Developmental pattern of cystine transport in isolated rat renal tubules

Isolated renal cortical tubule fragments from rats ranging in age from less than 48 h to 15 weeks were used to examine the pattern of cystine uptake with development. Immature tubules took up cystine with a faster initial rate than mature tubules and did not reach a steady state by 60 min. By eight weeks of age, the timed uptake of cystine began to approach a steady state and between 8 and 11 weeks the uptake pattern achieved its adult form of reaching a steady state by 30 min of incubation. Analysis of the intracellular metabolism of the cystine taken up by the newborn tubules revealed that the majority had been reduced to cysteine with the formation of small amounts of reduced glutathione. Cystine entered the renal cortical tubule cell from the newborn via two saturable transport systems similar to the mature animal. The kinetic parameters of initial uptake of these two transport systems were similar in the mature and newborn animal except for a higher maximum transport velocity for the low K_m, low capacity system in the newborn. Lysine inhibited cystine uptake by newborn tubules and this inhibition appeared to occur on the low K_m, low capacity transport system similar to the adult. Cystine uptake was sodium dependent with an apparent affinity for sodium of 36 mequiv./l. From this data, the physiologic cystinuria of the immature animal does not appear to be referrable to a lower rate of influx as previously observed with the cortical slice. Other mechanisms should be sought to explain this phenomenon of immaturity.     

Metabolism of cyclic AMP in isolated renal tubules: Effects of prostaglandins and parathyroid hormone

Concentrations of cyclic AMP (cAMP) were increased in isolated renal cortical tubules from hamsters by both parathyroid hormone (PTH) and prostaglandin E₁ (PGE₁) with maximal effects of PGE₁ being 6–8 fold greater than those of PTH during a 10 min period. However, cAMP concentrations in cells treated with 1-methyl-3-isobutylxanthine (MIX) were increased with maximal concentrations of either hormone to the same degree. Similar effects of both hormones were observed on adenylate cyclase activity in renal homogenates. Simultaneous addition of hormones produced changes in both cAMP concentrations in intact tubules as well as adenylate cyclase activity of homogenates which were not completely additive. Degradation of cAMP, estimated in intact tubules as the difference in cAMP levels in the presence and absence of MIX, was increased by both hormones, however, changes were 2–3 fold greater in tubules exposed to PTH than to PGE₁. Neither hormone directly altered cAMP phosphodiesterase (PDE) activity in either 30,000 x g supernatant or pellets from renal cortical homogenates.The results suggest that both hormones increase the production of cAMP in renal cortical tubules and may share a common target cell type in this response. Degradation of cAMP, however, is differentially effected by the two hormones, probably reflecting differences exerted on intracellular mechanisms regulating the enzymatic hydrolysis of cAMP.     

Metabolism of cyclic AMP in isolated renal tubules: effects of prostaglandins and parathyroid hormone.

Concentrations of cyclic AMP (cAMP) were increased in isolated renal cortical tubules from hamsters by both parathyroid hormone (PTH) and prostaglandin E1 (PGE1) with maximal effects of PGE1 being 6-8 fold greater than those of PTH during a 10 min period. However, cAMP concentrations in cells treated with 1-methyl-3-isobutylxanthine (MIX) were increased with maximal concentrations of either hormone to the same degree. Similar effects of both hormones were observed on adenylate cyclase activity in renal homogenates. Simultaneous addition of hormones produced changes in both cAMP concentrations in intact tubules as well as adenylate cyclase activity of homogenates which were not completely additive. Degradation of cAMP, estimated in intact tubules as the difference in cAMP levels in the presence and absence of MIX, was increased by both hormones, however, changes were 2-3 fold greater in tubules exposed to PTH than to PGE1. Neither hormone directly altered cAMP phosphodiesterase (PDE) activity in either 30,000 x g supernatant or pellets from renal cortical homogenates. The results suggest that both hormones increase the production of cAMP in renal cortical tubules and may share a common target cell type in this response. Degradation of cAMP, however, is differentially effected by the two hormones, probably reflecting differences exerted on intracellular mechanisms regulating the enzymatic hydrolysis of cAMP.     

Calcium compartmentation in isolated renal tubules in suspension

Substantial increases of total cell Ca2+ have been observed in suspensions of isolated rabbit proximal tubules subjected to hypoxic injury or treated with exogenous ATP followed by apparent recovery with reoxygenation of the hypoxic tubules or continued incubation of ATP-treated tubules. Ca2+ compartmentation studies using digitonin and metabolic inhibitors were done to clarify the basis for these changes. Digitonin, 40-90 micrograms/mg tubule protein, rapidly permeabilized the tubule cells and did not impair mitochondrial Ca2+ sequestration. Most of the increases of tubule cell Ca2+ produced by hypoxia and ATP were accounted for by pools which could be rapidly removed by exposure of tubules to EGTA and the uncoupler carbonyl cyanide m-chlorophenyl hydrazone without concomitant use of digitonin, suggesting that the changes of Ca2+ predominantly reflect sequestration by mitochondria in severely damaged cells or mitochondria already released to the medium from them. The time course of uptake followed by spontaneous release of mitochondrial Ca2+ from tubule cells deliberately permeabilized with digitonin, then incubated for prolonged periods, indicated that the decreases of tubule cell Ca2+ during reoxygenation of hypoxic suspensions and prolonged incubation of ATP-treated tubules were likely to be attributable to loss of Ca2+ from free mitochondria and those in damaged cells rather than to extrusion by intact cells.     

