Infradian biological variability in long-term salt and water homeostasis. In the 105-day Mars105 Study, we reduced the subjects’ average daily salt intake from 12 g/d (200 mmol/d sodium) to 9 g/d (150 mmol/d) and then to 6 g/d (100 mmol/d). In the 205-day Mars520 study, all subjects were additionally reexposed to the 12-g/d salt intake regime after salt intake had been reduced from 12 g/d to 6 g/d over the course of months. Long-term urinary Na+ excretion (UNaV), long-term urine volume, and long-term water intake (Figure 1A) are shown for a representative subject (see Supplemental Figure 1 for data on each individual subject; supplemental material available online with this article; https://doi.org/10.1172/JCI88530DS1). As expected, the average daily urinary Na+ excretion rapidly adjusted to the new salt intake levels. The subject exhibited additional rhythmical day-to-day variability in UNaV at each dietary salt intake level. This highly variable UNaV was not paralleled by obvious changes in urine volume. Furthermore, and contrary to our initial hypothesis, low UNaV in response to a reduction in dietary salt intake was accompanied by increased fluid intake in the subject.

Figure 1 Time series data presentation and mixed linear model analysis to visualize the effect of increasing salt intake and the resulting Na+ excretion on water intake and urine volume formation. (A) Time series of 24-hour sodium excretion (UNaV), urine volume, and water intake in the representative subject 54 during the 205-day experiment. (B) Average fluid intake per salt intake phase in all 10 subjects (n = 1,646). (C) Quantification of the changes in fluid intake per salt intake phase or per 24-hour UNaV tertile. (D) Average urine volume per salt intake phase in the same subjects (n = 1,644). (E) Quantification of the changes in urine volume per salt intake phase or per UNaV tertile. (F) Average 24-hour UNaV per salt intake phase in the same subjects (n = 1,646). (G) Quantification of the changes in UNaV per salt intake phase or per UNaV tertile. Data are expressed as the average ± SD (B, D, and F) or as the Δ change ± SEM (C, E, and G). Data were statistically analyzed by mixed linear model. Details on statistical analysis for Figure 1 are provided in the Supplemental Materials (page 28).

We next analyzed the effect of the prescribed dietary salt intake (dietary salt phase) and 24-hour UNaV on water intake and urine volume in all subjects. The rhythmical nature of urinary Na+ excretion led to a lagged elimination of dietary salt, resulting in periodically low and high levels of salt excretion at each dietary salt intake phase, which may have masked the diuretic effect of elevated salt intake. We therefore additionally classified UNaV values into 3 tertiles of low, medium, and high UNaV levels to test for the effect of renal Na+ elimination on water balance. We found that higher salt intake reproducibly reduced water intake (Figure 1B). Mixed linear model analysis showed that water intake was 293 ± 27 ml/d higher during the 6-g/d salt intake phase than during the 12-g/d salt intake phase (Figure 1C). Water intake in the lowest (first) tertile of UNaV was 237 ± 27 ml/d higher than in the highest (third) tertile of UNaV (Figure 1C). A 6-g/d increase in salt intake did not significantly increase the urine volume (Figure 1, D and E). In contrast, urine volume in the lowest (first) tertile of UNaV was 316 ± 24 ml/d lower than in the highest (third) tertile of UNaV (Figure 1E). We interpret this finding as showing that an increase in urinary salt excretion induced some diuresis. This salt-osmolyte–driven diuretic response was often prevented at the 12-g/d salt intake level. Finally, we confirmed the accuracy of our long-term Na+ balance approach. We found the expected increase of approximately 50 mmol/d in UNaV per salt phase when salt intake was increased by 3 g/d (Figure 1, F and G).

Urine osmolyte excretion induces osmolyte-free water generation in the kidneys. The renal concentration process is characterized by osmolyte excretion with parallel renal water reabsorption, resulting in a negative clearance of osmolyte-free water. We therefore next tested the hypothesis that the salt-driven increase in urine osmolyte excretion was coupled with enhanced free water reabsorption. We found that the sum of 2Na+ concentration (2-fold Na+ to account for unmeasured accompanying anions), 2K+ concentration (2-fold K+ to account for unmeasured accompanying anions), and urea concentration (U[2Na+2K+Urea]) amounted to almost 100% of the subjects’ urine solutes (Supplemental Figure 2). We show the relationship between the salt intake phase, osmolyte excretion, and free water clearance (FWC) in the same representative subject (Figure 2A). Any decrease or increase in the subject’s dietary salt intake resulted in a parallel decrease or increase in his urine osmolyte excretion. The salt intake–driven changes in urine osmolyte excretion, however, were coupled with antiparallel changes in the subject’s osmolyte-free water excretion in the urine. This finding, which indicates that increasing osmolyte excretion was associated with renal water conservation, was reproducible in 10 of 10 study subjects (see Supplemental Figure 3 for data on each individual subject). Quantitative analysis showed that increasing the salt intake level gradually decreased FWC (Figure 2B). A 6-g/d increase in salt intake (corresponding to ≈100 mmol/d Na+ and Cl–) decreased renal FWC by 540 ± 27 ml/d (Figure 2C), while urine osmolyte excretion was increased by 201 ± 8 mmol/d (Figure 2, D and E). Increasing osmolyte excretion and increasing Na+ intake reduced FWC (Figure 2, F and G) and reduced fluid intake (Figure 1, B and C). In contrast, increasing fluid intake increased FWC (Figure 2H), indicating excretion of surplus consumed water by an intact urine dilution process.

