Edited by: Peter Schemmer, Medical University of Graz, Austria
Reviewed by: Stanislaw Stepkowski, University of Toledo, United States; Silvio Nadalin, Tübingen University Hospital, Germany
This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
During the process of organ transplantation, the ischemia reperfusion injury (IRI) together with the systemic inflammatory response to brain death causes infrastructural organ injury which could lead to initial poor function and ultimately primary non-function (
Preconditioning with calcineurin inhibitors (CNIs) has been shown to have protective effects in a model of renal transplantation in rats compared to vehicle-treated animals (
The aim of this study has been to investigate the feasibility and the effects of oral preconditioning of DBD donors with CNI (Cyclosporine A) vs. mTORi (Everolimus) vs. conventional administration of steroid in a porcine model of kidney transplantation.
German landrace pigs (weight: 33.2 ± 3.9 kg) were given access to standard laboratory chow (ssniff R/M-H, ssniff Spezialdiäten, Soest, Germany) and tap water before experiments. The study protocol was reviewed and approved by the responsible animal welfare state authority (Regierungspräsidium Karlsruhe, Baden-Württemberg, Germany (file number: 35-9185.81/G-5/16) and were performed according to the institutional guidelines at the Ruprecht-Karls Univesity, Heidelberg, Germany in accordance with the guidelines of FELASA (Federation for Laboratory Animal Science Associations).
All operations and investigations were performed under general anesthesia. After premedication (azaperone 6 mg/kg intramuscularly (i.m.), ketamine 10 mg/kg i.m., and midazolamine hydrochloride 0.5 mg/kg i.m.), anesthesia was induced with ketamine [1 mg/kg intravenously (i.v.)], midazolamine hydrochloride (0.1 mg/kg i.v.), and atropine (0.04 mg/kg i.v.). During the operation, anesthesia was maintained with 1.5–2% isoflurane. Ventilation was pressure-controlled in a half-closed system. The ventilation parameters included a tidal volume of 240 ml, frequency of 17/min, maximum pressure of 24 cmH2O and positive end-expiratory pressure of 3–5 cmH2O. The pH, HCO3, pCO2, and pO2 concentrations were determined by routine analysis of arterial blood gases. The respiration parameters were then adapted to these values. During surgery, controlled infusion therapy was applied using 20 ml/kg/h Sterofundin (B. Braun, Melsungen, Germany). The mean arterial pressure (MAP) as well as the central venous pressure (CVP) and the heart rate (HR) were continuously monitored. For these reasons, the right common carotid artery and the jugular vein were first prepared, cannulated and connected to pressure transducers. The central venous catheter additionally served for volume substitution, for the administration of pharmaceuticals and for obtaining central venous blood samples.
Our standardized method for the induction of brain death in pigs has been published elsewhere (
Six hours the after induction of brain death (i.e., 2 h prior to organ procurement) preconditioning was performed with the oral administration of Cyclosporine suspension (Novartis Pharma GmbH, Nuremberg, Germany) (10 mg/kg body weight) (
Study design. Six hours after the induction of brain death, German landrace donor pigs (33.2 ± 3.9 kg) were randomly preconditioned with either Cyclosporine (
A full-length midline laparotomy was performed and abdominal aorta and inferior vena cava (IVC) were dissected at the level of iliac bifurcation. Subsequently supratruncal aorta was prepared just below the diaphragm. After the administration of 200 IU/Kg heparin, the perfusion catheter was inserted into the aorta. Renal artery was checked for possible lower pole arteries. Slight mobilization of adrenal gland was done for better exposure of renal vein. The aorta was cross-clamped and the cold perfusion was performed with HTK (histidine tryptophan ketoglutarate) solution (Custodiol®, Dr. F. Köhler Chemie GmbH, Alsbach-Hähnlein, Germany) and the infrarenal IVC was vented. The renal artery was cut without a patch; renal veins were cut with a short IVC cuff. After the procurement, renal artery was catheterized by a soft cannula and perfused again. The kidney was subsequently cold-stored in HTK for 18 h.
