Am J Physiol Renal Physiol 293: F961–F973, 2007.
First published July 18, 2007; doi:10.1152/ajprenal.00192.2007.
Invited Review
WNK4-mediated regulation of renal ion transport proteins
Ji-Bin Peng and David G. Warnock
Nephrology Research and Training Center, Division of Nephrology, Department of Medicine, University of Alabama
at Birmingham, Birmingham, Alabama
Submitted 20 April 2007; accepted in final form 13 July 2007
WNK1; kidney; hypertension; ion channel; transporter
nephron, including the distal convoluted tubule (DCT), the connecting tubule (CNT), and the
cortical collecting duct (CCT), is important for Na⫹, K⫹, and
Ca2⫹ homeostasis (65, 80). An array of ion transport proteins
located in this portion of the nephron plays important roles in
maintaining an electrolyte balance and, in turn, a normal blood
pressure. The roles of these ion transport proteins in maintaining a normal blood pressure have been demonstrated in genetic
disorders (59, 103). For examples, gain-of-function in the
amiloride-sensitive epithelial Na⫹ channel (ENaC) causes hypertension in Liddle’s syndrome (32, 90); loss-of-function
in the thiazide-sensitive Na⫹-Cl⫺ cotransporter (NCC;
SLC12A3; also known as TSC or NCCT) results in hypotension in Gitelman syndrome (91).
The linkage of familial hyperkalemic hypertension (FHH;
also known as pseudohypoaldosteronism type II, PHAII, or
Gordon’s syndrome) (30), an autosomal dominant form of
hypertension, to two members of the WNK [With No K
(lysine)] family of protein serine-threonine kinases has powered efforts to understand the roles of WNK kinases in regulating ion transporters in the distal tubule (104). The WNK
family consists of four members; they all lack an invariant
lysine residue in the catalytic site in subdomain II, which is
important for binding ATP (36, 99, 104, 109). Mutation in
THE DISTAL PORTION OF THE
Address for reprint requests and other correspondence: J.-B. Peng, Div. of
Nephrology, ZRB 625, Univ. of Alabama at Birmingham, 1900 Univ. Blvd.,
Birmingham, AL 35294-0006 (e-mail: [email protected]).
http://www.ajprenal.org
either the WNK1 or WNK4 gene causes FHH (104). The
FHH-causing gene mutations in WNK4 are missense mutations
clustered in a short “acidic motif” rich in negatively charged
amino acids downstream of the kinase domain (29, 104), with
the exception of R1185C, which is close to the COOH terminus of the kinase (104). FHH is also caused by overexpression
of WNK1, another member of the WNK family, due to deletions in the first intron of the WNK1 gene (104). The clinical
features of FHH, including hyperkalemia, hypertension, and
metabolic acidosis, are common among patients carrying
WNK1 and WNK4 mutations. Abnormal Ca2⫹ homeostasis
has been reported in some FHH patients (84, 89, 92). Interestingly, hypercalciuria was observed in patients carrying that
WNK4Q565E mutation (64), but not in patients carrying the the
WNK1 gene mutation (1). The association of the two WNK
kinases with hypertension due to imbalanced electrolyte homeostasis has stimulated studies on their regulation of renal ion
transport proteins. While these studies aimed at connections
between WNK kinases and the molecular pathogenesis of
FHH, insights obtained through these studies are very helpful
in understanding the regulation of renal handling of electrolytes.
The roles of WNK kinases in regulation of ion transport proteins
and in the pathogenesis of FHH have been subject of excellent
reviews (9, 22, 26, 30, 31, 42, 43, 45, 79, 95, 108, 110). Due to the
limited scope of this review, regulations mediated by WNK1 (2,
7, 10, 53, 68, 73, 101, 102, 111, 112, 123) and WNK3 (13, 41, 56,
81) will not be covered. Instead, we will focus on the WNK4mediated regulation of ion transport proteins.
0363-6127/07 $8.00 Copyright © 2007 the American Physiological Society
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Peng J-B, Warnock DG. WNK4-mediated regulation of renal ion transport proteins. Am J Physiol Renal Physiol 293: F961–F973, 2007. First published July 18, 2007;
doi:10.1152/ajprenal.00192.2007.—Point mutations in WNK4 [for With No K (lysine)], a serine-threonine kinase that is expressed in the distal nephron of the kidney, are
linked to familial hyperkalemic hypertension (FHH). The imbalanced electrolyte
homeostasis in FHH has led to studies toward an understanding of WNK4-mediated
regulation of ion transport proteins in the kidney. A growing number of ion transport
proteins for Na⫹, K⫹, Ca2⫹, and Cl⫺, including ion channels and transporters in the
transcellular pathway and claudins in the paracellular pathway, are shown to be
regulated by WNK4 from studies using models ranging from Xenopus laevis oocytes
to transgenic and knockin mice. WNK4 regulates these transport proteins in different
directions and by different cellular mechanisms. The common theme of WNK4mediated regulation is to alter the abundance of ion transport proteins at the plasma
membrane, with the exception of claudins, which are phosphorylated in the presence of
WNK4. The regulation of WNK4 can be blocked by the full-length WNK1, whose
action is in turn antagonized by a kidney-specific WNK1 variant lacking the kinase
domain. In addition, WNK4 also activates stress-related serine-threonine kinases to
regulate members of the SLC12 family members of cation-chloride cotransporters. In
many cases, the FHH-causing mutants of WNK4 exhibit differences from wild-type
WNK4 in regulating ion transport proteins. These regulations well explain the clinical
features of FHH and provide insights into the multilayered regulation of ion transport
processes in the distal nephron.
