Ivan Linares

Eight-Hour Continuous Normothermic Ex Vivo Kidney Perfusion Is a Safe Preservation Technique for Kidney Transplantation: A New Opportunity for the Storage, Assessment, and Repair of Kidney Grafts.

Background Hypothermic kidney storage causes preservation injury and is poorly tolerated by renal grafts. We investigated whether static cold storage (SCS) can be safely replaced with a novel technique of pressure-controlled normothermic ex vivo kidney perfusion (NEVKP) in heart-beating donor kidney transplantation. Methods Right kidneys were removed from 30 kg Yorkshire pigs in a model of heart-beating donation and either preserved in cold histidine-tryptophan-ketoglutarate solution for 8 hours (n = 5), or subjected to 8 hours of pressure-controlled NEVKP (n = 5) followed by renal heterotopic autotransplantation. Results During NEVKP, physiologic perfusion conditions were maintained with low intrarenal resistance and normal electrolyte and pH parameters. Aspartate aminotransferase and lactate dehydrogenase as injury markers were below the detectable analyzer range (<4 and <100 U/L, respectively). Perfusate lactate concentration decreased from baseline until the end of perfusion (10.38 ± 0.76 mmol/L vs 1.22 ± 0.26 mmol/L; P < 0.001). Posttransplantation, animals transplanted with NEVKP versus SCS grafts demonstrated similar serum creatinine peak levels (NEVKP, 2.0 ± 0.5 vs SCS 2.7 ± 0.7 mg/dL; P = 0.11) and creatinine clearance on day 10 (NEVKP, 65.9 ± 18.8 mL/min vs SCS 61.2 ± 15.6 mL/min; P = 0.74). After 10 days of follow-up, animals transplanted with NEVKP grafts had serum creatinine and blood urea nitrogen values comparable to their basal levels (P = 0.49 and P = 0.59), whereas animals transplanted with SCS grafts had persistently elevated serum creatinine and blood urea nitrogen when compared with basal levels (P = 0.01 and P = 0.03). Conclusions Continuous pressure-controlled NEVKP is feasible and safe in good quality heart-beating donor kidney grafts. It maintains a physiologic environment and excellent graft function ex vivo during preservation without causing graft injury.

Continuous Normothermic Ex Vivo Kidney Perfusion Improves Graft Function in Donation After Circulatory Death Pig Kidney Transplantation.

Background Donation after circulatory death (DCD) is current clinical practice to increase the donor pool. Deleterious effects on renal graft function are described for hypothermic preservation. Therefore, current research focuses on investigating alternative preservation techniques, such as normothermic perfusion. Methods We compared continuous pressure-controlled normothermic ex vivo kidney perfusion (NEVKP) with static cold storage (SCS) in a porcine model of DCD autotransplantation. After 30 minutes of warm ischemia, right kidneys were removed from 30-kg Yorkshire pigs and preserved with 8-hour NEVKP or in 4°C histidine-tryptophan-ketoglutarate solution (SCS), followed by kidney autotransplantation. Results Throughout NEVKP, electrolytes and pH values were maintained. Intrarenal resistance decreased over the course of perfusion (0 hour, 1.6 ± 0.51 mm per minute vs 7 hours, 0.34 ± 0.05 mm Hg/mL per minute, P = 0.005). Perfusate lactate concentration also decreased (0 hour, 10.5 ± 0.8 vs 7 hours, 1.4 ± 0.3 mmol/L, P < 0.001). Cellular injury markers lactate dehydrogenase and aspartate aminotransferase were persistently low (lactate dehydrogenase < 100 U/L, below analyzer range; aspartate aminotransferase 0 hour, 15.6 ± 9.3 U/L vs 7 hours, 24.8 ± 14.6 U/L, P = 0.298). After autotransplantation, renal grafts preserved with NEVKP demonstrated lower serum creatinine on days 1 to 7 (P < 0.05) and lower peak values (NEVKP, 5.5 ± 1.7 mg/dL vs SCS, 11.1 ± 2.1 mg/dL, P = 0.002). The creatinine clearance on day 4 was increased in NEVKP-preserved kidneys (NEVKP, 39 ± 6.4 vs SCS, 18 ± 10.6 mL/min; P = 0.012). Serum neutrophil gelatinase-associated lipocalin at day 3 was lower in the NEVKP group (1267 ± 372 vs 2697 ± 1145 ng/mL, P = 0.029). Conclusions Continuous pressure-controlled NEVKP improves renal function in DCD kidney transplantation. Normothermic ex vivo kidney perfusion might help to decrease posttransplant delayed graft function rates and to increase the donor pool.

Continuous Normothermic Ex Vivo Kidney Perfusion Is Superior to Brief Normothermic Perfusion Following Static Cold Storage in Donation After Circulatory Death Pig Kidney Transplantation.

Hypothermic preservation is known to cause renal graft injury, especially in donation after circulatory death (DCD) kidney transplantation. We investigated the impact of cold storage (SCS) versus short periods of normothermic ex vivo kidney perfusion (NEVKP) after SCS versus prolonged, continuous NEVKP with near avoidance of SCS on kidney function after transplantation. Following 30 min of warm ischemia, kidneys were removed from 30‐kg Yorkshire pigs and preserved for 16 h with (A) 16 h SCS, (B) 15 h SCS + 1 h NEVKP, (C) 8 h SCS + 8 h NEVKP, and (D) 16 h NEVKP. After contralateral kidney resection, grafts were autotransplanted and pigs followed up for 8 days. Perfusate injury markers such as aspartate aminotransferase and lactate dehydrogenase remained low; lactate decreased significantly until end of perfusion in groups C and D (p < 0.001 and p = 0.002). Grafts in group D demonstrated significantly lower serum creatinine peak when compared to all other groups (p < 0.001) and 24‐h creatinine clearance at day 3 after surgery was significantly higher (63.4 ± 19.0 mL/min) versus all other groups (p < 0.001). Histological assessment on day 8 demonstrated fewer apoptotic cells in group D (p = 0.008). In conclusion, prolonged, continuous NEVKP provides superior short‐term outcomes following DCD kidney transplantation versus SCS or short additional NEVKP following SCS.

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.

Normothermic ex vivo kidney perfusion for graft quality assessment prior to transplantation

Normothermic ex vivo kidney perfusion (NEVKP) represents a novel approach for graft preservation and functional improvement in kidney transplantation. We investigated whether NEVKP also allows graft quality assessment before transplantation. Kidneys from 30‐kg pigs were recovered in a model of heart‐beating donation (group A) after 30 minutes (group B) or 60 minutes (group C) (n = 5/group) of warm ischemia. After 8 hours of NEVKP, contralateral kidneys were resected, grafts were autotransplanted, and the pigs were followed for 3 days. After transplantation, renal function measured based on peak serum creatinine differed significantly among groups (P < .05). Throughout NEVKP, intrarenal resistance was lowest in group A and highest in group C (P < .05). intrarenal resistance at the initiation of NEVKP correlated with postoperative renal function (P < .001 at NEVKP hour 1). Markers of acid‐base homeostasis (pH, HCO3–, base excess) differed among groups (P < .05) and correlated with posttransplantation renal function (P < .001 for pH at NEVKP hour 1). Similarly, lactate and aspartate aminotransferase were lowest in noninjured grafts versus donation after circulatory death kidneys (P < .05) and correlated with posttransplantation kidney function (P < .001 for lactate at NEVKP hour 1). In conclusion, assessment of perfusion characteristics and clinically available perfusate biomarkers during NEVKP allows the prediction of posttransplantation graft function. Thus, NEVKP might allow decision‐making regarding whether grafts are suitable for transplantation.