Improving the quality of kidney grafts is currently one of the main challenges, as the increasing demand for donor organs clearly exceeds the availability. In 2016, there were 33,291 adult patients removed from the kidney waiting lists worldwide; with over one fourth deceased due to aggravated medical condition during the waiting time for transplantation, which reflects the donor organ shortage [1
]. Numerous organs, which have been rejected due to their extended criteria donors, or donation after cardiac death status in the past might have been rescued or will be rescued in future if improved preservation strategies become available. These organs suffer from extended warm ischemia time and the aggravated reperfusion injury.
The major challenge is to overcome the factors inducing kidney graft dysfunction, which is mainly related to Ischemia and reperfusion (I/R)-injury with oxidative stress and inflammation [2
]. During reperfusion after a period of ischemia, the amount of reactive oxygen species can increase dramatically thereby exceeding the natural antioxidant defenses, causing damage to macromolecules and thus significantly injuring cell structures and function on a local, as well as the systemic level [6
]. This results in a systemic inflammatory response syndrome, manifesting as pyrexia, tachycardia, leukocytosis, hypotension, edema, and organ failure [2
]. Moreover, hypoperfusion, over- or underhydration, usage of nephrotoxic drugs, endotoxemia and cholesterol emboli can add to tubular injury, edema and kidney injury.
During the last decades, several strategies for preservation of kidney grafts prior to transplantation have been developed. As a golden standard, cold storage is applied, and different research concepts, such as the combination with hypothermic machine perfusion preceding, following or in between cold storage have been focused [7
]. However, during recent years normothermic machine perfusion (NMP) of porcine [8
] or human kidneys [9
] emerged as the most promising strategy, enabling the successful transplantation of human kidney grafts that were previously declined [10
]. Yong et al. postulated that NMP might have the potential to increase the donor pool by improving the outcome after organ transplantation of organs from extended criteria donors or those from donations after cardiac death [11
Our working group recently published a study showing significant benefits of six hours direct NMP without previous cold flushing on porcine kidney function and damage (FABRY et al.). Kidneys preserved by NMP immediately after explanation were compared to cold flushed grafts according to clinical protocols before NMP was initiated. Regardless of the observed beneficial effects (i.e., improved creatinine clearance, reduced osmotic nephropathy, decreased urine protein concentration), we stated that the perfusate, which was in accordance to commonly used recipes [12
], needs further improvement to extend the benefits of NMP without cold flush- or storage beyond six hours of perfusion.
Vitamin C is an essential, pleiotropic, and water-soluble micronutrient required for more than 60 enzymatic reactions, among which are the synthesis of norepinephrine collagen and carnitine [14
]. Vitamin C is involved in iron absorption, peptide amination, tyrosine and steroid metabolism and cytochrome P450-driven hydroxylation of aromatic drugs and carcinogens [16
]. Vitamin C enhances cell differentiation from somatic cells to induced pluripotent stem cells [17
], which might play an important role for regenerating processes in patients undergoing major surgery (e.g., kidney transplantation), as well as in isolated organs. Vitamin C is known to restore vascular responsiveness to vasoconstrictors [18
], ameliorates microcirculatory blood flow, preserves endothelial barriers [19
], prevents apoptosis and augments the bacterial defense [20
]. Based on its redox-potential and powerful antioxidant capacity, vitamin C has been described as the most important antioxidant, especially in I/R injury [21
]. Vitamin C demonstrated organoprotective effects in the nervous, cardiovascular, respiratory, gastrointestinal, coagulation and immune systems in preclinical as well as in clinical studies [16
Therefore, we aimed to investigate the antioxidant capacity of vitamin C and its pleiotropic effects as an essential micronutrient for organ protection in an in vitro I/R-porcine kidney NMP model.
The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University Hospital and performed in accordance with German legislation governing animal studies following the ‘Guide for the Care and Use of Laboratory Animals’ (National Institute of Health publication, 8th edition, 2011) and the Directive 2010/63/EU on the protection of animals used for scientific purposes (Official Journal of the European Union, 2010).
