Pendrin: an apical Clminus /OHminus /HCO3minus exchanger in the kidney cortex

Manoocher Soleimani1,2, Tracey Greeley1, Snezana Petrovic1, Zhaohui Wang1, Hassane Amlal1, Peter Kopp3, and Charles E. Burnham1

1 Department of Medicine, University of Cincinnati and 2 Veterans Affairs Medical Center at Cincinnati, Cincinnati Ohio 45267-0585; and 3 Department of Medicine, Northwestern University, Chicago, Illinois 60611


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The identities of the apical Cl-/base exchangers in kidney proximal tubule and cortical collecting duct (CCD) cells remain unknown. Pendrin (PDS), which is expressed at high levels in the thyroid and its mutation causes Pendred's syndrome, is shown to be an anion exchanger. We investigated the renal distribution of PDS and its function. Our results demonstrate that pendrin mRNA expression in the rat kidney is abundant and limited to the cortex. Proximal tubule suspensions isolated from kidney cortex were highly enriched in pendrin mRNA. Immunoblot analysis studies localized pendrin to cortical brush-border membranes. Nephron segment RT-PCR localized pendrin mRNA to proximal tubule and CCD. Expression studies in HEK-293 cells demonstrated that pendrin functions in the Cl-/OH-, Cl-/HCO3-, and Cl-/formate exchange modes. The conclusion is that pendrin is an apical Cl-/base exchanger in the kidney proximal tubule and CCD and mediates Cl-/OH-, Cl-/HCO3-, and Cl-/formate exchange.

proximal tubule; cortical collecting duct; anion exchanger; apical membrane


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

THE KIDNEY IS THE MAJOR ORGAN responsible for maintaining electrolyte balance and acid-base homeostasis in mammals. This is accomplished predominantly by absorption of NaCl and secretion of acid or base equivalents in different segments of the nephron (2). The proximal tubule is responsible for the bulk of Na+, Cl-, and HCO3- reabsorption via an apical Na+/H+ exchanger and a Cl-/base exchanger acting in parallel (2, 4, 5, 37). Several functional studies have indicated that the apical Cl-/base exchanger in kidney proximal tubule likely functions in Cl-/formate exchange mode, with formate converting to formic acid in the lumen and diffusing back into the cell (2, 5). In beta -intercalated cells of the cortical collecting duct (CCD), an apical Cl-/base exchanger is predominantly responsible for Cl- reabsorption and HCO3- secretion and as a result is involved in acid-base and electrolyte homeostasis (2, 27, 35). Whereas functional studies have characterized these exchangers, their molecular identities remain unknown. None of the known AE exchangers (AE-1, -2 or -3) is found in the apical membrane of kidney nephron segments.

Recent studies have identified a new class of anion exchangers, including those downregulated in adenoma (DRA) and pendrin (PDS) (14, 17, 23, 30, 33). Neither PDS nor DRA are structurally related to the AE (AE-1, -2, and -3) family. Indeed, the homology at the amino acid level between DRA or PDS and AE family members is <15%. (The GenBank accession numbers NP000333, NP003031, and NP005061 were used for AE-1, AE-2, and AE-3, respectively.) PDS is expressed at extremely high levels in the thyroid, where it is thought to be involved in iodine transport across apical membranes of thyroid follicular epithelial cells (14, 26, 30). PDS is also expressed in the inner ear (15), but its function in this organ is unknown. Mutations in PDS cause Pendred's syndrome, an autosomal recessive hereditary disorder characterized by prelingual deafness, goiter, and impaired iodine organification as evidenced by a positive perchlorate test (12-15, 20, 21, 24).

Functional studies in Xenopus oocytes indicate that PDS transports Cl- (9) and can function in Cl-/formate exchange mode (30). Pendrin mRNA is found in the whole kidney RNA preparation (14). However, its renal distribution, membrane localization, and physiological function have not been characterized.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Proximal Tubular Suspension and Brush-Border and Basolateral Membrane Preparation

Proximal tubular suspensions were prepared with Percoll gradient centrifugation as described (36). Brush-border membrane (BBM) vesicles were isolated from rat kidney cortex by a Ca2+ aggregation method (34, 36). Basolateral membrane (BLM) vesicles were isolated by differential and Percoll gradient centrifugation (34, 36). The purification of BBM vesicles relative to the initial homogenate, as determined by alkaline phosphatase assay, was 8.2 ± 1.1-fold. The purification of BLM vesicles relative to the initial homogenate, as determined by Na+-K+-ATPase assay, was 9.2 ± 1.3-fold.

