Early polyuria and urinary concentrating defect in potassium deprivation

Hassane Amlal1, Carissa M. Krane2, Qian Chen1, and Manoocher Soleimani1,3

Departments of 1 Medicine, and 2 Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati School of Medicine, Cincinnati 45267-0585, and 3 Veterans Affairs Medical Center at Cincinnati, Cincinnati, Ohio 45220


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The time course of the onset of nephrogenic diabetes insipidus and its relationship to aquaporin-2 (AQP2) expression in K+ deprivation (KD) remains unknown. Rats were fed a K+-free diet and killed after 12 h, 1, 2, 3, 6, or 21 days. Serum K+ concentration was decreased only after, but not before, 3 days of a K+-free diet. Urine osmolality, however, decreased as early as 12 h of KD (1,061 ± 26 vs. 1,487 ± 102 mosmol/kgH2O in control, P < 0.01). It decreased further at 24 h (to 858 ± 162 mosmol/kgH2O in KD, P < 0.004) and remained low at 21 days of KD (436 ± 58 mosmol/kgH2O, P < 0.0001 compared with baseline). Water intake decreased at 12 h (P < 0.002) but increased at 24 h (P < 0.05) and remained elevated at 21 days of KD. Urine volume increased at 24 h of KD (8 ± 2 to 15 ± 2 ml/24 h, P < 0.05) and remained elevated at 21 days. Immunoblot analysis demonstrated that AQP2 protein abundance in the outer medulla remained unchanged at 12 h (P > 0.05), decreased at 24 h (~44%, P < 0.001), and remained suppressed (~52%, P < 0.03) at 21 days of KD. In the inner medulla the AQP2 protein abundance remained unchanged at both 12 and 24 h of KD. AQP2 protein abundance in the cortex, however, decreased at 12 h (~47%, P < 0.01) and remained suppressed at 24 h (~77%, P < 0.001) of KD. Northern blot analysis showed that AQP2 mRNA decreased as early as 12 h of KD in both cortex (P < 0.02) and outer medulla (P < 0.01) and remained suppressed afterward. In conclusion, the urinary concentrating defect in KD is an early event and precedes the onset of hypokalemia. These studies further suggest that the very early urinary concentrating defect in KD (after 12 but before 24 h) results primarily from the suppression of cortical AQP2, whereas the later onset of a urinary concentrating defect (after 24 h) also involves a downregulation of medullary AQP2.

antidiuretic hormone; aquaporin-2; kidney


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

K+ deprivation causes impairment of the urine-concentrating ability in humans (32, 33) and experimental animals (2, 5, 11, 12, 25, 31). The mechanism by which K+ deprivation causes a urinary concentrating defect is not fully understood. Earlier studies have shown that K+ deprivation-induced polyuria was not corrected by the administration of antidiuretic hormone (ADH) (16). Since then several mechanisms have been proposed to explain the defect in urinary concentrating ability in K+ depletion, including prostaglandin overproduction (10), blunted responsiveness to antidiuretic hormone (21), primary polydipsia (38), altered ADH release by the posterior pituitary gland (34), abnormal medullary oxidative metabolism (19), and reduced medullary solute (24).

ADH [or arginine vasopressin (AVP)] plays an essential role in fluid homeostasis by increasing the reabsorption of water in the collecting duct. This occurs predominantly in the principal cells via a transcellular pathway involving highly specialized water channels [aquaporin-2 (AQP2), AQP3, and AQP4]. AQP2, the AVP-regulated water channel (4, 14, 40), is expressed in the apical surface of the entire collecting duct system (9). Subcellular immunolocalization experiments demonstrate that AQP2 is exclusively restricted to the apical membrane of principal cells, with strong labeling in small subapical vesicles (8, 29, 30). This distribution is consistent with the rapid regulation of water transport through AQP2 by AVP (41, 42). In the outer and inner medulla, AQP2 is present in both glycosylated (~35 kDa) and nonglycosylated (~29 kDa) forms, whereas in the cortex it is detected only in the nonglycosylated form (25, 30). AQP3 and AQP4 are coexpressed with AQP2 in the principal cells and are located exclusively in the basolateral membrane domain (17, 38, 39). A number of pathophysiological states including K+ deprivation are associated with dysregulation of kidney water channels. A recent study reported that rats on a K+-free diet for 11 days exhibited a significant decrease in the expression of the AQP2 protein in the cortical and inner medullary collecting duct, along with a urinary concentrating defect (25).

