Ex-Vivo Kidney Perfusion With Hemoglobin-Based Oxygen Carriers, Red Blood Cells, or No Oxygen Carrier.

INTRODUCTION: Normothermic machine perfusion (NMP) of donor kidneys provides the opportunity to assess and improve organ viability prior to transplantation. This study explored the necessity of an oxygen carrier during NMP and whether the hemoglobin-based oxygen carrier (HBOC-201) is a suitable alternative to red blood cells (RBCs). METHODS: Porcine kidneys were perfused with a perfusion solution containing either no-oxygen carrier, RBCs, or HBOC-201 for 360 min at 37°C. RESULTS: Renal flow and resistance did not differ significantly between groups. NMP without an oxygen carrier showed lower oxygen consumption with higher lactate and aspartate aminotransferase levels, indicating that the use of an oxygen carrier is necessary for NMP. Cumulative urine production and creatinine clearance in the RBC group were significantly higher than in the HBOC-201 group. Oxygen consumption, injury markers, and histology did not differ significantly between these two groups. However, methemoglobin levels increased to 45% after 360 min in the HBOC-201 group. CONCLUSIONS: We conclude that HBOC-201 could be used as an alternative for RBCs, but accumulating methemoglobin levels during our perfusions indicated that HBOC-201 is probably less suitable for prolonged NMP. Perfusion with RBCs, compared to HBOC-201, resulted in more favorable renal function during NMP.

The association between hemoglobin levels and renal function parameters during normothermic machine perfusion: A retrospective cohort study using porcine kidneys.

BACKGROUND: Ex vivo normothermic machine perfusion (NMP) is a promising tool for assessing an isolated kidney prior to transplantation. However, there is no consensus on the perfusate's optimal oxygen-carrying capacity to support renal function. To investigate the association of hemoglobin levels with renal function parameters, a retrospective analysis of isolated, normothermically, perfused porcine kidneys was performed. METHODS: Between 2015 and 2021, a total of 228 kidneys underwent 4 h of NMP with perfusates that varied in hemoglobin levels. A generalized linear model was used to determine the association of hemoglobin levels with time-weighted means of renal function markers, such as fractional sodium excretion (FENa) and creatinine clearance (CrCl). Stratified by baseline hemoglobin level (<4.5, 4.5-6, or >6 mmol/L), these markers were modeled over time using a generalized linear mixed-effects model. All models were adjusted for potential confounders. RESULTS: Until a hemoglobin level of around 5 mmol/L was reached, increasing hemoglobin levels were associated with superior FENa and CrCl. Thereafter, this association plateaued. When hemoglobin levels were categorized, hemoglobin <4.5 mmol/L was associated with worse renal function. Hemoglobin levels were neither significantly associated with proteinuria during NMP nor with ATP levels at the end of NMP. Hemoglobin levels >6 mmol/L showed no additional benefits in renal function. CONCLUSION: In conclusion, we found an association between baseline hemoglobin levels and superior renal function parameters, but not injury, during NMP of porcine kidneys. Furthermore, we show that performing a retrospective cohort study of preclinical data is feasible and able to answer additional questions, reducing the potential use of laboratory animals.

Organ-specific metabolic profiles of the liver and kidney during brain death and afterwards during normothermic machine perfusion of the kidney.

