In vitro nephrotoxicity testing: Polarized approach of proximal tubular epithelium

PosterSession3B. Organ Toxicity experimental systemusing renal cortical slices preparedfrom rat and human. Urinary excretion of NAG and y-GTP were determined in 47 patients before and after drip infusion with 100 rnl of LOCM. Renal cortical slices prepared from rat kidney and human kidney were incubatedwith iopamodol(LOCM)or diatrizoate (HOCM)in 10 ml of buffer solution at 37°C for 120 min. NAG and y-GTP released from the slices into incubationmedium were determined. Urinary NAGand y-GTP significantly increasedby LOCM,especially in patientswith imparedrenal function.Dose-relatedincreases of NAG and y-GTP release by iopamidol were observedboth in rat and human renal slices. The effect of diatrizoate on NAG release from rat renal slices was clear, but NAG release from human renal slices was almost stable. Dose-depended increase of y-GTP caused by diatrizoate was demonstrated both in rat and human renal slices. In the experiment using human renal tissue, no significant difference was shown in NAG or y-GTP release between diatrizoate and iopamidol. The present clinical investigation suggested that LOCM injured renal tubular cells, while those renal damage can not be detected by serum Cr or Ccr. It is cotroversy whether or not LOCM is less nephrotoxic compared with HOCM. However, renal cortical slice techniques revealedthat LOCM was as nephrotoxic as HOCM.

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P3B63

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GLUCOSE HANDLINGBY AN IN VITRORABBIT PROXIMAL TUBULE (RPT) EPITHELIUM MODEL. A NEW ENDPOINT FOR NEPHROTOXICITY

I. Genestie *1, V.Keravee2, G. Lorenzoir', J.P. Morin1 • IINSERM U295, Universite de Rouen; 2 CEB, Mont-St-Aignan; 3 HMR, Romainville, France Glucose transepithelial transport and cellular uptake were studied on confluentmonolayers of RPf cells grown on porous membranes, with AMG ([14C]a-methyl-glucopyranoside). An unidirectional apicaVbasolateral transepithelial AMG net flux 29.5 ± 1.2 vs basolateral/apical 1.5 ± 0.2 j.tlfcm2/90 min was observed. Paracellular transportassessedby Inulinclearancewas ~ 1.5 j.tlfcm219O min. This transport was saturable with a KIn = 1.79 mM, compatible with the characteristics of the rabbit proximal convoluted segmentof isolated perfusedrabbit proximaltubules,a Vm =84.1 nmollcm2 /90 min and was selectively inhibited by phloridzin (0.4 mM). Uptake of AMG from apical compartment was 14 times greater than the basolateral one and specifically inhibited by phloridzin. Calculated kinetic parameters show that the culture on porous membranes exhibited a higher affinity (lower Km = 0.98 vs 1.58 mM) and a lower transport capacity (lower Vmax = 2.61 vs 3.63 nmollmg prol/min) than those in standard plastic wells. Compartimentalized exposure to nephrotoxic compoundsshowed side dependentqualitative and quantitative responses. Gentamicin inhibited both transport and uptake of AMG when applied by apical side (-20%, 2.5 mg/ml). Cephaloridine induced inhibition of AMG transport and uptake essentially by basolateral side (-30% and -20% respectively). Cyclosporineffect was solely seen on transport after apical (-25%, 0.25 j.tg/ml) or basolateral(-40%,5 JLg/rnl) exposure,while,only AMGuptake was affectedby Aspirin.Paracetamolinhibitedboth glucosetransportand uptake higher when applied from basolateral compartment (>0.63 mg/rnl). Cisplatin has no effect on AMG handling. In conclusion, beside the evaluationof Inulin paracellularpassage,glucose transepithelial trausport may be a very sensitive and useful endpoint for the assessmentof toxicological impact on RPT epithelialfunction. This approach will allow new insights in nephrotoxicity testings.

