Objectives: Organ damage due to long cold ischemia time remains a hurdle in transplantation. In this preliminary animal study, we compared the new Baskent University Preservation Solution (BUPS) with the University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate (HTK) solutions.

Materials and Methods: BUPS composition included electrolytes, raffinose, mannitol, N-acetylcysteine, taurine, adenosine, and ascorbic acid. In experiment 1, kidneys from 50 male Sprague-Dawley rats were placed into BUPS, HTK, or UW solution to assess cold ischemia injury, with biopsies taken at different time points for pathologic evaluation. In experiment 2, to investigate ischemia-reperfusion injury, 5 rats were renal transplant donors to 10 rats and 6 pigs were used as transplant donors-recipients among each other.

Results: In experiment 1, no significant cellular injury was shown at up to 3 hours of perfusion with any solution. At 6- to 48-hour perfusion, tubular injury was shown, with lowest injury in BUPS and HTK versus UW and control groups (P < .01). The BUPS group showed more moderate degree of tubular apoptosis and cytoskeletal rearrangement than the HTK and UW groups at 12-, 24-, and 48-hour perfusion (P < .01). In experiment 2, after ischemia-reperfusion injury, no significant differences were found between HTK and BUPS groups regarding tubular damage. Although no significant differences were shown regarding tubular cytoskeletal rearrangment and apoptosis in pig reperfusion group with BUPS versus HTK, significant differences were shown with these solutions in other groups.

Conclusions: Tubular damage during ischemia-reperfusion injury (cytoskeletal disruption, increased apoptosis) were lower with BUPS. BUPS can be a cost-effective perfusion solution in transplantation.

Key words : Cold ischemia time, Organ preservation solution, Preliminary experiment

Introduction

Since the first speculations about organ preservation over 200 years ago, there has been enormous progress in this field. The translation of solid-organ transplantation from the level of experimental studies to become the criterion standard is undoubtedly due to advances in organ retrieval from deceased donors and their preservation.^1,2 The objective of the preservation process is to preserve organ function and cellular integrity until the time of transplantation. Due to increased patient numbers on wait lists and a limited organ and tissue pool, marginal grafts from deceased donors have become more frequently used. In this regard, organ and tissue preservation techniques are of even greater importance in obtaining betters outcomes with marginal grafts.^3,4

Organ preservation is the most important step for all transplanted tissues and organs, including heart, lungs, liver, kidneys, cornea, pancreas, and small intestines. If metabolism can be slowed down during the ischemic process, which develops as a result of the cessation of an organ’s vascular supply, then the resulting tissue injury will be proportionately reduced. Preservation solutions are needed to protect organ viability and metabolism during organ transplant to the recipient.^5-7 An appropriate and effective preservation solution should consider the following: (1) reduce hypothermia-induced cellular swelling, (2) prevent intracellular acidosis, (3) not spread into the interstitial space during irrigation, (4) protect the organ from cellular injury caused by free oxygen radicals formed during reperfusion, and (5) provide necessary sources to reproduce adenosine triphosphate during reperfusion.

Today, various solutions with different contents, but with similar goals, are used for organ preservation. In general, preservation solutions contain electrolytes (sodium, potassium, chloride, gluconate, magnesium), acidity regulators (sulfate, bicarbonate, phosphate, lactobionate), sugars (glucose, trehalose, raffinose), colloids (hydroxyethyl starch, dextran), free oxygen radical scavengers (N-acetylcysteine, allopurinol, glutathione), and some other substances, albeit at variable proportions.^8,9 An organ must be preserved without being harmed during transport to the immunologically suitable recipient’s hospital. With suitable preservation solutions and methods, the heart can currently be preserved for more than 6 hours, the liver for 24 hours, the pancreas for 48 hours, and the kidney for 110 hours.¹⁰ The basic technique used for organ preservation is simple hypothermic preservation using preservative solutions.