Invited Review
F962
WNK4-MEDIATED REGULATION OF ION TRANSPORT PROTEINS
Distribution of WNK4 Along the Nephron
WNK4 Regulates Ion Transporters and Channels in the
Transcellular Pathway and Claudins in the
Paracellular Pathway
The distribution of WNK4 allows it to regulate a variety of
ion transport proteins along the distal tubule as tested in
experimental systems including Xenopus laevis oocytes and
mammalian cells. The regulations of ion transport proteins by
WNK4 are summarized in Table 1 and Fig. 1. These include
apical or basolateral ion transporters, ion channels in the
Table 1. WNK4-mediated regulation of ion transport proteins
Ion
Transport
Protein
Role
⫹
⫺
Renal Distribution
Effect of WNK4
Effect of
Kinase-Dead
Mutant
Effect of FHH
Mutants
NCC
Na -Cl
cotransport
DCT, apical
Inhibition
Not
effective
Less effective
ROMK
K⫹ secretion
CNT, CCD, apical
Inhibition
Effective
More effective
ENaC
Na⫹ reabsorption
CNT, CCD, apical
Inhibition
Effective
No effect
TRPV5
Ca2⫹ reabsorption
DCT, CNT, apical
Enhancing
Less effective with
NCC present
TRPV4
Osmolarity
sensing
Cl⫺ secretion
TAL, DCT, CNT,
CD, basolateral
Renal tubules and
extrarenal
PT, apical extrarenal
Inhibition
Inhibition
Inhibition or
not
effective
Not
effective
As effective
Inhibition
ND
ND
Extrarenal
Inhibition
ND
ND
Renal; extrarenal
Inhibition
PT, basolateral;
extrarenal
PT, TAL, DCT, CD,
basolateral;
extrarenal
Claudin 4 in CCD,
tight junction
Inhibition
Not
effective
Not
effective
Not
effective
Same as WT
WNK4
Same as WT
WNK4
Same as WT
WNK4
ND
More effective
CFTR
CFEX
NKCC1
KCC1
KCC3
KCC4
Claudins
⫺
Cl /anion
exchange
Na⫹, K⫹, 2Cl⫺
cotransport
K⫹, Cl⫺
cotransport
K⫹, Cl⫺
cotransport
K⫹, Cl⫺
cotransport
Paracellular
permeability
Inhibition
Enhancing
Less effective
More effective
Mechanism of Regulation
Reference(s)
Block of forward
transport from
Golgi; increased
lysosomal
degradation
Increased
clathrin-mediated
endocytosis
Increased degradation
mediated by
Nedd4-2
Likely increase in
secretory pathway
5, 28, 29, 52,
96, 105, 116,
118
Decreased surface
expression
Decreased surface
expression
ND
23
33, 44
21, 82
38
117
39
Decreased surface
expression
ND
39
ND
27
ND
27
Phosphorylation of
claudins
40, 114
27
WNK, With No K (lysine); NCC, Na⫹-Cl⫺ cotransporter; ROMK, renal outer medullary K⫹ channel; ENaC, epithelial Na⫹ channel; TRPV4 and TRPV5,
transient receptor potential vanilloid subfamily member 4 and member 5, respectively; CFEX, Cl⫺/formate exchanger; NKCC1, Na⫹-K⫹-2Cl⫺ cotransporter 1;
KCC, K⫹-Cl⫺ cotransporter; CFTR, cystic fibrosis transmembrane conductance regulator; PT, proximal tubule; TAL, thick ascending limb; DCT, distal
convoluted tubule; CNT, connecting tubule; CCD, cortical collecting duct; CD, collecting duct; WT, wild-type; ND, not determined.
AJP-Renal Physiol • VOL
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Early Northern blot analysis suggests that WNK4 is exclusively expressed in the kidney among 12 organs examined
(104). Later reports indicate that WNK4 is also expressed in
extrarenal tissues such as colon, brain, testis, heart, liver,
prostate, and lung of mice (39) or humans (99). Within the
kidney, the strongest WNK4 transcript signal was detected in
the DCT and CNT by in situ hybridization approach (71).
Moderate levels of WNK4 transcript in the macula densa,
medullary thick ascending limb, and collecting duct were also
detected (71). This is broader than the immunohistochemical
staining results showing WNK4 in the DCT and collecting duct
(CD) (104). Interestingly, renal WNK4 is upregulated by high
dietary K⫹ intake and is downregulated by low Na⫹ intake,
whereas aldosterone treatment does not significantly increase
the WNK4 mRNA level (71). This suggests that WNK4 is a
factor that mediates the body’s response to alterations in K⫹
and Na⫹ balance, in parallel to but distinct from aldosterone.
WNK4 is fairly restricted to epithelial cells, such as tubular
epithelial cells in the kidney (104), cuboidal epithelial cells in
the pancreatic and the biliary ducts, pseudostratified columnar
epithelial cells of epididymis, and colonic crypt epithelial cells
(39). WNK4 is also weakly detected in endothelial cells, such
as those comprising the blood-brain barrier (39). At the subcellular level, WNK4 is usually distributed in the cellular
junction and often extends to the lateral membrane. In the
kidney, WNK4 colocalizes with the tight junction marker ZO-1
in the DCT; in the CD, however, cytoplasm distribution of
WNK4 was also observed (104). When transfected into MadinDarby canine kidney (MDCK) cells, WNK4 localizes to the
intercellular junction as well as to the cytosol (40, 114). This
pattern of cellular distribution allows WNK4 to readily regulate proteins at the intercellular junction, e.g., the claudins
(114). However, it is somewhat puzzling how WNK4 regulates
ion transporters and channels in apical or basolateral membranes. In a transgenic mouse line carrying FHH-causing
WNK4D564A, the mutated WNK4 localizes to the apical membranes in the thick ascending limb of Henle’s loop and to
cytoplasm in the distal tubule (115).
Invited Review
WNK4-MEDIATED REGULATION OF ION TRANSPORT PROTEINS
F963
transcellular pathway, and claudins in the paracellular pathway, which will be summarized below.
Thiazide-sensitive Na⫹-Cl⫺ cotransporter NCC. NCC is the
target of thiazide diuretics and is localized to the DCT (25).
Loss-of-function of NCC results in Gitelman syndrome, featuring hypokalemia, hypotension, and hypocalciuria (91). The
clinical features of FHH, such as hyperkalemia, hypertension,
metabolic acidosis, and hypercalciuria, are opposite those of
Gitelman syndrome (64). Because FHH patients are responsive
to thiazide administration (20) and WNK4 colocalizes with
NCC in the DCT (104), two groups independently studied the
regulation of NCC by WNK4 (105, 116). Both groups found
that WNK4 inhibits NCC by decreasing its level at the plasma
membrane in the X. laevis system (105, 116). Yang et al. (116)
found that while WNK1 alone has no effect on NCC, it blocks
the inhibitory effect of WNK4. Wilson et al. (105) found that
WNK4 interacts with NCC and the kinase activity of WNK4 is
required for the regulation. Both groups found the FHHcausing WNK4Q562E mutant (mouse WNK4, equivalent to
Q565E) has impaired ability to inhibit NCC. Golbang et al.
(29) reported that a new FHH-causing WNK4 mutant, D564H,
also has decreased ability to inhibit NCC. In contrast, two other
FHH-causing mutants tested, E559K and D561A (equivalent to
E562K and D564A in human WNK4), inhibit NCC to the
extent comparable to wild-type WNK4 (116). Using the mammalian cell line Cos-7, Cai et al. (5) confirmed the inhibitory
effect of WNK4 on NCC surface expression. Reduced abilities
to inhibit NCC surface expression by FHH-causing E562K and
R1185C mutants were observed in this experimental system
(5). Using stable MDCK cells, Yang et al. (120) reported that
WNK4 overexpression decreases the expression of NCC at the
AJP-Renal Physiol • VOL
apical membrane. However, no difference between FHH-causing D564A and wild-type WNK4 in blocking the apical expression of NCC was observed (120).
The significance of the regulation of NCC by WNK4 in the
pathogenesis of FHH was elegantly illustrated using a transgenic mouse model. Latioti et al. (52) created transgenic mice
harboring an additional copy of either the wild-type Wnk4 gene
or a Wnk4 gene with the Q562E mutation (corresponding to
Q565E in FHH/PHAII patients), denoted as Tg(Wnk4WT)
and Tg(Wnk4PHAII), respectively. The expression level of
Wnk4Q562E is comparable to the wild-type Wnk4 in
Tg(Wnk4PHAII) mice, whereas there is a 50% increase in
total Wnk4 in Tg(Wnk4WT) mice. The Tg(Wnk4WT) mice
exhibit hypertension, hyperkalemia, and hypercalciuria similarly to the patients carrying WNK4Q565E (63, 64); whereas
the Tg(Wnk4WT) mice have opposite manifestations (52). Immunostaining using antibodies against tubular markers reveals
a marked reduction of NCC luminal surface area in
Tg(Wnk4WT) mice and a significant increase in NCC-positive
nephron segments in the Tg(Wnk4PHAII) mice. On the other
hand, renal outer medullary K⫹ channel (ROMK) and ENaC
staining are not altered (52). Targeted deletion of NCC in mice
results in a significant reduction of DCT structures (87).
