Effect of the blood lactate concentration on renal glutamine metabolism in dogs with chronic metabolic acidosis

It appears that glutamine and lactate are the principal substrates for the kidney in dogs with chronic metabolic acidosis. Accordingly, the purpose of this study was to determine if a higher or lower rate of renal lactate extraction would influence the rate of glutamine extraction at a constant rate of renal ATP turnover. The blood lactate concentration was 0.9 +/- 0.01 mM in 15 acidotic dogs. However, eight dogs with chronic metabolic acidosis had a spontaneous blood lactate concentration of 0.5 mM or lower. The kidneys of these dogs extracted considerably less lactate from the arterial blood (19 vs. 62 mumol/100 mL glomerular filtration rate (GFR]. Nevertheless, glutamine, alanine, citrate, and ammonium metabolism were not significantly different in these two groups of dogs. Renal ATP balance in acidotic dogs with a low blood lactate could only be achieved if a substrate other than additional glutamine were oxidized in that segment of the nephron which normally oxidized lactate; presumably a fat-derived substrate and (or) lactate derived from glucose was now the metabolic fuel at these more distal sites. When the blood lactate concentration was greater than 1.9 mM, lactate extraction rose to 219 mumol/100 mL GFR. Glutamine, alanine, citrate, and ammonium metabolism were again unchanged; in this case, ATP balance required substrate flux to products other than carbon dioxide, presumably, gluconeogenesis. It appears that renal ammoniagenesis is a proximal event and is independent of the rate of renal lactate extraction.     

Acidotic alterations in oxidative metabolism influencing rat renal slice ammoniagenesis

We believe that two findings are interconnected and help to comprehend a major mechanism behind the regulation of renal ammonia production during acidosis. First, slices from acidotic compared to control and alkalotic rats produce more ammonia from glutamine. Second, inhibition of renal oxidative metabolism at various points by metabolic inhibitors augments slice ammoniagenesis. Based on this, our purpose was to determine whether enhanced renal ammoniagenesis during acidosis could occur through the same mechanism as the metabolic inhibitors. However, metabolic inhibitors (malonate; arsenite; 2,4-dinitrophenol) usually decrease while acidosis increases slice gluconeogenesis. There is one known exception. Fluorocitrate, which blocks citrate metabolism, simulates the acidotic condition by enhancing both ammonia and glucose production. Accordingly, a block of oxidative metabolism if located prior to citrate oxidation in the tricarboxylic acid cycle could theoretically augment ammoniagenesis during acidosis. Lactate, is a major renal fuel whose oxidative metabolism would be blocked by fluorocitrate. There, we concentrated on the effects of acidosis on lactate as well as glutamine metabolism. Lactate decarboxylation decreases in the face of increased glucose production during acidosis, and lactate inhibition of glutamine decarboxylation decreases in slices from acidotic rats. Also, we found lesser oxygen consumption in the presence of lactate by kidney slices from acidotic rats compared to control and alkalotic rats. We postulate that relatively less incorporation of lactate into the TCA cycle, causing decreased citrate formation and citrate oxidation during acidosis, contributes, at least in part, to acidotic adaptation of ammoniagenesis.     

Metabolism of glutamine and glutamate by rat renal tubules. Study with 15N and gas chromatography-mass spectrometry

Gas chromatography-mass spectrometry was utilized to study the metabolism of [15N]glutamate, [2-15N]glutamine, and [5-15N]glutamine in isolated renal tubules prepared from control and chronically acidotic rats. The main purpose was to determine the nitrogen sources utilized by the kidney in various acid-base states for ammoniagenesis. Incubations were performed in the presence of 2.5 mM 15N-labeled glutamine or glutamate. Experiments with [5-15N]glutamine showed that in control animals approximately 90% of ammonia nitrogen was derived from 5-N of glutamine versus 60% in renal tubules from acidotic rats. Experiments with [2-15N]glutamine or [15N]glutamate indicated that in chronic acidosis approximately 30% of ammonia nitrogen was derived either from 2-N of glutamine or glutamate-N by the activity of glutamate dehydrogenase. Flux through glutamate dehydrogenase was 6-fold higher in chronic acidosis versus control. No 15NH3 could be detected in renal tubules from control rats when [2-15N]glutamine was the substrate. The rates of 15N transfer to other amino acids and to the 6-amino groups of the adenine nucleotides were significantly higher in normal renal tubules versus those from chronically acidotic rats. In tubules from chronically acidotic rats, 15N abundance in 15NH3 and the rate of 15NH3 appearance were significantly higher than that of either the 6-amino group of adenine nucleotides or the 15N-amino acids studied. The data indicate that glutamate dehydrogenase activity rather than glutamate transamination is primarily responsible for augmented ammoniagenesis in chronic acidosis. The contribution of the purine nucleotide cycle to ammonia formation appears to be unimportant in renal tubules from chronically acidotic rats.     

