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Am J Physiol Renal Physiol 291: F129-F139, 2006. First published February 21, 2006; doi:10.1152/ajprenal.00290.2005
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Increased NHE1 expression is associated with serum deprivation-induced differentiation in immortalized rat proximal tubule cells

Luciene Regina Carraro-Lacroix,1 Marco Antonio Ramirez,2 Telma M. T. Zorn,3 Nancy Amaral Rebouças,1 and Gerhard Malnic1

Departments of 1Physiology and Biophysics and 3Cell Biology and Development, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil; and 2Biomedical Department, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile

Submitted 15 July 2005 ; accepted in final form 10 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We studied the proton secretion mechanisms involved with pHi regulation in immortalized rat proximal tubule cells (IRPTC), a SV40-immortalized cell line derived from rat proximal tubule, and characterized the effects of serum deprivation on them. Using pHi measurements with the fluorescent probe BCECF, we demonstrated that the IRPTC express both Na+/H+ exchanger and H+-ATPase, but only NHE1 is modulated by serum deprivation. In these cells, 24 h of serum starvation increased pHi from 7.08 ± 0.008 (n = 34) to 7.18 ± 0.018 (n = 33) as well as the pH recovery rate from intracellular acidification with NH4Cl from 0.29 ± 0.022 pH U/min (n = 14) to 0.50 ± 0.024 pH U/min (n = 14), without modifying their buffering capacity. These effects were followed by several modifications in morphological features, indicating an increase in differentiation status. The altered activity of NHE1 was consistent with an increase of both transcription and translation of the antiporter, as the utilization of actinomycin D and cycloheximide significantly inhibited the upregulation of NHE1 induced by serum withdrawal. Inhibition of tyrosine phosphorylation by genistein blocked the serum deprivation-dependent activation of NHE. Moreover, the pharmacological inhibition of MEK1/2, the upstream activator of ERK1/2 by UO-126, significantly inhibited the stimulatory effect of serum starvation on Na+/H+ exchanger activity, whereas the putative p38 MAPK inhibitor SB-203580 failed to cause any effect on pHi recovery rates. Our findings indicate that during IRPTC differentiation by serum deprivation, there was a net enhancement of NHE1 activity. This upregulation of NHE by serum removal was consistent with an increase of RNA and protein synthesis of the exchanger, which depends on tyrosine kinase phosphorylation and ERK pathway activation.

cell pH; tyrosine kinase; MAPK

INTRACELLULAR pH (pHi) regulation is extremely important, because virtually every biological process is pH sensitive. Changes in pHi may be important in modulating transepithelial solute transport, in modifying biochemical or endocrinological function, and in controlling cell growth and differentiation (3). In general, pHi, in addition to being clamped in a narrow range by intracellular buffers, is regulated by transport systems located in the plasma membrane. Among these mechanisms, the most important are the Na+/H+ exchanger, the vacuolar H+-ATPase, the Cl–/HCO3– exchanger, and Na+/HCO3– cotransport (20, 27).

The mammalian proximal tubule (PT) is one of the most important sites of transepithelial acid secretion. The mechanisms of this transport have been detailed in a number of investigations (2, 21, 24). Several PT cell lines, such as LLC-PK1 and OK, have been extensively used to study such mechanisms in vitro. The findings show that these cell lines present several properties common to cells studied in vivo, indicating that they may be used as cell models for PT epithelium (16, 34, 50). However, as many physiological and molecular studies (using rat cDNA and cRNA probes) have been performed in rat species, the development of immortalized rat PT cell lines is of great benefit to study PT systems (38). Thus in 1995, an immortalized PT cell line was developed from primary cultures of rat kidney using simian virus (SV)-40, the immortalized rat proximal tubule cells (IRPTC) (38). This cell line expresses a large number of PT markers and seems to have much utility as a cell system in which multiple aspects of the cellular and molecular functions of the PT may be examined (17, 38).

It has been documented that increased pHi caused by the Na+/H+ exchanger (NHE1) provides either a permissive or an obligatory signal for cell proliferation (15, 18) and differentiation (32, 45). In some cell types, the elevation of intracellular pH has been shown to play an important role in cell growth (15). Cell differentiation results also in important alterations in growth patterns and involves activation of a number of genes, but the molecular mechanisms underlying these alterations remain poorly understood (13). Rao et al. (32) reported the regulation of Na+/H+ expression and its role in retinoic acid-induced differentiation of HL60 cells. Immediately before differentiation into granulocyte-like cells, the activity of the Na+/H+ exchanger increases and remains elevated into differentiation. Moreover, there was an 18-fold increase in mRNA levels as well as a 7-fold increase in protein levels for NHE1 (32). This dramatic rise in antiporter levels suggests that Na+/H+ exchanger gene expression and differentiation are closely linked.

