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Am J Physiol Cell Physiol 295: C1658-C1667, 2008. First published October 29, 2008; doi:10.1152/ajpcell.00419.2008
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Pendrin in the mouse kidney is primarily regulated by Cl– excretion but also by systemic metabolic acidosis

Patricia Hafner,1,* Rosa Grimaldi,1,2,* Paola Capuano,1 Giovambattista Capasso,2 and Carsten A. Wagner1

1Institute of Physiology and Center for Human Integrative Physiology, University of Zurich, Zurich, Switzerland; and 2School of Medicine, Second University of Naples, Naples, Italy

Submitted 14 August 2008 ; accepted in final form 23 October 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The Cl–/anion exchanger pendrin (SLC26A4) is expressed on the apical side of renal non-type A intercalated cells. The abundance of pendrin is reduced during metabolic acidosis induced by oral NH4Cl loading. More recently, it has been shown that pendrin expression is increased during conditions associated with decreased urinary Cl– excretion and decreased upon Cl– loading. Hence, it is unclear if pendrin regulation during NH4Cl-induced acidosis is primarily due the Cl– load or acidosis. Therefore, we treated mice to increase urinary acidification, induce metabolic acidosis, or provide an oral Cl– load and examined the systemic acid-base status, urinary acidification, urinary Cl– excretion, and pendrin abundance in the kidney. NaCl or NH4Cl increased urinary Cl– excretion, whereas (NH4)2SO4, Na2SO4, and acetazolamide treatments decreased urinary Cl– excretion. NH4Cl, (NH4)2SO4, and acetazolamide caused metabolic acidosis and stimulated urinary net acid excretion. Pendrin expression was reduced under NaCl, NH4Cl, and (NH4)2SO4 loading and increased with the other treatments. (NH4)2SO4 and acetazolamide treatments reduced the relative number of pendrin-expressing cells in the collecting duct. In a second series, animals were kept for 1 and 2 wk on a low-protein (20%) diet or a high-protein (50%) diet. The high-protein diet slightly increased urinary Cl– excretion and strongly stimulated net acid excretion but did not alter pendrin expression. Thus, pendrin expression is primarily correlated with urinary Cl– excretion but not blood Cl–. However, metabolic acidosis caused by acetazolamide or (NH4)2SO4 loading prevented the increase or even reduced pendrin expression despite low urinary Cl– excretion, suggesting an independent regulation by acid-base status.

transporter; collecting duct

THE KIDNEY plays a central role in regulating the systemic homeostasis for electrolytes and acid-base homeostasis, which are intricately linked together. The bulk reabsorption of electrolytes and bicarbonate is mediated by several transport proteins in the proximal tubule and thick ascending limb of the loop of Henle. The fine control, however, takes place along the connecting segment and collecting duct. There, the reabsorption of water and Na+ by principal or segment-specific cells is regulated, as well as the secretion of protons or bicarbonate, by the different subtypes of intercalated cells. The exact mechanisms underlying trans- and paracellular Cl– absorption in these segments have not yet been fully clarified (10, 11).

The Cl–/anion exchanger pendrin (SLC26A4) belongs to a large superfamily of anion transporters and is predominantly expressed in the inner ear, thyroid gland, and kidney (8, 16). Mutations in pendrin cause Pendred syndrome, which is associated with sensorineural deafness and goiter (Online Mendelian Inheritance in Man Database No. 274600 [OMIM] ) (7). In the kidney, pendrin expression is restricted to the apical side of non-type A intercalated cells (i.e., type B and non-type A/B intercalated cells) in the connecting segment and cortical collecting duct (13, 20, 29). There, pendrin has been implicated in bicarbonate secretion into urine stimulated during metabolic alkalosis (20). Indeed, pendrin can function as a Cl–/HCO3– exchanger, as evident from experiments in cells with exogenous expression of pendrin (21). Moreover, pendrin-deficient mice secrete less bicarbonate in isolated perfused cortical collecting ducts during metabolic alkalosis (20). Also consistent with a role in the regulated secretion of bicarbonate during metabolic alkalosis, we and several other groups have observed that pendrin abundance was reduced during metabolic acidosis induced by oral NH4Cl loading (9, 18, 27). These findings were interpreted as the downregulation of pendrin due to metabolic acidosis.

