Mapping the Transcriptome Underpinning Acute Corticosteroid Action within the Cortical Collecting Duct

Abstract

Key Points:

1. We report the transcriptomes associated with acute corticosteroid regulation of ENaC activity in polarized mCCD_cl1 collecting duct cells.

2. Nine genes were regulated by aldosterone (ALDO), 0 with corticosterone alone, and 151 with corticosterone when 11βHSD2 activity was inhibited.

3. We validated three novel ALDO-induced genes, Rasd1, Sult1d1, and Gm43305, in primary cells isolated from a novel principal cell reporter mouse.

Background: Corticosteroids regulate distal nephron and collecting duct (CD) Na⁺ reabsorption, contributing to fluid-volume and blood pressure homeostasis. The transcriptional landscape underpinning the acute stimulation of the epithelial sodium channel (ENaC) by physiological concentrations of corticosteroids remains unclear.

Methods: Transcriptomic profiles underlying corticosteroid-stimulated ENaC activity in polarized mCCD_cl1 cells were generated by coupling electrophysiological measurements of amiloride-sensitive currents with RNAseq. Generation of a principal cell-specific reporter mouse line, mT/mG-Aqp2Cre, enabled isolation of primary CD principal cells by FACS, and ENaC activity was measured in cultured primary cells after acute application of corticosteroids. Expression of target genes was assessed by qRT-PCR in cultured cells or freshly isolated cells after the acute elevation of steroid hormones in mT/mG-Aqp2Cre mice.

Results: Physiological relevance of the mCCD_cl1 model was confirmed with aldosterone (ALDO)-specific stimulation of SGK1 and ENaC activity. Corticosterone (CORT) only modulated these responses at supraphysiological concentrations or when 11βHSD2 was inhibited. When 11βHSD2 protection was intact, CORT caused no significant change in transcripts. We identified a small number of ALDO-induced transcripts associated with stimulated ENaC activity in mCCD_cl1 cells and a much larger number with CORT in the absence of 11βHSD2 activity. Principal cells isolated from mT/mG-Aqp2Cre mice were validated and assessment of identified ALDO-induced genes revealed that Sgk1, Zbtbt16, Sult1d1, Rasd1, and Gm43305 are acutely upregulated by corticosteroids both in vitro and in vivo.

Conclusions: This study reports the transcriptome of mCCD_cl1 cells and identifies a small number of ALDO-induced genes associated with acute stimulation of ENaC, including three previously undescribed genes.

Graphical abstract

Figure 1

Effects of acute corticosteroids on ENaC-mediated transport and SGK1 activity and expression in mCCD_cl1 cells. Amiloride-sensitive currents measured from polarized mCCD_cl1 cells after 3 hours (I_ami-3h) exposure to increasing concentrations of corticosterone (Ai) or aldosterone (Bi) in the absence (−CBX, solid line) or presence (+CBX, dashed line) of CBX (10 µM, 30 minutes preincubation) to inhibit 11βHSD2 activity. H₂O was used as vehicle control for CBX, and amiloride (10 µM, 10 minutes) was added after corticosteroid treatment. The activity (Aii) and expression (Aiii) of SGK1 was determined by measuring the abundance of P-NDRG1 or T-SGK1, respectively, in cell lysates after corticosteroid treatment and electrophysiological measurements. These are expressed relative to T-NDRG1 and β-actin, respectively. Upper panels show representative blots, and lower panels show densitometric data expressed as a fold change from the control (0) lane (log₁₀). Data are shown as mean ±95% confidence interval (95% CI) (n=7). Statistical significance was determined by a two-way ANOVA for the electrophysiological data, with a Dunnett's post hoc test, and a one-way ANOVA for Western blot data, because of separate blots for − and + CBX, with a Dunnett's post hoc test. CORT, corticosterone; CBX, carbenoxolone.

Figure 2

Differentially expressed transcripts in corticosteroid-treated mCCD_cl1 cells. (Ai) I_eq measured from cells treated with either solvent vehicle (Veh), 3 nM Aldo, or 100 nM Cort for 3 hours; arrow indicates addition of corticosteroid. An additional CORT group was pretreated CBX (10 µM, 30 minutes), all other groups received vehicle control for this period. Amiloride (10 µM) was added to the apical bath for a final 10 minutes, and Aii shows the amiloride-sensitive current (I_ami-3h). Data are shown as mean ±95% confidence interval (CI) (left panel) and as individual points and mean±95% CI (right panel). Statistical significance was determined by one-way ANOVA with a Tukey's post hoc test used to compare groups to vehicle control, **P<0.01 and ***P<0.001. (B) Differentially expressed transcripts are plotted as log₂ fold change versus −log₁₀ false discovery rate (FDR). The horizontal dashed line represents the specified FDR threshold (0.05) and the vertical dashed lines indicate the specified fold change threshold (2) in both positive and negative directions. Each treatment, (Bi) ALDO, (Bii) CORT or (Biii) CBX+CORT, is compared with vehicle-treated control. Points passing only the FDR threshold are shown in green, passing only the fold change threshold are shown in blue and those passing both thresholds are shown in red. Individual transcripts passing both thresholds in ALDO-treated cells are labeled.

