Rat Kidney Cortex Slices Research Articles

Effect of dexmedetomidine on the release of [ 3H]-noradrenaline from rat kidney cortex slices: characterization of α 2-adrenoceptor

The presynaptic modulation of [ 3H]-noradrenaline (NA) release from rat kidney cortex slices, a method used for the first time, was investigated. Rat kidney cortex slices were loaded with [ 3H]-NA and the release of radioactivity at rest and in response to field stimulation was determined. The α 2-adrenoceptor agonist, dexmedetomidine inhibited the stimulation-evoked release of NA from kidney slices in a concentration-dependent manner, whereas α 2-adrenoceptor antagonist CH-38083 (7,8-methyenedioxy-14-alpha-hydroxyalloberbane HCl), an α 2-adrenoceptor antagonists, enhanced it. When dexmedetomidine and BRL-44408, a selective α 2A antagonist, were added together, the effect of dexmedetomidine was significantly antagonized. In contrast, ARC-239 (2-(2,4-( o-piperazine-1-yl)-ethyl-4,4-dimethyl-1,3-(2H, 4H)disoguinolinedione chloride), a selective α 2B-antagonist, had no effect on the release and failed to prevent the effect of dexmedetomidine. Prazosin, an α 1- and α 2B/C-adrenoceptor antagonist enhanced the release evoked by field stimulation. It is therefore suggested that there is a negative feedback modulation of NA release at the sympathetic innervation of kidney cortex, and dexmedetomidine, a clinically used anesthetic adjunct inhibits the release via activation of α 2C-adrenoceptors.

Immunolocalization of Na/SO4-cotransport (NaSi-1) in rat kidney.

The proximal tubule is the major site for renal reabsorption of sulphate. A sodium-dependent transport system for sulphate (NaSi-1) has recently been identified from a rat kidney cortex cDNA library. Recent work demonstrated that NaSi-1 mRNA is expressed predominantly in proximal tubules. In the present work expression along the nephron of the Na/SO4-cotransporter NaSi-1 was studied by immunofluorescence. A polyclonal antibody was raised in rabbits against a fusion protein containing a 53-amino-acid polypeptide specific for the NaSi-1 sequence. The anti-NaSi-1 polyclonal antibody specifically detected a 68-kDa protein on Western blots and, by immunofluorescence specific staining, was observed in MDCK cells transfected with the NaSi-1 cotransporter. Using rat kidney cortex slices specific NaSi-1-related immunoreactivity was detected in proximal tubules and was restricted to the apical membrane. No immunoreactivity was observed in the other nephron segments. This was confirmed by Western blot analysis using proximal tubular apical and basolateral membranes isolated by free-flow electrophoresis. The results indicate that the Na/SO4-cotransporter NaSi-1 is expressed in the apical membrane of proximal tubular cells and is therefore likely to be involved in proximal reabsorption of sulphate.

Effect of Hippurate on Glucose Utilization in Rat Kidney Cortex Slices

Hippurate action on glucose utilization was evaluated in rat kidney cortex slices. Studies have shown the following. (1) Hippurate inhibits markedly basal as well as insulin-stimulated glucose utilization and basal gluconeogenesis. (2) Ca deficiency and specific Ca channel blockers diltiazem and isradipine abolish the hippurate inhibition of glucose utilization. (3) K+ channel blockers, i.e. the increased K+ concentration in incubation medium, procaine and sulfonylurea drugs also abolish the hippurate inhibition of glucose utilization. It is concluded that hippurate and benzoate operate through the ATP-dependent K+ channel.

Cisplatin-induced injury to calcium uptake by mitochondria in glutathione-depleted slices of rat kidney cortex.

Changes in functional activities of mitochondria from kidney cortical slices incubated with cisplatin and the glutathione depletor diethylmaleate were examined. Diethylmaleate, which decreased the glutathione level in the slices, enhanced the cisplatin-induced decreases in glutathione level and calcium uptake in the mitochondria. The movement of cisplatin into mitochondria in the slices was not affected by diethylmaleate. These results suggest that the depressions in glutathione level and calcium uptake by cisplatin in mitochondria are enhanced by a decrease in cytoplasmic glutathione.

