A tubule cell model for ifosfamide nephrotoxicity

Mechanisms leading to ifosfamide (IF)-induced renal damage have not been fully elucidated. Recent work suggests that localized renal tubular metabolism of IF and the production of the nephrotoxic chloroacetaldehyde may lead to nephrotoxicity. Presently no pharmacological method to reduce IF nephrotoxicity has been identified. The objectives of this study were to establish a tubule cell model for IF nephrotoxicity, to verify whether renal proximal tubular cells have the necessary cytochrome P450 (CYP) enzymes to oxidize IF, and whether they can metabolize IF to chloroacetaldehyde. CYP3A, and 2B mRNA and protein were identified in LLCPK-1 cells. The cells metabolized the R- and S-IF enantiomers to their respective 2- and 3-dechloroethylifosfamide metabolites, by-products of chloroacetaldehyde formation. Metabolite production was both time and concentration-dependent. IF did not affect cell viability. In contrast, glutathione-depleted cells showed time and dose-dependent damage. The presence of the relevant CYP enzymes in renal tubular cells along with their ability to metabolize IF to its 2- and 3-dechloroethylifosfamide metabolites suggests that nephrotoxic damage may result from the localized production of chloroacetaldehyde. Glutathione is a major defence mechanism against IF toxicity, thus pharmacological methods for replenishing intracellular glutathione may be effective in modulating IF-induced nephrotoxicity.     

Detection and quantification of (R) and (S)-dechloroethylifosfamide metabolites in plasma from children by enantioselective LC/MS/MS

Ifosfamide (IF), a potent chemotherapeutic agent for solid tumors, is known to cause high rates of nephrotoxicity in children with cancer, which is most likely due to the renal production of the metabolite chloroacetaldehyde. Using plasma samples obtained from pediatric oncology patients, we developed a simple nonderivatizing enantioselective liquid chromatography mass spectrometry method to detect the (R) and (S)-2- and 3-dechloroethylifosfamide metabolites. The (R) and (S)-enantiomers of the 2- and 3-DCEIF (N-3-dechlroethylifosfamide) were detectable in all 22 patients' samples with levels ranging from 9.9 to 238.7 ng/ml for (R)-2-DCEIF, 15.8 to 663.0 ng/ml for (S)-2-DCEIF, 20.8 to 852.8 ng/l for (R)-3-DCEIF and 28.0 to 862.0 ng/ml for (S)-3-DCEIF. In addition, the lower limit of quantification for this method is 1 ng/ml. Future studies should concentrate on (R) or (S) production of the 2-DCEIF and 3-DCEIF and subsequently chloroacetaldehyde formation with the aim of considering the administration of only the (R)-IF as its metabolism results in a lower production of chloroacetaldehyde.     

Evidence of renal metabolism of ifosfamide to nephrotoxic metabolites

Nephrotoxicity is a limiting factor in the use of ifosfamide in children. Despite the co-administration of uroprotective agents such as sodium 2-mercaptoethanesulfonate (mesna), ifosfamide chemotherapy is associated with nephropathy characterized by glomerular toxicity and Fanconi syndrome in many children treated with this drug. This is in distinction to cyclophosphamide, an analogue which differs solely by the position of a chloroethyl group, and which is not associated with nephrotoxicity. We hypothesized that ifosfamide is metabolized by cytochrome P450 (CYP) enzymes located in the renal tubular cell to the toxic metabolite chloroacetaldehyde; and, that the higher production of chloroacetaldehyde from ifosfamide than from cyclophosphamide explains the clinical differences in nephrotoxicity. We found that in both pig renal cortical microsomes and whole human kidney microsomes incubated with 1 mM ifosfamide for 3 hr, 2 and 3 dechloroethylifosfamide (DCEI) were produced. Our study provides evidence that porcine and human kidney microsomes are capable of biotransforming ifosfamide to DCEI metabolites that are produced in equimolar amounts with chloroacetaldehyde, indicating that chloroacetaldehyde is locally produced by renal cells as a possible mechanism for nephrotoxicity.     

