Naoto Matsuno

Application of machine perfusion preservation as a viability test for marginal kidney graft.

Background. This study evaluated the usefulness of machine perfusion preservation parameters as indicators of kidney graft viability. Methods. Eighty-eight cadaveric kidneys were analyzed in this study. Of these, 74 kidneys (84.1%) were procured from nonheartbeating donors. The criteria for an acceptable kidney for transplantation were a perfusion flow of more than 0.4 mL/min/g with a concurrent decreasing perfusion pressure. The average perfusion pressure was 30–50 mmHg. We divided the kidneys into three groups: group 1 (n=35), 0.45–0.65 mL/min/g machine perfusion flow (MPF); group 2 (n=30), 0.65–0.90 mL/min/g MPF; and group 3 (n=23), more than 0.9 mL/min/g MPF. Results. A higher rate of primary nonfunction (PNF; 25.7%) was found in group 1, compared with 6.7% in group 2 and 0% in group 3. A higher rate of 30.4% immediate function was found in group 3, compared with 16.7% in group and 8.6% in group 1. However, a longer period of acute tubular necrosis (ATN; 12.0 days) was found in group 1 compared with 8.6 days in group 2 and 8.7 days in group 3. PNF was detected in 7 (77.8%) cases with more than 16 hr of total ischemic time (TIT) in group 1. In contrast, all of nine cases with more than 16 hr of TIT in group 3 were functional. Conclusions. MPF is a reliable indicator of graft viability based on the rate of PNF and immediate renal allograft function, especially in marginal donors.

Immunosuppressant pharmacodynamics on lymphocytes from healthy subjects and patients with chronic renal failure, nephrosis, and psoriasis: Possible implications for individual therapeutic efficacy

In organ transplantation, patients with peripheral blood mononuclear cells (PBMCs) that exhibit resistance to cyclosporine (INN, ciclosporin) or glucocorticoids in vitro are refractory to therapy based on these drugs in vivo. However, detection or distribution of the resistant patients with immunologic disorders remains to be documented.

Glucocorticoids and cyclosporine induce apoptosis in mitogen-activated human peripheral mononuclear cells

Induction of apoptosis by immunosuppressive agents such as glucocorticoids (GCs) and cyclosporine (CsA) in cultured lymphoid cells has been suggested. However, there are few studies which demonstrate the induction of apoptosis by these agents in the activation process of human peripheral blood mononuclear cells (PBMCs). Here we show that potent immunosuppressive GCs and CsA induce apoptosis in concanavalin A (con A)-activated human PBMCs. In this study, GCs and CsA suppressed human PBMC-blastogenesis when activated by con A in a dose-dependent manner, where healthy PBMCs treated with > 100 ng/ml of each immunosuppressive agent exhibited a DNA-ladder structure in electrophoretic analysis. In three chronic renal failure (CRF) patients, dose-dependency of the PBMC-apoptosis induction was confirmed by our quantification of fragmented DNA using ELISA. Furthermore, the enrichment of DNA fragmentation was significantly associated with the rate of PBMC-blastogenesis when treated with GCs or CsA (r = -0.466, P < 0.01). These results suggested that suppression of the mitogen-induced PBMC-blastogenesis by the immunosuppressive agents should be correlated with the induction of apoptosis.

Impact of Rewarming Preservation by Continuous Machine Perfusion: Improved Post-Transplant Recovery in Pigs

BACKGROUND Utilization of grafts from donors after cardiac death (DCD) greatly expands the organ pool. However, implementation of such a strategy requires the development of novel preservation methods to achieve recovery from changes owing to warm ischemia. METHODS To assess potential methods, porcine livers harvested after 60 minutes of warm ischemic time (WIT) were perfused and preserved under the following conditions: Group 1 (n = 3), 2-hour simple cold storage and 2-hour machine perfusion (MP) at 8°C; group 2 (n = 3), 2 hours at 25°C and MP at 25°C and group 3 (n = 3), 2-hour simple cold storage and gradual rewarming to 25°C by MP. The preserved liver grafts were transplanted orthotopically into recipients. RESULTS The aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and hyaluronic acid (HA) levels in recipient blood at 2 hours after reperfusion were significantly lower among group 3: AST, 789 ± 258.8, 1203 ± 217.0, and 421 ± 55.8 IU/L; LDH, 1417 ± 671.2, 2132 ± 483.9, and 634 ± 263.9 IU/L; and HA, 1660 ± 556.5, 1463 ± 332.3, and 575 ± 239.0 ng/mL for groups 1, 2 and 3, respectively. Histologically, necrosis and swelling of hepatocytes were less severe among group 3 than groups 1 and 2. Group 3 animals showed better vital responses and started spontaneous breathing within 2 hours after reperfusion; 1 recipient survived for >24 hours, although all animals in groups 1 and 2 died within 2 to 3 hours after reperfusion. CONCLUSION Rewarming by MP preservation may facilitate recovery and resuscitation of DCD liver grafts.

Rewarming preservation by organ perfusion system for donation after cardiac death liver grafts in pigs.

