Regulation of sodium transporters in the thick ascending limb of rat kidney: res - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:39 |只看该作者 |倒序浏览 |打印

作者：Tae-Hwan Kwon, Jakob Nielsen, Young-Hee Kim, Mark A. Knepper, Jørgen Frøkiær,  Søren Nielsen作者单位：1 The Water and Salt Research Center, University of Aarhus, DK-8000 Aarhus C, Denmark; Department of Physiology, School of Medicine, Dongguk University, Kyungju 780-71 Korea; and Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health


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      【摘要】4 a+ g- W0 I. o% J, ~
   The effect of ANG II treatment of rats for 7 days was examined with respect to the abundance and subcellular localization of key thick ascending limb (TAL) Na transporters. Rats were on a fixed intake of Na and water and treated with 0, 12.5, 25, 50 (ANG II-50), 100 (ANG II-100), and 200 (ANG II-200) ng·min -1 ·kg -1 ANG II (sc). Semiquantitative immunoblotting revealed that Na /H exchanger 3 (NHE3) abundance in the inner stripe of the outer medulla (ISOM) of ANG II-treated rats was significantly increased: 179 ± 28 (ANG II-50, n = 5), 166 ± 23 (ANG II-100, n = 7), and 167 ± 19% (ANG II-200, n = 4) of control levels ( n = 6, P whereas lower doses of ANG II were ineffective. The abundance of the bumetanide-sensitive Na -K -2Cl - cotransporter (BSC-1) in the ISOM was also increased to 187 ± 28 (ANG II-50), 162 ± 23 (ANG II-100), and 166 ± 19% (ANG II-200) of control levels ( P in the abundance of Na -K -ATPase and the electroneutral Na -HCO 3 cotransporter NBCn1. Immunocytochemistry confirmed the increase in NHE3 and BSC-1 labeling in medullary TAL (mTAL). In the cortex and the outer strip of the outer medulla, NHE3 abundance was unchanged, whereas immunocytochemistry revealed markedly increased NHE3 labeling of the proximal tubule brush border, suggesting subcellular redistribution of NHE3 or differential protein-protein interaction. Despite this, ANG II-treated rats (50 ng·min -1 ·kg -1 for 5 days, n = 6) had a higher urinary pH compared with controls. NH 4 Cl loading completely blocked all effects of ANG II infusion on NHE3 and BSC-1, suggesting a potential role of pH as a mediator of these effects. In conclusion, increased abundance of NHE3 and BSC-1 in mTAL cells as well as increased NHE3 in the proximal tubule brush border may contribute to enhanced renal Na and HCO 3 reabsorption in response to ANG II.   w9 C6 L+ q2 N4 i! J! X$ M2 P
      【关键词】 bumetanidesensitive sodiumpotassiumchloride cotransporter NaKATPase type sodiumhydrogen exchanger bicarbonate transport sodium transport; y7 ?6 A% t; j0 w
   RENAL REABSORPTION AND EXCRETION of water and Na are critical to the regulation of extracellular fluid volume. The renin-angiotensin-aldosterone system has been demonstrated to play a critical role in the regulation of renal Na and water metabolism through a variety of physiological pathways. In particular, ANG II has known effects on the regulation of renal hemodynamics, glomerular filtration rate, aldosterone secretion, as well as more direct effects on renal tubule transport in the proximal tubule ( 13, 25 ). In the proximal tubules, the main site for renal water, salt, and HCO 3 reabsorption ( 4 ), it is well established that ANG II increases Na and HCO 3 reabsorption ( 13, 19, 45 ). Consistent with this, micropuncture and microperfusion studies have demonstrated that proximal tubule Na and water transport are stimulated by physiological concentrations of ANG II on the peritubular side ( 24 ). Moreover, ANG II stimulates the rate of Na uptake by the isolated proximal tubule cells through the amiloridesensitive Na /H exchanger, and this was inhibited by the receptor antagonist saralasin ( 45 ).  N8 a$ n, {7 _9 J" }* T4 }
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The type 3 Na /H exchanger (NHE3), which is expressed apically in the proximal tubules ( 2, 9 ), is believed to be the protein that mediates a major fraction of the transcellular Na and HCO 3 reabsorption in conjunction with both Na -K -ATPase and electrogenic Na -HCO 3 cotransporter (kNBC1) expressed in the basolateral plasma membrane ( 46 ). Consistent with the proposed roles of NHE3 in kidney tubules, the proximal convoluted tubules from NHE3 gene knockout mice have a marked decrease in fluid and HCO 3 absorption (by 69 and 61%, respectively) ( 47 ). These findings therefore indicate that NHE3 is critically involved in proximal tubule Na  , fluid, and HCO 3 reabsorption.- L$ X) ?2 |( q
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NHE3 is also apically expressed in the cortical and medullary thick ascending limb (mTAL) cells ( 2, 9 ), suggesting that the Na /H exchanger may also play an important role in the Na and HCO 3 reabsorption in this segment ( 23 ). Nevertheless, there is little information available regarding the effect of ANG II on the renal tubules distal to the proximal tubule, and it is not known whether ANG II has an effect on the regulation of Na transporters, including NHE3 in the TAL. Recently, several studies suggest that ANG II may have an important effect on the TAL in addition to the proximal tubules: 1 ) the TAL cells (particularly mTAL) have ANG II binding sites ( 38 ) and ANG II receptor mRNA and protein ( 11, 42 ); 2 ) ANG II was demonstrated to have an effect on the activity of the apical K channel, cotransporter, and bicarbonate transporter in isolated TAL ( 3, 21, 22, 36 ); 3 ) ANG II regulates several signaling pathways, including intracellular Ca 2 , protein kinase C activity, and metabolites of arachidonic acid in the TAL ( 3, 11, 22, 36 ).
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The TAL is importantly involved in NaCl reabsorption and urine concentration by generating a high osmolality in the renal medulla through the countercurrent multiplier ( 26, 28 ). Apically expressed NHE3 and the Na -K -2Cl - cotransporter (rat type 1 bumetanide-sensitive cotransporter: BSC-1 or NKCC2), in conjunction with basolaterally expressed Na -K -ATPase, are mainly responsible for Na reabsorption by the TAL ( 26 ). Moreover, a major fraction of ammonium ( ) is reabsorbed at the TALs mainly through the Na -K -2Cl - cotransporter by substituting for K ions for countercurrent multiplication of in the renal medulla ( 18 ). We have recently suggested that the electroneutral Na -HCO 3 cotransporter (NBCn1) ( 15 ), which is expressed in the basolateral plasma membrane of the mTAL cells, may also play an important role in HCO 3 transport into the cells and reabsorption ( 33 ).' A) l0 q( a' O8 ^. }& s

