Hypertension: The Missing WNKs

20 The With no Lysine [K] (WNK) family of enzymes are central in regulation of blood pressure. 21 WNKs have been implicated in hereditary hypertension disorders, mainly through control of the activity 22 and levels of ion cotransporters and channels. Actions of WNKs in the kidney have been heavily 23 investigated, and recent studies have provided insight not only into the regulation of these enzymes but 24 also how mutations in WNKs and their interacting partners contribute to hypertensive disorders. 25 Defining the roles of WNKs in in the cardiovascular system will provide clues into additional mechanisms 26 by which WNKs can regulate blood pressure. This review summarizes recent developments in the 27 regulation of the WNK signaling cascade and its role in regulation of blood pressure. 28

Dbouk HA, Huang CL, Cobb MH.Hypertension: the missing WNKs.Am J Physiol Renal Physiol 311: F16 -F27, 2016.First published March 23, 2016; doi:10.1152/ajprenal.00358.2015.-TheWith no Lysine [K] (WNK) family of enzymes are central in the regulation of blood pressure.WNKs have been implicated in hereditary hypertension disorders, mainly through control of the activity and levels of ion cotransporters and channels.Actions of WNKs in the kidney have been heavily investigated, and recent studies have provided insight into not only the regulation of these enzymes but also how mutations in WNKs and their interacting partners contribute to hypertensive disorders.Defining the roles of WNKs in the cardiovascular system will provide clues about additional mechanisms by which WNKs can regulate blood pressure.This review summarizes recent developments in the regulation of the WNK signaling cascade and its role in regulation of blood pressure.cardiovascular; hypertension; WNK HYPERTENSION IS PREVALENT in ϳ80 million adults in the US alone, accounting for nearly one-third of the adult population (97).Hypertension is a major risk factor for cardiovascular disorders and stroke, with ϳ65,000 deaths attributed to high blood pressure in the United States in 2011 alone (19,97).Salt reabsorption in the kidney is central in the regulation of blood pressure; the relationship between blood pressure and renal sodium excretion (i.e., pressure-natriuresis relationship curve) is well known (18,32,49,147).Consistent with this notion, monogenic hypertensive and low-normal blood pressure (hypotensive) disorders are commonly caused by mutations in the genes modulating renal salt balance, including channels and cotransporters or the proteins controlling their surface expression and activity (82).Besides renal Na ϩ reabsorption and extracellular fluid volume, vascular tone is also an important factor in the pathogenesis of hypertension.The With No Lysine [K] (WNK) pathway is a major player in the control of blood pressure, initially identified as mutated genes in the familial hypertensive disorder known as pseudohypoaldosteronism type II (PHAII) (10,22,52,113,158,169,179,180).The function of WNKs in essential hypertension in the general population has recently been suggested (73,99,153,165).This review will focus on the participation of WNK family members in hypertension, highlighting their modes of regulating Na ϩ transport in the kidney as well as exploring their actions in the vasculature.

The WNK Pathway
WNKs are a family of serine/threonine protein kinases first discovered during a screen for novel mammalian MAP2Ks (174).The four members of this family have a conserved, N-terminal kinase domain and an autoinhibitory segment but are otherwise divergent in sequence and length.Numerous splice isoforms are expressed in a tissue-specific manner (25,93,137,157), but regulation of the expression and splicing of these enzymes is not well studied.The control of WNK expression is particularly interesting in light of recent papers showing that clustered regularly interspaced short palindromic repeats-mediated knockout of WNK1 leads to compensatory expression of other isoforms (127), suggesting unknown cellular mechanisms regulating the levels of WNK pathway components to maintain a steady-state WNK expression.This family of kinases is characterized by a unique positioning of the catalytic lysine in the glycine-rich loop in ␤ strand 2 instead of the canonical position in ␤ strand 3 (92,174).The altered localization of the catalytic lysine may allow phosphorylation of otherwise inaccessible substrates (55), and a recent study has shown that this unusual active site positioning allows sensing of intracellular Cl Ϫ concentration (115).Cl Ϫ binds to a site formed by residues including leucine-369 and leucine-371 within the WNK kinase active site to maintain the inactive conformation (115).Thus decreases in intracellular Cl Ϫ activate the kinase, with the different WNK isoforms sensitive to different chloride concentrations (7,115,151).This places WNKs as chloride-sensing kinases in the cell, which may play central roles in chloride ion-dependent signaling (115,136).However, chloride binding is not the only mechanism for WNK activation, as the enzymes are also activated by hyperosmotic stress (185), which would induce cell shrinkage and lead to high intracellular Cl Ϫ , a condition where WNKs would be expected to be inhibited due to the increased intracellular Cl Ϫ but are in fact activated.One possible mechanism to explain increased WNK1 activity under conditions of high intracellular Cl Ϫ involves the fact that WNK4 is inhibited at lower chloride concentrations than WNK1 (151), which would block some of the antagonistic functions of WNK4 (discussed below) and relieve WNK1 inhibition, thereby indirectly activating the WNK1 signaling pathway (52).The exact mechanisms by which hyperosmotic stress leads to increased WNK activity are still unclear, suggesting additional mechanisms for WNK activity regulation that are still uncharacterized.