The present study was undertaken to characterize the HCO3–-independent mechanisms involved with pHi regulation in IRPTC and the influence of serum deprivation, a stimulus for cell differentiation, on these mechanisms. We found that IRPTC express both Na+/H+ exchanger and H+-ATPase, but only the first mechanism is modulated by serum withdrawal. In these immortalized cells, morphological differentiation could be initiated by removal of serum from the culture media, accompanied by an enhancement in NHE1 activity. In addition, this serum deprivation-induced NHE1 upregulation is consistent, at least in part, with an increase of both transcription and translation of the exchanger, which depends on tyrosine kinase and MEK1/2 activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials

IRPTC were provided by Dr. J. R. Ingelfinger (Pediatric Renal Research Laboratory, Massachusetts General Hospital, Boston, MA). DMEM, heat-inactivated fetal bovine serum, sodium pyruvate, nonessential amino acids, and penicillin/streptomycin were purchased from GIBCO (Grand Island, NY). Schering 28080 was obtained from Gerhard Giebisch (Yale University, New Haven, CT), BCECF-AM from Molecular Probes (Eugene, OR), actinomycin D from Calbiochem (San Diego, CA), N-ethylmaleimide from ICN Biomedicals (Irvine, CA), and S3226 and HOE-694 were provided by Jurgen Punter (Hoechst). Rabbit Na+/H+ exchanger-1 (NHE1) polyclonal antibody was purchased from Chemicon International (Temecula, CA). Anti-NHE1 (N1P1) was provided by J. Noel (Université de Montréal, Montreal, PQ). Anti-NHE3 (2B9) monoclonal antibody was kindly provided by D. Biemesderfer and P. Aronson (Yale University). The composition of the solutions used in this work is described in Table 1. The osmolality of solutions ranged from 290 to 310 mosmol/kgH2O. The Na+ concentration in nominally Na+-free solutions was less than 1 meq/l. Reagents were obtained from Sigma (St. Louis, MO) unless otherwise specified.


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  		Table 1. Composition of solutions

 
Methods

Cell culture. IRPTC were used from passage 33 to 43. Serial cultures were maintained in DMEM supplemented with 45 mM NaHCO3, 25 mM HEPES buffer, 0.1 mM sodium pyruvate, 0.01 mM nonessential amino acids, 5% (vol/vol) heat-inactivated fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells were grown at 37°C, 95% humidified air-5% CO2 (pH 7.4) in a CO2 incubator (Lab-Line Instruments, Melrose Park, IL). The cells were harvested with trypsin-EGTA (0.02%), seeded on sterile glass coverslips, and incubated again for 72 h in the same medium to become confluent. In certain studies, as noted, cells were placed in serum-free medium 24 h before the experiments.

Measurement of pHi by fluorescence microscopy. Intracellular pH was measured spectrofluorimetrically at 37°C with the fluorescent pH-sensitive probe, BCECF. Cells grown to confluence on glass coverslips were loaded with the dye by exposure for 5 min to 12 µM BCECF-AM in the control solution (solution 1; Table 1). The acetoxymethyl ester form of BCECF enters the cell and is rapidly converted to the anionic-free acid form by intracellular esterases. After the loading period, the glass coverslips were rinsed with the control solution to remove the BCECF-containing solution and placed in a thermoregulated chamber mounted on an inverted epifluorescence microscope (Nikon, TMD). The measured area under the microscope had a diameter of 260 µm and contained on the order of 40 cells. Bathing solutions were rapidly exchanged without disturbing the position of the coverslips. Fluorescence was monitored using alternately 440 nm (pH insensitive) or 495 nm (pH sensitive) as excitation wavelenghts utilizing a xenon light source. Emission was measured at 530 nm by a photomultiplier-based fluorescence system (Georgia Instruments, PMT-4000) at time intervals of 5 s. pHi was calculated from the fluorescence emission ratio of the two excitation wavelengths using a standard calibration procedure based on the use of 10 µM nigericin in high-potassium Ringer (solution 2; Table 1), at pH 5.91, 7.04, or 8.01 (39).

Cell pH recovery. Cell pH recovery was examined after the acidification of pHi with the NH4Cl pulse technique (6) after 2-min exposure to 20 mM NH4Cl (solution 3; Table 1), in the following situations: in the presence of external 145 mM Na+ (control; solution 1; Table 1) or in the absence of external Na+ (solution 4; Table 1), with or without several inhibitors, as described later. In all the experiments, we calculated the initial rate of pH recovery (dpHi/dt, pH U/min) from the first 2 min after the start of the pHi recovery curve by linear regression analysis.

Determination of intrinsic buffering capacity. Intracellular buffer capacity (betai) was determined using the technique described by Boyarsky et al. (7) and calculated as described by Weintraub and Machen (48). In our system, betai refers to the ability of intrinsic cellular components excluding HCO3–/CO2 to buffer changes in pHi, and thus estimations of betai were carried out using HEPES-buffered solutions. The H+ membrane transporters were blocked by a 0 mM Na+ solution plus 0.1 µM concanamycin (CCM), because betai is most precisely estimated in cells whose pHi-regulatory mechanisms are blocked. After removal of Na+ from the external medium, the cells were exposed to a HEPES-buffered solution containing 50 mM NH4Cl, which was then stepwise reduced to 1 mM (in 50, 20, 10, 5, 2.5, and 1 mM steps). Calculation of betai was performed according to the formula betai = {Delta}[NH4+]i/{Delta}pHi, where the intracellular NH4+ concentration ([NH4+]i) was calculated from the Henderson-Hasselbalch equation on the assumption that NH3i = NH3o.

