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Am J Physiol Renal Physiol 292: F914-F920, 2007. First published October 31, 2006; doi:10.1152/ajprenal.00361.2006
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EDITORIAL FOCUS

Angiotensin II increases chloride absorption in the cortical collecting duct in mice through a pendrin-dependent mechanism

Vladimír Pech,1 Young Hee Kim,1 Alan M. Weinstein,2 Lorraine A. Everett,3 Truyen D. Pham,1 and Susan M. Wall1,4

1Renal Division, Department of Medicine, and 4Department of Physiology, Emory University School of Medicine, Atlanta, Georgia; 2Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York; 3The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom

Submitted 8 September 2006 ; accepted in final form 13 October 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Pendrin (Slc26a4) localizes to type B and non-A, non-B intercalated cells in the distal convoluted tubule, the connecting tubule, and the cortical collecting duct (CCD), where it mediates apical Cl–/HCO3– exchange. The purpose of this study was to determine whether angiotensin II increases transepithelial net chloride transport, JCl, in mouse CCD through a pendrin-dependent mechanism. JCl and transepithelial voltage, VT, were measured in CCDs perfused in vitro from wild-type and Slc26a4 null mice ingesting a NaCl-replete diet or a NaCl-replete diet and furosemide. In CCDs from wild-type mice ingesting a NaCl-replete diet, VT and JCl were not different from zero either in the presence or absence of angiotensin II (10–8 M) in the bath. Thus further experiments employed mice given the high-NaCl diet and furosemide to upregulate renal pendrin expression. CCDs from furosemide-treated wild-type mice had a lumen-negative VT and absorbed Cl–. With angiotensin II in the bath, Cl– absorption doubled although VT did not become more lumen negative. In contrast, in CCDs from furosemide-treated Slc26a4 null mice, Cl– secretion and a VT of ~0 were observed, neither of which changed with angiotensin II application. Inhibiting ENaC with benzamil abolished VT although JCl fell only ~50%. Thus substantial Cl– absorption is observed in the absence of an electromotive force. Attenuating apical anion exchange with the peritubular application of the H+-ATPase inhibitor bafilomycin abolished benzamil-insensitive Cl– absorption. In conclusion, angiotensin II increases transcellular Cl– absorption in the CCD through a pendrin- and H+-ATPase-dependent process.

transepithelial voltage; bafilomycin; H+-ATPase; knockout mice; S1c26a4; intercalated cell

WITHIN THE CORTICAL COLLECTING DUCT (CCD), transepithelial transport of Cl– occurs through both paracellular and transcellular transport (29, 30). The driving force for paracellular transport-mediated Cl– absorption is the lumen-negative transepithelial voltage, VT, generated by ENaC-mediated Na+ absorption (29, 30). In rabbit CCD, only ~20% of net Cl– absorption occurs through paracellular transport, while the majority of net Cl– absorption occurs through transepithelial transport (30, 33). However, the molecular mechanism responsible for this transepithelial transport is poorly understood. With the recent explosion in genetically engineered mice, our ability to examine the mechanism of transepithelial Cl– absorption in the CCD has been greatly expanded. However, because perfusing mouse tubules in vitro requires greater technical skill than is needed with rat or rabbit, few studies have utilized this technique to examine ion transport in mouse CCD.

Within the CCD, transepithelial transport of Cl– occurs primarily across intercalated rather than principal cells (27). Intercalated cells are classified based on whether they express the basolateral Cl–/HCO3– exchanger AE1 and the localization of the H+-ATPase within the cell (29, 41). Within mouse CCD, the predominant intercalated cell subtypes are type A and type B cells (34). Type A cells secrete H+ equivalents through the H+-ATPase, which localizes to the apical plasma membrane. Type B cells secrete OH– equivalents and absorb Cl– through an apical Cl–/HCO3– exchanger, which operates in tandem with net H+ and Cl– efflux across the basolateral membrane mediated by the H+-ATPase and a Cl– channel (29, 41). Pendrin localizes to the apical regions of type B and non-A, non-B intercalated cells and likely mediates the apical Na+-independent, Cl–/HCO3– exchange observed in these cell types (25, 26, 31, 38, 41).

Within the type B cell, pendrin and apical anion exchange are upregulated with aldosterone analogs, which enhance secretion of OH– equivalents and absorption of Cl– (26, 43). Thus pendrin-mediated transport attenuates the alkalosis observed following the administration of aldosterone analogs. Moreover, the apparent vascular volume expansion and the hypertension which follow treatment with desoxycorticosterone pivalate and a high-NaCl diet are critically dependent on pendrin expression, likely through its effect on renal Cl– absorption (38). Thus, while pendrin has an important role in acid-base balance, it also plays a novel role in the maintenance of blood pressure and fluid and electrolyte balance (26, 38).

Pendrin is also upregulated in treatment models not associated with increased circulating aldosterone (37). For example, during selective dietary Cl– restriction, pendrin expression increases, although circulating aldosterone does not change (39). However, because plasma renin concentration is elevated in this treatment model, pendrin expression might increase from enhanced angiotensin II production.

