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J. Biol. Chem., Vol. 280, Issue 20, 19902-19910, May 20, 2005

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Thiazide-sensitive NaCl-cotransporter in the Intestine

POSSIBLE ROLE OF HYDROCHLOROTHIAZIDE IN THE INTESTINAL Ca²⁺ UPTAKE^*

Claudia Bazzini, Valeria Vezzoli, Chiara Sironi, Silvia Dossena, Andrea Ravasio, Silvia De Biasi, MariaLisa Garavaglia, Simona Rodighiero¶, Giuliano Meyer, Umberto Fascio¶, Johannes Fürst, Markus Ritter||, Guido Bottà**, and Markus Paulmichl¶

From the Department of Biomolecular Sciences and Biotechnology, Università degli Studi di Milano, Via Celoria 26, I-20133 Milan, Italy, the Department of Physiology and Medical Physics, Innsbruck Medical University, Fritz-Pregl Strasse 3, A-6020 Innsbruck, Austria, the ^¶Centro Interdipartimentale di Microscopia Aranzata and Centro Interdisciplinare Materiali e Interfacce Nanostruturali, Via Celoria 26, I-20133 Milan, Italy, and the ^||Paracelsus Private Medical University, Strubergasse 21, A-5020 Salzburg, Austria

Received for publication, October 21, 2004 , and in revised form, March 21, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Thiazides, such as hydrochlorothiazide (HCTZ), are used to control blood pressure and to reduce renal calcium excretion. These effects are a result of interactions with the NaCl-cotransporter (NCC). This is demonstrated by the fact that mutations within the NCC protein lead to salt-resistant hypotension and hypocalciuria, paralleled by an increase in bone mineral density. These symptoms are also known as Gitelman syndrome. It has become increasingly evident that the effect of HCTZ on blood pressure and calcium homeostasis cannot be attributed exclusively to kidney functions, where the primary action of HCTZ on NCC is postulated to occur. We demonstrated the presence of the NCC transporter in the rat small intestine (ileum and jejunum) and human HT-29 cells, by using reverse transcription-PCR, Northern blot, Western blot, and immunofluorescence. Furthermore, we show that HCTZ modulates Ca²⁺ uptake by intestinal cells, while affecting the electrical parameters of the cellular membrane, thus suggesting a functional interaction between NCC and the epithelial voltage-dependent calcium channel. The experiments presented here support the hypothesis of a direct involvement of the intestinal cells in the interaction between HCTZ and NaCl, as well as calcium homeostasis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Thiazide diuretics are used in the treatment of most patients with hypertension, as suggested by the seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC-7) (1). The unique role of thiazide diuretics in the management of high blood pressure is a result of their dual effect on the NaCl, as well as the Ca²⁺ transport. In the distal convolute tubule of the kidney they interact with the apical NaCl-cotransporter (NCC¹), which in turn is responsible for the majority of salt transport, and whose inhibition causes an increase in renal sodium excretion. In addition to reducing NaCl reabsorption, thiazide diuretics are also responsible for decreasing renal Ca²⁺ excretion. The involvement of the NCC in Ca²⁺ homeostasis has been demonstrated by the fact that mutations in the NCC gene are associated with Gitelman syndrome. This disease complex is characterized by hypernatriuria and hypocalciuria, paralleled by an increase in bone mineral density (2–5). All these symptoms can also be individuated in NCC-knock-out mice (6). The mechanism by which the apical NCC cotransporter in the kidney is functionally interconnected with the co-localized Ca²⁺ transport systems has not yet been understood in detail. However, it was shown that the inhibition of NCC by thiazide diuretics, such as hydrochlorothiazide (HCTZ) leads to hyperpolarization of the apical plasma membrane. This would facilitate luminal Ca²⁺ entry (7, 8), possibly through epithelial Ca²⁺ channels (ECaC). It is generally accepted that the kidney is the cardinal target of thiazides. However, it was postulated that the distinct action of thiazide diuretics in the management of high blood pressure and calcium homeostasis cannot be attributed exclusively to this organ, but that in addition an effect of these drugs on the intestinum appears to be relevant (5, 9). The thiazide-sensitive NaCl-cotransporter was characterized as a renal-specific gene (10, 11). However, information regarding the extra-renal expression of NCC has lately become increasingly available. These studies were all conducted at the level of mRNA transcription by using RT-PCR (12) and/or Northern-Blots (13), and more recently by using cDNA microarrays (Gene Expression Omnibus Data Bank, www.ncbi.nlm.nih.gov/geo/). In extra-renal cell types, NCC has only been investigated thus far at a functional and molecular level in osteoblasts (14), peripheral blood mononucleated cells (15) and, partially, in the pancreas (16). For the gastrointestinal tract only indirect evidence indicating the presence of NCC (5, 12, 17, 18) has been obtained. The goal of the present study was to investigate whether NCC and ECaC interact on a functional level in the intestine. Here we show that (i) the thiazide-sensitive NaCl-cotransporter NCC and ECaC are indeed present in the intestine (rat small intestine and human HT-29 cell line), and (ii) that HCTZ modulates Ca²⁺ entry into the intestinal cells, by affecting the electrical parameters of the cellular membrane. Therefore, the experiments presented here underline the hypothesis of a direct involvement of the intestinal cells in the effect of HCTZ on blood pressure and calcium homeostasis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Animal Care—Animal testing was performed according to the Italian Health Ministry animal care guidelines.