Cisplatin-Induced Injury to Calcium Uptake by Mitochondria in Glutathione-Depleted Slices of Rat Kidney Cortex

Changes in functional activities of mitochondria from kidney cortical slices incubated with cisplatin and the glutathione depletor diethylmaleate were examined. Diethylmaleate, which decreased the glutathione level in the slices, enhanced the cisplatin-induced decreases in glutathione level and calcium uptake in the mitochondria. The movement of cisplatin into mitochondria in the slices was not affected by diethylmaleate. These results suggest that the depressions in glutathione level and calcium uptake by cisplatin in mitochondria are enhanced by a decrease in cytoplasmic glutathione.

PERTUSSIS TOXIN ATTENUATES ANGIOTENSIN II BUT NOT β‐ADRENOCEPTOR FACILITATION OF NORADRENALINE RELEASE FROM RAT KIDNEY CORTEX

1. Angiotensin II (AII; 0.01 and 0.1 mumols/L), angiotensin I (AI, 0.1 mumols/L) and the beta-adrenoceptor agonist isoprenaline (0.1 mumols/L) all facilitated the stimulation-induced outflow of radioactivity from slices of rat kidney cortex incubated in [3H]-noradrenaline. 2. Treatment of rats with pertussis toxin (25 and 50 micrograms/kg i.v.) to inactivate G-proteins attenuated the facilitation caused by AII and AI, but not that caused by isoprenaline. 3. The hypothesis that isoprenaline enhances noradrenaline release by generating AII to activate facilitatory prejunctional AII receptors is not supported by the present study. The hypothesis predicts that pertussis toxin, by inactivating the G-proteins associated with AII receptors, should have inhibited the facilitatory effect of isoprenaline. This did not occur.

Pertussis toxin differentiates between α1- and α2-adrenoceptor-mediated inhibition of noradrenaline release from rat kidney cortex

In slices of rat kidney cortex incubated in [ 3H]noradrenaline, the α 1-adrenoceptor agonist methoxamine (10 μM), the α 2-adrenoceptor agonist clonidine (0.1 μM), as well as adenosine (10 μM), inhibited the electrical stimulation-induced (S-I) outflow of radioactivity, at a stimulation frequency of 1 Hz. Prior treatment of rats with pertussis toxin (25 μg/kg i.v.) which abolished the negative inotropic effect of carbachol (10 μM) on isolated atria, prevented the inhibition caused by methoxamine, but not caused by clonidine or adenosine. At a stimulation frequency of 5 Hz, the α 2-adrenoceptor antagonist idazoxan (0.1 μM) and the prostaglandin synthesis indomethacin (10 μM) both facilatated the S-I outflow of radioactivity, and neither of these effects were altered by pertussis toxin. These results suggest that a pertussis toxin sensitive G-protein is involved in α 1-adrenoceptor inhibition of noradrenaline release, but not in α 2-adrenoceptor, adenosine or prostaglandin inhibition.

Comparative studies of volume restoration following cold-stress induced swelling in renal tissues—I. Effects of ouabain, K+ free medium, colchicine and cytochalasin B on rat and rabbit kidney cortex slices

1. Cold-stress-induced swelling in rabbit and rat kidney cortex slices cannot be due to the sole inhibition of a Na+/K+ exchange system. In these tissues indeed, ouabain induces no swelling and an exchange of Na+ for K+ or a l/l basis. Inhibition of K+ extrusion at low temperature has also to be taken into consideration. 2. Volume restoration at 27 degrees C after cold-stress-induced swelling is inhibited by ouabain in rabbit slices, not in rat ones. The inhibition in rabbit slices is concomitant with an increase in Na+ at levels higher than equilibrium with the external medium. 3. Volume restoration does not seem to implicate colchicine or cytochalasin B sensitive processes.

