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Am J Physiol Regul Integr Comp Physiol 280: R1632-R1641, 2001;
0363-6119/01 $5.00
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Vol. 280, Issue 6, R1632-R1641, June 2001

Altered expression of renal aquaporins and Na+ transporters in rats treated with L-type calcium blocker

Weidong Wang1, Tae-Hwan Kwon1,2, Chunling Li3, Allan Flyvbjerg4, Mark A. Knepper5, Jørgen Frøkiær3, and Søren Nielsen1

1 Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus; 3 Department of Clinical Physiology, Institute of Experimental Clinical Research, Aarhus University Hospital, DK-8200 Aarhus N; 4 Laboratory of M (Diabetes and Endocrinology), Aarhus University Hospital, DK-8000 Aarhus C, Denmark; 2 Department of Physiology, School of Medicine, Dongguk University, Kyungju 780-714, Korea; and 5 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nifedipine, a calcium antagonist, has diuretic and natriuretic properties. However, the molecular mechanisms by which these effects are produced are poorly understood. We examined kidney abundance of aquaporins (AQP1, AQP2, and AQP3) and major sodium transporters [type 3 Na/H exchanger (NHE-3); type 2 Na-Pi cotransporter (NaPi-2); Na-K-ATPase; type 1 bumetanide-sensitive cotransporter (BSC-1); and thiazide-sensitive Na-Cl cotransporter (TSC)] as well as inner medullary abundance of AQP2, phosphorylated-AQP2 (p-AQP2), AQP3, and calcium-sensing receptor (CaR). Rats treated with nifedipine orally (700 mg/kg) for 19 days had a significant increase in urine output, whereas urinary osmolality and solute-free water reabsorption were markedly reduced. Consistent with this, immunoblotting revealed a significant decrease in the abundance of whole kidney AQP2 (47 ± 7% of control rats, P < 0.05) and in inner medullary AQP2 (60 ± 7%) as well as in p-AQP2 abundance (17 ± 6%) in nifedipine-treated rats. In contrast, whole kidney AQP3 abundance was significantly increased (219 ± 28%). Of potential importance in modulating AQP2 levels, the abundance of CaR in the inner medulla was significantly increased (295 ± 25%) in nifedipine-treated rats. Nifedipine treatment was also associated with increased urinary sodium excretion. Consistent with this, semiquantitative immunoblotting revealed significant reductions in the abundance of proximal tubule Na+ transporters: NHE-3 (3 ± 1%), NaPi-2 (53 ± 12%), and Na-K-ATPase (74 ± 5%). In contrast, the abundance of the distal tubule Na-Cl cotransporter (TSC) was markedly increased (240 ± 29%), whereas BSC-1 in the thick ascending limb was not altered. In conclusion, 1) increased urine output and reduced urinary concentration in nifedipine-treated-rats may, in part, be due to downregulation of AQP2 and p-AQP2 levels; 2) CaR might be involved in the regulation of water reabsorption in the inner medulla collecting duct; 3) reduced expression of proximal tubule Na+ transporters (NHE-3, NaPi-2, and Na, K-ATPase) may be involved in the increased urinary sodium excretion; and 4) increase in TSC expression may occur as a compensatory mechanism.

calcium-channel blocker; diuresis; hypertension; natriuresis; urinary concentration mechansim


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CALCIUM-CHANNEL BLOCKERS (CCBs) have gained wide acceptance in the treatment of hypertension primarily because of their efficacy and tolerability. Several in vivo studies (34) have provided evidence that nifedipine, one of the dihydropyridine derivatives of CCBs, produces significant diuresis and natriuresis with no changes in the glomerular filtration rate (GFR) and renal blood flow (RBF). This suggests that nifedipine has an inhibitory effect on the renal tubular reabsorption of water and sodium. Recently, studies have suggested the presence of L-type calcium channels in most renal tubule segments. Saunders and Isaacson (45) identified L-type calcium channels in the apical plasma membrane from rabbit proximal straight tubules, cortical thick ascending limbs, distal convoluted tubules, and cortical collecting tubules, and they concluded that there were L-type calcium channels throughout the nephron and collecting duct. More recent studies also have L-type calcium channels in TAL/DCT cells and PST cells (40, 50, 51). Several studies (25, 49) undertaken to localize the site of action of dihydropyridine on renal tubules suggest that both proximal and distal tubular sites were involved. However, the exact sites and molecular mechanisms responsible for the altered renal transport of water and sodium in response to nifedipine treatment have not yet been clearly elucidated.

