Mamoru Kusaka

Brain death and its influence on donor organ quality and outcome after transplantation.

Transplantation has evolved as the treatment of choice for many patients with end-stage organ disease. However, despite the .80% one-year functional survival rate of most transplanted organs at the present time, the ultimate goal—to provide long-term treatment for an irreversible process—has not been achieved; the rate of attrition over time has not changed appreciably throughout the entire experience (1). Although recurrent disease, de novo infections, malignancies, and other factors may contribute to late graft deterioration, chronic rejection remains the most important etiologic factor (2). Despite well-characterized functional and morphological changes, the mechanisms leading to this progressive state remain poorly understood. Its pathophysiology has been conceptualized as stemming from both antigendependent and -independent risk factors (3). Although immune-mediated events are considered to be primarily responsible for the late graft changes, it seems increasingly that the influence of nonimmunological events has been underestimated. This concept has been emphasized by recent pooled United Network of Organ Sharing data that show that the survival rates of kidneys from living-unrelated and one haplotype-matched living-related donors are identical despite potentially important differences in genetic relationship with the given recipient (4). In addition, organs from all living donors demonstrate consistently superior results to those from cadaver sources over both the shortand long-term. Various nonimmunological factors that might explain these discrepancies include the effects of initial ischemia/reperfusion injury, inadequate functioning nephron mass, viral infections, and drug toxicity. Brain death is a rarely considered risk factor uniquely relevant to the cadaver donor. Multivariate analysis has emphasized that both initial and long-term results of engrafted cadaver organs may be dependent upon donor demographics and the etiology of the central injury (5). In virtually all experimental studies of organ transplantation, young, healthy living animals are used as donors; in clinical practice, in contrast, a relatively low percentage of organs comes from living donors, as cadavers remain the primary source of supply. Amongst other variables, the difference between the two donor populations implies the effect of profound physiological and structural derangements that may occur during and subsequent to brain death and before the actual engraftment procedure.

Activation of the Heart by Donor Brain Death Accelerates Acute Rejection After Transplantation

BackgroundDonor brain death upregulates expression of inflammatory mediators in the heart. It is hypothesized that these nonspecific changes trigger and amplify acute rejection in unmodified recipients compared with hearts from normal living donors. We examined the inflammatory and immunological consequences of gradual-onset donor brain death on cardiac allografts after transplantation. Methods and ResultsFunctioning hearts were engrafted from normotensive donors after 6 hours of ventilatory support. Hearts from brain-dead rats (Fisher, F344) were rejected significantly earlier (mean±SD, 9.3±0.6 days) by their (Lewis) recipients than hearts from living donor controls (11.6±0.7 days, P =0.03). The inflammatory response of such organs was accelerated, with rapid expression of cytokines, chemokines, and adhesion molecules and brisk infiltration of associated leukocyte populations. Upregulation of major histocompatibility class II antigens increased organ immunogenicity. Acute rejection evolved in hearts from brain-dead donors more intensely and at a significantly faster rate than in controls. ConclusionsDonor brain death is deleterious to transplanted hearts. The resultant upregulation of inflammatory factors provokes host immune mechanisms and accelerates the acute rejection process in unmodified hosts.

Activation of inflammatory mediators in rat renal isografts by donor brain death

BACKGROUND Brain death (BD) has been thought to influence the early course of transplanted organs by triggering a series of nonspecific inflammatory events that in turn may increase the kinetics and intensity of the immunological host responses. In this study early nonspecific, cellular, and molecular changes occurring in kidney isografts from BD donors are compared with those from normal anesthetized, ventilated controls. METHODS After induction of brain death, the animals were mechanically ventilated for 6 hr before organ removal. Only rats with stable blood pressure (mean arterial pressure >80 mmHg) were included. Serum creatinines were measured daily. Representative grafts were harvested 6 hr after brain death and between 1 hr and 5 days after engraftment for morphology, immunohistology, and reverse transcriptase-polymerase chain reaction. The presence of serum cytokines was assessed by enzyme linked immunoabsorbant assay. RESULTS Serum creatinine levels rose slightly in recipients from BD donors. Serum interleukin-1beta levels increased within 6 hr versus controls (P<0.05). mRNA levels of interleukin-1beta and macrophage inhibitory protein-1 in the kidneys were up-regulated transiently before engraftment (6 hr after BD) and 1 hr after revascularization (P<0.05). By immunohistology, numbers of infiltrating polymorphonuclear leukocytes peaked at 24 hr in parallel with intragraft induction of P- and E-selectin, complement, and other proinflammatory chemokines and cytokines. At 5 days, the isografts from BD donors were highly infiltrated by host leukocyte populations associated with intense up-regulation of their products. In contrast, those from control donors remained relatively normal through this initial follow-up period. CONCLUSIONS The intense nonimmune inflammation produced in isografts after donor BD may represent the initial stages of a continuum between an initial nonspecific and later immune reactivity, when placed in the context of allotransplantation.