RNA Isolation and Northern Blot Hybridization

Total cellular RNA was extracted from kidney cortex, outer medulla, inner medulla, and proximal tubule suspensions using TriReagent (10), quantitated spectrophotometrically, and stored at -80°C. Hybridization was performed according to Church and Gilbert (11). The membranes were washed, blotted dry, and exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA). A 32P-labeled 488-bp PCR fragment (see section below) from the rat kidney PDS cDNA was used for Northern experiments.

RT-PCR of Rat Pendrin

Total RNA was prepared from rat renal cortex, poly(A)+-, selected using Oligotex latex beads (Qiagen) and then reverse transcribed at 47°C using SuperScript II RT (Life Technologies) and oligo(dT) primers. Oligonucleotide primers (5'-CAT TCT GGG GCT GGA CCT C, and 5'-CCT TCG GGA CAT TCA CTT TCA C) were designed based on rat pendrin cDNA (GenBank accession number AF167412). After PCR, the product was gel purified (revealing a single band of 488 bp) and used as a probe for Northern blot hybridizations. Sequence analysis of the PCR product verified the sequence as rat pendrin.

Nephron Segment RT-PCR

Single nephron segments (proximal tubule or CCD) were dissected from freshly killed rat kidney at 4-6°C. The dissection medium comprised of (in mM) 140 NaCl, 2.5 K2HPO4, 2 CaCl2, 1.2 MgSO4, 5.5 D-glucose, 1 Na-citrate, 4 Na-lactate, and 6 L-alanine, pH 7.4, and bubbled with 100% O2. Tubule length for proximal straight tubule (S2 segment) was ~0.7 mm and for CCD it was ~0.5 mm. The nephron segments were pooled in a small volume (5-10 µl) of ice-cold phosphate-buffered saline (PBS), three or four segments per pool. The tubules were centrifuged at 12,000 g for 1 min at room temperature, and the PBS was removed and replaced with 10 µl of a tubule lysis solution consisting of 0.9% Triton X-100, 5 mM dithiothreitol (DTT), and 1 U/µl rRNasin (Promega). After 5 min on ice the tubules were agitated gently by tapping the tube, and 1 µl (0.5 µg) oligo(dT) primer, 1 µl H2O, 4 µl 5× reverse transcription buffer, 2 µl DTT (0.1 M), and 1 µl dNTP (10 mM each) were added. The reaction was equilibrated to 42°C for 2 min, and 1 µl SuperScript II RT (Life Technologies) was added, mixed, and incubated for 1 h at 42°C. After reverse transcription, 30 µl of TE (10 mM Tris/Cl-, 1 mM EDTA, pH 8.0) were added, and the combined mixture was heated to 95°C for 5 min and placed on ice.

Amplification of the pendrin cDNA by the PCR was performed using parameters previously established using renal cortex RNA (see RT-PCR of Rat Pendrin). Briefly, each PCR contained 10 µl cDNA, 5 µl 10× PCR buffer (with 20 mM MgCl2), 1 µl 10 mM dNTP, 10 pmol each primer, and 2.5 units Taq DNA polymerase in a final volume of 50 µl. Cycling parameters were 95°C, 45 s; 47°C, 45 s; and 72°C, 2 min.

Antibody Generation and Immunoblot Analysis

Two peptides corresponding to amino acids 734 to 752 (KSREGQDSLLETVARIRDC) and amino acids 36 to 54 (RERRLPERRTRLDSLARSC) of mouse PDS (GenBank accession number AF136751) were synthesized and used for antibody generation. The preimmune and immune sera of the third bleed were purified by IgG purification kit (Sigma) and used for immunoblot analysis. BBM and BLM from rat kidney cortex and microsomal membranes from tranfected HEK-293 cells were resolved by SDS-PAGE (5 µg/lane) and transferred to nitrocellulose membrane. The membrane was blocked with 5% milk proteins and then incubated for 6 h with 40 µl of PDS-immune serum diluted at 1:1,000. The secondary antibody was a donkey anti-rabbit IgG conjugated to horseradish peroxidase (Pierce). The site of antigen-antibody complexation on the nitrocellulose membranes was visualized using chemiluminescence method (SuperSignal Substrate, Pierce) and captured on light-sensitive imaging film (Kodak).