Modulation of noradrenaline release in slices of rat kidney cortex through α1- and α2-adrenoceptors

Slices of rat kidney cortex were incubated in [ 3H]noradrenaline, then placed in a flow cell and subjected to electrical field stimulation. At a stimulation frequency of 5 Hz, both the α 2-adrenoceptor antagonist idazoxan (0.1 μM) and the α 1-adrenoceptor antagonist prazosin (0.1 μM) significantly enhanced the stimulation-induced (S-I) outflow of radioactivity from the slice. However, neither clonidine (0.1 μM) nor methoxamine (10 μM), α 2- and α 1-agonists respectively, affected the S-I outflow of radioactivity at this stimulation frequency. At a lower stimulation frequency (1 Hz), the S-I outflow of radioactivity was not affected by idazoxan or prazosin, but was inhibited by both clonidine and methoxamine. The effect of clonidine was prevented by idazoxan (0.1 μM), but not by prazosin (0.1 μM). The effect of methoxamine was abolished by prazosin (0.1 μM), but not by idazoxan (0.1 μM). The inhibitory effect of methoxamine was not prevented by the prostaglandin synthesis inhibitor indomethacin (10 μM) or the adenosine receptor antagonist 8-phenyltheophylline (1 μM) and thus was not mediated by either prostaglandins or adenosine. The results suggest that both prejunctional α 1- and α 2- adrenoceptors are directly involved in modulation of noradrenaline release from the renal sympathetic nerves of the rat.

The possible involvement of changes in phosphoinositol turnover in the responses of renal sodium transport to noradrenaline

Noradrenaline stimulated the formation of [ 3H]inositol monophosphates from 3H-inositol-phospholipids in rat kidney cortex slices. This response was inhibited by the α 1-adrenoceptor antagonist prazosin but not by the α 2-adrenoceptor antagonist yohimbine, suggesting that the response was mediated via an α 1 adrenoceptor. This is in keeping with the ability of noradrenaline to increase sodium transport in this tissue by α 1-adrenoceptor stimulation.

Localization and role of pyruvate kinase isoenzymes in the regulation of carbohydrate metabolism and pyruvate recycling in rat kidney cortex

This work was performed to gain more information on the role of pyruvate kinase isoenzymes in the regulation of renal carbohydrate metabolis. Immunohistochemically, pyruvate kinase type L is shown to be localized in the proximal tubule of the nephron and pyruvate kinase type M 2 in the distal tubule and the collecting duct. A tight relationship between gluconeogenesis and pyruvate recycling was found. The rate of gluconeogenesis (8 μmol/g wet wt. per 30 min) was of the same order of magnitude as the ratet of pyruvate recycling (10.92 μmol/g wet wt. per 30 min). Stimulation of gluconeogenesis from 20 mM lactate in kidney cortex slices of 24-h-starved rats by dibutyryl-cAMP, alanine and parathyroid hormone was connected with a decrease in pyruvate recycling; inhibition of gluconeogenesis due to a lack of Ca 2+ in the incubation medium was linked with an increase in pyruvate recycling. The degradation of [6- 14C]glucose to lactate, pyruvate, ketone bodies and CO 2 and of [2- 14C]lactate was unaffected by dibutyryl-cAMP, alanine, epinephrine, vasopressin or the omission of Ca 2+ from the incubation medium. 1 mM dibutyryl-cAMP or 5 mM alanine did not alter the activities of oxaloacetate decarboxylase, ‘malic’ enzyme and malate dehydrogenase from rat kidney cortex. Since aerobic glycolysis in the distabl tubules and the collecting ducts is not influenced by hormones, dibutyryl-cAMP and Ca 2+, pyruvate kinase type M 2 residing in this tissue is unlikely to be a control point of glycolysis. Since this tissue degrades only one-seventh of the glucose formed via gluconeogenesis, it does not contribute significantly to pyruvate recycling. Therefore, the decrease of pyruvate recycling in the presence of dibutyryl-cAMP and alanine in rat kidney cortex slices, leading to increased renal gluconeogenesis, has to be ascribed to the regulation of pyruvate kinase type L.

Morphological and physiological studies of rat kidney cortex slices undergoing isosmotic swelling and its reversal: a possible mechanism for ouabain-resistant control of cell volume.

