Tetraethylammonium Accumulation Research Articles

Interactions of antiretroviral drugs with the SLC22A1 (OCT1) drug transporter.

The SLC22A1 influx transporter is expressed on the basolateral membrane of hepatocytes and is involved in the excretion of numerous cations. Inhibition of SLC22A1 by several antiretrovirals, such as the protease inhibitor darunavir, has not previously been determined. In order to better understand and predict drug-SLC22A1 interactions, a range of antiretrovirals were screened for SLC22A1-associated inhibition and transport. Stable SLC22A1-expressing KCL22 cells were produced previously by nucleofection. Control KCL22 cells were transfected with the empty vector pcDNA3.1. Accumulation of tetraethylammonium (5.5 μM, 30 min) was determined in SLC22A1-expressing and mock-transfected cells with and without 50 μM of SLC22A1 inhibitor prazosin, or 50 μM of each antiretroviral drug. SLC22A1 IC50 values for efavirenz, darunavir, and prazosin were determined. Cellular accumulation of efavirenz and darunavir was also assessed in SLC22A1-expressing KCL22 cells and reversibility of this accumulation was assessed using prazosin. Tetraethylammonium accumulation was higher in SLC22A1-expressing cells compared to mock-transfected cells (10.6 ± 0.8 μM vs. 0.3 ± 0.004 μM, p = 0.009) and was significantly reduced in SLC22A1-expressing cells when co-incubated with all antiretrovirals tested except atazanavir, lamivudine, tenofovir, zidovudine, and raltegravir. Particularly noticeable was the predominance of SLC22A1 inhibitors in the protease inhibitor and non-nucleoside reverse transcriptase inhibitor classes. Absolute SLC22A1 IC50 values for efavirenz, darunavir, and prazosin were 21.8, 46.2, and 2.8 μM, respectively. Efavirenz accumulation was higher in SLC22A1-expressing cells compared to mock-transfected cells (17% higher, p = 0.009) which was reversed using prazosin, whereas no difference was observed for darunavir (p = 0.86). These data inform the mechanistic basis for disposition, drug-drug interactions and pharmacogenetic candidate gene selection for antiretroviral drugs.

Altered Pharmacokinetics of Cationic Drugs Caused by Down-Regulation of Renal Rat Organic Cation Transporter 2 (Slc22a2) and Rat Multidrug and Toxin Extrusion 1 (Slc47a1) in Ischemia/Reperfusion-Induced Acute Kidney Injury

In the proximal tubules of rat (r) kidney, the polyspecific organic cation transporters (OCTs), rOCT1 and rOCT2, mediate the baso-lateral uptake of various organic cations, including many drugs, toxins, and endogenous compounds, and the apical type of H(+)/ organic cation antiporter, rat multidrug and toxin extrusion 1 (rMATE1), mediate the efflux of organic cations. Renal clearances of H(2) receptor antagonists, including famotidine, were reported to be decreased in patients with kidney disease. Therefore, acute kidney injury (AKI) could influence renal excretion and disposition of organic cations accompanied by the regulation of organic cation transporters. The aim of this study was to investigate the pharmacokinetic alteration of cationic drugs and the expression of tubular organic cation transporters, rOCT1, rOCT2, and rMATE1, in ischemia/reperfusion (I/R)-induced AKI rats. I/R-induced AKI increased the plasma concentration of i.v. administrated famotidine, a substrate for rOCT1 and rOCT2, or tetraethylammonium (TEA), a substrate for rOCT1, rOCT2, and rMATE1. The areas under the plasma concentration curves for famotidine and TEA were 2- and 6-fold higher in I/R rats than in sham-operated rats, respectively. The accumulation of TEA into renal slices was significantly decreased, suggesting that organic cation transport activity at the basolateral membranes was reduced in I/R rat kidney. The protein expressions of basolateral rOCT2 and luminal rMATE1 were down-regulated in I/R rat kidneys. These data suggest that the urinary secretion of cationic drugs via epithelial organic cation transporters is decreased in AKI.

Reduced expression of organic cation transporters rOCT1 and rOCT2 in experimental diabetes.