Figure 2 Time series data presentation and mixed linear model analysis to visualize the effect of increasing salt intake and the resulting Na+ excretion on free water accrual by urine concentration. (A) Time series of the sum of 24-hour urine Na+ (with accompanying anions), K+ (with accompanying anions), and urea osmolyte excretion (U2Na2KUreaV) and FWC in the representative subject 54 during the 205-day experiment. (B) Average FWC per salt intake phase in all 10 subjects (n = 1,644). (C) Quantification of the changes in FWC per salt intake phase or per 24-hour UNaV tertile. (D) Average urine osmolyte excretion (U2Na2KUreaV) per salt intake phase in the same 10 subjects (n = 1,646). (E) Quantification of the changes in urine osmolyte excretion per salt intake phase or per 24-hour UNaV tertile. (F–H) Relationship among 24-hour osmolyte excretion, designated 24-hour Na+ intake, designated 24-hour fluid intake, and 24-hour FWC in the urine. Data are expressed as the average ± SD (B and D) or as the Δ change ± SEM (C and E). Data were statistically analyzed by mixed linear model (C and E) or simple linear regression (F–H). Details on statistical analysis for Figure 2 are provided in the Supplemental Materials (page 64).

Water intake was 32% higher than urine volume (Figure 1, B and D), indicating normal levels of insensible water loss in our subjects (25). On the assumption that 32% of the osmolyte-free water generated by the kidneys was similarly excreted by insensible water loss, the salt-driven 540-ml/d reduction in renal osmolyte–free water excretion increased our subjects’ osmolyte-free body water content by –540 ml/d × –1 × 0.68 = 367 ml/d (Figure 3A). This projected salt-driven increase in osmolyte-free body water by improved urine concentration (Figure 3B) can reduce thirst and corresponds well to the measured 293 ± 27 ml/d decrease in fluid intake in response to a 6-g/d increase in salt intake in our subjects. This state of affairs suggests that the physiological response to daily-life increases in salt intake in humans relies on selective renal osmolyte elimination by urine concentration, which allows maintenance of a body fluid balance in the absence of environmental water sources.

Figure 3 Salt-driven changes in FWC, fluid intake, urine osmolyte excretion, and urine osmolyte concentration. (A) Effect of a 6-g/d increase in salt intake on 24-hour FWC and 24-hour fluid intake in the 10 subjects. (B) Effect of 6-g/d, 9-g/d, and 12-g/d salt intake on 24-hour osmolyte excretion (U2Na2KUreaV; n = 1,646) and osmolyte concentration in the 24-hour urine samples (U[2Na2KUrea]; n = 1,636) for the 10 subjects. Data were statistically analyzed by mixed linear model and are expressed as the Δ change ± SEM. Details on statistical analysis for Figure 3 are provided in the Supplemental Materials (page 100). The projected insensible water loss was estimated from the measured difference between fluid intake and urine volume in all subjects and at all phases of salt intake.

Dietary salt is excreted within the renal concentration mechanism. Figure 4A shows how increasing UNaV led to a rise in urinary Na+ concentration in our representative subject (see Supplemental Figure 4 for data on each individual subject). We next questioned which alternative urinary osmolytes might be retained and accumulated in the kidney to provide the antidiuretic driving force necessary to concentrate dietary salt in the urine. We observed an antidromic reduction in the urea and K+ concentration when Na+ was concentrated in the subject’s urine (Figure 4A). In all subjects, the excretion of dietary salt increased the urinary Na+ and accompanying anion concentration by 98 ± 3 mmol/l (Figure 4, B and C). The excretion of dietary salt by urinary concentration was paralleled by a 37 ± 4 mmol/l reduction in the urinary urea concentration (Figure 4, D and E), while the K+ concentration and accompanying anion concentration showed no reproducible changes (Figure 4, F and G). We interpret these findings as suggesting that urea accumulation in the renal interstitium provides the osmotic driving force necessary for antidiuretic water movement when dietary salt is concentrated in the urine.

