Aluminum citrate inhibits cytotoxicity and aggregation of oxalate crystals

Calcium oxalate monohydrate (COM), which represents a major component of kidney stones, is an end metabolite of ethylene glycol. COM accumulation has been linked with acute renal toxicity in ethylene glycol poisoning. COM injures the kidney either by directly producing cytotoxicity to the kidney cells or by aggregating in the kidney lumen leading to the blockage of urine flow. The present studies were designed to examine whether aluminum citrate could reduce the toxicity of COM. Toxicity was determined in human proximal tubule cells by leakage of lactate dehydrogenase or uptake of ethidium homodimer and in erythrocytes by degree of hemolysis. Aluminum citrate significantly inhibited the leakage of lactate dehydrogenase from human proximal tubule cells and protected against cell death from COM. The inhibitory effect of aluminum citrate was greater than that of other citrate or aluminum salts such as sodium citrate, aluminum chloride, calcium citrate, ammonium citrate or potassium citrate. Aluminum citrate significantly inhibited the aggregation of COM crystals in vitro and decreased red cell membrane damage from COM. Aluminum citrate appeared to directly interact with COM, but not with the cell membrane. As such, aluminum citrate reduced the cytotoxicity by a physico-chemical interaction with the COM surface, and not by dissolving the COM crystals. These studies suggest that aluminum citrate may protect against tissue damage that occurs with high levels of oxalate accumulation, especially in ethylene glycol poisoning and possibly in hyperoxaluric states.     

Calcium oxalate, and not other metabolites, is responsible for the renal toxicity of ethylene glycol

Ethylene glycol (EG) is nephrotoxic due to its metabolism. Many studies suggest that the toxicity is due to oxalate accumulation, but others have conversely suggested that toxicity results from effects of metabolites such as glycolaldehyde or glyoxylic acid on proximal tubule cells. In vivo studies have indicated that accumulation of calcium oxalate monohydrate (COM) corresponds closely with development of toxicity in renal tissue. The present studies were therefore designed to clarify the roles of various metabolites in the mechanism for EG toxicity in vitro by comparing the relative cytotoxicity of EG metabolites using three measures of cell death, ethidium homodimer uptake, lactate dehydrogenase (LDH) release and the conversion of the tetrazolium salt XTT to a colorimetric dye. Human proximal tubule cells in culture were incubated in physiologic buffers for 6h at 37 degrees C with COM (147-735microg/ml, an oxalate equivalence of 1-5mM), glycolate (5-25mM), glyoxylate (0.2-5mM) and glycolaldehyde (0.2-2mM). To assess the effects of acidity on the cytotoxicity, incubations were carried out at pH 6-7.4. The results show that COM dose-dependently increased LDH release and ethidium homodimer uptake, while the other metabolites did not. Conversely, COM had no effect on the XTT assay, while high concentrations of glycolaldehyde and glyoxylate decreased XTT activity, but the latter only at acidic pH. The correlation between the uptake of ethidium homodimer and the release of LDH suggest that COM is cytotoxic to human kidney cells in culture, while the XTT assay does not validly measure cytotoxicity in this system. These results indicate that COM, and not glyoxylate or glycolaldehyde, is the toxic metabolite responsible for the acute tubular necrosis and renal failure that is observed in EG-poisoned patients.     

Calcium oxalate monohydrate, a metabolite of ethylene glycol, is toxic for rat renal mitochondrial function.

Ethylene glycol poisoning can produce acute renal failure, requiring long-term hemodialysis to restore function. The mechanism of the renal failure is unknown, but is associated with tubular cell necrosis and ethylene glycol metabolism. The end metabolite of ethylene glycol is oxalic acid, the precipitation of which as calcium oxalate monohydrate (COM) crystals in the tubular lumen has been linked with the renal toxicity. Our recent studies suggest that COM is an intracellular toxicant to normal human proximal tubule cells in culture. The present studies were designed to assess whether COM or ionic oxalate alters mitochondrial function so as to lead to renal cell death. In isolated rat kidney mitochondria, COM produced a dose-dependent decrease in State 3 respiration (40% decrease at 0.05 mM COM with either succinate or glutamate/malate as substrate), without affecting either State 4 respiration or the ADP/O ratio. COM, from 0.01-0.05 mM also dose-dependently increased mitochondrial swelling, which was completely blocked by cyclosporin A. The inhibition of State 3 respiration, however, was not reversed by cyclosporin A administration. Potassium oxalate, at concentrations up to 5 mM did not inhibit mitochondrial respiration or induce swelling. These results suggest that COM, and not the oxalate ion, damages rat kidney mitochondria and induces the mitochondrial permeability transition, which may then lead to renal cell death. Since COM is transported intracellularly by kidney cells, the renal toxicity of ethylene glycol may result from inhibition of mitochondrial respiratory function in proximal tubular cells by COM crystals.     