Figure 3

tdTom and GFP expression in kidney sections from mT/mG-Aqp2Cre mice. Longitudinal sections of fixed kidneys from adult mT/mG^+/+-Aqp2Cre^−/− mice (A) and mT/mG^+/+-Aqp2Cre^+/− mice (B) showing tdTom and GFP labeling. (i) Tiled images taken with ×40 objective across the section with 488-nm (gray, autofluorescence to show tissue morphology; green, GFP signal) and 555-nm (red, tdTomato signal) excitation light. (ii) Cortex and medullary regions are shown at higher magnification, regions of interest denoted in (i) by cyan and yellow boxes, respectively. (iii) Individual 555-nm and 488-nm channels for (ii) are shown. Scale bars: (i) 500 µm and (ii–iii) 50 µm.

Figure 4

Effects of acute addition of CORT on ENaC-mediated transport and SGK1 activity and expression in cultured primary principal cells. (i) Amiloride-sensitive currents were determined in primary principal cells after 3h (I_ami-3h) exposure to increasing concentrations of CORT in the absence (−CBX) or presence (+CBX) of CBX (10 µM, 30 minutes preincubation) to inhibit 11βHSD2 activity. H₂O was used as vehicle control for CBX, and amiloride (10 µM, 10 minutes) was added after corticosteroid treatment. (ii) The activity of SGK1 was determined by measuring the abundance of P-NDRG1 in cell lysates after corticosteroid treatment and electrophysiological measurements, expressed relative to T-NDRG1. Upper panels show representative blots, and lower panels show densitometric data expressed as a fold change from the control (0) lane (log₁₀). Data are shown as mean±95% confidence interval (CI) (n=7). Statistical significance was determined by a two-way ANOVA for the electrophysiological data and a one-way ANOVA for Western blot data, because of separate blots for − and + CBX, with a Dunnet's post hoc test, ***P<0.001.

Figure 5

Corticosteroids stimulate ENaC-mediated Na⁺ transport in cultured primary principal cells. I_eq was measured across polarized monolayers of primary principal cells after treatment with corticosteroids. (A) Cells were preincubated with H₂O (−CBX, left) or 10 µM CBX (+CBX, right) for 30 minutes. Cort (10 nM, dashed line) or solvent vehicle (solid line) was subsequently added for 3 hours; arrow denotes addition. (B) Aldo (3 nM, dashed line) and (C) DEX (100 nM, dashed line), or respective solvent vehicle (solid line), were added to cells for 3 hours. In all experiments, amiloride (10 µM) was subsequently applied for 10 minutes. Data shown in traces (Ai, Bi, Ci) are mean I_eq±95% confidence interval (CI) and in bar graphs (Aii, Bii, Cii) as individual points and mean I_ami-_3h±95% CI (n=8). Statistical significance in (A) was determined by two-way ANOVA and Tukey's post hoc test and in (B) and (C) by unpaired t test, ***P<0.001.

Figure 6

Expression of identified corticosteroid-induced transcripts in primary principal cells treated with CORT. Cells were treated with CBX or solvent vehicle for 30 minutes before addition of Cort (10 nM) or solvent vehicle for a further 3 hours. Amiloride (10 µM) was added for a final 10 minutes. Transcript expression of GOI is relative to the average expression of reference genes (log₁₀): Actb1 and Hprt. Data are shown as individual points and mean ±95% confidence interval (CI), and GOI is indicated in bold italics above each graph. Statistical significance was determined by one-way ANOVA with Tukey's post hoc test or Kruskal-Wallis test with Dunn's post hoc test, where appropriate: *P<0.05, **P<0.01, and ***P<0.001.

Figure 7

Expression of identified corticosteroid-induced transcripts in primary principal cells treated with ALDO or DEX. Polarized primary principal cells were treated with either (A) Aldo (3 nM) or (B) Dex (100 nM) for 3 hours. Amiloride (10 µM) was added for a final 10 minutes. Transcript expression of GOI is relative to the average expression of reference genes (log₁₀): Rn18S and Tbp (Aldo) or Rn18s, Tbp, and Hprt (Dex). Data are shown as individual points and mean ±95% confidence interval (CI) and GOI is indicated in bold italics above each graph. Statistical significance was determined by unpaired t test or Mann-Whitney test, where appropriate: **P<0.01 and ***P<0.001.

Figure 8

Steroid-induced transcript expression in isolated primary principal cells after acute injection of corticosteroids. Expression of selected target genes were quantified in cells isolated from mT/mG-Aqp2Cre mice 3 hours after ip injection of solvent vehicle (Ctl), Aldo, Cort, after 8 days ad libitum access to H₂O. Two further groups were concomitantly treated with CBX (2.5 mg/kg BW per day by mouth) for 8 days with subsequent ip injection of solvent vehicle (CBX) or CORT (CBX+Cort) for 3 hours. Target genes were selected from ALDO-induced genes identified in the RNA sequencing dataset. Transcript expression of GOI is relative to the average expression of reference genes (log₁₀): Rn18S and Tbp. Data are shown as individual points and mean ±95% confidence interval (CI), and GOI is indicated in bold italics above each graph. Statistical significance was determined by one-way ANOVA with Tukey's post hoc test or Kruskal-Wallis test with Dunn's post hoc test, where appropriate: *P<0.05, **P<0.01, and ***P<0.001.