Abstract
Solid organ transplantation was one of the greatest medical advances during the past few decades. Organ preservation solutions have been applied to diminish ischemic/hypoxic injury during cold storage and improve graft survival. In this article, we provide a general review of the history and advances of preservation solutions for kidney transplantation. Key components of commonly used solutions are listed, and effective supplementations for current available preservation solutions are discussed. At cellular and molecular levels, further insights were provided into the pathophysiological mechanisms of effective ingredients against ischemic/hypoxic renal injury during cold storage. We pay special attention to the cellular and molecular events during transplantation, including ATP depletion, acidosis, mitochondrial dysfunction, oxidative stress, inflammation, and other intracellular mechanisms.
Introduction
The 20th century had witnessed great advances in organ transplantation since the first human kidney transplant was attempted by Yuriy Voroniy in 1933 1 . Kidney transplantation offers the opportunity to replace failed kidneys that are unable to perform their normal physiological functions. It is vital to preserve kidneys in optimal condition before transplantation takes place. The majority of kidney donations are now from non-heart-beating donors (NHBD), and it often takes hours, or even days under some circumstances, to transport organs between medical centers, with high risk of ischemic/hypoxic damage 2,3 .
Numerous preservation solutions have been developed for organ storage to prevent ischemic/hypoxic injury, improve histocompatibility matches, and ensure immediate and normal organ function after implantation. Previous studies and clinical applications have demonstrated that the choices of different preservation solutions may affect both short-term and long-term transplant outcomes 4,5 . To minimize ischemic damage, preservation solutions have been designed to specifically target against the biological changes that may occur during the ischemic/hypoxic process 6 . Pharmacological, biochemical, hormonal, and immunological approaches have been applied in the development of preservation solutions (see previous reviews 7 –10 ).
Based on our research experience, we will discuss in this review the history of several “gold-standard” preservation solutions, recent advances, and future developments in kidney preservation solutions. We highlight the underlying molecular mechanisms of commonly used preservation solutions against ischemic/hypoxic injury at present. Literature searches were conducted of online databases, including PubMed and MEDLINE, using kidney transplantation, preservation solutions, additives, ischemia, and hypoxia as keywords to capture all relevant publications. The final date for literature searches was July 15, 2019.
History
The importance of ensuring the preservation of kidneys in optimal condition between retrieval and implantation has been long recognized. Cold storage, in which a preservation solution is infused into the organ and the organ is stored at hypothermic conditions, is a process used to reduce the metabolic requirements of organs and attenuate ischemic/hypoxic injury. It has been successfully applied in kidney transplantation for more than 50 years, and for decades has been the most widely used organ preservation method due to its simplicity and low cost 9,11 . Cold storage preservation solutions have continued to evolve since their early development. In the late 19th century, the concept of continuous perfusion and simple cooling with cold solutions was developed for organ preservation 11 . In the 1930s, it was believed that continuous hypothermic perfusion could help eliminate toxic metabolic products and provide nutrients to preserved organs 12 . In 1968, Belzer et al. performed a successful kidney perfusion preservation 13 . A year later, Geoffrey Collins developed the first cold storage preservation solution, named Collins Solution, which increased kidney preservation time up to 24 h 14 . Collins Solution was further modified by the Eurotransplant Foundation in 1980 into the Euro-Collins Solution (EC) 15 , which was commonly used in Europe for about 15 years. In the late 1970s, Ross et al. developed hyperosmolar citrate (HOC) solution (also known as Marshall’s solution), which is effective for 72 h of kidney preservation and is still in clinical use today 16,17 . In 1986, Belzer et al. reported a newly developed University of Wisconsin (UW) solution containing lactobionate and raffinose 18 . Another popular preservation solution in the 1980s was histidine–tryptophan–ketoglutarate (HTK) solution 19 . UW and HTK solutions were regarded as the “gold-standard” and accounted for the majority of preservation solutions used in the clinic 9,20 . Celsior is an extracellular type preservation solution for cold storage preservation developed in 1994, and has also been widely used in kidney transplantation 21 .
Here, we concentrate on the most widely used solutions (EC, UW, HTK, Celsior, HC-A and IGL-1) with comparable post-transplant outcomes, and discuss several recent advances for kidney preservation solutions.
Commonly used solutions for kidney preservation
Collins Solution
Collins Solution preserved kidneys successfully with simple ice storage before transplantation 14,22 . It was realized that kidneys can absorb water and sodium and lose potassium when deprived of oxygen or nutrients under hypothermic conditions. Therefore, Collins Solution was designed to have high potassium, high magnesium, and low sodium concentrations to mimic the composition of intracellular fluid. It is a phosphate-based solution with high glucose concentration that balances the osmolarity 14 . Later, several modifications were made based on the Collins Solution, which became known as EC Solution 15 (Table 1). EC was commonly used clinically for the next 15 years, until the “gold-standard” UW solution was introduced.
