Am J Physiol Renal Physiol. 2009 Oct; 297(4): F838–F848.

PMCID: PMC3350128
Published online 2009 May 27. doi: 10.1152/ajprenal.00159.2009: PMID: 19474192
10.1152/ajprenal.00159.2009

The thiazide-sensitive Na+-Cl− cotransporter: molecular biology, functional properties,

and regulation by WNKs
Gerardo Gamba

Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México,
Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, and Instituto Nacional de Cardiología Ignacio
Chávez, Mexico City, Mexico
Corresponding author.
Address for reprint requests and other correspondence: G. Gamba, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico
City, Mexico (e-mail: gamba@biomedicas.unam.mx or gamba@quetzal.innsz.mx).

Received 2009 Mar 16; Accepted 2009 May 21.

Copyright © 2009 American Physiological Society

Abstract

The thiazide-sensitive Na+-Cl− cotransporter is the major salt reabsorption pathway in the distal convo‐
luted tubule, which is located just after the macula densa at the beginning of the aldosterone-sensitive
nephron. This cotransporter was identified at the molecular level in the early 1990s by the pioneering
work of Steven C. Hebert and coworkers, opening the molecular area, not only for the Na+-Cl− cotrans‐
porter but also for the family of electroneutral cation-coupled chloride cotransporters that includes the
loop diuretic-sensitive Na+-K+-2Cl− cotransporter of the thick ascending limb of Henle's loop. This
work honoring the memory of Steve Hebert presents a brief review of our current knowledge about salt
and water homeostasis generated as a consequence of cloning the cotransporter, with particular empha‐
sis on the molecular biology, physiological properties, human disease due to decreased or increased ac‐
tivity of the cotransporter, and regulation of the cotransporter by a family of serine/threonine kinases
known as WNK. Thus one of the legacies of Steve Hebert is a better understanding of salt and water
homeostasis.

Keywords: distal tubule, ion transport, hypertension, diuretics

thiazide-type diuretics were developed in the middle of the twentieth century and quickly became
popular, since they were the first active agents with proven beneficial effects for lowering blood pres‐
sure in patients with arterial hypertension (47, 75). Fifty years later, thiazides are still recommended as
the first line of pharmacological therapy for the treatment of hypertension (13). Pioneering studies by
Kunau et al. (57) were the first to suggest that thiazide-type diuretics inhibited chloride reabsorption in
the distal nephron. Later, studies by Renfro (85, 86) demonstrated that the urinary bladder of the teleost
winter flounder (Pseudopleuronectes americanus) exhibited a salt transport mechanism in the apical
membrane that featured an interdependence between Na+ and Cl− transport, suggesting the existence of
an electroneutral Na+-Cl− cotransporter.1 A few years later, Stokes et al. (103) observed that this trans‐
port mechanism in flounder urinary bladder was specifically inhibited by thiazide diuretics in a dose-
dependent manner. This was followed by the work of Costanzo (15) and Ellison et al. (31), who estab‐
lished that in mammalian kidney the apical membrane of the distal convoluted tubule (DCT) possesses
a similar thiazide inhibitable Na+-Cl− transport pathway, and thus DCT is the site of action for thiazide
type diuretics. In the next few years, the Na+-Cl− cotransporter was studied as the “thiazide receptor”
by assessing the binding of the tracer [3H]metolazone to plasma membranes from the renal cortex (6,
32, 107). However, the molecular area for the renal Na+-Cl− cotransporter, and the rest of the SLC12
family of electroneutral cation chloride cotransporters, began with the pioneering work of Steven
Hebert and coworkers, who isolated a cDNA clone encoding the thiazide-sensitive Na+-Cl− cotrans‐
porter (NCC) from the winter flounder urinary bladder, following an expression cloning strategy in
Xenopus laevis oocytes (35).2

After several months of unsuccessful experiments that were designed to clone the rat outer medullary
bumetanide-sensitive Na+-K+-2Cl− cotransporter, based on the observations of Stokes et al. (103) and
probably inspired by the remarkable book of Homer Smith From Fish to Philosopher (102), Steven
Hebert suggested that we should pursue the expression cloning of the thiazide-sensitive Na+-Cl− co‐
transporter from the winter flounder urinary bladder, with the idea that cloning this fish cDNA would
probably be the best place to start to identify, not only the mammalian ortholog, but also the
bumetanide-sensitive Na+-K+-2Cl− cotransporters because, he said, “they probably are related.” He
was right. Cloning of the flounder cDNA cotransporter was quickly followed by the molecular identifi‐
cation of rat cDNAs encoding the thiazide-sensitive Na+-Cl− cotransporter, two isoforms of the
bumetanide-sensitive Na+-K+-2Cl− cotransporters (24, 34), and four isoforms of the K+-Cl− cotrans‐
porters (40, 73, 81). This is one of the many visionary projects that Steve Hebert often generated.

As shown in Fig. 1, the DCT mediates reabsorption of 5–10% of the glomerular filtrate and is the first
segment of the aldosterone-sensitive distal nephron. It is divided into early and late segments, also
known as DCT1 and DCT2, respectively. The major salt reabsorption pathway in the DCT is the thi‐
azide-sensitive NCC (15, 31, 57, 82, 109). In DCT1, salt transport is driven exclusively by NCC,
whereas in DCT2, the epithelial sodium channel (ENaC) also participates (10, 31, 62, 63, 78, 84). The
sodium gradient that drives transport from the lumen to the interstitium is generated and maintained by
the Na+-K+-ATPase that is polarized to the basolateral membrane (28). Part of the potassium entering
the cell at the basolateral membrane is secreted at the luminal membrane via K+ channels (120) and via
an apical K+-Cl− cotransporter (3). Thus the rate of Na+-Cl− reabsorption determines, in part, the rate
of K+ secretion. In addition, NCC modulates magnesium reabsorption in parallel with sodium reab‐
sorption, and it regulates calcium reabsorption inversely with sodium reabsorption. The higher the
sodium reabsorption, the lower the calcium reabsorption and vice versa (15). Because of its anatomical
localization just after the macular densa cells, the DCT is the first part of the nephron in which the salt
reabsorption rate is not subjected to compensation by the tubuloglomerular feedback mechanism, and
thus it affects the final concentration of salt in urine. Therefore, salt reabsorption in the DCT is ex‐
pected to affect extracellular fluid balance and arterial blood pressure.
Molecular Biology of NCC

The NCC belongs to solute carrier family 12 (SLC12; Human Genome Organization), known as the
electrically silent, cation-coupled chloride cotransporter family (36, 46). The SLC12 family of mem‐
brane transproters translocate chloride together with Na+ and/or K+ observing an stoichiometry of 1:1
(chloride:cation), and their activity is modulated by cell volume. SLC12A, the gene encoding the thi‐
azide-sensitive Na+-Cl− cotransporter is located on chromosome 16q13 in humans (65, 101), 19p12–14
in rat (105), and 8 in mouse (80). In humans, SLC12A3 is a 55-kb gene containing 26 exons (101). The
NCC has been identified at the molecular level in human (65, 101) rat (34), mouse (58), rabbit (110),
and eel kidney (17), as well as in flounder urinary bladder (35). The NCC is a membrane protein of
1,002 to 1,028 amino acid residues with a proposed topology featuring a central hydrophobic domain
made up of 12 putative transmembrane (TM) spanning regions. A hydrophilic loop connects TMs 7
and 8 and contains two glycosylation sites. The hydrophobic domain is flanked by a short amino-termi‐
nal domain and a long carboxy-terminal domain that are located within the cell. Soon after the molecu‐
lar identification of NCC, anti-NCC polyclonal antibodies were generated and used to demonstrate that
this cotransporter is specifically expressed in the apical membrane of the DCT of the kidney (4, 82).
The degree of identity among mammalian NCCs is ∼90% and that of any mammalian NCC with the
flounder NCC is ∼60%. Two putative NCC genes from eel have been reported, and interestingly, the
degree of identity shared with the mammalian or flounder NCCs is ∼50% for both (17). Two alterna‐
tively spliced isoforms differing in the length of the 3′-untranslated regions were discovered in rat (34),
but no alternative splicing isoform affecting the primary structure of NCC has been found in mammals.
Rabbit and human NCCs are longer than rat and mouse orthologs due to the presence of 17–26 amino
acid residues in the carboxy-terminal domain. These extra residues in humans are encoded by a sepa‐
rate exon (exon 20) that is not present in mouse or rat. Interestingly, this exon contains a putative PKA
site that is not present in mouse and rat NCCs.

Tissue distribution analysis by Northern blot in the rat revealed renal-specific expression of NCC (34).
However, recent studies have clearly demonstrated NCC protein expression in intestine (5) and bone
(30) tissues. Although NCC has been postulated to exist in brain (25), blood vessels (14), pancreas (7),
peripheral blood mononuclear cells (2), gallbladder (16), and heart (29), confirmation of its presence in
these tissues at the molecular level has been unsuccessful. The role of NCC in the intestine is not clear,
but it is probably related to salt and calcium absorption. It has been shown in many clinical studies that
the use of thiazide diuretics in elderly subjects promotes an increase in bone mineral density and helps
to prevent pathological fractures (49, 98). Consistent with this beneficial effect of thiazides, NCC is ex‐
pressed in rat and human bones. Addition of thiazides to osteoblasts in culture increases the formation
of mineralized nodules, an effect that was no longer present after decreasing NCC expression by trans‐
fecting cells with a NCC antisense plasmid (30).