Analysis of Na+/H+ exchanger isoform expression by RT-PCR. Total RNA was isolated from IRPTC by using the guanidine method (11) with some modifications. mRNA purification from total RNA was made with a Oligotex mRNA kit using the procedures described by the manufacturer (Qiagen). First-strand cDNA synthesis was carried out using the SuperScript Preamplification System as recommended by the manufacturer (Invitrogen). Primers for rat NHE1, NHE2, NHE3, and NHE4 were designed from published sequences (GenBank accession nos. NM_012652, NM_012653, NM_012654, and NM_173098, respectively; NHE1 sense, 5'-TCAGACCCACTGGCCTATGAA-3', antisense, 5'-TCACTTGGTCCGGGGGTGAA-3'; NHE2 sense, 5'-AAGCTTCCCGAGAAGTTGCAGA-3', antisense, 5'-GAGAGCAGGGGCTGCCTCAA-3'; NHE3 sense, 5'-ATTCCTAATGGGAAGCTGCCTA-3', antisense, 5'-AATGGAGTCAGGCGGCGGAA-3'; NHE4 sense, 5'-TTGAGGGAGAGCCTGCGGAAA-3', antisense, 5'-TTGGCCCCCAGGTCCCCAAA-3'). For NHE8, we used degenerated oligonucleotides, designed for mouse NHE8 [GenBank accession no. AF482993; NHE8 sense, 5'-AAGAC(T/C/A)GAGAAGATGGGIAA(T/C)GC-3', antisense, 5'-(A/G)TCCTC(A/G)(G/C)(T/A)GCCI(G/C)(A/T)IGG(T/G/C/A)CC-3']. In all cases, the primers were designed to amplify a segment of cDNA that corresponds to the cytoplasmic regions of Na+/H+ exchangers, because these segments are more divergent among the isoforms. RT-PCR was carried out using a PerkinElmer Gene Amp PCR System 2400 (Wellesley, MA). Amplification was performed for 35 cycles at 94°C/20 s, 56°C/60 s, and 70°C/60 s. PCR products were subsequently electrophoresed on a 1% agarose gel, and product sizes were determined against the 100-bp DNA Ladder (Invitrogen). Gels were stained with ethidium bromide, and the PCR products were analyzed with an Eagle Eye System (Stratagene, La Jolla, CA).

Western blotting. Protein samples were solubilized in SDS sample buffer, and proteins were separated by SDS-PAGE using 10% polyacrylamide gels according to Laemmli (19). For immunoblotting, proteins were transferred to a nitrocellulose membrane (Hybond-ECL; Amersham Biosciences, Little Chalfont, Bucks, UK) from polyacrylamide gels at 500 mA for 1 h and 30 min at 4°C with a Mini-PROTEAN transfer electrophoresis system (Bio-Rad, Hercules, CA). Entire sheets of Hybond-ECL membranes containing transferred proteins were incubated first in Blotto (5% nonfat dry milk and 0.1% Tween 20 in PBS, pH 7.4) for 1–2 h to block nonspecific binding of antibodies, followed by overnight incubation in primary antibodies (anti-NHE1 or anti-NHE3) diluted 1:1,000 in Blotto. The membranes were then washed 10 times in Blotto and incubated for 1 h with peroxidase-conjugated goat anti-rabbit IgG (from Jackson Immunoresearch Laboratories) or anti-mouse IgG (from Zymed Laboratories). Bound antibodies were detected with ECL-enhanced chemiluminescence (Amersham Biosciences) according to the manufacturer's protocols. Hyperfilm-ECL (Amersham Biosciences) exposed to membranes for 1–3 min were developed in an X-ray film processor.

Electron microscopy. To prepare samples for transmission electron microscopy, cultures were grown until the midexponential phase and were removed with Tyrode. The samples were then centrifuged at 2,000 rpm for 5 min, eliminating the supernatant. Samples were fixed with Karnowsky solution (2% glutaraldehyde, 4% paraformaldehyde) in 0.1 M cacodylate buffer, pH 7.2 for 24 h at 4°C. Cells were then washed four times for 15 min in the same buffer and postfixed with 1% osmium tetroxide for 2 h. The samples were dehydrated in increasing concentrations of ethanol (from 70, 95, and 100%) during 10 to 15 min. After dehydration, the cells were washed two times with propylene oxide for 15 min and subsequently were treated with a solution of 1:1 (vol/vol) propylene oxide and Spurr's resin (Electron Microscopy Sciences, Hatfield, PA) for 18 h. Finally, samples were embedded in Spurr's resin and polymerized for 5 days at 60°C. Thin sections (50 nm) were obtained with a diamond knife, contrasted with uranyl acetate and lead citrate, and observed with a JEM-1010 transmission electron microscope. Micrographs were obtained and enlarged for analysis.

Statistics. Data are presented as mean values ± SE. Statistical comparisions were made by ANOVA followed by the Bonferroni contrast test. For qualitative data (morphological results), the {chi}2-test was used. Differences were considered significant if P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characterization of the Proton Extrusion Systems Present in IRPTC and the Influence of Serum Deprivation

To study the H+ extrusion systems present in IRPTC and the influence of serum deprivation on them, we examined the intracellular pH recovery after acidification of pHi with the NH4Cl pulse technique. Figure 1A (A1 and A2) shows two representative experiments in which IRPTC were first bathed with a 141 mM Na+ solution, exhibiting the basal pHi. After 2 min of exposure to NH4Cl, during which the cell pHi increased transiently, NH4Cl removal caused a rapid acidification of pHi as a result of NH3 efflux. In nondeprived cells, the pHi recovery rate in the first 2 min was 0.29 ± 0.022 pH U/min (n = 14). Removal of serum from culture medium 24 h before the experiments resulted in a significant increase of the rate of pHi recovery to 0.50 ± 0.024 pH U/min (n = 14). Serum starvation for periods of 6 to 48 h before the experiments caused the alterations in dpHi/dt shown in Fig. 1B, indicating that the maximum increase occurred at 24 h deprivation. For this reason and to optimize the stimulatory response, in all experiments in which we studied the influence of serum deprivation on the proton extrusion systems of IRPTC, we performed serum deprivation for 24 h. Figure 1C shows the comparison between the first two situations, where the serum deprivation significantly stimulated the pHi recovery in IRPTC.


