Renal Allograft Preservation

Mitchell L. Henry

Ronald P. Pelletier

Division of Transplantation, Department of Surgery, The Ohio State University Medical Center, Columbus, Ohio 43210

HISTORICAL PERSPECTIVE

Living-related renal transplantation became an accepted clinical practice by the late 1960s. Attention subsequently was directed toward utilization of kidneys from cadaveric donors in an effort to meet the demand for organs that could not be met with the available pool of living donor organs. The definition of “brain death” in the United States in the late 1960s paved the way for the acquisition of kidneys from heart-beating cadaveric donors to be used for transplantation. Utilization of these cadaveric organs necessitated a period of preservation prior to engraftment. Thus, during this same time period, efforts were underway to design preservation methods to make the use of cadaveric organs feasible. In 1968, Belzer et al (1) reported a successful human kidney transplant following preservation with a hypothermic, cryoprecipitated-plasma perfusion method refined in the laboratory. Initially, the chilled preservation fluid used to flush the blood out of the preserved organ was designed to mimic extracellular fluid. However, Collins et al (2) reported in 1969 superior preservation using crystalloid solutions designed to mimic intracellular fluid. This and other such solutions contain high concentrations of phosphate and potassium. With the availability of these cheap, effective preservation fluids, the volume of kidney transplants in the United States dramatically increased. These events influenced the volume of renal transplantation worldwide, and efforts continued toward developing even better preservation fluids. For instance, EuroCollins’ solution was developed in Europe (3). This solution lacked magnesium that would interact with phosphate, forming undesirable insoluble crystals. Citrate solutions, such as Ross and Marshall hypertonic citrate, were subsequently developed that replaced phosphate with citrate and glucose with mannitol (4). The citrate acts as a buffer and chelates the magnesium, creating a cell membrane impermeable product. The mannitol is much less permeable than glucose, which can enter cells and promote anaerobic glycolosis leading to tissue acidosis.

In the late 1970s, transplantation of solid organs other than the kidney was becoming accepted due to the improved outcomes that resulted from the use of improved immunosuppressive drugs. Laboratory efforts to design preservation solutions better suited for nonkidney solid organs led to the clinical development of the University of Wisconsin (UW) solution (5). This solution contained impermeable solutes, buffering agents, colloids, and electrolytes in addition to other helpful adjuvants such as allopurinol, adenosine, and glutathione. The excellent clinical results following transplantation of nonrenal solid organs preserved using UW solution established this as the preferred preservation solution for use in multiple-organ cadaveric donors.

GOALS OF PRESERVATION

The objectives of organ preservation are to minimize cellular metabolic processes, thus reducing the rate of energy utilization leading to a requisite reduction in energy production. Uninterrupted metabolic cellular activity in the setting of oxygen and nutrient depravation as occurs with ischemia results in irreversible cell damage and death via a
cascade of cellular events. Additionally, accumulation of metabolic end-products as a result of ischemia-induced anaerobic metabolism results in reperfusion injury when blood flow is restored. Reaction of the oxygen delivered to the reperfused organ with the end-products of anaerobic metabolism leads to the formation of intracellular toxic compounds such as hydrogen peroxide, superoxide, and hydroxyl radicals that exacerbate ischemia-induced cellular, membrane, and microvascular (endothelial) injury. Preservation solutions represent the cornerstone of the overall preservation process of solid organs, the goal of which is to minimize the ischemia/reperfusion injury inherent in the transplant procedure.

COMPONENTS OF PRESERVATION

Hypothermia is one of the essential concepts of organ preservation. Reducing the temperature of an organ dramatically decreases the rate of cellular metabolism. However, some biologic cellular functions are not as significantly affected by cooling as others. For instance, ion transmembrane passive diffusion is not appreciably affected whereas active, energy-dependent, ion transmembrane transport mechanisms are inhibited below 10°C (6). An unfortunate result of this differential effect on transmembrane ion transport is that permeable substances equilibrate across the plasma membranes, leading to cellular swelling and injury. Preservation solutions have been designed to inhibit cell swelling by the addition of impermeants of various types. Although tissue oxygen consumption decreases dramatically with hypothermia (5% of normal at 5°C [7]), low-level metabolism persists. Thus, even at low temperatures the accumulation of damaging metabolic end-products will eventually occur. Some preservation solutions contain additives that inhibit metabolism either during hypothermia or upon reperfusion. Finally, preservation solutions are designed to control the pH of the organ extracellular fluid. An end result of ongoing low-level metabolism during hypothermia is the accumulation of waste products, such as lactic acid, that lower the pH. Adding buffers to the preservation solution counteracts the adverse effects of an acidotic local cellular environment.

