The Cl - /HCO 3 - exchanger pendrin in the rat kidney is regulated in response t - 《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 08:08 |显示全部帖子

作者：Fabienne Quentin, Régine Chambrey, Marie Marcelle Trinh-Trang-Tan, Marinos Fysekidis, Michèle Cambillau, Michel Paillard,, Peter S. Aronson, and Dominique Eladari,作者单位：1 Institut National de la Santé et de la Recherche Médicale Unité 35 Institut Fédératif de Recherche 5 Université René Descartes, 75006 Paris; 2 Institut National de la Santé et de la Recherche Médicale Unité 7 Institut National d ! n- n" C& p4 W, C' G
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      【摘要】: j9 V, v: z: M1 y
   Pendrin (Pds; Slc26A4) is a new anion exchanger that is believed to mediate apical Cl - /HCO 3 - exchange in type B and non-A-non-B intercalated cells of the connecting tubule and cortical collecting duct. Recently, it has been proposed that this transporter may be involved in NaCl balance and blood pressure regulation in addition to its participation in the regulation of acid-base status. The purpose of our study was to determine the regulation of Pds protein abundance during chronic changes in chloride balance. Rats were subjected to either NaCl, NH 4 Cl, NaHCO 3, KCl, or KHCO 3 loading for 6 days or to a low-NaCl diet or chronic furosemide administration. Pds protein abundance was estimated by semiquantitative immunoblotting in renal membrane fractions isolated from the cortex of treated and control rats. We observed a consistent inverse relationship between Pds expression and diet-induced changes in chloride excretion independent of the administered cation. Conversely, NaCl depletion induced by furosemide was associated with increased Pds expression. We conclude that Pds expression is specifically regulated in response to changes in chloride balance.
      【关键词】 anion exchanger extracellular volume blood pressure HTA acidbase6 @5 n# K8 G+ j0 C2 }
   IN THE KIDNEY, FINE REGULATION of acid-base and NaCl homeostasis occurs in the terminal part of the nephron, including the connecting tubule (CNT) and the collecting duct. Along these nephron segments, electrogenic sodium reabsorption is aldosterone dependent and occurs through principal cells via the sodium channel ENaC expressed at the apical membrane, functionally coupled with the basolaterally expressed Na -K -ATPase. In parallel with sodium, chloride is reabsorbed as the accompanying anion and NaCl reabsorption regulation is of critical importance in the regulation of renal NaCl balance, which in turn controls extracellular volume and blood pressure regulation ( 12 ). In contrast to sodium reabsorption, chloride reabsorption is not believed to occur via principal cells ( 11 ). The routes of chloride reabsorption are not completely defined, but chloride reabsorption appears to be linked in part to bicarbonate secretion ( 32 ). For example, in the rabbit collecting duct, one-third of chloride reabsorption is via the paracellular passive pathway driven by the lumen-negative transepithelial voltage generated by electrogenic sodium reabsorption, whereas two-thirds occur through a transcellular pathway mainly via apical Cl - /HCO 3 - exchange and Cl - conductances ( 11, 32 ).
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Secretion of base or acid has been studied extensively in the CNT and the cortical collecting duct (CCD) and is believed to occur through intercalated cells (for a review, see Refs. 14 and 31 ). At least three subtypes of intercalated cells have been described ( 1, 4, 37 ). Type A intercalated cells secrete net H equivalents and express the H -ATPase pump at the apical membrane and the Cl - /HCO 3 - exchanger kAE1 at the basolateral membrane, respectively. These cells are believed to be crucial for the secretion of ammonium needed to excrete the daily acid load generated by protein metabolism. Conversely, type B intercalated cells that secrete bicarbonate ( 31 ) express the H -ATPase at the basolateral membrane and a Cl - /HCO 3 - exchanger, distinct from kAE1, at the apical membrane ( 1, 14 ). Thus type B intercalated cells are suited to allow the excretion of an alkali load when needed. The physiological function of non-A-non-B intercalated cells remains unknown ( 14 ).
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Pendrin (Pds) is a recently cloned member of the SLC26 anion exchanger superfamily ( 8 ). Described initially in the thyroid, Pds is disrupted in Pendred syndrome, characterized by goiter and congenital sensorineural deafness ( 7, 21 ). Pds has been shown to mediate Cl - /iodide, Cl - /formate, Cl - /OH -, and Cl - /HCO 3 - exchange activity when expressed in Xenopus laevis oocytes and HEK-293 cells (33-35). It has been proposed that impaired iodide transport by thyrocytes and altered ion and fluid transport by the endolymphatic sac epithelium resulting from Pds disruption are responsible for the decreased thyroid hormone synthesis and goiter and the enlarged vestibule and deafness, respectively, observed in Pendred syndrome ( 10, 18, 25 ).1 L, S) }- |8 O" f& ?