Angiotensin II plays a critical role in the regulation of systemic blood pressure as well as fluid and electrolyte balance (8). Besides acting as a powerful vasoconstrictor, angiotensin II regulates Na+, Cl–, and H2O excretion through direct effects on renal epithelia (21, 46). In the proximal tubule, angiotensin II upregulates apical Na+/H+ exchange and basolateral Na+-HCO3– cotransport, which act in series to increase absorption of Na+ and secretion of H+ equivalents (7, 17). In principal cells of the CCD, angiotensin II also increases Na+ absorption by stimulating the epithelial Na+ channel (ENaC) (23). The effect of angiotensin II on Cl– transport is poorly understood. Weiner et al. (44) showed that angiotensin II application to the bath increases luminal alkalization and increases apical Cl–/HCO3– exchange in rabbit outer CCD through an AT1 receptor-dependent process (44). Because apical anion exchange of the type B intercalated cell is most likely the gene product of Slc26a4, the gene which encodes pendrin, we were prompted to study the effect of angiotensin II in vitro on pendrin-dependent transepithelial net Cl– transport (JCl).

In the present study, we asked following questions: 1) does angiotensin II increase JCl in mouse CCD?; and 2) does angiotensin II increase JCl through pendrin-dependent transcellular transport across intercalated cells? To answer these questions, JCl and transepithelial voltage (VT) were measured in CCDs perfused in vitro from wild-type and Slc26a4 null mice.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. All experiments were performed using male and female Slc26a4 (–/–) mice developed by Everett et al. (4) and in wild-type mice from the same strain (129S6/SvEv Tac; Taconic Farms), which were bred in parallel. Unless otherwise indicated, mice were fed either a standard, salt-replete rodent chow (LabDiet 5001; PMI Nutrition International, Richmond, IN) or a balanced diet (53881300; Zeigler Brothers) prepared as a gel (0.6% agar, 74.6% water, and 24.8% mouse chow) supplemented with NaCl (~1.13 meq NaCl/day) or NaCl plus furosemide (100 mg·kg–1·day–1) for 5 days.

Measurement of blood pressure, arterial pH, and serum electrolytes. Systolic blood pressure was measured in conscious mice by tail cuff using a BP-2000 (Visitech Systems). Arterial blood was collected through the abdominal aorta under isofluorane anesthesia. Arterial pH and PCO2 were measured using ABL5 analyzer (Radiometer America, Westlake, OH). Serum electrolytes were measured as described previously (38).

In vitro perfusion of isolated CCD. CCDs were dissected from medullary rays and perfused at flow rates of 2–3 nl·min–1·mm–1 in the presence of symmetric, physiological solutions containing (in mM) 125 NaCl, 2.5 K2HPO4, 24 NaHCO3/5% CO2, 2 CaCl2, 1.2 MgSO4, and 5.5 glucose bubbled with 95% air-5% CO2 (26, 43). Tubules were equilibrated at 37°C for 30 min before the start of the collections. As specified below, angiotensin II (10–8 M) and bafilomycin A1 (10–8 M) were applied to the bath, while benzamil hydrochloride (3 x 10–6 M) was added to the perfusate. Stock solutions of angiotensin II and benzamil hydrochloride (10–5 M, 3 x 10–3 M, respectively) were prepared in deionized water. A stock solution of bafilomycin (10–5 M) was prepared in absolute ethanol. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Measurement of JCl. Cl– concentration was measured in perfusate and collected samples using a continuous-flow fluorimeter and the Cl–-sensitive fluorophore, 6 methoxy-N-(3-sulfopropyl) quinolinium (SPQ; Molecular Probes, Eugene, OR), as described previously (5, 42, 43). JCl was calculated according to the equation

Formula
where Co and CL are perfusate and collected fluid Cl– concentrations, respectively, Q is flow rate (nl/min), and L is tubule length. Net fluid transport was taken to be zero since net fluid flux has not been observed in CCDs when perfused in vitro in the presence of symmetric solutions and in the absence of vasopressin (15, 16). JCl was expressed in picomoles per millimeter per minute.

VT. VT was measured in the perfusion pipette connected to a high-impedance electrometer through an agar bridge saturated with 0.16 M NaCl and a calomel cell, as described previously (40). The reference was an agar bridge from the bath to a calomel cell.

Calculation of chloride permeability, ionic conductance, and transepithelial electrical resistance. Chloride permeability (PCl) was calculated from the Nernst equation

Formula
where c is the mean Cl– concentration present in the lumen and bath and taken to be 0.129 M, Z is the valence of the ion, F is Faraday's constant, R is the gas constant, and T is temperature in degrees Kelvin. We assumed ZF to be 96.5 C/meq and RT to be 2.57 J/meq, respectively, at 37°C.

