NHE3 activity and trafficking depend on the state of actin organization in proximal tubule

C. Chalumeau1, D. Du Cheyron1, N. Defontaine1, O. Kellermann3, M. Paillard1,2, and J. Poggioli1

1 Institut National de la Santé et de la Recherche Médicale Unité 356, Institut Fédératif de Recherche 58; 2 Université Paris VI, Hôpital Broussais, Assistance Publique; and 3 Institut Pasteur, Laboratoire de Différenciation Cellulaire, Paris, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study was addressed to define the contribution of cytoskeleton elements in the kidney proximal tubule Na+/H+ exchanger 3 (NHE3) activity under basal conditions. We used luminal membrane vesicles (LMV) isolated from suspensions of rat cortical tubules pretreated with either colchicine (Colch) or cytochalasin D (Cyto D). Colch pretreatment of suspensions (200 µM for 60 min) moderately decreased LMV NHE3 activity. Cyto D pretreatment (1 µM for 60 min) elicited an increase in LMV NHE3 transport activity but did not increase Na-glucose cotransport activity. Cyto D pretreatment of suspensions did not change the apparent affinity of NHE3 for internal H+. In contrast, after Cyto D pretreatment of the suspensions, NHE3 protein abundance was increased in LMV and remained unchanged in cortical cell homogenates. The effect of Cyto D on NHE3 was further assessed with cultures of murine cortical cells. The amount of surface biotinylated NHE3 increased on Cyto D treatment, whereas NHE3 protein abundance was unchanged in cell homogenates. In conclusion, under basal conditions NHE3 activity depends on the state of actin organization possibly involved in trafficking processes between luminal membrane and intracellular compartment.

kidney; sodium/hydrogen exchanger 3 antiporter; protein trafficking


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MODULATION OF TRANSPORTER traffic between plasma membrane and internal compartments contributes to the regulation of vectorial transport across epithelia. In kidney proximal tubule, several apical transporters appear to be regulated, at least in part, by trafficking processes. Rats fed an acutely low-Pi diet exhibit a higher Na-Pi cotransport rate associated with an increase in type 2 Na-Pi cotransporter protein abundance in brush-border membrane vesicles (BBMV) and no change in cortical homogenate (20, 21). Colchicine (Colch), which disrupts the microtubule network, prevents this adaptation (21). Colch also prevents acute exocytic insertion of endosomal H+-ATPases in the apical membrane of straight rat proximal tubule perfused in vitro with high-PCO2 medium (25). Metabolic acidosis in rats is associated with an increase in enzymatic and transport activity of H+-ATPases in BBMV, without modification of the enzyme activity in cortical homogenates (8). Na+/H+ exchanger 3 (NHE3), which mediates 100% of NaCl absorption and two-thirds of HCO3 absorption in the proximal tubule, has been recently shown to be located in both apical membranes and intracellular vesicular compartments (3). Variations of NHE3 activity related to changes in the cellular location of the transport protein have recently been reported in experimental animal and in vitro models. After parathyroid hormone infusion in parathyroidectomized rats, a decrease in NHE3 activity and protein is observed in BBMV without modification of total cortical NHE3 protein abundance, and the decrease in BBMV NHE3 protein abundance is prevented by pretreatment of rats with Colch (11). A redistribution of NHE3 protein from cortex BBM into subapical compartments has been observed on natriuresis-induced acute hypertension in rats (31, 33). In the human colonic adenocarcinoma cell line Caco-2, expressing endogenous NHE3, confocal morphometric analysis complemented by cell surface protein biotinylation shows that the phorbol ester-induced decrease in NHE3 activity is partly due to redistribution of NHE3 protein from brush-border membrane to subapical cytoplasmic compartment (14).

The present study was addressed to define the contribution of cytoskeleton elements in the control of NHE3 activity present in the luminal membrane of rat kidney cortical tubules, under basal conditions. We used Colch to disrupt microtubule network and cytochalasin D (Cyto D) to depolymerize the actin cytoskeleton. Present data suggest that the state of actin organization plays a role in NHE3 activity and trafficking between intracellular sites and the luminal membrane.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Preparation of partially purified luminal membrane vesicles. A suspension of cortical tubules free of glomeruli was prepared as previously described (24). Five-milliliter samples of tubule suspension in a Ringer medium [(in mM) 116 NaCl, 3 KCl, 1 MgSO4, 0.2 KH2PO4, 0.8 K2HPO4, 10 HEPES, 1 CaCl2, 25 NaHCO3, 5 glucose, 5 alanine, and 10 Na pyruvate, as well as 0.1% bovine serum albumin] were equilibrated at 37°C under an atmosphere of 95% O2-5% CO2 for 15 min before the addition of the agent that had to be tested. The incubation was carried out for 60 min and stopped by adding 10 ml of ice-cold Ringer. After centrifugation, the tubules were resuspended in hypoosmotic homogenization medium [(in mM) 125 mannitol, 2 dithiothreitol (DTT), 5 trizma, pH 7.4, 10 EGTA-Tris, pH 7.4, 10 benzamidine, and 0.2 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochlorine with 0.1 µg/ml aprotinin and 12.5 µg/ml leupeptin]. Purified LMV were prepared by Mg2+ precipitation as described (2). LMV were suspended in a Na-free medium [(in mM) 200 mannitol, 3 EGTA, 50 tetramethylammonium-nitrate, and 50 Tris-Mes, pH 6], pelleted (30,000 g × 30 min, 4°C), resuspended in the same acidic medium, and used immediately or kept at -80°C until use. When 22Na uptake was measured as a function of intravesicular pH, the final pellet was resuspended in a medium containing (in mM) 200 mannitol, 6 EGTA, 120 N-methyl-D-glucamine nitrate, 80 N-methyl-D-glucamine gluconate, and 5 Tris-HEPES, pH 7. Each pellet (from control tubules and from Cyto D-treated tubules) was split into six fractions that were diluted two times to obtain pH values of 5.5, 5.83, 6.1, 6.5, 7.03, and 7.53, respectively, in the resuspended medium. Diluted LMV were kept at -80°C until the day for 22Na uptake experiments. The purity and yield of the LMV preparation were routinely followed by measuring the activity of enzyme markers maltase (9) and Na+-K+-ATPase (12). There was no difference between the membranes prepared from control tubules and those pretreated with Cyto D or Colch regarding the specific activity of the markers, yield, and enrichment. It is worth noting that Cyto D did not directly affect the protein yield in LMV and the activity of maltase as presented in Table 1.