We investigated metabolic changes during brain death (BD) using hyperpolarized magnetic resonance (MR) spectroscopy and ex vivo graft glucose metabolism during normothermic isolated perfused kidney (IPK) machine perfusion. BD was induced in mechanically ventilated rats by inflation of an epidurally placed catheter; sham-operated rats served as controls. Hyperpolarized [1-13 C]pyruvate MR spectroscopy was performed to quantify pyruvate metabolism in the liver and kidneys at 3 time points during BD, preceded by injecting hyperpolarized[1-13 C]pyruvate. Following BD, glucose oxidation was measured using tritium-labeled glucose (d-6-3H-glucose) during IPK reperfusion. Quantitative polymerase chain reaction and biochemistry were performed on tissue/plasma. Immediately following BD induction, lactate increased in both organs (liver: eµd 0.21, 95% confidence interval [CI] [-0.27, -0.15]; kidney: eµd 0.26, 95% CI [-0.40, -0.12]. After 4 hours of BD, alanine production decreased in the kidney (eµd 0.14, 95% CI [0.03, 0.25], P < .05). Hepatic lactate and alanine profiles were significantly different throughout the experiment between groups (P < .01). During IPK perfusion, renal glucose oxidation was reduced following BD vs sham animals (eµd 0.012, 95% CI [0.004, 0.03], P < .001). No differences in enzyme activities were found. Renal gene expression of lactate-transporter MCT4 increased following BD (P < .01). In conclusion, metabolic processes during BD can be visualized in vivo using hyperpolarized magnetic resonance imaging and with glucose oxidation during ex vivo renal machine perfusion. These techniques can detect differences in the metabolic profiles of the liver and kidney following BD.

Normothermic machine perfusion reduces bile duct injury and improves biliary epithelial function in rat donor livers.

Bile duct injury may occur during liver procurement and transplantation, especially in livers from donation after circulatory death (DCD) donors. Normothermic machine perfusion (NMP) has been shown to reduce hepatic injury compared to static cold storage (SCS). However, it is unknown whether NMP provides better preservation of bile ducts. The aim of this study was to determine the impact of NMP on bile duct preservation in both DCD and non-DCD livers. DCD and non-DCD livers obtained from Lewis rats were preserved for 3 hours using either SCS or NMP, followed by 2 hours ex vivo reperfusion. Biomarkers of bile duct injury (gamma-glutamyltransferase and lactate dehydrogenase in bile) were lower in NMP-preserved livers compared to SCS-preserved livers. Biliary bicarbonate concentration, reflecting biliary epithelial function, was 2-fold higher in NMP-preserved livers (P < 0.01). In parallel with this, the pH of the bile was significantly higher in NMP-preserved livers (7.63 ± 0.02 and 7.74 ± 0.05 for non-DCD and DCD livers, respectively) compared with SCS-preserved livers (7.46 ± 0.02 and 7.49 ± 0.04 for non-DCD and DCD livers, respectively). Scanning and transmission electron microscopy of donor extrahepatic bile ducts demonstrated significantly decreased injury of the biliary epithelium of NMP-preserved donor livers (including the loss of lateral interdigitations and mitochondrial injury). Differences between NMP and SCS were most prominent in DCD livers. Compared to conventional SCS, NMP provides superior preservation of bile duct epithelial cell function and morphology, especially in DCD donor livers. By reducing biliary injury, NMP could have an important impact on the utilization of DCD livers and outcome after transplantation. Liver Transplantation 22 994-1005 2016 AASLD.

Slow induction of brain death leads to decreased renal function and increased hepatic apoptosis in rats.

BACKGROUND: Donor brain death (BD) is an independent risk factor for graft survival in recipients. While in some patients BD results from a fast increase in intracranial pressure, usually associated with trauma, in others, intracranial pressure increases more slowly. The speed of intracranial pressure increase may be a possible risk factor for renal and hepatic graft dysfunction. This study aims to assess the effect of speed of BD induction on renal and hepatic injury markers. METHODS: BD induction was performed in 64 mechanically ventilated male Fisher rats by inflating a 4.0F Fogarty catheter in the epidural space. Rats were observed for 0.5, 1, 2 or 4 h following BD induction. Slow induction was achieved by inflating the balloon-catheter at a speed of 0.015 ml/min until confirmation of BD. Fast induction was achieved by inflating the balloon at 0.45 ml/min for 1 min. Plasma, kidney and liver tissue were collected for analysis. RESULTS: Slow BD induction led to higher plasma creatinine at all time points compared to fast induction. Furthermore, slow induction led to increased renal mRNA expression of IL-6, and renal MDA values after 4 h of BD compared to fast induction. Hepatic mRNA expression of TNF-α, Bax/Bcl-2, and protein expression of caspase-3 was significantly higher due to slow induction after 4 h of BD compared to fast induction. PMN infiltration was not different between fast and slow induction in both renal and hepatic tissue. CONCLUSION: Slow induction of BD leads to poorer renal function compared to fast induction. Renal inflammatory and oxidative stress markers were increased. Liver function was not affected by speed of BD induction but hepatic inflammatory and apoptosis markers increased significantly due to slow induction compared to fast induction. These results provide initial proof that speed of BD induction influences detrimental renal and hepatic processes which could signify different donor management strategies for patients progressing to BD at different speeds.

