The biosynthesis of 2-guanidinoethanol in intact mice and isolated perfused rabbit kidneys

The metabolic pathway for the synthesis of 2-guanidinoethanol (GEt) was studied in intact mice and isolated perfused rabbit kidneys. GEt excretions in 24-hr urine increased after the intraperitoneal injection of ethanolamine (EA) into mice. Perfusion of isolated rabbit kidneys with EA and L-arginine (Arg) enhanced the GEt excretion from the ureter. This enhancement was observed in an EA concentration-dependent manner under the presence of Arg. When glycine (Gly) was added to the perfusion medium together with EA and Arg, the enhancement of GEt excretion was inhibited, whereas, guanidinoacetic acid excretion was increased to the same extent as during the perfusion with Gly and Arg. These results indicate that GEt is synthesized from Arg and EA in the kidney and that this synthesis is catalyzed by Arg:Gly amidinotransferase (EC 2.1.4.1.). We also described the guanidino compound excretion levels, including levels of GEt, in the rabbit, mouse, rat, and cat. The levels varied considerably with mammalian species.     

Transplantation of rabbit kidneys perfused with glycerol solutions at 10 degrees C.

Experiments are described in which rabbit kidneys were perfused with solutions containing 2, 3, or 4 m glycerol and then were autografted in order to measure any functional impairment. The basic perfusate contained Haemaccel (17.5 g/litre) and mannitol (111 mm) and had a total osmolality of 400 mosmol/kg. The perfusion temperature was 10 °C. Glycerol concentration was slowly increased at a rate of 30 mm min^?1 to 2, 3, or 4 m, was held at the maximum concentration for 30 min, and was then decreased to < 0.1 m at 30 mm min^?1. An immediate contralateral nephrectomy was carried out, and the function of the perfused kidney was assessed by serial measurements of blood urea and serum creatinine, by the determination of 24-hr endogenous creatinine clearance at 1 month and by histological examination. It was found that a concentration of 3 m glycerol was readily tolerated.     

Flow distribution and cryoprotectant concentration in rabbit kidneys perfused with glycerol solutions

Rabbit kidneys were perfused at 10 °C with a solution containing gelatin polypeptides (Haemaccel), and glycerol was introduced, and then removed, using a technique that has previously been shown to result in viable kidneys. This involved increasing the concentration of glycerol in the perfusate from zero to a maximum of 3 , holding it at this level for 30 min, and then decreasing it at the same rate to < 0.1 . Measurements were made of the concentration of glycerol in cortex, cortico-medullary zone, and medulla at various stages of perfusion. During the experiments it was observed that vascular resistance increased dramatically toward the end of deglycerolization, and changes in regional perfusate flow were measured by the diffusable indicator method. It was found that renal tissue is effectively permeated by glycerol using this technique. The perfusate flow throughout all regions of the kidney was reduced during deglycerolization but the greatest effect was on cortico-medullary flow, which was found to be abnormally high during the initial stages of hypothermic perfusion, but was severely impaired when the glycerol was removed. The cryoprotectant was almost completely removed by the washout procedure adopted.     

Effect of piperonyl butoxide on organic anion and cation transport in rabbit kidneys

Piperonyl butoxide has been shown to reduce accumulation of cephaloridine in rabbit renal cortex; however, the mechanism responsible for this effect remains unclear. Cephaloridine is a zwitterion and its accumulation in renal cortex has been suggested to be regulated by both organic anion and cation transport systems. Thus, it was of interest to determine the effect of piperonyl butoxide on renal transport of p-aminohippurate (PAH, an organic anion) and tetraethylammonium (TEA, an organic cation). Although pretreatment with piperonyl butoxide markedly inhibited renal cortical uptake of cephaloridine, the same treatment had less inhibitory effect on either PAH or TEA uptake. Efflux of PAH from preloaded renal cortical slices was enhanced by pretreatment with piperonyl butoxide; however, TEA efflux was unaffected. Thus, piperonyl butoxide appears to have effects on renal membrane functions which result in differential effects on PAH, TEA, and cephaloridine transport.     

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.     