In addition to the kinase domain near its N terminus containing the unique catalytic lysine placement, all WNKs contain an autoinhibitory domain directly C terminal to the kinase domain as well as two to three coiled coils, but are otherwise largely structurally uncharacterized (87,175).Cursory analysis of these uncharacterized regions suggests that they are intrinsically disordered.Intrinsically disordered proteins (IDPs) are a group of proteins with low-complexity sequences and which can undergo numerous posttranslational modifications and may act as interaction hubs with an impact on many cellular processes (170).In WNKs, these low-complexity regions include many proline-rich regions that may interact with SH3 domain-containing proteins, as has been shown for intersectin and ArhGAP12 interaction with WNKs (55,106).Disordered regions of IDPs can undergo reorganization and folding upon binding to associated proteins or following posttranslational modification (6,45).This suggests that modifications and interactions of WNKs may cause structural changes that can be essential for function.The significance of WNK interactions in their role as signaling hubs is clear from the kinase-independent functions of WNKs, with WNKs functioning as scaffolds to mediate cellular roles, ranging from the endocytosis and plasma membrane localization of ion transporters and channels (55,176,177) to the regulation of splicing factor activity (80).Indeed, an Akt-mediated phosphorylation event on WNK1 has been shown to mediate kinase-independent activation of the serum-and glucocorticoid-induced protein kinase SGK1 (17,57,68,160,177).These apparently kinase-independent functions of WNKs need to be further explored, as the range of interacting partners identified suggests widespread roles for these enzymes in cells.One must note that the exact mechanisms behind many of these kinase-independent functions of WNKs are not clear, and whether the kinase function is still required along with the scaffolding function has not been unequivocally determined.One such example is regulation of Nedd4-2 by WNK1, which is purported to be via kinase-independent activation of SGK1 (57,176,177), but WNK1 and WNK4 can also directly phosphorylate Nedd4-2 (57), and WNK3 can regulate Nedd4-2 independently of SGK1 and SPAK (75).Furthermore, SGK1 can phosphorylate WNK4 at multiple C-terminal residues, regulating its activity (123,128,130).These observations suggest complexity in WNK functions, with both kinase-dependent and -independent mechanisms acting simultaneously to mediate functions.

