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(Circulation Research. 1997;80:853-860.)
© 1997 American Heart Association, Inc.

Articles

Activity and Expression of Na⁺-H⁺ Exchanger Isoforms 1 and 3 in Kidney Proximal Tubules of Hypertensive Rats

M. P. Kelly, P. A. Quinn, J. E. Davies, , L. L. Ng

From the Division of Clinical Pharmacology, Department of Medicine & Therapeutics, Leicester (UK) Royal Infirmary.

Correspondence to Dr Leong L. Ng, Division of Clinical Pharmacology, Department of Medicine & Therapeutics, Clinical Sciences Building, Leicester Royal Infirmary, Leicester, LE2 7LX, UK.

	Abstract

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Abstract Increased activity of the cellular Na⁺-H⁺ exchangers (NHEs), especially isoform 1 (NHE-1), is a recognized intermediate phenotype of hypertension. NHE activity has been demonstrated to be increased in proximal tubules of the spontaneously hypertensive rat (SHR). However, with the recent cloning of other members of this family of transporters, it is unclear which isoforms may contribute to this increased activity. We have used specific antibodies raised against glutathione-S-transferase fusion proteins of rat NHE-1 and NHE-3 to determine the relative contributions of these isoforms to the NHE activity in freshly isolated and cultured proximal tubule cells from SHR and Wistar-Kyoto (WKY) normotensive control rats. In freshly isolated proximal tubule cells, NHE activity was elevated almost 3-fold in SHR cells (P<.001), and in both rat strains, the contribution from NHE-1 and NHE-3 was approximately equal. Western blots of membranes from these cells showed equal amounts of NHE-1 protein in SHR and WKY cells. However, NHE-3 protein expression was increased 50% in SHR cells (P<.001), and this may account for the elevated activity of this isoform in SHR. The effect of culturing these cells in vitro was then examined. Although total NHE activity in both cell types was decreased during culture, this was mainly due to loss of expression of NHE-3 protein. NHE-1 activity was persistently elevated in the SHR cells in culture. These findings suggest that elevated NHE activity in SHR proximal tubules could be mediated by two mechanisms: (1) increased NHE-1 activity without any increased NHE-1 protein content that persists despite culture and may resemble those changes described for extrarenal tissues and (2) increased NHE-3 activity due to increased expression of NHE-3 protein. Disappearance of NHE-3 during culture implies that our culture conditions did not replicate the in vivo environment and may have removed the factors contributing to the increased NHE-3 expression in SHR cells.

Key Words: Na⁺-H⁺ antiport • proximal kidney tubule • hypertension

	Introduction

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The NHEs represent a growing family of membrane transporters that use the extracellular to intracellular Na⁺ gradient to drive H⁺ efflux from cells.^{1 2 3} Among the essential functions performed by this group of transporters are the regulation of cell volume and pH, a permissive role in cell proliferation, and the transepithelial transport of Na⁺.^{1 2 3} The cloning of the amiloride-sensitive, growth factor–activatable NHE isoform, called NHE-1,⁴ has enabled further isoforms to be identified. NHE-1 is present in most types of tissue⁵ and is likely to have a "housekeeping" role in regulating cell pH_i and volume. NHE-1 is not the only isoform present in Na⁺-reabsorbing epithelia, such as gut and kidney, where NHE-3 and NHE-4 have been described.^{5 6} Therefore, in the rat, it appears that kidney tissue contains mRNA of all four isoforms, but the abundance varies in the order NHE-3>NHE-1>>(NHE-2 and NHE-4).⁵ In hypertension, a variety of defects in the membrane transport of Na⁺ that may play a role in the genesis or maintenance of the raised blood pressure have been described. Activation of NHEs has been consistently documented in a wide variety of cells both in human essential hypertension and in a genetic rat model of hypertension, the SHR,^{7 8 9 10} and these changes persist in cultured cells.^{9 10}

Long-term control of blood pressure may depend on the pressure-natriuresis mechanism in the kidney, and in hypertension, the set point for maintenance of normal extracellular fluid volume is achieved at a higher renal perfusion or arterial pressure.¹¹ Evidence for the crucial role of the kidneys comes from transplantation experiments, in which an SHR kidney graft to the normotensive WKY rat leads to hypertension in the recipient¹² and SHR kidneys exhibit an altered pressure-natriuresis relationship.¹³ The proximal tubule is a major site for H⁺ secretion and Na⁺ and HCO^-₃ reabsorption and is therefore important for both acid-base and Na⁺ homeostasis.¹⁴ Recent evidence has pointed to an intrinsic difference in tubule function that could contribute to enhanced Na⁺ reabsorption in SHR and a resetting of the pressure-natriuresis mechanism. Brush border membranes¹⁵ and cultured proximal tubule cells^{16 17} from SHR exhibit enhanced NHE activity, which is present even in the prehypertensive rats.^{15 16} However, there was no analysis of which NHE isoforms were involved. The importance of NHEs in the pathophysiology of hypertension was stressed in the recent description of transgenic mice overexpressing NHE-1,¹⁸ in which increased NHE-1 expression in renal tubules was associated with the development of salt-sensitive hypertension.

