Summary<p>Renal proximal tubular (RPT) cells can be exposed apically to glomerulary filtrated and basolaterally to non-filtrated nephrotoxic compounds. To excrete these compounds via the urine, RPT cells are equipped with transport systems able to transport nephrotoxicants from the basolateral to the apical side of the tubule. Since the majority of <em>in vitro</em> models used so far to study nephrotoxicity concern suspensions of RPT cells or RPT cells cultured on solid supports, in which transport systems are only partially or no longer active, less attention has been paid to the role of these transport systems in renal toxicity. An adequate assessment of RPT toxicity of nephrotoxicants should include the role of functional transport systems. Therefore, the aim of the present thesis was to develop such an <em>in vitro</em> model. We cultured rat RPT cells to confluency on porous supports of tissue culture inserts. Monolayers of RPT cells remained confluent for 24h. After that time, they became leaky and cell viability gradually declined (Chapter 2).<p>The glutathione (GSH) conjugates of (hydro)quinones, nitrobenzene and ethacrynic acid were used as model compounds. <em>In vivo,</em> these compounds are targetted to RPT cells since these cells are equipped with both enzyme and transport systems that use these potentially toxic glutathione conjugates as substrate.<p>The glutathione conjugate of the quinoid compound menadione (MGNQ) was only toxic after basolateral challenge of confluent monolayers of RPT cells (Chapter 2). By preventing basolateral uptake of this compound via organic anion systems, an increase in cytotoxicity was observed. Similar effects were observed when basolateral uptake and subsequent deacetylation of M(NAC)NQ, the <em>N</em> -acetylcysteine conjugate of menadione, were inhibited. It is generally agreed upon that γglutamyltranspeptidase (γGT) and dipeptidase mediated metabolism of GSH conjugates into cysteine conjugates is a prerequisite for cellular uptake of the latter conjugates. MGNQ induced apical cytotoxicity only after inhibition of apical γGT. These observations suggest that cellular uptake of thioether conjugates is associated with detoxification. The postulated reaction mechanism resulting in detoxification of the GSH and <em>N</em> -acetyleysteine conjugate of menadione is drawn in Fig. 4 (Chapter 2).<p>Detoxification of quinone cystein(glycinyl) conjugates can occur via an intramolecular cyclization reaction which eliminates the reactive quinone function (Monks <em>et al.</em> , 1990). The identification and characterisation of the cyclization product of MGNQ provided evidence that this reaction is responsible for the detoxification of MGNQ by RPT cells in this <em>in vitro</em> system (Chapter 4).<p>In agreement with results obtained with MGNQ, both mono-substituted glutathione conjugates of 2- <em>tert</em> -butyl hydroquinone (5SG-TBHQ and 6SG-TBHQ) were only toxic after basolateral exposure of RPT cell monolayers (Chapter 3). Apparently, at this side of the monolayer γGT-mediated metabolism of GSH conjugates is probably not sufficient for complete detoxification of these conjugates in this <em>in vitro</em> system (Chapters 2 and 3). In addition, the di-substituted GSH conjugate of TBHQ (3,6SG-TBHQ) was not cytotoxic in our system. In line with this, 3,6SG-TBHQ has a lower ability to generate reactive oxygen species compared with both monosubstituted conjugates (Van Ommen <em>et al.</em> , 1992), suggesting that the (hydro)quinone induced proximal tubular toxicity mainly results from redox cycling (Chapter 3). Since superoxide dismutase and catalase, which cannot be transported into the cell, completely abolished SG- TBHQ induced cytotoxicity, quinol-thioether induced redox cycling is likely to take place extracellularly (Chapters 2 and 3).<p>When RPT cells were cultured on solid supports, apical exposure to 5SG-TBHQ induced a cytotoxic effect whereas 6SG-TBHQ did not. Inhibition of γGT completely abolished 5SG-TBHQ induced cytotoxicity, whereas a cytotoxic effect was observed with 6SG-TBHQ. This result underlines the concept that γGT on the one hand initiates detoxification of GSH-conjugated (hydro)quinones by removing the reactive quinone function (5SG-TBHQ) and on the other hand it gives rise to conjugates that can undergo redox cycling more easily (6SG-TBHQ) (Chapter 3).<p>No transepithelial transport of MGNQ or metabolites thereof could be observed across RPT cell monolayer. Most likely, transport occurred via paracellular leakage as a result of a reduction in monolayer integrity due to toxicity via extensive redox cycling of the quinone under the culture conditions. Also, no basolateral to apical transport of the GSH conjugate of ethacrynic. acid, an α,βunsaturated ketone (EASG), was observed across the RPT cell monolayer. Probably, the transporter responsible for transport of this conjugate lost its function in our model. In contrast, only apical to basolateral transport of EASG was observed. Since acivicin reduced this transport of EASG and inhibited the formation of free, unconjugated ethacrynic acid (EA), not the GSH conjugate is transported but rather the cysteine conjugate and/or free EA (Chapter 4).