The NCC is able to form dimers, and it is likely that it functions as a dimer (19). The NCC is glycosy‐
lated at two sites (N404 and N424) located in the long extracellular loop (48). Elimination of each site
reduced the rat NCC activity by 50%, and elimination of both sites reduced it by 95%. Decreased ex‐
pression in the plasma membrane accounts for most of the reduction in NCC activity. Thus the glycosy‐
lated loop between TM 7 and 8 must be oriented toward the extracellular space, and glycosylation of
NCC plays a key role in trafficking the protein to the cell surface. Interestingly, absence of glycosyla‐
tion in the rat NCC (48), but not in the flounder NCC (70), is associated with increased affinity for thi‐
azides, suggesting that in rat sugar moieties on NCC probably prevent thiazide from reaching its bind‐
ing site on the cotransporter.
Functional Properties of NCC

Robust and reproducible expression of the flounder, rat, mouse, and human NCCs has been achieved by
several groups using the heterologous expression system of X. laevis oocytes (18, 34, 35, 42, 58, 69,
93). Only two groups (20, 88) have reported some degree of functional NCC expression by transfecting
human NCC cDNA into MDCK or HEK-293 cells, respectively. These systems turned out to be better
tools for studying NCC at the biochemical level rather than at the functional level because robust ex‐
pression of NCC was observed by Western blot analysis; although protein expression was significant, at
the functional level, NCC expression was low and not high enough to analyze NCC functional proper‐
ties or regulation. Thus all our current knowledge on NCC function comes from studies in X. laevis
oocytes. As shown in Table 1, a number of interesting differences in functional properties between the
fish and mammalian NCCs have been observed. The apparent Km values for Na+ and Cl− in rat (69) or
mouse NCC proteins (93) are significantly lower than the Km values observed in the flounder NCC
(108). NCC activity is specifically inhibited by thiazide type diuretics, with the following potency pro‐
file: polythiazide > metolazone > bendroflumethiazide > trichloromethiazide > chlorthalidone. In all
cases, the flounder NCC exhibited lower affinity for each thiazide. While a 100-μM concentration of
the less potent thiazides trichloromethiazide and chlorthalidone inhibited the rat NCC by >95% (69),
the flounder NCC activity was reduced by only 68 and 46%, respectively (108).

Differences in functional properties between the rat and flounder NCCs have been exploited to design
and analyze chimeric and site-directed mutagenesis approaches to gain insight into NCC structure-
function relationships (70). Observations in this study revealed that the functional characteristics of
NCC are defined by the central hydrophobic domain, and it was possible to define different segments
within the central domain defining Cl− or thiazide affinity (Fig. 2). Chimeras in which the TM segment
1–7 was interchanged between flounder and rat demonstrated that affinity-defining residues for Cl− are
located within this segment. In contrast, the affinity-defining residues for thiazide inhibition are located
within TM segments 8–12. This information argued against a previous proposal that thiazide diuretics
and chloride compete for the same binding site (107). The observation that Cl− affinity is defined by
residues located within TM segments 1–7 is supported by another study from Moreno et al. (71) in
which the functional consequences of a single nucleotide polymorphism (SNP) that change a single
residue within NCC were analyzed. It was observed that a highly conserved glycine within TM 4 plays
a critical role in defining the level of cotransporter activity and the affinity for Cl−. Accordingly, a
glycine to alanine SNP at position 264 resulted in a 50% decrease in cotransporter activity associated
with an increased affinity for Cl− of one order of magnitude. This observation has been translated to
pharmacogenomics because it was later observed that people harboring the G264A SNP exhibit a
stronger diuretic response to the loop diuretic furosemide (112). Because of the decreased activity of
NCC harboring the G264A polymorphism, it is possible that the DCT cannot increase salt reabsorption
in response to increased salt delivery that follows furosemide administration, thus increasing the natri‐
uretic response to the loop diuretic. Finally, little is known about residues or domains defining speci‐
ficity for ion transport or thiazide inhibition. Chimeric constructs between NCC and the apical
bumetanide-sensitive Na+-K+-2Cl− cotransporter NKCC2 suggest that the key residues must be within
the central hydrophobic domain because the amino and carboxy terminals play no role in this issue
(106).
NCC and Human Disease

Inactivating mutations of the SLC12A3 gene encoding NCC are the cause of Gitelman's disease, an in‐
herited autosomal recessive disease featuring hypokalemic metabolic alkalosis, arterial hypotension,
hypocalciuria, and hypomagenesemia that are not usually recognized until the second decade of life.
More than 70 families have been studied and more than 100 different mutations of NCC have been re‐
ported (http://archive.uwcm.ac.uk/uwcm/mg; Ref. 36). Functional and biochemical analysis of human
or rat NCCs containing some of the reported point mutations revealed two major groups: one in which
NCC mutants cannot be glycosylated and are not functional and another in which NCC proteins can be
glycosylated and retain a certain level of activity but which have a lower level of activity than the wild-
type NCC. (18, 58, 93).

Gordon's disease, pseudohypoaldosteronism type II (PHAII), or familial hypertension with hyper‐

kalemia are the terms that have been assigned over the years for a rare inherited illness featuring arte‐
rial hypertension with hyperkalemia and metabolic acidosis, despite a normal glomerular filtration rate
(43, 66). Three loci for PHAII have been observed in humans in the following regions: 1q31-42 (64),
12p13 (26), and 17p11-q21 (64). Linkage for these loci has been excluded in at least two unrelated kin‐
dreds with autosomal dominant PHAII, indicating there is a fourth locus involved (27). The gene re‐
sponsible for this condition in families with Gordon's disease linked to chromosome 1 remains a mys‐
tery, but in chromosomes 12 and 17, PHAII cosegregates with mutations in two serine/threonine ki‐
nases known as WNK1 and WNK4, respectively (116). Intronic deletions in the first intron of WNK1
lead to an increased expression of a normal WNK1 protein, whereas missense mutations in a conserved
acidic region of WNK4 are responsible for the disease. Most of the pathophysiology of PHAII seems to
be explained by the effects of WNKs upon distal nephron ion transport proteins, particularly the NCC.
Thus the rest of the present work is devoted to an analysis of the regulation of NCC (and other distal
nephron transport proteins) by WNKs and the implication of this new knowledge in our understanding
of distal nephron physiology.

Effects of WNK4 on NCC

The WNK family of serine/threonine kinases is composed of four genes known as WNK1 to WNK4,
which are located on human chromosomes 12, 9, X, and 17, respectively (53). These kinases exhibit a
conserved serine/threonine kinase domain that lacks the typical catalytic lysine in subdomain II [hence,
the name WNK; with no lysine (K); Ref. 118] flanked by an unconserved short amino-terminal domain
and a long carboxy-terminal domain. The carboxy-terminal domain varies in size and contains an au‐
toinhibitory domain, two coiled-coil domains, and a highly conserved acidic region of 10 residues in
which most of the WNK4-PHAII-type missense mutations occur. WNK1, WNK3, and WNK4 are ex‐
pressed in several tissues, including chloride absorbing or secreting epithelia (22, 50, 52, 89, 116, 118).
In the nephron, WNK1 transcripts and WNK3 proteins have been shown to be expressed in all nephron
segments (76, 89), whereas WNK4 is present mainly in the aldosterone-sensitive nephron (76, 116).

The discovery that PHAII is due to mutations in WNK1 and WNK4 (116) suggested that WNKs are
potential regulators of ion transport in the kidney, particularly through the regulation of NCC. First,
PHAII patients feature a clinical condition that mirrors Gitelman's syndrome in which NCC is not func‐
tional (101). Second, clinical studies (44, 66) have shown that PHAII patients are particularly sensitive
to very low doses of thiazide diuretics, as 20% of the typical “thiazide” dose is sufficient to improve hy‐
pertension and the other clinical features. Third, the expression of both WNK1 and WNK4 proteins
were specifically localized to the DCT (116). Thus using X. laevis oocytes, we learned that wild-type
WNK4 reduces the activity of NCC, whereas WNK4 mutants lacking catalytic activity (obtained by in‐
troducing a D318A mutation in kinase domain of WNK4) or harboring the PHAII-type mutation
E562K no longer inhibit NCC (117). The reduction in NCC activity by WNK4 was at least in part due
to a decrease in the number of NCCs present at the cell surface. These observations were corroborated
by other groups also using X. laevis oocytes (42, 122) or epithelial cells (9) in which NCC expression
at the cell surface was assessed by confocal image techniques. Thus the proposed pathophysiological
mechanism of PHAII is that WNK4 is a natural inhibitor of NCC and that this effect is lost through
PHAII-type mutations in WNK4, increasing the activity of NCC, resulting in hypertension, hyper‐
kalemia, and metabolic acidosis.

The hypothesis described above has been confirmed in vivo by two groups using different strategies in
transgenic animals. Yang et al. (126) produced a knockin WNK4D561A/+ that imitates the genetics of
PHAII because this mouse model has one normal and one mutated WNK4 allele. The mice developed
the PHAII phenotype that was corrected by thiazide administration and exhibited increased expression
of phosphorylated NCC at Ser-71, which together with threonines 53 and 58 were previously shown to
be required for full activation of NCC by intracellular chloride depletion (79), and phosphorylation of
the STE-20 serine/threonine kinases SPAK/OSR1 that have been proposed to lie downstream of WNK1
and WNK4 (38, 72, 111). Additionally, it was demonstrated that activation of NCC by intracellular
chloride depletion requires NCC-SPAK/OSR1 interaction and that in this condition SPAK/OSR1 in‐
duces phosphorylation of human NCC at threonine residues 45, 55, and 60 and serine 91 (87, 88), cor‐
responding to threonines 43, 53, and 58, and serine 89 in rat NCC (79).

Lalioti et al. (59) produced BAC transgenic mice with four alleles of wild-type WNK4 (WNK4+/+/+/+)
and PHAII-type mutant mice with two alleles of wild-type WNK4 and two of mutant WNK4
(WNK4PHAII). Thus in addition of the two normal alleles of WNK4, these mice exhibit two extra alle‐
les of wild-type or mutant WNK4. WNK4+/+/+/+ mice developed a Gitelman's-like condition, with hy‐
poplasia and hypotrophy of the DCT, indicating that, as suggested by in vitro studies (117, 122), wild-
type WNK4 indeed inhibits NCC. In contrast, WNK4PHAII mice developed a clear PHAII-like pheno‐
type, with arterial hypertension and hypertrophy and hyperplasia of the DCT. When exposed to a high
potassium diet, WNK4PHAII mice actually died due to hyperkalemia. A very intriguing observation was
that crossing WNK4PHAII mice with NCC null mice (99) fully prevented the phenotype, including the
hyperkalemia during high potassium diet, unmasking the importance of NCC activity, not only in blood
pressure regulation but also in potassium and hydrogen regulation in the collecting duct.

