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.     

Stimulation of ATP synthesis in hypothermically perfused dog kidneys by adenosine and PO4

During continuous hypothermic perfusion of dog kidneys there occurs a gradual decrease in ATP from about 1.4 to 0.6 μmol/g wet wt after 5 days of preservation. The loss of ATP can be prevented by including both adenosine (10 mM) and PO₄ (25 mM) in the perfusate. Under these conditions kidney cortex ATP levels were more than double control values — 3.5 μmol/g wet wt. Both adenosine and PO₄ were necessary since omission of one substance resulted in no net synthesis of ATP. Furthermore, these high levels of ATP were obtained only if adequate concentrations of adenosine were maintained during perfusion. Following 3 days of perfusion the adenosine level in the perfusate decreased to about 1 mM and under this condition ATP levels were low. Adenosine levels were maintained in the perfusate by two methods: (1) addition of fresh perfusate or (2) pretreatment of the kidney with the adenosine deaminase inhibitor—deoxycoformycin. The increased levels of ATP appear directly related to the availability of nucleotide precursors and the presence of inhibitors of the enzymes involved in the catabolism of nucleotides and nucleosides (PO₄ and deoxycoformycin). Mitochondrial activity was similar in kidneys with high or low ATP levels following 5 days of preservation.     

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

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

Comparison of the effect of 3- and 5-day hypothermic perfusion of dog kidneys on metabolism of tissue slices

Hypothermic perfusion effectively preserves the viability of kidneys for 3 days. Long-term preservation (5 days or greater) has not been consistently obtained. In this study, the differences between kidneys perfused for 3 and 5 days were compared by determining the "integrated-metabolic" capabilities of tissue slices incubated in vitro at 30 degrees C. The "integrated-metabolic" parameters determined include (1) respiration rates, (2) cell volume regulation [total tissue water (TTW) and saccharide permeable space], (3) rate of reaccumulation of K+ and pumping of Na+, (4) maintenance of ATP concentrations, and (5) mitochondrial functions. Conditions that result in high and low concentrations of ATP following perfusion of kidneys for 5 days were also compared for effects on tissue slice metabolism. The results indicate that energy metabolism in tissue slices is well preserved under all conditions and times of perfusion of kidneys. This includes average respiration rates (315 +/- 50, 275 +/- 35, and 255 +/- 45 mumol O2/hr/g dry wt at 0, 3, and 5 days, respectively, mitochondrial function [respiratory control ratio (RCR) = 4.6, 4.0, and 4.1 for 0, 3, and 5 days, respectively], and steady-state concentration of ATP in slices after incubation (4.0 +/- 1.45, 3.9 +/- 1.28, and 3.3 +/- 0.81 mumol/g/dry wt, for 0, 3, and 5 days, respectively). The primary differences between 3- and 5-day perfused kidneys were the capability of the slices to regulate cell volume and reaccumulate K+. Slices from kidneys perfused for 3 days maintained the TTW at 3.8 kg/kg dry wt, a value similar to that of control tissue slices. However, slices from 5-day perfused kidneys remained swollen (TTW = 4.6 kg/kg dry wt). Also, slices from the 5-day perfused kidney pumped K+ at less than one-half the rate found in slices from control or 3-day preserved kidneys. No significant differences were apparent in the permeability properties of the tissue slices from kidneys perfused for 3 and 5 days to radiolabeled saccharides. The defects in membrane-linked transport functions, resulting from long-term kidney perfusion, were reduced in kidneys containing a high concentration of ATP. The results suggest that one factor which may limit successful preservation of kidneys is the increased membrane permeability (to electrolytes) which is partially prevented by maintaining elevated concentrations of tissue ATP during perfusion.     

Dextran is resistant to lysosomal digestion in kidney tubules

Low molecular weight dextran (Rheomacrodex) was infused into dextran resistant rats in a dose of 5 g/kg body weight. The kidneys were studied by electron microscopy at different time intervals after infusion using a special fixative for the demonstration of dextran. The lysosomes of proximal tubule cells gradually accumulated dextran which remained in small amounts even after 10 days. In separate kidney slice experiments the ability of dextran-loaded proximal tubule lysosomes to digest absorbed proteins was determined using 125I-labelled lysozyme. There were no changes in lysosomal protein digestion. Labelled dextran was resistant to digestion in vitro by homogenates of rat or rabbit kidney cortex or isolated rat lysosomal enzymes. It is concluded that the protein absorption pathway and lysosomal protein catabolism is unchanged after tubular uptake of dextran despite pronounced ultrastructural alterations to the lysosomal system and that dextran is resistant to lysosomal digestion in renal proximal tubules.     