Composition of Common Kidney Preservation Solutions.
University of Wisconsin Solution
UW solution is the most common cold storage solution in organ preservation. It was initially developed as a preservation solution for pancreas transplantation 18 , but has been widely used in preserving different kinds of organs, including the kidney, liver, and small bowel, since the late 1980s 24 –26 .
Like EC solution, UW is an intracellular preservation solution with low sodium and high potassium concentrations to mimic an intracellular environment 18 (Table 1). There are several advantages of UW compared with EC: a) hydroxyethyl starch (HES) serves as the colloid carrier to minimize interstitial edema during perfusion; b) metabolic substrates such as lactobionate and raffinose were introduced to replace glucose to prevent cell swelling; c) allopurinol and glutathione were added as oxygen radical scavengers to reduce oxidative stress 27 ; d) adenosine is used as an adenosine triphosphate (ATP) precursor 28 .
UW solution has proven superior to EC solution in both animal experiments and clinical trials 29 –31 . For example, in the dog kidney autotransplant model, all kidneys were viable when stored for 72 h in UW solution, while none were viable when stored for 72 h in EC solution, suggesting that UW solution effectively preserves kidneys for 72 h 29 . The European Multicenter Trial in the 1990s demonstrated better kidney preservation outcomes using UW solution compared with EC solution 30 , with fewer incidences of delayed graft function (DGF, 35.8% vs. 52.2%) and better 1-year graft survival (92% vs. 86%) 31 .
Despite its popularity, recent studies have suggested potential disadvantages of UW solution, including a) high viscosity generated by HES, which complicates tissue wash-out and saturation with preservation solution 32 ; b) addition of penicillin G, dexamethasone, and insulin before use 20 ; and c) the current cost per liter of UW solution is $282, and the total cost of UW solution per liter is over $300 when additives are included 20 . Therefore, further modifications have been made to create more effective or economical solutions, such as RPS-96 (UW lacking HES) 33 , dextran 40-based UW (replacing HES with dextran 40 in UW) 34 , perfluorocarbons (PFC)-based UW 35 , hyperbranched polyglycerol (HPG) solution (replacing HES with HPG in UW) 36 , and sodium lactobionate sucrose (SLS) solution (replacing raffinose with sucrose in UW) 37 .
Histidin-Tryptophan-Ketoglutarat Solution
HTK solution was originally developed and introduced by Bretschneider as a cardioplegia solution for open heart surgery in the 1970s 19 . Subsequently, it proved to be effective in preserving liver, kidney, and pancreas 38,39 . Unlike UW solution, HTK solution is an extracellular preservation solution with high sodium and low potassium concentrations. Histidine, tryptophan, and ketoglutarate are the three key components in HTK solution. Histidine serves as a buffer and retards the tissue pH decline during cold ischemic/hypoxic conditions. Tryptophan acts as a free radical scavenger and membrane stabilizer. Another amino acid, ketoglutarate, serves as an energy substrate 19 (Table 1). HTK solution has a viscosity similar to that of water. This lower viscosity provides a shorter cooling time to reach hypothermic conditions compared with UW solution, and also eliminates the need for pressurized perfusion.
The effectiveness of HTK solution was confirmed in clinical trials in the early 1990s. Eurotransplant conducted a randomized multicenter study comparing kidney graft preservation with HTK, UW, and EC solutions 40 . The results showed the equivalence of HTK compared with UW in preservative ability, with an equal incidence of initial non-function and similar 3-year kidney graft survival 24,41 . A minimal DGF rate was also observed in HTK as compare with EC solution (0 vs. 33.3%) 42 .
However, Stewart et al. reported that if there are additional graft factors such as donation after cardiac death, HTK preservation was associated with an increased risk of graft loss with cold ischemia time over 8 h 43 . Another potential disadvantage of HTK solution is the higher perfusion volume caused by its low viscosity. However, even with the greater volume of HTK needed, the overall cost is decreased compared with UW solution 20 ($181/L vs. $282/L, Table 1). Despite single critical papers, HTK solution is still one of the most popular kidney preservation solutions in use today.
Celsior Solution
Celsior is an extracellular type of preservation solution developed by Menasché et al. in 1994 for cold storage preservation of cardiac grafts 21 . Currently, it has been applied successfully to heart, lung, liver, pancreas, kidney, and small bowel preservation 44,45 . Celsior is a high-sodium, low-potassium solution. It adopts the key effective components of both UW and HTK solution. Lactobionate and mannitol were used as impermeants to limit cellular edema. Reduced glutathione was added to Celsior solution as a free-radical scavenger. Celsior solution has greater buffering capacity against acidosis and less viscosity than UW due to the absence of colloids (Table 1).
