Carnitine transport and its inhibition by sulfonylureas in human kidney proximal tubular epithelial cells.

The kidney plays an important role in the homeostasis of carnitine by its ability to reabsorb carnitine almost completely from the glomerular filtrate. The transport process responsible for this reabsorption has been investigated thus far only in laboratory animals. Here we report on the characteristics of carnitine uptake in a proximal tubular epithelial cell line derived from human kidney. The uptake process was found to be obligatorily dependent on Na+ with no involvement of anions. The process was saturable, with a Michaelis-Menten constant of 14 +/- 1 microM. The Na+:carnitine stoichiometry was 1:1. The same process also was found to be responsible for the uptake of acetylcarnitine and propionylcarnitine, two acyl esters of carnitine with potential for therapeutic use in humans. The uptake process was specific for carnitine and its acyl esters. Betaine, a structural analog of carnitine, interacted with the uptake process to a significant extent. The present studies also showed that sulfonylureas, oral hypoglycemic agents currently used in the management of type 2 diabetes, inhibited the carnitine uptake system. Among the sulfonylureas tested, glibenclamide was the most potent inhibitor. The inhibition was competitive. Glibenclamide inhibited the uptake not only of carnitine but also of acetylcarnitine and propionylcarnitine. The inhibition most likely was the result of direct interaction of the compound with the carnitine transporter because the inhibition could be demonstrated in purified rat kidney brush border membrane vesicles.     

Membrane transport function in primary cultures of human proximal tubular cells

To further develop primary cultures of human proximal tubular (hPT) cells for study of drug disposition, we determined kinetics and protein expression of several key transporters for organic anions and cations, peptides, and neutral amino acids. p-Aminohippurate uptake exhibited similar kinetics as published values, was inhibited by cephaloridine, cimetidine, methotrexate, and urate, consistent with function of both organic anion transporter 1 (OAT1) and OAT3. Transport rates by organic cation transporters (OCTs) were up to three-fold higher than those of OATs. Of the OCT substrates tested, triethanolamine exhibited the highest transport rates across the basolateral membrane (BLM). OCTN1 exhibited high-affinity, low-capacity BLM transport of l-carnitine. Glycylsarcosine transport by PepT2 was rapid and comparable to that of OCTs. Amino acid System L on the BLM exhibited comparable kinetic parameters for transport of l-leucine as the OATs. Efflux of verapamil across the brush-border membrane by P-glycoprotein was very rapid. Expression of carriers was generally maintained throughout 5 days of culture. Of the four OAT proteins studied (OAT1-4), expression of OAT1 and OAT3 was the most readily detected and exhibited interindividual variation. OCTN2 was the major OCT in hPT cells. Expression was also quantified for multidrug resistance-associated proteins 2 and 5 and P-glycoprotein. These results show that primary cultures of hPT cells express a diverse array of transporters for major classes of important drugs and are suitable for study of drug transport and disposition and assessment of potential drug-drug interactions in human kidney.     

Characterisation of human tubular cell monolayers as a model of proximal tubular xenobiotic handling

The aim of this study was to determine whether primary human tubular cell monolayers could provide a powerful tool with which to investigate the renal proximal tubular handling of xenobiotics. Human proximal and distal tubule/collecting duct cells were grown as monolayers on permeable filter supports. After 10 days in culture, proximal tubule cells remained differentiated and expressed a wide palette of transporters at the mRNA level including NaPi-IIa, SGLT1, SGLT2, OCT2, OCTN2, OAT1, OAT3, OAT4, MDR1, MRP2 and BCRP. At the protein level, the expression of a subset of transporters including NaPi-IIa, OAT1 and OAT3 was demonstrated using immunohistochemistry. Analysis of the expression of the ATP binding cassette efflux pumps MDR1, MRP2 and BCRP confirmed their apical membrane localisation. At the functional level, tubule cell monolayers retain the necessary machinery to mediate the net secretion of the prototypic substrates; PAH and creatinine. PAH secretion across the monolayer consisted of the uptake of PAH across the basolateral membrane by OAT1 and OAT3 and the apical exit of PAH by a probenecid and MK571-sensitive route consistent with actions of MRP2 or MRP4. Creatinine secretion was by OCT2-mediated uptake at the basolateral membrane and via MDR1 at the apical membrane. Functional expression of MDR1 and BCRP at the apical membrane was also demonstrated using a Hoechst 33342 dye. Similarly, measurement of calcein efflux demonstrated the functional expression of MRP2 at the apical membrane of cell monolayers. In conclusion, human tubular cell monolayers provide a powerful tool to investigate renal xenobiotic handling.     

