Protocols and Applications of Cellular Metabolomics in Safety Studies Using Precision-Cut Tissue Slices and Carbon 13 NMR.

Numerous xenobiotics are toxic to human and animal cells by interacting with their metabolism, but the precise metabolic step affected and the biochemical mechanism behind such a toxicity remain often unknown. In an attempt to reduce the ignorance in this field, we have developed a new approach called cellular metabolomics. This approach, developed in vitro, provides a panoramic view not only of the pathways involved in the metabolism of physiological substrates of any normal or pathological human or animal cell but also of the beneficial and adverse effects of xenobiotics on these metabolic pathways. Unlike many cell lines, precision-cut tissue slices, for which there is a renewed interest, remain metabolically differentiated for at least 24-48 h and allow to study the effect of xenobiotics during short-term and long-term incubations. Cellular metabolomics (or metabolic flux analysis), which combines enzymatic and carbon 13 NMR measurements with mathematical modeling of metabolic pathways, is illustrated in this brief chapter for studying the effect of insulin on glucose metabolism in rat liver precision-cut slices and of valproate on glutamine metabolism in human renal cortical precision-cut slices. The use of very small amounts of test compounds allows to predict their toxic effect and eventually their beneficial effects very early in the research and development processes. Cellular metabolomics is complementary to other omics approaches, but, unlike them, provides functional, mechanistic, and dynamic pieces of information by measuring enzymatic fluxes.

Cadmium chloride inhibits lactate gluconeogenesis in mouse renal proximal tubules: An in vitro metabolomic approach with (13)C NMR.

Using isolated mouse renal proximal tubules incubated with lactate as substrate, we have found that the addition of 1-50 µM cadmium chloride (CdCl2) caused a concentration-dependent decrease in lactate utilization, in glucose production and in the cellular level of ATP, coenzyme A, acetyl-coenzyme A and glutathione (reduced and oxidized forms). Combining enzymatic and (13)C NMR measurements in a cellular metabolomic approach, we have shown that, in the presence of 10 µM CdCl2, fluxes through the key-enzymes of gluconeogenesis, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were greatly depressed by cadmium. This was accompanied by a reduction in fluxes through the enzymes of the tricarboxylic acid cycle. Comparing the mouse and human renal metabolic responses to cadmium, it is interesting to observe that the mouse renal proximal tubule was much more sensitive than the human renal proximal tubule to the adverse effects of CdCl2. As far as renal gluconeogenesis is concerned, the mouse seems to be an appropriate and convenient animal model to study the mechanism of cadmium nephrotoxicity. However, the data obtained in the mouse should be extrapolated to humans with caution because the inhibition of fluxes through the enzymes of the tricarboxylic acid cycle in mouse tubules were not observed in human tubules.

Use of precision-cut renal cortical slices in nephrotoxicity studies.

1.Unlike cell lines and primary cells in culture, precision-cut tissue slices remain metabolically differentiated for at least 24-48 h and allow to study the effect of xenobiotics during short-term and long-term incubations. 2.In this article, we illustrate the use of such an experimental model to study the nephrotoxic effects of (i) chloroacetaldehyde, a metabolite of the anticancer drug ifosfamide, (ii) of cobalt chloride, a potential leakage product of the cobalt-containing nanoparticles, and (iii) of valproate, a widely used antiepileptic drug. 3.Since all the latter test compounds, like many toxic compounds, negatively interact with cellular metabolic pathways, we also illustrate our biochemical toxicology approach in which we used not only enzymatic but also carbon 13 NMR measurements and mathematical modelling of metabolic pathways. 4.This original approach, which can be applied to any tissue, allows to predict the nephrotoxic effects of milligram amounts of test compounds very early during the research and development processes of drugs and chemicals. This approach, combined with the use of cells that retain their in vivo metabolic properties and, therefore, are predictive, reduces the risk, the time and cost of such processes.

Brain slices from glutaminase-deficient mice metabolize less glutamine: a cellular metabolomic study with carbon 13 NMR.

