WNK4 kinase regulates surface expression of the human sodium chloride cotransporter in mammalian cells

original article

http://www.kidney-international.org & 2006 International Society of Nephrology

see commentary on page 2116

WNK4 kinase regulates surface expression of the human sodium chloride cotransporter in mammalian cells H Cai1,3, V Cebotaru2,4, Y-H Wang3,4, X-M Zhang2, L Cebotaru3, SE Guggino2,3 and WB Guggino3 1

Division of Nephrology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; 2Division of Gastroenterology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA and 3 Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Pseudohypoaldosteronism type II (PHA II) is caused by mutations of two members of WNK ((with no lysine (k)) kinase family. WNK4 wild type (WT) has been shown to inhibit the activity and surface expression of sodium chloride cotransporter (NCC) when expressed in Xenopus oocytes. Here, we have studied NCC protein processing in mammalian cells in the presence or absence of WNK4 WT and its mutants, E562K and R1185C, by surface biotinylation, Western blot, co-immunoprecipitation (Co-IP) and immunostaining. WNK4 WT significantly reduced NCC surface expression in Cos-7 cells (58.976.8% vs 100% in control, Po0.001, n ¼ 6), whereas its mutant E562K has no significant effect on NCC surface expression (92.975.3% vs 100%, P ¼ NS, n ¼ 6). Another mutant R1185C still partially reduces surface expression of NCC (76.2711.8% vs 100%, Po0.05, n ¼ 6). The reduction of NCC surface expression by WNK4 WT (62.973.3% of control group) is not altered by WT dynamin ((61.873.7% (P ¼ NS)) or its mutant K44A ((65.4714.1% (P ¼ NS)). A Co-IP study showed that both WNK4 WT and WNK4 E562K interact with NCC. Furthermore, a proton pump inhibitor, bafilomycin A1, partially reverses the inhibitory effect of WNK4 WT on NCC expression. Our data suggest that WNK4 WT significantly inhibits NCC surface expression, which is not owing to an increase in clathrin-mediated endocytosis of NCC, but likely results from enhanced degradation of NCC through a lysosomal pathway. Kidney International (2006) 69, 2162–2170. doi:10.1038/sj.ki.5000333; published online 10 may 2006 KEYWORDS: pseudohypoaldosteronism type II; WNK4 kinase; sodium chloride; cotransporter; lysosomal pathway; dynamin

Correspondence: WB Guggino or H Cai, Department of Physiology, WBSB Rm 210, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, Maryland 21205, USA. E-mail: [email protected] or [email protected] 4

These authors contributed equally to this work.

Received 22 July 2005; revised 26 October 2005; accepted 18 November 2005; published online 10 may 2006 2162

WNK (with no lysine (k)) kinase is a newly characterized member of the subfamily of serine/threonine kinases.1 Mutations in two members of this family, WNK1 and WNK4, result in pseudohypoaldosteronism type II (PHA II),2 also referred to as Gordon syndrome or familial hyperkalemia and hypertension. PHA II is a rare autosomal dominant disorder characterized by hypertension, hyperkalemia, hyperchloremic metabolic acidosis, and normal glomerular filtration rate. The clinical phenotype of PHA II is opposite to that reported for Gitelman syndrome, a disease caused by loss-of-function mutations in the thiazide-sensitive sodium chloride cotransporter (NCC).3 Previous studies on PHA II have shown that those clinical features are chloride dependent4,5 and correctable by a thiazide diuretic, a specific inhibitor of NCC, implicating a primary defect in sodium chloride handling in the distal nephron. Studies have shown that wild-type (WT) WNK4 inhibits sodium uptake by reducing NCC surface expression in Xenopus oocytes.6,7 A recent study further showed that the WNK4 carboxylterminus mediates NCC suppression.8 However, the molecular mechanism of how WNK kinases regulate NCC surface expression remains to be clarified, especially in mammalian cells. To this end, we report that WNK4 WT reduces NCC surface expression, whereas the PHA II-causing mutants either lose their ability entirely (WNK4 E562K) or partially reduce NCC surface expression (WNK4 R1185C). Coexpression of NCC with a dynamin 2 WT or its dominantnegative mutant K44A does not alter the ability of WNK4 WT to reduce NCC surface expression. Our data also suggest that the reduced NCC surface expression induced by WNK4 WT is likely owing to enhanced degradation of NCC through the lysosomal pathway. RESULTS Effects of WNK4 and its mutants on NCC surface expression in Cos-7 cells

Cos-7 cells were transiently transfected with green fluorescent protein (GFP)-NCC either alone or in combination with WNK4 WT or its mutants E562K or R1185C (Figure 1). Immunostaining and confocal microscopy were subsequently Kidney International (2006) 69, 2162–2170

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H Cai et al.: Sodium chloride cotransporter regulation by WNK4

