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.     

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.     

Perfusion of rabbit kidneys with glycerol solutions at 5 °C

The long-term preservation of whole organs will almost certainly require the use of subzero temperatures and cryoprotectants. An essential part of such a technique is the ability to add a cryoprotectant in adequate concentration and subsequently to remove it without damage to the organ. In this study rabbit kidneys have been perfused with solutions containing 3% dextran and 2 m glycerol at 5 °C, and their function has been measured after removal of the glycerol. The assay technique involved the measurement of glomerular filtration rate, protein leakage, and tubular reabsorption of sodium and glucose. The results indicate that the inclusion in the perfusate of an impermeant solute (mannitol) and limitation of the rate of change of glycerol concentration (to 30 mm min^?1) permits rabbit kidneys to retain a degree of function similar to that found in perfused control kidneys, although somewhat reduced in comparison with freshly isolated kidneys.     

Improved vitrification solutions based on the predictability of vitrification solution toxicity

Long-term preservation of complex engineered tissues and organs at cryogenic temperatures in the absence of ice has been prevented to date by the difficulty of discovering combinations of cryoprotectants that are both sufficiently non-toxic and sufficiently stable to allow viability to be maintained and ice formation to be avoided during slow cooling to the glass transition temperature and subsequent slow rewarming. A new theory of the origin of non-specific cryoprotectant toxicity was shown to account, in a rabbit renal cortical slice model, for the toxicities of 20 vitrification solutions and to permit the design of new solutions that are dramatically less toxic than previously known solutions for diverse biological systems. Unfertilized mouse ova vitrified with one of the new solutions were successfully fertilized and regained 80% of the absolute control (untreated) rate of development to blastocysts, whereas ova vitrified in VSDP, the best previous solution, developed to blastocysts at a rate only 30% of that of controls. Whole rabbit kidneys perfused at -3 degrees C with another new solution at a concentration of cryoprotectant (8.4M) that was previously 100% lethal at this temperature exhibited no damage after transplantation and immediate contralateral nephrectomy. It appears that cryoprotectant solutions that are composed to be at the minimum concentrations needed for vitrification at moderate cooling rates are toxic in direct proportion to the average strength of water hydrogen bonding by the polar groups on the permeating cryoprotectants in the solution. Vitrification solutions that are based on minimal perturbation of intracellular water appear to be superior and provide new hope that the successful vitrification of natural organs as well as tissue engineered or clonally produced organ and tissue replacements can be achieved.     

Factors influencing renal cryopreservation. II. Toxic effects of three cryoprotectants in combination with three vehicle solutions in nonfrozen rabbit cortical slices

The [K+]/[Na+] ratio of rabbit renal cortical slices was used to examine, at 25 degrees C, the effects on viability of three cryoprotectant agents (CPA) (dimethyl sulfoxide (Me2SO), ethylene glycol, and glycerol) in combination with three vehicle solutions (Krebs-Henseleit (K-H), solution A, and RPS-2). Viability assessment by [K+]/[Na+] for all test solutions was made after incubating the slices in modified Cross-Taggart solution (C-T). With K-H and solution A, all concentrations of ethylene glycol and glycerol resulted in lowered ratios, whereas with Me2SO, concentrations greater than 1.4 M are required to reduce [K+]/[Na+]. With RPS-2 no decrease in the ratios was found until concentrations greater than 2.8 M were reached for all three CPAs. Binding of Me2SO to albumin, studied using [14C]Me2SO, was inhibited by RPS-2 when compared to K-H. Introduction and removal of Me2SO at 10 degrees C allowed an improvement in viability, at higher Me2SO concentrations, as compared to 25 degrees C.     

Comparison of the effects of adenine-ribose with adenosine for maintenance of ATP concentrations in 5-day hypothermically perfused dog kidneys

The quality of preservation of kidneys is dependent upon a number of factors, one of which may be the concentration of adenine nucleotides in the tissue during long-term perfusion preservation. In this study we have investigated how adenine (5 mM) and ribose (5 mM) in combination affect the concentration of adenine nucleotides in dog kidney cortical tissue after 5 days of continuous hypothermic perfusion preservation. These results were compared to kidneys perfused with adenosine and without any added purine precursors of adenine nucleotide synthesis. Additionally, we investigated how these conditions affected renal tissue slice function after 5 days of preservation and how adenine plus ribose affected renal function after autotransplantation in the dog. Adenosine is nearly completely degraded during 5 days of perfusion but there was little loss of adenine (10%). The adenosine triphosphate concentration in kidney cortical tissue was higher in adenine/ribose-perfused kidneys (1.41 +/- 0.19 mumol/g) than in adenosine-perfused kidneys (0.71 +/- 0.1 mumol/g) after 5 days of preservation. Tissue slices prepared from kidneys preserved in the presence of adenine plus ribose were metabolically more functional (slice volume control and electrolyte pump activity) than slices from adenosine-perfused kidneys. Adenine plus ribose had no detrimental effects on kidneys preserved for 3 days as tested in the autotransplant model but did not yield successful 5-day preservation. Because of some potentially detrimental factors in using adenosine as an adenine nucleotide synthesis precursor, we have now switched to the combination of adenine and ribose for perfusion preservation of kidneys both in the laboratory and in the clinic.     

