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.     

Glycine metabolism in rat kidney cortex slices.

We have previously described a method for measuring the rotational diffusion of membrane proteins by using fluorescent triplet probes [Johnson & Garland (1981) FEBS Lett. 135, 252-256]. We now describe the criteria by which the suitability of such probes may be judged. In general, the greatest sensitivity is achievable with probes where the ratio of the quantum yields for prompt fluorescene (phi f) and triplet formation (phi t) are high, as with Rhodamine (phi f/phi t congruent to 10(3)). However, considerations of heat generation at the sample membrane, of time resolution of fast rotations and of irreversible bleaching of the fluorescent probe also apply. The immediate environment of a probe molecule at a membrane protein must also be important in determining the performance of a given probe. Nevertheless, we describe guidelines for evaluating the likely usefulness of fluorescent triplet probes in measurements of membrane protein rotation.     

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.     

Transport and metabolism of thiamine in rat brain cortex in vitro

1. Aerobic incubation at 37° of rat brain-cortex slices in Krebs–Ringer phosphate medium containing glucose and labelled thiamine results in accumulation in the tissue of labelled thiamine and labelled thiamine phosphates. The concentration of the labelled thiamine in the tissue cell water increases with increase of external labelled thiamine concentration in an approximately linear manner, the concentration ratio for labelled thiamine (tissue:medium) exceeding unity with low external thiamine concentrations (e.g. 0·2μm) and diminishing to about unity as the external thiamine concentration is increased to 1μm. The concentration of labelled phosphorylated thiamine in the tissue is at least double that of the labelled thiamine present and its amount increases with increase of external thiamine concentration. Labelled phosphorylated thiamine appears in the medium, its amount being about one-fifteenth of that in the tissue. Phosphorylation of thiamine in the tissue proceeds during incubation for 3hr. and, with an external labelled thiamine concentration of 0·2μm, about 48% conversion of thiamine takes place. 2. In the presence of ouabain (0·1mm), which does not inhibit thiamine phosphorylation in rat brain extract, there is a fall in the uptake of labelled thiamine by brain-cortex slices and the concentration ratio for the labelled thiamine (tissue:medium) falls to below unity. Anaerobiosis, lack of Na⁺ or the presence of Amprol (0·01mm) leads to marked inhibition of thiamine phosphorylation, and the concentration ratio for labelled thiamine (tissue:medium) falls to about unity. The facts lead to the conclusion that thiamine is conveyed into the brain cell against a concentration gradient by an energy-assisted process mediated by a membrane carrier. Pyri-thiamine is a marked inhibitor of thiamine phosphorylation in brain extract. 3. Thiamine monophosphate and thiamine diphosphate inhibit thiamine phosphorylation in brain extract. They diminish `total' thiamine (free and phosphorylated) uptake into brain-cortex slices and inhibit the transport of thiamine into the brain cell, possibly by competition for the carrier. 4. Phosphorylation of labelled thiamine in brain extract is brought about not only by adenosine triphosphate (in the presence of Mg²⁺) but apparently by adenosine diphosphate and uridine triphosphate.     

Transport and metabolism of acetate in rat brain cortex in vitro

1. [1-(14)C]Acetate undergoes metabolism when incubated aerobically at 37 degrees in the presence of rat brain-cortex slices, forming (14)CO(2) and (14)C-labelled amino acids (glutamate, glutamine, aspartate and relatively small quantities of gamma-aminobutyrate). In the absence of glucose the yield of (14)C-labelled aspartate exceeds that of (14)C-labelled glutamate and glutamine. The addition of glucose brings about a doubling of the rate of formation of (14)CO(2) and a greatly increased yield of (14)C-labelled glutamate or glutamine, whereas that of (14)C-labelled aspartate is diminished. 2. The addition of potassium chloride (100mm) to the incubation medium causes an increased rate of (14)CO(2) formation in the presence or absence of glucose and an increased rate of utilization of acetate. 3. The addition of 2,4-dinitrophenol (0.1mm) suppresses the rate of utilization of [1-(14)C]acetate. 4. The presence of ouabain (10mum) suppresses the rate of formation of (14)CO(2) from [1-(14)C]acetate and the rate of acetate utilization. Acetate conversion into carbon dioxide in the rat brain cortex is both Na(+)- and K(+)-dependent and controlled by operation of the active sodium-transport process. Only the Na(+)-stimulated rate is suppressed by ouabain. 5. Sodium fluoroacetate (1mm) decreases the rate of (14)CO(2) evolution from [1-(14)C]acetate in the presence of rat brain cortex without affecting the respiratory rate. The results are consistent with the conclusion that fluoroacetate competes with, or blocks, a transport carrier for acetate, so that in its presence only the passive diffusion rate of acetate takes place. 6. The presence of sodium propionate or sodium butyrate suppresses the utilization of [1-(14)C]acetate in rat brain cortex and leads to a concentration ratio (tissue/medium) of [1-(14)C]-acetate greater than unity. 7. The presence of NH(4) (+) diminishes acetate utilization, this being attributed to a diminished ATP concentration. Glycine is also inhibitory. It is concluded that acetate transport into the brain is carrier-mediated and dependent on the operation of the sodium pump.     

