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Hypertension. 2007;49:1186-1193
Published online before print February 26, 2007, doi: 10.1161/HYPERTENSIONAHA.106.083444
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(Hypertension. 2007;49:1186.)
© 2007 American Heart Association, Inc.

Original Articles

Activity and Regulation of Na+-HCO3– Cotransporter in Immortalized Spontaneously Hypertensive Rat and Wistar–Kyoto Rat Proximal Tubular Epithelial Cells

Rui Pedrosa; Nuno Gonçalves; Ulrich Hopfer; Pedro A. Jose; Patrício Soares-da-Silva

From the Institute of Pharmacology and Therapeutics Faculty of Medicine (R.P., N.G., P.S.d.S.), Porto, Portugal; the Department of Physiology and Biophysics (U.H.), Case Western Reserve School of Medicine, Cleveland, Ohio; and the Department of Pediatrics (P.A.J.), Georgetown University, Washington, DC.

Correspondence to Patrício Soares-da-Silva, Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200-319 Porto, Portugal. E-mail psoaresdasilva{at}netcabo.pt


*    Abstract
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 *Abstract
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 down arrowResults
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The present study evaluates the presence and functional proprieties of the Na+-HCO3– cotransporter (NBC) in immortalized renal proximal tubular epithelial cells from spontaneously hypertensive (SHR) and normotensive Wistar–Kyoto (WKY) rats. The expected size and nucleotide sequence of a 1031-bp fragment corresponding to type 1 NBC (NBC1) was identified in both cell lines. The expression of the NBC1 transcript was lower (P<0.05) in SHR than in WKY cells. After intracellular acidification and in the presence of amiloride (1 mmol/L), the addition of sodium (115 mmol/L) in the absence of chloride resulted in rapid intracellular pH recovery that was higher in WKY than in SHR cells. This was an Na+- and HCO3–-dependent process in both cell lines. 4,4'-Diisothiocyanatodihydrostilbene-2,2'-disulphonic acid inhibited NBC activity in both WKY and SHR cells; the inhibitory effect was, however, more pronounced in WKY than in SHR cells. Forskolin (10 µmol/L) and dibutyryl cAMP (0.5 mmol/L) did not alter NBC activity. Acidosis induced by a 24-hour treatment with NH4+ (20 mmol/L) increased NBC activity to a greater extent in SHR than in WKY cells, without changes in intracellular pH and cell viability. Treatment with acetazolamide (300 µmol/L) for 24 hours did not change NBC activity in both cell lines. In contrast to NBC, Na+-K+ ATPase activity and expression were higher in SHR than in WKY cells. It is concluded that SHR cells are endowed with lower NBC activity than WKY cells, but the former is more resistant to 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulphonic acid and responds better to acidosis.


Key Words: Na+-HCO3– • cotransporter • hypertension • acidosis • SHR • WKY


*    Introduction
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 *Introduction
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A considerable part of the filtered load of HCO3– at the kidney level is reabsorbed in the proximal tubules by anion exchangers.1,2 The Na+/HCO3– cotransporter, the Na+-dependent Cl–/HCO3– exchanger, and the Na+-independent Cl–/HCO3– exchanger have been described in the kidney.3–6 All of these types of anion exchange proteins facilitate the reversible electroneutral exchange of Cl– for HCO3– across the plasma membrane and regulate intracellular pH (pHi), intracellular chloride concentration, bicarbonate metabolism, and cell volume. After an intracellular acid load, the cell responds with stimulation of the Na+/H+ exchanger (NHE), the Na+/HCO3– cotransporter, and the Na+-dependent Cl–/HCO3– exchanger to mediate the recovery of pHi.7–9 In contrast, the Na+-independent Cl–/HCO3– exchanger usually mediates the recovery from an intracellular alkalinization.10 Notwithstanding the view that all transporters work strictly together, the sodium bicarbonate cotransporter in the kidney (kNBC1) has been identified as the main pathway for bicarbonate efflux across the basolateral membrane in the proximal tubule.11–13 On the other hand, in kidney proximal tubules, the main mechanism of apical sodium reabsorption is via type 3 NHE (NHE3) that works in concert with the Cl–/HCO3– exchanger (SLC26A6),14 this being essential to maintain electrolyte and acid–base homeostasis. We have recently reported an increased activity and expression of both NHE3 and SLC26A6 transporters in immortalized proximal tubular epithelial (PTE) cells from spontaneously hypertensive rats (SHRs) when compared with their normotensive controls (Wistar–Kyoto rats; WKY).15–17