In the brain, glutaminase is considered to have a key role in the provision of glutamate, a major excitatory neurotransmitter. Brain slices obtained from wild-type (control) and glutaminase-deficient (GLS1+/-) mice were incubated without glucose and with 5 or 1 mmol/L [3-(13)C]glutamine as substrate. At the end of the incubation, substrate removal and product formation were measured by both enzymatic and carbon 13 nuclear magnetic resonance ((13)C-NMR) techniques. Slices from GLS1+/- mice consumed less [3-(13)C]glutamine and accumulated less [3-(13)C]glutamate. They also produced less (13)CO(2) but accumulated amounts of (13)C-aspartate and (13)C-gamma-aminobutyric acid (GABA) that were similar to those found with brain slices from control mice. The newly formed glutamine observed in slices from control mice remained unchanged in slices from GLS1+/- mice. As expected, flux through glutaminase in slices from GLS1+/- mice was found diminished. Fluxes through all enzymes of the tricarboxylic acid cycle were also reduced in brain slices from GLS1+/- mice except through malate dehydrogenase with 5 mmol/L [3-(13)C]glutamine. The latter diminutions are consistent with the decreases in the production of (13)CO(2) also observed in the slices from these mice. It is concluded that the genetic approach used in this study confirms the key role of glutaminase for the provision of glutamate.

Effects of valproate on glutamate metabolism in rat brain slices: a (13)C NMR study.

Sodium valproate is a drug widely used for the treatment of epilepsy and mood disorders. We studied the effect of valproate on cerebral energy metabolism by incubating rat brain slices with 5 mM [3-(13)C]glutamate in the absence and the presence of 1 mM valproate. Substrate removal and product formation were measured by enzymatic and carbon 13 NMR methods. Fluxes through the enzymatic steps involved were calculated with an original mathematical model. We demonstrate that, in the presence of valproate, glutamate consumption and aspartate accumulation and labeling were inhibited, whereas GABA accumulation and labeling were increased. Consistent with these observations, this drug inhibited the unidirectional flux from glutamate to α-ketoglutarate and fluxes through several enzymes (gamma aminobutyric acid aminotransferase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, aspartate aminotransferase, malic enzyme, pyruvate dehydrogenase, pyruvate carboxylase and citrate synthase). By contrast, glutamic acid decarboxylase flux was increased. With 2 mM glutamate+1 mM valproate and with 5 mM glutamate+2 mM valproate, GABA and aspartate labelings were similarly altered. On the basis of the effects of valproate, it is concluded that our cellular model and our cellular metabolomic approach appear suitable to study the beneficial and adverse interactions of neurotropic compounds with the cerebral metabolic pathways.

Rat brain slices oxidize glucose at high rates: a (13)C NMR study.

Since glucose is the main cerebral substrate, we have characterized the metabolism of various (13)C glucose isotopomers in rat brain slices. For this, we have used our cellular metabolomic approach that combines enzymatic and carbon 13 NMR techniques with mathematical models of metabolic pathways. We identified the fate and the pathways of the conversion of glucose carbons into various products (pyruvate, lactate, alanine, aspartate, glutamate, GABA, glutamine and CO(2)) and determined absolute fluxes through pathways of glucose metabolism. After 60 min of incubation, lactate and CO(2) were the main end-products of the metabolism of glucose which was avidly metabolized by the slices. Lactate was also used at high rates by the slices and mainly converted into CO(2). High values of flux through pyruvate carboxylase, which were similar with glucose and lactate as substrate, were observed. The addition of glutamine, but not of acetate, stimulated pyruvate carboxylation, the conversion of glutamate into succinate and fluxes through succinate dehydrogenase, malic enzyme, glutamine synthetase and aspartate aminotransferase. It is concluded that, unlike brain cells in culture, and consistent with high fluxes through PDH and enzymes of the tricarboxylic acid cycle, rat brain slices oxidized both glucose and lactate at high rates.

Metabolic fate of a high concentration of glutamine and glutamate in rat brain slices: a ¹³C NMR study.