A multicenter randomized study of 187 renal transplants showed that the kidney preservation by Celsior solution was equivalent to that of UW solution in DGF rate (31.3% vs. 33.9%) and 2-year graft survival (84% vs. 75%) 46 . The incidence of acute tubular necrosis was much lower in the Celsior group than in the UW group (23% vs. 36%) after transplantation 47 . These results indicated equivalence, and even some advantages, of Celsior solution when compared with the traditional UW solution.
Hypertonic Citrate Adenine Solution
Hypertonic citrate adenine (HC-A) is the most widely used preservation solution for kidney transplantation in China. It was first developed in the late 1970s by Shanghai Changzheng Hospital and the Shanghai Blood Center 48 . Since then, HC-A solution has been extensively applied in China for over three decades. HC-A is a hyperosmolar citrate-based solution with additive adenine as nutrient to improve kidney function after circulation (Table 1). HC-A solution has a high mannitol concentration to act as an impermeant to limit cellular edema and oxidative stress.
Twenty years after the application of HC-A solution, HC-A II solution was developed (based on the formula of HC-A) by Shanghai Changzheng Hospital. HC-A II has a double-buffer system of citrate and phosphate to achieve enhanced buffer capacity. The concentration of magnesium was significantly reduced, and concentration of adenosine was increased compared with the HC-A solution. New ingredients of arginine, tryptophan, and ligustrazine were also added to stabilize cell membranes and protect mitochondrial function 49 . In a multi-center randomized controlled trial conducted from 2008 to 2012 in China, HC-A II solution showed similar efficacy and safety in kidney preservation compared with HTK solution, suggesting that HC-A II solution is a promising option for clinical application 49 .
Application of HC-A solution accounts for approximately 50% of total kidney transplantation in China nowadays due to its efficacy and low cost, although few specific studies on HC-A have been reported in the English literature. Indeed, the efficacy and safety of HC-A solution seem reasonably sound since it has been used for more than 100,000 grafts in clinical settings in China 50 . However, more studies are needed due to lack of detailed data from basic and clinical research.
Other Solutions
Apart from the above mentioned solutions, several other solutions are also utilized for cold storage preservation of kidneys, including phosphate buffered sucrose (PBS) 140 51,52 , HP16 53 , HBS 54 , B2 55 , Lifor 56,57 , Ecosol 58 , Biolasol 59,60 , renal preservation solution 2 (RPS-2) 61 , F-M 62 , AQIXRS-I 63 , WMO-II 64 , CZ-1 65 solutions, etc. Each solution may vary in composition; however, the key strategy with each solution is similar: use colloids, impermeants, electrolytes, antioxidants, and nutrients to minimize ischemic/hypoxic injury and maximize kidney function after reperfusion 66,67 .
Recent Advances in Kidney Preservation Solutions
Due to a deeper understanding of the process of ischemic/hypoxic injury and components of commonly used preservation solutions, efforts have been made to develop new preservation solutions. Particular components are now added to standard solutions to further prevent organs from ischemic/hypoxic injury. In the present review, we attempt to provide a summary of the effective additives available, with experimental evidence of animal models (Table 2).
Recent Advances in Kidney Preservation Solutions.
PEG, polyethylene glycol; CORM, carbon monoxide releasing molecules; BNP-1, bovine neutrophil peptide-1; SP, substance P; NGF, nerve growth factor; IGF-1, insulin-like growth factor-1; EGF, epidermal growth factor; HGF, hepatocyte growth factor; rh BMP-7, recombinant human bone morphogenetic protein-7; TNF-RFP, TNF-receptor fusion protein; MMP, matrix metalloproteinase; ICAM, intercellular adhesion molecule; TMZ, trimetazidine; lec-SOD, lecithinized superoxide dismutase; MitoQ, mitoquinone; IGL-1, Institute Georges Lopez-1 solution; SCOT, Solution de Conservation des Organes et des Tissus; PBSc, Phosphate-buffered sucrose solution; SBS, sucrose-based solution; EC, Euro-Collins solution; UW, University of Wisconsin solution; HTK, Histidin-Tryptophan-Ketoglutarat solution; CS, Celsior solution; HOC, hyperosmolar citrate solution; KPS-1, kidney perfusion solution-1.
Colloids
Several high molecular weight colloids have been applied in preservation solutions to sustain the intravascular oncotic pressure and prevent interstitial edema 131 . UW solution uses HES (50 kDa), which is a stable non-toxic oncotic agent, as the colloids 132 . Moreover, other colloids were also utilized to substitute HES to make solutions more cost-effective.
Dextran (40 kDa) is one of the most widely investigated replacements of HES 34 . It is a safe replacement of HES in UW solution for the purpose of clinical kidney preservation 34 . Dextran 40 may be beneficial during the graft reperfusion phase due to reduced vascular resistance and anti-thrombogenesis function 34,133 .
