Protective effect of an intrinsic antioxidant, HMH (5-hydroxy-1-methylhydantoin; NZ-419), against cellular damage of kidney tubules.

HMH (5-hydroxy-1-methylhydantoin; NZ-419) is a mammalian creatinine metabolite and an intrinsic antioxidant. HMH prevents the progression of chronic kidney disease in rats when a sufficient amount is taken orally. We assessed whether intrinsic and higher levels of HMH could protect tubular epithelial cells, LLC-PK(1) cells, against known cellular damage caused by xenobiotics, such as cisplatin and cephaloridine, or by hypoxia/reoxygenation treatment. Both cell damage and peroxidation, monitored as the leakage of lactate dehydrogenase (LDH) and malondialdehyde (MDA), respectively, from cells into the media, were inhibited by HMH in a concentration-dependent manner. The minimum effective concentration of HMH (2.5 µM) seemed to be too low for HMH to only be a direct hydroxyl radical scavenger. Additional antioxidant effect(s) inhibiting reactive oxygen species generation and/or modulating signal transduction pathways were suggested. The possibility that intrinsic HMH could be a protectant for the kidney was indicated. At the same time, for sufficient inhibition, higher concentrations than intrinsic HMH concentrations may be necessary. Patterns of efficacies of HMH on LDH and MDA against different kinds of cellular damage were compared with our reported data on those of corresponding, naturally occurring antioxidants. A common and specific inhibitory mechanism as well as common target(s) in kidney injuries were indicated.

Kidney injury molecule-1 expression in rat proximal tubule after treatment with segment-specific nephrotoxicants: a tool for early screening of potential kidney toxicity.

Dose-response expression of kidney injury molecule-1 (KIM-1) gene in kidney cortex and its correlation with morphology and traditional biomarkers of nephrotoxicity (plasma creatinine and blood urea nitrogen, BUN) or segment-specific marker of proximal tubule injury (kidney glutamine synthetase, GSK) were studied in male rats treated with proximal tubule segment-specific nephrotoxicants. These included hexachloro-1:3-butadiene (HCBD, S(3) segment-specific), potassium dichromate (chromate, S(1)-S(2) segment-specific), and cephaloridine (Cph, S(2) segment-specific). Rats were treated with a single intraperitoneal (ip) injection of HCBD 25, 50, and 100 mg/kg, subcutaneous (sc) injection of chromate 8, 12.5, and 25 mg/kg; or ip injection of Cph 250, 500, and 1,000 mg/kg. KIM-1 gene showed a dose-dependent up-regulation induced by all segment-specific nephrotoxicants. Interestingly, magnitude of the up-regulation reflected the severity of microscopic tubular changes (degeneration, necrosis, and regeneration). Even low-severity microscopic observations were evidenced by significant gene expression changes. Furthermore, KIM-1 showed significant up-regulation even in the absence of morphological changes. In contrast, traditional and specific markers demonstrated low sensitivity or specificity. In conclusion, this study suggested KIM-1 as a sensitive molecular marker of different levels of tubular injury, and it is likely to represent a potential tool for early screening of nephrotoxicants.

L-type fatty acid binding protein transgenic mouse as a novel tool to explore cytotoxicity to renal proximal tubules.

A novel biomarker of renal dysfunction, liver-type fatty acid binding protein (L-FABP), which is expressed in human proximal tubules, binds to lipid peroxidation products during renal injury and is excreted into the urine. Here, we examined the usefulness of human L-FABP transgenic (Tg) mice as a tool to explore nephrotoxicity, employing two model drugs, cephaloridine and cisplatin, which are taken up by renal tubules via organic anion and cation transporters, respectively. Urinary excretion of L-FABP increased after administration of cephaloridine in most of the Tg mice, whereas glomerular filtration markers such as blood-urea-nitrogen (BUN) and plasma creatinine (CRE) were almost unchanged. Thus, L-FABP is a highly sensitive detector of the nephrotoxicity of cephaloridine. Urinary excretion of L-FABP in the Tg mice also increased after administration of cisplatin, and this increase was reduced by coadministration of cimetidine. Both BUN and CRE also increased after the cisplatin treatment, but these parameters were minimally affected by coadministration of cimetidine, suggesting that cimetidine reduces cisplatin-induced renal tubular toxicity with only a minimal effect on the glomerulus. These results indicate that the L-FABP Tg mouse should be a useful drug screening system to evaluate specifically the toxicity of transporter substrates to renal tubules.