The mechanism by which WNK4 modulates the activity of NCC is not fully understood. Wild-type
WNK4 induces a decrease in NCC expression in the plasma membrane in both oocytes (42, 117, 122)
and WNK4-NCC-transfected Cos-7 cells (9). In these studies, decreased expression of NCC in plasma
membrane was not observed with a WNK4-harboring PHAII-type mutant. In addition, in transgenic
mice mimicking PHAII, mutant WNK4 is associated with increased expression of NCC in DCT apical
membrane (59, 126). The effect of wild-type WNK4 is not prevented by wild-type or mutant dynamin,
suggesting that clathrin-induced vesicle internalization is not involved. Treatment of Cos-7 cells with
bafilomycin A1, an inhibitor of the vacuolar-type H+-ATPas,e partially reduced the WNK4-induced de‐
crease in NCC expression, suggesting that at least part of the reduction of NCC expression is due to in‐
creased degradation of the cotransporter in lysosomal compartment (9).
Modulation of Distal Nephron Ion Transport Systems by WNK4

The clinical features of PHAII suggested that, in addition to NCC, other renal ion transport systems of
the distal nephron could be regulated by WNKs. Thus effects of WNK4 on these transport systems
have been extensively studied over the last few years (for review see Refs. 37, 54, 94). The results ob‐
tained (Table 2) using wild-type WNK4, PHAII-type WNK4, and a constitutively phosphorylated
WNK4 in a canonical site for the serum glucocorticoid kinase (SGK; WNK4-S1169D) positioned
WNK4 as a potential switch that can work in different functional states to modulate distal renal activity
from a basal state to one that either maximizes salt reabsorption or maximizes K+ secretion. These
functional states of WNK4 may help explain how the kidney achieves the balance between renal salt re‐
absorption and potassium secretion, which occurs even though both are stimulated by aldosterone, a
steroidal hormone that is released from the adrenal glands under two different physiological conditions:
intravascular volume depletion and hyperkalemia. In the former, aldosterone promotes maximal renal
salt reabsorption to preserve and restore intravascular volume, whereas in the latter, renal K+ secretion
is maximized.

Table 2 shows the effects of different types of WNK4 upon ion transport systems of the distal nephron.
In addition to the effects discussed above on NCC, wild-type WNK4 is a negative regulator of the api‐
cal potassium channel ROMK (45, 55, 60). However, unlike the regulation of NCC, WNK4 catalytic
activity is not required for the regulation of ROMK, and PHAII-type mutations in WNK4 result in fur‐
ther inhibition of ROMK. In contrast, WNK4-S1169D no longer inhibits ROMK (91). Wild-type
WNK4 reduces the activity of ENaC in a kinase-independent fashion, but like NCC and unlike ROMK,
PHAII-type mutations in WNK4 prevent the inhibitory effect of WNK4 on ENaC (90). Additionally,
WNK4-S1169D also loses its inhibitory effect on ENaC (91). Other important targets for WNK4 are
the claudins, which are believed to mediate paracellular Cl− flux in the distal nephron. Two indepen‐
dent groups (51, 121) observed that in MDCKII cells wild-type WNK4 increases the paracellular trans‐
port to Cl−. This effect requires WNK4 catalytic activity and is associated with phosphorylation of
claudin-4 in its carboxy-terminal domain. Similar to their effects on ROMK, PHAII-type mutations in
WNK4 are associated with further increases in Cl− permeability and phosphorylation of claudin-4. The
effect of WNK4-S1169D on paracellular chloride permeability or claudin phosphorylation has not been
addressed. Finally, wild-type WNK4 also inhibits the activity of the K+-Cl− cotransporters by a mecha‐
nism that requires catalytic activity (39). KCCs in the distal nephron are critical for potassium secretion
in the DCT (3) and acid secretion in the collecting duct (8). Thus PHAII-type mutations in WNK4 be‐
have as “loss of function” mutations toward NCC and ENaC but as “gain of function” mutations toward
ROMK and claudins, causing confusion about whether PHAII is a gain or loss of function type of in‐
herited disease.

As presented in Table 2, it is evident that WNK4 can perform in at least three different functional
states. One is the wild-type WNK4 that reduces the activities of NCC, ENaC, and ROMK and stimu‐
lates claudins. Therefore, salt reabsorption in the DCT by NCC is partial, and some of the sodium de‐
livered to the connecting tubule (CNT) is interchanged with potassium by simultaneous operation of
the ENaC and ROMK channels, whereas some is reabsorbed by ENaC with chloride passing through
the parecellular space. From here, WNK4 can move toward two different functional states. One is ob‐
tained by constitutive phosphorylation of the canonical site for SGK S1169, which is one of the well-
known mechanisms for aldosterone action because this hormone stimulates SGK (100). As shown in
the interactive Supplemental Fig. 1, the consequences of WNK4-S1169D resemble what would be ex‐
pected to occur during hyperkalemia when aldosterone is increases (supplemental data for this article
are available online at the Am J Physiol Renal Physiol website). The consequences of S1169 phospho‐
rylation on WNK4 are that ENaC and ROMK activities are increased (91). It is known, for example,
that these channel activities are modulated by SGK (11, 127). In contrast, NCC activity remains sup‐
pressed because WNK4-S1169D inhibits NCC similarly to wild-type WNK4 (95). Thus salt escapes
reabsorption in DCT1, and sodium delivered to DCT2 and CNT is interchanged with potassium due to
increased ENaC and ROMK activities. The combination of these effects maximizes potassium secre‐
tion without increasing salt reabsorption. In contrast, as explained in the interactive supplemental Fig.
2, the consequence of PHAII-type mutations in WNK4 resembles what would be expected to occur
during intravascular volume depletion. The activities of NCC, ENaC, and paracellular chloride trans‐
port are increased by WNK4-PHAII, whereas the activity of ROMK is further reduced. Thus DCT1
salt reabsorption is increased with the consequent reduction in salt delivery to DCT2 or the CNT, even
though the salt that reaches the CNT is reabsorbed due to increased ENaC and claudin activities.
Because transcellular sodium reabsorption is accompanied by paracelluar chloride transport, the lumi‐
nal negative potential that is required for potassium secretion is not generated, and additionally, ROMK
is further inhibited by WNK4-PHAII. Thus salt reabsorption is maximized without increasing potas‐
sium secretion. These observations suggest that the PHAII mutations mimic a natural state resulting
from volume depletion. The next question to pursue addresses what the upstream regulatory signal for
this natural state might be. Since the hallmark of intravascular volume depletion is the increase in the
peptide hormone ANG II circulating levels, which are not increased during hyperkalemia, signaling of
ANG II through its G-protein-coupled receptor AT1R is an attractive candidate to be the physiological
counterpart of the functional state generated by PHAII mutations in WNK4. Supporting this hypothe‐
sis, reconstitution experiments in Xenopus oocytes have recently demonstrated that ANG II signaling
acts through WNK4 and the kinase SPAK to increase NCC activity (95). ANG II increases NCC activ‐
ity only in the presence of WNK4, and this effect is completely prevented by the AT1R blocker losar‐
tan. In addition, WNK4-PHAII that no longer inhibits NCC also exhibited no response to ANG II, pro‐
viding evidence that the ANG II effect can be substituted in full by PHAII mutations in WNK4.
Conversely, ANG II signaling did not reverse WNK4 inhibition of ROMK. Interestingly, it was also ob‐
served that ANG II signaling increased phosphorylation at key regulatory sites in both SPAK and NCC
in mammalian cells. Several studies (33, 72, 83, 87, 88, 111) have suggested that WNKs lie upstream
of SPAK, which probably phosphorylates the cotransporters. These findings place WNK4-SPAK in the
signaling pathway between ANG II and NCC and reveal a key role for the ANG II-WNK4-SPAK-NCC
pathway in the renal response to intravascular volume depletion. Supporting these observations, experi‐
ments in vivo have shown that ANG II promotes the trafficking of NCC to the apical membrane of
DCT cells (97) and has an inhibitory effect on ROMK activity (115). Additionally, a recent genomic
wide association study analysis in the Amish population showed a strong association signal between
blood pressure levels and common variants within the SPAK gene (114). Taking all this information to‐
gether, our current understanding of the WNK4 modulation of NCC activity is as follows (Fig. 3).
During normovolemia (Fig. 3A), in which the renin-ANG system is suppressed, WNK4 behaves as a
natural inhibitor of NCC, reducing its activity by decreasing the number of NCCs present in the plasma
membrane (9, 97, 117). Although a protein-protein interaction between WNK4 and NCC has been ob‐
served in oocytes (117) and in mice (59), at this point it is not clear whether the negative effect of
WNK4 on NCC is direct. During hypovolemia (Fig. 3B), when the renin-ANG system is activated,
ANG II interacts with its AT1R, turning WNK4 into an activator of NCC by increasing NCC trafficking
to the plasma membrane, and this is likely mediated through SPAK (88, 95). Supporting this proposal,
eating a low-salt diet is associated with phosphorylation of SPAK and NCC (12) in similar phosphoa‐
ceptor sites to those phosphorylated after exposing oocytes to ANG II (95). The model shown in Fig. 3
depicts the location of AT1R at the basolateral membrane. However, renin and ANG-converting en‐
zymes have been localized within the distal nephron (56, 92), and apical AT1R was shown to be present
in the collecting duct (115), introducing the possibility that ANG II present in the distal tubular fluid
may have an effect on distal tubule transport. According to our observations, patients with PHAII (
Fig. 3C) have one allele with mutations that imitate the effect of ANG II. Thus there is a constitutive
activation of SPAK and NCCs, abnormally increasing salt reabsorption and decreasing potassium se‐
cretion, hence, producing arterial hypertension with hyperkalemia. Consistent with this proposal, the
PHAII-type knockin mouse exhibits increased phosphorylation of SPAK and NCC in the kidney (126).
These proposals imply that PHAII is a “gain of function” type of disease, which agrees with its domi‐
nant mode of inheritance. The gain of function is to make WNK4-SPAK-NCC behave as if there was a
continuous activation of AT1R in the DCT.

