Surplus osmolyte and water excretion by urine concentration in mice and humans. We fed C57/BL6 mice either a low-salt diet (0.1% NaCl chow and tap water to drink, referred to herein as LS) or a high-salt diet (4% NaCl chow and 0.9% saline water to drink; referred to herein as HS+saline) for 2 consecutive weeks and studied their 24-hour water balance in metabolic cages. To prevent artificial osmolyte contamination of the urine, the mice received their assigned fluids without food during the 24-hour urine collection period. We also measured the animals’ food and fluid intake in normal cages in the 24-hour period before their transfer to the metabolic cages. In the normal cages, daily average food intake was 154 ± 22 g/kg body weight, corresponding to 12 kg in an 80-kg human. The average fluid intake was 506 ± 281 ml/kg, corresponding to 40 liters in an 80-kg human. The HS+saline diet not only increased fluid intake by 3.1-fold (714 ± 170 ml/kg versus 228 ± 48 ml/kg; P < 0.001), but also led to a 1.2-fold increase in food intake (165 ± 21 g/kg versus 140 ± 15 g/kg; P < 0.05). The large nutrient intake suggests that not only surplus Na+ but also large amounts of K+ and urea osmolytes were excreted together with surplus water.

Food and water intake of the previous day dominated fluid intake behavior and osmolyte and water excretion in the subsequent 24-hour metabolic cage studies. It was not fluid intake on the same day, but fluid intake on the previous day that determined urine volume formation in the metabolic cage experiments (Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/JCI88532DS1). Furthermore, the higher fluid intake in the HS+saline mice in the normal cage determined lower fluid intake in the same mice during the subsequent metabolic cage experiment (Supplemental Figure 1C). This state of affairs confirmed the hypothesis that the surplus body fluid generated earlier was excreted in the metabolic cage experiments.

Despite a 10-fold increase in urine Na+ excretion (UNaV), HS+saline mice showed no significant increase in total osmolyte excretion (Table 1). Urea was the dominating urine osmolyte, being 40-fold higher than urine Na+ in LS diet–fed mice and 4-fold higher than urine Na+ in HS+saline diet–fed mice. The sum of 2Na+ (2-fold to account for accompanying anions), 2K+ (2-fold to account for accompanying anions), and urea excretion (U2Na2KUreaV; mmol/kg/d) was dependent on the previous day’s food intake (Supplemental Figure 2), indicating that Na+ salts, K+ salts, and predominantly protein intake determined surplus urine osmolyte excretion in mice in the metabolic cage 24 hours later.

Table 1 Effect of increasing salt intake on urine osmolyte, volume, and glucocorticoid-hormone excretion in balance studies in mice and humans

Despite a massive difference in UNaV, neither LS- nor HS+saline-treated mice showed a significant increase in urine volume (Table 1), indicating that surplus water excretion was not proportional to surplus Na+ excretion. This disproportional Na+ and water excretion was explained by the finding that HS+saline mice showed an 8-fold increase in their urine Na+ concentration. Excretion of surplus dietary salt and water thus occurred within the physiological regulatory pattern of the renal concentration mechanism. In the renal excretion process, proportional amounts of osmolytes and water are first filtered from the blood plasma into the primary urine. The hyperosmolal microenvironment in the renal interstitium then drives water reabsorption and, thereby, urine concentration. This antidromic movement of osmolytes and water can be assessed by calculating the negative free water clearance (FWC), which is an estimate of renal tubular water reabsorption in relation to osmolyte excretion. The average FWC in LS and HS+saline mice was –162 ml/kg/d (Table 1), which would correspond to water conservation of approximately 13 l/d by renal osmolyte free water reabsorption in an 80-kg human.

Dietary salt excretion did not change urine osmolality or FWC, because the 25.7% increase in urine 2Na+ concentration was balanced by a 11.7% reduction in urine 2K+ concentration and a 13.9% reduction in urine urea concentration (Table 1 and Figure 1A). This finding suggests that K+ and urea osmolytes provided the primary osmotic driving force necessary to reabsorb water and concentrate excess dietary Na+ osmolytes into the urine. Urine formation by total surplus osmolyte and water excretion showed the expected close relationship between U2Na2KUreaV and water excretion (Figure 1B). These findings indicate that in HS+saline mice, further increase in urine volume were prevented by K+- and urea-driven maintenance of the renal concentration mechanism, thereby preventing Na+-driven extracellular volume loss.