In addition to any genetic factors influencing the increased NHE activity phenotype of hypertension, a number of extracellular signals leading to activation of NHE activity could contribute to the increased NHE activity of SHR proximal tubule cells. Glucocorticoids stimulated NHE activity in renal brush border vesicles,¹⁹ increased NHE-3 mRNA transcripts in ileal tissue,²⁰ and may be necessary for the elevation of blood pressure in SHR.²¹ Furthermore, growth factors such as EGF and insulin may also stimulate NHE activity.^{17 22}

In the present investigation, we have determined the NHE activity of freshly isolated proximal tubule cells from SHR and WKY rats and attributed activity to NHE-1 by its sensitivity to a specific inhibitor, HOE 694.²³ NHE-1 and NHE-3 proteins were detected using specific polyclonal antibodies to these isoforms. The effect of culturing proximal tubule cells in vitro on NHE activity and expression was also examined. Our findings suggest that increased NHE activity in SHR proximal tubules was due to both NHE-1 and NHE-3, although only NHE-3 expression was increased in vivo.

	Materials and Methods

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Materials
Ham's F-12 medium and DMEM were obtained from ICN Flow. FCS was supplied by Advanced Protein Products. Percoll, collagenase type IV, hyaluronidase type 1S, soybean trypsin inhibitor, fluorescamine, bovine insulin, EGF, selenite, transferrin, triiodothyronine, hydrocortisone, prostaglandin E₁, p-nitrophenyl phosphate, L--glutamyl-p-nitroanilide, glycylglycine, dexamethasone, rabbit polyclonal antibody to -actin, 3-aminopropyltriethoxy-silane, proteinase K, and the Sigma fast 3,3'-diaminobenzidine/urea hydrogen peroxide tablet kits were supplied by Sigma Chemical Co. Aquamount was obtained from Merck Ltd. The proximal tubule growth medium contained 1% FCS, 2 mmol/L glutamine, 10⁵ IU penicillin/L, 100 mg streptomycin/L, 24 mmol/L NaHCO₃ (pH 7.4 with 5% CO₂ in air), and the following additives: 25 µg/L prostaglandin E₁, 10 mg/L insulin, 5 mg/L transferrin, 30 nmol/L selenite, 10 µg/L EGF, 5 µmol/L hydrocortisone, and 1 nmol/L triiodothyronine. Protein-A Sepharose CL4B, glutathione Sepharose 4B, and pGEX-5x3 were from Pharmacia LKB Biotechnology. Protogel acrylamide solution was obtained from National Diagnostics. X-ray film was from Genetic Research Instrumentation Ltd. Hybond C–extra supported nitrocellulose, horseradish peroxidase–conjugated donkey anti-rabbit antibody, enhanced chemiluminescence Western blotting reagents, ¹⁴C molecular weight markers, and Aurodye were obtained from Amersham International. Molecular weight markers, N,N,N',N'-tetramethylethylenediamine, and ammonium persulfate were from Bio-Rad. BCECF-AM was from Cambridge Bioscience. The NHE-1–specific inhibitor HOE 694²³ was obtained from Dr W. Scholz, Hoechst AG. EIPA was a gift from Merck, Sharpe and Dohme, Rahway, NJ. Transparent polyethylene terephthalate–permeable supports (0.4 µm) were obtained from Becton Dickinson Labware.

Preparation of Proximal Tubules
SHR and WKY rats were obtained from the colony maintained at the Biomedical Services Unit, Leicester University. Eight-week-old rats were anesthetized with pentobarbital, and the kidneys were rapidly dissected out. These were immediately immersed in ice-cold preoxygenated DMEM. After stripping off the perirenal fat, the cortex was separated from the medulla. The method for preparing proximal tubule segments was modified from Gesek et al.²⁴ Briefly, cortical tissue was finely minced and transferred to siliconized flasks containing an enzyme solution saturated with 95% O₂/5% CO₂ (DMEM with 1 g/L collagenase type IV, 1 g/L hyaluronidase type 1S, 0.5 g/L soybean trypsin inhibitor, 2 mmol/L glutamine, and 10 mmol/L pyruvate, pH 7.4, with HEPES/NaOH). The tissue was gently shaken over 45 minutes, and digestion was usually complete by this period. At periods of 10 to 15 minutes, the supernatant containing crude tubule segments was removed from the siliconized flasks and recovered by brief centrifugation (60g for 30 seconds). These pellets were washed with oxygenated DMEM containing 10% FCS. All pellets from the same rat were combined and layered onto a preformed 43% Percoll gradient. After centrifugation at 1100g for 20 minutes, the fraction of proximal tubule segments at a density of 1.076 to 1.088 g/mL was removed, washed free of Percoll, and either seeded onto collagen-coated tissue culture flasks and glass coverslips or used immediately for further studies of NHE activity or for Western blotting of cell membranes.

