Prolonged Normothermic Ex Vivo Kidney Perfusion Is Superior to Cold Nonoxygenated and Oxygenated Machine Perfusion for the Preservation of DCD Porcine Kidney Grafts

The increased usage of marginal grafts has triggered interest in perfused kidney preservation to minimize graft injury. We used a donation after circulatory death (DCD) porcine kidney autotransplantation model to compare 3 of the most frequently used ex vivo kidney perfusion techniques: nonoxygenated hypothermic machine perfusion (non-oxHMP), oxygenated hypothermic machine perfusion (oxHMP), and normothermic ex vivo kidney perfusion (NEVKP). Methods: Following 30 min of warm ischemia, grafts were retrieved and preserved with either 16 h of non-oxHMP, oxHMP, or NEVKP (n = 5 per group). After contralateral nephrectomy, grafts were autotransplanted and animals were followed for 8 d. Kidney function and injury markers were compared between groups. Results: NEVKP demonstrated a significant reduction in preservation injury compared with either cold preservation method. Grafts preserved by NEVKP showed superior function with lower peak serum creatinine (NEVKP versus non-oxHMP versus oxHMP: 3.66 ± 1.33 mg/dL, 8.82 ± 3.17 mg/dL, and 9.02 ± 5.5 mg/dL) and more rapid recovery. The NEVKP group demonstrated significantly increased creatinine clearance on postoperative day 3 compared with the cold perfused groups. Tubular injury scores on postoperative day 8 were similar in all groups. Conclusions: Addition of oxygen during HMP did not reduce preservation injury of DCD kidney grafts. Grafts preserved with prolonged NEVKP demonstrated superior initial graft function compared with grafts preserved with non-oxHMP or oxHMP in a model of pig DCD kidney transplantation

Mechanisms of Improved Renal Function Following Normothermic Ex-vivo Kidney Perfusion Preservation of Grafts Severely Damaged by Prolonged Warm Ischemia

Normothermic ex-vivo kidney perfusion (NEVKP) is an emerging technique for renal graft preservation. We investigated whether NEVKP could improve early function of severely injured donation-after-cardiovascular death (DCD) grafts to reduce the incidence of delayed graft function (DGF). First, we develop a large-animal model of significant renal dysfunction (SRD) that resembles clinical DGF following static cold storage (SCS). After comparing the function of these grafts following NEVKP-storage, we investigated potential mechanisms that may account for the observed outcomes by examining their gene expression pattern. To establish the SRD model, kidneys from 30kg-Yorkshire pigs were procured following increasing warm ischemic (WI) times of 0-120min (n=3-6/group) to mimic DCD. Following 8h of SCS and autotransplantation, animals were followed for 7-days. SRD was defined as oliguriaPh.D

Rapamycin-induced Allograft Tolerance: Elucidating Mechanisms and Biomarker Discovery

The long-term success of transplantation is limited by the need for immunosuppression; thus, tolerance induction is an important therapeutic goal. A 16-day treatment with rapamycin in mice led to indefinite graft survival of fully mismatched cardiac allografts, whereas untreated hearts were rejected after 8-10 days. Specific tolerance was confirmed through subsequent skin grafts and in vitro lymphocyte assays that showed recipient mice remained immunocompetent towards 3rd party antigens but were impaired in responding to donor antigens. Mechanisms that account for this tolerant state were then investigated. Splenic CD8+CD44+ memory T-cells were reduced in tolerant mice but had increased frequencies of the CD62LLO population. CD4+CD25+Foxp3+ regulatory T-cells were increased in tolerant mice. Through multiplex PCR, 4 regulatory T-cell related genes were found up-regulated and 2 proinflammatory genes were down-regulated in accepted hearts. This expression pattern may serve as a putative biomarker of tolerance in patients undergoing transplantation.MAS

Targeted deletion of FGL2 leads to increased early viral replication and enhanced adaptive immunity in a murine model of acute viral hepatitis caused by LCMV WE.

Mounting effective innate and adaptive immune responses are critical for viral clearance and the generation of long lasting immunity. It is known that production of inhibitory factors may result in the inability of the host to clear viruses, resulting in chronic viral persistence. Fibrinogen-like protein 2 (FGL2) has been identified as a novel effector molecule of CD4(+)CD25(+) Foxp3(+) regulatory T (Treg) cells that inhibits immune activity by binding to FCγRIIB expressed primarily on antigen presenting cells (APC). In this study, we show that infection of mice with Lymphocytic Choriomeningitis Virus WE (LCMV WE) leads to increased plasma levels of FGL2, which were detected as early as 2 days post-infection (pi) and persisted until day 50 pi. Mice deficient in FGL2 (fgl2(-/-)) had increased viral titers of LCMV WE in the liver early p.i but cleared the virus by day 12 similar to wild type mice. Dendritic cells (DC) isolated from the spleens of LCMV WE infected fgl2(-/-) had increased expression of the DC maturation markers CD80 and MHC Class II compared to wild type (fgl2(+/+)). Frequencies of CD8(+) and CD4(+) T cells producing IFNγ in response to ex vivo peptide re-stimulation isolated from the spleen and lymph nodes were also increased in LCMV WE infected fgl2(-/-) mice. Increased frequencies of CD8(+) T cells specific for LCMV tetramers GP33 and NP396 were detected within the liver of fgl2(-/-) mice. Plasma from fgl2(-/-) mice contained higher titers of total and neutralizing anti-LCMV antibody. Enhanced anti-viral immunity in fgl2(-/-) mice was associated with increased levels of serum alanine transaminase (ALT), hepatic necrosis and inflammation following LCMV WE infection. These data demonstrate that targeting FGL2 leads to early increased viral replication but enhanced anti-viral adaptive T & B cell responses. Targeting FGL2 may enhance the efficacy of current anti-viral therapies for hepatotropic viruses

Hepatic necrosis.