Salt-dependent binding of Escherichia coli initiation factor 3 to nucleic acids determined by sedimentation partition chromatography

The binding of 14CH3- initiation factor 3 (IF3) to polynucleotides is strongly dependent upon the concentration of added salt. The observed association constant, Kobs, increases by ca. a factor of 10(2) when the NaCl concentration is lowered from 200 to 100 mM for the binding of 14CH3-IF3 to all nucleic acids examined. This salt-dependent binding suggests that at physiological salt concentrations the formation of an IF3-polynucleotide complex is primarily driven by the release of cations from the nucleic acid, although anion effects are involved also. For single-stranded nucleic acids, nonelectrostatic interactions may contribute a factor of 10(2) to the value of Kobs, although accurate assessment of these interactions is complicated by anion effects. The binding of 14CH3-IF3 to the double helix, poly(A).poly(U), appears to be exclusively electrostatic. 14CH3-IF3 forms a maximum of 8 +/- 2 ion pairs with most single-stranded polynucleotides. The value of Kobs increases from ca. 10(3) to 10(5) M-1 when the NaCl concentration is lowered from 200 to 100 mM for the binding of 14CH3-IF3 to poly(A), poly(C), poly(U), and poly(A).poly(U). At physiological salt concentrations, IF3 shows no preference for any of these bases or for single or double-stranded structures. However, 14CH3-IF3 binds ca. 60 times greater to poly(A,G), at al NaCl concentrations examined, than to the other nucleic acids, indicating that IF3 has some preference for guanine-containing polynucleotides. The presence of 10 mM Mg2+ tends to reduce the value of Kobs at any given NaCl concentration, but to a smaller degree than predicted by simply a competition between Mg2+ and IF3 for the nucleic acid lattice.     

Translation initiation factor IF1 of Bacillus stearothermophilus and Thermus thermophilus substitute for Escherichia coli IF1 in vivo and in vitro without a direct IF1-IF2 interaction

Bacterial translation initiation factor IF1 is homologous to archaeal aIF1A and eukaryal eIF1A, which form a complex with their homologous IF2-like factors (aIF5B and eIF5B respectively) during initiation of protein synthesis. A similar IF1-IF2 interaction is assumed to occur in all bacteria and supported by cross-linking data and stabilization of the 30S-IF2 interaction by IF1. Here we compare Escherichia coli IF1 with thermophilic factors from Bacillus stearothermophilus and Thermus thermophilus. All three IF1s are structurally similar and functionally interchangeable in vivo and in vitro. However, the thermophilic factors do not stimulate ribosomal binding of IF2DeltaN, regardless of 30S subunits and IF2 origin. We conclude that an IF1-IF2 interaction is not universally conserved and is not essential for cell survival.     

Hepatic and renal conjugation (Phase II) enzyme activities in young adult, middle-aged, and senescent male Sprague-Dawley rats

Acetaminophen (APAP)-induced nephrotoxicity is age dependent in male Sprague-Dawley rats: nephrotoxicity occurs at lower dosages of APAP in 12- to 14-month olds compared with 2- to 3-month olds. The mechanisms responsible for enhanced nephrotoxicity in 12-month-old Sprague-Dawley rats are not entirely clear, but may be related to age-dependent differences in APAP metabolism in liver and/or kidney. Major pathways of hepatic APAP metabolism include sulfation and glucuronidation; glutathione conjugation represents a pathway for detoxification of reactive oxidative APAP metabolites. The present studies were designed to quantify in vitro activity of three Phase II enzyme activities: glutathione S-transferase using 1-chloro-2,4-dinitrobenzene as substrate, UDP-glucuronyl transferase using APAP as substrate, and sulfotransferase using APAP as substrate, in subcellular fractions of liver and kidney of 3-, 12-, 18-, and 30-month-old naive male Sprague-Dawley rats. In liver, glutathione S-transferase, UDP glucuronyl transferase, and sulfotransferase activities were not significantly different in rats from 3 through 30 months of age. Renal UDP glucuronyl transferase and sulfotransferase activities were similar in rats from 3 through 30 months of age. In contrast, renal glutathione S-transferase activity was characterized by a lower Km in 12- and 30-month olds when compared with 3-month olds. These data suggest that the reduced total systemic clearance of APAP in 12-month-old male Sprague-Dawley rats previously observed cannot be attributed to age-dependent differences in hepatic APAP metabolism. In addition, it is unlikely that differences in renal APAP metabolism contribute to age-dependent APAP nephrotoxicity.     