More recently, Quentin et al. (19), Vallet et al. (24), Verlander et al. (26), and Wall et al. (30) have described that pendrin expression was regulated by urinary Cl– excretion and suggested a role in transcellular Cl– absorption. In particular, they noted that maneuvers increasing urinary Cl– excretion decreased pendrin expression. Treatments inducing Cl– depletion and stimulating renal Cl– reabsorption were associated with higher pendrin protein levels (19, 24, 26, 30).

These data raised the question of whether the downregulation in pendrin abundance observed in NH4Cl-loaded animals was solely due to Cl– loading or was also, at least in part, due to the metabolic acidosis induced by NH4+ loading. To this end, in the present study, we examined the correlation between pendrin expression and urinary acid and Cl– excretion by Western blot analysis in several animal models. Systemic acid-base homeostasis and urinary acid excretion were manipulated by providing mice with different diets, including NH4Cl, (NH4)2SO4, the carbonic anhydrase inhibitor acetazolamide, and high protein (50%). All these treatments have been shown to induce metabolic acidosis or to provide an acid challenge leading to increased urinary acid excretion [i.e., high urinary ammonium and net acid excretion (NAE)] (6, 12, 22, 31). In addition, other groups of animals received either only sucrose (which was added to all diets), a diet containing a normal protein content (20%), or NaCl and Na2SO4 to provide a Cl– load, or a control for (NH4)2SO4 loading, respectively. The results indicate that pendrin expression is regulated by urinary Cl– excretion, but the abundance may also be reduced during metabolic acidosis despite low urinary Cl– excretion.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal experiments. All animal experiments were conducted according to Swiss Animal Welfare Laws and were approved by the local Veterinary Authority (Kantonales Veterinäramt, Zurich, Switzerland). NMRI mice (Charles River Laboratories, male, 12 wk, 35–45 g) were maintained on standard chow and had access to drinking water ad libitum. Animals were divided into two main groups. The first group was subdivided into the following six subgroups that received different additives in their drinking water: control (2% sucrose), NH4Cl (280 mM NH4Cl and 2% sucrose), NaCl (0.28 M NaCl and 2% sucrose), (NH4)2SO4 [0.28 M (NH4)2SO4 and 2% sucrose], Na2SO4 (0.28 M Na2SO4 and 2% sucrose), and azetazolamide (80 mg acetazolamide/100 ml water and 2% sucrose) for 7 days each. The second group of animals received normal drinking water and an isocaloric diet containing either normal protein content (20%) or high protein content (50%) for 7 or 14 days. Care was taken that all other constituents, particularly electrolytes, of the diets were kept constant (Kliba). Each group consisted of five animals for each time point and treatment. Animals were kept in normal cages for the first 5 days of the diet and were slowly adapted to metabolic cages for several hours every day. For the last 2 days, animals were kept constantly in metabolic cages (Tecniplast, Buguggiate, Italy); food and water intake, feces, and urine output were monitored, and 24-h urine was collected under mineral oil. At the end of the collection period, mice were anesthetized with ketamine-xylazine intraperitoneally, and heparinized venous blood samples were collected and analyzed immediately for blood gases and electrolytes on a Radiometer ABL 505 (Radiometer, Copenhagen, Denmark) blood gas analyzer. Urine was collected, and pH was measured immediately using a pH microelectrode connected to a Thermo Orion 290 pH meter. Serum and urine were frozen until further analysis. Creatinine in serum and urine were measured using an enzymatic reaction (WAKO Chemicals) and the Jaffe reaction, respectively. Electrolytes in diluted urine samples were determined using ion chromatography (Metrohm ion chromatograph). Phosphate in urine was measured using a commercial kit (Sigma), and ammonium in urine was measured using the Berthelot protocol (19). Titratable acids were measured in 200-µl urine samples diluted with an equal volume of 0.1 M HCl after samples had been boiled for 2 min at 37°C by titration with 0.1 M NaOH to pH 7.4 as previously described (5). NAE was calculated as the sum of the measured titratable acidity and ammonium.