The purine nucleotide cycle and ammonia formation from glutamine by rat kidney slices.

To test the significance of the purine nucleotide cycle in renal ammoniagenesis, studies were conducted with rat kidney cortical slices using glutamate or glutamine labelled in the alpha-amino group with 15N. Glucose production by normal kidney slices with 2 mM-glutamine was equal to that with 3 mM-glutamate. With L-[15N]glutamate as sole substrate, one-third of the total ammonia produced by kidney slices was labelled, indicating significant deamination of glutamate or other amino acids from the cellular pool. Ammonia produced from the amino group of L-[alpha-15N]glutamine was 4-fold higher than from glutamate at similar glucose production rates. Glucose and ammonia formation from glutamine by kidney slices obtained from rats with chronic metabolic acidosis was found to be 70% higher than by normal kidney slices. The contribution of the amino group of glutamine to total ammonia production was similar in both types of kidneys. No 15N was found in the amino group of adenine nucleotides after incubation of kidney slices from normal or chronically acidotic rats with labelled glutamine. Addition of Pi, a strong inhibitor of AMP deaminase, had no effect on ammonia formation from glutamine. Likewise, fructose, which may induce a decrease in endogenous Pi, had no effect on ammonia formation. The data obtained suggest that the contribution of the purine nucleotide cycle to ammonia formation from glutamine in rat renal tissue is insignificant.     

Metabolism of glutamine and glutamic acid by isolated perfused kidneys of normal and acidotic rats

1. When isolated kidneys from fed rats were perfused with glutamine the rate of ammonia release at pH7.4 (110–360μmol/h per g dry wt.) was one to two times that of glutamine removal. Glucose formation from 5mm-glutamine was 16μmol/h per g. If kidneys were perfused with glutamine at pH7.1 (10–13mm-sodium bicarbonate) there was no increase in glutamine removal or in the formation of ammonia or glucose. 2. When isolated kidneys from fed rats were perfused with glutamate at pH7.4, glucose formation was 59μmol/h per g, glutamine formation was 182μmol/h per g and ammonia release was negligible. At pH7.1 glutamine synthesis was inhibited and formation of ammonia and glucose were increased. 3. In perfused kidneys from acidotic rats, which had received 1.5% (w/v) NH₄Cl to drink for 7–10 days, gluconeogenesis from glutamine was enhanced (101μmol/h per g). Glutamine removal and ammonia formation were also increased, compared with the rates in perfused kidney from normal rats. The extra glutamine consumed was equivalent to the extra glucose formed. 4. When the kidney from the 7–10-day-acidotic rat was perfused with glutamate gluconeogenesis was increased (113μmol/h per g). Synthesis of glutamine was decreased, and ammonia release was approximately equal to the rate of glutamate removal. 5. The time-course of these metabolic alterations was investigated after the rapid induction of acidosis by infusion of 0.25m-HCl into the right side of the heart. The increase in gluconeogenesis from glutamine developed gradually over several hours. When kidneys from 6h-acidotic rats were perfused with glutamate, formation of glucose and glutamine were both rapid. 6. In acidotic rat kidneys perfused with glutamine, tissue concentrations of glutamate and glucose 6-phosphate were increased compared with those in control perfused kidneys from non-acidotic rats. 7. The results are discussed in terms of control of the renal metabolism of glutamine. In particular, it is suggested that in acidotic rats glucose formation is the major fate of the carbon of the extra glutamine utilized by the kidney, and that inhibition of glutamine synthetase could contribute to the increase in intracellular ammonia concentration in the kidney.     