Effect of repeated administration of 4-methylpyrazole on renal function and lipid peroxidation products in rat kidney after ethylene glycol poisoning

Toxic effects of ethylene glycol (EG) and its metabolites are mainly related to metabolic acidosis and kidney damage. EG biotransformation involving CYP2E1 affects the oxidant-antioxidant balance. The study assessed the effect of repeated administration of 4-methylpyrazole (4MP, 15 mg/kg b.w. after 2 h, followed by 10 mg/kg b.w. every 12 h) on renal function (creatinine, urea and urinary protein levels) as well as products of kidney’s lipid peroxidation (MDA and TBARS levels) in rats poisoned with EG (5745 mg/kg b.w.). Serum EG and glycolic acid (GA) concentrations were measured throughout the experiment. Repeated administration of 4MP reduced the rate of EG elimination, extended the period of EG persistence in serum and significantly limited formation of GA. The study showed the temporary intensification of kidney oxidative processes that correlated with changes in kidney function. It was found that the use of 4MP in EG poisoning inhibited its biotransformation to toxic metabolites, but simultaneously intensified oxidative damages in kidneys.     

Platinum complex-induced dysfunction of cultured renal proximal tubule cells

Platinum coordination complexes (PtCx) are potent against several types of cancer but are often nephrotoxic. With a view to developing a PtCx nephrotoxicity model, the toxicity of cisplatin (cDDP), transplatin (tDDP) and carboplatin (CBDCA) was studied in primary cultures of rabbit proximal tubule (RPT) cells and in the renal epithelial OK cell line. The cytotoxicity of these PtCx (10–3000 M) was assessed after 24 h exposure of confluent monolayers in terms of LDH release; their effects at non-cytotoxic concentrations (1–1000 M) on DNA and protein synthesis, glucose transport, marker enzymes and the total glutathione concentration were also determined, together with cellular platinum uptakes. The cytotoxicity ranking of the studied compounds differed for OK and RPT cells (cDDP>tDDP; cDDP>CBDCA and tDDP>cDDP; cDDP>CBDCA, respectively). Only results which were obtained in RPT cells corresponded to reported nephrotoxicity in vivo, making OK cells inappropriate for the study of PtCx nephrotoxicity in vitro. cDDP was about 10 times less cytotoxic for OK cells than for RPT cells because of lower cellular uptake. tDDP was unable markedly to inhibit biochemical and functional parameters in RPT cells below cytotoxic concentrations. At non-cytotoxic concentrations, cDDP and CBDCA depressed synthetic activity (mainly DNA) and, to a lesser extent, Na⁺-K⁺- ATPase activity and glucose transport in RPT cells. Total glutathione levels in RPT cells steadily increased during exposure to cDDP, tDDP and CBDCA, before the onset of cell death, arguing against an early role of glutathione depletion in PtCx toxicity. However, brush-border enzymes (-glutamyl transferase and alkaline phosphatase) and succinate dehydrogenase were insensitive to the action of either cDDP or CBDCA in RPT cells. On the basis of cytotoxicity and functional impairments, the toxicity of the three PtCx for RPT cells in vitro correlated with their reported nephrotoxicity in vivo, showing that RPT cells in primary culture are suitable for investigating the nephrotoxicity of PtCx.     

