Key words : Antioxidant, Caspase 3, Kidney preservation, Organ viability, Potassium channel opener

Introduction

Organ transplantation has become a sophisticated and integrated clinical service and is presently the only effective therapy for end-stage organ failure. The preservation of the organs and tissues during storage in a manner that facilitates restoration of function on reperfusion remains a crucial aspect of organ transplant. The key factor in organ transplant is maintenance of organ viability during recovery and preservation. Moreover, successful preservation outcomes require favorable hypothermic conditions to maintain the lowest possible metabolic load and proper exsanguination of microvasculature to facilitate optimal reperfusion. Presently there are 2 well-established approaches to organ preservation that are used for most organ transplants: static versus dynamic. Simple static cold storage (known as SCS) is the method for static storage under hypothermic conditions, whereas hypothermic machine perfusion (known as HMP), which ensures homogeneous and continuous supply of metabolic substrates to the graft, is the method for dynamic storage.^1,2 There are several commercially available preservation solutions, including the University of Wisconsin cold storage solution, Bretschneider solution, and histidine-tryptophan-ketoglutarate (HTK) solution.

Low levels of cellular ATP caused by inhibition of Na+/K+-ATPase represent an urgent concern for organ survival. Reduction in intracellular ATP accelerates degradation of adenosine, which leads to accumu­lation of hypoxanthine and xanthine oxidase.^3,4 The subsequent microvascular and parenchymal cell injury may lead to the production of reactive oxygen species (ROS), superoxide, and hydrogen peroxide in various pathology disorders associated with organ preser­vation. Release of proinflammatory cytokines, incidence of acidosis, increased expression of adhesion molecules (eg, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, P-selectin), effects of vasoactive agents such as endothelin and thromboxane A2, and disruption of calcium homeostasis are conditions induced by ischemia, and ischemia may be further intensified by the reduction in blood flow associated with loss of equilibrium between nitric oxide and endothelin.

The ATP-sensitive potassium (KATP) channels promote regulation of intracellular ATP by membrane hyperpolarization, which depletes Ca2+ concentration and subsequently increases in intracellular ATP levels. In contrast, regulation of nitric oxide may improve blood flow, which may promote more favorable conditions to combat ischemic damage.⁵ Therefore, our hypothesis is that organ survival can be enhanced during storage with a prepared solution of nicorandil-supplemented organ preservation fluid (OPF) for the regulation of intracellular ATP and nitric oxide-mediated ischemic recovery. Previous studies have reported the preservation efficiency of nicorandil in ischemic conditions during heart preservation.⁶ Yamashita and colleagues have shown the efficiency of nicorandil in lung preservation.⁷ Given that KATP receptors are expressed in cells in a variety of tissues, including muscle, pancreatic β cells, and the brain, it is logical to investigate the preservation efficiency of nicorandil in other organs as well.^8,9 Other studies have suggested rutin trihydrate (RTT) to be a powerful antioxidant that inhibits free radical-mediated cytotoxicity and lipid peroxidation.^10,11 With this background, our aim in the present study was to evaluate the efficacy of HTK solution supplemented with either nicorandil or RTT for kidney preservation.

Materials and Methods

Materials
We obtained nicorandil as a generous gift from Sun Pharma, and we purchased RTT from HiMedia Laboratories. Custodiol was purchased from Sandor Medicaids. Propidium iodide (PI) and the ATP bioluminescent assay kit were purchased from Sigma-Aldrich. Penicillin G, streptomycin sulfate, and 2',7'-dichlorofluorescin diacetate (DCFDA) were obtained from Invitrogen. Polyclonal antibodies to caspase 3 and to phospho-p42/p44 mitogen-activated protein kinases were purchased from Santa Cruz Biotechnology. All other reagents and materials were purchased from HiMedia Laboratories and Sigma Aldrich.

