In this study we examined whether
In this study, we examined whether zebrafish ionocytes might also share other types of similarity in transport function with kidney cells. In addition to ion transport, mammalian kidney Torin 2 also play key roles in xenobiotic elimination, through the action of ATP-Binding Cassette (ABC) transporters (Giacomini et al., 2010). ABC transporters with xenobiotic transport (XT) activity typically are associated with one of three subfamilies: ABCB (P-glycoprotein [P-gp]), ABCC (multidrug resistance protein [MRP]), and ABCG (breast cancer resistance protein [BCRP]) (Dean et al., 2001). These efflux transporters are highly enriched not only in the kidney but also at other major barrier epithelia including the liver, intestine, and blood-brain barrier (Giacomini et al., 2010; Leslie et al., 2005). They have also been demonstrated to be important for xenobiotic defense in aquatic organisms (Smital and Sauerborn, 2002; Hamdoun et al., 2004; Epel et al., 2008) including zebrafish. Functional assays of MRP-like and P-gp-like activities have previously been employed in zebrafish embryos as well as an embryonic cell line (Long et al., 2011; Fischer et al., 2013; Tian et al., 2017).
The specific goal of this study was to determine whether embryonic zebrafish ionocytes have ABC transporter-mediated xenobiotic efflux activity, similar to that observed in mammalian renal cells. Applying a well-characterized method for examining ABC transporter efflux activity (Epel et al., 2008), we used fluorescent small molecule substrates of the xenobiotic transporters (reviewed in Gökirmak et al., 2014) and confocal microscopy to characterize cell type specific patterns of efflux and uptake. The results were supported by analysis of an existing single-cell RNA sequencing dataset, indicating that an ABCB/P-gp-like gene, is enriched in the ionocytes. Collectively, the results demonstrate the presence of xenobiotic transporter activity in zebrafish ionocytes and lay the groundwork for future investigation of the role of these cells in protection of the embryo.
Materials and methods
Discussion In this study, we examined accumulation and efflux patterns of ABC transporter substrates in the zebrafish embryonic epidermis using confocal microscopy. The power of this approach is the ability to assign transporter activity to specific subtypes of cells within the embryo. Consistent with what has been previously reported for CAM and RhB (Fischer et al., 2013), one of the most striking patterns observed with these molecules is the selective retention of some small fluorescent molecules (such as calcein) in ionocytes, with the contrasting absence of accumualtion of other molecules (eg. RhB) in these same cells. Here, we showed here that this selective retention is not simply an artifact of how the substrates are used, such as differences in concentration (Fig. 2). We also considered whether distribution differences between dye were due to the physical characteristics of the substrates or due to unique features of the ionocyte itself. One of these alternatives we considered is that RhB could end up in the yolk simply because it is more lipophilic than calcein. However, we did not observe DiOC6 in yolk even though it is also lipophilic. In addition both FDA and CMFDA were detected in the yolk region, both of which are more hydrophilic than RhB (Fig. 1). Another alternative explanation for these observations could be that higher accumulation of AM dyes is the result of a higher level of esterase activity in HR cells, but esterase activity is typically not rate-limiting (Cole et al., 2013) and DiOC6, which is not cleaved by esterases, also accumulated in the ionocytes in a transporter-inhibitor sensitive manner. Thus, our results indicate that active uptake and efflux mechanisms in the ionocyte are the most plausible explanation for the observed susbtrate accumaultion patterns. This study sheds some preliminary light on the potential nature of the efflux mechanism in ionocytes. Zebrafish have at least 11 ABC transporter homologs that could participate in drug efflux (Annilo et al., 2006; Luckenbach et al., 2014). Homologs of P-gp (ABCB1) and MRP (ABCC1/C2) account for the major transport activities in most embryos studied (Toomey and Epel, 1993; Hamdoun and Epel, 2007; Epel et al., 2008). Consistent with this expectation, inhibitors of P-gp caused significant increases in accumulation of CAM and DiOC6, which are P-gp transporter substrates, in the ionocytes (Fig. 5A, B). Similarly, the accumulation of BCECF-AM, typically considered an MRP substrate, was increased by addition of MK571 (Fig. 5C).