Accelerated rejection of renal allografts from brain-dead donors.

ObjectiveTo define the potential influences of donor brain death on organs used for transplantation. Summary Background DataDonor brain death causes prompt upregulation of inflammatory mediators on peripheral organs. It is hypothesized that this antigen-independent insult may influence the rate and intensity of host alloresponsiveness after engraftment. MethodsThe rates of survival of unmodified Lew recipients sustained by kidney allografts from brain-dead, normal anesthetized, and anesthetized ventilated F344 donors were compared. Brain death was induced by gradually increasing intracranial pressure under electroencephalographic control. Tracheotomized brain-dead animals and anesthetized controls were mechanically ventilated for 6 hours before transplant nephrectomy. The rate and intensity of the acute rejection event were examined by histology, immunohistology, and reverse transcriptase–polymerase chain reaction. ResultsAnimals bearing kidneys from brain-dead donors died of renal failure secondary to acute rejection at a significantly faster rate than those from anesthetized living controls or anesthetized animals ventilated for 6 hours. Within 3 hours after placement and reperfusion of brain-dead donor grafts, significant neutrophil infiltration was observed, followed by increasing numbers of macrophages and T cells. mRNA of proinflammatory mediators detected in kidneys within 6 hours of brain death and upregulated even before transplantation increased thereafter and appeared to accelerate and amplify host alloresponsiveness, as manifested by the rapid expression of chemokines, cytokines, adhesion molecules, and major histocompatibility complex class II antigens in the engrafted organ. The process evolved in the controls less intensely and at a slower rate. ConclusionsDonor brain death is a significant risk factor for peripheral organs used for transplantation. The activated state of such organs appears to trigger host immune mechanisms that accelerate the process of acute rejection. The effects of this central injury may explain in part the less satisfactory performance of cadaver organs in human transplantation compared with those from living sources.

Sequential cellular and molecular kinetics in acutely rejecting renal allografts in rats.

The initial (0-24 hr), early (3-5 days), and late (7-14 days) events occurring in LBNF1 renal allografts transplanted into Lew recipients were examined to define precisely the sequential cellular and molecular kinetics during acute rejection. Grafts and spleens were harvested at 3, 6, 12, and 24 hr, and at 3, 5, 7, and 14 days and processed for morphology, immunohistology, and reverse transcriptase-polymerase chain reaction. Various factors (mRNA) were up-regulated sequentially in the allografts over time. In the initial phase, E-selectin and complement (C1 and C3) expression was noted within 6 hr, peaking by 24 hr. RANTES (regulated upon activation, normal T cell expressed and secreted) increased within 6 hr, and then again between 3 and 6 days. By immunohistology, MHC class II was up-regulated consistently after day 1. Intercellular adhesion molecule-1 expression increased after day 3; lymphocyte function-associated antigen-1+ infiltrating leukocytes peaked at day 5. Infiltrating CD8+ T lymphocytes increased strikingly between days 1 and 3, peaking at day 5; CD4+ cells infiltrated more slowly until day 5. The kinetics of ED1+ macrophages were similar to those of lymphocyte function-associated antigen-1+ cells. The CD4+ T cell-derived product, interleukin (IL)-2, peaked at 7 days. Interferon-gamma increased progressively up to 14 days. By 3 days, the macrophage-associated factor, transforming growth factor-beta, peaked; this was followed by increased IL-6 expression by day 5. IL-1, tumor necrosis factor-alpha, and inducible nitric oxide synthase increased slowly until day 7, declining thereafter. Endothelin increased progressively over the 14-day follow-up period. Cytokine dynamics occurring in host spleen were similar to those noted in the allografts. Although acute rejection is primarily T cell mediated, adhesion molecules, macrophages, and their associated products may influence initial and later changes. The brisk expression of complement, E-selectin, and RANTES within the first few hours after engraftment may occur secondary to ischemic injury and trigger subsequent immunological events. Macrophages and their products may play a larger role in the process than hitherto appreciated.