Recent reports have documented a functional deficit of organic cation transport in diabetic rats by an unknown mechanism. This study was designed to test the hypothesis that experimental diabetes decreases expression of organic cation transporters at the basolateral membrane. Streptozotocin-induced diabetic rats were maintained for varying durations after induction of diabetes. A second group of age-matched control rats was maintained in a parallel manner. Kinetic analysis of tetraethylammonium accumulation in freshly isolated proximal tubular cells indicated a significantly lower V(max) value for the diabetics versus controls with no statistical difference in K(m) values between the two groups. Cortex sections were processed by standard procedures for Northern and immunoblot analysis. Protein expression of the organic cation transporters rOCT1 and rOCT2 progressively decreased with increasing duration of diabetes. After 21 days of diabetes, rOCT1 and rOCT2 were maximally reduced by 50 and 70%, respectively. Quantification of mRNA expression revealed that the roct1 transcript remained unchanged, whereas the roct2 transcript was decreased by 50% after 14 days of diabetes. Treatment with insulin prevented the reductions in transporter levels. These results support the hypothesis by demonstrating that experimental diabetes decreased expression of both rOCT1 and rOCT2 protein and also of roct2 mRNA accumulation. On the other hand, roct1 mRNA levels were unaffected by the diabetic state. This suggests that differences in rOCT2 protein may result from transcriptional and/or translational changes, whereas rOCT1 deficits may be due to posttranscriptional alterations.

Functional impairment of renal organic cation transport in experimental diabetes.

This study was designed to determine the effect of diabetes on the function of the renal organic cation transport system that mediates the excretion of a wide variety of toxicants and drugs. The experiments compared the ability of renal cortex slices from streptozotocin-induced diabetic and non-diabetic rats to accumulate the model cation, 14C-tetraethylammonium under controlled conditions. Initial experiments demonstrated a progressive decline in tetraethylammonium accumulation with increasing duration of diabetes. The maximal decrease was observed at 21 days after streptozotocin injection. Time-dependent incubations revealed that tetraethylammonium uptake from both diabetic and non-diabetic rats followed a curvilinear pattern expected of an active process. However, at steady state the diabetic-derived slices accumulated a significant 38% less tetraethylammonium versus slices from non-diabetics. Concentration-dependent incubations of tetraethylammonium (0.01-10 mM, 60 min.) demonstrated saturable transport in both diabetic and non-diabetic slices with a significantly decreased capacity of diabetic-derived slices to accumulate tetraethylammonium. Cellular respiration rates in the two groups were not different. Insulin treatment of the diabetic rats prevented the transport decline. While the causative factor of the transport impairment in diabetes is unresolved, this study documents an aspect of diabetic nephropathy that has not been previously reported but which may have important implications for renal excretion of cationic drugs and toxicants. The results also provide a mechanism for the well-documented "protection phenomenon" by which the kidneys of diabetic rats are resistant to nephrotoxicity induced by the chemotherapeutic agent cisplatin.

Reduced hepatic uptake and intestinal excretion of organic cations in mice with a targeted disruption of the organic cation transporter 1 (Oct1 [Slc22a1]) gene.

The polyspecific organic cation transporter 1 (OCT1 [SLC22A1]) mediates facilitated transport of small (hydrophilic) organic cations. OCT1 is localized at the basolateral membrane of epithelial cells in the liver, kidney, and intestine and could therefore be involved in the elimination of endogenous amines and xenobiotics via these organs. To investigate the pharmacologic and physiologic role of this transport protein, we generated Oct1 knockout (Oct1(-/-)) mice. Oct1(-/-) mice appeared to be viable, healthy, and fertile and displayed no obvious phenotypic abnormalities. The role of Oct1 in the pharmacology of substrate drugs was studied by comparing the distribution and excretion of the model substrate tetraethylammonium (TEA) after intravenous administration to wild-type and Oct1(-/-) mice. In Oct1(-/-) mice, accumulation of TEA in liver was four to sixfold lower than in wild-type mice, whereas direct intestinal excretion of TEA was reduced about twofold. Excretion of TEA into urine over 1 h was 53% of the dose in wild-type mice, compared to 80% in knockout mice, probably because in Oct1(-/-) mice less TEA accumulates in the liver and thus more is available for rapid excretion by the kidney. In addition, we found that absence of Oct1 leads to decreased liver accumulation of the anticancer drug metaiodobenzylguanidine and the neurotoxin 1-methyl-4-phenylpyridium. In conclusion, our data show that Oct1 plays an important role in the uptake of organic cations into the liver and in their direct excretion into the lumen of the small intestine.

