We previously demonstrated that uroguanylin (UGN) significantly inhibits Na+/H+ exchanger (NHE)3-mediated bicarbonate reabsorption. In the present study, we aimed to elucidate the molecular mechanisms underlying the action of UGN on NHE3 in rat renal proximal tubules and in a proximal tubule cell line (LLC-PK1). The in vivo studies were performed by the stationary microperfusion technique, in which we measured H+ secretion in rat renal proximal segments, through a H+-sensitive microelectrode. UGN (1 μM) significantly inhibited the net of proximal bicarbonate reabsorption. The inhibitory effect of UGN was completely abolished by either the protein kinase G (PKG) inhibitor KT5823 or by the protein kinase A (PKA) inhibitor H-89. The effects of UGN in vitro were found to be similar to those obtained by microperfusion. Indeed, we observed that incubation of LLC-PK1 cells with UGN induced an increase in the intracellular levels of cAMP and cGMP, as well as activation of both PKA and PKG. Furthermore, we found that UGN can increase the levels of NHE3 phosphorylation at the PKA consensus sites 552 and 605 in LLC-PK1 cells. Finally, treatment of LLC-PK1 cells with UGN reduced the amount of NHE3 at the cell surface. Overall, our data suggest that the inhibitory effect of UGN on NHE3 transport activity in proximal tubule is mediated by activation of both cGMP/PKG and cAMP/PKA signaling pathways which in turn leads to NHE3 phosphorylation and reduced NHE3 surface expression. Moreover, this study sheds light on mechanisms by which guanylin peptides are intricately involved in the maintenance of salt and water homeostasis.
- renal microperfusion
- bicarbonate reabsorption
in the past decades, it has been postulated that Sta-like guanylin peptides participate in a control system regulating salt balance in response to oral salt intake, linking intestine and kidney in the process of salt and water homeostasis (8, 19, 22). These peptides are known as endogenous agonists for the Escherichia coli heat-stable toxin receptor, guanylate cyclase C (GC-C), uroguanylin (UGN) being its most potent member (12, 18, 20, 27).
UGN is a 16 amino acid peptide (27), expressed throughout the intestinal tract, secreted by enterochromaffin cells as prouroguanylin that undergoes postsecretory proteolytic cleavage, generating the active form (15, 28, 41). Recently, Qian and colleagues (44) demonstrated that the conversion of prouroguanylin to UGN occurs within the lumen of renal tubules.
Renal effects of UGN include natriuresis, kaliuresis, diuresis, and increased excretion of cGMP (17, 26). In a previous work using stationary in vivo microperfusion, our group demonstrated that UGN perfusion stimulates potassium secretion through Maxi-K channels in connecting and cortical collecting ducts (2). Furthermore, this study demonstrated that UGN perfusion also leads to an inhibition of bicarbonate reabsorption in proximal tubules by reducing the activity of the Na+/H+ exchanger, NHE3 isoform (2).
In renal proximal tubule cells, NHE3 is the principal mechanism of hydrogen secretion, which leads to filtered bicarbonate reabsorption (1, 2, 4, 47, 52). NHE3 is also responsible for the isotonic reabsorption of approximately two-thirds of the filtered NaCl and water (3, 43). Accordingly, Schultheis and co-workers (47) demonstrated that NHE3 knockout mice are hypovolemic, hypotensive, and present metabolic acidosis and reduced reabsorption of Na+, HCO3−, and volume. In addition, the mortality of these NHE3 knockout mice is increased when they are submitted to a low-salt diet (33). Considering NHE3 physiological roles, relatively small alterations of its activity may have significant consequences. Indeed, NHE3 is among the most extensively regulated transport proteins of cell membranes, being modulated by several physiological and pathological conditions (13, 24).
The mechanisms underlying UGN renal actions are not completely elucidated, especially regarding the natriuretic effect. High-salt diets increase the concentration of UGN and cGMP in the urine (22, 42), but in GC-C knockout mice, guanylin peptides still cause natriuresis (9). Our previous studies suggested an inhibitory effect of guanylin peptides on NHE3 activity in proximal tubules (2, 35). Thus, the present study was designed to investigate the signaling mechanisms involved in the natriuretic effect of UGN, focusing on the inhibition of NHE3 activity by the peptide.