Therefore, the hyperplasia of DCT is likely caused by the
increased expression of NCC in the Tg(Wnk4PHAII) mice due
to the impaired inhibition of NCC by Wnk4Q562E, although the
mechanism by which NCC controls the morphology of DCT is
unknown. Mice lacking NCC exhibit a phenotype similar to
Gitelman syndrome (67, 87), which is opposite that of FHH.
Thus increased NCC is likely the cause of the FHH-like
phenotype in the Tg(Wnk4PHAII) mice. This is confirmed by
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Fig. 1. Regulation of renal ion transport proteins by WNK4 [for With No K (lysine)] in health and familial hyperkalemic hypertension (FHH). Left: distribution
of key ion transport proteins and WNK kinases along the distal nephron. The distributions of respective ion transport proteins and WNK kinases are illustrated
based on publications (48, 60, 61, 65, 71). Each segment of the distal nephron is not proportional to the exact length. DCT1/DCT2, early/later segment of distal
convoluted tubule; CNT, connecting tubule; CCD/OMCD, cortical/outer medullary collecting duct. Right: regulation of ion transport proteins by WNK4 in health
and FHH. The direction of regulation is indicated by plus and minus signs; the relative strength of regulation is indicated by the thickness of the arrows. The
increased protein levels of the thiazide-sensitive Na⫹-Cl⫺ cotransporter (NCC) and epithelial Na⫹ channel are shown. ENaCs in FHH are supported by in vivo
studies using mouse models (52, 119), whereas increased Cl⫺ permeability and decreased renal outer medullary K⫹ channel (ROMK) protein level in FHH are
not evident in these in vivo studies. For details, see the text.
Invited Review
F964
WNK4-MEDIATED REGULATION OF ION TRANSPORT PROTEINS
AJP-Renal Physiol • VOL
nephron (6). A luminal negative potential created by ENaCmediated Na⫹ influx is necessary for K⫹ secretion and paracellular Cl⫺ flux. By measuring the nasal potential difference
in FHH patients carrying WNK4Q565E, Farfel and colleagues
(21) provided evidence that WNK4Q565E upregulates amiloride-sensitive Na⫹ transport likely mediated by ENaC in the
airway. By coexpression of ENaC subunits with WNK4 in
X. laevis oocytes, Ring and colleagues (82) found that wildtype WNK4 inhibits ENaC activity. The kinase-dead WNK4 is
capable of inhibiting ENaC to the extent of wild-type WNK4,
but the FHH-causing WNK4Q562E fails to inhibit ENaC. The
amiloride-sensitive Na⫹ transport in the distal colon in the
Tg(Wnk4PHAII) mice is elevated compared with that in wildtype mice, indicating that ENaC is regulated by WNK4 in vivo
(82). The increased ENaC activity could be due to increased
ENaC protein in the apical membrane, although no change in
the ENaC protein level in Tg(Wnk4PHAII) mice has been
reported so far. In the Wnk4D561A/⫹ knockin mice, the protein
levels of ENaC subunits are elevated and the luminal surface
area of the ENaC-positive segment appears to be increased
(119). Consistent with these observations, the amiloride-sensitive Na⫹ permeability is also increased in Wnk4D561A/⫹ mice
as determined by CCD microperfusion (119). However, in
contrast to NCC, the difference in ENaC expression between
wild-type and the Wnk4D561A/⫹ mice is not evident after
chronic thiazide treatment, suggesting the increase in ENaC
could be a secondary effect (119). Nevertheless, the regulation
of ENaC by WNK4 and the loss-of-function behavior of the
FHH-causing mutants of WNK4 are consistent with the hypertension manifestation in FHH, because the increased Na⫹
reabsorption by ENaC in patients carrying the Q565E mutant
will cause volume expansion and hypertension.
Epithelial Ca2⫹ channel TRPV5. TRPV5 (for transient receptor potential cation channel, vanilloid subfamily, member
5) is a Ca2⫹-selective channel that is expressed in the apical
membrane of DCT and CNT (34, 61, 76). TRPV5 plays an
important role in regulating urinary Ca2⫹ excretion. Mice
lacking TRPV5 exhibit a sixfold increase in Ca2⫹ excretion
(35). Because FHH patients carrying WNK4Q565E exhibit hypercalciuria (64), the possible regulation of TRPV5 by WNK4
was investigated (38). In contrast to the inhibitory effects of
WNK4 on other ion transporter proteins in the transcellular
pathway, WNK4 increases TRPV5-mediated Ca2⫹ uptake by
approximately twofold in X. laevis oocytes. The regulation
appears to have a certain level of specificity. On the one hand,
WNK1 has no effect on TRPV5; on the other, WNK4 has no
significant effect on TRPV6 (75, 77), which also participates in
renal reabsorption of Ca2⫹ in addition to its major role in
intestinal Ca2⫹ absorption (3). WNK4 increases the level of
mature TRPV5 at the plasma membrane without significantly
affecting the channel property. WNK4-mediated regulation of
TRPV5 likely involves the kinase activity of WNK4, because
the kinase-dead mutant of WNK4 significantly inhibits TRPV5
at 1 day after injection of cRNA instead of upregulating it.
This is likely due to a dominate negative effect of the
kinase-dead WNK4 on endogenous WNK4 (38). All the
FHH-causing mutants tested, including E562K, D564A,
Q565E, and R1185C, behave very much like wild-type
WNK4 in enhancing TRPV5 (38).
In the distal tubule, Ca2⫹ reabsorption is inversely related to
Na⫹ reabsorption (11). The opposite effect of WNK4 on
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the observation that the removal of NCC in Tg(Wnk4PHAII)
background corrects all the abnormalities observed in the
Tg(Wnk4PHAII) mice (52). It appears that all the features of
FHH could be explained by the impaired regulation of NCC by
WNK4. Nevertheless, altered regulations of other ion transport
proteins by WNK4FHH also explain the clinical features of
FHH.
The most recent results obtained from the Wnk4D561A/⫹
knockin mice confirmed the importance of NCC in the pathogenesis of FHH (119). The Wnk4D561A/⫹ knockin mouse is an
excellent model for FHH. In this model, mice develop hyperkalemia, hypertension, and metabolic acidosis at 3.5 mo of age,
and the abnormalities could be corrected by hydrochlorothiazide (119). In contrast to the lack of difference between
wild-type WNK4 and FHH-causing D561A (corresponding to
human D564A), a mutant of WNK4 as tested in X. laevis
oocytes (116) and MDCK cells (120), the NCC protein level in
the total lysates and the membrane fractions of the kidney is
elevated in the Wnk4D561A/⫹ mice (119). Consistent with the
observation in Tg(Wnk4PHAII) mice, the luminal surface area
of some NCC-positive segments appears to be increased. In
addition, the phosphorylation of NCC at serine 71 (Ser71) is
increased (119). Because the phosphorylation of NCC increases its activity (74), it is likely both the surface level of
NCC and the activity of individual NCC are increased in the
Wnk4D561A/⫹ mice. Although other changes were observed in
the Wnk4D561A/⫹ mice, such as elevated levels of ENaC,
maxi-K, NCC is the only one that remains elevated in
Wnk4D561A/⫹ mice after chronic thiazide treatment, suggesting
that alterations in NCC play a primary role in the pathogenesis
of FHH caused by the WNK4 gene mutation (119).
