Renal preservation by hypothermic perfusion. IV. The use of gelatin polypeptides as the sole colloid

Rabbit kidneys were perfused with a solution of extracellular electrolyte composition, rendered hypertonic with glucose and containing 1.75% Haemaccel (Hoechst) as the sole colloid. Perfusions were carried out at 10 °C for 24 and 48 hr using perfusion pressures of 20 or 40 mm Hg. Function, was tested by autografting with immediate contralateral nephrectomy. All the kidneys perfused at 20 mm Hg for 24 hr showed excellent life-sustaining function and 7 of 10 survived in the 40 mm Hg group; the difference was not statistically significant. However, all the kidneys perfused for 48 hr failed to sustain life, and histological examination revealed extensive breakdown of the microcirculation. The 24 hr results were similar to those previously obtained with an albumin-based perfusate, but the 48-hr results were inferior: However, the obvious advantages of a well-standardised, cheap, and easily stored perfusate are such as to justify further study of gelatin-derived colloids for organ preservation.     

Renal preservation by hypothermic perfusion. I. The importance of pressure-control

Rabbit kidneys were preserved by hypothermic perfusion at 5 °C using a perfusate containing an extracellular balance of ions, dextran and bovine serum albumin. Two groups were studied: in one, the pressure was kept constant at 40 mm Hg, while in the other the flow was maintained at 13 ml/min. The mean flows in the two groups were similar but the resistance of the kidneys perfused under constant-flow conditions was lower and more stable: the vascular resistance in the constant-pressure group showed considerable fluctuations throughout the 24 hr perfusion period. The function of the kidneys was assessed by autotransplantation with immediate contralateral nephrectomy. The constant-pressure group functioned better in all respects: the proportion of animals surviving was higher, the postoperative blood urea and creatinine levels were lower, and histological examination of the kidneys revealed less damage. It is concluded that constantpressure perfusion should be preferred to constant-flow perfusion. These experiments confirmed that there is a correlation between potassium release into the perfusate and subsequent function, and an unexpected inverse correlation was observed between the perfusate glucose level and subsequent function. Possible reasons for this are discussed.     

Effects of chlorpromazine and methylprednisolone on perfusion preservation of rabbit livers

The isolated perfused rabbit liver was used to determine how continuous hypothermic perfusion affected liver function. Rabbit livers were perfused for 0, 24, 48, and 72 hr at 5 degrees C with the UW perfusate containing hydroxyethyl starch (5 g%) dissolved in a solution containing gluconate (80 mM), adenosine (5 mM), glutathione (3 mM), phosphate (25 mM), and additives as described previously, and they were used successfully for kidney preservation. At the end of preservation the livers were perfused in an isolated circuit with a Krebs-Henseleit solution with addition of 4 g% bovine serum albumin and 10 mM glucose at 38 degrees C for 120 min. Bile was collected from the cannulated common duct. Biliary excretions of indocyanine green and liver enzymes lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase, were determined both in the cold perfusate and the normothermic perfusate. Livers were also studied after pretreatment of the donor with chlorpromazine (CPZ) and/or methylprednisolone (MP). Bile production (ml/120 min, 100 g liver) upon reperfusion produced the most interesting data and decreased from a control value of 10.3 +/- 2.6 to 9.3 +/- 1.0 (24 hr), 5.3 +/- 0.7 (48 hr), and 4.1 +/- 1.5 (72 hr). Enzyme release was not predictive of the degree of preservation-induced damage. Pretreatment of rabbits with a combination of CPZ/MP improved bile flow at 48 and 72 hr (8.3 +/- 3.0 and 7.0 +/- 1.3, P less than 0.05). Pretreatment with either drug alone also improved function after 72 hr of preservation (7.1 +/- 1.8, CPZ; 8.2 +/- 3.5, MP).(ABSTRACT TRUNCATED AT 250 WORDS)     