Renal extraction of glutamine from plasma and whole blood: studies in dogs and rats

The change in plasma and blood cell pools of L-glutamine during a single pass through the kidney was studied in dogs and rats. It was shown that the glutamine content of blood cells does not change following one passage through the renal vascular bed in normal or acidotic dogs. Furthermore, an infusion of L-glutamine elevating by 10-fold the plasma concentration of this amino acid only minimally changed the blood cells' glutamine content. Therefore within the time frame of acute experiments, the dog blood cells can be assumed to be impermeable to glutamine in vivo. Accordingly, renal glutamine extraction can be measured using either whole blood or plasma arteriovenous difference in this species. However, the latter value is larger and therefore can be measured more accurately. In normal rats, no net renal glutamine extraction is measured. In contrast, a considerable renal glutamine uptake occurs in acidotic rats, 23% of the extracted glutamine coming from the blood cell pool. A load of glutamine in vivo significantly elevates both the plasma and the blood cell concentration. It is concluded (i) that the renal extraction of glutamine is best estimated using plasma arteriovenous difference in the dog, especially when the renal extraction is small; (ii) that whole blood measurements should be obtained in the rat.     

Effect of acidosis on glutamine transport by isolated rat renal brush-border and basolateral-membrane vesicles.

Glutamine uptake was examined in isolated renal brush-border and basolateral-membrane vesicles from control and acidotic rats. In brush-border vesicles from acidotic animals, there was a significant increase in the initial rate of glutamine uptake compared with that in controls. Lowering the pH of the medium increased the initial rate of glutamine uptake in brush-border vesicles from acidotic, but not from control, rats. In brush-border vesicles from both groups of animals, two saturable transport systems mediated glutamine uptake. There was a 2-fold increase in the Vmax. of the low-affinity high-capacity system in the brush-border vesicles from the acidotic animals compared with that from control animals, with no alteration in the other kinetic parameters. There was no difference in glutamine uptake by the two saturable transport systems in basolateral vesicles from control and acidotic animals. Lowering the incubation-medium pH increased the uptake of glutamine by basolateral vesicles from both control and acidotic rats to a similar extent. The data indicate that during acidosis there are alterations in glutamine transport by both the basolateral and brush-border membrane which could enhance its uptake by the renal-tubule cell for use in ammoniagenesis.     

Hepatic glutaminase flux regulation of glutamine homeostasis. Studies in vivo

The role of hepatic glutaminase flux in regulating plasma glutamine homeostasis was studied in the intact rat. Interorgan glutamine flow during chronic metabolic acidosis was away from the splanchnic bed and to the kidneys. Hindquarter and hepatic glutamine release were the major sources of glutamine removed by the kidneys. Interorgan glutamate flow was from the liver to the hindquarters and kidneys. Chronic metabolic acidosis reduced arterial glutamine concentration 30%. Acute respiratory acidosis (pH 7.12 +/- 0.02) returned arterial glutamine concentration to normal values, increasing and decreasing hepatic glutamine and glutamate release respectively; renal and gut glutamine removal rates were not decreased. Hepatic unidirectional glutamine utilization measured isotopically was decreased 51% by acute acidosis; unidirectional glutamine production was unchanged. The results are consistent with the proposed role of ammonia-activated hepatic glutaminase in the regulation of glutamine homeostasis during acute acidosis.     

Comparison of AMP deaminase from skeletal muscle of acidotic and normal rats.