Hormonal regulation of organic cation transporter OCT2 expression in rat kidney

Rat (r) OCT2 was identified as the second member of the organic cation transporter (OCT) family, and is predominantly expressed in the kidney. We reported previously that rOCT2 was responsible for the gender differences in renal basolateral membrane organic cation transport activity. As renal rOCT2 expression in males is much higher than that in females, we hypothesized that rOCT2 expression may be under the control of sex hormones. Treatment of male and female rats with testosterone significantly increased the expression levels of rOCT2 mRNA and protein in the kidney, whereas estradiol treatment moderately decreased the expression levels of rOCT2. There was no regulation of renal rOCT1 mRNA expression by testosterone or estradiol. Treatment of male and female rats with testosterone significantly stimulated the tetraethylammonium (TEA) accumulation by renal slices, whereas estradiol treatment caused a decrease in the TEA accumulation by slices from male but not female rats. The present findings suggested that testosterone up-regulates renal rOCT2 expression and estradiol moderately down-regulates rOCT2.

Differential expression of the polyspecific drug transporter OCT1 in rat hepatocarcinoma cells

The polyspecific drug transporter OCT1 is a plasma transmembrane protein involved in the uptake of cationic drugs into hepatocytes. In order to determine whether hepatic OCT1 levels, like those of the other cationic drug transporter P-glycoprotein, may be altered during hepatocarcinogenesis, we have investigated OCT1 expression and activity in rat liver carcinoma cells. Similar levels of OCT1 mRNAs were evident in both normal liver and diethylnitrosamine-induced hepatocarcinomas by Northern blot analysis. In contrast, five hepatoma cell lines (Fao, Faza, H5, HTC and RHC1) showed either a decrease or an absence of OCT1 expression compared to normal hepatocytes; these hepatoma cells also displayed lower intracellular accumulation of tetraethylammonium (TEA), a well-known substrate for OCT1. However, among the hepatoma cell lines, the well-differentiated Fao cell line was found to retain substantial levels of OCT1 expression and of intracellular TEA uptake. Therefore, these data provide the first evidence that OCT1 expression is well-preserved in chemically-induced rat malignant neoplastic liver lesions, whereas it is either decreased or undetectable in hepatoma cell lines, which may be related to the loss of various liver functions usually occurring in these cell lines.

Nephrotoxicity of N-(3-bromophenyl)-2-hydroxysuccinimide: role of halogen groups in the nephrotoxic potential of N-(halophenyl)succinimides

Among N-(halophenyl)succinimides, N-(3,5-dichlorophenyl)succinimide (NDPS) is a potent nephrotoxicant as well as an agricultural fungicide. Although two chloride groups on the phenyl ring are essential to induce optimal nephrotoxicity, the role of halogen groups in NDPS nephrotoxicity is not clear. In this study, N-(3-bromophenyl)-2-hydroxysuccinimide (NBPHS) was prepared as a monohalophenyl derivative of N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS), an oxidative and nephrotoxicant metabolite of NDPS. The nephrotoxic potential of NBPHS was evaluated in vivo and in vitro to determine the role of halogen groups in N-(halophenyl)succinimide nephrotoxicity. Male Fischer 344 rats (four/group) were administered a single intraperitoneal (i.p.) injection of NBPHS (0.1, 0.4 or 0.8 mmol/kg) or vehicle (25% dimethyl sulfoxide in sesame oil) and renal function monitored for 48 h. Administration of NBPHS (0.8 mmol/kg) induced nephrotoxicity, while very mild changes or no changes in renal function were observed following administration of 0.4 mmol/kg or 0.1 mmol/kg of NBPHS, respectively. Nephrotoxicity induced by NBPHS (0.8 mmol/kg) was characterized by diuresis, transiently increased proteinuria, glucosuria and hematuria, elevated kidney weight, and reduced tetraethylammonium (TEA) uptake by renal cortical slices, and was not as marked as nephrotoxicity induced by NDHS (0.1 mmol/kg) or NDPS (0.4 mmol/kg). In the in vitro studies, the effects of NBPHS on organic ion accumulation, pyruvate-stimulated gluconeogenesis, and lactatc dehydrogenase (LDH) release were measured using renal cortical slices. NBPHS decreased p-aminohippurate (PAH) and TEA accumulation at NBPHS bath concentrations of 0.05 mM and 0.5 mM or greater, respectively. Renal gluconeogenesis was inhibited by NBPHS at 1 mM bath concentration, while LDH leakage was not increased at NBPHS bath concentrations up to 1 mM. The results demonstrate that NBPHS is a mild nephrotoxicant in vivo and in vitro, but does not have cytotoxic effects to renal tissues at the concentrations tested. From these results, it appears that halogen groups are essential to the nephrotoxic potential of N-(halophenyl)-2-hydroxysuccinimides or N-(halophenyl)succinimides and play an important role in the mechanism of NDPS nephrotoxicity following NDHS formation.