MATERIALS AND METHODS
Reagents and antibodies.
All chemicals were obtained from Sigma (St. Louis, MO) unless otherwise noted. UGN was purchased from Bachem (Philadelphia, PA). KT5823, a specific inhibitor of protein kinase G (PKG), was purchased from Calbiochem (San Diego, CA). S3226 was kindly donated by Dr. Jurgen Punter from Sanofi-Aventis Deutschland GmbH (Frankfurt, Germany). EZ-Link Sulfo-NHS-SS-Biotin as well as immunopure immobilized streptavidin were purchased from Thermo Fisher Scientific (Rockford, IL). A monoclonal antibody (mAb) raised to the renal brush-border Na+/H+ exchanger isoform 3 (NHE3), clone 3H3, was kindly provided by Drs. Daniel Biemesderfer and Peter Aronson (Yale University, New Haven, CT). Phosphospecific NHE3 mAbs to NHE3 (phospho-serines 552 and 605), anti-PS552, clone 14D5, anti-PS605, and clone 10A8 (32) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Life Technologies (Carlsbad, CA).
Animal procedures and protocols were followed in accordance with the ethical principles in animal research of the Brazilian College of Animal Experimentation and were approved by the institutional animal care and use committee. Experiments were performed using male Wistar rats (250 to 300 g) housed under standardized conditions (constant temperature of 22°C, 12:12-h dark-light cycle, and relative humidity of 60%) at the University of São Paulo Biomedical Institute animal facility. To perform stationary in vivo microperfusion, the animals were anesthetized with intramuscular ketamine (75 mg/kg) and xylazine (8 mg/kg). The left jugular vein was cannulated for infusion of 3% mannitol in isotonic saline at a rate of 0.1 ml/min. The kidney was exposed by a lumbar approach and prepared for “in vivo” micropuncture.
Stationary in vivo microperfusion.
The microperfusion procedure was performed as described previously (23). A proximal tubule was punctured by means of a double-barreled micropipette, one barrel being used to inject FDC-green colored Ringer perfusion solution (in mM: 80 NaCl, 5 KCl, 25 NaHCO3, 1 CaCl2, 1.2 MgSO4, and raffinose to reach isotonicity, at 0 Pco2), and the other to inject Sudan black-colored castor oil used to block the injected fluid columns in the lumen. A single micropipette containing the same Ringer solution plus the polypeptide agent was impaled into a neighboring proximal loop. A proximal segment of the same nephron, recognized by the colored perfusion and by its low transepithelial potential difference (PD), was impaled by a double-barreled asymmetric microelectrode to measure intratubular pH, the larger barrel containing at its tip the H+ ion-sensitive ion exchange resin (Fluka, Buchs, Switzerland) and the smaller, 1 M KCl reference solution colored by FDC-green. Properties of the microelectrode were described previously (23). The pH microelectrodes were calibrated before and after every impalement on the kidney's surface by perfusion with 20 mM phosphate-Ringer buffer solutions containing 130 mM NaCl, at 37°C. The pH values were adjusted to 6.5, 7.0, and 7.5 with 0.1 N NaOH or HCl. A luminal oil block was split by perfusions so that the solution was isolated and blocked by oil. The perfusion rate was sufficient to increase luminal pH to values near those of the perfusion fluid, i.e., 25 mM NaHCO3 or pH ∼8.0. After the luminal solution was blocked with oil, the increase in luminal H+ activities, representing bicarbonate reabsorption, was followed until a stable level was reached.