It must be emphasized that in all K+ restriction studies to date, experiments on water balance and/or AQP2 expression have been performed after 10 days of treatment. As such, the onset of polyuria and the possible signals involved in the generation of nephrogenic diabetes insipidus in K+ depletion remain unknown. The purpose of these studies was to examine the time course of the onset of the urine concentrating defect and its possible molecular basis in K+ deprivation.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
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Animal model. Male Sprague-Dawley rats (125-150 g) were placed in metabolic cages 3 days before starting a K+-deficient diet. Rats had free access to food and water. Animals were divided into two groups. The first group was fed a normal diet (control) for the entire duration of the experiment. The second group was switched to a K+-free diet (catalog no. 960189, ICN Biochemical) after 3 days on a normal diet. Animals were studied for up to 21 days. A third group was fed a K+-deficient diet but had its drinking water supplemented with 0.85% KCl. In both control and K+-deprived groups, rats were killed at different time points (12 h, 1, 2, 3, 6, and 21 days) by intraperitoneal injection of 50 mg of pentobarbital sodium. Water intake, urine output, urine osmolality, and body weight were measured daily. At the time of death, intracardiac blood was obtained for serum K+ ([K+]) and and HCO3- concentration ([HCO3-]) measurement. Kidneys were removed and cortex, inner stripe of outer medulla, and inner medulla were dissected and snap-frozen in liquid nitrogen.

RNA isolation and Northern hybridization. Total cellular RNA was extracted from renal cortex, inner stripe of outer medulla, and inner medulla by the method of Chomczynski and Sacchi (6), quantitated spectrophotometrically, and stored at -80°C. Total RNA samples (30 µg/lane) were fractionated on a 1.2% agarose-formaldehyde gel and transferred to nylon membranes by capillary transfer using 10× sodium chloride-sodium phosphate-EDTA buffer. Membranes were cross-linked by ultraviolet light and baked. Hybridization was performed according to Church and Gilbert (7). Membranes were washed, blotted dry, exposed to PhosphorImager screens at room temperature for 24-72 h, and scanned by the PhosphorImager. A 32P-labeled cDNA fragment corresponding to nucleotides 281-651 of the mRNA-encoding rat AQP2 (Gene Bank accession no. D13906) was used as a specific probe. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe was a generous gift from Drs. P. James and J. Lingrel at the University of Cincinnati.

AQP2-specific antibodies. Peptide-derived polyclonal antibodies specific to the AQP2 water channel was raised by using commercial services (Genosys Biotechnologies, The Woodlands, TX). As it has been used successfully by other investigators before (30), the rat AQP2 peptide NH2-CEVRRRQSVELHSPQSLPRGSKA-COOH, which corresponds to amino acid residues 250-271 of the COOH-terminal tail of the vasopressin-regulated AQP2 water channel, was used to develop the AQP2 antibody. A cysteine residue was added at the NH2-terminal end of the peptide to facilitate its conjugation with to a carrier protein. Two rabbits were immunized with the conjugate. Both rabbits developed ELISA titers greater than 1:100,000. The antisera were affinity purified by covalently immobilizing the immunizing peptide on commercially available columns (Sulfo-Link Immobilization Kit 2, Pierce, Rockford, IL).