Nephrotoxic potential of 2-amino-5-chlorophenol and 4-amino-3-chlorophenol in Fischer 344 rats: comparisons with 2- and 4-chloroaniline and 2- and 4-aminophenol

Nephrotoxicity occurs following intraperitoneal (i.p.) administration of 2-chloroaniline or 4-chloroaniline hydrochloride to Fischer 344 rats, but the nephrotoxicant chemical species and mechanism of nephrotoxicity are unknown. The purpose of this study was to evaluate the in vivo and in vitro nephrotoxic potential of 2-amino-5-chlorophenol and 4-amino-3-chlorophenol, metabolites of 4-chloroaniline and 2-chloroaniline. A comparison was also made between the nephrotoxic potential of the aminochlorophenols and the corresponding aminophenols to examine the effect of adding a chloride group on the nephrotoxic potential of the aminophenols. Male Fischer 344 rats (4/group) were given an i.p. injection of a chloroaniline or aminochlorophenol hydrochloride (1.5 mmol/kg), an aminophenol (1.0 or 1.5 mmol/kg), or vehicle, and renal function monitored at 24 and 48 h. Both aminochlorophenols induced smaller and fewer renal effects than the parent chloroanilines in vivo. Also, 4-aminophenol was markedly more potent as a nephrotoxicant than 4-amino-3-chlorophenol, while 2-aminophenol and 2-amino-5-chlorophenol induced only mild changes in renal function. In vitro, the phenolic compounds reduced p-aminohippurate accumulation by renal cortical slices at bath concentrations of 0.01 mM, while a bath concentration of 0.50 mM or greater was required for the chloroanilines. However, all compounds reduced tetraethylammonium accumulation at bath concentrations of 0.1–0.5 mM or greater. These results indicate that extrarenally-produced aminochlorophenol metabolites do not contribute to the mechanism of chloroaniline nephrotoxicity. Also, the reduced nephrotoxic potential of 4-amino-3-chlorophenol compared to 4-aminophenol could result from an altered ability of the aminochlorophenol to redox cycle or form conjugates.

Differential toxicity of cisplatin, carboplatin, and CI-973 correlates with cellular platinum levels in rat renal cortical slices.

The differential toxicity of the platinum-containing compounds, cisplatin, carboplatin, CI-973, and transplatin, was investigated in renal cortical slices over a 24-hr period. Platinum accumulation was measured to assess whether platinum levels correlated with inhibition of specific cell functions (accumulation of organic ions, protein synthesis) or potassium loss. The earliest indicator of toxicity was decreased accumulation of the organic anion, para-aminohippuric acid (PAH), which preceded a decrease in the accumulation of the organic cation, tetraethyl-ammonium (TEA), and protein synthesis. Cisplatin (1 mM) and CI-973 (3 mM) treatment reduced PAH accumulation within 2 hr; carboplatin (3 mM) decreased PAH at 6 hr. TEA accumulation was absent in slices incubated for 6 hr or longer in 1 mM cisplatin, or for 24 hr in 3 mM carboplatin or CI-973. Protein synthesis was inhibited 80, 75, and 87% in slices treated for 24 hr with 100 microM cisplatin, 1 mM carboplatin, and 1 mM CI-973, respectively, compared to control. Intracellular potassium levels, which decreased between 6 and 24 hr, were the least sensitive indicator of cell damage. Transplatin, which lacks antitumor activity and is nonnephrotoxic in vivo, effectively decreased protein synthesis, intracellular potassium, and organic ion accumulation in rat renal cortical slices in vitro. The concentration of slice-associated platinum following treatment with cisplatin, carboplatin, CI-973, and transplatin increased with time and concentration. Inhibition of protein synthesis and loss of intracellular potassium correlated with increased total cellular platinum, indicating that platinum compounds negatively affect cell function and viability. The relative toxicity of these compounds in rat renal cortical slices was cisplatin = transplatin > CI-973 > carboplatin.