Extension of a PBPK model for ethylene glycol and glycolic acid to include the competitive formation and clearance of metabolites associated with kidney toxicity in rats and humans

A previously developed PBPK model for ethylene glycol and glycolic acid was extended to include glyoxylic acid, oxalic acid, and the precipitation of calcium oxalate that is associated with kidney toxicity in rats and humans. The development and evaluation of the PBPK model was based upon previously published pharmacokinetic studies coupled with measured blood and tissue partition coefficients and rates of in vitro metabolism of glyoxylic acid to oxalic acid, glycine and other metabolites using primary hepatocytes isolated from male Wistar rats and humans. Precipitation of oxalic acid with calcium in the kidneys was assumed to occur only at concentrations exceeding the thermodynamic solubility product for calcium oxalate. This solubility product can be affected by local concentrations of calcium and other ions that are expressed in the model using an ion activity product estimated from toxicity studies such that calcium oxalate precipitation would be minimal at dietary exposures below the NOAEL for kidney toxicity in the sensitive male Wistar rat. The resulting integrated PBPK predicts that bolus oral or dietary exposures to ethylene glycol would result in typically 1.4-1.6-fold higher peak oxalate levels and 1.6-2-fold higher AUC's for calcium oxalate in kidneys of humans as compared with comparably exposed male Wistar rats over a dose range of 1-1000 mg/kg. The converse (male Wistar rats predicted to have greater oxalate levels in the kidneys than humans) was found for inhalation exposures although no accumulation of calcium oxalate is predicted to occur until exposures are well in excess of the theoretical saturated vapor concentration of 200 mg/m³. While the current model is capable of such cross-species, dose, and route-of-exposure comparisons, it also highlights several areas of potential research that will improve confidence in such predictions, especially at low doses relevant for most human exposures.     

Inhalation and epidermal exposure of volunteers to ethylene glycol: kinetics of absorption, urinary excretion, and metabolism to glycolate and oxalate

Ethylene glycol (EG) is a widely used liquid. Limited data are published regarding inhaled EG and no data regarding transdermal EG uptake in humans. In order to gain information on the quantitative fate of EG, four male volunteers inhaled between 1340 and 1610 micromol vaporous 13C-labeled EG (13C2-EG) for 4h. Separately, three of these subjects were epidermally exposed for up to 6h to liquid 13C2-EG (skin area 66 cm2). Plasma concentrations and urinary amounts of 13C2-EG were determined by gas chromatography with mass selective detection. Additionally, plasma was assayed for 13C-labeled glycolic acid 13C2-GA) and urine for 13C2-GA and 13C-labeled oxalic acid (13C2-OA). Both EG metabolites were nephrotoxic in animals and humans and embryotoxic in rodents. 13C-labels enabled to differentiate from also determined endogenous EG, glycolic acid (GA), and oxalic acid (OA). Of 13C2-EG inhaled, 5.5+/-3.0%, 0.77+/-0.15%, and 0.10+/-0.12% were detected in urine as 13C2-EG, 13C2-GA, and 13C2-OA, respectively. The skin permeability constant of liquid EG was 2.7 x 10(-5)+/-0.5 x 10(-5)cm/h. Of the dose taken up transdermally, 8.1+/-3.2% and up to 0.4% were excreted in urine as 13C2-EG and 13C2-GA, respectively. It is calculated that equally long-lasting exposure to 10 ppm vaporous EG or wetting of both hands by liquid EG leads to about the same body burden by EG and metabolites. The amounts of GA and OA excreted daily in urine as a result of exposure (8h/day) to 10 ppm EG are about 15% and 2%, respectively, of those excreted from naturally occurring endogenous GA and OA.     

Electrophoresis of phosphoglycerate kinase-2 to determine testicular damage induced by ethylene glycol monomethyl ether and sterility associated with chromosomal abnormality