Therefore, it can be hypothesized that ANG II treatment may regulate or be associated with significant alteration in the expression levels of NHE3, BSC-1, Na -K -ATPase, and NBCn1 in the TAL of rat kidneys, in addition to the previously observed increase in NHE activity in the proximal tubules ( 13, 19, 25 ). In the present study, we therefore aimed to examine 1 ) whether ANG II treatment affects the abundance of TAL Na transporters (NHE3, BSC-1, Na -K -ATPase, and NBCn1); 2 ) whether ANG II treatment in rats is associated with a change in urinary pH because the targets for ANG II regulation (e.g., NHE3) are known to be involved in renal acid-base metabolism; and 3 ) whether ANG II treatment affects the abundance of proximal tubule Na transporters (NHE3 and Na -K -ATPase). We used specific antibodies against major renal Na transporters to determine the changes in abundance of each transporter in rat kidneys treated with ANG II, and this was performed by semiquantitative immunoblotting, immunohistochemistry, confocal laser microscopy, and immunoelectron microscopy.
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METHODS* `) G9 s4 c% b0 o7 W* R; U; H

Experimental Protocols. s$ u( O" Y& G; o' k& i
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Protocol 1. Experiments were performed using male Munich-Wistar rats (250-300 g, Møllegard Breeding Centre), which were maintained on a standard rodent diet [15 g · 220 g body weight (BW) -1 · day -1; Altromin 1324, Altromin, Lage, Germany]. Control rats and ANG II-treated rats were chosen randomly and maintained in metabolic cages. Food mixed with water to ensure a fixed daily water intake (37 ml · 220 g BW -1 · day -1 ) and standard rat diet (15 g·220 g BW -1 ·day -1, Altromin 1324) was given to each rat, as described previously ( 28, 33 ). The estimated daily Na intake in food was 1.3 meq Na · 220 g BW -1 · day -1 in both control rats and ANG II-treated rats. 1 The rats were fed once daily in the morning and ate all of the offered food during the course of the day. For ANG II infusion to normal Munich-Wistar rats, osmotic minipumps (model 2002, Alzet, Palo Alto, CA) were implanted subcutaneously in the neck of each rat. For implantation, osmotic minipumps were filled with ANG II (Sigma) dissolved in Ringer lactate ( 35 ). The pumps were equilibrated with normal saline for 4 h before insertion. Three different dosages of ANG II ( groups 2, 3, and 4 ) were administered.$ m) ~  M8 H! {  ^4 F0 V1 A. D

In group 1 ( n = 6), control rats were treated with vehicle only (0.5 µl/min sc for 7 days). In group 2 ( n = 5), rats were treated with ANG II (50 ng · min -1 · kg -1 sc for 7 days; ANG II-50). In group 3 ( n = 7), rats were treated with ANG II (100 ng · min -1 · kg -1 sc for 7 days; ANG II-100). In group 4 ( n = 4), rats were treated with ANG II (200 ng · min -1 · kg -1 sc for 7 days; ANG II-200).

The rats were maintained in metabolic cages, and daily 24-h urine output and water intake were measured during the entire experimental period. Urinary volume, osmolality, creatinine, and Na and K concentration were measured. After 7 days of ANG II treatment, all rats were anesthetized under halothane inhalation, the right kidney was rapidly removed and processed for membrane fractionation for semiquantitative immunoblotting, and the left kidney was subjected to perfusion fixation for immunohistochemistry. Plasma was collected from the abdominal aorta at the time of death for measurement of Na and K concentration, creatinine, and osmolality./ ]& }9 F5 e/ g* k3 m. ?/ {
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Protocol 2. Another set of ANG II-treated rats and control rats was created. This protocol was identical to protocol 1 except that different dosages of ANG II ( groups 2, 3, and 4 ) were administered.5 K3 f* k: z4 k+ O
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In group 1 ( n = 5), control rats were treated with vehicle only (0.5 µl/min sc for 7 days). In group 2 ( n = 5), rats were treated with ANG II (12.5 ng · min -1 · kg -1 sc for 7 days; ANG II-12.5). In group 3 ( n = 5), rats were treated with ANG II (25 ng · min -1 · kg -1 sc for 7 days; ANG II-25). In group 4 ( n = 4), rats were treated with ANG II (200 ng · min -1 · kg -1 sc for 7 days; ANG II-200).

Protocol 3. This protocol was identical to protocol 1 except that both control rats and ANG II-treated rats received an NH 4 Cl-mixed diet for the last 2 days of the experiment to examine how the kidneys of control rats and ANG II-treated rats handle an acid load. Both control rats and ANG II-treated rats received a diet mixed with water to ensure a fixed daily water intake (37 ml · 220 g BW -1 · day -1 ) and a standard rat diet (15 g·220 g BW -1 ·day -1, Altromin 1324) identical to that in protocol 1. For the last 2 days of the experiment, rats in both groups received NH 4 Cl in their diet at 7.2 mmol · 220 g BW -1 · day -1 with the same amount of food (15 g·220 g BW -1 ·day -1, Altromin 1324) and water (37 ml · 220 g BW -1 · day -1 ) ( 28, 33 ).2 P  J, B+ m: }

In group 1 ( n = 6), control rats were treated with vehicle only (0.5 µl/min sc for 7 days). In group 2 ( n = 6), rats were treated with ANG II (50 ng · min -1 · kg -1 sc for 7 days). The rats were maintained in metabolic cages, and daily 24-h urinary output and water intake were measured during experimental periods. Urinary volume, osmolality, creatinine, Na  , K  , Cl -, and pH levels were measured. After 7 days of ANG II treatment, all rats were anesthetized under halothane inhalation, the right kidney was rapidly removed and processed for membrane fractionation, and the left kidney was subjected to perfusion fixation for immunohistochemistry. Blood was collected from the abdominal aorta at the time of death for the measurement of plasma Na and K concentration, creatinine, osmolality, and whole blood HCO 3 and total CO 2 levels., M7 F! B  c5 b" O! d8 A