Modulation of NCC Activity by Other WNKs

The effect of WNK1 upon NCC and upon the rest of the ion transport systems in the aldosterone-sensi‐
tive distal nephron is complex and still unclear. Two isoforms of WNK1 are expressed in the kidney
(23, 77): the complete long WNK1 (L-WNK1) and a shorter transcript that results from alternative
splicing of exons 1- 4 (KS-WNK1 that lacks the first 437 amino acid residues, including the entire ki‐
nase domain). The L-WNK1 transcript isoform is expressed along the entire nephron, whereas KS-
WNK1 transcripts are only present in the DCT and the CNT (76). WNK1 seems to be a key regulator
of other WNKs, either by phosphorylation processes (61) or by direct interactions at the protein level
(123, 125). For instance, there is no evidence so far that WNK1 has a direct effect on NCC.
Nevertheless, since NCC expression in mammalian cells has not been properly achieved, it is not
known if the absence of WNK1 effects on NCC using oocytes as the expression system can be ex‐
tended to mammalian cells. In this regard, Yang et al. (122) observed in oocytes that WNK1 prevents
the WNK4-induced inhibition of NCC. That is, in the presence of WNK1, WNK4 no longer inhibits
NCC. This interaction is modulated by the alternatively spliced KS-WNK1 isoform because KS-
WNK1, by interacting with L-WNK1 in a dominant negative fashion, eliminates the L-WNK1-induced
inhibition of WNK4 (104). Thus it has been proposed that in normal subjects KS-WNK1, by high-jack‐
ing L-WNK1, prevents its inhibitory effect upon WNK4, allowing WNK4 to inhibit NCC. In contrast,
in PHAII patients, the KS-WNK1-to-L-WNK1 ratio is reduced due to intronic deletions of the WNK1
gene that increase the expression of L-WNK1. As a consequence, L-WNK1 is able to inhibit WNK4
and thus NCC activity is increased, augmenting salt reabsorption in the DCT and thus arterial pressure.
Consistent with these observations, KS-WNK1 and L-WNK1 also interact with each other to regulate
ROMK activity. L-WNK1 decreases ROMK activity, an effect that requires catalytic activity and that is
prevented by KS-WNK1 (60, 113). L-WNK1 is also a modulator of the ENaC via SGK (119). WNK1
induces the phosphorylation of SGK, which in turn phosphorylates and inhibits a protein named Nedd4
that is known to reduce ENaC activity by promoting its endocytosis via a clathrin-dependent mecha‐
nism (1). In addition, in mouse cortical collecting duct cells in culture aldosterone seems to modulate
the L-WNK1-to-KS-WNK1 ratio of expression (74). Therefore, ENaC activity is enhanced. The in‐
creased expression of L-WNK1 in the DCT of a mouse model carrying a deletion of WNK1 intron 1
has been confirmed (22). However, increased expression of KS-WNK1 in DCT was also observed in
the same study. Thus the molecular mechanism for WNK1-induced NCC activity in PHAII patients
with WNK1 intron 1 deletion remains unknown.

WNK3, a member of the WNK family not associated with PHAII, is a powerful regulator of all the
members of the SLC12 family and is expressed along the entire nephron, as well as in several epithelial
and nonepithelial cells outside the kidney (53, 94). WNK3 is an activator of the Na+-driven cotrans‐
porters NKCC1/2 and NCC and is also an inhibitor of the K+-driven cotransporters KCC1-KCC4 (21,
52, 89). That is, WNK3 activates chloride influx pathways and inactivates chloride efflux pathways.
Since the movements of sodium into and potassium out of the cell are quickly resorted by the Na+-K+-
ATPase, what WNK3 seems to promote is the net chloride movement into the cells. Activation by
WNK3 is associated with phosphorylation of two amino-terminal domain threonines in NKCC1 and
NKCC2 (52, 89). Because threonines that are conserved in both NCC and the NKCC1/2 are phospho‐
rylated by similar stimuli, such as intracellular chloride depletion (72, 79, 88), it is highly likely that
these threonines in NCC also become phosphorylated by WNK3. WNK3 activation of NCC is associ‐
ated with increased numbers of NCC on the cell surface, suggesting that WNK3 promotes the insertion
of NCC vesicles into the plasma membrane (89). The WNK3-induced activation of NKCC1/2s or inhi‐
bition of KCCs occurs in oocytes even during cell swelling, which is a well-known inhibitor of
NKCC1/2 and NCC activities (34, 41, 69). Interestingly, elimination of WNK3 catalytic activity by the
D294A substitution in the kinase domain (WNK3-D294A) not only prevented the WNK3-induced acti‐
vation of NKCC1/2 and NCC and its inhibition of KCCs but also turned WNK3 into a powerful in‐
hibitor of NKCC1/2 and NCC and an activator of KCCs, even in isotonic or hypertonic conditions, in‐
ducing cell shrinkage, which is known to activate NKCC1/2 and inhibit KCCs. For example, microin‐
jection of cRNA encoding the K+-Cl− cotransporters KCC1, KCC3, or KCC4 induces expression of
these cotransporters that can be demonstrated by assessing the Cl−-dependent 86Rb+ influx but only
when oocytes are exposed to a hypotonic medium to induce cell swelling. When incubated in isotonic
medium, the cotransporters are completely inactive (67, 68). In contrast, when coinjected with WNK3-
D294A cRNA, KCCs are fully active when incubated in isotonic medium (21). Thus inactive WNK3
promotes net chloride movement out of the cells. Since the activation of KCCs by WNK3-D294A can
be prevented with calyculin A and/or cyclosporine A, it is likely due to a WNK3-D294A activation of
protein phosphatases 1 and 2B (21). Thus WNK3 modulates the activity not only of NCC but also of
all members of the SLC12 family and, in doing so, bypasses the changes in cell volume and/or intracel‐
lular chloride concentration that are usually required for their activation or inhibition. For these rea‐
sons, it has been proposed that WNK3 could be the intracellular kinase sensitive to cell volume and/or
intracellular chloride concentration (53).

From observations discussed above, it is evident that wild-type WNK3 and WNK4 have opposite ef‐
fects on NCC. Whereas WNK3 is an activator, WNK4 is an inhibitor. Because both kinases are ex‐
pressed in the DCT, it is possible that NCC activity at any given time is a result of the algebraic sum of
both effects. In this regard, Yang et al. (124) performed a series of experiments in X. laevis oocytes by
coinjecting NCC cRNA with different fragments of WNK3 and WNK4 cRNA and concluded that these
kinases interact and regulate each other by means of the carboxy-terminal domains. For instance, al‐
though the effects of WNK3 and WNK4 on NCC require kinase activity when full-length constructs
are used (9, 42, 89, 117, 124), it was observed that WNK3 activation or WNK4 inhibition of NCC was
reproduced using only the carboxy-terminal domain that lacks the entire kinase domain (124). These
types of interactions were not observed with wild-type full-length proteins or with full-length proteins
harboring a single point mutation that eliminates catalytic activity. Unlike what was proposed for L-
WNK1/KS-WNK1 regulation of NCC, there is no evidence that such truncated isoforms of WNK3 or
WNK4 are expressed in the DCT. In addition, the presence of endogenous WNK3 and WNK4 tran‐
scripts in X. laevis oocytes (96) and the known interaction between WNKs (61, 123, 124) make these
data difficult to interpret. Thus, at the moment, the physiological relevance of WNK3 and WNK4 car‐
boxy-terminal domain fragments interactions is unclear.

Several differences between WNK3 and WNK4 suggest that each kinase could serve different pur‐
poses. The effect of WNK4, but not of WNK3, on NCC can be modulated by ANG II (95), suggesting
that NCC activity is regulated by intravascular volume via WNK4, but not via WNK3. Additionally,
WNK1, the other WNK member clearly associated with blood pressure regulation, prevents WNK4 but
not WNK3 effects on NCC (122, 124). On the other hand, elimination of WNK4 catalytic activity abro‐
gates WNK4 inhibition of NCC, whereas elimination of WNK3 catalytic activity switches WNK3 from
an activator to an inhibitor. Thus it is possible that WNK3 and WNK4 respond to different upstream
signals for modulation of NCC activity, as it is possible that downstream pathways between WNKs and
NCC are also different. In this regard, we have constructed chimeric proteins between WNK3 and
WNK4 by swapping the low conserved amino- or carboxy-terminal domains (12–15% identity) that
flank the well-conserved kinase domain (>80% identity). Chimeras were functional and possessed cat‐
alytic activity. The chimera containing the amino-terminal domain of WNK3 swapped into the kinase
and carboxy-terminal domain of WNK4 activated NCC in a kinase-dependent fashion. In contrast, the
chimera containing the amino-terminal domain of WNK4 swapped into the kinase and carboxy-termi‐
nal domain of WNK3 inhibited NCC in a kinase-dependent fashion. These observations suggest that
WNK3 and WNK4 amino-terminal domains contain sequences defining the type of effects that they
have on NCC (96).

The understanding of NCC structure-function relationship and its regulation by WNKs and down‐
stream kinases like SPAK/OSR1 have advanced quickly in the past few years, but there is still a lot of
exciting discovering waiting for us to get there. Meanwhile, this little stop in our journey was done to
recognize the invaluable contribution and legacy of Steve C. Hebert. Without his passion for under‐
standing salt transport mechanisms in the kidney, most of what is discussed in the present work would
not be possible.

GRANTS

This work is supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT No. 59992),
National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-064635, and the
Foundation Leducq Transatlantic Network on Hypertension.

Supplementary Material

[Supplemental Figures]

1
This is to my knowledge the first study that demonstrated the existence of an electrically silent Na-coupled chlo‐
ride cotransport mechanism.

2The thiazide-sensitive Na-Cl cotransporter was the first member of the SLC12A family that was identified at the
molecular level.

