Search

Background: Donor lungs are often discarded, with gastric aspiration accounting for ~9% of lungs unsuitable for transplantation. To increase the donor pool, it is important to understand the pathophysiology of aspiration-induced lung damage (AILD) and to assess its treatment. Methods: Precision-cut lung slices (PCLS) were prepared from murine lungs and exposed to acid—pH 1.5 to 5.5—for 15 min. We also investigated whether acid-exposed slices (pH 3.5) could affect unexposed slices. In addition, we investigated whether dexamethasone (0.5 or 1 μM) could mitigate and treat the damage in each group. In each experiment (n = 3), we analyzed cell viability (ATP/protein content) and markers of inflammation (IL-1β, IL-6, TNF-𝛼, TRAIL). Results: PCLS subjected to pH 1.5–3.5 had a significantly reduced amount of ATP, albeit no increase in inflammation markers. There was no interaction of secretions from acid-exposed slices on unexposed slices. Dexamethasone had no beneficial effects in either group. Conclusion: Direct exposure to acid in the PCLS leads to a decrease in cell viability. Acid-exposed slices had no effect on the cell viability of unexposed slices. Treatment with dexamethasone offered no mitigation. More studies have to be performed to elucidate the pathophysiology of AILD and the possible treatment of aspiration-induced injury.

Background. Normothermic machine perfusion (NMP) is a promising modality for marginal donor kidneys. However, little is known about the effects of NMP on causing endothelial glycocalyx (eGC) injury. This study aims to evaluate the effects of NMP on eGC injury in marginal donor kidneys and whether this is affected by perfusion pressures and hematocrits. Methods. Porcine slaughterhouse kidneys (n = 6/group) underwent 35 min of warm ischemia. Thereafter, the kidneys were preserved with oxygenated hypothermic machine perfusion for 3 h. Subsequently, 4 h of NMP was applied using pressure-controlled perfusion with an autologous blood-based solution containing either 12%, 24%, or 36% hematocrit. Pressures of 55, 75, and 95 mm Hg were applied in the 24% group. Perfusate, urine, and biopsy samples were collected to determine both injury and functional parameters. Results. During NMP, hyaluronan levels in the perfusate increased significantly (P < 0.0001). In addition, the positivity of glyco-stained glycocalyx decreased significantly over time, both in the glomeruli (P = 0.024) and peritubular capillaries (P = 0.003). The number of endothelial cells did not change during NMP (P = 0.157), whereas glomerular endothelial expression of vascular endothelial growth factor receptor-2 decreased significantly (P < 0.001). Microthrombi formation was significantly increased after NMP. The use of different pressures and hematocrits did not affect functional parameters during perfusion. Conclusions. NMP is accompanied with eGC and vascular endothelial growth factor receptor-2 loss, without significant loss of endothelial cells. eGC loss was not affected by the different pressures and hematocrits used. It remains unclear whether endothelial injury during NMP has harmful consequences for the transplanted kidney.

Ischemia-reperfusion injury (IRI) is a hallmark for tissue injury in donation after circulatory death (DCD) kidneys. The implementation of hypothermic machine perfusion (HMP) provides a platform for improved preservation of DCD kidneys. Doxycycline administration has shown protective effects during IRI. Therefore, we explored the impact of doxycycline on proteolytic degradation mechanisms and the urinary proteome of perfused kidney grafts. Porcine kidneys underwent 30 min of warm ischemia, 24 h of oxygenated HMP (control/doxycycline) and 240 min of ex vivo reperfusion. A proteomic analysis revealed distinctive clustering profiles between urine samples collected at T15 min and T240 min. High-efficiency undecanal-based N-termini (HUNTER) kidney tissue degradomics revealed significantly more proteolytic activity in the control group at T-10. At T240, significantly more proteolytic activity was observed in the doxycycline group, indicating that doxycycline alters protein degradation during HMP. In conclusion, doxycycline administration during HMP led to significant proteomic and proteolytic differences and protective effects by attenuating urinary NGAL levels. Ultimately, we unraveled metabolic, and complement and coagulation pathways that undergo alterations during machine perfusion and that could be targeted to attenuate IRI induced injury.