This study was performed to analyze the metabolic fate of a high concentration (5 mM) of glutamine and glutamate in rat brain slices and the participation of these amino acids in the glutamine-glutamate cycle. For this, brain slices were incubated for 60 min with [3-¹³C]glutamine or [3-¹³C]glutamate. Tissue plus medium extracts were analyzed by enzymatic and ¹³C NMR measurements and fluxes through pathways of glutamine and glutamate metabolism were calculated. We demonstrate that both substrates were utilized and oxidized at high rates by rat brain slices and served as precursors of neurotransmitters, tricarboxylic acid (TCA) cycle intermediates and alanine. In order to determine the participation of glutamine synthetase in the appearance of new glutamine molecules with glutamine as substrate, brain slices were incubated with [3-¹³C]glutamine in the presence of methionine sulfoximine, a specific inhibitor of glutamine synthetase. Our results indicate that 36.5% of the new glutamine appeared was glutamine synthetase-dependent and 63.5% was formed from endogenous substrates. Flux through glutamic acid decarboxylase was higher with glutamine than with glutamate as substrate whereas fluxes from α-ketoglutarate to glutamate and through glutamine synthetase, malic enzyme, pyruvate dehydrogenase, pyruvate carboxylase and citrate synthase were in the same range with both substrates.

Protocols and applications of cellular metabolomics in safety studies using precision-cut tissue slices and carbon 13 NMR.

Numerous xenobiotics are toxic to human and animal cells by interacting with their metabolism, but the precise metabolic step affected and the biochemical mechanism behind such a toxicity often remain unknown. In an attempt to reduce the ignorance in this field, we have developed a new approach called cellular metabolomics. This approach, developed in vitro, provides a panoramic view not only of the pathways involved in the metabolism of physiologic substrates of any normal or pathologic human or animal cell but also of the beneficial and adverse effects of xenobiotics on these metabolic pathways. Unlike many cell lines, precision-cut tissue slices, for which there is a renewed interest, remain metabolically differentiated for at least 24-48 h and allow to study the effect of xenobiotics during short-term and long-term incubations. Cellular metabolomics (or cellular metabonomics), which combines enzymatic and carbon 13 NMR measurements with mathematical modeling of metabolic pathways, is illustrated in this brief chapter for studying the effect of insulin on glucose metabolism in rat liver precision-cut slices, and of valproate on glutamine metabolism in human renal cortical precision-cut slices. The use of very small amounts of test compounds allows to predict their toxic effect and eventually their beneficial effects very early in the research and development processes. Cellular metabolomics is complementary to other omics approaches, but, unlike them, provides functional and dynamic pieces of information by measuring enzymatic fluxes.

Cadmium chloride inhibits lactate gluconeogenesis in isolated human renal proximal tubules: a cellular metabolomic approach with 13C-NMR.

As part of a study on cadmium nephrotoxicity, we studied the effect of cadmium chloride (CdCl2) in isolated human renal proximal tubules metabolizing the physiological substrate lactate. Dose-effect experiments showed that 10-500 µM CdCl2 reduced lactate removal, glucose production and the cellular levels of ATP, coenzyme A, acetyl-coenzyme A and of reduced glutathione in a dose-dependent manner. After incubation with 5 mM L: -[1-(13)C]-, or L: -[2-(13)C]-, or L: -[3-(13)C] lactate or 5 mM L: -lactate plus 25 mM NaH(13)CO3 as substrates, substrate utilization and product formation were measured by both enzymatic and carbon 13 NMR methods. Combination of enzymatic and NMR measurements with a mathematical model of lactate metabolism previously validated showed that 100 µM CdCl2 caused an inhibition of flux through lactate dehydrogenase and alanine aminotransferase and through the entire gluconeogenic pathway; fluxes were diminished by 19% (lactate dehydrogenase), 28% (alanine aminotransferase), 28% (pyruvate carboxylase), 42% (phosphoenolpyruvate carboxykinase), and 52% (glucose-6-phosphatase). Such effects occurred without altering the oxidation of the lactate carbons or fluxes through enzymes of the tricarboxylic acid cycle despite a large fall of the cellular ATP level, a marker of the energy status and of the viability of the renal cells. These results that were observed at clinically relevant tissue concentrations of cadmium provide a biochemical basis for a better understanding of the cellular mechanism of cadmium-induced renal proximal tubulopathy in humans chronically exposed to cadmium.

Uranyl nitrate inhibits lactate gluconeogenesis in isolated human and mouse renal proximal tubules: a 13C-NMR study.

