High salt intake activates the hypothalamic–pituitary–adrenal axis, amplifies the stress response, and alters tissue glucocorticoid exposure in mice

Figures

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  Figure 1. Activation of HPA axis after high salt intake. (A) Plasma corticosterone measured at 7am (diurnal nadir) and 7pm (diurnal peak) in male C57BL/6 mice (n = 8) first on a 0.3% Na diet (control, open circles) and again after 14 days on 3% Na diet (high salt; closed circles). Individual values are shown with group mean ± SD; statistical comparison was two-way repeated measures ANOVA for the main effects of time of day (P < 0.0001) and diet (P = 0.071) and the interaction (P = 0.016); P-values for planned comparisons (Holm–Sidak) are given. (B) The effect of salt diet on diurnal peak plasma corticosterone was measured in separate groups of mice fed either control (open circles) or high salt diet (closed circles) for 3 days (n = 4/5), 1 week (n = 8/8), 3 weeks (n = 8/8) or 8 weeks (n = 8/8) of either. Individual values are shown with group mean ± SD; statistical comparison was two-way ANOVA for the main effects of ‘diet’ (P = 0.476) and ‘duration’ (P < 0.0001) and the interaction (P < 0.0001); P-values for planned comparisons (Holm–Sidak) are shown. (C) Schematic highlighting key components of HPA axis activation that were then measured at mRNA level in tissue taken from different…

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  Figure 2. High salt intake amplifies the stress response. (A) protocol for measuring the stress response in male C57BL/6 mice (n = 20) fed 0.3% Na diet (control; open circles) and again after 2 weeks on a 3% Na diet (high salt; closed circles). (B) Plasma corticosterone in samples taken at 8am (baseline) and again after 15 min of tube restraint (Stress). Analysis was two-way ANOVA with repeated measures for the main effects of ‘stress-response’ (P < 0.0001) and ‘diet’ (P = 0.0006) and the interaction (P = 0.0014); P-values for planned comparisons (Holm–Sidak) are given. (C) The paired peak stress response in each mouse (n = 20), analysed by paired t-test. (D) The corticosterone recovery from stress was measured at either 30 (n = 6/6), 60 (n = 8/7) or 90 minutes (n = 5/6) following release from the restraint tube. Analysis was two-way ANOVA with repeated measures for the main effects of ‘time’ and ‘diet’ and the interaction; P-values for planned comparisons (Holm–Sidak) are given. Individual measurements are shown with group mean ± SD.

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  Figure 3. Effect of high salt intake on corticosterone-binding globulin. Male C57BL/6 mice were fed either 0.3% (control; open circles) or 3% (high salt; closed circles), diet for 14 days and blood taken for measurement of (A) Corticosterone-binding globulin binding capacity (n = 7/7). (B) mRNA abundance of SerpinA6, encoding CBG, was measured in liver cDNA (n = 6/8). Expression was normalized to that of reference genes Gapdh and Tbp and log-transformed. Individual measurements are shown with group mean ± SD; statistical comparisons were made with Student’s unpaired t-test and two-tailed P-values are given.

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  Figure 4. Effect of high salt intake on mRNA abundance of Fkbp5, Nr3c1 and Nr3c2 in brain regions. Male C57BL/6 mice were fed either 0.3% (control; open circles; n = 8) or 3% (high salt; closed circles; n = 8), diet for 14 days and tissues taken at cull. Excluding samples that failed to yield sufficient material for analysis, mRNA abundance of FK506-binding protein 5 (Fkbp5), glucocorticoid receptor (Nr3c1) and mineralocorticoid receptor (Nr3c2) was measured in hippocampus (n = 5/8), hypothalamus (n = 5/8) and anterior pituitary (n = 5/6) Values were normalized to the expression of reference genes Gapdh and Tbp and log-transformed. Individual measurements for samples that passed QC are shown, along with group mean ± SD. Statistical comparisons were made with Student’s unpaired t-test and P-values are given.

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  Figure 5. Effect of high salt intake on fluid volume markers. Male C57BL/6 mice were fed 0.3% (Control; open circles n = 8) diet and then 3% (high salt; closed circles n = 8) diet for 14 days. (A) Food intake and body weight; (B) urine excretion of sodium, potassium, chloride and urea; (C) water turnover; (D) free water clearance and (E) plasma osmolarity. The following measurements were made from samples collected from other experiments: (F) mRNA abundance of tonicity-responsive enhancer-binding protein (Tonebp) in the hypothalamus; (G) plasma copeptin; (H) mRNA abundance of vasopressin V2 receptor (Avpr2) in kidney cortex and (I) mRNA abundance of vasopressin V1b receptor (Avpr1b) in anterior pituitary. Individual measurements are shown with group mean ± SD; statistical comparisons were made with Student’s unpaired t-test and two-tailed P-values are given.