The aquaporins are a family of membrane proteins that function as water channels (37). Aquaporin-1 (AQP1) is expressed in the proximal tubule and descending limb of Henle's loop, where it plays an important role in fluid absorption (1). Several studies, including in AQP1 gene-knockout mice, have now emphasized its critical role in the constitutive water reabsorption in these segments. The water permeability of the collecting duct is regulated by vasopressin, and vasopressin-regulated water transport across the apical plasma membrane of collecting duct cells is chiefly mediated by AQP2 (36). Water reabsorption in the collecting duct is regulated by both short- and long-term mechanisms, both of which have been shown to depend critically on AQP2 (37). Recently, we and others have demonstrated that altered expression and apical targeting of AQP2 play a critical role in water balance disorders using a series of experimental models (for recent review, see Ref. 37). Water transport across the basolateral plasma membrane of collecting duct principal cells is thought to be mediated by AQP3 (12) and AQP4. Consistent with this view, transgenic mice lacking AQP3 (32) are severely polyuric, and inner medullary collecting ducts from AQP4-deficient mice have a fourfold reduction in vasopressin-stimulated water permeability (7). Therefore, alterations in aquaporin expression in the kidney may be hypothesized to play a role in the development of increased diuresis in response to the treatment with CCBs.

Reabsorption of sodium in the proximal tubule occurs mainly via the type 3 Na/H exchanger (NHE-3) (3), but a variety of sodium-coupled cotransporters is also involved including the type 2 Na-Pi cotransporter (NaPi-2) (4). Importantly, it has been reported that dihydropyridine derivatives promote urinary excretion of sodium and also of phosphate and hence result in natriuresis and phosphaturia (30, 46). The Na-K-ATPase, present in the basolateral plasma membrane domains (20), is involved in establishing the driving force to promote sodium reabsorption. Thus it can be speculated that changes in NHE-3, Na-K-ATPase, and/or NaPi-2 expression may be involved in the altered sodium and phosphate reabsorption in the proximal tubule. In the thick ascending limbs and distal convoluted tubules, apically expressed diuretic inhibitable Na+-coupled cotransporters are responsible for apical Na+ entry from the tubule lumen. These are the Na-K-2Cl cotransporter [type 1 bumetanide-sensitive cotransporter (BSC-1) or NKCC2] (13) and the thiazide-sensitive Na-Cl cotransporter (TSC) (39), respectively. Both are recognized regulatory targets. For example, increased delivery of sodium to the distal nephron induces significant increases in the expression of BSC-1 (13), whereas aldosterone and sodium restriction induce TSC expression (24). We hypothesize that CCB treatment may be associated with alterations in the expression of one or both of these transporters. Moreover, recent studies have revealed dysregulation of proximal tubule and distal tubule/TAL sodium transporters in experimental rat models of nephrotic syndrome viz adriamycin-induced nephrosis (14), in experimental chronic renal failure (5/6 nephrectomy), and in ischemia-induced acute renal failure (27, 28) in conjunction with altered tubular sodium handling and decreased urinary concentration. These pathophysiological studies further underscore a critical role of renal sodium transporters in renal regulation of sodium metabolism. Thus we hypothesize that CCB treatment may elicit distinct changes in the expression of major renal sodium transporters (in addition to changes in aquaporins) resulting in the impairment of tubular sodium and water reabsorption as well as in the impairment of the urinary concentration.

The purposes of the present study are 1) to examine the changes in urinary water and sodium excretion in response to L-type CCB treatment (using nifedipine), 2) to examine whether there are changes in the levels of renal aquaporins and sodium transporter expression in response to nifedipine treatment, and 3) to examine whether the altered expression of renal aquaporins and major renal sodium transporters correlates with the changes in renal water and sodium metabolism in nifedipine-treated rats.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental Animals and Experimental Protocol

Studies were performed on 14 adult female Wistar rats initially weighing 190-195 g. The following protocols were followed, in which rats were fed powdered rat chow mixed with or without nifedipine.

Protocol 1: nifedipine-treated group. The food contained nifedipine in a dose of 700 mg/kg fodder. Rats were fed for 19 days (n = 7).

Protocol 2: control group. The rats were maintained on placebo food for 19 days, with free access to water (n = 7).