Purity of the proximal tubule segments was checked after each preparation. The segments were granular and had a prominent brush border. In addition, enzyme markers for the proximal tubule, such as -glutamyltranspeptidase (EC 2.3.2.2) and alkaline phosphatase (EC 3.1.3.1), were assayed using the substrates L--glutamyl-p-nitroanilide and p-nitrophenyl phosphate, respectively, using established methods.²⁵ The mean±SEM -glutamyltranspeptidase activities of SHR and WKY proximal tubule preparations were enriched at 543±89 and 495±53 µmol·min^-1·g protein^-1. Alkaline phosphatase activities were also enriched, being 55.6±6.2 µmol·min^-1·g protein^-1 in SHR and 48.3±6.8 µmol·min^-1·g protein^-1 in WKY proximal tubules. These values are within the reported range for these enzyme markers in proximal tubules.^{25 26}

Cultures of Proximal Tubular Cells and Measurement of NHE Activity
After seeding the proximal tubule segments onto collagen-coated tissue culture flasks and glass coverslips, the cells were fed every day with the growth medium, leading to confluent cultures of cells after 4 days. Freshly isolated proximal tubule segments or cultured cells adherent on the coverslips were loaded with 5 µmol/L BCECF-AM for 1/2 hour in serum-free DMEM at 37°C, washed twice, and then left to deesterify the dye at room temperature for a further 1/2 hour. pH_i was determined in suspensions of freshly isolated tubule segments or in cells on coverslips clamped at an angle of 60° to the incident light²⁷ in a 37°C thermostatted sample compartment holder within a dual grating fluorometer (Deltascan, Photon Technology International Inc), with dual-wavelength excitation (500 and 439 nm; slit widths, 5 nm) and emission at 530 nm (slit width, 5 nm). The buffer used was HBSS composed of (mmol/L) NaCl 130, KCl 5, CaCl₂ 1.8, MgSO₄ 1, glucose 5, and HEPES 20, along with 1 g/L BSA, pH 7.4. The 500/439 ratios were calibrated using nigericin and monensin (5 µmol/L of each) in isotonic KCl buffers (replacing the NaCl of HBSS with KCl and omitting the BSA) of various pH values in the range 6.0 to 8.0, as described previously.²⁷ In order to determine the V_max of the NHEs, pH_i was clamped to 6.0 (near the V_max of NHE-1 and NHE-3^{28 29} ) after a 5-minute incubation in isotonic KCl buffer (pH 6.0) containing 5 µmol/L of monensin and nigericin. After removing the ionophores with BSA, the non–Na⁺-dependent rate of change of pH_i (dpH_i/dt) was measured over the first 20 seconds of records, using Na⁺-free medium (replacing the NaCl in HBSS with N-methyl-D-glucamine chloride, pH 7.4). The total dpH_i/dt was determined in HBSS, and the Na⁺-dependent dpH_i/dt was calculated as the difference between total and non–Na⁺-dependent measurements. Furthermore, the Na⁺-dependent dpH_i/dt that was due to NHE-1 was obtained by measuring H⁺ efflux into HBSS containing 10 µmol/L HOE 694, the recently described specific inhibitor of NHE-1.²³ The amiloride derivatives were too nonspecific for this particular application, since even EIPA inhibits both NHE-1 and NHE-3 isoforms. The residual Na⁺-dependent dpH_i/dt that was not HOE 694 sensitive was attributed to NHE-3, since Northern blots of mRNA from proximal tubule cells revealed only the presence of these two isoforms of NHE. After the addition of a 50 mmol/L NH₄Cl pulse and measurement of the change in maximal pH_i resulting from this, intrinsic buffering capacity in these cells was calculated using the following:

where pH_o is the pH of the extracellular buffer, pH_b and pH_a are the pH_i values before and after adding the NH₄Cl, and pK is the dissociation constant for NH₄. Fluxes were calculated as the product of buffering capacity and the Na⁺-dependent dpH_i/dt in the presence and absence of HOE 694.

Production and Characterization of Polyclonal Antibodies to Rat NHE Isoforms 1 and 3
GST fusion proteins containing the regulatory C-terminal domains of NHE-1 and NHE-3 were constructed so that homology was minimal. These selected NHE domains were ligated to the polylinker site of pGEX-5X3. The GST/NHE-1 construct consisted of amino acids 627 to 820 of the cytoplasmic domain of NHE-1, and the GST/NHE-3 construct consisted of amino acids 528 to 636 of NHE-3. The original rat NHE-1 and NHE-3 plasmids were obtained from Dr J. Orlowski, McGill University, Montreal, Canada. The induction and purification of these fusion proteins were as detailed in the manufacturer's published protocols (Pharmacia). After purification, fusion proteins were resolved further on SDS gels, and the bands were eluted from the gels. Over 6 months, four rabbits were immunized with monthly intravenous injections of 100 µg of the GST fusion proteins. The NHE-1– and NHE-3–specific immunoglobulins (referred to as G116 and G110, respectively) were further purified from polyclonal sera of the rabbits by elution from protein-A Sepharose CL4B beads. The specificity of these antisera was checked for cross-reactivity by (1) determining the reaction of each antiserum against the two fusion proteins and (2) establishing that each fusion protein was able to specifically abolish the reactivity of the antiserum for that fusion protein on Western blots. Furthermore, the NHE-1–specific antiserum G116 detected the same 95-kD proteins on Western blots of vascular myocyte extracts as our previously characterized antiserum G252 against human NHE-1,²⁷ whereas the NHE-3 antiserum G110 showed no specific immunoreactivity toward an 85-kD protein in these vascular myocyte extracts, which are known not to contain NHE-3.³⁰