ROMK. ROMK is a K⫹ channel in the aldosterone-responsive collecting duct that mediates K⫹ secretion. The driving
force for K⫹ secretion is created by the Na⫹ influx via ENaC.
Thus a net Na⫹ uptake and K⫹ secretion in the collecting duct
are mediated by the concerted actions of ROMK and ENaC.
FHH features hypertension with hyperkalemia, suggesting a
possible involvement of disregulation of ROMK-mediated K⫹
secretion. In the X. laevis oocyte system, Kahle et al. (44)
found that ROMK-mediated K⫹ current is drastically inhibited
by WNK4 due to the reduction of the ROMK protein level at
the plasma membrane. In contrast to the WNK4-mediated
regulation of NCC, the action of WNK4 on ROMK is independent of kinase activity. The FHH-causing mutants of
WNK4 tested, including Q562E and E559K (44) and D564H
(29), further inhibited ROMK. This gain-of-function effect on
WNK4FHH mutants, which is very different from the loss-offunction effect on NCC (105), could explain the hyperkalemia
in FHH. However, the inhibitory effects of FHH-causing
WNK4 mutants were not observed in two transgenic mouse
models, carrying WNK4D564A (115) or WNK4Q562E (52), suggesting that the change in surface expression of ROMK is not
a primary regulatory event. In Wnk4D561A/⫹ knockin mice, the
abundance of maxi-K(␣) protein level is significantly elevated
in contrast to the unaltered ROMK level (119). Because the
elevation of maxi-K(␣) is not evident after chronic thiazide
treatment, the increase in maxi-K is likely a compensatory
mechanism for the need to increase K⫹ secretion in the
Wnk4D561A/⫹ mice (119).
ENaC. ENaC mediates electrogenic, amiloride-sensitive
Na⫹ influx in the aldosterone sensitive segment of the distal
Invited Review
WNK4-MEDIATED REGULATION OF ION TRANSPORT PROTEINS
AJP-Renal Physiol • VOL
KCC4, it is unlikely that the metabolic acidosis in FHH is
caused by the WNK4FHH-mediated regulation of KCC4.
CFTR. Mutations of the CFTR gene cause cystic fibrosis
(CF), a common hereditary disease with pathophysiological
abnormalities in the lung and other epithelial tissues (88).
WNK4 was shown to be well colocalized with CFTR in the
colon (39). More recently, Yang et al. (117) reported that
CFTR is inhibited by both WNK1 and WNK4. The effect of
WNK4 on CFTR is similar to that on ROMK (117). The
inhibition of CFTR by WNK4 is likely due to the reduction in
surface expression, and the inhibition is independent of WNK4
kinase activity. The FHH-causing WNK4Q562E exhibits a gainof-function effect and further reduces CFTR activity than does
wild-type WNK4 (117). In contrast to the moderate level of
CFTR in the lung, CFTR is abundantly expressed in the kidney
(12), in tubular cells including those in the CCD (18, 98).
However, CF patients have no significant abnormality in renal
function (106). CFTR functionally regulates ENaC (57, 94)
and ROMK (51, 62, 85), which are also regulated by WNK4.
How much the enhanced inhibition of CFTR by WNK4FHH
affects ENaC and ROMK, and to what extent the overall effect
contributes to the pathogenesis of FHH, are questions to be
addressed in future studies.
Transport proteins outside the kidney. Since WNK4 is rather
broadly expressed in many epithelial cells, its regulatory effects are not restricted to the ion transporters within the kidney.
In fact, WNK4 regulates amiloride-sensitive Na⫹ transport in
the airway (21) and the colon (82). In addition, the chlorideformate exchanger (CFEX) is inhibited by WNK4 (39). Since
WNK4 has not been detected in the proximal tubule, where
CFEX is expressed in the kidney (49), the regulation of CFEX
by WNK4 is likely significant outside the kidney, in many
Cl⫺-secreting epithelia where CFEX is expressed.
Claudins in the paracellular pathway. In addition to the
transporters in the transcellular pathways of ion transport,
WNK4 is capable of regulating the paracellular pathway of
Cl⫺ transport likely via claudins. Increased reabsorption ability
for Cl⫺ in the distal nephron, known as “chloride shunt,”
which results in a decreased driving force for K⫹ and H⫹
secretion later in the tubule, has been postulated by Schambelan et al. (86) as the cause of FHH. It is not clear whether the
increased Cl⫺ permeability is via a transcellular pathway or
paracellular pathway. Since the majority of WNK4 proteins are
in the tight junction of the distal tubule (104), it is possible that
the paracellular pathway is affected. Yamauchi et al. (114)
developed MDCK cell lines stably expressing wild-type
WNK4 or the FHH-causing WNK4D564A in a tetracyclineregulated manner. The paracellular Cl⫺ permeability of the
MDCK monolayer is increased on the induction of WNK4 and
is further increased by the FHH-causing WNK4D564A. On
induction of WNK4, protein levels and cellular distributions of
occludin and claudins 1-4 are not altered. However, the phosphorylation of claudins 1-4 but not occludin is increased. A
further increase in phosphorylation of claudins 1-4 was observed by the overexpression of WNK4D564A. WNK4 binds to
claudins via a YV motif in the COOH terminus of claudins,
and the phosphorylation of claudins occurs at the COOHterminal region as well (114).
Because claudins 1 and 2 are not expressed in the distal
nephron where WNK4 is expressed, the regulation of these two
claudins by WNK4 may not be of physiological importance in
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TRPV5 and NCC further suggests that WNK4 might be involved in the inverse relation of Na⫹ and Ca2⫹ transport.
Interestingly, the effect of WNK4 on TRPV5 is abrogated by
NCC in a dose-dependent fashion (38). In the presence of
NCC, WNK4Q565E fails to significantly increase TRPV5-mediated Ca2⫹ uptake, whereas wild-type WNK4 is capable of
enhancing TRPV5 significantly (38). This suggests that the
effect of WNK4 on TRPV5 depends on the protein level of
NCC in the later portion of the DCT, where the three proteins
are coexpressed. In the case of the Q565E mutation, the level
of NCC is increased due to the loss-of-function effect of
WNK4 on NCC. The increased NCC will further block the
enhancing effect of WNK4Q565E on TRPV5, resulting in increased Na⫹ reabsorption and decreased Ca2⫹ reabsorption.
Thus it is likely that this mechanism may contribute to the
increased urinary Ca2⫹ excretion in FHH patients carrying
WNK4Q565E.
Osmolarity-sensitive cation channel TRPV4. TRPV4 (transient receptor potential cation channel, vanilloid subfamily,
member 4) is a nonselective cation channel that could be
activated by reduction of osmolarity in the extracellular solution (16, 58, 93, 107). It is expressed in the basolateral
membrane of the water-impermeant nephron segments from
the thin ascending limb of Henle’s loop with the exception of
the macula densa (97). By coexpressing WNK4, WNK1, or
WNK4 and WNK1 with TRPV4 stably expressed in human
embryonic kidney (HEK) cells, Fu et al. (23) demonstrated that
both WNK4 and WNK1 decrease TRPV4 activity by reducing
the cell surface level of TRPV4. FHH-causing mutants E559K
and Q562E are less effective in blocking TRPV4. The effect is
likely kinase dependent as the kinase-dead mutant of WNK4 is
not effective in blocking TRPV4. Intriguingly, WNK4 constructs without portions in the NH2-terminal region, which
contains the kinase domain, are effective in blocking TRPV4.