Coenzyme A in dog kidneys during hypothermic perfusion

In kidneys perfused hypothermically for 48 hr, both major intracellular pools of CoA (present in cytosol and mitochondria) decreased when solutions with intracellular or extracellular electrolyte composition without CoA precursors were used. The presence of substrates such as glucose or caprylic acid in the perfusate did not counteract the depletion of CoA.The addition of the CoA precursors pantethine and adenosine together with respiratory substrates to the perfusion medium counteracted the loss of CoA. The best solution in this respect among 16 different perfusates tested contained palmitic acid and l-carnitine together with CoA precursors and dibutyryl cyclic AMP. With this solution, we obtained good preservation of the cytosolic and the mitochondrial pool of CoA.     

Forty-eight hours hypothermic pulsatile perfusion of canine hearts before transplantation.

Canine hearts preserved for 24 hr under hypothermic pulsatile perfusion had a good function after transplantation. The perfusate consisted of cryoprecipitated plasma that was modified by the addition of salt poor albumin, potassium chloride and glucose; the final osmolarity was 340 mOsm/L. Fresh allografts without perfusion survived for an average of two weeks after transplantation, and the 24 hr perfused hearts survived for more than 19 days after transplantation. When the perfusion was extended to 48 hr, the survival was decreased to 11 days. These data indicate that hypothermic pulsatile perfusion is completely safe and feasible for 24 hr without significant functional or histological impairment. The survival response of the hearts perfused for 48 hr was significantly decreased when compared to the hearts perfused for 24 hr.     

Effects of short-term hypothermic perfusion and cold storage on function of the isolated-perfused dog kidney

The isolated-perfused dog kidney was used as a model to measure the effects of short-term hypothermic preservation on renal function and metabolism. Kidneys were cold-stored in Collins' solution, hypotonic citrate, or phosphate-buffered sucrose for 4 and 24 hr, or were continuously perfused for 4 and 24 hr with a synthetic perfusate. Following preservation kidneys were perfused with an albumin-containing perfusate at 37 degrees C for 60 min for determination of renal function. The results indicate that many of the effects of short-term preservation on renal function in dog kidneys are similar to results reported for rat and rabbit kidneys. Cold storage for 4 hr resulted in a large decrease in GFR (57%), but only a small decrease in Na reabsorption (from 97 to 87%). Cold storage for 24 hr caused a further decline in renal function (GFR = 95% decrease, Na reabsorption = 49-64%). Results were similar for all cold storage solutions tested. Perfusion for 4 hr was less damaging to renal function than cold storage. The GFR decreased only 14% and urine formation and Na reabsorption were practically normal. After 24 hr of hypothermic perfusion, the GFR was reduced by 79%, urine flow was normal, and Na reabsorption was 78%. There were no obvious biochemical correlates (adenine nucleotides, tissue edema, or electrolyte concentration) with the loss of renal function during short-term preservation. The results suggest that the isolated-perfused dog kidney can be used to test the effects of preservation on renal function, and yields results similar to those obtained using small animal models.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rabbit kidney function in vitro: the effect of colloids, energy substrate, a vasodilator, perfusion pressure, and bovine serum albumin

An in vitro perfusion system at 37 degrees C for the assessment of rabbit kidney function is described. The purpose of this assay system is to evaluate the effects of cryobiological manipulation on kidney function. The effect of the colloids dextran (MW = 70,000, 80,000, and 180,000) in the perfusate at 110 mm Hg were compared to a reduced perfusion pressure, colloid-free perfusate. Better function was obtained at lower perfusion pressure with the colloid-free perfusate. Less damage was noted histologically on light and electron microscopy. Investigation of energy substrates on rabbit kidney function demonstrated that butyrate, or lactate, in addition to glucose resulted in increased sodium and glucose reabsorption over glucose alone. Substrate-free perfused kidneys exhibited depressed Na transport. Lactate, and to some extent butyrate, decreased net glucose utilization. An alpha-adrenergic blocking agent, isoxsuprine, in the initial flush solution did not appear to be beneficial. An increase of perfusion pressure from 50 to 75 mm Hg resulted in an increase in GFR. Tubular function was enhanced by inclusion of small amounts of BSA in the perfusate.     