End-ischemic machine perfusion reduces bile duct injury in donation after circulatory death rat donor livers independent of the machine perfusion temperature.

A short period of oxygenated machine perfusion (MP) after static cold storage (SCS) may reduce biliary injury in donation after cardiac death (DCD) donor livers. However, the ideal perfusion temperature for protection of the bile ducts is unknown. In this study, the optimal perfusion temperature for protection of the bile ducts was assessed. DCD rat livers were preserved by SCS for 6 hours. Thereafter, 1 hour of oxygenated MP was performed using either hypothermic machine perfusion, subnormothermic machine perfusion, or with controlled oxygenated rewarming (COR) conditions. Subsequently, graft and bile duct viability were assessed during 2 hours of normothermic ex situ reperfusion. In the MP study groups, lower levels of transaminases, lactate dehydrogenase (LDH), and thiobarbituric acid reactive substances were measured compared to SCS. In parallel, mitochondrial oxygen consumption and adenosine triphosphate (ATP) production were significantly higher in the MP groups. Biomarkers of biliary function, including bile production, biliary bicarbonate concentration, and pH, were significantly higher in the MP groups, whereas biomarkers of biliary epithelial injury (biliary gamma-glutamyltransferase [GGT] and LDH), were significantly lower in MP preserved livers. Histological analysis revealed less injury of large bile duct epithelium in the MP groups compared to SCS. In conclusion, compared to SCS, end-ischemic oxygenated MP of DCD livers provides better preservation of biliary epithelial function and morphology, independent of the temperature at which MP is performed. End-ischemic oxygenated MP could reduce biliary injury after DCD liver transplantation.

Prednisolone has a positive effect on the kidney but not on the liver of brain dead rats: a potencial role in complement activation.

BACKGROUND: Contradictory evidence has been published on the effects of steroid treatments on the outcomes of kidney and liver transplantation from brain dead (BD) donors. Our study aimed to evaluate this disparity by investigating the effect of prednisolone administration on BD rats. METHODS: BD induction was performed in ventilated rats by inflating a Fogarty catheter placed in the epidural space. Prednisolone (22.5 mg/kg) was administered 30 min prior to BD induction. After four hours of determination of BD: serum, kidney and liver tissues samples were collected and stored. RT-qPCR, routine biochemistry and immunohistochemistry were performed. RESULTS: Prednisolone treatment reduced circulating IL-6 and creatinine plasma levels but not serum AST, ALT or LDH. Polymorphonuclear influx assessed by histology, and inflammatory gene expression were reduced in the kidney and liver. However, complement component 3 (C3) expression was decreased in kidney but not in liver. Gene expression of HSP-70, a cytoprotective protein, was down-regulated in the liver after treatment. CONCLUSIONS: This study shows that prednisolone decreases inflammation and improves renal function, whilst not reducing liver injury. The persistence of complement activation and the negative effect on protective cellular mechanisms in the liver may explain the disparity between the effects of prednisolone on the kidney and liver of BD rats. The difference in the molecular and cellular responses to prednisolone administration may explain the contradictory evidence of the effects of prednisolone on different organ types from brain dead organ donors.

Histopathologic and molecular evaluation of the Organ Procurement and Transplantation Network selection criteria for intestinal graft donation.

