Actual doses of adenine ingested by the animals

Table 1 presents the actual doses of adenine ingested by the animals, calculated based on their food consumption at each dietary adenine level. As expected, adenine intake increased proportionally with the amount provided in the food. However, over time, adenine ingested declined due to reduced food consumption, particularly at the 2500 mg/kg dose, and was also influenced by changes in both food intake and body weight at the 2000 mg/kg dose after 7 weeks.

Table 1 Time course of the actual adenine ingested, as a function of the amount of adenine provided in food and expressed as mg/kg body weightBlood composition of control and adenine-fed rats

Blood chemistry results depicted in Table 2 show that rats fed lower doses of adenine (1500 to 2500 mg/kg) for 7 weeks have a normal blood electrolyte profile, normal glucose, Hct, and HB levels, and unchanged acid–base composition, as compared to the control (Table 2). However, a significant increase in BUN and creatinine levels was observed in rats fed 2500 mg/kg adenine for 7 weeks (Table 2). As expected, rats fed a higher dose (5000 mg/kg) of adenine for 7 weeks showed a significant disorder in almost all measured blood components compared to both control and lower doses of adenine (Table 2). These effects result from the development of renal failure initiated by massive volume depletion and reduced food intake, as previously described in our laboratory [12].

Table 2 Blood composition of control and adenine-fed rats for 7 weeksTime course effects of one week of adenine feeding on food intake, water balance, and urine osmolality

Figure 2 is a time course study of the effects of adenine at 2500 mg/kg for 6 days on these physiological parameters. As shown, 2500 mg/kg adenine feeding to rats did not alter food intake (Fig. 2A, P > 0.05) but caused a significant increase in water intake (Fig. 2B, P < 0.001, n = 4) and urine volume (Fig. 2C, P < 0.0002, n = 4) along a sharp reduction in urine osmolality (Fig. 2D, P < 0.0002, n = 5) within the first 24 h, as compared to control (n = 4). Water intake (Fig. 2B) and urine volume (Fig. 2C) increased further and remained elevated for up to 6 days. Inversely, urine osmolality decreased further to a nadir value and remained low for the duration of the treatment (Fig. 2D).

Fig. 2

Low dose of Adenine alters water balance and urine osmolality without affecting food intake. Rats were placed individually in metabolic cages and had free access to powdered rodent chow in the absence (Control, n = 4) or presence of 2500 mg/kg adenine (n = 4) with free access to distilled water. A Food intake was not significantly affected by adenine for the duration of the experiment (P > 0.05 vs. Control. B Daily water intake in control and adenine-containing rodent chow. After switching to an adenine diet, water intake increased significantly within 48 h (*P,0.02, n = 4) and remained elevated for the duration of adenine feeding (**P < 0.001, n = 4), as compared to Control(n = 4). C Corresponding daily urine output, which increased significantly during the first 24 h after switching to adenine diet (*P < 0.05, **P < 0.003; n = 4) but increased incrementally after that to a maximum level after 6 days (¶P < 0.0002, n = 4) of the treatment, vs. Control (n = 4). D Urine osmolality dropped sharply within 24 h (*P < 0.04, n = 4) after switching to adenine diet, dropped further after 48 h (P < 0.003, n = 4), and remained low for the duration of the experiment (*P < 0.0002, n = 4) vs. Control (n = 4). These physiologic parameters remained unchanged during the duration of the experiment in rats fed a control diet. Daily data points are mean ± SEM. Data points at time zero are the average of 2 data points for a 2-day baseline period

Time course and dose-dependent effect of adenine feeding on food intake, body weight, water balance, and urine osmolality

We have previously shown that a higher dose of adenine feeding (5000 mg/kg) to rats is associated with an early and sustained reduction in food intake with significant body weight loss within 1 week of adenine feeding [12]. Here, we demonstrate that lower doses of adenine (1500, 2000, and 2500 mg/kg) feeding to rats for up to 7 weeks did not significantly affect daily food intake (P > 0.05, n = 4, Fig. 3A, B, C) compared to control. However, adenine did lower body weight at all doses within the first week (P < 0.02, n = 4/group, Fig. 3D and F) and the body weight remained significantly low for up to 7 weeks (P < 0.01, n = 4/group, Fig. 3D and F) although some recovery of body weight was observed at the third week for 1500 and 2000 mg/kg adenine feeding (Fig. 3E), as compared to control.