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.     

Cryopreservation of organs by vitrification: perspectives and recent advances

The cryopreservation of organs became an active area of research in the 1950s as a result of the rediscovery of the cryoprotective properties of glycerol by Polge, Smith, and Parkes in 1949. Over the ensuing four decades of research in this area, the advantages of vitrification, or ice-free cryopreservation, have become apparent. To date, experimental attempts to apply vitrification methods to vascularized whole organs have been confined almost entirely to the rabbit kidney. Using techniques available as of 1997, it was possible to vitrify blood vessels and smaller systems with reasonable success, but not whole organs. Beginning in 1998, a series of novel advances involving the control of cryoprotectant toxicity, nucleation, crystal growth, and chilling injury began to provide the tools needed to achieve success. Based on these new findings, we were first able to show that an 8.4M solution (VMP) designed to prevent chilling injury at -22 degrees C was entirely non-toxic to rabbit kidneys when perfused at -3 degrees C and permitted perfusion-cooling to -22 degrees C with only mild additional damage. We next investigated the ability of the kidney to tolerate a 9.3M solution known as M22, which does not devitrify when warmed from below -150 degrees C at 1 degrees C/min. When M22 was added and removed at -22 degrees C, it was sometimes [corrected] fatal, but when it was perfused for 25min at -22 degrees C and washed out simultaneously with warming, postoperative renal function recovered fully. When kidneys loaded with M22 at -22 degrees C were further cooled to an average intrarenal temperature of about -45 degrees C (about halfway through the putative temperature zone of increasing vulnerability to chilling injury), all kidneys supported life after transplantation and returned creatinine values to baseline, though after a higher transient creatinine peak. However, medullary, papillary, and pelvic biopsies taken from kidneys perfused with M22 for 25min at -22 degrees C were found to devitrify when vitrified and rewarmed at 20 degrees C/min in a differential scanning calorimeter. It remains to be determined whether this devitrification is seriously damaging and whether it can be suppressed by improving cryoprotectant distribution to more weakly perfused regions of the kidney or by rewarming at higher rates. In conclusion, although the goal of organ vitrification remains elusive, the prospects for success have never been more promising.     

Cryoprotectant agent toxicity in porcine articular chondrocytes

Large articular cartilage defects have proven difficult to treat and often result in osteoarthritis of the affected joint. Cryopreservation of articular cartilage can provide an increased supply of tissues for osteochondral allograft but cryoprotective agents are required; however, few studies have been performed on the toxicity of these agents. This study was designed to determine the order of toxicity of five commonly used cryoprotectant agents as well as interactions that occur between them. Isolated porcine articular chondrocytes were exposed to individual cryoprotectant agents and combinations of these agents at 1 M and 3 M concentrations for 5 min and 120 min. Cell viability was determined using membrane integrity dyes and a metabolic activity assay. Subsequently, a regression analysis based study was undertaken to extract the maximum amount of information from this data. Results of this study demonstrated that all 1 M solutions were minimally toxic. The 3 M solutions demonstrated varying toxicity after 120 min. Ethylene glycol and glycerol were less toxic than propylene glycol, dimethyl sulfoxide, and formamide. Combinations of cryoprotectant agents were less toxic than single cryoprotectant agents at the same concentration. This is the most comprehensive study investigating cryoprotectant agent toxicity in articular chondrocytes and has resulted in important information regarding the order of toxicity and interactions that occur between these agents.     