Isolated proximal tubular cells from rat kidney as an in vitro model for studies on nephrotoxicity. I. An improved method for preparation of proximal tubular cells and their functional characterization by alpha-methylglucose uptake

Rat renal proximal tubular cells were isolated by successive EGTA and collagenase perfusions and purified by filtration and isopycnic centrifugation. The method is rapid and provides a much higher fraction of proximal tubular cells (90-95%) than comparable methods. The yield of viable (97 +/- 3%) cells is high (30 X 10(6) cells/g kidney). The intracellular ATP was 16 +/- 2 nmol/mg protein and remained essentially constant for at least 3 hr. The cells were characterized for transport of organic ions and glucose. Glucose transport was studied by alpha-[14C]methylglucose uptake; apparent Km and Vmax values were 3.4 +/- 0.5 mM and 4.1 +/- 0.9 nmol/min.mg protein, respectively. This transport could almost be completely inhibited by phloridzin, indicating that the uptake is mediated by the brush border glucose carrier.     

Moment analysis of drug disposition in kidney. V:In Vivo transepithelial transport ofp-aminohippurate in rat kidney

A new method that can assess the kinetics of in vivotransepithelial transport in rat kidney has been established. The method is based upon a multiple-indicator dilution experiment and the moment analysis theory. After simultaneous bolus injections of p-aminohippurate (PAH) and inulin into the right renal artery, blood samples were taken from the carotid artery and urine was separately collected from right and left ureters. The characteristic response for the first passage of drugs through the right kidney was evaluated by taking blood circulation into consideration. To determine the mean artery-tovein transit time and the extraction ratio in the kidney, an intravenous injection was also performed as a reference experiment for deconvolution. The urinary excretion curve corresponding to the first passage was obtained as the difference between both kidneys. The mean artery-tolumen transit time (mean transepithelial transit time, ¯T _cell )was computed by subtracting the mean urinary transit time of inulin from that of secreted PAH. Sinc transport across the luminal membrane into the lumen from tubular epithelial cells can influence the cellular residence time of drugs, ¯ T_cell and the single-pass mean residence time in epithelial cells (¯T _cell.sp )can be thought of describing luminal membrane transport. The value of ¯T _{cell obtained for 0. t mM PAH was 22 sec and it was prolonged to 61 sec in the presence of probenecid, suggesting an inhibitory effect on transport across the luminal membrane. On the other hand, antiluminal membrane transport into cells from blood is characterized by the volume of distribution in the kidney} (Vd _PAH ). Vd _{PAH was remarkably decreased by treatment with probenecid, indicating an inhibitory effect on antiluminal membrane transport. The effects of probenecid on both sides of epithelial cell membrane transport were first demonstrated} in vivo.The present method is useful for the analysis of in vivotransepithelial transport including antiluminal and luminal membrane transport for drugs excreted via tubular secretion.     

Moment analysis of drug disposition in kidney. V: In vivo transepithelial transport of p-aminohippurate in rat kidney

A new method that can assess the kinetics of in vivo transepithelial transport in rat kidney has been established. The method is based upon a multiple-indicator dilution experiment and the moment analysis theory. After simultaneous bolus injections of p-aminohippurate (PAH) and inulin into the right renal artery, blood samples were taken from the carotid artery and urine was separately collected from right and left ureters. The characteristic response for the first passage of drugs through the right kidney was evaluated by taking blood circulation into consideration. To determine the mean artery-to-vein transit time and the extraction ratio in the kidney, an intravenous injection was also performed as a reference experiment for deconvolution. The urinary excretion curve corresponding to the first passage was obtained as the difference between both kidneys. The mean artery-to-lumen transit time (mean transepithelial transit time, Tcell) was computed by subtracting the mean urinary transit time of inulin from that of secreted PAH. Since transport across the luminal membrane into the lumen from tubular epithelial cells can influence the cellular residence time of drugs, Tcell and the single-pass mean residence time in epithelial cells (Tcell,sp) can be thought of describing luminal membrane transport. The value of Tcell obtained for 0.1 mM PAH was 22 sec and it was prolonged to 61 sec in the presence of probenecid, suggesting an inhibitory effect on transport across the luminal membrane. On the other hand, antiluminal membrane transport into cells from blood is characterized by the volume of distribution in the kidney (VdPAH). VdPAH was remarkably decreased by treatment with probenecid, indicating an inhibitory effect on antiluminal membrane transport. The effects of probenecid on both sides of epithelial cell membrane transport were first demonstrated in vivo. The present method is useful for the analysis of in vivo transepithelial transport including antiluminal and luminal membrane transport for drugs excreted via tubular secretion.     