Although no specific renal alteration has been reported in patients with Pendred syndrome, it has been demonstrated, based on functional studies in Pds-null mice, that Pds could account for the apical Cl - /HCO 3 - exchange activity observed in the CCD ( 26 ). Moreover, Pds expression has been found on the apical membrane of both type B and non-A-non-B intercalated cells of CNT and CCD ( 15, 26, 39 ), and Pds expression has also been found to be regulated in response to changes in acid-base balance ( 9, 35, 36, 38 ). Taken together, these results suggest that Pds plays an important role in distal renal acidification and HCO 3 - secretion.2 C/ [$ Z* Y2 x8 @7 z1 d- c  k# V/ k
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However, in addition to its role in acid-base homeostasis, Pds may also be important for regulation of renal chloride reabsorption. Indeed, after the injection of deoxycorticosterone pivalate (DOCP), an aldosterone analog, Pds -/- mice did not develop mineralocorticoid-induced hypertension like control mice, suggesting that these animals failed to increase distal NaCl reabsorption, extracellular volume, and blood pressure ( 36 ).

Therefore, the goal of the present study was to test the hypothesis that Pds is regulated specifically in response to changes in chloride balance. To accomplish this goal, we measured changes in the abundance of Pds polypeptide in response to chronic changes in dietary Cl - intake or to furosemide-induced NaCl depletion. Our findings revealed a striking dependence of Pds expression on chloride balance.
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MATERIALS AND METHODS

Animal treatment. Male adult Sprague-Dawley rats (IFFA-CREDO, L'Arbresle, France) were used for these studies. In the first protocol (NaCl, NaHCO 3, and NH 4 Cl loadings), six independent series were performed. In each series, a control group and three treated groups were handled in parallel over a 6-day period. Each group consisted of two rats. Treated rats were given either 0.28 M NH 4 Cl, NaHCO 3, or NaCl in the drinking water and were fed ad libitum with standard laboratory rat chow containing 0.241% Na  , 0.398% Cl -, 0.678% K (Dietex). Control rats were fed ad libitum with standard laboratory rat chow and had free access to distilled water for the same period of time. In the second protocol (KCl and KHCO 3 loading), two independent series were performed. In each series, a control group and two treated groups were handled in parallel over a 6-day period. Each group consisted of three rats. Treated rats were given either 0.28 M KCl or KHCO 3 and were fed ad libitum with standard laboratory rat chow (Dietex). Control rats were fed ad libitum with standard laboratory rat chow and had free access to distilled water for the same period of time. In the third protocol (NaCl restriction), six independent series were performed. In each series, one NaCl-supplemented group and one NaCl-restricted group were handled in parallel over a 6-day period. Each morning, each NaCl-restricted rat (-Na or low-NaCl group) was fed with 15 g of a low (0.009% Na )-sodium gel diet (UAR no. 212 Na, UAR, Villemoisson, France) containing 35 ml H 2 O and 0.2 g agar. The NaCl-supplemented group consisted of rats that were pair-fed with the same gel diet but with addition of 2.5% NaCl. Rats in both groups ingested the totality of the food. In the fourth protocol (furosemide administration), furosemide (a loop diuretic; Sigma, Saint Quentin Fallavier, France) was given to five rats for 6 days at a dose of 10 mg·100 g body wt -1 ·day -1 mixed with 20 g standard rat food. Because of this slight food restriction, rats ingested the totality of the drug. Four rats that served as controls were given 20 g/day food without furosemide.8 z/ M% \0 v/ L9 ]8 z" B9 J
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All investigations involving animals were conducted in conformity with the Guiding Principles in the Care and Use of Animals of the American Physiological Society.
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At the end of the experimental period of each protocol, blood was collected by aortic puncture under anesthesia induced by peritoneal injection of pentobarbital sodium. The bladder was punctured just after anesthesia, and urine was withdrawn. Kidneys were rapidly removed and immersed into ice-cold Hanks' modified solution containing (in mM) 137 NaCl, 5.4 KCl, 25 NaHCO 3, 0.3 Na 2 HPO 4, 0.4 KH 2 PO 4, 0.5 MgCl 2, 10 HEPES, 5 glucose, and 1 leucine as well as 1 mg/ml BSA. Arterial pH, P CO 2, and P O 2 were measured with an AVL Compact 1 pH/blood-gas analyzer (AVL Instruments Médicaux, Eragny-sur-Oise, France). Serum and urine electrolytes and creatinine were measured by standard methods with a Beckman LX20 autoanalyzer (Coulter-Beckman, Villepinte, France) and Olympus AU 400 (Olympus, Rungis, France). Plasma and urinary osmolalities were measured with a Roebling osmometer (Bioblock, Rungis, France). Plasma aldosterone was measured by radioimmunoassay (DPC Dade Behring, La Défense, France). Urinary ammonia was measured by the method of Berthelot ( 3 ).
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Membrane fraction preparation. Kidneys were removed and cut into 5-mm slices. The renal cortex was excised under a stereoscopic microscope and placed into ice-cold isolation buffer (250 mM sucrose, 20 mM Tris-HEPES, pH 7.4) containing (in µg/ml) protease inhibitors: 4 aprotinin, 4 leupeptin, 1.5 pepstatin A, and 28 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF). Minced tissues were homogenized in a Dounce homogenizer (pestle A x 5) followed by five passes through a Teflon-glass homogenizer rotating at 1,000 rpm. The homogenate was centrifuged at 1,000 g for 10 min, and the supernatant was centrifuged at 100,000 g for 20 min at 4°C. The pellet was resuspended in isolation buffer. Protein contents were determined using the Bradford protein assay.

