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Am J Physiol Renal Physiol 293: F1178-F1186, 2007. First published July 18, 2007; doi:10.1152/ajprenal.00153.2007
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Ceramide mediates inhibition of the renal epithelial sodium channel by tumor necrosis factor-{alpha} through protein kinase C

Hui-Fang Bao,1 Zhi-Ren Zhang,2 You-You Liang,3 Joshua J. Ma,1 Douglas C. Eaton,1 and He-Ping Ma1,3

1Department of Physiology, Emory University School of Medicine, Atlanta, Georgia; 2Department of Medicine, Division of Nephrology, Medical University of South Carolina, Charleston, South Carolina; and 3Department of Medicine, Division of Nephrology, University of Alabama at Birmingham, Birmingham, Alabama

Submitted 2 April 2007 ; accepted in final form 17 July 2007


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To determine whether ceramide mediates regulation of the renal epithelial sodium channel (ENaC) by tumor necrosis factor-{alpha} (TNF-{alpha}), confocal microscopy and patch-clamp experiments were performed in A6 distal nephron cells. We found that TNF-{alpha} (100 ng/ml) had no effect on ENaC activity and ceramide level when the cells were grown in the presence of aldosterone, but significantly inhibited ENaC and induced ceramide production after the cells were pretreated with LY 294002, an inhibitor of phosphatidylinositol 3-kinase, for 24 h. The inhibition of ENaC induced by TNF-{alpha} was mimicked by exogenous sphingomyelinase (0.1 U/ml) and C2-ceramide (50 µM), but neither C2-dihydroceramide, a membrane-impermeable analog of C2-ceramide, nor choline, and abolished by pretreatment with GF109203X, a protein kinase C (PKC) inhibitor. C2-ceramide failed to affect ENaC in the cells pretreated with GF109203X, but not in the cells pretreated with PD-98059, a mitogen-activated protein kinase kinase inhibitor. C2-ceramide induced the externalization of phosphatidylserine (PS) in control A6 cells, but not in the cells pretreated with GF109203X. Together with our previous finding that cytosolic PS maintains ENaC activity in A6 cells, these data suggest that ceramide mediates TNF-{alpha} inhibition of the renal ENaC via a pathway associated with PKC-dependent externalization of PS.

A6 cells; single-channel recordings; confocal microscopy; sphingomyelinase; C2-ceramide; phosphatidylserine

THE EPITHELIAL SODIUM CHANNEL (ENaC) plays a very important role in sodium transport across the apical membrane of a variety of epithelia including the colon, lung, and kidney. ENaC must be tightly regulated to maintain sodium homeostasis because abnormal ENaC activity leads to several severe diseases. Inhibition of ENaC expression in colonic epithelial cells by proinflammatory cytokines, particularly tumor necrosis factor-{alpha} (TNF-{alpha}) (3), accounts for diarrhea in ulcerative colitis (2). The activity and expression of ENaC in alveolar epithelial cells are also inhibited by TNF-{alpha} (11). Since transgenic mice lacking functional ENaC die from fluid-filled airways within 40 h of birth (23), ENaC is certainly responsible for fluid clearance in the neonatal and probably in the adult lung. Thus proinflammatory cytokines may cause pulmonary edema by inhibiting ENaC. However, another study, also in alveolar epithelial cells, shows that ENaC is activated by TNF-{alpha} (15), which may mediate TNF-{alpha}-dependent increases in alveolar fluid clearance in rats with acute bacterial pneumonia (40). Therefore, it has been suggested that this cytokine has a dichotomous role in pulmonary edema: TNF-{alpha} inhibits fluid clearance via TNF receptors but stimulates fluid clearance via its lectin-like domain (5).

