Canine Kidneys Research Articles

Metabolism of canine kidneys in anaerobic ischemia and in aerobic ischemia by persufflation with gaseous oxygen

The changes in the status of the adenylic acid-creatine phosphate system and in glycogen, glucose, and lactate were investigated in canine kidneys during preservation in anaerobic and aerobic ischemia at normo- and hypothermia. For anaerobic ischemia, the kidneys were stored without additional measures; for aerobic ischemia, the ischemic kidneys were persufflated with gaseous O2 or gas mixtures containing O2 via the renal artery or vein (orthograde and retrograde aerobic ischemia respectively). Prior to preservation, the kidneys were flushed with cell-free perfusates. Changes in the metabolic status characteristic of anaerobic ischemia were a decrease in the tissue levels of ATP, creatine phosphate, glycogen, glucose, and the sum of adenine nucleotides, a transitory increase in ADP and AMP, and an increase in the tissue level of lactate. Hypothermia caused a retardation of the anaerobic alterations; the loss of ATP was slowed down by factors of 2 and 9.5 when the temperatures were lowered from 37° to 26° and 6°C, respectively. In aerobic ischemia with orthograde and retrograde persufflation of 100% O2 (persufflation pressure = PP 60 and 30 mm Hg), deviations from a regular metabolic status developed very slowly. While the ATP-level decreased to 50% of the control value within scarcely 30 min of anaerobic ischemia at 6°C, it was almost 48 h before a similar loss of ATP occurred during orthograde aerobic ischemia at 6°C. Reduction of O2 in the persufflation gas to 40 and 21 vol-% resulted in less adequate preservation of the metabolic status. Increasing PP to 100 mm Hg for orthograde persufflation had no effect. An increase in PP to 60 mm Hg for retrograde persufflation resulted in a further delay in the metabolic deterioration. The long-term preservation of a regular metabolic status or the slow deviation of the metabolic pattern from the normal range during ischemia with O2-persufflation indicates a significantly smaller energy deficit than during storage in anaerobic ischemia. It must be attributed to a sufficient aerobiosis in the aerobically ischemic kidney, during which organogenic substrates brought in by the initial perfusion are utilized.

Ultrastructure of Canine Renal Autografts Following 24-Hour Hypothermic Preservation

29 canine kidneys were preserved for 24 h by hypothermic, pulsatile or nonpulsatile perfusion with albumin or cryoprecipitated plasma and were autotransplanted. Electron microscopy of biopsies taken at 60 min posttransplantation revealed no significant ultra-structural differences between surviving kidneys preserved with pulsatile or nonpulsatile flow, or between organs preserved with plasma or the albumin solution. Comparison of surviving and nonsurviving grafts, however, revealed conspicuous glomerular ultrastructural changes and the presence of polymorphonuclear leukocytes in the capillaries of the nonsurvivors. These changes appear to be useful in determining graft viability following preservation.

Cryoprotective properties of various gases in kidney preservation (author's transl)

In order to improve cryopreservation of whole organs, possible cryoprotective properties of various gases and gas mixtures were studied in hyperbaric kidney preservation by freezing to ––32 °C at 5–7 atü. After a 12-day storage period the canine kidneys were connected to the neck vessels of the donor and function assessed by measuring urinary output, urea clearance and creatinine clearance after recirculation. In addition, microscopic examination was performed. Using various hyperbaric gases clear differences in function and renal structure were obtained. Xenon pure and xenon/carbogen mixtures demonstrated the best results.

Endothelial damage in perfused dog kidney and cold sensitivity of vascular NaK-ATPase

After prolonged perfusion of canine kidneys with cryoprecipitated plasma at 10 °C, endothelial damage begins between the fourth and sixth day. When aortic intimal strips were used as a model of the renal vascular endothelium, the ATPase activity of the intima was completely inhibited at 10 °C, but was 100% active at 37 °C. Because of the difficulty in isolating viable endothelial cells, strips of aortic intima were used, since this tissue is probably representative of renal vascular endothelium. The difference in cold sensitivity of the renal vasculature as compared to the renal parenchymal cells is probably an important factor in the inability to store kidneys consistently for 7 days or more by hypothermic perfusion.

Renal cortical transit times. A simple parameter for evaluation of renal perfusion.

Renal cortical transit times in dogs were measured by videodensitometry. The calculation of two, interdependent parameters gives normal values for canine kidneys. A relatively simple, inexpensive device, the videodensitometer should provide meaningful clinical information on renal vascular perfusion.