Ionic conductance (GCl) was calculated based on the relationship (33)

Formula
In the calculation of GCl, it was assumed that benzamil affects Cl– flux entirely through its effect on VT.

Immunohistochemistry. Kidney tissue from wild-type mice given 7 days of the NaCl-replete gel diet or diet plus furosemide was processed for preembedding peroxidase staining using the anti-pendrin antibody and embedded in the Epon as described previously (13). Tissue was cut into 1.5-µm sections, stained with hematoxylin, and examined by light microscopy.

Statistics. All data are presented as means ± SE. Data from two collections from each tubule were averaged to obtain a single value. Each n used in the statistical analysis represents data from separate mice. To test for statistical significance between two groups, an unpaired Student's t-test was used. When results from more than two groups were compared, an ANOVA was used followed by Tukey's protected t-test. The criterion for statistical significance was P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of angiotensin II on transepithelial Cl– transport, JCl, and VT. In CCDs from wild-type mice ingesting the NaCl-replete diet, JCl and VT were not different from zero in either the presence or absence of 10–8 M angiotensin II in the bath solution (Fig. 1). Thus, under basal conditions, no appreciable net transepithelial Cl– transport was observed.


Figure 1
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  		Fig. 1. Effect of angiotensin II (ANG II) on transepithelial net chloride transport (JCl) and transepithelial voltage (VT) under basal conditions. In cortical collecting ducts (CCDs) from wild-type mice ingesting a standard salt-replete rodent chow, JCl was not different from zero either in the absence (–2.3 ± 1.1 pmol·mm–1·min–1, bullet, n = 7) or in the presence of 10–8 M angiotensin II when applied to the bath [–0.3 ± 1.0 pmol·mm–1·min–1, {circ}, n = 7, P = not significant (NS)]. Each circle represents a single tubule. Values are means ± SE. Under the same conditions, VT was not different from zero either in the absence or in the presence of ANG II (1.0 ± 0.3, n = 3 vs. 0.8 ± 0.9 mV, n = 3, P = NS).

 
Effect of furosemide on pendrin expression. We sought a treatment model in which pendrin protein expression is increased. Figure 2 shows that pendrin immunostaining appears more prominent in both connecting tubule and CCD in mice given a NaCl-replete diet and furosemide relative to mice ingesting the NaCl-replete diet alone, as described previously (25). Moreover, the subapical cytoplasmic area was larger in pendrin-positive cells from furosemide-treated mice, which gave the appearance that these cells "bulge" into the lumen. Thus further studies examined Cl– transport in furosemide-treated mice.


Figure 2
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  		Fig. 2. Effect of furosemide on pendrin expression. Representative pendrin immunostaining in mouse cortex is shown. Following a NaCl-replete diet and furosemide (B and D), pendrin immunostaining increased in both connecting tubule (B) and CCD (D) relative to tubules from mice ingesting the NaCl-replete diet alone [connecting tubule (CNT; A), CCD (C)]. Moreover, the subapical cytoplasmic area appeared larger in pendrin-positive cells (arrows) from furosemide-treated mice, thus giving the appearance that the cells "bulge" into the lumen.

 
Because pendrin is critical in the renal absorption of Cl– (43), the effect of furosemide on apparent vascular volume in wild-type and Slc26a4 null mice was explored (Table 1). As shown, after loop diuretic treatment, Slc26a4 null mice developed greater weight loss, a higher blood urea nitrogen and lower blood pressure than wild-type mice over the treatment period. Thus with furosemide administration these mutant mice had greater apparent vascular volume contraction than wild-type mice. We conclude that pendrin is upregulated with loop diuretics, which makes pendrin critical for the conservation of apparent vascular volume in this treatment model.


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  		Table 1. Effect of furosemide on serum electrolytes, arterial pH, blood pressure, and body weight in wild-type and Slc26a4 null mice

 
Angiotensin II stimulates Cl– absorption in CCDs from furosemide-treated mice. The effect of angiotensin II on JCl and VT were studied in mice given the high-NaCl diet and furosemide (Fig. 3). Cl– absorption and a lumen-negative VT were observed in CCDs from wild-type mice (Fig. 3B). With the application of angiotensin II to the bath, JCl doubled (Fig. 3A), although VT did not become more lumen negative (Fig. 3B). We conclude that angiotensin II-induced Cl– absorption is not mediated by an increase in electromotive force.