Figure 1
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  		Fig. 1. Effect of serum deprivation on the intracellular pH (pHi) recovery after cellular acidification with NH4Cl pulse technique in immortalized rat proximal tubule cells (IRPTC). A1: typical tracing for nondeprived cells. A2: tracing for serum-deprived cells. B: time-dependent stimulation of pHi recovery by serum deprivation. C: comparision between nondeprived and deprived cells, showing the significant stimulation of pHi recovery after 24 h of serum deprivation. *P < 0.001 vs. control.

 
Table 2 summarizes the main values of pHi responses found in some of the studied experimental groups. Considering all the results from this work, IRPTC in pH 7.4 HCO3–-free solution have a mean baseline pHi of 7.08 ± 0.008 (n = 34) in nondeprived cells and 7.18 ± 0.018 (n = 33) in 24-h serum-deprived cells (P < 0.0001). In serum-deprived cells, the steady-state pHi in the Na+-HEPES-buffered solution is higher than in nondeprived cells, which was observed in all experimental groups.


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  		Table 2. Summary of pHi responses in IRPTC to the addition of different solutions after an acute acid load

 
Figure 2 shows the effect of the absence of external Na+ and of the Na+/H+ exchanger inhibitor, 5-(N,N-hexamethylene) amiloride (HMA; 0.1 mM), on the pHi recovery rates in nondeprived and serum-deprived cells. In the absence of Na+ or in the presence of HMA, the pHi recovery rates were significantly reduced compared with controls in both situations (and pHi recovery was not complete; Table 2), indicating that the pHi recovery is mostly dependent on the Na+/H+ exchanger. However, even in the absence of Na+, a significant rate of pHi recovery was still observed in both conditions, indicating the presence of at least one Na+-independent proton extrusion system in IRPTC. This behavior has been shown before in other PT cells, and evidence was presented suggesting that it was not due to the activity of the H+-K+-ATPase, because in the groups where HMA plus Schering 28080 (a specific inhibitor of H+-K+-ATPase) were used, the pHi recovery rates were similar to the HMA groups. The behavior observed in nondeprived and in serum-deprived cells was similar, showing that the cells express the Na+/H+ exchanger as their main H+ extrusion system, but also a Na+-independent mechanism. The pHi recovery by this Na+-independent system in serum-deprived cells was not significantly different from controls, indicating that the stimulatory effect of serum deprivation is on the Na+/H+ exchanger.


Figure 2
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  		Fig. 2. Initial rate of pHi recovery following acute intracellular acidification in nondeprived and serum-deprived cells in the presence of control solution, absence of Na+, or in the presence of the Na+/H+ exchanger inhibitor 5-N,N-hexamethylene amiloride (HMA; 0.1 mM) with or without Schering (10 µM). #*P < 0.001 vs. control. $P < 0.001 vs. control of serum-deprived cells.

 
Because several studies have shown the presence of H+-ATPase in PT cells (2, 8), the next step was to evaluate the presence of this H+ extrusion system in IRPTC. Figure 3 shows the effects of N-ethylmaleimide (NEM; 0.5 mM) and concanamycin (0.1 µM), inhibitors of H+-ATPase on Na+-independent pHi recovery from an acid pulse. In nondeprived cells, the utilization of these inhibitors reduced significantly the pHi recovery to the same extent. The addition of EGTA to NEM did not change the recovery rate compared with the group where the sulfhydryl agent was used alone, indicating that extracellular calcium has no effect on the Na+-independent mechanism. In serum-deprived cells, the pHi recovery was reduced in the presence of concanamycin, confirming the presence of H+-ATPase also in this experimental condition. In all these groups where Na+-free solutions were used to inhibit the Na+/H+ exchanger, the pHi after the acid load was lower than in control situations, because whereas the Na+/H+ exchanger starts acting immediately after the change of the solutions, increasing the measured pH, H+-ATPase is activated only approximatly 2 min after solution change. For this reason, in Na+-free conditions, we analyzed the rate of pH recovery in two moments: after 2 min of acid load, when H+-ATPase starts acting, and at the same pHi at which the dpHi/dt was measured in control situations. In both cases, the pHi recovery reached the same values, because the profile of pHi recovery by H+-ATPase is constant, following a straight line. Therefore, in the H+-ATPase experiments, dpHi/dt was measured 2 min after the start of recovery.


Figure 3
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  		Fig. 3. Initial rate of pHi recovery following acute intracellular acidification in nondeprived and serum-deprived cells in the absence of Na+ with or without N-ethylmaleimide (NEM; 0.5 mM), EGTA (2.5 mM), or concanamycin (0.1 µM). *P < 0.001 vs. Na+ (0 mM). $P < 0.001 vs. Na+ (0 mM) of serum-deprived cells.

 
Isoforms of Na+/H+ Exchanger Modulated By Serum Deprivation Present in IRPTC

To analyze the Na+/H+ exchanger isoforms present in IRPTC that are modulated by serum, we used two inhibitors, HOE-694 (5 µM) for NHE1 and S3226 (1 µM) for NHE3 (12, 46), in nondeprived and serum-deprived cells. Figure 4 shows the pHi recovery rates in serum-deprived cells. The utilization of S3226 did not affect the pHi recovery compared with control, as the pHi recovery rate in the first 2 min was 0.57 ± 0.035 pH U/min (n = 6), indicating the absence of NHE3 in IRPTC. However, with HOE-694, the pHi recovery was significantly reduced to a value similar to those of Na+-free solutions plus 0.1 µM concanamycin (0.019 ± 0.0070 pH U/min, n = 10). This finding shows that the only Na+/H+ exchanger isoform present in IRPTC and that is modulated by serum deprivation is NHE1. The same behavior was observed also in nondeprived cells (data not shown).