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.     

Changes in lysosome populations in the rat kidney cortex induced by experimental proteinuria

1. Experimental proteinuria (262.9 mg protein/24 hr urine) was induced in rats by repeated intraperitoneal injections of BSA. 2. Hypertrophy of the kidney cortex was significant 8 days after the start of the BSA injections, and the activities of lysosomal enzymes in kidney cortex and urine were significantly higher in proteinuric compared to nonproteinuric rats. 3. Lysosome populations in the kidney cortex were examined by rate sedimentation of the homogenate and by rate zonal and isopycnic centrifugation of the lysosome-rich ML fraction. 4. The activity of lysosomal enzymes in the kidney cortex increased slightly, essentially in the large, fragile lysosomes mainly recovered from the proximal tubule. 5. Proteinuria induced a shift/reduction in the density of small lysosomes from 1.235 and 1.20 g/ml to 1.225 and 1.185 g/ml, respectively. 6. Proteinuria induced a new population of small lysosomes (density 1.185 g/ml) enriched in cathepsin D.     

Effects of insulin on cardiac lysosomes and protein degradation

Hearts perfused in the absence of added insulin had 1) accelerated rates of protein degradation, as assessed by release of phenylalanine and tyrosine; 2) increased rates of release of seven other amino acids; 3) decreased lysosomal latency and sedimentable lysosomal enzyme activity; 4) increased numbers of autophagic vacuoles in cardiac muscle cells; and 5) decreased activity of beta-N-acetylglucosaminidase in dense lysosomes (1.06-1.09 g/ml), as compared to hearts perfused in the presence of the hormone. After 3 h of perfusion in the absence of insulin, the changes that developed in protein degradation, lysosomal latency, and sedimentability, and in enzyme activity in dense lysosomes, were reversed by insulin addition during 90 min of subsequent perfusion. These studies suggest a role for insulin in controlling the activity of the lysosomal system and the involvement of this system in protein degradation, particularly in insulin-deprived tissue.     

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)     

Comparison of the effect of temperature on kidney cortex mitochondria from rabbit,dog, pig,and human: Arrhenius plots of ADP-stimulated respiration

The effect of temperature on the rate of ADP-stimulated respiration of mitochondria from dog, rabbit, pig, and human kidney cortex mitochondria was plotted according to the Arrhenius relationship. The temperature at which the plot demonstrated a break was at 15 °C for mitochondria from dog, pig, and human kidneys. The discontinuity occurred at 10 °C or less for mitochondria from rabbit kidneys. This difference suggests that mitochondria from rabbit kidneys undergo a lipid-phase transition at lower temperatures than for other species commonly used in experimental renal preservation. The implications of this difference suggest caution in using results obtained with rabbit kidneys for comparison to results obtained from hypothermic renal preservation of other species kidneys. Apparent fluidization of dog kidney mitochondrial membranes with adamantine abolished the discontinuity in the Arrhenius plot.     

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.     

Effects of hypothermic perfusion of kidneys on tissue and mitochondrial phospholipids

The changes in the level of phospholipids in kidney tissue and isolated mitochondria from dog kidneys perfused hypothermically (6-8 degrees C) for 1, 3, and 5 days were compared. Following 1 day of perfusion there was no change in total tissue phosphatidylserine (PS), a 25% decrease in the level of phosphatidylethanolamine (PE), and a 16% decrease in phosphatidylcholine (PC). No further decrease was observed with longer perfusion times. In fact, an increase in the level of PE occurred between the third and fifth days. Mitochondria isolated from perfused kidneys also showed a slight decrease in PE and PC following 1 day, no further change at 3 days, and an increase at Day 5. The loss of tissue phospholipids does not appear related to the viability of perfused kidneys. The major loss occurs within 1 day of perfusion and kidneys perfused up to 3 days are fully viable. Five-day perfused kidneys are nonviable, but show no greater loss of phospholipids than the viable 1- or 3-day perfused kidneys.     