Animals
The protocol of animal treatment and surgery was duly approved by the institutional animal ethical committee at the Institute of Nuclear Medicine and Allied Sciences, Delhi, India (vide project No. INM/IAEC/2018/11). Male and female Sprague Dawley rats (200-250 g, n = 6 in each group) were housed and maintained at the institutional animal facility at the Institute of Nuclear Medicine and Allied Sciences. All methods in this study were in accordance with the guidelines of India’s Committee for the Purpose of Control and Supervision on Experiments on Animals. All animals were maintained with a 12:12-hour light-dark cycle, with free access to standard diet pellets and water.

Preparation of solutions of supplemented organ preservation fluid
The OPF solutions were prepared with standard aseptic conditions. Briefly, all components of HTK solution were dissolved in a specific quantity in water for injection, and the resulting solution was sterilized by membrane filtration (0.22 μm).⁶ To prepare the nicorandil-supplemented solution (OPF-NCD) and the RTT-supplemented solution (OPF-RTT), the standard OPF solution was supplemented with nicorandil (0.5 mmol/L) or RTT (0.1 mmol/L), respectively. The concentrations of nicorandil and RTT were optimized with a series of initial trials, and the solutions were evaluated for parameters such as clarity, pH, sterility, and pyrogen content.

Source of cell line and cell culture
We used HEK-293 human embryonic kidney cells for all in vitro studies, to simulate and approximate the conditions of the whole-organ kidney model. The HEK-293 cells were obtained from the National Centre for Cell Science at Savitribai Phule Pune University. Cells were cultured in a high-glucose preparation of Dulbecco’s modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics (penicillin, streptomycin, and nystatin). Stock cultures were passaged every third day with 0.05% trypsin and seeded with a density of 0.3 × 10⁶ cells in 60-mm tissue culture petri dishes (BD Falcon). All experiments were with cultures of exponentially growing cells.

Treatment procedure
Cells were seeded in 12-well plates at a density of 0.065 × 10⁶ cells/well and incubated at 37 °C in a CO₂ incubator for 24 hours. After 24 hours, the cells were treated with Custodiol solution (as control samples) or treated with the prepared formulations of OPF-NCD and OPF-RTT (as experimental samples). Cells were incubated in these media at various temperatures (4 °C, 15 °C, 25 °C, and 37 °C) to determine the effect of temperature on preservation efficacy. To determine the effect of treatment duration on preservation efficacy, incubation was terminated at 4 and 24 hours, and samples were taken for evaluation of various parameters by in vitro assays.

Temperature-induced oxidative stress
We used DCFDA probe to quantify the ROS present in the samples. At 4 and 24 hours after treatment, cells were incubated for 30 minutes in a probe buffer containing 1 mM MgCl₂, 1 mM CaCl₂, 5 mM glucose, and 20 μM DCFDA in phosphate-buffered saline (PBS) at 37 °C in a CO₂ incubator. After incubation, cells were washed with PBS followed by trypsinization and then resuspended in PBS. The cells were transferred to 96-well plates, and mean fluorescence intensity (MFI) was measured with a Varioskan fluorescence plate reader (Thermo Scientific) at an excitation/emission ratio of 485/535. The MFI values were quantified by normalization with the respective number of cells.

ATP determination
Cellular ATP levels were determined with an ATP bioluminescence assay kit (Sigma). Briefly, cells were seeded in a 12-well plate at a density of 0.075 × 106 cells/well and maintained at 37 °C in a CO₂ incubator. The next day, cells were treated with the OPF solutions, and plates were maintained at temperatures of 4 °C, 15 °C, 25 °C, and 37 °C, respectively. The ATP assay was performed 24 hours after treatment, with 0.01 × 10⁶ cells according to the manufacturer’s protocol. The ATP luminescence was measured with a Varioskan fluorescence plate reader, and the ATP levels were quantified by comparison with an ATP reference sample of known concentration and expressed as picomoles per cell.

Propidium iodide uptake assay
Cellular sensitization was analyzed by PI uptake assay. At 4 and 24 hours after treatment, cells were stained with PI (5 μM for 10 min at 37 °C). Cells were then washed with PBS and resuspended in PBS before analysis. The PI signals were recorded (in the phycoerythrin channel) on a flow cytometer (FACSAria III cell sorter, BD Biosciences). Data were analyzed with flow cytometry analysis software (FlowJo, Tree Star), corrected for the baseline fluorescence intensity derived from the isotype control, and expressed as a percentage of baseline (0 hours).