Slices of rat kidney cortex were induced to swell by preincubation at 1 degree C in an isotonic Ringer's solution, and their capacity to reverse swelling, by net extrusion of cellular water, was studied during subsequent incubation at 25 degrees C. The recovery from swelling was prevented by the respiratory inhibitor, antimycin A. On the other hand, extrusion of water was little affected by ouabain. The extrusion of water continuing in the presence of ouabain (but not that in its absence) was significantly reduced when furosemide was added or when medium Cl- was replaced by NO-3 or I-. There was substantial variability in the morphological appearance of cells within the cortical slices. Different segments of the nephron showed different structural changes during swelling and its reversal, the proximal tubules being most markedly affected. Proximal tubular cells of swollen slices showed disorganization of brush borders and expansion of their apical surfaces, and contained vesicles in their apical cytoplasm. Upon recovery at 25 degrees C, the apical portions of these cells showed reversal of the expansion, but some apical vesicles remained. These vesicles were much more numerous after recovery in the presence of ouabain, but they were much reduced in numbers, or totally absent, when recovery took place in the presence of furosemide or absence of Cl-, with or without ouabain. The vesicles seen in the presence of ouabain alone appeared to fuse with each other and with infoldings of the basolateral plasma membrane. Rather similar results were obtained with distal tubular cells in the slices. We suggest that volume regulation in the proximal and distal tubular cells proceeds by way of two mechanisms. The first consists of extrusion of water coupled to the ouabain-sensitive transport of Na+ and K+. The other proceeds by way of an ouabain-resistant entry of water into apical cytoplasmic vesicles, following furosemide-sensitive movements of Cl- and Na+; the vesicles then expel their contents by exocytosis at the basolateral cell borders.

Tamm-Horsfall glycoprotein release from rat kidney cortex slices in vitro.

Rat kidney cortex slices were incubated for 30 min at 37 degrees C in unmodified Krebs-Henseleit buffer containing aldosterone, vasopressin, theophylline, ethacrynic acid, frusemide, spironolactone or ouabain. Tamm-Horsfall glycoprotein (THG) released into the media was measured by radioimmunoassay and at the end of each experiment the slices were homogenized and assayed for THG content. Incubation of kidney cortex slices in unmodified buffer resulted in a significant increase in the slice THG content when compared with pre-incubation levels. The increase was prevented by puromycin or cycloheximide. Incubation in ethacrynic acid (1 mmol/l) or frusemide (10 mmol/l) resulted in a significant increase in release of THG when compared with unmodified buffer. Puromycin or cycloheximide failed to prevent the increased release. THG release induced by ethacrynic acid or frusemide is probably the result of an aggregation-disaggregation reaction on the cell membrane. It is suggested that the action of the chloride inhibiting diuretics, ethacrynic acid and frusemide, is mediated in some way via THG.

Effects of Pb 2+ added in vitro on Ca 2+ movements in isolated mitochondria and slices of rat kidney cortex

We have studied the effects of Pb 2+ added in vitro on the movements of Ca 2+ in renal cortical mitochondria and tissue slices. The isolated mitochondria rapidly accumulated 45Ca 2+ at 25° by a mechanism that was dependent on respiration and inhibited 96% by ruthenium red. A concentration of 10 μM Pb 2+ inhibited the Ca 2+ accumulation at least as effectively as did ruthenium red. About 20% of the Ca 2+ accumulation persisted at 1° with a similar sensitivity to inhibitors, including 60% inhibition by Pb 2+. Similar results were obtained when the accumulation of Ca 2+ at 25° was measured by means of a calcium-sensitive electrode, Pb 2+ inhibiting by 80%. Calcium that had been accumulated by mitochondria at 25° was released completely by the ionophore A23187 or by 10 μM Pb 2+. The release induced by Pb 2+ was greatly inhibited by ruthenium red. The Ca 2+ content of tissue slices of renal cortex increased 4-fold during incubation at 1° while the Ca 2+ content of mitochondria within the slices more than doubled, the latter being determined by isolation of mitochondria from the slices after incubation. The presence of Pb 2+ (200 μM) in the incubation medium of the slices substantially reduced the entry of Ca 2+ into the whole slices and into mitochondria within the slices. When the slices preincubated at 1° were warmed to 25° in oxygenated medium, they brought about a net extrusion of Ca 2+, some of which was derived from the mitochondria; Pb 2+ did not alter the final level of Ca 2+ then attained in the slices, but it caused a significant decrease in the quantity retained in the mitochondria. We conclude that Pb 2+ both inhibits the uptake of Ca 2+ by renal cortical mitochondria and displaces Ca 2+ from them, these effects occurring whether the mitochondria are isolated or in situ.