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Figure 1 | Wild-type human WNK4 and NCC and their predicted domains and WNK4 mutant constructs. P, proline-rich regions; KD, the kinase domain; AI, an autoinhibitory domain; Cc, a coiled-coil domain; N, the amino-terminus; and C, the carboxyl-terminus. All WNK4 constructs contain an myc tag at the N-terminus. WNK4 WT is a wild-type WNK4 construct; WNK4 E562K is a WNK4 PHA II-causing mutant with a point mutation in a highly conserved region just distal to the first coiled-coil domain; and WNK4 R1185C is another WNK4 PHA II-causing mutant with a point mutation in the region just distal to the second coiled-coil domain. All point mutations are indicated as black line inserts. GFP-NCC is a human WT NCC construct with an N-terminal GFP tag; HA-NCC is a human WT NCC construct with HA tag at the N-terminus; and TM indicates the 12 transmembrane domains of NCC.

performed to assess the distribution of NCC in Cos-7 cells. As shown in Figure 2, the cells transfected with NCC alone, the distribution of GFP-NCC (Figure 2a–c) was at the cell membrane and within the cytoplasm. When cells were cotransfected with WNK4 WT and GFP-NCC (Figure 2d–f), NCC was now detected primarily within the cytoplasm and notably the cell surface portion of NCC was reduced. In contrast, when Cos-7 cells were cotransfected with WNK4 E562K and GFP-NCC (Figure 2g–i), NCC could again be detected at the cell surface, similar to that observed in the cells transfected with NCC alone. A similar effect on NCC surface expression was also observed in the cells cotransfected with WNK4 R1185C (Figure 2j–l). Similar expression patterns of NCC, WNK4 WT, and its mutants were found in each group when Cos-7 cells were transfected with HANCC (data not shown), suggesting that the addition of a tag to NCC would not impact NCC expression pattern. These results suggested that WNK4 WT reduces NCC surface expression, whereas PHA II-causing mutants do not affect NCC surface expression. Effects of WNK4 and its mutants on NCC expression in renal epithelial cells

The effects of WNK4 WT and its mutants on NCC surface expression were subsequently evaluated in renal epithelial M-1 cells. As shown in Figure 3, NCC was expressed both in the apical membrane and cytoplasm in M-1 cells transfected with GFP-NCC alone (Figure 3a). As observed in Cos7 cells, surface NCC was reduced after cotransfection with GFP-NCC and WNK4 WT (Figure 3b). However, NCC surface expression was not affected in M-1 cells cotransfected with NCC and WNK4 E562K (Figure 3c), suggesting that WNK4 E562K lacked an inhibitory effect on NCC surface expression. Similarly, NCC surface expression was unaffected in M-1 cells cotransfected with NCC and WNK4 R1185C (Figure 3d). Kidney International (2006) 69, 2162–2170

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Figure 2 | WNK4 WT reduces, but PHA II-causing mutants do not affect NCC surface expression in Cos-7 cells. Cos-7 cells were transfected only with (a–c) GFP-NCC or doubly transfected with (d–f) GFP-NCC and WNK4 WT or its disease mutants, (g–i) E562K or (j–l) R1185C. Immunofluorescence and confocal microscopy were performed in Cos-7 cells 48 h after transfection. (a–c) Cos-7 cells were transfected with GFP-NCC as a control and NCC in (a) green is seen both in plasma membrane and cytoplasm. (d–f) Cos-7 cells were cotransfected with GFP-NCC and myc-tagged WNK4 WT. GFP-NCC in (d) green, WNK4 WT stained in (e) red, and the (f) merge picture are shown. NCC is distributed primarily in the cytoplasm and colocalized with WNK4 in the perinuclear region. (g–i) Cos-7 cells were cotransfected with GFP-NCC and myc-tagged WNK4 E562K. GFP-NCC in (g) green, E562K stained in (h) red, and the (i) merge picture are shown. (g) The surface expression of NCC is restored and its cytoplasmic distribution is similar to WNK4 WT group. (j–l) Cos-7 cells were cotransfected GFP-NCC and myc-tagged WNK4 R1185C. GFP-NCC again in (j) green, R1185C stained in (k) red, and the (l) merge picture are shown. The expression patterns of NCC and R1185C are similar to the E562K group.