Antibodies against pendrin. The antibodies against mouse pendrin were raised in the rabbit and guinea pig against the COOH-terminal sequence CKDPLDLMEAEMNAEELDVQDEAMRRLAS coupled to KLH (Pineda Antibody Service, Berlin, Germany). This sequence has been used previously to raise antibodies against pendrin (14). The guinea pig and rabbit antisera recognized only one major band of ~120 kDa, and the band was abolished by preincubation of serum with the immunizing peptide and absent in preimmune serum (see Supplemental Fig. 1).1 Both sera were also tested by immunohistochemistry and stained the luminal side of intercalated cells only in the late distal tubule, connecting segment, and cortical collecting duct, as previously described for other anti-pendrin antibodies (14, 20, 27, 29). No staining was observed when the preimmune serum was used or when the immune serum was preincubated with the immunizing peptide (see Supplemental Fig. 2). We used the guinea pig serum for immunoblot analysis and the rabbit serum for immunohistochemistry.

Western blot analysis. Mice were killed, and kidneys rapidly harvested and transferred into ice-cold K-HEPES buffer (200 mM mannitol, 80 mM K-HEPES, and 41 mM KOH; pH 7.5) with pepstatin, leupeptin, K-EDTA, and PMSF added as protease inhibitors. Samples were homogenized with a tip sonicator and centrifuged at 1,000 g for 10 min at 4°C, and the supernatant was saved. Subsequently, the supernatant was centrifuged at 100,000 g for 1 h at 4°C, and the resultant pellet was resuspended in K-HEPES buffer containing protease inhibitors. After measurement of the total protein concentration (Bio-Rad protein kit), 75 µg of membrane protein were solubilized in Laemmli sample buffer, and SDS-PAGE was performed on 10% polyacrylamide gels. For immunoblot analysis, proteins were transferred electrophoretically from unstained gels to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA). After being blocked with 5% milk powder in Tris-buffered saline and 0.1% Tween 20 for 60 min, blots were incubated with guinea pig anti-pendrin immune serum and mouse monoclonal anti-actin (42 kDa, Sigma) at 1:500 dilution for either 2 h at room temperature or overnight at 4°C. After being washed and subsequent blocked, blots were incubated with secondary antibodies conjugated with alkaline phosphatase or horseradish peroxidase [goat anti-rabbit: 1: 5,000, goat anti-guinea pig: 1:5,000, and donkey anti-mouse: 1:10,000 (Promega)] for 1 h at room temperature. Antibody binding was detected with an enhanced chemiluminescence kit (Pierce) in the case of horseradish peroxidase-linked antibodies and with the CDP Star kit (Roche) for alkaline phosphatase-linked antibodies before the detection of chemiluminescence with the Diana III Chemiluminescence detection system. Bands were quantified with Aida Image Analyzer software (Raytest).

Immunohistochemistry. Mice were anesthetized with ketamine-xylazine and perfused through the left ventricle with PBS followed by paraformaldehyde-lysine-periodate fixative (PLP) (15). Kidneys were removed and fixed overnight at 4°C by an immersion in PLP. Kidneys were washed three times with PBS and rapidly frozen with liquid propane, and 5-µm cryosections were cut after cryoprotection with 2.3 M sucrose in PBS.