As part of a study on uranium nephrotoxicity, we investigated the effect of uranyl nitrate in isolated human and mouse kidney cortex tubules metabolizing the physiological substrate lactate. In the millimolar range, uranyl nitrate reduced lactate removal and gluconeogenesis and the cellular ATP level in a dose-dependent fashion. After incubation in phosphate-free Krebs-Henseleit medium with 5 mM L-[1-13C]-, or L-[2-13C]-, or L-[3-13C]lactate, substrate utilization and product formation were measured by enzymatic and NMR spectroscopic methods. In the presence of 3 mM uranyl nitrate, glucose production and the intracellular ATP content were significantly reduced in both human and mouse tubules. Combination of enzymatic and NMR measurements with a mathematical model of lactate metabolism revealed an inhibition of fluxes through lactate dehydrogenase and the gluconeogenic enzymes in the presence of 3 mM uranyl nitrate; in human and mouse tubules, fluxes were lowered by 20% and 14% (lactate dehydrogenase), 27% and 32% (pyruvate carboxylase), 35% and 36% (phosphoenolpyruvate carboxykinase), and 39% and 45% (glucose-6-phosphatase), respectively. These results indicate that natural uranium is an inhibitor of renal lactate gluconeogenesis in both humans and mice.

Targets of chloroacetaldehyde-induced nephrotoxicity.

Chloroacetaldehyde, one of the main products of hepatic ifosfamide metabolism, contributes to its nephrotoxicity. However, the pathophysiology of this toxicity is not fully understood. The present work examined the time and dose effects of clinically relevant concentrations of chloroacetaldehyde (25-75microM) on precision-cut rat renal cortical slices metabolizing a physiological concentration of lactate. Chloroacetaldehyde toxicity was demonstrated by the decrease in total glutathione and cellular ATP levels. The drop of cellular ATP was linked to the inhibition of oxidative phosphorylation at the level of complex I of the mitochondrial respiratory chain. The large decrease in glucose synthesis from lactate was explained by the inhibition of some gluconeogenic enzymes, mainly glyceraldehyde 3-phosphate dehydrogenase. The decrease in lactate utilization was demonstrated not only by a defect of gluconeogenesis but also by the decrease in [(14)CO(2)] formation from [U-(14)C]-lactate. All the effects of chloroacetaldehyde were concentration and time-dependent. Finally, the chloroacetaldehyde-induced inhibition of glyceraldehyde 3-phosphate dehydrogenase, which is also a glycolytic enzyme, suggests that, under conditions close to those found during ifosfamide therapy, the inhibition of glycolytic pathway by chloroacetaldehyde might be responsible, at least in part, for the therapeutic efficacy of ifosfamide.

Ifosfamide metabolite chloroacetaldehyde inhibits cell proliferation and glucose metabolism without decreasing cellular ATP content in human breast cancer cells MCF-7.

Chloroacetaldehyde (CAA), a product of hepatic metabolism of the widely used anticancer drug ifosfamide (IFO), has been reported to decrease cancer cell proliferation. The basis of this effect is not completely known but has been attributed to a drop of cellular ATP content. Given the importance of glucose metabolism and of the 'Warburg effect' in cancer cells, we examined in the present study the ability of CAA to inhibit cancer cell proliferation by altering the glycolytic pathway. Cell proliferation, ATP content, glucose transport and metabolism as well as the activities of the main enzymes of glycolysis were determined in human breast cancer cells MCF-7 in the presence of various CAA concentrations (5-50 microm). Our results show that low CAA concentrations inhibited cell proliferation in a concentration-dependent manner. This inhibition was explained by a decrease in glucose utilization. Cellular ATP content was not reduced but even increased with 25 microm CAA. The inhibition of glucose metabolism was mainly explained by the decrease in glucose transport and hexokinase activity. The activity of glyceraldehyde-3-phosphate dehydrogenase, but not that of phosphofructokinase, was also inhibited. Glycolysis inhibition by CAA was effective in decreasing the proliferation of MCF-7 cells. Interestingly, this decrease was not due to ATP depletion; rather, it was linked to a drop of biosynthetic precursors from glycolytic intermediates. This CAA-induced inhibition of cell proliferation suggests that it might play a role in the antitumor activity of IFO.

