Oxalate accumulation in rat renal cortical slices

Tubular transport of oxalate is thought to be an energy-mediated process which may contribute to the renal deposition of calcium oxalate in a variety of pathologic states. In order to examine this possibility, the renal handling of oxalate was investigated in rat renal cortical slices in vitro. Slices incubated in vitro with 1 microM [14C]oxalate in Krebs-Ringer bicarbonate buffer at 25 degrees C for 180 min achieved a mean slice to medium ratio of 2.8 +/- 0.08 (SEM) and a mean tissue concentration of 7.7 +/- 0.2 mumol/kg dry wt (N = 64). Section freeze-dry autoradiographs demonstrated maximum uptake within proximal tubule cells but no crystals were evident. Substituting N2 for O2, adding KCN, or removing Ca2+ increased uptake of 14C-oxalate. Dinitrophenol (DNP) and iodoacetamide (IoAc), however, significantly decreased, and O degrees C eliminated slice uptake. Slices incubated with 100 microM [14C]oxalate showed a further increase in tissue accumulation and the appearance of [14C]oxalate crystals. Crystals formed in vitro were deposited throughout the tissue. Oxalic acid did not appear to share the organic acid by renal cortical slices in vitro is largely independent of energy-mediated mechanisms.     

Factors influencing renal cryopreservation. II. Toxic effects of three cryoprotectants in combination with three vehicle solutions in nonfrozen rabbit cortical slices

The [K+]/[Na+] ratio of rabbit renal cortical slices was used to examine, at 25 degrees C, the effects on viability of three cryoprotectant agents (CPA) (dimethyl sulfoxide (Me2SO), ethylene glycol, and glycerol) in combination with three vehicle solutions (Krebs-Henseleit (K-H), solution A, and RPS-2). Viability assessment by [K+]/[Na+] for all test solutions was made after incubating the slices in modified Cross-Taggart solution (C-T). With K-H and solution A, all concentrations of ethylene glycol and glycerol resulted in lowered ratios, whereas with Me2SO, concentrations greater than 1.4 M are required to reduce [K+]/[Na+]. With RPS-2 no decrease in the ratios was found until concentrations greater than 2.8 M were reached for all three CPAs. Binding of Me2SO to albumin, studied using [14C]Me2SO, was inhibited by RPS-2 when compared to K-H. Introduction and removal of Me2SO at 10 degrees C allowed an improvement in viability, at higher Me2SO concentrations, as compared to 25 degrees C.     

Factors influencing renal cryopreservation. I. Effects of three vehicle solutions and the permeation kinetics of three cryoprotectants assessed with rabbit cortical slices

A renal cortical slice model was used to assess the effects on viability of three vehicle solutions-Krebs-Henseleit (K-H), solution A, and RPS-2--at 25 degrees C. After 120 min incubation no differences in [K+]/[Na+] ratios were found. Tracer techniques were used to study the osmotic effects and permeation kinetics at 25 degrees C of three cryoprotectants (dimethyl sulfoxide (Me2SO), ethylene glycol, and glycerol) and the effect of the vehicle solution (K-H or RPS-2) on Me2SO kinetics. It was found that Me2SO was most permeable and ethylene glycol least, and that ethylene glycol had unusual effects which suggest that it may not act as a simple solute. Differences were found when Me2SO was introduced in K-H and RPS-2 that are believed to be related to the binding properties of Me2SO to cell constituents.     

Uptake of uric acid and p-aminohippurate (PAH) by renal cortical slices of various mammals.

1. Accumulation of uric acid and PAH was measured in renal cortical slices of various mammalian species. 2. The slice to medium ratio of uric acid was above unity in the rabbit, guinea pig, pig and cow, suggesting an active accumulation of uric acid, while it was near or below unity in the rat and mongrel dog. 3. Uric acid uptake in the rabbit, guinea pig and cow was significantly inhibited by PAH. 4. Uric acid was a potent inhibitor of PAH uptake in the rabbit, guinea pig, dog and pig, but much less potent in the rat and cow. 5. Kinetic analysis showed that uric acid inhibited PAH uptake in a competitive manner in all species studied except for the cow showing a noncompetitive type. 6. These results indicate that uric acid and PAH share a common transport mechanism at the basolateral membrane of the rabbit, guinea pig and pig.     