Changes in concentration of amino acids and other metabolites during hypothermic perfusion of the canine kidney

Changes in the concentration of amino acids and other metabolites were determined in the perfusate during 24 hr of ex vivo hypothermic perfusion of dog kidneys. There was an increase in concentration of most of the amino acids. Two patterns were identified. One showed an increase in concentrations up to 12 hr, and then a leveling off as exemplified by alanine, serine, and glutamate. The other pattern was one of persistent elevation as exemplified by phenylalanine, threonine, and methionine. Glucose, lactate, pyruvate, sodium, potassium, pH, and pO2 were also measured in the perfusate. The results suggest that a degradation of kidney protein may occur during the first 24 hr of perfusion. The levels of other metabolites measured support the fact that glycolysis is responsible for a considerable portion of the total energy production in the kidney under hypothermia.     

An improved apparatus for blood perfusion of the canine cerebral vasculature

An improved perfusion apparatus is described which consists of a membrane oxygenator, roller pump, reservoir, heat exchanger, blood filter, and inert tubing. Heparinized blood may be used and is delivered at flow rates from 10 to 250 ml/min. Dogs are anesthetized with halothane and their cerebral arterial blood supply isolated by the method of Gilboe et al. (8). When the canine brain is perfused for 5 hr using the described apparatus, the rates of cerebral oxygen and glucose consumption are 5.19±0.12 ml/100 g/min and 39.9±6.5 mol/100 g/min, respectively. Of the total glucose consumed by the brain, about 1/4 is contributed by the erythrocytes. An equivalent of about 9% of the consumed glucose is returned to the blood as lactate. Electron microscopic examination of cerebral cortex samples reveals no differences between 5-hr perfused brain and appropriate nonperfused controls. It is concluded that the apparatus is a useful system for organ perfusion and that the canine brain perfused by this method remains physiologically and metabolically active for at least 5 hr.With the technical assistance of Evelyn Townsend.     

Effects of hypothermic kidney preservation on the isolated perfused kidney: a comparison of reperfusion methods

Two isolated-perfused kidney methods were used to study the effects of hypothermic preservation on renal function in dog kidneys. The isolated-machine-perfused kidney (IMPK) used an in vitro perfusion technique--the perfusate was a Krebs-bicarbonate type delivered to the kidney at 37 degrees C by a mechanical pump at a constant pressure (100 mm Hg). The isolated-blood-perfused kidney (IBPK) utilized transplantation of the preserved kidney to the femoral vasculature. Renal function (urine analysis) was determined over a 1-hr reperfusion interval and included GFR (creatinine clearance), urine formation, and Na+ reabsorption. Kidneys preserved for only 24 hr by cold storage in either Collins'--C3 solution or in hypotonic citrate and kidneys hypothermically perfused for 24 hr demonstrated greater retention of renal function when reperfused by blood (IBPK) than with the in vitro perfusate (IMPK). The GFR was reduced by 38-58% when tested with the IBPK, but by 80-90% when tested with the IMPK. Na+ reabsorption was normal (97%) with blood reperfusion but was reduced to 36-50% in cold-stored kidneys and 82% in hypothermically perfused kidneys determined by machine reperfusion (IMPK). However, kidneys perfused for 72 hr demonstrated more similar renal functions when tested by either IMPK or IBPK. GFR was reduced to 20% (IBPK) and 11% (IMPK) and Na+ reabsorption averaged 76-85% (IBPK or IMPK). These results suggest that either reperfusion method is suitable for determining the effects of renal preservation on kidney function in kidneys preserved for 72 hr but, for short-term preserved kidneys (24 hr), the IBPK model may be preferred.     