Membrane Fractionation and Immunoblotting3 i% |& Q0 C0 @+ i2 u/ f
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All rats were killed under light halothane anesthesia, and the right kidney was rapidly removed. The kidney was dissected into cortex/outer stripe of the outer medulla (OSOM), inner stripe of outer medulla (ISOM), and inner medulla. Tissue samples were processed for membrane fractionation using the 200,000- g pellet of an initial 4,000- g spin of the homogenate ( 32, 33 ). Immunoblotting was performed as previously described using anti-rat NHE3 ( 28, 32 ), anti-rat BSC-1 ( 17, 32 ), the anti- 1 -subunit of Na -K -ATPase ( 32 ), anti-thiazide-sensitive Na -Cl - cotransporter (TSC) ( 29, 32 ), or anti-rat NBCn1 ( 33 ). Enhanced chemiluminescence films with bands within the linear range were scanned using an AGFA scanner (ARCUS II) and Corel Photopaint Software to control the scanner. The labeling density was corrected by densitometry of Coomassie-stained gels. After densitometry, the labeling density in the samples from the ANG II-treated rats was calculated as a fraction of the mean control value for that gel.8 A% g& I# Y* j0 H1 t/ x

Immunohistochemistry, Confocal Laser Microscopy, and Immunoelectron Microscopy. g; n9 Y: J- s
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The kidneys were fixed by retrograde perfusion via the aorta with 3% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4. Immunolabeling was performed on sections from paraffin-embedded preparation (2-µm thickness) using methods described previously in detail ( 33, 51 ).% s1 \. K% f7 I/ D0 M
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For immunoelectron microscopy, the frozen samples were freeze-substituted in a Reichert AFS freeze substitution unit and prepared for immunolabeling for BSC-1 ( 41 ). For immunoelectron microscopy of NHE3 in the mTAL of the kidney [ANG II-200 ( n = 3) and controls ( n = 3)], a preembedding method was used as described previously ( 30 ). Ultrathin sections were examined in Philips Morgagni and Philips CM100 electron microscopes.

Statistical Analyses. _# F( P- ^( U

Values are presented as means ± SE. Comparisons between two groups were made by unpaired t -test. P values were considered significant.0 F0 V7 u# N/ _( `8 [6 s* p
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RESULTS8 N, d' D( v* T) s$ a5 ?

Urinary Output and Na Excretion Were Unchanged, Whereas pH Was Significantly Altered in ANG II-Treated Rats& `2 s" O* e$ B% |. F9 @

In protocol 1, rats treated with ANG II for 7 days with controlled water and food intake had unchanged urinary output, osmolality, and fractional Na excretion (FE Na ) compared with control rats ( Table 1 ). In protocol 3 (identical to protocol 1 up to day 5 ), ANG II-treated rats had significantly increased urinary pH levels during the treatment (8.28 ± 0.31 vs. 7.16 ± 0.03 at day 2 and 8.05 ± 0.18 vs. 7.43 ± 0.04 at day 5, P Fig. 1 ).

Table 1. Changes in renal function (protocol 1)
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Fig. 1. Time course of the changes in urinary pH in ANG II-treated rats and control rats ( protocol 3 ). ANG II-treated rats (open bars) have significantly increased urinary pH during the treatment at days (d) 2 and 5 compared with control rats (filled bars). For the last 2 days of the experiment, both ANG II-treated rats and control rats received an NH 4 Cl-mixed diet, and both groups demonstrate decreased urinary pH. However, there is no difference between the 2 groups in urinary pH in response to the NH 4 Cl-mixed diet. Arrow indicates the time point of osmotic minipump insertion for ANG II treatment for 7 days. * P

Increased NHE3 Abundance in ISOM in Response to ANG II Treatment
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Semiquantitative immunoblotting of membrane fractions prepared from the ISOM of ANG II-treated rats and control rats ( protocol 1 ) demonstrated that ANG II treatment for 7 days was associated with a marked increase in NHE3 protein abundance: 179 ± 28 (ANG II-50, n = 5), 166 ± 23 (ANG II-100, n = 7), and 167 ± 19% (ANG II-200, n = 4) of control levels ( n = 6, P Fig. 2; see Table 3 ). In contrast, treatment with lower doses of ANG II ( protocol 2 ) was associated with unchanged NHE3 protein abundance in ISOM: 107 ± 25 (ANG II-12.5, n = 5, not significant) and 123 ± 18% (ANG II-25, n = 5, not significant) of control levels (see Table 3 ), whereas a high dose of ANG II (200 ng · min -1 · kg -1 in protocol 2 ) increased NHE3 abundance, corresponding to 167 ± 3% of control levels ( P Table 3 ), consistent with protocol 1.% ?+ K# ]. i, H
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Fig. 2. Semiquantitative immunoblotting of membrane fractions prepared from the inner stripe of the outer medulla (ISOM) of ANG II-treated rats [50, 100, and 200 ng · min -1 · kg -1 ANG II; ANG II-50 ( n = 5), ANG II-100 ( n = 7), and ANG II-200 ( n = 4), respectively] and control rats (Con; n = 6, protocol 1 ). A, C, and E : immunoblots are reacted with anti-type 3 Na/H exchanger (NHE3) and reveal a single 87-kDa band. B, D, and F : densitometric analyses reveal that ANG II treatment [ANG II-50 ( B ), ANG II-100 ( D ), and ANG II-200 ( F )] for 7 days is associated with a marked increase in NHE3 protein abundance in ISOM. * P
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Table 3. Densitometric analysis of immunoblots from ANG II-treated rats and control rats
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Immunoperoxidase microscopy demonstrated that NHE3 labeling was associated with the apical plasma membrane domains of mTAL (arrows in Fig. 3, C and D ) and cortical TALs (arrows in Fig. 4 A ) in control rats, whereas the apical plasma membrane domains of macula densa cells were not labeled (MD in Fig. 4 A ). This is consistent with previous observations ( 43 ). Immunoperoxidase ( Fig. 3, A, C, and E, n = 4/group in protocol 1 ) and immunofluorescence microscopic analyses (not shown) revealed that NHE3 labeling of the apical plasma membrane of mTAL cells was increased in response to ANG II treatment (arrows in Fig. 3, A and E, protocol 1 ) compared with control rats (arrows in Fig. 3 C ). This is consistent with the increased abundance of NHE3 observed in the ISOM by immunoblotting ( Fig. 2 ). Moreover, NHE3 labeling in the apical plasma membrane of cortical TAL cells was also enhanced in ANG II-treated rats (arrows in Fig. 4, B - D, protocol 1 ) compared with control rats ( Fig. 4 A ). There was no sign of subcellular redistribution of NHE3 in the mTAL and cortical TAL cells in response to ANG II treatment ( Fig. 3, A, C, and E, and Fig. 4, B - D ). In protocol 2, treatment with lower doses of ANG II was associated with unchanged NHE3 labeling in the mTAL (arrows in Fig. 3 B ), consistent with immunoblotting, whereas a high dose of ANG II was associated with increased NHE3 labeling (arrows in Fig. 3 F ) compared with control rats (arrows in Fig. 3 D ). This was confirmed by immunoelectron microscopy of NHE3 immunoperoxidase labeling using a preembedding method ( Fig. 5 ). Immunoperoxidase labeling of NHE3 was associated with the apical domains, including the apical plasma membrane, of mTAL cells in control rat kidney ( Fig. 5 A ) and was increased in response to ANG II treatment (arrows in Fig. 5 B ).