The rats were maintained in individual cages throughout the study, and water intake was measured daily. Nifedipine-treated rats and control rats had the same food intake (g/day), respectively: day 4 (20.9 ± 0.5 and 20.4 ± 0.6), day 7 (20.7 ± 0.6 and 20.7 ± 0.7), day 14 (21.6 ± 0.6 and 20.3 ± 0.7), and day 19 (21.2 ± 0.6 and 21.2 ± 0.8). At the end of the study, animals from each group were placed in metabolic cages to determine daily water intake and urine output. At day 19, rats were anesthetized with pentobarbital sodium (50 mg/kg), tail-cuff blood pressure was measured, and a blood sample was taken. Right kidneys were removed, frozen rapidly in liquid nitrogen, and maintained at -80°C for membrane fractionation of whole kidney. Left kidneys were dissected, and inner medulla was used for membrane fractionation. Osmolality, creatinine, sodium and potassium concentrations were measured in plasma and urine samples.

Primary Antibodies

For semiquantitative immunoblotting, previously characterized monoclonal antibodies or affinity-purified polyclonal antibodies were used. 1) AQP2 (LL127 serum, 1:3,000): immune serum to AQP2 has previously been described (9). 2) Phosphorylated-AQP2 (p-AQP2) (AN244-pp-AP): an affinity-purified rabbit polyclonal antibody to p-AQP2 has previously been described (8). 3) AQP3 (LL178, 1:300): an affinity-purified rabbit polyclonal antibody to AQP3 has previously been described (12). 4) AQP1 (LL266 serum, 1:3,000): immune serum to AQP1 has previously been described (47). 5) Polyclonal anti-calcium-sensing receptor (CaR) antibody (1:1,000) was obtained from Affinity Bioreagents. 6) NHE-3 (LL546, 1:300): an affinity-purified rabbit polyclonal antibody to NHE-3 has previously been characterized (14, 21). 7) NaPi-2 (LL696, 1:600): it was raised against synthetic peptide corresponding to the final 24 amino acids of the COOH-terminal tail of NaPi-2 (4). 8) Na-K-ATPase (1:5,000): a monoclonal antibody against the alpha -1 subunit of Na-K-ATPase has previously been characterized (20). 9) TSC (LL573, 1:300): an affinity-purified rabbit polyclonal antibody to the apical Na-Cl cotransporter of the distal convoluted tubule has previously been characterized (24). 10) BSC-1 (LL320, 1:300): an affinity-purified rabbit polyclonal antibody to the Na-K-2Cl cotransporter of the thick ascending limb has previously been characterized (13, 22).

Membrane Fractionation for Immunoblotting

Whole kidneys or inner medullas were homogenized (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, containing 8.5 µM leupeptin, 1 mM phenylmethyl sulfonylfluoride) using an ultra-turrax T8 homogenizer (IKA Labortechnik), and the homogenate was centrifuged in an Eppendorf centrifuge at 4,000 g for 15 min at 4°C to remove whole cells, nuclei, and mitochondria. The supernatant was then centrifuged at 200,000 g for 1 h to produce a pellet containing membrane fractions containing plasma membranes and intracellular vesicles. Gel samples (Laemmli sample buffer containing 2% SDS) were made from this pellet.

Electrophoresis and Immunoblotting

Samples of membrane fractions were run on 12% polyacrylamide minigels (BioRad Mini Protean II) for AQP2, p-AQP2, AQP3, AQP1, NaPi-2, NHE-3, and Na-K-ATPase, or 6-16% gradient polyacrylamide minigels for CaR, TSC, and BSC-1. For each gel, an identical gel was run parallel and subjected to Coomassie brilliant blue staining to assure identical loading within ±5% of the mean (determined by densitometry of major bands on gel). The other gel was subjected to immunoblotting. After transfer by electroelution to nitrocellulose membranes, blots were blocked with 5% milk in PBS-T (80 mM Na2HPO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 20 mM NaH2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h and incubated overnight at 4°C with affinity-purified primary antibodies. The labeling was visualized with horseradish peroxidase-conjugated secondary antibodies (P447 or P448, DAKO, Glostrup, Denmark; diluted 1:3,000) using enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Quantitation of Expression Levels of AQPs and Sodium Transporters

ECL 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 determined of blots where samples of kidneys from the nifedipine-treated group were run together with samples from control kidneys (33). The labeling density was corrected by densitometry of Coomassie brilliant blue-stained gels (i.e., to control for minor difference in protein loading). Values were presented in the text as means ± SE. Comparisons between groups were made by unpaired t-test. P values <0.05 were considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Urine Production Is Increased and Urinary Concentration Is Decreased in Nifedipine-Treated Rats

