Optimization of a vehicle solution for the introduction and removal of glycerol with rabbit kidneys

Previous studies with rabbit kidneys in our laboratories have used a plasma-like solution as the vehicle for the introduction and removal of glycerol. Other workers have usually employed high-potassium solutions. In this study we have assayed the function of rabbit renal cortical slices after incubation in a range of solutions, each of which contained 1 M glycerol, for 4 hr, followed by stepwise removal of the cryoprotectant. The functions measured were endogenous oxygen consumption, p-aminohippurate uptake, and the ability of the slices to accumulate potassium. Exposure to glycerol produced a considerable reduction of slice function, but, in the presence of glycerol, elevation of the potassium concentration was beneficial, whereas high concentrations of magnesium were detrimental. The optimum potassium concentration was 70-100 mM. Replacement of chloride by a range of anions of higher molecular weight was either without benefit (glycerophosphate) or detrimental (sulfate, citrate, and gluconate). Elevation of total osmolality from 300 to 400 mosmolal with glucose, mannitol, glycerophosphate, or Pipes reduced slice function, but when the same osmolality was achieved by raising the concentration of all the components of the solution in the same ratio, there was no significant loss of function. There was a weak optimum pH at ca. 7.0. These experiments led to the formulation of a bicarbonate-buffered perfusate containing 80 mM potassium and 17.5 g Haemaccel per liter, having a pH of 7.0 with 5% CO2 at 10 degrees C, and an osmolality of 400 mosmol/kg. This solution was used to preserve rabbit kidneys for 20 hr at 10 degrees C, by continuous perfusion, and was compared with our previous Haemaccel perfusate, HP5, which contained 4 mM K+, 111 mM mannitol, and had a pH of 7.4. The two solutions were equally effective.     

Distribution and removal of glycerol by vascular albumin perfusion in rabbit kidneys.

A simple perfusion circuit for gradual glycerolization and deglycerolization of rabbit kidneys is described; it has been used to study vascular resistance and glycerol distribution during perfusion at +10 °C with a perfusate of extracellular composition.An attempt was made to diminish the dramatic rise in vascular resistance during deglycerolization of rabbit kidneys, seen in previous experiments, by increasing the perfusate colloid osmotic pressure and decreasing the rate of change of cryoprotectant concentration during the last third of removal. These modifications of the method did not improve perfusion characteristics or post-transplant function.Melting points of perfusates and different parts of the kidney were determined by differential thermal analysis before glycerolization, after glycerolization to 3 m and after subsequent deglycerolization. The variations of tissue melting temperatures were found to be identical with variations in the perfusate, thus indicating a complete distribution and removal of cryoprotective concentrations of glycerol.     

Flow distribution and cryoprotectant concentration in rabbit kidneys perfused with glycerol solutions

Rabbit kidneys were perfused at 10 °C with a solution containing gelatin polypeptides (Haemaccel), and glycerol was introduced, and then removed, using a technique that has previously been shown to result in viable kidneys. This involved increasing the concentration of glycerol in the perfusate from zero to a maximum of 3 , holding it at this level for 30 min, and then decreasing it at the same rate to < 0.1 . Measurements were made of the concentration of glycerol in cortex, cortico-medullary zone, and medulla at various stages of perfusion. During the experiments it was observed that vascular resistance increased dramatically toward the end of deglycerolization, and changes in regional perfusate flow were measured by the diffusable indicator method. It was found that renal tissue is effectively permeated by glycerol using this technique. The perfusate flow throughout all regions of the kidney was reduced during deglycerolization but the greatest effect was on cortico-medullary flow, which was found to be abnormally high during the initial stages of hypothermic perfusion, but was severely impaired when the glycerol was removed. The cryoprotectant was almost completely removed by the washout procedure adopted.     

Cooling of rabbit kidneys permeated with glycerol to sub-zero temperatures.