A model of gradual onset brain death for transplant-associated studies in rats

BACKGROUND The relatively few studies that have examined the systemic events after brain death have primarily involved large animals. For more precise definition of the physiology of this central catastrophe and its influence on peripheral organs, we have established a reproduceable model of gradual onset brain death in rats. METHODS The central injury is induced by graded inflation of a Fogarty catheter placed intracranially under EEG and blood pressure monitoring. The rats were mechanically ventilated for 6 hr before removal of their kidneys. Complications and mortality are discussed. RESULTS The majority (83%) of the 100 experimental animals could be used as organ donors. After a transient period of autonomic storm, the mean arterial blood pressure remained consistently between 80-100 mmHg, not appreciably different from controls. Despite normotension, the transplanted kidneys from brain dead donors showed a significantly longer interval to regain uniform cortical color and turgor than kidneys from control animals. CONCLUSIONS We describe a controlled model of gradual onset brain death in the rat in which normotension can be sustained for several hours before the kidneys are removed for transplantation. Despite stable donor blood pressure, ischemia of peripheral organs may explain in part the increased incidence of delayed graft function of cadaver kidneys compared with those from living donors. This model is suitable for transplant-related studies involving organs from donors with irreversible central injury.

Serum neutrophil gelatinase-associated lipocalin as a predictor of organ recovery from delayed graft function after kidney transplantation from donors after cardiac death.

Because of a worldwide shortage of renal grafts, kidneys procured from donors after cardiac death (DCD) have recently become an important source of renal transplants. However, DCD kidneys often have complications with delayed graft function (DGF) and recipients require hemodialysis (HD) in the early period after kidney transplantation (KTx). This study evaluated serum NGAL as a potential specific parameter to predict early functional recovery of transplanted DCD kidneys. The average serum neutrophil gelatinase-associated lipocalin (NGAL) level in normal samples was 53 ± 30 ng/ml, while that in patients with chronic renal failure requiring HD was markedly raised at 963 ± 33 ng/ml. In patients undergoing a living-related KTx from a living donor (n = 11), serum NGAL level decreased rapidly after KTx, and only in two cases, with serum NGAL levels over 400 ng/ml on postoperative day 1 (POD1), was HD required due to DGF. In contrast, all patients undergoing a KTx from a DCD (n = 5) required HD due to DGF. Even in these cases, serum NGAL levels decreased rapidly several days after a KTx prior to the recovery of urine output and preceding the decrease in serum creatinine level. The pattern of decline in serum NGAL was biphasic, the decrease after the second peak indicating a functional recovery within the next several days. These data suggest that monitoring of serum NGAL levels may allow us to predict graft recovery and the need for HD after a KTx from a DCD.

Increased Urinary Neutrophil Gelatinase Associated Lipocalin Levels in a Rat Model of Upper Urinary Tract Infection

PURPOSE Recurrent upper urinary tract infection is a common complication of vesicoureteral reflux that often leads to irreversible renal scarring. In our previous study of a rat model of renal bacterial infection we performed global gene expression profiling of the kidney during the onset of renal scarring. We have further investigated the product of an up-regulated gene product, NGAL, in this animal model to evaluate its potential usefulness as a biomarker of renal scarring. MATERIALS AND METHODS Renal NGAL mRNA and protein levels were examined by real-time polymerase chain reaction, Western blot and immunohistochemistry. Urinary NGAL levels were monitored by direct enzyme-linked immunosorbent assay. RESULTS Rat renal NGAL mRNA and protein levels were found to be increased soon after bacterial injection. They then decreased rapidly but subsequently persisted at high levels until the 6-week time point after injection. On histological analysis we found that NGAL protein was overproduced in macrophages and renal tubular cells 2 weeks after injection. However, renal tubular cells continued to produce NGAL 6 weeks after injection, whereas this expression was lost in infiltrating cells. Rat urinary NGAL levels were also markedly increased at the early stages of infection and they persisted at high levels throughout the latter stages of the experiment. CONCLUSIONS Urinary NGAL may be a potential noninvasive diagnostic biomarker of renal scarring.