The details regarding operation procedures have been published elsewhere (
The recipients were monitored on the operating table for 4 h, after which blood samples and protocol biopsies were taken and the abdomen was closed. The indwelling central venous catheter was kept in order to draw blood samples as well as for the intravenous administration of analgesics, antibiotics and volume and substrate substitution. The catheter in the carotid artery was removed after surgery. The animals were then extubated and returned to the cage. Recipients received 0.05 mg/kg buprenorphine, 25–50 mg/kg Metamizole for analgesia as well as 200 mg ciprofloxacin and 125 mg metronidazole, over the remaining central venous catheter. Buprenorphine 0.02–0.05 mg/kg and Metamizole 25–50 mg/kg were given every 12 h for the first 48 h postoperatively.
When the animals were awake and had regained their physiological body temperature, they were taken to the holding area of the University's Interfaculty Biomedical Research Facility. The animals were under observation of the competent animal caretakers and veterinarians, immediately gaining free access to water. On the evening of the operating day, the animals received 500 ml glucose 5% + 500 ml lactated Ringer. On the 1st postoperative day, the animals received 1,000 mL glucose 10% + 1,000 mL ringer lactate. Solid food was allowed only after bowel sound was heard. Parenteral nutrition with Nutriflex peri was considered for animals unable to eat. After the surgery based on pigs' general performance, it was decided how they should be observed and kept. All the pigs were visited three times a day and checked in terms of weight change. Blood was drawn over the central venous catheter daily to measure complete blood count, blood urea nitrogen (BUN), creatinine (Cr) and electrolytes up to postoperative day (POD) 5. No immunosuppression was administered. Animals were sacrificed at the end of the study on POD 5 under deep anesthesia by intravenous injection of potassium chloride (2 mmol/kg).
To investigate early histopathological changes during kidney transplantation, wedge biopsies were obtained 4 h after reperfusion. Kidney samples were fixed in 10% buffered formalin, routinely embedded in paraffin, cut into 4 μm-thick sections for hematoxylin and eosin stain as well as for Periodic acid-Schiff reaction according to standard protocols. Qualitative assessment of samples was performed to determine and grade acute tubular injury (1 = mild, dilated tubules, partial brush border loss, 2 = moderate, dilated tubules, complete brush border loss, hyaline cylinders, 3 = severe, complete epithelial atrophy, tubule necrosis). Quantitative assessment of acute tubular damage was also performed and scored as quartiles (1 = 0–25, 2 = 26–50, 3 = 51–75, and 4 = 76–100%).
For immunohistochemical examination, sections were labeled with commercially available antibodies against cytochrome c (Abcam, Cambridge, UK, ab90529, dilution 1:200) and TNF-α (Abcam, Cambridge, UK, ab6671, dilution 1:50). After heat-induced antigen retrieval at pH 9 (Target Retrieval Solution, Agilent Technologies, Inc., Santa Clara, USA) for cytochrome c and pH 6 (Target Retrieval Solution, Agilent Technologies, Inc., Santa Clara, USA) for TNF-α, respectively, the slides were blocked with Dako REAL Peroxidase-Blocking Solution (Agilent Technologies, Inc., Santa Clara, USA) and incubated with the primary antibody. An anti-rabbit secondary antibody conjugated to HRP (Polyview plus HRP (anti-rabbit) reagent, ENZO Life Sciences GmbH, Lörrach, Germany) was applied. AEC solution (Dako REAL Substrate Solution, Agilent Technologies, Inc., Santa Clara, USA) was used to visualize the signal.