Poly-ethylene glycol (PEG), a neutral water soluble nontoxic polymer, has been used to substitute HES in the several newly developed preservation solutions 134 including Institute Georges Lopez-1 (IGL-1), Polysol, and “Solution de Conservation des Organes et des Tissus” (SCOT) solution. IGL-1 is a PEG 35 kD-based preservation solution with inverted Na+/K+ concentration compared with that of UW solution 135 (Table 1). Encouraging results of IGL in kidney transplantation have been demonstrated in both rat and pig transplantation studies 68,136 . In a multi-center study conducted in 2009, IGL-1 solution-preserved kidneys showed outcomes similar to those preserved in the UW solution in terms of DGF, rejection rates, and patient and graft survival 137 . Polysol is another PEG 35 kD-based preservation solution that contains antioxidants, amino acids, energy substrates, and vitamins 138 . In pig kidney transplant studies, Polysol displayed improved microcirculation and structural integrity following prolonged ischemia compared with UW and HTK solutions 139 –141 . Nonetheless, a pilot clinical study failed to demonstrate the advantage of Polysol in living kidney transplantation, with higher acute rejection rates observed in recipients compared with UW solution controls 142 . Therefore, the use of Polysol solution in the clinic is questionable. SCOT is another PEG-based preservation solution, which contains PEG 20kD as a colloid 143 . The effectiveness of SCOT in kidney transplantation was displayed in a preliminary clinical study, with comparable or better outcomes versus commonly used UW or EC solutions 144 .
PEG could spontaneously bind to cell surfaces to stabilize membrane lipids 145,146 and may further enhance immunoprotective properties 134,147 . It has vasodilatory, anti-fibrosis, and antioxidant effects in addition to the colloid properties 134,135 . Thus, PEG-based preservation solutions are considered promising alternatives for commonly used solutions in clinical applications.
Gases
The use of therapeutic gases as effective additives in the context of preservation solutions has become popular in recent years 148 . Several gases have been used to reduce ischemic/hypoxic injury in kidney transplantation, including oxygen (O2), hydrogen (H2), carbon monoxide (CO), nitric oxide (NO), hydrogen sulfide (H2S) and argon (Ar).
The retrograde venous application of humidified pure O2 during cold storage was developed by Isselhard et al. in 1972 149 . The effect was further confirmed by a small pilot clinical study 150 . In a large animal preclinical model, addition of M101 (an O2 carrier with high-oxygen affinity) into UW and HTK solutions provided significant benefits to grafts, both in early function recovery and long-term outcomes 83 .
H2 is a highly diffusible antioxidant gas, which can easily enter the cell. H2 could preserve mitochondrial function by attenuating reactive oxygen species (ROS) production, thus preventing apoptotic cell death 151 . A rat study has demonstrated the beneficial effect of H2-rich UW solution during kidney transplantation. H2-rich UW solution showed decreased oxidative stress, tubular apoptosis, interstitial fibrosis, improved renal function and survival rate compared with UW simple cold storage 78 .
CO, NO, and H2S are considered to be endogenous gas transmitters. CO has been implicated to have cytoprotective effects by attenuating proinflammatory cytokines, lipid peroxidation, and improving mitochondrial function
152,153
. Supplementation of UW solution with CO or CO-releasing molecules provided significant protection against renal ischemic/hypoxic injury in both rat and porcine transplantation studies
79
–81
. NO is mainly generated from the endogenous metabolism of
In addition, argon saturated Celsior solution (Ar-Celsior) decreased inflammation, interstitial fibrosis, tubular necrosis, and apoptosis in a porcine kidney transplantation model, resulting in improved early functional recovery, graft quality, and survival 84 . Therefore, adding gas at an appropriate level and composition into current preservation solutions is a promising approach to improve kidney preservation.
Amino Acids, Peptides and Proteins
Amino acids appear to be quite effective in providing nutrients or acting as metabolic substrates. The commonly used preservation solutions utilize amino acids, such as tryptophan in HTK solution, glutamic acid in Celsior solution, and histidine in both HTK and Celsior solution, as energy substrates (Table 1). Amino acids with antioxidant effects were also introduced into newly developed preservation solutions as additives.
Recent studies have focused on trophic factors as additives to improve storage efficiency of preservation solutions 93,94,159 . With the addition of a combination of bovine neutrophil peptide-1 (BNP-1), substance P (SP), nerve growth factor-β (NGF-β), insulin-like growth factor-1 (IGF-1), and epidermal-like growth factor (EGF) to the UW solution, McAnulty et al. successfully achieved 6-day kidney preservation in a canine model 93 . Moreover, addition of hepatocyte growth factor (HGF) to EC solution accelerated both renal blood flow recovery and glomerular filtration rate in dog kidney during cold ischemia 95 . Experimental evidence has suggested that trophic factors protect cells against inflammation, fibrosis and apoptosis, and improve mitochondrial function as well 160 .