                              
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Table 1.   Proteins, maltase activity, and Na-K-ATPase activity in homogenates and LMV fraction

Immunoblot analysis. Aliquots of 90 µl of LMV fractions were mixed with 30 µl of Laemmli buffer, heated at 90°C for 10 min and stored at -20°C until use. Samples were subjected to SDS-PAGE (7.5%) as described by Laemmli (19), and equivalent amounts of protein from controls or experimental media were run in parallel. Each sample was run in duplicate. Proteins on the gel were electrophoretically transferred onto nitrocellulose membranes (0.45 mm, Schleicher & Schuell) by using a Bio-Rad apparatus. The blots were rinsed and incubated overnight at 4°C with anti-NHE3 antiserum 1568 (generous gift of Dr. R. Alpern and Dr. O. Moe, Southwestern Medical Ctr., Dallas, TX, dilution 1:3,000). The nitrocellulose membranes were washed and probed with horseradish peroxidase-conjugated goat anti-rabbit antibody and then developed with an enhanced chemiluminescence kit (ECL) from Amersham. Polaroid pictures were taken with an Amersham apparatus. Apparent molecular masses were calculated on the basis of the mobility of a panel of molecular mass markers from Sigma. Quantitative data were obtained by scanning the photos (Scanjet, Hewlett Packard, using Deskan) and analyzed with National Institutes of Health Image sofware. Data were normalized to protein loading on the gel as described in (17).

Na and glucose uptake measurements. Na/H activity was assessed by proton gradient-stimulated initial rate of 22Na uptake using the rapid filtration method (pHin = 6.0, pHout = 8.0). Reaction was initiated by adding 20 µl of LMV (2-4 mg protein/ml in the Na-free acidic resuspension medium described above) to 80 µl of uptake medium [(in mM) 1 22NaCl (1.5 µCi/ml), 200 mannitol, 3 EGTA, 50 tetramethylammonium-nitrate, and 50 Tris-HEPES, pH 8] with or without ethylisopropyl amiloride (EIPA; 100 µM). It was stopped 10 s later by adding 2.5 ml of ice-cold washing solution [(in mM) 280 mannitol, 20 Tris-HEPES, pH 7.4, and 0.5 amiloride], and the mixture was filtered onto nitrocellulose filters (Millipore). The filters were rinsed three times with 5 ml of washing solution and counted by liquid scintillation. Each sample was assayed in triplicate at room temperature. 22Na uptake was linear with time until 30 s (not shown).

Na-glucose activity was assessed by Na gradient-stimulated initial rate of 14C-labeled alpha -methylglucopyranoside (AMG) uptake using the rapid filtration method (Nain = 0, Naout= 100 mM). Reaction was initiated by adding 20 µl of LMV (2-4 mg protein/ml in the Na-free resuspension medium, pH 7.4) to 80 µl of uptake medium {(in mM) 100 mannitol, 3 EGTA, 100 NaCl, and 50 Tris-HEPES, pH 7.4, as well as 80 µM [14C]AMG (1.3 µCi/ml)} with or without phlorizin (400 µM). It was stopped 10 s later by adding 2.5 ml of ice-cold washing solution [(in mM) 150 NaCl, and 1 Tris-HEPES, pH 7.4, and 10 µM phlorizin], and the mixture was filtered onto nitrocellulose filters. The filters were rinsed three times with 5 ml of washing solution and counted by liquid scintillation. Each sample was assayed in triplicate at room temperature. [14C]AMG uptake was linear with time until 1 min or more (not shown).