Determination of NHE-1 and NHE-3 Abundance by Western Blotting
Freshly isolated proximal tubule cells were suspended in homogenization buffer consisting of (mmol/L) EDTA 5, phenylmethyl sulfonyl fluoride 1, phenanthroline 1, and iodoacetamide 1 along with 1 µg/mL pepstatin 1 and 2 µg/mL leupeptin 2, in PBS. Flasks of confluent tubule cells were snap-frozen with liquid nitrogen, and the cells were scraped into the homogenization buffer.²⁷ These cell suspensions were then disrupted by nitrogen cavitation at a pressure of 10 bars for 15 minutes at 4°C. After depressurization, the homogenate was centrifuged at 600g for 10 minutes at 4°C to obtain a postnuclear fraction. This fraction was then spun on an ultracentrifuge at 50 000g for 60 minutes to obtain a crude membrane pellet. These membranes were solubilized by adding Laemmli gel sample buffer (125 mmol/L Tris [pH 6.8], 4% SDS, 20% glycerol, and 0.004% bromophenol blue), sonicated briefly, and then boiled for 5 minutes. Protein concentrations were determined with a Bio-Rad detergent-compatible protein assay kit and were also checked by the fluorescamine protein assay. Extracts were resolved on 7.5% SDS-PAGE gels followed by electrotransfer onto supported nitrocellulose. These membranes were then blocked overnight with 10% low-fat milk powder (Marvel) in 20 mmol/L Tris (pH 7.4), 137 mmol/L NaCl, and 0.1% Tween 20 (TBS-Tween), followed by the addition of 2 µg/mL of G110 or 1 µg/mL of G116 antibody in 5% Marvel in TBS-Tween for 2 hours. The polyclonal rabbit antibody to -actin was used at a dilution of 1:2000 for 2 hours. After extensive washes in TBS-Tween, blots were developed with horseradish peroxidase–linked donkey anti-rabbit second antibody (1:1500 dilution) with enhanced chemiluminescence detection according to manufacturer's instructions (Amersham). Bands corresponding to NHE-1 (molecular mass, 95 kD) and NHE-3 (molecular mass, 85 kD) were quantified on a Bio-Rad densitometer.²⁷

Immunohistochemistry of Kidney Sections Using NHE-1– and NHE-3–Specific Antibodies
Kidneys were removed immediately after the rats were killed, cut longitudinally into quarters, and fixed in 10% formol saline overnight at 4°C. The slices were then embedded in paraffin wax blocks using standard protocols.³¹ Sections (4 µm) were cut and individually mounted onto microscope slides that had been pretreated with 3-aminopropyltriethoxy-silane. The sections were allowed to dry onto the slides for 16 hours at 42°C before use.

Sections were dewaxed in xylene and gradually rehydrated, passing through two changes of 99% ethanol, 95% ethanol, and, finally, distilled water. The sections were then incubated with a 5 µg/mL solution of proteinase K (Sigma) in 10 mmol/L Tris-HCl (pH 7.8) for 30 minutes at 37°C to improve access of the antibody to the target. The sections were washed gently several times with TBS-Tween, and endogenous peroxidase activity was then blocked by incubating the sections with a 3% solution of hydrogen peroxide in water for 10 minutes at room temperature. The sections were then washed gently for 2 minutes with distilled water. The sections were preincubated with preimmune horse serum (1:20) in TBS-Tween for 20 minutes at room temperature. Antibodies G110 and G116 were then preincubated with purified GST (2 µg/mL), since proximal tubules express GST, which could have cross-reacted with these antibodies because they were raised against GST fusion proteins. The sections were then incubated overnight (18 hours) at 4°C in TBS-Tween with these preabsorbed antibodies (G110, 24 µg/mL; G116, 12 µg/mL). After the incubation, the sections were washed with PBS for 10 minutes (twice). They were then incubated with horseradish peroxidase–linked donkey anti-rabbit second antibody (1:150 dilution in TBS-Tween) for 30 minutes at room temperature and washed with PBS for 10 minutes (twice). Antibody-linked peroxidase activity was then demonstrated using a Sigma fast 3,3'-diaminobenzidine/urea hydrogen peroxide tablet kit. The sections were washed in running tap water for 5 minutes and counterstained with Mayer's hematoxylin solution (Sigma). The sections were then washed in running tap water for 10 minutes and rinsed in distilled water, the residual fluid was carefully soaked up with a tissue, and the sections were mounted with Aquamount and a coverslip before microscopic examination.

mRNA Isolation and Analysis
Total cellular RNA was extracted from proximal tubule preparations by a modification of a previously described method using selective precipitation in 6 mol/L urea and 3 mol/L LiCl.³² RNA concentrations were determined by spectrophotometry at 260 nm, and aliquots were resolved on agarose gels stained with ethidium bromide to exclude RNA degradation. Northern blotting was then performed using standard protocols.³³ Briefly, 60 µg of total RNA per sample was electrophoresed through 1.2% (wt/vol) agarose gel containing 2.2 mol/L formaldehyde and transferred to nylon membranes (Hybond, Amersham). Membranes were initially probed with a 1.3-kb Pst I–Pst I cDNA fragment from rat kidney NHE-3 cDNA (clone RKNHE2-1 from Dr J. Orlowski, McGill University, Montreal, Canada)⁵ to determine NHE-3 mRNA levels. This was followed by probing with a 1.2-kb human fetal liver cDNA for GAPDH, the mRNA of which is constitutively expressed in all cells. All probes (20 ng per reaction) were radioactively labeled with deoxy-[-³²P]CTP by the random primer method. Probes had specific activities higher than 1x10⁹ cpm/µg DNA, as determined by trichloroacetic acid precipitation and radioscintillation counting.