ATP-dependent transport of tetraethylammonium by endosomes isolated from rat renal cortex

During renal organic cation secretion by some species, intracellular concentrations greatly exceed the 10- to 15-fold ratio predicted by the potential-driven mechanism thought to mediate their basolateral uptake. Free cytoplasmic organic cation concentrations within the tubular cells might be decreased through sequestration within intracellular organelles. The data reported here show that endosomal vesicles isolated from rat renal cortex take up tetraethylammonium (TEA) by an ATP-dependent mechanism. Addition of 0.2-5 mM ATP to the medium stimulated uptake 5- to 10-fold at 5 min and 20-fold at 60 min. More than 80% of the ATP-dependent uptake was associated with an osmotically active space. The nonhydrolyzable ATP analogue, adenosine 5'-O-(3-thiotriphosphate), did not stimulate TEA uptake. Mg2+ and Cl- were required for stimulation. Uptake was inhibited by several organic cations, including TEA itself. Uptake was also inhibited by inhibitors of intravesicular acidification, e.g., monensin and N-ethylmaleimide. Furthermore, the ATP requirement could be bypassed by establishing a pH gradient (inside acidic). These data show that endosomal TEA accumulation is mediated by proton/TEA exchange and is driven by the pH gradient maintained by H(+)-adenosinetriphosphatase. This potent sequestration mechanism may play an important role in organic cation secretion.

Studies on the renal transport of trimethylpentanoic acid metabolites of 2,2,4-trimethylpentane in rat renal cortical slices.

2,2,4-Trimethylpentane (TMP), a nephrotoxic component of unleaded gasoline in male but not female rats, undergoes oxidative metabolism to yield 2,2,4- and 2,4,4-trimethylpentanol, pentanoic acid and 5-hydroxypentanoic acid. We have examined the effect of three of these pentanoic acid metabolites on the renal transport of the organic anion p-aminohippurate (PAH) and the organic cation tetraethylammonium (TEA) in renal cortical slices from male Fischer 344 rats. 2,4,4-Trimethylpentanoic acid, the major urinary metabolite in rats, produced a selective decrease in the accumulation of PAH without affecting TEA accumulation. Kinetic analysis showed that 2,4,4-trimethylpentanoic acid was a competitive inhibitor of the organic anion transport system, with a Ki of 4 mM. 2,4,4-Trimethyl-5-hydroxypentanoic acid also showed selective inhibition of PAH transport, while 2,2,4-trimethylpentanoic acid was less selective and reduced both PAH and TEA transport. Additional studies with radiolabeled 2,4,4-trimethylpentanoic acid showed that there was a time- and concentration-dependent accumulation of radioactivity into slices of renal cortex. However, experiments conducted at 4 degrees C and studies with metabolic inhibitors, or with an inhibitor of organic anion transport, indicated that little of the accumulated material was entering the cell. We conclude from these studies that the pentanoic acid metabolites formed from 2,2,4-trimethylpentane are not actively transported by the renal organic anion transport system. In summary, in vitro the pentanoic acid metabolites appear to bind to renal cortical tissue and thereby reduce the transport of PAH.