Fig. 3. Immunoperoxidase microscopy of NHE3 in the kidney ISOM [ protocol 1 ( A, C, and E ) and protocol 2 ( B, D, and F )]. In protocol 1, NHE3 labeling in the ISOM of control rats ( C ) is seen at the apical plasma membrane domains of medullary thick ascending (TAL; arrows) and thin limb structures (asterisk). In ANG II-treated rats [ANG II-50 ( A ) and ANG II-200 ( E )], NHE3 labeling of the apical plasma membrane domains of the medullary TAL (arrows) from ANG II-treated rats is increased compared with control rats ( C ), but no sign of subcellular redistribution of NHE3 in response to ANG II treatment is seen. In protocol 2, strong labeling of NHE3 is also seen at the apical domains of the medullary TAL of ANG II-treated rats (ANG II-200, arrows in F ), whereas NHE3 labeling is unchanged in rats treated with lower dose of ANG II [ANG II-25 ( B )]. CD, medullary collecting duct. Magnification: x 630 ( A - D ).
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Fig. 4. Immunoperoxidase microscopy of NHE3 in the kidney cortex ( protocol 1 ). A : in the cortex of normal control rats, NHE3 labeling is seen at the apical plasma membrane domains of cortical TAL (arrows) and proximal tubules, whereas macula densa cells (MD) are unlabeled. B - D : in ANG IItreated rats [ANG II-50 ( B ), ANG II100 ( C ), and ANG II-200 ( D )], NHE3 labeling of the apical plasma membrane domains of the cortical TAL (arrows) is increased compared with control rats (arrows in A ), but no sign of subcellular redistribution of NHE3 in response to ANG II treatment is seen. G, glomerulus; MD, macula densa; P, proximal tubule. Magnification: x 630 ( A - D ).

Fig. 5. Immunoelectron microscopy of NHE3 in the medullary TAL ( protocol 2 ). A : NHE3 immunolabeling is seen at the apical domains of medullary TAL in the ISOM of control rats (arrows). B : in ANG II-treated rats (ANG II-200), NHE3 immunolabeling in the apical domains of the mTAL cells (arrows) is increased. Magnification: x 10,800 ( A and B ).
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Unchanged NHE3 Abundance but Increased NHE3 Immunolabeling of Proximal Tubule Brush Border

Semiquantitative immunoblotting of membrane fractions prepared from the cortex and OSOM of ANG II-treated and control rats revealed that NHE3 abundance was unchanged in response to ANG II treatment for 7 days: 124 ± 8 (ANG II-50, n = 5), 134 ± 20 (ANG II-100, n = 7), and 94 ± 20% (ANG II-200, n = 4) of control levels ( n = 6, not significant, respectively) (see Table 3 ).

Immunoperoxidase microscopy in kidney cortex and OSOM of control rats demonstrated NHE3 immunolabeling of the apical plasma membrane domains of segment 1 and segment 2 (S1 and S2) of convoluted proximal tubules (arrows in Fig. 6 A ), with a weaker staining of the S3 straight proximal tubule segments (not shown). The labeling was exclusively confined to the apical domains, with labeling of endocytic invaginations and the initial parts of microvilli (arrows in Fig. 6 A ), whereas the distal tips of microvilli and basolateral plasma membranes were unlabeled ( Fig. 6 A ). Immunoperoxidase and confocal laser-scanning microscopy ( n = 4/group in protocol 1 ) demonstrated increased NHE3 immunolabeling of the brush border of the proximal tubules from ANG II-treated rats (arrows in Fig. 6, B - D ) compared with control rats (arrows in Fig. 6 A ), suggesting subcellular redistribution or increased antigenecity potentially due to secondary modifications of NHE3 or altered protein-protein interaction. Increased labeling was also demonstrated in protocol 2 (not shown).4 C6 `7 x# n* X% }/ [" Q2 Y3 p' S
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Fig. 6. Immunoperoxidase microscopy of NHE3 in the kidney cortex ( protocol 1 ). A : in the cortex of normal control rats, NHE3 labeling is seen at the apical plasma membrane domains of proximal tubules. The labeling is exclusively confined to the apical domains, whereas the distal tip of the microvilli (arrows) and basolateral plasma membranes are unlabeled. B - D : in ANG II-treated rats [ANG II-50 ( B ), ANG II-100 ( C ), and ANG II-200 ( D )], NHE3 immunolabeling in the brush border of the proximal tubules from ANG II-treated rats (arrows in B - D ) is increased. D, distal convoluted tubule. Magnification: x 1,000 ( A - D ).
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Increased BSC-1 Abundance in ISOM in Response to ANG II Treatment+ ~8 {5 [) U1 h; I. k7 {
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Semiquantitative immunoblotting of membrane fractions prepared from the ISOM of ANG II-treated rats and control rats revealed that ANG II treatment ( protocol 1 ) was associated with an increase in BSC-1 protein abundance: 187 ± 28 (ANG II-50, n = 5), 162 ± 23 (ANG II-100, n = 7), and 166 ± 19% (ANG II-200, n = 4) of control levels ( n = 6, P respectively) ( Fig. 7, see Table 3 ). In contrast, the abundance of BSC-1 in the membrane fractions of cortex and OSOM was unchanged in response to ANG II treatment: 121 ± 24 (ANG II-50, n = 5), 72 ± 13 (ANG II-100, n = 7), and 92 ± 16% (ANG II-200, n = 4) of control levels ( n = 6, not significant, respectively) (see Table 3 ).
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Fig. 7. Semiquantitative immunoblotting of membrane fractions prepared from the ISOM of ANG II-treated rats (ANG II-50, n = 5) and control rats ( n = 6, protocol 1 ). A : immunoblot is reacted with anti-bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1) and reveals a broad 146- to 176-kDa band of molecular mass centered at 161 kDa. B : densitometric analysis reveals that ANG II treatment (ANG II-50) for 7 days with controlled water and food intake is associated with a marked increase in BSC-1 protein abundance in the ISOM. * P