Transport and metabolism of pantothenic acid by rat kidney

Transport of [14C]pantothenic acid was studied using brush-border membrane vesicles prepared from rat kidney. In the presence of a Na+ gradient an accumulation of pantothenic acid 3-fold above equilibrium was observed. The Km and Vmax found were 7.30 microM and 23.8 pmol/mg protein per min, respectively. Isolated perfused rat kidneys were employed to study excretion of pantothenic acid at various concentrations in the perfusate. At physiological plasma concentrations, the filtered pantothenic acid was largely reabsorbed by the active process observed in the vesicles. At higher concentrations, pantothenic acid was found to undergo tubular secretion. Penicillin inhibited this secretory process indicating that both compounds share a secretory mechanism. Live animal studies indicated that the only compound excreted after injection of [14C]pantothenic acid was free pantothenic acid. After 1 week only 38% of the administered dose was excreted in the urine, indicating that effective conservation was taking place in the whole animal.     

Glutamine affects glutamate metabolism in isolated rat kidney cortex mitochondria.

The active state respiration of isolated rat kidney cortex mitochondria with 10 mM glutamate as single substrate is substantially increased by the addition of 10 mM glutamine. This increase in respiration was accompanied by a higher transamination rate and was found to be insensitive to the selective inhibition of either the transamination or the desamination pathway of glutamate oxidation. These data can be explained by an approximately 2-fold elevated intramitochondrial glutamate concentration observed in the additional presence of glutamine.     

Transport ATPase cytochemistry: ultrastructural localization of potassium-dependent and potassium-independent phosphatase activities in rat kidney cortex

A cytochemical method for the light and electron microscope localization of the K- and Mg-dependent phosphatase component of the Na-K-ATPase complex was applied to rat kidney cortex, utilizing p-nitrophenylphosphate (NPP) as substrate. Localization of K-N-ATPase activity in kidneys fixed by perfusion with 1% paraformaldehyde -0.25% glutaraldehyde demonstrated that distal tubules are the major cortical site for this sodium transport enzyme. Cortical collecting tubules were moderately reactive, whereas activity in proximal tubules was resolved only after short fixation times and long incubations. In all cases, K-NPPase activity was restricted to the cytoplasmic side of the basolateral plasma membranes, which are characterized in these neplron segments by elaborate folding of the cell surface. Although the rat K-NPPase appeared almost completely insensitive to ouabain with this cytochemical medium, parallel studies with the more glycoside-sensitive rabbit kidney indicated that K-NPPase activity in these nephron segments is sensitive to this inhibitor. In addition to K-NPPase, nonspecific alkaline phosphatase also hydrolyzed NPP. The latter could be differentiated cytochemically from the specific phosphatase, since alkaline phosphatase was K-independent, insensitive to ouabain, and specifically inhibited by cysteine. Unlike K-NPPPase, alkaline phosphatase was localized primarily to the extracellular side of the microvillar border of proximal tubules. A small amount of cysteine-sensitive activity was resolved along peritubular surfaces of proximal tubules. Distal tubules were unreactive. In comparative studies, Mg-ATPase activity was localized along the extracellular side of the luminal and basolateral surfaces of proximal and distal tubules and the basolateral membranes of collecting tubules.     

Enalapril maleate affects 2-oxoglutarate metabolism in mitochondria from the rat kidney cortex

Enalapril maleate (EM) is the salt of N-{(S)-1-(ethoxycarbonyl)-3-phenylpropyl}-L -alanyl-L -proline, used therapeutically as an anti-hypertensive agent. The effects of EM on some aspects of the energy metabolism and membrane properties of mitochondria from rat liver and kidney cortex were studied, but only the latter were significantly affected. With 0·8 mM of EM and 2-oxoglutarate as oxidizable substrate for isolated mitochondria from rat kidney cortex, the findings were: (a) inhibition of the respiratory rate in state III (37 per cent) and decrease (45 per cent) in respiratory control ratio (RCR), with only one addition of ADP; (b) reinforcement of the inhibition when a second addition of ADP was made; (c) no significant effect either on the rate of respiration in state IV or on the ADP/O ratio; (d) no effect on the ATPase activity of mitochondria from liver or kidney cortex; (e) inhibition of the transmembrane potential (Δψ) after a second addition of ADP; (f) inhibition of the 2-oxoglutarate dehydrogenase complex. It is suggested that in kidney mitochondria, EM interferes in the gluconeogenesis dependence of at least five substrates: 2-oxoglutarate, glutamine, glutamate, lactate, and pyruvate. Also EM may inhibit Na⁺/H⁺ exchange causing natriuresis.     