Preservation of renal function by adenosine-stimulated ATP synthesis in hypothermically perfused dog kidneys

Dog kidneys were hypothermically perfused for 1 to 5 days in the presence or absence of adenosine (5 mM). Following 1, 3, and 5 days, kidneys were reperfused at normothermia in an isolated perfusion system using a bovine serum albumin containing perfusate and renal function was determined. At the end of normothermic perfusion, kidney cortical slices were removed for biochemical analysis. Kidneys preserved in the presence of adenosine generated much higher concentrations of ATP during normothermic perfusion than kidneys preserved in the absence of adenosine at all time periods studied. In kidneys reperfused (37 degrees C) after 3 days of preservation, the ATP concentration averaged 9.15 mumol/g dry wt (+adenosine) vs. 4.75 mumol/g dry wt (-adenosine). After 5 days, the average was 12.65 mumol/g dry wt (+adenosine) vs. 4.00 mumol/g dry wt (-adenosine). The tissue concentration of K+ was higher in kidneys perfused in the presence of adenosine for all time periods studied. The presence of adenosine had little effect on the GFR (creatinine clearance) which was reduced by about 90% from control values at both 3 and 5 days of preservation. The primary effect of adenosine on renal function was a greater preservation of the capability of the isolated perfused kidney to reabsorb Na+ from the glomerular filtrate. In the absence of adenosine Na+ reabsorption was reduced from 97 to 50% whereas in the presence of adenosine was reduced to only 80% after 3 days of preservation. After 5 days of perfusion Na+ reabsorption was unaffected by the presence of adenosine and the amount resorbed was only 25-30% of the amount filtered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of four cryoprotectants in combination with two vehicle solutions on cultured vascular endothelial cells.

The necessary first step in successful organ cryopreservation will be the maintenance of endothelial cell integrity during perfusion of high concentrations of cryoprotective agents (CPAs). In this report we compare the effects of incubation on cultured porcine endothelial cells at 10 degrees C for 1 h with the CPAs glycerol, dimethyl sulfoxide (Me2SO), ethanediol (EG), and propane-1,2-diol (PG) in the vehicle solutions RPS-2 (high potassium, high glucose) and HP-5NP (low potassium, high sodium), both with and without added colloids. Tritiated adenine uptake and acid phosphatase estimation of cell number were used as indicators of cell viability. HP-5NP was superior to RPS-2 except with Me2SO when the differences in viability were not significant. Adding Haemaccel to HP-5NP improved the results, but adding albumin to RPS-2 was of no significant benefit. Osmotic stress appeared to be the major problem with glycerols use. Beyond 3.0 M the toxicity of Me2SO increased dramatically but it could not be determined if this was osmotic or chemical toxicity. PG was remarkably well tolerated to 3.0 M but a sharp decrease in cell viability beyond this concentration suggests that PG may be most useful with mixtures of other CPAs. Overall, EG appeared to be the least toxic CPA and in the context of vascular preservation warrants further investigation.     

Toxicity and protective efficiency of cryoprotectants to flounder (Paralichthys olivaceus) embryos

With the purpose of finding an ideal cryoprotectant or combination of cryoprotectants in a suitable concentration for flounder (Paralichthys olivaceus) embryo cryopreservation, we tested the toxicities, at culture temperature (16 degrees C), of five most commonly used cryoprotectants-dimethyl sulfoxide (Me2SO), glycerol, methanol (MeOH), 1,2-propylene glycol (PG) and ethylene glycol (EG). In addition, cryoprotective efficiency to flounder embryos of individual and combined cryoprotectants were tested at -15 degrees C for 60 min. Five different concentrations of each of the five cryoprotectants and 20 different combinations of these cryoprotectants were tested for their protective efficiency. The results showed that the toxicity to flounder embryos of the five cryoprotectants are in the following sequence: PG < MeOH < Me2SO < glycerol < EG (P < 0.05); whereas the protective efficiency of each cryoprotectant, at -15 degrees C for a period of 60 min, are in the following sequence: PG > Me2SO approximately MeOH approximately glycerol > EG (greater symbols mean P < 0.05, and approximate symbols mean P > 0.05). Methanol combined with any one of the other cryoprotectants gave the best protection, while ethylene glycol combined with any one of the other cryoprotectants gave the poorest protection at -15 degrees C. Toxicity effect was concentration dependent with the lowest concentration being the least toxic for all five cryoprotectants at 16 degrees C. For PG, MeOH and glycerol, 20% solutions gave the best protection at -15 degrees C; whereas a 15% solution of Me2SO, and a 10% solution of EG, gave the best protection at -15 degrees C.     