<p>Representative pictures of the left lateral lobe with H&E staining showing a less necrotic area in the RGZ vs control group (A) 24hrs after reperfusion (26.66±4.78 vs 45.62±4.57%; p = 0.032). Image software analysis demonstrated that this difference was significant (B). Abbreviations: CTRL Control, RGZ Rosiglitazone. Five experiments (n = 5) per group per time point were performed. Results are shown as mean ± SEM, Mann-Whitney U test.</p

Hepatic macrophages.

<p>Immunohistochemistry technique showing how the total percentage of F4/80 staining decreases progressively after the IRI event. No difference was found when RGZ treated group was compared against the control group. Abbreviations: RGZ Rosiglitazone, CTRL Control. Five experiments (n = 5) per group per time point were performed. Results are shown as mean ± SEM, Mann-Whitney U test.</p

Hepatic injury and apoptosis in RGZ treated mice following reperfusion.

<p>Analysis of hepatic injury showed no significant difference in the RGZ vs Control group for AST (14451±3518 vs 18231±3688U/L, p = 0.482), Inhibition of PPAR-γ with GW9662 increased further the levels of AST when compared with control group (A). Representative images of TUNEL immunohistochemistry showing a similar grade of apoptosis in RGZ, CTRL and RGZ+GW-9662 groups at 6hr post-reperfusion (B). TUNEL positive staining was found similar in RGZ vs control group at 6hr post-reperfusion (42±6 vs 48±3%, p = 0.442) (C). GW-9662 showed a reversal of the slight beneficial effect on apoptosis found in the RGZ group (C). Note: Staining was assessed with image software analysis. Abbreviations: AST-aspartate aminotransferase, RGZ-Rosiglitazone, CTRL-Control, TUNEL-Terminal deoxynucleotidyl transferase(TdT) dUTP Nick-End-Labeling. Five experiments (n = 5) per group per time point were performed. Results are shown as mean ± SEM, Mann-Whitney U test.</p

PPAR-γ antagonist-6hrs after reperfusion.

<p>Flow-cytometry shows how the effect on pro-inflammatory NO+ KC polarization was significantly blocked by the use of PPAR-γ antagonist (GW9662) (RGZ: 3.93±3.61%, RGZ+GW9662: 35.86±21.85%, Control: 31.12±21.42%, p = 0.009) (A). Serum AST levels were also significantly reversed when antagonist was included as an intervention (B). The grade of apoptosis was significantly increased with the use of antagonist in combination with RGZ when compared with RGZ alone (C). Abbreviations: DAF-FM 4-amino-5-methylamino-2’,7’-Difluoroflurescein, TUNEL-Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling, RGZ-Rosiglitazone, CTRL-Control. Five experiments (n = 5) per group per time point were performed. Results are shown as mean ± SEM, Mann-Whitney-U-test.</p

Hepatocellular injury after 60min of ischemia and 1, 6, 12 and 24hrs of reperfusion.

<p>Significant lower AST levels were found in the RGZ vs CTRL group at 1hr (3092±105 vs 4469±551; p = 0.042), 6hr (7041±1169 vs 12192±1443; p = 0.015) and 12hr (5746±328 vs 8609±1259; p = 0.049) after reperfusion (A). RGZ vs control group showed a trend to a significant difference at 6hrs post-reperfusion in ALT (9185±1754 vs 13823±1465; p = 0.054) (B). Abbreviations: AST-aspartate aminotransferase, ALT-alanine aminotransferase, RGZ-Rosiglitazone, CTRL-Control. Five experiments (n = 5) per group per time point were performed. Results are shown as mean ± SEM, Mann-Whitney U test.</p

Level of apoptosis.

<p>Representative images of TUNEL(A) and Cleaved Caspase-3(B) immunohistochemistry showing a lower grade of apoptosis in RGZ vs Control group at 1hr and 6hr post-reperfusion. Significant lower TUNEL-staining was found in the RGZ vs CTRL group at 1hr (2.46±0.49 vs 6.90±0.85%; p = 0.001) and 6hrs (26.40±2.93 vs 50.13±8.29%; p = 0.048) post-reperfusion by image analysis(C). Cleaved caspase-3 immunohistochemistry also demonstrated less staining in the RGZ group vs control group, the difference was assessed by image software analysis (D). Abbreviations: TUNEL-Terminal deoxynucleotidyl transferase(TdT) dUTP Nick-End-Labeling, RGZ-Rosiglitazone, Ctrl-Control. Five experiments (n = 5) per group per time point were performed. Results are shown as mean ± SEM, Mann-Whitney-U-test.</p