For all probes, prehybridization (4 hours) and hybridization (16 hours) were carried out at 45°C in a buffer containing 50% (vol/vol) formamide, 6x SSPE (20x SSPE consists of 3.6 mol/L NaCl, 0.2 mol/L sodium phosphate, and 20 mmol/L EDTA, pH 7.7), 5x Denhardt's solution (100x Denhardt's solution consists of 2% [wt/vol] each of BSA, Ficoll 400, and polyvinyl-pyrolidone), 0.5% (wt/vol) SDS, 6% polyethylene glycol 6000, and denatured salmon sperm DNA (200 µg/mL). After hybridization, membranes were washed to a stringency of 0.1x SSPE and 0.1% SDS at 65°C. Autoradiography was carried out at -70°C using X-OMAT AR film (Eastman Kodak) and intensifying screens. Molecular weights of bands were estimated from a concurrently run RNA molecular weight ladder. Before reprobing, membranes were stripped by immersing in a solution of 10 mmol/L Tris (pH 8.0) and 0.2% SDS at 100°C with agitation until the solution cooled to room temperature. Intensities of autoradiographic signals were quantified on a Bio-Rad densitometer. All samples to be compared were always run, hybridized, and autoradiographed together to allow for variabilities related to loading and transfer. Densitometric values for NHE-3 mRNA for each sample were normalized by dividing with the corresponding GAPDH mRNA values before comparisons were made.

Statistics
Results are expressed as mean±SEM, and comparisons were determined by Student's t test, performed on an Oxstat statistics package (Microsoft Corp). Two-tailed values of P<.05 were considered significant; n refers to the number of animals studied.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fig 1 shows the pH_i measurements in freshly isolated proximal tubule cells and the effect of culture in vitro for 4 days. In freshly isolated cells, pH_i was similar between SHR and WKY preparations. Culture resulted in a higher level of pH_i in both cell types (P<.001, n=12), with a significantly higher pH_i in cultured tubule cells from SHR compared with WKY rats (P<.001). When the freshly isolated proximal tubule cells were clamped to pH_i 6.0, addition of HBSS led to a rapid increase in pH_i (Fig 2A). In Na⁺-free media (NMG in Fig 2), the rate of increase of pH_i was much slower in both SHR and WKY cells. The identity of NHEs was further elucidated using the NHE-1–specific inhibitor HOE 694. This led to a reduction of the Na⁺-dependent component of pH_i recovery by about half. The mean H⁺ efflux data are presented in Fig 1, which shows that total Na⁺-dependent H⁺ efflux was increased in SHR compared with WKY freshly isolated tubules (P<.001, n=12) and that the components due to NHE-1 (HOE 694 sensitive) and NHE-3 (HOE 694 insensitive) were elevated in SHR cells (P<.001 for both). We have inferred that the HOE 694–insensitive component of H⁺ efflux was mediated by NHE-3, since these two NHE isoforms were the only ones detected on mRNA blots. By contrast, the non–Na⁺-dependent component of H⁺ efflux (which could be mediated by passive H⁺ permeability mechanisms or H⁺-ATPase^{15 16} ) was very similar in both cell types. The amiloride derivative EIPA (50 µmol/L) did not distinguish between NHE-1 and NHE-3, inhibiting both isoforms so that the remaining H⁺ efflux rate in SHR proximal tubules was 11.3±0.6 mmol·L^-1·min^-1 (n=6), very similar to the values obtained for Na⁺-independent H⁺ efflux (10.9±0.8 mmol·L^-1·min^-1). Very similar values were obtained for WKY proximal tubules.

View larger version (32K): [in this window] [in a new window]   Figure 1. Determinations of pHi, total NHE activity at pHi 6.0, HOE 694–sensitive H+ effluxes due to NHE-1, HOE 694–insensitive Na+-dependent H+ effluxes due to NHE-3, and Na+-independent H+ effluxes in freshly isolated proximal tubules and cultured tubule cells from SHR and WKY rats. Values are mean±SEM. *P<.001 for SHR compared with respective WKY measurements (n=18 experiments in each group).

View larger version (15K): [in this window] [in a new window]   Figure 2. A, A typical example of fluorometric traces from freshly isolated proximal tubule cells from SHR and WKY rats. The pHi was clamped to 6.0, and the rate of pHi recovery was measured in NaCl (HBSS) to determine total rate of dpHi/dt and in Na+-free media (NMG) to measure Na+-independent pHi recovery. The effect of HOE 694 on pHi was investigated using HBSS containing 10 µmol/L HOE 694. B, A typical example of pHi recovery traces from cultured tubule cells from SHR and WKY rats in the presence (NaCl) and absence (NMG) of extracellular Na+. Cultured cells incubated with HOE 694 in HBSS produced traces identical to those in the Na+-free NMG buffers.