Drugs and Chemicals—RPMI 1640 medium, fetal bovine serum, Trypsin-EDTA solution, glutamine, HEPES, EGTA, and all standard salts were purchased from Sigma.

HT-29 Cell Culture—Cell cultures were performed according to standard methods. The medium consisted of RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 mM HEPES buffer, 2 mM Na-pyruvate, 100 units/ml penicillin, 100 µg/ml streptomycin, and amphotericin 0.25 µg/ml. Cultures remained in a humidified atmosphere of 5% CO₂ at 37 °C. After cells had become confluent, a 10-min treatment with a trypsin-EDTA mixture (final concentration 0.5–0.2 g/liter) in phosphate-buffered saline (PBS) was used to detach the cells, which were subsequently reseeded at high density.

RT-PCR—Male albino rats (Wistar strain, Italian Charles River), weighing 200–300 g, were used for the experiments. Under 2,2,2-tribromoethanol anesthesia, the upper jejunum or ileum was removed, and enterocytes were collected by scraping off the mucosal layer from the excised small intestine. Rats were subsequently killed by exsanguination under anesthesia. Total RNA was isolated with the Qiagen RNeasy kit, as specified by the manufacturer. RNA (2 µg) was reverse transcribed by using Superscript II RT (Invitrogen). When poly(A)mRNA was used, it was purified from total RNA with the Qiagen mRNA easy kit. In this context 0.5 µg of poly(A)mRNA were used to induce the reverse-transcription reaction. The PCR reactions were performed using Taq polymerase (Bioline). Sequences of PCR primers and annealing temperatures are outlined below in Table I. The obtained amplification products were cloned into a pSTBlue1 vector to be sequenced subsequently (MWG, Germany).

Northern Blot Analysis—4 µg of kidney total RNA (obtained as described above) and 8 µg of small intestine poly(A)mRNA (purified from total RNA with the Qiagen Oligotex mRNA kit) were separated by 0.8% agarose-formaldehyde denaturizing electrophoresis, and transferred overnight by capillarity with the TurboBlotter system (Schleicher and Schuell) to a Hybond-N nylon membrane (Amersham Biosciences). Following transfer, the membrane was UV-cross-linked. Pre-hybridization and hybridization steps were performed as described (14). The membrane was kept at –80 °C up to 1 week for the final autoradiography procedure.

Western Blot—The small intestine and kidney cortex were homogenized in buffer A (0.25 M sucrose, 30 mM histidine, 1 mM EDTA, 300 µM phenylmethylsulfonyl fluoride, 100 µM N-tosyl-n-phenylalanine chloromethyl ketone, 1.5 µM pepstatin, 1.5 µM leupeptin, pH 7.4). Cellular debris was removed by centrifuging at 1,000 x g, 10 min, 4 °C. Supernatant was centrifuged at 7,000 x g, 15 min, 4 °C, and the resulting supernatant was centrifuged once more at 47,000 x g, 45 min, 4 °C. Membranes were resuspended in ice-cold PBS (50 mM phosphate buffer, 150 mM NaCl, pH 7) containing 1% octylphenylpolyethylene glycol and 0.5% SDS. HT-29 cells membrane proteins were obtained by Triton X-114-based lysis as reported previously (19). Deglycosylation was performed with the N-glycosidase F deglycosylation kit (Roche Applied Science) according to manufacturer instructions.