Transcriptomic analysis of nephrotoxicity induced by cephaloridine, a representative cephalosporin antibiotic.

Cephaloridine (CER) is a classical beta-lactam antibiotic that has long served as a model drug for the study of cephalosporin antibiotic-induced acute tubular necrosis. In the present study, we analyzed gene expression profiles in the kidney of rats given subtoxic and toxic doses of CER to identify gene expression alterations closely associated with CER-induced nephrotoxicity. Male Fischer 344 rats were intravenously injected with CER at three different dose levels (150, 300, and 600 mg/kg) and sacrificed after 24 h. Only the high dose (600 mg/kg) caused mild proximal tubular necrosis and slight renal dysfunction. Microarray analysis identified hundreds of genes differentially expressed in the renal cortex following CER exposure, which could be classified into two main groups that were deregulated in dose-dependent and high dose-specific manners. The genes upregulated dose dependently mainly included those involved in detoxification and antioxidant defense, which was considered to be associated with CER-induced oxidative stress. In contrast, the genes showing high dose-specific (lesion-specific) induction included a number of genes related to cell proliferation, which appeared to reflect a compensatory response to CER injury. Of the genes modulated in both manners, we found many genes reported to be associated with renal toxicity by other nephrotoxicants. We could also predict potential transcription regulators responsible for the observed gene expression alterations, such as Nrf2 and the E2F family. Among the candidate gene biomarkers, kidney injury molecule 1 was markedly upregulated at the mildly toxic dose, suggesting that this gene can be used as an early and sensitive indicator for cephalosporin nephrotoxicity. In conclusion, our transcriptomic data revealed several characteristic expression patterns of genes associated with specific cellular processes, including oxidative stress response and proliferative response, upon exposure to CER, which may enhance our understanding of the molecular mechanisms behind cephalosporin antibiotic-induced nephrotoxicity.

In vitro gene expression analysis of nephrotoxic drugs in rat primary renal cortical tubular cells.

Rat primary renal cortical tubular cells were exposed to seven test substances, some with, and some without, known direct renal tubular cell toxicity. Cells were exposed to the substances at either one-third or one-tenth of the TC50 for cytotoxicity for 6 h or 24 h, so as not to induce cytotoxicity but to cause some transcriptional changes. Transcriptional profiles were investigated by using the Affymetrix Rat Toxicology U34 arrays, containing probes for more than 850 genes and ESTs. Four direct toxicants, cisplatin (CDDP), its less nephrotoxic analogue carboplatin (CBDCA), cephaloridine and gentamicin, were grouped together in a hierarchical clustering. In addition, the four direct toxicants affected more than 32 transcripts at their subcytotoxic concentrations at either 6 h or 24 h exposure. On the other hand, diclofenac, cyclosporine A and zinc, which are not considered to be directly toxic to tubules, affected less than 12 transcripts. Decreased Map3k12 and increased Hmox1 were commonly observed among the four direct toxicants, which appeared to be responses to cellular damage. Two platinum complexes, CDDP and CBDCA, induced similar changes, regardless of exposure duration or concentration. The types of transcriptional changes observed in this study were consistent with previously reported in vivo data, although there were some differences. These observations suggest that an in vitro gene expression analysis approach using GeneChip is feasible for screening for direct tubular toxicity of drugs and may help to clarify the underlying mechanisms of tubular toxicity.

Protective effect of serum thymic factor, FTS, on cephaloridine-induced nephrotoxicity in rats.