Sites of lipase activation and prostaglandin synthesis in isolated, perfused rabbit hearts and hydronephrotic kidneys

The tissue lipids of isolated, perfused rabbit hearts and hydronephrotic kidneys were labelled with [¹⁴C]-arachidonic acid by two different techniques: direct infusion of [¹⁴C]-arachidonic acid in a protein free media into the perfused organ (method A), and recirculation of [¹⁴C]-arachidonic acid in a solution containing albumin (method B). Autoradiography of the labelled organs demonstrated that method A resulted in selective labelling of arteries and arterioles in both perfused organs as well as glomeruli in the kidney. Labelling with method B resulted in a non-specific radioisotope incorporation in both the vasculature and myocardial cells in the heart; and of the vasculature and renal tubules in the perfused kidneys. Analysis of the tissue lipids shows similar patterns of incorporation of radioactivity between methods A and B.Peptide hormone stimulation (bradykinin) and non-specific noxious stimulation (with transient ischemia) were employed to elicit lipase activation (i.e., release of [¹⁴C]-arachidonate) and prostaglandin (PG) synthesis. It was found that in both hearts and hydronephrotic kidneys, the radioactive PG release in response to bradykinin and ischemia was much higher with method A (vascular labelling) than with method B (diffuse labelling) despite the appearance of comparable amounts of bioassayable PG release, thus indicating the sites of PG synthesis in these organs is predominantly localized in the vascular tissue. Furthermore, the radioactive arachidonic acid release in response to bradykinin stimulation in the hydronephrotic kidneys was 3 times higher with method A than with method B, suggesting the predominant sites of hormone specific lipase activation in the renal cortex is also in the vasculature. However, the radioactive arachidonic acid release in response to ischemia was much higher with method B than with method A in both hearts and hydronephrotic kidneys, indicating the sites of non-specific lipase activation in these organs are more diffusely distributed, and present also in the myocardial cells and renal tubules.     

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.     

Relationship between cation accumulation and water content of salt-tolerant grasses and a sedge

Abstract Salt-tolerant grasses and a sedge were grown at three salinities in a controlled-environment greenhouse. They were measured for growth rate, ash content, water content and cations. Fourteen species from the genera Sporobolus, Aeluropus, Leptochloa, Paspalum, Puccinellia, Hordeum, Elymus, Distichlis and Spartina survived up to the highest salt treatment (540 mol m^?3 NaCl). These were designated halophytes. Eleven species from the genera Triticum, Phragmites, Dactylotenium, Cynodon, Polypogon, Panicum, Jovea and Heleocharis only survived up to 180 mol m^?3 NaCl and were designated salt-tolerant glycophytes. All species except Distichlis palmeri grew fastest on the non-saline control treatment. All species tended to have higher Na⁺ contents and lower K⁺ and water contents on saline treatments compared to control plants. Halophytes differed from glycophytes in having statistically significant lower water contents on the non-saline treatment, and lower ash contents and Na:K ratios on 180 mol m^?3. However, the range of values among species was greater than the differences between halophytes and glycophytes. All species appeared to use Na⁺ accumulation and loss of water as the main means of osmotic adjustment. Three halophytic species were grown for a longer period of time to check the above results. The osmolality of the cell sap was measured directly by the vapour pressure method and compared to calculated values based on Na⁺, K⁺ and water contents (and assuming a balancing anion such as Cl^?). Na⁺ and K⁺ alone could account for greater than 75% of the osmotic potential at all salinities. Hence, the accumulation of organic solutes did not appear to be an important factor in the osmotic adjustment of these species. The results support the conclusion that grasses coordinate Na⁺ uptake and water loss to maintain a constant osmotic potential gradient between the shoot tissues and the external solution. The results were compared to a previous study with dicotyledonous halophytes at the same location.     

The cation content of phospholipides from swine erythrocytes

Phospholipides from swine erythrocytes were isolated and separated into four reproducible fractions. One of the fractions seems to be pure phosphatidylserine. The others are almost certainly not single compounds, although the analytical data indicate that they represent mixtures considerably simpler than the parent mixture extracted from the cells. All four fractions contained Na⁺ and K⁺, but very little Ca²⁺. Sodium was the predominant cation in two of the fractions under all conditions although the major intracellular cation was potassium. In the other two fractions the ratio Na/K varied with the extraction procedure largely because the quantity of K⁺ seemed to depend on the solvent system used. There appear to be reasons to believe that the entire system of phospholipides binds Na⁺ preferentially. In addition, it was observed that the quantity of Na⁺ found in the lipide extracts varied when the extrusion of Na⁺ from the cells was made to vary. Both of these observations are consistent with the possibility that the phospholipides play some part in the extrusion of Na⁺ from these cells.     