Indirect immunohistochemistry was carried out as previously described (4, 27). Sections were incubated with 1% SDS for 5 min, washed three times with PBS, and incubated with PBS containing 1% BSA for 15 min before the primary antibodies. The primary antibodies [rabbit and guinea pig anti-pendrin, mouse monoclonal anti-calbindin D28k (SWANT, Bellinzona, Switzerland), and goat anti-aquaporin 2 (AQP2; Chemicon, now Millipore)] were diluted in PBS at 1:1,000, 1: 20,000, and 1:400, respectively, and applied for either 75 min at room temperature or overnight at 4°C. Sections were then washed twice for 5 min with high-NaCl PBS (PBS + 2.7% NaCl) and once with PBS and incubated with dilutions of the secondary antibodies [donkey anti-rabbit Alexa 594, donkey-anti guinea-pig Cy3, donkey anti-mouse Alexa 488, and donkey anti-goat 488 (Molecular Probes)] at 1:1,000; 1:400, 1:400, and 1:400, respectively, combined with 4',6-diamidino-2-phenylindole to counterstain cell nuclei (1:200) for 1 h at room temperature. Sections were again washed twice with high-NaCl PBS and once with PBS before being mounted with VectaMount (Vector Laboratories, Burlingame, CA). Sections were viewed with a confocal microscope (Leica CLSM). Images were processed (overlays) using Adobe Photoshop. Cells were counted as previously described (27) in at least three sections from four animals each for each treatment group. Omission of primary antibodies or preincubation of anti-pendrin sera with the immunizing peptide abolished all staining (see Supplemental Figs. 1 and 2).

Statistics. All data were tested for significance using Student's t-test and ANOVA followed by Bonferroni's multiple-comparison test. Only data with P < 0.05 were considered statistically significant. Data are reported as means ± SE.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Treatment of animals with NH4Cl for 7 days induced metabolic acidosis, as evident from blood gas analysis and urinary NAE (Table 1 and Fig. 1). Urinary total and fractional urinary Cl– excretion were strongly increased. Immunoblot analysis showed a reduction in relative pendrin protein expression levels to 62 ± 13% compared with control animals, as previously described in mice and rats (9, 18, 19, 27) (Fig. 1). In contrast, addition of NaCl to the drinking water provided a Cl– load, as evident from the urinary fractional Cl– excretion, but did not affect systemic acid-base status or urinary acid excretion (Table 1 and Fig. 1). As expected, pendrin protein abundance was reduced to 80 ± 16% (Fig. 1).


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  		Table 1. Blood and urine analysis for control, NH4Cl, NaCl, (NH4)SO4, Na2SO4, and acetazolamide groups

 

Figure 1
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  		Fig. 1. NH4Cl and NaCl decrease pendrin abundance. Mice were given 0.28 M NH4Cl + 2% sucrose or 0.28 M NaCl + 2% sucrose in drinking water, respectively, for 7 days. Control mice received only 2% sucrose. NH4Cl loading decreased relative pendrin abundance and increased blood and urinary Cl– and net acid excretion (NAE). The addition of NaCl also reduced the relative pendrin abundance without altering NAE and blood Cl– concentration but increased urinary Cl– excretion. n = 5 animals/group. *P < 0.05; **P < 0.01; ***P < 0.001.

 
In a second pair of animals, mice received (NH4)2SO4 to induce metabolic acidosis without providing a direct Cl– load. (NH4)2SO4 induced severe hyperchloremic metabolic acidosis with high NAE and low urinary fractional Cl– excretion but high blood Cl– concentrations, possibly due to dehydration (as also evident from the increased Na+ concentration and weight loss; Table 1 and Fig. 2). Relative pendrin expression levels were decreased to 49 ± 5% (Fig. 2). Na2SO4 was given to control for the effects of sulfate, which increases the lumen-negative potential in the collecting duct as an impermeable anion and facilitates H+ secretion. Na2SO4 did not affect net anion excretion but caused urinary Cl– retention, most likely due to dehydration. Pendrin expression was highly stimulated to 252 ± 12% (Fig. 2). Thus, low urinary Cl– excretion stimulates pendrin expression levels, as previously described (19, 30). However, concomitant metabolic acidosis, as in the case of (NH4)2SO4 loading, seems to reverse this effect and reduces pendrin expression.


Figure 2
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  		Fig. 2. (NH4)2SO4 decreases and Na2SO4 increases pendrin abundance. Mice were given 0.28 M (NH4)2SO4 + 2% sucrose or 0.28 M Na2SO4 + 2% sucrose in drinking water, respectively, for 7 days. Control mice received only 2% sucrose for 7 days. (NH4)2SO4 loading reduced the relative pendrin abundance and decreased urinary Cl–excretion while increasing blood Cl– concentration and urinary NAE. In contrast, Na2SO4 stimulated pendrin expression while reducing urinary Cl– and NAE and increased blood Cl– concentration due to dehydration. n = 5 animals/group. *P < 0.05; ***P < 0.001.