These experiments were repeated on proximal tubule cells cultured on coverslips, and typical H⁺ efflux traces are presented in Fig 2B. The total H⁺ efflux in SHR cells was markedly elevated compared with WKY cells, although the non–Na⁺-dependent component of H⁺ efflux was again similar in the two strains. Thus, the Na⁺-dependent component of H⁺ efflux was elevated in SHR cultured cells. However, HOE 694 inhibited H⁺ efflux so that in the presence of this inhibitor, traces were identical to that of the Na⁺-free (NMG) traces in Fig 2B. This implied that all the NHE activity in cultured cells was due to NHE-1. The mean data for cultured proximal tubule cells is presented in Fig 1, where the majority of Na⁺-dependent H⁺ efflux is attributed to NHE-1 with little NHE-3 activity. This was further substantiated by the presence of only NHE-1 message in mRNA blots of cultured cells. Both Na⁺-dependent H⁺ efflux and H⁺ efflux due to NHE-1 were significantly increased in cultured SHR proximal tubule cells compared with WKY cells (P<.001, n=18). Non–Na⁺-dependent H⁺ efflux was similar in both cell types.

The presence of these two isoforms was sought using Western blotting of crude membrane extracts from freshly isolated and cultured proximal tubule cells. Antibody G110 reacted with the GST/NHE-3 fusion protein on Western blots, and its reactivity against this fusion protein was abolished when the antibody (2 µg/mL) was coincubated with an excess of the fusion protein (4 µg/mL). In contrast, G116 reacted with the GST/NHE-1 fusion protein only, and this was neutralized by an excess of that fusion protein. Furthermore, G110 detected an 85-kD protein in crude kidney extracts (which are known to contain NHE-3) but not in vascular myocyte extracts (which only contain NHE-1³⁰ ). In contrast, G116 detected a 95-kD band in both vascular myocyte and crude kidney extracts, both of which contain NHE-1.^{5 6 30}

Using the NHE-3–specific antibody G110, Western blots revealed the presence of an 85-kD protein in SHR and WKY extracts (Fig 3A through 3C). This immunoreactivity was higher in SHR extracts and could be specifically attenuated by the inclusion of the GST/NHE-3 fusion protein (Fig 3B). The specifically immunoreactive band was well defined, in agreement with data suggesting that NHE-3 is not N-glycosylated.^{34 35 36} There were two nonspecific bands that flanked the NHE-3 immunoreactivity, and these were still detected in blots using G110 preabsorbed with the GST/NHE-3 fusion protein. These proteins were more abundant in the SHR extracts and were furthermore detected even with preimmune serum from the rabbit from which G110 was raised. Thus, they may represent nonspecific reactivity before the immunization protocol. These differences in NHE-3 content between SHR and WKY extracts from freshly isolated proximal tubules were not due to differences in protein loading (since equal amounts of protein were loaded in the tracks). Furthermore, on probing the nitrocellulose filters with a polyclonal anti–-actin antibody, very similar amounts of this cytoskeletal protein were present between SHR and WKY extracts (Fig 3C). Differences in NHE-3 content were therefore unlikely to be due to differences in protein loading between tracks.

View larger version (25K): [in this window] [in a new window]   Figure 3. Western blot of membrane extracts from SHR (S) and WKY (W) freshly isolated proximal tubule segments, detected by the NHE-3–specific antibody G110, is shown (A). Equal amounts of protein (25 µg) were loaded per lane. In the presence of the GST/NHE-3 fusion protein, the immunoreactivity of this membrane protein was abolished (B). Incubation with anti–-actin antibody indicated that there were insignificant differences in the amounts of this cytoskeletal protein between SHR and WKY extracts (C). A similar blot of membrane extracts was detected by the NHE-1–specific antibody G116 (D). An excess of GST/NHE-1 fusion protein abolished the immunoreactivity of NHE-1 (E).

In Fig 3, panels D and E show typical Western blots of extracts from freshly isolated cells, detecting NHE-1 with the antibody G116. A 95-kD protein was present in both SHR and WKY extracts, with approximately equal amounts of NHE-1 protein. Furthermore, the molecular mass heterogeneity of this NHE-1 immunoreactivity may be due to different patterns of N-linked glycosylation.^{2 3 22 27} However, patterns were similar between WKY and SHR extracts. NHE-1 immunoreactivity was abolished by preincubation with the GST/NHE-1 fusion protein (Fig 3E).

In cultured proximal tubule cells, only NHE-1 protein was present in Western blots of membrane extracts (Fig 4). We compared NHE-1 protein content of cultured cell extracts from SHR and WKY rats, and this indicated that the NHE-1 protein of SHR cultured cells was reduced compared with that of WKY cells extracted after the same period of time in culture. Comparing extracts from cultured cells with freshly isolated tubules, the NHE-1 immunoreactivity was reduced in the cultured cells of both strains (Fig 5). In both SHR and WKY cultured cell extracts, no immunoreactive NHE-3 could be detected (data not shown). NHE-3 immunoreactivity was not detectable even when cells were cultured on 0.4-µm permeable supports or when hydrocortisone (5 µmol/L) was substituted with the glucocorticoid dexamethasone (100 nmol/L).