By this technique, several (∼2 to 5 each) control, experimental, and recovery curves were obtained, the mean of control plus last recovery curves and experimental curves constituting the pair of values for this tubule. The value of N given for an experimental condition corresponds to the number of perfused tubules, ∼1 to 3 being perfused in one rat, and for each group at least 4 rats were used. Luminal HCO3− activity, initially starting with 25 mM, was then progressively reduced to a stationary level (HCO3−s) by H+ secretion. The voltage between the microelectrode barrels, representing the luminal H+ activity, was sampled every 0.5 s by an AD converter (Lynx, São Paulo, Brazil) in a microcomputer. At the same time, the potential difference between the reference barrel and ground (the rat tail) was recorded, giving the evolution of transepithelial PD with time during the perfusion. Luminal bicarbonate was calculated from luminal pH and blood Pco2 was measured by a Severinghaus electrode. The rate of tubular acidification was expressed as the half-time of the exponential reduction of the injected HCO3− concentration to its stationary level (t1/2). Net HCO3− reabsorption (JHCO3−) per cm2 of tubule epithelium was calculated from the equation: JHCO3− = ln 2/t1/2 ([HCO3−]0 − [HCO3−]s)*r/2, where t/2 is the half-time of bicarbonate reabsorption, r is the tubule radius measured by an ocular micrometer, and [HCO3−]o and [HCO3−]s are the concentrations of the injected HCO3− and HCO3− at the stationary level, respectively. The tubules were perfused with control solution, 10−6 M UGN, and/or the specific NHE3 inhibitor, 5 μM S3226. In the experiments designed to investigate the signaling mechanisms of UGN action, we used 10−6 M KT5823 (PKG inhibitor); 10−5 M H89 (PKA inhibitor).
Gene expression of GC-C receptor in the rat kidney and LLC-PK1 cells.
RNA was purified from rat kidney and LLC-PK1 cell cortices using TRIzol Reagent (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. Total RNA (2 μg) was reversed transcribed using Super Script III (Invitrogen) and random hexamer primers were followed by amplification of cDNA by PCR using Taq DNA Polymerase (Invitrogen). The specific primers to the rat GC-C receptor (NM_013170.1), forward (5′-ATGACGTCACTCCTGGGCTT-3′) and reverse (5′-GCGCTTTCGCACGATGTCCAGT-3′), were used for initial PCR with an expected PCR product of ∼200 bp plus multiple bands. Forward primer (5′-GACGTCACTCCTGGGCTTGGC-3′) and reverse primer (5′-GTGGCACTTCT-GCCTCACCT-3′) were used for nested PCR with an expected PCR product of ∼90 bp. Thermal cycling for initial rat GC-C analysis included a denaturation step at 95°C for 5 min followed by 30 cycles of 95°C for 30 s, annealing temperature of 58°C for initial PCR, and 60°C for nested PCR for 30 s and 72°C for 1 min. LLC-PK1 cell cDNA was subjected to PCR using two sets of oligonucleotide primers [5′-CCCGCTGTTGGCCTTGGCTT-3′ (forward) and 5′-GGTACACGAGGGCCCCAGGA-3′ (reverse)] and [5′-GCCTCCCTG-AAAGCCCAGAGC-3′ (forward) and 5′-TACACGAGGGCCCCAGGAGG-3′ (reverse)]. These two PCR primer pairs were designed according to the porcine guanylate cyclase 2C (heat-stable enterotoxin receptor) mRNA (NM_214105.1). The expected sizes of the amplified DNA bands were 367 and 438 bp. PCR products were resolved by electrophoresis at 100 V through 1% agarose gel and visualized with ethidium bromide.
LLC-PK1 cells were obtained from American Type Culture Collection and used from passages 2 to 7. Serial cultures were maintained in DMEM medium supplemented with 45 mM NaHCO3, 25 mM HEPES buffer, 0.1 mM sodium pyruvate, 0.01 mM nonessential amino acids, 10% 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 subcultured with trypsin-EGTA (0.02%) and seeded on tissue culture plates with sterile glass coverslips (for pH measurements) to become confluent. For all experiments, cells were placed in serum-free medium 24 h before the experiments.
Measurement of Na+/H+ exchanger activity by fluorescence microscopy.