However, without the extreme COOH-terminal region of
WNK4, the blocking effect of WNK4 on TRPV4 is abolished.
These complicated structural requirements for the blocking
effect of WNK4 on TRPV4 suggest that a certain conformation
of the WNK4 protein is necessary for the inhibition of TRPV4
to take place. Since the physiological role of TRPV4 is less
clear in the kidney (8), how much this WNK4-mediated regulation of TRPV4 is involved in the pathogenesis of FHH is yet
to be investigated.
Other members of the SLC12 family of cation-coupled Cl⫺
cotransporters. Besides NCC, WNK4 also inhibits some other
members of the SLC12 family. NKCC1, a Na⫹-K⫹-2Cl⫺
cotransporter in the basolateral membrane of Cl⫺-secreting
epithelia, is inhibited by WNK4 through the reduction of
expression at the plasma membrane (39). Three K⫹-Cl⫺ cotransporters, KCC1, KCC3 and KCC4, are also negatively
regulated by WNK4 (27). Coexpression of WNK4 with the
three KCCs reduces their activity in swollen oocytes, which is
necessary for the activation of the KCCs. The effect of WNK4
on these KCCs requires the kinase activity as the kinase-dead
WNK4 has a reduced effect on KCC1 and has no effect on
KCC3 and KCC4. Three FHH-causing mutants tested, including E559K, D561A, and Q562E, inhibit KCC4 to a similar
extent. Among the three KCCs, KCC4 is known to be expressed in the ␣-intercalated cells of the CD and KCC4 null
mice exhibit metabolic acidosis (4). Nonetheless, due to the
absence of any alterations of the FHH-causing mutants on
F965
Invited Review
F966
WNK4-MEDIATED REGULATION OF ION TRANSPORT PROTEINS
Downstream Effectors of WNK4: WNK4 Activates STE20
Kinases to Regulate Members of the SLC12 Family
WNK4 is able to activate the STE20-like kinases, which are
stress-related serine-threonine kinases, such as SPAK and
OSR1 (17), and in turn regulates members of the SLC12
family. SPAK and OSR1 interact with the cation-chloride
cotransporters, including NKCC1, NKCC2, and KCC3 (78).
Under both isosmotic (basal) and hyperosmotic (stimulated)
conditions, neither SPAK nor WNK4 alone regulates NKCC1;
in contrast, the coexpression of both kinases significantly
increased NKCC1 activity in isotonic conditions, and no further effect was observed when the oocytes were exposed to
hypertonicity (24). The regulation requires kinase activity of
both SPAK and WNK4. WNK4 interacts with SPAK through
an interacting motif, RFQV, in the COOH-terminal region of
WNK4 (24). Thus WNK4 likely activates SPAK, which in turn
enhances NKCC1. Indeed, Vitari and colleagues (100) demonstrated that both WNK1 and WNK4 interact with and phosphorylate SPAK and OSR1 at specific sites and markedly
increase their kinase activity (100). In addition, a robust phosphorylation of the NH2-terminal region of NKCC1 was observed only when WNK1 or WNK4 together with either
SPAK or OSR1 (100). Moriguchi et al. (66) also observed
the direct phosphorylation of NH2-terminal-regulatory regions of NKCC1, NKCC2, and NCC by SPAK and OSR1.
WNK4 could be phosphorylated by WNK1, which is activated
by hypertonic stress, and to a lesser extent by hypotonic stress
in kidney epithelial cells as well as in breast and colon cancer
AJP-Renal Physiol • VOL
cells (55). Thus WNK1 and WNK4 regulate ion flux mediated
by cation-chloride cotransporters via the STE20 kinases to
restore cell volume in response to hypertonic or hypotonic
stress.
The significance of WNK4-mediated regulation of SPAK/
OSR1 in the pathogenesis of FHH is highlighted by the study
using Wnk4D561A/⫹ mice (119). While the protein levels of
OSR1 and SPAK are not altered, their phosphorylated forms
(OSR1 at Ser325 or SPAK at Ser380) are increased in the
kidney of Wnk4D561A/⫹ mice (119). Phosphorylation of key
residues activates OSR1/SPAK (100), which in turn phosphorylates NCC (66) and increases its transport activity (74). Thus
the WNK4-OSR1/SPAK-NCC signaling pathway plays an
important role in blood pressure control and is involved in the
pathogenesis of FHH (119).
Upstream Modulators of WNK4
WNK4 interacts with long and kidney-specific WNK1. In one
of the two initial reports showing NCC is inhibited by WNK4,
Yang et al. (116) found that the effect of WNK4 is abrogated
by WNK1, which has no effect on NCC alone. The effect of
WNK1 on WNK4 involves a physical association between
WNK1 and WNK4 via their kinase domains, and the intact
kinase activity of WNK1 as well as the entire protein of
WNK1 (118).
Full-length WNK1 (noted as L-WNK1) is rather broadly
expressed in multiple tissues (109). A shorter splice variant
without the kinase domain is specifically expressed in the
kidney (designated as KS-WNK1) (15, 72, 113). L-WNK1 is
ubiquitously expressed throughout the kidney, whereas KSWNK1 is most abundant in the DCT (15, 71). KS-WNK1 but
not L-WNK1 is induced by aldosterone in a mouse cortical
collecting duct cell line (68). L-WNK1 is increased whereas
KS-WNK1 is decreased by dietary K⫹ restriction in the rat
kidney (53). KS-WNK1 is significantly upregulated by a
high-K⫹ diet, high-Na⫹ diet, and aldosterone in mice as well
(71). KS-WNK1 was reported to stimulate ENaC (68). Thus
KS-WNK1 appears to be an important regulatory factor responsive to K⫹ and Na⫹ balance and aldosterone.
In a follow-up study on the effect of WNK1 on WNK4mediated regulation of NCC, Subramanya et al. (96) found that
KS-WNK1 alleviates L-WNK1-mediated suppression of
WNK4 dose dependently. KS-WNK1 interacts with L-WNK1
and attenuates its kinase activity as demonstrated with in vitro
kinase assay (96). Similarly, KS-WNK1 also acts as an antagonist of the L-WNK1-mediated regulation on ROMK (53,
102). In response to high K⫹ intake or aldosterone release, the
KS-WNK1 level is increased, and the increased KS-WNK1
blocks NCC-mediated Na⫹-Cl⫺ reabsorption (96). This will
increase the availability of Na⫹ and Cl⫺ in the CCD and
promote luminal negative potential due to increased Na⫹ influx
mediated by ENaC and consequently result in increased K⫹
secretion. Thus this additional layer of regulation of NCC and
ROMK by KS-WNK4 modulates K⫹ secretion in response to
aldosterone and K⫹ intake.
SGK1 phosphorylates WNK4 to deliver the action of aldosterone. Na⫹ reabsorption and K⫹ secretion in the distal tubule
are modulated by aldosterone, which is produced in response to
either a reduction in intravascular volume or a high K⫹ level in
the circulation. Aldosterone regulates Na⫹ and K⫹ transport in
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the kidney. Claudins 3 and 4 are distributed in the distal
nephron (48), and a recent study suggests that claudin 4 is
involved in Cl⫺ permeability (37). Therefore, the regulation of
claudin 4 by WNK4 might be of pathophysiological significance.