The aims of this study were to investigate if kidney preservation could be enhanced by cooling of the organs to high sub-zero temperatures after depression of their freezing points by addition of glycerol, and to study whether the added amounts of this compound would confer protection to the organs during freezing and thawing at slow rates.Glycerol was added and removed gradually by continuous, hypothermic perfusion, and the post-preservation viability was assessed by autotransplantation.Brief cooling to ?5 °C of kidneys perfused with 3 m glycerol was found to be compatible with life-sustaining posttransplant function, whereas no kidneys stored at that temperature for 5 days survived.Slow cooling af kidneys glycerolized to 3 m to ?80 °C was associated with a marked increase in vascular resistance after thawing, and none of such frozen kidneys functioned after transplantation. They showed immediately after revascularization severe impairment of the circulation, and vascular damage was observed by light microscopy. The use of 5 m glycerol for cryoprotection attenuated this rise in vascular resistance and reduced the release of the endocellular enzyme, lactate dehydrogenase after thawing, indicating less cellular damage although no kidneys functioned after grafting.It is suggested that the mechanical effect of interstitial and intravascular ice formation is a major factor in damage to intact organs during freezing, and that further injury is produced by incomplete removal of the cryoprotectant before transplantation.     

Transplantation of rabbit kidneys perfused with glycerol solutions at 10 degrees C.

Experiments are described in which rabbit kidneys were perfused with solutions containing 2, 3, or 4 m glycerol and then were autografted in order to measure any functional impairment. The basic perfusate contained Haemaccel (17.5 g/litre) and mannitol (111 mm) and had a total osmolality of 400 mosmol/kg. The perfusion temperature was 10 °C. Glycerol concentration was slowly increased at a rate of 30 mm min^?1 to 2, 3, or 4 m, was held at the maximum concentration for 30 min, and was then decreased to < 0.1 m at 30 mm min^?1. An immediate contralateral nephrectomy was carried out, and the function of the perfused kidney was assessed by serial measurements of blood urea and serum creatinine, by the determination of 24-hr endogenous creatinine clearance at 1 month and by histological examination. It was found that a concentration of 3 m glycerol was readily tolerated.     

Introduction and removal of cryoprotective agents with rabbit kidneys: assessment by transplantation

Rabbit kidneys were perfused with up to 4 M glycerol or propane-1,2-diol (propylene glycol, PG) in three vehicle solutions: one normokalemic and made hypertonic with mannitol (HP5), one hyperkalemic but without mannitol (HP6), and one hyperkalemic and with mannitol (HP7). Subsequent function was assessed by autotransplantation. Up to 3 M glycerol in HP5 was well tolerated but not in HP6 or HP7. Conversely, up to 3 M PG in HP7 was compatible with excellent post-transplant function, but the same concentration in HP5 was severely damaging. PG (4 M) in either solution was severely injurious and no kidneys survived perfusion with this concentration. Vascular resistance was well controlled by the vehicle solutions with mannitol, but it was generally higher during perfusion with the hyperkalemic HP7 compared with the normokalemic HP5. No kidneys perfused with 3 M solutions of either of the cryoprotective agents and cooled briefly to -6 degrees C without freezing had any post-transplant function, and neither did kidneys perfused with 3 M PG or 4 M glycerol tolerate slow cooling to -80 degrees C and warming. The need to optimize perfusate composition for the CPA being used is clear, and the dramatic increase in toxicity of PG when the concentration exceeds 3 M supports the suggestion that mixtures of PG and glycerol should be considered. The observation of damage at high subzero temperatures, before freezing has occurred, requires further detailed study.     

The distribution and metabolism of glycerol in the rabbit.