Preliminary studies on the vitrification of red sea bream (Pagrus major) embryos

The objective was to identify an appropriate cryoprotectant and protocol for vitrification of red sea bream (Pagrus major) embryos. The toxicity of five single-agent cryoprotectants, dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycerol (GLY), and methyl alcohol (MeOH), as well as nine cryoprotectant mixtures, were investigated by comparing post-thaw hatching rates. Two vitrifying protocols, a straw method and a solid surface vitrification method (copper floating over liquid nitrogen), were evaluated on the basis of post-thaw embryo morphology. Exposure to single-agent cryoprotectants (10% concentration for 15 min) was not toxic to embryos, whereas for higher concentrations (20 and 30%) and a longer duration of exposure (30 min), DMSO and PG were better tolerated than the other cryoprotectants. Among nine cryoprotectant mixtures, the combination of 20% DMSO+10% PG+10% MeOH had the lowest toxicity after exposure for 10 min or 15 min. High percentages of morphologically intact embryos, 50.6+/-16.7% (mean+/-S.D.) and 77.8+/-15.5%, were achieved by the straw vitrifying method (20.5% DMSO+15.5% acetamide+10% PG, thawing at 43 degrees C and washing in 0.5M sucrose solution for 5 min) and by the solid surface vitrification method (40% GLY, thawing at 22 degrees C and washing in 0.5M sucrose solution for 5 min). After thawing, morphological changes in the degenerated embryos included shrunken yolks and ruptured chorions. Furthermore, thawed embryos that were morphologically intact did not consistently survive incubation.     

Studies in cryoprotection II: Propylene glycol and glycerol

Propylene glycol (PG), glycerol (G), or a combination of both were introduced into and eluted from 116 rabbit kidneys. The function of the kidneys was then studied on an ex vivo shunt by determining bloodflow and creatinine clearance. The cryoprotective agents (CPAs) were introduced and eluted at the rate of about 30 mM/min in an albumin-Ringer's lactate perfusate. In this system, 1.75 to 2.25 M cryoprotectant could be introduced and removed with no demonstrable loss of function. Significant injury was incurred at and above 2.5 M concentrations. When a combination of PG and G (equal proportions) was utilized, both 2.5 and 2.75 M total concentrations were tolerated by the kidney, with no loss of function. These studies demonstrate that in this very rigorous test system, cryoprotectant-induced injury can be significantly diminished by combining two agents of low toxicity. This suggests that osmotic injury may be enhanced by specific toxicities of cryoprotective agents, which can be minimized by using combinations, such as was done in these studies. Also, it has been shown that PG is at least as low in toxicity in the rabbit kidney as is G.     

Cryoprotectant agent toxicity in porcine articular chondrocytes

Large articular cartilage defects have proven difficult to treat and often result in osteoarthritis of the affected joint. Cryopreservation of articular cartilage can provide an increased supply of tissues for osteochondral allograft but cryoprotective agents are required; however, few studies have been performed on the toxicity of these agents. This study was designed to determine the order of toxicity of five commonly used cryoprotectant agents as well as interactions that occur between them. Isolated porcine articular chondrocytes were exposed to individual cryoprotectant agents and combinations of these agents at 1 M and 3 M concentrations for 5 min and 120 min. Cell viability was determined using membrane integrity dyes and a metabolic activity assay. Subsequently, a regression analysis based study was undertaken to extract the maximum amount of information from this data. Results of this study demonstrated that all 1 M solutions were minimally toxic. The 3 M solutions demonstrated varying toxicity after 120 min. Ethylene glycol and glycerol were less toxic than propylene glycol, dimethyl sulfoxide, and formamide. Combinations of cryoprotectant agents were less toxic than single cryoprotectant agents at the same concentration. This is the most comprehensive study investigating cryoprotectant agent toxicity in articular chondrocytes and has resulted in important information regarding the order of toxicity and interactions that occur between these agents.     

Effects of cadmium-metallothionein on renal organic ion transport and lipid peroxidation in rats

The effects of cadmium-metallothionein (Cd-MT) on organic ion uptake in renal cortical slices and lipid peroxidation in the kidney were studied in rats. For in vitro studies, slices were prepared from kidneys of control animals and incubated in buffer containing either cadmium chloride (CdCl₂) or Cd-MT in equimolar Cd concentrations ranging from 5 × 10^?6 to 2 × 10^?4 M. Uptake into the slices of the organic anion p-aminohippuric acid (PAH) was found to be inhibited by both forms of Cd in a dose-dependent manner. Although this inhibition was slightly greater in the presence of Cd-MT, accumulation of Cd into the slices was approximately 12 times greater with CdCl₂ than Cd-MT. Tetraethylammonium (TEA) uptake was less sensitive to the inhibitory effects of both CdCl₂ and Cd-MT, although a dose-dependent inhibition did occur with higher Cd concentrations. To study the in vivo effects of Cd-MT on transport function and lipid peroxidation in the kidney, rats were injected with Cd-MT (0.3 mg Cd per kilogram body weight [bw]) and sacrificed at specific time intervals. Similar to the in vitro studies, PAH uptake into the renal cortical slices was markedly inhibited within 12 hours after Cd-MT injection whereas inhibition of TEA uptake was less and not observed until 48 hours after injection. Only a small increase (1.4-fold) in lipid peroxidation, as measured by generation of malondialdehyde (MDA), in the kidney was detected at four hours postinjection, and no further increase was observed at later time periods. The results suggest that Cd-MT affects the transport of organic anions and cations during its renal uptake but that lipid peroxidation may play only a minor role in Cd-MT-induced renal toxicity.     