Figure 4 Effect of increasing 24-hour urine Na+ excretion on urine Na+, urea, and K+ concentration. To quantify the effect of increasing urine Na+ excretion on urine osmolyte concentration, the data are depicted and analyzed per tertile of urine Na+ excretion. (A) Twenty-four-hour UNaV tertiles and their relation to urine Na+ concentration (U[Na+]), urine urea concentration (U[Urea]), and urine K+ concentration (U[K+]) in the representative subject 16 during the 105-day experiment. (B) Average urine 2×Na+ concentration per salt intake phase in the 10 subjects (n = 1,644). (C) Quantification of the changes in urine Na+ concentration per salt intake phase or per 24-hour UNaV tertile. (D) Average urine urea concentration per salt intake phase (n = 1,636). (E) Quantification of the changes in urine urea concentration per salt intake phase or per 24-hour UNaV tertile. (F) Average urine 2×K+ concentration per salt intake phase (n = 1,644). (G) Quantification of the changes in urine K+ concentration per salt intake phase or per 24-hour UNaV tertile. Data are expressed as the average ± SD (B, D, and F) or as the Δ change ± SEM (C, E, and G). Data were statistically analyzed by mixed linear model. Details on statistical analysis for Figure 4 are provided in the Supplemental Materials (page 112). L, low; M, medium; H, high.

Mineralocorticoid and glucocorticoid release in relation to water balance. A 6-g/d reduction in salt intake increased 24-hour urine aldosterone excretion (UAldoV) by 5.1 ± 0.2 μg/d (P < 0.001) and reduced 24-hour urine cortisone excretion (UCortisoneV) by 11.4 ± 1.0 μg/d (P < 0.001). However, as reported earlier (11), UAldoV and UCortisoneV showed additional rhythmical half-weekly and weekly patterns of change that were independent of salt intake. This spontaneous endogenous variability led us to stratify our data into tertiles of low, medium, or high UAldoV or UCortisoneV across each salt intake level (Table 1) and to study the effect of hormone level on water balance (Table 2), as well as on osmolyte-driven urine volume formation (Table 3).

Table 1 Twenty-four-hour urine aldosterone excretion (UAldoV), cortisone excretion (UCortisoneV), and Na+ intake per tertile of aldosterone or cortisone excretion at 3 different levels of salt intake

Table 2 Urine volume, water intake, the resulting difference, i.e., water balance gap, body weight, and urine osmolality in response to the spontaneous changes in urine aldosterone and cortisone levels at 3 different levels of salt intake

Table 3 Osmolyte excretion and osmolyte concentration in response to the spontaneous changes in urine aldosterone and cortisone levels at 3 different levels of salt intake

The aldosterone-driven changes in body fluid balance occurred across all 3 levels of salt intake (Table 2). The spontaneously rhythmical 7.6 ± 0.2 μg/d UAldoV increase reduced urine volume by 219 ± 25 ml/d (P (aldosterone) < 0.001) and increased fluid intake by 95 ± 27 ml/d (P (aldosterone) < 0.01; Figure 5A). The resulting increase in water balance was coupled with a measurable body weight increase in the subjects (+0.41 ± 0.10 kg; P (aldosterone) < 0.001). We interpret these findings as indicating that the high mineralocorticoid levels induced body fluid retention.

Figure 5 Long-term rhythmical hormonal control of water balance and its modulation by dietary salt intake. (A) Effect of rhythmical mineralocorticoid release, independent of salt intake, on water intake (n = 1,646), urine volume (n = 1,644), renal water balance (n = 1,646), and body weight (n = 1,631). (B) Effect of rhythmical glucocorticoid release, independent of salt intake, on water intake (n = 1,646), urine volume (n = 1,644), renal water balance (n = 1,646), and body weight (n = 1,631). (C) Projected combined effect of salt-driven modulation of rhythmical mineralocorticoid and glucocorticoid release on water intake, urine volume, renal water balance, and body weight. (D) Measured effect of third tertile Na+ excretion in the urine on water intake (n = 1,646), urine volume (n = 1,644), renal water balance (n = 1,646), and body weight (n = 1,631). Data were statistically analyzed by mixed linear model and are expressed as the Δ change ± SEM (A, B, and D). The projected combined effect of mineralocorticoid suppression and glucocorticoid increase by dietary salt intake in C was calculated from the data presented in A and B and the measured suppression of mineralocorticoid and increase in glucocorticoid levels by a 6-g/d increase in salt intake. Details on the supplemental calculations and statistical analyses pertaining to Figure 5 are provided in the Supplemental Materials (pages 22 and 148, respectively).