In vivo efficacy of organ preservation fluid in rat model of renal ischemia
Clinically, kidney ischemia may be caused by a variety of conditions, such as reduced cardiac output, renal vascular obstruction, and kidney transplant. Therefore, this model is a good choice for evaluation of functional efficiency of prepared supplemented OPF solutions. The unilateral ischemia-reperfusion injury with immediate contralateral nephrectomy model was developed and previously published by Li and colleagues.9 Briefly, animals were divided into 3 groups of 6 animals. The sample size (n = 6) was determined by power analysis with α = .05 and β = 80%. The SD values were taken from the previous literature. Animals were anesthetized with intraperitoneal injection of pentobarbital sodium (2 mg/kg). Anesthesia was maintained with isoflurane (0.5%) when required. The abdominal cavity was exposed by a 3-cm ventral midline incision (Figure 1a). The right-side kidney was momentarily removed and ligated with nonabsorbable sutures. The renal artery of the left-side kidney was ligated from the distal and proximal ends (Figure 1b). A nontraumatic vascular curved clamp (Unimax Science Tools) was used at the renal vein (Figure 1c), and subsequently a tiny hole was created toward the proximal side of the kidney. A volume of 4 mL for each of the 2 supplemented solutions and the commercial OPF formulation (maintained at 4 °C) was administered at the proximal renal artery with a fine 22-gauge needle with a controlled flow rate of 1.25 mL/min (Figure 1d). The outlet of kidney perfusate was collected from the tiny hole in the renal vein. The perfused kidney was stored in the respective preservation solution at 4 °C for 1 hour (Figure 1e) in an incubator to control the change in body temperature. Finally, all the ligations were opened to allow blood reperfusion. The ventral midline incision was closed in 2 layers with polyglycolic acid sutures. A subcutaneous injection of buprenorphine (0.03 mg/kg) was administered as an analgesic for the complete recovery of the animal. At 48 hours after reperfusion, blood samples were collected for the determination of serum creatinine and blood urea nitrogen level as markers of kidney function.

Immunohistological examination
For determination of the extent of apoptosis, the rat kidney tissues were stored in respective OPF formulations for 24 hours, after which the expression of caspase 3 were determined according to the protocol of Zhou and colleagues.¹² The tissues were fixed in paraffin wax, and sections (5-mm thick) were mounted on glass slides. The sections were treated for antigen retrieval with 10.2 mmol/L sodium citrate buffer (pH 6.1) for at least 20 minutes at 95 °C. Sections were washed with 0.01 M PBS containing 0.3% Triton X-100 (pH 7.4; PBS-T) and immersed in 2% normal goat serum in PBS for 2 hours at 37 °C. The sections were then incubated at 4 °C for 24 hours with a polyclonal antibody to caspase 3 (1:50; Santa Cruz Biotechnology) in PBS containing 1% bovine serum albumin. The cells were washed with PBS and then further incubated in goat anti-rabbit immunog-lobulin G (1:200; Boster Biological) in PBS for 2 hours at room temperature, followed by a wash with PBS-T. Then, cells were incubated in avidin-biotin-peroxidase complex solution (1:100; Boster Biological) for 2 hours at room temperature, with a final rinse in PBS-T (3 rinse cycles, 5 minutes each). Immunolabeling was visualized with 0.05% 3,3'-diaminobenzidine plus 0.3% H₂O₂ in PBS. After the initial staining procedure, the sections were counterstained with hematoxylin, dehydrated with ethanol and xylene, and mounted to slides with coverslips and Permount.

Statistical analyses
All data are presented as mean values with SD.
One-way analysis of variance was applied to determine the significant differences (GraphPad Prism software, version 8). The level of significance for this study was 5% (P < .05).