Effect of WNK4 on NCC surface expression quantified in Cos-7 cells

To confirm the above immunofluorescent results, surface biotinylation experiments were performed to determine the effect of WNK4 WT and its mutants on NCC surface expression in Cos-7 cells. As shown in Figure 4, NCC surface expression was reduced significantly (41.1%) in Cos-7 cells cotransfected with WNK4 WT and NCC compared to the cells cotransfected with NCC and a CD4 plasmid (control group) (58.976.8% vs 100%, Po0.001, n ¼ 6) (Figure 4b). CD4, a membrane protein transcribed using the same cytomegalovirus promoter, was employed to equalize the amount of DNA in all experiments. There was no difference in NCC surface expression between the cells transfected with NCC alone and that cotransfected with NCC and CD4 plasmid (data not shown). In Cos-7 cells transfected with NCC and WNK4 E562K, NCC surface expression was not significantly diminished compared to control group 2163

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Figure 3 | WNK4 reduces the surface expression of NCC in polarized M-1 cells. M-1 cells were grown on TranswellTM cell culture inserts and were transiently transfected with a GFP-tagged NCC alone as a (a) control or in (b) combination with myc tagged WNK4 WT or (c) PHA II-causing mutant WNK4 E562K or (d) WNK4 R1185C after the cells reached 80–90% confluence. Immunostaining experiments were performed 2 days after transfection and confocal microscopic images of the cells were obtained. Mouse monoclonal anti-myc antibody and rabbit anti-ZO-1 antibody were used for immunostaining studies. The results of immunofluorescence and confocal microscopy are shown in M-1 cells. All images were captured in an identical manner. The image stacks at top were obtained at Z-axis and the image at the bottom was captured at apical level as indicted using ZO-1, a tight junction marker. ZO-1 stained in red, WNK4 stained in blue, and NCC in green are shown as indicated. In (a), NCC (green) was localized to both the apical membrane and cytoplasm. In (b), NCC appears to be distributed in the subapical region and cytoplasm as seen in a Z-axis view and the surface expression of NCC is reduced. In (c), NCC is localized to both the apical membrane and cytoplasm, a distribution similar to that in the (a) control, suggesting that surface expression of NCC is restored in the presence of the PHAII-causing mutant, WNK4 E562K. In (d), the surface expression of NCC in the WNK4 R1185C group is similar to the WNK4 E562K group, indicating that PHAII-causing mutants lost their inhibitory effect on NCC surface expression.

(92.975.3% vs 100%, P ¼ NS, n ¼ 6) (Figure 4b), indicating that WNK4 E562K lost its ability to reduce NCC surface expression. To our surprise, another mutant, WNK4 R1185C, could still reduce NCC surface expression albeit to a less extent compared to control group (76.2711.8% vs 100%, Po0.05, n ¼ 6) (Figure 4b). In addition, WNK4 D321A (dead kinase) had no effect on NCC surface expression, suggesting that WNK4 to function properly requires intact kinase activity (data not shown).

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Figure 4 | Quantitative analysis of the effect of WNK4 WT and its mutants on surface expression of NCC in Cos-7 cells. Cos-7 cells were cotransfected with NCC in combination with either CD4, or WNK4 WT or WNK4 mutants as indicated. (a) The top panel is a representative immunoblot measuring the surface biotinylated NCC. Bottom panel represents protein loading control with b-actin. (b) The data shown in the bar graph represent the combined results of luminescent analysis of six independent experiments of surface biotinylation studies. Biotinylated surface protein (NCC) and the correspondent amount (total NCC) of total cell lysate were quantified using Fujifilm LAS-1000. The fraction of cell surface NCC expression over total NCC in the cell lysates was calculated and the values shown reflect the relative percentage of that in NCC þ CD4 (control group). *Po0.001 in NCC þ WT group vs NCC þ CD4 group. #Po0.05 in NCC þ R1185C group vs to NCC þ CD4 group. NS indicates no statistical difference of NCC surface expression in NCC þ E562K group vs control group. The error bars are the s.d.

blocks clathrin-mediated endocytosis of many membrane proteins10 and is also known to affect the integrity of the Golgi in some cell types.11 To investigate whether the ability of WNK4 WT to reduce NCC surface expression involves a dynamin-mediated pathway, we quantified the NCC surface expression by surface biotinylation in Cos-7 cells in the presence or absence of Dyn WT or Dyn K44A. As shown in Figure 5, Cos-7 cells were cotransfected with HA-NCC in combination with either CD4 or WNK4 WT. NCC surface expression was significantly reduced in WNK4 WT group (62.973.3%, lane 4 vs 100% in CD4 control group, lane 1, Po0.001, n ¼ 4). When Cos-7 cells were cotransfected HANCC with WNK4 WT and Dyn WT or its dominant-negative mutant Dyn K44A, the reduction of NCC surface expression was not further altered by Dyn WT (61.873.7%, P ¼ NS compared to WNK4 group) or Dyn K44A (65.4714.1%, P ¼ NS, compared to WNK4 group). These data suggest that the reduction of NCC surface expression by WNK4 is independent of the clathrin-mediated endocytosis pathway.