Serum thymic factor (FTS), a thymic peptide hormone, has been reported to increase superoxide disumutase (SOD) levels in senescence-accelerated mice. In the present study, we examined the effect of FTS on cephaloridine (CER)-induced nephrotoxicity in vivo and in vitro. We previously reported that CER led to extracellular signal-regulated protein kinase (ERK) activation in the rat kidney. So, we also investigated whether FTS has an effect on ERK activation induced by CER. Treatment of male Sprague-Dawley rats with intravenous CER (1.2 g/kg) for 24 h markedly increased BUN and plasma creatinine levels and urinary excretion of glucose and protein, decreased creatinine clearance and also led to marked pathological changes in the proximal tubules, as revealed by electron micrographs. An increase in phosphorylated ERK (pERK) was detected in the nuclear fraction prepared from the rat kidney cortex 24 h after CER injection. Pretreatment of rats with FTS (50 microg/kg, i.v.) attenuated the CER-induced renal dysfunction and pathological damage. FTS also suppressed CER-induced ERK activation in the kidney. In vitro treatment of the established cell line, LLC-PK1 cells, with FTS significantly ameliorated CER-induced cell injury, as measured by lactate dehydrogenase (LDH) leakage. Our results, taken together with our previous report that MEK inhibitors ameliorated CER-induced renal cell injury and ERK activation induced by CER, suggest that FTS participates in protection from CER-induced nephrotoxicity by suppressing ERK activation induced by CER.

Protective effect of a protein kinase inhibitor on cellular injury induced by cephaloridine in the porcine kidney cell line LLC-PK(1).

We investigated the effects of a protein kinase C inhibitor and a tyrosine kinase inhibitor on the cellular injury induced by cephaloridine in an established renal epithelial cell line, LLC-PK(1). Cephaloridine increased the leakage of lactate dehydrogenase (LDH) from LLC-PK(1) cells into the medium and also caused an increase in the level of lipid peroxide (index of oxidative stress) in the cells. Treatment of the cells with a hydroxyl radical scavenger, dimethylthiourea (DMTU), inhibited the increases in LDH leakage and lipid peroxidation in LLC-PK(1) cells exposed to cephaloridine. A protein kinase C inhibitor, H-7, and tyrosine kinase inhibitors, genistein and lavendustinA, inhibited the increases in LDH leakage and lipid peroxidation in LLC-PK(1) cells exposed to cephaloridine. These results suggest that a signaling pathway which involves protein kinase C and tyrosine kinase plays a role in the generation of reactive oxygen species in LLC-PK(1) cells damaged by cephaloridine.

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.

Protective effect of resveratrol against 6-hydroxydopamine-induced impairment of renal p-aminohippurate transport.

In the present study, the effects of resveratrol on 6-hydroxydopamine (6-OHDA)-induced p-aminohippurate (PAH) transport impairment were investigated in vitro using rat renal cortical slices. Cisplatin and cephaloridine (CPH), known nephrotoxins, were used as positive controls. In one series of experiments, renal cortical slices were incubated in a cisplatin-containing medium or a cisplatin-free medium. In another series of experiments, renal cortical slices were incubated in a CPH-containing medium, in a CPH- and probenecid-containing medium, or in a CPH-free medium. Subsequently, for each series of experiments kidney slices were incubated in a media containing PAH or tetraethylammonium. In a further series of experiments, renal cortical slices were incubated in a 6-OHDA-containing medium and in a 6-OHDA-free medium. In another series of experiments, renal cortical slices were incubated in a medium containing 50 micro M 6-OHDA, in a 6-OHDA- and resveratrol-containing medium or in a 6-OHDA- and resveratrol-free medium. Subsequently, for each series of experiments kidney slices were incubated in media containing PAH. The results of this study in which slices were incubated in 6-OHDA-containing media indicate that 6-OHDA induced a time- and concentration-dependent decrease in PAH accumulation by renal cortical slices. Resveratrol inhibited the 6-OHDA-induced time-dependent decrease of PAH accumulation in a concentration-dependent manner. Therefore, 6-OHDA causes functional injuries of renal proximal tubule cell membrane, thus leading to impairment of transport processes across the cell membrane and to nephrotoxicity. Resveratrol has a nephroprotective effect.

Enhancement of protein kinase C activity and chemiluminescence intensity in mitochondria isolated from the kidney cortex of rats treated with cephaloridine.