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.

We next tested the reproducibility of our findings in additional experiments in mice with dietary salt loading and free access to water (4% NaCl chow and tap water; HS+tap) and compared the responses with results from 10 human subjects whose salt intake was increased by 6 g/d during two 105-day and 205-day ultra-long-term salt and water balance studies (4). Surplus Na+ and K+ excretion was 5-fold higher and surplus urea excretion was 10-fold higher in the mice than in the human subjects (Table 1). Accounting for unmeasured anions, surplus osmolyte excretion was 7-fold higher in mice than in humans, while surplus water excretion was 3-fold higher. The mice thus had a 2-fold higher urine osmolality than did the human subjects. Endogenous free water accrual by negative FWC in the kidney was therefore 15-fold higher in the mice than in the human subjects (Table 1). Despite these substantial differences in body osmolyte and water homeostasis and across the different dietary salt interventions, the mice and human subjects shared the natriuretic-ureotelic regulatory principle of excreting surplus dietary salt by concentrating Na+ into the urine and in parallel reducing the urine urea concentration (Table 1).

Independent of the salt intake level, mice and humans eliminated surplus Na+, K+, and urea osmolytes with increasing amounts of surplus water (Figure 1C). Across the species and the different salt-intake protocols increased surplus osmolyte excretion was characterized by increased urine concentration and, thus, by reduced clearance of free water (Figure 1D). This finding indicates that the biological and physiological pattern of extracellular volume control during surplus osmolyte excretion substantially relies on water conservation by urine concentration. Extracellular volume control in the mice and human subjects with increasing osmolyte excretion was therefore not dependent on a parallel adjustment of fluid intake (Figure 1E). A 6-g/d increase in salt intake reduced FWC in humans, and the resulting increase in endogenous free water accrual resulted in surplus water excretion and reduced fluid intake (see Table 1 and the accompanying article [ref. 4] for details). This water intake and excretion pattern was best simulated in our metabolic cage experiments involving HS+saline mice (Table 1).

Glucocorticoid-coupled surplus water excretion and its association with dietary salt loading in mice and humans. In our ultra-long-term water balance study in humans, spontaneous rhythmical cortisone release was coupled with increased urine volume formation and increased free water excretion without increasing fluid intake, indicating a rhythmical release of endogenously generated surplus water (see the accompanying article [ref. 4] for details). This finding was reproducible in mice. In humans, increasing cortisol excretion augmented urine volume (Figure 2A) without increasing water intake (Figure 2B), resulting in a negative water balance (water balance gap: difference between water intake and urinary output; Figure 2C) and indicating excretion of surplus water. Relative to body mass, corticosterone levels in the mice in metabolic cages were higher than the cortisol levels detected in the human subjects. These higher glucocorticoid levels were coupled with further increases in urine volume (Figure 2A), while water intake was not similarly increased (Figure 2B), resulting in a negative water balance across the species (Figure 2C).

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.

We hypothesized that dietary salt loading not only promotes urea-driven free water accrual within the renal urine concentration process to prevent a negative water balance, but additionally induces glucocorticoid-driven catabolism for metabolic urea osmolyte generation and increased metabolic water production. We tested these hypotheses in HS+saline mice, which, like the human subjects, showed increased glucocorticoid levels in response to the dietary salt–loading protocol (Table 1).

