in this process because of the profound changes experienced during ischemia and reperfusion. Understanding the mechanisms that lead to mitochondrial damage may be important for developing strategies aimed at improving graft outcome. In this review, we examine the role of mitochondria in ischemia/reperfusion injury and the possible mechanisms that may contribute to organ dysfunction.

Ischemia/Reperfusion Injury in Human Kidney Transplantation: An Immunohistochemical Analysis of Changes after Reperfusion

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.

Cell adhesion molecules in clinical renal transplantation.

Leukocyte adhesion molecules are critically involved at a number of stages in immune and inflammatory responses, and their importance in the response to a renal allograft has been recognized for some years. They are involved in antigen presentation, in the cascade of events leading to extravasation of leukocytes into the allograft, in the subsequent migration of leukocytes through the extracellular matrix, and in the interactions between effector and target cells. Thus the adhesion molecules are highly attractive targets for therapeutic intervention in organ transplantation. Strategies have been explored to exploit the involvement of adhesion molecules in ischemia/reperfusion injury, allograft rejection, and the induction of immunological tolerance. Furthermore, the expression of a number of adhesion molecules is regulated by cytokines, and elevated levels may be detected both in transplant biopsies and as soluble forms measured in serum and urine. It has been proposed that these changes in levels might provide useful information in the diagnosis of allograft rejection and differentiation from other causes of graft dysfunction.

Leukocyte infiltration and inflammatory antigen expression in cadaveric and living-donor livers before transplant1

Background. There is evidence to indicate that organs obtained from cadaveric donors may be injured as a result of inflammatory events occurring at around the time of brain death. The aim of this study was to investigate whether there are differences in the expression of proinflammatory molecules between cadaveric and living-donor livers before transplant and to determine whether there is any association with donor factors and posttransplant graft function. Methods. A comparison of biopsies obtained before implantation from cadaveric (n=22) and living-related donor (LRD) (n=10) livers was performed. Cryostat tissue sections were stained with antibodies to leukocyte subpopulations, adhesion molecules, and human leukocyte antigen class II antigens. Results. Significantly higher levels of CD3+ lymphocytes (1.5%±0.8% vs. 0.5%±0.3%; P =0.00004), CD68+ monocytes and macrophages (4.0%±1.2% vs. 2.7%±0.6%; P =0.0003), and Fas-ligand staining (4.2%±2.6% vs. 1.5%±1.1%; P =0.0003) were detected in cadaveric livers compared with LRD livers before transplantation. Furthermore, higher levels of intercellular adhesion molecule-1 expression were detected in cadaveric donor livers and found to be associated with longer periods of ventilation (P =0.01), infection in the donor (P =0.013), and administration of dopamine (P =0.03). Although there were no differences in neutrophil infiltration between cadaveric and LRD livers, significantly higher levels were found in cadaveric donors with infection (P =0.01). Conclusion. This study demonstrates that inflammatory changes occur in cadaveric donor livers and are associated with events occurring during the period of intensive care. These proinflammatory changes did not seem to affect the short-term clinical outcome of cadaveric liver allografts but may contribute to alloimmune responses and impairment of graft function in the long term.

Cadaveric versus living-donor livers: differences in inflammatory markers after transplantation

Background. Prolonged cold storage of organs for transplantation may lead to inflammatory damage upon reperfusion. The aim of this study was to investigate whether organs from living donors experience less damage upon reperfusion than those retrieved from cadaver donors, where cold ischemia times are significantly longer. MethodS. Biopsies were obtained from cadaveric (n=23) and living-related donor (LRD) (n=10) liver transplants before and 2 hours after reperfusion. Cryosections were stained with antibodies against neutrophils, platelets, activated platelets, and endothelium. Results. LRD liver allografts showed minimal changes postreperfusion. In contrast, after reperfusion of cadaver allografts, neutrophil infiltration was detected in 22% and increased expression of von Willebrand factor (vWF), CD41, and P-selectin in 48%, 30%, and 13% of allografts, respectively. In cadaver allografts with deposition of activated platelets expressing either P-selectin or vWF, the cold ischemia time was significantly longer (885±123 min vs. 608±214 min, P =0.04; 776.8±171 min vs. 559.3±216 min, P =0.01, respectively). Increases in neutrophils and platelets after reperfusion were not significantly associated with clinical events posttransplant. However, in cadaver transplants that experienced early acute rejection, the mean cold ischemia time was significantly longer than in allografts with no rejection (732±174 min vs. 480±221 min, P =0.006). Conclusions. This study demonstrates that in the clinical situation, cold ischemia causes platelet deposition and neutrophil infiltration after reperfusion of cadaveric liver allografts. These early inflammatory events may contribute to make the graft more susceptible to acute rejection.

Expression of MHC class I-related Chain B (MICB) molecules on renal transplant biopsies.