Capsaicin Ameliorates Cisplatin-Induced Renal Injury through Induction of Heme Oxygenase-1

Cisplatin is one of the most potent chemotherapy agents. However, its use is limited due to its toxicity in normal tissues, including the kidney and ear. In particular, nephrotoxicity induced by cisplatin is closely associated with oxidative stress and inflammation. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in the heme metabolism, has been implicated in a various cellular processes, such as inflammatory injury and anti-oxidant/oxidant homeostasis. Capsaicin is reported to have therapeutic potential in cisplatin-induced renal failures. However, the mechanisms underlying its protective effects on cisplatin-induced nephrotoxicity remain largely unknown. Herein, we demonstrated that administration of capsaicin ameliorates cisplatin-induced renal dysfunction by assessing the levels of serum creatinine and blood urea nitrogen (BUN) as well as tissue histology. In addition, capsaicin treatment attenuates the expression of inflammatory mediators and oxidative stress markers for renal damage. We also found that capsaicin induces HO-1 expression in kidney tissues and HK-2 cells. Notably, the protective effects of capsaicin were completely abrogated by treatment with either the HO inhibitor ZnPP IX or HO-1 knockdown in HK-2 cells. These results suggest that capsaicin has protective effects against cisplatin-induced renal dysfunction through induction of HO-1 as well as inhibition oxidative stress and inflammation.     

Renal brush border membrane bound intrinsic factor

A highly active receptor for intrinsic factor (IF)-cobalamin (Cbl) complex has been detected and reported in mammalian kidney earlier (Seetharam, B., et al. (1988) J. Biol. Chem. 263, 4443-4449). The physiological role of this receptor in normal Cbl homeostasis is not known. In addition to binding of exogenously added IF-[57Co]Cbl, the renal apical membranes contain endogenous IF or IF-Cbl. Washing with pH 5/EDTA buffer enhanced the binding of exogenously added IF-[57Co]Cbl to renal apical but not basolateral membranes. The pH 5/EDTA extract from renal apical membranes bound [57Co]Cbl. The complex also bound to rat ileal brush border membrane and promoted ileal transport of [57Co]Cbl. On immunoblots using monospecific antiserum to IF a 62 kDa protein was identified in renal and intestinal apical membranes, serum and in tissue extracts of unperfused rat liver, kidney and heart. The 62 kDa band was eliminated from the renal apical membranes following pH 5/EDTA wash. Rat urine demonstrated unsaturated [57Co]Cbl binding (0.2 to 0.4 pmol/day) of which only 30-40% was immunoprecipitated with anti IF and could be identified on immunoblots. The identification of IF in rat renal apical membranes (160-200 ng/mg protein) and secretion of only traces of IF in urine suggest that the renal IF-Cbl receptor may play a role in sequestering IF/IF-Cbl and prevent urinary loss of Cbl.     

Carvedilol: a beta blocker with antioxidant property protects against gentamicin-induced nephrotoxicity in rats

Gentamicin is an antibiotic effective against gram negative infections, whose clinical use is limited by its nephrotoxicity. Since the pathogenesis of gentamicin-induced nephrotoxicity involves oxygen free radicals, the antioxidant carvedilol may protect against gentamicin-induced renal toxicity. We therefore tested this hypothesis using a rat model of gentamicin nephrotoxicity. Carvedilol (2 mg/kg) was administered intraperitoneally 3 days before and 8 days concurrently with gentamicin (80 mg/kg BW). Estimations of urine creatinine, glucose, blood urea, serum creatinine, plasma and kidney tissue malondialdehyde (MDA) were carried out, after the last dose of gentamicin. Kidneys were also examined for morphological changes. Gentamicin caused marked nephrotoxicity as evidenced by increase in blood urea, serum creatinine and decreased in creatinine clearance. Blood urea and serum creatinine was increased by 883% and 480% respectively with gentamicin compared to control. Carvedilol protected the rats from gentamicin induced nephrotoxicity. Rise in blood urea, serum creatinine and decrease in creatinine clearance was significantly prevented by carvedilol. There was 190% and 377% rise in plasma and kidney tissue MDA with gentamicin. Carvedilol prevented the gentamicin induced rise in both plasma and kidney tissue MDA. Kidney from gentamicin treated rats, histologically showed necrosis and desquamation of tubular epithelial cells in renal cortex, whereas it was very much comparable to control with carvedilol. In conclusion, carvedilol with its antioxidant property protected the rats from gentamicin-induced nephrotoxicity.     