Brush border or basolateral membranes were prepared as previously described by Mg²⁺ precipitation, followed by differential centrifugation (20, 21). Proteins from the kidney (25 µg), intestines, brush border membranes, basolateral membranes, or HT-29 cells (100 µg) were analyzed by 8% SDS-PAGE followed by Western blotting. The proteins, transferred to polyvinylidene difluoride membranes, were first incubated with rabbit polyclonal anti-rNCC2–112 (raised against region 2–112 of the rat kidney NCC) (22) 1:1500 or anti-rNCC1–98 (raised against region 1–98 of the rat kidney NCC) (23) 1:2,500 overnight at 4 °C, followed by incubation for 1 h with a 1:2,500 dilution of peroxidase-coupled goat anti-rabbit antibody. Antibody-protein complexes were then detected using the ECL detection kit (Amersham Biosciences) followed by autoradiography.

HT-29 Immunofluorescence—Cells were grown on coverslips to 70% of confluence, fixed in 0.1 M PBS containing 3% paraformaldehyde, and permeabilized with PBS containing 0.1% Triton X-100 for 3 min at room temperature. After inhibition by means of 3% BSA in PBS, cells were incubated overnight at 4 °C with anti-NCC1–98 antibody at 1:600 dilution in PBS containing 0.1% BSA. After washing out the excessive primary antibody, cells were incubated for 1 h with Cy^TM2-conjugated anti-rabbit antibody (Jackson ImmunoResearch Laboratories) diluted 1:100. 4',6-Diamidino-2-phenylindole dihydrochloride staining of nuclei was performed with standard methods. Cells were imaged using a Leica TCS-NT (Leica Microsystems, Heidelberg, Germany) confocal laser-scanning microscope, equipped with laser argon/krypton, 75-milliwatt multiline. A focal series of horizontal planes of section were monitored for fluorescein isothiocyanate by means of the 488 nm laser lines, and a fluorescein isothiocyanate band pass 530/30 nm filter, for 4',6-diamidino-2-phenylindole dihydrochloride with the Coherent Enterprise U.V. argon ion laser (Laser Innovations, Santa Paula, CA). No signal was detectable in negative controls performed by omitting the primary antibody.

Immunohistochemistry—The intestine was dissected from adult rats and fixed by immersion in 4% paraformaldehyde in 0.1 M PBS, pH 7.6 (5 h at 4 °C), subsequently dehydrated in ethanol, and embedded in paraffin. Sections (10-mm thick) were dewaxed and processed for the immunoenzymatic detection of the NCC transporter using a standard avidin-biotin-peroxidase complex procedure. Briefly, after quenching of free aldehyde groups with 0.05 M NH₄Cl in 0.01 M PBS, pH 7.4 (30 min), sections were treated with 1% H₂O₂ (15 min) to inactivate endogenous peroxidase, as well as preincubated in PBS containing 1% BSA and 0.2% Triton X-100 (30 min). After this, the sections were incubated with the polyclonal antiserum anti-NCC1–98 diluted 1:200 in PBS containing 0.1% BSA (48 h at 4 °C in a humid chamber). After multiple rinses in PBS, sections were incubated at room temperature in biotinylated goat anti-rabbit IgG (Vector, Burlingame, CA) diluted 1:200 in PBS containing 0.1% BSA (75 min), rinsed in PBS, and incubated in the avidin-biotin-peroxidase complex (ABC kit, Vector) diluted 1:100 in PBS. After rinsing in PBS and in 0.05 M Tris-HCl buffer (pH 7.6), sections were reacted in a fresh solution of 0.05% diaminobenzidine tetrahydrochloride (Sigma) and 0.002% H₂O₂ in Tris-HCl buffer, and subsequently dehydrated and covered with a coverslip. Control slides were incubated in the same solutions without the primary antiserum and processed as above. As positive control, the polyclonal antiserum anti-NCC1–98 was also tested on paraffin sections of kidney, fixed, and processed as those of the intestine. Negative control sections of the intestine and kidney were processed without including the primary or secondary antiserum. All sections were examined and photographed with a Zeiss III photomicroscope. No immunoreactivity was detected when sections were incubated without the primary or secondary antiserum.