The immunohistochemical scoring was performed according to Allred et al. (
Statistical analysis was performed using IBM SPSS Statistics for Windows, Version 22.0 (IBM Corp. Released 2013. Armonk, NY). Continuous data are expressed as mean values ± standard deviation (SD) and differences between groups were analyzed using the one-way ANOVA test. Categorical data were compared using the chi-square test of association. Histopathological data were analyzed using the Kruskal-Wallis test followed by the Bonferroni
There was no difference in preoperative hemodynamic parameters, hemoglobin/hematocrit, electrolytes as well as intraoperative blood loss between the groups (
Baseline data.
Weight (kg) |
32.76 ± 1.88 | 34.07 ± 2.20 | 32.25 ± 1.28 | 0.130 |
BUN (mg/dl) |
18.67 ± 7.45 | 19.37 ± 5.55 | 25.87 ± 7.95 | 0.098 |
Cr (mg/dl) |
1.50 ± 0.34 | 1.39 ± 0.24 | 1.36 ± 0.26 | 0.581 |
K (mmol/L) |
5.19 ± 1.47 | 4.75 ± 2.14 | 3.89 ± 0.70 | 0.243 |
Ca (mmol/L) |
2.01 ± 0.27 | 2.20 ± 0.17 | 2.19 ± 0.15 | 0.122 |
Hemoglobin (g/dl) |
10.38 ± 1.85 | 11.03 ± 1.47 | 12.24 ± 1.61 | 0.087 |
mean arterial pressure (mmHg) |
62.78 ± 3.53 | 63.67 ± 3.24 | 65.00 ± 3.50 | 0.421 |
heart rate |
101.22 ± 4.92 | 97.56 ± 3.50 | 99.00 ± 5.26 | 0.255 |
Temperature (°C) |
35.33 ± 0.22 | 35.34 ± 0.19 | 35.47 ± 0.17 | 0.271 |
Blood loss (ml) | 130 ± 27 | 137 ± 21 | 147 ± 17 | 0.318 |
Operative times.
Cyclosporine | 6.6 ± 2.1 | 18.4 ± 2.0 | 48.9 ± 7.7 |
Everolimus | 7.8 ± 0.8 | 20.9 ± 3.6 | 48.9 ± 10.5 |
Methylprednisolone | 6.8 ± 1.6 | 18.5 ± 2.9 | 43.8 ± 8.3 |
All groups | 7 ± 1.6 | 19.3 ± 2.9 | 47.7 ± 8.7 |
0.39 | 0.24 | 0.48 |
Posttransplant blood urea nitrogen (BUN) in recipients in different study groups.
Posttransplant creatinine (Cr) in recipients in different study groups.
Posttransplant calcium (Ca) in recipients in different study groups.
Posttransplant potassium (K) in recipients in different study groups.
Histological assessment revealed no significant differences between the groups (
Quantitative and qualitative histopathological assessment of acute tubular injury.
Cyclosporine | 1 (1–2) | 1 (1–2) |
Everolimus | 1 (1–2) | 1 (1–2) |
Methylprednisolone | 1 (1–3) | 1 (1–2) |
0.825 | 0.491 |
TNF-α antibody staining after preconditioning with Cyclosporine. Arrows show different intensities; blue: intensity 0, orange: intensity 1, brown: intensity 2, and black: intensity 3.
TNF-α antibody staining after preconditioning with Everolimus. Arrows show different intensities; blue: intensity 0, orange: intensity 1, brown: intensity 2, and black: intensity 3.
TNF-α antibody staining after preconditioning with Methylprednisolone. Arrows show different intensities; blue: intensity 0, orange: intensity 1, brown: intensity 2, and black: intensity 3.
Cytochrome c antibody staining after preconditioning with Cyclosporine. Arrows show different intensities; blue: intensity 0, orange: intensity 1, brown: intensity 2, and black: intensity 3.
Cytochrome c antibody staining after preconditioning with Everolimus. Arrows show different intensities; blue: intensity 0, orange: intensity 1, brown: intensity 2, and black: intensity 3.
Cytochrome c antibody staining after preconditioning with Methylprednisolone. Arrows show different intensities; blue: intensity 0, orange: intensity 1, brown: intensity 2, and black: intensity 3.
