1 The Water and Salt Research Center, University of Aarhus, DK-8000 Aarhus C, Denmark; 2 Department of Physiology, School of Medicine, Dongguk University, 780-714 Kyungju, Korea; and 3 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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The purpose of this study was to
examine whether hypokalemia is associated with altered abundance of
major renal Na+ transporters that may contribute to the
development of urinary concentrating defects. We examined the changes
in the abundance of the type 3 Na+/H+ exchanger
(NHE3), Na+-K+-ATPase, the bumetanide-sensitive
Na+-K+-2Cl cotransporter (BSC-1),
the thiazide-sensitive Na+-Cl
cotransporter
(TSC), and epithelial sodium channel (ENaC) subunits in kidneys of
hypokalemic rats. Semiquantitative immunoblotting revealed that the
abundance of BSC-1 (57%) and TSC (46%) were profoundly decreased in
the inner stripe of the outer medulla (ISOM) and cortex/outer stripe of
the outer medulla (OSOM), respectively. These findings were confirmed
by immunohistochemistry. Moreover, total kidney abundance of all ENaC
subunits was significantly reduced in response to the hypokalemia:
-subunit (61%),
-subunit (41%), and
-subunit (60%), and
this was confirmed by immunohistochemistry. In contrast, the renal
abundance of NHE3 in hypokalemic rats was dramatically increased in
cortex/OSOM (736%) and ISOM (210%). Downregulation of BSC-1, TSC, and
ENaC may contribute to the urinary concentrating defect, whereas
upregulation of NHE3 may be compensatory to prevent urinary
Na+ loss and/or to maintain intracellular pH levels.
hypokalemia; sodium transport; kidney; urine concentration
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INTRODUCTION |
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HYPOKALEMIA, A COMMON ELECTROLYTE disturbance frequently encountered in clinical medicine, is often associated with several distinct renal functional defects, including nephrogenic diabetes insipidus presented by polyuria and urinary concentrating defect. Consistent with this, our laboratory previously demonstrated that rats with hypokalemia have a significant vasopressin-resistant polyuria with reduced expression of inner medullary aquaporin-2 (AQP2) levels (33).
Urinary concentration and dilution depend on the presence of a distinct segmental distribution of transport properties along the renal tubule, and urinary concentration depends on 1) the hypertonic medullary interstitium, driven by active NaCl reabsorption as a consequence of countercurrent multiplication in water-impermeable nephron segments (35); and 2) osmotic equilibration in water-permeable renal tubular segments, which chiefly depends on aquaporin water channels (38). Thus defects in any of these mechanisms would be predicted to be associated with urinary concentrating defects.
Over the years, physiological and biochemical investigations have
identified the major proteins involved in physiological processes of
transepithelial salt and water transport along the nephron and
collecting duct. Cloning of cDNAs and determination of the amino acid
sequence of these proteins have made it possible to produce selective
polyclonal or monoclonal antibodies to them. This has allowed
investigators to use comprehensive sets of antibodies against proteins
relevant to a given physiological process and to investigate entire
pathways through simultaneous assessment of the regulatory state of all
members of the pathway. Recently, a series of studies have identified
that dysregulated sodium transporters play an important role in animal
models of various disorders of sodium and water balance, including
chronic renal failure (28), ischemia-induced acute
renal failure (29), cirrhosis (13), lithium-induced nephrogenic diabetes insipidus (31),
syndrome of inappropriate antidiuretic hormone secretion
(10), primary aldosteronism (25, 34), and
vitamin D-induced hypercalcemia (49). In the kidney
proximal tubule, the type 3 Na+/H+ exchanger
(NHE3) is expressed apically and participates in sodium reabsorption in
this segment (3) . The loop of Henle generates a high
osmolality in the renal medulla by driving the countercurrent multiplier, which is dependent on NaCl reabsorption by the thick ascending limb (TAL). In this segment, the apically located
Na+-K+-2Cl cotransporter [rat
type 1 bumetanide-sensitive cotransporter (BSC-1 or
NKCC2)] (15-17, 19) and NHE3, in conjunction with
the Na+-K+-ATPase in the basolateral membranes,
are mainly responsible for sodium reabsorption by the TAL
(3). In the distal convoluted tubule, the
thiazide-sensitive Na+-Cl
cotransporter (TSC
or NCC) is involved in apical sodium reabsorption (21,
25). Epithelial sodium channel (ENaC) subunits are known to be
expressed in the connecting tubule and collecting duct, including the
inner medullary collecting duct (18). ENaC participates in
sodium reabsorption and is regulated by the hormones controlling sodium
and water balance, e.g., the mineralocorticoid aldosterone and
vasopressin (10, 34).