In renal epithelial cells, increased ENaC activity caused by mutations in the COOH terminus of either beta- or {gamma}-ENaC subunit accounts for volume-expended hypertension in Liddle's syndrome. Conversely, epidermal growth factor (EGF)-induced decreases in ENaC activity may contribute to the formation of fluid-filled cysts in polycystic kidney disease (PKD) (46, 47). Besides EGF, TNF-{alpha} is also found in the cyst fluid (16), so that TNF-{alpha} may also be partially responsible for the decreased ENaC activity found in PKD collecting duct cells. However, like its effect on alveolar epithelium, TNF-{alpha} may also cause contrasting effects in the distal nephron. Instead of inhibiting ENaC, TNF-{alpha} can stimulate ENaC-mediated sodium reabsorption in distal tubule cells isolated from diabetic rats but not in cells from normal rats (12). As discussed above, the stimulatory effect of TNF-{alpha} on pulmonary fluid clearance is mediated by its lectin-like domain while the inhibitory effect is through TNF receptors, which are termed TNF receptor type 1 (TNF-R1) and type 2 (TNF-R2) (1, 29). It has long been recognized that a TNF receptor can exist in a soluble form (17, 37). Interestingly, TNF-{alpha} inhibits pulmonary fluid clearance but activates the clearance when its receptor binding site is associated with and blocked by the soluble TNF-R1 (5). Since soluble TNF receptors are elevated in diabetes (6, 52), the soluble receptors may play a permissive role in diabetic rats to allow TNF-{alpha} stimulation of sodium reabsorption. Conversely, in the absence of soluble TNF receptors, TNF-{alpha} may inhibit renal ENaC via TNF receptors in the cell membrane.

TNF-{alpha} binding to TNF-R1 initiates a signal transduction cascade by stimulating both neutral and acidic sphingomyelinases (49), enzymes that cleave membrane sphingolipids to produce choline and ceramide. Several lines of evidence indicate that ceramide can act as an important second messenger to activate several protein kinases (41) including protein kinase C-Raf (24), kinase suppressor of Ras (51), mitogen-activated protein kinases (39), and different isoforms of protein kinase C (PKC) (25, 38, 42). Since activation of PKC inhibits ENaC (14, 31), one hypothesis is that TNF-{alpha}-induced ceramide generation may inhibit ENaC through activation of PKC. In the present study, we found that TNF-{alpha} downregulates ENaC in A6 distal nephron cells and that ceramide mediates TNF-{alpha} downregulation of renal ENaC via a pathway associated with PKC-dependent externalization of phosphatidylserine (PS).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. A6 distal nephron cells were purchased from American Type Culture Collection (Rockville, MD). The cells were cultured in plastic flasks in a modified media containing 3 parts Coon's F-12 medium (Invitrogen), 7 parts Leibovitz's L-15 medium (Invitrogen), 103 mM NaCl, 25 mM NaHCO3, 25 U/ml penicillin, 25 U/ml streptomycin, 2 mM L-glutamine, 10% fetal bovine serum (Invitrogen), and, otherwise indicated, 1 µM aldosterone (Sigma, St. Louis, MO) at 26°C and 4% CO2. Cells were removed from the flasks and plated on permeable supports attached to Snapwell inserts (Corning Costar). The cells were cultured on permeable supports for 10 to 14 days to allow them to be fully polarized before patch-clamp and confocal microscopy experiments.

Chemicals and solutions. Most chemicals including TNF-{alpha}, sphingomyelinase, N-acetyl-D-sphingosine (C2-ceramide), C2-dihydroceramide, phorbol 12-myristate 13-acetate (PMA), mouse monoclonal anti-ceramide antibody (15B4), and annexin V-FITC were purchased from Sigma. Alexa Fluor 488 goat anti-mouse IgM was purchased from Invitrogen. NaCl solution contained (in mM) 100 NaCl, 3.4 KCI, 1 CaCl2, 1 MgCl2, and 10 HEPES, at a pH of 7.4. All the concentrations shown in this article are the final concentrations.

Patch-clamp cell-attached recordings. Immediately before use, a Snapwell insert was thoroughly washed with NaCl solution (see Chemicals and solutions) and transferred into the patch chamber mounted in the stage of a Nikon inverted microscope. Using patch-clamp techniques, the cell-attached configuration was established on the apical membrane of A6 cells with polished micropipettes. The tip resistance of the micropipettes was from 5 to 7 M{Omega} when filled with NaCl solution. Under the above culture conditions, a patch seal (seal resistance >10 G{Omega}) was usually formed after releasing positive pressure in the patch pipette and applying a slightly negative pressure. After the cell-attached mode was established, only patches containing channel activity without obvious baseline drift were used for further experiments. Single-channel currents were obtained with zero applied pipette potential (i.e., the electrical driving force was voltage-clamped at the resting membrane potential), filtered at 1 kHz, and recorded on a computer hard disk. Before digitization with pClamp 9 software (Molecular Devices), single-channel records were low-pass filtered at 30 Hz. The total numbers of active channels (N) in the patch were estimated by observing the number of peaks detected on all-point amplitude histograms. Open and closed current levels were first identified manually, and then transition analysis using 50% cut-off between open and closed levels was employed during formation of all-points amplitude histograms. As a measure of channel activity, NPo [number of channels (N) x the open probability (Po)] was calculated by using 3 min of a single-channel record, as we previously described (32). Only records from patches with low noise and containing four or less active channels were used for the calculation to minimize possible errors. A single-channel record before application of each reagent served as a control and was paired with that after application of the reagent. Experiments were conducted at 22–23°C.

