Volume 67, Issue 1, Pages 201-216 (January 2005) Vasopressin-independent regulation of collecting duct aquaporin-2 in food deprivation  Catherine Wilke, Sulaiman Sheriff, Manoocher Soleimani, Hassane Amlal, Ph.D.  Kidney International  Volume 67, Issue 1, Pages 201-216 (January 2005) DOI: 10.1111/j.1523-1755.2005.00071.x Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 1 Effects of food deprivation on water balance and urine osmolality. Teen rats were placed in metabolic cages, with free access to food and water. At time 0, rats were deprived of food but had free access to water. Water intake (A), urine volume (B), and urine osmolality (C) were monitored every 24 hours for the duration of the experiment. Rats were sacrificed after 24 (N = 5) and 72 (N = 5) hours of food deprivation. *P < 0.0001, **P < 0.001, §P < 0.002 vs. baseline. Kidney International 2005 67, 201-216DOI: (10.1111/j.1523-1755.2005.00071.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 2 Expression of cortical AQP2 in food deprivation (FD). The expression of AQP2 was examined in the cortex harvested from control and food-deprived rats. Immunoblotting experiments showing AQP2 protein abundance in membrane fractions isolated from cortex (A, upper panel). Corresponding densitometric analysis showing a biphasic effect of food deprivation on AQP2 protein abundance (*P < 0.0001 vs. control, **P < 0.001 vs. 24 hours of food deprivation, N = 5 rats in each group) (lower panel). Densitometric analysis showing the mean expression of AQP2 mRNA-to-28S rRNA in the cortex of control and food deprived rats (B). Food deprivation did not alter AQP2 mRNA at 24 hours but increased its expression levels after 72 hours (§P < 0.01 vs. control, N = 5 rats in each group). Twenty micrograms of total protein and 30 μg of total RNA from different rats were loaded per lane in the immunoblotting (A) and Northern hybridization experiments (B), respectively. Kidney International 2005 67, 201-216DOI: (10.1111/j.1523-1755.2005.00071.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 3 Expression of outer medullary AQP2 in food deprivation (FD). Outer medulla was isolated from kidneys of control and food-deprived rats and used for total membrane proteins and total RNA isolation (A). Immunoblotting experiments showing AQP2 protein abundance in the outer medulla (upper panel). Corresponding densitometric analysis showing dual effect of food deprivation on AQP2 protein abundance (§P < 0.0001 vs. control, *P < 0.0001 vs. 24 hours of food deprivation, §§P < 0.01 vs. control, **P < 0.001 vs. 24 hours of food deprivation, N = 5 rats in each group) (lower panel). Densitometric analysis showing the mean expression of AQP2 mRNA-to-28S rRNA in the outer medulla of control and food deprived rats (B). AQP2 mRNA expression levels correlates with its protein abundance in food deprivation (*P < 0.05 vs. control, §P < 0.01 vs. 24 hours of food deprivation, N = 5 rats in each group). Twelve micrograms of total protein and 30 μg of total RNA from different rats were loaded per lane in the immunoblotting (A) and Northern hybridization experiments (B), respectively. Kidney International 2005 67, 201-216DOI: (10.1111/j.1523-1755.2005.00071.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 4 Expression of inner medullary AQP2 in food deprivation (FD). Inner medulla was isolated from kidneys of control and food-deprived rats and used for total membrane proteins and total RNA isolation (A). Immunoblotting experiments showing AQP2 protein abundance in the inner medulla (upper panel). Corresponding densitometric analysis showing that AQP2 protein abundance did not change at 24 hours of food deprivation but increased significantly after 72 hours of food deprivation (*P < 0.004 vs. control, §P < 0.05 vs. control, N = 5 rats in each group) (lower panel). Densitometric analysis showing the mean expression of AQP2 mRNA-to-28S rRNA in the inner medulla of control and food deprived rats (B). Food deprivation did not alter AQP2 mRNA at 24 hours but increased its expression levels after 72 hours (**P < 0.05 vs. control, N = 3 different RNA samples isolated from pooled IM tissues of different rats in each group). Three micrograms of total protein and 30 μg of total RNA from different rats were loaded per lane in the immunoblotting (A) and Northern hybridization experiments (B), respectively. Kidney International 2005 67, 201-216DOI: (10.1111/j.1523-1755.2005.00071.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 5 Expression of cortical AQP1 in food deprivation (FD). The same membrane fractions used above for cortical AQP2 Figure 2. were used here for AQP1 immunoblotting. Immunoblotting experiment showing AQP1 protein abundance in control and food-deprived rats (upper panel). Corresponding densitometric analysis showing that AQP1 protein abundance remained unchanged during the entire period of food deprivation (lower panel). Seven micrograms of total cortical protein from different rats were loaded per lane. N = 5 rats in each group. Kidney International 2005 67, 201-216DOI: (10.1111/j.1523-1755.2005.00071.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 6 Water balance and urine osmolality in response to sodium deprivation. Five rats were placed in metabolic cages and fed control diet with free access to distilled water for four days. At time 0, rats were switched to a Na+-free diet but remained on distilled water for another 5 days. Water intake (A), urine volume (B), and urine osmolality (C) were monitored every 24 hours for the duration of the experiment. Kidney International 2005 67, 201-216DOI: (10.1111/j.1523-1755.2005.00071.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 7 Water balance and urine osmolality in response to chloride deprivation. Five rats were placed in metabolic cages and fed control diet with free access to distilled water for three days. At time 0, rats were switched to a chloride-free diet but remained on distilled water for another 5 days. Water intake (A), urine volume (B), and urine osmolality (C) were monitored every 24 hours for the duration of the experiment. Kidney International 2005 67, 201-216DOI: (10.1111/j.1523-1755.2005.00071.