The exit of sodium across the basolateral membrane of proximal tubule occurs mainly through the Na+-K+-ATPase.18 However, the activity of basal electrogenic kNBC1, of which the main function is extrusion of HCO3– at the basal cell side of proximal tubular cells, also contributes to Na+ reabsorption and electrolyte homeostasis and may be directly linked or regulated by changes in NHE3 and SLC26A6 activity and/or expression. The present study was designed to evaluate the presence and functional proprieties of the kNBC1 transporters in immortalized renal PTE cells from SHR and WKY rats.


*    Methods
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 *Methods
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Cell Culture
Immortalized renal PTE cells from 4- to 8-week–old WKY rats and SHRs19 were maintained in a humidified atmosphere of 5% CO2––95% air at 37°C. WKY and SHR cells were grown in DMEM nutrient mixture F-12 Ham (Sigma Chemical Company) supplemented with 100 U/mL of penicillin G, 0.25 µg/mL of amphotericin B, 100 µg/mL of streptomycin (Sigma), 4 µg/mL of dexamethasone (Sigma), 5 µg/mL of transferrin (Sigma), 5 µg/mL of insulin (Sigma), 5 ng/mL of selenium (Sigma), 10 ng/mL of epidermal growth factor (Sigma), 5% FBS (Sigma), and 25 mmol/L of HEPES (Sigma). For subculturing, the cells were dissociated with 0.10% trypsin-EDTA, split 1:4 and subcultured in Costar plates with 21-cm2 growth areas (Costar). For pHi measurement experiments, cells were grown in 96-well plates (Costar) or glass coverslips. The cell medium was changed every 2 days, and the cells reached confluence after 3 to 5 days of incubation. For 24 hours before each experiment, the cells were maintained in FBS-free medium. Experiments were generally performed 1 to 2 days after cells reached confluence and 4 to 5 days after the initial seeding; each squared centimeter contained {approx}50 µg of cell protein.

Animals
SHRs and WKY rats (Harlan-Inferfauna, Barcelona, Spain), 12 weeks old and weighing 284 to 287 g, respectively, were used in the experiments. Animals were kept under controlled environmental conditions (12-hour light/dark cycle and room temperature 22±2°C). All of the animal interventions were performed in accordance with the European Directive number 86/609, and the rules of the "Guide for the Care and Use of Laboratory Animals." Rats were euthanized by decapitation, and the kidneys were removed.

pHi Measurements
For pHi measurement experiments, WKY and SHR PTE cells were grown in 96-well plates, as described previously.15 At days 4 to 5 after seeding SHR and WKY PTE cells cultured in 96-well plates, pHi measurements were performed after loading the cells with 5 µmol/L acetoxymethyl ester of 2',7'-bis(carboxyethyl)-56-carboxyfluorescein at 37°C for 30 minutes. Cells were placed in the sample compartment of a dual-scanning microplate spectrofluorometer (Spectramax Gemini XS, Molecular Devices), and fluorescence was measured every 17 s alternating between 440- and 490-nm excitation at 535-nm emission, with a cutoff filter of 530 nm. The ratio of intracellular 2',7'-bis(carboxyethyl)-56-carboxyfluorescein fluorescence at 490 and 440 nm was converted to pHi values by comparison with values from an intracellular calibration curve using the nigericin (10 µmol/L) and high-K+ method.20

Na+-HCO3– Cotransporter Activity
The NBC activity was determined as the initial rate of the Na+-dependent pHi recovery (dpHi/dt, pH/s) in an HCO3–-containing solution after an acid load induced by NH4+ loading, as described previously.21 The experiments were performed in the presence of 1 mmol/L of amiloride to inhibit the NHE activity. To define the initial rate of pHi recovery dependence for Na+ or Cl–, the apical side of the monolayers was bathed with a modified Krebs–Hensleit solution containing choline or gluconate, respectively, without affecting the concentrations of other ions.