Short-circuit Experiments—Approximately 5 cm of ileum was removed and placed in oxygenated, ice-cold Ringer solution, opened flat and washed for mounting in standard Ussing chambers (0.25-cm² exposed area). Both chambers were filled (5 ml each) for control experiments with the following solution (mM): 145.3 Na⁺, 6.0 K⁺, 125.3 Cl^–, 2.5 Ca²⁺, 1.2 Mg²⁺, 13.7 , 12.5 mannitol, 0.7 H₂PO₄^–, 2.5 , pH 7.4. Test solutions contained also HCTZ (1 mM) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) 50 µM. Bathing solutions were gassed with 100% O₂. Short-circuited conditions were obtained by using an automatic voltage clamp device manufactured in our workshop as previously described (24). A chart recorder was used to register short-circuit current and transepithelial electric potential difference (V_ms).

Fura-2 Experiments—Experiments were performed on subconfluent HT-29 cells cultured on a glass coverslip in Petri dishes. Cytoplasmic Ca²⁺ concentration was measured with Fura-2 as outlined further above (25). Briefly, cells on a glass coverslip were loaded with 1 µM Fura-2-pentakis(acetoxymethyl) ester and incubated for 20 min at 37 °C. The cells were mounted into a recording chamber and placed on the stage of a microscope (Olympus IX70) equipped with a monochromator (TILL Photonics Polychrome IV) and a charge-coupled device camera (TILL Photonics IMAGO) controlled by the imaging software TILLvisION 4.01. After rinsing the cells for at least 20 min with solution A (control solution, mM: NaCl 135.0, KCl 5.0, CaCl₂ 2.0, MgCl₂ 1.2, glucose 5.0, and HEPES 10.0, pH 7.4), the dye was alternatively excited by 340 and 380 nm wavelengths through a 40x oil immersion objective, and the emitted fluorescence was measured at 510 nm from regions of interest within the cells. Intracellular Ca²⁺ concentration variations were evaluated by analyzing the 340/380 ratio with Microsoft Excel following background subtraction. Ca²⁺-free solutions were obtained by omitting CaCl₂ from solution A in the presence of 1 mm EGTA. HCTZ (1 mM) was freshly dissolved 1–2 h prior to the experiment in solution A.

Patch-clamp Experiments—HT29 cells for electrophysiological recordings were seeded on round coverslips (10-mm diameter), cultured as usual and employed 16–48 h after splitting. Before sealing the patch pipette to the cell, cells were kept in a nominally Ca²⁺-free solution containing (mM): 150 NaCl, 6 CsCl, 1 MgCl₂, 10 HEPES, 10 glucose, and 50 mannitol, pH 7.4 with CsOH to prevent Ca²⁺ overload. The extracellular divalent cation free solution (bath) contained (mM) 150 NaCl, 6 CsCl, 10 HEPES, 5 EGTA, 10 glucose, and 50 mannitol, pH 7.4, with CsOH. The hyperosmolarity of the extracellular solution is routinely used to prevent activation of volume-regulated anion channels. The internal (pipette) solution contained (mM): 20 CsCl, 100 cesium aspartate, 1 MgCl₂, 10 EGTA, 4 Na₂ATP, and 10 HEPES, pH 7.2 with CsOH.

Whole cell currents were measured under voltage clamp conditions, with an EPC-9 amplifier (HEKA Elektronik, Germany; sampling rate 100 µs; four-pole Bessel filter, 2.9 kHz) interfaced with a Macintosh computer running the Pulse software (HEKA Elektronik). The step protocol consisted of a series of 50-ms long voltage steps applied from a holding potential of +20 mV to voltages between –100 and +100 mV, at increments of 20 mV. To generate current voltage relation, the mean current between two cursors shown in Fig. 8 was calculated with the Pulse Fit software. Electrode resistances were between 7 and 8 M; the total cell capacitance was routinely compensated, and measured 15 ± 1 picofarads (n = 8); series resistances were also compensated and currents were leak-corrected; all experiments were performed at room temperature.