Immunoperoxidase microscopy demonstrated that BSC-1 immunolabeling was associated with the apical domains of mTAL (arrows in Fig. 8 A ) and cortical TALs (arrows in Fig. 8 C ) in control rats. Kidneys ( n = 4/group in protocol 1 ) were studied using immunoperoxidase and immunoelectron microscopy. Consistent with the increased abundance of BSC-1 in the ISOM observed by immunoblotting ( Fig. 7, see Table 3 ), BSC-1 labeling in the apical domains of mTAL cells from ANG II-treated rats (arrows in Fig. 8 B ) was increased compared with control rats (arrows in Fig. 8 A ). In contrast, BSC-1 labeling in the apical domains of the cortical TAL cells was unchanged in ANG II-treated rats (arrows in Fig. 8 D ) compared with control rats ( Fig. 8 C ), consistent with immunoblotting data (see Table 3 ). Immunoelectron microscopy further demonstrated the increased BSC-1 labeling in mTAL cells from ANG II-treated rats ( Fig. 9 ).

Fig. 8. Immunoperoxidase microscopy of BSC-1 in the kidney ISOM and cortex ( protocol 1 ). A : in the ISOM of normal control rats, BSC-1 labeling is seen at the apical plasma membrane domains of medullary TAL cells (arrows). B : in ANG II-treated rats (ANG II-200), BSC-1 immunolabeling of the mTAL cells (arrows) is increased. C : in the cortex of normal control rats, BSC-1 labeling is seen at the apical plasma membrane domains of cortical TAL cells (arrows) and macula densa cells (MD). D : in ANG II-treated rats (ANG II-200), BSC-1 immunolabeling of the cortical TAL cells (arrows) is unchanged. G, glomerulus; MD, macula densa; P, proximal tubule. Magnification: x 630 ( A and B ) and x 1,000 ( C and D ).) P. `: b, a  y! T/ {: @# N
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Fig. 9. Immunoelectron microscopy on ultrathin Lowicryl HM20 sections from the ISOM tissues of control ( A ) and ANG II-treated rats ( B ). A : in control rats, BSC-1 immunogold labeling is associated with the apical plasma membrane (arrows) and intracellular vesicles (arrowheads) in medullary TAL cells. B : in ANG II-treated rats (ANG II-200), immunogold labeling of BSC-1 associated with the apical plasma membrane of the medullary TAL cells (arrows) is increased. M, mitochondria; N, nucleus. Magnification: x 26,000 ( A and B ).. \; v) a5 b$ `  a  y2 l; w
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Na -K -ATPase and NBCn1 Abundance in ISOM Was Unchanged in Response to ANG II Treatment6 i3 B! h/ y. u& S5 ~$ Q
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Semiquantitative immunoblotting of membrane fractions prepared from the ISOM of ANG II-treated rats and control rats revealed that ANG II treatment ( protocol 1 ) was not associated with changes in Na -K -ATPase abundance: 130 ± 10 (ANG II-50, n = 5), 88 ± 16 (ANG II-100, n = 7), and 107 ± 27% (ANG II-200, n = 4) of control levels ( n = 6, not significant, respectively) (see Table 3 ). Moreover, the abundance of NBCn1 in ISOM was not altered: 100 ± 8 (ANG II-50, n = 5), 118 ± 7 (ANG II-100, n = 7), and 125 ± 19% (ANG II-200, n = 4) of control levels ( n = 6, not significantly, respectively) (see Table 3 ).0 s0 Y5 ^; t: y/ Q* t
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Na -K -ATPase and TSC Abundance in the Cortex and OSOM Was Unchanged in Response to ANG II Treatment: e1 w& Z" r$ k& }
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Semiquantitative immunoblotting of membrane fractions prepared from the cortex and OSOM of ANG II-treated rats and control rats revealed that ANG II treatment ( protocol 1 ) was associated with unchanged Na -K -ATPase abundance: 112 ± 23 (ANG II-50, n = 5), 130 ± 19 (ANG II-100, n = 7), and 129 ± 36% (ANG II-200, n = 4) of control levels ( n = 6, not significant, respectively) (see Table 3 ). Moreover, the abundance of TSC in the membrane fractions of cortex and OSOM was not altered: 110 ± 29 (ANG II-50, n = 5), 98 ± 26 (ANG II-100, n = 7), and 96 ± 11% (ANG II-200, n = 4) of control levels ( n = 6, not significant, respectively) (see Table 3 ).

Abundance of NHE3 and BSC-1 in the ISOM Was Not Affected by ANG II Infusion in the Presence of NH 4 Cl Loading

In protocol 3, which was identical to protocol 1 except that both control rats and ANG II-treated rats were treated with a NH 4 Cl-mixed diet for the last 2 days of the experiment, the urinary output, urinary osmolality, and FE Na were not different between the two groups ( Table 2 ). The plasma K levels, however, were significantly decreased in ANG II-treated rats that received the NH 4 Cl-mixed diet compared with vehicleinfused rats that received NH 4 Cl: 4.2 ± 0.1 vs. 4.8 ± 0.2 meq/l ( P protocol 3, Table 2 ).
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Table 2. Changes in renal function (protocol 3)( P+ H- D* q2 x
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Figure 1 shows the measurements of urinary pH as a function of time in the protocol 3 experiments. Before NH 4 Cl administration, ANG II-treated rats had significantly increased urinary pH (8.28 ± 0.31 vs. 7.16 ± 0.03 at day 2 and 8.05 ± 0.18 vs. 7.43 ± 0.04 at day 5, P respectively) ( Fig. 1 ). For the last 2 days of the experiment, both groups demonstrated decreased urinary pH, although there was no difference in the urinary pH between the two groups ( Fig. 1 ). Moreover, at the end of the experiment, blood HCO 3 levels (26.5 ± 1.4 in ANG II-treated rats vs. 24.7 ± 0.7 mmol/l in controls, not significant) were not different between NH 4 Cl-loaded, ANG II-treated rats and NH 4 Cl-loaded control rats.