Cell surface biotinylation. Cell surface biotinylation was performed on mouse kidney cortical cells [MKCCs; (7)] as described (14). MKCCs were grown on 6-cm petri dishes at 5-6 days postconfluency. Cells were incubated in serum-free medium without hormones or growth factors for 24 h before the test. Cells were incubated in culture medium at 37°C under 5% CO2-95% air (pH 7.4) for 45 min in the presence of 3 µM Cyto D or its vehicle (0.1% dimethylsulfoxide, vol/vol) for controls. Cells were then washed three times with PBS at 4°C. The surface plasma membrane proteins were then biotinylated for 1 h at 4°C [500 µl/dish of sulfo-NHS-SS-biotin (0.5 mg/ml)] in borate buffer [(in mM) 154 NaCl, 10 boric acid, 7.2 KCl, and 1.8 CaCl2, pH 9]. The biotinylation solution was then discarded, and the cells were washed three times with PBS at 4°C. Five hundred microliters of solubilization medium were added on the monolayer for 45 min at 4°C [(in mM) 150 NaCl, 3 KCl, 5 EDTA, and 60 HEPES-Tris, pH 7.4, containing 3 µM aprotinin, 20 µM phosphoramidon, 200 µM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochlorine, and 10 µM leupeptin, as well as 1% Triton X-100 (vol/vol)]. After that, the cells were sonicated and the resulting homogenate was centrifuged (15 min at 16,000 g, 4°C). The supernatant was incubated with streptavidin-agarose to separate the biotinylated proteins from nonbiotinylated proteins by binding the former to streptavidin-agarose. After washes, biotinylated proteins were processed as described above for Western blot analysis.

Immunocytochemistry. Cells were fixed with 3% paraformaldehyde in PBS for 30 min at room temperature, washed for 5 min with 50 mM NH4Cl, permeabilized with 0.1% Triton X-100 for 1 min, and incubated with Dako antibody diluent for 10 min to block nonspecific binding. Actin filaments were labeled with phalloidin-tetramethylrhodamine isothiocyanate (0.1 µg/ml, Sigma). The cells were washed with PBS and mounted with Vectashield. The relative distributions of actin in cortex cells were observed with a Zeiss LSM 510 confocal microscope using a ×63 objective.

Materials. Collagenase was obtained from Boeringher, Mannheim, Germany. BSA, Cyto D, Colch, EIPA, and streptavidin-agarose were from Sigma (St. Louis, MO). Sulfo-NHS-SS-biotin was from Pierce (Rockford, IL). Nitrocellulose filters were from Millipore (Bedford, MA). Vectashield was from Vector Laboratories (Burlingame, CA), and Dako diluent was from Dako (Carpinteria, CA). 22Na and [14C]AMG were from Amersham (Buckinghamshire, UK).

Statistics. Results are expressed as means ± SE. Statistical significance was assessed by paired t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Microtubule disruption by colchicine moderately reduced NHE3 activity in LMV isolated from the cortical tubule suspension. Na/H exchange activity in the apical membrane of rat proximal tubule is due to NHE3, as previously shown (15, 30). Na/H exchange activity in LMV was assessed by the initial rate of 22Na uptake in the presence of an outward H+ gradient. We used 100 µM EIPA to completely inhibit the NHE3 activity as previously documented (15, 30). After pretreatment of the tubule suspension with Colch (200 µM for 60 min), which disrupted microtubule network, LVM total 22Na uptake was reduced by 10.8 ± 4.6% (3.09 ± 0.15 vs. 3.51 ± 0.14 nmol · mg protein-1 · 10 s-1, n = 11, P < 0.02), and the residual EIPA-resistant component of 22Na uptake did not change. EIPA-sensitive 22Na uptake decreased by 11.8 ± 4.7% (2.76 ± 0.14 vs. 3.18 ± 0.15 nmol · mg protein-1 · 10 s-1, n = 11, P < 0.02; Fig. 1A). Colch pretreatment of the tubule suspension did not modify LMV 22Na uptake at equilibrium (1.57 ± 0.15 vs. 1.59 ± 0.13 nmol/mg protein for controls, n = 7).


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Fig. 1.   Effect of microtubule disruption on Na+/H+ exchanger (NHE3) activity in luminal membrane vesicles (LMV) isolated from cortical tubules in suspension. A: cortical tubule fragments were isolated, incubated for 60 min with colchicine (Colch; 200 µM) or vehicle (0.1% dimethylsulfoxide, vol/vol, control), and LMV were subsequently isolated. H+-stimulated 22Na uptake was determined with or without ethylisopropyl amiloride (EIPA; 100 µM). Values are means ± SE of 11 experiments. * P < 0.02. B: cortical tubule fragments were isolated, and LMV were subsequently isolated. LMV were incubated for 60 min at room temperature with Colch (200 µM) or vehicle (1% dimethylsulfoxide, vol/vol, control). H+-stimulated 22Na uptake was determined with or without EIPA (100 µM). Values are means ± SE of 5 experiments. A and B: open bars, total 22Na uptake; solid bars, EIPA-resistant; striped bars, EIPA-sensitive.* P < 0.02. C: cortical tubule fragments were isolated, incubated for 60 min with Colch (200 µM) or vehicle [0.1% dimethylsulfoxide, vol/vol, controls (Cont)], and LMV were subsequently isolated. Ten micrograms of protein from each sample of LMV (top) or from each homogenate (bottom) were subjected to 7.5% SDS-PAGE and immunoblotting with anti-NHE3 antiserum (serum 1568, 1:3,000 dilution). Figure represents 1 Western blot representative of 11 membrane preparations. Left: molecular mass standards.