Effect of WNK4 on NCC surface expression is not affected by dynamin

Dynamin 2 and its dominant-negative mutant K44A do not affect the distribution of NCC in the presence or absence of WNK4

Dynamin 2 wild type (Dyn WT) controls the pinching of clathrin-coated endocytic vesicles from the plasma membrane and the budding of vesicles from the Golgi apparatus.9 A dominant-negative dynamin mutant K44A (Dyn K44A)

NCC is expressed both at the plasma membrane and within the cytoplasm in Cos-7 cells transiently transfected with HANCC (Figure 6b). Dyn WT is expressed uniformly in Cos-7 cells (Figure 6a). Dyn K44A, on the other hand, has a distinct

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H Cai et al.: Sodium chloride cotransporter regulation by WNK4

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Figure 5 | The influence of WNK4 WT on NCC surface expression is not altered by presence of dynamin WT or its dominant-negative mutant K44A. (a) Cos-7 cells were cotransfected with (1) either HAtagged NCC and CD4 plasmid (as control), or (2) HA-tagged NCC and WNK4 WT, in combination with either pEGFP vector or GFP-tagged dynamin 2 wild-type (Dyn WT) or its dominant-negative mutant K44A (Dyn K44A) as indicated. Forty-eight hours post-transfection, the cell surface proteins were labeled with membrane-impermeable sulfoNHS-SS-biotin at 41C. The cell surface proteins were isolated by NeutrAvidin beads, fractionated on SDS-polyacrylamide gel electrophoresis, and the surface portion of NCC was detected by Western blotting using an NCC antibody as indicated. Total lysate dynamin was detected by Western blotting using a GFP polyclonal antibody as indicated. b-Actin is shown here as protein loading control. (b) The data shown in the bar graph represent the combined results of luminescent analysis of four independent experiments of surface biotinylation studies. Biotinylated surface protein NCC and the corresponding total NCC in cell lysate were quantified using Fujifilm LAS-1000. The fraction of cell surface over total NCC expression in the cell lysates was calculated and the values shown are the relative percentage of that in CD4 þ Vect group (control). *Po0.001 in WNK4 þ Vect group or WNK4 þ Dyn WT or WNK4 þ K44A group compared to CD4 þ Vect group, respectively. There is no statistical difference between NCC surface expression in CD4 þ Dyn WT or CD4 þ K44A group compared to CD4 þ Vect group (control group) or in WNK4 þ Dyn WT or WNK4 þ K44A group compared to WNK4 þ Vect group. The error bars are the s.d.

punctate expression pattern (Figure 6c), consistent with a previous report that this mutant blocks clathrin-mediated endocytosis and is arrested at the clathrin-coated pits at the plasma membrane.12 To further investigate whether Dyn WT or Dyn K44A affects the localization of NCC in the presence or absence of WNK4 WT, we conducted immunofluorescent studies to examine the distribution of NCC in Cos-7 cells. As shown in Figure 6, NCC retains a similar distribution in the Kidney International (2006) 69, 2162–2170

Figure 6 | Dynamin WT or its dominant-negative mutant dose not change NCC expression pattern. Cos-7 cells were (1) singly transfected (top row) with (a) Dynamin 2 wild-type-GFP (Dyn WT), (b) HA-NCC (NCC) or (c) Dynamin 2 K44A-GFP (Dyn K44A) or (2) doubly transfected with either (d–f, second row) HA-NCC and Dyn WT or (g–i, third row) HA-NCC and Dyn K44A or (3) triply transfected with either (j–l, fourth row) HA-NCC, Dyn WT, and myc-tagged WNK4 WT or (m–o, bottom row) HA-NCC, Dyn K44A, and myc-tagged WNK4 WT. Immunofluorescence was performed 48 h after transfection. HA-NCC was detected by a polyclonal antibody to (red) HA epitope, myc-WNK4 WT was detected by a monoclonal antibody against (blue) myc epitope and (green) Dyn WT and Dyn K44A were visualized by green fluorescence on a confocal microscope. Surface NCC expression is reduced in the presence of WNK4 WT, irrespective of presence of either Dyn WT or Dyn K44A. Scale bar ¼ 20 mm.

plasma membrane and cytoplasm in the cells cotransfected with HA-NCC and Dyn WT (Figure 6e) or Dyn K44A (Figure 6h) in the absence of WNK4 WT. Whereas in the cells cotransfected with WNK4 WT and HA-NCC in combination with either Dyn WT (Figure 6k) or Dyn K44A (Figure 6n), NCC expression displayed a punctate pattern, primarily distributed in the cytoplasm. These data again suggest that WNK4 WT reduces NCC membrane expression and this effect is not altered in the presence of either Dyn WT or its dominant-negative mutant K44A. WNK4 and its mutant E562K interact with NCC in Cos-7 cells

To elucidate whether the loss of function of the PHA IIcausing mutant, WNK4 E562K, is owing to a disruption of the interaction between WNK4 and NCC, co-immunoprecipitation (Co-IP) experiments were performed in Cos-7 cells transiently expressing NCC and either WNK4 WT or WNK4 2165