Previously several papers have been reported that ischemia-reperfusion (I/R) injury causes cytoskeletal disruption and reorganization in various tissues. Cytoskeletal organization compromises numerous microfilaments and proteins such as actin, tubulin, vinculin, poly(ADP‐ribose) polymerase 1 (PARP-1), and paxillin.¹¹ In studies on the influence of preservation on the cytoskeleton, hypothermia induced actin and tubulin filament disorganization in pulmonary endothelial cells.¹² Cytoskeletal rearrangement (CSKR) with shortening of micro-tubules has also been reported in kidney epithelial cells under hypothermia.^13,14 However, the conditions in some of these studies were different from those of organ preservation and I/R injury.

Early after the ischemic period, basolateralapical protein polarity is disturbed, and the microvilli brush border is lost in conjunction with the loss of cytoskeletal integrity.¹¹ During rearrangement of the cytoskeleton, increased expression of actin, vinculin, tubulin PARP-1, and paxillin have been reported in some experimental studies.^11,13-15

Apoptosis is permanent cell death in which the nucleus undergoes fragmentation; however, during necrosis, cellular changes may be reversible. In previous studies that used the terminal deo-xynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, approximately 45% to 75% of tubular epithelial cells died via apoptosis during the first 3 to 6 hours of reperfusion.^16,17

Despite significant advances in organ trans-plantation, I/R damage and long cold ischemia time remain serious hurdles. In this preliminary experimental animal study, we compared the efficacy of the new Baskent University Preservation Solution (BUPS) with the University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate (HTK) solutions. We investigated the influence of all three organ preservation solutions on tubular cell cytoskeleton changes and attempted to identify the influence of each of these solutions on the development of apoptosis, tubular degeneration, and tubular necrosis during I/R injury.

Materials and Methods

Animal experiments
This study was performed at the Baskent University Experimental Research Center. In experiment 1, we used 50 male Sprague-Dawley rats (350 to 400 g) to assess I/R injury. In experiment 2, we used 15 rats and 6 pigs (50-60 kg). Both animal experiments were approved by our University’s Animal Research Ethics Committee (DA5/18), and all surgical procedures were done in accordance with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals.”

Composition of BUPS
The composition of BUPS was as follows (g/100 mL): NaCl (0.7), KCl (0.034), NaH₂PO₄ (dibasic; 0.01), NaH₂PO₄ (monobasic; 0.018), NaHCO₃ (0.07), MgSO4 (0.1), MgCl2 (0.00468), raffinose (0.7), mannitol (0.7), taurine (0.1), N-acetylcysteine (0.03), adenosine (0.2), and ascorbic acid (0.003).

All of the ingredients, except for magnesium salts, were added to sterile distilled water and mixed until they dissolved. A mixture was then created in a carbon-oxygen gas environment (95% O2/5% CO2) for 15 minutes for pH adjustment (otherwise, pH of the solution would shift to alkaline values due to the bicarbonate content and lead to precipitation of bivalent cations). Magnesium salts were dissolved in sterile distilled water in a separate beaker and added to the solution afterward.

In addition to the electrolytes (sodium, potassium, chloride, magnesium), BUPS also contained raffinose (a trisaccharide composed of galactose, glucose, and fructose) used as an energy source; mannitol as an osmoregulator; N-acetylcycteine (NAC) as an antioxidant, an antiapoptotic, and a microsomal glutathione transferase substrate to increase the cellular pools of free radical scavengers; taurine(a sulfonated amino acid) as a membrane stabilizer, an antioxidant to protect against I/R injury, an intracellular calcium regulator, and an osmoregulator; adenosine(a purine riboside composed of adenine molecule attached to a ribose sugar molecule) as an energy source, and an anti-inflammatory agent; and ascorbic acid (a cofactor for multiple enzymes) as an electron donor for mono- and dioxygenases and as a strong antioxidant (Table 1).