Lidocaine-containing Euro-Collins solution prevents renal injury in the isolated perfused canine kidney exposed to prolonged cold ischemia

Previous studies have reported a decreased incidence of delayed graft function after cadaveric transplantation with the use of lidocaine pretreatment of the donor. We evaluated the effects of lidocaine on prolonged cold ischemia and reperfusion injury in a canine model of isolated kidney perfusion (IPK). The purpose of this study was to evaluate the renal function of isolated perfused canine kidneys after 48 h of cold storage with Euro-Collins (EC) solution or EC solution plus lidocaine. Isolated perfused canine kidneys were randomized into four groups which contained six kidneys: I) cold flush with EC solution and immediately reperfused, II) cold flush with EC solution plus lidocaine and immediately reperfused, III) 48 h of cold storage with EC and reperfusion, IV) 48 h of cold storage with EC solution plus lidocaine and reperfusion. The measured renal functions were glomerular filtration rate, urine production, perfusate flow, urinary lactic dehydrogenase (ULDH), Na reabsorptive capacity, and tissue MDA levels. Histological examination was performed after reperfusion. The tubular functions of kidneys preserved with EC solution containing lidocaine were better when compared with the kidneys preserved with EC alone. Tubular injury marker levels (ULDH) in group IV were significantly lower than in group III and lidocaine also reduced lipid peroxidation during reperfusion. This is in agreement with the histological results. The results of the present study can be taken as evidence of the cytoprotective effect of lidocaine, which may therefore be accepted as a useful agent for kidney preservation.     

An airlift pump device for low pressure perfusion storage of the isolated heart

A portable apparatus for the continuous hypothermic perfusion of the isolated heart is described. The system has been used successfully to store pig and baboon hearts for periods of up to 48 hr, and to store human donor hearts for periods of 7 to 17 hr before being transplanted. The perfusate is both oxygenated and circulated by gas flow from a pressurized oxygen cylinder, using the air-lift pump principle. The apparatus has no moving parts and requires no electrical energy supply; malfunction is, therefore, extremely unlikely. A regulator has been incorporated which can be adjusted to increase or decrease the myocardial perfusion pressure. The system and environmental variables which can affect flow and pressure within the apparatus are discussed. The storage time allowed by this system will enable transportation of donor hearts between most of the world's major cities.     

The assessment of renal preservation by normothermic bloodless perfusion.

A method of estimating renal function by normothermic perfusion in vitro has been developed. In this paper, its application to the study of different methods of hypothermic renal preservation in the rabbit is described. Groups of kidneys were stored at 4 °C for 24 hr by surface cooling alone, by initial perfusion followed by storage (washout perfusion), and by continuous perfusion. Renal function was found to be severely compromised after surface cooling alone or after washout perfusion with an isotonic solution resembling extracellular fluid. Washout with a solution containing sufficient additional glucose to raise the osmolality to 400 mosm/kg gave greatly improved function, but increasing the concentration of magnesium from 2 to 72 mequiv/litre failed to confer any additional benefit, and increasing the concentration of potassium from 4 to 74 mequiv/ litre depressed function. Continuous perfusion with a solution containing albumin and dextran gave results that were inferior to the best washout method, but increasing the osmolality of the perfusate with glucose again resulted in a very significant improvement in function, which however was still inferior to the best washout method of storage. The further use of this test system to study methods of renal preservation is advocated.     

Effect of PGE1 on gluconeogenesis and glycerol esterification in perfused liver of fasted rats

Using perfused livers of rats fasted for 48 hours, glucose production and incorporation of 2-¹⁴C pyruvate (trace dose) into perfusate glucose were studied. Both were found to be inhibited by PGE₁ (infused at a concentration of 0.5 μg/min) by about 60 %. The incorporation of 1-¹⁴C glycerol into perfusate glucose and into glycerol-glyceride part of the liver glycerides were also studied, using the same test conditions. The former incorporation was significantly inhibited (56%) and the latter strongly stimulated (360 %) by PGE₁. PGE₁ had no effect on glucose production in a perfusate overloaded with sodium pyruvate, nor on pyruvate carboxylase and phospho-enolpyruvate carboxykinase activity. This was in contrast with the results obtained in perfusions with a trace dose of 2-¹⁴C pyruvate. The results showed that PGE₁, at the physiological concentration used, stimulated the incorporation of 1-¹⁴C glycerol into glycerol-glyceride part of liver glycerides and, when there was no overload of pyruvate present in the perfusion medium, inhibited gluconeogenesis at some point, possibly, but perhaps not exclusively, between the glycerol and glucose steps.     

