The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α- and γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule.

Introduction

Hypertension affects ∼1 billion individuals worldwide [1]. In the USA, almost one-third of the adult population has hypertension [2]. The key role of kidney sodium reabsorption in the development of hypertension is emphasized by the fact that all presently identified forms of Mendelian hypotension or hypertension involve genetic defects in sodium reabsorption [3]. The thiazide-sensitive sodium chloride cotransporter (NCC) is the salt reabsorptive pathway localized to the apical membrane of the mammalian distal convoluted tubule (DCT). The epithelial sodium channel (ENaC) is the sodium reabsorptive pathway localized to the second part of the DCT (DCT2), connecting tubule and collecting duct. It is composed of three subunits (α, β, and γ). Genetic disorders of blood pressure have informed us that increases in activity of either protein result in hypertension and decreases in activity result in hypotension [49]. These proteins are the main sodium reabsorptive pathways of the aldosterone-sensitive distal nephron, the final regulator of sodium and volume homeostasis in the kidney. In rats, mice and humans NCC and ENaC are coexpressed in one segment of the distal nephron, the DCT2 [1016]. As this is the only part of the DCT that expresses the aldosterone-specifying enzyme, 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) [17,18], it is presumably the only part of the DCT that is responsive to aldosterone. Not surprisingly, both of these proteins are quite sensitive to aldosterone and are regulated by key intracellular mediators of aldosterone's actions.

Aldosterone stimulation of ENaC has been established for decades and its actions have been well described [19]. The determination that NCC is also a key regulatory target for aldosterone has only been recognized more recently. There are now many studies that have demonstrated stimulation of NCC by low-salt diets or infused aldosterone [2024]. Additionally, two of the key intracellular mediators of aldosterone's actions, neural precursor cell-expressed developmentally down-regulated protein 4-2 (NEDD4-2) and serum and glucocorticoid-regulated kinase 1 (SGK1), regulate both NCC and ENaC. These are the two primary sodium transporters that are utilized by aldosterone to maintain sodium homeostasis and thus blood pressure. Alterations of either result in clear changes in blood pressure. Despite their role in regulating blood pressure, shared regulatory pathways, and coexpression in an aldosterone-sensitive nephron segment, interactions between ENaC and NCC have not previously been reported.

Now we report that these two critical proteins are bound together in the DCT2. Understanding how these proteins interact and affect the function of each other could lead to a significant alteration of our understanding of salt handling in the kidney and thus blood pressure homeostasis.

Experimental procedures

Materials

Materials were purchased from Sigma-Aldrich (St. Louis, MO), unless stated otherwise.

Confocal microscopy in mouse kidney cortex

To determine if γ-ENaC colocalizes with NCC, kidney slices from mice were stained with primary antibodies to NCC and γ-ENaC. Following treatment with fluorescent secondary antibodies, γ-ENaC (red) and NCC (green) were examined using confocal microscopy on an Olympus Fluoview 1000 confocal microscope. To examine colocalization, we first determined that there was no fluorescence bleed-through from the green channel to the red channel or vice versa. Then, we merged images from the green channel and the red channel; yellow in the merged image was an indication of colocalization. Primary antibodies were rabbit anti-NCC antibody raised against a mouse peptide antigen [25] and donkey anti-γ-ENaC antibody (F-20, Santa Cruz Biotechnology, cat # sc-22245). Secondary antibodies were Alexa Fluor 488 rabbit anti-goat (1:600) and Alexa Fluor 594 donkey anti-rabbit (1:600).

Cell culture and treatments

mDCT15 cells were plated on cell culture dishes and grown in growth medium containing a 50:50 mixture of DMEM/F12, 5% heat-inactivated fetal bovine serum (FBS), and 1% penicillin/streptomycin/neomycin (P/S/N), at 37°C. Experiments were conducted when the cells reached 90–95% confluence.

Electrophysiology

Gigaohm seals were obtained on individual mDCT15 cells grown on permeable supports and single-channel activity recorded for up to 30 min [26]. Channel density (N), channel open probability (Po), and the current/voltage (I/V) relationship were calculated as previously reported [27]. To examine the amiloride sensitivity of the current, the tips of patch pipettes were filled with saline (∼5 mm from the tip). The rest of the pipette was filled with a saline solution containing 50 nM amiloride. After the patch was formed, normal channel activity was recorded prior to the diffusion of amiloride to the cell surface at the pipette tip (typically 5–10 min). Current tracings were recorded with no applied potential.

For the electrophysiology studies examining the effects of thiazides on ENaC, the following methodology was used: mDCT15 cells were grown on cover-glasses to confluence in DMEM/F12 medium (Cellgro, USA), supplemented with 5% FBS, 100 IU/ml penicillin and 100 µg/ml streptomycin, and 50 nM dexamethasone. Single-channel activity of ENaC on the membrane of mDCT15 cells was determined under voltage-clamp conditions in outside-out configuration. Gap-free single-channel current data from gigaohm seals were acquired with an Axopatch 200B (Molecular Devices) patch-clamp amplifier interfaced via a Digidata 1440 (Molecular Devices). Currents were low-pass filtered at 1 kHz with an eight-pole Bessel filter (Warner Instruments). Events were inspected visually prior to acceptance. Recording pipettes had resistances of 5–8 MΩ. Bath and pipette solutions were (in mM): 140 NaCl, 10 EDTA, 10 Hepes (pH 7.31) and 150 mM CsCl, 10 BAPTA, 10 Hepes (pH 7.3). Channel activity (NPo) and open probability (Po) were assessed using Clampfit 10.5 software (Molecular Devices). For calculating Po in paired experiments, N was fixed as the greatest number of active channels observed in control or experimental conditions. For representation, current traces were filtered at 200 Hz. For studies in CHO cells, recombinant mouse ENaC subunits (αβγ) were overexpressed in CHO cells by transfecting 0.2 µg/subunit/9.6 cm2 of the appropriate plasmid cDNAs using the Polyfect reagent (Qiagen, Valencia, CA) as described previously [28,29]. Single channels were recorded as detailed above.