Machine perfusion techniques are becoming standard care in the clinical donation and transplantation setting. However, more research is needed to understand the mechanisms of the protective effects of machine perfusion. For preservation related experiments, porcine kidneys are acceptable alternatives to human kidneys, because of their size and similar physiology. In this experiment, the use of slaughterhouse kidneys was evaluated with normothermic kidney perfusion (NKP), thereby avoiding the use of laboratory animals. Porcine kidneys were derived from two local abattoirs. To induce different degrees of injury, different warm ischemic times and preservation techniques were used. After preservation, kidneys were reperfused for 4 h with two different NKP solutions to test renal function and damage. The effect of the preservation technique or a short warm ischemic time was clearly seen in functional markers, such as creatinine clearance and fractional sodium excretion levels, as well as in the generic damage marker lactate dehydrogenase (LDH). Porcine slaughterhouse kidneys are a useful alternative to laboratory animals for transplantation- and preservation-related research questions. To maintain kidney function during NKP, a short warm ischemic time or hypothermic machine perfusion during the preservation phase are mandatory.

Diminishing ischemia-reperfusion injury (IRI) by improving kidney preservation techniques offers great beneficial value for kidney transplant recipients. Mitochondria play an important role in the pathogenesis of IRI and are therefore interesting targets for pharmacological interventions. Hypothermic machine perfusion (HMP), as a preservation strategy, offers the possibility to provide mitochondrial–targeted therapies. This study focuses on the addition of a mitochondrial protective agent SUL—138 during HMP and assesses its effect on kidney function and injury during normothermic reperfusion. In this case, 30 min of warm ischemia was applied to porcine slaughterhouse kidneys before 24 h of non–oxygenated HMP with or without the addition of SUL—138. Functional assessment was performed by 4 h normothermic autologous blood reperfusion. No differences in renal function or perfusion parameters were found between both groups. ATP levels were lower after 30 min of warm ischemia in the SUL–138 group (n.s, p = 0.067) but restored significantly during 24 h of HMP in combination with SUL—138. Aspartate aminotransferase (ASAT) levels were significantly lower for the SUL—138 group. SUL—138 does not influence renal function in this model. Restoration of ATP levels during 24 h of HMP with the addition of SUL in combination with lower ASAT levels could be an indication of improved mitochondrial function.

BackgroundThe gap between demand and supply of kidneys for transplantation necessitates the use of kidneys from extended criteria donors. Transplantation of these donor kidneys is associated with inferior results, reflected by an increased risk of delayed graft function. Inferior results might be explained by the higher immunogenicity of extended criteria donor kidneys. Normothermic machine perfusion (NMP) could be used as a platform to assess the quality and function of donor kidneys. In addition, it could be useful to evaluate and possibly alter the immunological response of donor kidneys. In this study, we first evaluated whether complement was activated during NMP of porcine and human discarded kidneys. Second, we examined the relationship between complement activation and pro-inflammatory cytokines during NMP. Third, we assessed the effect of complement activation on renal function and injury during NMP of porcine kidneys. Lastly, we examined local complement C3d deposition in human renal biopsies after NMP.MethodsNMP with a blood-based perfusion was performed with both porcine and discarded human kidneys for 4 and 6 h, respectively. Perfusate samples were taken every hour to assess complement activation, pro-inflammatory cytokines and renal function. Biopsies were taken to assess histological injury and complement deposition.ResultsComplement activation products C3a, C3d, and soluble C5b-9 (sC5b-9) were found in perfusate samples taken during NMP of both porcine and human kidneys. In addition, complement perfusate levels positively correlated with the cytokine perfusate levels of IL-6, IL-8, and TNF during NMP of porcine kidneys. Porcine kidneys with high sC5b-9 perfusate levels had significantly lower creatinine clearance after 4 h of NMP. In line with these findings, high complement perfusate levels were seen during NMP of human discarded kidneys. In addition, kidneys retrieved from brain-dead donors had significantly higher complement perfusate levels during NMP than kidneys retrieved from donors after circulatory death.ConclusionNormothermic kidney machine perfusion induces complement activation in porcine and human kidneys, which is associated with the release of pro-inflammatory cytokines and in porcine kidneys with lower creatinine clearance. Complement inhibition during NMP might be a promising strategy to reduce renal graft injury and improve graft function prior to transplantation.