Preparation of membrane fractions from renal cortex, outer medulla and inner medulla. A crude total membrane fraction containing plasma membrane and intracellular membrane vesicles was prepared as described (20) with minor modifications. Briefly, the tissue samples were homogenized in ice-cold isolation solution (250 mM sucrose and 10 mM triethnolamine, pH 7.6) containing protease inhibitors (phenazine methylsulfonyl fluoride, 0.1 mg/ml; leupeptin, 1 µg/ml) by using a Polytron homogenizer. The homogenates were centrifuged at low speed (1,000 g) for 10 min at 4°C to remove nuclei and cell debris. After this, the supernatants were spun at 150,000 g for 90 min at 4°C. The pellets containing plasma membrane and intracellular vesicles were suspended in isolation solution with protease inhibitors. The total protein concentration was measured, and the membrane fractions were solubilized at 60°C for 20 min in Laemmli sample buffer.

Electrophoresis and immunoblotting. Experiments were carried out as described (20). Briefly, the solubilized membrane proteins were size fractionated on 12% polyacrylamide minigels (Novex, San Diego, CA) under denaturing conditions. By using a Bio-Rad transfer apparatus (Bio-Rad Laboratories, Hercules, CA), the separated proteins were electrophoretically transferred to nitrocellulose membranes. The membranes were blocked with 5% milk proteins and then probed with affinity-purified anti-AQP2 immune serum at an IgG concentration of 0.14 µg/ml. The secondary antibody was donkey anti-rabbit IgG conjugated to horseradish peroxidase (0.16 µg/ml; Pierce). The sites of antigen-antibody complexation on the nitrocellulose membranes were visualized by using the chemiluminescence method (SuperSignal Substrate, Pierce) and captured on light-sensitive imaging film (Kodak). Bands corresponding to 29- and 35-kDa AQP2 protein were quantitated by densitometric analysis (ImageQuant 5.0, Molecular Dynamics, Sunnyvale, CA) and expressed as percentage of control.

Materials. [32P]dCTP was purchased from New England Nuclear (Boston, MA). Nitrocellulose filters and other chemicals were purchased from Sigma (St. Louis, MO). The RadPrime DNA labeling kit was purchased from GIBCO-BRL.

Statistics. Results are expressed as means ± SE. Statistical significance between experimental groups was determined by Student's t-test, and P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Serum [K+] and [HCO3-] in K+ deprivation. Rats fed a K+-free diet developed significant hypokalemia at 6 days and after, but not before, 3 days. A decline in serum [K+ ] was observed at 3 days (5.1 ± 0.2 meq/l in K+-deprived vs. 5.4 ± 0.2 meq/l in control, n = 8, P >=  0.05) but became statistically significant only after 6 days of K+ deprivation (4.9 ± 0.1 meq/l in K+-deprived vs. 5.3 ± 0.1 meq/l in control, n = 6, P < 0.05). Serum [K+] was further decreased after 2 wk of K+-free diet (2.9 ± 0.3 meq/l in K+-deprived vs. 5.3 ± 0.4 meq/l in control, n = 4, P < 0.01). Serum [HCO3-] in rats on a K+-free diet for 2 wk was not significantly different from control (27 ± 1.2 meq/l in K+-deprived vs. 25 ± 1.1 meq/l in control, n = 4, P > 0.05).

Time course effect of K+ deprivation on water balance and urine osmolality. In these experiments, rats were placed in metabolic cages and fed a normal diet. After 3 days, a group of rats was switched to a K+-free diet whereas the other group remained on a normal diet for the entire experiment (time control). Water intake, urine output, and urine osmolality were measured daily. The results are summarized in Fig. 1 and show that water intake increased from a baseline level of 23 ± 2 to 33 ± 3 ml/24 h after 24 h on a K+-free diet (P < 0.05, n = 8, Fig. 1A). The water intake further increased and plateaued at 36 ± 2 ml/24 h (n = 8) after 4 days of K+ deprivation (P < 0.001 compared with baseline, n = 8, Fig. 1A). The urine output also increased from a baseline level of 10 ± 2 to 16 ± 3 ml/24 h after 24 h of K+ deprivation (P < 0.05, n = 8, Fig. 1B). The urine volume further increased and plateaued at 24 ± 3 ml/24 h after 4 days of K+ deprivation (P < 0.0001 compared with baseline, n = 8, Fig. 1B). The urine osmolality decreased from a baseline level of 1,692 ± 105 to 897 ± 210 mosmol/kgH2O as early as 24 h of K+ deprivation (P < 0.004, n = 8, Fig. 1C). The urine osmolality further decreased and plateaued at 465 ± 135 mosmol/kgH2O after 4 days of K+ deprivation (P < 0.0001 compared with baseline, n = 8, Fig. 1C). Water intake (Fig. 1A), urine output (Fig. 1B), and urine osmolality (Fig. 1C) in the control group remained stable during the entire experiment (Fig. 1).