Effects of cryoprotectants in combination on the survival of frozen sugarcane cells

Cryoprotection of suspension cultures of sugarcane cells (Saccharum sp.) during freezing to various temperatures was tested using glucose, dimethylsulfoxide, and ethylene glycol at various concentrations, alone and in combinations. Viability of the thawed cells was assessed by triphenyltetrazolium chloride reduction, cell growth, and microscopic examination. Enhanced cryoprotection—as much as a doubling in viability value—was achieved by employing glucose and dimethylsulfoxide in mixtures, as compared with the lesser cryoprotective effect of either compound alone, at 1.9 molar total concentration in all cases; the mixture was most effective at a concentration of about 1.9 molar, with a molar ratio of the two components of about 1:3, respectively. Much of the increase in viability value arose from a decrease in toxic effect that came about through mixing the cryoprotective agents. Binary mixtures containing ethylene glycol and either glucose or dimethylsulfoxide were less effective and more toxic than comparable glucose-dimethylsulfoxide mixtures. Use of the optimized latter mixture allowed freezing of these tropical cells to −23 C with little decrease in survival, or to −40 C, still with the capability for delayed growth.     

The effect of norethandrolone on organic ion transport by rat renal cortical slices.

Norethandrolone (NE) and other androgenic steroids have been shown to be renotropic in various species and have also been reported to have salutary effects in patients with diminished renal function. Renal cortical slices prepared from rats pretreated with NE showed an increased capability to concentrate p-aminohippuric acid (PAH). Pretreatment with NE failed to stimulate the transport of the organic base tetraethylammonium and the organic acid benzylpenicillin. Stimulation of PAH transport was observed after eight daily subcutaneous injections of NE. No stimulation was observed with shorter pretreatment intervals. When NE was given subcutaneously for 14 days at doses of 2.6 or 20 mg kg-1 day-1, significant stimulation of PAH transport was seen at all three dose levels but no dose-effect relationship was apparent. Stimulation of PAH transport was seen in female rats as well as castrated and intact males. In addition to its general anabolic properties, NE induces the synthesis of hepatic microsomal drug-metabolizing enzymes. For comparative purposes, therefore, the effect of pregnenolone-16 alpha-carbonitrile (PCN) was also investigated. This agent is a potent inducer of drug metabolism but is neither anabolic nor renotropic. When rats were pretreated with an inducing dose of PCN (75 mg kg-1 day-1 for 3 days), there was no significant stimulation of PAH transport. It would seem, then, that the stimulatory effect of NE on PAH transport is more closely associated with its generalized anabolic effect than with its ability to induce hepatic microsomal enzymes.     

Effects of impermeant medium ions on the composition of rabbit renal cortical slices

When incubated in isosmotic oxygenated medium in which chloride was completely replaced by gluconate, rabbit renal cortical slices lost chloride with sodium, potassium and water before reaching a new steady-state composition after 15-30 min. When corrected for extracellular space, there was an electroneutral loss of alkali metal cations (Na + K) with chloride, accompanied by isosmotic loss of water from the cells. The losses of chloride and water were independent of medium pH over the range of 6.4-8.2, and were the same with potassium rather than sodium as the dominant medium cation. Incubation in isosmotic sodium chloride medium restored tissue composition of slices transferred from gluconate medium. This recovery was not dependent specifically upon medium chloride, for slice water content also recovered when nitrate rather than chloride was substituted for medium gluconate. With sodium completely replaced by n-methyl d-glucamine (nmdG+), cells in slices lost far more sodium and potassium than chloride before reaching a new steady-state composition after some 30 min. However, the loss of water was as predicted from the total losses of measured inorganic ions. With sodium and chloride completely replaced by nmdG+ and gluconate, there was a greater loss of water than found with unilateral substitutions. Again, the combined loss of diffusible inorganic cations exceeded the loss of chloride but the water loss was that expected for isosmotic loss accompanying the measured losses of ions. These results reveal that both gluconate and nmdG+ behave as impermeant ions in this tissue preparation. It is suggested that, in the absence of medium sodium, sodium-hydrogen exchange is inhibited. Retained hydrogen ions are buffered on charged cellular non-diffusible solutes and the associated hydroxyl (or bicarbonate) ions are lost from the cells accompanied by the inorganic univalent cations lost in excess of chloride in nmdG+ medium.     