Potassium depletion has been demonstrated to be associated with altered
sodium reabsorption in distinct segments of the renal tubules.
Soleimani et al. (44) demonstrated that potassium
depletion was associated with an increase in luminal
Na+/H+ exchange and basolateral
Na+-HCO cotransporter in the
medulla and the Na+-Cl
cotransporter in the
cortex were decreased in kidneys of rats with hypokalemia along with an
increased urinary chloride loss (5). Micropuncture studies
also have shown that potassium depletion was associated with reductions
in the delivery of sodium and water to early and late regions of the
distal tubule (47). Moreover, plasma aldosterone levels,
which are critically involved in controlling sodium balance, are
suppressed by potassium depletion (27). These findings
therefore strongly indicate that changes in external potassium balance
may affect the transport of sodium and fluid along the nephron and that
this may be caused by changes in the expression of major renal sodium
transport proteins.
Therefore, the purposes of the present study were 1) to examine whether hypokalemia affects the protein abundance of major renal sodium transporters and 2) to examine whether the changes in the abundance of sodium transporters are associated with alterations in urinary concentration and urinary sodium excretion in hypokalemic rats. The abundance and cellular/subcellular distribution of sodium transporters were determined by semiquantitative immunoblotting and immunohistochemistry using antibodies against major renal sodium transporters (NHE3, Na+-K+-ATPase, BSC-1, TSC, and ENaC subunits).
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METHODS |
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Experimental Animals
Studies were performed in male Wistar rats (M & B, Lille-Skensved, Denmark) that were kept in metabolic cages for at least 48 h before the experiment was started and had free access to control chow and tap water (C1000, Altromin, Lage, Germany). During the entire experiment, the rats were kept in individual metabolic cages, with a 12:12-h light-dark cycle and a temperature of 21 ± 2°C.Experimental Protocol
After a period of acclimation, the animals were randomized into two groups matched for body weight: the hypokalemia group (n = 12) and the control group (n = 12). To produce hypokalemia, rats were fed a potassium-deficient diet (C1037, potassium content: 0.18 g/kg chow, Altromin) for 4 days. In the control group, rats were fed control chow (C1000, potassium content: 7 g/kg chow, Altromin) and offered the amount of food corresponding to the mean intake of food in the hypokalemia group of rats during the previous day. Thus the food intake was matched between the two groups. Rats in both groups had free access to water throughout the experiment.Clearance Studies
Daily urinary output and water intake were determined throughout the study. Urinary volume, osmolality, creatinine, sodium, and potassium concentration were measured. Plasma was collected from the abdominal aorta at the time of death for measurement of potassium, sodium, creatinine, and plasma osmolality.Measurement of Plasma Aldosterone
Blood samples were drawn in lithium-heparin glass vials. Immunoreactive aldosterone was measured by a radioimmunoassay method (Diagnostic System Laboratories, Webster, TX). Using a rabbit anti-aldosterone antibody and 125I-aldosterone, a radioimmunoassay was performed by incubation of plasma samples in precoated tubes.Primary Antibodies
For semiquantitative immunoblotting and immunocytochemistry, previously characterized polyclonal and monoclonal antibodies were used and summarized below.NHE3 (LL546AP). An affinity-purified polyclonal antibody against NHE3 has previously been characterized (12, 23).
TSC (LL573AP). An affinity-purified polyclonal antibody against apical TSC in the distal convoluted tubule has previously been characterized (25).
BSC-1 (LL320AP). An affinity-purified polyclonal antibody against apical BSC-1 in the TAL has previously been characterized (11, 23, 39).
-ENaC (Q3560-2 and 92472).
An affinity-purified polyclonal antibody and polyclonal immuneserum
against
-ENaC has previously been characterized (18, 34).
-ENaC (Q3755).
An affinity-purified polyclonal antibody against
-ENaC has
previously been characterized (34).