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Fig. 1.   Effect of K+ deprivation (KD) on water balance and urine osmolality. Each time point in A-C is the mean ± SE of indicated n rats. A: water intake decreased at 12 h (n = 9) and then increased above the baseline after 24 h of KD (n = 8). B: urine output was increased slightly at 12 (n = 9, P > 0.05) but significantly after 24 h of K+ deprivation and remained elevated (P < 0.05 vs. control, n = 8 for each). C: urine osmolality was decreased at 12 h of KD (n = 9, P < 0.01 vs. control, n = 13) and remained low for the duration of the experiment.

The above experiments indicate that the generation of a urinary concentrating defect is present at 24 h of K+ deprivation. The purpose of the next series of experiments was to study urine osmolality and urine output at time points earlier than 24 h. As indicated in Fig. 1, urine osmolality was significantly decreased as early as 12 h of K+ deprivation (from 1,692 ± 105, n = 13, to 1,235 ± 77 mosmol/kgH2O, n = 9, P < 0.01, Fig. 1C). At this early time point, water intake was actually decreased from 23 ± 2.0 (n = 13) to 16 ± 2 ml/24 h (n = 9) (P < 0.002), whereas urine output was slightly increased (from 10 ± 2, n = 13, to 12 ± 2 ml/24 h, n = 9, P > 0.05). These results indicate that a K+ deprivation-induced urinary concentrating defect is developed as early as 12 h of K+ deprivation and that the syndrome of polyuria-polydipsia follows after 24 h of K+ restriction.

Effect of K+ deprivation on AQP2 protein abundance in the kidney outer medulla. Marples et al. (25) have previously shown that rats on a K+-free diet for 11 days develop hypokalemia and demonstrate downregulation of AQP2 protein in the cortex and inner medulla (25). In this study, effect of K+ depletion on the abundance of AQP2 protein in the outer medulla was not examined (25). Therefore, we examined the effect of K+ deprivation on outer medullary AQP2 at 12 h, 1, 2, 3, 6, and 21 days of K+ deprivation. Microsomes were prepared from the inner stripe of outer medulla of experimental animals and examined for AQP2 abundance. Immunoblot analysis studies demonstrated that the AQP2 expression remained unchanged at 12 of K+ deprivation [100 ± 4.6 vs. 90 ± 3%, for control (n = 13) and 12 h K+-deprived (n = 7), respectively, P >=  0.05, Fig. 2, A and D].1 The expression of AQP2 was, however, significantly decreased after 24 h of K+ deprivation compared with control [100 ± 4.6 vs. 53 ± 7% for control (n = 13) and 24 h KD (n = 7), respectively, P <=  0.01, Fig. 2, A and D].1 After 2 days of K+ deprivation, AQP2 abundance further decreased by 89% (P <=  0.0001, n = 6, Fig. 2, B and D). Interestingly, there was a significant recovery of the AQP2 protein abundance at 3 days and up to 21 days of K+ deprivation (compared with 2 days) (Fig. 2D). However, the protein expression levels remained suppressed at any given time point vs. control, with AQP2 protein abundance decreasing to 31 ± 4, 52 ± 6, and 48 ± 5 at 3, 6, and 21 days of K+ deprivation, respectively [n = 6, P <=  0.0001; n = 5, P <=  0.03; and n = 7, P <=  0.03 for each group vs. control (n = 13), Fig. 2, B-D]. The equity in protein loading was verified by gel staining (data not shown).