Study of a new indirect method based on absolute norms of variation to detect autologous blood transfusion.

The aim of this study was to investigate an indirect method based on a determination of absolute norms of variation in biological markers that could be used to identify autologous blood transfusion within the framework of the fight against doping. The selection of markers was made from experimental variations obtained during different phases including an increase in training volume at sea level, high altitude training, blood withdrawal and autologous blood reinfusion. The global statistical method was then developed in order to fix absolute norms of variation for each selected marker. The markers selected were haematocrit (Hct), haemoglobin concentration ([Hb]), stimulation index (Off-hr) and the absolute norms of variation (normDelta) established for a maximal 15 days period were normDeltaHct(0-15) >6%, normDelta[Hb](0-15) >4% and normDeltaOff-hr(0-15) >20%. From analyses between two blood samples spaced at an interval of maximum 15 days, this method allows to show "abnormal" variation when a variation for one of the selected markers is strictly superior to the absolute norms of variation established. The legal framework for an immediate application of this method could be that of the internal regulations implemented by each international federation in accordance with the health policy in vigour.

In vivo mesna and amifostine do not prevent chloroacetaldehyde nephrotoxicity in vitro.

Chloroacetaldehyde (CAA) is the putative metabolite responsible for ifosfamide-induced nephrotoxicity. Whereas evidence suggests that sodium 2-mercaptoethanesulfonate (mesna) and amifostine protect renal cells against CAA toxicity in vitro, their efficacy in clinical studies is controversial. To better understand the discrepancy between in vivo and in vitro results, we combined the in vivo intraperitoneal administration of either saline or mesna (100 mg/kg) or amifostine (200 mg/kg) in rats and the in vitro study of CAA toxicity to both proximal tubules and precision-cut renal cortical slices. The measured renal cortical concentrations of mesna and amifostine were 0.6+/-0.1 micromol/g and 1.2+/-0.2 micromol/g, respectively; these drugs did not cause renal toxicity. Despite this, none of the adverse effects of 0.5 mM CAA was prevented by the previous in vivo administration of mesna or amifostine. Toxicity of 0.5 mM CAA to rat proximal tubules was shown by the fall of cellular adenosine triphosphate (ATP), total glutathione and coenzyme A + acetyl-coenzyme A levels and by the altered metabolic viability of renal cells. Long-term exposure of cortical slices to CAA concentrations > or =30 microM caused severe cell toxicity (i.e. decrease in cellular ATP, total glutathione, and coenzyme A + acetyl-coenzyme A levels), which was not prevented by the in vivo administration of mesna or amifostine.

Mechanisms of the ifosfamide-induced inhibition of endocytosis in the rat proximal kidney tubule.

The Fanconi syndrome is a common side effect of the chemotherapeutic agent ifosfamide. Current evidences suggest that chloroacetaldehyde (CAA), one of the main metabolites of ifosfamide activation, contributes to its nephrotoxicity. However, the pathophysiology of CAA-induced Fanconi syndrome is not fully understood. The present work examined the adverse effects of CAA on precision-cut rat renal cortical slices, which allowed studying the toxic effect of CAA on proximal endocytosis. We demonstrated that clinically relevant concentrations of CAA (< or =200 microM) are able to inhibit the uptake of horseradish peroxidase, a marker of proximal tubular cell endocytosis in renal tubular proximal cells. CAA > or =75 microM has adverse effects, both on viability parameters and on energy metabolism, as shown by the great decrease in total glutathione and ATP levels. In addition, the V-ATPase, which plays a crucial role in intracellular vesicle trafficking, was inhibited by 100 microM of CAA. By contrast, the slight decrease in Na-K-ATPase activity observed for CAA> or = 125 microM (maximum inhibition: 33%) could not totally explain the inhibition of the reabsorption processes. In conclusion, the addition of the two main adverse effects of CAA (decrease in ATP levels and inhibition of the V-ATPase) could explain the inhibition of endocytosis and the Fanconi syndrome observed during ifosfamide treatments.

[Renal and urinary tract disease national research program]. / Programme national de recherche sur les maladies du rein et des voies urinaires.