Antibodies. Rabbit polyclonal antibody to COOH-terminal rat Pds amino acids 630-643 has been previously characterized ( 16, 38 ). Rabbit polyclonal antibody to the thiazide-sensitive Na-Cl cotransporter (NCC) was a gift of Dr. D. H. Ellison (Oregon Health and Science Univ., Portland, OR) and has also been previously extensively characterized ( 6, 29, 30 ).
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Immunoblot analyses. Membrane proteins were solubilized in SDS loading buffer (10 mM Tris·HCl, pH 6.8, 1% SDS, 2% dithiothreitol, 13% glycerol, and bromophenol blue) incubated at 20°C for 30 min. Electrophoresis was initially performed for all samples on 7.5% polyacrylamide minigels (XCell SureLock Mini-cell, Invitrogen Life Technologies), which were stained with Coomassie blue to provide quantitative assessment of loading. Figure 1 A shows one representative experiment of Coomassie blue-stained control gels. Densitometry analyses ( Fig. 1 B ) revealed no variation in the Coomassie blue band densities, indicating operationally identical protein loading of the different samples. For immunoblotting, proteins were transferred electrophoretically (XCell II Blot Module, Invitrogen Life Technologies) for 1.5 h at 4°C from unstained gels to nitrocellulose membranes (Amersham, Arlington Heights, IL) and then stained with 0.5% Ponceau S in acetic acid to check uniformity of protein transfer onto the nitrocellose membrane. Posttransfer staining of gels with Coomassie blue confirmed complete protein transfer. Initial electrophoretic transfers used two membranes in tandem, and the absence of transferred protein on the membrane placed further away from the gel indicated that significant amounts of protein did not pass through the membrane to be probed. Membranes were first incubated in 5% nonfat dry milk in phosphate-buffered saline, pH 7.4, for 1 h at room temperature to block nonspecific binding of antibody, followed by the primary antibody (anti-rat Pds 1:30,000; anti-rat NCC 1:20,000) in PBS containing 1% nonfat dry milk overnight at 4°C. After four 5-min washes in PBS containing 0.1% Tween 20, membranes were incubated with 1:10,000 dilution of goat anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA) in PBS containing 5% nonfat dry milk for 2 h at room temperature. Blots were washed as above, and luminol-enhanced chemiluminescence (New England Nuclear Life Science Products, Boston, MA) was used to visualize bound antibodies before exposure to Hyperfilm ECL (Amersham). The autoradiography was digitized with use of a laser scanner (Epson Perfection 1650, Epson), and quantification of each band was performed by densitometry using National Institutes of Health Image software. Densitometric values were normalized to the mean for the control group, which was defined as 100%, and results were expressed as means ± SE.) s9 p3 M) \, I0 R