In contrast to protocol 1, the abundance of NHE3 and BSC-1 in the ISOM was not affected by NH 4 Cl loading in protocol 3. Semiquantitative immunoblotting of membrane fractions prepared from the ISOM of NH 4 Cl-loaded, ANG II-treated rats ( n = 6) and NH 4 Clloaded control rats ( n = 6) showed no effect of ANG II infusion in the presence of NH 4 Cl loading (95 ± 7 vs. 100 ± 11% in controls, not significant, Fig. 10, A and C ). It was also demonstrated by immunoperoxidase microscopy that apical NHE3 labeling in the mTAL cells of control rats was similar in NH 4 Cl-loaded, ANG II-treated rats and NH 4 Cl-loaded, vehicle-infused rats (arrows in Fig. 11, A and B ). Moreover, the NHE3 labeling in the brush border of the proximal tubules, which was markedly enhanced in response to ANG II treatment ( Fig. 6 ), was also similar in NH 4 Cl-loaded, ANG II-treated and NH 4 Cl-loaded, vehicle-infused rats (arrows in Fig. 11, C and D ). Furthermore, a comparison of the NHE3 labeling in proximal tubules seen for NH 4 Cl-loaded, vehicle-infused rats ( Fig. 11 C ) vs. non-NH 4 Cl-loaded, vehicle-infused rats ( Fig. 6 A ) suggests that the NH 4 Cl loading itself has a substantial effect of increasing immunoreactive NHE3 in the brush-border membrane. Finally, the abundance of BSC-1 in the membrane fractions of ISOM was not altered by ANG II infusion in the presence of NH 4 Cl loading (106 ± 7 vs. 100 ± 12% in controls, not significant; Fig. 10, B and D ). Thus NH 4 Cl loading appears to abolish all effects of ANG II on NHE3 and BSC1 in rats, pointing to a potential role of ANG II-mediated systemic or intracellular pH changes in these responses.
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Fig. 10. Semiquantitative immunoblotting of membrane fractions prepared from the ISOM of ANG II-treated rats and control rats ( protocol 3 ). A : immunoblot is reacted with anti-NHE3 and reveals a single 87-kDa band. B : immunoblot is reacted with anti-BSC-1 and reveals a broad 146- to 176-kDa band of molecular mass centered at 161 kDa. C and D : densitometric analyses reveal that ANG II-treated rats and control rats that received the same amount of NH 4 Cl loading (7.2 mmol · 220 g body wt -1 · day -1 ) for the last 2 days of the experiment have unchanged NHE3 and BSC-1 abundances.
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Fig. 11. Immunoperoxidase microscopy of NHE3 in the kidney ISOM and cortex ( protocol 3 ). A and B : in the ISOM of normal control ( A ) and ANG II-treated rats ( B ) that received an NH 4 Cl-mixed diet for the last 2 days of the experiment, NHE3 labeling is seen at the apical plasma membrane domains of medullary TAL cells (arrows) and thin limb structures (asterisks). The apical NHE3 labeling in the medullary TAL cells of control rats is similar to that of ANG II-treated rats. C and D : in the cortex of normal control ( C ) and ANG II-treated rats ( D ), the NHE3 labeling in the brush border of the proximal tubules (arrows in C and D ), which was markedly enhanced in response to ANG II treatment ( Fig 5 ), was similar in these 2 groups after acid loading for the last 2 days of the experiment. CD, collecting duct. Magnification: x 630 ( A and B ) and x 1,000 ( C and D ).
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DISCUSSION
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We demonstrated that ANG II treatment of rats was associated with significantly increased abundance and apical expression level of the Na /H exchanger NHE3 and Na -K -2Cl - cotransporter BSC-1 in mTAL, whereas Na -K -ATPase and electroneutral Na -HCO 3 cotransporter NBCn1 levels remained unchanged. Functionally, ANG II treatment was associated with a significant increase in urinary pH. Previous studies have demonstrated increased Na and HCO 3 reabsorption in response to ANG II treatment. Thus the present results suggest that upregulation of the Na /H exchanger NHE3 and the Na -K -2Cl - cotransporter BSC-1 is likely to play a significant role in the increased Na and HCO 3 reabsorption in this renal tubular segment in response to ANG II. In the proximal tubule of ANG II-treated rats, the overall abundance of NHE3 and Na-K-ATPase remained unchanged. In contrast, immunoperoxidase microscopy and confocal laser microscopy demonstrated that NHE3 immunolabeling in the brush border of the proximal tubules was markedly enhanced in ANG II-treated rats. Immunoblotting and immunoperoxidase microscopy revealed that NH 4 Cl loading prevented the ANG II-induced changes in the expression levels of the NHE3 and BSC-1. Furthermore, previous studies have shown that that the abundance of outer medullary NHE3 ( 28 ) and BSC-1 ( 5 ) is significantly upregulated by NH 4 Cl loading in normal rats. Thus it appears that NH 4 Cl loading and ANG II infusion have similar effects to increase NHE3 and BSC-1 expression. This raises the possibility that the two stimuli may have regulatory components in common. For example, it is possible that the ANG II effects are mediated by changes in intracellular or extracellular pH or that the acid-loading effects could be mediated by altered local production of ANG II in the proximal tubule and TAL. Furthermore, it is of particular interest that acid loading produces the same increase in NHE3 proximal brush-border labeling as does ANG II treatment. Thus increased brush-border labeling of NHE3 is seen in two conditions known to be associated with increased HCO 3 reabsorption in the proximal tubule ( 1, 19, 44 ). Below we have discussed the different key findings.4 [% E: `4 m% V' K; ^( W

NHE3 Abundance in the ISOM and Apical Expression in TAL Cells Were Increased in Response to ANG II Treatment( d0 Y& O' W8 w8 H+ f# o

We demonstrate that the abundance of NHE3 in the ISOM and the apical expression of NHE3 in the mTAL and cortical TAL cells were significantly increased in response to ANG II treatment in rats ( Table 3 ). The TAL is the site where Na and Cl - are actively reabsorbed via apically expressed the Na -K -2Cl - cotransporter BSC-1 and Na /H exchanger NHE3, but water is impermeable ( 31 ). Therefore, the loop of Henle generates a high osmolality in the renal medulla via the countercurrent multiplier, which is dependent on NaCl reabsorption by the TAL. The TAL also participates in the regulation of acid-base balance by reabsorbing most of the filtered HCO 3 that is not reabsorbed by the proximal tubules ( 20 ). H secretion is necessary for HCO 3 reabsorption, and this is believed to be medicated by apically expressed NHE3 in TAL cells ( 2, 9, 28 ). Therefore, the increased abundance, as well as increased expression, of apical membrane NHE3 of TAL cells seen in the present study in response to ANG II treatment is likely to contribute to increased Na and HCO 3 absorption. Consistent with this, several observations have suggested that apical NHE3 is responsible for HCO 3 reabsorption in TAL cells. First, NHE3 immunolabeling is present at the apical domains of mTAL and cortical TAL cells, in addition to the proximal tubules in the cortex and thin limb structures in the outer medulla ( 2, 9 ). Second, chronic metabolic acidosis increases apical Na /H exchange activity and HCO 3 absorption in the mTAL ( 23 ), in association with upregulation of the NHE3 abundance in the ISOM ( 28 ) and in whole kidney ( 32 ). Third, intravenous infusion of ANG II into rats was associated with an increase in HCO 3 absorption by the loop segment and this was inhibited by the type 1 ANG II receptor (AT 1 ) antagonist ( 14 ). This suggests that ANG II increases apical Na /H exchange activity as well as HCO 3 reabsorption in the mTAL. Thus the present observation that ANG II increases NHE3 expression may provide the molecular explanation for these functional observations.( F8 d6 w0 F! U$ T0 b0 S; |