Detection of NBC1 Transcripts in WKY and SHR Cells
Cells were homogenized (Diax, Heidolph) in Trizol reagent (75 mg/mL; Invitrogen), and total RNA was extracted according to the manufacturer’s instructions. The RNA obtained was dissolved in diethyl pyrocarbonate–treated water and quantified by spectrophotometry at 260 nm. One microgram of total RNA was reverse transcribed to cDNA with SuperScript First Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer’s instructions. The reverse transcription was performed at 50°C, using 5 µg/µL of random hexamers. Rat expressed sequence tag database was blast-searched against the rat NBC1 sequence (GenBank accession No. NM_053424). On the basis of the NBC1 cDNA sequence, the following oligonucleotide primers (forward: 5'-CCA AGC GAA AGA TAG ACA CGA-3' and reverse: 5'-CCA GGA AGA GGA TGA AGG AC-3') corresponding with nucleotides 1074 and 2085 of the rat cDNA were designed and used for RT-PCR on RNA isolated from immortalized renal PTE cells and kidney cortex from WKY rats and SHRs. PCR was performed with Platinum TaqPCRx DNA Polymerase (Invitrogen). Amplification conditions were as follows: hot start of 3 minutes at 95°C; 30 cycles of denaturing (95°C for 30 s), annealing (60°C for 1 minute), and extension (72°C for 45 s) and a final extension of 7 minutes at 72°C. The PCR products were separated by electrophoresis in a 2% agarose gel and visualized under UV light in the presence of ethidium bromide. Sequencing of amplified transcripts was performed in both directions by GATC Biotech AG.

Real-Time PCR Quantification of NBC1
Cells were homogenized (Diax, Heidolph) in Trizol reagent (75 mg/mL; Invitrogen), and total RNA was extracted according to the manufacturer’s instructions. The RNA obtained was dissolved in diethyl pyrocarbonate–treated water and quantified by spectrophotometry at 260 nm. One microgram of total RNA was reverse transcribed to cDNA with the SuperScript First Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer’s instructions. The reverse transcription was performed at 50°C, using 5 µg/µL of random hexamers. Standards for NBC1 and GAPDH were obtained by conventional PCR amplification, using Platinum TaqPCRx DNA Polymerase (Life Technologies) and the following rat specific primers: rNBC1 forward primer 5'-TCCTCAAGCCGCTCATCTCC-3' and reverse primer 5'-CTCCCCACCCTGTTCCACTTT-3' (positions 239 and 409 bp in rat NBC1 sequence NM_ 053424); rat GAPDH forward primer 5'-GGC ATC GTG GAA GGG CTC ATG AC-3' and reverse primer 5'-ATG CCA GTG AGC TTC CCG TTC AGC-3' (positions 1348 and 1512 bp in rat GAPDH sequence M17701). PCR products were gel purified with Qiaex II (Qiagen), quantified by spectrophotometry at 260 nm, and further diluted accordingly in serial steps. Real-time PCR was carried out using a LightCycler (Roche). Each RT-PCR mixture (50 µL) included reverse transcription products corresponding with 50 ng of total RNA or standard DNA, 1x SYBR Green I master mix (LightCycler FastStart DNA MasterPLUS SYBR Green I, Roche), and 0.5 µmol/L of each forward and reverse primers, mentioned above. Cycling conditions were as follows: denaturation (95°C for 1 minute), amplification and quantification (95°C for 10 s, 62°C for 10 s, and 72°C for 5 s, with a single fluorescence measurement at the end of the 72°C for 5 s segment) repeated 35 times, a melting curve program (65°C to 95°C with a heating rate of 0.1°C/s and continuous fluorescence measurement), and a cooling step to 40°C. Amplification specificity was checked using melting curves following the manufacturer’s instructions. In addition, PCR products were separated by electrophoresis in a 2% 90 mM Tris/64.6 mM boric acid/2.5 mM EDTA (pH 8.3) agarose gel to confirm that correct band sizes were obtained. Target mRNAs were quantified by measuring the threshold cycle (when fluorescence is statistically significantly above background) and reading against a calibration curve. Results were analyzed with LightCycler Software version 3.5 (Roche Applied Science) using the second derivate maximum method. The relative amount of each mRNA was normalized to a housekeeping gene (GAPDH) mRNA. Each sample was tested in duplicate.