The development of nephrotoxicity induced by cephaloridine (CER) has been reported to be due to reactive oxygen species (ROS). Protein kinase C (PKC) has been suggested to modulate the generation of ROS. We investigated the possible participation of ROS generation assessed by chemiluminescence (CL) and PKC activity in rat kidney cortical mitochondria in the development of CER-induced nephrotoxicity. We first evaluated the magnitude of the nephrotoxic damage caused by CER in rats. The plasma parameters and ultrastructural morphology changes were increased markedly 24hr after the treatment of rats with CER. We demonstrated that the treatment of rats with CER clearly evoked not only enhancement of Cypridina luciferin analog (CLA)-dependent CL intensity, but also the activation of PKC in mitochondria isolated from the kidney cortex of rats 1.5 and 3.5 hr after injection of the drug. These changes were detected in advance of those observed in plasma and by electron microscopy. The increase in CLA-dependent CL intensity detected in the kidney cortical mitochondria 1.5 and 3.5 hr after injection of CER was inhibited completely by the addition of superoxide dismutase, suggesting the generation of superoxide anion in these mitochondria during the early stages of CER-induced nephrotoxicity. These results suggest that the activation of PKC and the enhancement of superoxide anion generation in kidney cortical mitochondria precede the increases in plasma parameters and the electron micrographic changes indicative of renal dysfunction in rats treated with CER. Additionally, they suggest a possible relationship between PKC activation in mitochondria and free radical-induced CER nephrotoxicity in rats.

Involvement of rat organic anion transporter 3 (rOAT3) in cephaloridine-induced nephrotoxicity: in comparison with rOAT1.

This study was performed to elucidate the possible involvement of organic anion transporter 3 (OAT3) in cephaloridine (CER)-induced nephrotoxicity and compare the substrate specificity between rOAT3 and rat OAT1 (rOAT1) for various cephalosporin antibiotics, using proximal tubule cells stably expressing rOAT3 (S2 rOAT3) and rOAT1 (S2 rOAT1). S2 rOAT3 exhibited a CER uptake and a higher susceptibility to CER cytotoxicity than did mock, which was recovered by probenecid. Various cephalosporin antibiotics significantly inhibited both estrone sulfate uptake in S2 rOAT3 and para-aminohippuric acid uptake in S2 rOAT1. The Ki values of CER, cefoperazone, cephalothin and cefazolin for rOAT3- and rOAT1-mediated organic anion transport ranged from 0.048 to 1.14 mM and from 0.48 to 1.32 mM, respectively. These results suggest that rOAT3, at least in part, mediates CER uptake and CER-induced nephrotoxicity as rOAT1. There was some difference of affinity between rOAT3 and rOAT1 for cephalosporin antibiotics.

[Nephrotoxicity of piperacillin combined with furosemide in rats].

Nephrotoxicity of piperacillin (PIPC) was evaluated in rats after combined administration with furosemide. After intravenous administration of PIPC (1600 mg/kg), the rats showed no change in urinalysis, biochemical analysis of plasma and histopathological analysis. The rats receiving furosemide (100 mg/kg) showed elevation of urinary NAG, BUN and creatinine concentrations, and showed slight degeneration of the renal proximal tubules. The rats receiving PIPC (1600 mg/kg) and furosemide (100 mg/kg) showed elevation of BUN and creatinine concentrations, and showed slight degeneration of the proximal tubules. These changes were comparable to those in rats receiving furosemide alone. The rats receiving cephaloridine (1600 mg/kg) showed elevation of urinary protein, BUN and creatinine concentrations, and showed moderate degeneration and necrosis of the proximal tubules. The nephrotoxicity was enhanced by combination with furosemide. In conclusion, no enhanced effect of nephrotoxicity was observed by combination of PIPC with furosemide.

Roles of oxygen radical production and lipid peroxidation in the cytotoxicity of cephaloridine on cultured renal epithelial cells (LLC-PK1).