Standard immunoblotting

mDCT15 cells were incubated as above. The cells were harvested, lysed using lysis buffer containing protease inhibitor, and homogenized by sonication on ice. The cell lysates were centrifuged briefly and supernatant was collected. Proteins were transferred electrophoretically to PVDF membranes. After blocking with 3% BSA, the membranes were probed with corresponding primary antibodies: anti-NCC [25] 1:4000–1:8000 Actin (Santa Cruz, 1:1000) overnight at 4°C. All ENaC antibodies (1:200–1:1000 dilution) utilized for immunoblotting were from the laboratory of Douglas Eaton and have been validated [3032]. The NCC antibody (1:5000–1:8000) was developed and validated in the Hoover Laboratory [25]. The blots were washed in Tris-buffered saline (TBS)–tween (TBST). Secondary antibodies were donkey anti-rabbit HRP-linked antibody (Amersham, 1:5000). Supersignal West Pico was used for chemiluminescence (Thermo Scientific). Chemiluminescence was detected with G:Box (gelbox) and analysis was performed by Genetools software (Syngene).

Immunoprecipitation

mDCT15 cells or mouse kidney cortex was lysed and then immunoprecipitated with the indicated antibody according to the manufacturer's instructions (Pierce Co-Immunoprecipitation Kit, cat # 26149). Antibodies for immunoprecipitation were the same as those used above for immunoblotting. Then, lysates and immunoprecipitated protein were immunoblotted for the indicated proteins.

Blue native PAGE

Blue native (BN)-PAGE was performed using the Novex Native-PAGE Bis–Tris gel system (Invitrogen) according to the manufacturer's directions. Briefly, total cellular proteins were extracted from mDCT15 cells using nondenaturing lysis buffer in the presence of n-dodecyl-β-d-maltoside at a final concentration of 1%. Samples were prepared with 2.5 µl of G250 Coomassie additive. Samples were electrophoresed on native PAGE Novex 3–12% Bis–Tris gels using ‘light blue’ cathode buffer for 1 h at 150 V, followed by 1 h at 250 V and transferred to PVDF membranes in NuPAGE Transfer Buffer for 2 h at 25 V. Membranes were washed using TBST and blocked with 5% milk (Bio-Rad) in TBST prior to immunoblotting. Primary antibodies were allowed to incubate overnight (4°C, constant rocking), with secondary incubation for 2 h at RT.

Animal preparation and experimentation

All animal protocols were approved by the Emory University Institutional Animal Care and Use Committee. Mice were kept in cages with autoclaved bedding and received free access to water and a standard diet (Diet 5001; Purina, 0.4% Na). Mice were killed using carbon dioxide, as approved through Emory IACUC (IACUC protocol # DAR-2002607-012417BN). Kidneys were then harvested and the cortex was dissected.

Preparation of kidney cortex

Mouse kidney cortex was homogenized in a glass tissue grinder in ice-cold RIPA buffer (1× protease inhibitors cocktail and 1× phosphatase inhibitor). After centrifugation at 13 000 rpm for 20 min at 4°C, the protein concentration was determined using a BCA protein assay kit. The appropriate amount of each sample was diluted in a Tris-glycine/SDS sample buffer [125 mM Tris (pH 6.8), 4% SDS, 10% glycerol, 3% β-mercaptoethanol, and 0.02% bromophenol blue] and heated at 70°C for 15 min prior to loading for SDS–PAGE.

Mammalian two-hybrid assay

We utilized the Matchmaker Mammalian Two-Hybrid Assay Kit (Clontech). A full-length clone for mouse NCC was inserted into the DNA-binding domain (DNA-BD) plasmid vector, pM. Full-length clones of each subunit of ENaC were inserted into the activation domain (AD) plasmid, pVP16. These constructs were transfected into CHO cells along with the pG5CAT reporter plasmid. Each ENaC subunit was tested singly with NCC. The reporter gene will only be expressed if the protein encoded in the DNA-BD vector binds to the protein encoded in the AD vector. Cells were harvested 48 h after transfection and binding was assessed by measuring reporter gene expression (chloramphenicol acetyltransferase, CAT). Experiments were analyzed for the presence of CAT with a commercially available UV assay (FAST CAT Assay Kit, Thermo-Fisher, Molecular Probes, cat # F-6616). Blots were visualized with a Kodak 2000 M camera system (Eastman Kodak). The intensity of the signal was detected with Image J.

Fluorescence resonance energy transfer acceptor photobleaching

NCC-enhanced green fluorescent protein (EGFP) and α-, β-, and γ-ENaC-mCherry constructs were generated and cotransfected into COS-7 cells (50–60% confluency) using the Xfect system (Clontech, Mountain View, CA). These chromophores are an excellent donor/acceptor pair, which have been previously used in fluorescence resonance energy transfer (FRET) studies [33,34]. FRET was measured by the acceptor photobleaching method. Live cell images were acquired during incubation at 37°C, 5% CO2. The donor (EGFP) excitation/emission (488/509 nm) and the acceptor (mCherry) excitation/emission (587/610 nm) spectra were used for all images. Three single images were taken before and after 20 rounds of photobleaching; these were used for before and after image analysis, where increases in EGFP fluorescence intensity were assessed after depletion of mCherry. Images were acquired using the Nikon1AR TE2000 laser scanning/live cell confocal microscope (60×, 1.4 NA, oil) and acquired/analyzed using Elements by Nikon (Nikon Instruments, Mellville, NY). Fluorescence images were acquired by a Nikon A1R TE2000 laser scanning confocal microscope. The FRET efficiency (%E) was calculated as: [(donorpostbleach − donorprebleach)/donorpostbleach)] × 100.