Measurement of Cell Viability
Cell viability was measured using calcein-AM (Molecular Probes). The membrane permeant calcein-AM, a nonfluorescent dye, is taken up and converted by intracellular esterases to membrane impermeant calcein, which emits green fluorescence. After treatment, cells were washed twice with Hanks’ medium (medium composition, in mM: NaCl 137, KCl 5, MgSO4 0.8, Na2HPO4 0.33, KH2PO4 0.44, CaCl2 0.25, MgCl2 1.0, Tris HCl 0.15, and sodium butyrate 1.0; pH 7.4) and loaded with 2 µmol/L of calcein-AM in Hanks’ medium, at room temperature for 30 minutes. Fluorescence was measured at 485-nm excitation and 530-nm emission wavelengths in a multiplate reader (Spectromax Gemini, Molecular Devices). To determine minimum staining for calcein-AM (calceinmin), 6 wells were treated with ethanol 15 minutes before calcein-AM addition. The percentage of viability was then calculated as [(calceinsample–calceinmin)/(calceincontrol–calceinmin)]x100.

Na+-K+ ATPase Expression
WKY and SHR PTE cells cultured to 90% confluence were washed twice with PBS and then lysed by brief sonication (15 s) in lysis buffer with protease inhibitors (150 mmol/L of NaCl, 50 mmol/L of Tris-HCl [pH 7.4], 5 mmol/L of EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/mL of PMSF, and aprotinin and leupeptin 2 µg/mL each) and incubated on ice for 1 hour. After centrifugation (16 000 g, 30 minutes, 4°C), the supernatant was collected and protein concentration determined using the method of Bradford.22 Twenty micrograms of protein were mixed in 6x sample buffer (0.35 mol/L of Tris-HCl, 4% SDS, 30% glycerol, 9.3% dithiothreitol [pH 6.8], and 0.01% bromphenol blue). Proteins were subjected to SDS-PAGE (10% SDS-polyacrylamide gel) and electrotransfered onto nitrocellulose membranes. The transblot sheets were blocked with 5% of nonfat dry milk in Tris.HCl 25 mmol/L (pH 7.5), NaCl 150 mmol/L, and 0.1% Tween 20 overnight at 4°C. Then, the membranes were incubated with mouse monoclonal anti-Na+-K+ ATPase antibody (1:1000; Santa Cruz Biotechnology) and anti-ß-actin primary antibody (1:10 000; Upstate Biotechnologies) in 5% nonfat dry milk in PBS-Tween 20 overnight at 4°C. The immunoblots against Na+-K+ ATPase and ß-actin were subsequently washed and incubated with fluorescent-labeled goat anti-mouse secondary antibody (1:5000; AlexaFluor 680, Molecular Probes), respectively, for 60 minutes at room temperature and protected from light. The membrane was washed and imaged by scanning at 700 nm with an Odyssey Infrared Imaging System (LI-COR Biosciences).

Na+-K+-ATPase Activity
Cell monolayers were continuously monitored for changes in short circuit current (Isc; microamps per centimeter squared) after the addition of amphotericin B to the apical-side reservoir to increase the sodium delivered to Na+-K+-ATPase at the saturating level. Under short-circuit conditions, the resulting current is because of the transport of sodium across the basolateral membrane mediated by Na+-K+-ATPase.8 WKY and SHR PTE cells grown on polycarbonate filters (Snapwell, Costar) were mounted in Ussing chambers (window area: 1.0 cm2) equipped with water-jacketed gas lifts bathed on both sides with 10 mL of Krebs–Hensleit solution, gassed with 95% O2 and 5% CO2 and maintained at 37°C. The Krebs–Henseleit solution contained (in mM): NaCl 118, KCl 4.7, NaHCO3 25, KH2PO4 1.2, CaCl2 2.5, and MgSO4 1.2; pH was adjusted to 7.4 after gassing with 5% CO2 and 95% O2. Monolayers were continuously voltage clamped to 0 potential differences by application of external current, with compensation for fluid resistance, by means of an automatic voltage current clamp (DVC 1000, World Precision Instruments). Transepithelial resistance (Ohm per centimeter squared) was determined by altering the membrane potential stepwise (±3 mV) and applying the ohmic relationship. The voltage/current clamp unit was connected to a PC via a BIOPAC MP1000 data acquisition system (BIOPAC Systems, Inc). Data analysis was performed using AcqKnowledge 2.0 software (BIOPAC Systems, Inc).