REFERENCES

1. Abriel H, Staub O. Ubiquitylation of ion channels. Physiology 20: 398–407, 2005 [PubMed: 16287989]

2. Abuladze N, Yanagawa N, Lee I, Jo OD, Newman D, Hwang J, Uyemura K, Pushkin A, Modlin RL, Kurtz I. Peripheral blood
mononuclear cells express mutated NCCT mRNA in Gitelman's syndrome: evidence for abnormal thiazide-sensitive NaCl
cotransport. J Am Soc Nephrol 9: 819–826, 1998 [PubMed: 9596079]

3. Amorim JB, Bailey MA, Musa-Aziz R, Giebisch G, Malnic G. Role of luminal anion and pH in distal tubule potassium
secretion. Am J Physiol Renal Physiol 284: F381–F388, 2003 [PubMed: 12529275]
4. Bachmann S, Velázquez H, Obermuller N, Reily RF, Moser D, Ellison DH. Expression of the thiazide-sensitive Na-Cl
cotransporter by rabbit distal convoluted tubule cells. J Clin Invest 96: 2510–2514, 1995 [PMCID: PMC185908] [PubMed:
7593642]

5. Bazzini C, Vezzoli V, Sironi C, Dossena S, Ravasio A, Debiasi S, Garavaglia M, Rodighiero S, Meyer G, Fascio U, Furst J,
Ritter M, Botta G, Paulmichl M. Thiazide-sensitive NaCl cotransporter in the intestine: possible role of HCTZ in the intestinal
Ca2+ uptake. J Biol Chem 280: 19902–19910, 2005 [PubMed: 15781471]

6. Beaumont K, Vaughn DA, Fanestil DD. Thiazide diuretic receptors in rat kidney: identification with [3H]metolazone. Proc
Natl Acad Sci USA 85: 2311–2314, 1988 [PMCID: PMC279981] [PubMed: 3353380]

7. Bernstein PL, Zawalach W, Bartiss A, Reilly R, Palcso M, Ellison DH. The thiazide-sensitive Na-Cl cotransporter is expressed
in rat endocrine pancreas. Journal Amer Soc Nephrology 6, 732 1995

8. Boettger T, Hubner CA, Maier H, Rust MB, Beck FX, Jentsch TJ. Deafness and renal tubular acidosis in mice lacking the K-Cl
co-transporter Kcc4. Nature 416: 874–878, 2002 [PubMed: 11976689]

9. Cai H, Cebotaru V, Wang YH, Zhang XM, Cebotaru L, Guggino SE, Guggino WB. WNK4 kinase regulates surface
expression of the human sodium chloride cotransporter in mammalian cells. Kidney Int 69: 2162–2170, 2006 [PubMed:
16688122]

10. Campean V, Kricke J, Ellison D, Luft FC, Bachmann S. Localization of thiazide-sensitive Na(+)-Cl(−) cotransport and
associated gene products in mouse DCT. Am J Physiol Renal Physiol 281: F1028–F1035, 2001 [PubMed: 11704553]

11. Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, Pearce D. Epithelial sodium
channel regulated by aldosterone-induced protein sgk. Proc Natl Acad Sci USA 96: 2514–2519, 1999 [PMCID: PMC26816]
[PubMed: 10051674]

12. Chiga M, Rai T, Yang SS, Ohta A, Takizawa T, Sasaki S, Uchida S. Dietary salt regulates the phosphorylation of
OSR1/SPAK kinases and the sodium chloride cotransporter through aldosterone. Kidney Int 74: 1403–1409, 2008 [PubMed:
18800028]

13. Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL, Jr, Jones DW, Materson BJ, Oparil S, Wright JT, Jr,
Roccella EJ. The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High
Blood Pressure: the JNC 7 Report. JAMA 289: 2560–2571, 2003 [PubMed: 12748199]

14. Clader JA, Schacheter M, Sever PS. Direct vascular actions of hydrochlorothaizide and indapamide in isolated small vessels.
Eur J Pharmacol 220: 19–26, 1992 [PubMed: 1425977]

15. Costanzo LS. Localization of diuretic action in microperfused rat distal tubules: Ca and Na transport. Am J Physiol Renal
Fluid Electrolyte Physiol 248: F527–F535, 1985 [PubMed: 3985160]

16. Cremaschi D, Porta C, Botta G, Bazzini C, Baroni MD, Garavaglia M. Apical Na(+)-Cl(−) symport in rabbit gallbladder
epithelium: a thiazide-sensitive cotransporter (TSC). J Membr Biol 176: 53–65, 2000 [PubMed: 10882428]

17. Cutler CP, Cramb G. Differential expression of absorptive cation-chloride-cotransporters in the intestinal and renal tissues of
the European eel (Anguilla anguilla). Comp Biochem Physiol B Biochem Mol Biol 149: 63–73, 2008 [PubMed: 17950642]

18. De Jong JC, Van Der Vliet WA, van den Heuvel LP, Willems PH, Knoers NV, Bindels RJ. Functional expression of
mutations in the human NaCl cotransporter: evidence for impaired routing mechanisms in Gitelman's syndrome. J Am Soc
Nephrol 13: 1442–1448, 2002 [PubMed: 12039972]

19. De Jong JC, Willems PH, Mooren FJ, van den Heuvel LP, Knoers NV, Bindels RJ. The structural unit of the thiazide-
sensitive NaCl cotransporter is a homodimer. J Biol Chem 278: 24302–24307, 2003 [PubMed: 12704198]

20. De Jong JC, Willems PH, van den Heuvel LP, Knoers NV, Bindels RJ. Functional expression of the human thiazide-sensitive
NaCl cotransporter in Madin-Darby canine kidney cells. J Am Soc Nephrol 14: 2428–2435, 2003 [PubMed: 14514720]
21. De Los Heros P, Kahle KT, Rinehart J, Bobadilla NA, Vazquez N, San Cristobal P, Mount DB, Lifton RP, Hebert SC, Gamba
G. WNK3 bypasses the tonicity requirement for K-Cl cotransporter activation via a phosphatase-dependent pathway. Proc Natl
Acad Sci USA 103: 1976–1981, 2006 [PMCID: PMC1413675] [PubMed: 16446421]

22. Delaloy C, Elvira-Matelot E, Clemessy M, Zhou XO, Imbert-Teboul M, Houot AM, Jeunemaitre X, Hadchouel J. Deletion of
WNK1 first intron results in misregulation of both isoforms in renal and extrarenal tissues. Hypertension 52: 1149–1159, 2008
[PubMed: 18955660]

23. Delaloy C, Lu J, Houot AM, Disse-Nicodeme S, Gasc JM, Corvol P, Jeunemaitre X. Multiple promoters in the WNK1 gene:
one controls expression of a kidney-specific kinase-defective isoform. Mol Cell Biol 23: 9208–9221, 2003 [PMCID:
PMC309643] [PubMed: 14645531]

24. Delpire E, Rauchman MI, Beier DR, Hebert SC, Gullans SR. Molecular cloning and chromosome localization of a putative
basolateral Na+-K+−2Cl− cotransporter from mouse inner medullary collecting duct (mIMCD-3) cells. J Biol Chem 269:
25677–25683, 1994 [PubMed: 7929272]

25. Deng L, Chen G. Cyclothiazide potently inhibits gamma-aminobutyric acid type A receptors in addition to enhancing
glutamate responses. Proc Natl Acad Sci USA 100: 13025–13029, 2003 [PMCID: PMC240738] [PubMed: 14534329]

26. Disse-Nicodeme S, Achard JM, Desitter I, Houot AM, Fournier A, Corvol P, Jeunemaitre X. A new locus on chromosome
12p13.3 for pseudohypoaldosteronism type II, an autosomal dominant form of hypertension. Am J Hum Genet 67: 302–310,
2000 [PMCID: PMC1287179] [PubMed: 10869238]

27. Disse-Nicodeme S, Desitter I, Fiquet-Kempf B, Houot AM, Stern N, Delahousse M, Potier J, Ader JL, Jeunemaitre X.
Genetic heterogeneity of familial hyperkalaemic hypertension. J Hypertens 19: 1957–1964, 2001 [PubMed: 11677360]

28. Doucet A. Function and control of Na-K-ATPase in single nephron segments of the mammalian kidney. Kidney Int 34: 749–
760, 1988 [PubMed: 2850394]

29. Drewnowska K, Baumgarten CM. Regulation of cellular volume in rabbit ventricular myocytes: bumetanide, chlorthiazide,
and ouabain. Am J Physiol Cell Physiol 260: C122–C131, 1991 [PubMed: 1987774]

30. Dvorak MM, De Joussineau C, Carter DH, Pisitkun T, Knepper MA, Gamba G, Kemp PJ, Riccardi D. Thiazide diuretics
directly induce osteoblast differentiation and mineralized nodule formation by interacting with a sodium chloride co-transporter
in bone. J Am Soc Nephrol 18: 2509–2516, 2007 [PMCID: PMC2216427] [PubMed: 17656470]

31. Ellison DH, Velazquez H, Wright FS. Thiazide-sensitive sodium chloride cotransport in early distal tubule. Am J Physiol
Renal Fluid Electrolyte Physiol 253: F546–F554, 1987 [PubMed: 3631283]

32. Fanestil DD, Tran JM, Vaughn DA, Maciejewski AR, Beaumont K. Investigation of the metolazone receptor. In: Diuretics
III: Chemistry, Pharmacology and Clinical Applications, edited by Puschett JB, Greenberg A. New York: Elsevier Science
Publication, 1990, p. 195–204

33. Gagnon KB, England R, Delpire E. Volume sensitivity of cation-Cl− cotransporters is modulated by the interaction of two
kinases: Ste20-related proline-alanine-rich kinase and WNK4. Am J Physiol Cell Physiol 290: C134–C142, 2006 [PubMed:
15930150]

34. Gamba G, Miyanoshita A, Lombardi M, Lytton J, Lee WS, Hediger MA, Hebert SC. Molecular cloning, primary structure
and characterization of two members of the mammalian electroneutral sodium-(potassium)-chloride cotransporter family
expressed in kidney. J Biol Chem 269: 17713–17722, 1994 [PubMed: 8021284]

35. Gamba G, Saltzberg SN, Lombardi M, Miyanoshita A, Lytton J, Hediger MA, Brenner BM, Hebert SC. Primary structure
and functional expression of a cDNA encoding the thiazide-sensitive, electroneutral sodium-chloride cotransporter. Proc Natl
Acad Sci USA 90: 2749–2753, 1993 [PMCID: PMC46173] [PubMed: 8464884]
36. Gamba G. Molecular physiology and pathophysiology of the electroneutral cation-chloride cotransporters. Physiol Rev 85:
423–493, 2005 [PubMed: 15788703]