Intracellular pH was examined in cells loaded with the fluorescent pH-sensitive probe BCECF by measuring the fluorescence intensity at alternating excitation wavelengths of 440 and 495 nm using a method described previously (7). Briefly, cells were grown to confluence on glass coverslips and were placed in a thermoregulated chamber mounted on an inverted epifluorescence microscope (Nikon, TMD) and loaded with 10 μM BCECF-AM (in control solution, containing in mM: 141 NaCl, 5.4 KCl, 1.0 CaCl2, 0.4 KH2PO4, 0.5 MgCl2, 0.4 MgSO4, 0.3 Na2HPO4, 10 HEPES, 0.6 glucose) for 5 min. After several washes, the BCECF-loaded cells were prepulsed with 20 mM NH4Cl for 2 min for subsequent acid loading and then exposed to control solution in the presence of UGN with or without several inhibitors, as described later. Measurements of NHE3 activity were performed in the presence of 10 μM HOE694 to obviate the contribution by NHE1 (7). 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. Fluorescence was monitored using alternately 440 or 495 nm as excitation wavelengths using 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 at different pHs.
cGMP and cAMP assay.
LLC-PK1 cells were cultured to 100% confluence in 96-well plates. Cells were incubated for 10 min with culture medium containing 1 mM 3-isobutyl-1-methylxanthine (IBMX) or IBMX and 10−6 M UGN or 10−6 M ANP (used as positive control in cGMP assay) or 10−4 M forskolin (used as positive control in cAMP assay). cGMP and cAMP were measured by using the Amersham enzyme immunoassay Biotrak (EIA) System (GE Healthcare) according to specifications of the manufacturer.
Determination of PKA activity in cell lysates.
LLC-PK1 grown to confluence in 24-well plates were treated or not with UGN and subsequently solubilized in lysis buffer containing 20 mM MOPS, 50 mM β-glycerolphosphate, 50 mM NaF, 1 mM sodium vanadate, 5 mM EGTA, 2 mM EDTA, 1% NP-40, 1 mM DTT, 1 mM benzamidine, 1 mM PMSF, 10 μg/ml aprotinin, and 10 μg/ml leupeptin. PKA activity was measured in this cell lysate using a nonradioactive PKA Kinase Activity Assay (Enzo Life Sciences, Farmingdale, NY) according to the manufacturer's instructions.
Determination of PKG activity in cell lysates.
LLC-PK1 grown to confluency in 24-well plates were treated or not with UGN and subsequently solubilized in extraction buffer containing 20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM PMSF, 1 μg/ml pepstatin, 0.5 μg/ml leupeptin, 2 mM NaF, 0.2 mM Na3VO4, 5 mM β-mercaptoethanol. PKG activity was measured in this cell lysate using a single-site and semiquantative CycLex cGK Assay Kit (CycLex, Nagano, Japan) according to the manufacturer's instructions.
Cell surface biotinylation.
The assay was performed as described previously (25). Cells were rinsed twice in ice-cold PBS-Ca-Mg (PBS with 0.1 mM CaCl2, 1.0 mM MgCl2). Surface membrane proteins were then biotinylated by incubating the cells twice for 25 min with 2 ml of ice-cold biotinylation buffer (150 mM NaCl, 10 mM triethanolamine, 2 mM CaCl2, and 2 mg/ml EZ-Link sulfo-NHS-SS-biotin). Cells were then rinsed twice for 20 min with a quenching buffer (PBS-Ca-Mg, 100 mM glycine), washed twice with ice-cold PBS-Ca-Mg, and strapped into ice-cold solubilization buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate, 1% Triton X-100, pH 7.4) containing protease inhibitors (0.7 μg/ml pepstatin A, 0.5 μg/ml leupeptin, and 40 μg/ml PMSF). After lysis on ice for 60 min, extracts were centrifuged for 10 min at 14,000 g and 4°C. The protein concentration of the supernatants was measured (38) and equal protein amounts of cell lysate (500 μg) were equilibrated with streptavidin-agarose beads at 4°C. The beads were then washed five times in ice-cold solubilization buffer. Biotinylated proteins were released by incubation in Laemmli buffer and subjected to SDS-PAGE and immunoblotting.
SDS-PAGE and immunoblotting.