Data Analysis
Arithmetic means are given with SEM. Statistical analysis was performed by 1-way ANOVA followed by Student’s t test or the Newman–Keuls test for multiple comparisons. A P<0.05 was assumed to denote a significant difference.

Drugs
Dibutyryl cAMP, 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulphonic acid (DIDS), forskolin, acetazolamide, amiloride hydrochloride hydrate, and tetramethylammonium-Cl were purchased from Sigma Chemical Co. Acetoxymethyl ester of 2',7'-bis(carboxyethyl)-56-carboxyfluorescein, ethylisopropylamiloride, and nigericin were obtained from Molecular Probes. 3(54)-N-isopropylidene-2-methyl-acrylamide dihydrochloride (S3226) and cariporide were kindly provided by Dr H. J. Lang (Aventis Pharma Deutschland).


*    Results
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 up arrowIntroduction
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 *Results
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Detection of NBC1 Transcripts in WKY and SHR Immortalized PTE Cells
The presence of NBC1 transcripts in WKY and SHR PTE cells and kidney cortices of 12-week–old SHRs and WKY rats was examined by RT-PCR with specific primers for NBC1 rat cDNA sequences as described in the Methods section. The kidney cortex from 12-week–old SHRs and WKY rats was used as a positive control. The expected size and nucleotide sequence of 1031-bp fragment corresponding to NBC1 was identified in total RNA from the kidneys and immortalized cells of both SHRs and WKY rats (available in a data supplement online at http://hyper.ahajournals.org). Transcript abundance of NBC1 was measured by quantitative real-time PCR in WKY and SHR PTE cells. The expression of NBC1 transcript was normalized to that of the housekeeping gene GAPDH, which was identical in WKY and SHR PTE cells. Data are presented as the ratio of NBC1/GAPDH. As depicted in Figure 1, the expression of the NBC1 transcript in SHR PTE cells was much lower than in WKY PTE cells.


Figure 1
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  		Figure 1. Abundance of NBC1 transcript in WKY and SHR renal PTE cells. Results are expressed as ratio to GAPDH, as determined by quantitative real-time PCR. Columns represent mean of 4 to 6 independent experiments; vertical lines indicate SEM. Significantly different from SHR PTE cells (*P<0.05) using Student’s t test.

Na+-HCO3– Cotransporter Activity in WKY and SHR Immortalized PTE Cells
The activity of the basolateral Na+/HCO3– cotransporter was assayed as the initial rate of Na+-dependent pHi recovery measured in the presence of CO2/HCO3– after an acid load imposed by 20 mmol/L of NH4Cl. To exclude the contribution of NHE activity, all of the experiments were performed in the presence of 1 mmol/L of amiloride. As shown in Figure 2A, the addition of Na+ induces a rapid pHi recovery after the acidification imposed by NH4Cl with a subsequent return of pHi toward basal values in WKY and SHR PTE cells. The NBC activity in WKY PTE cells was greater than that in SHR cells (Figure 2B). It should be emphasized that basal pHi in SHR PTE cells (7.72±0.09 pH units) was higher (P<0.05) than that in WKY PTE cells (7.39±0.06 pH units), which correlates well with the differences in NBC activity between WKY and SHR PTE cells. Assuming that pHi recovery from intracellular acidification involves basically the NBC, then removal of Na+ or HCO3– should inhibit pHi recovery. To test this hypothesis, Na+ in the perfusion medium was replaced by choline. The pHi recovery was Na+ and HCO3– dependent, as shown by lack of significant pHi recovery in the absence of Na+ or HCO3– (please see the data supplement). DIDS is a classical inhibitor of anionic transporters, such as the Na+/HCO3– cotransporter, the Na+-dependent Cl–/HCO3–, and the Cl–/HCO3– exchanger, in a variety of cell types, including renal cells.5,16,23–26 To test this classic inhibitor, we changed the standard design of the pHi recovery experiments, because DIDS precipitated in the presence of amiloride. For such a reason, amiloride, an nonspecific inhibitor of NHE, was replaced by 2 selective inhibitors of NHE3 and NHE1, S3226 (10 µmol/L) and cariporide (10 µmol/L), respectively.27,28 As shown in Figure 3, DIDS markedly reduced the pHi recovery rate in a concentration-dependent manner; however, this inhibitory effect was more pronounced in WKY PTE cells than in SHR PTE cells.