Statistics—Unless otherwise specified, the experimental values are expressed as mean ± S.E. of n experiments. Student's t or analysis of variance tests were used (with unpaired data unless otherwise specified) for the statistical analysis. The value of p > 0.05 is indicated by ns.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Molecular Characterization and Localization of the Small Intestine NCC Cotransporter
Expression of the NCC Protein in the Rat Small Intestine—We verified the expression of NCC mRNA in the rat small intestine by performing RT-PCRs with three different sets of primer pairs (Table I) and by using enterocyte cDNA as a template. In all PCRs a band at the expected molecular weight was obtained (Fig. 1). With the primer pair rTSC3 the PCR was performed on cDNA from ileum and jejunum, and in both experiments a positive result was obtained. The amplified fragments were sequenced and aligned with the corresponding amino acid sequence of the rat kidney NCC (GenBank^TM accession number NP_062218 [GenBank] ), revealing that, within the tested regions, the intestinal and renal rat NCC sequences are identical.

We confirmed the expression of the NCC mRNA by Northern blot. A ³²P-labeled probe corresponding to the region 1598–1834 of the renal NCC isoform (GenBank^TM accession number NM_019345 [GenBank] ) was used to identify the full-length transcript of small intestine enterocyte NCC mRNA. Cortical kidney total RNA was used as positive control, where (Fig. 2) two distinct signals corresponding to 4.4 and 3.3 kb could be identified, in good agreement with earlier reports (26) showing that both bands correspond to NCC and varying solely by the 3'-untranslated regions. Using the intestine mRNA only one band at 4.4 kb could be observed. Even a prolonged exposure (1 week) did not reveal a second band at 3.3 kb.

View larger version (52K): [in this window] [in a new window]   FIG. 1. RT-PCR experiments demonstrate that the NCC mRNA is transcribed in the small intestine. RT-PCRs performed with three different primer sets (rTSC1, rTSC2, and rTSC3, see Table I) on jejunum or ileum mRNA. PCR with rTSC3 primers was also performed on the intestinal sub-mucosa.

View larger version (46K): [in this window] [in a new window]   FIG. 2. Northern blot experiment performed on small intestine RNA. Rat intestinal poly(A)RNA was probed with a 32P-labeled cRNA. Rat kidney total RNA was used as a positive control. Two autoradiography exposure times (24 h and 1 week) are shown for the intestine.

View larger version (56K): [in this window] [in a new window]   FIG. 3. Western blot experiments performed with anti-rNCC-antibodies. a, Western blot (anti-NCC2–112 antibody) of membrane protein preparations from rat kidney (positive control), jejunum, and ileum. b, Western blot (anti-NCC2–112 antibody) of F-glycosidase-treated small intestine or kidney protein preparations (+ glyc F). Negative control was performed without including the F-glycosidase (– glyc F) in the deglycosylation procedure. c, Western blot of membrane protein preparations from rat kidney (positive control) and small intestine performed with the anti-rNCC1–98 antibody. d, Western blot of intestinal brush border (BBM) and basolateral (BLM) membrane protein preparations (anti-rNCC1–98 antibody).

View larger version (164K): [in this window] [in a new window]   FIG. 4. Immunohistochemical localization of NCC in sections of rat small intestine (a– c) and kidney (d). NCC immunoreactivity is selectively localized on the apical membrane (arrows) of intestinal epithelial cells (epi), located at the tip of the villi (a–c) and of distal tubular segments in the kidney (d); glo, unlabeled glomeruli. Original magnifications: a = 370x; b and c = 640x; d = 270x.

The HCTZ-induced depolarization of V_ms could only be observed if the drug was added to the mucosal side, whereas the addition of the drug to the serosal side had no effects on V_ms (data not shown), consistent with an apical localization of the cotransporter.

Given that the NCC cotransporter is electroneutral, the observed rapid and immediate depolarization cannot be attributed to the inhibition of the NCC cotransporter itself. In analogy to what has been proposed for the kidney distal convoluted tubule (DCT) (7, 8), we propose that HCTZ hyperpolarizes the apical membrane, thus causing an overall decrease of the transepithelial potential (V_ms = V_m – V_s; V_s is normally greater than V_m) by reducing the driving force for chloride to exit the cell through a chloride conductance (7, 32). According to this hypothesis, the inhibition of chloride channels in the absence of HCTZ should also lead to a depolarization of V_ms by blocking the depolarizing resting chloride current. We indeed measured a depolarization of V_ms by 7.4 ± 1.7% (n = 9) after the addition of 50 µM NPPB, a known blocker of epithelial chloride channels, to the mucosal side of the epithelium. Accordingly, the addition of HCTZ in the presence of NPPB did not further depolarize V_ms (6.3 ± 0.7%; n = 9; this value is not statistically different from the 7.4 ± 1.7%, mentioned above).