COMPONENTS OF SOLUTIONS

Inhibitors of Cell Swelling

Preservation solutions inhibit cell swelling by including impermeants that increase the extracellular oncotic force. The increased extracellular oncotic force counteracts the normally higher intracellular oncotic force that, in the setting of hypothermia-caused absence of energy-dependent transmembrane transport, would drive water into the cells. Impermeants that have been used include saccharides (glucose, sucrose, mannitol, and raffinose), anions (lactobionate, phosphate, citrate, sulphate, and gluconate), and colloids (dextrans, hydroxyethyl starch, and polyethylene glycols).

Electrolytes

Initially, preservation solutions were created that contained a sodium concentration approaching that found in extracellular fluid. However, it was quickly realized that solutions with a higher potassium and lower sodium concentration were better at preventing the loss of cellular potassium. Smaller amounts of magnesium and/or calcium are also included in many preservation solutions. Magnesium has been eliminated from a number of preservation solutions because of the development of undesirable precipitates.

Metabolic Inhibitors

Additives to preservation solutions meant to interfere with hypothermic metabolism during preservation include adenosine, allopurinol, glutathione, dexamethasone, glycine, histidine, chlorpromazine, trifluoperazine, calcium channel blockers, phospholipase inhibitors, and prostanoids. The clinical efficacy of these various metabolic inhibitors remains controversial. For these agents to be effective, they must attain an effective intracellular concentration. Whether many of these compounds reach the intracellular milieu in the hypothermic environment is not generally known. Additionally, many of these chemicals are meant to counteract the generation of toxic molecules following reperfusion, a time when the preservation solution will already have been flushed from the organ.

Buffers

Phosphate is the most commonly used preservation solution buffer. However, histidine, included in some solutions, may also provide some buffering capacity.

Belzer’s Early Solution

Belzer’s solution is a 305 mOsm solution that mimicked extracellular fluids in regard to the electrolyte content, containing 140 mmol/L sodium and 10 mmol/L potassium (Table 6.1). Magnesium was also present in this solution. HEPES, a biological buffer, was used as the buffer. Glutathione and dexamethasone were added as inhibitors of metabolic end-products. Small amounts of glucose were also added to support the low level of ongoing glucose utilization during hypothermia.

The cryoprecipitated plasma initially used in the Belzer’s solution as the cellular swelling inhibitor was difficult and time-consuming to prepare. Additionally, the low-density lipoproteins (LDLs) present in plasma have a propensity to precipitate at low temperatures and with changes in pH and ionic strength. Thus, microfiltration and avoidance of exposure to gas (also promotes LDL precipitation) were avoided. These manipulations made the preparation of this solution both complex and costly. Substitution of human serum albumin for cryoprecipitated plasma (8,9) or the use of silica gel treatment (10,11) eliminates lipoproteins as well as fibrinogen
and other entities. These maneuvers obviated the need for microfiltration and provided a stable perfusate of predictable composition.

TABLE 6.1. Composition of Belzer’s early solution

Component	Concentration
Gluconic acid Na salt	80 mmol/L
Gluconic acid Mg salt	5 mmol/L
K₂PO₄	10 mmol/L
Glucose	10 mmol/L
Glutathione	3 mmol/L
HEPES	20 mmol/L
Albumin	37.5 gm/L
Penicillin	6×10⁵ U/L
Phenosulphothalien	12 mg/L
Dexamethasone	12 mg/L
NaCl	10-20 mmol/L
NaOH	Adjusted to pH 7.5
Na, sodium; Mg, magnesium; K, potassium; P, phosphorus; O, oxygen; Cl, chlorine; H, hydrogen.