Figure 2
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  		Figure 2. Assessment of (A) pHi recovery and (B) Na+-HCO3– cotransporter activity under maximum velocity conditions as the initial rate of pHi recovery after acid load induced by NH4+ in presence of HCO3–, Na+, and amiloride. Traces and columns represent mean of 12 to 15 independent experiments; vertical lines indicate SEM. Significantly different from WKY PTE cells (*P<0.05) using Student’s t test.


Figure 3
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  		Figure 3. Assessment of pHi recovery in the presence of DIDS (0.5 and 1.0 mmol/L) in WKY (A) and SHR (B) renal PTE cells. (C) Effect of DIDS (0.5 and 1.0 mmol/L) on the Na+-HCO3– cotransporter activity in the presence of S3226 (10 µmol/L) and cariporide (10 µmol/L) in WKY and SHR PTE cells. Traces and columns represent means of 3 to 6 experiments per group; vertical lines show SEM; significantly different from corresponding control values (*P<0.05) using the Newman–Keuls test.

Previous studies have demonstrated that protein kinase A (PKA), a second messenger that is linked to several receptors, is involved in the regulation of Na+/HCO3– cotransporter activity by shifting the HCO3–:Na+ coupling ratio from 3:1 to 2:1 form.29–31 To evaluate whether or not this was the case in WKY and SHR PTE cells, we examined the effect of forskolin, an adenylyl cyclase agonist, and dibutyryl cAMP, a membrane-permeable cAMP analog, which indirectly and directly activate PKA, respectively. However, treatment with forskolin (10 µmol/L) and dibutyryl cAMP did not change the Na+/HCO3– cotransporter activity in both WKY and SHR PTE cells (please see the data supplement).

Because 1 of the most important roles of the Na+/HCO3– cotransporter is related to responses to intracellular acidification, and the loss of function in NBC1 gene results in a severe ocular and renal phenotype characterized by blindness, cataracts, glaucoma, and renal proximal tubular acidosis,32,33 it was considered worthwhile to evaluate the response of WKY and SHR PTE cells to acidosis induced by treatment with NH4+ (20 mmol/L) over 24 hours. Acidosis increased NBC activity in both WKY and SHR PTE cells (Figure 4), with no changes in pHi and cell viability (please see the data supplement). However, the increase in NBC activity induced by treatment with NH4+ (20 mmol/L) was greater in SHR PTE cells than in WKY PTE cells (Figure 4). By contrast, 24-hour treatment with acetazolamide (300 µmol/L), a potent inhibitor of the type II of carbonic anhydrase (CAII) that is highly expressed in renal PTE cells34 and strongly linked to Na+/HCO3– cotransporter activity,35 did not affect Na+-HCO3– activity in WKY and SHR PTE cells (Figure 4).


Figure 4
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  		Figure 4. Effect of NH4Cl (20 mmol/L; 24 hour) and acetazolamide (300 µmol/L; 24 hour) on Na+-HCO3– cotransporter activity in WKY and SHR renal PTE cells. Columns represent means of 6 to 8 experiments per group; vertical lines indicate SEM. Significantly different from corresponding control values (#P<0.05) or WKY from SHR (*P<0.05) using the Newman–Keuls test.

Na+-K+ ATPase Expression
The expression of Na+-K+ ATPase was evaluated in immortalized renal PTE cells from WKY rats and SHRs. Immunoblot analysis showed that the monoclonal anti-Na+-K+ ATPase antibody stained only 1 band in immortalized renal PTE cells from WKY rats and SHRs. As shown in Figure 5A, the level of expression of Na+-K+ ATPase was greater in SHR PTE cells than in WKY PTE cells. These results correlate well with the view that SHR PTE cells have an increased activity and expression of the apical NHE3 and Cl–/HCO3– exchanger,15–17 and the exit of Na+ across the basolateral membrane of the renal proximal tubule occurs mainly through the Na+-K+-ATPase.18


Figure 5
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  		Figure 5. (A) Expression of Na+-K+ ATPase and ß-actin in immortalized WKY and SHR renal PTE cells. Representative immunoblots are depicted on top of the bar graphs. (B) Representative trace of changes in Isc induced by amphotericin B (3.0 µg/mL) in monolayers of SHR and WKY PTE cells under control conditions. (B) Changes in Isc induced by amphotericin B (3 µg/mL) in monolayers of WKY and SHR PTE cells. Columns represent mean of 3 or 4 independent determinations; vertical lines indicate SEM. Significantly different from corresponding values in WKY PTE cells (*P<0.05) using Student’s t test.