Isolated proximal tubular cells from rat kidney as an in vitro model for studies on nephrotoxicity. II. Alpha-methylglucose uptake as a sensitive parameter for mechanistic studies of acute toxicity by xenobiotics

Many nephrotoxic agents exert their effect primarily on the cells of the proximal tubules. We isolated these cells and investigated whether the uptake of alpha-methylglucose (alpha-MG) could serve as a parameter to assess effects of nephrotoxins on the functional integrity of the cells. Agents that are acutely nephrotoxic in vivo, CD2+, Hg2+, UO22+, p-aminophenol, and bis-2,3-dibromopropylphosphate, inhibited alpha-MG uptake at low concentrations. Most agents that exert their effect in vivo with delay or only when used chronically (gentamicin, cephaloridine, phenacetin, and acetaminophen) inhibited alpha-MG uptake only at much higher concentrations; cisplatin, however, inhibited alpha-MG uptake at a low concentration. S-(1,1-Difluoro-2,2-dichloroethyl)-L-cysteine and its N-acetyl derivate, two metabolites of the nephrotoxic agent 1,1-dichloro-2,2-difluoroethylene, inhibit alpha-MG uptake. Aminooxyacetic acid, which prevents the formation of the ultimate toxic metabolite by inhibition of beta-lyase, abolished almost completely the toxicity of both compounds. The nephrotoxic conjugate of hexachlorobutadiene, S-(1,2,3,4,4-pentachlorobutadienyl)-glutathione, also inhibited alpha-MG uptake. The selective inhibitor of gamma-glutamyltranspeptidase, anthglutin, completely prevented this inhibition. These results indicate that the uptake of alpha-methylglucose by isolated proximal tubular cells from rat kidney is a useful parameter for the study of nephrotoxicity, since in vitro results reflect acute nephrotoxicity as observed in vivo.     

Moment analysis of drug disposition in rat kidney: role of basolateral membrane transport in renal transepithelial transport of p-aminohippurate.

The determining step in transepithelial transport of p-aminohippurate (PAH) in renal tubular secretion has been elucidated in the rat isolated perfused kidney. The method was based upon a multiple indicator dilution experiment and non-compartmental moment analysis. Single-pass dilution curves were obtained from venous and urinary effluents after simultaneous intra-arterial injections of Evans blue with albumin, [3H]inulin and [14C]PAH. Probenecid was used as a transport inhibitor and dissolved in both perfusate and injection solution. The urinary excretion of PAH decreased depending on the probenecid concentration, while that of inulin was not affected. No correlation was observed between the amount of secretion and the mean residence time of secreted PAH in renal epithelial cells (Tcell). Since Tcell ought to be affected by the rate of secretion from cells to lumen, it was suggested that the secretion rate was independent of the amount secreted. In contrast, the amount of PAH excreted via tubular secretion showed a linear correlation with the volume of distribution in the kidney and the apparent rate constant for tubular uptake of PAH. Since these kinetic parameters reflect the transport from blood into cells across the basolateral membranes of renal epithelial cells, the present results demonstrated that basolateral membrane transport is a determining step in the transcellular transport of PAH and that the major effect of probenecid is the inhibition of transport from blood into cells.     

Cocaine-induced kidney toxicity: an in vitro study using primary cultured human proximal tubular epithelial cells

Renal failure resulting from cocaine abuse has been well documented, although the underlying mechanisms remain to be investigated. In the present study, primary cultured human proximal tubular epithelial cells (HPTECs) of the kidney were used to investigate its ability to metabolize cocaine, as well as the cytotoxicity induced by cocaine and its metabolites benzoylecgonine (BE), ecgonine methyl ester (EME) and norcocaine (NCOC). Gas chromatography/ion trap-mass spectrometry (GC/IT-MS) analysis of HPTECs exposed to cocaine (1 mM) for 72 h confirmed its metabolism into EME and NCOC, but not BE. EME levels increased along the exposure time to cocaine, while NCOC concentration diminished after reaching a maximum at 6 h, indicating a possible secondary metabolism for this metabolite. Cocaine promoted a concentration-dependent loss of cell viability, whereas BE and EME were found to be non-toxic to HPTECs at the tested conditions. In contrast, NCOC revealed to have higher intrinsic nephrotoxicity than the parent compound. Moreover, cocaine-induced cell death was partially reversed in the presence of ketoconazole (KTZ), a potent CYP3A inhibitor, supporting the hypothesis that NCOC may play a role in cocaine-induced nephrotoxicity. Cocaine-induced cytotoxicity was found to involve intracellular glutathione depletion at low concentrations and to induce mitochondrial damage at higher concentrations. Under the present experimental conditions, HPTECs death pathway followed an apoptotic pattern, which was evident for concentrations as low as 0.1 mM.     