BACKGROUND: The Organ Procurement and Transplantation Network (OPTN) has formulated criteria for the selection of donors for intestinal transplantation. To date, however, no study has correlated histologic findings of intestinal injury with the OPTN criteria. We aimed to describe histopathologic and molecular features of allograft injury in relation to donor conditions defined by the OPTN criteria. MATERIALS AND METHODS: Graft histology (Park Score), Claudin-3 staining, systemic inflammatory markers (C-reactive protein/lipopolysaccharide-binding protein) and expression of heat shock protein 70, heme oxygenase 1, and interleukin 6 were evaluated in multiorgan deceased donors (donation after brain death [DBD] and donation after cardiac death [DCD]). RESULTS: Ninety-seven samples (52 jejunum/45 ileum) were recovered from 59 donors (46 DBD/13 DCD). The OPTN criterion cold ischemia time correlated with histologic injury (Park score) to which the jejunum appeared more susceptible than the ileum. Claudin-3 staining was higher, and heat shock protein 70 expression lower in donors meeting the OPTN criteria compared with donors not meeting the criteria and in DBD versus DCD. In DBD donors, interleukin 6 expression was higher compared with DCD donors and inversely related to C-reactive protein. CONCLUSIONS: Our multiparameter analysis suggests that the OPTN criteria can be discriminative concerning intestinal graft quality. Our data suggest that DCD intestinal allografts are qualitatively inferior and that the jejunum is more sensitive to ischemia than the ileum.

Male versus female inflammatory response after brain death model followed by ex vivo lung perfusion.

BACKGROUND: Ex vivo lung perfusion (EVLP) is a useful tool for assessing lung grafts quality before transplantation. Studies indicate that donor sex is as an important factor for transplant outcome, as females present higher inflammatory response to brain death (BD) than males. Here, we investigated sex differences in the lungs of rats subjected to BD followed by EVLP. METHODS: Male and female Wistar rats were subjected to BD, and as controls sham animals. Arterial blood was sampled for gas analysis. Heart-lung blocks were kept in cold storage (1 h) and normothermic EVLP carried out (4 h), meanwhile ventilation parameters were recorded. Perfusate was sampled for gas analysis and IL-1ß levels. Leukocyte infiltration, myeloperoxidase presence, IL-1ß gene expression, and long-term release in lung culture (explant) were evaluated. RESULTS: Brain dead females presented a low lung function after BD, compared to BD-males; however, at the end of the EVLP period oxygenation capacity decreased in all BD groups. Overall, ventilation parameters were maintained in all groups. After EVLP lung infiltrate was higher in brain dead females, with higher neutrophil content, and accompanied by high IL-1ß levels, with increased gene expression and concentration in the culture medium (explant) 24 h after EVLP. Female rats presented higher lung inflammation after BD than male rats. Despite maintaining lung function and ventilation mechanics parameters for 4 h, EVLP was not able to alter this profile. CONCLUSION: In this context, further studies should focus on therapeutic measures to control inflammation in donor or during EVLP to increase lung quality.

As there is a shortage of viable lungs for transplantation, methods of lung preservation, such as ex vivo perfusion, are important. This method is a good alternative, as it will not only preserve the lungs, but also enable lung function assessment and treatment of the organs. Studies have showed that lungs from donors of the female sex have greater risk of being rejected, when transplanted to male receptors. However, it's not certain if sex differences in anatomy, physiology and specially in immune response could interfere with the transplant result. Females do present a greater and more efficient immune response to any hazard, however after brain death this control is lost, producing a great inflammatory response as a result. Therefore, in this study we have investigated in more detail the influence of sex on the effects of brain death followed by the preservation method. Thus, we performed a brain death model in males and females rats and placed their lungs in an ex vivo lung perfusion machine. At the end of the experiment, we analyzed lung ventilation, gas exchange, and inflammatory parameters. The obtained data indicated that overall the lung ventilation and gas exchange is maintained by the ex vivo perfusion machine. Also, that lung inflammation is influenced by the sex of the donor; where the lungs from females present greater inflammation compared to the lungs from males.