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Fig. 2.   Time course effect of KD on aquaporin-2 (AQP2) protein abundance in the outer medulla (OM). Eight micrograms of total protein from OM were loaded per lane. Representative immunoblots of AQP2 protein level in the OM after 12 h (0.5 day) and 1 day (A), 2 and 3 days (B), and 6 and 21 days (C) of K+ deprivation are shown as are 29- and 35-kDa AQP2 bands. D: densitometry of AQP2 protein (29- and 35-kDa) expression in the OM at time 0 (control, n = 13) and 12 h (n = 7), 1 day (n = 7), 2 days (n = 6), 3 days (n = 6), 6 days (n = 5), and 21 days (n = 7) of KD. A significant decrease in AQP2 protein became evident only after 1 day of KD. ** P <=  0.01, * P <=  0.0001, and § P <=  0.03 compared with pooled controls (n = 13).

Effect of K+ deprivation on mRNA expression of AQP2 in the outer medulla. We next examined the expression of AQP2 mRNA in K+ deprivation to correlate the results with immunoblot studies. Total RNA was isolated from the inner stripe of outer medulla of rats at 12 h, 1, 2, 3, 6, or 21 days after the start of the K+-free diet and utilized for Northern hybridization. As shown in Fig. 3, and compared with control (n = 9), AQP2 mRNA expression decreased significantly (~42%, n = 7, P <=  0.01) as early as 12 h of K+ deprivation (Fig. 3, A and C) and further decreased (~57%, n = 7, P <=  0.01) after 24 h of K+ deprivation (Fig. 3, A and C). The AQP2 mRNA levels remained suppressed at 2 (70 ± 4%, n = 7, P <=  0.04 vs. control), 3 (45 ± 6%, n = 7, P <=  0.03), and up to 21 days of K+ deprivation (Fig. 3, B and C). Taken together, these results indicate that the downregulation of AQP2 expression in the outer medulla is an early event and precedes the onset of hypokalemia.


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Fig. 3.   Time course effect of KD on AQP2 mRNA expression in the OM. Representative Northern hybridization of AQP2 mRNA level in the OM at 12 and 24 h (A) and 2 and 3 days (B) of KD (top) are shown. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression is shown as the control for RNA loading (bottom). C: mean AQP2 mRNA-to-GAPDH mRNA ratio. Mean expression levels declined by 42% after 12 h (n = 7, P <=  0.01) and by 67% (n = 7, P <=  0.01) after 24 h of KD. AQP2 mRNA levels decreased by 30 (n = 7, P <=  0.03) and 55% (n = 7, P <=  0.01) after 2 and 3 days of KD, respectively, and remained suppressed up to 21 days of KD. Thirty micrograms total RNA were loaded per each lane. * P <=  0.01 and § P <=  0.03 compared with pooled controls (n = 9).

Effect of K+ deprivation on AQP2 protein abundance in the cortex and inner medulla. We next examined the expression of AQP2 protein in the inner medulla and cortex at 12 and 24 h of K+ deprivation to determine whether AQP2 dysregulation plays a role in the generation of polyuria at the early time points of 12 and 24 h. Immunoblot analysis studies demonstrated that in the inner medulla, AQP2 protein expression remained unchanged at 12 and 24 h of K+ deprivation (Fig. 4, A and B). The expression of AQP2 protein in the cortex was, however, significantly decreased at 12 and 24 h of K+ deprivation by ~42 (n = 7, P < 0.01) and 77% (n = 7, P < 0.001), respectively (Fig. 5, A and B). The equal protein loading for these experiments was verified by gel staining (gel picture not shown). The AQP2 mRNA expression in the cortex was examined at these early time points. As shown in Northern blot experiments depicted in Fig. 5, C and D, AQP2 mRNA levels were decreased by 28 (n = 7, P < 0.02) and 33% (n = 7, P < 0.04) at 12 and 24 h of K+ deprivation, respectively (Fig. 5D). These results indicate that the abundance of AQP2 protein in the cortex correlated with its mRNA levels in the cortex at 12 and 24 h of K+ deprivation.