1. The effective volume of distribution of labelled glycerol was studied in conscious young adult rabbits provided with in-dwelling cannulae in the femoral blood vessels. This could be estimated after sampling arterial blood throughout an intravenous infusion of [2-3H]glycerol. The volume was calculated by using an algebraic method of graphical area analysis over 100 min of equilibration, and is symbolized 100V e or 100V e%. It occupied 34.1 +/- 2.2% (mean +/- S.E.M.; n = 13) of the body weight. The pool of endogenous glycerol occupying this space is distinguished in the present paper by calling it the transit pool, symbolized 100Me. 2. The median time of transit of glycerol through this pool was approx. 6 min in these conscious rabbits with normal (less than 0.2 mM) blood glycerol concentrations. 3. The metabolism of glycerol was also studied in rabbits while anaesthetized with urethane or while conscious. On average, half of the change in glycerol concentration that occurred on overnight starvation could be attributed to a decrease in clearance, whereas half was due to an increase in lipolysis. 4. The correlation between the reciprocal of glycerol concentration and clearance showed that in these animals about a quarter of the variation in concentration was due to an association with clearance. The remainder of the variation was attributed to variations in the rate of glycerol formation (lipolysis). 5. The regression of glycerol turnover rate on concentration implied that turnover was positive at zero glycerol concentration. This confirms previous findings from studies on other species. The explanation offered for this phenomenon is that the well-known physiological changes induced by feeding (decreased lipolysis, increased splanchnic blood flow) may independently decrease the glycerol concentration by both decreasing its release into the blood and simultaneously increasing its clearance.     

Acute renal insufficiency in the rabbit by glycerol.

The appearance of an acute renal insufficiency in the rabbit, after glycerol injection (10, 13 or 15 ml/kg of a 50% solution) is investigated. After a 24 hours of intoxication, especially in the ten following days, cylinders, erythrocytes and renal cells appear in the urine sediment. Proteinuria appears after 24 hours and practically disappears after 72 h. Glucosuria persists from 24 hours to 6 days. Haemoglobinuria is intense after 24 and 48 hours and persists slightly about 6 days. Na, K and Cl elimination in urine diminishes clearly in all animals. Plasma K increases in non-surviving animals and does not change in those surviving. Plasma Na does not change in the dying ones, and decreases in those surviving. In non-surviving animals, pH, pCO2 and CO3H minus decrease sharply. In the surviving ones pCO2 decreases clearly after 24 hours, increasing afterwards slowly to normal values. pH increases, slightly during the first 48 hours, and then neatly during approximately 6 days. Standard CO2H minus does not change during the first 48 hours, increasing afterwards during 6 to 7 days. Histologically, the chief lesion is a vacuolar degeneration of the proximal tubule. The possible mechanisms of such alterations are discussed.     

Hypothermic preservation of rabbit kidneys for 48 hours using low ionic strength solutions

Microwaves offer the prospect of rapid and uniform heating of frozen organs. This is significant in the context of cryopreservation, and particularly of vitrification, because microwave heating may help to avoid crystallization or recrystallization of ice during warming, minimize any effects of high cell density, and reduce thermal-mechanical stresses. Previous work has established a rationale for reducing the ionic strength of solutions used to prepare tissues for microwave heating, since this permits the use of lower frequencies, which makes heating more uniform, without increasing the risk of thermal runaway (T. P. Marsland, S. Evans, and D. E. Pegg, Cryobiology 24, 311-323, 1987). In this paper we report a study of two possible low ionic strength perfusates, in rabbit kidneys, using 48 hr of hypothermic storage and autotransplantation as the test system. This model was chosen because there is a great deal of basic information about it. Both a single-pass "flush" preservation solution and a solution designed for continuous perfusion gave excellent results. The continuous perfusion system, which would be the more suitable for introducing cryoprotectants, gave five of five surviving animals with peak serum creatinine levels of 353-555 mumol/liter normal histology in three cases, and only very minor damage in the other two. There would therefore seem to be no obstacle to the use of perfusates having a low ionic strength in renal cryopreservations studies.     

Stabilization of flavobacterium meningosepticum glycerol kinase by introduction of a hydrogen bond

The thermostability of Flavobacterium meningosepticum glycerol kinase was increased by the change from Ser329 to Asp [Protein Eng., 14, 663-667 (2001)]. Based on a three-dimensional structure model of the mutant, we have postulated that a new charged-neutral hydrogen bond was formed between Asp329 and Ser414, and the formation of the hydrogen bond contributed to the stabilization of the tertiary structure and increased thermostability of the mutant enzyme. If the postulation is the case, FGK thermostabilization would be possible similarly by the single amino acid substitution from Ser414 to another amino acid which could form the hydrogen bond with Ser329. We did a single amino acid substitution of the wild-type enzyme from Ser414 to Asn. As we expected, S414N showed comparable thermostability to that of S329D. On the other hand, a difference in kinetic properties for ATP between S414N and S329D was observed.     

