Background: The kidney is responsible for the long-term control of blood pressure (BP) and this is achieved by maintaining total body Na⁺ balance. The kidneys match Na⁺ excretion with Na⁺ intake with fine-tuning of Na⁺ balance occurring in the collecting duct (CD). In response to decreased BP, the key volume-regulating corticosteroid aldosterone (ALDO) increases Na⁺ reabsorption via the epithelial sodium channel (ENaC) by upregulating its expression and activity, driving fluid reabsorption and thus normalising BP. ALDO exerts its effects specifically in principal cells (PC) of the CD due to expression of the enzyme 11βHSD2 which inactivates the related hormone cortisol (CORT). Corticosteroids are typically thought to passively diffuse into target cells owing to their lipophilicity. However, preliminary studies in the lab have provided the first evidence that CORT may be actively transported in the CD. This study aimed to determine if ALDO is also actively transported in PCs and to identify which transporters may be responsible.

 

Methods: Murine cortical collecting duct cells (mCCD_cl1) were cultured on permeable supports for 9 days [1]. Cells were treated with ALDO (3nM, 3h) in the basolateral bath. Amiloride-sensitive ENaC-mediated Na⁺ transport was assessed by measuring the equivalent short-circuit current. Samples of media were collected from the apical and basolateral baths and the monolayer lysed in 0.1% SDS. ALDO concentration was determined by solid-liquid phase (SLE) extraction and targeted LC-MS/MS analysis using an assay optimised to detect physiological concentrations of ALDO. The apical membrane was isolated from polarised mCCD_cl1 cells by biotin labelling and streptavidin pulldown. Isolation was validated by western blot using antibodies against streptavidin and α-ENaC. Unbiased proteomic analysis of the apical fraction was carried out by LC-MS/MS enabling assessment of changes in protein expression in response to ALDO.

 

Results:

The stimulatory effect of ALDO on ENaC activity was confirmed where ALDO increased I_eq by ‑3.0±1.3 μA∙cm^-2 after 3h compared to control, where the change in I_eq was 0.1±0.2 μA∙cm^-2. Interestingly, after 3h, ALDO was unevenly distributed across the monolayer of cells. 16.5±2.8% was detected in the apical bath, 72.3±10.2% detected in the basolateral bath, and a small proportion 2.7±1.1% in cell lysates (n=6-9).

Following separation of the apical membrane from the remaining protein lysate (supernatant) in vehicle or ALDO-treated cells, streptavidin was detected in the apical fraction but not the supernatant, confirming successful isolation (n=2). α-ENaC was also detected in both fractions, the presence in the apical fraction correlating with functional channel activity. Subsequent proteomic analysis of the apical membrane detected a total of 3188 proteins. Changes in expression of 9 members of the ABC and SLC transporter superfamilies was seen in response to ALDO treatment; 6 up-regulated and 3 down-regulated.

 

Conclusions: Together these data suggest an active mechanism contributes to ALDO transport into and/or out of PCs of the CD. This has important implications for the control of ALDO bioavailability within target PCs, and may present a possible therapeutic target. Ongoing work is being carried out to interrogate which transporter(s) are responsible.