High salt intake reprioritizes osmolyte and energy metabolism for body fluid conservation

Abstract

Natriuretic regulation of extracellular fluid volume homeostasis includes suppression of the renin-angiotensin-aldosterone system, pressure natriuresis, and reduced renal nerve activity, actions that concomitantly increase urinary Na+ excretion and lead to increased urine volume. The resulting natriuresis-driven diuretic water loss is assumed to control the extracellular volume. Here, we have demonstrated that urine concentration, and therefore regulation of water conservation, is an important control system for urine formation and extracellular volume homeostasis in mice and humans across various levels of salt intake. We observed that the renal concentration mechanism couples natriuresis with correspondent renal water reabsorption, limits natriuretic osmotic diuresis, and results in concurrent extracellular volume conservation and concentration of salt excreted into urine. This water-conserving mechanism of dietary salt excretion relies on urea transporter-driven urea recycling by the kidneys and on urea production by liver and skeletal muscle. The energy-intense nature of hepatic and extrahepatic urea osmolyte production for renal water conservation requires reprioritization of energy and substrate metabolism in liver and skeletal muscle, resulting in hepatic ketogenesis and glucocorticoid-driven muscle catabolism, which are prevented by increasing food intake. This natriuretic-ureotelic, water-conserving principle relies on metabolism-driven extracellular volume control and is regulated by concerted liver, muscle, and renal actions.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Natriuretic-ureotelic generation of urine volume by surplus osmolyte and water excretion in mice and in humans.

(A) Relative contribution of 24-hour Na+ (2UNaV), K+ (2UKV), and urea (UUreaV) excretion to total 24-hour Na+, K+, and urea osmolyte excretion in mice on a LS diet (n = 6) or a HS diet with isotonic saline to drink (HS+saline; n = 8), and in 10 men consuming a 6-g/d or 12-g/d salt diet. Two-fold values of UNaV and UKV are given to account for their unmeasured accompanying anions. (B) Relationship among surplus 2Na+, 2K+, and urea osmolyte excretion (U2Na2KUreaV) and surplus water excretion in mice on a 0.1% NaCl diet with tap water (LS) (n = 6) or a 4% NaCl diet with 0.9% saline (HS+saline) (n = 8) for 2 consecutive weeks. (C) Relationship between surplus U2Na2KUreaV and surplus water excretion in all mice and human subjects presented in Table 1. (D) Relationship between surplus U2Na2KUreaV and FWC in the same mice and human subjects. (E) Relationship between surplus U2Na2KUreaV and water intake in the same mice and human subjects. We performed regression analysis in humans, and across the species. Mouse experiment 1: HS+saline study; mouse experiment 2: HS+tap study.

Figure 2. Glucocorticoid levels and water balance in mice and humans.

(A) Relationship between glucocorticoid (UGlucocorticoidV) levels and water excretion in the urine for all mice and human subjects presented in Table 1. (B) Relationship between glucocorticoid levels and water intake in the same mice. (C) Relationship between glucocorticoid levels and water balance in the same mice. Regression analysis was performed for humans and across the species. Mouse experiment 1: HS+saline study; mouse experiment 2: HS+tap study.

Figure 3. Renal urea accumulation, plasma Na+ and urea concentration, and plasma osmolality in response to experimental salt loading.

(A) Urea content in the renal medulla in mice that received less than 0.1% salt and tap water (LS; n = 6) or 4% salt and 0.9% saline (HS+saline; n = 7). (B) Representative UT-A1 and UT-A2 expression in the outer medulla and inner medulla of mice on a LS (n = 3), HS+saline (n = 3), or HS+saline+NOHA (n = 3) diet. (C) Quantification of UT-A1 and UT-A2 expression in the outer and inner medullae of mice on a LS (n = 8), HS+saline (n = 8), or HS+saline+NOHA (n = 8) diet. (D) Plasma urea concentration in mice on a LS (n = 14), HS+saline (n = 16), or HS+saline+NOHA (n = 14) diet. (E) Plasma Na+ concentration in mice on a LS (n = 12), HS+saline (n = 15), or HS+saline+NOHA (n = 14) diet. (F) Plasma osmolality in mice on a LS diet (n = 13), HS+saline diet (n = 16), or a HS+saline diet plus NOHA treatment (HS+saline+NOHA; n = 14). Data were determined by multivariate analysis (general linear model) and Bonferroni’s post-hoc subgroup comparisons.

Figure 4. Relationship among tissue urea content, tissue arginase activity, and plasma urea concentration.