Release of alkaline phosphodiesterase I from rat kidney plasma membrane produced by the phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis.

The release of plasma-membrane-bound enzymes by phosphatidylinositol-specific phospholipase C obtained from Bacillus thuringiensis was investigated. Among the ectoenzymes of plasma membrane tested, alkaline phosphodiesterase I was released markedly from rat kidney cortex slices, in addition to alkaline phosphatase and 5'-nucleotidase. Other membrane-bound enzymes; alanine aminopeptidase, leucine aminopeptidase, dipeptidyl peptidase, leucine aminopeptidase, dipeptidyl peptidase IV, esterase and gamma-glutamyl transpeptidase could not be liberated from the treated slices. Alkaline phosphodiesterase I was released linearly from rat kidney slices with the concentration of phosphatidylinositol-specific phospholipase C, but little enzyme was released from rat liver slices. Alkaline phosphodiesterase I separated from kidney tissue with n-butanol still retained phosphatidylinositol and was transformed into a lower molecular weight form by phosphatidylinositol-specific phospholipase C. This suggests an important function for phosphatidylinositol in the binding of alkaline phosphodiesterase I to the plasma membrane of rat kidney cells. The alkaline phosphodiesterase I released from rat kidney had a molecular weight of about 240,000 and an isoelectric point (pI) of 5.4. The enzyme hydrolyzed the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate at pH 8.9 and had a Km value of 0.3 mM. The enzyme was activated by Mg2+ and Ca2+, but was inhibited by EDTA. Strong inhibition took place on the addition of adenosine 5'-phosphosulfate or the nucleotide pyrophosphates, i.e., UDP-galactose and alpha, beta-methylene ATP.

Use of beta-methylene-D,L-aspartate to assess the role of aspartate aminotransferase in cerebral oxidative metabolism.

Several inhibitors of aspartate aminotransferase, a key enzyme of the malate-aspartate shuttle, were investigated for their effects on cerebral oxidative metabolism in vitro. beta-Methylene-D,L-aspartate (2 mM), aminooxyacetate (0.1 mM), and D,L-vinylglycine (20 mM) all significantly reduced the activity of aspartate aminotransferase and the rate of oxygen consumption of rat cerebral cortex slices respiring on glucose. In the presence of beta-methyleneaspartate, a one-to-one correlation was found between the degree of inhibition of tissue respiration and the degree of inhibition of transaminase activity. Slices of rat liver incubated in the presence of glucose and beta-methyleneaspartate showed a similar one-to-one relationship between inhibition of oxygen comsumption and inhibition of aspartate aminotransferase activity, whereas with rat kidney cortex slices, the inhibition of aspartate aminotransferase activity was greater than the inhibition of oxygen consumption. Structural analogs of beta-methyleneaspartate (D,L-beta-methyl-D,L-aspartate, gamma-methyl-D,L-glutamate, and alpha-methyl-D,L-didehydroglutamate) that did not inhibit the activity of aspartate aminotransferase similarly did not inhibit the rate of oxygen consumption by cerebral cortex slices. In the presence of beta-methyleneaspartate, pyruvate oxidation by cerebral cortex slices was inhibited to almost the same extent as was glucose oxidation, and the oxidation of succinate was decreased by approximately 20%. The artificial electron acceptor phenazine methosulfate (0.1 mM) only partially overcame the beta-methyleneaspartate-mediated inhibition of respiration with glucose as substrate. The content of ATP and phosphocreatine declined steadily in slices incubated with glucose and beta-methyleneaspartate. At 1 h the concentration of lactate and the lactate/pyruvate ratio, an indicator of the cytoplasmic redox state, increased threefold, whereas the concentrations of malate, citrate, and aspartate decreased. The findings are interpreted in the context of the hypothesis that enzymes common to the malate-aspartate shuttle and the tricarboxylic acid cycle are physically complexed in brain, so that inhibition of aspartate aminotransferase, a component of the complex, impedes the flow of carbon through both metabolic pathways.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibitory effect of unconjugated bilirubin on [formula omitted] transport in rat kidney cortex slices