Exploitation of the continuum between early ischemia/reperfusion injury and host alloresponsiveness: indefinite kidney allograft survival by treatment with a soluble P-selectin ligand and low-dose cyclosporine in combination.

BACKGROUND We have shown previously that sPSGL, a soluble glycoprotein ligand for P and E selectins, reduces the events associated with ischemia/reperfusion injury of the kidney. In the present study, we have attempted to modulate differentially early inflammatory influences and later host alloresponsiveness in an LBNF1-Lewis renal graft model by treatment with sPSGL in combination with a marginally effective dose of cyclosporine (CsA). METHODS Four experimental groups were studied: group 1=control animals receiving vehicle only; group 2=sPSGL monotherapy alone; group 3=low-dose CsA; group 4=sPSGL plus low-dose CsA. Grafts were removed at 1, 3, 5, and 7 days (n=3/time point) and assessed by histology, immunohistology, and reverse transcriptase-polymerase chain reaction. Long-surviving grafts in recipients of groups 3 and 4 were followed functionally for more than 28 weeks. RESULTS Graft function was prolonged indefinitely in recipients in group 4, all of which survived for more than 200 days. In contrast, survival of animals in groups 1 and 2 was not increased substantially, whereas only 4 of 17 animals in group 3 (23.5%) survived more than 24 days (P<0.01). Five days after engraftment, necrosis was relatively minimal in group 4 organs but pronounced in those of the other groups. By immunohistology, numbers of infiltrating CD4+ and CD8+ T cells and ED1+ macrophages were significantly diminished in group 4 allografts compared with those of the other groups. Serial assessment of chemokine and cytokine mRNA expression confirmed these findings. The long-term effects of CsA treatment alone were compared with those of sPSGL in combination with CsA. Proteinuria remained virtually absent in group 4 recipients. Morphologically, the few long-surviving grafts in group 3 showed signs of chronic rejection; those in group 4 remained relatively normal. CONCLUSIONS Although treatment with sPSGL alone showed no apparent influence on the acutely rejecting transplants, at least by the parameters examined in this study, it produced indefinite survival of kidney grafts when used in combination with low-dose CsA. The data support the influence of early nonspecific injury on later immunological rejection.

Genomewide expression profiles of rat model renal isografts from brain dead donors

Background. It has been well documented that two factors, brain death (BD) and ischemia/reperfusion (I/R) injury, have distinct but overlapping adverse influences on the clinical outcome of renal transplantation. Method. We previously established a rat model of renal isografting from brain dead donors. In the present study, we performed genomic expression profiling with a high-density oligonucleotide microarray to identify genes that were upregulated or downregulated by BD and/or I/R injury. Results. Among a total of 20,550 genes, most of those upregulated by BD were genes for adhesion molecules and cytokines or for chemokines such as Gro1 and IP-10. When overexpression of these genes was assessed by real-time reverse transcriptase–polymerase chain reaction, it was only observed one hr after the engraftment of kidneys from BD donors and returned to baseline thereafter, indicating the presence of an acute systemic inflammatory response to BD. Analysis of biologic networks demonstrated the activation of specific pathways that were clearly different for BD and I/R injury. The p53 and NF&kgr;B pathway was involved in the acute response to BD, whereas the Myc, Jun, and c-fos pathway was involved in I/R injury. Investigation of secretory protein genes identified LCN2 and SPP1 as candidate genes for biologic markers. Conclusion. Because our experimental system is a good model of renal transplantation from brain dead or living human donors, our data may be useful for elucidating the pathologic processes involved and for identification of novel markers for graft dysfunction of renal transplantation.