ARA290, a non-erythropoietic EPO derivative, attenuates renal ischemia/reperfusion injury.

BACKGROUND: In contrast with various pre-clinical studies, recent clinical trials suggest that high dose erythropoietin (EPO) treatment following kidney transplantation does not improve short-term outcome and that it even increases the risk of thrombotic events. ARA290 is a non-erythropoietic EPO derivative and does not increase the risk of cardiovascular events, but potentially has cytoprotective capacities in prevention of renal ischemia/reperfusion injury. METHODS: Eight female Dutch Landrace pigs were exposed to unilateral renal ischemia for 45 minutes with simultaneous cannulation of the ureter of the ischemic kidney. ARA290 or saline was administered by an intravenous injection at 0, 2, 4 and 6 hours post-reperfusion. The animals were sacrificed seven days post-reperfusion. RESULTS: ARA290 increased glomerular filtration rate during the observation period of seven days. Furthermore, ARA290 tended to reduce MCP-1 and IL-6 expression 15 minutes post-reperfusion. Seven days post-reperfusion ARA290 reduced interstitial fibrosis. CONCLUSIONS: The improvement in renal function following renal ischemia/reperfusion and reduced structural damage observed in this study by ARA290 warrants further investigation towards clinical application.

Renoprotective capacities of non-erythropoietic EPO derivative, ARA290, following renal ischemia/reperfusion injury.

BACKGROUND: ARA290 is a non-erythropoietic EPO derivative which only binds to the cytoprotective receptor complex (EPOR2-ßcR2) consisting of two EPO-receptors (EPOR) and two ß common receptors (ßcR). ARA290 is renoprotective in renal ischemia/reperfusion (I/R). In a renal I/R model we focussed on timing of post-reperfusional administration of ARA290. Furthermore, we investigated the anti-inflammatory properties of ARA290. METHODS: Twenty-six male Lewis/HanHsd rats were exposed to unilateral ischemia for 30 minutes, with subsequent removal of the contralateral kidney. Post-reperfusion, ARA290 was administered early (one hour), late (four hours) or repetitive (one and four hours). Saline was used as vehicle treatment. Rats were sacrificed after three days. RESULTS: Early ARA290 treatment improved renal function. Late- or repetitive treatment tended to improve clinical markers. Furthermore, early ARA290 treatment reduced renal inflammation and acute kidney injury at three days post-reperfusion. Late- or repetitive treatment did not affect inflammation or acute kidney injury. CONCLUSIONS: ARA290 attenuated renal ischemia/reperfusion injury. This study showed the anti-inflammatory effect of ARA290 and suggests early administration in the post-reperfusional phase is most effective. ARA290 is a candidate drug for protection against ischemic injury following renal transplantation.

Comparison of acute kidney injury following brain death between male and female rats.

BACKGROUND: Clinical reports associate kidneys from female donors with worse prognostic in male recipients. Brain Death (BD) produces immunological and hemodynamic disorders that affect organ viability. Following BD, female rats are associated with increased renal inflammation interrelated with female sex hormone reduction. Here, the aim was to investigate the effects of sex on BD-induced Acute Kidney Injury (AKI) using an Isolated Perfused rat Kidney (IPK) model. METHODS: Wistar rats, females, and males (8 weeks old), were maintained for 4h after BD. A left nephrectomy was performed and the kidney was preserved in a cold saline solution (30 min). IPK was performed under normothermic temperature (37°C) for 90 min using WME as perfusion solution. AKI was assessed by morphological analyses, staining of complement system components and inflammatory cell markers, perfusion flow, and creatinine clearance. RESULTS: BD-male kidneys had decreased perfusion flow on IPK, a phenomenon that was not observed in the kidneys of BD-females (p < 0.0001). BD-male kidneys presented greater proximal (p = 0.0311) and distal tubule (p = 0.0029) necrosis. However, BD-female kidneys presented higher expression of eNOS (p = 0.0060) and greater upregulation of inflammatory mediators, iNOS (p = 0.0051), and Caspase-3 (p = 0.0099). In addition, both sexes had increased complement system formation (C5b-9) (p=0.0005), glomerular edema (p = 0.0003), and nNOS (p = 0.0051). CONCLUSION: The present data revealed an important sex difference in renal perfusion in the IPK model, evidenced by a pronounced reduction in perfusate flow and low eNOS expression in the BD-male group. Nonetheless, the upregulation of genes related to the proinflammatory cascade suggests a progressive inflammatory process in BD-female kidneys.