At the end of the experiment, tail-cuff blood pressure was measured, and the urine output was determined. There was no significant difference in blood pressure between the two groups of rats (Table 1). The urine output was significantly increased to 120 ± 14 µl · min-1 · kg-1 in nifedipine-treated rats, compared with control rats (50 ± 4.7 µl · min-1 · kg-1, P < 0.05, Table 1). Parallel to this, there was a significant increase in water intake in nifedipine-treated rats (168 ± 16 µl · min-1 · kg-1), when compared with control rats (92 ± 7.3 µl · min-1 · kg-1, P < 0.05, Table 1). This marked increase in urine output was accompanied by a significant decrease in urine osmolality. Urine osmolality in nifedipine-treated rats was 482 ± 53 vs. 1,161 ± 52 mosmol/kgH2O in control rats (P < 0.05, Table 1). Consistent with changes in urine output and urine osmolality, the urine-to-plasma osmolality ratio (U/P osm) was declined to 1.5 ± 0.2 in nifedipine-treated rats vs. 3.5 ± 0.2 in control rats (P < 0.05, Table 1), and the solute-free water reabsorption (TcH2O) was reduced to 49 ± 19 vs. 124 ± 9 µl · min-1 · kg-1 in control rats (P < 0.05, Table 1).

                              
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  		Table 1.   Functional data

AQP2 Abundance Is Decreased in Nifedipine-Treated Rats

The anti-AQP2 antibodies exclusively recognize the 29-kDa and the 35- to 50-kDa bands (Fig. 1), corresponding to nonglycosylated and glycosylated AQP2. As shown in Fig. 1 and Table 2, semiquantitative immunoblotting of all samples from nifedipine-treated and control rats revealed a marked decrease in total kidney AQP2 abundance to 47 ± 7%, compared with control rats (100 ± 16%, P < 0.05). In kidney inner medulla, AQP2 abundance was also significantly reduced to 60 ± 7% in nifedipine-treated rats, compared with control rats (100 ± 16%, P < 0.05, Fig. 2).


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  		Fig. 1.   Immunoblot of membrane fractions of whole kidney prepared from control (Control; n = 7) and nifedipine-treated rats (Nifedipine; n = 7). A: the immunoblot was labeled with anti-aquaporin-2 (AQP2) and revealed 29- and 35- to 50-kDa AQP2 bands, representing nonglycosylated and glycosylated forms of AQP2. The labeling intensities were reduced in Nifedipine. B: densitometry revealed that AQP2 levels were significantly reduced in Nifedipine, to 47 ± 7%, compared with Control (100 ± 16%, *P < 0.05).


                              
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  		Table 2.   Total kidney abundance of aquaporins and sodium transporters in nifedipine-treated rats



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  		Fig. 2.   Immunoblot (A) and corresponding densitometric analysis (B) of AQP2 protein expression in membrane fractions from kidney inner medulla from Control (n = 7) and Nifedipine (n = 7). Compared with levels in Control, expression was reduced in Nifedipine. Densitometry revealed a significant reduction in AQP2 expression in Nifedipine compared with Control (60 ± 7 vs. 100 ± 12%, *P < 0.05).

Moreover, semiquantitative immunoblotting with antibodies that selectively recognize AQP2 phosphorylated in the protein kinase A (PKA) consensus site (Serine 256) (8) demonstrated that p-AQP2 was also markedly reduced with nifedipine treatment. As shown in Fig. 3, densitometric analysis of immunoblots revealed a dramatic decrease in p-AQP2 abundance to 17 ± 6%, compared with control rats (100 ± 23%, P < 0.05).


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  		Fig. 3.   Immunoblot of membrane fractions of kidney inner medulla prepared from Control (n = 7) and Nifedipine (n = 7). A: the immunoblot was labeled with anti-phosphorylated (p)-AQP2 and revealed 29- and 35- to 50-kDa AQP2 bands, representing nonglycosylated and glycosylated forms of p-AQP2. The labeling intensities were reduced in Nifedipine. B: densitometry revealed that p-AQP2 levels were significantly reduced in Nifedipine, to 17 ± 6%, compared with Control (100 ± 23%, *P < 0.05).

AQP3 and AQP1 Abundance Was Not Reduced in Nifedipine-Treated Rats

As shown in Fig. 4 and Table 2, densitometric analysis of all samples from nifedipine-treated rats and control rats revealed a marked increase in total kidney AQP3 abundance to 219 ± 28% in nifedipine-treated rats, compared with control rats (100 ± 18%, P < 0.05). However, inner medullary AQP3 abundance was unchanged in nifedipine-treated rats (85 ± 9%), compared with control rats (100 ± 9%, P > 0.05, not shown), demonstrating that the increase in AQP3 occurs only in cortical and outer medullary collecting duct where AQP3 is the predominant basolateral water channel (12). Moreover, semiquantitative immunoblotting demonstrated that the abundance of proximal nephron water channel AQP1 was not altered in nifedipine-treated rats (94 ± 8%), compared with control rats (100 ± 9%, P > 0.05, Fig. 5 and Table 2).