Protein samples were solubilized in Laemmli sample buffer and separated by SDS-PAGE using 7.5% polyacrylamide gels. For immunoblotting, proteins were transferred to PVDF (Millipore Immobilon-P, Millipore, Bedford, MA) at 350 mA for 8–10 h at 4°C with a TE 62 transfer electrophoresis unit (GE HealthCare). Sheets of PVDF containing transferred proteins were incubated first in Blotto (5% nonfat dry milk and 0.1% Tween 20 in PBS, pH 7.4) for 1 h to block nonspecific binding of antibody, followed by overnight incubation in primary antibody. Primary antibodies, diluted in Blotto, were used at dilutions ranging from 1:200 to 1:50,000. The sheets were then washed in Blotto and incubated for 1 h with an appropriate horseradish peroxidase-conjugated secondary antibody diluted 1:2,000 in Blotto. After being washed 5× in Blotto and 2× PBS (pH 7.4), an enhanced chemiluminescence detection (ECL) system (GE HealthCare) with Kodak Biomax imaging film (Kodak) was used for visualization of the bound antibodies. The visualized bands were digitized using the ImageScanner III (GE HealthCare) and quantified using the Scion Image Software (Scion, Federick, MD). In some experiments, PVDF blots were reprobed with additional primary antibodies after the first antibody was stripped away. This was accomplished by incubating the PVDF sheets in Restore Western Blot Stripping Buffer (Thermo Scientific, Rockford, IL).
The data were analyzed by a Visual Basic program in Excel software. Statistical comparisons were made by the unpaired t-test, taking the probability of 0.05 (5%) as the limit of significance. When more than two groups were compared, one-way ANOVA followed by Tukey's post hoc test, taking 0.05 (5%) as limit of significance, was performed. In microperfusion experiments, a minimum of six tubules was used (n = number of perfused tubules).
Signaling mechanisms mediating the inhibitory effect of UGN on NHE3-mediated bicarbonate reabsorption in rat renal proximal tubules.
The perfusion of proximal tubules with 1 μM UGN promoted a significant reduction, ∼46%, of net bicarbonate reabsorption (Fig. 1). No significant differences were observed between the group treated with 5 μM S3226 (specific NHE3 inhibitor) (48) or S3226 plus UGN. These results suggest that UGN inhibits NHE3-mediated bicarbonate reabsorption in proximal tubules, in agreement with our previous work (2).
Several studies showed that guanylin peptides regulate the intestinal transport of electrolytes and water via GC-C (20, 27). The activation of this receptor promotes an increase of cGMP intracellular content, which activates PKG. Although several studies demonstrated that the guanylin classic receptor GC-C is expressed in rat renal cortex (10, 18), a recent study suggested otherwise (45). As seen in Fig. 2A, we were able to detect the presence of GC-C mRNA in renal cortex by means of nested PCR. Thus, we next sought to examine whether activation of the cGMP/PKG signaling pathway mediates inhibition of NHE3 promoted by UGN. To investigate the involvement of PKG in the UGN action, we perfused proximal tubules with UGN associated with the PKG inhibitor KT5823. There was no significant alteration in net proximal bicarbonate reabsorption when the segments were perfused with UGN + 10−6 μM KT5823 (Fig. 2B). These findings suggest that inhibition of NHE3 by UGN is mediated, at least in part, by the cGMP/PKG signaling pathway.
Inhibition of NHE3 by a PKA-dependent mechanism has also been well-demonstrated (13, 24, 31, 32). Thus, we next evaluated whether the PKA signaling pathway was also involved in mediating the effects of UGN in renal proximal tubule. As illustrated in Fig. 2C, the inhibitory effect of UGN on proximal bicarbonate reabsorption was prevented by the addition of the PKA inhibitor H89 (10−5 M). These results suggest the involvement of PKA activation in the UGN action.
Considering the possibility of PKA activation and the findings demonstrated by another study, which suggested the involvement of a G protein activation by UGN in kidney cells (50), we next examined whether adenylyl cyclase (AC) activation would be involved in mediating the UGN effect in rat renal proximal tubule. By using the stationary microperfusion technique, we found that the inhibitory effect of UGN on NHE3 activity persisted in the presence of 100 μM 2′,5′-dideoxyadenosine (adenylyl cyclase inhibitor; Fig. 2D). This result suggests that the reduction of bicarbonate reabsorption promoted by UGN in rat proximal tubule does not depend on AC activation.
UGN treatment inhibits NHE3 activity in LLC-PK1 cells.