Figure 4
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  		Fig. 4. Na+/H+ exchanger isoforms in serum-deprived IRPTC. Effects of the NHE1 inhibitor HOE-694 (5 µM) and the NHE3 inhibitor S3226 (1 µM) on Na+-dependent pHi recovery after an acid load. The NHE3 isoform inhibitor S3226 had no effect on pHi recovery rates, indicating that the Na+-dependent pHi recovery was not due to NHE3. The NHE1-specific inhibitor HOE-694 reduced pHi recovery significantly to the same extent as did Na+ (0 mM), demonstrating that NHE1 is the only NHE isoform present in IRPTC. *P < 0.001 vs. control of serum-deprived cells.

 
To confirm these data, we performed RT-PCR in nondeprived and serum-deprived cells, using primers to several Na+/H+ exchanger isoforms commonly expressed in epithelial tissues (NHE1–4 and NHE8). Figure 5A shows the results obtained from serum-deprived cells, where PCR yielded products of the expected molecular weight only with the NHE1 primer (248 bp), confirming the presence of only this isoform in IRPTC. Similar results were obtained also in nondeprived cells (data not shown). Bands of the predicted sizes were detected for isoforms NHE1 (248 bp), NHE3 (329 bp), and NHE4 (213 bp) in cDNA samples from both the cortex and medulla of rats, illustrating the quality of the primers utilized.


Figure 5
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  		Fig. 5. RT-PCR and Western blot experiments in IRPTC. A: RT-PCR analysis of NHE isoforms in IRPTC, cortex, and medulla of rat. The primers selectively designed to detect NHE1–4 and NHE8 isoforms were used to amplify the cDNAs prepared from IRPTC, cortex, and medulla of rats. In IRPTC, only NHE1 was detected, confirming the functional data. In contrast, in cortex and medulla, NHE1, NHE3, and NHE4 were observed, attesting to the quality of primers. The positive control is beta-actin. Molecular weight standard was a 100-bp DNA ladder. Arrows indicate the standards, that are, from top to bottom, 2,072, 600, and 100 bp. B: Western blot analysis using antibodies against NHE1 and NHE3 in cell lysates from IRPTC. The rabbit antibody to rat NHE1 detected an ~85-kDa protein in IRPTC, confirming the presence of NHE1 in this cell line. Anti-NHE3 failed to label the protein in IRPTC, but recognized an ~80-kDa protein in rat kidney, indicating the efficiency of the antibody. Standards for NHE1 are, from top to bottom, 120 and 85 kDa, and for NHE3, 80 kDa (Arrows).

 
To confirm the Na+/H+ exchange proteins expressed in IRPTC, cell lysates were immunoblotted for NHE1 and NHE3 (Fig. 5B). NHE1 was revealed with a polyclonal antibody, which recognized an ~85-kDa protein, whereas NHE3 was not detected, confirming the previous results. In rat kidney, anti-NHE3 labeled an ~80-kDa protein, showing the quality of the antibody.

Effect of Serum Deprivation on betai

betai is an important mechanism to maintain pHi at a constant level during acute pHi displacements. In Fig. 6, calculated betai is plotted vs. pHi at intervals of 0.2 U, each point representing the mean buffer capacity in these intervals. The solid line drawn through the points is the result of a fit to an exponential curve. betai depends significantly on pHi over the tested pHi range, increasing as pHi is lowered. The data show that nondeprived and serum-deprived cells possess a similar betai, because we found no significant diference between corresponding points at the curves.


Figure 6
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  		Fig. 6. Effect of serum deprivation on the intrinsic buffering capacity (betai) in bicarbonate-free conditions. Intrinsic buffering capacity was determined as described in MATERIALS AND METHODS. Calculated betai is plotted vs. pHi at intervals of 0.2 pH U, each point representing the mean buffer capacity in these intervals. Values are means ± SE from 6 independent experiments (of each experimental situation) and show that nondeprived and serum-deprived cells have a statistically similar betai.

 
Influence of Serum Deprivation on the Morphology of IRPTC

Observations made on semiconfluent monolayers of IRPTC, which were cultured in the presence or absence of serum, suggested that morphological characteristics of the cells could be greatly influenced by medium composition. This was already visible at optical microscopy examination (data not shown). Electron microscopic studies of IRPTC grown in the presence of serum were poorly differentiated, as illustrated in Fig. 7, A and B. In general, the cells grown in medium with serum were characterized by lack of clear-cut polarization, rarity of microvilli, and of junctional complexes (Fig. 7B). Our criteria of differentiation were the presence of apical microvilli, basally located nuclei, and evident junctional complexes between cells. Apical microvilli were always coexistent with basal nuclei, but junctional complexes were not always concomitant with the other two criteria. In a total of 22 nondeprived cells, 36.4% had such well-defined characteristics of differentiation. In contrast, cells grown in the absence of serum exhibited varying degrees of epithelial differentiation (Fig. 7, C, D, and E). IRPTC presented polarization properties, with the nucleus localized at the basolateral side of the cells, with microvilli covering the apical membrane, and with more developed tight junctions (Fig. 7C). Out of 21 analyzed cells, 66.7% had such properties. Furthermore, serum-deprived cells showed more developed endoplasmic reticulum as well as Golgi apparatus (Fig. 7D) and also a large amount and more developed nuclear pores (Fig. 7E). However, some serum-deprived cells, as noted, displayed signs of degeneration, such as a large amount of vacuoles and lysosomes.