37. Gamba G. Role of WNK kinases in regulating tubular salt and potassium transport and in the development of hypertension.
Am J Physiol Renal Physiol 288: F245–F252, 2005 [PubMed: 15637347]

38. Gamba G. WNK lies upstream of kinases involved in regulation of ion transporters. Biochem J 391: e1–e3, 2005 [PMCID:
PMC1237149] [PubMed: 16173916]

39. Garzon-Muvdi T, Pacheco-Alvarez D, Gagnon KB, Vazquez N, Ponce-Coria J, Moreno E, Delpire E, Gamba G. WNK4
kinase is a negative regulator of K+-Cl− cotransporters. Am J Physiol Renal Physiol 292: F1197–F1207, 2007 [PubMed:
17182532]

40. Gillen CM, Brill S, Payne JA, Forbush B., III Molecular cloning and functional expression of the K-Cl cotransporter from
rabbit, rat and human. A new member of the cation-chloride cotransporter family. J Biol Chem 271: 16237–16244, 1996
[PubMed: 8663127]

41. Gimenez I, Forbush B. Regulatory phosphorylation sites in the N-terminus of the renal Na-K-Cl cotransporter (NKCC2). Am
J Physiol Renal Physiol 289: F1341–F1345, 2005 [PubMed: 16077079]

42. Golbang AP, Cope G, Hamad A, Murthy M, Liu CH, Cuthbert AW, O'Shaughnessy KM. Regulation of the expression of the
Na/Cl cotransporter (NCCT) by WNK4 and WNK1: evidence that accelerated dynamin-dependent endocytosis is not involved.
Am J Physiol Renal Physiol 291: F1369–F1376, 2006 [PubMed: 16788137]

43. Gordon RD. Syndrome of hypertension and hyperkalemia with normal glomerular filtration rate. Hypertension 8: 93–102,
1986 [PubMed: 3002982]

44. Gordon RD, Hodsman GP. The syndrome of hypertension and hyperkalaemia without renal failure: long term correction by
thiazide diuretic. Scott Med J 31: 43–44, 1986 [PubMed: 3961473]

45. He G, Wang HR, Huang SK, Huang CL. Intersectin links WNK kinases to endocytosis of ROMK1. J Clin Invest 117: 1078–
1087, 2007 [PMCID: PMC1821066] [PubMed: 17380208]

46. Hebert SC, Mount DB, Gamba G. Molecular physiology of cation-coupled Cl(−) cotransport: the SLC12 family. Pflügers
Arch 447: 580–593, 2004 [PubMed: 12739168]

47. Hollander W, Chobanian AV, Wilkins RW. Studies on the antihypertensive action of chlorothiazide. Clin Res 6: 21–22, 1958

48. Hoover RS, Poch E, Monroy A, Vazquez N, Nishio T, Gamba G, Hebert SC. N-glycosylation at two sites critically alters
thiazide binding and activity of the rat thiazide-sensitive Na(+):Cl(−) cotransporter. J Am Soc Nephrol 14: 271–282, 2003
[PubMed: 12538726]

49. Jones G, Nguyen T, Sambrook PN, Eisman JA. Thiazide diuretics and fractures: can meta-analysis help? J Bone Miner Res
10: 106–111, 1995 [PubMed: 7747616]

50. Kahle KT, Gimenez I, Hassan H, Wilson FH, Wong RD, Forbush B, Aronson PS, Lifton RP. WNK4 regulates apical and
basolateral Cl− flux in extrarenal epithelia. Proc Natl Acad Sci USA 101: 2064–2069, 2004 [PMCID: PMC357052] [PubMed:
14769928]

51. Kahle KT, MacGregor GG, Wilson FH, Van Hoek AN, Brown D, Ardito T, Kashgarian M, Giebisch G, Hebert SC, Boulpaep
EL, Lifton RP. Paracellular Cl− permeability is regulated by WNK4 kinase: insight into normal physiology and hypertension.
Proc Natl Acad Sci USA 101: 14877–14882, 2004 [PMCID: PMC522037] [PubMed: 15465913]

52. Kahle KT, Rinehart J, De Los HP, Louvi A, Meade P, Vazquez N, Hebert SC, Gamba G, Gimenez I, Lifton RP. WNK3
modulates transport of Cl− in and out of cells: implications for control of cell volume and neuronal excitability. Proc Natl Acad
Sci USA 102: 16783–16788, 2005 [PMCID: PMC1283843] [PubMed: 16275911]
53. Kahle KT, Rinehart J, Ring A, Gimenez I, Gamba G, Hebert SC, Lifton RP. WNK protein kinases modulate cellular Cl− flux
by altering the phosphorylation state of the Na-K-Cl and K-Cl cotransporters. Physiology 21: 326–335, 2006 [PubMed:
16990453]

54. Kahle KT, Ring AM, Lifton RP. Molecular physiology of the WNK kinases. Annu Rev Physiol 70: 329–355, 2008 [PubMed:
17961084]

55. Kahle KT, Wilson FH, Leng Q, Lalioti MD, O'Connell AD, Dong K, Rapson AK, MacGregor GG, Giebisch G, Hebert SC,
Lifton RP. WNK4 regulates the balance between renal NaCl reabsorption and K+ secretion. Nat Genet 35: 372–376, 2003
[PubMed: 14608358]

56. Komlosi P, Fuson AL, Fintha A, Peti-Peterdi J, Rosivall L, Warnock DG, Bell PD. Angiotensin I conversion to angiotensin II
stimulates cortical collecting duct sodium transport. Hypertension 42: 195–199, 2003 [PubMed: 12835330]

57. Kunau RT, Weller DR, Webb HL. Clarification of the site of action of chlorothiazide in the rat nephron. J Clin Invest 56:
401–407, 1975 [PMCID: PMC436599] [PubMed: 1150878]

58. Kunchaparty S, Palcso M, Berkman J, Velázquez H, Desir GV, Bernstein P, Reilly RF, Ellison DH. Defective processing and
expression of thiazide-sensitive Na-Cl cotransporter as a cause of Gitelman's syndrome. Am J Physiol Renal Physiol 277: F643–
F649, 1999 [PubMed: 10516289]

59. Lalioti MD, Zhang J, Volkman HM, Kahle KT, Hoffmann KE, Toka HR, Nelson-Williams C, Ellison DH, Flavell R, Booth
CJ, Lu Y, Geller DS, Lifton RP. Wnk4 controls blood pressure and potassium homeostasis via regulation of mass and activity of
the distal convoluted tubule. Nat Genet 38: 1124–1132, 2006 [PubMed: 16964266]

60. Lazrak A, Liu Z, Huang CL. Antagonistic regulation of ROMK by long and kidney-specific WNK1 isoforms. Proc Natl
Acad Sci USA 103: 1615–1620, 2006 [PMCID: PMC1360592] [PubMed: 16428287]

61. Lenertz LY, Lee BH, Min X, Xu BE, Wedin K, Earnest S, Goldsmith EJ, Cobb MH. Properties of WNK1 and implications
for other family members. J Biol Chem 280: 26653–26658, 2005 [PubMed: 15883153]

62. Loffing J, Kaissling B. Sodium and calcium transport pathways along the mammalian distal nephron: from rabbit to human.
Am J Physiol Renal Physiol 284: F628–F643, 2003 [PubMed: 12620920]

63. Loffing J, Loffing-Cueni D, Valderrabano V, Klausli L, Hebert SC, Rossier BC, Hoenderop JG, Bindels RJ, Kaissling B.
Distribution of transcellular calcium and sodium transport pathways along mouse distal nephron. Am J Physiol Renal Physiol
281: F1021–F1027, 2001 [PubMed: 11704552]

64. Mansfield TA, Simon DB, Farfel Z, Bia M, Tucci JR, Lebel M, Gutkin M, Vialettes B, Christofilis MA, Kauppinen-Makelin
R, Mayan H, Risch N, Lifton RP. Multilocus linkage of familial hyperkalaemia and hypertension, pseudohypoaldosteronism type
II, to chromosomes 1q31-42 and 17p11-q21. Nat Genet 16: 202–205, 1997 [PubMed: 9171836]

65. Mastroianni N, DeFusco M, Zollo M, Arrigo G, Zuffardi O, Bettinelli A, Ballabio A, Casari G. Molecular cloning,
expression pattern, and chromosomal localization of the human Na-Cl thiazide-sensitive cotransporter (SLC12A3). Genomics
35: 486–493, 1996 [PubMed: 8812482]

66. Mayan H, Vered I, Mouallem M, Tzadok-Witkon M, Pauzner R, Farfel Z. Pseudohypoaldosteronism type II: marked
sensitivity to thiazides, hypercalciuria, normomagnesemia, and low bone mineral density. J Clin Endocrinol Metab 87: 3248–
3254, 2002 [PubMed: 12107233]

67. Mercado A, Song L, Vazquez N, Mount DB, Gamba G. Functional comparison of the K+-Cl− cotransporters KCC1 and
KCC4. J Biol Chem 275: 30326–30334, 2000 [PubMed: 10913127]

68. Mercado A, Vazquez N, Song L, Cortes R, Enck AH, Welch R, Delpire E, Gamba G, Mount DB. Amino-terminal
heterogeneity in the KCC3 K+- Cl− cotransporter. Am J Physiol Renal Physiol 289: F1246–F1261, 2005 [PubMed: 16048901]
69. Monroy A, Plata C, Hebert SC, Gamba G. Characterization of the thiazide-sensitive Na(+)-Cl(−) cotransporter: a new model
for ions and diuretics interaction. Am J Physiol Renal Physiol 279: F161–F169, 2000 [PubMed: 10894798]

70. Moreno E, San Cristobal P, Rivera M, Vazquez N, Bobadilla NA, Gamba G. Affinity defining domains in the Na-Cl
cotransporter: different location for Cl− and thiazide binding. J Biol Chem 281: 17266–17275, 2006 [PubMed: 16624820]