Cloning of Pendrin cDNA

The human wild-type PDS cDNA was generated by RT-PCR using total RNA from normal human thyroid tissue and Pfu polymerase (Stratagene, LaJolla, CA). Primer sequences were based on the sequence published by Everett et al. (14). The sense primer was 5'-CGCGAGCAGAGACAGGTCATGGCAGCG-3' and the antisense primer was 5'-TCTGGATCCCGGATGCAAGTGTACGCATAGCCTC-3'. The primers contained appropriate linkers for subcloning the PDS cDNA into the XhoI and BamH I sites of pSVL (Amersham Pharmacia, Piscataway, NJ).

Stable Transfection With the Cloned Pendrin cDNA

For stable transfection in HEK-293 cells, the PDS cDNA was released from the pSVL vector by digestion with Xho1 and BamH I, blunt ended, and subcloned into a pCIneo vector. HEK-293 cells, grown in 60-mm dishes, were transfected with 8 µg of the full length PDS cDNA construct according to established methods (9). Transfected cells were selected by the resistance to G418. The cells were grown in DMEM supplemented with 10% fetal calf serum, 50 U/ml penicillin, and 200 µg/ml of G418. The cells were maintained at 37°C in a 5% CO2:95% O2 air incubator. Six clones of putative transfectants were analyzed by Northern blot using full length PDS cDNA as probe. For experiments, two clones were extensively analyzed.

Cell pH Measurement

Changes in intracellular pH (pHi) were monitored using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) (3, 8). HEK 293 cells were grown to confluence on a glass coverslip and incubated in the presence of 5 µM BCECF in a Cl--containing, HCO3--free solution consisting of (in mM) 115 NaCl, 25 Na-gluconate, 4 KCl, 1 K-gluconate, and 10 HEPES, at pH 7.4, and gassed with 100% O2. The monolayer was then perfused with the appropriate solutions at 37°C in a Delta Scan dual excitation spectrofluorometer (PTI, South Brunswick, NJ). For the Cl--free solution, all Cl--containing salts were replaced with gluconate salts. For formate-containing solutions, 1 mM K-gluconate was replaced with 1 mM K-formate. For HCO3--containing solution, 25 mM Na-gluconate was replaced isosmolarly with NaHCO3- and gassed with 5% CO2:95% O2. All solutions contained (in mM) 0.8 K2HPO4, 0.2 KH2PO4, 1 CaCl2, and 1 MgCl2. For Cl--free solutions, CaCl2 and MgCl2 were replaced with the gluconate salts.

To determine pHi values, the fluorescence ratio at excitation wavelengths of 500 and 450 nm was used. Calibration curves were established by KCl/nigericin. HCO3--free or HCO3--containing solutions were used to determine the HCO3- dependence of the transporter. To examine the Cl-/OH- exchanger activity, cells were switched to a Cl--free medium. This maneuver causes cell alkalinization via reversal of the Cl-/OH- exchanger. On pHi stabilization in Cl--free medium, cells were switched back to Cl--containing solution, resulting in recovery of pHi to baseline levels due via Cl-/OH- exchange. To examine Cl-/HCO3- exchange activity, the above experiments were repeated in HCO3--containing solutions. The rate of cell acidification immediately on switching to the Cl--containing solution was used as the initial rate of Cl-/OH- or Cl-/HCO3- exchanger activity. To examine the Cl-/formate exchange activity, experiments were performed in the presence of 1 mM potassium formate.