Primary culture of proximal tubular cells from normal rat kidney as an in vitro model to study mechanisms of nephrotoxicity. Toxicity of nephrotoxicants at low concentrations during prolonged exposure

The aim of this study was to set up an in vitro system to study nephrotoxicity of xenobiotics which allows exposure at low concentrations for long periods (1-5 days). A very pure preparation of isolated proximal tubular cells (PTC) from rat kidney (Boogaard et al., Toxicol Appl Pharmacol 101: 135-143, 1989) was brought into primary culture. Cells grew to confluence in 3 days and could be maintained up to 8 days in a modification of Dulbecco's modified Eagle's medium Ham F12 nutrient mixture supplemented with fetal calf serum. Fibroblast growth was completely suppressed by replacement of L-valine by D-valine and of L-arginine by L-ornithine. Polarity was retained: in cells grown on filters organic anions were transported at the basolateral membrane while D-glucose transport was located at the apical membrane. Inhibition of the latter was used to assess the functional integrity of the cells after exposure to nephrotoxins. The newly grown cells expressed gamma-glutamyltranspeptidase activity since incubation with the glutathione-conjugate of 1,1-dichloro-2,2-difluoroethylene (DCDFE) induced cytotoxicity. Both beta-lyase and acylase activities were expressed because the cysteine-S-conjugate and the corresponding mercapturate of DCDFE showed cytotoxicity. Cultured cells showed toxicity on prolonged exposure to very low concentrations of gentamicin, cephaloridine, cisplatin and the cysteine-S-conjugate of chlorotrifluoroethylene. The lowest concentrations at which toxicity can be observed are 1-3 orders of magnitude lower in primary cultures than in freshly isolated PTC in suspension. This indicates that this cell model is suitable to investigate mechanisms of nephrotoxicity in vitro, at prolonged exposure to the low concentrations that are relevant in vivo levels.     

Predicting tubular reabsorption with a human kidney proximal tubule tissue-on-a-chip and physiologically-based modeling

Kidney is a major route of xenobiotic excretion, but the accuracy of preclinical data for predicting in vivo clearance is limited by species differences and non-physiologic 2D culture conditions. Microphysiological systems can potentially increase predictive accuracy due to their more realistic 3D environment and incorporation of dynamic flow. We used a renal proximal tubule microphysiological device to predict renal reabsorption of five compounds: creatinine (negative control), perfluorooctanoic acid (positive control), cisplatin, gentamicin, and cadmium. We perfused compound-containing media to determine renal uptake/reabsorption, adjusted for non-specific binding. A physiologically-based parallel tube model was used to model reabsorption kinetics and make predictions of overall in vivo renal clearance. For all compounds tested, the kidney tubule chip combined with physiologically-based modeling reproduces qualitatively and quantitatively in vivo tubular reabsorption and clearance. However, because the in vitro device lacks filtration and tubular secretion components, additional information on protein binding and the importance of secretory transport is needed in order to make accurate predictions. These and other limitations, such as the presence of non-physiological compounds such as antibiotics and bovine serum albumin in media and the need to better characterize degree of expression of important transporters, highlight some of the challenges with using microphysiological devices to predict in vivo pharmacokinetics.     

Convective diffusional analysis for drug transport through a tubular polymeric membrane.

The transport of three p-aminobenzoate esters (ethyl, butyl, and hexyl) through a tubular dimethyl polysiloxane membrane into a flowing liquid was investigated. The tubular configuration permits the exact determination of the convective diffusional contribution to membrane transport with models that account for fluid hydrodynamics. The observed transport behavior ranged from complete convective diffusion control for the hexyl ester to complete membrane control for the ethyl ester; the butyl ester exhibited a change in control with flow rate. The implications of convective diffusional considerations to intestinal absorption and dissolution studies are discussed.     