BACKGROUND Use of grafts from donors after cardiac death (DCD) would greatly contribute to the expansion of the donor organ pool. However, this requires the development of novel preservation methods to recover the organ from changes due to warm ischemia time (WIT). METHODS Porcine livers were perfused with a newly developed machine perfusion (MP) system. The livers were perfused with modified University of Wisconsin solution (UW) - gluconate. All grafts were procured after acute hemorrhagic shock with the ventilator off. For group 1 (n = 6), grafts were procured after WIT of 60 minutes and preserved by hypothermic MP (HMP) for 3 hours. For group 2 (n = 5), grafts were preserved with 2 hours of simple cold storage (SCS) and HMP for 2 hours. For group 3 (n = 6), grafts were preserved with 2 hours of SCS and rewarming up to 25°C by MP for 2 hours (RMP). The preserved liver grafts were transplanted orthotopically. RESULTS The alanine aminotransferase level in perfusate in RMP during perfusion preservation was maintained at less than that of HMP. The levels of aspartate aminotransferase and lactate dehydrogenase in the 2 hours after reperfusion were significantly lower in group 3. Histologically, the necrosis of hepatocytes was less severe in group 3. The survival rate in group 3 was 2/4, but 0/4 in the other group. CONCLUSION RMP is expected to facilitate the recovery of the DCD liver grafts.

Pretransplant Screening and Evaluation of Liver Graft Viability Using Machine Perfusion Preservation in Porcine Transplantation

A novel method using machine perfusion for pretransplant screening and evaluation of the viability of liver grafts has been proposed, seeking to prevent severe ischemia-reperfusion injury and to reduce the risk of primary graft nonfunction. This study sought to evaluate the viability of critical grafts, which were obtained from expanded criteria donors or donation after cardiac death donors during preservation with a new machine preservation perfusion system (NES-01). The normalized pressure transition in the hepatic artery was employed as an evaluation index for liver viability. As a result, the normalized pressure (p/p(0)) in the hepatic artery showed a distinctive transition under each experimental conditions controlled by warm ischemic time (WIT). The high viability graft, obtained under the condition of WIT as 0 minutes (WIT0), showed a quick response to hepatic artery pressure after initiating perfusion, whereas the normalized pressure showed a sudden decrease. In contrast, the normalized pressure among WIT60, which may cause the graft to lose viability, showed a poor hepatic artery response. These findings corresponded to the cumulative release of enzymes. The findings of our study suggest that monitoring of the pressure drop rate in the hepatic artery during machine perfusion can be used to evaluate liver graft viability.

Functional Recovery of Donation After Cardiac Death Liver Graft by Continuous Machine Perfusion Preservation in Pigs

INTRODUCTION Grafts from donation after cardiac death (DCD) will greatly contribute to the expand the donor pool. However, these grafts may require the development of the preservation methods because of primary nonfunction and severe ischemic bile duct injury. METHODS Porcine livers were perfused with a newly developed machine perfusion (MP) system. Each system for the portal vein or the hepatic artery had a roller pump, a flow meter, and a pressure sensor. The livers were perfused with University of Wisconsin (UW)-gluconate at 4°C-6°C for 3 hours after 2 hours simple cold storage (CS). The portal vein flow rate was 0.5 mL/min/g liver (pressure, 10 mm Hg) and the hepatic artery flow rate was 0.2 mL/min/g liver (pressure, 30 mm Hg). Orthotopic liver transplantation was performed in pigs comparing Group 1 (n = 4) procured after acute hemorrhagic shock preserved by MP, Group 2 (n = 3) procured after warm ischemia time (WIT) of 30 minutes with CS preservation, and Group 3 (n = 4) procured with 30 minutes of WIT and MP preservation. RESULTS Collected effluent aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels in the perfusion solution and serum AST and LDH were significantly lower in Group 1. AST and LDH results were lower in Group 3 than Group 2. Survival rates in Groups 1 and 3 were 3/4, but 0/3 in Group 2. CONCLUSION MP preservation was a useful promising preservation mode for DCD liver grafts.

Importance of DQB as an indicator in living-related kidney transplant

Southern blot hybridization was performed in 16 pairs of living-related kidney transplant patients and donors, using DNA samples extracted from peripheral blood lymphocytes. The number of HLA-DNA-mismatched bands was used as an indicator for graft survival. The total number of DNA-mismatched bands seemed to be a valuable parameter. This was further analyzed and it was found that DQB in particular could be used to predict graft survival. Especially, usefulness of HLA-DNA typing was found in positive MLR, where good prognosis was strongly related to DQB matching. This method can therefore be applied in selecting a suitable living-related donor with the best chance of graft survival.