Dietary salt induces urea transporter–driven urea osmolyte accumulation in the kidney. After 6 weeks on their specified diets, the LS and HS+saline mice were studied to determine the inner medullary urea content and expression of the outer medullary urea transporter UT-A2 and the inner medullary urea transporter UT-A1 (Figure 3, A–C, and see Supplemental Figure 3, A and B, for the complete, unedited Western blots). We found a marked increase in medullary urea content in the HS+saline mice, which was paralleled by increased UT-A1 expression. We interpret these findings as showing that UT-A1–driven urea accumulation in the renal medulla provides the osmotic gradient necessary to reabsorb water when dietary salt is excreted. This water-saving effect of urea osmolyte accumulation is the basis of the concentration mechanism, whereby the kidney excretes salt without major changes in fluid intake or urinary water loss. HS+tap-treated mice showed a similar urine osmolyte excretion and concentration profile (Table 1) and similar increases in UT-A1 expression in the renal medulla (Supplemental Figure 3, C and D), but no increased glucocorticoid excretion. This finding indicates that the renal natriuretic-ureotelic response was also triggered when free access to water was offered in the diet and that increasing glucocorticoid levels was not necessary to induce the urea-driven renal water conservation process.

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.

Urea and Na+ hold water in the plasma space. We next tested the hypothesis that renal medullary urea accumulation not only facilitates renal water reabsorption but also changes plasma urea concentration. We found that HS+saline treatment significantly increased the plasma urea concentration (Figure 3D). Treatment with the unspecific arginase inhibitor N-ω-hydroxy-L-norarginine (NOHA) left plasma urea levels unchanged. Conversely, HS+saline decreased plasma Na+ concentrations, while NOHA treatment left plasma Na+ levels unchanged (Figure 3E). Plasma osmolality was not different between HS+saline- and HS+saline+NOHA-treated mice (Figure 3F). We conclude that both urea and Na+ act as osmolytes to conserve plasma water and thereby contribute to extracellular volume regulation. NOHA did not significantly reduce renal UT-A1 or UT-A2 expression (Figure 3, B and C, and see Supplemental Figure 3, A and B, for the complete, unedited Western blots). This finding suggests that, besides transporter-driven renal urea recycling, extrarenal urea osmolyte production may play an important role in extracellular volume control.

Extrarenal urea generation is a determinant of plasma urea concentration. We thus tested the relationship among arginase activity, tissue urea content, and plasma urea levels. HS+saline increased renal medullary urea content and plasma urea concentration (Figure 4A); however, this increase was not dependent on renal arginase activity, suggesting that the observed decrease in medullary urea content and plasma urea concentration in NOHA-treated mice was most likely due to reduced extrarenal urea production. Therefore, we next studied the role of urea production in the liver (Figure 4B). HS+saline increased hepatic arginase activity and thereby increased urea content in the liver. We found that liver urea content was a better predictor of plasma urea levels than was renal medullary urea content. In contrast to what we observed in kidney, liver arginase activity strongly predicted liver urea content and plasma urea concentration. NOHA treatment reduced liver arginase activity and, in parallel, reduced liver urea content as well as the plasma urea concentration. We interpret this finding as showing that liver and kidney jointly control body osmolyte content and thereby determine extracellular volume homeostasis. We also found that HS+saline increased arginase activity and urea content in skeletal muscle (Figure 4C). NOHA treatment reduced muscle urea content back to the control values. This finding suggests that the increase in muscle urea content was due to local urea production in the muscle, indicating additional extrahepatic urea generation.

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.

Salt leads to catabolic muscle mass loss. We next tested the hypothesis that salt-driven urea osmolyte generation induces a catabolic state and studied the effect of dietary salt loading on the respiratory quotient (RQ) in the mice. A 4% NaCl diet did not significantly decrease RQ as long as mice had free access to tap water (Figure 5A). Immediately upon replacement of tap water with isotonic saline, HS+saline-treated mice had decreased RQ during the inactive period to a value of 0.7. We interpret these findings as showing that HS+saline induces catabolism with predominant fat oxidation for energy generation in mice. The time course of the changes in food intake and body weight in LS- and HS+saline-treated mice over 4 weeks of ad libitum feeding, followed by 2 weeks of pair-feeding (Figure 5, B and C), revealed that with ad libitum feeding, HS+saline mice showed a 20%–30% increase in food intake. However, body weights remained similar between the groups. We then pair-fed the mice and reduced the food intake of HS+saline diet–fed mice to that of LS diet–fed mice. With pair-feeding, HS+saline mice lost approximately 10% of their total body weight within 1 week. In contrast to salt loading with the HS+saline diet, the HS diet with free access to water neither increased glucocorticoid levels (Table 1), nor increased food intake in the ad libitum food intake phase, nor decreased body weight in the pair-feeding phase (Supplemental Figure 4).