Electron microscopy

For this mouse, kidneys were fixed by in vivo perfusion of 3% PFA/PBS, followed by post-fixation of kidney slices with 3% PFA/0.1% glutaraldehyde/PBS for 2 h and cryoembedding. Ultrathin sections were blocked with serum-free protein block (Dako), followed by double staining with guinea pig anti-NCC antibody and rabbit anti-γ-ENaC antibody (gifts of Johannes Loffing) [35]. Secondary antibodies were colloidal gold-labeled anti-guinea pig (12 nm) and anti-rabbit (6 nm), both from Jackson laboratories. Images were captured with a Phillips CM-100 TEM.

Statistical analysis

Statistical analysis was performed using either SigmaPlot (Systat, San Jose) or GraphPad Prism (Graphpad Software, San Diego, CA). Error bars represent SEM. Data were analyzed for statistical significance using a paired t-test, ANOVA (Holm–Sidak), or Mann–Whitney rank sum test where appropriate. A P-value of <0.05 was taken as statistically significant.

Results

NCC and ENaC colocalize to the apical membrane of DCT2 by confocal microscopy in mouse kidney cortex

Coexpression of NCC and all three subunits of ENaC in the DCT2 of mouse, rat, and human kidney cortex has been shown many times [1016]. In fact, the DCT2 is often defined by the coexpression of NCC and ENaC. Those studies all used staining of consecutive sections, but they all demonstrated apically oriented NCC and apically oriented ENaC. The lack of studies utilizing antibodies derived from different species has resulted in the absence of images demonstrating colocalization by confocal microscopy. Here, utilizing our rabbit anti-NCC antibody [25] and goat anti-γ-ENaC antibody (Santa Cruz), we examined the coexpression of NCC and ENaC in mouse kidney cortex by confocal microscopy (Figure 1). The merged images demonstrate colocalization of NCC and γ-ENaC in the apical membrane of mouse DCT2. This confirms the previous reports of apically oriented NCC and ENaC as described above.

γ-ENaC colocalizes with NCC in mouse kidney cortex by confocal microscopy.

Figure 1.
γ-ENaC colocalizes with NCC in mouse kidney cortex by confocal microscopy.

(A) Merged image of NCC (from panel B, green) and γ-ENaC (from panel C, red) immunostaining. (B) NCC immunostaining in mouse kidney cortex with rabbit anti-NCC antibody (1:1000). (C) γ-ENaC immunostaining with donkey anti-γ-ENaC antibody (1:200). (D) Brightfield image. Similar images were obtained on two other occasions.

Figure 1.
γ-ENaC colocalizes with NCC in mouse kidney cortex by confocal microscopy.

(A) Merged image of NCC (from panel B, green) and γ-ENaC (from panel C, red) immunostaining. (B) NCC immunostaining in mouse kidney cortex with rabbit anti-NCC antibody (1:1000). (C) γ-ENaC immunostaining with donkey anti-γ-ENaC antibody (1:200). (D) Brightfield image. Similar images were obtained on two other occasions.

mDCT15 cells express functional ENaC channels

We have established a model of the DCT2 (mDCT15 cells) that natively expresses NCC, all three subunits of ENaC, NEDD4-2, SGK1, mineralocorticoid receptor, and many of the other key proteins expressed in the second part of the DCT [24,25,36]. This model has been extensively validated [25] and provides an excellent model to study the interaction of NCC and ENaC. Having previously established that these cells express functional NCC [24,25,37], we examined the function of ENaC in these cells by a cell-attached patch clamp. Single-channel current recordings demonstrated channels with long mean open and closed times characteristic of ENaC (Figure 2A). Six of 25 patches had similar channels and patches with channels usually had multiple channels (Figure 2A). The current/voltage (I/V) curve inwardly rectified with a conductance of 6 pS near zero applied voltage (Figure 2B), consistent with ENaC. Amiloride sensitivity of the current was then demonstrated (Figure 2C). These studies demonstrate the presence of functional ENaC channels in mDCT15 cells and establish our ability to measure ENaC function.

mDCT15 cells express functional ENaC.

Figure 2.
mDCT15 cells express functional ENaC.

(A) Single-channel records from a cell-attached patch with no applied potential. ‘c’ marks the closed levels and ‘o1–o5’ mark the levels of one to five channels open. (B) IV relationship for the channel. X-axis voltages are minus the pipette potential; that is, the values are displacements from the resting potential of the cell. The current/voltage (I/V) curve inwardly rectified with a conductance of 6 pS near zero applied voltage, consistent with ENaC. The current is fit by the Goldman current equation. The fit predicts a membrane potential between −10 and −20 mV and a relatively high intracellular sodium (∼20 mM), consistent with NCC activity. (C) Current tracing with no applied potential demonstrating amiloride sensitivity. To examine the amiloride sensitivity of the current, the tips of patch pipettes were filled with saline (∼5 mm from the tip). The rest of the pipette was filled with a saline solution containing 50 nM amiloride. After the patch was formed, normal channel activity was recorded prior to the diffusion of amiloride to the cell surface at the pipette tip (typically 5–10 min).

Figure 2.
mDCT15 cells express functional ENaC.

(A) Single-channel records from a cell-attached patch with no applied potential. ‘c’ marks the closed levels and ‘o1–o5’ mark the levels of one to five channels open. (B) IV relationship for the channel. X-axis voltages are minus the pipette potential; that is, the values are displacements from the resting potential of the cell. The current/voltage (I/V) curve inwardly rectified with a conductance of 6 pS near zero applied voltage, consistent with ENaC. The current is fit by the Goldman current equation. The fit predicts a membrane potential between −10 and −20 mV and a relatively high intracellular sodium (∼20 mM), consistent with NCC activity. (C) Current tracing with no applied potential demonstrating amiloride sensitivity. To examine the amiloride sensitivity of the current, the tips of patch pipettes were filled with saline (∼5 mm from the tip). The rest of the pipette was filled with a saline solution containing 50 nM amiloride. After the patch was formed, normal channel activity was recorded prior to the diffusion of amiloride to the cell surface at the pipette tip (typically 5–10 min).