Using a similar experimental system, Kahle et al. (40) also
demonstrated that wild-type WNK4 (but not the kinase-dead
D318A mutant) decreases the transepithelial resistance of
MDCK cells by increasing Cl⫺ permeability without altering
the tight junction structure. The FHH-causing mutants Q562E
and E559K further increased Cl⫺ permeability. Thus both
studies indicate a gain-of-function effect of WNK4FHH in
regulating paracellular Cl⫺ permeability. Interestingly, WNK1
overexpression in MDCK cells also causes the same effect as
WNK4 mutants, including increased paracellular Cl⫺ permeability and increased phosphorylation of claudin 4 (73). Coexpression of WNK4 could not further increase the WNK1induced increase in Cl⫺ permeability, suggesting a common
pathway of the WNK4- and WNK1-mediated increase in the
paracellular route of Cl⫺ transport (73).
Although an increase in paracellular Cl⫺ permeability by
FHH-causing mutations of WNK4 is evident in MDCK cell
models (40, 114), the involvement of this mechanism in the
pathogenesis of FHH is not supported by results from the
Wnk4D561A/⫹ mice (119). A significant difference in the Cl⫺/Na⫹
permeability ratio (PCl/PNa) of the CCD was observed between
Wnk4D561A/⫹ and wild-type mice; this difference was eliminated in the presence of the ENaC blocker amiloride (119).
This indicates that the Na⫹ permeability mediated by ENaC,
rather than paracellular Cl⫺ permeability, is increased in the
Wnk4D561A/⫹ mice (119).
Invited Review
WNK4-MEDIATED REGULATION OF ION TRANSPORT PROTEINS
regulated. Second, the regulation differs in the dependence of
the WNK4 kinase activity, as tested with the kinase-dead
mutant. The effects of WNK4 on NCC (28, 105), TRPV5 (38),
and KCCs (27) are kinase dependent, whereas those on ROMK
(44), ENaC (82), and CFTR (117) are kinase independent.
Third, the effects of WNK4FHH on ion transport proteins are
different. The effects of WNK4FHH are indistinguishable to
wild-type WNK4 on TRPV5 (in the absence of NCC) (38) and
KCCs (27); loss-of-function effects were observed on NCC (5,
29, 105, 116), TRPV4 (23), and ENaC (82); and gain-offunction effects were observed for ROMK (33, 44) and CFTR
(117), and perhaps claudins (114). Thus WNK4 is capable of
regulating ion transport proteins through different mechanisms,
which are not yet fully understood. Some details of the WNK4mediated regulations of ROMK (33, 44), ENaC (82), NCC (5,
28), and TRPV5 (38) are illustrated in Fig. 2 and will be
discussed below.
WNK4 enhances the endocytosis of both ROMK (33, 44)
and ENaC (82), however, in different ways. The removal of
ROMK from the plasma membrane is mediated by clathrindependent endocytosis, and this process involves a NPXY
internalization motif in the COOH terminus of ROMK (44,
124). Removal or mutation of this NPXY motif abolished
WNK4-mediated inhibition on ROMK (44). Furthermore, disruption of clathrin-dependent endocytosis by a dominant-negative mutant of dynamin (K44R) prevents the action of WNK4
on ROMK. Thus the WNK4-mediated regulation on ROMK is
to facilitate the removal of ROMK through the clathrin-mediated endocytosis (44). The molecular mechanism of the regulation of ROMK by WNK4 involves the interaction between
WNK4 and ROMK and intersectin (ITSN) (33). ITSN is a
linker protein that is involved in the clathrin-dependent endocytosis (70). Three PXXP motifs adjacent to the FHH-causing
mutations in WNK4 interact with the SH3 domains of ITSN,
and FHH-causing WNK4E559K increases this interaction (33).
Furthermore, the interaction of ROMK with WNK4 is increased by the FHH-causing mutations. Thus the increased
interactions between WNK4FHH and ROMK and ITSN coincide with the strengthened inhibition of ROMK by
WNK4FHH (33).
Cellular Mechanisms of WNK4-Mediated Regulation
Many studies, such as WNK4-mediated regulation on NCC
(28, 29, 105, 116), ROMK (44), TRPV5 (38), ENaC (82),
KCCs (27), and CFTR (117), were carried out using the X.
laevis oocyte system; however, the mechanisms underlying
these regulations differ. The common feature of WNK4-mediated regulation on ion transporters or channels in the transcellular pathway is that WNK4 alters the abundance of a transport
protein at the plasma membrane but does not significantly alter
its transport function. However, the regulation differs in three
aspects (Table 1). First, the direction of WNK4-mediated
regulation differs. TRPV5 is upregulated by WNK4 (38),
whereas the rest of the ion transport proteins tested are downAJP-Renal Physiol • VOL
Fig. 2. Cellular mechanisms of WNK4-mediated regulation. Left: WNK4
enhances endocytosis of ROMK and ENaC and degradation of NCC in the
lysosomal pathway. Right: WNK4 likely blocks the forward trafficking of
NCC and enhances that of TRPV5 (for transient receptor potential cation
channel, vanilloid subfamily, member 5). The direction of regulation is
indicated by plus and minus signs. PM, plasma membrane; TGN, trans-Golgi
network; ER, endoplasmic reticulum.
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part by rapid induction of a serum- and glucocorticoid-inducible kinase (SGK1). SGK1 phosphorylates Nedd4-2, an ubiquitin ligase which interacts with the PY domain in the ␤- and
␥-subunits of ENaC and mediates their retrieval from the
plasma membrane (47). The phosphorylated Nedd4-2 does not
bind to ENaC and therefore makes ENaC stay in the plasma
membrane for a longer time period (14). ROMK is also
positively regulated by SGK1 by increasing its abundance at
the surface (121, 122). Thus SGK1 is an important mediator of
aldosterone action. In a recent study, Ring and colleagues (83)
showed that WNK4 can be phosphorylated by SGK1, and a
SGK1 phosphorylation site (Serine 1169) was identified close
to the COOH-terminal region of WNK4. The S1169D WNK4
mutant mimics the phosphorylated state of WNK4 and alleviates the inhibition of WNK4 on both ENaC and ROMK (83).
Increased ENaC activity will promote the luminal negative
potential for ROMK-mediated K⫹ secretion. Thus the phosphorylation of WNK4 by SGK1 promotes Na⫹ reabsorption
and K⫹ secretion to restore intravascular volume and to reduce
the serum K⫹ level, respectively. Therefore, WNK4 is likely a
mediator of aldosterone action in response to volume depletion
and hyperkalemia (see Fig. 3).