Regulation of WNK Activity in Cells
As depicted in Fig. 1, there are multiple modes of regulation of WNK activity in cells.The catalytic activity of the enzymes is altered by osmotic stress, with both hyperosmotic and hyposmotic conditions resulting in increased activation of the enzymes (81,174).The mechanism of activation by hyposmolality is likely due to decreases in intracellular Cl Ϫ resulting from cell swelling and Cl Ϫ efflux when exposed to a hypotonic Cl Ϫ extracellular milieu.Cl Ϫ binding controls WNK kinase activity by blocking autophosphorylation (147).As mentioned above, hypertonic stress likely activates WNKs through a different mechanism, and many questions remain regarding activation by Cl Ϫ -independent mechanisms.The second mode of regulation of WNK signaling is through control of their abundance, a major factor in patients with PHAII (10,104,149,155,169). WNK1 mutations in PHAII, mainly deletions within intron 1, result in increased WNK1 mRNA abundance likely from enhanced gene transcription (10,158).Mutations in WNK4 are missense mutations resulting in decreased degradation (104,132,163).While mutations of WNK1 and WNK4 are recognized as important causes of PHAII, they only account for mutations in ϳ70% of affected patients (51).Recent findings on additional genes mutated in PHAII patients have shed light on how amounts of WNKs are regulated in normal physiology.Tight control of WNKs is thought to be mediated by the ubiquitin-proteasome system through cullin3 and Kelchlike 3 (KLHL3), mutations of which in PHAII result in increased WNK accumulation (11,84).KLHL3-mediated regulation of WNK4 is also mediated by autophagy, in a p62dependent manner and independent of the proteasome (94), while KLHL2 stability is also regulated by autophagy (186), pointing to additional mechanisms for control of WNK levels and degradation.WNKs bind to the E3 ubiquitin ligase cullin3-KLHL2/3 complex via an acidic motif located adjacent to the autoinhibitory region and conserved in all four WNK family members (95,163).Interaction with cullin3 mediates ubiquitination of WNKs and enhances degradation, leading to reduced WNK signaling and maintaining the steady-state levels of the enzymes (11,84,88,95,131,132,163,186).These new findings explain why missense mutations of WNK4 in the acidic motif and loss-of-function mutations in the cullin3-KLHL2/3 complex lead to increased WNKs and similar phenotypes (4,11,43,84,85,88,95,104,131,140,146,154,155,163,186).The interaction of WNKs with KLHL2/3-cullin3 is a regular target of stimuli affecting WNK signaling such as angiotensin II, underlining the significance of this regulatory mechanism (139,184,186).Other less-understood mechanisms of WNK regulation include control of their oligomerization and their subcellular localization (Fig. 1).Many enzymes are regulated by subcellular localization, with signaldependent targeting to cellular compartments that places them in proximity to their activators, inhibitors, or substrates.In the case of WNKs, the enzymes are typically localized in the cytosol with a punctate distribution, possibly on vesicular structures (134,174).Through fluorescence recovery after photobleaching experiments, WNK mobility in the cytosol has been shown to decrease upon hyperosmotic or hyposmotic stresses (134), possibly due to relocalization of WNKs to vesicles derived from the trans-Golgi network or recycling endosomes (135,185).While this is another potential mechanism of modulation of WNK activity and function, the subcellular localizations of WNK are still not clear and need to be determined, and furthermore some studies on localization and mobility have used overexpressed, tagged constructs that do not always replicate endogenous WNK behavior.An additional level of regulation is the ability of WNKs to homo-and hetero-oligomerize (81).Oligomerization can lead to inhibition or activation of kinases in the multimer and may be essential for proper WNK downstream signaling (15,152,166).Oligomerization may also regulate the localization of WNKs in a tissue-specific manner based on which family members and splice forms are expressed and their isoform-specific interactions with different proteins, thus providing a deeper level of complexity to WNK signaling (Fig. 1).

WNK Targets and Effectors
Activation of WNKs leads to phosphorylation and activation of their substrates, two sterile 20 (ste20)-related kinases, oxidative stress responsive-1 (OSR1), and ste20/SPS1-related proline/alanine-rich kinase (SPAK; also known as PASK) (39,96,116,161,162).These enzymes are closely related, with SPAK originating from gene duplication of OSR1 (28), and they display overlapping but also distinct tissue-specific expression (37).Both enzymes contain an N-terminal kinase domain, with SPAK having a stretch of Ͼ70 amino acids rich in proline and alanine residues preceding the kinase domain compared with the fewer than 20 amino acid stretch ahead of the kinase domain in OSR1 (28).Both OSR1 and SPAK have two other shared domains, PASK/Fray homology domains 1 and 2 (PF1 and PF2).The PF2 