Effects of temperature on kidneys preserved by hypothermic perfusion.

The feasibility of renal function studies as viability assays during isolated in vitro perfusion at 25 C has already been described. The present study demonstrates the applicability of this assay system to currently improved perfusion protocols at lower temperatures. Carefully isolated canine kidneys were perfused for 2 hr at 5 C with homologous pooled plasma exposed to 95% 02/5% CO2 in a membrane oxygenator. One-half-hour samples of urine and plasma were collected for urine: plasma (U:P) ratios and clearance determinations. Temperatures were then returned to 25 C for 1 hr to allow comparison between retained function and previously established base line values. Randomly chosen kidneys were autografted to nephrectomized donors as a final test of functional capacity. Although decreases in mean perfusion pressure and renal plasma flow (RPF) of 20 mm Hg and 50% were respectively noted, a 66% increase in renal resistance occurred at 5 C. A 75% drop in renal oxygen consumption was accompanied by low U:PPAE, U:PNa, and U:PK values, indicative of diffuse suppression of activity throughout the nephron. Marked decreases in creatinine and para-aminohippuric acid (PAH) clearance effected a falloff in mean urine flow. This metabolic and physiological inhibition was completely reversible at 25 C, and autografted kidneys functioned normally after minimal elevations of blood urea nitrogen and creatinine occurred during the 1st postoperative week. While 5 C temperatures appear to preclude most studies of nephron physiology, values are still obtainable to permit in vitro preoperative assessment of functional integrity.

Hypothermic pulsatile perfusion of canine kidneys. The course of the lactate-pyruvate ratio in various experimental conditions

Hypothermic pulsatile perfusion of canine kidneys. The course of the lactate-pyruvate ratio in various experimental conditions

Studies on renin. II. Continuous infusion of homologous renin at very low rates in intact or nephrectomized, conscious or anesthetized dogs.

We have devised a method of evaluation, in terms of Goldblatt units (GU), of the data obtained in various laboratories for the excretion of renin from canine and human kidneys. An average value of 1.3 x 10 -4 GU/min g -1 normal renal tissue resulted for both dog and man. A blood pressure increase of 18-45 mm Hg was induced in normal or nephrectomized, conscious or anesthetized dogs by the continuous infusion of homologous renin at even lower rates (0.25-0.50 x 10 -4 GU/min g -1 renal tissue), i.e., at 20-40% of the rate excreted by the kidney. This suggests that renin, even at its normal rate of excretion from the kidney, participates in the maintenance of normal blood pressure. The increase of blood pressure and of concentration of renin in the dog's blood was measured at infusion rates ( C ) of 0.0042, 0.0083, 0.0167, 0.0333, and 0.0667 GU/min. At each rate, after a fixed period of infusion ( t = 60 minutes), a constant fraction (54% of the infused renin) was retained in the circulation. From this and the results of infusion experiments carried out for 30, 60, and 120 minutes, the total amount of exogenous renin, R , present in the circulation at time t can be expressed as R = C/K (1 - e -Kt ). K = 0.0232 minutes -1 and represents the fraction of exogenous and, presumably, also endogenous renin that is being eliminated from the circulation of the normal dog per minute. Thus, the time required for removal of 50% of the renin from the blood is t ½ = log e 2/ K = 30 minutes.

Attempted antigenic alteration of canine kidneys by 24- or 48-hr perfusion with immunosuppressive agents.

Attempted antigenic alteration of canine kidneys by 24- or 48-hr perfusion with immunosuppressive agents.

Experimental studies on the preservation of the canine kidney. Histochemical enzyme activities and mitochondrial respiration