View larger version (23K): [in this window] [in a new window]   Figure 4. Western blot of membrane extracts from 7-day cultures of WKY (W) and SHR (S) proximal tubule cells, using G116 to detect NHE-1 protein, is shown (A). Excess of the GST/NHE-1 fusion protein abolished the NHE-1 immunoreactivity (B). There was no significant NHE-3 immunoreactivity with the antiserum G110.

View larger version (37K): [in this window] [in a new window]   Figure 5. Western blot of membrane extracts from freshly isolated WKY (W) and SHR (S) tubules and identical tubule cells cultured for 7 days, using the NHE-1–specific antibody G116 to detect this NHE isoform. P0 indicates extracts from 7 day cultures ("primary cultures").

The immunoblot data are summarized in Fig 6, where all immunoblot densities have been normalized to a value of 1 in the WKY freshly isolated cell data. Thus, the mean NHE-3 protein content of SHR freshly isolated cells significantly exceeds that of WKY cells (P<.001, n=18), whereas the NHE-1 protein contents are similar in these cells. The NHE-3/NHE-1 protein ratios are significantly elevated (P<.001) in freshly isolated SHR cells (1.54±0.09) compared with WKY cells (1.00±0.06, n=18). In contrast, there was no significant NHE-3 expression in cultured cells of either type, and the expression of NHE-1 was reduced in both cell types during culture, but more markedly so in the SHR cells.

View larger version (16K): [in this window] [in a new window]   Figure 6. Histograms showing the relative abundance of NHE-1 (column A) and NHE-3 (column B) in freshly isolated and cultured proximal tubule cells from SHR and WKY rats. All data have been normalized to a value of 1 in the NHE-1 or NHE-3 data of freshly isolated WKY cells. Values are mean±SEM. *P<.001 for SHR compared with respective WKY measurements (n=18 experiments in each group).

Immunohistochemistry of formalin-fixed sections of SHR and WKY kidneys with the NHE-1– and NHE-3–specific antibodies confirmed the basolateral localization of NHE-1 and the brush border localization of NHE-3 in proximal tubule cells that have been reported by others^{34 36 37 38} in renal and gut epithelia. There was no evidence of any abnormal localization of the NHE isoforms in SHR proximal tubule cells.

Since protein content of NHE-3 was significantly elevated in freshly isolated SHR cells, we investigated the NHE-3 mRNA abundance in these cells from SHR and WKY. Fig 7 illustrates a Northern blot of total mRNA extracts of both cell types, detected with an NHE-3–specific probe. There was increased NHE-3 mRNA abundance in SHR cells. Normalized to the GAPDH signal, the mRNA abundance in SHR tubule cells was increased by 62±5% compared with WKY tubule cells (P<.001, n=4). In agreement with the protein immunoblot findings, there was no detectable NHE-3 mRNA in extracts from cultured cells of either type. NHE-1 mRNA levels were similar in SHR and WKY fresh proximal tubule extracts and were reduced in cultured cell extracts (data not shown).

View larger version (31K): [in this window] [in a new window]   Figure 7. A Northern blot of mRNA extracts from SHR (S) and WKY (W) cells, showing an 5-kb message when membranes were detected with an NHE-3–specific probe. The GADPH probe demonstrated an equivalent signal for mRNA from both rat strains.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The absorption of Na⁺ and HCO₃^- by the proximal tubules is mediated to a large extent by the operation of NHEs.¹⁴ In hypertension, there is evidence that NHE-1, the isoform that is most frequently studied, exhibits increased activity in a large variety of cell types (reviewed in Reference 7⁷ ). Moreover, when NHE-1 is overexpressed in the brush border of kidney tubules of transgenic mice, salt-sensitive hypertension results from the increased renal Na⁺ reabsorption.¹⁸ In the SHR, freshly isolated proximal tubules^{16 17} and brush border membranes¹⁵ have increased NHE activity, although the isoform involved has not been clarified. Recent evidence suggests that NHE-1 is expressed predominantly on the basolateral membrane of renal tubules,^{34 37} whereas NHE-3 is expressed on the brush border membrane of proximal renal tubules,^{34 38} and we confirmed this distribution in the SHR and WKY proximal tubules. This pattern of expression also conforms to that in gut epithelium.³⁶ However, Weinman et al³⁷ have suggested that NHE-1 is also expressed on the brush border membranes of renal tubules in addition to its basolateral localization. In view of these findings, it is likely that NHE-1 predominantly plays a housekeeping pH_i-regulatory role and that NHE-3 (and possibly NHE-1) is responsible for the transepithelial reabsorption of Na⁺. Since the phenomenon of pressure natriuresis is blunted in SHR kidneys,¹³ there could be an enhanced tubular reabsorption of Na⁺.¹¹ Although a predominant basolateral localization of NHE-1 may make its involvement in Na⁺ reabsorption less likely, recent evidence has indicated an interaction between basolateral and brush border NHE activity, so that there is a functional coupling between these two activities.³⁹ Increases in basolateral NHE activity may lead to increased brush border NHE activity and result in HCO₃^- reabsorption.³⁹