Figure 3
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  		Fig. 3. Effect of ANG II on JCl and VT in CCDs from furosemide-treated mice. A: CCDs from furosemide-treated wild-type mice (CON; bullet, Pds +/+) absorbed Cl– (JCl = 4.2 ± 0.5 pmol·mm–1·min–1, n = 4), which doubled with 10–8 M angiotensin II (ANG II; {circ}) when applied to the bath (JCl = 8.8 ± 1.4 pmol·mm–1·min–1, n = 5, P < 0.05). In contrast, CCDs from furosemide-treated Slc26a4 null mice (Pds –/–) secreted Cl– (JCl = –5.2 ± 1.0 pmol·mm–1·min–1, n = 4), which was unaffected by ANG II (JCl = –6.6 ± 1.6 pmol·mm–1·min–1, respectively, n = 4, P = NS). B: in CCDs from furosemide-treated wild-type mice, VT was lumen negative (VT = –3.8 ± 1.1 mV, n = 11) but did not become more lumen negative with the application of ANG II (VT = –1.9 ± 1.0 mV, n = 11, P = NS). In CCDs from Slc26a4 null mice, VT was not different from zero in either the absence or presence of ANG II (VT = –0.2 ± 0.4 mV, n = 4 vs. –0.5 ± 0.5 mV, n = 5, respectively, P = NS). *P < 0.05 vs. CON. #P < 0.05 vs. Pds +/+.

 
In contrast, in CCDs from furosemide-treated Slc26a4 null mice, Cl– secretion was observed, which was unaffected by angiotensin II (Fig. 3A). VT in CCDs from furosemide-treated Slc26a4 null mice was not different from zero either in the presence or absence of angiotensin II (Fig. 3B). We conclude that angiotensin II-induced Cl– absorption observed in wild- type mice is dependent on pendrin expression.

Angiotensin II does not increase Cl– absorption through paracellular transport. Because the lumen-negative VT does not increase with angiotensin II, the driving force for paracellular Cl– absorption does not increase with application of the hormone. However, paracellular Cl– transport might increase with angiotensin II by augmenting paracellular permeability, such as through changes in tight junction properties. To test this hypothesis, the effect of angiotensin II on JCl was tested when the H+-ATPase was inhibited with application of bafilomycin (10–8 M) to the bath. Basolateral plasma membrane H+-ATPase inhibition should reduce net H+ efflux in type B cells, thus reducing intracellular HCO3– concentration and hence reducing the driving force for apical anion exchange (3, 19, 36). The effect of H+-ATPase inhibition on JCl is shown in Fig. 4. In the presence of bafilomycin, angiotensin II did not increase JCl (Fig. 4A). Because the lumen-negative VT persisted following bafilomycin application (Fig. 4B), failure of angiotensin II to stimulate JCl did not occur from a reduced electromotive force. Thus the angiotensin-induced increase in JCl does not involve increased paracellular permeability.


Figure 4
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  		Fig. 4. Effect of H+-ATPase inhibition on ANG II-sensitive JCl. A: with bafilomycin present in the bath (BAF; 10–8 M, bullet, JCl = 6.4 ± 2.4 pmol·mm–1·min–1, n = 7), application of ANG II (10–8 M) to the bath (BAF+ANG II, {circ}) did not increase JCl (6.4 ± 1.3 pmol·mm–1·min–1, n = 6, P = NS). Failure of ANG II to stimulate JCl did not occur from a reduced electromotive force since the lumen-negative VT (B) was not reduced with bafilomycin (–4.1 ± 1.3 mV, n = 6, BAF vs. –8.0 ± 1.7 mV, n = 6, BAF+ANG II, P = 0.09).

 
Cl– absorption occurs in the absence of an electromotive driving force. We asked whether Cl– absorption occurs in the absence of an electromotive force, such as following application of the ENaC inhibitor benzamil (Fig. 5). With benzamil (3 x 10–6 M) present in the perfusate and with angiotensin II (10–8 M) present in bath, the lumen-negative VT was abolished, although Cl– absorption was reduced by only 50%. Thus significant Cl– absorption is observed in the absence of an electromotive driving force. Further experiments examined the mechanism of this "active," benzamil-insensitive component of Cl– absorption. Following application of bafilomycin to the bath, where reduced apical Cl–/HCO3– exchange is expected, benzamil-insensitive Cl– absorption was abolished. We conclude that active Cl– absorption is H+-ATPase dependent and does not occur through paracellular transport.


Figure 5
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  		Fig. 5. JCl measured in the absence of an electromotive force. With ANG II (10–8 M) present in the bath, Cl– absorption (JCl = 8.8 ± 1.6 pmol·mm–1·min–1) and a lumen-negative VT (VT = –2.3 ± 0.8 mV) were observed (ANG II, bullet, n = 5). With the addition of benzamil hydrochloride (3 x 10–6 M) to the perfusate ({circ}, ANG II+BENZ), the lumen-negative VT was abolished (VT = 0.9 ± 0.6 mV, n = 5), although Cl– absorption was reduced by only 50% (JCl = 4.3 ± 1.1 pmol·mm–1·min–1, n = 5). Thus significant Cl– absorption occurs in the absence of an electromotive driving force. Addition of bafilomycin (10–8 M) to the bath ({triangleup}, ANG II+BENZ+BAF) completely abolished the benzamil-insensitive JCl without changing VT (JCl = 0.4 ± 0.7 pmol·mm–1·min–1, n = 5; VT = –1.0 ± 0.3 mV, n = 3). *P < 0.05, **P < 0.01 vs. ANG II.