Effect of aminoglycosides on proximal tubular membranes of the human kidney

Summary The effect of the aminoglycosides amikacin, gentamicin, netilmicin sisomicin and tobramycin on the proximal tubule of the human kidney was investigated in 78 healthy subjects. Fifteen adults were each given gentamicin, sisomicin or tobramycin 3 mg/kg bodyweight, 10 subjects received netilmicin 3 mg/kg or amikacin 15 mg/kg additionally seven subjects amikacin 10 mg and six subjects netilmicin 6 mg on three consecutive days. The principal enzyme of the brush border membrane of the proximal tubule, alanine aminopeptidase (AAP), was determined enzymatically and immunologically in 24-hour urines. The effects of the various aminoglycosides on the membranes were different. Less of membrane AAP was greatest after amikacin and was least after tobramycin. There was no difference between gentamicin, netilmicin, and sisomicin, which had an effect intermediate between the other two compounds. The elimination of AAP occurred at intervals whicht might possibly have been caused by impairment of cell cycles.     

Cadmium, vectorial active transport, and MT-3-dependent regulation of cadherin expression in human proximal tubular cells.

Previous studies from this laboratory have implicated the expression of the third isoform of metallothionein (MT-3) in the maintenance of proximal tubular vectorial active ion transport. It was shown that HK-2 cells have no expression of MT-3 and do not form domes in culture; whereas, the human proximal tubular (HPT) cells and HK-2 cells stably transfected with MT-3 [HK-2(MT-3)] form these structures. In the present study, this association was further explored by determining the effect of MT-3 expression on the expression of the E -, P -, N -, K -, and Ksp-cadherins. It was demonstrated that the HPT cells and HK-2(MT-3) cells had significant elevations in the expression of messenger RNA and protein for the E -, P -, and Ksp-cadherins compared with that of the HK-2 cells transfected with the blank vector [HK-2(blank vector)]. In contrast, the HK-2(blank vector) cells had significantly elevated expression of N- and K-cadherin compared with both the HPT and HK-2(MT-3) cell lines. These patterns of cadherin expression provide strong evidence that MT-3 might be involved in epithelial to mesenchymal transition that is postulated to occur during several disease states and in the mesenchymal to epithelial transition that occurs during normal kidney morphogenesis. A final goal of the study was to determine if Cd(+2) exposure influenced vectorial active transport of the proximal tubular cells and if this might occur through alterations in the expression of MT-3. It was shown that exposure to Cd(+2) eliminated vectorial active transport by the proximal tubular cell lines, but that Cd(+2) exposure did not reduce the expression of the MT-3 protein. The study shows that the level of MT-3 expression in HPT cells influences transepithelial resistance and cadherin expression but does not influence the Cd(+2)-induced loss of vectorial active transport.     

Evaluation of human nasal RPMI 2650 cells grown at an air-liquid interface as a model for nasal drug transport studies

This study tests the hypothesis that human nasal RPMI 2650 cells grown at an air-liquid interface is a feasible model for drug transport studies via the nasal route. RPMI 2650 cells were cultured in Eagle's minimal essential medium (MEM) at both air-liquid and liquid-liquid interfaces. For each culture regimen, monolayer integrity was tested by measuring the transepithelial resistance (TEER) as well as the transport of paracellular and transcellular markers across the monolayer. The expression of tight junction proteins-differentiation markers-in cells of the different monolayers was studied by western blot analysis and confocal microscopy. The highest TEER values (192 +/- 3 Omega . cm2) were observed for RPMI 2650 cells seeded onto collagen-coated permeable polytetrafluoroethylene inserts and grown at an air-liquid interface for 10 days; a seeding density of 4 x 10(5)/cm2 generated and maintained a cell monolayer with suitable barrier properties at days 9-12. Microscopic examination showed that RPMI 2650 cells grown on filter inserts formed a fully confluent monolayer. The apparent permeability coefficients of the paracellular marker, [14C] mannitol, and the transcellular marker, [3H] propranolol, were 5.07 +/- 0.01 x 10(-6) cm/s and 16.1 +/- 0.1 x 10(-6) cm/s, respectively. Western blot analysis indicated the presence of four tight junction proteins: ZO-1, occludin, claudin-1 and E-cadherin; and the quantities of ZO-1, occludin, and E-cadherin were significantly higher in cells grown at an air-liquid interface than in cells grown at a liquid-liquid interface. Confocal microscopic studies showed ZO-1, F-actin, occludin and claudin-1 proteins at cell-cell contacts and revealed significant differences in the distributions and densities of ZO-1 protein in cells grown at the two types of interface. The data indicate that RPMI 2650 cells grown at an air-liquid interface form polarized monolayers with the cells interconnected by tight junction proteins. This human nasal cell line model could provide a useful tool for in vitro screening of nasal drug candidates.     