Contribution of acetone and osmotic-diuresis by streptozotocin-induced diabetes in attenuation of cephaloridine nephrotoxicity

Previous studies have indicated that cephaloridine nephrotoxicity was reduced in streptozotocin (STZ)-induced diabetic rats. Experiments were performed to investigate if a shorter duration of diabetes would reduce cephaloridine nephrotoxicity. Studies were also conducted to examine the contribution of osmotic diuresis and ketone accumulation to the mechanism for reduced toxicity. Male Fischer 344 (F344) rats were injected with 30 mg/kg STZ or vehicle. Seven days after STZ or vehicle administration, the animals were treated (i.p.) with 1500 mg/kg cephaloridine. Increased kidney weight, blood urea nitrogen (BUN) level and decreased renal cortical slice accumulation of p-aminohippurate (PAH) and tetraethyl-ammonium (TEA) were measured in the normoglycemic group. No differences in renal function were detected between diabetic groups treated with cephaloridine or vehicle (PFC). Pretreatment of euglycemic rats with 0 or 10% dextrose in the drinking water and by oral gavage failed to prevent the renal damage produced by 1500 mg/kg cephaloridine despite glucosuria and urine output comparable to diabetic animals. However, dextrose-diuresis afforded a slight reduction in toxicity as indicated by changes in kidney weight and renal cortical slice accumulation of PAH and TEA. Pretreatment (oral) with 0 or 1.5 ml/kg acetone had no effect on cephaloridine toxicity (1000 mg/kg, i.p.). These findings suggested that attenuation of cephaloridine toxicity may be independent of the duration of diabetes. These results also indicated that glucose-mediated osmotic diuresis and acetone accumulation cannot account for reduced cephaloridine toxicity in diabetic rats.

Acute nephrotoxicity induced by isomeric dichloroanilines in Fischer 344 rats

Chlorinated anilines are widely used as chemical intermediates in the manufacture of numerous dyes, pesticides, drugs and industrial compounds. The purpose of this study was to examine the nephrotoxic potential of the six dichloroaniline (DCA) isomers in vivo and in vitro. In the in vivo studies, male Fischer 344 rats (4–8 rats/group) were administered a single, intraperitoneal injection of a DCA isomer (0.4, 0.8 or 1.0 mmol/kg) as the hydrochloride salt or given vehicle (0.9% saline, 2.5 ml/kg), and renal function monitored at 24 and 48 h. Renal effects induced by DCA were characterized by decreased urine volume, increased proteinuria, hematuria, modest elevations in blood urea nitrogen (BUN) concentrations, decreased accumulation of p-aminohippurate (PAH) by renal cortical slices, and no change or a slight decrease in kidney weight. Renal morphological changes were observed as proximal tubular necrosis with lesser effects on distal tubular cells and collecting ducts. Based on the overall effects on renal function and morphology, the decreasing order of nephrotoxic potential was found to be 3,5-DCA > 2,5-DCA > 2,4-, 2,6- and 3,4-DCA > 2,3-DCA. The ability for the DCA to induce nephrotoxicity correlated well with the lipophilic properties of the DCA isomers and Hammett constants (σ) for the various chloro substitutions. In the in vitro studies, renal cortical slices from naive male Fischer 344 rats were co-incubated with a DCA isomer (0–10 −3 M) and PAH or tetraethylammonium (TEA). All DCA isomers decreased PAH and TEA accumulation at 10 −3 M DCA concentration in the media with 3,5-DCA inducing the largest decrease at this concentration. These results indicate that DCA are capable of altering renal function in vivo and in vitro and that 3,5-DCA possesses the greatest nephrotoxic potential in vivo and in vitro.

Nephrotoxicity of cisplatin, carboplatin and transplatin

The present study was designed to compare the nephrotoxicity induced by the three platinum compounds cisplatin (CDDP), carboplatin (CBDCA) and transplatin (TDDP) in vitro and to obtain information to elucidate the mechanism of platinum compound-induced nephrotoxicity. Rat or rabbit renal cortical slices were incubated for different periods of time in platinum compound-containing media (0.42 or 1.67 mM) and thereafter monitored for platinum content, tetraethylammonium(TEA) and paraaminohippurate(PAH) accumulation and gluconeogenesis. Malondialdehyde(MDA) content of slices was determined as a parameter of lipid peroxidation. Activity of glucose-6-phosphatase of rat renal microsomes was investigated after platinum-compound exposure. In all series of experiments the effect of the antioxidant N,N'diphenyl-p-phenylenediamine (DPPD) was tested. CBDCA showed no effects on all parameters of renal cell function at all concentrations and all time points investigated, except for the activity of glucose-6-phosphatase, which was slightly affected by CBDCA. CBDCA-induced MDA production was lower, compared to CDDP, which showed marked toxic effects on TEA and PAH accumulation, gluconeogenesis and glucose-6-phosphatase activity. The onset of CDDP-induced alterations was dependent on drug concentration. MDA production was reduced by DPPD. Protection against the platinum compound-induced decrease in TEA and PAH accumulation was observed after the use of DPPD. DPPD had no protective effect on CDDP-induced inhibition of gluconeogenesis and glucose-6-phosphatase, which might indicate an effect on gluconeogenesis by direct inhibition of glucose-6-phosphatase. DPPD did not alter uptake of platinum compounds in rat renal cortical slices. TDDP showed different in vitro properties compared to in vivo conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of streptozotocin (STZ)-induced diabetes, dextrose diuresis and aceton on cisplatin nephrotoxicity in Fischer 344 (F344) rats