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.

We therefore next hypothesized that the observed weight loss in HS+saline mice after pair-feeding was due to corticosterone-driven loss of muscle mass. We first tested whether elevated corticosterone levels in HS+saline mice led to glucocorticoid receptor (GR) activation in the muscle (Figure 5D, upper panel, Figure 5E, and see Supplemental Figure 5 for the complete, unedited Western blots). HS+saline did not increase GR protein levels in the cytoplasm, the membrane, or in the soluble nuclear fraction. In contrast, nuclear chromatin–bound GR protein expression increased with HS+saline, showing GR activation. In parallel with GR activation, microtubule-associated protein 1A/1B chain 3 (LC3, structural precursor protein within the autophagic pathway) was preferentially lipidated from the cytosolic form (LC3-I, cleaved from LC3) to the derivate associated with autophagosome membrane expansion, LC3-II. The resulting increase in the LC3-II/LC3-I ratio (Figure 5D, lower panel, and see Supplemental Figure 6 for the complete, unedited Western blots), which was paralleled by a tendency toward higher levels of autophagy-related protein 5 (ATG5, involved in the lipidation of LC3-I to LC3-II) and by reduced levels of the autophagosome carrier protein p62 (SQSTM1, sequestosome 1, which targets polyubiquitinated proteins for degradation), indicates increased autophagosome formation (see Supplemental Figure 7, A–C, for the complete, unedited Western blots). Additionally, skeletal muscle in HS+saline mice showed increased 1-methyl histidine and 3-methyl histidine levels, indicating muscle protein breakdown (Supplemental Figure 7D).

Mice given HS+saline had increased chromatin-bound GR protein levels, increased autophagosome formation markers, and increased muscle protein breakdown and showed higher corticosterone levels in urine (Table 1) and plasma (Figure 5F). We therefore investigated the relative contribution of muscle mass to body weight loss by nuclear magnetic resonance spectroscopy. Fifty-nine percent of the variability of body weight was explained by changes in lean body mass, which was consistent with salt-induced muscle wasting (Figure 5G). We further confirmed the autophagy component of muscle wasting in vivo in mice expressing GFP under the control of the LC3 promoter. We found large increases in LC3 promoter activity in LC3-GFP mice with HS+saline treatment after pair-feeding (Figure 5H). These data support the notion that HS+saline induces a catabolic state for urea osmolyte generation when excess salt is excreted.

Salt promotes nitrogen transfer from muscle to liver. We next hypothesized that muscle protein serves as a nitrogen source for accelerated ureagenesis in HS+saline mice. Metabolomic analysis of free amino acids in skeletal muscle showed a substantial reduction of serine, threonine, methionine, alanine, and tyrosine in HS+saline-treated mice (Figure 6). These amino acids are nitrogen donors for hepatic ureagenesis (Supplemental Figure 8). We found no decrease in free amino acid content in liver. However, liver glutamine and aspartate levels were selectively increased, suggesting a mobilization and redistribution of nitrogen for urea production from muscle to liver.

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.