The deamination of AMP by AMP aminohydrolase (EC 3.5.4.6.) serves as the major source of ammonia production in skeletal muscle. It has been suggested that the ammonia may serve either in a buffering capacity to combat acidosis due to the accumulation of lactic acid produced during prolonged muscular activity, or as a substrate for glutamine formation which can ultimately be utilized by the kidney in adapting to metabolic acidosis. In view of this proposal, the properties of the enzyme obtained from skeletal muscle of acidotic rats have been compared with the enzyme from normal muscle. The specific activity of AMP deaminase in crude homogenates of acidotic muscle was not significantly different from normal levels. The enzyme from acidotic muscle was purified to homogeneity and was found to be identical to the enzyme obtained from normal muscle by the criteria of electrophoretic mobility, pH optimum, molecular weight, sedimentation coefficient, subunit composition, amino acid composition, monovalent cation requirement, substrate saturation, and inhibition by ATP, Pi and creatine-P. Thus, if the enzyme functions to prevent acidosis, the ability to respond to changes in the intracellular environment which accompany acidosis must be built into the structure of the enzyme normally found in skeletal muscle. Three lines of evidence strongly support this viewpoint: (a) the rate of deamination is approximately 2-fold higher at pH 6.5 than at pH 7.0, (b) the activity increases linearly with a decrease in the adenylate energy charge, and (c) within the normal physiological range of the adenylate energy charge, the enzyme is operating at only 10--20% of its maximum capacity.     

The transport and metabolism of glutamine by kidney-cortex mitochondria from normal and acidotic rats.

1. The oxidation of glutamine by kidney-cortex mitochondria from normal and acidotic rats was not inhibited by avenaciolide, which did inhibit glutamate uptake and oxidation. The oxidation of glutamine by these mitochondria was always greater than that of glutamate. Direct measurements of the metabolism of [1-14C]glutamine in the presence of glutamate, and of [1-14C]glutamate in the presence of glutamine, demonstrated that the uptake and metabolism of external glutamate is insufficient to account for the observed rate of glutamine uptake and metabolism. Thus the postulated glutamine/glutamate antiport does not play a quantitatively important role in the metabolism of glutamine by renal mitochondria. 2. Rapid swelling of these mitochondria was observed in iso-osmotic solutions of L-glutamine and L-glutamyl-gamma-monohydroxamate but not in D-glutamine or L-isoglutamine (1-amido-2-aminoglutaric acid). Thus a relatively specific glutamine uniport exists in these mitochondria. 3. The utilization of glutamine was increased about 3-fold in mitochondria from chronically acidotic rats. Thus mitochondrial adaptations play an important part in the renal response to metabolic acidosis.     

Renal ammoniagenesis in rats made acutely acidotic by swimming

Rats develop metabolic acidosis acutely after exercise by swimming. Renal cortical slices from exercised rats show an increase in both ammoniagenesis and gluconeogenesis from glutamine. In addition, plasma from the exercised rats also stimulates ammoniagenesis in renal cortical slices from normal rats. In exercised rats renal phosphate dependent glutaminase shows a 200% activation when the enzyme activity is measured at subsaturating concentration of glutamine (1 mM) while only an increase of 12% in Vmax is observed. When kidney slices from normal rats are incubated in plasma from exercised rats an activation of phosphate dependent glutaminase is obtained with a 1.0 mM (100%) but not with 20 mM glutamine as substrate. This activation of phosphate dependent glutaminase at subsaturating levels of substrate may indicate a conformational change in PDG effected by a factor present in the plasma of exercised acidotic rats.     

The mechanism of inhibition by acidosis of gluconeogenesis from lactate in rat liver.

1. Gluconeogenesis from lactate or pyruvate was studied in perfused livers from starved rats at perfusate pH7.4 or under conditions simulating uncompensated metabolic acidosis (perfusate pH6.7-6.8). 2. In 'acidotic' perfusions gluconeogenesis and uptake of lactate or pyruvate were decreased. 3. Measurement of hepatic intermediate metabolites suggested that the effect of acidosis was exerted at a stage preceding phosphoenolpyruvate. 4. Total intracellular oxaloacetate concentration was significantly decreased in the acidotic livers perfused with lactate. 5. It is suggested that decreased gluconeogenesis in acidosis is due to substrate limitation of phosphoenolypyruvate carboxykinase. 6. The possible reasons for the fall in oxaloacetate concentration in acidotic livers are discussed; two of the more likely mechanisms are inhibition of the pyruvate carboxylase system and a change in the [malate]/[oxaloacetate] ratio due to the fall in intracellular pH.     