 
Based on these data (Table 2, Fig. 5), we calculated PCl to be 0.81 x 10–5 cm/s in mouse CCD, similar to the PCl estimated in rat CCD by Weinstein (0.60 x 10–5 cm/s) (45). Based on this PCl, a chloride conductance, GCl, of 3.8 mS/cm2 was calculated. If the epithelial Na+ and Cl– conductances are comparable, then the overall CCD electrical resistance is 132 {Omega}·cm2 in mouse CCD, compared with a resistance of 266 {Omega}·cm2 observed in rabbit CCD under basal conditions (33) and 51 {Omega}·cm2 observed in CCDs from desoxycorticosterone-treated rats (28).


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  		Table 2. Net transepithelial chloride transport and transepithelial voltage used in the calculation of chloride permeability and ionic conductance in mouse cortical collecting duct

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates 1) mouse CCDs secrete or absorb Cl– on demand; 2) Cl– absorption in mouse CCD is increased by angiotensin II; and 3) angiotensin II-induced Cl– absorption occurs through transcellular transport across intercalated cells, through a pendrin- and H+-ATPase-dependent mechanism(s).

Previous studies in rabbit and rat demonstrated that net Cl– and HCO3– transport in the CCD are highly regulated by in vivo conditioning (9, 18). While no significant net Cl– movement is observed in rabbit CCD under basal conditions, substantial net Cl– absorption is detected in CCDs from rabbits treated with the aldosterone analog DOCA (9). Following aldosterone analogs, Cl– absorption and HCO3– secretion increase in tandem, mediated by electroneutral, Na+-independent, apical Cl–/HCO3– exchange (6, 32). This process likely occurs through increased pendrin expression in the apical plasma membrane of type B and non-A, non-B cells (26, 38, 43). Similarly, while we observed little net Cl– flux in CCDs from mice fed a NaCl-replete diet alone, with increased pendrin expression achieved with furosemide treatment in vivo (25), substantial Cl– absorption was observed. However, Cl– absorption was not observed in CCDs from furosemide-treated Slc26a4 null mice, as expected, due to the absence of pendrin-mediated Cl–/HCO3– exchange and the absence of a lumen-negative VT, which drives conductive absorption of Cl–. The absence of a lumen-negative VT in these mutant mice likely results from the reduction in ENaC expression and the low ENaC-mediated current reported previously in preliminary form within CCDs of Slc26a4 null mice (14, 22). We conclude that total and angiotensin II-stimulated Cl– absorption are pendrin dependent.

In rat and dog renal cortex, interstitial angiotensin II concentration is 3–50 nM (20). Thus 10 nM (10–8 M) falls within the physiological range of angiotensin II concentrations observed in vivo within the cortical interstitium. This angiotensin II concentration (10–8 M) was selected since Weiner et al. (44) observed that 10–7 M, but not 10–10 M angiotensin II, increased B cell apical anion exchange when applied to the bath. Since the effect of angiotensin II on JCl in the CCD has not been tested at concentrations <10–8 M, we cannot exclude the possibility that angiotensin II has a biphasic effect on JCl in the CCD as was observed in the proximal tubule (10).

The present study extends the previous study of Weiner and colleagues (44) to show that angiotensin II directly regulates JCl in the CCD through a pendrin- and H+-ATPase-dependent mechanism(s). We also show that angiotensin II stimulates intercalated cell-dependent Cl– absorption through a direct effect on the CCD, rather than through other systemic effects of angiotensin II observed in vivo, such as changes in blood pressure or changes in the distal delivery of Cl– (37).

Angiotensin II likely modulates apical anion exchange through AT1a receptors, since they represent the dominant AT1 receptor expressed in the collecting duct (1). However, the distribution of the AT1a receptor along the collecting duct is controversial. One study localized AT1 receptors along the collecting duct primarily to the apical regions of intercalated cells (12), whereas others noted diffuse localization of the receptor within both principal and intercalated cells (11). Thus angiotensin II might increase apical anion exchange directly through stimulation of intercalated cell AT1a receptors or could act on principal cell receptors, thereby changing intercalated cell transporter function through cell-cell interaction. In this regard, we cannot exclude the possibility that angiotensin II, when applied to the bath, modulates JCl in mouse CCD by diffusing across tight junctions and acting on apical plasma membrane AT1 receptors.

Since pendrin transport inhibitors are not available, we studied the role of this protein in angiotensin II-induced Cl– absorption using mice with genetic disruption of Slc26a4 (4). H+-ATPase inhibitors were also employed, which should reduce the driving force for apical anion exchange.1 While angiotensin II doubled Cl– absorption in CCDs from wild-type mice, it had no effect in tubules from Slc26a4 null mice. Similarly, inhibiting H+-ATPase-mediated transport eliminated the angiotensin II-induced increase in JCl in wild-type mice, although it did not make VT less lumen negative. Thus angiotensin II increases JCl through changes in transcellular transport across intercalated cells, rather than through changes in paracellular permeability or through changes in electromotive force.