Five female German Landrace pigs with 64.4 ± 0.8 kg body weight (BW, mean ± SEM) were housed in fully air-conditioned rooms with 22 °C room temperature, and a relative humidity of 50%. After arrival, the pigs were allowed to acclimatize to their surroundings for a minimum of seven days and fasted for 12 h before surgery with free access to water. As premedication the animals received intramuscular injections of 8 mg/kg BW azaperone (Stresnil, Janssen-Cilag GmbH, Neuss, Germany), 15 mg/kg BW ketamine (Ceva GmbH, Duesseldorf, Germany) and 10 mg atropine (1 mL/1% atropine sulfate, Dr. Franz Köhler Chemie GmbH, Bensheim, Germany). The femoral vein of the anaesthetized animal was cannulated, and 600 mL of venous blood were withdrawn into two heparinized blood bags (5000 IU/bag, B. Braun Melsungen AG, Melsungen, Germany), before the animals were euthanized by an intravenous administration of 1 mL/kg BW pentobarbital (Narcoren, Merial GmbH, Hallbergmoss, Germany). Immediately after cardiac arrest, a midline laparotomy was performed, and both kidneys were explanted simultaneously to achieve an equal warm ischemic time. The warm ischemic time was defined as the duration from cardiac arrest until explanation. In compliance with the 3R principle (Replacement, Reduction and Refinement of animal experiments) [23
], the kidneys for this study were obtained from animals which were initially used by another in-house working group and furthermore, the other organs of the animals were also used for different in vitro research purposes in different in-house institutes.
2.2. Test Circuits
Two identical in-vitro test-circuits for normothermic machine perfusion of porcine kidneys were used to investigate the effects of vitamin C in porcine kidneys. The perfusate was collected from the renal vein into a hard shell-reservoir (Capiox CR10NX, Terumo Deutschland GmbH, Eschborn, Germany), and circulated by a centrifugal pump through an oxygenator (Deltastream DP2, HILITE 800®; both MEDOS Medizintechnik AG, Stolberg, Germany) into the renal artery. A continuous pressure of 75 mmHg was maintained by a computer-controlled custom-built pump controller. The temperature was kept at 38 °C by a water bath thermostat. Perfusate flow and pressures were monitored continuously, using an ultrasonic flow probe (SonoTT, em-tec GmbH, Finning, Germany) and pressure transducers (DATEX AS/3, GE Healthcare; Solingen, Germany).
The test circuit was primed using 300 mL of autologous blood and 300 mL of modified Ringer’s solution. After connection of the kidney graft, the first 200 mL drainage from the renal vein were discarded, leaving a full circuit volume of 500 mL.
2.3. Kidney Perfusion and Vitamin C Administration
The kidney blood vessels and the ureter were cannulated immediately after explanation (renal artery catheter: retrograde cardioplegia catheter, 14 French, Edwards Life Sciences; Unterschleißheim, Germany/renal vein catheter: ¼” tube connector, ¼” tubing, free life medical GmbH, Aachen, Germany/ureter catheter: 14 French catheter; Convatec Germany GmbH, Munich, Germany). The kidneys were then connected to the test circuit and perfused at 75 mmHg mean arterial pressure for 6 h. Both kidneys of one animal were perfused simultaneously in two identical test circuits. One circuit received vitamin C for intravenous use (WOERWAG Pharma GmbH & Co. KG, Boeblingen, Germany) diluted in Ringer’s solution, while in the control circuit an equal amount of Ringer’s solution was administered; all other parameters, interventions and handling procedures were identical in both circuits. Kidneys were allocated to experimental groups (vitamin C or control) and test-circuits in a randomized manner, to prevent a bias. The vitamin C was stored at 7 °C and administered to the circuit with ultraviolet (UV)-protected infusion lines and syringes. The vitamin C circuit was primed with an initial bolus injection of 500 mg vitamin C into the perfusate immediately before connecting the kidneys, followed by continuous infusion of 60 mg/h. This dosage was chosen in line with the dose-finding study by Fowler et al., which was observed it to be most effective. As high-dose vitamin C administration—especially for longer times—may also have negative effects on kidneys, for example occurrence of kidney stones [24
], our research group abstained from higher vitamin C dosages, such as 66 mg/kg/h [25
] or 125 mg/kg [26
], which have previously described by other authors in other clinical settings.