Buffering Power

The intrinsic buffering power (beta i; mMH+/pH unit) was measured in cultured cells (transfected and nontransfected) using the NH4+ pulse method according to the formula beta i = Delta [NH4+]i/Delta pHi, and as described by Roos and Boron (25). Cells were initially incubated in Na+- and HCO3--free solution and monitored for pHi recording. The Na+- and HCO3--free solution was similar to the HCO3--free solution that was used for the pH studies except that the NaCl was replaced with isosmolar concentration of TMACl. At steady-state pHi, addition of 20 mM NH4Cl (20 mM TMACl was replaced with 12 mM NH4Cl, where TMAC1 is tetramethylammonium chloride) caused a rapid initial increase in cell pH due to the influx of NH3 and subsequent generation of NH4+. This alkalinization was followed by a plateau (no acidification was observed in the presence or absence of HCO3-). The buffering capacity in the presence of HCO3- (total buffering power) was determined in both transfected and nontransfected cells according to established formula (25).

Materials

[32P]dCTP was purchased from New England Nuclear (Boston, MA). Nitrocellulose filters and other chemicals were purchased from Sigma Chemical (St. Louis, MO). RadPrime DNA labeling kit was purchased from GIBCO-BRL. BCECF was from Molecular Probes (Eugene, OR).

Statistical Analyses

Values are expressed as means ± SE. The significance of difference between mean values were examined using ANOVA. P < 0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Pendrin mRNA Expression in the Kidney

To examine the distribution of pendrin mRNA in rat kidney, Northern blots were prepared using total RNA from whole kidney, cortex, outer medulla, and inner medulla, and then probed with rat pendrin cDNA probe. The results were as shown in Fig. 1A. Pendrin mRNA is expressed exclusively in the cortex under normal conditions. Within the cortex both proximal and distal tubules are present. Figure 1B shows a Northern blot prepared from RNA isolated from proximal tubule suspensions and probed with pendrin cDNA. As indicated, pendrin mRNA is abundantly expressed in proximal tubules.


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Fig. 1.   A: Northern hybridization of pendrin in rat kidney. Representative Northern blots demonstrating pendrin (PDS) transcript levels in whole kidney, cortex, outer medulla, and inner medulla. PDS transcript size was ~4.8 kb. rRNA levels (28S) are shown as constitutive controls. RNA (30 µg) were loaded on each lane. B: Northern hybridization of pendrin in rat kidney cortex and proximal tubule (PT). Representative Northern blots demonstrating PDS transcript levels in cortex and PT. The expression of 28S rRNA is shown as a constitutive control. RNA (30 µg) were loaded on each lane. C: nephron segment RT-PCR using primers specific for pendrin. Two representative ethidium bromide stainings of agarose gel (left and right panels) demonstrate a PCR product of expected size (488 bp) for rat pendrin (PDS = pds) in PT and cortical collecting duct (CCD). The right lane (left and right panels) is the 488-bp pds cDNA fragment, which was amplified from rat kidney cortex using the same primers that were used for nephron segment RT-PCR (see EXPERIMENTAL PROCEDURES). Left panel: 2 left lanes show CCD and PT, respectively. The right panel demonstrates both the negative (no RT) and positive (with RT) reactions for PT and CCD.

In the next series of experiments we investigated the expression of pendrin in two distinct cortical nephron segments (proximal tubule and CCD) using nephron segment RT-PCR (see EXPERIMENTAL PROCEDURES). Figure 1C shows two representative ethidium bromide gel pictures from nephron segment RT-PCR experiments and demonstrates that in addition to proximal tubule, PDS mRNA is also expressed in CCD.

Immunoblotting of Pendrin in the Kidney

In this series of experiments the membrane localization of pendrin was examined. We first determined the specificity of pendrin immune serum in HEK-293 cells stably transfected with PDS cDNA. As shown in Fig. 2A, the pendrin immune serum labeled an ~100-kDa band in transfected HEK-293 cells. This reaction was specific because it was prevented by immune preadsorption (Fig. 2A). The PDS mRNA and protein were not detected in nontransfected cells (data not shown). To determine the expression of pendrin in the kidney cortex and examine its membrane domain localization, proteins from cortical microsomes, BBM and BLM, were resolved by SDS-PAGE and probed with the immune serum. As indicated in Fig. 2B, a ~100-kDa protein was strongly labeled in the cortical microsomes, consistent with the expression of pendrin in the cortex. Pendrin localized to the BBM but not the BLM domain (Fig. 2B). This reaction was specific because it was prevented by immune preadsorption (Fig. 2B). These results are consistent with the localization of pendrin in cortical BBM. Repeated attempts in our laboratory in performing immunocytochemical studies with PDS-specific polyclonal antibodies have not been successful. As such, we could not examine the protein expression of PDS in CCDs.