Kidney extraction time has a detrimental effect on post-transplantation outcome. This study aims to improve the flush-out and potentially decrease ischemic injury by the addition of hydrogen sulphide (H2S) to the flush medium. Porcine kidneys (n = 22) were extracted during organ recovery surgery. Pigs underwent brain death induction or a Sham operation, resulting in four groups: donation after brain death (DBD) control, DBD H2S, non-DBD control, and non-DBD H2S. Directly after the abdominal flush, kidneys were extracted and flushed with or without H2S and stored for 13 h via static cold storage (SCS) +/− H2S before reperfusion on normothermic machine perfusion. Pro-inflammatory cytokines IL-1b and IL-8 were significantly lower in H2S treated DBD kidneys during NMP (p = 0.03). The non-DBD kidneys show superiority in renal function (creatinine clearance and FENa) compared to the DBD control group (p = 0.03 and p = 0.004). No differences were seen in perfusion parameters, injury markers and histological appearance. We found an overall trend of better renal function in the non-DBD kidneys compared to the DBD kidneys. The addition of H2S during the flush out and SCS resulted in a reduction in pro-inflammatory cytokines without affecting renal function or injury markers.

Metformin may act renoprotective prior to kidney transplantation by reducing ischemia‐reperfusion injury (IRI). This study examined whether metformin preconditioning and postconditioning during ex vivo normothermic machine perfusion (NMP) of rat and porcine kidneys affect IRI. In the rat study, saline or 300 mg/kg metformin was administered orally twice on the day before nephrectomy. After 15 minutes of warm ischemia, kidneys were preserved with static cold storage for 24 hours. Thereafter, 90 minutes of NMP was performed with the addition of saline or metformin (30 or 300 mg/L). In the porcine study, after 30 minutes of warm ischemia, kidneys were preserved for 3 hours with oxygenated hypothermic machine perfusion. Subsequently, increasing doses of metformin were added during 4 hours of NMP. Metformin preconditioning of rat kidneys led to decreased injury perfusate biomarkers and reduced proteinuria. Postconditioning of rat kidneys resulted, dose‐dependently, in less tubular cell necrosis and vacuolation. Heat shock protein 70 expression was increased in metformin‐treated porcine kidneys. In all studies, creatinine clearance was not affected. In conclusion, both metformin preconditioning and postconditioning can be done safely and improved rat and porcine kidney quality. Because the effects are minor, it is unknown which strategy might result in improved organ quality after transplantation.

Background. Donation after unexpected circulatory death (uDCD) donors are often suggested to increase the number of donor organs. In 2014, a uDCD protocol was implemented in three transplant centers in the Netherlands which unfortunately did not result in additional transplantations. This study was initiated to identify demographic factors influencing the potential success of uDCD programs. Methods. Dutch resuscitation databases covering various demographic regions were analyzed for potential donors. The databases were compared with the uDCD implementation project and successful uDCD programs in Spain, France, and Russia. Results. The resuscitation databases showed that 61% of all resuscitated patients were transferred to an emergency department. Age selection reduced this uDCD potential to 46% with only patients aged 18–65 years deemed eligible. Of these patients, 27% died in the emergency department. The urban region of Amsterdam showed the largest potential in absolute numbers (52 patients/y). Comparison with the uDCD implementation project showed large similarities in the percentage of potential donors; however, in absolute numbers, it showed a much smaller potential. Calculation of the potential per million persons and the extrapolation of the potential based on the international experience revealed the largest potential in urban regions. Conclusions. Implementation of a uDCD program should not only be based on the number of potential donors calculated from resuscitation databases. They show promising potential uDCD percentages for large rural regions and small urban regions; however, actual numbers per hospital are low, leading to insufficient exposure rates. It is, therefore, recommendable to limit uDCD programs to large urban regions.

