Dagmar Kollmann

PPAR-gamma activation is associated with reduced liver ischemia-reperfusion injury and altered tissue-resident macrophages polarization in a mouse model

Background PPAR-gamma (γ) is highly expressed in macrophages and its activation affects their polarization. The effect of PPAR-γ activation on Kupffer cells (KCs) and liver ischemia-reperfusion injury (IRI) has not yet been evaluated. We investigated the effect of PPAR-γ activation on KC-polarization and IRI. Materials and methods Seventy percent (70%) liver ischemia was induced for 60mins. PPAR-γ-agonist or vehicle was administrated before reperfusion. PPAR-γ-antagonist was used to block PPAR-γ activation. Liver injury, necrosis, and apoptosis were assessed post-reperfusion. Flow-cytometry determined KC-phenotypes (pro-inflammatory Nitric Oxide +, anti-inflammatory CD206+ and anti-inflammatory IL-10+). Results Liver injury assessed by serum AST was significantly decreased in PPAR-γ-agonist versus control group at all time points post reperfusion (1hr: 3092±105 vs 4469±551; p = 0.042; 6hr: 7041±1160 vs 12193±1143; p = 0.015; 12hr: 5746±328 vs 8608±1259; p = 0.049). Furthermore, liver apoptosis measured by TUNEL-staining was significantly reduced in PPAR-γ-agonist versus control group post reperfusion (1hr:2.46±0.49 vs 6.90±0.85%;p = 0.001; 6hr:26.40±2.93 vs 50.13±8.29%; p = 0.048). H&E staining demonstrated less necrosis in PPAR-γ-agonist versus control group (24hr:26.66±4.78 vs 45.62±4.57%; p = 0.032). The percentage of pro-inflammatory NO+ KCs was significantly lower at all post reperfusion time points in the PPAR-γ-agonist versus control group (1hr:28.49±4.99 vs 53.54±9.15%; p = 0.040; 6hr:5.51±0.54 vs 31.12±9.58%; p = 0.009; 24hr:4.15±1.50 vs 17.10±4.77%; p = 0.043). In contrast, percentage of anti-inflammatory CD206+ KCs was significantly higher in PPAR-γ-agonist versus control group prior to IRI (8.62±0.96 vs 4.88 ±0.50%; p = 0.04). Administration of PPAR-γ-antagonist reversed the beneficial effects on AST, apoptosis, and pro-inflammatory NO+ KCs. Conclusion PPAR-γ activation reduces IRI and decreases the pro-inflammatory NO+ Kupffer cells. PPAR-γ activation can become an important tool to improve outcomes in liver surgery through decreasing the pro-inflammatory phenotype of KCs and IRI.

Comparison of Continuous Normothermic Ex Vivo Kidney Perfusion to Dynamic and Static Hypothermic Preservation Techniques in Porcine Kidneys Donated after Cardiac Death

Background Strategies to decrease preservation-related injury in renal grafts donated after circulatory death (DCD) urgently needed. This highlights the importance of direct comparison of dynamic preservation technologies for kidneys. Our prior work has suggested superior graft function of continuous normothermic ex vivo kidney perfusion (NEVKP) over static cold storage. Here we investigated whether NEVKP could promote functional recovery compared to hypothermic machine perfusion (HMP). Methods 15 porcine kidneys were exposed to 30min of warm ischemia, then subjected to either 16hrs of SCS, 16hrs of HMP (LifePort® 1.0) or 16hrs of NEVKP prior to autotransplantation (n=5 each group). Animals were followed for 8 days. Graft function and histology were assessed. Results Grafts preserved by NEVKP demonstrated improved graft function, and more rapid graft recovery, with the mean peak serum creatinine of 3.66 ± 1.33mg/dl, occurring on day 1, compared with 8.82 ± 3.17mg/dl in the HMP group, occurring on day 2, and 10.5 ± 5.38mg/dl in the SCS group, occurring on day 3. Differences between daily serum creatinine levels reached significance between NEVKP and HMP on day 1 (p=0.002), day 2 (p=0.004) and day 3 (p=0.024), and between HMP and SCS on day 3 (p=0.016) and day 4 (p=0.046). Injury scores [scaled from 0 to 3] were lower in both perfused groups (NEVKP, HMP): score: 1 (1-1.5) compared to SCS group: score: 1.5(1-3) in wedge biopsies taken on postoperative day 8, however this did not reach statistical significance (p>0.05). Similarly, inflammation scores were not statistically different between any groups. Figure. No caption available. Conclusion In this DCD model of renal autotransplantation, we demonstrated that NEVKP significantly improved initial graft function compared to either HMP or SCS. Thus, normothermic perfusion may provide superior preservation options for DCD renal grafts than conventional hypothermic methods. The biologic mechanisms underlying normothermic preservation present new opportunities to advance these findings.