Anesthesia
For anesthesia, rats were intraperitoneally injected with 50 mg/kg ketamine hydrochloride and 8 mg/kg xylazine hydrochloride. In pigs, oral nutrition was stopped 12 hours before anesthesia. After intra-muscular sedation using atropine (0.03 mg/kg), ketamine (10 mg/kg), and xylazine (1-3 mg/kg), the rats were brought to the research unit. They were intubated endotracheally in the supine position with direct laryngoscopy using special blades (30 cm) and No. 7-9 cuffed intubation tubes. General anesthesia was applied using an anesthesia circuit (synchronized intermittent mandatory ventilation mode), with 1% to 1.5% isoflurane, 2 L/min oxygen, and 2 L/min nitric oxide.

Experiment 1
Sprague-Dawley rats were randomized into the three solution groups (BUPS, HTK, or UW solution). After laparotomy, the infrarenal aorta and inferior vena cava (IVC), the subdiaphragmatic aorta, and the infrahepatic IVC were suspended in all rats with 4/0 Vicryl. The distal and proximal aorta and the distal IVC were ligated. The intra-abdominal organs were perfused with 50 cm³ of perfusion solution at +4ºC from the distal aorta. The suprarenal IVC was cut to drain the perfusion solution (Figure 1). Both kidneys were removed and placed in the same solution to assess cold ischemia injury. Biopsy samples were taken from these kidneys at 0, 1, 3, 6, 12, 24, and 48 hours for histopathologic evaluation.

Experiment 2
Simple kidney transplant technique in rats
Our experiment to assess I/R injury during kidney transplant included 15 rats and 6 pigs. Of 5 rats used as donors, 3 rats were perfused with BUPS and 2 rats were perfused from the aorta with HTK solution (Figure 2). Rats underwent renal transplantation using the same technique as previously described in the literature.¹⁸ However, we used 4 magnification loops (Rose 4X-S CE) for anastomosis (Figure 3). After 3 hours, transplanted kidneys were removed for evaluation of I/R injury
Simple kidney transplant technique in pigs
Under general anesthesia, the right kidneys of 6 pigs were removed with right crescentic incision.¹⁹ Three of the kidneys were perfused with BUPS, and the other 3 were perfused with HTK and stored in the same solution at +4 C for 48 hours. At the end of this period, kidney transplant was performed on the right side of a different pig with right crescentic incision (Figure 4). The right kidney was retroperitoneally transplanted by anastomosis of the renal vein to the IVC and the renal artery to the aorta in an end-to-side manner (7/0 prolene). Vascular clamps were opened. After kidney perfusion had finished, the graft ureter was anastomosed to the recipient ureter in an end-to-end manner (6/0 prolene). After hemostasis, the fascia was closed with continuous Vicryl in 2 layers, and the skin was closed with 2/0 continuous silk suture. After surgery, we monitored the activity, nutritional status, defecation, and other behaviors of pigs. After 72 hours, the transplanted kidneys were removed from the pigs under general anesthesia and sent to pathology to assess I/R injury.

Histopathology
Retrieved grafts were fixed with 10% neutral buffered formalin and embedded in paraffin blocks. The 4-µm sections were stained with hematoxylin-eosin and evaluated by a renal pathologist (BHO), who was blinded to the experimental groups, for hallmarks of tubular degeneration and tubular necrosis. The percentage of tubular degeneration and the presence or absence of tubular necrosis were noted for each kidney.

Tubular degeneration is often an early indicator of necrosis. Tubular degeneration, in some cases, is preceded by vacuolization. In general, tubular degeneration is characterized by several morphologic and variable cell features, such as cell swelling with or without cytoplasmic vacuolization, pale staining or eosinophilic tinctorial change, and fragmented cytoplasm. Tubular necrosis generally includes cell swelling, nuclear pyknosis and/or karyorrhexis, and cellular sloughing.

In experiment 2, in order to evaluate the histopathologic changes of the transplanted organs and I/R injury, biopsies were taken after cold ischemia and after reperfusion. Reperfusion injury and regeneration of tubules were studied in all transplanted kidneys from both rats and pigs.