View larger version (54K): [in this window] [in a new window]   FIG. 5. Short-circuit experiments performed on excised intestine. a and b, original tracings obtained in short-circuit experiments showing the time course of the transepithelial electric potential (Vms). The upper trace corresponds to an experiment performed under control conditions; the arrows indicate the substitution of the mucosal solution with an identical solution. In the lower trace, three treatment periods (10 min each) with 1 mM HCTZ are shown. "C" (control) or "HCTZ" indicate the substitution of the mucosal solution with an identical one or with the solution containing HCTZ. c, summary of the HCTZ-induced depolarization (12.0 ± 2.4%, n = 56), the depolarization induced by NPPB (7.4 ± 1.7%, n = 9), and the effect of HCTZ in the presence of NPPB (6.3 ± 0.7%, n = 9). In the presence of NPPB, HCTZ is not able to further depolarize Vms.

ECaC are inwardly rectifying Ca²⁺-permeable channels that can be activated by negative voltages, can be blocked by ruthenium red, and show a prominent monovalent permeability in divalent cation-free solutions (34). According to protocols that exploit the characteristic fingerprints for ECaC (35, 36), we carried out patch-clamp experiments aimed to investigate whether ECaC-related currents can be shown in HT-29 cells.

As shown in Fig. 8, native HT-29 cells, exposed to a divalent cation-free solution and stimulated via a step protocol from –100 mV to +100 mV (+20-mV steps; the holding potential was +20 mV), exhibited a strong inwardly rectifying current, which could be blocked reversibly (75% at –100 mV) by the addition of 10 µM ruthenium red to the bath solution. The observed biophysical characteristics of the currents correspond to ECaC currents observed in other systems (34, 37). It is not clear at the moment, why, in the presence of 10 µM ruthenium red, in three of five experiments an outward current could be measured. Further work is necessary to elucidate whether these currents are the result of an unspecific effect of ruthenium red on the membrane at potentials more positive than +40 mV, or if ruthenium red is able to activate or unmask an outwardly rectifying anionic current at the respective positive holding potentials.

View larger version (50K): [in this window] [in a new window]   FIG. 6. NCC and ECaC are expressed in human HT-29 cells. a, RT-PCR performed with specific NCC and ECaC1/2 primers (Table I) using total RNA of HT-29 cells. b, Western blot of membrane protein preparations from rat small intestine and HT-29 cells (anti-NCC1–98 antibody).