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Fig. 2.   Immunoblot analysis of pendrin. A: HEK cells transfected with pendrin cDNA. Microsomal membrane proteins (5 µg/lane) were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and blotted against PDS-immune serum. Left panel: PDS immunoblot analysis; right panel: preadsorbed immune serum. A ~100-kDa protein was detected with the immune serum. B: membrane vesicles from kidney cortex. Cortical microsomes and brush-border membrane (BBM) and basolateral membrane (BLM) proteins (5 µg/lane) were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and blotted against pendrin immune serum. Left panel: PDS immunoblot analysis against BBM, BLM, and cortical microsomes; right panel: preadsorbed immune serum. A ~100-kDa protein was localized to the BBM.

Functional Identity of Pendrin

In these series of experiments the functional identity of pendrin was examined in HEK-293 cells stably transfected with PDS cDNA.

Cl-/OH- exchange. In the first series of functional studies we examined whether pendrin can operate in Cl-/OH- exchange mode. Cells were loaded with BCECF in the presence of a NaCl-containing, HCO3--free solution (EXPERIMENTAL PROCEDURES) and monitored for pHi. The representative pHi tracings in Fig. 3 demonstrate that switching to a Cl--free solution resulted in a rapid intracellular alkalinization in cells expressing pendrin. Switching back to the Cl--containing solution caused a rapid pHi return to normal. Nontransfected HEK-293 cells did not demonstrate any pHi alteration in response to exposure to the Cl--free medium (Fig. 3A). DIDS, at 0.5 mM, almost abolished the pHi alteration in transfected cells (Fig. 3A). These results are consistent with pendrin functioning as a Cl-/OH-(hydroxyl) exchanger. The averaged results of multiple experiments (Fig. 3B) indicated that the rate of Cl-/OH- exchange activity was 0.16 ± 0.02 in transfected cells (n = 11) and 0.01 ± 0.002 in nontransfected cells (n = 7, P < 0.001). The Cl-/OH- exchanger activity was inhibited by ~88% in the presence of 0.5 mM DIDS (n = 5), with the exchanger activity decreasing to 0.02 ± 0.002 (P < 0.001 vs. no DIDS).


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Fig. 3.   Functional identity of pendrin. A: Cl-/OH- exchange activity (representative tracings). Representative tracings demonstrating Cl-/OH- exchanger activity in HEK-293 cells stably transfected with pendrin cDNA. HEK-293 cells grown on glass coverslips were loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and perfused with solutions corresponding to the figure labels (see EXPERIMENTAL PROCEDURES for details). Solutions were gassed with 100% O2. B: Cl-/OH- exchange activity (summary of the results). Cells transfected with pendrin cDNA show Cl-/OH- exchange activity. C: Cl-/HCO3- exchange activity (summary of the results). Cl-/base exchanger activity was assayed by subsequent removal and addition of Cl-. Buffering capacity (beta i) was calculated in normal or transfected cells in the absence of HCO3- and according to EXPERIMENTAL PROCEDURES. The beta i was 47.1 ± 1.7 mM H+/pH unit (at pHi = 7.31 ± 0.026) in nontransfected cells and 44.4 ± 1.9 (at pH 7.29 ± 0.028) in transfected cells. The total buffering power (beta i in the presence of HCO3-) was calculated according to established formula (EXPERIMENTAL PROCEDURES). The transport of Cl--dependent, base equivalent in the presence of HCO3- was determined by multiplying the total buffering power by the transport rate (dpHi/dt) and expressed as millimole base equivalent/min. The rate of pendrin-mediated base equivalent transport rate was then calculated by subtracting the base equivalent transport rate in nontransfected cells (endogenous Cl-/HCO3- activity) from transfected cells. As shown, base equivalent transport rate by pendrin was increased in the presence of HCO3- vs. HCO3--free media.