Fig. 1. Representative Coomassie blue-stained polyacrylamide gels used to control protein loading. The gels presented here show aliquots of the same samples presented in Fig. 2. A : control rats (c) and NaCl-loaded rats (na). B : densitometric analyses of total electrophoresed protein separated on these gels established that the accompanying immunoblots (loaded identically; Fig. 2 A ) were uniformly loaded. Values are means ± SE.0 W" _; |% a" w

Statistical methods. All data are represented as means ± SE. Comparisons among groups were assessed by ANOVA followed by the Bonferroni post hoc test or unpaired Student's t -test. Differences were considered significant at P + q6 N& a3 S7 e( n

RESULTS$ M. u; I% `$ o; ?/ d
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Pds abundance was decreased in response to NaCl and NH 4 Cl loading but not in response to NaHCO 3 loading. Two different studies have previously reported conflicting results regarding changes in Pds protein abundance in response to NaHCO 3 -induced metabolic alkalosis ( 9, 38 ). However, these studies differed by the amount of NaCl administrated to the control rats, so that the conflicting findings concerning Pds expression may have resulted from an independent effect of the NaCl load on transporter expression. To test simultaneously the effect of Na per se and alteration of the acid-base status, we first studied in parallel the effects of chronic NaCl, NaHCO 3, and NH 4 Cl loading compared with a group of control rats without increased Na intake. The main functional data obtained in these animals are summarized in Table 1. As expected, rats subjected to NaHCO 3 loading developed metabolic alkalosis with a significant increase in plasma HCO 3 - concentration from the control value of 27.8 ± 0.4 to 31.2 ± 0.7 mmol/l ( P

Table 1. Functional data in rats after chronic NaCl, NaHCO 3, and NH 4 Cl loading
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Figures 2 A and 3 A show that Pds protein abundance was markedly decreased in response to chronic NaCl loading (46 ± 5% in NaCl-loaded rats vs. 100 ± 7% in control group, P

Fig. 2. Effects of chronic NaHCO 3, NH 4 Cl, and NaCl loading on pendrin (Pds) protein abundance in the rat kidney cortex. A : immunoblots of membrane fractions from the cortex obtained from 6 series of NaCl-loaded rats (na) and their respective controls (c) showing downregulation of Pds abundance in response to high-NaCl intake. B : immunoblots of membrane fractions from the cortex obtained from 6 series of NH 4 Cl-loaded rats (ac) and their respective controls (c) showing downregulation of Pds abundance in response to NH 4 Cl loading-induced metabolic acidosis. C : immunoblots of membrane fractions from the cortex obtained from 6 series of alkalotic rats (al) and their respective controls (c) showing that Pds protein abundance is not altered by NaHCO 3 loading-induced metabolic alkalosis. Each lane was loaded with 10 µg protein. Immunoblots were reacted with anti-Pds antibody and revealed the expected 120-kDa band. Densitometric analyses of the data are summarized in Fig. 3.! j3 q& [1 H0 u! w( q( k: D& s

Fig. 3. Effects of NaCl, NH4Cl, and NaHCO 3 loading on Pds expression and on urinary chloride excretion (Cl u ) in the rat kidney. A : summary of densitometric analyses of Pds protein abundance in response to NaCl, NH 4 Cl, and NaHCO 3 loading expressed as % of control values. B : urinary chloride excretion in response to the same treatments. Creat u, urinary creatinine excretion.Values are means ± SE. *Statistically significant difference vs. control; $ statistically significant difference vs. NaHCO 3 loading values; # statistically significant difference vs. NH 4 Cl loading values (all P