71. Moreno E, Tovar-Palacio C, De Los HP, Guzman B, Bobadilla NA, Vazquez N, Riccardi D, Poch E, Gamba G. A single
nucleotide polymorphism alters the activity of the renal Na+:Cl− cotransporter and reveals a role for transmembrane segment 4 in
chloride and thiazide affinity. J Biol Chem 279: 16553–16560, 2004 [PubMed: 14766743]

72. Moriguchi T, Urushiyama S, Hisamoto N, Iemura S, Uchida S, Natsume T, Matsumoto K, Shibuya H. WNK1 regulates
phosphorylation of cation-chloride-coupled cotransporters via the STE20-related kinases, SPAK and OSR1. J Biol Chem 280:
42685–42693, 2005 [PubMed: 16263722]

73. Mount DB, Mercado A, Song L, Xu J, George AL, Jr, Delpire E, Gamba G. Cloning and characterization of KCC3 and
KCC4, new members of the cation-chloride cotransporter gene family. J Biol Chem 274: 16355–16362, 1999 [PubMed:
10347194]

74. Naray-Fejes-Toth A, Snyder PM, Fejes-Toth G. The kidney-specific WNK1 isoform is induced by aldosterone and stimulates
epithelial sodium channel-mediated Na+ transport. Proc Natl Acad Sci USA 101: 17434–17439, 2004 [PMCID: PMC536044]
[PubMed: 15583131]

75. Novello FC, Sprague JM. Benzothiadiazine dioxides as novel diuretics. J Am Chem Soc 79: 2028–2029, 1957

76. O'Reilly M, Marshall E, Macgillivray T, Mittal M, Xue W, Kenyon CJ, Brown RW. Dietary electrolyte-driven responses in
the renal WNK kinase pathway in vivo. J Am Soc Nephrol 17: 2402–2413, 2006 [PubMed: 16899520]

77. O'Reilly M, Marshall E, Speirs HJ, Brown RW. WNK1, a gene within a novel blood pressure control pathway, tissue-
specifically generates radically different isoforms with and without a kinase domain. J Am Soc Nephrol 14: 2447–2456, 2003
[PubMed: 14514722]

78. Obermuller N, Bernstein P, Velázquez H, Reilly R, Moser D, Ellison DH, Bachman S. Expression of the thiazide-sensitive
Na-Cl cotransporter in rat and human kidney. Am J Physiol Renal Fluid Electrolyte Physiol 269: F900–F910, 1995 [PubMed:
8594886]

79. Pacheco-Alvarez D, San Cristobal P, Meade P, Moreno E, Vazquez N, Munoz E, Diaz A, Juarez ME, Gimenez I, Gamba G.
The Na-Cl cotransporter is activated and phosphorylated at the amino terminal domain upon intracellular chloride depletion. J
Biol Chem 281: 28755–28763, 2006 [PubMed: 16887815]

80. Pathak BG, Shaughnessy JD, Jr, Meneton P, Greeb J, Shull GE, Jenkins NA, Copeland NG. Mouse chromosomal location of
three epithelial sodium channel subunit genes and an apical sodium chloride cotransporter gene. Genomics 33: 124–127, 1996
[PubMed: 8617496]

81. Payne JA, Stevenson TJ, Donaldson LF. Molecular characterization of a putative K-Cl cotransporter in rat brain. A neuronal-
specific isoform. J Biol Chem 271: 16245–16252, 1996 [PubMed: 8663311]

82. Plotkin MD, Kaplan MR, Verlander JM, Lee WS, Brown D, Poch E, Gullans SR, Hebert SC. Localization of the thiazide
sensitive Na-Cl cotransporter, rTSC1, in the rat kidney. Kidney Int 50: 174–183, 1996 [PubMed: 8807586]

83. Ponce-Coria J, San Cristobal P, Kahle KT, Vazquez N, Pacheco-Alvarez D, De Los HP, Juarez P, Munoz E, Michel G,
Bobadilla NA, Gimenez I, Lifton RP, Hebert SC, Gamba G. Regulation of NKCC2 by a chloride-sensing mechanism involving
the WNK3 and SPAK kinases. Proc Natl Acad Sci USA 105: 8458–8463, 2008 [PMCID: PMC2448858] [PubMed: 18550832]

84. Reilly RF, Ellison DH. Mammalian distal tubule: physiology, pathophysiology, and molecular anatomy. Physiol Rev 80: 277–
313, 2000 [PubMed: 10617770]
85. Renfro JL. Water and ion transport by the urinary bladder of the teleost Pseudopleuronectus americanus. Am J Physiol 228:
52–61, 1975 [PubMed: 1147028]

86. Renfro JL. Interdependence of active Na+ and Cl− transport by the isolated urinary bladder of the teleost, pseudopleuronectes
americanus. J Exp Zool 199: 383–390, 1977 [PubMed: 850117]

87. Richardson C, Alessi DR. The regulation of salt transport and blood pressure by the WNK-SPAK/OSR1 signalling pathway. J
Cell Sci 121: 3293–3304, 2008 [PubMed: 18843116]

88. Richardson C, Rafiqi FH, Karlsson HK, Moleleki N, Vandewalle A, Campbell DG, Morrice NA, Alessi DR. Activation of the
thiazide-sensitive Na+-Cl− cotransporter by the WNK-regulated kinases SPAK and OSR1. J Cell Sci 121: 675–684, 2008
[PubMed: 18270262]

89. Rinehart J, Kahle KT, De Los HP, Vazquez N, Meade P, Wilson FH, Hebert SC, Gimenez I, Gamba G, Lifton RP. WNK3
kinase is a positive regulator of NKCC2 and NCC, renal cation-Cl− cotransporters required for normal blood pressure
homeostasis. Proc Natl Acad Sci USA 102: 16777–16782, 2005 [PMCID: PMC1283841] [PubMed: 16275913]

90. Ring AM, Cheng SX, Leng Q, Kahle KT, Rinehart J, Lalioti MD, Volkman HM, Wilson FH, Hebert SC, Lifton RP. WNK4
regulates activity of the epithelial Na+ channel in vitro and in vivo. Proc Natl Acad Sci USA 104: 4020–4024, 2007 [PMCID:
PMC1805455] [PubMed: 17360470]

91. Ring AM, Leng Q, Rinehart J, Wilson FH, Kahle KT, Hebert SC, Lifton RP. An SGK1 site in WNK4 regulates Na+ channel
and K+ channel activity and has implications for aldosterone signaling and K+ homeostasis. Proc Natl Acad Sci USA 104: 4025–
4029, 2007 [PMCID: PMC1803763] [PubMed: 17360471]

92. Rohrwasser A, Morgan T, Dillon HF, Zhao L, Callaway CW, Hillas E, Zhang S, Cheng T, Inagami T, Ward K, Terreros DA,
Lalouel JM. Elements of a paracrine tubular renin-angiotensin system along the entire nephron. Hypertension 34: 1265–1274,
1999 [PubMed: 10601129]

93. Sabath E, Meade P, Berkman J, De Los HP, Moreno E, Bobadilla NA, Vazquez N, Ellison DH, Gamba G. Pathophysiology
of functional mutations of the thiazide-sensitive Na-Cl cotransporter in Gitelman disease. Am J Physiol Renal Physiol 287:
F195–F203, 2004 [PubMed: 15068971]

94. San Cristobal P, De Los HP, Ponce-Coria J, Moreno E, Gamba G. WNK kinases, renal ion transport and hypertension. Am J
Nephrol 28: 860–870, 2008 [PMCID: PMC2820349] [PubMed: 18547946]

95. San Cristobal P, Pacheco-Alvarez D, Richardson C, Ring AM, Vazquez N, Rafiqi FH, Chari D, Kahle KT, Leng Q, Bobadilla
NA, Hebert SC, Alessi DR, Lifton RP, Gamba G. Angiotensin II signaling increases activity of the renal Na-Cl cotransporter
through a WNK4-SPAK-dependent pathway. Proc Natl Acad Sci USA 106: 4384–4389, 2009 [PMCID: PMC2647339]
[PubMed: 19240212]

96. San Cristobal P, Ponce-Coria J, Vazquez N, Bobadilla NA, Gamba G. WNK3 and WNK4 amino-terminal domain defines
their effect on the renal Na+-Cl− cotransporter. Am J Physiol Renal Physiol 295: F1199–F1206, 2008 [PMCID: PMC2576145]
[PubMed: 18701621]

97. Sandberg MB, Riquier AD, Pihakaski-Maunsbach K, McDonough AA, Maunsbach AB. ANG II provokes acute trafficking of
distal tubule Na+-Cl− cotransporter to apical membrane. Am J Physiol Renal Physiol 293: F662–F669, 2007 [PubMed:
17507603]

98. Schoofs MW, van der KM, Hofman A, de Laet CE, Herings RM, Stijnen T, Pols HA, Stricker BH. Thiazide diuretics and the
risk for hip fracture. Ann Intern Med 139: 476–482, 2003 [PubMed: 13679324]

99. Schultheis PJ, Lorenz JN, Meneton P, Nieman ML, Riddle TM, Flagella M, Duffy JJ, Doetschman T, Miller ML, Shull GE.
Phenotype resembling Gitelman's syndrome in mice lacking the apical Na+-Cl− cotransporter of the distal convoluted tubule. J
Biol Chem 273: 29150–29155, 1998 [PubMed: 9786924]
100. Shigaev A, Asher C, Latter H, Garty H, Reuveny E. Regulation of sgk by aldosterone and its effects on the epithelial Na(+)
channel. Am J Physiol Renal Physiol 278: F613–F619, 2000 [PubMed: 10751222]

101. Simon DB, Nelson-Williams C, Johnson-Bia M, Ellison D, Karet FE, Morey-Molina A, Vaara I, Iwata F, Cushner HM,
Koolen M, Gainza FJ, Gitelman HJ, Lifton RP. Gitelman's variant of Bartter's syndrome, inherited hypokalaemic alkalosis, is
caused by mutations in the thiazide-sensitive Na-Cl cotransporter. Nature Genetics 12: 24–30, 1996 [PubMed: 8528245]