-ENaC (LL550).
An affinity-purified polyclonal antibody against
-ENaC has
previously been characterized (18, 34).
Na+-K+-ATPase.
A monoclonal antibody against the 1-subunit of
Na+-K+-ATPase has previously been characterized
(22).
Membrane Fractionation for Immunoblotting
The kidneys from six rats in the control group and seven rats in the hypokalemia group were used for membrane fractionation, which was followed by immunoblotting. One kidney from each rat was used as the total kidney, and the other kidney was dissected into the cortex/outer stripe of the outer medulla (OSOM), inner stripe of the outer medulla (ISOM), and inner medulla (IM). The tissue was homogenized in dissection buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH = 7.2, containing and 8.5 µM leupeptin, 0.4 mM pefabloc, and 1 mM phenylmethylsulfonyl fluoride) using an Ultraturrax T8 homogenizer (IKA Labortechnik), and the homogenate was centrifuged in an Eppendorf 5403 centrifuge at 4,000 g for 15 min at 4°C to remove whole cells, nuclei, and mitochondria. The supernatant was either used (ISOM) or centrifuged at 200,000 g for 1 h to produce a pellet containing membrane fractions enriched for both plasma membrane and intracellular vesicles (total kidney and cortex/OSOM). Gel samples were made using Laemmli sample buffer containing 2% SDS.Electrophoresis and Immunoblotting
Samples of membrane fractions together with molecular markers were run on 9 or 12% polyacrylamide minigels (Bio-Rad Mini Protean II). For each gel, an identical gel was run in parallel and subjected to Coomassie staining to ensure identical loading. The other gel was subjected to immunoblotting. After transfer by electroelution to nitrocellulose membranes, blots were blocked with 5% milk in 80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, and 0.1% Tween 20, pH = 7.5 (PBS-T) for 1 h and incubated overnight at 4°C with affinity-purified primary antibodies (NHE3, TSC, BSC-1, Na+-K+-ATPase andQuantitation of Expression Levels of Sodium Transporters
ECL films with bands within the linear range were scanned using an AGFA scanner (Arcus II) and Microsoft software to control the scanner. The labeling density was determined on blots where samples from hypokalemic rats were run on each gel with samples from respective control rats. The labeling density was corrected by densitometry of Coomassie-stained gels.Preparation of Tissue for Immunohistochemistry and Immunoelectron Microscopy
Four hypokalemic and four control rats were anesthetized with halothane inhalation, and their kidneys were fixed by retrograde perfusion through the abdominal aorta with 3% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH = 7.4). The kidneys were used for paraffin embedding for immunohistochemistry. For immunoperoxidase labeling, kidney blocks containing all kidney zones were dehydrated and embedded in paraffin. The paraffin-embedded tissue was cut at 2 µm on a rotary microtome (Leica). The staining was carried out using indirect immunoperoxidase. The sections were dewaxed and rehydrated, and the endogenous peroxidase was blocked by 0.5% H2O2 in absolute methanol for 10 min at room temperature. To reveal antigens, sections were incubated with 1 mmol/l Tris solution (pH 9.0) supplemented with 0.5 mM EGTA and heated using a microwave oven for 10 min. Nonspecific binding of immunoglobulin was prevented by incubating the sections in 50 mM NH4Cl for 30 min, followed by blocking in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2% gelatin. Sections were incubated overnight at 4°C with primary antibodies diluted in PBS supplemented with 0.1% BSA and 0.3% Triton-X-100. After a rinsing with PBS supplemented with 0.1% BSA, 0.05% saponin, and 0.2% gelatine for 3 × 10 min, the sections were incubated in horseradish peroxidase-conjugated secondary antibodies (P0448 goat anti-rabbit or P0447 goat anti-mouse, DAKO) diluted 1:200 in PBS supplemented with 0.1% BSA and 0.3% Triton-X-100, followed by incubation with diaminobenzidine. The light microscopy was carried out using a Leica DMRE light microscope.Statistical Analyses
Values are presented as means ± SE. Comparisons between groups were made by unpaired t-test with equal or unequal variances. P values <0.05 were considered significant. ![]() |
RESULTS |