Confocal microscopy analysis of ceramide formation and PS externalization. To be comparable with the results obtained from patch-clamp experiments, A6 cells used for these experiments were also cultured on Snapwell inserts for 10 to 14 days. Immediately before the experiments, Snapwell inserts were washed three times with NaCl solution. To label ceramide, mouse monoclonal anti-ceramide antibody (15B4) was used. The procedure for immunostaining of ceramide was according to that previously used by others (19). Briefly, control or treated A6 cells were fixed with 1% paraformaldehyde in NaCl solution for 10 min, washed two times, and then incubated in a staining buffer containing mouse monoclonal anti-ceramide antibody (15B4; 1:30), 1% FBS, and 0.1% NaN3 at room temperature for 45 min. The cells were secondarily stained with Alexa Fluor 488 goat anti-mouse IgM (5 µg/ml) at room temperature for 30 min. Direct staining with Alexa Fluor 488 goat anti-mouse IgM alone served as controls in both untreated and treated cells and showed no detectable fluorescence.

Annexin V is a protein that binds to phospholipids, but with a relatively higher affinity for PS. FITC-conjugated annexin V has been extensively used to label externalized PS (13). Therefore, annexin V-FITC was used to detect the appearance of PS on the luminal surface of the apical membrane of A6 cells. Immediately before the experiments, Snapwell inserts were washed three times with NaCl solution. Control and treated A6 cells were stained with annexin V-FITC in the binding buffer provided by the manufacturer for 10 min and then washed twice. Unstained cells served as controls in both untreated and treated cells and showed no detectable fluorescence. The support membranes attached to Snapwell inserts were then excised and mounted on glass slides. Control and treated A6 cells stained either by anti-ceramide antibody (detected by the fluorescent secondary antibody) or by FITC-conjugated annexin V were, respectively, analyzed by a Leica confocal microscopy.

Statistical analysis. Data are reported as means ± SE. Statistical analysis was performed with SigmaPlot and SigmaStat software (Jandel Scientific). Paired t-tests were used to determine statistical significance between two groups before and after experimental manipulations. Results were considered significant if P < 0.05, as we described previously (32).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
TNF-{alpha} inhibits ENaC in A6 distal nephron cells. To determine whether and how TNF-{alpha} regulates ENaC single-channel activity in renal epithelial cells, cell-attached patches were formed on the apical membrane of A6 cells. To mimic the in vivo environment, the cells were cultured in Snapwell inserts which separated the luminal compartment from basolateral compartment (see EXPERIMENTAL PROCEDURES). Because ENaC activity is highly variable from patch to patch, a protocol was designed to use the same patch as a control, as we reported previously (33). Control ENaC activity in A6 cells was recorded before application of any reagents. We found that application of TNF-{alpha} at a final concentration of 100 ng/ml, as used by others (11), to the luminal bath had no effect on ENaC activity when A6 cells were under control culture conditions in the presence of 1 µM aldosterone, as shown in Fig. 1A (top traces). The mean NPo, which is the number of active channels times open probability, was unchanged by TNF-{alpha}, 0.63 ± 0.18 (before) vs. 0.61 ± 0.16 (after TNF-{alpha}; P = 0.5; n = 6), as shown in Fig. 1B, left. We hypothesize that phosphatidylinositol 3-kinase (PI3K) may be responsible for the failure of TNF-{alpha} in regulating ENaC in aldosterone-conditioned A6 cells because aldosterone stimulates PI3K (4, 22) the products of which can inhibit sphingomyelinase (44, 45), a downstream molecule in the TNF-R1-initiated signaling pathway. Indeed, TNF-{alpha} significantly reduced ENaC activity after the cells were pretreated with 1 µM LY 294002, a PI3K inhibitor, for 24 h, as shown in Fig. 1A (bottom traces). The mean NPo of ENaC was decreased in response to TNF-{alpha}, from 0.45 ± 0.12 (before) to 0.20 ± 0.11 (after TNF-{alpha}; P < 0.01; n = 6), as shown in Fig. 1B, right.