To clarify the mechanism of cephalosporin nephrotoxicity, the cytotoxic effects of cephaloridine (CER), a nephrotoxic cephalosporin antibiotic, on the pig kidney proximal tubular epithelial cell line (LLC-PK1) were studied in culture. CER increased the content of hydrogen peroxide and decreased the activity of catalase in the treated cells, followed by an increase in the content of lipid peroxide and decreases in both glutathione peroxidase activity and in the non-protein sulfhydryl content. The levels of NADPH-dependent hydrogen peroxide and superoxide anion production by microsomes prepared from LLC-PK1 cells, and by NADPH-cytochrome P-450 reductase purified from the rat renal cortex were significantly increased by paraquat. The production of these molecules was antagonized by p-chloromer-curibenzoate, an inhibitor of NADPH-cytochrome P-450 reductase. On the other hand, CER did not significantly affect the production of hydrogen peroxide or superoxide anions. These results suggested that the cytotoxic effect of CER on cultured LLC-PK1 cells was due to the increases in hydrogen peroxide and lipid peroxide levels and not microsomal oxygen radical production, and that the mechanism of this cytotoxicity is very different from that of paraquat which induces microsomal oxygen radical production.

Role of organic anion transporter 1 (OAT1) in cephaloridine (CER)-induced nephrotoxicity.

UNLABELLED: Role of organic anion transporter 1 (OAT1) in cephaloridine (CER)-induced nephrotoxicity. BACKGROUND: Cephaloridine (CER) has been used to elucidate the mechanisms of cephalosporin antibiotic-induced nephrotoxicity. Organic anion transporters have been thought to mediate CER uptake by the proximal tubule. The purpose of this study was to elucidate the possible involvement of organic anion transporter 1 (OAT1) in CER-induced nephrotoxicity. METHODS: A mouse terminal proximal straight tubule (S3) cell line stably expressing rat OAT1 (S3 rOAT1) was established and used in this study. The cellular uptake of [14C]-para-aminohippuric acid (PAH), a prototype organic anion, and that of [14C]-CER were measured. The effects of CER on the viability of the cells and the amount of lipid peroxidation were estimated. RESULTS: S3 rOAT1 expressed a functional organic anion transporter in the cytoplasmic membrane, and exhibited CER uptake activity. CER treatment resulted in a more significant decrease in the viability and a more significant increase in the amount of lipid peroxidation in S3 rOAT1 than in S3 cells transfected with an expression vector lacking the rOAT1 insert. Probenecid, an inhibitor of organic anion transport, and probucol, an antioxidant, significantly suppressed the decrease in viability and increase in the amount of lipid peroxidation in S3 rOAT1 treated with CER. The effects of various cephalosporin antibiotics on the uptake of [14C]PAH were correlated significantly with the effects of these drugs on cell viability. CONCLUSIONS: These results suggest that rOAT1 is, at least in part, responsible for the cellular uptake of CER and therefore CER-induced nephrotoxicity.

Cephaloridine in vitro toxicity and accumulation in renal slices from normoglycemic and diabetic rats.

Previous work has shown a reduction in cephaloridine nephrotoxicity in a diabetic rat model. The following studies examined in vitro cephaloridine toxicity in renal slices from normoglycemic and diabetic Fischer 344 rats. Diabetes was induced by acute intraperitoneal injection of 35 mg/kg streptozotocin. Renal cortical slices were isolated from normoglycemic and diabetic animals. Tissues were exposed to 0-5 mM cephaloridine for 15-120 min. Pyruvate-directed gluconeogenesis was diminished in all groups exposed to 2-5 mM cephaloridine for 60-120 min. Leakage of lactate dehydrogenase (LDH) was apparent only in the normoglycemic group in the presence of 4-5 mM cephaloridine for 120 min. LDH leakage was not increased at any cephaloridine concentration in the diabetic tissue. Total glutathione levels were compared in renal cortical slices exposed to cephaloridine for 30-120 min. Baseline values for glutathione were comparable between normoglycemic and diabetic tissue suggesting that the mechanism for reduced toxicity was not due to higher glutathione levels in diabetic tissue. Total glutathione levels were diminished more rapidly in normoglycemic than diabetic tissue by incubation with 5 mM cephaloridine. Comparison of cephaloridine accumulation indicated that diabetic tissue accumulated less cephaloridine than the normoglycemic group when tissues were incubated with 0-2 mM cephaloridine. However, renal slice accumulation was similar between normoglycemic and diabetic groups following in vitro incubation with 4-5 mM cephaloridine. These results suggest that the mechanism for reduced in vitro cephaloridine toxicity in diabetic tissue cannot be limited to differences in accumulation and must include an unidentified cellular component.

Advantages of a two-chamber culture system to test drug nephrotoxicity: the example of cephaloridine.

