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)     

Decrease of Extracellular Taurine in the Rat Dorsal Hippocampus After Central Nervous Administration of Vasopressin

The extracellular amino acid concentrations in the left and right dorsal hippocampus of male rats were studied before and during application of vasopressin into the right hippocampus. The method of intracerebral microdialysis was used for both arginine vasopressin administration and monitoring of the composition of the extracellular fluid. The concentrations of 16 amino acids were measured by HPLC in the perfusate samples. The level of taurine declined 20% in the right hippocampus during perfusion with vasopressin, whereas o-phosphoethanolamine decreased in both sides, the left 20% and the right 24%. These alterations may be related to cerebral osmoregulation. Also, the levels of tyrosine and phenylalanine increased 15% and 35%, respectively, during administration of vasopressin. No changes of other amino acids were observed.     

Kinetic Analysis of Cerebrovascular Isoleucine Transport from Saline and Plasma

The concentration dependence of regional isoleucine transport across the blood-brain barrier was determined in anesthetized rats with the in situ brain perfusion technique of Takasato et al. [Am. J. Physiol. 247, H484-493 (1984)]. This technique allows, for the first time, accurate measurements of cerebrovascular amino acid transport in the absence of competing amino acids using saline perfusate, and in the presence of physiological concentrations of amino acids using plasma perfusate. Cerebrovascular isoleucine transport from saline perfusate followed Michaelis-Menten saturation kinetics where Vmax = 9 - 11 X 10(-4) mumol X s-1 X g-1 and Km = 0.054-0.068 mumol X ml-1 in six brain regions. A component of nonsaturable transport was not detected in any brain region even though perfusate isoleucine concentration was increased to greater than or equal to 150 times the normal plasma concentration. Isoleucine influx during plasma perfusion was only 8% of that predicted from the saline perfusion data due to transport inhibition by competing amino acids in plasma. Competitive inhibition increased the apparent Km for isoleucine transport from plasma by greater than or equal to 24-fold to 1.5-1.7 mumol X ml-1. These data provide accurate new estimates of the kinetic constants that describe amino acid transport across the blood-brain barrier. In addition, they indicate that the cerebrovascular transfer-site affinity (1/Km) for isoleucine is approximately fivefold greater than previously reported with the brain uptake index technique.     

Metabolism of valine and 3-methyl-2-oxobutanoate by the isolated perfused rat kidney.

Metabolism of branched-chain amino and 2-oxo acids was studied in the isolated perfused kidney. Significant amounts of 2-oxo acids were released by perfused kidney with all concentrations of amino acids tested (0.1-1.0 mM each), despite the high activity of branched-chain 2-oxo acid dehydrogenase in kidney. As perfusate valine concentration was increased from 0.2 to 1.0 mM, [1-14C]valine transamination (2-oxo acid oxidized + released) increased roughly linearly; [1-14C]valine oxidation, however, increased exponentially. Increasing perfusate concentration of 3-methyl-2-oxo[1-14C]butanoate from 0 to 1.0 mM resulted in a linear increase in the rate of its oxidation and a rise in perfusate valine concentration; at the same time significant decreases occurred in perfusate isoleucine and leucine concentrations, with corresponding increases in rates of release of their respective 2-oxo acids. Comparison of rates of oxidation of [1-14C]valine and 3-methyl-2-oxo[1-14C]butanoate suggests that 2-oxo acid arising from [1-14C]valine transamination has freer access to the 2-oxo acid dehydrogenase than has the 2-oxo acid from the perfusate. The observations indicate that, when branched-chain amino and 2-oxo acids are present in perfusate at near-physiological concentrations, rates of transamination of the amino and 2-oxo acids by isolated perfused kidney are greater than rates of oxidation.     