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Rats Treated with a Potassium-Deficient Diet Had Low Plasma and Urinary Potassium Levels Along with Polyuria and Decreased Urinary Concentration
Rats treated with a potassium-deficient diet for 4 days developed significant hypokalemia with a decrease in plasma potassium levels to 2.5 ± 0.04 vs. 3.7 ± 0.06 mmol/l in controls (P < 0.05) (Table 1). Urinary excretion of potassium decreased significantly to 2.7 ± 0.3 vs. 9.5 ± 0.2 µmol/min in controls (P < 0.05), and the fractional excretion of potassium also decreased markedly to 15 ± 2.3 vs. 31 ± 1.1% in controls (P < 0.05). Along with this, the hypokalemic rats developed significant polyuria and increased water intake. In the basal period before treatment with a potassium-deficient diet, urinary output was not different between rats subjected to hypokalemia and controls: 49 ± 4 vs. 45 ± 3 µl · min
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Rats Treated with a Potassium-Deficient Diet Had No Changes in Plasma and Urinary Sodium Levels
In contrast to the marked alteration in water and potassium balance, no significant changes in plasma and urinary sodium levels were observed in the hypokalemic rats. Plasma sodium levels were unchanged in both groups of rats: 137 ± 0.4 mmol/l in hypokalemic rats and 137 ± 0.4 mmol/l in controls. Urinary excretion of sodium was 5.9 ± 0.2 µmol · kgRats Treated with a Potassium-Deficient Diet Had No Changes
in Blood pH or HCO
Rats with Hypokalemia Had Low Plasma Aldosterone Levels
An identical set of hypokalemic and control rats was set up for measurement of plasma aldosterone levels, and there were significantly lower plasma aldosterone levels in the hypokalemic rats compared with controls: 140 ± 12 vs. 234 ± 40 pg/ml (P < 0.05).Rats with Hypokalemia Had a Marked Increase in Renal NHE3 Abundance
Semiquantitative immunoblotting of kidneys from hypokalemic rats showed a significant increase in the protein abundance of NHE3 in the kidney cortex/OSOM (736 ± 45 vs. 100 ± 15%, P < 0.05) and in the ISOM (210 ± 28 vs. 100 ± 25%, P < 0.05) (Fig. 1, C-F, Table 2). Moreover, there was a parallel increase in total kidney NHE3 abundance in hypokalemic rats: 441 ± 37 vs. 100 ± 5% (P < 0.05) (Fig. 1, A and B, Table 2). Immunohistochemical analysis also showed profoundly increased NHE3 labeling in kidneys from hypokalemic rats (Fig. 2). In control rats, anti-NHE3 antibody labeled the apical plasma membrane domains of the proximal tubules, whereas basolateral plasma membranes were unlabeled (Fig. 2A). Furthermore, an intense labeling of the apical plasma membrane domains of cortical medullary TAL was also seen (Fig. 2, C and E). In kidney sections of hypokalemic rats, NHE3 labeling was significantly increased in the apical part of the proximal tubules in the cortex (Fig. 2B, arrow) and OSOM (not shown) as well as in the apical part of the TAL in the cortex (Fig. 2D, arrow), OSOM (not shown), and ISOM (Fig. 2F, arrow).
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In contrast to the dramatic upregulation of the NHE3, small but
significant changes were observed in the expression levels of the
Na+-K+-ATPase, with an upregulation in total
kidney to 126 ± 3 vs. 100 ± 2% (P < 0.05)
(Fig. 3, A and
B, Table 2) and in the
cortex/OSOM to 113 ± 2 vs. 100 ± 3% in control
(P < 0.05) (Fig. 3, C and D, Table 2). No significant changes were observed in ISOM (87 ± 4 vs. 100 ± 8%, NS) (Fig. 3, E and F, Table
2).
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Rats with Hypokalemia Had a Marked Decrease in Renal BSC-1 and TSC Abundance
Figure 4 shows immunoblots of BSC-1 using membrane preparations from ISOM in hypokalemic rats and control rats. Affinity-purified anti-BSC-1 antibody recognized a broad band of molecular mass 146-176 kDa centered at ~161 kDa (Fig. 4), consistent with previous observations (11). In contrast to the significantly increased kidney levels of NHE3 in hypokalemic rats, densitometric analysis revealed a marked decrease in BSC-1 abundance in ISOM from rats with hypokalemia corresponding to 57 ± 6% of levels in control rats (100 ± 16%, P < 0.05) (Fig. 4B, Table 2). Immunohistochemistry also showed a marked downregulation of BSC-1 in ISOM (Fig. 5). In control rats, distinct BSC-1 labeling was seen in the apical part of TAL cells (Fig. 5, A, C, and E), whereas in hypokalemic rats the labeling in the apical part of TAL cells was much weaker (Fig. 5, B, D, and F).