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Figure 7 | Co-IP showed an interaction between WNK4 WT or its mutant WNK4 E562K and NCC. Cos-7 cells were cotransfected with GFP-NCC in combination with either myc-tagged WNK4 WT (lanes 1 and 2) or PHA II-causing mutant, WNK4 E562K (lanes 3 and 4) or vector (lanes 5 and 6). The cells were lysed in lysis buffer and lysates for Co-IP were incubated with mouse monoclonal anti-myc antibody overnight, followed by protein A–agarose beads for 2 h at 41C. After washing with phosphate-buffered saline, beads were eluted with SDS Laemmli buffer (Bio-Rad Laboratories, Hercules, CA, USA) and the eluted proteins were separated by SDS-polyacrylamide gel electrophoresis, blotted, and probed with an anti-GFP antibody. After stripping, the membrane was blotted and probed with an anti-myc antibody in immunoprecipitation (IP) experiments. (a) IP experiments: Anti-myc antibody was used for precipitating WNK4 as well as probing. Lane 1, lysate protein input; lane 2, IP for the WNK4 WT group; lanes 3 and 4, lysate protein input and IP, respectively, for the WNK4 E562K group; lanes 5 and 6, lysate protein input and IP, respectively, for the vector group (negative control). As shown in (a) anti-myc antibody immunoprecipitates both WNK4 WT and its mutant, E562K. (b) Co-IP experiment: Anti-myc antibody was used for precipitating NCC and the anti-GFP antibody was used for probing. The lanes were indicated as in (a). As seen in (b), NCC cannot be co-immunoprecipitated in the vector group lacking WNK4 expression. However, NCC can be immunoprecipitated by both WNK4 WT and the PHA II-causing mutant, WNK4 E562K, suggesting that (1) WNK4 interacts with NCC either directly or indirectly and (2) WNK4 E562K does not alter this interaction.

E562K. As shown in Figure 7, vector alone (negative control) could not pull down NCC (Figure 7b, lane 6). WNK4 WT coimmunoprecipitated NCC (Figure 7b, lane 2) and so does its mutant, WNK4 E562K (Figure 7b, lane 4). These results demonstrated that WNK4 WT interacted with NCC and that the disease mutant did not alter this interaction. This excludes the possibility that loss of function of the WNK4 mutant was owing to altered protein–protein interaction. Instead, it might be attributed to other mechanisms such as alteration of phosphorylation of NCC that ultimately affects NCC processing. A role for lysosomal degradation in the regulation of NCC surface expression by WNK4

To determine more closely how WNK4 affects NCC, we investigated the effect of V-type proton pump inhibition on NCC protein expression in Cos-7 cells. Bafilomycin A1 (Baf A1) specifically inhibits the vacuolar-type H þ -ATPase and thereby affects acidic proteases by disturbing the pH of endocytic organelles, including lysosomes.13 As shown in 2166

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Figure 8 | The role of WNK4 on NCC surface expression is owing to enhanced degradation of NCC via a lysosomal pathway in Cos7 Cells. One day after transfection of the GFP-NCC with CD4 (control) or WNK4 as indicated, Cos-7 cells were treated with 0.5 and 1.0 mM of the V-type proton pump inhibitor Baf A1 for 16 h at 371C. Lysates were subjected to 5% SDS-polyacrylamide gel electrophoresis followed by immunoblotting with rabbit anti-GFP and mouse anti b-actin antibodies. Protein expression change was quantified after the Baf A1 treatment. Plots are presented as the ratio change from non-treated cells from control group (CD4). In (a), the top band indicates a representative Western blot showing steady-state protein level of NCC in the presence or absence of WNK4 WT after Baf A1 treatment. b-Actin indicates protein loading control, suggesting that Baf A1 treatment does not affect actin expression level. The histogram at bottom represents a summary result of three independent experiments. In (b), top two bands indicate a representative Western blot. The graph at the vbottom represents a summary result of three independent experiments. Note that steadystate amount of NCC in the presence of WNK4 WT was increased in a dose-dependent manner after Baf A1 treatment, whereas NCC expression in the presence of WNK4 mutant E562K was not significantly changed, suggesting that the inhibitory effect of WNK4 WT on NCC surface expression is at least partially owing to increase of degradation of NCC via lysosomal pathway. #Po0.01 compared to control group (NCC þ CD4). *Po0.05 compared to the group of NCC þ WNK WT without Baf A1 treatment.