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Fig. 4.   Effect of KD on AQP2 protein abundance in the inner medulla (IM). Three micrograms of total protein from IM were loaded per each lane. A: representative immunoblots of AQP2 protein level in the IM of control, 12 and 24 h of KD. B: densitometry of AQP2 protein expression in the IM at time 0 (control, n = 7), 12 (n = 7), and 24 h (n = 7) of K+ deprivation. KD did not alter the 29-kDa AQP2 protein in the IM at these early time points. NS, not significant. A 28% decrease in 35-kDa AQP2 was observed at 24 h of KD (* P < 0.001, compared with control).



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Fig. 5.   Effect of KD on AQP2 abundance in the cortex: Twelve micrograms of total protein from cortex were loaded per each lane. A: representative immunoblots of AQP2 protein level in the cortex of control, 12 and 24 h of KD. Only the 29-kDa AQP2 band is detected in the cortex. Top: short exposure. Bottom: long exposure. B: densitometry of AQP2 protein abundance in the cortex at time 0 (control, n = 7), 12 (n = 7), and 24 h (n = 7) of KD. As indicated, KD decreased the abundance of the 29-kDa band in the cortex by 40 and 77% at 12 (P < 0.01) and 24 h (P < 0.001), respectively. C, top: representative Northern hybridization of AQP2 mRNA level in the cortex. Bottom: GAPDH mRNA expression (the control for RNA loading). D: AQP2 mRNA-to-GAPDH mRNA ratio. Thirty micrograms of total RNA were loaded per each lane. Mean expression levels declined by 28% after 12 h (n = 7, P < 0.02) and by 33% (n = 7, P < 0.04) after 24 h of KD.

Effect of K+ intake on K+ deprivation-induced urinary concentrating defect. To determine whether the urinary concentrating defect in K+-restricted rats is exclusively due to the lack of K+ in the diet (or due to the presence of other substituting chemicals in the K+-free diet), rats were placed on a K+-free diet and supplemented with 0.85% KCl added to their drinking water. The rats on a normal diet (control) also received KCl in their drinking water. Both groups were on a normal diet and received KCl in their drinking water for 3 days before switching to the appropriate diets (See Fig. 6). The animals were studied for 6 days after the switch to the K+-free diet. Water intake, urine output, and urine osmolality were measured daily. As depicted in Fig. 6, the presence of KCl in the drinking water prevented the drop in urine osmolality of rats on K+-free diet (Fig. 6C). Water intake (Fig. 6A) and urine output (Fig. 6B) were actually decreased in rats switched to K+-free diet. It is worth mentioning that the presence of KCl in the drinking solution increased both water intake and urine volume compared with no KCl (see control group in Fig. 1). Water intake was 33 ± 1.7 (n = 4) in the presence and 23 ± 2 ml/24 h (n = 5) in the absence of KCl (Fig. 6A vs. Fig. 1A, P < 0.05). Urine output was 15 ± 1 (n = 4) in the presence and 8 ± 2 ml/24 h (n = 5) in the absence of KCl in the drinking water (Fig. 6B vs. Fig. 1B, P < 0.04). The increase in water intake and urine output in rats on 0.85% KCl is likely due to increased solute load.


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Fig. 6.   Effect of KD on water balance and urine osmolality in the presence of KCl in the drinking water. When rats were fed a regular diet, the presence of KCl in their drinking water increased the baseline of both water intake (P < 0.05, n = 4; A), and urine output (P < 0.04, n = 4; B) compared with baseline values in the absence of KCl (Fig. 1). Switching rats to K+-free diet was associated with an immediate return of water intake and urine production to baseline values (compared with baseline values in the absence of KCl, Fig. 1). C: urine osmolality was not altered by KCl intake, and the latter prevented its decrease by KD. Each time point is the mean ± SE of n = 4 rats in both KD and control.