NCC and ENaC run in the same band in BN gel

BN-PAGE utilizes mild detergents to preserve protein–protein interactions, allowing the ability to assess native oligomeric structure and associated proteins. Therefore, we sought to determine if NCC and ENaC are present in the same oligomeric band in BN gels. NCC has recently been shown to run as an ∼720 kDa band on BN gels [38]. ENaC was previously shown to run in a band somewhere above 600 kDa [39]. We ran membrane protein samples from mDCT15 cells on BN gels and probed the blots with antibodies for NCC and α-ENaC. These studies confirmed that NCC and α-ENaC both run at ∼720 kDa (Figure 3). This demonstrated that in cells natively coexpressing NCC and ENaC, we see α-ENaC in the same band as NCC. Interestingly, in contrast with NCC, α-ENaC seems to run in multiple bands. This could be consistent with differential binding of NCC and ENaC depending on conditions such as phosphorylation or cleavage. These studies indicate that all of the NCC and at least a major fraction of the ENaC protein appear to exist in the same macromolecular complex in the native nondenatured conformation.

BN-PAGE detects NCC and ENaC in the same macromolecular complex.

Figure 3.
BN-PAGE detects NCC and ENaC in the same macromolecular complex.

mDCT15 cell lysate run on nondenaturing Bis–Tris gel (Invitrogen) and immunoblotted with anti-NCC (1:5000) and anti-α-ENaC (1:500) antibodies. Representative image of three replicates.

Figure 3.
BN-PAGE detects NCC and ENaC in the same macromolecular complex.

mDCT15 cell lysate run on nondenaturing Bis–Tris gel (Invitrogen) and immunoblotted with anti-NCC (1:5000) and anti-α-ENaC (1:500) antibodies. Representative image of three replicates.

NCC coimmunoprecipitates with ENaC in mDCT15 cells and rodent kidney cortex

Since these proteins are similarly regulated, expressed in the same location, and appear to exist in the same macromolecular complex, we decided to assess whether they were bound together. In initial studies, lysate from mDCT15 cells was immunoprecipitated with anti-NCC antibody and immunoblotted for γ- and α-ENaC. These studies demonstrate coimmunoprecipitate (co-IP) of the 65 kDa band of γ-ENaC and the 65 kDa band of α-ENaC in mDCT15 cells and mouse kidney cortex (Figure 4A). IgG and resin-only (no antibody) negative controls are included as well, demonstrating the specificity of this interaction. Then, using mouse kidney cortex, we immunoprecipitated with γ-ENaC antibody and probed for NCC, demonstrating that NCC associates with γ-ENaC whether we IP with NCC antibody or γ-ENaC antibody (Figure 4B). In Figure 4C, we focus on α-ENaC in mDCT15 cells to closely examine the characteristics and specificity of this interaction. Here, we demonstrate that when we immunoprecipitated with anti-NCC antibody, α-ENaC associates with NCC and that no association is found in the absence of NCC antibodies. Additionally, neither the α-subunit of the sodium–potassium ATPase nor GAPDH associates with NCC. Having established that immunoprecpitated NCC associates with α-ENaC, we then went on to test whether immunoprecipitating with anti-αENaC antibody brought down NCC. As seen in Figure 4C, NCC does associate with immunoprecipitated α-ENaC, while neither GAPDH nor Na-KATPase does. Thus, NCC and ENaC remain in a specific complex whether we immunoprecipitate with NCC or ENaC. We then went on to demonstrate that the same holds true in mouse kidney cortex (Figure 4D). The controls demonstrate that sodium–potassium ATPase appears to have a small amount of nonspecific binding to the Pierce Co-IP kit resin, unchanged in the presence of antibody. Interestingly, when we IP with NCC antibody, we seem to preferentially pull down cleaved ENaC. This is consistent with the native PAGE data, indicating that a portion of ENaC runs in the same band as NCC, but a portion runs in other bands. This extensive examination of the association of α- and γ-ENaC with NCC in DCT2 cells and in rodent kidney cortex reveals a strong and consistent interaction between these proteins.

Coimmunoprecipitation reveals association of NCC and ENaC.

Figure 4.
Coimmunoprecipitation reveals association of NCC and ENaC.

(A) Lysate of mDCT15 cells was immunoprecipitated with anti-NCC antibody and probed with anti-α and anti-γ-ENaC antibodies. IgG and resin-only (no antibody) negative controls are also displayed. (B) IP of mouse kidney cortex lysate with γ-ENaC antibody probed with NCC antibody. (C) mDCT15 lysate immunoprecipitated with NCC antibody and probed with α-ENaC antibody (first panel) or NCC antibody (second panel). mDCT15 lysate immunoprecipitated with α-ENaC antibody and probed with NCC antibody (third panel). No antibody lanes contain lysate with resin only. Blots of NaK-ATPase and GAPDH are included as negative controls (panels 4 and 5). (D) Experiments as done in C, but on mouse kidney cortex lysate instead of mDCT15 cells. Each of the demonstrated Co-IP experiments was replicated a minimum of three times.

Figure 4.
Coimmunoprecipitation reveals association of NCC and ENaC.

(A) Lysate of mDCT15 cells was immunoprecipitated with anti-NCC antibody and probed with anti-α and anti-γ-ENaC antibodies. IgG and resin-only (no antibody) negative controls are also displayed. (B) IP of mouse kidney cortex lysate with γ-ENaC antibody probed with NCC antibody. (C) mDCT15 lysate immunoprecipitated with NCC antibody and probed with α-ENaC antibody (first panel) or NCC antibody (second panel). mDCT15 lysate immunoprecipitated with α-ENaC antibody and probed with NCC antibody (third panel). No antibody lanes contain lysate with resin only. Blots of NaK-ATPase and GAPDH are included as negative controls (panels 4 and 5). (D) Experiments as done in C, but on mouse kidney cortex lysate instead of mDCT15 cells. Each of the demonstrated Co-IP experiments was replicated a minimum of three times.