102. Smith HW. From Fish to Philosopher. Boston, MA: Little Brown and Co., 1953

103. Stokes JB, Lee I, D'Amico M. Sodium chloride absorption by the urinary bladder of the winter flounder. A thiazide-
sensitive, electrically neutral transport system. J Clin Invest 74: 7–16, 1984 [PMCID: PMC425179] [PubMed: 6736252]

104. Subramanya AR, Yang CL, Zhu X, Ellison DH. Dominant-negative regulation of WNK1 by its kidney-specific kinase-
defective isoform. Am J Physiol Renal Physiol 290: F619–F624, 2006 [PubMed: 16204408]

105. Taniyama Y, Sato K, Sugawara A, Uruno A, Ikeda Y, Kudo M, Ito S, Takeuchi K. Renal tubule-specific transcription and
chromosomal localization of rat thiazide-sensitive na-cl cotransporter gene. J Biol Chem 276: 26260–26268, 2001 [PubMed:
11313351]

106. Tovar-Palacio C, Bobadilla NA, Cortes P, Plata C, De Los HP, Vazquez N, Gamba G. Ion and diuretic specificity of
chimeric proteins between apical Na+-K+-2Cl− and Na+-Cl− cotransporters. Am J Physiol Renal Physiol 287: F570–F577, 2004
[PubMed: 15149970]

107. Tran JM, Farrell MA, Fanestil DD. Effect of ions on binding of the thiazide-type diuretic metolazone to kidney membrane.
Am J Physiol Renal Fluid Electrolyte Physiol 258: F908–F915, 1990 [PubMed: 2330985]

108. Vazquez N, Monroy A, Dorantes E, Munoz-Clares RA, Gamba G. Functional differences between flounder and rat thiazide-
sensitive Na-Cl cotransporter. Am J Physiol Renal Physiol 282: F599–F607, 2002 [PubMed: 11880320]

109. Velazquez H, Good DW, Wright FS. Mutual dependence of sodium and chloride absorption by renal distal tubule. Am J
Physiol Renal Fluid Electrolyte Physiol 247: F904–F911, 1984 [PubMed: 6507630]

110. Velazquez H, Naray-Fejes-Toth A, Silva T, Andujar E, Reilly RF, Desir GV, Ellison DH. Rabbit distal convoluted tubule
coexpresses NaCl cotransporter and 11 beta-hydroxysteroid dehydrogenase II mRNA. Kidney Int 54: 464–472, 1998 [PubMed:
9690213]

111. Vitari AC, Deak M, Morrice NA, Alessi DR. The WNK1 and WNK4 protein kinases that are mutated in Gordon's
hypertension syndrome, phosphorylate and active SPAK and OSR1 protein kinases. Biochem J 391: 17–24, 2005 [PMCID:
PMC1237134] [PubMed: 16083423]

112. Vormfelde SV, Sehrt D, Toliat MR, Schirmer M, Meineke I, Tzvetkov M, Nurnberg P, Brockmoller J. Genetic variation in
the renal sodium transporters NKCC2, NCC, and ENaC in relation to the effects of loop diuretic drugs. Clin Pharmacol Ther 82:
300–309, 2007 [PubMed: 17460608]

113. Wade JB, Fang L, Liu J, Li D, Yang CL, Subramanya AR, Maouyo D, Mason A, Ellison DH, Welling PA. WNK1 kinase
isoform switch regulates renal potassium excretion. Proc Natl Acad Sci USA 103: 8558–8563, 2006 [PMCID: PMC1482529]
[PubMed: 16709664]

114. Wang Y, O'Connell JR, McArdle PF, Wade JB, Dorff SE, Shah SJ, Shi X, Pan L, Rampersaud E, Shen H, Kim JD,
Subramanya AR, Steinle NI, Parsa A, Ober CC, Welling PA, Chakravarti A, Weder AB, Cooper RS, Mitchell BD, Shuldiner AR,
Chang YP. Whole-genome association study identifies STK39 as a hypertension susceptibility gene. Proc Natl Acad Sci USA
106: 226–231, 2009 [PMCID: PMC2629209] [PubMed: 19114657]

115. Wei Y, Zavilowitz B, Satlin LM, Wang WH. Angiotensin II inhibits the ROMK-like small conductance K channel in renal
cortical collecting duct during dietary potassium restriction. J Biol Chem 282: 6455–6462, 2007 [PMCID: PMC2822470]
[PubMed: 17194699]
116. Wilson FH, Disse-Nicodeme S, Choate KA, Ishikawa K, Nelson-Williams C, Desitter I, Gunel M, Milford DV, Lipkin GW,
Achard JM, Feely MP, Dussol B, Berland Y, Unwin RJ, Mayan H, Simon DB, Farfel Z, Jeunemaitre X, Lifton RP. Human
hypertension caused by mutations in WNK kinases. Science 293: 1107–1112, 2001 [PubMed: 11498583]

117. Wilson FH, Kahle KT, Sabath E, Lalioti MD, Rapson AK, Hoover RS, Hebert SC, Gamba G, Lifton RP. Molecular
pathogenesis of inherited hypertension with hyperkalemia: the Na-Cl cotransporter is inhibited by wild-type but not mutant
WNK4. Proc Natl Acad Sci USA 100: 680–684, 2003 [PMCID: PMC141056] [PubMed: 12515852]

118. Xu B, English JM, Wilsbacher JL, Stippec S, Goldsmith EJ, Cobb MH. WNK1, a novel mammalian serine/threonine protein
kinase lacking the catalytic lysine in subdomain II. J Biol Chem 275: 16795–16801, 2000 [PubMed: 10828064]

119. Xu BE, Stippec S, Chu PY, Lazrak A, Li XJ, Lee BH, English JM, Ortega B, Huang CL, Cobb MH. WNK1 activates SGK1
to regulate the epithelial sodium channel. Proc Natl Acad Sci USA 102: 10315–10320, 2005 [PMCID: PMC1177404] [PubMed:
16006511]

120. Xu JZ, Hall AE, Peterson LN, Bienkowski MJ, Eessalu TE, Hebert SC. Localization of the ROMK protein on apical
membranes of rat kidney nephron segments. Am J Physiol Renal Physiol 273: F739–F749, 1997 [PubMed: 9374837]

121. Yamauchi K, Rai T, Kobayashi K, Sohara E, Suzuki T, Itoh T, Suda S, Hayama A, Sasaki S, Uchida S. Disease-causing
mutant WNK4 increases paracellular chloride permeability and phosphorylates claudins. Proc Natl Acad Sci USA 101: 4690–
4694, 2004 [PMCID: PMC384808] [PubMed: 15070779]

122. Yang CL, Angell J, Mitchell R, Ellison DH. WNK kinases regulate thiazide-sensitive Na-Cl cotransport. J Clin Invest 111:
1039–1045, 2003 [PMCID: PMC152590] [PubMed: 12671053]

123. Yang CL, Ellison DH. WNK1 interacts physically with WNK4. J Am Soc Nephrol 14: 77A, 2003

124. Yang CL, Zhu X, Ellison DH. The thiazide-sensitive Na-Cl cotransporter is regulated by a WNK kinase signaling complex.
J Clin Invest 117: 3403–3411, 2007 [PMCID: PMC2045602] [PubMed: 17975670]

125. Yang CL, Zhu X, Wang Z, Subramanya AR, Ellison DH. Mechanisms of WNK1 and WNK4 interaction in the regulation of
thiazide-sensitive NaCl cotransport. J Clin Invest 115: 1379–1387, 2005 [PMCID: PMC1074678] [PubMed: 15841204]

126. Yang SS, Morimoto T, Rai T, Chiga M, Sohara E, Ohno M, Uchida K, Lin SH, Moriguchi T, Shibuya H, Kondo Y, Sasaki
S, Uchida S. Molecular pathogenesis of pseudohypoaldosteronism type II: generation and analysis of a Wnk4(D561A/+) knockin
mouse model. Cell Metab 5: 331–344, 2007 [PubMed: 17488636]

127. Yoo D, Kim BY, Campo C, Nance L, King A, Maouyo D, Welling PA. Cell surface expression of the ROMK (Kir 1.1)
channel is regulated by the aldosterone-induced kinase, SGK-1, and protein kinase A. J Biol Chem 278: 23066–23075, 2003
[PubMed: 12684516]
Figures and Tables

Fig. 1.

Salt reabsorption pathways at the beginning of the aldosterone-sensitive distal nephron. NCC, Na+-Cl− cotransporter. ENaC, ep‐
ithelial sodium channel. DCT, distal convoluted tubule; CNT, connecting tubule. Inset: mechanisms for salt and calcium reab‐
sorption in DCT1. Arrow shows macula densa.

Table 1.

Functional properties of rat, mouse, and flounder NCC

Rat NCC Mouse NCC Flounder NCC

Na+Km, mM 5.5±1.0 7.2±0.4 30.0±6.0

Cl−Km, mM 2.6±0.6 5.6±0.5 15.0±2.0

Polythiazide IC50, μM 3 × 10−7 4 × 10−7 7 × 10−6

Data are means ± SE. Table uses information from Refs. 69, 70, 93, 108. NCC, thiazide-sensitive Na+-Cl− cotransporter.

Fig. 2.

Structure-function relationship model for NCC [from Moreno et al. (69)].

Table 2.

Effect of wild-type and mutant WNK4 or wild-type WNK1 on distal nephron transport systems

WNK4 S1169D WNK4 PHAII WNK4 WNK1

NCC ⇓ ⇓ ⇑

ROMK ⇓ ⇑ ⇓⇓ ⇓

ENaC ⇓ ⇑ ⇑ ⇑

Claudin 4 ⇑ ND ⇑⇑ ⇑

NKCC ⇓ ND ⇓ ND

ENaC, epithelial sodium channel. NKCC, apical bumetanide-sensitive Na+-K+-2Cl− cotransporter, PHAII, pseudohypoaldostero‐
nism type II.

Fig. 3.

Model for ANG II modulation of WNK4-SPAK-NCC interaction in normovolemia (A), in hypovolemia (B), and in patients with
PHAII type mutations (C) [from San Cristobal et al. (95)].