Beneficial effect of low concentrations of cryoprotective agents on short-term rabbit kidney perfusion

This report describes observations concerning the influence of the addition of 0.3 M cryoprotective agents (propylene glycol or glycerol) to Ringer's albumin solution used for rabbit kidney perfusion for periods of less than 4 and 24 hr. Endogenous creatinine clearance during short-term parabiotic perfusion on a shunt was used to evaluate function after perfusion. The findings were (A) Perfusion for less than 4 hr resulted in a significant loss of function by comparison with 1 hr ice-stored controls. (B) Continuing the perfusion for 24 hr resulted in a further significant fall in function. Much of the early perfusional injury seen in these two groups could be avoided by including 0.3 M cryoprotective agents in the perfusate.     

Lack of effect of amino acid concentration on protein synthesis in the perfused rat liver.

The effect of increasing the perfusate concentration of amino acids on the incorporation of labelled valine into protein was followed in perfusions of rat livers lasting for 2h. A fixed amount of labelled and unlabelled valine was added to the perfusate as the other amino acids were increased in multiples of the concentrations normally found in rat plasma. Under these conditions no increase in valine incorporation was observed, which appeared to be in conflict with results published by other workers, However, a different method of labelling from that used here was used in the earlier studies. An increasing amount of a labelled amino acid was added as the concentrations of the unlabelled amino acids were increased in the perfusate. An experiment directly comparing to the two labelling methods produced results that indicated that the apparent increase in liver protein synthesis observed by the other workers could have been due to the method of radioisotope addition. It is therefore concluded that increasing the perfusate concentration of amino acids does not increase amino acid incorporation into liver protein.     

Effects of differences in substrate supply on the energy metabolism of hypothermically perfused canine kidneys

Experiments were performed on the effects of differences in substrate supply on canine kidneys. Following 2 min of ischemia and flush perfusion for 5 min the kidneys were continuously perfused at 6 °C using albumin perfusate containing free fatty acids and glucose or Haemaccel perfusate without substrates.During 120 hr of perfusion neither potassium nor LDH nor GOT accumulation differed between the two perfusates and up to the 48th hr the tissue contents of adenine nucleotides as well as the energy charge potential were almost identical. The results show that in canine kidneys glucose or FFA supply during hypothermic continuous perfusion does not influence the overall cellular integrity and energetic capacity of the renal cortex at least up to the 48th hr of preservations.     

Prediction of viability of rabbit kidneys preserved by hypothermic perfusion

The results of a range of tests carried out during renal preservation by hypothermic perfusion were examined to see whether they could be used to predict the viability of rabbit kidneys. Kidneys were perfused at +5 °C for 24 hr and measurements were made of weight gain and change in vascular resistance as well as changes in the composition of the perfusate. Renal function was assessed by autografting and postoperatively measurements of blood urea and serum creatinine were made; an index of renal function was obtained by integrating the blood urea concentration with respect to time during the first 14 postoperative days. Two sets of statistical correlations were made, the first was between the tests and survival or nonsurvival, and the second was between the test results and the excellence of function in the surviving group. The traditional indices of weight gain and resistance change during perfusions were not found to have any predictive value. Of the biochemical parameters measured only the GOT activity of the perfusate proved to be of significance both in terms of prediction of viability and of subsequent renal function. The results obtained, however, were not decisive enough to support a firm recommendation that GOT measurements should be used in clinical practice to decide whether or not to transplant a particular kidney. Nevertheless, a high GOT concentration is a feature of damaged kidneys and it may be that with longer perfusion periods and longer warm ischaemia times, the test may have greater discriminatory value.     

Partial ischemia and proteinuria during isolated kidney perfusion is accompanied by the release of vascular [35S]heparan sulfate

The isolated kidney perfused with modified Krebs-Henseleit buffer with amino acids yields heavy proteinuria associated with reduced ATP levels characteristic of partial ischemia. These conditions are associated with a similar perfusion time dependent release of degraded vascular [35S]heparan sulfate proteoglycan into the perfusate solution which included a 60% loss of [35S]macromolecular material from the glomerulus after 2h of perfusion. Small amounts of [35S]macromolecular material were found in the urine and lymph. These results demonstrate that partial ischemia promotes a specific response in the overall renal vasculature, probably involving oxygen reactive metabolites, that results in the preferential release of heparan sulphate from the basement membrane and endothelial cells on the luminal side of the capillary wall.     