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  		Fig. 4.   Immunoblot of membrane fractions of rat whole kidney prepared from Control (n = 7) and Nifedipine (n = 7). A: the immunoblot was labeled with anti-AQP3 and revealed 27- and 33- to 40-kDa AQP3 bands, representing nonglycosylated and glycosylated forms of AQP3. The intensities were increased in Nifedipine. B: densitometry performed on the immunoblot of kidney membrane fractions from all Nifedipine and Control. AQP3 levels were significantly increased in Nifedipine, to 219 ± 28%, compared with Control (100 ± 18%, *P < 0.05).



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  		Fig. 5.   Immunoblot of membrane fractions of rat whole kidney prepared from Control (n = 7) and Nifedipine (n = 7). A: immunoblot was reacted with anti-AQP1 and revealed 29- and 35- to 50-kDa AQP1 bands, representing nonglycosylated and glycosylated forms of AQP1. B: densitometric analysis of all Nifedipine and Control showed no differences in AQP1 expression (densities were 94 ± 8 and 100 ± 9%, respectively, P > 0.05).

Inner Medullary Abundance of CaR Is Markedly Increased in Nifedipine-Treated Rats

The CaR has been implicated to be involved in modulating AQP2 expression (43). We therefore investigated if the receptor expression was altered. The antibody against CaR recognizes a 138-kDa band that corresponds to CaR from the rat kidney (42, 43). Densitometric analysis demonstrated a significant increase in the abundance of CaR in nifedipine-treated rats to 294 ± 25% of the levels in the control rats (100 ± 25%, P < 0.05, Fig. 6).


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  		Fig. 6.   Immunoblot of membrane fractions of rat kidney inner medulla prepared from Control (n = 7) and Nifedipine (n = 7). A: immunoblot was reacted with anti-calcium-sensing receptor (CaSR) protein and revealed 138-kDa bands. B: densitometric analysis of all Nifedipine and Control showed a significant increase of CaSR abundance in Nifedipine to 294 ± 25% of Control levels (100 ± 25%, *P < 0.05).

Urinary Sodium Excretion Is Increased in Nifedipine-Treated Rats

As shown in Table 1, the fractional excretion of sodium (FENa) was significantly increased to 1.5 ± 0.2%, compared with control rats (0.5 ± 0.1%, P < 0.05). The urinary sodium excretion rate was also increased to 0.63 ± 0.02 in nifedipine-treated rats, compared with control rats (0.53 ± 0.04 µmol · min-1 · 100 g body wt-1, P < 0.05, Table 1).

Abundance of NHE-3 and NaPi-2 Is Decreased in Nifedipine-Treated Rats

The changes in abundance of the proximal tubule sodium transporters were analyzed including the major Na-reabsorption pathway via NHE-3 and a minor pathway involving NaPi-2. An additional reason for examining NaPi-2 expression is the known disturbance in phosphate metabolism in response to CCB treatment (19, 31).

The affinity-purified anti-NHE-3 antibody recognized approximately an 87-kDa band in membrane preparations from the whole kidney, consistent with previous studies (2, 5, 6). As shown in Fig. 7, densitometric analysis revealed a marked decrease in total kidney NHE-3 band density to 3 ± 1% in nifedipine-treated rats, compared with control rats (100 ± 12%, P < 0.05, Fig. 7 and Table 2). As shown in Fig. 8, the affinity-purified anti-NaPi-2 antibodies recognized an ~85-kDa main band in membrane preparations from the whole kidney, consistent with previous studies (4). Densitometric analysis revealed a significant decrease in the density of this band in total kidney to 53 ± 12% in nifedipine-treated rats, compared with control rats (100 ± 12%, P < 0.05, Fig. 8 and Table 2).


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  		Fig. 7.   Immunoblot of membrane fractions of rat whole kidney prepared from Control (n = 7) and Nifedipine (n = 6). A: the immunoblot was reacted with anti-type 3 Na/H exchanger (NHE-3) and revealed an ~87-kDa band. B: densitometry performed on immunoblots of whole kidneys from all Nifedipine and Control. NHE-3 levels were significantly decreased in Nifedipine, to 3 ± 1%, compared with Control (100 ± 12%, *P < 0.05).