The canine kidneys were preserved at a low temperature (2°-4°C) in a hyperbaric oxygen chamber (3ATM) up to 168 hours. These kidneys had been removed few minutes after autotransplantation and examined for histochemical enzyme activities and mitochondrial respiration. The following results were obtained.1. Histological findings (H. E. stain)Although great individual differences are seen in the histological findings, the changes such as dissociation of the epithelium of the proximal tubuli begin to appear at about 24 hours of preservation. Generally hyperemia of interstitial capillaries, especially those in the renal medulla, is intense. The interstitium is somewhat edematous. At about 72 hours of preservation, dilatation and hyperemia of the glomerular arterioles are markedly intensified and the presence of vascular disturbance can be inferred. However, even at 168 hours of kidney preservation some healthy nephrons persist.2. Histochemical enzyme activitiesEnzyme activities of LAP, SDH, ATP-ase, β-G, Al-p-ase and Acid-p-ase were studied. There was a great variation in enzyme activities in each specimens. However, almost no change was noted in ATP-ase. Other histochemical enzyme activities decreased in parallel with degeneration of the renal tubular epithelium. As the time of preservation lengthens, the areas of decreasing activities progress in extent. However, even at 168 hours some nephrons retain enzyme activities and some healthy nephrons persist. Changes during this period are continuous and it is difficult to judge at what degree of disappearance the viability of the whole kidneic is irretrievably lost.3. Electron microscopic findings of the mitochondrial fractionsMitochondria were separated by the Hogeboom and Schneider method and confirmed through the electron microscope. However, even mitochondria obtained from a healthy control kidney showed marked swelling and rupture of their outer membranes. No characteristic differences in parallel with the preservation time were seen from the morphological findings by the electron microscope.4. Respiratory control function of the mitochondriaThe average values for the ADP/0 ratio and RCI (Respiratory Control Index) for the healthy control kidneys were 1.246 and 1.99 respectively. The ADP/0 ratio and RCI of mitochondria of preserved kidneys was calculated on a percentage basis based on the healthy control kidneys. Up to 24 hours, excluding those in which Indigo carmine excretion was not observed within 10 minutes, the ADP/0 ratio was more than 80%, and RCI was at the 80% level. In the 48 hours preserved kidney two groups were established, one group with the ADP/0 ratio maintained at 70-80% and the other group with a decrease and eventual disappearance being noted. The number in the two groups was the same, being 37.8% on the average. RCI was 70% on the average when unmeasurable cases were made to represent 0. As for the kidneys preserved more than 72 hours, the ADP/0 ratio was 0 in all cases and it was inferred that oxidative phosphorylation was disturbed. In the 168 hours preserved kidney the respiratory control function was extinguished. From the above findings it can be accepted that preservation of the kidney at 2°-4°C under 3ATM oxygen for 48 hours is the limit of viability. Preservation for more than 72 hours is extremely difficult.

Metabolism of canine kidneys in anaerobic ischemia and in aerobic ischemia by persufflation with gaseous oxygen.

The changes in the status of the adenylic acid-creatine phosphate system and in glycogen, glucose, and lactate were investigated in canine kidneys during preservation in anaerobic and aerobic ischemia at normo- and hypothermia. For anaerobic ischemia, the kidneys were stored without additional measures; for aerobic ischemia, the ischemic kidneys were persufflated with gaseous O2 or gas mixtures containing O2 via the renal artery or vein (orthograde and retrograde aerobic ischemia respectively). Prior to preservation, the kidneys were flushed with cell-free perfusates.

Trickle perfusion for organ preservation.

THE two most successful methods of preserving canine kidneys are those described by Collins et al.1 and Belzer et al.2. In the Collins system, which is extremely simple, the organ is flushed with a solution (“Collins C4”) similar to intracellular fluid with a high potassium, phosphate and magnesium content. This solution is left in the kidney stored at 4° C, which will function well when re-implanted after 24 h. It is essential, however, that there should be no warm ischaemic damage to the kidney3. In Collins's experiments, the animals were very carefully maintained so that the kidneys were not maltreated until they were removed and rapidly cooled.

Effects of radiopaque dyes on renal vascular resistance.

Abstract Radiopaque contrast medium (diatrizoate, 50 per cent, or iodopyracet, 45 per cent) was injected into the renal artery; renal vascular resistance and urine flow were measured in naturally perfused and pump-perfused canine kidneys. Bolus injections of hyperosmotic solutions contrast medium, NaCl (sodium chloride), or urea increased renal vascular resistance for 2 to more than 20 minutes in both preparations. Blood flow was decreased in naturally perfused kidneys and perfusion pressure was increased in pump-perfused kidneys. The magnitude and duration of this response varied directly with the control resistance. The greater the control resistance, the greater was the response. The rise in renal resistance was accompanied regularly by a rise in urine flow and less frequently by gross hematuria and a transient increase in aortic pressure. Injections of isosmotic solutions of the contrast medium or NaCl did not alter renal resistance. Slow infusion of hyperosmotic contrast medium lowered renal resistance. These studies indicate that the rise in renal vascular resistance was dependent on the rate and amount of contrast medium injected and on the control resistance. It is suggested that renal arteriography be done with caution in patients whose renal vascular resistance is likely to be high.