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As shown in Fig. 6, affinity-purified
anti-TSC antibody recognized a broad band centered at ~165 kDa. TSC
abundance in the membrane fractions of cortex/OSOM corresponded to
46 ± 6% of levels in control rats (100 ± 20%,
P < 0.05) (Fig. 6D, Table 2). Consistent with this, a significant decrease in TSC abundance was also seen in
membrane fractions from total kidney: 58 ± 7 vs. 100 ± 9%
in controls (P < 0.05) (Fig. 6B). This was
confirmed by immunohistochemistry showing reduced labeling in the
distal convoluted tubule of kidneys from potassium-deficient rats (Fig.
7, B and D),
whereas control rats demonstrated intense TSC labeling in the apical
part of the distal convoluted tubules (Fig. 7, A and
B). The downregulation of TSC is consistent with the
observed reduction in plasma aldosterone.
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Rats with Hypokalemia Had a Marked Decrease in Renal -,
-, and
-ENaC Abundance
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DISCUSSION |
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The present study demonstrated that experimentally induced
hypokalemia is associated with substantial upregulation of NHE3 in the
proximal tubules and TALs of rat kidney. In contrast, the abundance of
the BSC-1 in the TALs was markedly decreased. This suggests that
downregulation of BSC-1 is likely to play a role in the urinary
concentration defect associated with hypokalemia, in addition to the
previously demonstrated downregulation of AQP2. Moreover, the protein
abundances of TSC and ENaC subunits were markedly reduced in
hypokalemia. Specifically, reduced TSC and -ENaC abundances are
consistent with the reduced plasma aldosterone levels associated with
hypokalimia. The reduced abundances of TSC and ENaC may well contribute
to the urinary concentration defect associated with hypokalemia.
Increased NHE3 Abundance in Proximal Tubules and TALs in Hypokalemia
NHE3, which is expressed apically in proximal tubule cells, is believed to be the protein that mediates transcellular sodium and HCOThese findings are also supported by a previous observation that
chronic potassium depletion was associated with increased apical
Na+/H+ exchange and basolateral
Na+-HCO
Hypokalemia also stimulates H+ secretion and
HCO
Upregulation of NHE3 in volume-contracted states has also been demonstrated (14). Hypokalemic rats in the present study had polyuria, polydipsia, and reduced urine osmolality. Although there were virtually no differences in plasma sodium and plasma osmolalities between hypokalemic and control rats, it cannot be excluded that the upregulation of NHE3 may be indirectly caused by a direct effect of volume depletion.
Finally, the previous demonstration that potassium depletion leads to intracellular acidification (1) should be mentioned, and it is possible that this could increase the abundance of the NHE3 to maintain intracelluar pH levels. Although this remains speculative, it would be consistent with previous observations that enhanced renal NHE3 protein abundance is associated with chronic metabolic acidosis (23, 28). Moreover, Amemiya et al. (4) demonstrated that exposure of opossum kidney cells (clone P) that express NHE3 to low extracellular potassium caused a transient decrease in intracellular pH, which was followed by activation and upregulation of NHE3 after 24 h of incubation. Thus it is possible that the observed increase in the expression of NHE3 in hypokalemic rats could be caused by, or is associated with, indirect effects of intracellular acidification related to hypokalemia. This may contribute to the maintenance of intracellular pH levels, in conjunction with NBC1 in the proximal tubules (37, 40) and the electroneutral NBCn1 in TALs (30).