Determination of apoptosis by terminal deoxy-nucleotidyl transferase-mediated dUTP nick-end labeling assay
Renal biopsy sections (2 μm thick) were prepared, deparaffinized, and immersed in phosphate-buffered saline containing 0.3% hydrogen peroxide for 10 minutes at room temperature. All biopsies were incubated with 20 mg/mL proteinase K for 15 minutes, and 75 μL of equilibration buffer were then added to the specimens and kept for 10 minutes at room temperature. This step was followed by addition of 55 mL of terminal deoxynucleotidyl transferase enzyme and incubation at 37°C for 1 hour, followed by addition of dUTP at 37°C and incubation in a moist chamber for 60 minutes. The reaction was terminated by transferring the slides to prewarmed wash buffer for 30 minutes. Sections were next incubated with a peroxidase-labeled antibody at 37°C for 30 minutes and then soaked in Tris buffer containing 0.02% diaminobenzidine and 0.02% hydrogen peroxide for 1 minute to achieve color development. Specimens were counterstained with hematoxylin. TUNEL-positive cells showed clear nuclear labeling corresponding to apoptotic cells.

Histologic examination of apoptosis was performed by a renal pathologist in a blinded fashion. Apoptotic tubular epithelial cells were quantitatively assessed per high-power field (×400). At least 10 high-power fields per slide were counted. The presence of apoptosis was determined in biopsies that were taken after cold ischemia and after reperfusion in both rats and pigs.

Determination of tubular epithelial cell cytoskeleton rearrangement by cytoskeleton proteins poly (ADP‐ribose) polymerase‐1 and paxillin immuno-histochemistry
We immunostained 3-μm sections with the cytoskeleton proteins PARP-1 and paxillin (Dako, OMNIS, automated immunohistochemistry). Tubular expression using both PARP-1 and paxillin was semiquantitatively graded from grade 1 to 3. Degree of CSKR of tubular epithelial cells was graded according to the sum of two cytoskeletal proteins as follows: PARP-1 + paxillin ≤ 3 was determined as CSKR grade 1; PARP-1 + paxillin = 4 to 5 was determined as CSKR grade 2; and PARP-1 + paxillin = 6 was determined as CSKR grade 3. Rearrangement of tubular epithelial cell cytoskeleton was determined in biopsies that were taken after cold ischemia and after reperfusion in both rats and pigs.

Results

Experiment 1
In experiment 1, no solution groups showed significant tubular injury or tubular necrosis at 0, 1, or 3 hours after perfusion. As shown in Table 2, the percent of tubular degeneration was not significantly different among the groups. At 6 hours after perfusion, the percent of tubular degeneration was found to be moderate in all groups. Although we observed no significant differences among the groups regarding tubular degeneration, 2 of 4 rats (50%) in the UW group showed tubular necrosis. This was in contrast to none of the kidneys in the BUPS or HTK groups showing tubular necrosis (Figures 5 and 6).

In grafts that were stored in UW and HTK solutions, tubular degeneration and tubular necrosis in renal tissue samples were more pronounced and occurred earlier than in grafts stored in BUPS. At 12, 24, and 48 hours after perfusion, percent tubule degeneration was found to be lowest in the BUPS group versus that shown in the HTK and UW groups.

Prolonged perfusion time (12, 24, and 48 hours) greatly increased the presence of tubular vacuoles, nuclear pyknosis, karyorrhexis, protein cast formation, and cellular sloughing, especially in grafts preserved in UW and HTK solutions. The presence of these characteristics was already apparent 12 hours after preservation and became much more severe at 24 and 48 hours, with a strong incidence of necrotic tubular epithelial cells. In contrast, only a few necrotic cells in a small number of cases could be observed in BUPS-preserved grafts (Table 2).

Table 3 shows comparisons among the 3 perfusion solution groups for the development of tubular CSKR and apoptosis. Compared with that shown in the HTK and UW groups, grade of apoptosis was more moderate in the BUPS group at 12, 24, and 48 hours of perfusion. The BUPS group showed the lowest degree of development of CSKR compared with the HTK and UW groups at each time point. However, significant differences between the BUPS solution group and the HTK and UW groups were only shown at the 12-, 24-, and 48-hour perfusion time points. In addition, kidneys with higher degrees of tubular CSKR tended to show higher degrees of apoptosis (r = 0.871, P < .001) and tubular degeneration (r = 0.861, P < .001).