Validation of transport measurements in skeletal muscle with N-13 amino acids using a rabbit isolated hindlimb model

We are studying the transport of C-11 and N-13 labeled amino acids in tumor-bearing rabbits to determine the role of amino acid transport in the pathogenesis of muscle wasting in cancer. To validate a new, in vivo, method for measuring transport in skeletal muscle with these compounds, an isolated hindlimb model was developed in rabbits. The limb was perfused with a non-recirculating, normothermic, constant pressure system and a cell-free perfusate. Hemodynamic and metabolic parameters were measured during the first 75 min. of perfusion and found to remain normal and stable. Flow varied directly with perfusion pressure over the normal range of resting flows in the intact rabbit hindlimb. Time-activity curves (TAC's) were recorded from the medial thigh following bolus co-injection of L-[amide N-13] glutamine or N-13 L-glutamate with Tc-99m human serum albumin (HSA) into the femoral artery. Regional plasma flow was determined from the Tc-99m data. The N-13 TAC's consistently manifested a three-phased washout with half times of approximately 30 sec., 5 min. and 2 hr. Capillary and cellular transport parameters were computed from the N-13 data using a double barrier, single capillary model of capillary and cellular transport and assuming that the three washout components result, respectively, from tracer throughput, extraction into the interstitial space and extraction into the intracellular space. This interpretation was validated and the sensitivity of the technique to transport processes demonstrated by examining the effects on the N-13 TAC's and computed transport parameters of several factors known to influence cellular transport of amino acids, viz., the insulin concentration, amino acid concentration and pH of the perfusate. Time-activity curves and transport parameters for N-13 L-glutamine in the isolated limb were very similar to those observed in the intact rabbit hindlimb, suggesting that studies in the perfused model are indicative of amino acid transport in vivo. The methodology described here is especially well suited for studying the specific effects on transport of factors which influence amino acid metabolism in skeletal muscle (e.g., hormones and monokines).     

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.     

Resuscitation of Ischemic Donor Livers with Normothermic Machine Perfusion: A Metabolic Flux Analysis of Treatment in Rats

Normothermic machine perfusion has previously been demonstrated to restore damaged warm ischemic livers to transplantable condition in animal models. However, the mechanisms of recovery are unclear, preventing rational optimization of perfusion systems and slowing clinical translation of machine perfusion. In this study, organ recovery time and major perfusate shortcomings were evaluated using a comprehensive metabolic analysis of organ function in perfusion prior to successful transplantation. Two groups, Fresh livers and livers subjected to 1 hr of warm ischemia (WI) received perfusion for a total preservation time of 6 hrs, followed by successful transplantation. 24 metabolic fluxes were directly measured and 38 stoichiometrically-related fluxes were estimated via a mass balance model of the major pathways of energy metabolism. This analysis revealed stable metabolism in Fresh livers throughout perfusion while identifying two distinct metabolic states in WI livers, separated at t = 2 hrs, coinciding with recovery of oxygen uptake rates to Fresh liver values. This finding strongly suggests successful organ resuscitation within 2 hrs of perfusion. Overall perfused livers regulated metabolism of perfusate substrates according to their metabolic needs, despite supraphysiological levels of some metabolites. This study establishes the first integrative metabolic basis for the dynamics of recovery during perfusion treatment of marginal livers. Our initial findings support enhanced oxygen delivery for both timely recovery and long-term sustenance. These results are expected to lead the optimization of the treatment protocols and perfusion media from a metabolic perspective, facilitating translation to clinical use.     