View larger version (19K): [in this window] [in a new window]   FIG. 7. Immunofluorescence of butyrate-treated (right) or untreated (left) HT-29 cells probed with the antirNCC1-98 antibody (green signal). Nuclei were stained by 4',6-diamidino-2-phenylindole dihydrochloride (blue signal).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The remarkable role the kidney plays in controlling blood pressure is explained by the fact that this organ is responsible for mediating the sodium handling of the organism. Reabsorption and secretion of sodium is tightly balanced along the renal tubules. The fine-tuning of the sodium balance is performed at the level of the distal tubular segment, where NCC is one of the key players for sodium transport. This is demonstrated by the fact that mutations within the NCC protein, able to reduce its activity, lead to salt-resistant hypotension, also known as the Gitelman syndrome (10, 45). Whereas the cardinal role of NCC in renal Ca²⁺ absorption is generally accepted, little is known whether or not NCC is also important for Ca²⁺ absorption in the intestine. Our results demonstrate the presence of the NCC cotransporter in rat small intestine (ileum and jejunum) and human HT-29 cells. RT-PCRs and Northern blot experiments conducted on rat small intestine suggest that the intestinal NCC isoform is identical to the kidney isoform. In the kidney, a probe corresponding to the last three highly conserved transmembrane segments recognized three bands at 11, 4.4, and 3.3 kb, a pattern similar to the one reported by other authors (13, 14, 26). It has been shown, that the 4.4-kb band as well as the 3.3-kb band code for NCC, and the sequence differences are limited to the 3'-untranslated region (26, 46). The nature of the 11-kb transcript is, although described also in UMR-106 osteosarcoma cells (14), still elusive. In intestine we identified only one main band at 4.4 kb, however, at a lower level of expression versus that of the kidney, as was also shown by Chang and co-workers in the human small intestine and colon (13). It has to be mentioned that no signal was found by Northern blot in rat small intestine by Gamba and co-workers (26). The differing finding may be a result of more sensitive conditions we introduced, because we used poly(A)RNA and radioactive labeled probes instead of total RNA and digoxigenin-labeled probes. Confirming RT-PCR data, the NCC protein was also identified by Western blot experiments, both in the jejunum and ileum. In accordance with the Northern blot experiments, the intensity of the signal was lower than in the kidney. The band appeared less broad and frequent, depending on the protein preparations, at a slightly lower molecular weight level. This may be a consequence of different glycosylation levels, because the Northern blot experiments indicate that the core protein is likely to be of the same molecular weight as the kidney isoform. Experiments performed with F-glycosidase-treated or -untreated membrane protein preparations indicate that native NCC is a glycosylated protein and the molecular mass of the core protein (about 110 kDa) is comparable to that of the kidney isoform (27, 28). A different level of glycosylation could be due to a different preservation of the integrity of the transporter during membrane preparation or due to a tissue-specific glycosylation pattern, as it has been proposed for other transport proteins (47). Western blot and immunohistochemistry experiments demonstrate that NCC expression in the intestine is mainly confined to the enterocytes in the apical region of the villi, whereas the majority of the signals seem to be located at the apical membranes of the cells. The localization of the transporter suggests its involvement in NaCl absorption from the intestinal lumen, in analogy to the sodium reabsorption from the DCT lumen in the kidney. Whereas in the DCT the NCC cotransporter represents the main pathway for NaCl absorption, in the intestine additional transporters, such as the Na⁺/H⁺-Cl^–/HCO^–₃ double exchange (48, 49), are likely to interact with NCC. This redundancy of the NaCl transport systems could justify the lower expression of NCC compared with that of the kidney. Nevertheless, the contribution of NCC to sodium and chloride entry into the cells in our experimental setting appears to be substantial, given that HCTZ leads to significant changes of V_ms. Our hypothesis on how HCTZ facilitates the reduction of V_ms implies the presence of an active Cl^– conductance, and our data obtained by using NPPB, a known Cl^– channel inhibitor, are in agreement with this assumption. In addition, the inhibition of NCC by HCTZ may reduce the uptake of Na⁺ into the cell, lowering the intracellular sodium concentration, and thus causing an increase in the activity of the basolateral Na⁺/Ca²⁺ exchanger (49–51) alongside a decrease in that of the Na⁺-K⁺ pump, and consequently contributing to the depolarization of V_ms. As mentioned earlier, apart from its diuretic effect, HCTZ is also applied in a therapeutic context based on its action on Ca²⁺ homeostasis related to its hypocalciuric effects (8, 52–54). This mechanism is the result of an increased Ca²⁺ reabsorption by DCT cells; however, it must be considered also that the intestine could be a target of HCTZ, a commonly discussed scenario ever since 1970 (55, 56). An increased intestinal Ca²⁺ absorption in response to thiazides treatment has been reported (17, 18). More recently, in a clinical study, it has been proposed that the Ca²⁺ balance in thiazide-treated subjects may be more positive, not only due to reduced renal loss but also as a result of a possible concomitant increase in intestinal calcium reabsorption (5).

View larger version (25K): [in this window] [in a new window]   FIG. 8. Voltage clamp experiments (whole cell configuration) performed on HT-29 cells. a, example of whole cell current traces obtained in control conditions (bath solution is free of divalent cations) when voltage increments from –100 to +100 mV were applied (increment is 20 mV, holding potential is +20 mV). b, current to voltage relationship obtained in control condition, or in the presence of the blocker ruthenium red. The effect of ruthenium red is reversible (washout). The outward current in the presence of ruthenium red was only observed in three out of five experiments. Histograms represent % currents measured at –100 mV in control condition or in the presence of the blocker.