Arginase utilizes water to convert arginine to urea and ornithine (Supplemental Figures 8 and 9). While increased arginase activity coincided with increased urea levels (Figure 4), ornithine levels were reduced in muscle in HS+saline mice (Figure 7A, left). This finding suggests that other enzyme systems downstream of arginase further converted ornithine, which harbors 2 amino groups. We thus focused on the enzyme ornithine-aminotransferase (OAT) that transfers the C5-amino group from ornithine to 2-oxo-carbon acids, ultimately generating glutamate or proline. Glutamate is the amino source for urea synthesis in the liver. We found a substantial increase in muscle OAT expression in HS+saline mice (Figure 7B, and see Supplemental Figure 10 for the complete, unedited Western blots), but reduced proline levels (Figure 7A, left). These results suggest that the ornithine generated by elevated arginase activity was likely further metabolized to glutamate. Glutamate is not actively transported out of muscle. OAT-initiated provision of muscle nitrogen therefore requires additional transfer of the amino group from glutamate to pyruvate to generate alanine (Supplemental Figure 9). This alanine-glucose-nitrogen shuttle transfers glucose-derived carbon skeletons (pyruvate) and glutamate-derived nitrogen from muscle to liver by way of the transamination to alanine. We found reduced alanine levels (Figure 7A, left), together with unchanged expression of the Na+/alanine cotransporters SLC38A1 and SLC38A2 (Figure 7B, and see Supplemental Figure 10 for the complete, unedited Western blots) in the muscle of HS+saline mice. In contrast, the expression of SLC38A1 and SLC38A2 was increased in liver (Figure 7B, and see Supplemental Figure 11 for the complete, unedited Western blots), suggesting uptake of muscle alanine in the liver. Urea cycle analysis in liver showed increased arginase activity with increased urea levels (Figure 4) and elevated argininosuccinate levels (Figure 7A, right). The enzyme argininosuccinate lyase converts argininosuccinate to arginine and fumarate (Supplemental Figure 9). We found that liver arginine levels were reduced, presumably as a result of high arginase activity, while fumarate levels were elevated (Figure 7A, right). Despite high fumarate levels, we found no changes in malate or oxaloacetate levels in liver. However, the alternative metabolite aspartate, which ultimately transfers nitrogen from the alanine-glucose-nitrogen shuttle into the urea cycle, was elevated (Figure 7A, right, and Supplemental Figure 9). We interpret these results as indicating that muscle in HS+saline mice increases urea production and transfers nitrogen and glucose via the alanine-glucose-nitrogen shuttle to the liver, where alanine is taken up by increased active transport and preferentially metabolized to urea.

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.

Salt induces ketogenesis, reduces gluconeogenesis, and entails fatty acid oxidation. We next studied the content of glycogen and intermediates for energy metabolism in skeletal muscle and liver (Figure 7A). HS+saline reduced the levels of glucose, glucose-6-phosphate, fructose-6-phosphate, and 6-phosphoguconate in muscle and in liver. The glycogen branch sugars maltotriose, maltotetratose, and maltopentatose were predominantly reduced in the liver. Both muscle and liver showed increased ketone body content. This finding suggests that liver switched pyruvate substrate utilization from energy-intense gluconeogenesis to energy-neutral ketogenesis and thereby reprioritized its energy expenditure in favor of urea osmolyte production (Supplemental Figure 12).

Liver ketogenesis deprives muscle of glucose. Muscle in HS+saline mice showed increased fatty acid carnitine esters and their respective 3-hydroxy isoforms (Figure 7A). This finding, corroborated by the observed RQ reduction (Figure 5A), indicates preferential mitochondrial β-oxidation in HS+saline mice. We conclude that muscle in HS-saline mice utilized ketone bodies together with fatty acids as energy fuels, while the availability of glucose was reduced.

However, the reduced availability of glucose also reduces glycolytic pyruvate generation (Supplemental Figure 12). Pyruvate is an essential substrate that initiates nitrogen transfer from muscle to liver via the alanine-glucose-nitrogen shuttle (Supplemental Figure 9). Liver-driven glucose/pyruvate deprivation may therefore explain the autophagy and muscle protein breakdown observed in HS+saline mice, because muscle protein remained the major available source of glutamate and pyruvate.