Na+-K+ ATPase Activity
To evaluate Na+-K+-ATPase activity in WKY and SHR PTE cells, it was decided to use an electrophysiological method in which cell monolayers were continuously monitored for changes in Isc after the addition of amphotericin B to the apical cell side to increase the sodium delivered to Na+-K+-ATPase to the saturating level.8 In SHR cells, the addition of amphotericin B to the apical cell side induced an increase in Isc, this effect being dependent on the concentration of amphotericin B used (data not shown) with a maximum effect attained at 0.5 µg/mL of amphotericin B. However, in WKY cells, which had a very low electrical resistance (11.8±1.3 {Omega}cm2) when compared with SHR cells (588.9±97.7 {Omega}cm2), only the highest concentration of amphotericin B produced a slight increase in Isc (Figure 5B). As shown in Figure 5C, the amphotericin B (3 µg/mL)–induced increase of Isc in SHR PTE cells was greater than that in WKY PTE cells followed by recovery to baseline.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The results presented here indicate that immortalized renal PTE cells from WKY and SHRs are endowed with the Na+/HCO3– cotransporter, which accepts Na+ and HCO3– and is sensitive to DIDS. Moreover, the Na+/HCO3– cotransporter expressed in WKY and SHR PTE cells may be mainly related to the type 1 NBC that is predominantly expressed in renal proximal tubules.36 The results further indicate that NBC activity is lower in SHR PTE cells than in WKY PTE cells. This observation correlates well with the view that SHR PTE cells are endowed with lower NBC1 mRNA levels. A direct consequence of the low NBC1 activity in SHR PTE cells seems to be the high pHi observed when compared with WKY PTE cells. However, this result does not conflict with the view that SHR PTE cells have an increased Na+ reabsorptive capacity at the level of the renal proximal tubule, as a consequence of high activity of the main mechanisms for Na+ transport, namely, the NHE3 and Na+/K+ ATPase.15,37–39

The major role of NBC in renal proximal tubules is concerned with the regulation of pHi, bicarbonate metabolism, and cell volume.25 One particular and important function of the NBC is related with cell responses to intracellular acidosis with stimulation of the NBC activity that mediates the pHi recovery.25,40 After induction of acidosis by 24-hour treatment with NH4+, both WKY and SHR PTE cells responded with increases in NBC activity, with no changes in pHi and cell viability. Interestingly, increases in NBC activity in SHR PTE cells were greater than in WKY cells. Because the main function of NBC is not related to Na+ reabsorption, it is interesting to note that the activity and expression of Na+/K+ ATPase, which is the major mechanism of basal proximal tubular Na+ transport,18 is increased in SHR PTE cells. This result correlates well with the observation that SHR PTE cells have an increased activity and expression of the apical NHE3 and Cl–/HCO3– exchanger (SLC26A6)15–17 that work in parallel in the apical membrane of proximal tubules to promote the Na+ reabsorption.