Background. MICA and MICB (MHC class I-related chain A and B) are polymorphic genes that encode molecules related to MHC class I and are expressed on epithelial cells in response to stress. Incompatible donor MIC antigens can stimulate antibody production in transplant recipients. This study was designed to determine MICB expression in kidney pretransplant and any subsequent changes in expression following transplantation and to correlate changes with inflammatory markers and clinical events. Methods. Paired renal biopsies obtained from living donor (n=10) and cadaveric allografts (n=50) before and 7 days posttransplant were stained for MICB, leukocytic infiltration, and HLA class II antigens. Results. Variable tubular MICB expression was evident in donor biopsies [high 6/60 (10%), low/negative 13/60 (22%), intermediate 41/60 (68%)]. Following transplantation, MICB was up-regulated on renal tubules of 17/60 (28%) biopsies and was associated with MHC class II antigen induction (P=0.02) and leukocyte infiltration (P=0.01). Acute tubular necrosis leading to delayed graft function (DGF) and acute rejection (AR) cause cellular stress within the transplanted kidney. We found a strong association between up-regulation of MICB and cellular stress, 15/17 biopsies with up-regulated MICB expression had AR and/or DGF (P=0.003). Conclusions. This is the first study demonstrating variable levels of MICB expression in kidneys before transplantation and induction of MICB expression following renal transplantation. MICB expression is associated with HLA class II antigen induction, leukocytic infiltration of the graft and cellular stress in the transplanted kidney. Expression of MICB could contribute significantly to the alloimmune response in mismatched donors and recipients.

Non-heart-beating versus cadaveric and living-donor livers: differences in inflammatory markers before transplantation.

Background. Liver transplantation from non–heart-beating donors (NHBD) has been reintroduced into clinical practice to increase the donor pool; however, little is known about the immune status of NHBD livers. The aim of this study was to assess intragraft cell populations and inflammatory markers in NHBD and to compare the findings with cadaveric and living-related donor (LRD) livers. Methods. Biopsy specimens were obtained from controlled NHBD (n=9), conventional cadaveric (n=22), and living-donor (n=10) livers at the end of cold storage. Cryostat sections were stained for monocytes-macrophages, T lymphocytes, and intercellular adhesion molecule (ICAM)-1. Results. The levels of leukocyte infiltration in NHBD reflected those found in conventional cadaver donors and were significantly higher than in LRD livers. Similar levels of CD68+ monocytes-macrophages were detected in cadaver (4.0±1.2%) and NHBD livers (4.6±1.2%) and were significantly greater than in the LRD livers (2.6±0.5%, P <0.01). Furthermore, the levels of T lymphocytes in NHBD (1.1±0.6%) and cadaver donors (1.5±0.8%) were similar, and were higher than in LRD (vs. 0.47±0.3%, P <0.05). Twelve of 22 (60%) cadaver livers had high levels of ICAM-1 expression (grade 3), compared with only 1 of 10 (10%) LRD livers (P =0.02). Four of nine (44%) controlled NHBD livers expressed high levels of ICAM-1. Conclusions. The results demonstrate that livers obtained from controlled NHBD before transplantation are similar to conventional cadaver donors regarding the level of leukocyte infiltration. Nevertheless, lower levels of ICAM-1 were detected in NHBD, suggesting less exposure to inflammatory mediators than conventional cadaver donor livers.

Late reflush in clinical renal transplantation : Protection against delayed graft function not observed

Mechanical flushing of cadaveric kidneys with organ preservation fluid immediately before transplantation has been reported to be associated with improved early graft function. We report here the results of a prospective randomized controlled study of cadaveric renal transplantation after late reflush with organ preservation fluid in which no benefit with respect to delayed graft function was observed and, indeed, the protocol may have been harmful. The study was terminated after recruitment of only 18 patients (9 to each arm) because postreperfusion biopsies of reflushed kidneys contained unusual features, including abnormal cellular debris within the tubules or eosinophilic proteinaceous material within Bowmans capsule. These features were not present in the control kidneys. Acute tubular necrosis and biopsy-proven acute rejection episodes were more frequently seen in the reflushed kidneys, but at 1 year there was no significant difference in the function of the surviving grafts.

Chemokines in ischemia/reperfusion injury

Ischemia/reperfusion injury is an unavoidable consequence of organ transplantation that results in the generation of reactive oxygen species and the initiation of an inflammatory response. The characteristic features of ischemia/reperfusion injury involve the activation of endothelium by reactive oxygen species and inflammatory cytokines, induction of adhesion molecules (for example, P-selectin and E-selectin), adherence of platelets, and infiltration by leukocytes consisting initially of neutrophils with subsequent infiltration by monocytes, macrophages, and T lymphocytes. In this review, we consider the current evidence for involvement of chemokines during the period after ischemia/reperfusion injury and examine the temporal relation between chemokine expression and leukocyte infiltration. Changes in the profile of chemokines after ischemia/reperfusion injury, in association with upregulation of endothelial adhesion molecules, may contribute to the immunogenicity of a transplanted organ by increasing its ability to recruit particular leukocyte subsets at specific time points after ischemia/reperfusion injury.