Table 2. Summary of densitometric analysis of data for pendrin protein expression# A1 `' J) _. q
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Pds was downregulated in response to chronic KCl loading but not to chronic KHCO 3 loading. Our results confirm the inhibition of Pds expression by NH 4 Cl loading and the absence of effect on Pds of NaHCO 3 loading as previously reported ( 9, 38 ). We also extend these data by directly demonstrating an inhibitory effect of NaCl loading as suggested by Frische et al. ( 9 ). Taken together, our findings could be interpreted to indicate that Pds is regulated by both acid-base status and Na intake, but an alternative conclusion is that Cl - loading downregulates Pds independently of acid-base or Na balance. Indeed, as summarized in Fig. 3, downregulation of Pds expression in response to NH 4 Cl and NaCl loading correlated with the increase in chloride excretion (NH 4 NaCl loading), whereas there was no effect on Pds expression when sodium loading was achieved with NaHCO 3. To test this hypothesis that Pds expression is regulated by chloride balance, we next studied the effects of the same chloride or HCO 3 - loads as in the previous experiments, but now administered with K as the accompanying cation rather than Na  .& L0 i( ?( J6 {5 ?3 B9 n
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Table 3 shows functional data obtained in rats subjected to either chronic KCl loading or chronic KHCO 3 loading. As expected, high dietary KCl intake led to a marked increase in urinary chloride excretion from the control value of 17 ± 0.7 to 59 ± 2 mmol/l ( P

Table 3. Functional data in rats after chronic administration of 0.28 M KCl or KHCO 3
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Table 2 and Fig. 4 show that Pds protein abundance was markedly decreased in response to chronic KCl loading (44 ± 6% in KCl-loaded rats vs. 100 ± 15% in control group, P

Fig. 4. Effects of chronic KCl loading on Pds protein abundance in the rat kidney cortex. A : immunoilots of membrane fractions from the cortex obtained from 6 KCl-loaded rats (K) and their respective controls (C). Each lane was loaded with 10 µg protein. Immunoblots were reacted with anti-Pds antibody and revealed the expected 120-kDa band. B : densitometric analyses from 6 independent experiments showing that the abundance of Pds in KCl-loaded rats is decreased in the cortex compared with control rats. Values are means ± SE. *Statistically significant increase vs. control ( P 9 F% o' v, |9 s9 L7 p* D
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Fig. 5. Effects of chronic KHCO 3 loading on Pds protein abundance in the rat kidney. A : immunoblots of membrane fractions from the cortex obtained from 6 KHCO 3 -loaded rats (K) and their respective controls (C). Each lane was loaded with 10 µg protein. Immunoblots were reacted with anti-Pds antibody and revealed the expected 120-kDa band. B : densitometric analyses from 6 independent experiments showing that the abundance of Pds in KHCO 3 -loaded rats was not significantly altered in the cortex compared with control rats. Values are means ± SE.
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Pds expression demonstrated a significant inverse correlation with the level of chloride loading. Summarizing all of the previous experiments, Fig. 6 shows the plot of Pds abundance against urinary chloride excretion. The same strong inverse correlation between both values was found when data originating from either the first protocol series (i.e., NaCl, NaHCO 3, and NH 4 Cl loading) ( Fig. 6 A ) or the second protocol series (i.e., KCl and KHCO 3 loading) ( Fig. 6 B ) were analyzed. The relationship between both values was linear after logarithmic transformation of urinary chloride excretion values (see insets in Fig. 6, A and B ) and was highly significant in both sets of experiments ( r 2 = 0.81, P

Fig. 6. Relationship between chloride urinary excretion and Pds expression in vivo, determined by immunoblot from control, NaCl-, NH 4 Cl-, NaHCO 3 -, KCl-, and KHCO 3 -loaded rats. A : individual densitometric analysis of immunoblots reacted with the anti-Pds antibody originating from the series of NaCl, NH 4 Cl, and NaHCO 3 loading relative to their respective controls plotted against urinary chloride ([Cl - ] u ) excretion. B : individual densitometric analysis of immunoblots reacted with the anti-Pds antibody originating from the series of KCl and KHCO 3 loading relative to their respective controls plotted against urinary chloride excretion. Line graphs in A and B show the relationship between both values when data are analyzed using logarithmic regression is shown. Insets : Pds protein abundance plotted against log-transformed urinary chloride excretion values. In both sets of experiments, a strong linear inverse correlation was found ( r 2 = 0.81, P

Rats with chronic NaCl restriction did not experience changes in Pds abundance. The preceding studies demonstrated that equivalent sodium loading did not affect Pds abundance unless chloride was the accompanying anion, suggesting that chloride rather than sodium balance is a key regulator of Pds expression. To definitively rule out the role of sodium balance in Pds regulation, we next investigated whether chronic reduction in sodium intake would affect Pds expression. For that purpose, Pds expression was compared in Na -restricted vs. NaCl-supplemented rats.