NCC directly binds ENaC in mammalian two-hybrid assay

Since NCC is bound in a complex with ENaC in mammalian tissue and in mDCT15 cells, we decided to test whether ENaC subunits directly bind NCC utilizing a mammalian two-hybrid assay. This is a variation of the yeast two-hybrid system used to assess direct binding between proteins. However, in this system, cDNAs encoding mammalian proteins are transfected into mammalian cells (as opposed to yeast), allowing for native conformation and post-translational modification. The mammalian two-hybrid system produces quantitative estimates of the strength of interaction between two proteins. NCC–DNA-BD plasmid and ENaC–AD plasmids were transfected into CHO cells along with the pG5CAT reporter plasmid and binding was assessed by measuring reporter gene expression (Figure 5). Each ENaC subunit was tested singly with NCC. Table 1 indicates what is loaded in each lane. The HA tag on NCC appears to nonspecifically enhance binding, so only untagged NCC was analyzed. A graphical representation of multiple replicates of the experimentation indicates that γ-ENaC and α-ENaC bind NCC directly, with β-ENaC trending toward binding when compared with control.

Protein–protein interaction between NCC and ENaC subunits in mammalian two-hybrid assay.

Figure 5.
Protein–protein interaction between NCC and ENaC subunits in mammalian two-hybrid assay.

NCC–DNA-BD construct and each ENaC–AD construct were transfected into CHO cells. Each ENaC subunit was tested singly with NCC. After 48 h, cells were harvested and reporter gene expression (CAT) was assessed. Table 1 indicates the transfected components for each lane. Lanes 1–4: appropriate controls as indicated in Table 1. Lanes 2 and 3 are the negative controls, indicating that the protein encoded by DNA-BD plasmid does not bind the protein encoded by the AD plasmid. Lane 4 is a positive control. Lanes 5–10: each ENaC subunit with wild-type (WT) and HA-tagged NCC as indicated in Table 1. Graphical representation (n = 3, *P < 0.05, error bars are SEM).

Figure 5.
Protein–protein interaction between NCC and ENaC subunits in mammalian two-hybrid assay.

NCC–DNA-BD construct and each ENaC–AD construct were transfected into CHO cells. Each ENaC subunit was tested singly with NCC. After 48 h, cells were harvested and reporter gene expression (CAT) was assessed. Table 1 indicates the transfected components for each lane. Lanes 1–4: appropriate controls as indicated in Table 1. Lanes 2 and 3 are the negative controls, indicating that the protein encoded by DNA-BD plasmid does not bind the protein encoded by the AD plasmid. Lane 4 is a positive control. Lanes 5–10: each ENaC subunit with wild-type (WT) and HA-tagged NCC as indicated in Table 1. Graphical representation (n = 3, *P < 0.05, error bars are SEM).

Table 1
Transfection protocol for mammalian 2 hybrid
Transfection DNA-BD (5 µg) AD plasmid (5 µg) Reporter (2 µg) 
1. Untransfected None None None 
2. Basal control pM pvP16 pG5CAT 
3. Negative control pM pvP16-CP pG5CAT 
4. Positive control pM-53 pvP16-T pG5CAT 
5. Experiment 1 pM-NCC pvP16-α-ENaC pG5CAT 
6. Experiment 2 pM-NCC-HA pvP16-α-ENaC pG5CAT 
7. Experiment 3 pM-NCC pvP16-β-ENaC pG5CAT 
8. Experiment 4 pM-NCC-HA pvP16-β-ENaC pG5CAT 
9. Experiment 5 pM-NCC pvP16-γ-ENaC pG5CAT 
10. Experiment 6 pM-NCC-HA pvP16-γ-ENaC pG5CAT 
Transfection DNA-BD (5 µg) AD plasmid (5 µg) Reporter (2 µg) 
1. Untransfected None None None 
2. Basal control pM pvP16 pG5CAT 
3. Negative control pM pvP16-CP pG5CAT 
4. Positive control pM-53 pvP16-T pG5CAT 
5. Experiment 1 pM-NCC pvP16-α-ENaC pG5CAT 
6. Experiment 2 pM-NCC-HA pvP16-α-ENaC pG5CAT 
7. Experiment 3 pM-NCC pvP16-β-ENaC pG5CAT 
8. Experiment 4 pM-NCC-HA pvP16-β-ENaC pG5CAT 
9. Experiment 5 pM-NCC pvP16-γ-ENaC pG5CAT 
10. Experiment 6 pM-NCC-HA pvP16-γ-ENaC pG5CAT 

NCC and ENaC FRET

To further demonstrate the close association of these proteins, we examined their relationship in living cells through FRET. NCC-EGFP and α-, β-, and γ-ENaC-mCherry constructs were generated and cotransfected into COS-7 cells (Figure 6). FRET was measured by the acceptor photobleaching method [33]. The positive control was a fused EGFP–mCherry construct (Figure 6A,D). Negative controls are tagged proteins cotransfected with untagged fluorophore only (Figure 6A,B). FRET performed on COS-7 cells expressing EGFP-tagged NCC and mCherry-tagged α-, β-, and γ-ENaC demonstrated increased EGFP fluorescence after acceptor photobleaching when compared with respective α-, β-, and γ-ENaC–mCherry + EGFP negative controls (Figure 6A,C). α- and β-ENaC also demonstrate increased EGFP fluorescence after acceptor photobleaching compared with the NCC-EGFP + mCherry negative control. While this does not prove direct binding, this does indicate that the proteins are close enough (within 10 nm) to directly bind to each other [39,40]. Negative controls do not exhibit significant FRET. Also shown are representative images of a negative control (Figure 6B), an experimental group (Figure 6C), and the positive control (Figure 6D).

FRET acceptor photobleaching demonstrates close proximity of NCC and ENaC subunits.