Good et al. ( 22 ) recently demonstrated that acute ANG II treatment of isolated, perfused TAL inhibits HCO 3 absorption in the mTAL of rat kidney. The inhibition was mediated via a cytochrome P -450-dependent signaling pathway that likely involved the production of 20-HETE ( 22 ). Because apically expressed Na /H exchange mediates virtually all of the H secretion necessary for HCO 3 reabsorption in the mTAL ( 23 ), the study by Good and associates suggests that ANG II may inhibit apical Na /H exchange activity in the mTAL. However, this finding was opposite the previous observations showing stimulated apical Na /H exchange and HCO 3 absorption by ANG II in the proximal tubule and early distal tubule ( 19, 34, 50 ). Our data strongly indicate that the abundance of NHE3 in the ISOM and the apical expression of NHE3 in the mTAL and cortical TAL cells are markedly increased in response to ANG II treatment, in association with increased urinary pH compared with control rats. Therefore, if ANG II directly inhibits HCO 3 reabsorption in mTAL cells ( 22 ) despite increased NHE3 abundance, it seems likely that ANG II may be associated with signal pathways which could be coupled to an inhibition of other transporters that are involved in HCO 3 reabsorption in the mTAL. In particular, HCO 3 transporters in the basolateral membrane of the mTAL are not well understood; thus further studies of the effects of ANG II on the basolateral membrane HCO 3 efflux pathways are needed to define the altered mechanism of HCO 3 transport in the mTAL.

Here, we demonstrated that the abundance of electroneutral NBCn1, which is mainly located in the basolateral plasma membrane of the mTAL cells and is involved in basolateral HCO 3 transport into the TAL cells (and unlikely to be involved in the HCO 3 efflux) ( 33 ), was unchanged in response to ANG II treatment. It previously has been shown that the protein abundance of NBCn1 is dramatically enhanced in response to chronic metabolic acidosis induced by oral NH 4 Cl loading, suggesting that NBCn1 may play a role in supporting reabsorption in the mTAL ( 33 ). The absence of changes in NBCn1 in response to ANG II treatment is consistent with the observation that ANG II-treated rats had no apparent sign of metabolic acidosis.. K; i- _) [5 D% W/ O

ANG II Treatment Is Associated with Increased NHE3 Brush-Border Labeling in the Proximal Tubule4 y7 R" b  n) b: J: n

The NHE3 abundance in the cortex and OSOM remained unchanged in response to ANG II treatment, whereas immunoperoxidase microscopy and confocal laser microscopy demonstrated a marked increase in NHE3 immunolabeling in the brush border of the proximal tubules in ANG II-treated rats. In control rats, NHE3 is exclusively present at the apical domains of the proximal tubule cells ( Fig. 6 ). Immunolocalization of NHE3 in the proximal tubule is notably difficult because the different compartments are difficult to separate using straightforward morphological techniques. For example, it is virtually impossible to distinguish between cross-sectioned invaginations and small vesicles in the apical part of the proximal tubule cell ( 10 ). There would be a need for a surface membrane marker. However, it has been proposed that NHE3 is present in vesicles in the subapical cytoplasm of proximal tubule cells, as well as in the apical plasma membrane of the proximal tubule ( 9 ). The potential presence of NHE3 in an intracellular vesicular compartment in the subapical part of the proximal tubule cells could potentially represent a site for recruitment of NHE3 by regulation of subcellular trafficking. Trafficking may also occur from plasma membrane invaginations or the proximal part of the microvillus membrane to more distal parts of the microvillus membrane of the brush border. The increased labeling of NHE3 in the brush border of the proximal tubule cells in response to ANG II treatment would be consistent with subcellular redistribution of NHE3. Interestingly, a previous study ( 52 ) showed redistribution of NHE3 immunofluorescence labeling from the brush border to the base of microvilli of renal proximal tubule cells in response to acute hypertension in rats. This also supports the view that subcellular trafficking may occur. Alternatively (or in combination), the present results showing increased immunoreactivity in the brush border may reflect changes in the access of the antibody to the epitope potentially by changes in protein-protein interaction between NHE3 and other proteins. In fact, Biemesderfer et al. ( 8 ) have suggested that megalin binds NHE3 in renal proximal tubule in a portion of the COOH-terminal tail of NHE3. This binding appears to occur in the intermicrovillar clefts of the apical brush-border membrane, and this binding has been shown to inactivate NHE3 transport activity ( 7 ). Therefore, these findings raise the possibility that inactivation of NHE3 may involve the binding of NHE3 to megalin in the intermicrovillar clefts. However, this remains to be established. Similarly, further studies are necessary to evaluate whether the increased NHE3 brush-border labeling is due to changes in trafficking or in protein-protein interaction (or other undefined mechanisms such as secondary modification of NHE3 due to phosphorylation or other modifications). It should be noted that we also found an increase in both NHE3 expression and brush-border labeling in response to NH 4 Cl loading as previously found by Alpern and associates ( 1 ), which supports the validity of the methods used in the present study. A major fraction of proximal tubule HCO 3 reabsorption is mediated by apical Na /H exchange ( 4 ). ANG II receptors are present in both apical and basolateral sides of the proximal tubules ( 16 ), and ANG II has been known to stimulate HCO 3 and Na reabsorption in the proximal tubule ( 4, 19, 24, 25, 34, 45 ). More direct evidence for the functional importance of NHE3 has demonstrated that proximal tubule HCO 3 reabsorption is significantly reduced to 60% in NHE3 null mice ( 47 ).+ U6 m, M4 b$ {4 Y, l, |7 a! r