Additional experiments were performed to distinguish between an inhibitory effect on NHE3 activity resulting from a direct effect or from a change in protein trafficking. When Colch was added directly to LMV, a statistically significant reduction of total 22Na uptake was observed; the residual EIPA-resistant component of 22Na uptake did not change. The EIPA-sensitive 22Na uptake decreased by 6.7 ± 1.6% (1.98 ± 0.16 vs. 2.12 ± 0.17 nmol · mg protein-1 · 10 s-1, n = 5, P < 0.02; Fig. 1B). Then, the relative distribution of NHE3 protein in the homogenate and LMV was estimated by Western blots. As shown in Fig. 1C, NHE3 protein abundance in LMV was not different from controls on Colch treatment. Thus Colch moderately reduced NHE3 activity in LMV, at least in part, by a direct effect on the membranes not related to any change in protein trafficking.

Actin cytoskeleton disruption by Cyto D increased NHE3 activity in LMV isolated from the cortical tubule suspension. After pretreatment of the tubule suspension with Cyto D, which depolymerizes actin filaments (1 µM for 60 min), LMV total 22Na uptake was increased by 39.4 ± 10.5% (4.99 ± 0.13 vs. 3.66 ± 0.27 nmol · mg protein-1 · 10 s-1, n = 5, P < 0.001), and the residual EIPA-resistant component of 22Na uptake did not change. EIPA-sensitive 22Na uptake increased by 41.9 ± 10.6% (4.55 ± 0.13 vs. 3.27 ± 0.23 nmol · mg protein-1 · 10 s-1, n = 5, P < 0.001; Fig. 2). Cyto D pretreatment of the tubule suspension did not modify LMV 22Na uptake at equilibrium (2.58 ± 0.12 vs. 2.41 ± 0.13 nmol/mg protein for controls, n = 11). It was verified that a direct application of Cyto D to LMV (1 µM Cyto D at room temperature for 60 min before 22Na uptake measurements) had no effect on NHE3 activity. Neither total (3.12 ± 0.26 vs. 3.13 ± 0.29 nmol · mg protein-1 · 10 s-1 for controls, n = 3), EIPA-sensitive (2.69 ± 0.4 vs. 2.61 ± 0.5 nmol · mg protein-1 · 10 s-1 for controls, n = 3), nor EIPA-resistant uptake (0.43 ± 0.02 vs. 0.51 ± 0.04 nmol · mg protein-1 · 10 s-1 for controls, n = 3) was stimulated by direct addition of Cyto D to LMV. Thus actin microfilament disruption by Cyto D stimulated NHE3 activity.


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Fig. 2.   Effect of actin cytoskeleton disruption on NHE3 activity in LMV isolated from cortical tubules in suspension. Open bars, total 22Na uptake; solid bars, EIPA-resistant; striped bars, EIPA-sensitive. Cortical tubule fragments were isolated, incubated for 60 min with Cytochalasin D (Cyto D; 1 µM) or vehicle (dimethylsulfoxide, 0.1% vol/vol, control) and LMV subsequently isolated. H+-stimulated 22Na uptake was determined with or without EIPA (100 µM). Values are means ± SE of 5 experiments, * P < 0.001.

Actin cytoskeleton disruption by Cyto D did not increase Na-glucose cotransporter activity in LMV isolated from the cortical tubule suspension. To address whether the effect of actin cytoskeleton disruption on NHE3 activity was specific for this transporter, experiments were performed to study the effect of Cyto D pretreatment on the activity of another luminal transport protein, namely, the Na-glucose cotransporter.

Na-glucose cotransport activity was assessed by the initial rate of [14C]AMG uptake in the presence of an inward Na+ gradient, with or without 400 µM phlorizin, a specific inhibitor of the cotransporter. After Cyto D pretreatment of the tubule suspension, LMV total AMG uptake was moderately decreased from 54.45 ± 6.31 to 46.04 ± 8.44 pmol · mg protein-1 · 10 s-1 (n = 4, P < 0.001), and the residual phlorizin-resistant component of AMG uptake did not change. Phlorizin-sensitive AMG uptake decreased from 44.24 ± 5.43 to 36.26 ± 6.10 pmol · mg protein-1 · 10 s-1 (n = 4, P < 0.001; Fig. 3). Cyto D pretreatment of the tubule suspension did not modify AMG uptake at equilibrium in LMV (190.12 ± 16.40 vs. 174.03 ± 22.68 pmol/mg protein for controls, n = 4). Thus the stimulation of NHE3 activity did not result from a generalized increase in luminal transport activities after Cyto D pretreatment of the tubules.


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Fig. 3.   Effect of actin cytoskeleton disruption on Na-glucose cotransporter activity in LMV isolated from cortical tubules in suspension. Cortical tubule fragments were isolated, incubated for 60 min with Cyto D (1 µM) or vehicle (0.1% dimethylsulfoxide, vol/vol, control), and LMV were subsequently isolated. Na+-stimulated 14C-labeled alpha -methylglucopyranoside (AMG) uptake was determined with or without phlorizin (Phlor; 400 µM). Open bars, total [14C]AMG uptake; solid bars, Phlor-resistant; striped bars, Phlor-sensitive.Values are means ± SE of 4 experiments, * P < 0.001.

