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  • br Material and methods br

    2019-10-09


    Material and methods
    Results
    Discussion Isolated DGAT-1 inhibitor pathway have several potential advantages over other in-vitro approaches, such as expression of properties similar to their site of origin including the possibility of bidirectional transmembrane transport and exposure. Furthermore, they make it possible to investigate site-selective toxicity in a chemically defined environment. Bringing renal cells into culture allows for maintenance of polarity and the examination of subchronic effects of toxins that cannot be readily examined in other short-term in-vitro systems [32]. The isolation procedure used in this study to obtain PT cells has been used previously in several studies and has been shown to result in an enriched population of viable proximal tubular cells [2], [13]. These findings were confirmed by measuring PT-specific parameters in freshly isolated as well as primary cultured PT cells under the conditions described in Section 2. Cummings and colleagues recently studied the cellular distribution of CYP450s in freshly isolated rat kidney cells, including PT cells [10]. The same authors also showed that human PT cells express several relevant biotransformation enzymes, including CYP450 [33]. These authors focused mainly on CYP4A11 expression, as the only CYP450s found at significant levels in human kidney microsomes are CYP4A11, CYP4F2 and CYP3A isoforms [34]. In the present study, we show that the primary cultured rat PT cells metabolize the different marker substrates associated with CYP450 isoenzyme activity. EROD activity is indicative for the expression of CYP1A1 [35], while caffeine is mainly metabolized by CYP1A1 and CYP1A2 in rat liver [36], [37]. The activities seen with 7-ethoxyresorufin and caffeine as substrates suggest CYP1A1 activity in primary PT cells, which is in agreement with the previous observation that CYP1A1 is DGAT-1 inhibitor pathway expressed at higher levels than CYP1A2 in rat renal tissue [38]. Expression of CYP1A1 in the renal samples was confirmed by Western blot analysis, revealing a protein of a molecular weight of about 56 kDa. The band observed in the liver microsomes migrated as a protein of 54 kDa represents CYP1A2, which is expressed predominantly in the liver of control rats [36]. It is worthwhile mentioning that EROD activity was very low after 24 h of culture, whilst caffeine seems to be metabolized for at least 24 h. This may indicate that there is no major contribution of CYP1A1 to the metabolism of caffeine in primary PTC. Our results support the observation that other biotransformation enzymes than CYP1A may contribute considerably to caffeine metabolism [39], [40]. Testosterone 16β-hydroxylation, which is associated with CYP2B1 activity [41], was not detected in PT cells. This observation is in agreement with the lack of dectecting immunoreactive CYP2B protein in microsomes from PT cells. Cummings et al. [10] also showed minimal CYP2B1/2 expression in PT cells of rat kidney. Tolbutamide is hydroxylated by CYP2C9 in man, and several studies suggest the involvement of a CYP2C member, probably CYP2C6/7 in rat liver microsomes [42], [43]. Hydroxylation of tolbutamide was also measurable in primary PTC. Furthermore, testosterone, which is metabolized regio- and stereoselectively by several different CYP450 isoenzymes, is 2α- and 16α-hydroxylated in primary PTC and kidney microsomes, indicating CYP2C11 activity [28]. Western blot analysis using the monoclonal antibody against rat CYP2C11 further confirmed expression of CYP2C11 by rat PT cells. Dextromethorphan-O-demethylation is associated with CYP2D activity in several species, such as man [44] and rat [45]. Although very low, the detected dextromethorphan-O-demethylase activity combined with the results obtained by kidney microsomal incubations and Western blotting strongly suggests the expression of a CYP2D family member in primary cultured rat PTC. Hiroi and colleagues [46] have recently shown mRNA expression of four CYP2D isoforms (CYP2D1/5, 2D2, 2D3 and 2D4/18) in rat kidney. A strong relationship between rat CYP2D1 and dextromethorphan-O-demethylation was shown by Miksys and colleagues [47]. However, the anti-CYP2D1 antibody was polyclonal, so it may have cross-reacted with either of the corresponding proteins.