In addition, the observed rise in urinary pH may be attributed to several mechanisms, which may include: 1 ) a marked increase in buffer excretion; this would most likely be ammonium because Nagami ( 39 ) has demonstrated that ANG II markedly increases ammonium production and secretion in the proximal tubule; and 2 ) a decrease in proton secretion in the collecting duct. Consistent with this, Tojo et al. ( 49 ) demonstrated that preincubation of cortical collecting duct segments with ANG II (10 -10 to 10 -5 M) caused a dose-dependent decrease in H -ATPase activity and the inhibitory effect of ANG II was abolished when the tubules were incubated with ANG II in the presence of AT 1 -receptor antagonist losartan. Thus additional studies are required to fully describe the effects of ANG II on net acid excretion.$ o0 C$ r3 }5 o" g4 ?9 k( w% J9 o

BSC-1 Abundance in the ISOM and Apical Expression in mTAL Cells Were Increased in Response to ANG II Treatment
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The Na -K -2Cl - cotransporter BSC-1 (or NKCC2), which is localized at the apical plasma membrane domains of mTAL and cortical TAL segments ( 17 ), mediates apical NaCl transport in these water-impermeable segments. This pathway is critical for the generation of the hypertonic medullary interstitium for concentrating urine. Consistent with this, we have previously demonstrated that downregulation of BSC-1 is associated with a marked reduction of urinary concentration in several well-characterized animal models, e.g., vitamin D-induced hypercalcemia ( 51 ). Moreover, it was demonstrated that the abundance of the Na -K -2Cl - cotransporter in the TAL is increased in response to DDAVP. Because the vasopressin V 2 receptor is coupled to activation of adenylyl cyclase, it is possible that the upregulation of BSC-1 by vasopressin is a result of elevated levels of cAMP. Consistent with this, a cAMP-regulatory element (CRE) was identified in the 5'-flanking region of the mouse NKCC2 gene ( 27 ).( @4 a* o2 A: F0 z; M* L8 k
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Because specific ANG II binding sites ( 38 ) or mRNA and protein for the AT 1 receptor ( 11, 42 ) are present in mTAL cells, it is possible that ANG II has an effect via AT 1 receptors in the mTAL. However, the effects of ANG II on the TAL are not well understood and have only been addressed in a few studies. Recent work by Amlal et al. ( 3 ) demonstrated a biphasic response of Na -K -2Cl - cotransport activity in the mTAL to ANG II, with low concentrations (10 -16 to 10 -12 M) inhibiting the activity and high concentrations (10 -11 to 10 -6 M) stimulating the activity in mTAL cells. We demonstrated in the present study that the abundance, as well as the apical plasma membrane expression, of BSC-1 in mTAL cells was markedly increased in response to ANG II treatment in vivo. Because plasma or intrarenal ANG II concentrations are ≤10 -11 M ( 3 ), an increase in the plasma or medullary ANG II concentrations could stimulate Na -K -2Cl - cotransporter activity and possibly increase BSC-1 abundance in mTAL cells. Consistent with our finding, a recent study in rats with congestive heart failure, which have high plasma renin levels and presumably high plasma ANG II levels ( 48 ), demonstrated significantly increased renal BSC-1 abundance. Moreover, treatment of rats with congestive heart failure with the AT 1 -receptor antagonist losartan normalized the abundance of BSC-1, indicating that renal BSC-1 abundance is partly regulated in response to plasma ANG II levels (in addition to the known effects of vasopressin) and this is mediated by the AT 1 receptor.) H( d+ m' S( g- y/ H4 k

Possible Mediators for the Physiological Effects of ANG II on NHE3 and BSC-1 Regulation

It is well known that the AT 1 receptor mediates the recognized effects of ANG II, including its effects in increasing Na reabsorption in the kidney. For example, treatment with the AT 1 -receptor antagonist candesartan decreased the abundance of NHE3 in the brush-border membranes from rat kidney proximal tubule ( 6 ). Moreover, treatment with the AT 1 -receptor antagonist losartan normalized the increased abundance of BSC-1 in rats with congestive heart failure ( 48 ). These studies strongly indicate that ANG II regulation of NHE3 and BSC-1 abundance is mediated by the AT 1 receptor. Several studies, however, suggest that other signaling pathways may participate as well.

First, we have previously characterized changes in the abundance of several key renal Na transporters in ANG II type 1a (AT 1a ) receptor knockout mice ( 12 ). In knockout mice maintained on a low-NaCl diet, the abundances of the two aldosterone-regulated transporters (TSC and -ENaC) were markedly lower, whereas there were no changes in the abundance of NHE3 and BSC-1 in the knockout mice. This raises the possibility that the expression of NHE3 and BSC-1 may also be controlled by factors other than AT 1a, although it does not rule out AT 1a regulation. Other factors could be ANG II-mediated expression via other isoforms of ANG II receptors or by different regulating factors. Second, the effect could potentially be mediated by aldosterone, which is also increased in response to ANG II. However, several lines of evidence from our laboratory argue against a direct effect of aldosterone on the abundances of NHE3 and BSC-1. High plasma aldosterone induced by 10 days of Na restriction was associated with unchanged abundances of NHE3 and BSC-1 in rats ( 37 ). Moreover, oral administration of the aldosterone-receptor antagonist spironolactone for 7 days to NaCl-restricted rats did not significantly alter the abundances of NHE3 and BSC-1 ( 40 ). In the present study, the abundance of TSC was not changed, which would have been expected in case there were major changes in plasma aldosterone. Thus the absence of changes in this study makes it unlikely that aldosterone directly regulates the abundance of NHE3 and BSC-1 in the present protocol. Taken together, these three studies argue against a role of aldosterone in regulating expression and point to a direct effect of ANG II.; d+ C2 v! ~% n  C+ y& Q) }9 |" K

ACKNOWLEDGMENTS
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The authors thank Zhila Nikrozi, Helle Høyer, Inger Merete Paulsen, Lotte V. Holbech, Mette Vistisen, Ida M. Jalk, and Gitte Kall for technical assistance.
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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 (Danmarks Grundforskningsfond). Support for this study was provided by the Karen Elise Jensen Foundation, the Human Frontier Science Program, the European Commission (QRLT 2000 00778 and QRLT 2000 00987), the Novo Nordic Foundation, the Danish Medical Research Council, the University of Aarhus Research Foundation, the Korea Science and Engineering Foundation (THK; R05-2001-000-00630-0), the University of Aarhus, and the intramural budget of the National Heart, Lung, and Blood Institute.
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