Corticosteroids are volume-regulating hormones which modulate Na+ reabsorption via the epithelial sodium channel (ENaC) in principal cells (PCs) of the collecting duct (CD). Classically, these lipophilic hormones are thought to passively diffuse into target cells, however there is evidence that mediated transport can occur via transporters including the ABC and OCT/OAT superfamilies [1]. This has been shown in several tissues including adrenal glands, brain and adipose tissue [2,3] and may reflect a mechanism underpinning intracellular bioavailability. The role of mediated transport in the CD remains unclear. Recent transcriptomic analysis of corticosteroid-treated murine mCCDcl1 collecting duct cells [4] identified several transporters with modulated expression including: Abce1, Abcc1, Abca2, Slc22a5. The aim of this study was to determine if corticosteroid hormones are actively transported in a cellular model of the CD and to determine localisation of putative transporters in mouse kidney.
mCCDcl1 cells [5] were cultured on permeable supports for 9-11 days to form polarised, transporting monolayers. Steroid hormone concentration in media samples was measured following solid-liquid phase extraction and LC-MS/MS analysis, data are mean±SD. Immunofluorescence labelling of putative transporters was carried out on transverse sections of wild-type male and female mouse (C57BL/6) kidneys with antibodies against SLC22A5, ABCA2, ABCE1, ABCC1, as well as 11ßHSD2 as a marker of PCs of the CD. Sections were imaged using a Zeiss AxioScan slidescanner.
mCCDcl1 cells were treated with corticosterone (CORT, 150nM, 3h, basolateral bath) following preincubation with carbenoxlone (10µM, 30min) to inhibit 11ßHSD2 activity. Previous work has shown that this treatment causes a robust stimulation of ENaC activity [4]. Media collected at time 0, revealed [CORT] was 144.7±9.9nM in the basolateral bath and below the limit of detection (BLD) in the apical bath. After 3h30, [CORT] was 100.0±3.5nM and 43.2±1.3nM in the basolateral and apical baths, respectively, and BLD in the cell lysate (n=8). Assessment of putative steroid transporters in murine kidney sections (n=3), revealed SLC22A5 and ABCA2 colocalised with 11bHSD2. SLC22A5 localised to the apical membrane of tubular cells, whereas ABCA2 showed nuclear expression. ABCE1 was localised to tubules expressing 11ßHSD2 cells, but in adjacent 11bHSD2 negative cells – likely intercalated cells of the CD. Finally, ABCC1 did not co-localise to tubules expressing 11bHSD2 in either male or female wild-type kidney sections. Further co-labelling studies were carried out and revealed ABCC1 partially co-localised with NaPi-2a, a marker of the proximal tubule.
Together these data provide evidence that mediated transport of a physiological concentration of CORT occurs across the apical membrane of polarised CD epithelia. The lack of detectable CORT in cell lysates suggests this likely occurs across the basolateral membrane as well, though further experiments are required. Of the identified putative transporters, only SLC22A5 and ABCA2 localised to PCs of the CD in mouse kidney, the apical localisation of SLC22A5 suggests this transporter may play a role in steroid transport. Further work aims to assess the localisation of these transporters following manoeuvres to alter plasma concentrations of corticosteroids, as well as assessing the potential for transport of physiological concentrations of aldosterone.