(A) Relationship among renal medullary urea content, renal medullary arginase activity, and plasma urea concentration in mice fed a LS (n = 14), HS+saline (n = 15), or HS+saline+NOHA (n = 14) diet. (B) Relationship among liver urea content, liver arginase activity, and plasma urea concentration in the same mice. (C) Relationship among muscle urea content, muscle arginase activity, and plasma urea concentration in the same mice. Data were determined by linear regression, multivariate analysis (general linear model), and Bonferroni’s post-hoc subgroup comparisons.

Figure 5. Catabolic muscle wasting by experimental salt loading.

(A) RQ in mice fed a 0.1% NaCl chow (LS) (n = 8) or a 4% NaCl chow (n = 8) diet for 7 consecutive days. To test the effect of additional isotonic saline, mice received tap water for 2 days (HS+tap, orange), followed by isotonic saline (HS+saline, red). The activity period at night is shown in gray, and the inactivity period during the daytime is shown in white. Food intake (B) and body weight (C) over a 28-day period of ad libitum feeding, followed by 14 days of pair-feeding with a LS (n = 8) or HS+saline (n = 8) diet. (D) Upper panel: GR binding in the cytoplasm (CP), membrane (M), soluble nuclear fraction (SN), chromatin-bound GR (CB), and cytoskeletal (CS) GR in the subcellular fraction in skeletal muscle in mice fed a LS (n = 5) or HS+saline (n = 5) diet. Lower panel: Protein expression of LC3 in its cytosolic form (LC3-I) and as its LC-3-phosphatidylethanolamine conjugate (LC3-II) in the muscle of mice fed a LS (n = 4) or HS+saline (n = 4) diet. (E) Quantification of chromatin-bound GR protein expression and ratio of LC3-II/LC3-I protein expression in mice fed a LS (n = 5) or HS+saline (n = 5) diet. (F) Plasma corticosterone levels in mice fed a LS (n = 8) or HS+saline (n = 8) diet. (G) Relationship between changes (Δ) in body weight and muscle mass, as measured by magnetic resonance lean tissue mass, in mice fed a LS (n = 8) or HS+saline (n = 8) diet. (H) In vivo detection of LC3 expression (green) in skeletal muscle of LC3-GFP mice after pair-feeding on a LS or HS+saline diet. Data were determined by multivariate analysis of repeated measurements, by Student’s t test for independent samples, or by linear regression.

Figure 6. LC-MS/MS free amino acid analysis in skeletal muscle and liver in mice after pair-feeding.

Effect of HS+saline on free amino acid levels (reductions in blue; increases in green) after pair-feeding in mice fed a LS (n = 6) or HS+saline (n = 6) diet. Data were analyzed by Student’s t test for independent samples.

Figure 7. LC-MS/MS metabolite analysis in liver and skeletal muscle of mice after pair-feeding.

(A) Effect on key metabolites of energy metabolism (AMP and ADP), the TCA cycle, ketone body formation, fatty acid oxidation, glycogen storage, glycolysis/gluconeogenesis, the alanine-glucose-nitrogen shuttle, and the urea cycle in muscle and liver in pair-fed mice given a LS (n = 6) or HS+saline (n = 6) diet (reductions in blue; increases in green). (B) Protein expression of OAT and the Na+-alanine cotransporters SLC38A1 and SLC38A2 in the same mice. (C) Protein expression of phosphorylated and unphosphorylated AMPK ACC in the same mice. Expression of GAPDH (37 kDa) and β-actin (42 kDa) proteins served as a loading control. Data were analyzed by Student’s t test for independent samples.

Figure 8. Cardiovascular responses to dietary salt loading.

Dietary salt levels were as follows: black = LS diet; orange = 4% NaCl diet with tap water (HS+tap); red = 4% NaCl diet with additional isotonic saline (HS+saline); green = 4% NaCl diet with isotonic saline and calorie intake restriction (HS+saline, pair-fed). (A) Locomotor activity, heart rate, and SBP in 6 mice over a 21-day period. (B) R-R interval distribution in the same mice after 1 (Day 9) and 4 (Day 12) days of a 4% NaCl diet with isotonic saline. (C) Relationship between heart rate (bpm) and SBP (mmHg) in the same mice. (D) Mean arterial BP (MAP) in acutely restrained mice that were fed a LS (n = 20) or HS+saline (n = 18) diet ad libitum for 2 weeks. *P < 0.05 and **P < 0.01. P(diet): effect of dietary intervention versus LS level; P(day): effect of intervention day; P(inter): interaction between dietary intervention and day of intervention. Data were determined by multivariate analysis of repeated measurements, Student’s t test for paired samples, Student’s t test for independent samples, and by linear regression.