Carbon 13 NMR spectroscopy: a powerful tool for studying renal metabolism.

Using precise examples, this paper shows that carbon 13 NMR spectroscopy in conjunction with radioactive and enzymatic methods as well as with adequate mathematical modeling of metabolic pathways allows not only to identify but also to quantify fluxes through enzymes involved in substrate and drug metabolism. Carbon 13 NMR spectroscopy is a tool of unprecedented power to unravel the complexity of renal metabolism. Currently it plays a major role in what is nowadays called metabolomics.

Acetate stimulates flux through the tricarboxylic acid cycle in rabbit renal proximal tubules synthesizing glutamine from alanine: a 13C NMR study.

Although glutamine synthesis has a major role in the control of acid-base balance and ammonia detoxification in the kidney of herbivorous species, very little is known about the regulation of this process. We therefore studied the influence of acetate, which is readily metabolized by the kidney and whose metabolism is accompanied by the production of bicarbonate, on glutamine synthesis from variously labelled [(13)C]alanine and [(14)C]alanine molecules in isolated rabbit renal proximal tubules. With alanine as sole exogenous substrate, glutamine and, to a smaller extent, glutamate and CO(2), were the only significant products of the metabolism of this amino acid, which was removed at high rates. Absolute fluxes through the enzymes involved in alanine conversion into glutamine were assessed by using a novel model describing the corresponding reactions in conjunction with the (13)C NMR, and to a smaller extent, the radioactive and enzymic data. The presence of acetate (5 mM) led to a large stimulation of fluxes through citrate synthase and alpha-oxoglutarate dehydrogenase. These effects were accompanied by increases in the removal of alanine, in the accumulation of glutamate and in flux through the anaplerotic enzyme pyruvate carboxylase. Acetate did not alter fluxes through glutamate dehydrogenase and glutamine synthetase; as a result, acetate did not change the accumulation of ammonia, which was negligible under both experimental conditions. We conclude that acetate, which seems to be an important energy-provider to the rabbit renal proximal tubule, simultaneously traps as glutamate the extra nitrogen removed as alanine, thus preventing the release of additional ammonia by the glutamate dehydrogenase reaction.

The rabbit kidney tubule simultaneously degrades and synthesizes glutamate. A 13C NMR study.

The rabbit kidney does not readily metabolize but synthesizes glutamine at high rates by pathways that remain poorly defined. Therefore, the metabolism of variously labeled [13C]- and [14C]glutamates has been studied in isolated rabbit kidney tubules with and without acetate. CO2, glutamine, and alanine were the main carbon and nitrogenous end products of glutamate metabolism but no ammonia accumulated. Absolute fluxes through enzymes involved in glutamate metabolism, including enzymes of four different cycles operating simultaneously, were assessed by combining mainly the 13C NMR data with a new model of glutamate metabolism. In contrast to a previous conclusion of Klahr et al. (Klahr, S., Schoolwerth, A. C., and Bourgoignie, J. J. (1972) Am. J. Physiol. 222, 813-820), glutamate metabolism was found to be initiated by glutamate dehydrogenase at high rates. Glutamate dehydrogenase also operated at high rates in the reverse direction; this, together with the operation of the glutamine synthetase reaction, masked the release of ammonia. Addition of acetate stimulated the operation of the "glutamate --> alpha-ketoglutarate --> glutamate" cycle and the accumulation of glucose but reduced both the net oxidative deamination of glutamate and glutamine synthesis. Acetate considerably increased flux through alpha-ketoglutarate dehydrogenase and citrate synthase at the expense of flux through phosphoenolpyruvate carboxykinase; acetate also caused a large decrease in flux through alanine aminotransferase, pyruvate dehydrogenase, and the "substrate cycle" involving oxaloacetate, phosphoenolpyruvate, and pyruvate.

Model applicable to NMR studies for calculating flux rates in five cycles involved in glutamate metabolism.

Based on the same principles as those utilized in a recent study for modeling glucose metabolism (Martin, G., Chauvin, M. F., Dugelay, S., and Baverel, G. (1994) J. Biol. Chem. 269, 26034-26039), a method is presented for determining metabolic fluxes involved in glutamate metabolism in mammalian cells. This model consists of five different cycles that operate simultaneously. It includes not only the tricarboxylic acid cycle, the "oxaloacetate --> phosphoenolpyruvate --> pyruvate --> oxaloacetate" cycle and the "oxaloacetate --> phosphoenolpyruvate --> pyruvate --> acetyl-CoA --> citrate --> oxaloacetate" cycle but also the "glutamate --> alpha-ketoglutarate --> glutamate" and the "glutamate --> glutamine --> glutamate" cycles. The fates of each carbon of glutamate, expressed as ratios of integrated transfer of this carbon to corresponding carbons in subsequent metabolites, are described by a set of equations. Since the data introduced in the model are micrograms of atom of traced carbon incorporated into each carbon of end products, the calculation strategy was determined on the basis of the most reliable parameters determined experimentally. This model, whose calculation routes offer a large degree of flexibility, is applicable to data obtained by 13C NMR spectroscopy, gas chromatography - mass spectrometry, or 14C counting in a great variety of mammalian cells.

Advantages and limitations of the use of isolated kidney tubules in pharmacotoxicology.