Decreased Abundance of BSC-1 in TALs in Hypokalemia
BSC-1 (15, 51), which is localized at the apical plasma membrane domains of medullary and cortical TAL segments (39), mediates apical NaCl transport in these water-impermeable segments. Several factors have previously been demonstrated to regulate the abundance of BSC-1 levels. An increase in the delivery of NaCl to the loop of Henle by chronic oral saline loading is known to upregulate BSC-1 levels (11). Moreover, expression of BSC-1 in the TAL is also known to be regulated by dDAVP (a vasopressin V2-receptor-selective agonist), and this regulation may be involved in the long-term regulation of the countercurrent multiplication system (24). Because expression of the Na+-K+-2ClTakahashi et al. (45) recently demonstrated the importance of BSC-1 for overall NaCl and fluid reabsorption. They showed that transgenic mice lacking BSC-1 suffer from dehydration and have renal insufficiency and die within 2 wk after birth, with signs of severe volume depletion. This is thought to be due to salt wasting by TAL malabsorption (45). Interestingly, a small fraction of these mice could be kept alive by the administration of indomethacin, although as adults they exhibited severe polyuria (45). Thus BSC-1 is important for urinary concentration. This supports our conclusion that the decrease in BSC-1 abundance observed in response to hypokalemia in this study plays a significant role in the hypokalemia-induced polyuria and urinary concentrating defect.
Because the abundance of the vasopressin-regulated AQP2 in kidney was also decreased in hypokalemic rats (33), it is possible that the vasopressin-adenylyl cyclase pathway is affected in both the TAL and in the collecting duct in response to hypokalemia. Indeed, Kim et al. (26) demonstrated that the increase in cAMP levels as well as adenylate cyclase activity in the isolated inner medullary collecting duct from hypokalemic rats were significantly blunted in response to vasopressin. This and the observation that BSC-1 expression is regulated by vasopressin are also consistent with the view that downregulation of BSC-1 is likely to play a role in the urinary concentration defects associated with hypokalemia (in addition to the downregulation of AQP2 abundance). However, further studies are needed to determine the underlying mechanisms involved in the downregulation of TAL sodium transporter BSC-1 expression in hypokalemia-induced nephrogenic diabetes insipidus.
Decreased Abundance of TSC and -,
-, and
-ENaC in
Hypokalemic Rats
Mice lacking TSC have only mild symptoms of disturbances of fluid and sodium homeostasis, but renal handeling of magnesium and calcium are altered, as observed in Gitelman's syndrome (42).
In this study, hypokalemia in rats was associated with a significantly decreased abundance of TSC and all three ENaC subunits in the kidney. Because potassium depletion is known to reduce plasma aldosterone levels (27), which was confirmed in this study, it is likely that the decreased abundance of TSC and ENaC subunits is directly caused by low plasma aldosterone levels. It is likely that the reduced abundances of TSC and EnaC subunits may contribute to the reduced urinary concentration mechanism.
Summary
Hypokalemia is associated with substantial upregulation of NHE3 in the proximal tubules and TALs of rat kidney, whereas BSC-1 in the TALs, TSC in the distal convoluted tubules, and ENaC subunits in the connecting segments and collecting ducts are downregulated. The decreased expression of BSC-1, TSC, and ENaC may contribute to the urinary concentrating defect in hypokalemia, in addition to the previously demonstrated decreased AQP2 levels. In contrast, upregulation of NHE3 is likely to be compensatory to prevent urinary sodium loss and/or to maintain intracellular pH levels. ![]() |
ACKNOWLEDGEMENTS |
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The authors thank Helle Høyer, Inger Merete Paulsen, Merete Pedersen, Zhila Nikrozi, Mette Vistisen, Lotte V. Holbech, and Gitte Christensen for technical assistance.
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FOOTNOTES |
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The Water and Salt Research Center at the University of Aarhus is established and supported by the Danish National Research Foundation. Support for this study was provided by the Karen Elise Jensen Foundation, the Human Frontier Science Program, the European Commission (KA 3.1.2 and KA 3.1.3. programs), the Novo Nordic Foundation, the Danish Medical Research Council, the Korea Science and Engineering Foundation (R05-2001-000-00630-0), the Dongguk University, the University of Aarhus, and the intramural budget of the National Heart, Lung, and Blood Institute.
Address for reprint requests and other correspondence: S. Nielsen, The Water and Salt Research Center, Univ. of Aarhus, Bldg. 233, DK-8000 Aarhus, Denmark (E-mail: sn{at}ana.au.dk).
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.
July 30, 2002;10.1152/ajprenal.00186.2002
Received 13 May 2002; accepted in final form 24 July 2002.
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