domain is central in the WNK signaling pathway (37).PF2 domains in OSR1 and SPAK can interact with RFxV motifs present in WNKs, which facilitates their activation, and in downstream targets (5,159), which allows them to bind, phosphorylate, and regulate the activity of the electroneutral cation-chloride cotransporters of the SLC12 family, including the Na ϩ -Cl Ϫ cotransporter (NCC), the Na ϩ -K ϩ -2Cl Ϫ cotransporters 1 and 2 (NKCC1 and NKCC2), and K ϩ -Cl Ϫ cotransporters (KCCs) (5,26,39,96,116,133,134,161,162).Members of this family of cotransporters are important regulators of physiological ion homeostasis, and deregulation of their function via direct mutations or disrupted regulation causes various pathological disorders (38,56).Recently, calcium binding protein 39 (Cab39)/mouse protein 25 (MO25) has been identified as a novel player in the WNK signaling pathway, which can act as a potentiator of the pathway by enhancing OSR1/SPAK activation, possibly via dimerization (35,40,117).Cab39 can also bind to WNK4 and allows direct phosphorylation of NKCCs by WNK4, independently of SPAK (118).While there is general consensus on the signaling pathways following WNK activation, there is still some controversy and discrepancy regarding the actions of WNK4 (62).Early in vitro and in vivo studies supported the notion that WNK4 inhibited NCC activation and cell surface amounts (12,41,77,180,181).The mechanism for this inhibitory impact of WNK4 was mediated by antagonizing WNK1 activity (180,181), which could be through heteromerization between WNK4 and WNK1 (15).However, later studies showed that both WNK1 and WNK4 can activate OSR1/ SPAK and increase the activity of NKCC1/2 (5), while WNK4 knockout mice were hypotensive and display decreased SPAK levels and phosphorylation as well as decreased NCC abundance (13,150), in contrast to the previously observed data from mice expressing WNK4 from a bacterial artificial chromosome (77).As such, the exact role of WNK4 is still debated, even though more recent evidence from WNK4 knockout mice suggests that it plays a similar stimulatory role in NCC signaling (62).In addition to the classic kinase-dependent activity, WNKs can also regulate ion homeostasis through kinaseindependent functions, as seen through their intersectin-dependent control of endocytosis of the renal K ϩ channel ROMK (55).Another example of kinase-independent WNK functions involves the control of SGK1 activation, which then results in SGK1-dependent inhibition of Nedd4-2 activity to block ubiquitination and internalization of the epithelial Na ϩ channel (ENaC) (57,75,176).While the focus has centered on the role of this pathway in the kidney, more recent studies have looked at WNK functions in extrarenal tissues (142), most notably the cardiovascular system (see below).
Inputs from multiple stimuli can control WNK pathway activity through different mechanisms.These include hormones such as angiotensin II, aldosterone, insulin and even ovarian hormones which can activate WNK, both WNK1 and WNK4, signaling pathway to regulate the Na ϩ , Cl Ϫ cotransporter (NCC) (13,14,34,71,72,100,124,126,128,129,145,156).One mechanism is through regulation of WNK expression and protein stability, with angiotensin II modulating KLHL2 expression in vascular smooth muscle cells (139,186).Phosphorylation of KLHL3 by PKC in Cos-7 cells is also regulated by angiotensin II and prevents binding to WNK4, thereby controlling the levels of WNKs in cells and subsequently pathway activity (139,186).The same site on KLHL3 which is phosphorylated by PKC can also be phosphorylated by PKA and Akt, providing additional inputs that can regulate WNK protein levels and stability (184) and suggesting mechanisms of WNK regulation by insulin and other growth factors.Furthermore, hormones can regulate the expression and splicing of different WNK isoforms as well as downstream signaling mediators such as SPAK, adding to the complexity of mechanisms by which WNKs can be regulated (33,98,124,126).Hormones can also directly activate WNK signaling, with angiotensin II, leading to SPAK phosphorylation and NCC activation, through direct activation of WNK1 and WNK4, and possibly through inhibition of the negative impact of WNK4 on WNK1 signaling (13,14,34,71,129).Interestingly, the activation of SPAK and NCC by angiotensin II is abrogated by WNK4 knockdown (13).Similarly, aldosterone can activate the WNK-SPAK-NCC pathway (72,156) as well as regulate WNK signaling via SGK1, which can lead to WNK4 phosphorylation and can result in increased ENaC membrane levels and activity due to its effects on Nedd4-2 (120,123,128).Aldosterone can also control the expression of WNK1 through microRNA (miRNA) and alternative splicing (33,98,126), suggesting multiple layers of input of this hormone on the WNK pathway.It is clear that WNK signaling is intricately regulated at expression and activity levels by the action of different hormones and growth factors, and the exact mechanisms need to be further elucidated to provide a complete overview of WNK activity and signaling in cells.