Hypothermic perfusion of rabbit livers: effect of perfusate composition (Ca and lactobionate) on enzyme release and tissue swelling

Rabbit livers were preserved by continuous hypothermic (5 degrees C) perfusion at a flow rate of 1 ml/min-1 g-1 for as long as 72 hr. Cell swelling (total tissue water, TTW) and the rate at which intracellular enzymes were released into the perfusate were measured. Livers perfused with a simple NaCl-based solution containing hydroxyethyl starch as a colloid released relatively large amounts of aspartate aminotransferase (AST, 442 +/- 224 u/liter-1 100 g-1) and lactic dehydrogenase (LDH, 1580 +/- 688 u/liter-1 100 g-1) into the perfusate during 72 hr of perfusion. The addition of Ca (0.5 mmol/liter) to the perfusate reduced the leakage of enzymes into the perfusate (AST, 70 +/- 30 u; LDH, 450 +/- 50 u) and reduced cell swelling (TTW, 3.1 kg/kg dry mass vs 4.4 kg/kg dry mass without added Ca). But the use of a higher concentration of Ca (1.5 mmol/liter) caused membrane damage (AST, 4000 +/- 1500 u; LDH, 10,000 +/- 2222 u) and increased cell swelling (TTW, 3.7 kg/kg dry mass). The release of intracellular enzymes caused by continuous perfusion with a chloride-based perfusate also could be reduced by replacing the chloride with lactobionate (AST, 100 +/- 30 u; LDH, 400 +/- 100 u, at 72 hr). In the lactobionate-containing perfusate, the addition of Ca (0.5 or 1.5 mmol/liter) did not alter the rate at which intracellular enzymes were released. There was no tissue swelling after 72 hr of preservation with the lactobionate-containing perfusate, and the TTW (2.1 kg/kg dry mass) was similar to the TTW of freshly harvested rabbit livers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Utilization of fatty acids in perfused hypothermic dog kidney

Utilization of oleic acid in whole dog kidneys perfused in vitro for 24 hr at 10 degrees C was studied, and the data were correlated with results on the utilization of oleic acid in kidney slices incubated in the same perfusate at 10 degrees C. Kidneys perfused without added oleate lost 35% of their total lipid content and 27% of their phospholipids. Addition of serum albumin-bound oleate to the perfusate prevented the loss of neutral lipid and reduced the loss of phospholipid to 8%. The kidney slices incorporated 29% of the added oleate into lipid and oxidized 3.2% to CO(2). Oleate apparently largely replaces endogenous fatty acids which are oxidized to meet the energy requirements of the kidney. The loss of phospholipid from the perfused organ is taken as an indication of cell damage, which may be reduced but is not prevented by the addition of oleate to the perfusate.     

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.     

Metabolic and mitochondrial disturbances in streptozotocin-treated Sprague-Dawley and Sherman rats

This study provides explanation for conflicting evidence in the literature relating to changes in mitochondrial function and metabolic parameters during chemically induced diabetes. Diabetes of 3 days' duration (early ketosis) did not alter heart, kidney, or liver mitochondrial respiratory rates with glutamate or succinate even though serum glucose and triglycerides were elevated. Diabetes of 5 weeks' duration did not alter kidney or liver mitochondrial function in the fed adult rat although weight gain was depressed. The amount of kidney mitochondrial protein isolated per gram of tissue was increased by 30% in the diabetic. This increase was reversed by insulin treatment as were the other biochemical modalities measured. Superimposition of a 24-hr fast resulted in enhanced gluconeogenesis as measured by an animal weight loss of 17% within 24 hr (liver weight loss, 21%) and an elevation of serum urea nitrogen by 180% compared to fasted control. Respiratory rates of diabetic kidney mitochondria with glutamate were unaffected in the fasted animal whereas diabetic liver mitochondrial respiratory rates during succinate oxidation were reduced by 43%. Respiratory control was unchanged in the fasted diabetic rat. All the observed changes were reversed by insulin. Variation in the serum and liver metabolic indices (urea nitrogen, creatinine, glycerol, free fatty acids, free amino acids, triglycerides, and glucose) and liver mitochondrial responses to 7 weeks of chemically induced diabetes was affected by the rat strain, Sprague-Dawley versus Sherman, and rat weight, 72 g versus 222 g. Liver mitochondrial respirations in fed Sherman rats were not depressed by diabetes. Both rat strains had elevated liver free fatty acids and glutamate dehydrogenase activity in the diabetic state. Serum leucine, isoleucine, and valine were more elevated and serum lysine and arginine were more depressed in the diabetic Sprague-Dawley rat than in the Sherman rat. Conjectures on these results are presented in the text.     