Normothermic Ex-vivo Kidney Perfusion Improves Function of Marginal Renal Grafts that were Subjected to Prolonged Ischemia Prior to Preservation

Background Normothermic ex-vivo kidney perfusion (NEVKP) is an emerging technique for renal graft preservation. We investigated whether NEVKP could promote improved marginal graft function compared to cold storage in a model of donation after cardiac death. Methods Kidneys from 30kg Yorkshire pigs were removed following 30, 60, 90, or 120 minutes of warm ischemia (WI). These grafts were then preserved in either cold histidine-tryptophan-ketoglutarate solution (CS) or subjected to pressure-controlled NEVKP for 8 hours prior to heterotopic autotransplantation. Results Prolonging WI time prior to kidney retrieval and subsequent storage in CS resulted in grafts that demonstrated incremental posttransplant increases in serum creatinine with grafts subjected to 120min of WI having persistent elevation (POD7: 13.45±3.50mg/dl vs baseline: 1.1±0.33mg/dl p<0.01, n=4). During NEVKP perfusion, 120min WI grafts cleared lactate from perfusion solution (0hr: 10.48±0.93mmol/L vs 7hr: 1.48±0.85mmol/L, p<0.01, n=5), had decreasing intra-renal resistance (0hr: 2.26±0.9mmHg/mL/min vs 7hr: 0.37±0.6mmHg/mL/min, p<0.01), and continuous urine production. Posttransplantation, 120min WI grafts with NEVKP, compared to CS, demonstrated significantly decreased serum creatinine peak values (POD4: 12.62±2.34mg/dl vs POD5: 18.95±1.11mg/dL, p=0.001) and higher creatinine clearance (POD4: 6.61±4.03mL/min vs 0.35±0.30mL/min, p=0.02 and POD7: 26.31±11.54mL/min vs 9.78±4.6mL/min, p=0.03). On POD7, serum creatinine returned to baseline values in the NEVKP group (POD7: 4.88±5.57mg/dL vs baseline: 1.02±0.16mg/dL, p=0.16) but not the CS group (POD7: 13.45±3.50mg/dl vs baseline: 1.1±0.33mg/dl p<0.01, n=4). Histology from 120min WI NEVKP grafts at POD7 demonstrated decreased tubular injury scores compared to cold CS grafts (1.8+/-0.8 vs. 3.0+/-0.0, p=0.03) as assessed by a blinded pathologist. Conclusion Kidney grafts subjected to 120min of WI before retrieval showed significant improvement in function following 8hrs of continuous pressure-controlled NEVKP compared to CS. This suggests NEVKP could be utilized to expand the donor pool through the consideration of extreme marginal grafts for transplantation.

Normothermic Ex-Vivo Kidney Perfusion Restores the Genetic Profile of Marginal Kidney Grafts Subjected to Warm Ischemia

