The aldosterone (ALDO)-sensitive distal nephron is typically defined as: the late distal convoluted tubule (DCT), connecting tubule (CNT) and cortical collecting duct (CD). ALDO-sensitivity is defined by 11βHSD2 (11β) activity (which inactivates corticosterone, CORT); mineralocorticoid (MR) rather than glucocorticoid receptor (GR) expression; and the target epithelial sodium channel (ENaC). Recent studies have reported ALDO-insensitive, but MR-dependent ENaC activity in DCT2/CNT [1,2] and that CORT-stimulated ENaC may be relevant in MR antagonism as 4th line anti-hypertensive therapy [3]. Whilst functional evidence of ALDO-insensitive ENaC activity is compelling, corticosteroid-regulated Na+ transport along the distal nephron is not fully defined. AIM: To develop a method enabling an unbiased measurement of 11βHSD2, MR and GR expression alongside Na+ transporter localisation in murine distal nephron.
Male C57BL/6 mouse kidney sections were subject to multiplex immunofluorescence (11β, MR and GR) and immunohistochemical labelling (NCC, TRPV5, γ-ENaC and AQP2), Hoechst was used as a nuclear counterstain with the former. All primary antibodies were recognised by either anti-rabbit or anti-mouse polymer HRP and visualised either by tyramide signal amplification with associated fluorophores (Fluorescein, Cy3 or Cy5) for multiplex IF, or respective chromagens (red, green, blue, brown) for multiplex IHC. Images were co-registered, annotated and analysed using Halo® software. At least 2 users annotated each kidney section. Data are shown as mean±SD.
Distal nephron segments were defined by the presence of transporter/channel: DCT – NCC; DCT/CNT ‑ NCC and TRPV5; CNT – TRPV5; CNT/CCD – TRPV5 and γ-ENaC/AQP2; CCD – γ-ENaC/AQP2. We did not detect NCC and γ-ENaC co-localisation. Average cells/section: DCT – 1165±131; DCT/CNT ‑ 329±81; CNT – 242±50; CNT/CCD – 878±246; CCD – 2247±341 (n=3). 2 additional annotations were made as controls: 11β/MR negative and GR negative cells where at least 100 cells of each criteria were identified from each section. Raw signal intensity for 11β, MR and GR were plotted and thresholds for a positive signal were set using the negatively labelled cells, for each section analysed.
DCT was predominantly 11β- (~80%) and GR dominant (GR:MR ratio ~8:1). In 11β+ cells (~20%), GR was also dominant (GR:MR ratio ~7:1). DCT/CNT and CNT were mainly 11β- (~75 and ~60%, respectively), where MR and GR were similar. In 11β+ cells, MR and GR were similar in DCT/CNT but MR was greater in CNT (MR:GR ratio ~4:1). CNT/CCD contained a greater proportion of 11β+ (~65%) cells. MR and GR were similar in 11β- cells, but 11β+ cells were almost exclusively MR (MR:GR ratio ~12:1). In CCD, ~30% of cells were 11β-, GR were greater (GR:MR ratio ~4:1), and ~70% cells were 11β+, these were predominantly MR (MR:GR ratio ~15:1).
This methodology has enabled an unbiased assessment of the molecular machinery underpinning corticosteroid-regulated Na+ transport in the distal nephron. Our data suggest that the DCT is a glucocorticoid-responsive segment, with 11β+ cells becoming progressively more dominant from CNT to CCD. The appearance of ENaC in the late CNT and particularly CCD, alongside 11β and MR, suggest these segments reflect the mineralocorticoid-responsive, and thus aldosterone-sensitive distal nephron.