Effective use of renal cortical slices in transport and metabolic studies

The uptake and metabolism of two water-soluble vitamins were measured in rat renal cortical slices, isolated tubules, and vesicles of the brush-border and basolateral cell membranes to determine (a) whether it is possible to produce slices that have open tubules and, (b) whether slices and tubules metabolize vitamins similarly. Transport of ascorbic acid is sodium-dependent in slices and in brush-border vesicles but is sodium-independent in basolateral vesicles, suggesting that the brush-border membrane of slices is accessible to components of the bathing solution. Nicotinic acid was metabolized similarly (97-98%) in both slices and isolated tubules. Oxygen consumption by slices maintained in a closed chamber was constant as pO2 decreased from 88% to 58%. Slices are concluded to be a suitable model for transport and metabolic studies providing that care is taken in their preparation and use.     

Effective use of renal cortical slices in transport and metabolic studies

The uptake and metabolism of two water-soluble vitamins were measured in rat renal cortical slices, isolated tubules, and vesicles of the brush-border and basolateral cell membranes to determine (a) whether it is possible to produce slices that have open tubules and, (b) whether slices and tubules metabolize vitamins similarly. Transport of ascorbic acid is sodium-dependent in slices and in brush-border vesicles but is sodium-independent in basolateral vesicles, suggesting that the brush-border membrane of slices is accessible to components of the bathing solution. Nicotinic acid was metabolized similarly (97–98%) in both slices and isolated tubules. Oxygen consumption by slices maintained in a closed chamber was constant as pO₂ decreased from 88% to 58%. Slices are concluded to be a suitable model for transport and metabolic studies providing that care is taken in their preparation and use.     

Developmental capacity of bovine oocytes frozen in different cryoprotectants

Iso-osmolar concentrations of 1.6 M of 1,2-propanediol, glycerol and dimethylsulfoxide were compared for their effectiveness as cryoprotectants for in vitro-matured bovine oocytes. Survival, defined as the number of morphologically normal oocytes, after freeze-thawing in 1,2-propanediol (49.8%) was significantly higher (P<0.01) than in glycerol (28.6%) or dimethylsulfoxide (32.6%). After insemination, the fertilization rate of morphologically normal oocytes frozen-thawed in 1,2-propanediol (57.9%) was higher (P<0.05) than that of those frozen in dimethylsulfoxide (38.3%). However, the proportion of surviving oocytes developing to blastocysts was not different among the 3 groups.     

The influence of cooling rate and warming rate on the response of renal cortical slices frozen to -40 degrees C in the presence of 2.1 M cryoprotectant (ethylene glycol, glycerol, or dimethyl sulfoxide)

Renal cortical slices were treated with 2.1 M cryoprotectant in RPS-2 vehicle solution, cooled at one of four rates to -40 degrees C, then immediately warmed at one of four rates to 25 degrees C for determination of the [K+]/[Na+] after a standard incubation period. Results are presented in the form of survival "topographical maps" or surfaces with the x axis representing [K+]/[Na+]; the y axis, cooling rate; and the z axis, warming rate. The rate of temperature change fell in the range of 0.5 to 10 degrees C/min. The results suggest that when RPS-2 vehicle solution is used for 2.1 M cryoprotectants, Me2SO offers the prospect for greatest post-thaw recovery. With this vehicle-cryoprotective agent combination, the greatest post-thaw recovery is attained with cooling-warming combinations of -3, +4, and -0.5, +10 degrees C/min.     