Fig. 3

Dose–response and time course effects of adenine on food intake and body weight. Sprague Dawley Rats (n = 5) were placed individually in metabolic cages and fed control or adenine-containing diet at different doses with free access to distilled water. A: Adenine feeding did not affect food intake at any of the doses (n = 4/dose) tested vs. pooled controls (P > 0.05, n = 12). B: Adenine at all doses tested decreased body weight after 1 (P < 0.02, n = 4/dose) and 7 (P < 0.01, n = 4/dose) weeks feeding with some recovery at 3 weeks for 1500 and 2000 mg/kg adenine (P > 0.05, n = 4/dose), as compared to pooled controls (n = 12). The data presented are from the last day of control or adenine feeding

Adenine feeding at lower dose of 1500 mg/kg to rats did not alter water balance or urine osmolality for the entire duration of the treatment, as compared to control (P > 0.05, n = 4, Fig. 4). However, a significant dose-dependent increase in water intake (P < 0.04 and P < 0.01, n = 4/group, Fig. 4A, B, C) and urine volume (P < 0.02 and P < 0.001, n = 4/group, Fig. 4D, E, F) with a parallel reduction in urine osmolality (P < 0.03 and P < 0.001, n = 4/group, Fig. 4G, H, I) were observed in response to adenine feeding at 2000 and 2500 mg/kg within the first week and remained as such for up to the seventh week of the treatment, as compared to control. Of note, the effect on these parameters is more profound with 2500 mg/kg adenine feeding, as compared to control or to the other lower doses.

Fig. 4

Dose–response and time course effects of adenine on water balance and urine osmolality. Rats were placed individually in metabolic cages with free access to distilled water and control or adenine-supplemented diet at 1500, 02000, or 2500 mg/kg and euthanized after 1 (n = 4/dose), 3 (n = 4/dose), or 7 (n = 4 to 5) weeks of treatment. As shown, adenine at 1500 mg/kg did not affect water intake (A, B, C), urine output (D, E, F), or urine osmolality (G, H, I) for any of the durations of the feeding (P > 0.05). However, adenine at 2000 and 2500 mg/kg significantly increased water intake (A, B, C) and urine volume (D, E, F) and reduced urine osmolality (G, H, I) in a time- and dose-dependent manner, as compared to pooled controls (n = 12). The data presented are from the last day of control or adenine feeding. (*) vs. Control; (§) vs. 1500 mg/kg adenine; (¥) vs. 2000 mg/kg adenine

Time course and dose-dependent effect of adenine feeding on AQP2 protein abundance in the kidney

To determine the molecular mechanism underlying polyuria, polydipsia, and reduced urine osmolality in adenine-fed rats, we examined the protein abundance of the collecting duct apical water channel AQP2 in different regions of the kidneys harvested from rats fed different doses of adenine or a control diet for up to 7 weeks.

AQP2 protein abundance in the kidney after 1 week of adenine feeding at 0.15%

As shown in Fig. 4, adenine feeding at a lower dose of 1500 mg/kg did not alter water intake, urine volume, or urine osmolality at any treatment period (Figs. 4A to I). As expected, AQP2 protein abundance was not changed by 1500 mg/kg adenine for any feeding duration in the kidney regions (Data not shown).

AQP2 protein abundance in the kidney after 1 week of adenine feeding at 2000 and 2500 mg/kg

After 1 week of 2000 mg/kg adenine feeding, AQP2 protein was not sharply altered throughout the kidney regions. In fact, AQP2 protein abundance is somewhat increased in the cortex (Figs. 5A and B, P< 0.02), whereas its expression was not affected in the outer medulla (Figs. 5A and C), vs. control (P > 0.05, n = 4 in each group). However, a slight but significant reduction in the glycosylated 35 kDa form is observed in the inner medulla of 2000 mg/kg adenine-fed rats (Figs. 5A and D, P< 0.01) vs. control (n = 4 in each group). This indicates that the increased water intake (Fig. 4A, P< 0.04) and increased urine volume (Fig. 4D, P< 0.02) along with reduced urine osmolality (Fig. 4G, P< 0.03) observed in rats fed 2000 mg/kg adenine is likely due to the inhibition of AQP2 trafficking to the apical membrane, as a result of adenine inhibition of vasopressin-induced cAMP stimulation in the collecting duct principal cells, as previously demonstrated in our laboratory [12]. The inhibition of vasopressin-induced cAMP production in the thick ascending limb likely resulted in NKCC2 activity reduction, which led to the increased salt excretion in rats fed adenine at 1500 and 2000 mg/kg for 7 weeks (Table 4), as NKCC2 protein abundance was not significantly altered under these conditions (data not shown).

