Adenine at lower doses acts in the kidney as an aquaretic agent and prevents hyponatremia

Abstract

We have previously reported that adenine at high doses interferes with the vasopressin signaling pathway, causes massive diuresis and volume depletion, and ultimately leads to renal failure. In the present study, we examined the effects of adenine on renal salt and water handling in a time course and dose-response study in rats housed in metabolic cages and fed control or adenine-containing diet at 1500, 2000, 2500 mg/kg and euthanized after 1, 3, and 7 weeks. Adenine at 2000 and 2500 mg/kg caused early and significant polyuria, polydipsia, and decreased urine osmolality in a dose-dependent manner without significantly affecting food intake, blood volume, blood electrolyte levels, or acid-base composition. The impaired water balance resulted from the downregulation of apical water channel AQP2 in the outer and inner medulla but not in the cortex. Adenine did not alter electrolytes (Na⁺, K⁺, Cl^-) excretion at these doses for up to 3 weeks. However, a slight but significant increase in salt excretion was observed in adenine-fed rats for 7 weeks, which correlates with a significant downregulation of NKCC2, mostly in rats fed 2500 mg/kg adenine. Adenine-fed rats exhibited a substantial resistance to vasopressin in response to water deprivation or vasopressin treatment. Lastly, 2500 mg/kg adenine prevented the development of hyponatremia in a rat experimental model of the syndrome of inappropriate secretion of antidiuretic hormone (SIADH). In conclusion, adenine acts as an aquaretic agent in the kidney at lower doses and during a short feeding period. It can be used as a vasopressin antagonist in conditions associated with hyponatremia.

Keywords: AQP2; Adenine; Aquaresis; Hyponatremia; Vasopressin resistance.

Fig. 1

Study design diagram. Sprague Dawley rats were switched to a liquid diet prepared using rodent chow as described in Methods in the absence (n = 5) or presence (n = 5) of 2500 mg/kg adenine with free access to distilled water. After 3 days, rats in both groups were then injected daily with dDAVP (3 µg/100 g BW, SC.) and euthanized after 3 days. The consumption of a liquid diet allows the animals to be water-loaded while they satisfy their hunger and their needs in calorie intake

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

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

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

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

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

Fig. 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

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)