We next studied the role of the rhythmical effect of aldosterone on the urinary concentrating mechanism. Across all levels of salt intake, the highly rhythmical UAldoV levels reduced 2UNaV by 58.9 ± 6.1 mmol/d (P (aldosterone) < 0.001). However, this aldosterone-driven reduction in urine Na+ excretion did not significantly reduce the sum excretion of Na+, K+, and urea osmolytes in the urine (–16.0 ± 8.9 mmol/d, P = 0.07), because high aldosterone levels increased 2UKV by 22.6 ± 2.1 mmol/d (P (aldosterone) < 0.001) as well as urinary urea excretion by 20.7 ± 3.3 mmol/d (P (aldosterone) < 0.001). High excretory aldosterone levels instead were associated with an increase in the urine concentration of 53 ± 7 mOsm/kg (P (aldosterone) < 0.001). The well-known antidiuretic effect of aldosterone thus became clinically visible within the context of the urine concentration mechanism by reduced FWC (Table 2). This effect resulted in predominant osmolyte-free water reabsorption (see the supplemental calculations in the Supplemental Materials and Figure 5A).

The glucocorticoid-associated changes in body fluid balance occurred across all 3 levels of salt intake (Table 2). Rhythmical elevation of urinary cortisone excretory levels by 33.8 ± 0.7 μg/d (P < 0.001) was linked with increases in urine volumes of 627 ± 20 ml/d (P (cortisone) < 0.001), without quantifiable changes in fluid intake (P (cortisone) = 0.67; Figure 5B). However, the resulting negative renal fluid balance was not accompanied by changes in body weight (P (cortisone) = 0.25). This state of affairs suggests that the water surplus that was excreted when glucocorticoid levels were high had been generated endogenously. The renal water conservation mechanism did not contribute to endogenous water generation when glucocorticoid levels were high. The rhythmical increase in UCortisoneV was associated with increased urinary 2Na+, 2K+, and urea excretion across all 3 levels of salt intake (Table 3), resulting in an increase of 114.0 ± 8.5 mmol/d in the sum of 2Na+, 2K+, and urea osmolyte excretion in the urine (P (cortisone) < 0.001). High rhythmical glucocorticoid levels were additionally linked with a reduction of 126 ± 7 mmol/l in the sum of 2[Na+], 2[K+], and [Urea] osmolyte concentrations (P (cortisone) < 0.001). Rhythmical glucocorticoid release was therefore linked to increased diuresis in the context of the urine dilution mechanism by increasing FWC (Table 2). The clinical readout was predominant osmolyte-free water excretion (see the supplemental calculations in the Supplemental Materials and Figure 5B).

Projected and measured salt-induced change in water balance. A 6-g/d increase in salt intake reduced UAldoV excretion by 5.1 ± 0.2 μg/d. The high salt intake thus reduced the level of spontaneously rhythmical 7.6 ± 0.2 μg/d UAldoV release by 73%, resulting in a projected –5.1/7.6 = –0.67-fold change in water intake, urine excretion, water balance, and body weight (Supplemental Figure 5). In contrast, a 6-g/d increase in salt intake increased UCortisoneV excretion by 11.4 ± 1.0 μg/d. The high salt intake thus increased the level of spontaneously rhythmical 33.8 ± 0.7 μg/d UCortisoneV release by 38%, resulting in a projected 11.4/33.8 = 0.34-fold change in water intake, urine excretion, water balance, and body weight (Supplemental Figure 5).

Calculation of the combined effect of salt-driven mineralocorticoid suppression and glucocorticoid activation (Supplemental Figure 6) showed a projected 358-ml/d increase in urine volume formation in response to a 6-g/d increase in salt intake, which was associated with a projected –60 ml/d decrease in fluid intake (Figure 5C). These results suggest that increased glucocorticoid activity was associated with excretion of an endogenously generated water surplus at the high salt intake level. This projected salt-driven modulation of mineralocorticoid- and glucocorticoid-mediated adjustment of body water balance corresponded well with the measured 316 ± 24 ml/d increase in urine volume and the 546 ± 34 ml/d reduction in the water balance gap between fluid intake and urine volume in the third tertile of UNaV excretion (Figure 5, C and D). The 24-hour negative water balance in the third tertile of UNaV excretion was preceded by an increase in body weight of 882 ± 99 g on the morning before the start of the 24-hour collection period (Figure 5D). This state of affairs suggests that increased urine volume formation and reduced fluid intake in the third tertile of UNaV excretion characterize the release of surplus salt osmolytes, together with a preexisting endogenous water surplus within the following 24-hour urine collection period.