Rabbit renal proximal tubular cells, cultured to confluency on a permeable collagen film in a two-chamber system, were exposed for 72 h to various concentrations of the nephrotoxic antibiotic, cephaloridine (CLD). A decrease in cellular proteins, leakage of lactate dehydrogenase and morphological changes appeared at CLD concentrations of 0.1, 1.0, and 0.5 mg/ml, respectively. The permeability of the monolayer to Lucifer yellow (LY), a dye that does not cross cell membranes, was increased by 1 or 2 mg/ml but not by lower concentrations of CLD. The large basolateral/apical glucose concentration gradient established by the cells was decreased by CLD. However, the fact that, at the CLD concentration of 1 mg/ml, LY totally equilibrated by diffusion across the monolayer, whereas the injured monolayer was still able to maintain a detectable glucose gradient, shows that damage caused by CLD to the diffusion barrier prevails over that affecting glucose uptake. Consistent with the data in the literature concerning the mechanism of CLD accumulation in renal cells, our results show that CLD was more toxic when it was added at the basolateral than at the apical side of the cultured cells. These results illustrate the advantages of using a two-chamber system of cell culture in nephrotoxicity studies.

Magnesium lithospermate B ameliorates cephaloridine-induced renal injury.

To determine whether magnesium lithospermate B ameliorates renal injury induced by cephaloridine, the effect of cephaloridine was investigated in rats given magnesium lithospermate B for 20 days preceding cephaloridine administration and in control rats given no magnesium lithospermate B. In the control rats, blood and urinary parameters and the activity of radical-eliminating enzymes in the renal tissue deviated from the normal range, indicating damage to the kidneys. In contrast, rats given magnesium lithospermate B showed decreased urine volume, increased urinary osmotic pressure, and decreased urinary levels of glucose, protein, sodium and potassium, denoting less damage to the kidney. In this group, the urinary nitrite/nitrate ratio, and the activities of superoxide dismutase and catalase in the renal tissue were increased, while the malondialdehyde levels were decreased, suggesting the involvement of radicals in the normalizing of kidney function. The increased levels of urea nitrogen in the blood of rats with induced renal failure were also lowered by administering magnesium lithospermate B.

Comparison of cephaloridine renal accumulation and urinary excretion between normoglycemic and diabetic animals.

The renal toxicity of cephaloridine is reduced in a streptozotocin diabetic rat model. This study tested the hypothesis that renal cortical cephaloridine accumulation was diminished in diabetic rats. The following studies also investigated whether renal excretion was enhanced in diabetic rats. Male Fischer 344 rats were randomly divided into normoglycemic or diabetic groups. Diabetes was induced by injection (intraperitoneal, i.p.) of 35 mg/kg streptozotocin. Normoglycemic and diabetic rats were injected (i.p.) with 1500 mg/kg cephaloridine. Peak plasma cephaloridine levels were similar in both groups. Renal cortical accumulation was diminished (P < 0.05) in the diabetic group 1 and 4 h after cephaloridine injection. Urinary cephaloridine excretion was enhanced (P < 0.05) in the diabetic group relative to the normoglycemic animals during the first 4 h after cephaloridine injection. Comparisons between normoglycemic and diabetic groups indicated renal cortical cephaloridine accumulation was lower in the diabetic group. These findings would support the hypothesis that reduced cephaloridine toxicity in diabetic animals was due to reduced renal cortical accumulation of the toxin. These data also demonstrate that cephaloridine excretion was enhanced in the diabetic group and may contribute to the diminished renal accumulation.

[Studies on the mechanisms of renal damages induced by nephrotoxic compounds].