Figure 6.
FRET acceptor photobleaching demonstrates close proximity of NCC and ENaC subunits.

(A) COS-7 cells were transfected with fluorescently labeled NCC-EGFP and/or ENaC (α, β, or γ)–mCherry (experimental groups), or empty vectors EGFP and mCherry (control groups). A fused EGFP–mCherry vector was used for positive control. Single color images taken before and after 20 rounds of acceptor (mCherry) photobleaching were assessed for an increase in fluorescence of the donor fluorophore, EGFP. Graph represents FRET efficiency as fluorescence intensity [((donorpostbleach − donorprebleach)/donorpostbleach) × 100)]. Negative controls do not exhibit significant FRET. Data are mean ± SEM, n = 6–8 (*P < 0.05, ***P < 0.0001). (B) Representative image of a negative control (empty vector EGFP + αENaC-mCherry) before and after acceptor (mCherry) photobleaching. Before and after single-channel EGFP and single-channel mCherry images are shown. Pre-photobleaching merged image is also displayed. (C) Representative image of an experimental group (NCC-EGFP + αENaC-mCherry), demonstrating an increase in EGFP fluorescent intensity following mCherry photobleaching. (D) Representative image of the positive control (fused EGFP–mCherry).

Figure 6.
FRET acceptor photobleaching demonstrates close proximity of NCC and ENaC subunits.

(A) COS-7 cells were transfected with fluorescently labeled NCC-EGFP and/or ENaC (α, β, or γ)–mCherry (experimental groups), or empty vectors EGFP and mCherry (control groups). A fused EGFP–mCherry vector was used for positive control. Single color images taken before and after 20 rounds of acceptor (mCherry) photobleaching were assessed for an increase in fluorescence of the donor fluorophore, EGFP. Graph represents FRET efficiency as fluorescence intensity [((donorpostbleach − donorprebleach)/donorpostbleach) × 100)]. Negative controls do not exhibit significant FRET. Data are mean ± SEM, n = 6–8 (*P < 0.05, ***P < 0.0001). (B) Representative image of a negative control (empty vector EGFP + αENaC-mCherry) before and after acceptor (mCherry) photobleaching. Before and after single-channel EGFP and single-channel mCherry images are shown. Pre-photobleaching merged image is also displayed. (C) Representative image of an experimental group (NCC-EGFP + αENaC-mCherry), demonstrating an increase in EGFP fluorescent intensity following mCherry photobleaching. (D) Representative image of the positive control (fused EGFP–mCherry).

NCC and γ-ENaC colocalize by electron microscopy in DCT2

Finally, to further examine the association of NCC and ENaC in native tissue, we utilized immunogold labeling and electron microscopy (Figure 7). Ultrathin sections from mouse kidney cortex were examined for coexpression of NCC and ENaC to identify DCT2. Images were taken from the apical portion of DCT2 cells. We found 12 nm gold-labeled NCC and 6 nm gold-labeled γ-ENaC to be coexpressed on the apical surface. Electron microscopy (EM) immunogold labels only a portion of expressed proteins. Given that only a subset of ENaC and NCC proteins are labeled, a smaller subset of associations of NCC and ENaC will be captured. Nonetheless, the magnified insert shows the larger immunogold-labeled NCC and the smaller ENaC-labeled immunogold particle separated by about the diameter of the NCC-labeled particle. Interestingly, the NCC-labeled particle appears closer to the ENaC-labeled particle than it does to its probable dimer partner NCC-labeled particle. This demonstrates that NCC and γ-ENaC appear to be within ∼12 nm of each other in the apical membrane of the DCT2. While this does not prove direct binding, this proximity is consistent with it.

EM detects close association of NCC and ENaC.

Figure 7.
EM detects close association of NCC and ENaC.

Immunogold labeling of NCC (12 nm particles) and γ-ENaC (6 nm particles) in mouse DCT2. The insert shows a magnified view of closely associated NCC and γ-ENaC. Similar associations were observed on at least three other occasions.

Figure 7.
EM detects close association of NCC and ENaC.

Immunogold labeling of NCC (12 nm particles) and γ-ENaC (6 nm particles) in mouse DCT2. The insert shows a magnified view of closely associated NCC and γ-ENaC. Similar associations were observed on at least three other occasions.

Inhibition of NCC results in decreased ENaC function

To ascertain whether this interaction has any functional impact on these transport proteins, we assessed the effect of inhibiting NCC on ENaC function in mdct15 cells (Figure 8). We began to examine the baseline functional implications of this relationship by measuring amiloride-sensitive uptakes as a measurement of ENaC activity with and without inhibitors of NCC. Here, we modified our well-established protocol for measuring NCC function by thiazide-sensitive 22Na+ uptakes in mDCT15 cells [24,25,41] to assess amiloride-sensitive 22Na+ uptakes. We simply substituted amiloride into the protocol instead of metolazone. Amiloride-sensitive uptakes were then performed in the presence and absence of 0.1 mM metolazone (Figure 8A). This indicated that amiloride-sensitive uptakes were significantly decreased in the presence of thiazide. While this could indicate a decrease in ENaC function with inhibition of NCC, it could also reflect a change in amiloride sensitivity in the presence of thiazides. Additionally, this does not address whether the number of channels or the open probability is altered. Therefore, we went on to examine this more closely through the use of electrophysiology. Using mDCT15 cells, single-channel recordings were obtained in excised outside-out patches with and without hydrochlorothiazide (HCTZ) applied to the apical surface. In Figure 8B, we show a representative continuous experiment demonstrating ENaC activity in the control and after HCTZ. Area 1 is a channel recording without HCTZ and area 2 is with HCTZ. ENaC open probability clearly significantly decreases with HCTZ. Figure 8C shows the compilation of data from six separate experiments, clearly demonstrating a consistent effect of decreased open probability of ENaC with NCC inhibition. While it is historically accepted, both clinically and experimentally, that thiazides have no significant effect on ENaC when expressed alone, we examined whether this was true in our hands. Heterologous expression of ENaC in a model system that does not express NCC (CHO cells) demonstrated no effect of 100 µM HCTZ on ENaC function (Figure 9). These studies confirm that the effect of thiazides on ENaC is an NCC-specific effect.