The present study confirms and extends previous work by Terada and Knepper (35), which showed that JCl is reduced 50% with the application of amiloride to the luminal fluid and eliminated with the application of both amiloride and hydrochlorothiazide (HCTZ). While HCTZ inhibits the NaCl cotransporter in the DCT, it also inhibits carbonic anhydrase (24), which markedly reduces type B cell apical anion exchange (19). Thus apical anion exchange should fall with reduced intracellular HCO3– concentration, as expected following carbonic anhydrase inhibition, such as with HCTZ, or following H+-ATPase inhibition with bafilomycin.

In the present study, net fluid flux was assumed to be zero in the CCD (15, 16). However, furosemide-treated Slc26a4 null mice have marked apparent vascular volume contraction, which might increase vasopressin secretion. If CCDs from furosemide-treated mice absorb fluid, such as through increased circulating vasopressin, JCl reported above would underestimate true Cl– absorption.2

Pendrin-mediated Cl– absorption and HCO3– secretion might generate net ion movement into the renal interstitium, such as through changes in VT or by buffering H+ secreted into the luminal fluid. However, because pendrin is a Cl–/HCO3– exchanger, it might not generate any net ion movement. Resolving this question will require measurement of net Na+, K+, and HCO3– flux under identical experimental conditions.

In conclusion, angiotensin II increases net Cl– absorption in mouse CCD through a transcellular pathway, which is pendrin and H+-ATPase dependent, rather than through conductive paracellular pathways. The H+-ATPase provides an "active step" for transcellular Cl– transport across type B cells.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52935 (to S. M. Wall). V. Pech is the recipient of an American Heart Association Postdoctoral Fellowship Award (0525384B).


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. Pech, Emory Univ. School of Medicine, Renal Div., 1639 Pierce Dr., NE, WMB Rm. 338, Atlanta, GA 30322 (e-mail: vpech{at}emory.edu)

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.

1 Application of bafilomycin (10 nM) to the bath reduces net H+ efflux in type B intercalated cells in mouse CCD, thereby reducing intracellular pH (3). However, bafilomycin is a membrane-permeable compound (2); thus we cannot exclude the possibility that application of bafilomycin to the bath also inhibits the apical H+-ATPase, which should increase luminal HCO3– concentration (36). Either of these expects should reduce the driving force for apical anion exchange of the type B intercalated cell. Back

2 It is unlikely that our failure to detect Cl– absorption in CCDs from Slc26a4 null mice occurs exclusively from vasopressin-stimulated fluid absorption, since Cl– absorption was not observed in CCDs from Slc26a4 null mice following administration of DOCP and NaHCO3 ingestion (43). Circulating vasopressin levels are not likely elevated in Slc26a4 null mice following this treatment protocol since increased serum osmolality and apparent vascular volume contraction are not observed (38). Back