Experiment 2
As shown in Table 4, I/R damage was correlated between BUPS and HTK perfusion solutions in both rats and pigs. The degree of tubular damage after cold ischemia was found to be lower in the BUPS group than in the HTK solution group. However, after reperfusion, no significant differences were found between the 2 perfusion solutions with regard to tubular damage and tubular necrosis. Although we observed no significant difference between the BUPS and the HTK group with regard to tubular CSKR and apoptosis in the pig reperfusion group, significant differences were found between the BUPS and HTK groups with regard to tubular CSKR and apoptosis in the other groups (Table 4, Figure 7).

Table 5 shows the influence of both BUPS and HTK perfusion solutions on the development of CSKR in rats and pigs with I/R damage. As shown in Table 5, no significant differences were found between BUPS and HTK on the development of CSKR during cold I/R injury. In experiment 2, only pig kidneys showed differences in perfusion solutions after cold ischemia with regard to the development of CSKR, with lower development of CSKR with BUPS than with HTK solution.

We also noted that kidneys with higher degrees of tubular CSKR tended to show higher degrees of apoptosis (r = 0.882, P < .001) and tubular degeneration (r = 0.887, P < .001).

Cost-effective considerations
When we compared all solutions with regard to cost-effectiveness, BUPS was about 5- to 7-fold less expensive than the other solutions. The actual price of 1 L is about US $358 for UW, US $258 for HTK,and US $31 to $41 for BUPS. Although the cost-effectiveness of our experimental BUPS was better than the other tested solutions, the real cost has yet to be estimated for production scenarios.

Discussion

Organ preservation plays an important role in solid-organ transplantation. It has a significant influence on I/R injury and early graft function as well as long-term graft survival. Both UW and HTK solutions are presently used as routine preservation solutions in many transplant centers worldwide. Both have shown the same efficacy in a remarkable number of studies over the years.²⁰

The need for optimization of graft quality has resulted in intensive experimental and clinical efforts to improve the ischemic tolerance of organs. One of the most important goals is to improve the quality of current preservation solutions or to invent new ones. In our preliminary study, we compared BUPS with the established preservation solutions (UW and HTK) in rat and pig models. Because of physiologic and anatomic similarities between rats, pigs, and humans, these animal models have been considered suitable for preliminary experimental studies.²¹

Preservation solutions can reduce substance traffic between intracellular and extracellular spaces during ischemia. For this purpose, high con-centrations of potassium, magnesium, phosphate, and glucose and lower levels of sodium and bicarbonate are added to the solution prepared with intracellular properties. Preservation solutions have been shown to reduce delayed graft function when used for kidney preservation, and HTK and UW solutions have been used for both living-donor and deceased-donor transplant procedures.⁷

For the present study, we tested whether our developed tissue preservation solution (BUPS) was as efficacious and cost-effective as the current formulations used for the same purpose. BUPS was designed to mimic extracellular fluid enriched with supplements. Except for calcium salts, its electrolyte content was inspired by the physiological Krebs-Henseleit solution. Several supplements, different from those used in standard commercial organ preservation solutions, were used in BUPS. Among them, taurine, a sulfonated amino acid, was chosen as a membrane stabilizer, an antioxidant to protect against I/R injury, an intracellular calcium regulator, and an organic osmolyte involved in cell volume regulation. These properties of taurine make it an ideal ingredient to be used in an organ preservation solution. Taurine is also a substrate for the formation of bile salts. It is involved in the modulation of intracellular free calcium concentration. Taurine is not incorporated into proteins; however, it is one of the most abundant amino acids in the body.²² Taurine has been used in long-term peritoneal dialysis patients as a membrane stabilizer.²³ Addition of taurine to St. Thomas cardioplegic solution has been shown to improve cardiac function recovery for prolonged hypothermic rat heart preservation.²⁴ The authors have suggested that taurine exerts its effect by suppressing DNA oxidative stress and cell apoptosis.