Figure 1
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  		Fig. 1. Inhibition of epithelial sodium channel (ENaC) by tumor necrosis factor-{alpha} (TNF-{alpha}) in A6 cells treated with a phosphatidylinositol 3-kinase (PI3K) inhibitor. A: representative single-channel current in cell-attached patches before and 5 min after application of TNF-{alpha} (100 ng/ml) to the luminal bath, which was recorded from either a cell under control conditions (top traces) or a cell pretreated with a PI3K inhibitor LY 294002 (1 µM) for 24 h (bottom traces). In all the figures showing single-channel records, upward events represent channel openings; "C–" indicates the baseline when channels are closed; patch pipettes were filled with NaCl solution (see Chemicals and solutions). B: summary plots of NPo values before ({circ}) and 5 min after TNF-{alpha} (100 ng/ml; bullet) in control A6 cells (left) and the cells pretreated with LY 294002 (1 µM) for 24 h (right).

 
Exogenous sphingomyelinase also inhibits ENaC in A6 cells. Activation of TNF-R1 by TNF-{alpha} stimulates both neutral and acidic sphingomyelinases (49). Therefore, if TNF-{alpha} inhibits ENaC by stimulating sphingomyelinases through TNF receptors, then addition of exogenous sphingomyelinase should also inhibit ENaC activity. As we hypothesized, after sphingomyelinase (0.1 U/ml) was added to the luminal bath, ENaC activity was significantly reduced (Fig. 2A). The mean NPo of ENaC was decreased, from 0.64 ± 0.12 (control) to 0.14 ± 0.03 (after sphingomyelinase; P < 0.01; n = 6), as shown in Fig. 2B. We hypothesized that sphingomyelinase, as a downstream molecule activated by TNF-R1, mediates the inhibitory effect of TNF-{alpha} on ENaC activity.


Figure 2
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  		Fig. 2. Inhibition of ENaC in A6 cells by exogenous sphingomyelinase under control conditions in the absence of LY 294002. A: representative single-channel current before and 5 min after application of a sphingomyelinase (0.1 U/ml) to the luminal bath, recorded from a cell-attached patch. B: summary plots of NPo values before ({circ}) and 5 min after sphingomyelinase (0.1 U/ml; bullet).

 
TNF-{alpha} induces ceramide production in A6 cells. Since sphingomyelinase cleaves sphingomyelin to produce choline and ceramide, whether TNF-{alpha} activates sphingomyelinase can be determined by detecting the product of sphingomyelinase, ceramide. Therefore, confocal microscopy experiments were performed to examine ceramide formation with monoclonal anti-ceramide antibody. There was no detectable ceramide in untreated A6 cells (Fig. 3A, left). TNF-{alpha} failed to induce formation of ceramide in A6 cells under control conditions (Fig. 3A, middle), even though application of sphingomyelinase, as a positive control, induced formation of ceramide in these untreated A6 cells (Fig. 3A, right). However, TNF-{alpha} did induce formation of ceramide in A6 cells pretreated with 1 µM LY 294002 for 24 h (Fig. 3B, right), compared with control A6 cells pretreated with LY 294002 in the absence of TNF-{alpha} (Fig. 3B, left). Since the PI3K-dependent effect of TNF-{alpha} on ceramide formation is very consistent with that of TNF-{alpha} on ENaC activity, these data indicate that TNF-{alpha} inhibits ENaC in A6 cells probably by inducing ceramide formation.


Figure 3
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  		Fig. 3. Production of ceramide induced by TNF-{alpha} in A6 cells treated with a PI3K inhibitor. A: representative images taken by confocal microscopy from either control A6 cells (left) or the cells treated with either TNF-{alpha} (100 ng/ml; middle) or sphingomyelinase (0.1 U/ml; right) for 5 min; 3 sets of such an experiment were performed and showed similar results. B: representative images taken by confocal microscopy from either control A6 cells (left) or the cells pretreated with LY 294002 (1 µM) for 24 h and then treated with TNF-{alpha} (100 ng/ml; right) for 5 min; 4 sets of such an experiment were performed with similar results.