The National Institute of Health and Medical Research (Inserm), the Society of Nephrology, and the French Kidney Foundation recognized the need to create a National Research Program for kidney and urinary tract diseases. They organized a conference gathering 80 researchers to discuss the state-of-the art and evaluate the strengths and weaknesses of kidney and urinary tract disease research in France, and to identify research priorities. From these priorities emerged 11 of common interest: 1) conducting epidemiologic studies; 2) conducting large multicenter cohorts of well-phenotyped patients with blood, urine and biopsy biobanks; 3) developing large scale approach: transcriptomics, proteomics, metabolomics; 4) developing human and animal functional imaging techniques; 5) strengthening the expertise in renal pathology and electrophysiology; 6) developing animal models of kidney injury; 7) identifying nontraumatic diagnostic and prognostic biomarkers; 8) increasing research on the fetal programming of adult kidney diseases; 9) encouraging translational research from bench to bedside and to population; 10) creating centers grouping basic and clinical research workforces with critical mass and adequate logistic support; 11) integrating and developing european research programs.

Glutamine gluconeogenesis in the small intestine of 72 h-fasted adult rats is undetectable.

Recent reports have indicated that 48-72 h of fasting, Type 1 diabetes and high-protein feeding induce gluconeogenesis in the small intestine of adult rats in vivo. Since this would (i) represent a dramatic revision of the prevailing view that only the liver and the kidneys are gluconeogenic and (ii) have major consequences in the metabolism, nutrition and diabetes fields, we have thoroughly re-examined this question in the situation reported to induce the highest rate of gluconeogenesis. For this, metabolically viable small intestinal segments from 72 h-fasted adult rats were incubated with [3-13C]glutamine as substrate. After incubation, substrate utilization and product accumulation were measured by enzymatic and NMR spectroscopic methods. Although the segments utilized [13C]glutamine at high rates and accumulated 13C-labelled products linearly for 30 min in vitro, no substantial glucose synthesis could be detected. This was not due to the re-utilization of [13C]glucose initially synthesized from [13C]glutamine. Arteriovenous metabolite concentration difference measurements across the portal vein-drained viscera of 72 h-fasted Wistar and Sprague-Dawley rats clearly indicated that glutamine, the main if not the only gluconeogenic precursor taken up, could not give rise to detectable glucose production in vivo. Therefore we challenge the view that the small intestine of the adult rat is a gluconeogenic organ.

Intrinsic gluconeogenesis is enhanced in renal proximal tubules of Zucker diabetic fatty rats.

Recent studies indicate that renal gluconeogenesis is substantially stimulated in patients with type 2 diabetes, but the mechanism that is responsible for such stimulation remains unknown. Therefore, this study tested the hypothesis that renal gluconeogenesis is intrinsically elevated in the Zucker diabetic fatty rat, which is considered to be an excellent model of type 2 diabetes. For this, isolated renal proximal tubules from diabetic rats and from their lean nondiabetic littermates were incubated in the presence of physiologic gluconeogenic precursors. Although there was no increase in substrate removal and despite a reduced cellular ATP level, a marked stimulation of gluconeogenesis was observed in diabetic relative to nondiabetic rats, with near-physiologic concentrations of lactate (38%), glutamine (51%) and glycerol (66%). This stimulation was caused by a change in the fate of the substrate carbon skeletons resulting from an increase in the activities and mRNA levels of the key gluconeogenic enzymes that are common to lactate, glutamine, and glycerol metabolism, i.e., mainly of phosphoenolpyruvate carboxykinase and, to a lesser extent, of glucose-6-phosphatase and fructose-1,6-bisphosphatase. Experimental evidence suggests that glucocorticoids and cAMP were two factors that were responsible for the long-term stimulation of renal gluconeogenesis observed in the diabetic rats. These data provide the first demonstration in an animal model that renal gluconeogenesis is upregulated by a long-term mechanism during type 2 diabetes. Together with the increased renal mass (38%) observed, they lend support to the view so far based only on in vivo studies performed in humans that renal gluconeogenesis may be stimulated by and crucially contribute to the hyperglycemia of type 2 diabetes.