Actin cytoskeleton disruption did not affect the apparent affinity of NHE3 for internal H+ in LMV isolated from the cortical tubule suspension. The activation of NHE3 by Cyto D may be due to increase in apparent affinity of NHE3 for internal H+. Thus the rate of NHE3 exchange in LMV was studied as a function of intravesicular pH values ranging from 5.55 to 7.53, the extravesicular medium being maintained at pH 8. As shown in Fig. 4, Cyto D pretreatment of suspensions did not significantly change the apparent affinity of NHE3 for internal H+ (pKHi; 6.44 ± 0.03 vs. 6.47 ± 0.05 for controls, n = 6).


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Fig. 4.   Absence of effect of actin cytoskeleton disruption on the apparent affinity for internal H+ of NHE3 in LMV isolated from cortical tubules in suspension. Cortical tubule fragments were isolated, incubated for 60 min with Cyto D (1 µM) or vehicle (0.1% dimethylsulfoxide, vol/vol, control), and LMV were subsequently isolated. H+-stimulated 22Na uptake was determined with or without EIPA (100 µM) as a function of extravesicular pH. Values are means ± SE of 6 experiments. * P < 0.001 by 2-way ANOVA.

Actin cytoskeleton disruption modified NHE3 subcellular distribution between the intracellular compartment and luminal membrane. Cyto D may increase NHE3 activity in the luminal membrane by stimulating movements of NHE3 protein from the internal compartment to luminal membrane. Two approaches were used to test this possibility. First, the relative distribution of NHE3 protein in homogenate and LMV was estimated by Western blots. As shown in Fig. 5, NHE3 protein abundance in LMV was increased on Cyto D treatment (70 ± 12%, n = 6, P < 0.001). The inhibitor did not modify NHE3 protein abundance in cortical tubule homogenates. These data are consistent with a Cyto D-mediated insertion of NHE3 protein in the luminal membrane leading to a rise in luminal NHE3 activity.


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Fig. 5.   Effect of actin cytoskeleton disruption on NHE3 protein abundance in LMV isolated from cortical tubules in suspension (left) and in homogenates (right). A: cortical tubule fragments were isolated, incubated for 60 min with Cyto D (CD; 1 µM) or vehicle [0.1% dimethylsulfoxide, vol/vol, control (Cont)], and LMV were subsequently isolated. B: 10-15 µg of protein from each sample of LMV or 10-20 µg of protein from each homogenate were subjected to 7.5% SDS-PAGE and immunoblotting with anti-NHE3 antiserum (serum 1568, 1:3,000 dilution). Figure represents 1 Western blot representative of 6 membrane preparations [protein gel loading (A); immunoblots of NHE3 (B)]. Left: molecular mass standards.

Second, we used a differentiated kidney cortical tubular cell culture (MKCCs) that expresses functional NHE3 protein and was developed in our laboratory from mice transgenic for the large T antigen of SV40 (7, 16) to quantify surface biotinylated NHE3 as a function of the presence or absence of Cyto D treatment. As shown in Fig. 6, by immunofluorescence labeling and confocal microscopy, the F-actin network appeared regular in controls (Fig. 6A) but appeared disorganized and cut into short actin filaments in Cyto D-treated cells (3 µM, 60 min at 37°C, Fig. 6B). For biotinylation studies, MKCCs were incubated as described above in the presence of Cyto D or its vehicle for 60 min and washed, and cell surface proteins were biotinylated at 4°C before Western blot analysis as described in MATERIALS AND METHODS. In agreement with data obtained on LMV of pretreated cortical tubules, Cyto D increased the abundance of cell surface biotinylated NHE3 (20 ± 1.0%, n = 5, P < 0.001) whereas NHE3 protein levels in the cell homogenates remained identical to controls (Fig. 7). Altogether, these data strongly support that Cyto D stimulated NHE3 activity, at least in part, by protein trafficking processes.


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Fig. 6.   Effect of Cyto D on actin network. Images are from control (A) or Cyto D-treated cells (B). Mouse kidney cortical cells (MKCCs) were starved for 24 h of hormones and growth factors and incubated for 45 min with 3 µM Cyto D. They were processed as described in MATERIALS AND METHODS for indirect immunofluorescence, actin was visualized by using tetramethylrhodamine isothiocyanate-phalloidin, and they were analyzed by confocal fluorescence microscopy.