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IP3B64 I IN VITRONEPHROTOXICITY TESTING: PQLARIZED APPROACH OF PROXIMAL TUBULAR EPITHELIUM

I. Genestie*1 , V. Keravec2 , J.P. Morin1 , J.P.Fillastre1 . IINSERM U295, Universite de Rouen;2C.E.B., Mont-St-Aignan, France Primary cultures of rabbit renal proximal tubules grown on bicompartimentalized microporous supportswere exposedat confluence to test compounds: Cephaloridine (CPH: 0.31 to 5 mg/rnl),Gentamicin (GEN: 0.08 to 5 mg/ml), Lauryl Sulphate (LS: 1.25 to 40 JLg/ml), and Paracetamol (PAR: 0.156 to 5 mg/rnl) either from apical (a) or basolateral (b) compartments. GST (glutathione-S-transferases), yGT (y-glutamyl-transpeptidase), GPX (glutathione peroxidase) and NaK (NaIK-ATPase) were relevant of biochemical markers. LS showed no specific effect on biochemical parametersbefore cell dissolution (>20 j.tg/ml a = b). Glutathione related enzymes were not altered by GEN, but transitorily increasedby CPH and PAR (b > a). NaK activitywas decreased by GEN (>0.08 mg/rnl a = b). Results show that epithelium integrity, assessed by paracellular Inulin diffusion, was selectively disturbed by CPH (>2.5 mg/ml b), GEN (>2.5 mg/rnl a), LS (>20 j.tg/ml a =b) and PAR(>2.5 mg/ml b). Basal to apical PAR and apical to basal glucose transepithelial transports were assessed as functional parameters. PAH clearance was decreased by CPH (0.63 mg/rnl b > 2.5 mg/ml a), GEN (1.25 mg/rnl b > 2.5 mg/rnl a), LS (5j.tg/ml a = b), and PAR (0.63 mg/ml b > a). Glucose transepithelial clearance and uptake were only disturbed by GEN concomitant with the loss of monolayer integrity. CPH decreased both glucose transport (0.63 mg/rnl a = b) and uptake (0.31 mg/rnl b > 1.25 mg/ml a). LS has no effect on glucose clearance but significantly decreased glucose uptake (5 j.tg/mla = b). Both clearanceand uptakeof glucose were reducedby PAR(1.25 mg/rnl b > a). In conclusion, this in vitro bicompartmentalized approach represents a useful tool for the evaluation and comprehension of renal pharmacotoxicologic mechanisms. Indeed, it allows the observation of different sensitivity patterns to the compounds, related to the occurrence of variable cascades of events as a function of proximal tubuleepitheliumside of exposure.

IP3B6S!INCREASE IN RENALMETABOLIZING ENZYME ACTlVmES INDUCED BY HEPATlC DAMAGE

Y.c. Kim *, H.K. Yim. College of Pharmacy, SeoulNational University, Seoul, Korea The effect of hepatic damage induced either by a hepatotoxicant or partial hepatectomy on extrahepatic drug metabolizing enzyme activities was examined using adult ICR mice. A hepatotoxic dose of carbon tetrachloride (CT; 0.05 mllkg, ip) administered to mice 24 hr prior to acetaminophen (APAP; 350 mglkg, ip) or chloroform (CF; 0.025 or 1.0 mlIkg, ip) challenge decreased the hepatotoxicity, but elevated the renal toxicity significantly as shown by increases in activitiesof aspartate amino-transferase, alanine aminotransferase and sorbitol dehydrogenase, and in levels of blood urea nitrogen and creatinine in serum. The CT pretreatment did not affect the glutathionelevels in liver or in renal cortex. The hepatic microsomal metabolizing enzyme activities were decreased by a 24 hr prior dose of CT significantly, however, the renal metabolizing activities were induced as indicated by the increases in p-nitrophenol hydroxylase, p-nitroanisole demethylase, and aminopyrine N-demethylase activities. In partially hepatectomized mice, the renal toxicity of CF or APAP was significantly elevated and corresponding increases in renal metabolizing activity were observed. The pulmonary metabolizing activities were not affected. In female or castrated male mice the effects of CT or hepatectomy on renal metabolizing activity and renal toxicity of APAPfCF were reduced significantly. The results above indicate that testosterone may play an important role in the