Fig. 5

Expression of AQP2 protein in the kidneys of control and adenine-fed rats for 1 week. A, E: Immunoblotting of water channel AQP2 protein using membrane protein fractions isolated from cortex, outer medulla (OM) and inner medulla (IM) dissected from kidneys of rats fed control (n = 4) or adenine containing diet at 2000 mg/kg (A, n = 4) or 2500 mg/kg (E, n = 4) for 1 week. Right bar graphs are the corresponding average ± SEMs of the densitometry analysis of AQP2/actin or AQP2/GAPDH bands of control (dark bars) and adenine-fed rats (gray bars) at 2000 mg/kg (B, C, D) and 2500 mg/kg (F, G, H). At 2000 mg/kg, adenine downregulated the glycosylated form (35 kDa, P < 0.01, D) of AQP2 protein in the OM vs. Control. Interestingly, adenine at 2000 mg/kg increased AQP2 protein (P < 0.02, B) in the cortex and did not affect the abundance of AQP2 native band (29 kDa) in the IM vs. Control (D, n = 4 in each). At 2500 mg/kg, adenine decreased the abundance of AQP2 glycosylated (P < 0.05, G), but did not affect the native band (G, P > 0.05) in the OM. However, adenine at 2500 mg/kg downregulated both the glycosylated (35 kDa, P < 0.003, H) and native form (29 kDa, P < 0.001, H) in the IM vs. control. Each lane was loaded with 40, 20 and 3 μg of membrane proteins from cortex, OM, and IM, respectively, from different rats

After 1 week of 2500 mg/kg adenine feeding, the abundance of AQP2 protein is not altered in the cortex (Figs. 5E and F, P> 0.05). Only the 35 kDa band was slightly but significantly reduced in the outer medulla (Figs. 5E and G, P< 0.05). In contrast, both the glycosylated (35 kDa) and native (29 kDa) bands of AQP2 protein are sharply reduced (P < 0.003 and P < 0.001, respectively) in the inner medulla of 2500 mg/kg adenine feeding vs. Control (Figs. 5E and 5H, n= 4 in each group). The downregulation of AQP2 in the inner medulla (~ 60%) accounts for the significant polyuria and polydipsia along with reduced urine osmolality exhibited by rats fed 2500 mg/kg adenine (Figs. 2, 4A, D, and G).

AQP2 protein abundance in the kidney regions after 3 and 7 weeks of adenine feeding

In the cortex, AQP2 protein abundance was not significantly altered in response to 2000 or 2500 mg/kg of adenine feeding for 3 or 7 weeks, as compared to the control adenine-free diet (Table 3, n = 4 in each group). In the outer medulla, AQP2 protein abundance is significantly downregulated only in response to 2500 mg/kg adenine feeding for both 3 weeks (−35%, P < 0.04, n = 4) and 7 weeks (−57%, P < 0.001, n = 5), as compared to control diet (Table 3). In the inner medulla, the AQP2 protein abundance is significantly downregulated in response to both 2000 mg/kg (P < 0.04) and 2500 mg/kg (P < 0.001) adenine during both 3 (n = 4) and 7 weeks (n = 5) of adenine feeding vs. control diet (Table 3). The downregulation of AQP2 protein in the outer and inner medulla accounts for the increased urine volume and water intake along with reduced urine osmolality caused by 2000 and 2500 mg/kg adenine during both periods of 3 and 7 weeks of feeding (Figs. 4B to I).

Table 3 AQP2 protein abundance expressed as AQP2/actin or AQP2/GAPDH ratios in the kidney regions of control and adenine-fed rats for 3 and 7 weeksResponse of rats fed 2500 mg/kg adenine or control diets to water deprivation and exogenous vasopressin treatment

The kidney adapts to water deprivation by increasing water reabsorption through the upregulation of AQP2 in the collecting duct system. To test whether this function is conserved or impaired in rats fed 2500 mg/kg adenine, we subjected rats to water deprivation for up to 48 h. Accordingly, rats were fed a control or 2500 mg/kg adenine-containing diet for 6 days and then subjected to water deprivation for up to 48 h. As shown, 2500 mg/kg adenine feeding significantly increased urine output (Fig. 6A, P< 0.05) and decreased urine osmolality (Fig. 6B, P< 0.01) compared to the control diet (n = 5 in each group) at baseline. In response to water deprivation, urine output decreased sharply in both Control and adenine-fed rats. However, adenine-fed rats exhibited a significantly higher water loss after both 24 (Fig. 6A, P< 0.002), and 48 (Fig. 6A, P< 0.0005) hours, as compared to control rats (n = 5 in each group). In response to water deprivation, urine osmolality increased slightly with similar magnitude in both groups during the first 24 h (Fig. 6B). However, the increase in urine osmolality was sharply reduced in adenine-fed rats vs. controls during the 48 h period of water deprivation (Fig. 6B, P < 0.002, n = 5 in each group). This indicates that adenine at a lower dose of 2500 mg/kg causes a significant urinary concentrating defect.