2.4. Sampling and Measurements
Blood samples were drawn at distinct points in time during 6 h of perfusion (0, 30, 60, 120, 240, 300 and 360 min) for blood gas analyses including hemoglobin (HB) and electrolytes (ABL800; Radiometer GmbH, Willich, Germany) and subsequent analysis. Markers of inflammation were assessed using appropriate ELISA kits (interleukin 6 (IL6), interleukin 10 (IL10), tumor necrosis factor-α (TNF-α); all from R&D Systems, Wiesbaden, Germany). The measurement of serum chemistry (serum and urine creatinine) was performed in the inhouse central laboratory. Hemolysis was detected using a colorimetric assay (hemoglobin FS (flüssig, stabil/liquid, stable) reagent; Diasys Inc, Holzheim, Germany). 25 µl of plasma was mixed with 85 µl of the kit-reagent in a 96 well plate format, and the absorption was detected on a microplate reader (iMark; BioRad Laboratories GmbH, Munich, Germany) at 540 (A1) and 680 nm (A2) wavelength. The concentration of free HB was calculated according to the formula (A1-A2) × 733 in mg/dL. Oxidative stress was assessed using the oxidation-reduction potential (ORP) and Antioxidant Capacity (AC), measured with the RedoxSYS Diagnostic SystemTM (Aytu BioScience, Inc., Englewood, CO, USA). A low ORP being a sum of all oxidants and reductants in the blood indicates low oxidative stress, while a high AC is a measure of good antioxidant defense. Hemodynamic parameters and urine excretion were monitored continuously. Renal tissue samples were processed and Periodic acid-Schiff (PAS) staining was performed as described previously [27
], to analyze tubular- and glomerular-injury, using a scoring system (0 = no; 1 = mild; 2 = moderate; 3 = severe).
2.5. Statistical Analysis
Statistical analysis was performed using GraphPad Prism 8 software package (GraphPad Software Inc, La Jolla, CA, USA). A two-way analysis of variance (ANOVA) and multiple Comparison were used followed by Bonferroni post-test correction for all measurements during perfusion, after performing a Shapiro-Wilk normality test. The effects of time were calculated by multivariate analysis for repeated measurements. For the comparsion of histological damage scores between the groups one sample t-test was applied. Data are presented as mean ± SEM and a p value < 0.05 was considered statistically significant.
In this study, porcine kidneys were perfused in an extracorporeal pressure-controlled normothermic perfusion system, and vitamin C administration to the perfusate was compared to placebo treatment in two identical perfusion circuits. We could demonstrate a strong effect of vitamin C on oxidative stress and antioxidant capacity. However, vitamin C did not improve the creatinine clearance, renal blood flow, or oxygen consumption. The proinflammatory cytokines IL6 and TNF-α increased in both groups, whereas the anti-inflammatory cytokine IL10 was slightly increased in the vitamin C group. Interestingly, we found significantly higher hemoglobin concentrations at the end of the experiments in the vitamin C group, as the result of significantly higher red blood cells counts. The electrolyte concentrations, potassium, chloride and calcium did not differ between groups, but a significant hypernatremia was observed in the vitamin C group, resulting from decreased fractional sodium excretion. Histological examination showed acute tubular injury and osmotic vacuolization in both groups as typical signs for in vitro perfused kidneys without significant differences between groups.
The vitamin C group had a significantly lower oxidation reduction potential throughout the whole experiment, indicating lower oxidative stress, as well as higher antioxidant capacity during the early phase of reperfusion, indicating better defense against oxidative stress. These results confirm the well-described antioxidant capacities of vitamin C, however to our knowledge, it is the first time that these effects are described in an in vitro organ perfusion setting for kidney grafts. As the greatest ischemia- and reperfusion injury is expected shortly after reperfusion, it is not surprising that the antioxidant capacity recovers in both groups during the next hours. The recovery of the antioxidant capacity in the control group could possibly be explained via mobilization of intracellular reserves of antioxidant molecules into the bloodstream as reaction to the consumption of these molecules. However, the transport of antioxidants between cells and extracellular remains yet to be elucidated [26
]. Whether the significant reduction of oxidative stress early after reperfusion will lead to improved kidney function reflected by creatine clearance and reduced kidney damage reflected in histologically observed damage remains unclear in our experiments, which might be due to the short duration of six hours.
The recovery of the antioxidant capacity in the control group could possibly be explained via mobilization of intracellular reserves of antioxidant molecules into the bloodstream as reaction to the consumption of these molecules. However, the transport of antioxidants between cells and extracellular remains yet to be elucidated [28
The pronounced hypernatremia we detected in the vitamin C group, together with lowered sodium excretion might be explained by increased vitamin C storage, which is initiated via renal sodium-dependent vitamin C transporters (SVCT1, SVCT2). Vitamin C and sodium are co-transported into the cells in the proximal renal tubes, leading to reduced sodium excretion and higher arterial sodium content [29
]. Thus, the treatment with a high vitamin C bolus dosage directly from the onset of organ perfusion, or systemic administration of vitamin C to the organ donor seem to be beneficial strategies to prepare the kidney for the detrimental effects of reperfusion after ischemia, by enhancing the intracellular vitamin C reserve.