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 8 Plasma vasopressin levels and vasopressin precursor mRNA expression in food deprivation (FD). Plasma vasopressin (AVP) levels measured by radioimmunoassay (N = 6 rats in each group) (A). Representative Northern hybridization of vasopressin preprohormone mRNA and 28S rRNA levels in control and food deprived rats (B, upper panels). Each lane was loaded with 30 μg total RNA isolated from a different rat. Corresponding densitometric analysis of mean AVP mRNA-to-28S rRNA ratio (N = 6 rat for each group) (lower panel). Food deprivation did not alter vasopressin synthesis or secretion. Kidney International 2005 67, 201-216DOI: (10.1111/j.1523-1755.2005.00071.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 9 Plasma vasopressin levels and vasopressin precursor mRNA expression in water deprivation. Plasma vasopressin (AVP) levels measured by radioimmunoassay in control and food deprived rat (N = 6 rats in each group) (A). Representative Northern hybridization of vasopressin precursor mRNA and 28S rRNA levels in control and water deprived rats (B, upper panels). Each lane was loaded with 30 μg total RNA isolated from a different rat. Corresponding densitometric analysis of mean AVP mRNA-to-28S rRNA ratio (N = 6 rat for each group) (lower panel). Water deprivation increased both the synthesis and secretion of vasopressin. Kidney International 2005 67, 201-216DOI: (10.1111/j.1523-1755.2005.00071.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 10 Effects of food deprivation (FD) on the circulating levels of adrenal steroids. Corticosterone (A) and aldosterone (B) levels were measured in the serum of control and food deprived rats by radioimmunoassay. FD increased corticosterone levels but did not affect the circulating levels of aldosterone. Kidney International 2005 67, 201-216DOI: (10.1111/j.1523-1755.2005.00071.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 11 Effects of food deprivation on water balance and urine osmolality in Brattleboro rats. Eight rats were placed in metabolic cages, with free access to food and water. At time 0, rats were deprived of food but had free access to water. Water intake (A), urine volume (B), and urine osmolality (C) were monitored every 24 hours for the duration of the experiment. Rats were sacrificed after 24 (N = 4) and 72 (N = 5) hours of food deprivation. *P < 0.0.05, **P < 0.02§P < 0.001, §§P < 0.0001 vs. baseline. Kidney International 2005 67, 201-216DOI: (10.1111/j.1523-1755.2005.00071.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 12 Expression of cortical AQP2 in food deprived Brattleboro rats. Immunoblotting experiments of AQP2 protein abundance (A, upper panel). Corresponding densitometric analysis showing a biphasic effect of food deprivation (FD) on AQP2 protein abundance (*P < 0.001 vs. control, **P < 0.001 vs. 24hrs of FD, N = 4 rats in each group) (lower panel). Northern hybridization of AQP2 mRNA and 28S rRNA levels in control and food-deprived rats (B, upper panel). Densitometric analysis showing the mean expression of AQP2 mRNA-to-28S rRNA in the cortex of control and food-deprived rats (lower panel). Food deprivation did not alter AQP2 mRNA at 24 hours but increased its expression levels after 72 hours (§P < 0.05 vs. 24hrs of FD, N = 3 to 4 rats in each group). Thirty micrograms of total protein and 30 μg of total RNA from different rats were loaded per lane in the immunoblotting (A) and Northern hybridization experiments (B), respectively. Kidney International 2005 67, 201-216DOI: (10.1111/j.1523-1755.2005.00071.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 13 Expression of outer medullary AQP2 in food-deprived Brattleboro rats. Immunoblotting experiments showing AQP2 protein abundance in the outer medulla (A, upper panel). Corresponding densitometric analysis showing dual effect of food deprivation (FD) on AQP2 protein abundance (**P < 0.001 vs. control, *P < 0.05 vs. 24 hours of FD, §P < 0.0003 vs. control, N = 4 rats in each group) (lower panel). Northern hybridization of AQP2 mRNA and 28S rRNA levels in control and food-deprived rats (B, upper panel). Densitometric analysis showing the mean expression of AQP2 mRNA-to-28S rRNA in the outer medulla of control and food-deprived rats (lower panel). AQP2 mRNA expression levels correlates with its protein abundance in food deprivation (**P < 0.05 vs. control, *P < 0.05 vs. 24 hours of FD, N = 3 to 4 rats in each group). Twenty micrograms of total protein and 30 μg of total RNA from different rats were loaded per lane in the immunoblotting (A) and Northern hybridization experiments (B), respectively. Kidney International 2005 67, 201-216DOI: (10.1111/j.1523-1755.2005.00071.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 14 Effect of food deprivation (FD) on inner medullary AQP2 expression in Brattleboro rats. Immunoblotting experiments of AQP2 protein abundance in the inner medulla (upper panel). Corresponding densitometric analysis showing AQP2 protein abundance in control and food-deprived rats (**P < 0.0001 vs. control, *P < 0.05 vs. control, N = 4 rats in each group) (lower panel). Food deprivation did not alter AQP2 protein at 24 hours but increased its abundance after 72 hours. Twelve micrograms of total protein from different rats were loaded per lane. Kidney International 2005 67, 201-216DOI: (10.1111/j.1523-1755.2005.00071.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 15 Effect of food deprivation on body weight and plasma osmolality in Sprague-Dawley rats. The figure shows the time course changes in body weight (A) and plasma osmolality (B) before and after food deprivation. The number (N) indicates the number of animals. **P < 0.001 vs. control group and *P < 0.05 vs. control or vs. 24 hours of fasting. Kidney International 2005 67, 201-216DOI: (10.1111/j.1523-1755.2005.00071.x) Copyright © 2005 International Society of Nephrology Terms and Conditions