Fig. 6

Adenine causes urinary concentrating defect and vasopressin resistance in rats. Rats were placed individually in metabolic cages and fed control or adenine (2500 mg/kg) containing diet for 6 days, and then both control and adenine-fed rats were deprived of water for 48 h. Urine volume (A) and urine osmolality (B) were measured daily before and after water deprivation. As shown, urine volume decreased sharply after water removal in control rats. However, adenine-fed rats exhibited a significantly higher water wasting at 24 (P < 0.002, n = 5) and 48 (P < 0.0005, n = 5) hours after water deprivation, as compared to control rats (n = 5). Urine osmolality increased with the same magnitude in the first 24 h but significantly less in adenine-fed rats during the second day (P < 0.0005, n = 5) of water deprivation, as compared to Control rats (n = 5). #P < 0.02, ¥P < 0.05, *P < 0.01, §P < 0.002, ¶P < 0.0005 vs. Control. Another set of rats was fed control or 2500 mg/kg adenine for 5 days and then injected with a single dose of vasopressin V2 receptor agonist dDAVP. Urine volume (C) and urine osmolality (D) were measured before and 24 h after dDAVP. In response to dDAVP, urine volume decreased significantly (P < 0.0001, n = 5) in Control but not (P > 0.05, n = 5) in adenine-fed rats, and urine osmolality increased significantly in Control (P < 0.0001, n = 5) but remained unchanged (P > 0.05, n = 5) in adenine-fed rats. NS: not significant

To ascertain that adenine-induced urinary concentrating defect involves a renal resistance to vasopressin during dehydration, we tested the response of rats fed 2500 mg/kg adenine or control diets to exogenous V2 receptor agonist (dDAVP), as described in Methods. The results depicted in Fig. 6 show that dDAVP decreased urine output significantly in control (Fig. 6C, P< 0.00, n = 5) but not in adenine-fed rats (Fig. 6C, P> 0.05, n = 5). Similarly, dDAVP increased urine osmolality significantly in control (Fig. 6D, P< 0.0001, n = 5) but not in adenine-fed rats (Fig. 6D, P> 0.05, n = 5). These results suggest that a lower dose of adenine feeding causes a significant renal resistance to vasopressin and impairs the ability of the kidney to conserve water and concentrate urine in response to both dehydration and exogenous vasopressin treatment.

Electrolytes excretion and NKCC2 expression: time course effects and adenine dose–response

Urinary excretion of Na+, K+, and Cl− was measured in urine collected during the last 24 h of 1, 3, and 7 weeks from rats fed control or adenine-containing diet at different doses (1500, 2000, or 2500 mg/kg). As shown in Table 4, the adenine diet, at all doses tested, did not affect the urinary excretion of Na+, K+, or Cl− for up to 3 weeks, as compared to the control diet (Table 4, P > 0.05, n = 4 in each group). However, after long-term feeding (7 weeks), adenine did significantly increase the excretion of these electrolytes at 1500 mg/kg (Na+ and K+) and 2000 mg/kg (Na+, K+, Cl−) doses, as compared to the control diet (Table 4, P < 0.01, n = 4 in each). At 2500 mg/kg, adenine did not alter the absolute excretion of these electrolytes (Table 4, P > 0.05, n = 5 in each); however, at this dose adenine did reduce food intake and thus the electrolytes intake, and when the results are expressed as a ratio of electrolytes excretion over food intake, a significant wasting of Na+ (P < 0.02), K+ (P < 0.001) and Cl− (P < 0.04) is observed in rats-fed 2500 mg/kg adenine vs. Control (Fig. 7A, n = 5 in each). The increase in Na+, K+, or Cl− wasting in rat-fed adenine diet for 7 weeks (Table 4) likely results from the inhibition of NKCC2 activity at 1500 and 2000 mg/kg adenine feeding, as its protein abundance was not significantly altered (Data not shown), and from the downregulation of NKCC2 in response to 2500 mg/kg adenine, as demonstrated by the immunoblot depicted in Fig. 7C and corresponding densitometry scanning shown in Fig. 7D (P < 0.003 vs. Control, n = 5 in each). Adenine feeding at 2500 mg/kg did not significantly alter the expression of Na+/H+ exchanger NHE3 in the medullary thick ascending limb (P > 0.05, Figs. 7C and D), and similar results were observed for 1500 and 2000 mg/kg adenine feeding vs. control (data not shown).