Inhibition of NCC results in decreased ENaC function.

Figure 8.
Inhibition of NCC results in decreased ENaC function.

(A) Amiloride-sensitive 22Na+ uptakes were performed in mDCT15 cells with and without 0.1 mM metolazone (n = 14, *P < 0.05). (B) A representative single-channel recording of ENaC in excised outside-out patches with and without 100 nM HCTZ in mDCT15 cells. Area 1 is a channel recording without HCTZ and area 2 is with HCTZ. (C) Graph of data from six separate single-channel recording experiments (n = 6, *P < 0.05 vs. control).

Figure 8.
Inhibition of NCC results in decreased ENaC function.

(A) Amiloride-sensitive 22Na+ uptakes were performed in mDCT15 cells with and without 0.1 mM metolazone (n = 14, *P < 0.05). (B) A representative single-channel recording of ENaC in excised outside-out patches with and without 100 nM HCTZ in mDCT15 cells. Area 1 is a channel recording without HCTZ and area 2 is with HCTZ. (C) Graph of data from six separate single-channel recording experiments (n = 6, *P < 0.05 vs. control).

Thiazides have no effect on ENaC function in the absence of NCC.

Figure 9.
Thiazides have no effect on ENaC function in the absence of NCC.

(A) Graph of data from four separate single-channel recording experiments (n = 4, not significantly different from control). (B) A representative single-channel recording of ENaC in excised outside-out patches with and without 100 nM HCTZ in CHO cells transfected with mouse ENaC (αβγ). Area 1 is a channel recording without HCTZ and area 2 is with HCTZ.

Figure 9.
Thiazides have no effect on ENaC function in the absence of NCC.

(A) Graph of data from four separate single-channel recording experiments (n = 4, not significantly different from control). (B) A representative single-channel recording of ENaC in excised outside-out patches with and without 100 nM HCTZ in CHO cells transfected with mouse ENaC (αβγ). Area 1 is a channel recording without HCTZ and area 2 is with HCTZ.

These findings could indicate that when bound together, inhibiting activity of one inhibits activity of the other. Perhaps, when bound together they operate only in parallel. Interestingly, this effect is not explained by decreased NCC sodium transport resulting in decreased intracellular sodium, as that would increase the driving force for ENaC. Alternatively, this could indicate that ENaC function is lessened when thiazide binds to the complex through NCC. Perhaps, this engenders a conformational change that results in more closed ENaC channels. Either way, an NCC-specific signal is being transmitted, which is resulting in functional changes in ENaC. Having now examined the impact of NCC inhibition on ENaC activity with two different methodologies, it is clear that there are significant functional implications of the association of NCC and ENaC.

Discussion

Using multiple techniques, we have now demonstrated a novel and previously unsuspected interaction between ENaC and NCC, both key proteins for maintaining blood pressure homeostasis. This interaction has a clear impact on the function of these transport proteins and occurs in the only part of the DCT that is presumably sensitive to aldosterone. Although the sensitivity of the DCT1 and the DCT2 to aldosterone has never been explicitly investigated, the expression pattern of 11βHSD2, the aldosterone-sensitizing enzyme, would suggest that only the DCT2 is aldosterone-sensitive. While mineralocorticoid receptors are expressed in DCT1 and DCT2 [17,4244], 11βHSD2 is only expressed in DCT2 [17,18]. While there has recently been reported some species variation concerning the expression of 11βHSD2 in the DCT2 [45], it is clear that in all species there is no 11βHSD2 in the DCT1. The localization of 11βHSD2 to DCT2 only would suggest that the reported large changes in NCC protein expression induced by chronic aldosterone [2123] are only taking place in the DCT2. These studies were largely done with immunoblotting of samples containing both segments of the DCT, so segment-specific changes remain unknown. But if a segment that makes up one-third of the DCT is causing a 3-fold change in protein expression of NCC in the whole DCT, this would imply that there are changes on the order of 9-fold in this segment. This presumably exquisitely aldosterone-sensitive segment is also the only segment that coexpresses NCC and ENaC. Interestingly, recent data have suggested that DCT1 and DCT2 may have quite different physiological roles. As detailed above, the DCT2 appears to be largely responsible for mediating the effects of aldosterone in the DCT. However, many recent studies have suggested that the primary physiological role of the DCT1 may be related to potassium homeostasis. Acute potassium loading rapidly dephosphorylates NCC, resulting in kaliuresis [4648]. This effect is independent of aldosterone and is probably predominantly occurring in DCT1. Conversely, inhibiting NCC acutely by thiazides (which inhibits NCC in DCT1 and DCT2) does not appear to promote kaliuresis [49], perhaps suggesting that NCC inhibition must be confined to the DCT1 to induce acute kaliuresis. Additionally, the expression of NCC in the DCT1 has recently been determined to be dependent on the presence of a basolateral K channel [50]. This apparent interdependence of potassium handling and NCC regulation in DCT1 may suggest a primary role for DCT1 in aldosterone-independent potassium regulation. In contrast, the DCT2 appears to have the primary role of mediating the DCT effects of aldosterone. As we have accumulated more data, it has now become clear that these two DCT segments are distinct in expression of proteins and perhaps even physiological role.

