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.

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.

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.

Metabolic viability and pharmaco-toxicological reactivity of cryopreserved human precision-cut renal cortical slices.

We have tested the suitability of cryopreserved human precision-cut renal cortical slices for metabolic and pharmaco-toxicological studies. The viability of these slices and their pharmaco-toxicological reactivity were assessed using intracellular ATP and protein contents, lactate dehydrogenase (LDH) leakage, lactate and glutamine metabolism and the ammoniagenic effect of valproate. Despite a decrease in ATP and protein contents when compared with those of fresh slices, cryopreserved slices did not show any LDH leakage and retained the capacity to metabolize glutamine and lactate. Glutamine removal and ammonia, lactate and alanine production were similar in fresh and cryopreserved slices; by contrast, cryopreserved slices accumulated more glutamate as a result of decreased flux through glutamate dehydrogenase which catalyses an oxygen-dependent reaction. Valproate markedly and similarly stimulated glutamine metabolism in fresh and cryopreserved slices. Cryopreservation did not alter lactate removal but inhibited lactate gluconeogenesis. In conclusion, these results demonstrate that, although their mitochondrial oxidative metabolism seems to be diminished, cryopreserved human precision-cut renal cortical slices remain metabolically viable and retain the capacity to respond to the ammoniagenic effect of valproate. Thus, this experimental model may be helpful to optimize the use of human renal tissue for metabolic and pharmaco-toxicological studies.

Toxicity of chloroacetaldehyde is similar in adult and pediatric kidney tubules.

The nephrotoxicity of chloroacetaldehyde (CAA), one of the main products of hepatic ifosfamide metabolism, was compared in isolated human pediatric and adult renal tubules. Tubules metabolizing lactate were incubated in the presence of various concentrations of CAA (0.1-0.5 mM). Both at low, clinically relevant (0.2 mM), and at higher concentrations (0.3, 0.4 and 0.5 mM), CAA induced a cellular depletion of thiol compounds, i.e. glutathione, coenzyme A and acetyl-coenzyme A that are involved in CAA detoxication and cellular energy metabolism, respectively. The toxicity to renal cells was clearly observed in the presence of 0.4 and 0.5 mM CAA, which led to a fall of the cellular ATP level, to the accumulation of pyruvate and the inhibition of glucose synthesis from lactate. Inhibition of lactate uptake and an increase in the release of lactate dehydrogenase were observed only in the presence of 0.5 mM CAA. The sensitivity of pediatric tubules to the toxic effects of CAA and the rate of their CAA uptake were not statistically different from those found in adult tubules. It is concluded that an increased susceptibility of pediatric tubules to CAA toxicity cannot be put forward to explain the increased risk for ifosfamide-induced nephrotoxicity in children relative to adults.

Human kidney tubules detoxify chloroacetaldehyde, a presumed nephrotoxic metabolite of ifosfamide.

The nephrotoxic effects of the antineoplastic drug ifosfamide have been attributed to its hepatic metabolite chloroacetaldehyde. The effects of chloroacetaldehyde on isolated human kidney cortex tubules metabolizing lactate (a physiologic substrate in human kidneys) were investigated. At concentrations of > or =0.5 mM, chloroacetaldehyde was toxic to the human kidney tubules, as demonstrated by a dramatic decrease in cellular ATP levels and a large increase in lactate dehydrogenase release; chloroacetaldehyde also stimulated pyruvate accumulation and inhibited lactate removal and glucose synthesis. These effects, which were associated with incomplete disappearance of chloroacetaldehyde and extensive depletion of the cellular CoA, acetyl-CoA, and glutathione contents, were prevented by the addition of thiol-protecting drugs (mesna and amifostine). Human kidney tubules were demonstrated to metabolize chloroacetaldehyde at high rates, presumably via aldehyde dehydrogenase, which is very active in human kidneys. Carbon-13 nuclear magnetic resonance spectroscopy measurements indicated that human kidney tubules converted [2-(13)C]chloroacetaldehyde to [2-(13)C]chloroacetate, the further metabolism of which was very limited. At equimolar concentrations, chloroacetate was much less toxic than chloroacetaldehyde, indicating that chloroacetate synthesis from chloroacetaldehyde by human kidney tubules represents a detoxification mechanism that could play a role in vivo in preventing or limiting the nephrotoxic effects observed during ifosfamide therapy.