Recombinant human bone morphogenetic protein-7 (rh BMP-7) perfused rat kidneys had elevated superoxide dismutase (SOD) activity and decreased lipid peroxidation level versus the UW-treated group 98 . The same effect was also achieved by direct introduction of lecithinized superoxide dismutase (lec-SOD) into the preservation solution 118 . TNF-receptor fusion protein (TNF-RFP) was able to suppress inflammation and promote organ survival in xenoperfused porcine kidneys 99 .
Signaling Pathway Blockers
The use of siRNAs, oligonucleotides, specific inhibitors or antagonists represented effective approaches to specifically target signaling pathways involved in ischemic/hypoxic injury. Exogenous administration of naked siRNAs via preservation solutions have been successfully tested in animal studies. Introducing naked caspase-3 siRNA in preservation solution resulted in reduced caspase-3 expression levels, cell apoptosis, and better acid-base homeostasis in a porcine kidney transplantation model 102 . Similarly, supplementation of preservation solution with matrix metalloproteinase (MMP)-2 siRNA protected transplant rat kidneys from preservation injury by inhibiting MMP-2 expression and tissue fibrosis 103 .
The adhesion of immune competent cells to the endothelial wall represents an important aspect of ischemic/hypoxic injury. Thus, phosphorothioate intercellular adhesion molecule (ICAM)-1 antisense oligodeoxynucleotide was added to EC solution to reduce ICAM-1-related inflammation response 104 . There was also experimental evidence of the benefit of blocking the vasoconstrictor mediators in improving kidney function. Studies in rats have shown that endothelial receptor antagonists could ameliorate renal ischemic/hypoxic injury after transplantation 85 –88 . Supplementation of Rho-kinase inhibitor HA1077 could improve renal function by antagonizing vasoconstriction 89 . Supplementing UW solution with a thrombin inhibitor melagatran in a porcine kidney transplantation model resulted in strong protection against both acute renal injury 90 and chronic lesions, including interstitial fibrosis, apoptosis, and inflammation 91,92 . Calcium channel blocker verapamil 108 –110 and platelet-activating factor (PAF) receptor antagonist 161 were also candidate additives in preservation solutions, the effect of which were confirmed in animal models. The postulated benefits include reduction of calcium overload, increase in tissue ATP concentration, and sodium re-absorption. Application of proteasome inhibitor is a potential strategy to reduce ischemic/hypoxic injury in both liver and heart transplantation, which lead to the up-regulation of AMPK activity, down-regulation of mTOR signaling, and influence autophagy 162,163 . Thus, supplying proteasome inhibitors into common preservation solutions can become a strategy to decrease renal ischemic/hypoxic injury as well. In conclusion, pathway-specific blockers that are based on well-defined ischemic/hypoxic mechanisms offer good opportunities to improve commonly used preservation solutions.
Other Pharmacological Additives
Apart from above-mentioned supplements, other pharmacological additives including radical scavengers, mitochondrial protectors and drugs have also been added to preservation solutions. The addition of radical scavengers (nicaraven 126 , trolox 117 , edaravone 119 ) could trap free radicals and protect cells against oxidative stress directly, contributing to reduced lipid peroxidation and higher survival rate of the preserved kidneys. In addition, exogenous administration of selenium 124,125 , propofol 127 (a widely used anesthetic), and tanshinone IIA 130 (an effective component of the traditional Chinese medicine Danshen) in preservation solutions tended to have antioxidant effects to attenuate malondialdehyde concentration, followed by the improvement in long-term renal function after transplantation.
Mitoquinone (MitoQ) 120,121 and quinacrine 122 act as mitochondrial targeted antioxidant compounds to reduce the damage of mitochondrial electron transport chain and improve renal cell viability in rat and porcine studies. Trimetazidine (TMZ) is another drug with mitochondrial-protective 164 functions. Supplementation of TMZ in UW solution was beneficial in porcine models, with improved functional recovery of preserved kidneys, higher survival rate, as well as reduced cellular and mitochondrial swelling, immune cell invasion, and interstitial fibrosis 111 –116 .
Other pharmacological additives that were beneficial in animal studies including prostaglandin E1 128,129 , taurine 105,106 , ranolazine 107 , etc. (Table 2). They also have the potential to be used in clinical transplantation. However, the underlying mechanisms and the specific signaling pathways involved are not well understood yet. Thus, more experimental studies and further clinical trials are still needed.
Mechanisms of Preservation Solutions Against Ischemic/Hypoxic Renal Injury
Ischemia and hypoxia are inevitable events during cold preservation of kidney transplantation. Ischemic/hypoxic renal injury is caused by a sudden cessation of blood flow along with the immediate oxygen deprivation to the kidneys 165 . Tissues are starved of oxygen and nutrients, leading to the accumulation of metabolic waste products. The imbalance in metabolic supply and demand within the ischemic organ results in profound tissue hypoxia and dysfunction. Subsequent reperfusion further enhances the activation of immune responses and cell death programs. A wide range of pathophysiological processes have been identified to be related to ischemic/hypoxic renal injury during cold storage, including ATP depletion, calcium overload, acidosis, mitochondrial dysfunction, oxidative stress, inflammatory response, and eventual cell death programs 166,167 (Fig. 1).