Low-NaCl intake (0.009% Na ) led to a marked decrease in urinary sodium excretion from the value in NaCl-supplemented rats of 29 ± 1 to 0.4 ± 0.1 mmol/mmol creatinine ( P 9 h: `  \# f6 n! B. F( Y+ J# A
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Fig. 7. Effects of a low-NaCl diet on Na-Cl cotransporter (NCC) protein abundance in the rat kidney cortex. A : immunoblots of membrane fractions from the cortex obtained from 6 rats receiving a low-sodium (0.009% Na ) diet (-na) and their respective control (c; i.e., NaCl-supplemented) rats. (These samples are aliquots from the same membrane preparations shown in Fig. 8.) Each lane was loaded with 10 µg protein. Immunoblots were reacted with anti-NCC antibody and revealed the expected 165- to 170-kDa band. B : densitometric analyses from 6 independent experiments revealed a significant increase in NCC abundance in the cortex of sodium-restricted rats compared with control rats. Values are means ± SE. *Statistically significant increase vs. control ( P

Fig. 8. Effects of low-NaCl diet on Pds protein abundance in the rat kidney cortex. A : immunoblots of membrane fractions from the cortex obtained from 6 rats receiving a low-sodium (0.009% Na ) diet (-na) and their respective control rats (c). (These samples are aliquots from the same membrane preparations shown in Fig. 9.) Each lane was loaded with 10 µg protein. Immunoblots were reacted with anti-Pds antibody and revealed the expected 120-kDa band. B : densitometric analyses from 6 independent experiments showing that the abundance of Pds in NaCl-restricted rats was not significantly altered in the cortex compared with NaCl-supplemented rats. Values are means ± SE.
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It should be noted that the low-Na diet also reduced urinary chloride excretion significantly from the NaCl-supplemented group value of 59 ± 2 to 27 ± 2 mmol/mmol creatinine ( P ' }  X' d6 Y* C+ ^' H% K% i
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Chronic furosemide administration led to a marked increase in Pds abundance The results of our experiments indicated that Pds is strongly downregulated by an increase in chloride intake. However, our experiments do not distinguish whether the effect of chloride loading to decrease Pds expression is due to the chloride loading of the organism or to the increase in tubular fluid chloride delivery to the distal nephron. Therefore, to distinguish the effect of total body chloride balance vs. the effect of urinary chloride, we next studied the effect of chronic furosemide administration. Furosemide exerts its diuretic action by binding to the Na -K -2Cl - cotransporter (NKCC2) in the thick ascending limb and blocking ion transport ( 24 ). In our protocol, furosemide-induced NaCl depletion was not compensated by NaCl supplementation, because we wanted to study the effect of the treatment at steady state when the treated rats have developed mild extracellular volume contraction (i.e., mild NaCl depletion) while maintaining the same urinary chloride excretion as control rats. Functional data are shown in Table 4. Furosemide treatment increased the urine output from the control value of 8.2 ± 1.1 to 31.5 ± 5.7 ml/day. At steady state, as expected, urinary Na  , K  , and Cl - excretions of treated rats were identical to controls. Furosemide-treated rats developed extracellular volume depletion, attested by an increase in plasma protein concentration from the control value of 50 ± 0.9 to 54 ± 1.4 g/l ( P : b- L5 c" k0 p0 G
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Table 4. Functional data during chronic administration of furosemide
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Fig. 9. Effects of chronic furosemide administration on Pds protein abundance in the rat kidney cortex. A : immunoblots of membrane fractions from the cortex obtained from 5 rats after 6 days furosemide administration (furosemide) and 4 control rats (control). Each lane was loaded with 10 µg protein. Immunoblots were reacted with anti-Pds antibody and revealed the expected 120-kDa band. B : densitometric analyses from 5 independent experiments revealed a significant increase in Pds abundance in the cortex of furosemide-treated rats compared with control rats. Values are means ± SE. *Statistically significant increase vs. control ( P
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However, in this set of experiments we cannot exclude that the combined effect of two other parameters, namely, secondary hyperaldosteronism and metabolic alkalosis, may have contributed to the observed upregulation of Pds. The preceding experiments showed that downregulation of Pds is independent of aldosterone and that Pds expression is unaffected by the mild metabolic alkalosis after NaHCO 3 loading. However, chronic furosemide administration most likely causes an increase in plasma aldosterone level in combination with a more severe metabolic alkalosis than in our preceding experiments because of the volume depletion. Although acid-base parameters were not measured in this experimental series, we did find ( Table 4 ) that furosemide administration caused a larger fall in plasma chloride than the preceding series with bicarbonate loading, suggesting a more severe metabolic alkalosis.5 ]8 @; R; {- l' f4 k  _( _