Results

Effects of variables on cellular oxidative stress
We used DCFDA probe to investigate the effect of temperature variance (4 °C, 15 °C, 25 °C, and 37 °C) on the status of cellular redox balance in HEK-293 cells at 4 and 24 hours after incubation in different preservation solutions. Our predominant obser-vation was that OPF-RTT was more effective than OPF-NCD or Custodiol to prevent temperature-induced endogenous production of ROS, and the commercially produced solution (Custodiol) was the least effective. Reduction of MFI per cell at 24 hours was approximately 0.25-fold for OPF-RTT versus approximately 0.45-fold for OPF-NCD. The for cells treated with Custodiol was 791.5 ± 61, whereas it was 171 ± 11.5 for OPF-RTT and 319.5 ± 23.6 for OPF-NCD. As seen in Figure 2, the levels of ROS generation in the 2 prepared OPF solutions (OPF-NCD MFI and OPF-RTT) were significantly low (P < .05) compared with Custodiol solution. Also, the ROS generation in cells in all 3 solutions noticeably increased in response to higher temperature or longer incubation time. These results indicate that OPF-NCD and OPF-RTT solutions are able to substantially regulate the temperature-induced oxidative stress in HEK-293 cells.

Effects of variables on energy metabolism (ATP production)
Next, we examined the effects of various incubation temperatures on cellular energy met-abolism as indicated by ATP production at 24 hours after incubation in HEK-293 cells. Intracellular ATP levels were assessed in HEK-293 cells incubated in the various OPF solutions and at various tempe-ratures. Figure 3 shows the respective correlation of intracellular ATP levels in HEK-293 cells. We observed an ATP level of 32 999 ± 1454 pmol/cell for OPF-NCD solution at 4 °C, which is significantly high (P < .05) compared with Custodiol solution (23 216 ± 1315 pmol/cell), and the ATP level in OPF-RTT solution (26 200 ± 1672 pmol/cell) at the same temperature was similar to the ATP level for Custodiol solution. As predicted, there was negative correlation between temperature and ATP production in HEK-293 cells; that is, an increase in temperature (from 4 °C to 37 °C) led to a linear decrease in ATP production. When the temperature was increased from 4 °C to 37 °C, the ATP counts for cells preserved in OPF-NCD, OPF-RTT, and Custodiol solutions, respectively, dropped from 32 999 ± 1454 to 20 893 ± 189, from 26 200 ± 1672 to 13 195 ± 1035, and from 23 216 ± 1315 to 14 033 ± 2785 pmol/cell. However, at normothermic conditions (25 °C and 37 °C), intracellular ATP counts were approximately 1.5-fold higher for HEK-293 cells preserved in OPF-NCD solution compared with Custodiol solution or OPF-RTT solution, as plotted in Figure 3.

Effects of variables on cell death
We studied the PI uptake by HEK-293 cells to validate whether the cellular redox status and energy metabolism in OPF-NCD solution was able to regulate cell death compared with Custodiol and OPF-RTT solutions. We found positive correlation between increased temperature and necrotic cell population in HEK-293 cells incubated in all 3 of the OPF solutions. However, the highest rates of cell death were in HEK-293 cells incubated in Custodiol solution at all 4 temperatures and at both time points. The PI positivity rate (necrotic cell death) signi-ficantly reduced in OPF-NCD and OPF-RTT solutions at both 4 hours and 24 hours compared with Custodiol solution (Figure 4) at 4 °C after 24-hour incubation. Furthermore, at 25 °C incubation, OPF-RTT solution showed a significant increase in PI positivity at 4 and 24 hours compared with OPF-NCD solution. The data suggest that OPF-NCD preservation solution has the best preservation efficiency of the 3 solutions, followed by OPF-RTT solution and Custodiol solution, respectively.

In vivo preservation efficiency
Functional recovery of rat kidney was determined after perfusion and cold storage in the OPF solutions. Data are illustrated in Figure 5. Mean values of serum creatinine were 1.18 ± 0.122, 0.548 ± 0.05, and 0.956 ± 0.05 mg/dL when kidney was perfused and stored in Custodiol, OPF-NCD, and OPF-RTT solutions, respectively. Similarly, mean values of blood urea nitrogen were 103.53 ± 10.24, 85.36 ± 4.64, and 92.85 ± 3.15 mg/dL for Custodiol, OPF-NCD, and OPF-RTT solutions, respectively.