Complement Is Activated During Normothermic Machine Perfusion of Porcine and Human Discarded Kidneys.

Background: The 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. Methods: NMP 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. Results: Complement 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. Conclusion: Normothermic 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.

Ex Vivo Perfusion With Methylprednisolone Attenuates Brain Death-induced Lung Injury in Rats.

The onset of brain death (BD) leads to the deterioration of potential donor lungs. Methylprednisolone is considered to increase lung oxygenation capacity and enhance the procurement yield of donor lungs, when applied in situ, during donor management. However, whether BD-induced lung damage is ameliorated upon treatment with methylprednisolone during acellular ex vivo lung perfusion (EVLP), remains unknown. We aimed to investigate whether the quality of lungs from brain-dead donors improves upon methylprednisolone treatment during EVLP. METHODS: Rat lungs were randomly assigned to 1 of 3 experimental groups (n = 8/group): (1) healthy, directly procured lungs subjected to EVLP; (2) lungs from brain-dead rats subjected to cold storage and EVLP; and (3) lungs from brain-dead rats subjected to cold storage and EVLP with 40 mg methylprednisolone added to the perfusate. Ventilation and perfusion parameters, histology, edema formation, metabolic profile, and inflammatory status of lungs were investigated. RESULTS: Methylprednisolone treated lungs from brain-dead donors improved positive inspiratory pressures needed to maintain tidal volumes of 7 mL/kg of body weight, which was 25.6 ± 5.8 cm H2O in untreated lungs and 18.0 ± 3.0 cm H2O in methylprednisolone treated lungs, after 6 h EVLP. Furthermore, dynamic lung compliance increased upon methylprednisolone treatment, with values of 0.11 ± 0.05 mL/cm H2O versus 0.18 ± 0.04 mL/cm H2O after 6 h of EVLP. Methylprednisolone treatment ameliorated the amount of lung edema, as corroborated by a reduction of 0.7 in the wet/dry ratio. Although glucose consumption levels were comparable, the BD-induced cumulative lactate production decreased from 0.44 ± 0.26 to 0.11 ± 0.16 mmol/L upon methylprednisolone treatment. Finally, BD-induced inflammatory status was reduced upon methylprednisolone treatment compared to untreated lungs from brain-dead donors, as reflected by lower proinflammatory gene expression levels of IL-1ß, IL-6 and MCP-1, and IL-6 perfusate levels. CONCLUSIONS: We showed that methylprednisolone treatment during EVLP attenuates BD-induced lung injury.

Methylprednisolone Treatment in Brain Death-Induced Lung Inflammation-A Dose Comparative Study in Rats.