Collins’ Solutions

Geoff Collins developed a preservation solution simpler than the then available Belzer solution (12). Collins’ solution employs glucose rather than cryoprecipitated plasma or human serum albumin for inhibition of cellular swelling, making it simpler and less expensive to prepare (Table 6.2). This solution mimicked intracellular fluids with high potassium and low sodium concentrations. Magnesium, sulphate, phosphate, and glucose were included in high concentrations. The use of this solution with its “intracellular” electrolyte composition extended the allowable cold preservation time for kidneys (12, 13, 14, 15). Magnesium was excluded from the extensively used EuroCollins’ solution because of problems with forming precipitates with phosphate. EuroCollins’ solution contains higher concentrations of glucose than Collins’ C2 solution. In the hypothermic setting, glucose is slowly permeable across cell cytoplasmic membranes, thus fueling anaerobic glycolysis and promoting development of tissue lactic acidosis. Replacement of glucose with either sucrose or mannitol, which are much less permeable and are not metabolized, has been shown to improve outcomes after renal preservation in animals (16,17) and humans (18).

TABLE 6.2. Composition of Collins’ solutions

Component	Collins’ (C2)	EuroCollins’	EuroCollins’ with sucrose	EuroCollins’ with mannitol
Na (mmol/L)	10	10	10	10
K (mmol/L)	115	110	110	110
Mg (mmol/L)	30	—	—	—
Cl (mmol/L)	15	15	15	15
HCO₃ (mmol/L)	10	10	10	10
SO₄ (mmol/L)	30	—	—	—
KH₂PO₄ (mmol/L)	15	15	15	15
K₂HPO₄ (mmol/L)	42.5	42.5	42.5	42.5
Glucose (mmol/L)	140	180	—	—
Sucrose (mmol/L)	—	—	180	—
Mannitol (mmol/L)	—	—	—	180
Na, sodium; K, potassium; Mg, magnesium; Cl, chlorine; H, hydrogen; C, carbon; O, oxygen; S, sulfur; P, phosphorus.

Citrate Solutions

Subsequent to the development of Collins’ and EuroCollins’ solutions was the development of citrate solutions (Table 6.3). These solutions, like Collins’ solution, have a high potassium and magnesium concentration. However, citrate is used in place of phosphate, and glucose is abandoned in favor of mannitol. The citrate acts as a buffer and chelates magnesium, making it impermeable to cell cytoplasmic membranes. The benefit of replacing glucose with mannitol has previously been discussed. Preservation with either the hypertonic or the isotonic citrate solutions resulted in transplant outcomes similar to that obtained with Collins’ solutions (4,19).

Sucrose Solutions

Evidence for improved efficacy of a solution that substituted sucrose for glucose as the cellular swelling inhibitor in a EuroCollins’ type solution promoted the production of sucrose-based solutions (16) (Table 6.4). These simple solutions use phosphate for buffering capacity and mimic extracellular fluid with a high sodium concentration and absence of potassium. Reported outcomes using this solution for preservation of animal and human kidneys have been excellent (20, 21, 22, 23).

Bretschneider’s HTK Solution

This solution, while originally created for cardioplegia, was successfully employed clinically as a preservation solution (24) (Table 6.5). Histidine-tryptophan-ketoglutarate (HTK), and mannitol are largely impermeable molecules that act as inhibitors of cellular swelling. Histidine acts as
both a buffer and an inhibitor of toxic metabolic products (free-radical scavenger). One clinical study found this solution superior to EuroCollins’ solution in kidney transplantation (25).

TABLE 6.3. Composition of citrate solutions

Component	Hypertonic	Isotonic
Na (mmol/L)	78	78
K (mmol/L)	84	84
Mg (mmol/L)	40	40
Citrate (mmol/L)	54	54
SO₄ (mmol/L)	40	40
Mannitol (mmol/kg)	200	100
Osmolality (mmol/kg)	400	300
Na, sodium; K, potassium; Mg, magnesium; S, sulfur; O, oxygen.