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Fig. 7.   Effect of Cyto D on cell surface expression of NHE3. MKCCs were starved for 24 h of hormones and growth factors and incubated for 45 min with 3 µM Cyto D, after which biotinylation was performed using sulfo-NHS-SS-biotin (0.5 mg/ml) as described in MATERIALS AND METHODS. Biotinylated proteins (A) and homogenates (B) were subjected to 7.5% SDS-PAGE and immunoblotting with anti-NHE3 antiserum [serum 1568 1:5,000 dilution plus or minus peptide (100 µg/ml) or 1:3,000 dilution in other experiments]. Figure shows 2 Western blots representative of 5 experiments on 5 different passages. Right: molecular mass standards.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study was designed to define the contribution of cytoskeleton elements in NHE3 activity present in the luminal membrane of proximal tubule, under basal conditions. The main informative points are the following: 1) LMV NHE3 activity was moderately reduced by Colch pretreatment of cortical tubule suspension; 2) Cyto D pretreatment selectively increased NHE3 activity in LMV; 3) Cyto D increased NHE3 protein abundance in LMV but not in cortical tubule homogenates; and 4) in differentiated murine cortical tubule cell cultures (MKCCs), expressing endogenous NHE3 protein, Cyto D increased the abundance of luminal cell surface biotinylated NHE3, without modification of the total amount of NHE3 protein in cell homogenates.

The microtubule network is involved in movements of numerous transporters to or from the plasma membrane of epithelial cells essentially after stimuli (see Ref. 13 for a review). In contrast, under basal conditions, the microtubule network does not play an important role in the regulation of NHE3 exchange activity. Indeed, Colch pretreatment of cortical tubule suspensions had no effect on the apical Na/H exchange activity in rabbits (4). The small inhibitory effect described in the present work is not related to NHE3 trafficking because inhibition was also observed when Colch was added directly to LMV. Whether this results from a pharmacological effect of 200 µM Colch on NHE3 protein or on the lipidic environment is unknown.

The role of the actin cytoskeleton in the regulation of ion channel and transporter activities has been previously examined in some studies. In excised inside-out patches of apical membranes of epithelial cells, Cyto D applied to the cytosolic side affected channel activities. Cyto D inhibits K+ channel activity in rat cortical collecting duct (27), Cl- channel activity in rabbit proximal tubule in primary culture (26), and activates Na+ channels in A6 epithelial cells (6). The relationship between the state of actin organization and Na+ channel activity in A6 cells has been the most extensively studied. Interestingly, only short actin filaments, generated by acute incubation with Cyto D or by capping actin with gelsolin, but not monomeric G actin or long F-actin, activate Na+ channels (1). Altogether, these results obtained in excised patches suggest an interaction between the state of actin organization and ion channel activities remaining in the apical membrane. In Caco-2 cells displaying only basolateral Na/H exchange, maximal Na/H exchange activity was reduced after deprivation of serum for 4 h, and Cyto D but not Colch prevented this effect (28). In the Cl--secreting intestinal cell line T84, Cyto D activates the basolateral Na-K-2Cl cotransporter (23), and jasplakinolide or phalloidin, which stabilizes long F-actin, inhibits activation of the cotransporter by cAMP (22). In the two latter studies, carried out in entire cells, the role of the drugs on the intrinsic activity of the proteins or on the protein-trafficking process has not been systematically explored.

The present study shows, for the first time to our knowledge, that Cyto D increases NHE3 activity in the luminal membrane of the proximal tubule, at least in part, via movements of NHE3 protein between the intracellular compartment and apical membrane. There is growing evidence supporting the presence of NHE3 recycling. NHE3 protein is present in subapical vesicles in kidney cortex (3) and in early endosomes of NHE3-transfected activator protein-1 fibroblasts (10).

The possibility that the effect of Cyto D on NHE3 reflects a reduction in the content of other proteins present in the apical membrane rather than a selective increase in NHE3 protein content is very unlikely. Indeed, there was no difference in the specific activity of the apical marker maltase between LMV isolated from controls and treated tubules. The specificity of the effect of Cyto D was also supported by the absence of effect of the drug on NHE3 activity when directly applied to LMV. The present results obtained in native cells of rat proximal tubule were also found in MKCCs, a differentiated mouse cortical tubule cell culture expressing endogenous NHE3 protein. Indeed, Cyto D increased abundance of luminal cell surface biotinylated NHE3 and cut F-actin into short actin filaments. Taken together, these results strongly support that Cyto D stimulates NHE3 activity by protein-trafficking processes. However, in a recent study, Cyto D, which disrupts F-actin, but also jasplakinolide, which stabilizes F-actin, has been shown to inhibit NHE3 activity in the transfected NHE-deficient activator protein-1 cell line, which exhibits NHE3 in recycling endosomes. The inhibition of the Na/H exchange rate has been attributed to changes in the intrinsic activity of NHE3, because the number of transporters at the cell surface appeared to be unaltered (18). There is no readily apparent explanation for the different effects observed in the presence of Cyto D on the transport activity and trafficking of NHE3 in that study and the present data. It is possible that the proximal tubule provides a unique environment, leading to a tissue-specific response of NHE3 to Cyto D. Our study has been performed in native and immortalized proximal tubule cells possessing endogenous NHE3, whereas an NHE3-deficient fibroblast cell line transfected with the antiporter was used in the other study.