<p>In Chapter 5 it was observed by means of <sup><font size="-2">19</font></SUP>F-NMR that monolayers of both LLCPK <sub><font size="-2">1</font></sub> cells and primary RPT cells were not able to metabolise the GSH conjugate of 2,5-difluoronitrobenzene (FGNB) into its <em>N</em> -acetylcysteine derivative (FNAcNB). Consequently, the cysteine conjugate of 2,5- difluoronitrobenzene (FCysNB) was the major metabolite formed. However, freshly isolated RPT cells were able to form mercapturic acids but, upon cultivation, quickly lose this capacity. In addition, no apical to basolateral transport of the cysteine conjugate of 2,5-difluoronitrobenzene (FCysNB) was observed across monolayers of primary RPT cells (Chapter 5).<p>In Chapter 6 we studied the ability of another <em>in vitro</em> renal cell system i.e. freshly isolated renal cortical slices to metabolise xenobiotics by measuring their <em>N</em> -acetylation capacity. We observed that these slices metabolised F <em>Cys</em> NB into the mercapturate F <em>N</em> AcNB. To find out whether cryopreserved rat renal cortical slices could be used as an alternative for freshly isolated tissue in metabolism studies, we compared their biotransformation capacity. For up to 4 h, the <em>N</em> -acetylation capacity of both type of slices is comparable. Thereafter, the <em>N</em> -acetylation capacity is substantially lower in cryopreserved tissue. Since cryopreserved rat renal cortical slices did not metabolise F <em>G</em> NB into a lower amount of F <em>Cys</em> NB than freshly isolated tissue, cryopreservation did not affect the activity of γGT and dipeptidases.<p>Since uptake of xenobiotics via organic anion transporters is another determinant of biotransformation capacity of RPT cells, we studied organic anion uptake of both type of slices using para-aminohippuric (PAH) acid as model compound. Compared with freshly isolated slices, cryopreserved rat renal cortical slices exhibited similar PAH uptake characteristics.<p><strong><em>Concluding remarks</em></strong><p>Selective exposure of confluent monolyers of rat renal proximal tubular cells to glutathione conjugates of the tested quinone and hydroquinones revealed that these compounds were more toxic after basolateral exposure than after apical exposure. In addition, the observed cytotoxicity appeared to be the result of mainly extracellular redox cycling of tested (hydro)quinones. The physiological significance of the observed effects remains obscure since our <em>in vitro</em> model suffers from several artefacts.<p>For instance, we could not observe basolateral uptake of GSH conjugated menadione and ethacrynic acid. In addition, the results obtained with GSH conjugated 2,5-difluoronitrobenzene (FGNB) suggest that apical uptake of cysteine- <em>S</em> -conjugates is also absent in our RPT cell monolayer. Probably as a result of the fact that the latter characteristic is a prerequisite for <em>N</em> -acetylation of these cysteine-S-conjugates by RPT cells, we were not able to measure <em>N</em> -acetylation of these conjugates. As a consequence of non- functional cysteine transport, large amounts of (hydro)quinone-thioethers remain outside the RPT cell. Since hydroquinones readily autoxidize under culture conditions, cytotoxicity emerges as a result of extracellular redox cycling.<p>In contrast to cultured RPT cells, the <em>N</em> -acetylation capacity was present in freshly isolated RPT cells. In another <em>in vitro</em> model it was shown that freshly isolated rat renal cortical slices also metabolised FGNB into its <em>N</em> -acetylcysteine conjugate. However, cryopreservation of rat renal cortical slices affected its <em>N</em> -acetylation capacity. Similar effects of cryopreservation were observed for organic anion transport by renal cortical slices.<p>In summary, RPT cell monolayers are useful for studying γGT and dipeptidase mediated metabolism of glutathione conjugates. With respect to metabolic capacity, freshly isolated renal cortical slices provide a more appropriate tool for investigating xenobiotic metabolism <em>in vitro.</em> However, the results obtained from both <em>in vitro</em> models underline the necessity to characterise renal biotransformation and transport systems involved in renal metabolism of xenobiotics. In addition, functional limitations of the in <em>vitro</em> model used have to be discerned. In order to improve the usability of RPT cell cultures it is of paramount importance to evaluate the influence of culture environment on the expression of differentiated cellular characteristics.
|Qualification||Doctor of Philosophy|
|Award date||2 Oct 1996|
|Place of Publication||S.l.|
|Publication status||Published - 1996|
- toxic substances