In addition to being localized to the aldosterone-sensitive DCT segment, ENaC and NCC also share a similar aldosterone-induced regulatory network. NEDD4-2 and SGK1 have been identified as key mediators of aldosterone's effects on ENaC. The E3 ubiquitin ligase NEDD4-2 binds to the PY (proline–tyrosine) motif in ENaC subunits and ubiquitinates it, resulting in endocytosis and degradation of ENaC [19]. SGK1 phosphorylates NEDD4-2, resulting in accumulation of ENaC at the cell surface. A previous publication examined the regulation of NCC by NEDD4-2, demonstrating that an inducible renal tubule-specific NEDD4-2 knockout mouse had increased NCC protein [36]. Additionally, it was shown that NEDD4-2 binds and ubiquitinates NCC, resulting in decreased NCC protein. Gene silencing of NEDD4-2 in mDCT15 cells resulted in increased total and cell surface NCC protein [36]. A study detailing the phenotype of this mouse surprisingly reported that while NCC protein expression was increased, functional ENaC was not [51]. While β-ENaC and γ-ENaC expression is increased, they are mostly cytoplasmically located and α-ENaC expression is unchanged. Thiazides, but not amiloride, corrected the salt-sensitive hypertension in these animals, suggesting that the hypertension was mostly due to increased NCC function. This finding was in contrast with the constitutive global NEDD4-2 knockout, which showed increased ENaC expression and unchanged NCC [52]. Recent evidence also points to NCC being regulated by SGK1 similarly to ENaC. Phosphorylation of NEDD4-2 by SGK1 appears to prevent the association of NCC and NEDD4-2, resulting in increased NCC protein [36]. SGK1 also appears to regulate ENaC and NCC independently of NEDD4-2 [53]. An inducible renal tubule-specific SGK1 knockout mouse was hypotensive and had decreased expression of NCC and all three subunits of ENaC [54]. All of these findings led us to hypothesize that these proteins were bound together in a complex. Our discovery that this cotransporter and channel directly bind to one another may suggest that their association is critical to the regulation of sodium homeostasis in this nephron segment.

Although the association of these particular proteins is novel, direct interaction between other ion channels and cotransporters is also rare. While it is reasonably common for different channels to bind, interact and even combine to form heteromeric channels, it is rare for this to happen with channels and transporters (exchangers, cotransporters, or other nonchannel transport proteins). Most of the reported direct interactions of transporters with channels involve the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR binds to and alters the function of several members of the SLc26a family of anion exchangers [5557]. An extensive review of the literature did not reveal any other reported direct interactions of transporters with channels. As implied by its name, it has been thought that CFTR's size and many regulatory domains make it equal parts regulatory element and channel. Others have felt that CFTR was more transporter than channel. Given these properties, it may have been unique in its ability to form complexes with transporters. Therefore, to our knowledge, this is the first reported direct association of a ‘true’ channel with a nonchannel transport protein.

While the novelty and somewhat surprising nature of these findings has necessitated the use of multiple techniques and systems to simply prove the interaction and demonstrate functional significance, this has left many intriguing questions to investigate. For example, examination of the IP blots (Figure 4) reveals that ENaC associated with NCC typically has a molecular weight of below 75 kDa. This would seem to suggest that NCC may preferentially bind the cleaved, activated form of ENaC. The appearance of ENaC in multiple bands in the native PAGE with NCC running only in the higher band could also suggest that the binding of the two proteins is conditional. A full investigation of the factors that weaken or strengthen the association is an important area of future study. In particular, determining the role of aldosterone in strengthening or weakening the association will be a critical study. Another important study is examining the importance of the interaction to the transmission of aldosterone signaling via intracellular signaling proteins such as NEDD4-2 and SGK1. NEDD4-2 binds NCC, but NCC does not have the necessary PY motif to directly bind NEDD4-2 [36]. ENaC directly binds NEDD4-2 and now we know that ENaC directly binds NCC. Is ENaC necessary for the NEDD4-2 effect on NCC? The presence of ENaC in the only part of the DCT that is presumably aldosterone-sensitive may not be happenstance. It is possible that ENaC is necessary to mediate aldosterone effects on NCC. So, examining the impact of the relationship of NCC and ENaC on aldosterone signaling and the impact of aldosterone signaling on the relationship will be an intriguing and likely complicated puzzle to unravel. We feel that understanding the relationship between these two key proteins could significantly alter our understanding of salt handling in the kidney and thus blood pressure.

Abbreviations

11βHSD2, 11β-hydroxysteroid dehydrogenase type 2; AD, activation domain; BN, blue native; CFTR, cystic fibrosis transmembrane conductance regulator; DCT, distal convoluted tubule; DNA-BD, DNA-binding domain; EGFP, enhanced green fluorescent protein; EM, electron microscopy; ENaC, epithelial sodium channel; FBS, fetal bovine serum; FRET, fluorescence resonance energy transfer; HCTZ, hydrochlorothiazide; NCC, sodium chloride cotransporter; NEDD4, neural precursor cell-expressed developmentally down-regulated protein 4; SGK1, serum and glucocorticoid-regulated kinase.

Author Contribution

A.C.M. and B.M.W. conducted much of the experimentation, analyzed the results, interpreted the data, and did some of the writing. L.Y., V.T., Q.Y., Y.Z., O.A.-K., R.M., A.A.A., A.M., H.-F.B., and F.T. also conducted experimentation and interpreted results. B.K., H.C., O.P., and D.C.E. helped in interpretation of data and writing the paper. D.C.E. also helped conceive the paper. R.S.H. conceived the idea for the project and wrote most of the paper.

Funding

This work was supported by grants from the National Institutes of Health R01 DK-085097 (to R.S.H.), T32 DK07656 (B.M.W.), 1K01DK099617 (A.A.A.), R37 DK037963 (D.C.E.), and RO1 DK095029 (O.P.); by the Department of Veteran Affairs VA Merit Awards I01BX002322-01 (R.S.H.) and I01BX000994 (H.C.); by the Swiss National Foundation (F.T.), AHA GIA 13GRNT16220002 (O.P.); and by the Russian Foundation for Basic Research #15-04-00938. This research project was also supported in part by the Emory University Integrated Cellular Imaging Microscopy Core of the Emory University Department of Physiology.

Acknowledgments

The authors thank Jan Loffing for providing antibodies for the EM studies.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Author notes

*

These authors contributed equally to this study.