Ammonia formation from glutamine isomers by nonacidotic and acidotic perfused rat kidneys.

The following points summarize these findings: (i) there are 2 glutamine utilizing enzyme systems in the rat kidney; (ii) the cytoplasmic glutamyltransferase system hydrolyzes either glutamine isomer while the mitochondrial localized glutaminase 1 is specific for the L-isomer; (iii) the cytoplasmic pathway contributes 70% of the total renal ammonia production in the normal kidney; (iv) chronic metabolic acidosis results in a 20-fold activation of the mitochondrial glutaminase 1 pathway.     

Lymphocyte monovalent cation metabolism: cell volume, cation content and cation transport

Mechanisms which determine sodium and potassium content and volume of rat thymic and human chronic lymphocytic leukemia (CLL) lymphocytes have been studied. The deleterious effect of cell isolation on monovalent cation content was proven by comparing thymus sodium and potassium concentration to that of thymocytes prepared from autologous hemithymus. In vivo distribution ratios of sodium-24 and potassium-42 between thymus water and plasma water were very similar to the distribution ratios of non-radioactive isotopes (sodium-23 and potassium-39). The similar lymphocyte: thymocyte ratio of (a) cell volume (1.48), (b) cell sodium plus potassium (1.47) and (c) cell water (1.50) demonstrated the close correlation of lymphocyte volume with monovalent cation content and water content. Steady-state CLL lymphocyte sodium (32 ± 1.9 mM) and potassium (131 ± 5.1 mM) and thymocyte sodium (31 ± 1.2 mM) and potassium (136 ± 3.9 mM) were similar; however, these steady-state levels were maintained by quantitatively different membrane functions. Radiopotassium and radiosodium uptake by thymocytes was more rapid than by CLL lymphocytes. Ouabain-sensitive potassium influx was 2.4 times greater in thymic (8.70 ± 2.28 mmoles/cm²/min × 10^?8) than in CLL (3.24 ± 0.45 mmoles/cm²/min × 10^?8) lymphocytes. Potassium exodus was also slower in CLL lymphocytes as compared to thymocytes. Ouabain-sensitive sodium accumulation and ouabain-insensitive sodium accumulation were also slower in CLL lymphocytes than in rat thymocytes. Half-maximal ouabain inhibition of sodium entry was 7.5 × 10^?3 M in thymic and CLL lymphocytes. The inhibitory effect of ouabain on sodium and potassium transport was easily reversible. Oligomycin inhibited ouabain-sensitive potassium accumulation in both lymphocyte types. Four lines of evidence indicate the presence in the lymphocyte of a system of leaks and pumps, the latter subserved by a ouabain and oligomycin-sensitive (sodium-potassium) ATPase: (a) steady-state monovalent cation gradient (K ～ 20:1, Na ～ 5:1), (b) the inability to maintain normal sodium and potassium gradients at cold temperature and in the presence of ouabain, (c) the effect of ouabain and oligomycin on active potassium influx and (d) the restitution of steady-state sodium and potassium concentration after cell isolation, ouabain treatment and cold exposure. CLL lymphocytes as compared to rat thymocytes have a decreased rate of ouabain-insensitive sodium uptake and potassium exodus requiring a reduced rate of active sodium extrusion and potassium accumulation to maintain steady-state cation content. Ouabain-sensitive ATPase is difficult to locate in lymphocytes in vitro possibly because it comprises a very small proportion of membrane ATPase since magnesium activated ecto-ATPase in intact lymphocytes is 1500 to 2500 times that of the intact erythrocyte. The inhibition by ouabain of blast transformation, mitosis, amino acid accumulation and nucleic acid synthesis in vitro, may reflect the importance of ouabain-sensitive ATPase and monovalent cation transport in the function of lymphoid cells.     