Schematic representation of effective supplementations in preservation solutions against ischemic/hypoxic renal injury.
ATP Depletion and Loss of Electrolytes Homeostasis
Nearly 95% of cellular ATP is depleted within 4 h as a consequence of organ cold storage during transplantation 6 . On the one hand, ATP deficits lead to a reduced activity of Na+/K+ ATPase, thereby contributing to increased intracellular sodium concentration 11 . Large amounts of water enter the cytoplasm due to the hyperosmolar intracellular environment, resulting in cell edema 168 . On the other hand, Na+/Ca2+ exchangers begin to work in the reverse direction and stop pumping calcium out of the cell, leading to calcium accumulation in cells 169 . Increased intracellular calcium levels can activate calcium-dependent phospholipase and proteases, promoting cell apoptosis 166 (Fig. 1).
To combat the above-mentioned ischemic/hypoxic renal injury during cold storage, a number of approaches have been used in preservation solutions. Energy substrates such as adenosine were often provided in solutions to allow for rapid ATP regeneration during preservation. The calcium channel blocker verapamil 108 –110 and other pharmacological reagents, which can be beneficial to Ca2+ homeostasis 105 –107 , are added into preservation solutions to prevent calcium overload (Table 2). H2 S could inhibit Na+/H+ exchanger activity via the PI3K/Akt/PKG-dependent pathway, hence reducing Ca2+ overload 170 . Impermeants and colloids are added to preservation solutions to prevent cell swelling 171 . The effectiveness of impermeants in preventing cell swelling is determined by their molecular weight (MW), with larger molecules being more effective. The monosaccharide glucose (MW 180) was applied in EC solution in the early stages but was soon abolished because it could pass through the cell membrane and become a source of lactate under hypoxic conditions 10 . The slightly larger monosaccharide mannitol (MW 182), which also has antioxidant potential, was used to replace glucose in HTK, Celsior, and HA-C solutions. Sucrose (MW 342) is a disaccharide and was used in phosphate-buffered sucrose (PBS) solution 172 . Raffinose (MW 504) was added to UW solution, along with lactobionate (Table 1). Colloids such as HES 18 , PEG 134,68,136,142 , and dextran 34,133 were also common components in preservation solutions, with convincing evidence of anti-edema function (see section Colloids for details).
Acidosis
Under ischemic/hypoxic conditions, energy metabolism in cells switches from fatty acid oxidation to more oxidation-efficient anaerobic glycolysis, allowing organs to sustain cellular viability 173 . Decreased intracellular pH and acidosis are the immediate results of anaerobic glycolysis, and are often observed in ischemic tissues. Mild acidosis has been suggested to favor cell survival by inhibiting activated proteases and phospholipases via the rate-limiting step in glycolysis. However, strong activation of proteases and phospholipases by severe acidosis could lead to protein and lipid breakdown, lysosomal damage, and eventual cell death 174 . Therefore, adequate control of cellular pH has become an important function of preservation solutions 175 . To achieve this goal, different buffering systems were applied. EC and UW solutions used phosphate as a buffer, while HTK and Celsior solutions used histidine (Table 1). Among these commonly used preservation solutions, HTK has the highest buffering capacity due to a high concentration of histidine 175 . The pH of common preservation solutions is between 7.0 and 7.4.
Mitochondrial Dysfunction
Mitochondrial dysfunction has been regarded as a critical event during ischemia. Under ischemic/hypoxic conditions, oxidative phosphorylation in the mitochondria is suppressed by a lack of oxygen, leading to impaired ATP synthesis and further severe ATP depletion 176 . There is experimental evidence that mitochondrial Ca2+ uptake was dramatically increased during cold ischemia, especially when it is challenged by high extra-mitochondrial Ca2+ concentrations 177 , resulting in impaired mitochondrial structure and function. Mitochondrial dysfunction may influence energy regeneration at the re-oxygenation stage 178 . Mitochondria are the major source of intracellular ROS production 176 . The uncoupling of the mitochondrial respiratory chain induces the formation of ROS, a process which is enhanced during the reperfusion stage. This could contribute to the denaturation of proteins, nucleic acids, and lipids, which induce cell apoptosis or necrosis 176 .
It is thus clear that maintaining mitochondrial integrity and protecting mitochondria function are key principles in developing preservation solutions. The supplementation of preservation solutions with mitochondrial-protective reagents H2S, MitoQ 120,121 , quinacrine 122 , and TMZ 111 –116 has proven to be effective (Table 2). AP39, a novel mitochondria-targeted H2S donor, can also stimulate cellular bioenergetics, and protect against the loss of mitochondrial DNA integrity 179 .