Figure 7
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  		Fig. 7. Transmission electron microscopic analysis of serum-starved and nondeprived IRPTC. A and B: cells grown in the presence of serum displayed an apical surface devoid of well-defined microvilli and poor intercellular contacts. C-E: cells adapted to serum-free media formed a well-organized epithelium, with clear polarization: cells displayed an apical domain bearing microvilli and a basolateral domain showing tight junctions, well-developed Golgi apparatus, and nuclear pores (arrows). Magnifications: A, x5,000; B, x6,000; C, x4,000; D, x20,000; E, x15,000.

 
The role of NHE1 for cell differentiation was also evaluated by analyzing morphological changes when the cells were cultured in the presence of the NHE1 blocker HOE-694 (5 µM). When this experiment was performed, we observed, both with light and electron microscopy, that in 62.2% of serum-deprived cells (total of 37 cells) apical microvilli were apparent, whereas in similarly deprived cells but in the presence of HOE-694, only 36.2% (n = 47) of cells had such properties. Similarly, basally located nuclei were found in 70.3% of serum-deprived cells, whereas only 29.8% of cells had basal nuclei in HOE-694-treated cells. When these differentiation criteria were analyzed statistically by the {chi}2-test, it was seen that differentiation was significantly (P < 0.01) associated with the treatments used, i.e., serum deprivation, no deprivation, and HOE-694. In addition, HOE-694-treated cells had large cytoplasmic vacuolization, extensive expansion of the perinuclear space, and widespread lack of intercellular junctions, impairing the formation of a continuous monolayer.

Transcriptional and Translational Modulation of NHE1 by Serum Deprivation

To determine whether RNA and protein synthesis are involved in the NHE1 stimulation by serum deprivation, IRPTC were treated with 0.1 µM actinomycin D (a transcription inhibitor) or 40 µM cycloheximide (a translation inhibitor) in the last 6 h of serum starvation, before the experiments. Unfortunately, we could not perform experiments in cells treated with actinomycin or cycloheximide for 24 h, which was the period of time used during starvation, because only a few cells could survive the treatment. Actinomycin D and cycloheximide did not cause any effect on pHi recovery rates from acid load in nondeprived cells, but significantly inhibited the stimulatory effect of serum starvation on NHE1 activity (reaching 0.35 ± 0.043, n = 6 and 0.34 ± 0.042, n = 6 pH U/min, respectively, against a control value of 0.50 ± 0.025, n = 5 pH U/min, as shown in Fig. 8A). When these agents were added 30 min before the experiments, values of dpHi/dt were also reduced, but did not reach the control level (data not shown). Figure 8B shows that both NHE1 mRNA and protein levels are increased after serum deprivation. beta-Actin and villin were used as internal controls. Taken together, these results suggest a role of RNA and protein synthesis in regulating the net rate of pHi recovery mediated by NHE1 stimulated by serum deprivation.


Figure 8
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  		Fig. 8. Transcriptional and translational modulation of NHE1 by serum deprivation. A: effects of actinomycin D (0.1 µM) and cycloheximide (40 µM) on initial rate of pHi recovery following acute intracellular acidification in nondeprived and serum-deprived cells. *P < 0.001 vs. control. #P < 0.05 vs. control of serum-deprived cells. B: effect of serum deprivation on NHE1 mRNA abundance (B1) and protein expression (B2). The levels of NHE1 mRNA were observed by RT-PCR in comparision to beta-actin. NHE1 protein levels were detected by Western blotting, using specific antibodies for NHE1 (N1P1) in comparision to villin. ND and D, nondeprived and deprived cells, respectively.

 
Involvement of Tyrosine Kinase and the MAPK Pathway in the Modulation of NHE1 by Serum Starvation

To determine whether tyrosine kinase is involved with the stimulatory effect of serum deprivation on NHE1 activity, the tyrosine kinase inhibitor genistein (10 µM) was used. Cells were pretreated with the inhibitor for 30 min before the experiments. The data (see Fig. 9) show that in nondeprived cells, genistein has no intrinsic effects on pHi responses. Surprisingly, in serum-deprived cells, genistein completely blocked the stimulatory effect of serum starvation (reaching 0.32 ± 0.043 pH U/min, n = 7, compared with the control of 0.50 ± 0.026 pH U/min, n = 9). These findings suggest that phosphotyrosine accumulation is required for the NHE1 stimulation induced by serum deprivation.


Figure 9
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  		Fig. 9. Signaling pathways involved with the stimulatory effect of serum deprivation on NHE1 activity of IRPTC. Effects of genistein (10 µM), UO-126 (10 µM), and SB-203580 (10 µM). *P < 0.001 vs. control. #P < 0.01 and $P < 0.05 vs. control of serum-deprived cells.