Doxycycline Alters the Porcine Renal Proteome and Degradome during Hypothermic Machine Perfusion

Ischemia-reperfusion injury (IRI) is a hallmark for tissue injury in donation after circulatory death (DCD) kidneys. The implementation of hypothermic machine perfusion (HMP) provides a platform for improved preservation of DCD kidneys. Doxycycline administration has shown protective effects during IRI. Therefore, we explored the impact of doxycycline on proteolytic degradation mechanisms and the urinary proteome of perfused kidney grafts. Porcine kidneys underwent 30 min of warm ischemia, 24 h of oxygenated HMP (control/doxycycline) and 240 min of ex vivo reperfusion. A proteomic analysis revealed distinctive clustering profiles between urine samples collected at T15 min and T240 min. High-efficiency undecanal-based N-termini (HUNTER) kidney tissue degradomics revealed significantly more proteolytic activity in the control group at T-10. At T240, significantly more proteolytic activity was observed in the doxycycline group, indicating that doxycycline alters protein degradation during HMP. In conclusion, doxycycline administration during HMP led to significant proteomic and proteolytic differences and protective effects by attenuating urinary NGAL levels. Ultimately, we unraveled metabolic, and complement and coagulation pathways that undergo alterations during machine perfusion and that could be targeted to attenuate IRI induced injury.     

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)     

Latency of acid hydrolases in rat kidney cortex

1. Some lysosomal populations in the rat kidney cortex appear to be mechanically weak and are readily disrupted by gentle homogenization, while other populations remain intact even after repeated homogenization. 2. Lysosomes in the rat kidney cortex appear to be resistant to hypertonic media but are readily disrupted under hypotonic conditions. 3. Lysosomes in rat kidney cortex are readily disrupted when incubated in isotonic sucrose at 37 degrees C. 4. Measurement of total and free activity of three acid hydrolases: N-acetyl-beta-D-glucosaminidase (NAG), acid beta-galactosidase and acid beta-glycerophosphatase, indicates that the latency of these enzymes is relatively low in the homogenate (10-29%) and the ML-fraction (14-42%), but high (60-95%) in the purified large lysosomes (protein droplets). 5. The latency of purified small lysosomes is relatively lower (30-60%) than that of large lysosomes, suggesting that small lysosome populations are relatively permeable to the acid hydrolase substrates. 6. Latency variations of acid hydrolases amongst subcellular fractions appear to reflect the heterogeneity of lysosomal populations present in the kidney cortical homogenate.     

Evidence for degradation of intracellular protein in liver lysosomes of fasted rats

Evidence that intracellular protein degradation occurs in lysosomes has been indirect and derived from liver perfusion (1) or the inhibitor studies (2,3). We report here that liver lysosomes of greater purity are obtained from fed rats than from fasted rats. Lysosomes of less purity may contain an enlarged pool of partially degraded intracellular protein; on the other hand, less purity could be due to less marker enzyme, NAβGase. Measurements of NAβGase activity and lysosomal protein of rat livers showed that both NAβGase and lysosomal protein increased upon fasting but protein more so (3.5 and 6.5x, in 2 days). The increase in lysosomal protein is direct evidence that liver lysosomes are involved in intracellular protein degradation during fasting of rats.     

Ultrastructural localization of lysosomal enzymes in the egg cortex of Brachydanio

The localization of acid phosphatase (E.C. 3.1.3.2), inorganic trimetaphosphatase (E.C. 3.6.1.2), and aryl sulfatase (E.C. 3.1.6.1) in the cortex of unactivated and activated eggs of Brachydanio was examined by ultrastructural cytochemistry. Using a lead capture method, activity for all three acid hydrolases was demonstrated in organelles of the cortex before and after egg activation. Acid phosphatase (AcPase) reaction product was consistently present in primary lysosomes, secondary lysosomes, multivesicular bodies, and yolk bodies. AcPase activity was absent from mitochondria, profiles of the endoplasmic reticulum, coated pits of exocytosed cortical granules, and coated vesicles. Although most cortical granules of the mature, unactivated egg were unreactive for this enzyme, a few showed AcPase reaction product. It is not clear whether the AcPase-positive granules might be an immature form of cortical granules or a subpopulation of these organelles with lysosomal properties. Most cisternae of the Golgi apparatus did not stain for AcPase; however, reaction product was occasionally localized in a single cisterna as well as several small vesicles at the inner face of the Golgi. The intensity of the reaction product and the pattern of distribution of trimetaphosphatase (Tm-Pase) activity was very similar to that of AcPase. However, TmPase was never observed in cortical granules. Cortices of unactivated and activated eggs showed less overall aryl sulfatase (ArSase) activity when compared with AcPase and TmPase. The presence of ArSase reaction product in lysosomes and multivesicular bodies confirmed the acid hydrolytic nature of these organelles. AcPase and TmPase, and to a lesser extent ArSase, are adequate markers of a cortical lysosomal system in the danio egg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Defective lysosomal enzyme secretion in kidneys of Chediak-Higashi (beige) mice