Effect of K+ intake on K+ deprivation-induced downregulation of AQP2 mRNA expression in the outer medulla. To examine whether the presence of KCl in the drinking water blocked the effect of K+ restriction on AQP2 expression, Northern hybridization of AQP2 was performed on RNA from the inner stripe of outer medulla. As indicated in Fig. 7, the presence of KCl in drinking water prevented the downregulation of AQP2 mRNA expression in rats on K+-free diet. Taken together, these results indicate that the effect of K+ restriction on water balance, urine osmolality, and AQP2 expression is exclusively due to the decreased K+ intake.


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Fig. 7.   Effect of KD on AQP2 mRNA expression in the presence of KCl in the drinking water. A, top: representative Northern hybridization of AQP2 mRNA level in the OM. Bottom: GAPDH mRNA expression is shown as the control for RNA loading. B: AQP2 mRNA-to-GAPDH mRNA ratio. Thirty micrograms total RNA were loaded per each lane. Mean expression of AQP2 mRNA levels was not altered in KD (P > 0.05 vs. control, n = 4 for each bar).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The studies described above demonstrate that the polyuria-polydipsia syndrome associated with K+ deprivation is an early event (Fig. 1) and occurs before the onset of hypokalemia. The time course studies revealed that the urinary concentrating ability is impaired as early as 12 h of K+ deprivation (Fig. 1C). The alterations in water balance and urine osmolality in K+ deprivation were prevented when the animals were provided with KCl in their drinking water (Fig. 6), suggesting that K+ deprivation-induced nephrogenic diabetes insipidus is exclusively due to K+ restriction. Immunoblot analysis studies demonstrated that AQP2 abundance in outer medulla remained unchanged at 12 h (Fig. 2, A and D) but decreased after 24 h of K+ deprivation (Fig. 2, A and D). The inner medullary AQP2 abundance remained unchanged at 12 and 24 h of K+ deprivation (Fig. 4, A and B). Interestingly, the early urinary concentrating defect in K+ deprivation (Fig. 1C) correlated with the downregulation of AQP2 in the cortex (Fig. 5).

Along with the finding of Marples et al. (25), these results indicate that the urinary concentrating defect in K+ deprivation involves a decrease in the expression of AQP2 protein along the entire collecting duct system. Several recent studies have demonstrated that the expression of AQP2 protein in the collecting duct is also decreased in several acquired forms of nephrogenic diabetes insipidus such as lithium treatment, hypercalcemia, ureteric obstruction, and models of nephrotic syndrome (reviewed in Ref. 26). The very early downregulation of cortical, but not medullary, AQP2 indicates that the cortical distal nephron is very sensitive to K+ deprivation. This is in agreement with recent observations from our laboratory, demonstrating that the downregulation of cortical Na-Cl cotransporter (thiazide-sensitive Na-Cl cotransporter) in K+ deprivation precedes the suppression of the outer medullary type 1 bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1) (1).

The mechanism by which K+ deprivation decreases AQP2 protein expression is not clear. Beck et al. (3) have reported that chronic K+ depletion (3 wk) causes a decrease in adenylate cyclase sensitivity in response to vasopressin, which should lead to a decrease in the production of cAMP, the signaling pathway by which AVP activates AQP2, and facilitates water absorption in the collecting duct. Güllner et al. (13) have studied the time course effect of K+ deprivation on production of renal prostaglandins. Their studies indicate that the urinary excretion of immunoreactive 6-keto-PGF1a, PGE2, and 13,14-dihydro-15-keto-PGF2a were increased only after 48 h of a K+-deficient diet (13). On the basis of these studies, it is unlikely that prostaglandins or adenylate cyclase plays an important role in early urinary concentrating defect (12-24 h) in K+ deprivation.