Cl-/HCO3- exchange. To determine whether pendrin can also function in Cl-/HCO3- exchange mode, the experiments were repeated in the presence of 25 mM HCO3-. The results of experiments demonstrated that in the presence of HCO3-, the rate of Cl-/base exchange activity was 0.22 ± 0.02 and 0.05 ± 0.01 pH/min in transfected and nontransfected cells, respectively (n = 6 for each group, P < 0.01). When adjusted for the minimal endogenous Cl-/HCO3- exchange activity in nontransfected cells (which was around 0.05 pH/min), it appears that the rate of Cl-/base exchange activity (expressed as dpHi/dt, pH/min) in PDS-expressing cells is not affected by the presence of HCO3-. However, after adjusting for the total buffering power in cultured cells (see EXPERIMENTAL PROCEDURES and legend to Fig. 3C), it becomes evident that in the presence of HCO3-, base equivalent transport rate is increased in cells transfected with pendrin (Fig. 3C). Taken together, these results indicate that pendrin can also function in Cl-/HCO3- exchange mode.

Cl-/formate exchange. We lastly examined whether pendrin can function in Cl-/formate exchange mode, as has been demonstrated in oocytes (29). The experiments were performed in the presence of formate and the absence of a formate gradient (all solutions had 1 mM formate). The purpose of having no formate gradient was to prevent the diffusion of formate (in the form of formic acid) across the plasma membrane, which in turn can alter the cell pH. Figure 4 demonstrates representative tracings in the presence of formate in control or transfected cells. In the presence of 1 mM formate, switching to a Cl--free solution resulted in a significant intracellular alkalinization only in transfected cells. The pHi returned to baseline on switching back to the Cl--containing solution. Nontransfected cells showed minimal pHi alterations in response to switching to the Cl--free solution (or back to the normal solution) (Fig. 4A). The results further indicated that the intracellular alkalinization was significantly increased in the presence of formate (vs. Fig. 3). The averaged results of multiple experiments (Fig. 4B) indicated that Cl-/base exchange activity was 0.45 ± 0.04 in transfected cells (n = 10) and 0.03 ± 0.003 pH/min in nontransfected cells (n = 7, P < 0.001). Comparison of the results indicates that the rate of Cl-/base exchange activity is approximately three times faster in the presence of formate than in its absence in PDS transfected cells (dpHi/dt was 0.16 ± 0.02 in the absence of formate and 0.45 ± 0.04 in the presence of formate, P < 0.01). The formate-mediated Cl-/base exchanger activity in transfected cells decreased to 0.13 ± 0.02 (n = 4) in the presence of 0.5 mM DIDS (P < 0.01 vs. no DIDS).


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Fig. 4.   Functional identity of pendrin. A: Cl-/formate exchange activity (representative tracings). Representative tracings demonstrating the effect of Cl- removal and re-addition on pHi in the presence of 1 mM formate in HEK-293 cells stably transfected with pendrin cDNA. HEK-293 cells (transfected or nontransfected) were grown on glass coverslips, loaded with BCECF, and perfused with solutions corresponding to the figure labels (see EXPERIMENTAL PROCEDURES for details). Solutions were gassed with 100% O2. B: Cl-/formate exchange activity (summary of the results). Cells transfected with pendrin cDNA show Cl-/formate exchange activity. When adjusted for the buffering power in transfected cells in the absence or presence of 1 mM formate (45.1 ± 1.5 and 43.4 ± 1.8 mM H ± pH unit, respectively) the base transport rate mediated via pendrin was calculated to be 7.2 ± 0.65 mM/min in the absence of formate and 19.35 ± 1.68 mM/min in the presence of 1 mM formate (P<= 0.001). These results indicate that formate increases the base equivalent transport rate via pendrin.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The results of our experiments indicate that pendrin is expressed in the kidney cortex and is absent in the medulla (Fig. 1A). Proximal tubules isolated from kidney cortex demonstrated high levels of pendrin mRNA expression (Fig. 1B). Nephron segment RT-PCR localized this exchanger to the proximal tubule and CCD (Fig. 1C). Immunoblotting studies in membrane proteins isolated from kidney cortex localized pendrin to the BBM domain (Fig. 2). Expression studies in cultured HEK-293 cells demonstrated that pendrin functions in Cl-/OH-/HCO3- and Cl-/formate exchange modes (Figs. 3 and 4).