 
To investigate the potential functional significance of the activation of the ERK pathway by serum deprivation, we investigated the consequences of pharmacological inhibition of MEK1/2, the upstream activator of ERK1/2, using the inhibitor UO-126 (10 µM). Once again, exposure of nondeprived cells to the agent for 30 min before the experiments did not affect the pHi recovery rates, but significantly inhibited the stimulatory effect of serum starvation on Na+/H+ exchanger activity (0.36 ± 0.047 pH U/min, n = 7). These results show that the stimulation of NHE1 activity by serum deprivation is mediated by activation of the ERK pathway. To detect whether p38 MAPK was involved in this effect, we preincubated the cells with SB-203580 (10 µM), a potent inhibitor of p38 MAPK. We found that the p38 MAPK inhibitor failed to affect the pH recovery rates in both nondeprived or deprived cells (0.33 ± 0.028, n = 7 and 0.47 ± 0.025, n = 6 pH U/min, respectively), indicating that this protein is not involved in the stimulatory effect of NHE1 by serum deprivation. All these results are summarized in Fig. 9.


   DISCUSSION
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The purpose of this work was to determine the HCO3–-independent proton extrusion systems present in IRPTC and their regulation by serum deprivation, a stimulus for cellular differentiation. The use of a HCO3–-independent system permits to limit the H+ ion transporters to be studied, eliminating HCO3– cotransporters and exchangers. Furthermore, the mechanisms underlying the different response in serum-deprived cells and the role of NHE in this process were analyzed. As most cell lines available are PT-like rather than PT per se and primary PT cells in culture have proven difficult to grow and passage, beyond dedifferentiating rapidly, a development of immortalized rat PT cell lines, such as IRPTC, is very useful. Therefore, IRPTC are a permanent cell line originated from the rat renal PT through transformation with temperature-sensitive A mutant virus of the simian virus 40 (tsA SV40) (38). According to Ingelfinger et al. (17), these cells polarize and express enzymes and proteins that are normally present in PT cells, thus displaying a highly differentiated phenotype. However, little is known about the regulation of cell pH in this cell line.

Our results concerning stationary cell pH and recovery from an ammonium pulse in nondeprived cells are comparable to data obtained with other proximal tubular cell lines, such as LLC-PK1 (22). However, steady-state pHi in pH 7.4 HCO3–-free solution increased with serum starvation, from 7.08 ± 0.008 (n = 34) in nondeprived cells to 7.18 ± 0.018 (n = 33) in 24-h serum-deprived cells. The pHi recovery rate in the first 2 min after the ammonium pulse was also higher in serum-starved cells, reaching 0.50 ± 0.024 pH U/min (n = 14) after 24 h of serum deprivation, in contrast to 0.29 ± 0.022 pH U/min (n = 14) in nondeprived cells. This indicates that serum deprivation stimulates mechanisms involved with pH regulation in IRPTC. It is not clear why the activation of NHE1 decreases after the peak at 24 h deprivation, but the cells, e.g., in the 48-h group, seem to suffer some degeneration and lose their ability to form a complete monolayer, suggesting that the prolonged incubation in serum-free medium might impair their viability.

The present study reports functional evidence for the presence of different proton secretion mechanisms, including the Na+/H+ exchanger and H+-ATPase in both nondeprived and serum-deprived IRPTC. The most important of these is the Na+/H+ exchanger, because removal of extracellular Na+ or utilization of HMA (a Na+/H+ exchanger inhibitor) led to an ~70% reduction in the rate of pHi recovery (Table 2) in nondeprived cells. These results are comparable with a number of studies by different authors (2, 3, 20, 30). As the present study was performed using impermeant glass supports, it was not possible to define the polarity of this proton transporter.

We were also able to show Na+-independent pHi recovery after an acute acid load in our preparation (Fig. 3) in both nondeprived and deprived cells. Considering that the epithelium used in our study contains cells with characteristics of proximal cells, H+ ion-transporting ATPases were probably responsible for the Na+-independent pHi recovery. In fact, with the use of a Na+-free solution to which 0.5 mM NEM or 0.1 µM concanamycin was added, the Na+-independent pH recovery was almost completely inhibited, indicating the presence of a vacuolar H+-ATPase. This observation agrees with the biochemical and functional results obtained by other authors (2, 24, 41). Moreover, in agreement with Wang et al. (47), we were unable to detect a component of Sch-28080-sensitive H+ extrusion, arguing against a significant role for H+-K+-ATPase in mediating PT acidification by IRPTC, although Sch-28080-sensitive K+-ATPase activity has been detected in this nephron segment (49).

The Na+-independent pHi recovery was apparently lower in serum-deprived cells than in nondeprived cells. However, the difference among the groups was not significant, indicating that the proton extrusion system stimulated by serum withdrawal in IRPTC is only the Na+/H+ exchanger. The isoform involved in this case is NHE1, because 1 µM S3226, a powerful inhibitor of NHE3 (35, 46), failed to inhibit the isoform in these cells, whereas 5 µM HOE-694 (a NHE1 inhibitor at this concentration) significantly inhibited the dHi/dt by the same magnitude as did the 0 Na+ group. This was further substantiated by the presence of only NHE1 in RT-PCR and Western blot analysis.

Besides the ubiquitous NHE1, two additional isoforms were detected by other authors in PTs, NHE3 (the most abundant) and NHE8 (5, 14). NHE2 is also expressed in other proximal tubular cell lines, MCT and RKPC2 (9, 23), but not in plasma membrane fractions isolated from rat renal cortex (10). Studies in NHE3 null mice suggest that NHE3 is responsible for almost all HCO3– reabsorption mediated by Na+/H+ exchange in the PT (47). However, the expression of NHE3, which is rapidly lost in primary culture as a consequence of the removal of the cells from their proper surrounding, appears to be also supressed by immortalization (9). The present study suggests that IRPTC, an immortalized cell line, have also lost NHE3 expression, even in serum-deprived cells, where cells are expected to be more differentiated. Apparently, differentiated functions that may not be needed for survival in culture are likely to be lost in immortalized cells (9). Indeed, MCT and RKPC-2, other proximal-like tubule cell lines established from primary cultures using SV40, also do not express NHE3 (9, 23).