Energetic consequences of salt in muscle and liver. In line with the catabolic nature of urea production and nitrogen mobilization in muscle, we found increased AMP and ADP levels in skeletal muscle in HS+saline mice (Figure 7A, left). AMPK, which acts as a sensor of cellular energy status, is considered a key enzyme in conditions of cellular energy deficit. AMPK is able to inhibit metabolic pathways that consume energy and increases mechanisms that produce energy (5). Binding of AMP to AMPK inhibits dephosphorylation of the kinase (6, 7). Phosphorylated AMPK (p-AMPK) facilitates fatty acid oxidation in mitochondria (8) and promotes autophagy in skeletal muscle (9, 10). HS+saline mice with increased AMP levels in muscle showed increased p-AMPK protein and an increased p-AMPK/AMPK ratio (Figure 7C, and see Supplemental Figure 13 for the complete, unedited Western blots). p-AMPK promotes mitochondrial fatty acid oxidation (11, 12) by phosphorylation of its downstream target acetyl CoA carboxylase (ACC). ACC promotes carboxylation of acetyl CoA to generate malonyl-CoA and initiate fat storage (13). p-AMPK–driven phosphorylation of ACC inhibits its carboxylase activity and thereby promotes mitochondrial β-oxidation (14). We found that HS+saline increased p-ACC levels and the p-ACC/ACC ratio (Figure 7C, and see Supplemental Figure 13 for the complete, unedited Western blots). These results indicate that a HS diet leads to an energy deficit in skeletal muscle, which induces an AMP-driven increase of fatty acid oxidation by p-AMPK–mediated phosphorylation of ACC.

In contrast to muscle, liver in HS+saline mice showed reduced AMP levels (Figure 7A, right), suggesting no organ-specific energy deficit, despite massively increased urea production and reduced availability of glucose or glycogen fuels. In line with low AMP levels, p-AMPK and p-ACC were not elevated in the liver of HS+saline mice (Figure 7C). We interpret these results as indicating that reduced gluconeogenesis and a switch to ketogenesis (Supplemental Figure 12) help prevent an energy deficit in liver, despite the energy-intense urea osmolyte generation that occurs in catabolic HS+saline mice.

Salt decreases cardiovascular energy expenditure. The observed reprioritization of energy metabolism in our HS+saline mice represents a typical adaptation pattern for water conservation in organisms, termed aestivation (15). In aestivators, the energy-intense nature of urea osmolyte generation for body water conservation also includes reduced cardiovascular energy expenditure (16). We therefore hypothesized that HS+saline not only reprioritized energy metabolism, but also induced reduced cardiovascular energy expenditure in mice. We therefore used radiotelemetry to study the cardiovascular response to our 4 different salt-intake regimens in 6 mice (Figure 8). The diet consisting of 4% NaCl chow with tap water increased systolic BP (SBP) from 119 ± 13 mmHg to 124 ± 11 mmHg (P < 0.05), without significant changes in heart rate (Figure 8A). Replacement of water with isotonic saline (HS+saline diet) not only led to a catabolic state with reprioritization of urea osmolyte and energy metabolism (Figures 5–7), but also induced the expected changes in cardiovascular energy expenditure. The initial response to HS+saline exposure was characterized by a steep increase in heart rate, with a monophasic short R-R interval distribution pattern (Figure 8B), and an increase in locomotor activity, suggesting an alarm reaction with predominant sympathetic drive. Within 4 days of HS+saline treatment, mice had markedly lower heart rates that were even below the initial baseline rate and now showed a biphasic distribution of short and prolonged R-R intervals, consistent with high parasympathetic tone (Figure 8B). SBP levels were intimately coupled with the cardiovascular energy expenditure level. Thirty-two percent of the variability in SBP was explained by changes in heart rate, suggesting that cardiovascular energy expenditure and sympathetic nerve drive during activity and inactivity were important determinants of the salt-induced elevation and reduction in BP (Figure 8C). Additional pair-feeding not only induced total body energy deficit and catabolism (Figures 5–7), but also resulted in a robust reduction in cardiovascular energy expenditure, with a low heart rate, low BP, and low locomotor activity, despite massive salt intake. Finally, we tested the hypothesis that the initial BP increase we observed in HS+saline-fed animals was inducible by an acute stressor. We found that intra-arterial BP was significantly elevated in mice fed a HS+saline diet for 2 consecutive weeks when we measured BP acutely in restrained animals (Figure 8D). We interpret these findings as indicating that HS+saline not only leads to catabolic urea production with reprioritization of energy metabolism, but also induces a reduction in cardiovascular energy expenditure with a reduced heart rate and low BP. This negative energy balance–driven cardiovascular response in HS+saline mice was rapidly reversible during acute alarm responses triggered by an external stressor. We conclude that the metabolic and cardiovascular response to HS+saline we observed in our mice is typical for water conservation by aestivation.