Oxidative Stress
The reperfusion stage, which occurs hours or days after the initial ischemic/hypoxic insult, is regarded as the final stage of ischemic injury 168 . It has a profound influence not only on the short-term but also the long-term recovery outcome of a transplanted kidney 168,180 . During this stage, blood flow and oxygen are re-introduced into organs, leading to a burst of ROS 181 . Hydrogen peroxide (H2O2) and the superoxide anion (O2–) are mainly generated by xanthine oxidase, and further lead to hydroxyl radical (OH–) formation. Meanwhile, cold storage itself has been suggested to promote ROS production via mitochondrial dysfunction 182,183 . ROS react rapidly with other molecules, leading to lipid peroxidation and oxidative damage of nucleic acids and proteins 180 , and eventually contribute to cell apoptosis 176 (Fig. 1).
Thus, inhibiting ROS production, especially at times of reperfusion, has become a key strategy to protect organs during transplantation. The beneficial effects of antioxidants and radical scavengers against ischemic/hypoxic injury have been confirmed by numerous studies. In UW solution, allopurinol (a xanthine oxidase inhibitor) and reduced glutathione (a thiol containing amino acid) were included to reduce ROS formation. Similarly, HTK solution contains tryptophan and histidine as ROS-scavenging amino acids (Table 1). Experimental additions of antioxidant agents into common preservation solutions have proven to be effective. Direct introduction of lecithinized superoxide dismutase (lec-SOD), a catalyzer of ROS degradation, into preservation solution demonstrated a long-term benefit of reducing oxidant stress
184
. H2S has also been studied for its anti-oxidative properties. The benefits were possibly related to lowering the generation of free radicals, inducing antioxidant gene expression and anti-apoptotic functions
82,185
. N-acetylcysteine contains a thiol group, which is readily accessible to the then intracellular compartment and could scavenge free radicals
123
. As a widely used anesthetic, propofol displays its antioxidant activity through reduced lipid peroxidation and increased SOD levels
127
. Moreover, several other compounds have also been added to preservation solutions, and were described to protect grafted kidneys against ischemic/hypoxic injury through direct or indirect anti-oxidative stress properties, including TMZ
186
, rh-BMP-7
98
,
Inflammation Process
The inflammatory response is another profound consequence of blood flow restoration and re-oxygenation in the reperfusion stage 171 . ROS generation and lipid peroxidation process seem to be major factors in the activation of innate immunity 166,187 . Inflammatory cells express NADPH oxidase, which in turn leads to the formation of additional ROS, further potentiating renal damage 188 (Fig. 1).
During reperfusion, activation of endothelial cells trigger the downstream signaling pathways, including mitogen-activated protein kinase (MAPK) and NF-κB, leading to the release of pro-inflammatory cytokines (e.g. IL-6 and TNF-α) and chemokines 166,189 . Chemokines are regarded as major mediators of inflammation that regulate adhesion molecules expression as well as leukocyte infiltration and activation 176 . Increased expression of adhesion molecules and Class II MHC proteins result in vasoconstriction, exacerbating tissue damage. Increased platelet activation and adhesion to the injured vascular endothelium also reduces blood flow and exacerbates ischemia 167,190 .
Multiple inflammatory pathways and factors take part in this process. Therefore, specific antibodies or pathway inhibitors have been applied in preservation solutions to modulate the inflammatory responses and attenuate post-ischemic injury. Direct inhibition of endothelial cell activation and vasoconstriction can be achieved by using endothelial receptor antagonists in preservation solutions 85 –88 . The pro-inflammatory effects of TNF-α were effectively blocked by application of TNF-receptor fusion protein (TNF-RFP) 99 . Administration of ICM-1 antisense deoxynucleotides ameliorated inflammatory injury 94 . Supplementation of UW solution with FR167653, a specific p38MAPK inhibitor, showed important benefits to graft function by reducing inflammation 94 . Administration of exogenous CO or CO-releasing molecules has cytoprotective effects involving anti-inflammatory response and hypoxia-inducible factor (HIF)-1α stabilization 191 . In addition, agents like H2S 156,76 , O2 83 , Ar 84 , melagatran 90,91 , cyclic helix B peptide 100 , TMZ 111 –116 , and lec-SOD 118 have also been implicated in preventing tissue inflammation during ischemia and reperfusion in various experimental models (Table 2 and Fig. 1).