The present study was designed to evaluate the relationship between renal lipid peroxidation and acute renal damage induced by six nephrotoxic compounds: mercuric chloride (MC), glycerol (GL), maleic acid (MA), cephaloridine (CER), gentamicin (GM) and cisplatin (CDDP) in rats. Urine and blood biochemical analyses, determination of renal lipid peroxidation and its scavengers, and histopathological examination were performed in the time or day course after a single dose, or during consecutive administration of these compounds. Moreover, the effects of the antioxidant N,N'-diphenyl-p-phenylene-diamine (DPPD) on the renal damage induced by each compound were studied. 1. MC was administered subcutaneously once at doses of 2 or 4 mg/kg. At a dose of 4 mg/kg, the increase of renal malondialdehyde (MDA) as an index of lipid peroxidation was observed 12 hours after administration. The increase of renal MDA was associated with the development of mild necrosis in proximal straight tubules (PST). Pretreatment of rats with DPPD 600 mg/kg, i.p. ameliorated MC-induced nephrotoxicity. These results indicate that lipid peroxidation plays a significant role in MC-induced renal damage. 2. GL was administered subcutaneously once at doses of 2.5 or 5.0 ml/kg. The increase of MDA was observed on and after 24 hours at a dose of 2.5 ml/kg, 12 hours at a dose of 5.0 ml/kg. These changes were associated with the development of mild necrosis in proximal convoluted tubules (PCT). Pretreatment of rats with DPPD ameliorated GL-induced nephrotoxicity. These results indicate that lipid peroxidation plays a significant role in GL-induced renal damage. 3. MA was administered intraperitoneally once at doses of 100 and 200 mg/kg. Renal MDA did not increase at any observation times. At both doses, vacuole formation, mitochondrial swelling and condensation in PCT were observed 3 hours, and tubular necrosis occurred 3 and 6 hours after administration, which were associated with a decrease of renal glutathione. Pretreatment of rats with DPPD did not ameliorate MA-induced nephrotoxicity. These results suggest that lipid peroxidation does not play a significant role in MA-induced renal damage. 4. CER was administered intravenously once at doses of 500 and 1000 mg/kg. Renal MDA did not increased at any observation times. At both doses, vacuole formation and mitochondrial swelling in PCT were observed 1 hour, and mild necrosis in PCT was induced 6 hours after administration. On the other hand, DPPD ameliorated CER-induced renal histopathological changes. These results indicate that lipid peroxidation play a possible role in CER-induced renal damage. 5. GM was administered subcutaneously on 7 consecutive days at a dose of 40, 80 and 120 mg/kg. Renal MDA increased from first to 7th day at a dose of 120 mg/kg. At all doses, lysosomes which contained many myeloid bodies in PCT were observed 1st day after administration. Daily pretreatment of rats with DPPD 300 mg/kg, i.p. did not affect GM-induced histopathological changes, but ameliorated a part of the urinary biochemical parameters. These results suggest that lipid peroxidation plays a possible role in GM-induced renal damage. 6. CDDP was administered intraperitoneally once at a dose of 4 or 8 mg/kg. Renal MDA increased on 7th day, but at both doses, necrosis in PST had observed 3rd day after administration. Daily pretreatment of rats with DPPD 300 mg/kg, i.p. did not ameliorate CDDP-induced nephrotoxicity. These results suggest that lipid peroxidation does not play a significant role in CDDP-induced renal damage. From these results, lipid peroxidation will be a possible toxicological mechanism of acute renal damage induced by well established nephrotoxic compounds.

A study of the nephrotoxicity of three cephalosporins in rabbits using 1H NMR spectroscopy.

Male New Zealand White rabbits received a single intravenous injection of 125 mg/kg cephaloridine, 500 mg/kg cefoperazone or 1000 mg/kg cephalothin. Histological examination of kidneys at 48 h post-dose confirmed the presence of bilateral necrosis of the proximal convoluted tubules in the cephaloridine-treated animals. 1H-NMR urinalysis of cephaloridine-treated rabbits detected drug-related resonances, decreased hippurate and increased glucose at 0-24 h post-dose accompanied by elevated levels of lactate, glycine, citrate, glutamine/glutamate and alanine at 24-48 h post-dose. No histopathological changes were observed following administration of cefoperazone or cephalothin. 1H-NMR spectra of urine collected from these animals showed drug-related resonances and decreased hippurate levels at 0-24 h post-dose, and increased glucose levels at 24-48 h post-dose. Analysis of urine by conventional clinical-chemistry failed to reveal any statistically significant differences between the treatment groups. Under the conditions of this study, the nephrotoxic effects of cephaloridine and the minimal effects of cefoperazone and cephalothin could be clearly distinguished by 1H-NMR urinalysis but not by conventional urinalysis.