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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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EXPERIMENTAL PROCEDURES |
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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.
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RESULTS |
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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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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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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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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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The authors acknowledge the contributions of Dr. Bellamkonda Kishore.
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FOOTNOTES |
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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.
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