To investigate the signaling pathways and molecular mechanisms mediating the inhibitory effect of UGN on NHE3 transport activity, we performed the next series of experiments in LLC-PK1 cells. At first, we examined whether UGN would affect LLC-PK1 pH recovery following intracellular acidification by using the NH4Cl pulse technique (5). As illustrated in Fig. 3, in control condition, the pHi recovery rate in the first 2 min was 0.33 ± 0.02 pH U/min (n = 10). This recovery was significantly reduced in the presence of 1 μM UGN, reaching 0.23 ± 0.02 pH U/min (n = 11; P < 0.01). To specifically analyze the modulation of the Na+/H+ exchanger isoform NHE3 by UGN, we next used 10 μM HOE-694, a selective NHE1 and NHE2 inhibitor. We observed that UGN significantly reduced the pH recovery mediated by the HOE694-resistant Na+/H+ isoform (0.093 ± 0.011 pH vs. 0.14 ± 0.014 pH U/min in control; Fig. 3). These findings indicate that UGN treatment decreases Na+-dependent pHi recovery rates from cell acidification mediated by NHE3 in LLC-PK1 cells.
To maintain NHE1 and NHE2 inhibited, all subsequent functional experiments in LLC-PK1 cells were performed in the presence of 10 μM HOE-694.
Role of PKG and PKA in the inhibition of NHE3 by UGN.
To verify the involvement of the PKG pathway in the UGN action, LLC-PK1 cells were treated with KT5823. The rate of pHi recovery in the presence of KT5823 and UGN was higher than the average obtained in the UGN group (0.11 ± 0.02 pH U/min UGN + KT5823, n = 15 vs. 0.093 ± 0.011 pH U/min UGN, n = 12; Fig. 4A), but this difference did not reach statistical significance. On the other hand, addition of H89 (PKA inhibitor) completely abolished the UGN effect (0.14 ± 0.017 pH U/min UGN + H89, n = 7 vs. 0.093 ± 0.011 pH U/min UGN, n = 12; P < 0.05; Fig. 4B). Collectively, these data suggest that PKA and maybe PKG mediate the inhibitory effect on NHE3 promoted by UGN in LLC-PK1 cells.
UGN incubation induces NHE3 phosphorylation in LLC-PK1 cells.
Since the NHE3 exchanger is normally phosphorylated in basal conditions and its level of phosphorylation is often affected as part of acute regulatory mechanisms (13, 32), we next investigated whether the inhibitory effect of UGN would involve changes on NHE3 phosphorylation. It is well-established that the serines 552 and 605 are PKA consensus sites for NHE3 phosphorylation (32). We therefore evaluated whether UGN would affect the NHE3 phosphorylation state at these PKA consensus sites by using phosphospecific antibodies specifically generated toward NHE3 phosphorylated at serine 552 or at serine 605 (32). The data shown in Fig. 5 demonstrate that inhibition of NHE3 activity by UGN is associated with increased levels of NHE3 phosphorylation at the PKA consensus sites. The presence of H89 or KT5823 completely blocked this effect (Fig. 5).
UGN incubation stimulates intracellular increment of cGMP, cAMP, PKG, and PKA activities.
Considering the possible involvement of PKG and PKA pathways on UGN action, we sought to evaluate the effect of this peptide on intracellular concentration of cGMP and cAMP. As illustrated in Fig. 6A, the treatment of LLC-PK1 cells with 1 μM UGN for 10 min increased the intracellular level of cGMP by 236 ± 0.31%. The UGN-induced increases in cGMP in LLC-PK1 cells suggest that GCC mRNA could be detected in these cells. We, therefore, examined the expression of mRNA encoding this receptor by means of RT-PCR and DNA sequencing analyses. As demonstrated in Fig. 6B, GC-C-mRNA is expressed in LLC-PK1 cells. Additionally, as demonstrated in Fig. 6C, incubation of LLC-PK1 cells with 1 μM UGN also induced an increment of 209 ± 10% in the intracellular content of cAMP.