Ventriculo–cisternal perfusion of twelve amino acids in the rabbit

The clearances of twelve amino acids from the ventricles during ventriculocisternal perfusion in the rabbit have been measured; uptake by the brain was also measured and this permitted the separate computation of loss to brain and loss to blood during the perfusion. Clearance under carrier-free conditions was greater than when a concentration of 5mM unlabeled amino acid was present in the perfusion fluid. Brain uptake was also usually reduced by the presence of unlabeled amino acid due presumably to suppression of accumulation by brain cells. Reduction of transport across the blood-brain barrier would tend to increase brain uptake, and there was some evidence for a balance between the two opposing tendencies. Inhibition of clearance of a given labeled amino acid could be brought about by unlabeled amino acids of different molecular species. In general, the amino acids fell into three categories: neutral, acidic, and basic, and there was some overlap between them; of the neutral amino acids the A- and L-classification of Christensen was valid, although once again there was some overlap. If, during ventriculo-cisternal perfusion of a labeled amino acid, the activity of this labeled amino acid in the blood was raised well above that in the inflowing perfusion fluid, the labeled amino acid continued to be cleared from the perfusion fluid, suggesting uphill transport. On this basis it was suggested that the normally low concentrations of amino acids in the cerebrospinal fluid (CSF), by comparison with those in plasma, were due to an active transport from the CSF to the blood. Substrate-facilitated transport, whereby the penetration of labeled amino acid into the perfusion fluid from blood could be accelerated by adding unlabeled amino acid to the perfusion fluid, or vice versa, was demonstrated.     

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.     

Protein synthesis in isolated perfused rat skeletal muscle. Contribution of intra- and extracellular amino acid pools

Protein synthesis was measured in perfused rat skeletal muscle using [14C]lysine in the presence of increasing amounts of lysine in the perfusate. The results obtained clearly indicated that both the extracellular and intracellular pools of amino acids act as direct precursors for protein synthesis and analysis of the data, according to O'Hara et al. (J. molec. cell. Cardiol. 13, 925-940, 1981) further indicated that extracellular sources of lysine supplied 64 and 81% of the lysine incorporated in the absence and presence of insulin, respectively. The rate of protein synthesis were assessed to be 0.22 mumol lysine incorporated g-1 muscle per 3 hr of perfusion increasing to 1.0 mumol g-1 3 hr-1 in the presence of insulin.     

Tyrosine and phenylalanine concentrations in haemolymph and tissues of the American cockroach, Periplaneta americana, during metamorphosis

Phenylalanine and tyrosine concentrations were measured in the haemolymph, fat body, and abdominal integument of the American cockroach, Periplaneta americana, during the pre- and post-ecdysial periods of cuticle formation and sclerotization.Gas-liquid chromatography of trimethylsilyl derivatives of phenylalanine, tyrosine, and their metabolites provided a very sensitive and rapid method for determining those amino acids in small haemolymph and tissue samples.Haemolymph tyrosine increased in two stages: initially near apolysis and 16 to 25 hr pre-ecdysis, reaching its highest concentration at ecdysis (3·5 μg tyrosine/mg haemolymph). During that time, total haemolymph tyrosine increased by approximately 700 μg/insect. Fat body and abdominal integument began to accumulate tyrosine near apolysis. Fat body tyrosine peaked between ecdysis and 3·3 hr post-ecdysis whereas abdominal integument tyrosine peaked at ecdysis. Maximum concentrations were 6·0 μg and 4·1 μg tyrosine/mg wet wt. of tissue, respectively. Between ecdysis and 24 hr post-ecdysis, the period of maximum sclerotization, total tyrosine in haemolymph and fat body decreased by approximately 600 μg and 420 μg/insect, respectively. Phenylalanine concentrations did not change significantly in the haemolymph, fat body, or abdominal integument during the pre- and post-ecdysial periods.The cockroach apparently does not store free phenylalanine or tyrosine in the fat body during larval development as compared to tyrosine storage in some Diptera. The rapid increase of haemolymph, fat body, and integument tyrosine just prior to ecdysis suggests another form of storage for this important amino acid.