 
In an alternative model of metabolic acidosis, acidosis was induced in mice treated with the carbonic anhydrase inhibitor acetazolamide. Acetazolamide causes metabolic acidosis by inducing urinary bicarbonate wasting due to inhibition of proximal tubular bicarbonate reabsorption (3, 6). This type of metabolic acidosis is thus associated with high urinary pH (Table 1) and high net anion excretion and allows us to distinguish between the effects of urinary and systemic pH. Acetazolamide caused metabolic acidosis with high net anion excretion, as expected. Urinary Cl– excretion was low, possibly due to dehydration (high blood Na+ and Cl– concentrations and weight loss), and relative pendrin expression levels were not changed (106 ± 12%; Fig. 3 and Table 1). Hence, metabolic acidosis with high urinary pH prevents the increase in pendrin protein abundance despite low urinary Cl– excretion.


Figure 3
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  		Fig. 3. Inhibition of carbonic anhydrase with acetazolamide does not alter pendrin abundance. Mice were given the carbonic anhydrase inhibitor acetazolamide in drinking water for 7 days, which caused metabolic acidosis, increased urinary NAE and blood Cl– concentration, and decreased urinary Cl– excretion. Relative pendrin abundance was not affected by acetazolamide treatment. n = 5 animals/group. *P < 0.05; **P < 0.01; ***P < 0.001.

 
Immunohistochemistry was performed on kidneys from control mice or mice treated with NH4(SO4)2, Na2SO4, or acetazolamide. Kidneys were stained for pendrin and the principal cell markers calbindin D28k and AQP2. Cells were counted for the absence or presence of these cell-specific markers separately along the connecting tubule and cortical collecting duct. In kidneys from control mice, the relative abundance of pendrin-positive or -negative cells and AQP2/calbindin D28k-expressing cells was similar to results previously described in the mouse kidney (22, 23, 27). Treatment with NH4(SO4)2 or acetazolamide significantly reduced the relative abundance of pendrin-positive cells and increased the abundance of cells lacking both pendrin and AQP2/calbindin D28k both in the connecting tubule and cortical collecting duct (Figs. 4 and 5 and Table 2). Application of Na2SO4 had no effect on the relative abundance of the different cell types.


Figure 4
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  		Fig. 4. Abundance of pendrin-expressing cells in the kidneys from mice with different treatments. Kidney sections were stained with antibodies against pendrin (red) and calbindin (Calb) D28k/ aquaporin 2 (AQP2; green), and cells were counted in the connecting tubule (CNT) and cortical collecting duct (CCD). A–D: pendrin-positive cells in the CNT. A: control group; B: (NH4)2SO4 treatment group; C: Na2SO4 treatment group; D: acetazolamide treatment group. E–H: pendrin-positive cells along the CCD. E: control; F: (NH4)2SO4 treatment group; G: Na2SO4 treatment group; H: acetazolamide treatment group. Arrows indicate pendrin-positive cells. *Pendrin-negative, calbindin D28k/AQP2-negative cells. Original magnification: x630.

 

Figure 5
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  		Fig. 5. Ammonium sulfate and acetazolamide reduce the number of pendrin-expressing intercalated cells. Kidney sections from 5 mice/group treated with sucrose (control), NH4(SO4)2, Na2SO4, or acetazolamide were stained for pendrin, calbindin D28k, and AQP2, and cells along the CNT and CCD were counted for the expression of these cell markers. Data are percentages of total cells counted; values are means ± SE. *P < 0.05.