 
Ceramide inhibits ENaC in A6 distal nephron cells. To determine whether ceramide, a TNF-{alpha}-induced product of sphingomyelin hydrolysis, contributed to the TNF-{alpha}-mediated reduction in ENaC activity, we applied a membrane-permeable analog of ceramide, C2-ceramide. Application of C2-ceramide (50 µM) to the apical bath significantly decreased ENaC activity, as shown in Fig. 4A (top traces). The mean NPo of ENaC was decreased in response to C2-ceramide, from 0.64 ± 0.10 (control) to 0.30 ± 0.08 (after C2-ceramide; P < 0.01; n = 8), as shown in Fig. 4B, top left. In contrast, luminal application of an inactive analog, C2-dihydroceramide, at the same concentration (50 µM) had no effect on ENaC activity, as shown in Fig. 4A (middle traces). The mean NPos before and after application of C2-dihydroceramide were not significantly different, 0.68 ± 0.14 (before) vs. 0.53 ± 0.08 (after C2-dihydroceramide; P = 0.2; n = 5), as shown in Fig. 4B, top right. Since sphingomyelin hydrolysis also produces choline, whether choline could inhibit ENaC was determined. Choline, even at a concentration (1 mM) which is 20 times higher than that we used for C2-ceramide, did not affect ENaC activity, as shown in Fig. 4A (bottom traces). The mean NPos before and after application of 1 mM choline were not significantly different, 0.51 ± 0.08 (before) vs. 0.50 ± 0.07 (after choline; P = 0.6; n = 5), as shown in Fig. 4B, bottom left. Together with confocal microscopy data, these results suggest that ceramide mediates the inhibitory effect of TNF-{alpha} on ENaC activity.


Figure 4
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  		Fig. 4. Inhibition of ENaC in A6 cells by C2-ceramide, but neither C2-dihydroceramide nor choline. A: representative single-channel current before and 5 min after application of C2-ceramide (50 µM; top traces), C2-dihydroceramide (50 µM; middle traces), or choline (1 mM; bottom traces) to the luminal bath, which was also recorded from cell-attached patches. B: summary plots of NPo values before ({circ}) and 5 min after application of C2-ceramide (50 µM; bullet in top left), C2-dihydroceramide (50 µM; bullet in top right), or choline (1 mM; bullet in bottom left) to the luminal bath.

 
Inhibition of PKC abolishes the effects of ceramide and TNF-{alpha} on ENaC activity. Since ceramide binds to different isoforms of PKC (24, 25) and activation of PKC inhibits ENaC (14, 31), we hypothesized that ceramide might inhibit ENaC by stimulating PKC. To determine whether PKC mediates the inhibitory effect of ceramide on ENaC activity, A6 cells were pretreated with a PKC inhibitor, GF109203X (0.4 µM), for 15 min. To completely block PKC, this PKC inhibitor has been previously used at concentrations of 1 µM (10) or 10 µM (21). Inhibition of PKC abolished the effect of C2-ceramide on ENaC activity, as shown in Fig. 5A (top traces). The mean NPo of ENaC in the cells pretreated with GF109203X was no longer altered by C2-ceramide, 0.77 ± 0.15 (before) vs. 0.70 ± 0.15 (after C2-ceramide; P = 0.1; n = 7), as shown in Fig. 5B, top left. Furthermore, PD-98059, a potent inhibitor of mitogen-activated protein (MAP) kinase kinase, was used to test whether ceramide inhibition of ENaC is through a MAP kinase-associated pathway. As shown in Fig. 5A (middle traces), C2-ceramide still inhibited ENaC activity in A6 cells treated with 10 µM PD-98059 for 15 min. The mean NPo of ENaC was reduced by C2-ceramide, from 0.47 ± 0.10 (before) to 0.14 ± 0.05 (after C2-ceramide; P < 0.01; n = 5; Fig. 5B, top right). These data suggest that the inhibitory effect of ceramide on ENaC activity is through a PKC-dependent pathway. To test whether PKC also mediates TNF-{alpha} inhibition of ENaC, first, A6 cells were pretreated with 1 µM LY 294002 for 24 h to allow TNF-{alpha} to initiate the signal transduction. Then, the cells were treated with 0.4 µM GF109203X for 15 min. Inhibition of PKC abolished the effect of 100 ng/ml TNF-{alpha} on ENaC activity, as shown in Fig. 5A (bottom traces). The mean NPo of ENaC in the cells treated with GF109203X was no longer altered by TNF-{alpha}, 0.52 ± 0.07 (before) vs. 0.49 ± 0.08 (after TNF-{alpha}; P = 0.3; n = 5; Fig. 5B, bottom left).