Phenylalanine hydroxylation in isolated rat kidney tubules

1. Phenylalanine hydroxylation has been demonstrated to occur in isolated rat kidney tubules under physiological conditions. 2. The hydroxylation flux response is hyperbolic with apparent Km and Vmax values of ca 85 microM phenylalanine and 49 nmol tyrosine formed/mg dry wt per hr respectively. 3. Hydroxylation in kidney tubules is substantially less sensitive to effectors of cyclic AMP turnover and Ca2+ mobilization than phenylalanine hydroxylation in isolated liver cells.     

Metabolism of acetaldehyde in human and baboon renal cortex. Ethanol synthesis by isolated baboon kidney-cortex tubules

Acetaldehyde (1-20 mM) was metabolized at high rates and in a dose-dependent manner in isolated human and baboon kidney-cortex tubules. Acetaldehyde removal was accompanied by a large accumulation of acetate in both human and baboon tubules. By contrast, a large synthesis of ethanol was observed only in baboon tubules. Consistent with the latter finding, ethanol was found to be metabolized at significant rates in baboon but not human tubules. In the tubules from both species, a significant fraction of the acetaldehyde removed was also completely oxidized to CO2 and H2O. These results suggest that, in both man and baboon, the kidneys participate in the in vivo metabolism of acetaldehyde; they also suggest that, in contrast with the human kidneys, the baboon kidneys contribute to the detoxication of circulating ethanol.     

Dopamine production by isolated glomeruli and tubules from rat kidneys

To locate the sites of dopamine (D) production in rat renal cortex, we separated glomeruli and proximal tubules by sieving or centrifugation in Percoll after collagenase digestion. After centrifugation layer I contained 60-80% glomeruli and 20-40% tubule fragments, half of which did not stain with alkaline phosphatase, layer II contained 0-5% glomeruli, 10-25% tubule fragments other than proximal tubules, and 70-85% proximal tubule fragments. Layer IV contained 85-95% proximal tubules. Gluconeogenic rates were (micromoles per hour per gram wet weight) as follows: I, 4 +/- 1; II, 7 +/- 2; and IV, 16 +/- 1. Norepinephrine (NE) content was (picomoles per gram wet weight) I, 310 +/- 30; II, 540 +/- 40; IV, 195 +/- 60. D content was (picomoles per gram wet weight) I, 26 +/- 6; II, 46 +/- 13; IV, 33 +/- 7. Surgical denervation 4-6 days previously reduced the norepinephrine content of layers I and II to 35 +/- 10 (p less than 0.001) and of IV to 60 +/- 20 (p less than 0.05) and the D content of layers I and II to 13 +/- 6 and 6 +/- 6 pmol/g, respectively (p less than 0.01); D content of layer IV was unchanged. D production from 10(-7) M 3,4-dihydroxyphenylalanine (DOPA) was (nanomoles per gram per minute) I, 0.2 +/- 0.03; II, 0.7 +/- 0.1; IV, 1.0 +/- 0.04. DOPA consumption was (nanomoles per gram per minute) I, 0.6 +/- 0.1; II, 1.4 +/- 0.3; and IV, 1.8 +/- 0.2. Denervation did not change D production or DOPA consumption.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carnitine metabolism in isolated rat kidney cortex tubules

Renal carnitine metabolism was studied in isolated kidney cortex tubules from fed rats. The tubular distribution of free carnitine (C), acid-soluble short chain acylcarnitine (AcC), and total acid-soluble carnitine was measured. The content of the last-mentioned in rat cortical tubule suspensions was 2.85 +/- 0.15 nmol/mg protein, 46% representing AcC. In the absence of metabolic substrates the AcC/C ratio declined from 0.84 to 0.48 during incubation. The administration of 2mM acetoacetate or 2mM 3-hydroxybutyrate caused an increase in AcC by 45% and 51%, respectively. The rise in AcC was paralleled by a decrease in C, resulting in an increase of the tubular AcC/C ratio to 1.69 and 1.85, respectively. In the presence of 1 mM exogenous L-carnitine 35 +/- 6 nmol AcC/(mg protein X h) was formed. The addition of acetoacetate and 3-hydroxybutyrate led to a 3.5 to 3.8-fold rise in AcC formation. Other substrates which are likewise metabolized by proximal tubules were less effective. More than 90% of the formed AcC was recovered in the extracellular fluid. The results suggest that proximal renal tubule cells are the intrarenal site of carnitine acylation and may be involved in the regulation of blood and/or urinary carnitine acylation state.     