Expression of caspase 3 by immunohistology
Caspase 3 (apoptosis marker) expression after 24-hour storage of rat kidney in respective OPF solutions is shown in Figure 6, which shows the expression of caspase 3 in Custodiol solution in the form of yellow brown granules (Figure 6a) and in OPF-NCD (Figure 6b) and OPF-RTT solutions (Figure 6c). We observed that the number of caspase 3-positive cells and the optical density were highest in kidney tissue preserved in Custodiol solution (0.274) followed by OPF-RTT solution (0.233) and OPF-NCD solution (0.184).

Discussion

Numerous organ preservation fluids such as Collins solution, University of Wisconsin solution, and HTK solution have been developed to regulate ischemic injury during cold storage and reperfusion. Several clinical studies demonstrated that the proper selection of OPF formulation is a crucial factor for successful short-term and long-term transplant outcomes. Collins solution was developed to mimic the intracellular fluid composition with high potassium, high magnesium, and low sodium content. Later, the University of Wisconsin solution was considered as the gold standard for organ preservation, with low-sodium and high-potassium content to mimic the intracellular fluid composition. This was superior to Collins solution because it consisted of more specific substances to prevent interstitial edema, cell swelling, and oxidative stress. Further development produced effective and affordable solutions such as HTK. Unlike the previous OPF solutions (Collins solution and University of Wisconsin solution), HTK is an extracellular preservation fluid with high sodium and low potassium content. The comparatively lower viscosity of HTK solution allows shorter cooling periods to maintain hypothermic conditions, with a comparatively lower financial cost. Despite the popularity of the organ preservation fluids discussed here, it remains clear that additional development is needed to further improve organ preservation within the normothermic conditions.^9,14

With the same objectives, the supplemented OPF solutions were prepared (OPF-NCD and OPF-RTT) and assessed in contrast to Custodiol solution by means of in vitro HEK-293 cell preservation study at 4 °C, 15 °C, 25 °C, and 37 °C. Various parameters such as ROS, intracellular ATP levels, and cell viability were determined under these conditions. A vital implication of this study is that preservation efficiency can be significantly increased with KATP channel openers and an antioxidant under hypothermic and even normothermic conditions compared with the use of Custodiol solution alone. This was the basis for our decision to use of nicorandil and RTT as the supplements for the prepared OPF solutions.

Production of various ROS (superoxide radicals, hydroxide radicals, and hydrogen peroxide) is a continuous process in living cells, and ROS are generated as byproducts of several cellular mec-hanisms and in parallel neutralized by intracellular antioxidant defense. However, high levels of ROS (during ischemia and even reperfusion) may cause several cytotoxic conditions.¹⁵ Therefore, measure-ment of intracellular ROS is an essential parameter for assessment of the preservative efficacy of OPF solutions. Here, we show that incubation of HEK-293 cells in OPF-RTT solution resulted in the lowest percentage of ROS generation. This result may be correlated to the antioxidant property of RTT, which is known to scavenge myeloperoxidase-dependent ROS and release ROS-dependent neutrophil extracellular traps. Generally, RTT scavenges superoxide radicals, which undergoes redox reaction to release hydrogen peroxide, capable of activating the formation of neutrophil extracellular traps. Moreover, the released myeloperoxidase converts hydrogen peroxide to hypochlorite and leads to formation of singlet oxygen, which is reported to be necessary in the formation of neutrophil extracellular traps. Thus the previous studies show that the antioxidant property of RTT not only acts on the hydroxyl and hypochlorite radicals (myeloperoxidase-derived species) but also targets superoxide radicals.^15-17

We also observed that ROS levels gradually increased with increasing temperature. This result is in accordance with previously published reports by Davidson and Schiestl in 2001 and Vacca and colleagues in 2004. These 2 studies state that heat stress can possibly cause impairments in mitoc-hondrial functions and result in the induction of oxidative damage that could manifest into lipid peroxidation and result in increased level of ROS such as hydrogen peroxide and superoxide anions.^18,19