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  		Fig. 8.   Immunoblot of membrane fractions of rat whole kidney prepared from Control (n = 7) and Nifedipine (n = 7). A: the immunoblot was reacted with anti-type 2 Na-Pi cotransporter (NaPi-2) and revealed an ~85-kDa band. B: densitometry performed on immunoblots of whole kidneys from all Nifedipine and Control. NaPi-2 levels are significantly decreased in Nifedipine, to 53 ± 12%, compared with Control (100 ± 12%, *P < 0.05).

Na-K-ATPase Abundance Is Decreased in Nifedipine-Treated Rats

The monoclonal antibody to the alpha -1 isoform of Na-K-ATPase recognized a band of ~96 kDa (20). This isoform is expressed in all renal tubule segments. Semiquantitative immunoblotting revealed a modest but significant decrease in total kidney Na-K-ATPase abundance to 74 ± 5% in response to nifedipine treatment, compared with control rats (100 ± 8%, P < 0.05, Fig. 9 and Table 2).


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  		Fig. 9.   Immunoblot of membrane fractions of rat whole kidney prepared from Control (n = 7) and Nifedipine (n = 7). A: the immunoblot was reacted with Na-K-ATPase and revealed an ~96-kDa band. B: densitometry performed on immunoblots of whole kidneys from all Nifedipine and Control. Na-K-ATPase levels were decreased in Nifedipine, to 74 ± 5%, compared with Control (100 ± 8%, *P < 0.05).

Increased TSC and Unchanged BSC-1 Expression in Response to Nifedipine Treatment

As shown in Fig. 10, the affinity-purified anti-TSC antibody recognized a broad band centered at ~165 kDa, consistent with previous studies (24). In contrast to the significant reductions in total kidney abundances of NHE-3, NaPi-2, and Na-K-ATPase in response to nifedipine treatment, semiquantitative immunoblotting revealed a significant increase of total kidney abundance of TSC in the distal convoluted tubule to 240 ± 29%, when compared with control rats (100 ± 17%, P < 0.05, Fig. 10 and Table 2). In contrast, the total kidney abundance of BSC-1 in the thick ascending limb was not changed in nifedipine-treated rats (104 ± 33%), compared with control rats (100 ± 21%, P > 0.05, Table 2).


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  		Fig. 10.   Immunoblot of membrane fractions of rat whole kidney prepared from Control (n = 7) and Nifedipine (n = 7). A: the immunoblot was reacted with anti-thiazide-sensitive Na-Cl cotransporter (TSC) and revealed an ~160-kDa band. B: densitometry performed on immunoblots of whole kidneys from all Nifedipine and Control. TSC levels were significantly increased in Nifedipine, to 240 ± 29%, compared with Control (100 ± 21%, *P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CCBs have diuretic and natriuretic properties in normal animals and humans. Injection or infusion of dihydropyridine derivatives into the renal artery demonstrated that CCBs have an inhibitory effect on the renal tubular reabsorption of water and sodium, irrespective of hemodynamic changes (30). Here, we have demonstrated that long-term treatment of rats with nifedipine (a dihydropyridine derivative) was associated with significant changes in transporter abundances in both the proximal and distal portions of the nephron that may provide explanations for the observed increases in salt and water excretion.

The approach taken in this paper is an example of a relatively new strategy for investigation of integrative problems in kidney physiology using molecular tools (10, 15, 27, 29). Most studies in kidney physiology that apply antibody and cDNA probes have focused on in vitro systems such as cultured cells and have investigated the regulation of one protein target at a time. Here, we exploit a number of antibody probes simultaneously in the same experiments to obtain a broad profile of Na+ transporter and water channel regulation in response to a distinct physiological stimulus in intact rats. Such data can be interpreted in the rich context of the experimental literature in integrative renal physiology that has defined the important mediators of renal sodium excretion regulation and blood pressure control. A drawback of the approach is that, because the experiments are done in intact animals, abundance changes in individual transporters could theoretically be due to direct effects of the stimulus (here nifedipine administration) or to indirect effects that may be compensatory in nature. Hence it is important to examine the data in the context of the foregoing experimental literature to take full advantage of it. In this discussion, we analyze the proximal and distal effects in turn.