Triacylglycerol metabolism in isolated rat kidney cortex tubules

Triacylglycerol metabolism has been studied in kidney cortex tubules from starved rats, prepared by collagenase treatment. Triacylglycerol was determined by a newly developed fully enzymic method. Incubation of tubules in the absence of fatty acids led to a decrease of endogenous triacylglycerol by about 50% in 1h. Addition of albuminbound oleate or palmitate resulted in a steady increase of tissue triacylglycerol over 2h. The rate of triacylglycerol synthesis was linearly dependent on oleate concentration up to 0.8mm, reaching a saturation at higher concentrations. Triacylglycerol formation from palmitate was less than that from oleate. This difference was qualitatively the same when net synthesis was compared with incorporation of labelled fatty acids. Quantitatively, however, the difference was less with the incorporation technique. Gluconeogenic substrates, which by themselves had no effect on triacylglycerol concentrations, stimulated neutral lipid formation from fatty acids. Glucose and lysine did not have such a stimulatory effect. Inhibition of gluconeogenesis from lactate by mercaptopicolinic acid likewise inhibited triacylglycerol formation. This inhibitory effect was seen with oleate as well as with oleate plus lactate. When [2-¹⁴C]lactate was used the incorporation of label into triacylglycerol was found in the glycerol moiety exclusively. Addition of dl-β-hydroxybutyrate (5mm) to the incubation medium in the presence of oleate or oleate plus lactate led to a significant increase in triacylglycerol formation. In contrast with the gluconeogenic substrates, dl-β-hydroxybutyrate had no stimulatory effect on fatty acid uptake. The results suggest that renal triacylglycerol formation is a quantitatively important metabolic process. The finding that gluconeogenic substrates, but not glucose, increase lipid formation, indicates that the glycerol moiety is formed by glyceroneogenesis in the proximal tubules. The effect of ketone bodies seems to be caused by the sparing action of these substrates on fatty acid oxidation. The decrease of triacylglycerol in the absence of exogenous substrates confirms previous conclusions that endogenous lipids provide fatty acids for renal energy metabolism.     

Characterization of primar cell cultures derived from rat renal proximal tubules

Summary Proximal tubules were prepared from rat kidney cortex by collagenase digestion and purified by percoll gradient centrifugation. Their enrichment was estimated by comparing the specific activities of various cell-specific enzymes in homogenates of renal cortex and of the isolated tubules. The tubules were cultured in a 50:50 mixture of Dulbecco’s modified Eagle’s and Ham’s F12 media supplemented with insulin, transferrin, epidermal growth factor, hydrocortisone, and prostaglandin E₁. After 2 to 3 d an extensive outgrowth of epithelial cells developed from the attached tubules. After 5 to 7 d near confluent monolayers were obtained. Hormonal responsiveness, marker enzyme activities, and transport properties were determined to further characterize the primary cultures. The cultured cells exhibited increased cyclic AMP production in response to parathyroid hormone but not calcitonin or vasopressin, consistent with the absence of cells derived from distal and collecting tubules. The cells also retained significant levels of 25-hydroxyvitamin D₃-lα-hydroxylase, alkaline phosphatase, and ψ-glytamyltranspeptidase, three enzymes that are primarily associated with the proximal tubule. The cultured epithelial cells also exhibit a Na⁺-dependent phosphate and glucose transport systems. Therefore, the cells retain many functional properties that are characteristic of proximal tubules. Thus, the primary cultures should be suitable for the study of processes that occur specifically within this segment of the rat nephron. This work was supported in part by the Veterans Administration (JBP), Washington, DC, by grant DK-37124 (NPC) from the National Institutes of Health, Bethesda, MD, and by grant BNS-86-17004 (CFL) from the National Science Foundation, Washington, DC.     

Glutamic acid decarboxylase in tubules and glomeruli isolated from rat kidney cortex

4-Aminobutyric acid (GABA) synthesis was examined in purified glomeruli and tubules of rat kidney cortex that were incubated in the presence of [2,3-3H2]glutamate. The GABA that was formed was separated from glutamate using anion-exchange resin, and identified by means of an automatic amino acid analyser. In the renal cortex only the tubules were able to form GABA (35.0 nmol mg-1 h-1); the remaining GABA synthesis found in the glomerular preparations can most probably be attributed to a contamination by cortical tubules (9%), as shown by determination of a known tubular marker enzyme (L-gamma-glutamyltransferase). Hydroxylamine (1 mM) and ethanolamine-O-sulfate (10 mM), well-known inhibitors of cerebral GABA formation and GABA catabolism respectively, inhibited renal tubular GABA formation at 100% and 44% respectively.     