We also used N-acetylcysteine in BUPS. N-acetylcysteine is an antioxidant, an anti-apoptotic, and a microsomal glutathione transferase substrate that increases the cellular pools of free radical scavengers. N-acetylcysteine has been recently reported to exhibit tissue-specific protective activity against intestinal I/R injury.²⁵

Adenosine was used as an energy source, a blood-flow regulator, an antiplatelet, and an anti-inflammatory agent in BUPS. Adenosine, a purine riboside composed of an adenine molecule attached to a ribose sugar molecule, has been shown to increase islet cell viability during mechanical perfusion when added to UW solution in a porcine model of pancreatic islet transplantation.²⁶ In addition, adenosine monophosphate-activated protein kinase has been reported to be a master regulator of energy metabolism and to protect against cold I/R injury in a rat model of cardiac transplantation.²⁷

Ascorbic acid is a crucial cofactor for several enzymes and serves as an electron donor for mono- and dioxygenases. Additionally, ascorbic acid is a potent antioxidant. It is the primary water-soluble antioxidant in human plasma. Furthermore, it has been reported to improve mitochondrial functionality toward oxidative phosphorylation.²⁸

Raffinose, the trisaccharide composed of galactose, glucose, and fructose, was used as an energy source in BUPS. Raffinose has also been shown to exert a cytoprotective effect on pulmonary grafts during preservation.²⁹

We also used mannitol as an osmoregulator in BUPS. Mannitol has long been known to act as a hyperosmolar agent and to be an active scavenger of the cytotoxic hydroxyl radical. It has been reported to improve postischemic function in a rabbit model of cardiac transplantation.³⁰

Our study presented initial histologic findings regarding the use of BUPS in comparison with the criterion standard preservation solutions. Our BUPS solution was prepared with several considerations. First, because trisaccharides, including raffinose, have been reported to exert more cytoprotection than glucose, we decided to use raffinose both as an energy source and an impermeant in BUPS. Second, we considered that NAC, shown to scavenge reactive oxygen species in similar experiments, provides a functional beneficial effect. Third, our rationale for adding ascorbic acid but not other water-soluble cofactors into BUPS was not only its use as a cofactor and an antioxidant but also for its pH-lowering effect. That is, it can be used to adjust the otherwise slightly basic pH of BUPS into the physiological range, in addition to the effect of buffers such as phosphate and bicarbonate, which are already present in BUPS.

In the present study, in rat kidneys after 0,1, 2, 3, 6, 12, 24, and 48 hours of cold ischemia, pathologic evaluation showed that BUPS seemed to be as protective as the standard HTK and UW solutions used in many transplant centers worldwide. We showed that BUPS had cytoprotective effects with lower degrees of tubular damage, tubular apoptosis, and tubular epithelial CSKR.

In this study, our histopathologic findings showed that both BUPS and HTK were better than UW solution. In experiment 1, both BUPS and HTK had similar histopathologic findings. Because of these close histopathologic findings, we only compared BUPS and HTK with each other in the I/R models in experiment 2.

In experiment 2, in which we evaluated I/R injury, we found that use of BUPS in rat and pig kidney transplant models subjected to long periods of ischemia was as effective in preserving kidneys as HTK solution. Rates of tubular damage, tubular apoptosis, and development of CSKR were found to be lower in the BUPS group than the HTK group.

Conclusions

Use of BUPS improved the preservation efficiency by reducing the risk of uncontrolled free radicals, the possible toxicity of degradation products with taurine, the fast energy deficit, cell edema, and therefore risk of development of tubular CSKR, apoptosis, and tubular necrosis.

Both functional and pathologic advantages in rat organ preservation were shown with BUPS. Other advantages of BUPS compared with standard solutions (HTK and UW) may become measurable in preclinical settings, where additional factors such as short and long ischemia time and cost-effectiveness of a preservation solution are considered. Further studies are justifiable in view of the preliminary results obtained in our rat organ preservation solution model and in view of the lower cellular injury obtained with BUPS during ischemic conditions compared with other preservation solutions. We believe BUPS to be a promising substitute, which so far is at least as effective as the already existing alternatives and more cost-effective.

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