The present studies on freshly isolated proximal tubules show that NHE activity is enhanced in SHR and that both the HOE 694–sensitive and –insensitive moieties of Na⁺-dependent H⁺ efflux are elevated. The HOE 694–insensitive fraction is likely to be mediated by NHE-3, since these were the only two isoforms detectable in mRNA blots from these cells. The increased NHE-3 activity is reflected in an increased expression of NHE-3 protein in SHR proximal tubules, and this in turn reflected increased NHE-3 mRNA content (although it remains to be determined if the increase in NHE-3 mRNA is due to increased transcription of the gene or reduced transcript turnover). The present findings that NHE-3 was an 85-kD protein concur with other reports of the size of this protein in rat tissue,^{34 36 38} in which the size has been estimated to range between 80 and 87 kD. Using our antibody G110, two nonspecific protein bands that flank the NHE-3 were evident even after preabsorption with the GST/NHE-3 fusion protein, and although these proteins appear more prevalent in the SHR extracts, this was not due to differences in protein loading. The nature of these proteins is at present unknown.

In contrast, even with an increased NHE-1 activity in SHR tubules, there was no concomitant increased expression of NHE-1 protein. The finding of increased NHE-1 activity without an enhanced NHE-1 protein content was also described in SHR vascular myocytes⁴⁰ and in lymphoblasts from patients with hypertension.⁴¹ These findings are also consistent with the documentation of no differences in NHE-1 mRNA levels in vascular myocytes of SHR and WKY rats.^{30 42} Thus, the NHE-1 phenotype may result from a more generalized derangement of NHE-1 regulation that was not specific to the kidney and that could be secondary to altered NHE-1 phosphorylation.^{40 41} These changes in NHE-1 and NHE-3 activity in SHR proximal tubules are of a sufficient magnitude to lead to hypertension over a period of time, since recent research on modeling the Na⁺ reabsorption of proximal tubules has suggested that only a 14% increase in NHE activity may increase Na⁺ reabsorption (by 8%) and raise blood pressure.⁴³ However, because of the possible functional coupling of basolateral and brush border NHE activity,³⁹ it is uncertain at present whether the increased basolateral NHE-1 activity in SHR proximal tubules could secondarily lead to apical NHE-3 activation.

When proximal tubule cells are cultured in vitro, the expression of NHE-1 protein falls in both cell types. pH_i was higher in these proliferating cultured cells. However, NHE-1 activity remains similar to that in freshly isolated cells, and the difference between the elevated NHE-1 activity in SHR and that in WKY cells persists. The set point of NHE-1 in both cell types may have increased in culture in which the cells had undergone phenotypic modulation from quiescence to proliferation, a finding that agrees with previous data demonstrating that proliferating cells had an elevated pH_i, a lower NHE-1 protein content, and a higher turnover number than nonproliferating cells.⁴⁴ In both fresh and cultured cells, NHE-1 showed molecular weight heterogeneity expected of a glycoprotein.²² Thus, even in cultured tubule cells, the phenotype of elevated NHE-1 activity without an increased NHE-1 protein content persists in SHR cells, implying the dependence of this particular phenotype on the genetic program, as has been suggested for other cell types in SHR and human hypertension.^{7 27 30 40 41} In contrast, there was no evidence of any HOE 694–insensitive Na⁺-dependent H⁺ efflux in either cultured cell type, and this was supported by the absence of detectable NHE-3 mRNA or NHE-3 protein in these extracts. When freshly isolated proximal tubule cells are compared with cultured cells on a substrate in vitro, it is unclear whether this reduction in NHE-3 expression resulted from a transition from quiescence to active proliferation and, hence, loss of differentiation of specialized function. Substitution of the hydrocortisone with 100 nmol/L dexamethasone or growing the cells on permeable supports to maintain their polarity did not lead to NHE-3 expression. Other possibilities could be the removal of paracrine, neuroendocrine, or other environmental factors that are present in vivo that could have contributed to the higher NHE-3/NHE-1 protein ratio in the SHR and may have been responsible for sustaining the enhanced NHE-3 expression in SHR tubules.

In summary, our investigations have provided evidence that the activities of both NHE-1 and NHE-3 are elevated in the proximal tubule of SHR, although the mechanisms for this increased activity differ. NHE-3 activity may be elevated from an increased expression of its protein, whereas NHE-1 activity may be elevated from a posttranslational processing mechanism that may be more generalized and present in a wide variety of SHR tissues. Only the latter housekeeping NHE-1 phenotype is preserved in cultured tubule cells from SHR and may be determined by the genetic program, and the specialized NHE-3 protein may be more susceptible to loss of environmental factors that are permissive for its continued expression. The nature of these permissive factors is currently unknown. Nevertheless, the cultured tubule cells represent a model for further investigations defining the mechanism underlying the NHE-1 phenotype of SHR.

	Selected Abbreviations and Acronyms

 

EGF = epidermal growth factor EIPA = ethylisopropyl amiloride GST = glutathione-S-transferase HOE 694 = 3-methylsulfonyl-4-piperidinobenzoyl guanidine NHE = Na+-H+ exchanger SHR = spontaneously hypertensive rat(s) WKY = Wistar-Kyoto

	Acknowledgments

 
We are grateful for the generous support of the British Heart Foundation. We are especially grateful to H.R. Patel, Division of Vascular Medicine, University of Leicester, for sharing his expert knowledge of immunohistochemistry.

Received August 5, 1996; accepted March 10, 1997.

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