The following studies examined the impact of the diabetic state on cisplatin nephrotoxicity. This study also investigated the potential mechanisms for diabetes mediated reduction of cisplatin toxicity. A diabetic state was induced in male Fischer 344 (F344) rats after intraperitoneal (i.p.) injection of 27–35 mg/kg STZ. Cisplatin (5 mg/kg, i.p.) nephrotoxicity was examined in normoglycemic and diabetic rats after 48 and 96 h. Cisplatin was nephrotoxic within 96 h to normoglycemic animals as indicated by an increased kidney weight, marked elevations in serum BUN levels as well as significant P < 0.05) decrease in renal cortical slice accumulation of p-aminohippurate (PAH) and tetraethylammonium (TEA). Cisplatin failed to depress renal cortical slice accumulation of PAH and TEA in the diabetic rats. Cisplatin was also less effective in increasing BUN levels or kidney weight in diabetic rats. Further studies investigated the impact of glycosuric diuresis and ketone bodies on cisplatin nephrotoxicity. Dextrose diuresis of normoglycemic rats failed to reduce the effect of cisplatin on BUN levels, kidney weight and renal cortical slice uptake of PAH and TEA. Acetone pretreatment of normoglycemic rats also did not reduce cisplatin nephrotoxicity. These results indicate: (1) cisplatin nephtotoxicity is attenuated in the experimental diabetic state, (2) diabetes does not reduced cisplatin nephrotoxicity through glycosuric diuresis and (3) ketone body accumulation does not modulate cisplatin nephrotoxicity.

Effect of Cyclosporine a on Accumulation of Tetraethylammonium and p-Aminohippurate, and on Lipid Peroxidation in Rat Renal Microsomes and Cortical Slices

The effect of cyclosporine A (CsA) on lipid peroxidation (LPO) was assessed in renal cortical slices and renal microsomes. Cortical slices were incubated with 1500 micrograms/ml CsA and microsomes with 0.5-20 micrograms/ml under identical conditions (pH 7.4, 37 degrees C) for 3 hours, and LPO monitored by the formation of malondialdehyde (MDA). CsA at concentrations of 3 micrograms/ml and higher caused a significant increase MDA in microsomes and renal cortical slices showed a time dependent release of MDA into the incubation medium. The influence of CsA on tetraethammonium (TEA) and p-aminohippurate (PAH) accumulation in renal cortical slices was investigated for up to 3 hours with concentration of CsA from 10 to 1000 micrograms/ml. CsA caused a time- and concentration-dependent decrease of TEA accumulation and higher concentrations of CsA decreased PAH accumulation in renal cortical slices. The results add further evidence to the suggestion that lipid peroxidation participate in CsA-induced impairment of kidney function.

Cephaloridine nephrotoxicity in streptozotocin induced diabetic fischer 344 (F344) rats

The purpose of this study was to determine if cephaloridine nephrotoxicity is attenuated in streptozotocin (STZ)-induced diabetic rats. Fischer 344 (F344) rats (205–250 g) were given a single injection (i.p.) of STZ (27–35 mg/kg) or citrate buffer. The nephrotoxicity of (750 mg/kg) cephaloridine (i.p.) was then compared with normoglycemic and 14-day diabetic rats. Increased blood urea nitrogen (BUN) levels as well as diminished renal cortical slice accumulation of tetraethylammonium (TEA) and lactate-stimulated p-aminohippurate (PAH) were measured ( P < 0.05) in normoglycemic rats 48 h after cephaloridine administration. Cephaloridine failed to alter BUN levels and organic ion accumulation in diabetic rats. Diabetes did not totally protect against cephaloridine toxicity since kidney weights were elevated in normoglycemic and diabetic rats 48 h after administration of 750 mg/kg cephaloridine. A series of experiments also measured BUN levels, kidney weight and renal cortical slice uptake of PAH and TEA 24, 48 and 72 h after (1500 mg/kg) cephaloridine administration. Cephaloridine increased ( P < 0.05) kidney wt and decreased PAH and TEA uptake ( P < 0.05) in the normoglycemic group at 24–72 h. No change in kidney wt, PAH or TEA uptake was observed in the diabetic rats. These data indicate diabetes reduces cephaloridine nephrotoxicity.