The increase in water reabsorption by AVP in the medullary collecting duct is contingent on the preservation of a hypertonic medullary interstitium, which is generated and maintained predominantly by countercurrent multiplication. This requires the coordinated action of several nephron segments as well as the medullary interstitium and involves active reabsorption of NaCl in the thick ascending limb (TAL) of Henle's loop. The majority of NaCl absorption in the TAL segment is mediated by apical BSC-1. Several studies have reported that chronic K+ depletion decreases NaCl reabsorption in the TAL of Henle's loop (14, 23). Recent studies from our laboratory indicated that K+ deprivation downregulates the expression and activity of BSC-1 in rat medullary TAL tubules (1). The effect of K+ deprivation on this transporter became significant only after 3 days of K+ restriction (1). Taken together, these observations suggest that the decrease in NaCl transport in the TAL, with subsequent reduction in the medullary osmotic gradient, is more likely involved in the maintenance, rather than the generation, of polyuria and urinary concentration defect.

A decrease in the ability of the kidney to excrete highly concentrated urine in K+ depletion has also been attributed to primary polydipsia (37). Our results indicate that at 12 h of K+ restriction, polyuria and diluted urine were associated with a significant decrease in water intake. Water intake increased only after 24 h of K+ restriction, indicating that the increase in urine output and coordinate decrease in the ability of the kidney to concentrate urine precede primary polydipsia. The polydipsia that is observed in K+ depletion is a secondary event and may play a major role in the prevention of volume depletion in nephrogenic diabetes insipidus.

It is noteworthy to mention that the downregulation of AQP2 in K+ deprivation is not the result of an unspecific downregulation of proteins. Several studies have demonstrated that the mRNA expression and protein abundance of colonic H-K-ATPase are increased in outer medullary collecting duct of K+-deprived rats and mediate increased HCO3- reabsorption (reviewed in Ref. 36). These results indicate that the suppression of AQP2 in the collecting duct is a specific response to K+ deprivation.

Studies by Linas et al. (22) reported a significant decrease in rat serum [K+] at 24 h of K+ deprivation. Our results, however, indicate that serum [K+] remained unchanged at 3 days of K+ deprivation. One possible explanation with respect to this discrepancy may be related to the age of animals used in these studies. The rats that were used in the present studies were younger (body wt 125-150 g) compared with those used by Linas et al. (body wt 200-300 g). It is likely that younger animals may be more resistant to hypokalemia than older ones. The fact that the plasma K+ remained unchanged despite zero K+ intake can be attributable to transcellular K+ shift and intracellular K+ depletion. We therefore suggest that the early changes in renal function in response to K+ deprivation (12-24 h) is likely due to intracellular K+ depletion.

In conclusion, the syndrome of polyuria associated with the urinary concentrating defect in K+ deprivation is an early event that precedes the onset of hypokalemia. The very early urinary concentrating defect in K+ deprivation (after 12 but before 24 h) likely results primarily from the suppression of cortical AQP2, whereas the later onset of the urinary concentrating defect (after 24 h) also involves a downregulation of medullary AQP2.


    ACKNOWLEDGEMENTS

The authors acknowledge the contributions of Dr. Bellamkonda Kishore.


    FOOTNOTES

These studies were supported by National Institute of Diabetes and Digestive and Kidney Disease Grants RO1-DK-46789, RO1-DK-52821, and RO1-DK-54430 and by a grant from Dialysis Clinic, Inc.

Address for reprint requests and other correspondence: H. Amlal/M. Soleimani, Univ. of Cincinnati Medical Center, 231 Bethesda Ave., MSB 5502, Cincinnati, OH 45267-0585.

1  All the values given in RESULTS refer only to the nonglycosylated form of the AQP2 protein (29 kDa) and are expressed as percentage of pooled controls.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5 April 2000; accepted in final form 8 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 279(4):F655-F663