Proximal Effects of Long-Term Nifedipine Administration

The proximal tubule reabsorbs two-thirds of the NaCl and water filtered by the glomerulus. Thus inhibition of proximal NaCl and water absorption could provide an explanation for increased NaCl and water excretion in response to nifedipine. Animal experiments and clinical trials have previously provided evidence for a proximal tubular natriuretic effect of nifedipine (16, 26, 49). Consistent with these observations, our present studies demonstrated that rats treated with nifedipine had increased urinary sodium excretion as well as increased FENa (Table 1). We observed marked decreases in the abundances of two apical sodium transporters in the proximal tubule (NHE-3 and NaPi-2) as well as the basolateral sodium pump (Na-K-ATPase). Although the Na-K-ATPase is found throughout the renal tubule, the observed decrease of alpha -1 subunit abundance is presumably reflective of a decrease in the proximal tubule owing to the sheer bulk of proximal tubules compared with other tubule segments. These changes in protein abundance provide a potential explanation for the natriuretic effect of nifedipine.

The major route of water absorption in the proximal tubule is the molecular water channel AQP1 (37). We did not find any changes in AQP1 abundance in response to nifedipine treatment. Therefore, it is likely that a decrease in water absorption induced by nifedipine in the proximal tubule is secondary to a decline in the expression of sodium transporter expression (including the major apical pathway involving NHE-3) resulting in a reduced driving force for the osmotic flow of water across the proximal tubule epithelium.

In addition to its role in sodium reabsorption (3), NHE-3 is believed to be responsible for proximal bicarbonate absorption via its role in proton secretion. On the basis of this view, it might be expected that the marked decrease in NHE-3 abundance in response to nifedipine demonstrated in this study would lead to severe metabolic acidosis owing to a failure of proximal bicarbonate absorption. This prediction was belied by the relatively benign physiological state in the rats receiving nifedipine. Interestingly, recent studies have shown that only a mild metabolic acidosis is present in NHE-3-knockout mice (35, 48). Possibly, renal proximal tubules have another (unidentified) transporter that mediates bicarbonate reabsorption (35, 48). Thus the picture is emerging that NHE-3 is the main transporter for absorption of sodium in the proximal tubule, but not critical to acid-base transport.

NaPi-2 is expressed in the apical domain of the proximal tubule cells and contributes to the sodium and phosphate reabsorption in this segment (4). In the present study, we found a significant reduction in NaPi-2 expression in response to nifedipine treatment, indicating that this may participate in the decreased proximal tubular sodium and phosphate reabsorption. This is consistent with previous studies demonstrating that nifedipine exerts significant inhibitory effects on the proximal tubule sodium and phosphate and water reabsorption (31, 46), hence resulting in natriuresis, increased urinary phosphate excretion, and polyuria. Moreover, our data revealed a marked decrease in the plasma concentration of phosphate, which is consistent with reduced NaPi-2 expression.

It is interesting that the pattern of proximal transporter abundance changes seen in response to nifedipine was similar to that seen in a rat model of hepatic cirrhosis induced by long-term CCl4 inhalation (15) with marked decreases in renal NHE-3 and NaPi-2 abundances and no decrease in AQP1 abundance. The decrease in proximal sodium transporters in the cirrhosis model was viewed as a possible compensatory response following hemodynamically mediated sodium retention. Furthermore, cirrhotic rats also manifested a marked increase in AQP3 abundance in the collecting ducts as seen here with nifedipine administration. It appears possible, therefore, that L-type calcium channels, the target for nifedipine's action, play a role in some of the renal tubular changes seen in cirrhosis.

Distal Effects of Long-Term Nifedipine Administration

Our results revealed that nifedipine treatment in rats was associated with a significant decrease in total kidney AQP2 as well as inner medullary AQP2 expression. The reduced AQP2 expression was associated with polyuria, decreased urine osmolality, and decreased TcH2O. Moreover, semiquantitative immunoblotting with antibodies that selectively recognize AQP2, which is phosphorylated in the PKA consensus site (Serine 256) (8), demonstrated that phosphorylation of AQP2 was also dramatically reduced with nifedipine treatment. Thus it appears likely that nifedipine treatment results in polyuria and decreased urinary concentration by both 1) inhibition of short-term regulation of vasopressin-regulated water channel AQP2 by decreasing phosphorylation and 2) inhibition of long-term adaptational regulation by decreasing AQP2 abundance.