Figure 5
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  		Fig. 5. PKC inhibitor eliminated ceramide- and TNF-{alpha}-induced ENaC inhibition. A: representative single-channel current in cell-attached patches before and 5 min after application of either C2-ceramide (50 µM) or TNF-{alpha} (100 ng/ml) to the luminal bath. Ceramide had no effect on ENaC activity in an A6 cell pretreated with a PKC inhibitor GF109203X (0.4 µM) for 15 min (top traces), but still inhibited ENaC in an A6 cell pretreated with a MAP kinase kinase inhibitor PD-98059 (10 µM) for 15 min (middle). TNF-{alpha} also had no effect on ENaC activity in an A6 cell pretreated with GF109203X (0.4 µM) for 15 min, even though the cell was pretreated with LY 294002 (1 µM) for 24 h (bottom traces). B: summary plots of NPo values before ({circ}) and 5 min after C2-ceramide (50 µM; bullet) in A6 cells pretreated with either GF109203X (top left) or PD-98059 (top right) or after TNF-{alpha} (100 ng/ml; bullet) in A6 cells pretreated with GF109203X (the cells were pretreated with 1 µM LY 294002 for 24 h; bottom left).

 
Ceramide induces externalization of PS via a PKC-dependent process. We previously reported that PS from the cytosolic side maintains ENaC activity (34). PKC activation is known to induce externalization of PS (13, 27). Therefore, we hypothesized that ceramide might inhibit ENaC by causing PS externalization through activation of PKC. To test this hypothesis, confocal microscopy experiments were performed to examine the localization of PS using FITC-conjugated annexin-V. There was no detectable PS on the surface of untreated A6 cells. However, PS was detected on the luminal surface of A6 cells treated with either C2-ceramide (50 µM) or PMA (100 ng/ml), a PKC activator, for 5 min (Fig. 6A). To determine whether ceramide induces PS externalization by stimulating PKC, A6 cells were pretreated with a PKC inhibitor, GF109203X (0.4 µM), for 15 min before ceramide treatment. We found that PKC inhibition abolished PS externalization induced by ceramide (Fig. 6B). These results indicate that ceramide inhibits ENaC in A6 cells via a PKC-dependent externalization of PS.


Figure 6
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  		Fig. 6. Externalization of phosphatidylserine (PS) induced by C2-ceramide and phorbol 12-myristate 13-acetate (PMA) was abolished by a protein kinase C (PKC) inhibitor. A: representative images taken by confocal microscopy from either control A6 cells (left) or the cells treated with either C2-ceramide (50 µM; middle) or PMA (100 ng/ml; right) for 5 min; 3 sets of such experiments were performed and showed similar results. B: representative images taken by confocal microscopy from either A6 cells treated with GF109203X (0.4 µM) alone for 15 min (left) or the cells pretreated with GF109203X (0.4 µM) for 15 min and then treated with C2-ceramide (50 µM; right) for 5 min; 3 sets of such experiments were performed and showed similar results. PS was detected by annexin V-FITC.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
As discussed earlier, TNF-{alpha} can either stimulate (15) or inhibit (11) ENaC in alveolar epithelial cells. The opposing effects of TNF-{alpha} on ENaC activity fit with its dichotomous role in pulmonary edema, in which TNF-{alpha} can either stimulate or inhibit fluid clearance via two pathways: one associated with its lectin-like domain and the other mediated by TNF receptors (5). There are two types of TNF receptors: TNF-R1 and TNF-R2 (1, 29). Thus the stimulatory effect of TNF-{alpha} on ENaC may be mediated by its lectin-like domain when TNF receptor binding sites are blocked by the soluble TNF-R1 (5). However, in the absence of soluble TNF receptors, TNF-{alpha} initiates different signaling cascades through TNF-R1 and TNF-R2 (35), which may account for its inhibitory effect on ENaC. TNF-R2 is an inducible receptor that plays a role in several inflammatory diseases and cancers (7). Therefore, TNF-R2 may not be important for the acute inhibition of ENaC by TNF-{alpha} under resting conditions. In contrast, TNF-R1 is a well-characterized receptor that can acutely stimulate both neutral and acidic sphingomyelinases (49). Using patch-clamp techniques, we demonstrated that TNF-{alpha} acutely reduced ENaC activity in A6 renal epithelial cells. However, the reduction occurred only when the cells were pretreated with a PI3K inhibitor, LY 294002. This is not very surprising because PI3K, which is stimulated by aldosterone (4, 22) in A6 cell culture medium, can inhibit sphingomyelinase (44, 45) which is required for TNF-R1 signaling (49). We also found that TNF-{alpha}-induced reduction of ENaC activity was reproduced by exogenous sphingomyelinase and the sphingomyelinase product, ceramide. These studies together suggest that TNF-{alpha} inhibits ENaC via a pathway associated with sphingomelinase and that the inhibition can be antagonized by activation of PI3K.