Atherosclerosis decreased prostacyclin formation in rabbit lungs and kidneys

Metabolism of arachidonic acid (AA) was studied in perfused lungs and kidneys of normal and atherosclerotic rabbits by determination of PGE₂, PGF_2α and the stable metabolites of PGI₂ (6-keto-PGF_1α) and TXA₂ (TXB₂). PGI₂ was the main AA metabolite formed by normal lungs and kidneys. Atherosclerosis reduced the formation of PGI₂ by about 50 % in both organs. TXA₂ formation was similarily decreased in lungs. In kidneys, the decrease in PGI₂ formation was accompanied by an increase in PGE₂ formation.     

Development of a rabbit pleural cancer model by using VX2 tumors

Primary and secondary pleural cancer remains an important clinical problem, with research progress limited by the lack of a suitable moderate- to large-sized (3 to 4 kg) animal model of pleural cancer. Many potential pleura-based imaging and treatment modalities cannot be investigated sufficiently by using currently available small murine animal models because their pleural space is not comparable to that of humans and therefore does not allow for the use of standard thoracoscopic techniques. Here we describe the development of a reproducible model of pleural malignancy in moderate-sized immunocompetent rabbits. Under thoracoscopic guidance, 9-15 x 10(6) VX2 carcinoma cells were inoculated into the plural space of 3 to 4 kg New Zealand white rabbits that had undergone gentle pleural abrasion. Malignant tumor involvement developed on the visceral and parietal pleural surfaces in an average of 2 to 4 wk. This novel pleural tumor model induction method likely will facilitate a broad range of investigations of pleural cancer diagnostics and therapeutics.     

Voltage-dependent Ca2+ fluxes in skeletal myotubes determined using a removal model analysis

The purpose of this study was to quantify the Ca2+ fluxes underlying Ca2+ transients and their voltage dependence in myotubes by using the "removal model fit" approach. Myotubes obtained from the mouse C2C12 muscle cell line were voltage-clamped and loaded with a solution containing the fluorescent indicator dye fura-2 (200 microM) and a high concentration of EGTA (15 mM). Ca2+ inward currents and intracellular ratiometric fluorescence transients were recorded in parallel. The decaying phases of Ca2+-dependent fluorescence signals after repolarization were fitted by theoretical curves obtained from a model that included the indicator dye, a slow Ca2+ buffer (to represent EGTA), and a sequestration mechanism as Ca2+ removal components. For each cell, the rate constants of slow buffer and transport and the off rate constant of fura-2 were determined in the fit. The resulting characterization of the removal properties was used to extract the Ca2+ input fluxes from the measured Ca2+ transients during depolarizing pulses. In most experiments, intracellular Ca2+ release dominated the Ca2+ input flux. In these experiments, the Ca2+ flux was characterized by an initial peak followed by a lower tonic phase. The voltage dependence of peak and tonic phase could be described by sigmoidal curves that reached half-maximal activation at -16 and -20 mV, respectively, compared with -2 mV for the activation of Ca2+ conductance. The ratio of the peak to tonic phase (flux ratio) showed a gradual increase with voltage as in rat muscle fibers indicating the similarity to EC coupling in mature mammalian muscle. In a subgroup of myotubes exhibiting small fluorescence signals and in cells treated with 30 microM of the SERCA pump inhibitor cyclopiazonic acid (CPA) and 10 mM caffeine, the calculated Ca2+ input flux closely resembled the L-type Ca2+ current, consistent with the absence of SR Ca2+ release under these conditions and in support of a valid determination of the time course of myoplasmic Ca2+ input flux based on the optical indicator measurements.