Petra J. Ottens

Distinct transcriptional changes in donor kidneys upon brain death induction in rats : Insights in the processes of brain death

Brain death affects hormone regulation, inflammatory reactivity and hemodynamic stability. In transplant models, donor organs retrieved from brain dead (BD) rats suffer from increased rates of primary non‐function and lower graft survival. To unravel the mechanisms behind brain death we have performed DNA microarray studies with kidney‐derived RNA from normo‐ and hypotensive BD rats, corresponding with optimal and marginal BD donors, respectively. In kidneys from normotensive donors 63 genes were identified as either up‐ (55) or down‐regulated (8), while 90 genes were differentially expressed (67 up‐regulated) in hypotensive BD donor kidneys. Most genes were categorized in different functional groups: metabolism/transport (including the down‐regulated water channel Aqp‐2), inflammation/coagulation (containing the largest number (16) of up‐regulated genes including selectins, Il‐6, α‐ and β‐fibrinogen), cell division/fibrosis (including KIM‐1 involved in tubular regeneration) and defense/repair (with the cytoprotective genes HO‐1, Hsp70, MnSOD2). Also, genes encoding transcription factors (including immediate early genes as Atf‐3, Egr‐1) and proteins involved in signal transduction (Pik3r1) were identified. Summarizing, the use of DNA microarrays has clarified parts of the process of brain death: Brain‐death‐induced effects ultimately lead, via activation of transcription factors and signal transduction cascades, to differential expression of different “effector” genes. Not only deleterious processes such as inflammation and fibrosis occur in brain dead donor kidneys but genes involved in protection and early repair processes are activated as well. These findings can be used to introduce specific cytoprotective interventions in the brain dead donor to better maintain or even increase organ viability.

Time-dependent changes in donor brain death related processes

Donor brain death (BD) affects kidney function and survival after transplantation. Studies on brain dead kidney donors indicate that, besides inflammation and coagulation, cytoprotective gene expression is activated as well. Here, we evaluated in a time‐course experiment progression of these renal BD‐related processes. Animals were sacrificed 0.5, 1, 2 or 4 h after BD and compared to sham‐operated controls. Proinflammatory genes (E‐selectin, MCP‐1, Il‐6) were massively up‐regulated (p < 0.05) already 0.5 h after BD. Inducers of proinflammatory gene expression were either activated (NF‐κB) or induced in expression (Egr‐1) after 0.5 h of BD. Increased numbers of infiltrating granulocytes were seen in the interstitium from 0.5 h on. Also, expression of protective genes HO‐1 and HSP70 were increased within 0.5 h. Remarkably, reactive oxygen species formation was detectable only in the later phase of BD. Among 14 measured serum cytokines, MCP‐1 and KC‐protein were significantly elevated from 0.5 h on. In conclusion, a fast induction of proinflammatory and stress‐induced protective processes in brain dead donor kidneys was demonstrated, probably triggered by changes occurring during BD induction. Importantly, hypoxia appeared not to be one of the initial triggers, and early increased systemic levels of chemokines MCP‐1 and KC may be regarded as the starting point for the inflammatory cascade in brain dead donor kidneys.

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.

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

BackgroundIn 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.MethodsEight 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.ResultsARA290 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.ConclusionsThe improvement in renal function following renal ischemia/reperfusion and reduced structural damage observed in this study by ARA290 warrants further investigation towards clinical application.

Brain death induces inflammation in the donor intestine

Background. Brain death donors are frequently used for transplantation. Previous studies showed that brain death (BD) negatively affects the immunological and inflammatory status of both liver and kidney. Because the intestine is increasingly used as a donor organ and no information on effects of BD on small intestine is available we performed this study. Methods. We studied the inflammatory and apoptotic changes in donor intestine after BD induction. Brain death was induced in rats by inflation of a balloon catheter. Three groups (n=6) were compared: 1-hr BD, 4-hr BD, and sham-operated controls. Results. An increased polymorphonuclear cell influx in ileum, as a measure of inflammation, was observed in 1- and 4-hr BD group compared with controls. Jejunum showed a significant increase at the 4-hr BD group compared with the control group. Intercellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, and interleukin-6 were upregulated after 1- and 4-hr BD. Caspase-3 positive cells were found in jejunum and ileum after 4-hr BD on the top of the villi. Serum interleukin-6 was severely elevated in the 1- and 4-hr brain dead rats. Conclusion. These data show the early occurrence of intestinal inflammation and apoptosis after BD induction. These events may ultimately have a negative influence on the outcome of intestinal transplantation.

The gradual onset brain death model: a relevant model to study organ donation and its consequences on the outcome after transplantation

Organs used for transplantation are usually derived from heart-beating brain dead donors. However, brain death is known to have negative effects on donor organ quality, previously studied using a difficult to control sudden onset experimental model. We have now developed a reproducible gradual onset brain death model in rats without requiring inotropic support. Fisher inbred rats weighing 260–300 g were used. Brain death was induced by a gradual inflation of a subdurally placed balloon catheter. During induction and the period following brain death, the animals were mechanically ventilated and blood pressure was continuously monitored. The blood pressure registration showed a characteristic pattern during brain death induction, in which a decrease in blood pressure, a hypotensive period in which the Cushing response occurred, and a sharp peak were consistent findings. After brain death was induced, blood pressure was maintained at normotensive levels up to 4 h. After the experiments, neuropathological evaluation of the brain located haemorrhagic cerebral parenchyma, and immunocytochemistry of liver tissue revealed a significant influx of polymorph nuclear cells, as was previously observed as well. This improved model allows the study of brain death on donor organ quality without the use of inotropic support.

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. Liver Transpl 21:1300‐1311, 2015.

End‐Ischemic Machine Perfusion Reduces Bile Duct Injury In Donation After Circulatory Death Rat Donor Livers

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. Liver Transpl 21:1300‐1311, 2015.

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

BackgroundContradictory 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.MethodsBD 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.ResultsPrednisolone 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.ConclusionsThis 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.

Gradual Rewarming with Gradual Increase in Pressure during Machine Perfusion after Cold Static Preservation Reduces Kidney Ischemia Reperfusion Injury

In this study we evaluated whether gradual rewarming after the period of cold ischemia would improve organ quality in an Isolated Perfused Kidney Model. Left rat kidneys were statically cold stored in University of Wisconsin solution for 24 hours at 4°C. After cold storage kidneys were rewarmed in one of three ways: perfusion at body temperature (38°C), or rewarmed gradually from 10°C to 38°C with stabilization at 10°C for 30 min and rewarmed gradually from 10°C to 38°C with stabilization at 25°C for 30 min. In the gradual rewarming groups the pressure was increased stepwise to 40 mmHg at 10°C and 70 mmHg at 25°C to counteract for vasodilatation leading to low perfusate flows. Renal function parameters and injury biomarkers were measured in perfusate and urine samples. Increases in injury biomarkers such as aspartate transaminase and lactate dehydrogenase in the perfusate were lower in the gradual rewarming groups versus the control group. Sodium re-absorption was improved in the gradual rewarming groups and reached significance in the 25°C group after ninety minutes of perfusion. HSP-70, ICAM-1, VCAM-1 mRNA expressions were decreased in the 10°C and 25°C groups. Based on the data kidneys that underwent gradual rewarming suffered less renal parenchymal, tubular injury and showed better endothelial preservation. Renal function improved in the gradual rewarming groups versus the control group.