Ammonia production and pathways of glutamine utilization in rat kidney slices.

Ammonia production from glutamine was studied in slices from non-acidotic and acidotic rat kidneys. Slices from non-acidotic kidneys made 53% as much ammonia from D-glutamine as from L-glutamine during the initial 15 min of incubation. Thereafter the production rate from the L-isomer accelerated while that from the D-isomer remained constant. The accelerated rate of ammonia production from L-glutamine was dependent upon tissue swelling since prevention of swelling reduced the production rate. Swelling activates the mitochondrial glutaminase I pathway as evidenced by the rise in ammonia produced per glutamine utilized ratio as well as by the accelerated rate of CO2 production derived from the oxidative disposal of glutamin's carbon skeleton. Cortical slice swelling activates the mitochondrial pathway in a manner not unlike that seen in vivo during chronic acidosis and may reflect increased permeability to glutamine. Acidotic rat kidneys are not swollen in vivo while cortical slices initially produce 4-fold more ammonia than do non-acidotic slices. After 15 min, this 4-fold difference in total ammonia production drops to only a 2-fold difference due to the swelling-induced activation of the mitochondrial pathway. Consequently, slice swelling obliterates the important fact that ammonia production by the mitochondrial pathway is 15-fold greater in acidotic than in non-acidotic kidneys.     

Comparison of AMP deaminase from skeletal muscle of acidotic and normal rats

The deamination of AMP by AMP aminohydrolase (EC 3.5.4.6) serves as the major source of ammonia production in skeletal muscle. It has been suggested that the ammonia may serve either in a buffering capacity to combat acidosis due to the accumulation of lactic acid produced during prolonged muscular activity, or as a substrate for glutamine formation which can ultimately be utilized by the kidney in adapting to metabolic acidosis. In view of this proposal, the properties of the enzyme obtained from skeletal muscle of acidotic rats have been compared with the enzyme from normal muscle. The specific activity of AMP deaminase in crude homogenates of acidotic muscle was not significantly different from normal levels. The enzyme from acidotic muscle was purified to homogeneity and was found to be identical to the enzyme obtained from normal muscle by the criteria of electrophoretic mobility, pH optimum, molecular weight, sedimentation coefficient, subunit composition, amino acid composition, monovalent cation requirement, substrate saturation, and inhibition by ATP, P_i and creatine-P. Thus, if the enzyme functions to prevent acidosis, the ability to respond to changes in the intracellular environment which accompany acidosis must be built into the structure of the enzyme normally found in skeletal muscle. Three lines of evidence strongly support this viewpoint: (a) the rate of deamination is approximately 2-fold higher at pH 6.5 than at pH 7.0, (b) the activity increases linearly with a decrease in the adenylate energy charge, and (c) within the normal physiological range of the adenylate energy charge, the enzyme is operating at only 10–20% of its maximum capacity.     

Proteomic profiling of the effect of metabolic acidosis on the apical membrane of the proximal convoluted tubule

The physiological response to the onset of metabolic acidosis requires pronounced changes in renal gene expression. Adaptations within the proximal convoluted tubule support the increased extraction of plasma glutamine and the increased synthesis and transport of glucose and of NH(4)(+) and HCO(3)(-) ions. Many of these adaptations involve proteins associated with the apical membrane. To quantify the temporal changes in these proteins, proteomic profiling was performed using brush-border membrane vesicles isolated from proximal convoluted tubules (BBMV(PCT)) that were purified from normal and acidotic rats. This preparation is essentially free of contaminating apical membranes from other renal cortical cells. The analysis identified 298 proteins, 26% of which contained one or more transmembrane domains. Spectral counts were used to assess changes in protein abundance. The onset of acidosis produced a twofold, but transient, increase in the Na(+)-dependent glucose transporter and a more gradual, but sustained, increase (3-fold) in the Na(+)-dependent lactate transporter. These changes were associated with the loss of glycolytic and gluconeogenic enzymes that are contained in the BBMV(PCT) isolated from normal rats. In addition, the levels of γ-glutamyltranspeptidase increased twofold, while transporters that participate in the uptake of neutral amino acids, including glutamine, were decreased. These changes could facilitate the deamidation of glutamine within the tubular lumen. Finally, pronounced increases were also observed in the levels of DAB2 (3-fold) and myosin 9 (7-fold), proteins that may participate in endocytosis of apical membrane proteins. Western blot analysis and accurate mass and time analyses were used to validate the spectral counting.     