Abnormal tubular permeability in hypothermic perfused kidneys.

Isolated canine kidneys perfused with cryoprecipitated plasma at 15 degrees C exhibit unexpectedly low inulin clearance (CIn) and creatinine clearance (CCr) rates. CIn and CCr, as well as p-aminohoppurate (PAH) clearance, varied linearly with urine flow rate, whether the variations in urine flow were spontaneous or induced, either by elevating perfusion pressure or by adding mannitol to the perfusate. Retrograde intraureteral injection (RII) of an isotonic fluid containing dextran, inulin, and PAH, followed by a period of ureteral occlusion and subsequent serial recollection of the injected fluid, revealed that inulin and PAH, relative to dextran, were lost from distal tubular fluid. Similar experiments in anesthetized dogs indicated no loss of inulin or PAH from tubules of in situ kidneys. Renal venous perfusate, collected from isolated kidneys during the low pressure phase of the RII, contained the following percentages of the quantities injected intraluminally: dextran, 9.22%; inulin, 11.0%; and PAH, 22.0%. These data indicate that a low measured glomerular filtration rate in hypothermic perfused kidneys is partly due to diffusion of inulin or creatinine out of the tubular lumen.     

Ascaris suum: continuous perfusion of the pseudocoelom and nutrient absorption

A double-cannulation apparatus was constructed for continuous perfusion of the pseudocoelom of adult Ascaris suum while maintaining the intact parasite in a controlled incubation chamber. Peristaltic pumps maintained a constant flow rate of artificial perienteric fluid through the incubation chamber (1 ml/min) and through the parasite (100 microliters/min). Based on protein determinations, perienteric fluid was removed from the pseudocoelom within 35 min of initiation of perfusion (3.5 ml). A nonabsorbable dye, Blue Dextran, was detected first in the perfusate 4 min (400 microliters) after initiation of infusion into the pseudocoelom, and was maintained at a constant concentration in the perfusate by 8 min after initiation of dye infusion. Removal of the dye from the pseudocoelom was accomplished within 8 min (800 microliters) after the cessation of dye infusion. Occlusion of the digestive tract had no effect (P less than 0.05) on the short-term (3 hr) absorption of 3H-labeled cholesterol, [14C]-3-o-methylglucose or [14C]glucose from the incubation medium into the perienteric cavity. Concentrations of isotopes in the pseudocoelom reached steady-state levels within 60 min of the initiation of incubation, but remained low (greater than 0.5%) when compared to medium concentration. Similarly, the time course of the accumulation of [14C]glucose into individual tissue components did not differ in intact worms with or without patent intestinal tracts. Thus, the cuticular/muscle tissue largely appears to be the primary route of absorption of cholesterol and glucose in adult A. suum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Continuous hypothermic perfusion of rabbit kidneys.

Addition of cryoprotective agents to whole organs is possible only by vascular perfusion with the cryoprotectant dissolved in a suitable perfusion fluid.Vascular resistance, organ weight gain, release of lactate dehydrogenase (LDH), and post-transplant function was studied during and after hypothermic perfusion at +6 °C of rabbit kidneys with six different perfusion fluids. A mixture of dextran and bovine serum albumin (BSA), BSA alone in various concentrations, and human serum albumin were tested as colloids, and the effect of perfusate osmolality was investigated.The dextran-BSA mixture was found to be superior to 4.5 and 6.0% BSA alone in terms of better perfusion characteristics, better post-transplant function, and lower LDH release. Perfusion characteristics during perfusion with human serum albumin and subsequent graft function were not different from those observed in experiments with dextran-BSA, but the LDH release was lower.Perfusate osmolality was increased by the addition of glucose or mannitol. Perfusion characteristics during perfusion with the hypertonic perfusates were not different from those observed during isotonic perfusion, but post-transplant function seemed to be better after perfusion with the fluid made hypertonic with glucose, whereas addition of mannitol seemed to be deleterious.Thus a perfusion fluid of extracellular electrolyte composition, containing human serum albumin as a colloid and made hypertonic with glucose, can be used as a vehicle for cryoprotectants during their addition to rabbit kidneys.     