View larger version (24K): [in this window] [in a new window]   FIG. 9. Fura-2 experiments performed on HT-29 cells demonstrate that HCTZ induce Ca2+ entry in HT-29 cells. Cyan line, cells were exposed to a bicarbonate-free solution containing 2 mM Ca2+, followed by exposure to 1 mM HCTZ in the same Ca2+ containing buffer for 6 min and by subsequent removal from the drug by replacement with the control solution. Red line, the same protocol was used with butyrate-treated cells. Green line, butyrate-treated cells were exposed to Ca2+ free extracellular buffer for 4 min before exposure to 1 mM HCTZ in the same Ca2+-free buffer. Washing out of the drug was achieved by repeatedly exchanging the bath solution with the Ca2+ free extracellular buffer. Blue line, control experiments: cells were exposed to three subsequent changes of the Ca2+ containing bath solution with an identical one. Vertical lines represent standard error.

View larger version (31K): [in this window] [in a new window]   FIG. 10. Single cell Fura-2 experiments in the presence of HCTZ. a, average intracellular Ca2+ response of butyrate-treated HT-29 cells to HCTZ. Cells were exposed to a bicarbonate-free solution containing 2 mM Ca2+, followed by exposure to 1 mM HCTZ in the same buffer and by the subsequent washing out of the drug by replacement with the control solution. Dotted lines represent the standard error (this trace was taken from Fig. 9). b, single cell responses. Two different cells (red line, cell A; blue line, cell B) elicit responses of different intensity as a reaction to the treatment with HCTZ.

HCTZ leads to a transient increase of the intracellular Ca²⁺ of HT-29 cells, and the Ca²⁺ peak is abolished in the absence of extracellular Ca²⁺ or the presence of ruthenium red, indicating that calcium enters from the outside. On the single cellular level we observed that the Ca²⁺ entry was effected by multiple and irregular calcium oscillations. It is important to note that in butyrate-treated cells the first Ca²⁺ transient was followed by a series of minor oscillations, showing a higher frequency as compared with untreated cells, and leading to a sustained increase of the intracellular Ca²⁺ concentration of an entire cell population. These results are in agreement with the observation that the use of sodium butyrate induces the differentiation of cultured intestinal cells toward a mature and more homogeneous enterocyte phenotype (33), showing the characteristics of absorptive cells. In HT-29 cells, the butyrate-induced differentiation is followed by a selective down-regulation of the NKCC1 cotransporter, typical of crypt cells, and an up-regulation of brush border markers (59); in other intestinal cell lines the differentiation is accompanied by a decrease in the Cl^– secretion (59). This suggests a physiological, biochemical, and morphological differentiation of the cells from a crypt-type cell involved in secretion, to a typical villous cell involved in reabsorption. Interestingly, butyrate impacts also the intact intestine by stimulating electroneutral NaCl absorption (60). In accordance with these data, our immunofluorescence experiments show that the number of HT-29 cells expressing the NCC cotransporter is actually increased by sodium butyrate treatment, confirming the role of the transporter in intestinal NaCl absorption.

In conclusion, our data demonstrate that the functional combination of sodium and Ca²⁺ transport by ECaC and NCC, originally described for the kidney (53, 61), may also hold true for the intestine. This suggests that the role of HCTZ in Ca²⁺ homeostasis should be extended from the kidney to a more comprehensive perspective, including the intestine.

	FOOTNOTES

 
^* This research was supported by the Italian Ministry of Instruction, University and Research (Fondo per gli Investimenti della Ricerca di Base (FIRB) RBAU01RANB_003). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence may be addressed. Tel.: 39-02-5031-4947; Fax: 39-02-5031-4948; E-mail: guido.botta{at}unimi.it. To whom correspondence may be addressed. Tel.: 43-512-507-3756; Fax: 43-512-577-656; E-mail: markus.paulmichl{at}uibk.ac or markus.paulmichl{at}unimi.it.

¹ The abbreviations used are: NCC, NaCl-cotransporter; DCT, distal convoluted tubule; ECaC, epithelial Ca²⁺ channels; HCTZ, Hydrochlorothiazide; RT, reverse transcription; PBS, phosphate-buffered saline; BSA, bovine serum albumin; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoic acid.

	ACKNOWLEDGMENTS

 
We wish to express our gratitude to Prof. D. Mount and Prof. S. Hebert for supplying anti-rNCC2–112 antibodies. Furthermore, we would like to extend our gratitude to Prof. D. Ellison for supplying anti-rNCC1–91 antibodies. We wish to also thank Dr. Igea D'Agnano, CNR, Milan, for her advice on HT 29 cell line treatment

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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