An important functional property of these electrogenic transporters is their HCO3–:Na+ coupling ratio, which sets the transporter reversal potential and determines the direction of sodium bicarbonate flux.41 kNBC1 mediates basolateral sodium bicarbonate efflux in proximal tubules in a 3:1 stoichiometry ratio.11,13 By contrast, pancreatic NBC1 mediates basolateral sodium bicarbonate influx in pancreatic ducts in a 2:1 stoichiometry ratio.42 However, when kNBC1 is heterologously expressed in various systems, the HCO3–:Na+ stoichiometry is 2:1 in Xenopus oocytes43 and mouse collecting duct cells41 or 3:1 in mouse renal proximal convoluted tubule cells.41 Moreover, in studies using isolated renal proximal tubules, the stoichiometry of basolateral sodium bicarbonate cotransport is 2:1 or 3:1 depending on the experimental condition used.44,45 Although we have not studied the HCO3–:Na+ coupling ratio, several pieces of evidence suggest that NBC activity in immortalized renal PTE cells from WKY rats and SHRs requires 2 HCO3– anions to be cotransported with 1 Na+ cation (2:1 stoichiometry). First, there is evidence that inhibition of NBC activity during PKA activation is associated with changes in the HCO3–:Na+ stoichiometry.30,46 In fact, the PKA-mediated inhibition of kNBC1 is accompanied by a shift from 3:1 to 2:1 stoichiometry in response to phosphorylation of Ser982 in its carboxy terminus (kNBC1-Ser982) by cAMP-dependent PKA.29 However, activation of PKA by forskolin, an activator of adenylyl cyclase, and the membrane-permeable cAMP analog dibutyryl cAMP did not change the Na+/HCO3– cotransporter activity in both WKY and SHR PTE cells. This result suggests that NBC in WKY and SHR PTE cells works in 2:1 HCO3–:Na+ stoichiometry mode. Second, inhibition of CAII is normally associated with a reduction in HCO3– reabsorption through NBC inhibition.47 However, treatment with acetazolamide, an inhibitor of CAII, over 24 hours did not change the Na+/HCO3– cotransporter activity in both WKY and SHR PTE cells. This fits well the findings of Gross et al48 that clearly showed that acetazolamide inhibited the NBC1 activity only when the latter operated in 3:1 mode but had no effect on the HCO3– transport in the 2:1 stoichiometry mode. However, CAII may play an important role in enhancing the flux through the transporter when kNBC1-Ser982 is unphosphorylated. Finally, we used an experimental procedure to evaluate the Na+/HCO3– cotransporter activity that was associated with the NBC influx mode activity.40 Altogether, these findings suggest that immortalized renal PTE cells from WKY rats and SHRs are endowed with a kNBC that works in 2:1 stoichiometry mode.

In conclusion, the findings reported here show that SHR PTE cells are endowed with lower NBC activity than WKY PTE cells, but the former is more resistant to DIDS and responds better to acidosis. Moreover, the lack of response of NBC to PKA activation or to CAII inhibition is possibly related to the fact that NBC activity in both WKY and SHR PTE cells works in a 2:1 HCO3–:Na+ stoichiometry mode.

Perspectives
The physiological response to acidosis is 1 of the most important roles of the Na+/HCO3– cotransporter. The present study clearly demonstrated that the response to acidosis in WKY and SHR PTE cells is accompanied by increases in NBC1 activity. However, this effect was more pronounced in the SHR PTE cells. The molecular mechanism involved in the upregulation of NBC1 by acidosis was not investigated in this article. However, responses of NBC1 to acidosis appear to be linked to the classical mitogen-activated protein kinase (MAPK) through extracellular signal regulated kinase 1/2 activation.49–52 In line with the view that the regulation of HCO3– transport and pHi is related to MAPK, we have shown recently that angiotensin II–induced stimulation of Cl–/HCO3– exchanger involves the classical MAPK activation in both WKY and SHR PTE cells.17 Recent studies have demonstrated a role for renal hydrogen peroxide (H2O2) in renal function and hypertension.53–56 Moreover, H2O2 is an indirect product of the reduced nicotinamide-adenine dinucleotide phosphate oxidase, of which activity and expression are increased in the SHR.57 Immortalized SHR PTE cells have an increased H2O2 generation that is abrogated by the presence of apocynin, a reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor (R. Pedrosa, P. A. José, and P. Soares-da-Silva, unpublished data, 2006). Another interesting observation was that by Tabet et al,58 which linked vascular smooth muscle cell H2O2 to MAPK activation. Moreover, the activation of MAPK by H2O2 was upregulated in SHRs. All together it is likely that MAPKs are involved in the regulation of acidosis in WKY and SHR PTE cells, which needs to be confirmed by future studies.


*    Acknowledgments
 
Sources of Funding

This work was supported by Fundação para a Ciência e a Tecnologia, Programa Operacional Ciência e Inovação, and Fundo Europeu de Desenvolvimento Regional (POCI/SAU-FCF/59207/2004).

Disclosures

None.

Received October 22, 2006; first decision November 8, 2006; accepted February 1, 2007.


*    References
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