Riboflavin transport by isolated perfused rabbit renal proximal tubules

Rabbit renal proximal tubular transport of riboflavin(RF) was examined by using the in vitro isolated tubule perfusiontechnique. We found that proximal tubules actively reabsorbed(J_lb) and secreted (J_bl)RF. At 0.1 µM RF concentration, J_bl wassignificantly higher than J_lb, resulting in anet secretion. This net secretion of RF was decreased at 0.01 µM RFconcentration and increased at 1 µM RF concentration. BothJ_lb and J_bl wereinhibited by lowering temperature or by adding iodoacetate, a metabolicinhibitor, and lumichrome, an RF analog, suggesting the involvement ofcarrier-mediated transport mechanisms. J_bl wasinhibited by probenecid, an anion transport inhibitor, and bypara-aminohippuric acid, an organic anion, suggesting therelevance of RF secretion to renal organic anion transport.J_bl was also inhibited by alkaline pH (8.0) and by the calmodulin inhibitor trifluoperazine, indicating the influence of pH and Ca²⁺/calmodulin-dependent pathway on RFsecretion. Finally, we found that addition of chlorpromazine, aphenothiazine derivative, inhibited both J_lb andJ_bl, raising the concern about the nutritionalstatus in patients receiving such a type of medication.

     

Effect of maleic acid on the kinetics of alpha-methyl-D-glucoside uptake by isolated rat renal tubules.

ALPHA-Methyl-D-glucoside has been used to study the invitro mechanism of the effect of maleic acid on sugar transport, using isolated rat renal tubule fragments. 6 mM maleate maximally inhibits the ability of the tubule to establish a concentration gradient for this model sugar with no evidence of ultrastructural changes. This inhibition is due to a 100% increase in efflux, as well as to a 50% decrease in influx with more prolonged incubation. The data presented here are consistent with those of other workers, but their work does not explain our results, which therefore deserve further investigation by other techniques.     

Metabolism of C-arachidonate in rat isolated lung

Metabolism of ¹⁴C-arachidonate was investigated in rat isolated lungs perfused via the pulmonary circulation with Krebs solution. Only 10% of the radioactivity derived from an infusion of ¹⁴C-arachidonate through the pulmonary circulation of rat isolated lungs appeared in the effluent by 10 minutes. At 10 min, the major component of effluent radioactivity and 20–40% of that retained in lung was unchanged arachidonate. Between 10 and 20 min of perfusion, a further small amount of radioactivity was lost in lung effluent and at 20 min the retained radioactivity showed a decrease in the proportion present as free arachidonate. Between 20 and 60 min, there was no further loss of radioactivity in effluent and no further change in the distribution in lung. Addition of albumin to the Krebs solution perfusate during the infusion of ¹⁴C-arachidonate increased effluent radioactivity to 80%, but albumin added after 10 min only caused the efflux of a small amount of radioactivity (10%). Treatment of labelled lung at 20 min with the calcium ionophore A23187 released biologically active metabolites of arachidonate but very little radioactivity. Metabolism of arachidonate, either during the infusion or after retention in lung, in rat lung was closer to that in human lung than to that in guinea-pig lung.     

Metabolic regulation of organic osmolytes in tubules from rat renal inner and outer medulla

Glycerophosphorylcholine (GPC), sorbitol and inositol were quantitated in renal tubule suspensions from inner and outer medulla of untreated Sprague-Dawley rats to study the regulation of organic osmolyte concentrations under different metabolic conditions and varying extracellular osmolalities in vitro. Inner medullary tubules prepared in hypertonic saline (550 mosm/kg) contained osmolyte concentrations comparable to those found in the kidney in vivo. Incubation for up to 8 h at 5 mmol/l glucose increased sorbitol in the inner medullary tubules and medium in an osmolality-dependent fashion, whereas GPC and inositol remained constant. At a given glucose concentration the rate of sorbitol formation decreased linearly with increasing tubular sorbitol concentration, which was regulated by an osmolality-dependent export mechanism. Perturbation of tubular mechanisms by inhibition of glycolysis or oxidative phosphorylation did not change the tubular osmolyte content. In contrast to papilla outer medullary tubules contained only inositol. Lactate added as a metabolic substrate to the outer medullary tubules did not change the cellular inositol levels. In outer medullary tubules osmolality changes (320-710 mosm/kg) had no effect on tubular inositol. Addition of furosemide was without effect, when added in vitro. The results indicate that tubular sorbitol formation is regulated by glucose concentration, the level of tubular sorbitol, and an osmolality-dependent export mechanism. In contrast, cellular inositol and GPC levels seem to be independent of acute changes in tubular metabolism.