Lack of effect on human lipoprotein-triacylglycerol on the function of perfused rat kidney

We determined whether addition of human lipoprotein-TG to the perfusate for the isolated rat kidney would increase net Na+ reabsorption or maintain renal tissue K+ content. Rat kidneys (n = 6) were perfused for 75 min with a perfusate containing 6 g% of substrate-free albumin in Krebs-Ringer bicarbonate and a mixture of human chylomicrons and very low density lipoproteins (human lipoprotein-triacylglycerol (HL-TG]. Control kidneys (n = 6) were perfused in the substrate-limited state, i.e., without any exogenous substrates added to the perfusate. Means (n = 6) for function of control kidneys were GFR = 808 +/- 50 microliter g-1 X min-1; %T-Na+ = 63.3 +/- 1.3%. A significant loss of tissue K+ occurred: tissue K+ remaining after 75 min of perfusion = 79.1 +/- 1.9%. Although kidney tissue contains lipoprotein lipase, HL-TG (n = 6) did not increase %Na+ reabsorption (64.3 +/- 2.6%) or maintain tissue K+ content (80.6 +/- 2.0%). Therefore, the TG might have been hydrolyzed and taken up for biosynthesis, the rat kidney lipoprotein lipase might have been inactive, or the rat kidney might not use lipoprotein-TG for biosynthesis or oxidation.     

Functional preservation of the mammalian kidney. IV. Functional effects of perfusion with dimethyl sulfoxide (DMSO) at normothermia

Rabbit kidneys were perfused at 37 °C with various concentrations of DMSO in a K⁺-Mg²⁺-rich perfusate. The effects of DMSO on various functional parameters of the rabbit kidney perfused for 60 min were compared with the functional effects of perfusion without DMSO under the same conditions. DMSO produced deviations in vascular resistance and perfusate flow rate from control values. In kidneys perfused with 1.4 and 2.8 m DMSO these vascular changes resulted in changes in GFR at relatively unchanged filtration fractions. The closely parallel relationship between changes in GFR and urine flow rate in all groups indicates that perfusion per se or perfusion with DMSO may shift the regulation of urine flow rate from tubular reabsorption, which obtains in the in vivo situation, to glomerular filtration. This view was supported by the relatively unchanged parameters of Na⁺ reabsorption and fractional water excretion during perfusion with all concentrations of DMSO. Additionally, DMSO perfusion resulted in significantly greater weight gains than those observed in kidneys perfused without DMSO, and significantly depressed clearances of PAH, with 2.1 and 2.8 m DMSO.     

Some determinants of quality and yield in the isolation of adult heart cells from rat

We investigated the effect of changes in perfusate substrate and Ca content on the quality and yield of isolated adult rat heart cells. When 1 mM Ca was added to the recirculating perfusate 15 min after collagenase addition, the ATP level of cells in the heart 15 min later, and their morphology in histological section, was no different from when no Ca was added back. The cells subsequently isolated were of similar yield, but a greater percentage were rod-shaped, compared with cells isolated without Ca restoration to the perfusate. Increased yield could be obtained by including substrates in the perfusate in addition to glucose. Either fatty acids or amino acids were effective. We conclude that: (1) all cells in the heart are Ca tolerant at the end of enzyme perfusion; (2) the presence of substrates in addition to glucose can help cells survive the isolation process.