Angiotensin II increases chloride absorption in the cortical collecting duct in - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 09:43 |只看该作者 |倒序浏览 |打印

作者：Vladimír Pech, Young Hee Kim, Alan M. Weinstein, Lorraine A. Everett, Truyen D. Pham, and Susan M. Wall,作者单位：1 Renal Division, Department of Medicine, and 4 Department of Physiology, Emory University School of Medicine, Atlanta, Georgia; 2 Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York; 3 The Wellcome Trust Sanger Institute, Wellcome Trust Genome Ca 2 v2 R, _5 t7 g% }+ q! |
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      【摘要】/ t; j. n: x1 q9 l3 W0 V7 i" n
   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 - /HCO 3 - exchange. The purpose of this study was to determine whether angiotensin II increases transepithelial net chloride transport, J Cl, in mouse CCD through a pendrin-dependent mechanism. J Cl and transepithelial voltage, V T, 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, V T and J Cl 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 V T and absorbed Cl -. With angiotensin II in the bath, Cl - absorption doubled although V T did not become more lumen negative. In contrast, in CCDs from furosemide-treated Slc26a4 null mice, Cl - secretion and a V T of 0 were observed, neither of which changed with angiotensin II application. Inhibiting ENaC with benzamil abolished V T although J Cl 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.
      【关键词】 transepithelial voltage bafilomycin H ATPase knockout mice Sca intercalated cell% F1 I4 |3 q( B  _
   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, V T, 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 - /HCO 3 - 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 - /HCO 3 - 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 - /HCO 3 - 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 )./ R' u  t; C5 S9 `5 ~

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 H 2 O excretion through direct effects on renal epithelia ( 21, 46 ). In the proximal tubule, angiotensin II upregulates apical Na /H exchange and basolateral Na -HCO 3 - 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 - /HCO 3 - exchange in rabbit outer CCD through an AT 1 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 ( J Cl ).- T7 x! x& i0 [8 E8 |1 V; [1 P
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In the present study, we asked following questions: 1 ) does angiotensin II increase J Cl in mouse CCD?; and 2 ) does angiotensin II increase J Cl through pendrin-dependent transcellular transport across intercalated cells? To answer these questions, J Cl and transepithelial voltage ( V T ) were measured in CCDs perfused in vitro from wild-type and Slc26a4 null mice.
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METHODS

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.( E8 L! }% g) x4 ]

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 P CO 2 were measured using ABL5 analyzer (Radiometer America, Westlake, OH). Serum electrolytes were measured as described previously ( 38 )., @! U$ S" N4 _5 ]  n0 {

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 K 2 HPO 4, 24 NaHCO 3 /5% CO 2, 2 CaCl 2, 1.2 MgSO 4, and 5.5 glucose bubbled with 95% air-5% CO 2 ( 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).
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Measurement of J Cl. 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 ). J Cl was calculated according to the equation
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where C o and C L 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 ). J Cl was expressed in picomoles per millimeter per minute.: u4 C. }5 R1 I1 A
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V T. V T 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.* }: k; A. S+ O+ T' ]# b1 y0 o

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

where 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.2 G* a/ x& F2 y) C* [6 |( \

Ionic conductance ( G Cl ) was calculated based on the relationship ( 33 )
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In the calculation of G Cl, it was assumed that benzamil affects Cl - flux entirely through its effect on V T.2 \7 {& X8 E7 S- o; W& N& R5 C

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.; e: N8 |3 L1 {+ _0 K6 E7 n: f" l( L5 H

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
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RESULTS
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Effect of angiotensin II on transepithelial Cl - transport, J Cl, and V T. In CCDs from wild-type mice ingesting the NaCl-replete diet, J Cl and V T 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., t" P/ t0 ~# v4 `7 h% @- V
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Fig. 1. Effect of angiotensin II (ANG II) on transepithelial net chloride transport ( J Cl ) and transepithelial voltage ( V T ) under basal conditions. In cortical collecting ducts (CCDs) from wild-type mice ingesting a standard salt-replete rodent chow, J Cl was not different from zero either in the absence (-2.3 ± 1.1 pmol·mm -1 ·min -1,, 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,, n = 7, P = not significant (NS)]. Each circle represents a single tubule. Values are means ± SE. Under the same conditions, V T 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.. Y. @. N/ a0 V8 U$ g: m$ x  T

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.; y1 R% D2 M* J9 s0 C5 F  z