The mechanisms whereby the state of actin organization influences the activity and trafficking of NHE3 remain largely unsettled, but some possibilities can be considered. Regulatory proteins (NHERF, E3KARP, EBP50) bind to NHE3 and ezrin (32). Ezrin itself is able to bind F-actin and is a link between actin and apical membrane. Bretscher (5) suggested that protein-protein interactions may retain transport proteins within the apical membrane and restrict their trafficking. Regarding NHE3, the increased abundance of the protein in the apical membrane observed in the presence of Cyto D might be related to alterations of protein-protein interactions secondary to disruption of F-actin. In a recent study with PS120 fibroblasts transfected with NHE3, when the normal binding of ezrin to NHERF was suppressed by using a truncated NHERF lacking the COOH-terminal 30 amino acids, NHE3 activity is rather enhanced (29), supporting the role of protein-protein interactions in regulating NHE3 activity.

In summary, the present data suggest strongly that the state of the actin cytoskeleton modulates NHE3 activity and trafficking between subapical intravesicular compartment and the luminal membrane in the proximal tubule.


    ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. R. Alpern and Dr. O. Moe for providing us with anti-NHE3 antisera, Dr. M. Froissart for statistical analysis, and C. Klein for confocal microscopy imaging.


    FOOTNOTES

Address for reprint requests and other correspondence: J. Poggioli, Institut National de la Santé et de la Recherche Médicale U356, 15 rue de l'École de Médecine 75270 Paris Cédex 06 France (E-mail: poggioli{at}ccr.jussieu.fr).

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.

Received 29 November 1999; accepted in final form 23 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Berdiev, B, Prat A, Cantiello D, Fuller C, Jovov B, Benos D, and Ismailov I. Regulation of epithelial sodium channels by short actin filaments. J Biol Chem 271: 17704-17710, 1996[Abstract/Free Full Text].

2.   Biber, J, Stieger B, Hasse W, and Murer H. A high yield preparation for rat kidney brush border membranes. Different behaviour of lysosomal markers. Biochim Biophys Acta 647: 169-176, 1981[ISI][Medline].

3.   Biemesderfer, D, Rutherford PA, Nagy T, Pizzonia JH, Abu-Alfa AK, and Aronson P. Monoclonal antibodies for high-resolution localization of NHE3 in adult and neonatal rat kidney. Am J Physiol Renal Physiol 273: F289-F299, 1997[Abstract/Free Full Text].

4.   Bloch, RD, Zikos D, Fisher KA, Schleicher L, Oyama M, Cheng JC, Skopicki HA, Sukowski EJ, Cragoe EJ, and Peterson DR. Activation of proximal tubular Na+-H+ exchange by angiotensin II. Am J Physiol Renal Fluid Electrolyte Physiol 263: F135-F143, 1992[Abstract/Free Full Text].

5.   Bretscher, A. Regulation of cortical structure by the ezrin-radixin-moesin protein family. Curr Opin Cell Biol 11: 109-116, 1999[ISI][Medline].

6.   Cantiello, H, Stow J, Prat A, and Ausiello D. Actin filaments control epithelial Na+ channel activity. Am J Physiol Cell Physiol 261: C882-C888, 1991[Abstract/Free Full Text].

7.   Chalumeau, C, Lamblin D, Bourgeois S, Borensztein P, Chambrey R, Bruneval P, Duong Van Huyen JP, Froissart M, Biber J, Paillard M, Kellermann O, and Poggioli J. Kidney cortex cells derived from SV40 transgenic mice retain intrinsic properties of polarized proximal tubule cells. Kidney Int 56: 559-570, 1999[ISI][Medline].

8.   Chambrey, R, Paillard M, and Podevin RA. Enzymatic and functional evidence for adaptation of the vacuolar H-ATPase in proximal tubule apical membranes from rats with chronic metabolic acidosis. J Biol Chem 269: 3243-3250, 1994[Abstract/Free Full Text].

9.   Dahlqvist, A. Assay of intestinal disaccharidases. Anal Biochem 22: 99-116, 1968[ISI][Medline].

10.   D'Souza, S, Garcia-Cabado A, Yu F, Teter K, Lukacs G, Skorecki K, Moore HP, Orlowski J, and Grinstein S. The epithelial sodium-hydrogen antiporter Na+/H+ exchanger 3 accumulates and is functional in recycling endosomes. J Biol Chem 273: 2035-2043, 1998[Abstract/Free Full Text].

11.   Fan, L, Wiederkehr MR, Collazo R, Wang H, Crowder L, and Moe OW. Dual mechanisms of regulation of Na/H exchanger NHE-3 by parathyroid hormone in rat kidney. J Biol Chem 274: 11289-11295, 1999[Abstract/Free Full Text].

12.   Forbush, BI. Assay of Na,K ATPase in plasma membrane preparations: increasing the permeability of membrane vesicles using sodium dodecyl sulfate buffered with bovine serum albumin. Anal Biochem 128: 159-163, 1983[ISI][Medline].

13.   Hamm-Alvarez, S, and Sheetz M. Microtubule-dependent vesicle transport: modulation of channel and transporter activity in liver and kidney. Physiol Rev 78: 1109-1129, 1998[Abstract/Free Full Text].

14.   Janecki, A, Montrose M, Zimniak P, Zweibaum A, Tse C, Khurana S, and Donowitz M. Subcellular redistribution is involved in acute regulation of the brush border Na+/H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. J Biol Chem 273: 8790-8798, 1998[Abstract/Free Full Text].