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Bouby N, Hus-Citharel A, Marchetti J, Bankir L, Corvol P, Llorens-Cortes C. Expression of type 1 angiotensin II receptor subtypes and angiotensin II-induced calcium mobilization along the rat nephron. J Am Soc Nephrol 8: 1658–1667, 1997.[Abstract]
  2. Drose S, Altendorf K. Bafilomycins and concanamycins as inhibitors of V-ATPases and P-ATPases. J Exp Biol 200: 1–8, 1997.[Abstract]
  3. Emmons C. H+ extrusion in mouse cortical collecting duct (CCD) intercalated cells (ICs) (Abstract). J Am Soc Nephrol 8: 5A, 1997.
  4. Everett LA, Belyantseva IA, Noben-Trauth K, Cantos R, Chen A, Thakkar SI, Hoogstraten-Miller SL, Kachar B, Wu DK, Green ED. Targeted disruption of mouse Pds provides insight about the inner-ear defects encountered in Pendred syndrome. Hum Mol Genet 10: 153–161, 2001.[Abstract/Free Full Text]
  5. Garcia NH, Plato CF, Garvin JL. Fluorescent determination of chloride in nanoliter samples. Kidney Int 55: 321–325, 1999.[CrossRef][ISI][Medline]
  6. Garcia-Austt J, Good DW, Burg MB, Knepper MA. Deoxycorticosterone-stimulated bicarbonate secretion in rabbit cortical collecting ducts: effects of luminal chloride removal and in vivo acid loading. Am J Physiol Renal Fluid Electrolyte Physiol 249: F205–F212, 1985.[Abstract/Free Full Text]
  7. Geibel J, Giebisch G, Boron WF. Angiotensin II stimulates both Na+-H+ exchange and Na+/HCO3– cotransport in the rabbit proximal tubule. Proc Natl Acad Sci USA 87: 7917–7920, 1990.[Abstract/Free Full Text]
  8. Hall JE, Brands MW. The renin-angiotensin-aldosterone systems: renal mechanisms circulatory homeostasis. In: The Kidney: Physiology and Pathophysiology, edited by Seldin DW and Giebisch G. Philadelphia, PA: Lippincott Williams & Wilkins, 2000, p. 1009–1045.
  9. Hanley MJ, Kokko JP. Study of chloride transport across the rabbit cortical collecting tubule. J Clin Invest 62: 39–44, 1978.[ISI][Medline]
  10. Harris PJ. Regulation of proximal tubule function by angiotensin. Clin Exp Pharmacol Physiol 19: 213–222, 1992.[ISI][Medline]
  11. Harrison-Bernard LM, Navar LG, Ho MM, Vinson GP, el-Dahr SS. Immunohistochemical localization of ANG II AT1 receptor in adult rat kidney using a monoclonal antibody. Am J Physiol Renal Physiol 273: F170–F177, 1997.[Abstract/Free Full Text]
  12. Joly E, Nonclercq D, Caron N, Mertens J, Flamion B, Toubeau G, Kramp R, Bouby N. Differential regulation of angiotensin II receptors during renal injury and compensatory hypertrophy in the rat. Clin Exp Pharmacol Physiol 32: 241–248, 2005.[CrossRef][ISI][Medline]
  13. Kim YH, Kwon TH, Christensen BM, Nielsen J, Wall SM, Madsen KM, Frøkiær J, Nielsen S. Altered expression of renal acid-base transporters in rats with lithium-induced NDI. Am J Physiol Renal Physiol 285: F1244–F1257, 2003.[Abstract/Free Full Text]
  14. Kim YH, Shin W, Everett LA, Green ED, Hummler E, Rossier B, Wall SM. Interdependence of Pendrin (Slc26a4) and ENaC expression (Abstract). J Am Soc Nephrol 16: 62A, 2005.
  15. Knepper MA, Good DW, Burg MB. Mechanism of ammonia secretion by cortical collecting ducts of rabbits. Am J Physiol Renal Fluid Electrolyte Physiol 247: F729–F738, 1984.[Abstract/Free Full Text]
  16. Knepper MA, Good DW, Burg MB. Ammonia and bicarbonate transport by rat cortical collecting ducts perfused in vitro. Am J Physiol Renal Fluid Electrolyte Physiol 249: F870–F877, 1985.[Abstract/Free Full Text]
  17. Liu FY, Cogan MG. Angiotensin II stimulation of hydrogen ion secretion in the rat early proximal tubule. Modes of action, mechanism, and kinetics. J Clin Invest 82: 601–607, 1988.[ISI][Medline]
  18. McKinney TD, Burg MB. Bicarbonate transport by rabbit cortical collecting tubules. Effect of acid and alkali loads in vivo on transport in vitro. J Clin Invest 60: 766–768, 1977.[ISI][Medline]
  19. Milton AE, Weiner ID. Regulation of B-type intercalated cell apical anion exchange activity by CO2/HCO3–. Am J Physiol Renal Physiol 274: F1086–F1094, 1998.[Abstract/Free Full Text]
  20. Navar LG, Nishiyama A. Why are angiotensin concentrations so high in the kidney? Curr Opin Nephrol Hypertens 13: 107–115, 2004.[ISI][Medline]
  21. Olsen ME, Hall JE, Montani JP, Guyton AC, Langford HG, Cornell JE. Mechanisms of angiotensin II natriuresis and antinatriuresis. Am J Physiol Renal Fluid Electrolyte Physiol 249: F299–F307, 1985.[Abstract/Free Full Text]
  22. Pech V, Zheng W, Pham TD, Verlander JW, Wall SM. Angiotensin II increases chloride absorption in the cortical collecting duct in mice by a pendrin-dependent mechanism (Abstract). FASEB J 20: A1225, 2006.[Free Full Text]
  23. Peti-Peterdi J, Warnock DG, Bell PD. Angiotensin II directly stimulates ENaC activity in the cortical collecting duct via AT1 receptors. J Am Soc Nephrol 13: 1131–1135, 2002.[Abstract/Free Full Text]