(1) The effects of unconjugated bilirubin on the accumulation of p- aminohippurate , kinetics of p- aminohippurate uptake, the efflux of pre-accumulated p- aminohippurate and water and electrolyte distribution were investigated in the rat kidney cortical slice. (2) The addition of unconjugated bilirubin to the incubation medium decreased the 60 min slice-to-medium concentration ratio of p- aminohippurate . (3) The decrease in p- aminohippurate accumulation by unconjugated bilirubin was found to be more pronounced by increasing the concentration of pigment in the medium. (4) The rate of uptake of p- aminohippurate as a function of p- aminohippurate concentration differed in aerobiosis and anaerobiosis, and unconjugated bilirubin decreased only the uptake of p- aminohippurate in aerobic conditions. (5) The efflux of pre-accumulated p- aminohippurate decreased when unconjugated bilirubin concentration in the medium was low (10–20 μM) but the efflux increased when the concentration of pigment was much higher (100 μM). (6) The addition of unconjugated bilirubin to the medium (40–100 μM) increased intracellular sodium and total tissue water content, and decreased intracellular potassium and oxygen consumption of tissue. However the slices incubated with low concentration of pigment (20 μM) did not exhibit significative changes in cellular functional parameters. (7) These findings suggest that unconjugated bilirubin impairs p- aminohippurate transport by a complex mechanism that might involve binding of pigment to sites necessary for anion transport, although effects related to pigment toxicity or to its oxidative decomposition are not excluded.

Na+-gradient-dependent stimulation of renal transport of rho-aminohippurate.

Transport of rho-aminohippurate was studied by the use of a preparation of rabbit kidney basolateral-membrane vesicles and in rat kidney-cortex slices under anaerobic conditions. With both preparations clear evidence of Na+-gradient stimulation of rho-aminohippurate transport ('overshoot') was obtained. These results thus indicate that a significant aspect of active rho-aminohippurate transport is by co-transport with Na+, and they appear to resolve previous disagreements concerning the role of Na+. Vesicle studies with a potential-sensitive dye suggested that rho-aminohippurate may be transported electroneutrally, i.e. in a 1:1 complex with Na+.

Glycine metabolism in rat kidney cortex slices.

When rat kidney cortex slices were incubated with glycine or [1-14C]glycine, after correcting for metabolite changes with control slices, product formation and glycine utilization fitted the requirements of the equation: 2 Glycine leads to ammonia + CO2 + serine. Evidence is presented that degradation via glyoxylate, by oxidation or transamination, is unlikely to have any significant role in kidney glycine catabolism. It is concluded that glycine metabolism in rat kidney is largely via glycine cleavage closely coupled with serine formation. 1-C decarboxylation and urea formation with glycine in rat hepatocyte suspensions were somewhat greater than decarboxylation or ammonia formation in kidney slices, showing that in the rat, potentially, the liver is quantitatively the more important organ in glycine catabolism. There was no evidence of ammonia formation from glycine with rat brain cortex, heart, spleen or diaphragm and 1-C decarboxylation was very weak.

Effect of potassium on p-aminohippurate transport in rat kidney cortex slices.

The effects of medium K+ concentration on the tissue uptake of p-aminohippurate (PAH) were investigated in rat kidney cortex slices. K+ concentration in the incubation medium was changed in the absence of Na+. Increase in K+ concentration up to 145 mM revealed an augmenting effect on PAH uptake when tissue water content was controlled at about 72% by elevating medium osmolarity with mannitol. The K+-induced enhancement of PAH uptake was almost completely abolished by the simultaneous presence of extremely high concentration of hippurate. Anoxia reduced this augmenting effect. Pyruvate added to the medium scarcely affected this augmenting effect. The uptake rate during the initial 10-min incubation increased linearly in proportion to the increase in log [K]o/[K]i. The results suggest that high medium K+ concentration stimulates PAH transport by a carrier-mediated process which is dependent on oxidative metabolism. However, the mode of action of K+ on PAH uptake seems to be different from that of Na+. It seems likely that K+ exerts its effect on PAH transport by the alteration of the peritubular membrane potential.