Earlier studies have demonstrated the depletion of intracellular ATP levels during ischemia. Lower intracellular ATP may impair various energy-dependent cellular metabolic pathways. A number of experimental and clinical studies have shown the importance of intracellular ATP levels on the survival rate of transplanted organs.^20,21 Therefore, we chose to maintain the intracellular ATP level during cold and even normothermic preservation of kidney. Our results indicate that intracellular ATP levels increase 1.5-fold in Custodiol and OPF-RTT solutions compared with OPF-NCD solution. This could be attributed to the fact that presence of nicorandil in OPF solution causes hyperpolarization mediated by SUR2B/Kir6.2 channel activation. Hyperpolarization, which is responsible for the reduction of the action potential duration and the increase in outward current, leads to a rise in intracellular ATP levels.²¹

The effect of nicorandil on the intracellular ATP level was in accordance with the results of a study by Mamprin and colleagues in 2005, which demonst-rated how different concentrations of ATP in the University of Wisconsin solution may regulate intracellular ATP levels and thus cell viability during the cold preservation of hepatocytes.²² They observed loss of intracellular ATP in the hepatocytes preserved without nucleosides after 72 hours of cold preservation, whereas significant improvement of intracellular ATP was recorded when solution was supplemented with extracellular ATP.

We observed that in all 3 groups the intracellular ATP level exponentially decreased with an increase in temperature. This inverse relationship is of particular interest in the context of a study from 1996 by Hubley and colleagues.²³ They demonstrated how temperature augmentation of media reduces the overall intracellular ATP level. According to their theory, intracellular diffusive transport of ATP is regulated by certain parameters such as viscosity, ionic strength, and pH of the intracellular diffusion media (cytoplasm). Among these parameters, viscosity of the cytoplasm is directly regulated by change in temperature, thus changing the diffusion coefficient of ATP.23 Sidell and Hazel demonstrated a 2-fold change in the viscosity of thermodynamic artificial model of cytoplasm between 25 °C and 5 °C to explain the ATP diffusion mechanism.²⁴

Cell viability was observed after 4 hours and 24 hours preservation in the 2 prepared OPF solutions and the Custodiol solution. Our results suggest that cell viability significantly improved with OPF-NCD and OPF-RTT solutions compared with the Custodiol solution. The improvement in cell viability in OPF-NCD solution may be due to nicorandil-mediated improvement of mitochondria function and structure, which can produce high levels of energy moieties (ATP molecules) that could help regulate various metabolic pathways for cell survival. The effect of KATP channel openers on the cellular energy and thus cell survival during ischemic conditions was helpfully explained by Yang and Yu.²⁵ They reported that mitochondrial depolarization provided better ATP preservation and that the KATP channel openers also activate fatty acid oxidation and electron transfer, which in turn results in ATP production.

Recurrent ischemia-reperfusion may also impair mitochondrial function, which may further exac-erbate the apoptosis. Previous studies have successfully demonstrated that the commencement of the K_ATP channel by nicorandil inhibits the depolarization of the mitochondrial membrane and the release of cytochrome c into the cytosol, thus inhibiting apoptosis. Protection of the mitochondrial structure and integrity is also an important factor to consider.²⁶ In a different study, nicorandil was shown to enhance differentiation capacity through preconditioning of neural stem cells to support cell survival under ischemic conditions. Nicorandil provides this protection through the activation of the apurinic/apyrimidinic endonuclease 1 pathway in the neurons when subjected to oxidative stress.²⁷

In vivo preservation efficacy of the OPF-NCD and OPF-RTT solutions was determined in a rat model of renal ischemia, in accordance with the central purpose objective of newly prepared preservation fluid, which is to maintain organ viability and enhance survival time during recovery and preservation. It is essential for any preservation fluid to provide better protection to the preserved organ with minimum cellular damage and controlled functionality. We chose the unilateral ischemia-reperfusion injury with immediate contralateral nephrectomy model because this model is associated with improved physiological mechanisms and thus renal recovery.²⁸ Data from our in vivo study showed that both preservation solutions (OPF-NCD and OPF-RTT) were effective, as shown by the improved functional recovery of the ischemic rat kidney. Serum creatinine and blood urea nitrogen levels were significantly (P < .05) lower in kidneys treated with OPF-NCD and OPF-RTT solutions compared with Custodiol solution. These results are in accordance with our in vitro cell viability study on HEK-293 cells. However, OPF-NCD was found to be superior to OPF-RTT and Custodiol solutions.

