Interrogation of Novel Aldosterone‐induced Genes Associated With Acute Stimulation Of ENaC In Primary Collecting Duct Cells
Authors: Loughlin Struan R. , Bailey Matthew A. , Mansley Morag K. 
Published: May 2022

Aldosterone (ALDO) is a key volume‐regulating hormone which promotes Na+ reabsorption in the distal nephron. ALDO sensitivity is conferred by the enzyme 11βHSD2 which breaks down the related more abundant steroid, cortisol. Mutations in Hsd11b2 give rise to the syndrome of apparent mineralocorticoid excess, where hypertension is associated with excessive renal Na+ reabsorption. In the distal nephron corticosteroid‐induced Na+ transport, via the epithelial Na+ channel ENaC, is due to transcriptional processes and previous studies have identified key genes including Sgk1. Our lab carried out transcriptomic analysis of mCCDcl1 cells following acute corticosteroid induced ENaC‐mediated Na+ transport, identifying several novel ALDO induced genes. The present study aimed to elucidate the effects of corticosteroids on these target genes in the collecting duct (CD), utilising an in vitro and in vivo approach.

A CD‐specific reporter mouse was generated using Aqp2Cre to express eGFP. Primary cells were isolated by FACS and either grown in culture or RNA was extracted immediately. Cells were grown on filter membranes to allow development of polarised monolayers and electrometric properties were assessed using an epithelial volt‐ohm‐meter. Target gene expression was determined by qRT‐PCR.

Assessment of nephron segment‐specific gene expression revealed an enrichment of genes encoding α‐ENaC, ROMK and 11βHSD2 but not NHE3 or NKCC2. NCC was detected but parvalbumin was absent, demonstrating isolation of cells from the late distal tubule onwards. In addition to genes associated with principal cells we also detected genes encoding pendrin and AE1, indicative of intercalated cells therefore this population reflects CD cells. When grown in culture for 9‐11 days, these cells exhibited Vt and Rt of ‑20.3±3.1 mV and 5.1 ±0.1 kΩ∙cm2, respectively. This gave rise to an Ieq of ‑3.9±0.6 μA·cm‑2 which was near abolished with 10 µM amiloride – indicating Na+ transport via ENaC.

Acute application of steroid hormones to cells for 3h: ALDO (3 nM), DEX (100 nM) and CORT (10 nM) – the latter only when 11βHSD2 activity was inhibited using carbenoxolone (CBX, 10 µM) – stimulated amiloride‐sensitive currents by 2.0‐, 3.6‐ and 2.4‐fold, respectively. Analysis of ALDO‐induced genes revealed both ALDO and CORT upregulated Sgk1, Zbtb16 and Rasd1. ALDO also upregulated Sult1d1 whereas CORT also upregulated an unannotated gene Gm43305. DEX upregulated all of these as well as 2 additional genes previously detected: Defb1 and Gm16178.

To determine the relevance of these findings in vivo reporter mice were administered ALDO, vehicle or CORT, by ip injection, the latter under control conditions or following CBX treatment for 8 days. Cells were isolated after 3h. The previously identified Zbtb16, Rasd1, Gm43305 and Sult1d1 were upregulated in both ALDO and CBX+CORT groups, though no change was detected in either Sgk1 or Defb1. Scnn1a was also upregulated in both groups and additionally Per1 and Sult1a1 were upregulated in mice treated with CBX‐CORT.

Together these data confirm 4 novel corticosteroid‐induced genes in primary CD cells associated with acute stimulation of ENaC activity.