Glucocorticoid-resistance in peripheral-blood lymphocytes does not correlate with number or affinity of glucocorticoid-receptors in chronic renal failure patients

Glucocorticoid (GC) resistance in patients with chronic renal failure (CRF) seriously impairs successive GC therapy after renal transplantation. We examined the relationship between GC-receptor (GC-R) parameters in peripheral-blood mononuclear cells (PBMC) and PBMC resistance to GC in 21 CRF patients and 18 healthy subjects. Each subject group was divided into two subgroups according to PBMC sensitivity to prednisolone in a mitogen assay procedure; i.e., sensitive (IC50 < 381 ng/mL) and resistant (IC50 > 381 ng/mL) groups. In healthy subjects, the mean GC-R Bmax and Kd in quiescent PBMC of the GC-sensitive group were 2.89 +/- 1.23 fmol/10(6) cells and 4.00 +/- 2.24 nM, respectively. The Bmax in these subjects significantly increased to 6.61 +/- 2.02 (257.7 +/- 107.8%) after 24 h stimulation with concanavalin A (p < 0.01), while the Kd change was not significant. The GC-R Bmax and Kd in quiescent PBMC of the GC-resistant group were 5.33 +/- 1.37 fmol/10(6) cells and 3.20 +/- 1.39 nM, respectively. Both of these parameters, however, did not change significantly after mitogen stimulation. There was a significant negative correlation between IC50S of prednisolone and increase-ratios (post/pre ratio) of Bmax after mitogen stimulation (p < 0.05). In CRF patients, Bmax and Kd in quiescent PBMC of the GC-sensitive group were 6.04 +/- 2.35 fmol/10(6) cells and 3.49 +/- 1.72 nM, respectively, while those in PBMC of the GC-resistant group were 5.13 +/- 2.31 fmol/10(6) cells and 4.04 +/- 1.62 nM, respectively. The Bmax and Kd were not significantly changed after mitogen stimulation in both subgroups of CRF. Moreover, in contrast to healthy subjects, there was no correlation between IC50 and GC-R parameters in CRF. We concluded that, in healthy subjects, decreased PBMC capacity to amplify GC-R numbers in response to mitogen is correlated with GC resistance, whereas in CRF patients the resistant mechanism is not correlated with GC-R parameters. An unknown event might be involved in GC-resistance of CRF.

Serum cholesterol levels and kidney transplantation outcome: attenuation of cyclosporine efficacy?

The role of high serum cholesterol levels in the clinical outcome of kidney-transplanted patients has been described in a recent report by Roodnat et al. (1). Their study of 676 kidney graft recipients showed that serum cholesterol levels have an independent influence on the graft, patient, and over-all graft failure. Because hyperlipidemia occurs in 60 – 80% of kidney transplant recipients (2), their findings might be of clinical value for the future improvement of kidney transplantation outcome. Although the underlying mechanisms of their findings are not clear, high serum cholesterol levels may increase the risks of cardiovascular disease and impair renal function, which may influence graft and patient survivals. In this letter, we will highlight another aspect of the role of high serum cholesterol levels on graft outcome in kidney transplanted patients. Serum cholesterol levels are reported to have an influence on cyclosporine blood disposition (3). In blood, about 40% of cyclosporine are bound to erythrocytes and 50 – 60% to cholesteroland triglyceride-containing lipoproteins (4). Cyclosporine is suggested to be incorporated into peripheral blood lymphocytes (PBLs) through low density lipoprotein(LDL) receptors on the cell surface such as the LDL-cyclosporine complex (5). High blood levels of LDL possibly increase the amount of LDL-bounded cyclosporine, but this may in turn down-regulate LDL-receptors on the cell surface (6), and subsequently reduce cellular uptake of LDL-cyclosporine complex. Indeed, hypercholesterolemia is reported to inhibit cyclosporine efficacy in nephrotic syndrome (7). In our retrospective pilot study in patients with nephrosis, we found that both total serum cholesterol and LDL-cholesterol levels are significantly correlated with individual IC50 values of cyclosporine against PBL blastogenesis in these patients (r50.733; P,0.002; n516 and r50.830; P,0.03; n57, respectively; unpublished results). A high serum cholesterol level is one of the typical clinical symptoms of nephrotic syndrome. Thus, these observations suggest that high serum cholesterol levels attenuate suppressive efficacy of cyclosporine against blastogenesis of patients’ PBLs in nephrosis. Of 676 recipients in the study of Roodnat et al.(1), 456 were treated with cyclosporine, and therefore serum cholesterol levels may possibly have had an influence on pharmacokinetics and/or pharmacodynamics of cyclosporine in these 456 recipients. One possible explanation for the increased incidence of graft failure in the recipients with high serum cholesterol levels is cyclosporine efficacy attenuation through an intervention of cyclosporine uptake into T lymphocytes via LDL-receptor down-regulation. From these points of view, a comparative evaluation of the effects of serum cholesterol levels on clinical outcome, especially incidences of rejection episodes and graft failure, between recipients treated with azathioprine and those treated with cyclosporine could be both interesting and informative in the study of Roodnat et al. (1). In addition, careful monitoring of immunological status and signs of rejection due to attenuation of cyclosporine immunosuppressive efficacy appears to be important to the recipients treated with cyclosporine when their serum cholesterol levels are unusually high.