The renal toxicity of (R)-3-chlorolactate in the rat

The renal toxicity of (R,S)-3-chlorolactate has been shown to be due to the (R)-isomer which, when administered to rats, induces diuresis and glucosuria. The metabolic activity of isolated tubule cells, prepared from rat kidney, was inhibited by (R)-3-chlorolactate and the action of the compound was localised as affecting mitochondrial metabolism. Studies with kidney mitochondria pin-pointed the site of action as being involved with the oxidative metabolism of malate but not the inhibition of mitochondrial malate dehydrogenase. The effects of oxalate, a metabolite of (R)-3-chlorolactate, and of (R,S)-3-chlorolactaldehyde on renal tubule cells was investigated. While some degrees of inhibition of metabolic activity were evident, these compounds were not responsible for the toxic effects produced by (R)-3-chlorolactate.     

The effect of treatment with the medicinal plant Rhazya stricta decne on gentamicin nephrotoxicity in rats.

Generation of oxygen free radicals in kidney cortex plays an important role in the pathogenesis of gentamicin (GM) nephrotoxicity, and the leaf extract of the medicinal plant Rhazya stricta has been shown to have an anti-oxidant action in rats. Therefore, in the present work we aimed at testing, in this species, the possible protective effect of R. stricta extract on GM nephrotoxicity. Crude water extract of R. Stricta leaves (0.25, 0.5 and 1 g/Kg) was given orally to rats four days before GM treatment, and thereafter, concomitantly with GM (80 mg/Kg/day) for another six days. Nephrotoxicity was evaluated histopathologically by light microscopy, and biochemically by measuring the concentrations of urea and creatinine in serum, reduced glutathione (GSH), lipid peroxidation and superoxide dismutase (SOD) activity in kidney cortex. The results suggested that the plant extract (0.25 g/Kg) was ineffective in significantly altering the indices of GM-induced nephrotoxicity. However, a dose-related amelioration in the indices of toxicity was noted when the two higher doses of the plant extract were given. The plant extract, at the three doses used, had no significant adverse effect on the body weight of treated rats or on the measured hepatic and renal functions in serum. However, the two higher doses, significantly and dose-dependently increased SOD activity and GSH concentration, and decreased that of lipid peroxides in the kidney cortex. These results suggest that R. stricta water extract may contain compounds that could potentially ameliorate GM nephrotoxicity in rats.     

Equilibrium between monomeric and dimeric mitochondrial F1-inhibitor protein complexes.

Mg-ATP particles from bovine heart mitochondria have more than 95% of their F1 in complex with the inhibitor protein (IF1). The F1-IF1 complex was solubilized and purified. The question addressed was if this naturally occurring complex existed as monomers or dimers. Size exclusion chromatography and electron microscopy showed that most of the purified F1-IF1 complex was a dimer of two F1-IF1. As determined by the former method, the relative concentrations of dimeric and monomeric F1-IF1 depended on the concentration of protein that was applied to the column. Apparently, there is an equilibrium between the two forms of F1-IF1.     

Modulation of metal toxicity by metallothionein

Toxic properties of several metals may be modified, since they are bound to metallothionein in vivo. Such modulation is particularly well known for cadmium (Cd), whose acute effects are prevented by metallothionein induction, whereas chronic effects on the kidney are partly explained on the basis of transport of cadmium-metallothionein (CdMt) into the kidney. Although intracellular Mt synthesis is induced by Cd, offering partial protection, nephrotoxicity may occur at times when such protection is insufficient. Pertubations in renal calcium metabolism may be an important basis for membrane dysfunction leading to proteinuria.     