The beige mouse is an animal model for the human Chediak-Higashi syndrome, a disease characterized by giant lysosomes in most cell types. In mice, treatment with androgenic hormones causes a 20-50-fold elevation in at least one kidney lysosomal enzyme, beta-glucuronidase. Beige mice treated with androgen had significantly higher kidney beta-glucuronidase, beta-galactosidase, and N-acetyl-beta-D-glucosaminidase (hexosaminidase) levels than normal mice. Other androgen-inducible enzymes and enzyme markers for the cytosol, mitochondria, and peroxisomes were not increased in kidney of beige mice. No significant lysosomal enzyme elevation was observed in five other organs of beige mice with or without androgen treatment, nor in kidneys of beige females not treated with androgen. Histochemical staining for glucuronidase together with subcellular fractionation showed that the higher glucuronidase content of beige mouse kidney is caused by a striking accumulation of giant glucuronidase-containing lysosomes in tubule cells near the corticomedullary boundary. In normal mice lysosomal enzymes are coordinately released into the lumen of the kidney tubules and appreciable amounts of lysosomal enzymes are present in the urine. Levels of urinary lysosomal enzymes are much lower in beige mice than in normal mice. It appears that lysosomes may accumulate in beige mice because of defective exocytosis resulting either from decreased intracellular motility of lysosomes or from their improper fusion with the plasma membrane. A similar defect could account for characteristics of the Chediak-Higashi syndrome.     

Subcellular distribution of histone-degrading enzyme activities from rat liver.

Chromatin prepared from liver tissue contains a histone-degrading enzyme activity with a pH optimum of 7.5-8.0, whereas chromatin isolated from purified nuclei is devoid of it. The histone-degrading enzyme activity was assayed with radioactively labelled total histones from Ehrlich ascites tumor cells. Among the different subcellular fractions assayed, only lysosomes and mitochondria exhibited histone-degrading enzymes. A pH optimum around 4.0-5.0 was found for the lysosomal fraction, whereas 7.5-8.0 has been found for mitochondria. Binding studies of frozen and thawed lysosomes or mitochondria to proteinase-free chromatin demonstrate that the proteinase associated with chromatin isolated from frozen tissue originates from damaged mitochondria. The protein degradation patterns obtained after acrylamide gel electrophoresis are similar for the chromatin-associated and the mitochondrial proteinase and different from that obtained after incubation with lysosomes. The chromatin-associated proteinase as well as the mitochondrial proteinase are strongly inhibited by 1.0 mM phenylmethanesulfonyl fluoride. Weak inhibition is found for lysosomal proteinases at pH 5. Kallikrein-trypsin inhibitor, however, inhibits lysosomal proteinase activity and has no effect on either chromatin-associated or mitochondrial proteinases. The higher template activity of chromatin isolated from a total homogenate compared to chromatin prepared from nuclei may be due to the presence of this histone-degrading enzyme activity.     

The osmotic stability of lysosomes from adult and foetal guinea-pig liver tissue

1. Lysosome-rich fractions were obtained from foetal liver tissues as early as 35 days uterine age. Foetal lysosomes showed the same ;structure-linked latency' and acid hydrolytic potentiality characteristic of their adult counterparts. 2. The osmotic stability of lysosome-rich fraction from foetal guinea-pig liver tissue was greater than that of the corresponding adult lysosome fractions, p-nitrophenyl-phosphatase being used as marker enzyme. 3. The observation was confirmed by using beta-glycerophosphatase and phenolphthalein beta-glucuronidase as alternative marker enzymes. p-Nitrophenyl phosphate and beta-glycerophosphate appear to act as substrates for the same enzyme. 4. By using p-nitrophenylphosphatase activity measurements it was shown that the osmotic stability of foetal lysosomal fractions decreased with increasing foetal age, but at no time achieved the degree of osmotic instability characteristic of adult lysosomal fractions. 5. The correlation of these findings with the intracellular environment of lysosomes is discussed.