Ammonia production and pathways of glutamine utilization in rat kidney slices

Ammonia production from glutamine was studied in slices from non-acidotic and acidotic rat kidneys. Slices from non-acidotic kidneys made 53% as much ammonia from d-glutamine as from l-glutamine during the initial 15 min of incubation. Thereafter the production rate from the l-isomers accelerated while that from the d-isomers remained constant. The accelerated rate of ammonia production from l-glutamine was dependent upon tissue swelling since prevention of swelling reduced the production rate. Swelling activates the mitochondrial glutaminase I pathway as evidenced by the rise in ammonia produced per glutamine utilized ratio as well as by the accelerated rate of CO₂ production derived from the oxidative disposal of glutamine's carbon skeleton. Cortical slice swelling activates the mitochondrial pathway in a manner not unlike that seen in vivo during chronic acidosis and may reflect increased permeability to glutamine.Acidotic rat kidneys are not swollen in vivo while cortical slices initially produce 4-fold more ammonia than do non-acidotic slices. After 15 min, this 4-fold difference in total ammonia production drops to only a 2-fold difference due to the swelling-induced activation of the mitochondrial pathway. Consequently, slice swelling obliterates the important fact that ammonia production by the mitochondrial pathway is 15-fold greater in acidotic than in non-acidotic kidneys.     

Variations in the kinetic response of several different phosphate-dependent glutaminase isozymes during acute metabolic acidosis

Summary We describe the kinetic modifications to mitochondrial-membrane-bound phosphate-dependent glutaminase in various types of rat tissue brought about by acute metabolic acidosis. The activity response of phosphate-dependent glutaminase to glutamine was sigmoidal, showing positive co-operativity, the Hill coefficients always being higher than 2. The enzyme from acidotic rats showed increased activity at subsaturating concentrations of glutamine in kidney tubules, as might be expected, but not in brain, intestine or liver tissues. Nevertheless, when brain and intestine from control rats were incubated in plasma from acutely acidotic rats enzyme activity increased at 1 mM glutamine in the same way as in kidney cortex. The enzyme from liver tissue remained unaltered. S_0.5 and n_H values decreased significantly in kidney tubules, enterocytes and brain slices preincubated in plasma from acidotic rats. The sigmoidal curves of phosphate-dependent glutaminase shifted to the left without any significant changes in V_max. The similar response of phosphate-dependent glutaminase to acute acidosis in the kidney, brain and intestine confirms the fact that enzymes from these tissues are kinetically identical and reaffirms the presence of an ammoniagenic factor in plasma, either produced or concentrated in the kidneys of rats with acute acidosis.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid - EDTA NN-1,2-Ethane-diylbis [N-(carboxymethyl)glycyne] - Tris 2-amino-2-hydroxymethyl-1,3-propanediol - PDG phosphate dependent glutaminase Publication No. 145 from Drogas, Tóxicos Ambientales y Metabolismo Celular Research Group. Department of Biochemistry and Molecular Biology, University of Granada, Spain     

Metabolic fate of glutamate carbon in rat renal tubules. Studies with 13C nuclear magnetic resonance and gas chromatography-mass spectrometry.