Mutations in pendrin have been shown to be the cause of Pendred's syndrome, an autosomal recessive hereditary disorder characterized by goiter, positive perchlorate test, and deafness (16). Using linkage analysis and positional cloning approaches, the gene mutated in Pendred's syndrome (PDS) was identified (12-14, 20, 21). Pendrin encodes an mRNA of ~5 kb in humans and is abundantly expressed in the thyroid (14). Lower levels of pendrin mRNA are expressed in the kidney (14). cDNA analysis indicates that pendrin is closely related to a family of anion transport proteins that include the rat sulfate-anion transporter (Sat-1), the human diastrophic dysplasia sulfate transporter (DTD), and the downregulated in adenoma gene (DRA) (7, 16, 17, 23, 28, 33). Sequence comparison revealed that pendrin has 29, 32, and 45% homology with Sat-1, DTD, and DRA, respectively, at the amino acid level (7, 14, 16, 28). Pendrin encodes a protein of ~95- to 100-kDa size, is localized on the apical domain of thyroid follicular cells (26), and is thought to be responsible for the transport of iodide (14, 30).

Our studies indicate that the mRNA expression of pendrin is abundantly present in the kidney proximal tubule (and CCD) but is absent in the medulla. This does not conflict with the studies in human kidney, where mRNA levels of pendrin were found to be low (14). Those studies utilized RNA isolated from the whole kidney, rather than the cortex (14). Immunoblot analysis localized pendrin to the apical membrane of kidney cortex, and functional studies demonstrated that it can exchange Cl- for OH- (HCO3-) or formate. Taken together, these results indicate that pendrin is an apical Cl-/base exchanger in the kidney cortex.

Studies in BBM vesicles isolated from rabbit kidney cortex indicate that the apical Cl-/base exchanger in the kidney proximal tubule can function in Cl-/formate exchange mode (5, 18, 19). In perfused rabbit proximal tubule, the rate of fluid reabsorption increased significantly in the presence of formate (5). This process was inhibited in the presence of DIDS, indicating that enhanced fluid reabsorption by formate is mediated via Cl-/formate exchanger. Taken together, these results indicate that Cl-/formate exchanger is responsible for the bulk of Cl- reabsorption in proximal tubule. It has been proposed that the formate, which is secreted into the lumen via Cl-/formate exchange, is recycled back to the cell, thereby providing substrate for the transporter (5).

In addition to the proximal tubule, beta -intercalated cells of CCD express a Cl-/base exchange on their apical membrane (2, 27, 38). The identity of this exchanger has remained unknown. The expression of PDS mRNA in CCD raises an intriguing possibility that this exchanger might be expressed in beta -intercalated cells. We have not examined the subcellular localization (apical vs. basolateral) of pendrin in CCD in detail. However, based on the apical localization of pendrin in the proximal tubule and our preliminary immunohistochemical studies in CCD,1 we suggest that this exchanger is located apically in beta -intercalated cells of CCD.

An intriguing aspect of the current studies is the functioning of pendrin in Cl-/OH- exchange mode. Several investigators have examined the presence of Cl-/OH- exchange in BBM vesicles or intact proximal tubule. Studies in BBM vesicles have been conflicting; whereas one study found the presence of Cl-/OH- exchange, the other did not (31, 41). In perfused intact proximal tubule, however, two reports have identified the presence of apical Cl-/OH- exchange (22, 32). Our studies are in full agreement with these latter two studies and confirm that the apical Cl-/base exchanger in kidney proximal tubule can function in Cl-/OH- exchange mode.