Figure 8, when Cos-7 cells were cotransfected with NCC and WNK4 WT, the steady-state protein expression of NCC was significantly reduced in presence of WNK4 WT (63.773.1% vs 10070% in NCC þ CD4 control group, Po0.01, n ¼ 3). After Baf A1 treatment, the ability of WNK4 WT to reduce the steady-state level of NCC was significantly diminished in a dose-dependent manner (63.773.1% in non-Baf A1treated group vs 71.974.1% in 0.5 mM Baf A1-treated groups and 63.773.1% in non-Baf A1-treated group vs 79.473.1% in 1.0 mM Baf A1-treated group, Po0.05, n ¼ 3) (Figure 8a). As expected, the steady-state level of NCC surface expression was not altered in the presence of the WNK4 E562K after Baf A1 treatment (Figure 8b). Immunostaining further showed that NCC, WNK4, and its mutant E562K colocalize with cathepsin D, a lysosomal marker (Figure 9). These results suggest that one of the mechanisms of action of WNK4 WT is to promote the degradation of NCC via a lysosome-mediated pathway. The observation that WNK4’s ability to reduce Kidney International (2006) 69, 2162–2170

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H Cai et al.: Sodium chloride cotransporter regulation by WNK4

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Figure 9 | WNK4 WT or its mutant E562K and NCC colocalize with cathepsin D, a lysosomal marker. Cos-7 cells were transfected with either (a–c) GFP-NCC or (d–f) WNK4 WT or its disease mutant, (g–i) E562K. Immunofluorescence experiments were performed 48 h after transfection. (a) GFP-NCC was visualized by green fluorescence. (d) Myc-WNK4 WT and its mutant (g) E562K were detected by a monoclonal antibody against myc epitope followed by (green) fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin (Ig) G. The endogenous cathepsin D, (b, e, and h) a lysosomal marker was detected by a polyclonal antibody followed by (red) Cy-3-labeled goat anti-rabbit IgG. (c, f, and i) The merged pictures were shown for the respective row in the third columns. This finding showed that WNK4 or its mutant E562K and GFP-NCC colocalize with cathepsin D, (in yellow) a lysosomal marker, suggesting that WNK4 directs mature NCC into a lysosomal pathway for degradation. Scale bar ¼ 20 mm.

NCC surface expression is not altered in the presence of dominant-negative dynamin mutant K44A (Figure 5) supports the conclusion that WNK4 promotes the direct degradation of NCC via the lysosome without it first translocating to the plasma membrane. DISCUSSION

The kinase, WNK4, plays an important role in renal function by regulating the activity of several renal transporters and ion channels including NCC,6,7 the outer medullary potassium ion channel (ROMK),14 the Na þ –K þ –2Cl– cotransporter (NKCC1)15 and the Cl/base exchanger SLC26A6 (CFEX).15 WNK4 is also known to regulate paracellular chloride ion flux.16 In this paper, we report that WNK4 WT reduces the surface expression of NCC in mammalian cells, especially in renal epithelial cells. The data are consistent with experiments published by others using the Xenopus oocyte heterologous expression system.6,7 Furthermore, we show that the reduction of NCC surface expression by WNK4 WT is not affected by the presence of a dominant-negative dynamin mutant but is affected by Baf A1, suggesting that one mechanism of action of WNK4 is to enhance degradation of NCC through a lysosome-mediated pathway without NCC first transiting to the plasma membrane. The PHAII-causing mutants, E562K and R1185C, either lose their functional effect totally or retain a partial ability to reduce NCC surface expression. Again to take this further, we Kidney International (2006) 69, 2162–2170

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showed that both WT and the WNK4 mutant E562K bind to NCC. Experiments using only the C-terminal region of NCC7 showed that WNK4 interacts with the C-terminus of NCC, a location in NCC known to play an essential role in the regulation of its protein processing17,18 and function.19,20 A recent study also showed that C-terminus of WNK4 is responsible for its interaction with NCC.8 Our observation that the WNK4 mutant E562K still binds to NCC demonstrates that the functional differences among WT and the PHAII-causing mutants are not owing to altered protein–protein interactions between WNK4 mutant and NCC. NCC function is known to be regulated by deamino-Cys21 D-Arg-vasopression, aldosterone,22 estradiol,23 loop diure24 tics, or low sodium diet,25 and is decreased by chronic hypokalemia.26 Structural analysis of NCC suggests that NCC contains putative protein kinase A and protein kinase C sites.27 Activation of protein kinase C with a phorbol ester results in significant reduction of NCC function.27 However, no effect of cAMP, cGMP, or 3-isobutyl-1-methylxanthine, an activator of protein kinase A, on NCC function was observed.27 Our observation that the WNK4 D321A (dead kinase mutant) cannot reduce NCC surface expression was also confirmed by others in oocytes.7 This finding is further consistent with the notion that the kinase domain of WNK4 plays a role in the regulation of NCC function and processing. One might speculate that WNK4 regulates NCC function through a phosphorylation-dependent mechanism. However, a recent study showed that C-terminus of WNK4 without kinase domain remained interaction with NCC and was still able to inhibit NCC function,8 which is inconsistent with our and others observations.7 Whether WNK4 regulates NCC function through its phosphorylation of NCC remains to be established. Our observation that the PHAII-causing mutant, E562K, does not decrease NCC surface expression is also consistent with experiments published by other using the Xenopus oocyte expression system.6,7 Interestingly, a mutation in WNK1, another member of WNK kinase family, also causes PHA II as result of the overexpression of WNK1 mRNA.2 WNK1 was shown to prevent WNK4 from reducing NCC activity and surface expression in Xenopus oocytes.6 It has been shown that WNK1 inhibition requires an intact WNK4 kinase domain, the region that binds to WNK1. WNK1 inhibition of WNK4 is dependent on WNK1 catalytic activity and an intact WNK1 protein.8 These findings suggest that WNK4 is a downstream target of WNK1 kinase and an intact kinase domain of WNK4 remains critical in the regulation of NCC function and NCC trafficking as well. Whether WNK4 possesses kinase activity and directly or indirectly phosphorylates downstream targets such as NCC is still unclear. NCC function, like other membrane transporters or channels, could be regulated by several possible mechanisms through its transport pathway. Reduction or abolishment of NCC transporter activity might be owing to its impaired protein synthesis, impaired protein processing, decreased 2167