ATP in the cortex of canine kidneys undergoing hypothermic storage

Adenosine-triphosphate (ATP) was determined in the cortex of canine kidneys undergoing hypothermic storage in vitro for 24–72 hours. After a rapid initial decrease during normathermia, continuous hypothermic perfusion resulted in partial restoration of the ATP level. Hypothermic storage with only initial perfusion resulted in a much slower decrease in ATP than did normothermia. The rate of disappearance was slower for an intracellular than an extracellular type of electrolyte solution. The efficacy of the various preservation methods as reflected in the results of functional studies, seems to be correlated with the ATP level in the renal cortex.

Timed Sequential Ultrastructural Changes of Canine Kidneys Unflushed and After Flushing with Plasma

Several methods and solutions have been used to preserve kidneys for transplantation. Among these are Collins C4; Ringer's Lactate and by perfusion by the (Belzer) LI-400 preservation system using plasma. In this experiment we hope to demonstrate ultrastructural changes of kidneys which were flushed with plasma, compared to kidneys which were unflushed.Eight dogs were used in our experiments. Each dog was anesthesized, the kidneys exposed, biopsied and then removed. Four dogs were sacrificed. One kidney from each dog was flushed with 150 ml of cold plasma, the other was left unflushed. They were then immersed in iced saline and kept at 0° to 4°C for the duration of the experiment. Biopsy times were 0, 1/2, 1, 2, 4, 8, 16 and 24 hours. The kidneys of the four remaining dogs were autografted at various intervals to compare ultrastructural changes with viability. All biopsy specimens were prepared for electron microscopy.

Ultrastructural Changes of Canine Kidneys During 24-Hour Perfusion on a LI-400 Preservation System

Short term experimental and clinical preservation of kidneys is presently best accomplished by hypothermic continuous pulsatile perfusion with cryoprecipitated and millipore filtered plasma. This study was undertaken to observe ultrastructural changes occurring during 24-hour preservation using the above mentioned method.A kidney was removed through a midline incision from healthy mongrel dogs under pentobarbital anesthesia. The kidneys were flushed immediately after removal with chilled electrolyte solution and placed on a LI-400 preservation system and perfused at 8-10°C. Serial kidney biopsies were obtained at 0-½-1-2-4-8-16 and 24 hours of preservation. All biopsies were prepared for electron microscopy. At the end of the preservation period the kidneys were autografted.

Effect of choline and hypothermia upon H + production and K + loss from the isolated canine kidney

Potassium loss and venous effluent pH were determined in four canine kidneys subjected to 4 hr of intermittent hypoxic perfusion with isotonic NaCl or choline chloride. Although often used as an inert cation, choline may alter sodium flux and metabolic activity of tissue slices. Approximately 60% of estimated renal potassium was lost after NaCl or choline chloride perfusion at 37 °C whereas 18–24% of potassium was lost at 5 °C. During normothermic perfusion with NaCl or choline chloride, venous effluent pH fell to 6.17 and 5.40, respectively. Fall in pH and differential effect of NaCl and choline chloride were diminished at 5 °C. A beneficial effect of hypothermia upon potassium loss and hydrogen ion production in the isolated kidney is demonstrated. Choline is a metabolically active substance but probably has no role in organ preservation.

Successful three- to seven-day preservation of canine kidneys. Immediate life-sustaining function.

In 12 experiments dog kidneys have been preserved for periods of 72, 96, 120, and 168 hours. The preservation system employed pulsatile perfusion, membrane oxygenation, hypothermia, and an acellular, cryoprecipitated plasma perfusate high in steroid content. Success, while consistent at 72 hours in six dogs, was less predictable when preservation time was increased. One of each pair preserved for the longer periods was a long-term survivor with immediate life-sustaining function and eventual return to normal or near normal function.

48-hour storage of canine kidneys after brief perfusion with Collins' solution.

Liu, Wen-Pen M.D.; Humphries, Arthur L. Jr. M.D.; Russell, Rufus; Stoddard, Leland D. M.D.; Moretz, William H. M.D. Author Information

The kidney and the calcium paradox

Isolated canine kidneys were perfused with either calcium-containing or calcium-free media. These kidneys did not show appreciable biochemical or functional changes. Renal arteriograms at the conclusion of the perfusion revealed no abnormalities. Introduction of calcium ions following a calcium-free perfusion period resulted in immediate biochemical and functional alterations. This is termed “calcium paradox.” Arteriograms of these kidneys showed extensive extravasation of radiopaque dye, clearly illustrating damage to vessel walls. The importance of the presence of calcium ions in all kidney perfusion media is stressed.