13C-n.m.r. spectroscopy and g.c.-m.s. were used to determine the metabolic fate of glutamate carbon in rat kidney. The main purpose was to characterize the effect of chronic metabolic acidosis on the utilization of glutamate carbon. Renal tubules obtained from normal and chronically acidotic rats were incubated in Krebs buffer, pH 7.4, in the presence of 2.5 mM-[3-13C]glutamate. During the course of incubation the concentrations of total glucose and NH3 were significantly (P less than 0.05) higher in tissue from acidotic rats. The levels of some tricarboxylic-acid-cycle intermediates were higher (P less than 0.05) in control tissue. In control tissue, 13C-n.m.r. spectra demonstrated a significantly higher rate of 13C appearance of aspartate, glutamine and [2,4-13C]glutamate. However, in acidosis the resonances of [13C]glucose carbon atoms were significantly higher. In the control, approx. 15% of glutamate carbon was accounted for by [13C]glucose formation as against 30% in chronic acidosis. However, in control tissue, 44% of glutamate carbon utilization was accounted for by recycling to glutamate and formation of aspartate, glutamine and GABA. In acidosis, only 11% was so recovered. Analysis of 15NH3 formation during the course of incubation with 2.5 mM-[15N]glutamate demonstrated a positive association between the appearance of [13C]glucose and 15NH3 both in the control and in acidosis. The data suggest that the control of gluconeogenesis and ammoniagenesis in acidosis is, in part, referable to a diminution in the rate of the reductive amination of alpha-oxoglutarate, that of the transamination reaction and that of glutamine synthesis.     

Substrate utilization by the distal nephron in dogs with chronic metabolic acidosis: studies with ethacrynic acid

Glutamine and lactate oxidations provide the bulk of ATP required for sodium reabsorption in the dog kidney during chronic metabolic acidosis. Indirect evidence has suggested that glutamine is oxidized in the proximal convoluted tubule; if this is true, lactate should be the major fuel of the more distal nephron sites. The purpose of these experiments was to determine which substrates were metabolized by the acidotic dog kidney when a significant proportion of sodium chloride reabsorption was inhibited in the thick ascending limb of the loop of Henle. Ethacrynic acid, a loop diuretic, caused the fractional excretion of sodium to increase from 1 to 34%. The glomerular filtration rate declined somewhat, but there was no significant change in the renal blood flow rate. Renal oxygen consumption declined in conjunction with the natriuresis. However, when the data were examined at a constant filtered load of sodium (a constant rate of ATP turnover), there was no reduction in glutamine uptake or glutamine conversion to ATP in the presence of this natriuretic agent. The major change observed concerned lactate metabolism, in the presence of ethacrynic acid, there was no longer a significant rate of lactate extraction. These data are best explained by assuming that glutamine is the fuel of the proximal convoluted tubule of the acidotic dog kidney, whereas lactate oxidation occurs principally in the nephron sites where sodium reabsorption was inhibited by ethacrynic acid.     

The 1986 Borden award lecture. The role of the kidney in amino acid metabolism and nutrition

Measurement of the arteriovenous differences for free amino acids across rat kidney reveals that glycine and citrulline are removed and serine and arginine are added to the circulation. In addition, glutamine is taken up in large quantities by kidneys of animals that need to excrete large quantities of acid (e.g., diabetic animals, NH4Cl-fed animals, and animals fed a high protein diet). Glutamine is the major precursor of urinary ammonia and thus renal glutamine metabolism plays a key role in acid-base homeostasis. This process occurs primarily in the cells of the convoluted proximal tubule. Glutamine carbon is converted to glucose in acidotic rats and is totally oxidized in dogs. Regulation of glutamine metabolism occurs at two levels: acute regulation and chronic regulation. Acute regulation is, in part, mediated through a fall in intracellular [H+]. This activates alpha-ketoglutarate dehydrogenase and, ultimately, glutaminase. Chronic regulation involves induction of key enzymes, including, in the rat, glutaminase, glutamate dehydrogenase, and phosphoenolpyruvate carboxykinase. During the acidosis of prolonged starvation, the kidneys' requirement for glutamine must be met from muscle proteolysis and thus becomes a drain on lean body mass. Serine synthesis occurs by two separate pathways: from glycine by the combined actions of the glycine cleavage enzyme and serine hydroxymethyltransferase and from gluconeogenic precursors using the phosphorylated-intermediate pathway. Both pathways are located in the cells of the proximal tubule. Conversion of glycine to serine is ammoniagenic and the activity of the glycine cleavage enzyme is increased in acidosis. The function of serine synthesis by the phosphorylated-intermediate pathway is not apparent.(ABSTRACT TRUNCATED AT 250 WORDS)