• 2018-07
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  • Judging from the densitometric analysis


    Judging from the densitometric analysis of the Western blots, CYP2E1 protein was expressed, although at very low levels, by PT cells. This corresponds to previous observations [10], showing that CYP2E1 was expressed in the PT, although expression was higher in the distal tubule. Testosterone 6β-hydroxylation, a marker for CYP3A activity [20], could not be detected in primary PT cell cultures. In addition, expression of CYP3A apoprotein was detected neither in freshly isolated nor in 7 day old cultured PT cells. Again, this is in agreement with previous findings [10]. Expression of CYP4A was very high in the PT cells (Fig. 4), which corresponds to the observations of Ito et al. indicating that the PT is the major site of expression of CYP4A mRNA and protein in the kidney of rats [48]. Strong CYP4A expression was also observed in whole kidney microsomes. As judged by densitometric analysis of the Western blot, at least two different proteins reacting with our CYP4A antibody may be expressed in the PT. Previous studies have identified the CYP4A isoforms expressed in the rat PT as CYP4A2 and CYP4A3 [10], [48], [49]. All results with PT cells were compared with CYP450 activity measured in liver and kidney microsomes of the same animals (Table 3). Comparison between metabolism of the described substrates in liver and kidney microsomes seemed necessary as the enzyme assays used in this study had been validated for liver microsomes [17], [18], [19], [20], [21]. The liver microsomal data thus served as a positive control. Likewise, the ephrin receptor applied were tested predominantly with rat liver microsomes [20]. The approach with whole kidney microsomes, however, does have the limitation that the kidney is a highly heterogeneous organ, and thus microsomal fractions from different renal cell types may influence substrate metabolism, as other NADPH-dependent enzymes may contribute to the biotransformation of marker substrates [41]. However, the preliminary results obtained with whole kidney microsomes, microsomes from the renal cortex or from PT cells did not differ significantly (data not shown). The obtained results enabled us to conclude that the marker substrates were indeed metabolized by CYP450 enzymes in the kidney. The detection of immunoreactive CYP450 protein confirmed this assumption. Primary PT cells metabolized substrates associated with the activity of several phase II enzymes, indicating expression of GST, GGT, beta-lyase and UGT. In addition, expression of beta-lyase apoprotein in freshly isolated PT cell-homogenates was detected by Western blot analysis (data not shown). These enzymes play an important role in either the nephrotoxicity of numerous GSH-conjugates (GST, GGT and beta-lyase) and/or are predominantly expressed in the proximal tubule (GGT, beta-lyase and UGT) [8]. Therefore, the measured activities of these enzymes provide further evidence for expression of proximal tubular specific differentiated functions. Rapid loss of CYP450 activity has also been reported previously in primary hepatocyte cultures: rat hepatocytes lose 50–80% of their CYP450 activity in the first 24 h of primary culture (for a review, see Paine [50]). Therefore, the time-dependent change in both phase I and II enzyme activities in PT cells during primary culture was determined. Metabolism of all marker substrates showed a strong time dependency. The decrease in CYP450 activity appeared to be isoform-dependent, as especially EROD (CYP1A1) and dextromethorphan hydroxylation (CYP2D) decreased rapidly, while caffeine demethylation and tolbutamide hydroxylation decreased less rapidly. In contrast, our results illustrate also that phase II biotransformation levels remain more stable than phase I biotransformation levels during primary PT cell culture. Similar observations have been reported previously for GST and 1-naphthol-UGT activity in rat hepatocyte cultures [51], [52]. In this study, we present the same observation for β-lyase activity. GGT activity, however, decreased continuously during culture. In contrast, Lash and coworkers [11] found a constant GGT activity in primary PT cells throughout 9 days of culture. Furthermore, the culture conditions, in particular serum deprivation following the initial attachment period, may have contributed to the decrease in enzyme activity [53]. The extent to which the culture conditions used in this study have influenced expression of differentiated functions of primary PT cells needs to be explored.