Introduction Metformin can accumulate and cause lactic acidosis in patients with renal insufficiency. Metformin is known to inhibit mitochondria, while renal secretion of the drug by proximal tubules indirectly requires energy. We investigated whether addition of metformin before or during ex vivo isolated normothermic machine perfusion (NMP) of porcine and rat kidneys affects its elimination.Research design and methods First, Lewis rats were pretreated with metformin or saline the day before nephrectomy. Subsequently, NMP of the kidney was performed for 90 min. Metformin was added to the perfusion fluid in one of three different concentrations (none, 30 mg/L or 300 mg/L). Second, metformin was added in increasing doses to the perfusion fluid during 4 hours of NMP of porcine kidneys. Metformin concentration was determined in the perfusion fluid and urine by liquid chromatography-tandem mass spectrometry.Results Metformin clearance was approximately 4–5 times higher than creatinine clearance in both models, underscoring secretion of the drug. Metformin clearance at the end of NMP in rat kidneys perfused with 30 mg/L was lower than in metformin pretreated rats without the addition of metformin during perfusion (both p≤0.05), but kidneys perfused with 300 mg/L trended toward lower metformin clearance (p=0.06). Creatinine clearance was not different between treatment groups. During NMP of porcine kidneys, metformin clearance peaked at 90 min of NMP (18.2±13.7 mL/min/100 g). Thereafter, metformin clearance declined, while creatinine clearance remained stable. This observation can be explained by saturation of metformin transporters with a Michaelis-Menten constant (95% CI) of 23.0 (10.0 to 52.3) mg/L.Conclusions Metformin was secreted during NMP of both rat and porcine kidneys. Excretion of metformin decreased under increasing concentrations of metformin, which might be explained by saturation of metformin transporters rather than a self-inhibitory effect. It remains unknown whether a self-inhibitory effect contributes to metformin accumulation in humans with longer exposure times.

BACKGROUND:Since the start of organ transplantation, hypothermia-forced hypometabolism has been the cornerstone in organ preservation. Cold preservation showed to protect against ischemia, although post-transplant injury still occurs and further improvement in preservation techniques is needed. We hypothesize that hydrogen sulphide can be used as such a new preservation method, by inducing a reversible hypometabolic state in human sized kidneys during normothermic machine perfusion. METHODS:Porcine kidneys were connected to an ex-vivo isolated, oxygen supplemented, normothermic blood perfusion set-up. Experimental kidneys (n = 5) received a 85mg NaHS infusion of 100 ppm and were compared to controls (n = 5). As a reflection of the cellular metabolism, oxygen consumption, mitochondrial activity and tissue ATP levels were measured. Kidney function was assessed by creatinine clearance and fractional excretion of sodium. To rule out potential structural and functional deterioration, kidneys were studied for biochemical markers and histology. RESULTS:Hydrogen sulphide strongly decreased oxygen consumption by 61%, which was associated with a marked decrease in mitochondrial activity/function, without directly affecting ATP levels. Renal biological markers, renal function and histology did not change after hydrogen sulphide treatment. CONCLUSION:In conclusion, we showed that hydrogen sulphide can induce a controllable hypometabolic state in a human sized organ, without damaging the organ itself and could thereby be a promising therapeutic alternative for cold preservation under normothermic conditions in renal transplantation.