The next step was to evaluate whether UGN could stimulate the enzymatic activity of PKA and PKG in LLC-PK1 cells treated with UGN. The incubation of cells with the peptide stimulated an increase of PKA activity of ∼230% after 2 min of treatment. This increment remained at this level along 30 min of incubation with UGN (Fig. 7A). PKG activity was also stimulated by UGN. However, after 2 min of incubation, PKG activity was increased by 47%, and the maximal effect was reached (238%) after 10 min of treatment with UGN. From 10 to 30 min of incubation, the PKG activity decayed gradually (Fig. 7B).
UGN incubation affects the surface abundance of NHE3.
It has been shown that besides variations in the level of NHE3 phosphorylation, alterations in the surface expression of the transporter also represent a mechanism of acute regulation (for review, see Ref. 13). The next step of our study was to investigate whether UGN treatment could change the expression level of NHE3 in the membrane of LLC-PK1 cells. Thus, surface proteins were biotinylated and precipitated with streptavidin. The precipitate was subjected to SDS-PAGE and to immunoblotting using an antibody directed to NHE3. Figure 8 shows that UGN treatment caused a decrease in the NHE3 surface expression. The percentage of inhibition was 33.0 ± 2.6% after treatment with 1 μM UGN for 30 min.
In the past half century, much has been discovered about the regulation of renal sodium handling; however, there are still several mechanisms involved in this process that require a better understanding. The kidneys exhibit a diurnal rhythm of sodium excretion that persists despite the constant intake (40). Sodium balance is connected to the control of extracellular fluid volume sensors involving arterial and venous pressure and volume. However, it has been difficult to demonstrate this relationship under conditions of single changes in sodium intake. The hypothesis of a regulatory mechanism linking the gastrointestinal tract and kidney is not new. Evidence of a gastrointestinal monitor for sodium balance was considered from the observation that an equivalent sodium load is more rapidly excreted after oral administration, compared with intravenous administration (6, 34). Otherwise, it has been suggested that guanylin peptides are responsible for this regulatory mechanism linking intestinal and renal transport of water and electrolytes (19).
We previously demonstrated the inhibitory effect of guanylin peptides on NHE3 activity, by stationary in vivo microperfusion (2, 35). In the present study, we aimed to investigate the mechanisms underlying the UGN-regulated natriuretic response, regarding the signaling pathways involved in the inhibition of NHE3 activity by this peptide. Reinforcing our previous studies, UGN perfusion inhibited the NHE3-mediated in vivo bicarbonate reabsorption in proximal tubules of rats. Moreover, our in vitro experiments, using LLC-PK1 cells, showed that the NHE3-mediated pHi recovery after an acid load is inhibited in the presence of UGN. These findings indicate that UGN inhibits the major route of sodium reabsorption in renal proximal tubules. Our findings also corroborate other studies that suggested the proximal tubule as a target for the UGN natriuretic effect (14, 36, 49, 51).
Regarding the signaling mechanisms of UGN action, it is well-known that the guanylate cyclase pathway is involved in the intestinal effect of UGN (16, 30). This peptide interacts with the GC-C receptor, increasing the intracellular content of cGMP, which stimulates Cl− and HCO3− secretion and inhibits Na+ absorption by the enterocytes (16, 21, 30). In the kidney, it has been documented that UGN increases the excretion of Na+, K+, water, and cGMP, without promoting changes either in glomerular filtration rate or in renal plasma flow or in osmolality (17, 18, 26). Although several studies indicate the presence of GC-C in nephron segments, especially in proximal tubules (10, 18, 42), others have suggested that the GC-C receptor is not expressed throughout the kidney (45). Additionally, studies with GC-C knockout mice showed that the intestinal effect of guanylin peptides is eliminated, but the renal effects remain, suggesting the existence of another receptor pathway in the kidney (9, 49). The current study demonstrated that GC-C is expressed in both rat renal cortex and in LLC-PK1 cells, suggesting that this receptor may mediate, at least in part, the renal actions of UGN.
Several studies have described a natriuretic action and an inhibition of NHE3 by cGMP or cGMP-dependent agonists (11, 29, 39, 46). These studies also demonstrated the involvement of PKG in the inhibition of Na transport by cGMP (11, 29). In our study, UGN failed in inhibiting bicarbonate reabsorption in proximal tubules of rats in the presence of a PKG inhibitor. These findings suggest the involvement of the guanylate cyclase pathway in the signaling mechanism of the UGN effect. On the other hand, our in vitro experiments, using measurement of NHE3-mediated pHi recovery after an acid pulse with NH4Cl, suggested that PKG might not be the only kinase involved in the inhibition of NHE3 by UGN, since incubation with the PKG inhibitor did not completely prevent the UGN effect in LLC-PK1 cells.