Background Increasing evidence demonstrates the superiority of normothermic ex-vivo kidney perfusion (NEVKP) over cold storage (CS) for grafts sensitive to ischemia-reperfusion injury, including kidneys procured following donation-after-cardiac-death (DCD). To account for this improvement, we investigated whether NEVKP-preservation returns the genetic profiles of DCD grafts to that of unmanipulated naïve kidneys. Methods Kidneys from 30kg Yorkshire pigs were removed following 30 minutes of warm ischemia in a model of DCD. These grafts were then stored in either static cold histidine-tryptophan-ketoglutarate solution (CS) or subjected to pressure-controlled NEVKP for 8 hours prior to heterotopic autotransplantation. Kidney biopsies were collected on POD3 and gene expression was compared with naïve porcine kidneys utilizing the Affymetrix GeneChip® Porcine Gene 1.0 ST Array platform examining over 23,000 transcripts. Validation was performed using quantitative real-time polymerase chain reaction. Results During NEVKP storage, DCD grafts demonstrated favourable perfusion characteristics including progressive lactate clearance (0hr: 10.29 +/-0.48 mmol/L vs 8hr: 1.67+/-0.67 mmol/L, n=5, P<0.01), decreasing intra-renal resistance (0hr:1.63+/-0.20 mmHg/mL/min vs 8hr:0.41+/-0.13 mmHg/mL/min, n=5, p<0.01), and continuous urine production. Post-transplantation graft function significantly improved with NEVKP compared to CS with decreased peak serum creatinine (POD1: 4.0+/-1.15mg/dL vs POD3: 12.0+/-0.78mg/dL, n=5, p<0.01) and higher creatinine clearance on POD3 (39.6+/-11.8mL/min vs 2.6+/-0.9ml/min, n=5, p<0.01). Genomic comparison of grafts subjected to NEVKP on POD3 showed significant differences in only 27 genes when compared to naïve porcine kidneys (Table 1, >±2-fold changes in expression over naïve, n=3, P<0.05, FDR p-value<0.20). In contrast, 668 genes were significantly differentially expressed between grafts stored with CS on POD3 and naïve kidneys (Table 2, >±2-fold changes in expression over naïve, n=3, P<0.05, FDR p-value<0.20). Table. No title available. Table. No title available. Conclusions NEVKP restored damaged kidney grafts in a model of DCD with a genomic profile closely resembling naïve kidneys. Conversely, ongoing injury was evident in DCD grafts following CS through increased expression of genes related to inflammation, apoptosis, and repair. Together, these findings support the use of NEVKP for storage of DCD grafts that are more susceptible to ischemia-reperfusion injury.

Live Donor Liver Transplantation Using selected Grafts with Two Bile Ducts compared to One Bile Duct Does not Impact on Patient Outcome

The outcome after living donor liver transplantation (LDLT) using grafts with multiple bile ducts (BDs) remains unclear. We analyzed 510 patients who received an adult‐to‐adult right lobe LDLT between 2000 and 2015 and compared outcome parameters of those receiving grafts with 2 BDs (n = 169) with patients receiving grafts with 1 BD (n = 320). Additionally, patients receiving a graft with 3 BDs (n = 21) were analyzed. Demographic variables and disease severity were similar between the groups. Roux‐en‐Y reconstruction was significantly more common in the 2 BD group (77% versus 38%; P < 0.001) compared with the 1 BD group. No difference was found in biliary complication rates within 1 year after LDLT (1 BD versus 2 BD groups, 18% versus 21%, respectively; P = 0.46). In the 2 BD group, 82/169 (48.5%) patients were reconstructed with 2 anastomoses. The number of anastomoses did not negatively impact biliary complication rates. Recipients’ major complication rate (Clavien ≥ 3b) was similar between both groups (1 BD versus 2 BD groups, 21% versus 24%, respectively; P = 0.36). Furthermore, no difference could be found between the 1 BD, the 2 BD, and the 3 BD groups in the frequency of developing biliary complications within 1 year (18%, 21%, 14%, respectively; P = 0.64), BD strictures (15%, 15%, 5%, respectively; P = 0.42), or BD leaks (10%, 11%, 10%, respectively; P = 0.98). In addition, the 1‐year (90% versus 91%), 5‐year (82% versus 77%), and 10‐year (70% versus 66%) graft survival rates as well as the 1‐year (92% versus 93%), 5‐year (84% versus 80%), and 10‐year (75% versus 76%) patient survival rates were comparable between the 1 BD and the 2 BD groups (P = 0.41 and P = 0.54, respectively). In conclusion, this study demonstrates that selected living donor grafts with 2 BDs can be used safely without negatively impacting biliary complication rates and graft or patient survival rates.