Background: The process of brain death (BD) leads to a pro-inflammatory state of the donor lung, which deteriorates its quality. In an attempt to preserve lung quality, methylprednisolone is widely recommended in donor lung management. However, clinical treatment doses vary and the dose-effect relation of methylprednisolone on BD-induced lung inflammation remains unknown. The aim of this study was to investigate the effect of three different doses methylprednisolone on the BD-induced inflammatory response. Methods: BD was induced in rats by inflation of a Fogarty balloon catheter in the epidural space. After 60 min of BD, saline or methylprednisolone (low dose (5 mg/kg), intermediate dose (12.5 mg/kg) or high dose (22.5 mg/kg)) was administered intravenously. The lungs were procured and processed after 4 h of BD. Inflammatory gene expressions were analyzed by RT-qPCR and influx of neutrophils and macrophages were quantified with immunohistochemical staining. Results: Methylprednisolone treatment reduced neutrophil chemotaxis as demonstrated by lower IL-8-like CINC-1 and E-selectin levels, which was most evident in rats treated with intermediate and high doses methylprednisolone. Macrophage chemotaxis was attenuated in all methylprednisolone treated rats, as corroborated by lower MCP-1 levels compared to saline treated rats. Thereby, all doses methylprednisolone reduced TNF-α, IL-6 and IL-1ß tissue levels. In addition, intermediate and high doses methylprednisolone induced a protective anti-inflammatory response, as reflected by upregulated IL-10 expression when compared to saline treated brain-dead rats. Conclusion: We showed that intermediate and high doses methylprednisolone share most potential to target BD-induced lung inflammation in rats. Considering possible side effects of high doses methylprednisolone, we conclude from this study that an intermediate dose of 12.5 mg/kg methylprednisolone is the optimal treatment dose for BD-induced lung inflammation in rats, which reduces the pro-inflammatory state and additionally promotes a protective, anti-inflammatory response.

Metformin Preconditioning and Postconditioning to Reduce Ischemia Reperfusion Injury in an Isolated Ex Vivo Rat and Porcine Kidney Normothermic Machine Perfusion Model.

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.

Donor pretreatment with carbamylated erythropoietin in a brain death model reduces inflammation more effectively than erythropoietin while preserving renal function.

OBJECTIVE: We hypothesized that donor treatment of deceased brain dead donors would lead to a decrease in inflammatory responses seen in brain death and lead to a restoration of kidney function. DESIGN: A standardized slow-induction rat brain death model followed by evaluation of kidney function in an isolated perfused kidney model. SETTINGS: Surgery Research Laboratory, University Medical Center Groningen, the Netherlands. SUBJECTS: Male Fisher rats. INTERVENTIONS: Donor treatment with erythropoietin, carbamylated erythropoietin, which lacks erythropoietic activity, or vehicle. MEASUREMENTS AND MAIN RESULTS: In brain death, carbamylated erythropoietin and, to a lesser extent, erythropoietin were able to decrease the expression of several proinflammatory genes and to decrease the infiltration of polymorphonuclear cells in the kidney. No effect on tubular injury parameters was seen. Kidney function decreased almost by 50% after brain death but was fully restored after treatment with both carbamylated erythropoietin and erythropoietin. CONCLUSIONS: Carbamylated erythropoietin can inhibit the inflammatory response caused by brain death more effectively than erythropoietin, whereas both substances can restore kidney function after brain death.

Metformin Preconditioning Improves Hepatobiliary Function and Reduces Injury in a Rat Model of Normothermic Machine Perfusion and Orthotopic Transplantation.

BACKGROUND: Preconditioning of donor livers before organ retrieval may improve organ quality after transplantation. We investigated whether preconditioning with metformin reduces preservation injury and improves hepatobiliary function in rat donor livers during ex situ normothermic machine perfusion (NMP) and after orthotopic liver transplantation. METHODS: Lewis rats were administered metformin via oral gavage, after which a donor hepatectomy was performed followed by a standardized cold storage period of 4 hours. Graft assessment was performed using NMP via double perfusion of the hepatic artery and portal vein. In an additional experiment, rat donor livers preconditioned with metformin were stored on ice for 4 hours and transplanted to confirm postoperative liver function and survival. Data were analyzed and compared with sham-fed controls. RESULTS: Graft assessment using NMP confirmed that preconditioning significantly improved ATP production, markers for hepatobiliary function (total bile production, biliary bilirubin, and bicarbonate), and significantly lowered levels of lactate, glucose, and apoptosis. After orthotopic liver transplantation, metformin preconditioning significantly reduced transaminase levels. CONCLUSIONS: Preconditioning with metformin lowers hepatobiliary injury and improves hepatobiliary function in an in situ and ex situ model of rat donor liver transplantation.

Increasing metformin concentrations and its excretion in both rat and porcine ex vivo normothermic kidney perfusion model.

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.