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insertion of an otherwise functional protein into the plasma membrane, impaired functional properties of the cotransporter, or accelerated protein removal or degradation. WNK4 WT has been shown to inhibit NCC activity and surface expression in Xenopus oocytes.6,7 We also showed that WNK4 WT inhibits NCC surface expression in mammalian cells, whereas the WNK4 mutant E562K loses its inhibitory effect. We further showed that reduction of NCC surface expression by WNK4 WT was not altered by the dominantnegative dynamin mutant K44A, indicating that the inhibitory effect of WNK4 WT on NCC surface expression is not owing to an increase in a clathrin-mediated endocytic pathway. A novel finding to our study is that steady-state protein levels of NCC are reduced in the presence of WNK4 WT, but not its PHA II-causing mutant E562K, and the reduction of NCC expression is partially reversed after treatment with V-type proton pump, Baf A1, indicating that WNK4 WT facilitates NCC degradation through a lysosomemediated pathway, which results in decreased insertion of NCC into the plasma membrane. These findings demonstrate that reduction of NCC surface expression by WNK4 WT is partially attributed to enhanced degradation of NCC through a lysosomal pathway, which provides insight into the molecular mechanism underlying PHA II. However, these data could not exclude the possibility that WNK4 may also change overall NCC degradation, which ultimately affects NCC surface expression. Other potential mechanisms of how WNK4 regulates NCC function and trafficking remains to be established. Another important finding to this study is that the PHAIIcausing mutant, R1185C, retains partial function while still reducing NCC surface expression, but the reduction is less than WT. The differing effects of WNK4 mutants, E562K and R1185C, on NCC surface expression could explain why the clinical phenotypes are different among the PHA II-affected families with these two different mutations. In the WNK4 E562K-affected family members, hypertension usually occurs at an early age of their lives and hyperkalemia is also present,28,29 whereas in the WNK4 R1185C-affected family, the affected members usually present with hyperkalemia associated with hyperchloremic acidosis and normal blood pressure.30 The relatively mild phenotype is certainly consistent with our observation that the R1185C retains a partial effect on NCC surface expression. NCC is predominantly expressed in the distal convoluted tubule and is responsible for 5% of the sodium reabsorption in the kidney.31,32 Current available data support the notion that WNK4 WT has a basal inhibitory effect on NCC activity and surface expression preventing sodium retention. On the other hand, the WNK4 mutant E562K is ineffective, ultimately leading to enhanced NCC activity and sodium reabsorption in distal nephron. The net result is hypertension consistent with Gordon’s hypothesis that is intended to explain the pathogenesis underlying PHAII.33 In the WNK4 R1185Caffected patients, blood pressure is usually normal as this mutant retains partial inhibitory effect on NCC. 2168