Splenectomy as Flow Modulation Strategy and Risk Factors of De Novo Portal Vein Thrombosis in Adult‐to‐Adult Living Donor Liver Transplantation

Portal vein thrombosis (PVT) is a severe complication after liver transplantation that can result in increased morbidity and mortality. Few data are available regarding risk factors, classification, and treatment of PVT after living donor liver transplantation (LDLT). Between January 2004 and November 2014, 421 adult‐to‐adult LDLTs were performed at our institution, and they were included in the analysis. Perioperative characteristics and outcomes from patients with no‐PVT (n = 393) were compared with those with de novo PVT (total portal vein thrombosis [t‐PVT]; n = 28). Ten patients had early portal vein thrombosis (e‐PVT) occurring within 1 month, and 18 patients had late portal vein thrombosis (l‐PVT) appearing later than 1 month after LDLT. Analysis of perioperative variables determined that splenectomy was associated with t‐PVT (hazard ratio [HR], 3.55; P = 0.01), e‐PVT (HR, 4.96; P = 0.04), and l‐PVT (HR, 3.84; P = 0.03). In contrast, donor age was only found as a risk factor for l‐PVT (HR, 1.05; P = 0.01). Salvage rate for treatment in e‐PVT and l‐PVT was 100% and 50%, respectively, without having an early event of rethrombosis. Mortality within 30 days did not show a significant difference between groups (no‐PVT, 2% versus e‐PVT, 10%; P = 0.15). No significant differences were found regarding 1‐year (89% versus 92%), 5‐year (79% versus 82%), and 10‐year (69% versus 79%) graft survival between the t‐PVT and no‐PVT groups, respectively (P = 0.24). The 1‐year (89% versus 96%), 5‐year (82% versus 86%), and 10‐year (79% versus 83%) patient survival was similar for the patients in the no‐PVT and t‐PVT groups, respectively (P = 0.70). No cases of graft loss occurred as a direct consequence of PVT. In conclusion, the early diagnosis and management of PVT after LDLT can lead to acceptable early and longterm results without affecting patient and graft survival.

Expanding the donor pool: Donation after circulatory death and living liver donation do not compromise the results of liver transplantation

Because of the shortfall between the number of patients listed for liver transplantation (LT) and the available grafts, strategies to expand the donor pool have been developed. Donation after circulatory death (DCD) and living donor (LD) grafts are not universally used because of the concerns of graft failure, biliary complications, and donor risks. In order to overcome the barriers for the implementation of using all 3 types of grafts, we compared outcomes after LT of DCD, LD, and donation after brain death (DBD) grafts. Patients who received a LD, DCD, or DBD liver graft at the University of Toronto were included. Between January 2009 through April 2017, 1054 patients received a LT at our center. Of these, 77 patients received a DCD graft (DCD group); 271 received a LD graft (LD group); and 706 received a DBD graft (DBD group). Overall biliary complications were higher in the LD group (11.8%) compared with the DCD group (5.2%) and the DBD group (4.8%; P < 0.001). The 1‐, 3‐, and 5‐year graft survival rates were similar between the groups with 88.3%, 83.2%, and 69.2% in the DCD group versus 92.6%, 85.4%, and 84.7% in the LD group versus 90.2%, 84.2%, and 79.9% in the DBD group (P = 0.24). Furthermore, the 1‐, 3‐, and 5‐year patient survival was comparable, with 92.2%, 85.4%, and 71.6% in the DCD group versus 95.2%, 88.8%, and 88.8% in the LD group versus 93.1%, 87.5%, and 83% in the DBD group (P = 0.14). Multivariate Cox regression analysis revealed that the type of graft did not impact graft survival. In conclusion, DCD, LD, and DBD grafts have similar longterm graft survival rates. Increasing the use of LD and DCD grafts may improve access to LT without affecting graft survival rates. Liver Transplantation 24 779–789 2018 AASLD.