Monolayers of porcine alveolar epithelial cells in primary culture as an in vitro model for drug absorption studies.

Filter-grown monolayers of porcine alveolar epithelial cells (pAEpC) in primary culture have been characterized as an in vitro model for pulmonary absorption screening of xenobiotics, including substrates of efflux systems. Experimental conditions and a protocol for transport experiments were optimized using transepithelial electrical resistances (TEER) and permeability of marker compounds as acceptance criteria. Since new drugs often feature poor water solubility, monolayer integrity in the presence of a solubilizer (dimethyl sulfoxide) was tested. Transport studies were carried out with budesonide and triamcinolone acetonide, i.e., two drugs commonly administered to the lungs. Furthermore, expression of P-glycoprotein (P-gp) was assessed by immunofluorescence microscopy and transport studies employing the substrates rhodamine 123 and digoxin. Hydrocortisone-supplemented (0.5 microg/ml) small airway basal medium as transport buffer and a maximal solubilizer concentration of 1.5% dimethyl sulfoxide were found to provide suitable conditions for drug transport studies across pAEpC, as reflected, e.g., by a minimum TEER of 600 Omega cm(2). Permeation of marker compounds was reproducible throughout several cell preparations and proved the model successful in distinguishing between low- and high-permeable drugs. P-gp expression was confirmed by immunocytochemistry, even though transport studies revealed no polarity in transepithelial marker transport. In conclusion, our results demonstrate that filter-grown monolayers of pAEpC can be used to study drug transport across alveolar epithelial barrier and thus, may represent a suitable in vitro model for pulmonary drug absorption and delivery.     

Moment analysis of drug disposition in kidney. VI: Assessment ofin vivo transmembrane transport ofp-aminohippurate in tubular epithelium

This paper describes a novel method to assess the antiluminal membrane (ALM) and luminal membrane (LM) transport in vivo across renal tubular epithelial cells. The method is based upon a noncompartmental moment analysis of the plasma concentration and urinary excretion rate curves following renal artery injection. Quantitative relationships are represented between the noncompartmental parameters (clearance, volume of distribution, and the mean transit time) and the first-order rate constants associated with transmembrane transport processes. The in vivo transepithelial transport of [¹⁴C]p -aminohippurate (PAH) was examined using the rat kidney in the absence or presence of various plasma concentrations of unlabeled PAH, cefazolin, and methotrexate. The tubular secretion intrinsic clearance was reduced with an increase in the plasma concentration of concurrent unlabeled organic onions. The distribution volume of PAH in the kidney decreased in association with a decrease in the amount of PAH secreted, whereas the mean transepithelial (artery-to-lumen) transit time (¯T_cell) remained constant. These findings indicate that ALM transport is a capacity-limited process determining the amount of tubular secretion, and that LM transport is linear over the concentration range examined and independent of the amount of secretion. The contribution of ALM and LM transport to transcellular transport was first clarified in vivo. The present method will be useful for analyzing the transmembrane transport processes in vivo for highly diffusible substances in the kidney.     

Autoradiographical studies on the localization of metallothionein in proximal tubular cells of the rat kidney

The renal distribution of ¹²⁵I-labelled metallothionein (¹²⁵I-MT), administered as a tracer dose to female Wistar rats, has been investigated by light and electron microscopical autoradiography. ¹²⁵I-MT was absorbed rapidly and 80% of the administered dose was observed in the kidney within 60 min after dosing. At 30 min, a high density of silver grains due to ¹²⁵I, was observed by light microscopy in the convoluted proximal tubules. Electron microscopical autoradiography showed that silver grains were located in large apical vacuoles and cytoplasmic bodies (heterolysosomes) of the endocytotic system in the apical or middle regions of the cells lining convoluted proximal tubules. The results suggest that endocytotic systems are associated with ¹²⁵I-MT absorption by epithelial cells lining the renal convoluted proximal tubules. The apparent inconsistency on the distribution pattern of silver grains due to ¹⁰⁹Cd-labelled MT and ¹²⁵I-labelled MT in these is also discussed.