Effect of oral administration of Arabic gum on cisplatin-induced nephrotoxicity in rats

It has been recently postulated from our laboratory that Arabic gum (AG) offers a protective effect in the kidney of rats against nephrotoxicity induced by gentamicin via inhibiting lipid peroxidation. It has also recently shown a powerful antioxidant effect through scavenging superoxide anions. In this study we utilized a rat model of cisplatin (CP)-induced nephrotoxicity to determine its peak time following (1, 2, 5, and 7 days) of a single CP (7.5 mg/kg, i.p.) injection. Also, a possible protective effect of cotreatment with AG (7.5 g/kg/day p.o.) on CP-induced nephrotoxicity was investigated. Biochemical as well as histological assessments were carried out. CP-induced nephrotoxicity was manifested by significant elevations of the functional parameters blood urea, serum creatinine, and kidney/body weight ratio. Maximum toxic effects of CP were observed 5 days after its injection, while it started after day 1 in the biochemical parameters, such as glutathione depletion in the kidney tissue with concomitant increases in lipid peroxides and platinum content. Additionally, severe necrosis and desquamation of tubular epithelial cells in renal cortex as well as interstitial nephritis were observed after 5 days in CP-treated animals. Five days after AG cotreatment with CP did not protect the kidney from the damaging effects of CP. However, it significantly reduced CP-induced lipid peroxidation. These findings suggest that lipid peroxidation is not the main cause of CP-induced nephrotoxicity but it is rather more dependent on other factors such as platinum disposition in renal interstitial tubules.     

Protective effect of fosfomycin on gentamicin-induced lipid peroxidation of rat renal tissue

Fosfomycin is clinically recognized to reduce the aminoglycoside antibiotics-induced nephrotoxicity. However, little has been clarified why fosfomycin protects the kidney from the aminoglycosides-induced nephrotoxicity. Gentamicin, a typical aminoglycoside, is reported to cause lipid peroxidation. We focused on lipid peroxidation induced by gentamicin as a mechanism for the aminoglycosides-induced nephrotoxicity. The aim of this study is to investigate the effect of fosfomycin on the gentamicin-induced lipid peroxidation. In rat renal cortex mitochondria, fosfomycin was shown to depress the gentamicin-induced lipid peroxidation, which was evaluated by formation of thiobarbituric acid reactive substances (TBARS). Interestingly, this effect was observed in rat renal cortex mitochondria, but not in rat liver microsomes. However, fosfomycin did not affect lipid peroxidation of arachidonic acid caused by gentamicin with iron. Fosfomycin inhibited the gentamicin-induced iron release from rat renal cortex mitochondria. These results indicated that fosfomycin inhibited the gentamicin-induced lipid peroxidation by depressing the iron release from mitochondria. This may possibly be one mechanism for the protection of fosfomycin against the gentamicin-induced nephrotoxicity.     

Protective actions of soy isoflavones and n-3 PUFAs on bone mass in ovariectomized rats

Ovariectomy (OVX) in female rats precipitates a marked reduction in endogenous estrogen concentrations and induces bone remodeling abnormalities that augment bone loss and increase the risk of developing osteopenia. This research examined the combined effects of two levels of soy isoflavones (IFs), trace (-IF) and high (+IF) (0.03 and 3.43 mg/g protein, respectively), and two levels of n-3 polyunsaturated fatty acids (PUFAs) on bone conservation in 2-month-old sexually mature OVX Sprague-Dawley rats. All dietary treatments provided 110.4 g/kg of fat from either safflower oil (N6) or a blend of safflower oil and menhaden oil (N3). OVX rats were randomly assigned to the N6-IF, N6+IF, N3-IF and N3+IF groups. The OVX and sham rats were euthanized after 12 weeks of feeding. Data for sequential femoral and tibial in vivo bone mineral density and bone mineral content (BMC) measurements were determined every 4 weeks. The hindlimb mineral data indicated a trend toward a positive bone mineral-sparing effect related to +IF. Among the OVX rats, those fed the N3+IF diet had a significantly higher value for tibial BMC. The concentration of serum pyridinoline cross-links was significantly lower in the N3+IF group. These findings indicate a complementary action of soy IFs and n-3 PUFAs for attenuating bone mineral reduction in OVX rats.     