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.: [8 o: v4 }* c0 |0 P7 @

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

Fig. 3. Effect of ANG II on J Cl and V T in CCDs from furosemide-treated mice. A : CCDs from furosemide-treated wild-type mice (CON;, Pds  / ) absorbed Cl - ( J Cl = 4.2 ± 0.5 pmol·mm -1 ·min -1, n = 4), which doubled with 10 -8 M angiotensin II (ANG II; ) when applied to the bath ( J Cl = 8.8 ± 1.4 pmol·mm -1 ·min -1, n = 5, P ) q3 \% e6 G5 d; y+ x
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In contrast, in CCDs from furosemide-treated Slc26a4 null mice, Cl - secretion was observed, which was unaffected by angiotensin II ( Fig. 3 A ). V T 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.
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Angiotensin II does not increase Cl - absorption through paracellular transport. Because the lumen-negative V T 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 J Cl 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 HCO 3 - concentration and hence reducing the driving force for apical anion exchange ( 3, 19, 36 ). The effect of H -ATPase inhibition on J Cl is shown in Fig. 4. In the presence of bafilomycin, angiotensin II did not increase J Cl ( Fig. 4 A ). Because the lumen-negative V T persisted following bafilomycin application ( Fig. 4 B ), failure of angiotensin II to stimulate J Cl did not occur from a reduced electromotive force. Thus the angiotensin-induced increase in J Cl does not involve increased paracellular permeability.3 N4 H% S4 g, Z  \) G  _
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Fig. 4. Effect of H -ATPase inhibition on ANG II-sensitive J Cl. A : with bafilomycin present in the bath (BAF; 10 -8 M,, J Cl = 6.4 ± 2.4 pmol·mm -1 ·min -1, n = 7), application of ANG II (10 -8 M) to the bath (BAF ANG II, ) did not increase J Cl (6.4 ± 1.3 pmol·mm -1 ·min -1, n = 6, P = NS). Failure of ANG II to stimulate J Cl did not occur from a reduced electromotive force since the lumen-negative V T ( 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).
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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 V T 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 - /HCO 3 - 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.) H1 @, U: D; a* O8 ?

Fig. 5. J Cl measured in the absence of an electromotive force. With ANG II (10 -8 M) present in the bath, Cl - absorption ( J Cl = 8.8 ± 1.6 pmol·mm -1 ·min -1 ) and a lumen-negative V T ( V T = -2.3 ± 0.8 mV) were observed (ANG II,, n = 5). With the addition of benzamil hydrochloride (3 x 10 -6 M) to the perfusate (, ANG II BENZ), the lumen-negative V T was abolished ( V T = 0.9 ± 0.6 mV, n = 5), although Cl - absorption was reduced by only 50% ( J Cl = 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 (, ANG II BENZ BAF) completely abolished the benzamil-insensitive J Cl without changing V T ( J Cl = 0.4 ± 0.7 pmol·mm -1 ·min -1, n = 5; V T = -1.0 ± 0.3 mV, n = 3). * P

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

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' W0 i$ u, b7 j$ N( l; F6 p2 H
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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).
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Previous studies in rabbit and rat demonstrated that net Cl - and HCO 3 - 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 HCO 3 - secretion increase in tandem, mediated by electroneutral, Na -independent, apical Cl - /HCO 3 - 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 - /HCO 3 - exchange and the absence of a lumen-negative V T, which drives conductive absorption of Cl -. The absence of a lumen-negative V T 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 J Cl in the CCD has not been tested at concentrations . i! P: M0 Y, Z( E" W

The present study extends the previous study of Weiner and colleagues ( 44 ) to show that angiotensin II directly regulates J Cl 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 ).* G( \& {2 R. n5 h" u# J* z# r6 x  I

Angiotensin II likely modulates apical anion exchange through AT 1a receptors, since they represent the dominant AT 1 receptor expressed in the collecting duct ( 1 ). However, the distribution of the AT 1a receptor along the collecting duct is controversial. One study localized AT 1 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 AT 1a 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 J Cl in mouse CCD by diffusing across tight junctions and acting on apical plasma membrane AT 1 receptors.- B1 b1 B5 X5 B

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 J Cl in wild-type mice, although it did not make V T less lumen negative. Thus angiotensin II increases J Cl through changes in transcellular transport across intercalated cells, rather than through changes in paracellular permeability or through changes in electromotive force.( ^$ W, T1 }% J' f# h
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The present study confirms and extends previous work by Terada and Knepper ( 35 ), which showed that J Cl 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 HCO 3 - 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, J Cl reported above would underestimate true Cl - absorption. 2
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Pendrin-mediated Cl - absorption and HCO 3 - secretion might generate net ion movement into the renal interstitium, such as through changes in V T or by buffering H secreted into the luminal fluid. However, because pendrin is a Cl - /HCO 3 - exchanger, it might not generate any net ion movement. Resolving this question will require measurement of net Na  , K  , and HCO 3 - flux under identical experimental conditions.+ N- H9 }. i& b, W% Q# Y: B
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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.& @' h: @: w+ a5 L4 E6 r% s

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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).
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