Effect of ovulation on the ionic and water content of rabbit oviduct

The water content, extracellular space, intracellular water, potassium, sodium and chloride content of oviduct and uterus removed from rabbits in estrus and 24 and 72 h following hCG injection have been determined. Following ovulation, there was an increase in water content of the region of the oviduct corresponding to the ampullary -isthmic junction which, at 24 h, is probably due to increased intracellular water. In the ampullary-isthmic junction, ovulation decreases the potassium content and, at 24 h in the isthmic region of the oviduct, chloride is reduced. The water and ionic content of mucosa and smooth muscle cells has been calculated and mucosa cells have greater intracellular water and chloride and sodium content and less potassium and extracellular space than the smooth muscle cells of the oviduct. The significance of the changes in ionic and water content is discussed.     

Measuring glomerular number and size in perfused kidneys using MRI

The goal of this work was to nondestructively measure glomerular (and thereby nephron) number in the whole kidney. Variations in the number and size of glomeruli have been linked to many renal and systemic diseases. Here, we develop a robust magnetic resonance imaging (MRI) technique based on injection of cationic ferritin (CF) to produce an accurate measurement of number and size of individual glomeruli. High-field (19 Tesla) gradient-echo MR images of perfused rat kidneys after in vivo intravenous injection of CF showed specific labeling of individual glomeruli with CF throughout the kidney. We developed a three-dimensional image-processing algorithm to count every labeled glomerulus. MRI-based counts yielded 33,786 ± 3,753 labeled glomeruli (n = 5 kidneys). Acid maceration counting of contralateral kidneys yielded an estimate of 30,585 ± 2,053 glomeruli (n = 6 kidneys). Disector/fractionator stereology counting yielded an estimate of 34,963 glomeruli (n = 2). MRI-based measurement of apparent glomerular volume of labeled glomeruli was 4.89 × 10(-4) mm(3) (n = 5) compared with the average stereological measurement of 4.99 × 10(-4) mm(3) (n = 2). The MRI-based technique also yielded the intrarenal distribution of apparent glomerular volume, a measurement previously unobtainable in histology. This work makes it possible to nondestructively measure whole-kidney glomerular number and apparent glomerular volumes to study susceptibility to renal diseases and opens the door to similar in vivo measurements in animals and humans.     

Metabolism of bile alcohols in the perfused rabbit liver.

The mechanism and sequence of side chain hydroxylation of cholesterol in bile acid synthesis was studied in the isolated perfused rabbit liver. A comparison was made between the importance of 26- and 25-hydroxylation in cholic acid biosynthesis in the rabbit. The formation of [G-3H]cholic acid was observed when the liver was perfused with 5beta-[G-3H]cholestane-3alpha, 7alpha-diol, 5beta-[G-3H]cholestane-3alpha, 7alpha-12alpha-triol, and 5beta-[G-3H]cholestane-3alpha, 7alpha, 26-triol. No [G-3H]chenodeoxycholic acid was detected in the bile. These findings indicate that potential precursors of chenodeoxycholic acid were hydroxylated at position 12alpha either subsequent to or before hydroxylation of the cholesterol side chain. In addition, no other intermediates (tetrahydroxy or pentahydroxy bile alcohols) were found in the bile when these compounds were perfused in the liver. Bile acid precursors were detected in bile when the rabbit liver was perfused with 5beta-[24-14C]cholestane-3alpha, 7alpha, 25-triol. The 5beta-[24-14C]cholestane-3alpha, 7alpha, 25-triol was hydroxylated in the liver at the 12alpha position to yield the corresponding 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol. The tetrol was further metabolized to a series of pentols (5beta-cholestane-3alpha, 7alpha, 12alpha, 22, 25-pentol; 5beta-cholestane-3alpha, 7alpha, 12alpha, 23, 25-pentol; 5beta-cholestane-3alpha, 7alpha, 12alpha, 24, 25-pentol; and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25, 26-pentol). The major bile acid obtained from the perfusion of the 5beta-cholestane-3alpha, 7alpha, 25-triol was cholic acid. The experiments indicated that in the rabbit liver 12alpha-hydroxylation can occur after hydroxylation of the cholesterol side chain at either C-25 (5 beta-cholestane-3alpha, 7alpha, 25-triol) or C-26 (5beta-cholestane-3alpha, 7alpha-26-triol). Apparently, the rabbit can form cholic acid via the classical 26-hydroxylation pathway as well as via 25-hydroxylated intermediates.     

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.