Inhibition of 2-oxoglutarate metabolism by lithium in the isolated rat kidney cortex tubules

2-Oxoglutarate metabolism in the isolated rat kidney cortex tubules was inhibited by lithium at 5 mM concentration, and at pH increased from 7.1 to 7.6. The metabolism of pyruvate and acetylcarnitine to citrate and 2-oxoglutarate was enhanced by lithium and the increased pH value. The content of 2-oxoglutarate in the renal tubular cells was lowered by lithium but increased at elevated pH values. Both the intracellular pH value and bicarbonate ion concentrations in renal tubular cells were increased by lithium in the medium containing 2-oxoglutarate. The results obtained indicate that lithium disturbs renal metabolism by intracellular alkalization, with a simultaneous inhibition of the inflow of dicarboxylic substrates to the renal tubular cells.     

Gluconeogenesis in developing rat kidney cortex

1. Gluconeogenesis in developing rat kidney cortex was studied by assaying the activities of two enzymes, glucose 6-phosphatase and phosphoenolpyruvate carboxykinase, and by measuring glucose formation in tissue slices. 2. Glucose 6-phosphatase and phosphoenolpyruvate carboxykinase are present in late foetal (21-22-day-old) tissue and increase rapidly postnatally. Maximum activity of phosphoenolpyruvate carboxykinase occurs at 7 days of age, followed by a decline to the adult level. Glucose 6-phosphatase activity rises during the first 2 postnatal weeks and then declines. 3. Late foetuses synthesize glucose from both pyruvate and l-glutamate. The rate increases during the first 2 weeks to above adult levels. Synthesis is always higher from pyruvate than from glutamate. 4. The effect of 24hr. starvation was studied in perinatal animals. The results indicate that the ability to increase the rate of glucose synthesis as a result of starvation is not present at birth, but develops some time after the second postnatal day.     

Transport and metabolism of pyridoxine in rat liver

Evidence, obtained with in situ perfused rat liver, indicated that pyridoxine is taken up from the perfusate by a non-concentrative process, followed by metabolic trapping. These conclusions were reached on the basis of the fact that at low concentrations (0.125 μM), the ³H of [³H]pyridoxine accumulated against a concentration gradient, but high concentrations (333 μM) of pyridoxine or 4-deoxypyridoxine prevented this apparent concentrative uptake. Under no conditions did the tissue water : perfusate concentration ratio of [³H]pyridoxine exceed unity.The perfused liver rapidly converted the labeled pyridoxine to pyridoxine phosphate, pyridoxal phosphate and pyridoxamine phosphate and released a substantial amount of pyridoxal and some pyridoxal phosphate into the perfusate. Since muscle and erythrocytes failed to oxidize pyridoxine phosphate to pyridoxal phosphate, it is suggested that the liver plays a major role in oxidizing dietary pyridoxine and pyridoxamine as their phosphate esters to supply pyridoxal phosphate which then reaches to other organs chiefly as circulating pyridoxal.     

Distribution of enzymes of cGMP metabolism in glomeruli and tubules isolated from normal and nephrotic rat kidney cortex

• 1.1. Puromycin aminonucleoside (PA) nephrosis may constitute a good experimental model to investigate the involvement of the cGMP system in the regulation of several kidney functions and especially glomerular permeability.
• 2.2. After a single intravenous injection of PA we studied the evolution of the guanylate cyclase (GCase) and cGMP-phosphodiesterase (G-PDE) activities in pure preparations of glomeruli (Gl) and tubules (TU).
• 3.3. Both Gl and TU homogenates showed a strong increase of the GCase activity 12 days after PA injection.
• 4.4. In the presence of Triton X-100, TU homogenate GCase showed the same pattern as in absence of this detergent while the Gl enzyme decreased unexpectedly. On the other hand, the only G-PDE change was observed in the TU where this activity decreases progressively.
• 5.5. Gl pellets and TU supernatants showed similar changes as in total homogenates. But, compared to the total homogenate, both Gl and TU supernatant GCases were strongly activated and in the Gl these activation rates were not the same in normal and 12 days-nephrotic rats.
• 6.6. These results could be explained by the existence of a membrane bound GCase “effector” involved in the physiopathological evolution of the disease.
• 7.7. In conclusion it seems to be clear that the cGMP-system is involved in the evolution of PA-nephrosis. But the precise relation between variations in the cGMP-system and the disease remains unclear and needs further investigation.

     

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.