Ischemia and hypothermia during preservation disrupts the normal cycle of cell metabolism and causes depletion of intracellular ATP levels. As a consequence, Na⁺/K⁺-ATPase suppression causes cellular edema and ROS production.²⁰ The commercially available OPF solutions generally regulate these secondary effects by maintaining osmolarity or ROS level or by providing an alternative energy source to the preserved organ, but these solutions are less effective to maintain the ATP levels. Because OPF-NCD acts directly on the ATP production pathway, it effectively controls various energy-dependent cellular metabolic process and thus improves recovery rates for cells and organs while also affecting secondary effects. Yang and Yu have demonstrated the similar effect on heart preservation with another KATP channel opener (pinacidil).²⁵ Significant improvement in various parameters (heart function, coronary flow, myocardial ultrastructure, and cardiac troponin I release) was reported.²⁵ In another study, Ozturk and colleagues confirmed that nicorandil can inhibit renal tubular damage and tubule interstitial fibrosis by reducing the oxidative stress in the rat kidney model of partial unilateral ureteral obstruction.²⁶

Caspase 3 (cysteine-aspartic acid protease) is a common protein (among 10 caspase molecules) in the apoptotic pathway and plays a crucial role in the cell apoptosis execution phase. Our present study demonstrated the expression of caspase 3 as an apoptosis marker after the preservation of kidney tissue in the prepared OPF solutions. It was reported in previous publications that, as a response of ischemia, deprivation of intracellular glucose and ATP may occur, which further aggravates the protein misfolding, thereby inducing the unfolded protein response. Furthermore, reperfusion of organs may cause oxidative stress and protein folding. Caspase 3 is a proapoptotic marker that starts induction within 4 to 8 hours of ischemia.^27-29 Our results confirm that proteins were expressed even after storage of rat kidney in the 2 prepared OPF solutions. However, the order of expression of proteins was the least in the kidney preserved in OPF-NCD, followed by OPF-RTT and Custodiol solutions. Least expression in OPF-NCD could be due to ATP maintenance ability of nicorandil, which overcomes the overall effect of ischemia and enhances cell survival. The supplement RTT was also found to effectively control the expression of apoptotic proteins due its free radical scavenger activity.

Our in vitro data from this experimental study show that OPF-NCD and OPF-RTT may provide better protection from the ischemia-reperfusion injuries. Data showed reduced levels of endogenous ROS and enhanced ATP production, which led to comprehensively less incidence of cell death when HEK-293 cells were preserved in OPF-NCD or OPF-RTT. The in vivo animal model clearly demonstrated that OPF-NCD and OPF-RTT possessed lower expression of necrotic biomarkers with improved kidney function. However, in vitro data suggest that the OPF-NCD and OPF-RTT are effective even in the normothermic conditions, but the confirmation step with the in vivo animal model at normothermic condition is still recommended, and this is a major limitation of our study. Further investigation of prepared OPF solutions in human subjects is needed to support the superiority and effectiveness reported here.

Conclusions

Our in vitro data from this experimental study show that OPF-NCD and OPF-RTT may provide better protection from the ischemia-reperfusion injuries. The endogenous ROS level in HEK-293 cells was found lowest in OPF-RTT; however, ATP production was highest with OPF-NCD solution. The OPF-NCD solution showed the lowest incidence of HET-293 cell death, even at normothermic conditions. Finally, the in vivo rat perfusion model explicitly demonstrated that OPF-NCD solution possessed the lowest expression of necrotic biomarkers and the highest improvement in kidney function among the 3 solutions that we tested, followed by OPF-RTT solution and Custodiol. Further investigation of prepared OPF solutions in human subjects is needed to support these results.

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