Table 4 Time course and adenine (mg/kg) dose–response of urinary Na+, K+, and Cl− excretionFig. 7

Electrolytes (Na+, K+, and Cl−) excretion, creatinine excretion, and expression of NKCC2 and NHE3 in the kidney outer medulla. Rats were fed a control or 2500 mg/kg adenine diet with free access to distilled water for 7 weeks. A Rats were placed individually in metabolic cages for food intake measurement and 24-h urine collection. Urinary Na+, K+, and Cl− excretion was measured and adjusted for food intake in control vs. 2500 mg/kg adenine feeding. As shown, electrolyte excretion/food intake increase significantly in adenine-fed (*P < 0.02, **P < 0.001, and ¶P < 0.04, n = 5) vs. Control (n = 5) rats. B Urinary creatinine excretion in control and adenine-fed rats at 2000 or 2500 mg/kg for 3 or 7 weeks. A significant reduction (−30%) in creatinine excretion is observed only in rats fed adenine at 2500 mg/kg for 7 weeks. C Immunoblots of NHE3, NKCC2, and actin in membrane fractions isolated from the kidney outer medulla of Control and 2500 mg/kg adenine-fed rats for 7 weeks. D: Densitometry of NHE3 and NKCC2 normalized to actin. As shown, adenine feeding for 7 weeks significantly downregulated NKCC2 protein abundance (§P < 0.003, n = 5) but did not affect NHE3 protein expression (P > 0.05, n = 5) in the kidney outer medulla, as compared to Control (n = 5). Each lane was loaded with 20 μg (NHE3) or 10 μg (NKCC2) membrane proteins from the outer medulla of kidneys harvested from different rats. NS: Not significant

Figure 7B shows that adenine feeding at doses of 2000 and 2500 mg/kg for 3 weeks did not significantly alter creatinine excretion compared to control rats (P > 0.05, Fig. 7B; n = 5 per group). However, long-term adenine administration (7 weeks) at 2500 mg/kg resulted in a significant 30% reduction in creatinine excretion (P < 0.01, Fig. 7B; n = 5), while the 2000 mg/kg dose had no significant effect (P > 0.05, Fig. 7B; n = 5), relative to control. Creatinine excretion was consistent with serum creatinine levels, which increased significantly (~ 38%) after 7 weeks of adenine feeding at 2500 mg/kg compared to control (Table 2). In contrast, 3-week adenine feeding did not significantly affect serum creatinine levels at either 2000 mg/kg (27 ± 1.61 µmol/L, n = 5, P > 0.05) or 2500 mg/kg (25 ± 0.90 µmol/L, n = 5, P > 0.05) compared to control (25 ± 1.71 µmol/L, n = 5).

Adenine at a lower dose prevents hyponatremia in a rat model of SIADH

We have previously shown that adenine inhibits vasopressin-induced cAMP production in vitro and urinary cAMP excretion in rats [12]. The above studies clearly demonstrate that adenine feeding at lower doses of 2000 and 2500 mg/kg in rats causes salt-free water wasting (aquaresis), at least for up to 3 weeks of treatment. Hence, the rationale behind this experiment is to test whether adenine can prevent the development of hyponatremia in a rat experimental model of SIADH. Indeed, the results depicted in Fig. 8 show that rats fed a liquid diet and treated with daily injections of dDAVP developed significant dilutional hyponatremia, as shown by a sharp reduction in serum [Na+] (Fig. 8, P < 0.001, n = 5), as compared to either control or 2500 mg/kg adenine-fed rats without dDAVP treatment. Interestingly, feeding rats with a liquid diet containing 2500 mg/kg adenine before daily injection of dDAVP prevented the development of hyponatremia (Fig. 8, P > 0.05, n = 5).

Fig. 8

Adenine feeding prevents the development of hyponatremia in a rat model of SIADH. Serum [Na+] was measured in rats fed rodent chow alone (Control) or rodent chow supplemented with 2500 mg/kg adenine for 1 week. Another set of rats was fed a liquid diet alone (dDAVP) or a liquid diet supplemented with 2500 mg/kg adenine (Adenine + dDAVP) for 3 days, and then both groups were injected with dDAVP (3 µg/100 g, SC) for an additional 3 days. As shown, liquid diet feeding + dDAVP caused a significant reduction in serum [Na.+] or hyponatremia (*P < 0.001, n = 5), as compared to Control (n = 4) or adenine feeding alone (n = 4). Hyponatremia is prevented in the presence of 2500 mg/kg adenine (P > 0.05, n = 5) vs. Control (n = 4) or adenine feeding alone (n = 4)