Identification of novel targets of cephaloridine in rabbit renal proximal tubules synthesizing glutamine from alanine.

Cephaloridine, which accumulates in the renal proximal tubule, is a model compound used for studying the toxicity of antibiotics towards this nephron segment. Several studies have demonstrated that cephaloridine alters renal intermediary and energy metabolism, but the mechanism by which this compound interferes with renal metabolic pathways remains incompletely understood. In an attempt to improve our knowledge in this field, we have studied the influence of cephaloridine on the synthesis of glutamine, which represents a key metabolic process involving several important enzymatic steps in the rabbit kidney. For this, suspensions of rabbit renal proximal tubules were incubated for 90 and 180 min in the presence of 5 mM alanine, an important glutamine precursor, both in the absence and the presence of 10 mM cephaloridine. Glutamate accumulation and glutamine synthesis were found to be inhibited by cephaloridine after 90 and 180 min of incubation, and cephaloridine accumulation in the renal proximal cells occurred in a time-dependent manner. The renal proximal tubule activities of alanine aminotransferase and glutamate dehydrogenase, which initiates alanine removal and releases the ammonia needed for glutamine synthesis, respectively, were inhibited to a significant degree and in a concentration-dependent manner by cephaloridine concentrations in the range found to accumulate in the renal proximal cells. Citrate synthase and glutamine synthetase activities were also inhibited by cephaloridine, but to a much lesser extent. The above enzymatic activities were not found to be inhibited when they were measured after successive dilutions of renal proximal tubules incubated for 180 min in the presence of 5 mM alanine and 10 mM cephaloridine. When microdissected segments (S1-S3) of rabbit renal proximal tubules were incubated for 180 min with 5 mM alanine with and without 5 and 10 mM cephaloridine, glutamate accumulation and glutamine synthesis were also inhibited in the three renal proximal segments studied; the latter cephaloridine-induced inhibitions observed were concentration-dependent except for glutamine in the S3 segment. These results are consistent with the view that cephaloridine accumulates and is toxic along the entire rabbit renal proximal tubule. They also demonstrate that cephaloridine interferes in a concentration-dependent and reversible manner mainly with alanine aminotransferase and glutamate dehydrogenase, which are therefore newly-identified targets of the toxic effects of cephaloridine in the rabbit renal proximal tubule.

Characteristics of glutamine metabolism in human precision-cut kidney slices: a 13C-NMR study.

The metabolism of glutamine, a physiological substrate of the human kidney, plays a major role in systemic acid-base homoeostasis. Not only because of the limited availability of human renal tissue but also in part due to the lack of adequate cellular models, the mechanisms regulating the renal metabolism of this amino acid in humans have been poorly characterized. Therefore given the renewed interest in their use, human precision-cut renal cortical slices were incubated in Krebs-Henseleit medium (118 mM NaCl, 4.7 mM KCl, 1.18 mM KH2PO4, 1.18 mM MgSO4*7H2O, 24.9 mM NaHCO3 and 2.5 mM CaCl2*2H2O) with 2 mM unlabelled or 13C-labelled glutamine residues. After incubation, substrate utilization and product formation were measured by enzymatic and NMR spectroscopic methods. Glutamate accumulation tended to plateau but glutamine removal and ammonia, alanine and lactate production as well as flux through GLDH (glutamate dehydrogenase) increased to various extents with time for up to 4 h of incubation indicating the metabolic viability of the slices. Valproate, a stimulator of renal glutamine metabolism, markedly and in a dose-dependent fashion increased ammonia production. With [3-13C]glutamine as a substrate, and in the absence and presence of valproate, [13C]glutamate, [13C]alanine and [13C]lactate accounted for 81 and 96%, 34 and 63%, 30 and 46% of the glutamate, alanine and lactate accumulations measured enzymatically respectively. The slices also metabolized glutamine and retained their reactivity to valproate during incubations lasting for up to 48 h. These results demonstrate that, although endogenous metabolism substantially operates in the presence of glutamine, human precision-cut renal cortical slices are metabolically viable and strongly respond to the ammoniagenic effect of valproate. Thus, this experimental model is suitable for metabolic and pharmaco-toxicological studies.