Systemic and localized ischemia and reperfusion injury remain clinically relevant issues after organ transplantation and contribute to organ dysfunctions, among which acute kidney injury is one of the most common. An in vitro test-circuit for normothermic perfusion of porcine kidneys after warm ischemia was used to investigate the antioxidant properties of vitamin C during reperfusion. Vitamin C is known to enhance microcirculation, reduce endothelial permeability, prevent apoptosis, and reduce inflammatory reactions. Based on current evidence about the pleiotropic effects of vitamin C, we hypothesize that the antioxidant properties of vitamin C might provide organ-protection and improve the kidney graft function in this model of ischemia and reperfusion. Methods: 10 porcine kidneys from 5 Landrace pigs were perfused in vitro for 6 h. For each experiment, both kidneys of one animal were perfused simultaneously with a 1:1 mixture of autologous blood and modified Ringer’s solution at 38 °C and 75 mmHg continuous perfusion pressure. One kidney was treated with a 500 mg bolus injection of vitamin C into the perfusate, followed by continuous infusion of 60 mg/h vitamin C. In the control test circuit, an equal volume of Ringer’s solution was administered as a placebo. Perfusate samples were withdrawn at distinct points in time during 6 h of perfusion for blood gas analyses as well as measurement of serum chemistry, oxidative stress and antioxidant capacity. Hemodynamic parameters and urine excretion were monitored continuously. Histological samples were analyzed to detect tubular- and glomerular-injury. Results: vitamin C administration to the perfusate significantly reduced oxidative stress (49.8 ± 16.2 vs. 118.6 ± 23.1 mV; p = 0.002) after 6 h perfusion, and increased the antioxidant capacity, leading to red blood cell protection and increased hemoglobin concentrations (5.1 ± 0.2 vs. 3.9 ± 0.6 g/dL; p = 0.02) in contrast to placebo treatment. Kidney function was not different between the groups (creatinine clearance vit C: 2.5 ± 2.1 vs. placebo: 0.5 ± 0.2 mL/min/100 g; p = 0.9). Hypernatremia (187.8 ± 4.7 vs. 176.4 ± 5.7 mmol/L; p = 0.03), and a lower, but not significant decreased fractional sodium excretion (7.9 ± 2 vs. 27.7 ± 15.3%; p = 0.2) were observed in the vitamin C group. Histological analysis did not show differences in tubular- and glomerular injury between the groups. Conclusion: Vitamin C treatment increased the antioxidant capacity of in vitro perfused kidney grafts, reduced oxidative stress, preserved red blood cells as oxygen carrier in the perfusate, but did not improve clinically relevant parameters like kidney function or attenuate kidney damage. Nevertheless, due to its antioxidative properties vitamin C might be a beneficial supplement to clinical kidney graft perfusion protocols.

Normothermic machine perfusion (NMP) of kidneys in combination with an optimized perfusate composition may increase donor organ preservation quality, especially in the case of marginal donor grafts. Optimization of currently employed perfusates is still a subject of present research. Due to the advantages of being cell-free, easy to store, and having minimal antigenicity, hemoglobin-based oxygen carriers, such as HBOC-301 (Oxyglobin^®, Hemoglobin Oxygen Therapeutics LLC, Souderton, PA, USA), offer an alternative to the commonly used perfusates based on packed red blood cells (pRBC). As previously described, using HBOC results in formation of methemoglobin (metHb) as an adverse effect, inducing hypoxic conditions during the perfusion. As a potential counterpart to metHb formation, the application of the antioxidant ascorbic acid (VitC) is of high interest. Therefore, this study was conducted in four experimental groups, to compare the effect of NMP with (1) HBOC or (3) pRBC, and additionally examine a beneficial effect of VitC in both groups (2) HBOC + VitC and (4) pRBC + VitC. All groups were subjected to NMP for 6 h at a pressure of 75 mmHg. Kidneys in the HBOC groups had a significantly lower renal blood flow and increasing intrarenal resistance, with reduced renal function in comparison to the pRBC groups, as demonstrated by significantly lower creatinine clearance and higher fractional sodium excretion rates. Clinical chemistry markers for tissue damage (LDH, lactate) were higher in the HBOC groups, whereas no significant histological differences were observed. Although the application of VitC decreased oxidative stress levels, it was not able to significantly increase the outcome parameters mentioned above in either group. This study demonstrated that HBOC-301 is inferior to pRBCs in our porcine kidney NMP model, independent of additional VitC administration. Oxidative stress and fragmentation of the hemoglobin polymers could be detected as a possible reason for these results, hence further research, focusing on the use of cell-free oxygen carriers that do not exhibit this complex of issues, is required.

Donor organ-shortage has resulted in the increased use of marginal grafts; however, normothermic machine perfusion (NMP) holds the potential for organ viability assessment and restoration of marginal grafts prior to transplantation. Additionally, cell-, oxygen carrier-free and antioxidants-supplemented solutions could potentially prevent adverse effects (transfusion reactions, inflammation, hemolysis), associated with the use of autologous packed red blood cell (pRBC)-based perfusates. This study compared 6 h NMP of porcine kidneys, using an established pRBC-based perfusate (pRBC, n = 7), with the novel cell- and oxygen carrier-free organ preservation solution Ecosol, containing taurine (Ecosol, n = 7). Despite the enhanced tissue edema and tubular injury in the Ecosol group, related to a suboptimal molecular mass of polyethylene glycol as colloid present in the solution, functional parameters (renal blood flow, intrarenal resistance, urinary flow, pH) and oxygenation (arterial pO₂, absence of hypoxia-inducible factor 1-alpha) were similar to the pRBC group. Furthermore, taurine significantly improved the antioxidant capacity in the Ecosol group, reflected in decreased lactate dehydrogenase, urine protein and tubular vacuolization compared to pRBC. This study demonstrates the feasibility of 6 h NMP using a taurine containing, cell- and oxygen carrier-free perfusate, achieving a comparable organ quality to pRBC perfused porcine kidneys.