 

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  		Table 2. Relative cell abundance of pendrin-expressing cells in the CNT and CCD

 
A second group of animals was given either low-protein diet (20% protein) or high-protein diet (50% protein), which provide a mild and more physiological acid load without altering Cl– balance. Control mice received the low-protein diet for 7 days, and two groups were given the high-protein diet for 7 or 14 days, respectively. The high-protein diet increased urinary NAE strongly without altering otherwise systemic acid-base status, urinary Cl– excretion, and blood Cl– concentrations (Table 3). Relative pendrin abundance levels were not affected by the high-protein diet (high-protein diet for 7 days: 105 ± 4% and high-protein dief for 14 days: 107 ± 10%; Table 2 and Fig. 6). Thus, increasing urinary acid excretion without inducing metabolic acidosis does not alter pendrin protein abundance.


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  		Table 3. Blood and urine analysis for groups on a low-protein diets for 7 days, a high-protein diet for 7 days, and a high-protein diet for 14 days

 

Figure 6
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  		Fig. 6. Dietary protein content and acid load does not affect pendrin expression. Mice received a dietary acid load by varying the dietary protein content from low protein (LP; 20%) to high protein (HP; 50%) for 7 or 14 days, respectively. Diets were otherwise identical in their fat and electrolyte composition. The HP diet caused strongly increased NAE without altering pendrin expression, blood, and urine Cl– concentration. n = 5 animals/group. *P < 0.05.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The connecting segment and collecting duct are the main sites where the fine regulation of excretion or reabsorption of several ions, including Na+, K+, and Cl–, and acid-base equivalents, such as H+ and HCO3–, take place. These highly regulated transport processes are mediated by a set of specific ion channels and transport proteins expressed in principal cells and different subtypes of intercalated cells. The transport of particular ions is often linked directly or indirectly to the transport of other ions, as exemplified by the reabsorption of Na+ and the secretion of K+ or the direct coupling of H+ and K+ in H+/K+-ATPase. Another example of mutual transport coupling is provided by the Cl–/anion exchanger pendrin, which is thought to couple the uptake of Cl– from urine to the secretion of bicarbonate into urine at the apical side of non-type A intercalated cells along the connecting tubule and cortical collecting duct (20, 21). Thus, pendrin could provide for bicarbonate secretion as well as for Cl– absorption and thereby contribute to acid-base status and/or systemic Cl– balance. Obviously, both processes need to be regulated, even in opposite directions. Thus, the abundance of pendrin may be influenced by more than one factor, and regulation by different factors may require coordination.

Previous experiments have provided evidence for the regulation of pendrin during NH4Cl loading at the level of protein abundance and subcellular localization (9, 18, 19, 27). These results implied that pendrin is regulated by acid-base status, focusing on its function as a bicarbonate secretory protein. More recently, it was shown that pendrin regulation directly correlates with urinary Cl– absorption, suggesting that pendrin is an important determinant for Cl– conservation and thus blood pressure regulation (19, 30). However, these findings raised the question of whether pendrin is only regulated by urinary Cl– excretion and load or if metabolic acidosis is a second determinant of pendrin abundance.

Our data demonstrate that 1) pendrin expression is reduced during Cl– loading with NH4Cl or NaCl, as previously demonstrated (9, 18, 27, 30); 2) pendrin expression is increased when urinary Cl– excretion is reduced, as in the case of Na2SO4 loading, similar to what has been demonstrated for furosemide treatment and NaCl restriction (19, 30); 3) severe metabolic acidosis with low urinary Cl– excretion, as induced by (NH4)2SO4 loading, is associated with reduced pendrin expression and the relative abundance of pendrin-expressing cells; 4) mild metabolic acidosis with low urinary Cl– excretion, as induced by acetazolamide treatment, prevents the expected stimulation of pendrin expression and even reduced the relative abundance of pendrin-expressing cells; 5) increased urinary acidification and stimulated net anion excretion does not influence pendrin expression, as evident from high-protein diet experiments; and 6) the systemic acid-base status but not urinary pH or acid excretion are associated with the downregulation of pendrin abundance, as demonstrated by the fact that (NH4)2SO4 caused an acidic urinary pH, whereas acetazolamide, alkaline urine, and both prevented the expected increase in pendrin expression. The reduction in pendrin expression during (NH4)2SO4 loading may be most likely caused by the more severe metabolic acidosis compared with the acetazolamide treatment. Interestingly, these results and previous reports have suggested that pendrin abundance in the kidney may be regulated either on the cellular level (as seen in cases where Cl– was primarily manipulated) (19, 24) or on the level of the whole collecting duct system altering the number of pendrin-expressing cells (as in cases where primarily acid-base status was altered). The underlying mechanisms of this potentially differential regulation should be addressed in future studies.