The influence of various cryoprotectants on semen quality of the rainbow trout (Oncorhynchus mykiss) before and after cryopreservation

The influence of permeating and of non-permeating cryoprotectants on motility, eosin permeability (sperm cells viability test) and leaking lactate dehydrogenase activity of spermatozoa of the rainbow trout ( Oncorhynchus mykiss ) was investigated. Correlations between sperm motility rate, percentage of eosinimpermeable (viable) cells and LDH activities established the three parameters as indicators for damages to rainbow trout spermatozoa. High sperm motility rates, velocities, and numbers of linear swimming sperm cells, high numbers of eosinimpermeable spermatozoa and a low extracellular LDH activity were evaluated as positive quality characteristics of semen. With a buffered physiological saline solution as basic extender we found a mixture of 0.67 mol L (5%) dimethylsulfoxide (DMSO) and 0.13 mol/L (1%) glycerol to be the most effective cryoprotectant, followed by DMSO (0.67 mol/L [5%]-1.85 mol/L [15%]) and glycerol (0.65 mol/L [5%]-1.78 mol/L [15%]) and propanediol (1.24mol L [10%]-1.78 mol/L [15%]). Addition of hen egg yolk (7%, 20%) and of 15 mmol/L (0.5%) sucrose significantly increased the semen quality in comparison to the same extender without these additives. Bovine serum albumin (1.5%, 3%) had no influence on the investigated semen parameters.     

Prostacyclin-independence in beta-adrenoceptor mediated renin release from dog renal cortical slices

There is some controversy regarding whether stimulation of renin release by the beta-adrenergic system is dependent on prostaglandin (PG) production. We have examined this problem in renal cortical slices of the dog and have obtained the following results: (1) Isoproterenol (4 X 10(-6) M) stimulated renin release, but had no effect on the formation of 6-keto PGF1 alpha, a stable metabolite of PGI2; (2) Indomethacin (2 X 10(-5) M) had no effect on isoproterenol stimulated renin release, but inhibited 6-keto PGF1 alpha formation; (3) Dibutyryl cyclic AMP (10(-3) M) stimulated both renin release, and 6-keto PGF1 alpha release. Indomethacin (2 X 10(-5) M) did not inhibit dibutyryl cyclic AMP-stimulated renin release, but did inhibit the production of 6 keto PGF1 alpha. These results indicate that the beta-adrenoceptor mediated renin release does not depend on the formation of PGI2, but renin release is dependent on cyclic AMP formation.     

The effect of prostaglandin synthesis inhibition on the direct stimulation of renin release from rabbit renal cortical slices

Prostaglandins have been hypothesized to have several mechanistic functions in sympathetically mediated release of renin. The rabbit renal cortical slice system was chosen to examine the prostaglandin dependency of renin release directly stimulated by either a direct adenylate cyclase activator, forskolin, or a β-agonist, isoproterenol. In this study, we demonstrate that with forskolin (1 × 10⁻⁵M) or isoproterenol (1 × 10⁻⁶M), renin release was elevated 2–3 fold above control, and that this increase was shown to accompany a substantial increase in the tissue levels f cAMP (19.5 fold and 3.5 fold respectively). We also demonstrate that the increase in renin release produced by these compounds was not inhibited by cyclooxygenase inhibitors, indomethacin (25 uM) or eicosatetraynoic acid (30 ug/ml), nor was it inhibited by the selective prostacyclin synthesis inhibitor, U-51605 (30 ug/ml). Each of these inhibitors was demonstrated to block the synthesis of prostaglandins in the cortical slices at the concentrations used. Thus we propose that prostaglandins do not play a role in the induction of renin release resulting from elevated cyclic nucleotide levels or β-adrenergic stimulation.