It has been demonstrated that the acute regulation of NHE3, which ensures the maintenance of volume and acid-base homeostasis in the face of rapid physiological challenges, occurs via changes in phosphorylation, trafficking, or membrane localization, acting on the existing cellular pool of NHE3 (13). The cytoplasmic loop of the NHE3 molecule contains several phosphorylation sites that are targeted by several kinases and areas of interaction with many regulatory factors (13). The cAMP-dependent PKA phosphorylates NHE3 in its COOH terminal. In has been shown that the phosphorylation of both serine 605 and 552 is required for maximum inhibition of NHE3 by cAMP, since the mutation of these serines individually reduced the inhibitory effect on NHE3 promoted by cAMP (54).
We found that UGN treatment can increase both intracellular content of cGMP and cAMP and the enzymatic activities of PKG and PKA in LLC-PK1 cells. Indeed, the rate of PKA activity increased faster and kept constant until the end of the experimental period, while PKG activity declined toward the basal level, which suggests a major contribution of PKA in the UGN effect. The involvement of the PKA pathway in the action mechanism of UGN was observed in our study, since, in the presence of a PKA inhibitor, UGN failed to inhibit NHE3 activity both during bicarbonate reabsorption in rat proximal tubules and during pHi recovery in LLC-PK1 cells. Interestingly, our microperfusion results demonstrated that the activation of PKA does not depend on activation of adenylyl cyclase by UGN. Furthermore, the in vitro results showed that UGN increases NHE3 phosphorylation at PKA consensus sites and that this effect was inhibited by PKG and PKA inhibitors. Thus, future work needs to be done to further examine the interaction between the cAMP/PKA and the cGMP/PKG signaling pathways that are triggered by UGN in the renal proximal tubule.
Besides changes on NHE3 phosphorylation, UGN also induced a reduction of NHE3 cell surface expression in LLC-PK1. This change on NHE3 surface expression does not precisely correlate with our functional experiments, since the time of exposure of proximal tubule cells to UGN in both microperfusion and pHi recovery measurements was much faster than 30 min. However, this finding represents an additional mechanism by which NHE3 is modulated by UGN. In this regard, a study using UGN knockout mice showed impairment of subcellular redistribution of NHE3, which could explain the high-Na+ reabsorption in response to a salt load observed in these animals (14, 37).
Numerous studies have demonstrated that NHE3 plays an adaptive role in response to changes during salt intake (33, 53). UGN has been postulated as an intestinal natriuretic factor, which would be synthesized and activated after an oral salt load. The present study describes a component of the well-documented UGN natriuretic effect (19), showing that NHE3 is downregulated by the peptide through alterations in the phosphorylation status and in the subcellular distribution of the transporter within the renal proximal tubule cell.
The authors thank Fundacão de Amparo a Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Pesquisas (CNPq), and Fund. C. Fortaleza (Funcap) for support.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: L.M.A.L., L.R.C.-L., A.C.C.G., M.C.F., and G.M. conception and design of research; L.M.A.L., L.R.C.-L., R.O.C., C.N.A.B., R.D., A.C.C.G., and G.M. performed experiments; L.M.A.L., L.R.C.-L., C.N.A.B., R.D., A.C.C.G., M.C.F., and G.M. analyzed data; L.M.A.L., L.R.C.-L., A.C.C.G., M.C.F., and G.M. interpreted results of experiments; L.M.A.L., L.R.C.-L., C.N.A.B., R.D., and A.C.C.G. prepared figures; L.M.A.L. drafted manuscript; L.M.A.L., L.R.C.-L., R.O.C., C.N.A.B., R.D., A.C.C.G., M.C.F., and G.M. edited and revised manuscript; L.M.A.L., L.R.C.-L., R.O.C., C.N.A.B., R.D., A.C.C.G., M.C.F., and G.M. approved final version of manuscript.
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