Aldosterone promotes ENaC-mediated Na⫹ reabsorption
and ROMK-mediated K⫹ secretion through at least four layers
of regulation. 1) Aldosterone induces SGK1 expression, which
increases the activity of ENaC and ROMK, respectively,
through phosphorylation of Nedd4-2 (14) and ROMK channel
protein itself (121). 2) Aldosterone decreases NCC-mediated
Na⫹-Cl⫺ reabsorption through upregulating KS-WNK1, which
promotes WNK4-mediated inhibition on NCC by abrogating
the blocking effect of L-WNK1 on WNK4 (96). This allows
Na⫹ to reach a more distal portion of the nephron to be
reabsorbed by ENaC. The resulted luminal negative potential
due to Na⫹ influx via ENaC will promote ROMK-mediated
K⫹ secretion. 3) In addition, elevated KS-WNK1 in response
to aldosterone also increases ROMK-mediated K⫹ secretion by
abrogating L-WNK1-mediated ROMK inhibition (53, 102). 4)
The phosphorylation of the SGK1 site in WNK4 by SGK1
abrogates the WNK4-mediated inhibition of both ENaC and
ROMK, resulting in a net increase in Na⫹ reabsorption and K⫹
secretion (83). Because L-WNK1 activates SGK1 (111),
WNK4 is a target of SGK1 (83); in addition, WNK1, WNK4,
and SGK1 interact with each other (83, 111, 118), and it is
likely that the three kinases form a protein complex at some
points of the process to deliver the effect of aldosterone on Na⫹
reabsorption and K⫹ secretion.
F967
Invited Review
F968
WNK4-MEDIATED REGULATION OF ION TRANSPORT PROTEINS
AJP-Renal Physiol • VOL
tory pathway via which mature TRPV5 reaches the plasma
membrane.
WNK4-mediated regulation involves protein-protein interaction. WNK4 associates with its downstream targets, including NCC (5, 105, 118), ROMK (33, 44), ITSN (33), claudins
(114), and SPAK/OSR1 (100) and upstream modulators, including WNK1 (118) and SGK1 (83). WNK4 interacts with
NCC and ROMK via the cytoplasmic COOH termini of the
channels (33, 44, 105, 118). While the role of the interaction
between WNK4 and NCC in the regulation of NCC by WNK4
is unclear, the interaction between WNK4 and ROMK alone
appears to be insufficient to mediate the inhibition of ROMK
by WNK4. A segment of WNK4 (amino acid residues 1– 444)
interacts with ROMK, but this segment alone is incapable of
inhibiting ROMK (33). Rather, the interaction between WNK4
and ITSN through a stretch of WNK4 (amino acids 473–584)
containing a proline-rich motif is necessary in mediating the
inhibition of ROMK via a clathrin-dependent endocytosis
pathway (33). The interaction of WNK4 with the conserved
COOH-terminal YV motif of claudins is likely involved in the
WNK4-mediated phosphorylation of the COOH terminal of
claudins (114). Similarly, the interaction between WNK4 and
SPAK via the interacting motif RFQV in the COOH-terminal
region of WNK4 (24) could be involved in the phosphorylation
of SPAK by WNK4 (100). The association of WNK4 with
WNK1 via their kinase domain is necessary for the modulation
of WNK4 by WNK1 (118). The association of WNK4 with
SGK1 could be involved in the phosphorylation on Ser1169 of
mouse WNK4, which abrogates the inhibitory effects of
WNK4 on ENaC and ROMK (83). Because WNK1, WNK4,
and SGK1 associate with each other (83, 111, 112, 118), it is
possible that they may coexist in a protein complex that
interacts with their targets such as SPAK and OSR1 and
relevant ion transport proteins they regulate. It should be noted
that not all ion transport proteins regulated by WNK4 directly
interact with it. TRPV4 does not coimmunoprecipitate with
WNK4 (23). There is no evidence that a direct interaction is
involved in the regulation of TRPV5 by WNK4.
An interesting question is how WNK4 manages to regulate
a variety of ion transport proteins using different mechanisms,
such as those observed for NCC, ENaC, ROMK, and TRPV5?
It is likely that WNK4 affects multiple pathways via its kinase
activity and/or specific domains, and the specificity of the
regulation lies in the ion transporter side. For instance, plasma
membrane retrieval and degradation of ENaC depend on the
interaction with the E3 ubiquitin ligase Nedd4-2 through the
PY motif in ENaC ␤- and ␥-subunits (82). On the other hand,
the endocytosis of ROMK is through a clathrin-dependent
pathway, which is dependent on a NPXY internalization motif
in the COOH terminal of ROMK (124). Although WNK4 may
affect both pathways of endocytosis, the effect is observed only
when the channel protein is affected by one of the pathways. If
the protein is no longer going through the pathway, e.g., ENaC
without the PY motif or ROMK without NPXY motif, the
regulatory effect of WNK4 will be abolished. Similarly, the
differential effects of WNK4 on TRPV5 and TRPV6 also
depend on the mechanisms by which they are delivered to the
plasma membrane as discussed earlier.
It is still unclear why WNK4 affects NCC and TRPV5 in
different directions in the same experimental system and
WNK4 appears to prevent the forward trafficking of NCC (28)
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The mechanism of WNK4-mediated regulation of ENaC
involves its COOH-terminal PY motif, which is responsible for
the interaction of ENaC with E3 ubiquitin ligase Nedd4-2 (82).
Each ENaC unit possesses a PY motif that interacts with WW
domains 3 and 4 in Nedd4-2 (46). Ubiquitinylation by Nedd4-2
facilitates the removal of ENaC from the plasma membrane for
degradation. A truncating mutation that removes the PY motif
in the ␤- or ␥-unit of ENaC causes Liddle’s syndrome, a
genetic form of hypertension due to the increased ENaC
activity (32, 90). WNK4 no longer inhibits ENaC mutants of
Liddle’s syndrome, indicating that WNK4 facilitates the removal of ENaC through the Nedd4-2-mediated retrieval and
degradation of ENaC (82).
With regard to the cellular mechanism by which WNK4
regulates NCC, a novel mechanism involving enhanced degradation of NCC via a lysosomal pathway by WNK4 was
reported by Cai and colleagues (5) using mammalian cell
models. They found that WNK4-mediated inhibition on NCC
surface expression is independent of the clathrin-mediated
endocytosis, as indicated by the lack of effect of dominantnegative dynamin mutant K44A. In contrast, bafilomycin, a
vacuolar proton pump inhibitor which disrupts the acidification
of lysosomes, dose dependently abrogates the inhibitory effect
of WNK4 on the steady-state level of NCC (5). This indicates
that the inhibitory effect of WNK4 on NCC surface expression
results from enhancing degradation of NCC via a lysosomal
pathway, suggesting that WNK4 interferes with the forward
trafficking of NCC to the plasma membrane. Golbang and
colleagues (28) subsequently reported the similar result confirming the findings of Cai and colleagues (5) and further
support the notion that WNK4 appears to block the forward
trafficking of NCC. When the forward trafficking from the
Golgi apparatus is blocked by brefeldin-A, the rate of NCC
endocytosis is no longer affected by WNK4 (28). In the
WnkD561A/⫹ knockin mice, the NCC protein level in the kidney
lysate and that in the membrane fractions are increased, indicating either the synthesis of NCC is increased or the degradation of NCC is decreased by the mutant WNK4 (119), which
is consistent with finding of Cai and colleagues (5). In addition,
the phosphorylation of Ser71, one of the potential phosphorylation sites for SPAK/OSR1 in NCC (66), is increased in
WnkD561A/⫹ mice (119). Because of the concomitant increase
in phosphorylated SPAK/OSR1 and NCC, it is likely
WNK4D561A increases the phosphorylation of SPAK/OSR1,
which in turn phosphorylates NCC. The phosphorylation of
NCC at Ser71 and other sites increase its transport activity
(74). Therefore, both the protein abundance and transport
activity of NCC are likely increased in FHH.