Among the cellular models used in in vitro renal pharmacotoxicology, isolated kidney tubules, used as suspensions mainly of proximal tubules, offer important advantages. They can be prepared in large amounts under nonsterile conditions within 1-2 h; thus, it is possible to employ a great number of experimental conditions simultaneously and to obtain rapidly many experimental results. Kidney tubules can be prepared from the kidney of many animal species and also from the human kidney; given the very limited availability of healthy human renal tissue, it is therefore possible to choose the most appropriate species for the study of a particular problem encountered in man. Kidney tubules can be used for screening and prevention of nephrotoxic effects and to identify their mechanisms as well as to study the renal metabolism of xenobiotics. When compared with cultured renal cell, a major advantage of kidney tubules is that they remain differentiated. The main limitations of the use of kidney tubules in pharmacotoxicology are (1) the necessity to prepare them as soon as the renal tissue sample is obtained; (2) their limited viability, which is restricted to 2-3 h; (3) the inability to expose them chronically to a potential nephrotoxic drug; (4) the inability to study transepithelial transport; and (5) the uncertainty in the extrapolation to man of the results obtained using animal kidney tubules. These advantages and limitations of the use of human and animal kidney tubules in pharmacotoxicology are illustrated mainly by the results of experiments performed with valproate, an antiepileptic and moderately hyperammonemic agent. The fact that kidney tubules, unlike cultured renal cells, retain key metabolic properties is also shown to be of the utmost importance in detecting certain nephrotoxic effects.

Unusual Microsporum canis infections in adult HIV patients.

Tinea capitis in men, even if infected with HIV, is infrequent. Microsporum species nail infections are extremely rare. In most cases Microsporum canis infection is usually easy to treat with antifungal agents. We describe two HIV-infected men with an unusual M. canis infection. Both patients had tinea capitis, presenting as alopecia in one and scaling of the scalp in the other. One patient also had tinea unguium caused by M. canis. Ketoconazole was ineffective in both patients; terbinafine was tried in one patient without benefit; itraconazole was effective in both, but treatment took many months and only one patient was cured.

Non-steady state model applicable to NMR studies for calculating flux rates in glycolysis, gluconeogenesis, and citric acid cycle.

We present a mathematical model for calculating most reaction rates of glycolysis, gluconeogenesis and citric acid cycle in mammalian cells. The model also includes cycles such as the "phosphoenolpyruvate (PEP)-->pyruvate-->oxaloacetate-->PEP" cycle and the "pyruvate-->acetyl-CoA-->citrate-->citric acid cycle-->oxaloacetate-->PEP--> pyruvate" cycle. The model, which does not require steady state conditions, is based on a set of equations, each one describing the fates of a given carbon of a selected intermediate. These fates are expressed as ratios of integrated transfer of this carbon to corresponding carbons in subsequent metabolites. At each bifurcation, the sum of all proportions adds up to 1. Among several calculation routes to determine a proportion value, we chose the one that was based on the most reliable parameter determined experimentally. The data introduced in the model are the micrograms of atom of traced carbon measured on each carbon of a number of products (corrected for natural tracer abundance). These incorporations can be measured by 13C NMR, gas chromatography-mass spectroscopy, or 14C counting. Thanks to its flexibility, this model can be applied to data obtained with substrates other than glucose under many experimental conditions.

The rabbit kidney tubule utilizes glucose for glutamine synthesis. A 13C NMR study.

The metabolism of variously labeled [13C]- and [14C]glucoses, used at a physiological concentration (5 mM), has been studied in isolated rabbit kidney tubules both in the absence and the presence of NH4Cl. When present as sole exogenous substrate, glucose was metabolized at high rates and converted not only into CO2 and lactate but also, in contrast to a previous conclusion of Krebs (Krebs, H.A. (1935) Biochem. J. 29, 1951-1969), into glutamine. Absolute fluxes through enzymes of glycolysis and gluconeogenesis and of enzymes of three different cycles operating simultaneously were assessed by using a novel model describing reactions of glucose metabolism in conjunction with the 13C NMR and, to a lesser extent, the radioactive data obtained. The presence of NH4Cl (5 mM) caused a large stimulation of glucose removal and a large increase in lactate, glutamine, and glycerol 3-phosphate accumulation. Under this condition, the stimulation of glutamine synthesis was accompanied not by an activation of citrate synthesis but by an inhibition of flux through alpha-ketoglutarate dehydrogenase. The resulting depletion of citric acid cycle intermediates was compensated by anaplerosis at the level of pyruvate carboxylase. The "futile" cycle involving oxaloacetate, phosphoenolpyruvate, and pyruvate, which was intense in the presence of glucose alone, was greatly stimulated by the addition of NH4Cl.

Glutamine synthesis from glucose and ammonium chloride by guinea-pig kidney tubules.

1. At a physiological concentration (5 mM), glucose was found to be metabolized by isolated kidney cortex tubules prepared from fed guinea pigs. 2. The release of 14CO2 from [U-14C]glucose indicated that oxidation of the glucose carbon skeleton represented about 50% of the glucose removed; significant amounts of lactate and glutamine also accumulated. 3. Addition of 0.1-10 mM NH4Cl led to a dose-dependent stimulation of glucose metabolism which was accompanied by a large increase in lactate and glutamine accumulation and, to a lesser extent, in glucose oxidation. 4. Comparison of the release of 14CO2 from [1-14C]- and [6-14C]glucose indicates that, in both the absence and the presence of NH4Cl, the pentose phosphate shunt was only a minor pathway of glucose metabolism. 5. The central role of pyruvate carboxylase in the conversion of glucose carbon into glutamine carbon was demonstrated by using a bicarbonate-free medium and measuring the fixation of 14CO2 from [14C]bicarbonate, which was recovered mostly at C-1 of glutamine plus glutamate. 6. The NH4Cl-induced stimulation of glucose removal was secondary not only to increased glutamine synthesis, as shown by the effect of methionine sulphoximine, an inhibitor of glutamine synthetase, but also to the stimulation of phosphofructokinase activity by NH4Cl. 7. Renal arterio-venous difference measurements revealed that, in vivo, the guinea-pig kidney removed glucose from the circulating blood, which suggests that glucose carbon may contribute to the carbon skeleton of the glutamine released by this organ.