DISCUSSION$ e2 q8 h6 W! Z0 \$ Z0 K9 s

The present study demonstrates that chronic alterations in chloride balance are associated with adaptive changes in protein abundance of the Cl - /HCO 3 - exchanger Pds in the rat kidney cortex. To study these regulations, rats were submitted to different treatments, leading to alterations in urinary chloride or sodium excretion for 6 days, and then Pds protein abundance was assessed by immunoblotting. We choose to submit the rats to these different challenges for 1 wk because the same duration has been previously used by others to study Pds regulation in response to acid-base status alterations or changes in potassium intake ( 9, 38 ). Moreover, in a previous study we have assessed the time course of sodium excretion after similar protocols of NaCl or NaHCO 3 loading and NaCl restriction, and we have shown that sodium excretion was significantly altered (increased or decreased, respectively) and stable after 3 days of these challenges, indicating that by day 6 the animals are very likely at steady state ( 6 ). Similarly, we also found that ammonium excretion after NH 4 Cl loading rapidly increases to stabilize 2 days after the beginning of this treatment (unpublished observations). Finally, at the end of chronic furosemide treatment, control and treated rats exhibited the same levels of urinary sodium and chloride excretion (see Table 3 ), also indicating that the animals were in steady-state balance with respect to these ions.% M; `# ^" R; ]* k2 ]. n- B5 J% j

We document decreased Pds protein abundance in the cortex in response to high Cl intake as well as increased Pds abundance in response to chloride depletion. Moreover, our results suggest that this effect occurs independently of changes in sodium balance, in plasma aldosterone level, and of alteration in acid-base status. We propose a model in which this protein could play an important role in specific renal regulation of chloride balance. Indeed, downregulation of Pds is likely to play a role in the increased chloride excretion observed during high-chloride intake and, conversely, during chloride depletion, upregulation of Pds may participate in renal chloride reclamation.- U8 p) y# O0 u! q6 X! X0 `' w. N; y

Several lines of evidence indicate that Pds represents the apical Cl - /HCO 3 - exchanger of type B and non-A-non-B intercalated cells in the CCD and the CNT and thus should play a key role in the adaptation of transepithelial bicarbonate transport in the CNT and CCD in response to changes in acid-base status ( 9, 26, 35, 36, 38 ). Accordingly, it has been previously reported that Pds is appropriately downregulated in response to NH 4 Cl-induced chronic metabolic acidosis ( 9, 23, 38 ). The effects of chronic metabolic alkalosis on Pds are more complex. Indeed, Wagner et al. ( 38 ) reported the absence of effect of 6 days of NaHCO 3 loading on Pds abundance compared with control rats with a lower Na intake, whereas a second study by Frische et al. ( 9 ) did find a marked stimulation of alkalosis on Pds expression. However, in the latter study, to assess specifically the effect of alkalosis after NaHCO 3 loading, the controls consisted of rats drinking an equimolar NaCl solution to balance sodium intake between the control and the alkalotic rats ( 9 ). Taken together, these results were interpreted to indicate that metabolic alkalosis exerts a potent stimulatory effect on Pds, masked in the first study by an independent inhibitory effect of the accompanying sodium load. However, because the study by Frische et al. did not include a control group with no increased Na intake, it was not possible to exclude in the latter study that the observed effect was limited to the inhibitory effect of NaCl loading with no effect of the base load. The results we report here in Figs. 2 and 3 are in agreement with the results reported in these previous studies, but our findings extend these data by demonstrating that it is the increase in chloride intake that decreases kidney Pds abundance and that NaHCO 3 -induced metabolic alkalosis has indeed no direct regulatory effect on Pds abundance.