15.   Karim, Z, Chambrey R, Chalumeau C, Defontaine N, Warnock D, Paillard M, and Poggioli J. Regulation by PKC isoforms of Na+-H+ exchanger in luminal membrane vesicles isolated from kidney cortical tubules. Am J Physiol Renal Physiol 277: F773-F778, 1999[Abstract/Free Full Text].

16.   Kellermann, O, and Kelly F. Immortalization of early embryonic cell derivatives after the transfer of the early region of simian virus 40 into F9 teratocarcinoma cells. Differentiation 32: 74-81, 1986[ISI][Medline].

17.   Klein, D, Kern R, and Sokol R. A method for quantification and correction of proteins after transfer to immobilization membranes. Biochem Mol Int 36: 59-66, 1995[ISI].

18.   Kurashima, K, D'Souza S, Szaszi K, Ramjeesingh R, Orlowski J, and Grinstein S. The apical Na+/H+ exchanger isoform NHE3 is regulated by actin cytoskeleton. J Biol Chem 274: 29843-29849, 1999[Abstract/Free Full Text].

19.   Laemmli, UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[ISI][Medline].

20.   Levi, M, Lötscher M, Sorribas V, Custer M, Arar M, Kaissling B, Murer H, and Biber J. Cellular mechanisms of acute and chronic adaptation of rat renal Pi transporter to alterations in dietary Pi. Am J Physiol Renal Fluid Electrolyte Physiol 267: F900-F908, 1994[Abstract/Free Full Text].

21.   Lötscher, M, Kaissling B, Biber J, Murer H, and Levi M. Role of microtubules in the rapid regulation of renal phosphate transport in response to acute alterations in dietary phosphate content. J Clin Invest 99: 1302-1312, 1997[Abstract/Free Full Text].

22.   Matthews, J, Awtrey C, and Madara J. Microfilament-dependent activation of Na+/K+/2Cl- cotransport by cAMP in intestinal epithelial monolayers. J Clin Invest 90: 1608-1613, 1992[ISI][Medline].

23.   Matthews, JB, Smith JA, and Hrnjez BJ. Effects of F-actin stabilization or disassembly on epithelial Cl- secretion and Na-K-2Cl cotransport. Am J Physiol Cell Physiol 272: C254-C262, 1997[Abstract/Free Full Text].

24.   Poggioli, J, Lazar G, Houillier P, Gardin JP, and Paillard M. Effects of angiotensin II and nonpeptide receptor antagonists on transduction pathways in rat proximal tubule. Am J Physiol Cell Physiol 263: C750-C758, 1992[Abstract/Free Full Text].

25.   Schwartz, G, and Al-Awqati Q. Carbon dioxide causes exocytotis of vesicles containing H+ pumps in isolated perfused proximal and collecting tubules. J Clin Inv 75: 1638-1644, 1985[ISI][Medline].

26.   Susuki, M, Miyazaki K, Ikeda M, Kawaguchi Y, and Sakai O. F-actin network may regulate a Cl- channel in renal proximal tubule cells. J Membr Biol 134: 31-39, 1993[ISI][Medline].

27.   Wang, W, Cassola A, and Giebish G. Involvement of actin cytoskeleton in modulation of apical K channel activity in rat collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 267: F592-F598, 1994[Abstract/Free Full Text].

28.   Watson, AJM, Levine S, Donowitz M, and Montrose MH. Serum regulates Na+-H+ exchange in Caco-2 cells by a mechanism which is dependent on F-actin. J Biol Chem 267: 956-962, 1992[Abstract/Free Full Text].

29.   Weinman, E, Steplock D, Donowitz M, and Shenolikar S. NHERF associations with sodium-hydrogen exchanger 3 (NHE3) and ezrin are essential for cAMP-mediated phosphorylation and inhibition of NHE3. Biochemistry 39: 6123-6129, 2000[ISI][Medline].

30.   Wu, MG, Biemersderfer D, Giebisch G, and Aronson PS. Role of NHE3 in mediating renal brush border Na+-H+ exchange. Adaptation to metabolic acidosis. J Biol Chem 271: 32749-32752, 1996[Abstract/Free Full Text].

31.   Yip, K, Tse C, McDonough A, and Marsh D. Redistribution of Na+/H+ exchanger isoform NHE3 in proximal tubules induced by acute and chronic hypertension. Am J Physiol Renal Physiol 275: F565-F575, 1998[Abstract/Free Full Text].

32.   Yun, CHC, Lamprech G, Forster DV, and Sidor A. NHE3 kinase A regulatory protein E3KARP binds the epithelial brush border Na+/H+ exchanger NHE3 and the cytoskeletal protein ezrin. J Biol Chem 273: 25856-25863, 1998[Abstract/Free Full Text].

33.   Zhang, Y, Mircheff AK, Hensley CB, Magyar CE, Warnock DG, Chambrey R, Yip KP, Marsh DJ, Holstein-Rathlou NH, and McDonough AA. Rapid redistribution and inhibition of renal sodium transporters during acute pressure natriuresis. Am J Physiol Renal Fluid Electrolyte Physiol 270: F1004-F1014, 1996[Abstract/Free Full Text].


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