Direct cross-links between initiation factors 1, 2, and 3 and ribosomal proteins promoted by 2-iminothiolane

Complexes were prepared containing 30S ribosomal subunits from Escherichia coli and the three initiation factors IF1, IF2, and IF3. In different experiments, each of the factors was radiolabeled with the others unlabeled. The complexes were allowed to react with 2-iminothiolane and then oxidized to promote the formation of intermolecular disulfide bonds, some of which were between factors and ribosomal proteins. Each of the labeled factors becomes covalently cross-linked to the complex as judged by its failure to dissociate when centrifuged in a sucrose gradient containing a high salt concentration. Proteins from the complexes were extracted and analyzed on two-dimensional polyacrylamide gels by nonequilibrium isoelectric focusing and sodium dodecyl sulfate gel electrophoresis. Spots corresponding to cross-linked dimers that contained initiation factors, as indicated on autoradiographs, were cut out and analyzed further. The following cross-linked dimers between factors and ribosomal proteins were identified: IF1-S12, IF1-S18, IF2-S13, IF3-S7, IF3-S11, IF3-S13, and IF3-S19. Cross-links between factors IF1-IF2 and IF3-IF2 were also identified. A model integrating these findings with others on the protein topography of the ribosome is presented.     

Functional expression of intrinsic factor-cobalamin receptor by renal proximal tubular epithelial cells.

Previous studies from our laboratory (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449; Fyfe, J. C., Ramanujam, K. S., Ramaswamy, K., Patterson, D. F., and Seetharam, B. (1991) J. Biol. Chem. 266, 4489-4494) have identified and isolated a 230-kDa receptor from rat and canine kidney which binds with high affinity [57Co]cyanocobalamin (Cbl) complexed to gastric intrinsic factor (IF). Although these studies have identified a renal receptor which binds intrinsic factor-cobalamin (IFCR), it is not known whether the binding is specific for IF-Cbl and whether renal cells internalize [57Co]Cbl bound to IF and transport [57Co]Cbl across the cell. Using a variety of renal cells, our results show that IF-[57Co]Cbl binding activity is detected in proximal tubular-derived epithelial cells from opossum (OK) and porcine kidney (LLC-PK1) but not in distal tubular-derived cells from canine kidney cells (MDCK). Metabolic labeling studies with Tran 35S-label confirmed the presence of a 230-kDa IFCR in OK and LLC-PK1 cells. Cell surface labeling and binding studies demonstrated that IFCR is targeted to the apical membrane. This apical expression of IFCR in OK cells is inhibited by the microtubule-disruptive drugs, colchicine and nocodazole. Opossum kidney cells when grown on culture inserts are polarized and transport [57Co]Cbl only when bound to IF and not to other Cbl binders. Furthermore, the transport of [57Co]Cbl occurred unidirectionally from the apical to the basolateral surface. Treatment of cells with colchicine or nocodazole inhibited the surface binding of IF-[57Co]Cbl as well as the transcytosis of [57Co]Cbl by 70-75%. IFCR retained intracellualarly by incubation of cells with colchicine or nocodazole is degraded by leupeptin-sensitive proteases. Based on these results, we suggest that proximal tubular-derived epithelial cells transport [57Co]Cbl bound to IF in a saturable way via receptor-mediated endocytosis.     

Determination of the enantiomers of mianserin,desmethylmianserin, and 8-hydroxymianserin in the plasma and urine of mianserin-treated patients

An HPLC method is presented which allows the measurement in the same run of the enantiomers of mianserin, desmethylmianserin, and 8-hydroxymianserin in plasma and urine of mianserin-treated patients. Limits of quantitation for the (S)- and (R)- enantiomers of mianserin and desmethylmianserin were 4 and 2.5 ng/ml, respectively, in plasma, and for the (S)- and (R)-enantiomers of mianserin, desmethylmianserin, and 8-hydroxymianserin 5, 2.5, and 5 ng/ml, respectively, in urine. The measured ratios of (S)-mianserin/(R)-mianserin and (S)-desmethylmianserin/(R)-desmethylmianserin in the plasmas of 10 mianserin-treated patients, all extensive metabolizers of debrisoquine as determined by CYP2D6 genotyping, varied, respectively, from 1.0 to 4.06 and from 0.19 to 0.64. As the enantiomers of mianserin differ in their pharmacological profile, these results could partially explain why, until now, no consistent relationship has been established between the therapeutic response and total [(S) + (R)] plasma levels of this antidepressant. © 1994 Wiley-Liss, Inc.