Cell Apoptosis, Necrosis and Autophagy
Ischemic/hypoxic and subsequent reperfusion injury leads to the activation of cell death programs, which can be categorized as apoptosis, necrosis, and autophagy-associated cell death 192 . Apoptosis is a process of programmed cell death that is characterized by blebbing, cell shrinkage, loss of mitochondrial integrity, and nuclear fragmentation 193 . Cell apoptosis may be induced by mitochondrial dysfunction. Cytochrome c was released into the cytosol from damaged mitochondria to induce the formation of apoptosomes. Subsequent apoptotic nuclear DNA damage was induced by the release of apoptosis-inducing factor (AIF) and endonuclease G 168,194 . Ischemic/hypoxic mechanisms such as oxidative stress and inflammatory signaling may contribute to apoptosis as well. Thus, blocking these pathways to inhibit apoptosis may have promises as therapeutic strategies for ischemic/hypoxic renal injury during cold storage. In addition, apoptosis can be blocked by direct target apoptosis effectors. The glucocorticoid, dexamethasone, has an inhibitory effect against apoptosis by upregulating Bcl-XL, inhibiting Bax production, and activating caspases-3/9 in renal ischemia-reperfusion injury rodent models 195 . Administration of naked caspase-3 siRNA in a preservation solution resulted in reduced caspase-3 expression level and cell apoptosis, elucidating the protective effect in isolated ischemic porcine kidneys 102 .
Necrosis, characterized by progressive cell swelling, plasma membrane rupture, and leakage of proteases and lysosomes into the extracellular compartment, is a frequent outcome of ischemic/hypoxic renal injury during cold storage 192 . Strong innate inflammatory signaling leads to cell necrosis, while necrosis in turn promotes inflammation through leakage of cellular contents, inflammatory cell infiltration, and cytokine production 196 .
Unlike necrosis, autophagy is a regulated, programmed process that disassembles unnecessary or dysfunctional components of the cell 192 . Autophagy-associated cell death is also a common outcome after renal cells are exposed to ischemic/hypoxic conditions. However, whether renal cells undergo necrosis or programmed cell death appears to be ATP-concentration dependent. Studies have shown that ATP is required for programmed cell death, whereas complete ATP depletion would lead to necrosis 186,197 . Experimentally, the effectiveness of pharmacological additives that may reduce cell swelling or ATP depletion on preventing necrosis have been recently illustrated (Table 2). AMP-activated protein kinase (AMPK) is an energy sensor that regulates cellular metabolism and homeostasis. Renal ischemia was reported to induce AMPK phosphorylation, and sequentially promotes autophagy through phosphorylation of downstream effectors. Treatment of rat or mouse models with AMPK activator attenuates renal ischemia/reperfusion injury, with decreased tubular necrosis and expression of apoptosis-related proteins, improved glomerular filtration rate, and increased expression of autophagy-related proteins 198,199 .
Conclusion and Perspectives
There has been major progression in developing preservation solutions for kidney transplantation in the past decades. To date, thousands of kidney transplants have been successfully performed using conventional preservation solutions. The main principle of organ preservation solution is to maintain the organ viability to the greatest extent during storage to achieve better transplant outcomes after the reperfusion stage 200 . The principal ingredients of preservation solutions, colloids, buffers, nutrients, and antioxidants are responsible for counteracting cellular edema, acidosis, ATP depletion, and the production of ROS, respectively. With a better understanding of the mechanisms of ischemic/hypoxic renal injury, it is now well recognized that supplementation of preservation solutions with pharmacological molecules has the great potential to improve graft quality. Pharmacological additives that specifically target ROS, inflammation, and apoptosis signaling pathways have proven to be effective.
UW, HTK, and Celsior solutions displayed a much lower risk of DGF after kidney transplantation compared with traditional EC solutions. The incidence of DGF is equal among these three solutions according to randomized clinical trials 9 . Thus, these well-established and commonly used solutions account for the majority of preservation solution used in the clinic. Meanwhile, numerous new preservation solutions with supplemental pharmacological agents have shown promising effects in ameliorating ischemic/hypoxic renal injury and DGF in animal studies, but clinical evidence is still lacking. Challenges remain in their translation from the bench to the bedside, including the specific dosage of additives, method of drug delivery, and potential off-target effects.
Since ischemic/hypoxic renal injury involves multiple mechanisms, targeting a single molecule or a single signaling pathway is unlikely to provide a complete amelioration of ischemic/hypoxic renal injury during cold storage. Instead, multi-drug approaches or a single drug targeting multiple molecules and/or pathways will be a more reliable strategy for better preservation of renal graft. The utilization of multi-drugs may activate protective signaling pathways and inhibit damaging ones at the same time. Indeed, several studies have explored the combination effects of compounds, each targeted against a specific pathway, and have demonstrated additivity of the approaches 94,123 . It still remains urgent to gain additional mechanistic insights into the molecular events and the underlying mechanisms related to ischemic/hypoxic renal injury during cold storage. The future development of new preservation solutions would be accelerated by the modern drug industry, with the systematic analyzing of dysregulated genes during ischemic/hypoxic damage and the screening of new drugs. Despite the challenges ahead, we are hopeful that new preservation solutions for kidney transplantation against ischemic/hypoxic renal injury will soon be applied for clinical practice through joint efforts of worldwide basic and clinical researchers, including our research team.
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: YX was supported by Science and Technology Commission of Shanghai Municipality (18401970100) and the National Natural Science Foundation of China (81873361).