Effect of cryoprotectants on the viability and function of unfrozen human polymorphonuclear cells

High concentrations of membrane permeable cryoprotectants are necessary to protect human polymorphonuclear leukocytes from osmotic stress injury during freezing, but there are reports that some cryoprotectants are chemically toxic. Cells were exposed to various concentrations of glycerol, dimethyl sulfoxide, or ethylene glycol for 5 min to 2 hr at 37, 22 or 0 degree C, adding or removing the cryoprotectant either slowly or rapidly. Assays included cell number recovery, membrane integrity, phagocytosis, microbicidal ability, and chemotaxis. We conclude that (1) 1 and 2 M concentrations generally are not toxic if they are added and removed slowly at 22 degrees C; (2) addition and removal of glycerol at 0 degree C was injurious even at 1 M; (3) slow addition and removal allowed better recovery than rapid addition or removal; (4) salt concentration in cryoprotectant solutions should be adjusted to isotonic on the basis of moles per liter of solution, rather than moles per kilogram of water; (5) the toxicity reported by other investigators can be largely explained by osmotic stress or dilution shock rather than chemical toxicity; and (6) ethylene glycol is the easiest cryoprotectant to add to and remove from these cells.     

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.     

Nitric oxide synthase inhibitor L-NAME has no effect on 86Rb accumulation in rat renal cortical slices

The aim of present study was to investigate the effect of the nitric oxide synthase inhibitor L-NAME on the 86Rb uptake in rat renal cortical slices. Rats were divided into three groups: 1. Control. 2. Acute: L-NAME (10 mg/kg i.v.) as a bolus 15 min before the excision of the kidneys. 3. Sub-chronic: L-NAME (10 mg/kg/day) per os for 4 days. Renal cortical slices were incubated for 10, 20, 30, 60, 90, 180 seconds in Krebs-Ringer solution containing 50 kBq 86Rb/100 ml (T = 37 degrees C, PO2 approximately 159 mm Hg). 56Rb accumulation (S/M) was calculated as the ratio of the radioactivity of the cortical slices (S) and the radioactivity of the incubating medium (M). The S/M ratio can be described as a function of time by the following equations. Control: y = 0.265 ln(x) - 0.220, r(xy) = 0.886; acute L-NAME: y = 0.224 ln(x) - 0.171, r(xy) = 0.921; sub-chronic L-NAME: y = 0.331 ln(x) - 0.496, r(xy) = 0.942. (y = S/M, x = t). p < 0.001 in all of the groups, but there is no difference between the groups. In conclusion, L-NAME administered in vivo failed to influence the in vitro 86Rb accumulation in rat renal cortical slices.     

The influence of temperature on the function of renal cortical slices frozen in various cryoprotectants

Renal cortical slices were frozen to various subzero temperatures after treatment with 2.1 M of one of three cryoprotectants, dimethyl sulfoxide (Me2SO), ethylene glycol, or glycerol. The effects on tissue [K+]/[Na+] of cooling to these temperatures were tested (using identical procedure times, cooling rates, and warming rates) by holding the slices at each experimental temperature for appropriate periods of time prior to rewarming. The effects of the holding time were assessed by comparison with slices which were cooled and rewarmed with no intermediate holding time. Slices treated with ethylene glycol or glycerol were found to exhibit a continuous decrease in [K+]/[Na+] with lowered temperatures, in contrast to those treated with Me2SO. Slices treated with Me2SO actually experienced a continuous increase in [K+]/[Na+] with lowered temperature (-12 to -33 degrees C). Me2SO does exhibit toxic effects at subzero temperatures. Adverse effects of holding time on viability are seen for Me2SO-treated slices at higher subzero temperatures. These effects were alleviated as the temperature is reduced, suggesting that temperature has a greater effect on survival of renal cortical tissue than Me2SO concentration. However, the toxicity observed at higher subzero temperatures is expected to be of importance, particularly for slowly cooled tissues which are exposed to these temperatures for relatively long periods of time.