Phosphoglycerate kinase (PGK, EC 2.7.2.3), which is expressed specifically in sperm and spermatids, is an enzyme in the Embden-Meyerhof pathway that converts glucose to pyruvate. We developed an electrophoresis method to determine relative PGK-2 quantity and applied it to evaluate spermatogenesis activity. In the ethylene glycol monomethyl ether (EGME)-induced testicular toxicity, relative PGK-2 quantity had not decreased until 4 weeks of exposure. Mean relative PGK-2 quantities, defined as PGK-2 quantity over PGK-1 quantity in a pooled spleen sample (±SD) were: 1.43±0.32 for control animals (N=10); 1.67±0.24 for the group exposed at 500 mg/kg for 5 days (N=6); 1.85±0.58 for the group exposed at 500 mg/kg for 2 weeks (N=6); 0.09±0.06 for the group exposed at 500 mg/kg for 4 weeks (N=6); not detectable in animals exposed at 500 mg/kg for 5 weeks (N=7); 0.208±0.103 for the group exposed at 250 mg/kg for 5 weeks (N=6); and 1.35±0.38 for the group exposed at 125 mg/kg for 5 weeks (N=6). These relative quantities showed a good correlation with sperm/spermatid counts (r=0.823,p<0.01) and histological findings. These findings suggest that EGME has toxicity on primary spermatocytes and spermatogonia. In the case of sterility associated with a chromosomal abnormality (chromosomal translocation between chromosome X and 16), relative PGK-2 quantity was not detected in any of the seven adult (12 weeks of age) mice, although many primary spermatocytes were detected by histological examination. Those findings suggest that cellular differentiation is arrested at meiosis due to the chromosomal abnormality. It was thus concluded that relative PGK-2 quantity provides information on testicular development and is therefore useful as an indicator of testicular function.     

Disruption of mitochondrial complexes,cytotoxicity, and apoptosis results from Mancozeb exposure in transformed human colon cells

Ethylene bisdithiocarbamate pesticides, including Mancozeb (MZ), are used as fungicides. Effects of MZ on apoptosis induction and mitochondrial activity of HT-29 colon cells were investigated. MZ exposed cells exhibited blebbing and cellular membrane disruption in scanning electron micrographs. Positive fluorescent staining with Annexin V at doses of 60–140 μM supports apoptosis as the mechanism of cell death. Activity of all electron transport chain complexes were evaluated. Mitochondrial Complex I activity was decreased in 100 μM treated cells. Mitochondrial Complex III activity was decreased in 60 and 100 μM MZ treated cells. Mitochondrial Complex II and Complex IV activities were decreased in cells treated with 60, 100, and 140 μM. Cells treated with 60 μM exhibited a decrease in Complex V enzyme activity. It is concluded that MZ exposure inhibits all mitochondrial complexes of HT-29 cells and that positive fluorescent microscopy and blebbing support previous studies of cell death via apoptosis.     

Role of ARF6 in internalization of metal-binding proteins, metallothionein and transferrin, and cadmium-metallothionein toxicity in kidney proximal tubule cells

Filtered metal-protein complexes, such as cadmium-metallothionein-1 (CdMT-1) or transferrin (Tf) are apically endocytosed partly via megalin/cubilin by kidney proximal tubule (PT) cells where CdMT-1 internalization causes apoptosis. Small GTPase ARF (ADP-ribosylation factor) proteins regulate endocytosis and vesicular trafficking. We investigated roles of ARF6, which has been shown to be involved in internalization of ligands and endocytic trafficking in PT cells, following MT-1/CdMT-1 and Tf uptake by PT cells. WKPT-0293 Cl.2 cells derived from rat PT S1 segment were transfected with hemagglutinin-tagged wild-type (ARF6-WT) or dominant negative (ARF6-T27N) forms of ARF6. Using immunofluorescence, endogenous ARF6 was associated with the plasma membrane (PM) as well as juxtanuclear and co-localized with Rab5a and Rab11 involved in early and recycling endosomal trafficking. Immunofluorescence staining of megalin showed reduced surface labelling in ARF6 dominant negative (ARF6-DN) cells. Intracellular Alexa Fluor 546-conjugated MT-1 uptake was reduced in ARF6-DN cells and CdMT-1 (14.8 microM for 24 h) toxicity was significantly attenuated from 27.3+/-3.9% in ARF6-WT to 11.1+/-4.0% in ARF6-DN cells (n=6, P<0.02). Moreover, reduced Alexa Fluor 546-conjugated Tf uptake was observed in ARF-DN cells (75.0+/-4.6% versus 3.9+/-3.9% of ARF6-WT cells, n=3, P<0.01) and/or remained near the PM (89.3+/-5. 6% versus 45.2+/-14.3% of ARF6-WT cells, n=3, P<0.05). In conclusion, the data support roles for ARF6 in receptor-mediated endocytosis and trafficking of MT-1/Tf to endosomes/lysosomes and CdMT-1 toxicity of PT cells.