The specific mediator of Pds regulation by chloride remains unidentified in our study. All along the nephron, chloride is known to be cotransported with sodium either through proteins of the cation-chloride cotransporter family or through functional coupling between sodium and chloride fluxes (i.e., chloride fluxes are driven by sodium reabsorption through tertiary active transport processes). Thus chloride transport is thought to be passively regulated and to follow the changes in sodium reabsorption. However, we did not find in our study a clear independent relationship between endogenous aldosterone secretion, the main regulatory hormone of sodium balance, and Pds expression. Moreover, our data demonstrating 1 ) that high- Cl - intake administrated with either Na  , NH 4  , or K specifically decreases Pds abundance, 2 ) that increased sodium intake administered with bicarbonate has no effect on Pds expression, and 3 ) that dietary sodium restriction has no effect on Pds expression together support the hypothesis that chloride transport is specifically regulated by changes in chloride balance through Pds regulation and not by changes in Na balance. Despite the fact that we found a strong inverse correlation between urinary chloride excretion and Pds expression levels, the upregulation of Pds with furosemide treatment excludes that a change in urine Cl - is required for the upregulation of Pds and is consistent with the concept that it represents a response to body Cl - depletion. However, the lower plasma Cl - concentration suggests that furosemide-treated rats had metabolic alkalosis, and they would also be expected to have very high aldosterone levels. Although we excluded that alkali loading causes upregulation of Pds and did not find a correlation between Pds expression with aldosterone levels in the preceding studies, we cannot rule out the possibility that the combination of metabolic alkalosis and elevated aldosterone together resulting from furosemide treatment may have contributed to the upregulation of Pds observed in this case. Thus furosemide treatment may have resembled the effect of administering the mineralocorticoid DOCP, which has been reported to enhance Pds expression ( 36 ).+ p$ ?2 C# u& p: P! M- v
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However, it should be noted that changes in chloride delivery from our different chloride-loading protocols and the administration of furosemide, a specific blocker of NKCC2, are both expected to modulate the rate of BSC1 (NKCC2)-mediated chloride transport across the apical membrane of macula densa (MD) cells. Such changes in the rate of Cl - transport into MD cells have been demonstrated to alter a complex signaling cascade that allows these cells to act as a biosensor sensitive to changes in chloride (or NaCl) content, which in turn controls the glomerular filtration rate and renin secretion ( 17, 19, 27 ). Interestingly, this regulation involves modulation of the secretion of various paracrine and endocrine factors, including ATP and PGE 2 ( 2, 22 ). Therefore, to test whether MD cells or thick ascending limbs cells, which also express BSC1 (NKCC2), secrete an unidentified factor that regulates Pds expression in CNT and CCD cells in response to changes in chloride balance would be of great interest.

Chloride is the most abundant anion of the extracellular fluid of the organism, and in various secreting or absorbing epithelia chloride transport is crucial for a wide range of physiological processes including clearance of water from the lung and inner ear, exocrine pancreatic function, and secretion of sweat and semen. Thus chloride transport is specifically regulated. Our data demonstrate for the first time that a specific chloride transport protein in the most distal part of the nephron is downregulated in response to high chloride intake and is upregulated in response to chloride depletion. Several lines of evidence indicate that this new regulatory process may be of pathophysiological interest. In the kidney, Cl - absorption or secretion needs to be tightly regulated to allow, in turn, proper regulation of extracellular volume and blood pressure. For example, it has been suggested on the basis of physiological studies that the inherited syndrome of hypertension and hyperkalemia, pseudohypoaldosteronism type II, involves increased distal nephron Cl - reabsorption ( 28 ). Interestingly, the serine-threonine kinases WNK1 and WNK4 (with no lysine kinase 1 and 4) involved in some cases of this syndrome ( 40 ) are also expressed in various chloride transporting epithelia ( 5, 13 ) and have been shown to modulate the surface expression of various Cl - transporters ( 13, 41, 42 ). Pds has been demonstrated to be insensitive to WNK4-mediated regulation and thus is unlikely to participate in WNK4-related pseudohypoaldosteronism type II ( 13 ), but it is possible that alternative pathways causing excessive Pds-mediated chloride could be involved in other forms of this disease. Conversely, the observation that Pds -/- mice are resistant to hypertension induced by the injection of DOCP, an aldosterone analog ( 36 ), also suggests that Pds is involved in the regulation of extracellular volume and in blood pressure regulation in mice ( 36 ).

In summary, this study demonstrates that polypeptide abundance of the Cl - /HCO 3 - exchanger Pds is specifically regulated in response to changes in chloride balance independent of sodium and acid-base balance. The mediators of this novel regulation remain to be elucidated.
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GRANTS

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-17433 and DK-33793 (to P. Aronson).8 I2 L( r- a8 o
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