Peroxidative damage and nephrotoxicity of dichlorovinylcysteine in mice.

Male NMRI mice were treated i.p. with dichlorovinylcysteine (DCVC) in a dosage of 2.5-500 mg/kg-1 and renal cortical slices from naive mice were incubated with 0-125 micrograms/ml-1 DCVC. The effects of DCVC on blood urea nitrogen (BUN), reduced glutathione (GSH) content, malondialdehyde (MDA) production, p-aminohippuric acid (PAH)- and tetraethylammonium (TEA)-accumulation and glucose synthesis (gluconeogenesis) were measured. DCVC depleted GSH in a time- and dose-dependent manner. Depletion of renal cortical GSH by DCVC was more pronounced in the kidney cortex than in the liver. DCVC caused a dose-dependent increase of ethane exhalation and of MDA production in the renal cortex. When animals were kept in a closed system, decrease in oxygen concentration increased the peroxidative damage. No increase of MDA concentration was observed in the liver. Treatment of mice with DCVC induced a dose-dependent increase in BUN and decreased the accumulation of PAH and TEA in renal cortical slices. Pretreatment of mice with aminooxyacetic acid (AOAA) and (+) cyanidanol-3 (CY) caused a significant reduction in DCVC-induced lipid peroxidation and nephrotoxicity. In vitro incubation of renal cortical slices of naive mice with DCVC resulted in a concentration-dependent increase in MDA and a concentration-dependent decrease in the accumulations of PAH, TEA and of gluconeogenesis. In conclusion, the interaction of DCVC and/or its metabolites with membrane lipids may be responsible for lipid peroxidation and nephrotoxicity. The formation of lipid peroxidation products was greater under hypoxic conditions and appeared to be related to the DCVC-induced nephrotoxicity. This data suggests lipid peroxidation as a possible mechanism of DCVC-induced nephrotoxicity.

Attenuation of cisplatin nephrotoxicity by streptozotocin-induced diabetes

The therapeutic use of cisplatin is associated with acute renal failure. The purpose of this study was to determine (a) if streptozotocin (STZ) was toxic to renal proximal tubules and (b) the nephrotoxicity of cisplatin in STZ-diabetic rats. Male Sprague-Dawley rats were injected with STZ (55 mg/kg, ip) to induce a diabetic state. BUN and renal cortical slice uptake of p-aminohippurate (PAH) and tetraethylammonium (TEA) were not altered, relative to normoglycemic rats, 3, 16, and 28 days following STZ treatment. These results indicate that STZ is not toxic to renal proximal tubules. Cisplatin nephrotoxicity studies were then conducted in STZ-diabetic and normoglycemic rats. Cisplatin nephrotoxicity was also evaluated in diabetic rats pretreated for 8 days with insulin. Diabetic and normoglycemic rats were administered 5 mg/kg cisplatin or water (ip). Increased kidney weight, BUN levels, glucosuria, and proteinuria were measured in normoglycemic rats 4 days after cisplatin administration. Renal cortical TEA and lactate-stimulated PAH uptake ( p < 0.05) were diminished in the normoglycemic rats 4 days after cisplatin injection. No change in kidney weight, BUN levels, or renal cortical slice accumulation of PAH and TEA was observed in diabetic rats treated with cisplatin. However, cisplatin administration to diabetic rats pretreated with insulin resulted in increased mortality, proteinuria, glucosuria and elevated kidney weight. These results indicate that the diabetic state attenuates cisplatin nephrotoxicity. Additionally, these results indicate that diabetes attenuation of cisplatin nephrotoxicity is dependent on the severity of the diabetic state.