Several lines of evidence indicate that ceramide acts as an important second messenger which regulates a variety of ion channels including a voltage-dependent potassium channel, Kv1.3 (20), Ca2+ channels (9, 28), a chloride channel (43), a Ca2+-activated potassium channel (30), and the HERG potassium channel (8). Our experiments demonstrated that a membrane-permeable analog of ceramide, C2-ceramide, but not choline, mimicked inhibition of ENaC by TNF-{alpha}. These data suggest that ceramide, but not the other product of sphingomyelin hydrolysis (choline), mediates the inhibition of ENaC by TNF-{alpha}. In contrast, a membrane-impermeable analog, C2-dihydroceramide, had no effect on ENaC activity, indicating that an intracellular signaling cascade associated with ceramide may be initiated. Since it is generally accepted that ceramide activates PKC (26, 48) and activation of PKC inhibits ENaC (14, 31), we examined whether ceramide could inhibit ENaC via PKC. The data demonstrated that inhibition of PKC abolished ENaC inhibition induced by ceramide. For several years, how PKC inhibits ENaC remained obscure because activation of PKC is unable to phosphorylate ENaC itself (50). However, now we show, as others have done in other types of cells (13, 27), that PKC activation induces externalization of PS in renal epithelial cells. We also showed that ceramide induces PS externalization in a PKC-dependent manner. PS externalization is often associated with apoptosis, but it has also been suggested that externalization can be a signaling mechanism in its own right (e.g., in the degranulation process of mast cells) (36). Together with our previous findings that only PS in the cytosolic leaflet of the apical membrane is sufficient to maintain ENaC activity (34), the present findings suggest that ceramide, as a downstream molecule of a TNF-{alpha}-initiated signaling cascade, inhibits ENaC by causing removal of PS from the inner leaflet with subsequent PS externalization via a PKC-dependent pathway.

It has been suggested that decreased ENaC activity contributes to the formation of fluid-filled cysts in PKD (46, 47). Since TNF-{alpha} is found in the cyst fluid (16), TNF-{alpha} inhibition of ENaC via a pathway associated with ceramide may play an important role in facilitating cyst formation in the kidney with PKD. Besides renal pathology, ceramide-induced inhibition of ENaC may also be important for lung diseases since ceramide is responsible for the pulmonary edema triggered by platelet-activating factor (PAF) (18). Since ENaC also plays a critical role in facilitating lung fluid clearance (23), whether inhibition of ENaC induced by ceramide accounts, in part, for PAF-induced pulmonary edema would be another interesting area to explore.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by Department of Health and Human Services and National Institutes of Health Grants R01-DK-067110 (to H.-P. Ma), T32-DK-007656 (to H.-F. Bao), and R37-DK-037963 (to D. C. Eaton).


   FOOTNOTES
 

Address for reprint requests and other correspondence: H.-P. Ma, Dept. of Medicine, Division of Nephrology, Univ. of Alabama at Birmingham, 1530 Third Ave. South, ZRB 510, Birmingham, AL 35294 (e-mail: hepingma{at}uab.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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