In contrast to the marked reduction in AQP2 expression, our data revealed that total kidney AQP3 expression was increased and inner medullary AQP3 expression was maintained, compared with the control levels. The mechanisms underlying regulation of AQP3 expression are not fully understood so far. Immunoblotting has shown that thirsting of rats for 48 h (12) [desamino-Cys1,D-Arg8]vasopressin (dDAVP) treatment of Brattleboro rats for 5 days (47) induces a marked increase in AQP3 expression, presumably secondary to increased intracellular cAMP levels. Thus there is clear evidence that AQP3 regulation is related to changes in vasopressin levels and water balance. However, several conditions revealed a decoupling of AQP2 and AQP3 expression [i.e., congestive heart failure (38), hepatic cirrhosis (15), escape from the water-retaining action of vasopressin (11), and low-protein normal caloric diet (44)], indicating that factors other than vasopressin-mediated changes in intracellular cAMP levels are involved in controlling AQP3 expression.

The abundance of TSC of the distal convoluted tubule (3, 24) was markedly increased following long-term nifidipine administration. This response is unlikely to be directly associated with the natriuresis caused by nifedipine and is likely instead to be a compensatory response. Recently, it has been demonstrated that aldosterone induces the expression of TSC (24) parallel with its effects to activate the epithelial sodium channel (23). Because we observed an increased urinary sodium excretion despite unchanged intake (food intake was identical between control and nifedipine-treated rats), it is likely that nifedipine treatment resulted in some degree of extracellular fluid volume depletion associated with increased aldosterone levels. Recently, upregulation of TSC was also reported in the setting of vasopressin escape providing a means of limiting or correcting the hyponatremia seen with simultaneous water loading and vasopressin treatment (10). The increase was not associated with a change in the plasma level of aldosterone. The similarity of the pattern of distal transporter abundance changes between the vasopressin escape phenomenon (decreased AQP2, increased AQP3, and increased TSC abundance) and nifedipine treatment (same changes, this study) raises the possibility that activation of nifedipine-sensitive calcium channels could be involved in vasopressin escape.

Nifedipine Treatment Is Associated with Increased Expression of CaR in the Inner Medulla

We demonstrated that CaR expression in the inner medulla was significantly increased in response to nifedipine treatment. Several studies have focused on the potential role of CaR as the mediator of the effects of extracellular Ca2+ on several aspects of renal function (17, 18, 41). CaR has been identified at the apical domains of rat terminal collecting ducts, where increases in tubular calcium concentrations reduce arginine-vasopressin (AVP)-stimulated water reabsorption (17, 43). Thus increased CaR expression in the inner medulla in response to nifedipine treatment may be speculated to contribute to the reduction of AVP-stimulated water reabsorption in this segment, hence resulting in a marked polyuria, although additional studies are necessary to support this view.

Perspectives

This study illustrates a relatively new approach to the study of complex physiological processes involved in regulation of renal sodium and water excretion in intact animals using an array of antibodies to major renal Na+ transporters and aquaporins. Here, the antibodies are employed to survey the nephron with regard to changes in transporter abundance using quantitative immunoblotting to assess protein amount. The results have provided potentially new insights into the role of L-type calcium channels in the renal tubule. The generally accepted view of the pathogenesis of hypertension recognizes that any form of sustained hypertension depends on a defect in renal tubule sodium transport regulation. The efficacy of nifedipine and its congeners in the treatment of some forms of hypertension therefore implies an effect on renal tubule sodium and water transport. Here, long-term nifedipine treatment caused marked changes in the renal abundances of several aquaporins and sodium transporters. Some of these changes provide plausible explanations for the observed ability of nifedipine to increase salt and water excretion. Others appear to be compensatory responses. Comparison of the responses obtained with findings of previous studies also points to a possible role of L-type calcium channels in the renal response to hepatic cirrhosis and in the vasopressin escape phenomenon. Further elucidation of the molecular mechanisms underlying the effect of nifedipine and other antihypertensive drugs can be expected to provide a better understanding of the renal role in blood pressure control.


    ACKNOWLEDGEMENTS

The authors thank M. Vistisen, H. Høyer, G. Christensen, I. M. Paulsen, and Z. Nikrozi for expert technical assistance.


    FOOTNOTES

Support for this study was provided by the Karen Elise Jensen Foundation, Novo Nordisk Foundation, Danish Medical Research Council, University of Aarhus Research Foundation, the University of Aarhus, the EU Commission (EU-Biotech, TMR and KA3.1.3 programmes), the Dongguk University, and the Intramural Budget of the National Heart, Lung, and Blood Institute.

Address for reprint requests and other correspondence: S. Nielsen, Dept. of Cell Biology, Institute of Anatomy, Univ. of Aarhus, DK-8000 Aarhus C, 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.

Received 10 August 2000; accepted in final form 10 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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