Distal Cl– delivery has been suggested to be a major regulator of pendrin expression (24). In our set of data, no clear correlation between total Cl– excretion, fractional excretion of Cl–, and pendrin expression could be established if the acid-base status was not taken into consideration. However, the actual distal Cl– delivery rates are difficult to estimate in our mouse models since we did not investigate other transport proteins affecting transcellular or paracellular Cl– transport, such as Na+/Cl– cotransport, epithelial Na+ channels, claudin 2, WNK kinases, or ClC-k channels.

Aldosterone, angiotensin II, and vasopressin have been implicated in the regulation of pendrin expression and activity (2, 17, 25). Verlander et al. (25, 26) and Wall et al. (30) have suggested that NaCl restriction and aldosterone increase pendrin expression and translocation into the luminal membrane. In contrast, Adler et al. (1) recently demonstrated that aldosterone decreased endogenous pendrin mRNA abundance in HEK-293 cells. Moreover, aldosterone reduced the pendrin promotor-driven luciferase activity in HEK-293 cells (1). Thus, the role of aldosterone in pendrin regulation in the kidney remains controversial. Even though we did not measure aldosterone levels in our experiments, we hypothesize that aldosterone is not a major regulator of pendrin expression since we imposed various conditions where aldosterone levels were either increased (NH4Cl and acetazolamide) or decreased (NaCl loading) but pendrin abundance was found to be decreased in all conditions. The vasopressin analog desmopressin increased in Brattleboro rat pendrin mRNA, an effect possibly mediated by V2 receptors (2). We did not measure vasopressin levels in our mouse models, but serum osmolarity was increased in two groups, (NH4)2SO4 and acetazolamide, and associated with lower pendrin expression levels. Thus, vasopressin may be an additional and independent regulator of pendrin expression.

Our data are consistent with the recent report from Adler et al. (1), where a pH-dependent regulation of pendrin promoter activity in HEK293 cells was demonstrated. There, extracellular acidification reduced and alkalinization increased promoter activity (1).

Taken together, the data suggest that pendrin expression is primarily correlated to urinary Cl– excretion and may function there in the regulation of transcellular Cl– absorption. However, pendrin abundance may also be regulated by systemic acid-base status despite low urinary Cl– excretion. Metabolic acidosis associated with low urinary Cl– excretion downregulates pendrin expression or at least prevents its stimulation. The sensing mechanism for metabolic acidosis must be located either intracellularly or at the basolateral side of the cells as only metabolic acidosis but not urinary acidification per se influence pendrin expression, as evident from the (NH4)2SO4, acetazolamide, and protein diet experiments. Alternatively, the signal(s) for pendrin expression could be generated in other cells or organs but must involve sensing of urinary Cl– excretion and the systemic acid-base status.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Swiss National Research Foundation Grant 31-068318 (to C. A. Wagner), the 7th Framework Coordinated European Union project of the European Network of the Study of Orphan Nephropathies (to C. A. Wagner), and a travel fellowship from the Faculty of Medicine, University of Naples, Naples, Italy (to R. Grimaldi). G. Capasso was supported by grants from the Italian Ministry of Research (Programmi di Ricerca di Rilevante Interesse Nazionale and Fond für Investitionen in der Grundlagenforschung).


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. A. Wagner, Institute of Physiology and Zurich Center for Integrative Human Physiology, Univ. of Zurich, Winterthurerstrasse 190, Zurich CH-8057, Switzerland (e-mail: Wagnerca{at}access.unizh.ch)

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.

* P. Hafner and R. Grimaldi contributed equally to this work. Back

1 Supplemental material for this article is available online at the American Journal of Physiology-Cell Physiology website. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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