The current studies indicate that pendrin functions as Cl-/OH-, Cl-/HCO3-, and Cl-/formate exchanger, suggesting that the apical Cl-/base exchanger in kidney cortex can function in several distinct modes. This is in agreement with studies in perfused rat kidney proximal tubule, where the presence of Cl-/formate and Cl-/OH-/HCO3- exchangers was identified in apical membrane domain (1). Those experiments were performed in the presence of HCO3- only and therefore could not discriminate between Cl-/OH- and Cl-/HCO3- exchange. Interestingly and similar to our current experiments, the rate of cell alkalinization in response to luminal Cl- removal (an index of Cl-/base exchange activity) was approximately threefold higher in the presence of formate than in its absence (1), suggesting that formate is a better substrate for the exchanger.

Studies have shown that formate increases Cl- reabsorption in distal tubule, presumably via an apical Cl-/base exchanger (5, 39). However, whether formate can be transported via the CCD apical Cl-/base exchanger remains speculative. Based on the presence of pendrin mRNA and protein in CCD and based on the observation that pendrin can function in Cl-/formate exchange mode (Fig. 4 and Ref. 30), we suggest that the apical Cl-/base exchanger in beta -intercalated cells might also function in Cl-/formate exchange mode. It is worth mentioning that the apical Cl-/base exchanger in beta -intercalated cells functions predominantly in Cl-/HCO3- exchange mode (and not Cl-/OH- exchange mode) (2, 27, 38). This raises the possibility that the affinity of pendrin for OH- vs. HCO3- is tissue specific.

Studies in our laboratories had demonstrated that liposomes reconstituted with BBM proteins displayed Cl-/formate and Cl-/HCO3-/OH- exchange activities (35). A 162-kDa protein was identified as a likely candidate to mediate both processes (35). A partial amino acid sequence analysis shows that the 162-kDa protein is a novel protein and shows no significant homology to any known protein (data not shown). We have not obtained the full length cDNA of the 162-kDa protein; however, the current results raise the possibility that Cl-/formate exchange may be mediated by more than one transporter. Additional studies are needed to address this issue.

It is not clear whether patients with Pendred's syndrome have enhanced renal Cl- excretion, as urinary electrolyte profiles have not been studied in these patients. Such a conclusion would be plausible based on the Cl--absorbing ability and the location of pendrin. However, downstream Cl- absorbing transporters (the apical Na-K-2Cl cotransporter in the thick ascending limb and the apical Na-Cl cotransporter in distal convoluted tubules) may be upregulated in response to increased delivery of Cl- from proximal tubule to more distal nephron segments. Indeed, in animals with metabolic acidosis, which demonstrate downregulation of proximal tubule apical Cl-/base (formate) exchanger (40), the apical Na-K-2Cl cotransporter is upregulated (6). Such a compensatory defense mechanism would increase the reabsorption of Cl- and attenuate the loss of Cl- into the urine. With respect to the role of pendrin as the apical Cl-/HCO3- exchanger in beta -intercalated cells, it is likely that patients with Pendred's syndrome might show an impairment in urine alkalinization. To unmask such a defect, however, patients need to be subjected to HCO3- loading or metabolic alkalosis.

In conclusion, pendrin is expressed in the kidney, with mRNA expression limited to the proximal tubule and CCD. Immunoblotting localized pendrin to the apical domain of cortical membranes. Functional studies demonstrated that pendrin functions in Cl-/OH-, Cl-/HCO3-, and Cl-/formate exchange modes. We propose that pendrin is an apical Cl-/base exchanger in the kidney proximal tubule and beta -intercalated cells of CCD.


    ACKNOWLEDGEMENTS

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46789, DK-52281, and DK-54430, a Merit Review grant, a Cystic Fibrosis Foundation grant and grants from Dialysis Clinic to M. Soleimani and a grant from Northwestern Memorial Hospital to P. Kopp.


    FOOTNOTES

Address for reprint requests and other correspondence: M. Soleimani, Div. of Nephrology and Hypertension, Dept. of Medicine, Univ. of Cincinnati, 231 Bethesda Ave., MSB 5502, Cincinnati, OH 45267-0585 (E-mail: manoocher.soleimani{at}uc.edu).

1 Preliminary immunocytochemical studies in our laboratory indicate specific apical staining in a subpopulation of CCD cells, consistent with the expression of pendrin in beta -intercalated cells.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 22 July 2000; accepted in final form 23 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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