The rapid increase in blood flow and oxygen consumption during the first hour of the perfusion initiates a strong I/R injury and inflammation cascade by inflow of immune cells into the warm-ischemia damaged tissue. Therefore, it would be of particular relevance in the initial phase of reperfusion to achieve a sufficient vitamin C supplementation in order to increase antioxidant defenses, by saturating the perfusate prior to reperfusion, as we applied in this study. A trend towards increased urine production was observed in the vitamin C group (data not shown), which might be clinically relevant when considering that delayed graft function is a predictor for impaired transplantation outcomes [31
]. Although the vitamin C treatment, especially as a bolus at the beginning of perfusion, seems to improve kidney function during the later phase of the experiments, it did not confine the inflammatory response. The increase of TNF-α as proinflammatory marker during the initial phase was significant in comparison to baseline levels in both groups. Even though there was a more prominent peak of TNF-α in the control group, the increase of IL6 in the follow-up phase of inflammatory cytokine release was not stronger compared to the vitamin C group.
The most prominent effect of vitamin C we observed, aside from the increased antioxidant capacity, was the preservation of the red blood cell (RBC) count, which was significantly decreased in the control group. Stabilizing effects of vitamin C on packed whole blood was recently described [32
], as well as beneficial effects on the rheology of RBCs, which were able to pass constricted blood vessels during impaired microcirculation when treated with vitamin C [33
]. We assume that the effect of vitamin C on the RBCs is one of the main effects which might contribute to improved kidney function during in vitro kidney perfusion, especially as a counterpart to the impaired microcirculation in I/R-settings, but the exact mechanisms have to be further elucidated. A possible negative effect of vitamin C is hemolysis, which was shown in glucose-6-phosphate dehydrogenase (G6DP)-deficient patients [34
], however, we did not detect increased hemolysis in comparison to the control group.
It remains to be determined whether the observed benefits of vitamin C treatment of kidney grafts are enhanced if application of vitamin C is started before the ischemic period. However, while there is no legal limitation to add medication to normothermic machine perfusion systems or apply vitamin C to organ recipients, there might be restrictions for the medication of donors prior to organ retrieval.
Our study had several limitations, the first being the small number of animals and kidneys. Nevertheless, the sample size was sufficient to observe statistically significant effects in some minor parameters. The extrapolation of the results to clinical relevance remains debatable, although we could show effects which were also described in clinical applications of vitamin C, such as hypernatremia. A real baseline value for the ORP and AC measured in the donor animals, prior to kidney explanation is missing in our experiments and will be included in following studies. Furthermore, we used UV-protected syringes and infusion lines for the application of vitamin C to the extracorporeal circuit, however the system itself consisted of non-UV-protective material.
In clinical practice, it is still debated by experts, whether vitamin C treatment is beneficial for kidney function. In a meta-analysis including 1.536 patients performed in 2013 by Sadat et al., vitamin C decreased the risk for acute kidney injury by 33% (risk ratio 0.672, confidence interval 0.466–0.969, p
= 0.034) [35
]. In contrast, excessive and long-term vitamin C consumption might lead to oxalate nephropathy, as described in several case reports [22
A high-dose intravenous vitamin C regimen of 66 mg/kg/hour for 24 h reduced fluid requirements, improved oxygenation and shortened duration of mechanical ventilation in 37 severe thermally injured patients [25
]. In a retrospective analysis, the same intervention led to a reduction of resuscitation fluid volume, increased urine output and trends towards decreased and shortened overall vasopressor requirement [38
] However, this therapy was associated higher urine output per day and per hour, but also with an increased risk of renal failure in a retrospective case-control study [39
]. No influence of vitamin C on kidney function was observed in two small clinical pilot trials [40
Additionally, it is still debated, which concentration of vitamin C is beneficial, as in theory, it can act as pro-oxidant in presence of redox-active metal ions, which was postulated to lead to the formation of hydroxyl radicals and thus negate any beneficial effects. However, this phenomenon is unlikely to occur in human biology under physiological circumstances [42