  24. Pickkers P, Garcha RS, Schachter M, Smits P, Hughes AD. Inhibition of carbonic anhydrase accounts for the direct vascular effects of hydrochlorothiazide. Hypertension 33: 1043–1048, 1999.[Abstract/Free Full Text]
  25. Quentin F, Chambrey R, Trinh-Trang-Tan MM, Fysekidis M, Cambillau M, Paillard M, Aronson PS, Eladari D. The Cl–/HCO3– exchanger pendrin in the rat kidney is regulated in response to chronic alterations in chloride balance. Am J Physiol Renal Physiol 287: F1179–F1188, 2004.[Abstract/Free Full Text]
  26. Royaux IE, Wall SM, Karniski LP, Everett LA, Suzuki K, Knepper MA, Green ED. Pendrin, encoded by the Pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion. Proc Natl Acad Sci USA 98: 4221–4226, 2001.[Abstract/Free Full Text]
  27. Schlatter E, Greger R, Schafer JA. Principal cells of cortical collecting ducts of the rat are not a route of transepithelial Cl– transport. Pflügers Arch 417: 317–323, 1990.[CrossRef][ISI][Medline]
  28. Schlatter E, Schafer JA. Electrophysiological studies in principal cells of rat cortical collecting tubules. ADH increases the apical membrane Na+-conductance. Pflügers Arch 409: 81–92, 1987.[CrossRef][ISI][Medline]
  29. Schuster VL. Function and regulation of collecting duct intercalated cells. Annu Rev Physiol 55: 267–288, 1993.[CrossRef][ISI][Medline]
  30. Schuster VL, Stokes JB. Chloride transport by the cortical and outer medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 253: F203–F212, 1987.[Abstract/Free Full Text]
  31. Soleimani M, Greeley T, Petrovic S, Wang Z, Amlal H, Kopp P, Burnham CE. Pendrin: an apical Cl–/OH–/HCO3– exchanger in the kidney cortex. Am J Physiol Renal Physiol 280: F356–F364, 2001.[Abstract/Free Full Text]
  32. Star RA, Burg MB, Knepper MA. Bicarbonate secretion and chloride absorption by rabbit cortical collecting ducts. Role of chloride/bicarbonate exchange. J Clin Invest 76: 1123–1130, 1985.[ISI][Medline]
  33. Stoner LC, Burg MB, Orloff J. Ion transport in cortical collecting tubule; effect of amiloride. Am J Physiol 227: 453–459, 1974.[Free Full Text]
  34. Teng-umnuay P, Verlander JW, Yuan W, Tisher CC, Madsen KM. Identification of distinct subpopulations of intercalated cells in the mouse collecting duct. J Am Soc Nephrol 7: 260–274, 1996.[Abstract]
  35. Terada Y, Knepper MA. Thiazide-sensitive NaCl absorption in rat cortical collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 259: F519–F528, 1990.[Abstract/Free Full Text]
  36. Tsuruoka S, Schwartz GJ. Mechanisms of HCO3– secretion in the rabbit connecting segment. Am J Physiol Renal Physiol 277: F567–F574, 1999.[Abstract/Free Full Text]
  37. Vallet M, Picard N, Loffing-Cueni D, Fysekidis M, Bloch-Faure M, Deschenes G, Breton S, Meneton P, Loffing J, Aronson PS, Chambrey R, Eladari D. Pendrin regulation in mouse kidney primarily is chloride-dependent. J Am Soc Nephrol 17: 2153–2163, 2006.[Abstract/Free Full Text]
  38. Verlander JW, Hassell KA, Royaux IE, Glapion DM, Wang ME, Everett LA, Green ED, Wall SM. Deoxycorticosterone upregulates PDS (Slc26a4) in mouse kidney: role of pendrin in mineralocorticoid-induced hypertension. Hypertension 42: 356–362, 2003.[Abstract/Free Full Text]
  39. Verlander JW, Kim YH, Shin W, Pham TD, Hassell KA, Beierwaltes WH, Green E, Everett LA, Matthews SW, Wall SM. Dietary Cl– restriction upregulates pendrin expression within the apical plasma membrane of type B intercalated cells. Am J Physiol Renal Physiol 291: F833–F839, 2006.[Abstract/Free Full Text]
  40. Wall SM. NH4+ augments net acid secretion by a ouabain-sensitive mechanism in isolated perfused inner medullary collecting ducts. Am J Physiol Renal Fluid Electrolyte Physiol 270: F432–F439, 1996.[Abstract/Free Full Text]
  41. Wall SM. Recent advances in our understanding of intercalated cells. Curr Opin Nephrol Hypertens 14: 480–484, 2005.[ISI][Medline]
  42. Wall SM, Fischer MP, Mehta P, Hassell KA, Park SJ. Contribution of the Na+-K+-2Cl– cotransporter NKCC1 to Cl– secretion in rat OMCD. Am J Physiol Renal Physiol 280: F913–F921, 2001.[Abstract/Free Full Text]
  43. Wall SM, Kim YH, Stanley L, Glapion DM, Everett LA, Green ED, Verlander JW. NaCl restriction upregulates renal Slc26a4 through subcellular redistribution: role in Cl– conservation. Hypertension 44: 982–987, 2004.[Abstract/Free Full Text]
  44. Weiner ID, New AR, Milton AE, Tisher CC. Regulation of luminal alkalinization and acidification in the cortical collecting duct by angiotensin II. Am J Physiol Renal Fluid Electrolyte Physiol 269: F730–F738, 1995.[Abstract/Free Full Text]
  45. Weinstein AM. A mathematical model of rat cortical collecting duct: determinants of the transtubular potassium gradient. Am J Physiol Renal Physiol 280: F1072–F1092, 2001.[Abstract/Free Full Text]
  46. Xie MH, Liu FY, Wong PC, Timmermans PB, Cogan MG. Proximal nephron and renal effects of DuP 753, a nonpeptide angiotensin II receptor antagonist. Kidney Int 38: 473–479, 1990.[ISI][Medline]



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