Immunohistochemical scoring after preconditioning with Cyclosporine, Everolimus, and Methylprednisolone 4 h following kidney transplantation. (A1) PS in TNF-α, (A2) IS in TNF-α, (A3) TS in TNF-α, (B1) PS in cytochrome c, (B2) IS in cytochrome c and (B3) TS in cytochrome c.
Brain death triggers an inflammatory response in the donor organs with T lymphocyte and macrophage infiltration and release of multiple proinflammatory cytokines, among all TNF-α, Interleukin-6, and Interleukin-10, which has been shown to enhance the immunogenicity of the organs and potentiate the deleterious effects of IRI after organ transplantation (
To our knowledge, there has been no study on the oral preconditioning of DBD donor in a big animal transplant model. Our present work showed that oral preconditioning with Cyclosporine or Everolimus in DBD pig kidney transplantation is feasible and down-regulates TNF-α expression. The reduction in TNF-α expression seems to be plausible in our model, as an increase of intragraft TNF-α expression is documented in the organs of DBD donors, and after organ reperfusion. TNF-α aggravates the adherence of leukocytes to vascular endothelium leading to enhancement of IRI and acceleration of acute allograft rejection after organ transplantation (
In the present work, in order to stimulate the actual clinical practice, we induced hypotensive brain death in our donors, and allowed 6 h' time for the inflammatory response following brain death to develop. Moreover, we kept an average of 18 h cold ischemia time to enhance IRI. No posttransplant immunosuppression therapy in recipients was administered to avoid confounding bias.
In order to detect the early allograft changes after IRI, the protocol biopsies were performed 4 h after reperfusion. Kusaka et al. have shown that the early changes including the expression of the inflammatory proteins could take place as early as 1 h after the implantation of the kidneys after DBD (
The present work has its own limitations. Although we administered the routine immunosuppressive doses, data on the appropriate oral doses of Cyclosporine and Everolimus for the purpose of oral preconditioning is lacking. Furthermore, the best time points for the administration of the oral preconditioning agents as well as the frequency of medication are not known and vary widely among different studies. Furthermore, the best time point to look for the early innate host immune response triggered synergistically by IRI and DBD in allografts is still unclear.
In summary, our findings suggest the feasibility of the oral preconditioning with CNI or mTORi in DBD donors in pig kidney transplantation. A reduced expression of TNF-α in transplanted organs in the early post-transplant phase was seen after oral preconditioning with these agents. Our data can serve as a platform for future experimental and clinical studies to evaluate the protecting role of donor oral preconditioning against IRI and its clinical relevance.
All datasets generated for this study are included in the article/supplementary material.
The animal study was reviewed and approved by Regierungspräsidium Karlsruhe, Baden-Württemberg, Germany (file number: 35-9185.81/G-5/16).
AN, AMe, and SA developed the original concept of the study. AN, SA, MN, OG, and EK developed the design and methodology. SA, OG, EK, MN, AA, SM, AMa, MS, MK, and AY participated in the operations and data collection. CE and TP participated in the pathological assessment. MN, EK, OG, and AA performed the statistical assessments and developed the analysis plan. SA, AN, MN, EK, OG, and AA contributed to drafting the article. AN, NG, MG, SM, AMa, MS, MK, AY, and AMe contributed to the revision of the final report. All authors read and approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
donors after brain death
ischemia-reperfusion injury
calcineurin inhibitors
inhibitors of mammalian target for Rapamycin
Histidine-Tryptophane-Ketoglutarate
tumor necrosis factor alpha
interleukin 6
interleukin 10
Glomerular filtration rate
post-operative day
Standard deviation
intramuscularly
intravenously
mean arterial pressure
central venous pressure
heart rate
inferior vena cava
proportion score
intensity score
total score
blood urea nitrogen
creatinine
acute tubular injury.