NHE1 was the first isoform to be cloned and plays a major role in intracellular pH homeostasis and cell volume regulation. An outstanding feature of NHE1 is that its activity can be modulated by a remarkably wide variety of stimuli, including growth factors, tumor promoters, hormones, and changes in cell volume or spreading (for reviews, see Refs. 25, 28, 43). A variety of signaling molecules is involved with NHE1 regulation by these stimuli, such as Ca2+/calmodulin (42), low-molecular-weight GTPases Ras, and Rho (40), ERK1/2 kinases (4), calcineurin B homologous protein (29), and phosphatidylinositol 4,5 bisphosphate (1). A great deal of effort has been made to determine the interrelations among the functions of these signaling molecules leading to NHE1 activation, but the complete understanding of these processes remains unclear.

The Na+/H+ exchanger is involved in cell proliferation and differentiation (15, 32). Our results, obtained from an immortalized cell line, suggest a possible involvement of serum withdrawal with increase of differential status of these cells. This was confirmed by ultrastructural analysis made by electron microscopy. Because IRPTC maintain their differentiation state without serum, it is possible that they may produce autocrine or paracrine factors that support their own survival. Among the immortalized cell lines that have been studied, several can be induced to differentiate by altering growth conditions or by adding trophic factors. In the catecholaminergic cell line Cath.a, differentiation could be initiated by serum deprivation (31). Moreover, P19 cells, an embryonal carcinoma cell line, can be induced to differentiate morphologically and biochemically by retinoic acid (45). In primary cultures of PT cells, cell differentiation is also modulated by medium composition (33). A number of studies have suggested a causal link between increased NHE1 activity and subsequent differentiation (13). In fact, the absence of Na+/H+ exchanger as a pH-regulatory system can result in deficiencies in cell differentiation (45). We were able to show that cultivation of IRPTC in the presence of HOE-694, a specific blocker of NHE1 at the concentration of 5 µM, caused marked reduction of several differentiation criteria, such as the presence of apical microvilli, basally located nuclei, and formation of junctional complexes. Because differentiation may require either intracellular alkalinization or increased Na+/H+ exchange activity, it is possible that the mechanism to fulfill this requirement is increased Na+/H+ exchanger gene expression followed by increased protein expression and activity (13).

Our findings suggest an involvement of RNA and protein synthesis in the NHE1 upregulation by serum deprivation in IRPTC, because in serum-deprived cells both NHE1 mRNA and protein were shown to be markedly increased. In addition, the inhibitors actinomycin and cycloheximide impaired the NHE1 stimulation of serum-deprived cells. Both these agents are antibiotics, actinomycin blocking RNA synthesis by complexing with DNA via deoxyguanosine residues, therefore inihibiting DNA-primed RNA polymerase, and cycloheximide blocking the translation of messenger RNA in cytosolic 80S ribosomes (26, 37). These results are in agreement with those from a number of other investigators (13, 32). These findings, together with an increase of the number of nuclear pores, as well as of their size, evidenced by ultrastructural analysis, suggest a more pronounced communication between nucleus and cytoplasm, agreeing with more protein synthesis. Nevertheless, in our system, we cannot exclude the involvement of other components, independent of NHE1 expression, in this modulation, such as regulatory proteins interacting with the cytoplasmic domain of NHE1 or direct phosphorylation of the antiporter.

In this study, we also examined the putative role of MAPKs on NHE1 modulation by serum deprivation. MAP kinases constitute a large family of Ser/Thr protein kinases activated by separate cascades, which in turn regulate multiple processes stimulated by extracellular agents. In mammalian cells, the first and best-characterized MAPK cascade is the ERK1/2. It involves activation of ERK1/2 by direct phosphorylation by MEK1 and MEK2, which are themselves phosphorylated and activated by the serine/threonine kinase Raf. Raf is recruited to the membrane by Ras on activation of either G protein-coupled receptors or tyrosine kinase receptors (4, 36). In our case, genistein completely blocked the stimulatory effect of serum deprivation on NHE1 activity, indicating the involvement of tyrosine kinase phosphorylation in this regulation. Moreover, UO-126 also inhibited the stimulation of NHE1 by serum deprivation, showing that MEK1/2, the upstream activator of ERK1/2, is related to the observed effect. Bianchini et al. (4, 44) as well as other authors also found an involvement of the MAPK cascade in regulation of NHE1, mediated, however, by growth factors. On the other hand, we found no evidence in favor of the involvement of p38 MAPK, as SB-203580 failed to inhibit the stimulatory effect of serum deprivation on the Na+/H+ exchanger.

In conclusion, our results suggest that serum deprivation results in an upregulation of the NHE present in IRPTC, NHE1, together with an increase in their differentiation status. This positive regulatory pathway depends on the activation of tyrosine kinases, on the ERK1/2 cascade and finally regulation of gene expression.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants and fellowships from Fundação de Amparo a Pesquisa do Estado de São Paulo (99/128663) and CNPq.


   ACKNOWLEDGMENTS
 
The authors thank Dr. M. Oliveira-Souza, C. N. Alves Bezerra, and F. A. Correia Barrence for helpful support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Malnic, Departamento de Fisiologia e Biofísica, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes 1524, 05508-900 São Paulo, SP, Brazil (e-mail: gemalnic{at}usp.br)

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


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