In contrast to the action on NCC, WNK4 appears to facilitate
the forward trafficking of TRPV5 because the complex glycosylated mature form of TRPV5 is increased by WNK4 (38).
Abolishing the N-linked glycosylation of TRPV5 by sitedirected mutation abrogates the effect of WNK4 on TRPV5
(unpublished observations). The complex glycosylation in the
Golgi apparatus is a step in the secretory pathways of protein
delivery to the plasma membrane. TRPV6 is not well complex
glycosylated in X. laevis oocytes and likely goes to the plasma
membrane via an alternative route because the majority of
TRPV6 in the plasma membrane is in core-glycosylated form
(unpublished observations). Since WNK4 selectively acts on
TRPV5, WNK4 likely enhances a certain step(s) of the secre-
Invited Review
WNK4-MEDIATED REGULATION OF ION TRANSPORT PROTEINS
but to promote that of TRPV5 (38). What causes this difference? In addition to the difference in channel proteins between
NCC and TRPV5, the ions that NCC and TRPV5 transport are
different. Expression of TRPV5 leads to an increase in intracellular Ca2⫹, an important intracellular messenger which is
involved in the interaction of WNK1 and synaptotagmin 2
(54). Thus Ca2⫹ ions may have a role in switching the action
mode of WNK4, as WNK4 is proposed as a molecular switch
in regulating diverse pathways of ion transport (45). However,
the inhibitory effect of WNK4 on TRPV4 (23), which is a
Ca2⫹-permeable cation channel, argues against the role of
Ca2⫹ in determining the regulation direction of WNK4.
Summary
AJP-Renal Physiol • VOL
Investigations of WNK4-mediated regulation are progressing very rapidly. The list of ion transport proteins regulated by
WNK4 will likely continue to grow in the next few years. A
clearer understanding of the WNK4-mediated regulation will
likely be achieved at the cellular and molecular levels. Identifying cellular processes regulated by WNK4, and proteins that
are either targets or modulators of WNK4 action, will certainly
help in decoding the regulatory processes. In addition, a target
of WNK4 may act as a modulator of the regulations of WNK4
on other ion transport proteins, e.g., NCC in WNK4-mediated
regulation of TRPV5 (38). The cross talk between WNK4 and
its targets adds more layers in WNK4-mediated regulation.
Compared with all the progress made in the actions of
WNK4 on its downstream targets, very little is known on how
WNK4 responds to physiological cues. WNK4 responds to
aldosterone indirectly through the action of SGK1, which
phosphorylates WNK4 and in turn abrogates the regulatory
effect of WNK4 on ENaC and ROMK (Fig. 3) (83). Lalioti
et al. (52) suggest that the renin-angiotensin system likely
provide upstream signals to shape the action of WNK4. Investigations in these areas are likely to provide further insight into
how WNK4 reacts in different pathophysiological conditions.
The role of WNK4 in the molecular pathogenesis of FHH
suggests that the genetic variations in the WNK4 gene are
possibly involved in the common forms of hypertension. Indeed, associations of single-nucleotide polymorphisms in the
WNK4 gene with the risk of essential hypertension in general
populations have been reported (19, 50), although further
studies are needed to verify these preliminary results and to
establish the role of the WNK4 gene in essential hypertension.
WNK4 could become a novel drug target for diseases related to
imbalanced ion homeostasis, such as hypertension. If specific
Fig. 3. WNK4 in the aldosterone signaling pathway. Aldosterone is produced
in response to either hypovolemia or hyperkalemia. Aldosterone induces the
expression of SGK1, which in turn phosphorylates WNK4. The phosphorylation of WNK4 alleviates the inhibition of ENaC and ROMK by WNK4. SGK1
also enhances ENaC and ROMK directly or indirectly. The increased Na⫹
influx mediated by ENaC creates a favorable luminal potential for ROMKmediated K⫹ secretion. The increased ENaC-mediated Na⫹ absorption and
ROMK-mediated K⫹ secretion will lead to volume expansion and decreased
K⫹ level in the circulation, respectively.
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WNK4 is a protein kinase with the exceptional ability to
regulate an array of ion transport proteins in the kidney. The
majority of the information was obtained in the X. laevis
system; thus the question arises as to what extent these in vitro
results reflect the in vivo situation. First, the results obtained
are consistent with the clinical features of FHH. In most cases,
the in vitro results well explain the clinical manifestations of
FHH (Fig. 1). The hypertension in FHH could be caused by the
impaired inhibitory effects of WNK4FHH on NCC and ENaC,
leading to increased Na⫹ reabsorption and in turn intravascular
volume expansion. Hyperkalemia in FHH could be explained
by the decreased K⫹ secretion due to three effects of
WNK4FHH action: 1) the strengthened inhibition of ROMK by
WNK4FHH reduces the secretion of K⫹; 2) the increased Cl⫺
flux likely mediated by claudin 4 in the CCD by WNK4FHH
diminishes the luminal negative potential for K⫹ secretion; and
3) the increased Na⫹ reabsorption by NCC limits Na⫹ delivery
to the CCD for ENaC-mediated Na⫹ reabsorption and in turn
reduces the secretion of K⫹ and H⫹ capabilities in the CCD.
The NCC and ENaC activity and the surface area expressing
NCC and ENaC are increased in the mouse models of FHH
(52, 82, 119), supporting the third mechanism. Metabolic
acidosis in FHH is likely secondary to the decreased K⫹
secretion. Hypercalciuria of FHH could be caused by the
diminished enhancing effect of WNK4 on TRPV5 through
the increased level of NCC. Alternatively, due to the inverse
relationship of Ca2⫹ and Na⫹ reabsorption in the distal tubule
(11), an increased Na⫹ reabsorption by NCC and ENaC may
reduce the reabsorption of Ca2⫹, likely through enhancing
passive Ca2⫹ transport in the proximal tubule (69). In addition,
a direct effect of WNK4 on the Na⫹/Ca2⫹ exchanger and/or the
Ca2⫹ pump in the basolateral membrane could not be ruled out.
Second, the regulation of NCC by WNK4 observed in transgenic mice is consistent with the in vitro results obtained using
oocytes (52). Although the effect of increased DCT mass in
Tg(Wnk4PHAII) mice could not be demonstrated in the oocyte
system, it is a logical result of increased NCC due to the
alleviated inhibition by WNK4FHH, given the fact that the
removal of NCC results in diminished DCT structure (87).
Third, the increased ENaC-mediated Na⫹ transport as demonstrated in oocyte and transgenic mice is also evident in human
subjects carrying the WNK4Q565E mutation (21). Thus it is
reasonable to assume that the effects observed in the in vitro
system largely reflect the reality in vivo, which is expected to
be illustrated in future studies.
F969
Invited Review
F970
WNK4-MEDIATED REGULATION OF ION TRANSPORT PROTEINS
delivery of drugs to the distal nephron becomes achievable,
drugs may be developed to temporally lock WNK4 in a certain
mode, acting on a specific ion transport protein. Understanding
the mechanisms of the WNK4-mediated regulation of ion
transport processes is an essential step in this direction.
GRANTS
This research was generously supported by grants from the American Heart
Association (0430125N) and the National Institute of Diabetes and Digestive
and Kidney Diseases (DK-072154).
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