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Importantly, the mutation, E562K, is located in the highly conserved region just distal to the first coiled-coil domain, whereas the mutation, R1185C, resides in the conserved region just distal to the second coiled-coil domain.2 The demonstration of the differing impact of these two missense mutations on WNK4 function and ultimate disease severity is the first step in understanding the functional importance of the individual domains of WNK4. MATERIALS AND METHODS Plasmids and constructs Human WT WNK4 was amplified by polymerase chain reaction technique using a human kidney cDNA library and expressed sequence tag clone from Incyte Corporation (Wilmington, DE, USA) as template. The polymerase chain reaction product matched the human WNK4 sequence (GenBank Accession no.: AF390018). The N-terminal myc-tagged WNK4 WT construct was generated by subcloning the WNK4 cDNA into pCMV-taq 3B vector (Stratagene, La Jolla, CA, USA). WNK4 disease mutants, E562K and R1185C, were generated using Quickchange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). Human WT NCC (GenBank Accession no.: NM_000339) was also amplified from a human kidney cDNA library using polymerase chain reaction technique and subcloned into pEGFP-C1 (Clontech, Palo Alto, CA, USA) and pcDNA 3.1 (Invitrogen, Carlsbad, CA, USA) vectors. Thus, the N-terminal GFP- and HA-tagged NCC constructs were generated. All constructs as shown in Figure 1 were confirmed by DNA sequencing. Dynamin 2 (aa) constructs (Dyn2-GFP (WT) and Dyn2K44A-GFP) were the gift of Dr MA McNiven (Mayo Clinic, Rochester, MN, USA). Cell culture and transfection Cos-7 and M-1 cells obtained from American Type Tissue Culture (ATCC) (Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (Cos-7) or Dulbecco’s modified Eagle’s medium/F12 (M-1) medium with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 mg/ml), and 10% fetal calf serum and 0.005 M dexamethasone (M-1 only). LipofectAMINE 2000 (Invitrogen) was used for transfection according to the manufacturer’s instructions. Forty-eight hours after transfection, cells were used for Western blot, immunostaining, and surface biotinylation. Antibodies and immunofluorescent staining The monoclonal antibody (9E10) for myc was obtained from Zymed Laboratories (South San Francisco, CA, USA). The polyclonal antibody for GFP was purchased from Clontech. The rabbit antiNCC antibody was a gift from Dr Mark Knepper (NIH/NHLBI, Bethesda, MD, USA). The polyclonal antibody against cathepsin D was obtained from Upstate (Charlottesville, VA, USA). For immunostaining, the fixed cells were blocked with 5% normal donkey serum in phosphate-buffered saline for 1 h. The cells were then incubated with the primary antibody for 1 h, followed by the appropriate secondary antibody conjugated to fluorescein isothiocyanate, Cy 3, or Cy 5 fluorescent dye (Jackson ImmunoResearch Lab, West Grove, PA, USA) for 1 h. No antibodies from the same species were used in any of the double-stained specimens. After staining, the coverslips (for Cos-7) or transwells (for M-1) were washed, mounted with antiquenching medium (Vector Lab, Burlingame, CA, USA), and the slides were sealed. Kidney International (2006) 69, 2162–2170

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H Cai et al.: Sodium chloride cotransporter regulation by WNK4

Western blotting analysis Cells were harvested and processed as described previously.34. Briefly after transfection, cells were lysed in lysis buffer containing 20 mM Hepes, pH 7.5, 120 mM NaCl, 5.0 mM ethylenediamine tetraacetic acid, 1.0% Triton X-100, 0.5 mM dithiothreitol, and the complete protease inhibitor (Roche Diagnotics, Mannheim, Germany). The lysates were spun at 6000 g for 5 min and the proteins from supernatant were quantified by BCA Protein Assay kit (Pierce, Rockford, IL, USA). The protein sample was then separated by SDSpolyacrylamide gel electrophoresis. After transferring, the membrane was probed with specific antibodies and detected using ECL plus system (Amersham Biosciences Corp., Piscataway, NJ, USA) or Super signal (Pierce) as described previously.35

6. 7.

8.

9. 10.

11.

12.

Confocal laser microscopy The fluorescence label was examined with UltraView confocal imaging system (Perkin-Elmer Life Sciences, Boston, MA, USA). Images were acquired using the manufacturer’s software. To obtain three-dimensional images, each XY plane of the sample with a depth of 0.4 mm in Z-direction was scanned by the confocal laser, and the picture serials along the Z-axis were combined, reconstructed, and presented as XZ and YZ cross-section images using the Velocity software (Improvision Ltd., Lexington, MA, USA). Images were prepared for publication with Adobe Photoshop. Surface biotinylation Biotinylated NCC at the plasma membrane was precipitated as described previously with some modifications.34 Lysates were incubated with immobilized NeutrAvidin beads (Pierce) overnight at 41C, and bound proteins were eluted with 2  Laemmli sample buffer supplemented with 100 mM dithiothreitol at 421C for 30 min. The eluted proteins were subjected to SDS-polyacrylamide gel electrophoresis and Western blot. GFP-NCC was detected with GFP antibody (1:3000).

13. 14. 15.

16.

17.

18.

19.

20.

21.

Statistical analysis The data are presented as the means7s.e. Statistical significance was determined by Student’s t-test and analysis of variance. We assigned significance at Po0.05.

22.

23.

ACKNOWLEDGMENTS

This work was supported by American Heart Association 0530222N (HC), National Institutes of Health Grants DK068226-01A1 (HC), and DK32753 (WBG). We thank Drs Xuhang Li, Peying Fong, Anne Fischer, and Deanne Dryciw for their helpful suggestions and discussions in this research projects. We also thank Dr Sang-Ho Kwon for the domain analysis of WNK4.

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