WNKs in the Kidney
The kidney is the major site of regulation of total body ion and fluid homeostasis.Following glomerular filtration of blood, ϳ60 -70% of filtered sodium is reabsorbed in the proximal tubule primarily by the action of the Na ϩ /H ϩ exchanger isoform 3 (NHE3), 20 -30% in the thick ascending limb (TAL) of the loop of Henle by NKCC2, and ϳ10% in the distal convoluted tubule (DCT) and collecting duct by NCC and ENaC, respectively.Most studies on the role of the WNK pathway in regulating renal Na ϩ reabsorption have focused on NCC, NKCC2, and ENaC.The significance of Na ϩ reabsorption in these segments in blood pressure regulation is highlighted by several Mendelian disorders altering the function and regulation of ion channels and cotransporters, leading to hypotension or hypertension.These include loss-of-function mutations in NKCC2 and NCC leading to Bartter's syndrome and Gitelman's syndrome, while gain-of-function mutations of ENaC lead to Liddle's syndrome (141,143,144).The regulation of the diverse ion channels and cotransporters in the kidney is mediated by many signaling pathways, with WNKs playing important roles in controlling their activities and functions.Mutations affecting the WNK pathway have been extensively studied in terms of their renal functions.Mutations in WNK1 and WNK4 or in their regulatory interacting partners, KLHL3 and cullin3, cause PHAII (11,84,169).PHAII, also referred to as familial hyperkalemic hypertension (FHHt) or Gordon's syndrome, is characterized by hypertension, hyper-kalemia, hyperchloremic metabolic acidosis, and a normal glomerular filtration rate (44).All the mutations in PHAII seem to converge on a single mechanism, increasing WNK1 and/or WNK4 proteins in cells.As discussed above, mutations in WNK1 are intronic, causing large deletions (41 or 22 Kb) in intron 1, leading to overexpression of full-length WNK1 relative to kidney-specific WNK1 (a shorter alternatively spliced isoform with antagonistic function to the full-length isoform; see below) in the DCT in PHAII patients (15,23,158,169).In contrast, mutations in WNK4 are in the coding sequence, causing missense mutations (169).These mutations cluster in an acidic region following the kinase domain, which is conserved in all WNK isoforms, and is responsible for interaction with ubiquitin ligases cullin3/KLHL3 that target WNKs for degradation (104).Interestingly, mutations in KLHL3 and cullin3 inhibiting interaction with WNKs impair degradation, thus increasing WNK levels (11,84,104,149,155,163,171).The cullin3-KLHL3 complex can also autoregulate to indirectly alter WNKs.Some activating mutations in cullin3 increase WNK ubiquitination but decrease its degradation due to simultaneous ubiquitination and degradation of KLHL3 (88).Some KLHL3 mutations lead to decreased stability of the KLHL3 protein itself or to decreased association with cullin3, thereby increasing WNK indirectly (95).Overall, in patients with PHAII mutations in WNK1, WNK4, cullin3, or KLHL3, the abundance of WNK1 and/or WNK4 is increased (155).Increased WNKs increase Na ϩ reabsorption via NCC, NKCC2, ENaC, and potentially other transport pathways (such as paracellular transport via regulation of claudin phosphorylation) via OSR1/SPAK or other kinase-independent mechanisms, and lead to hypertension (113,147).
The four WNKs in mammals are each encoded by separate genes (70).Over three dozen alternatively spliced variants of the WNK1 gene have been identified in mice and humans, one being kidney-specific KS-WNK1 (25,137).WNK2 is not present in the kidney.Under normal conditions, WNK1 variants, including KS-WNK1, WNK3, and WNK4, are expressed in the kidney tubules, including the DCT.WNK3 has been suggested to have a minimal role in the kidney in vivo (105), despite its ability to regulate NCC and ROMK in vitro (75,121).It is possible that deletion of WNK3 in mice is compensated by upregulation of WNK1 and WNK4 and activation of the WNK-SPAK pathway (90,105).KS-WNK1 is initiated from a start site within an alternatively spliced promoter within intron 4 in the WNK1 gene, which results in a shorter protein lacking the majority of the kinase domain and is therefore catalytically inactive (25,108,174,178).The function of KS-WNK1 is best viewed in the context of its effects on other WNK isoforms, with its ability to bind to substrates and compete with and inhibit the functions of other WNKs (148).Via inhibition of other WNK isoforms, KS-WNK1 can indirectly increase ROMK activity and inhibit NCC (16,79,148).However, some data suggest that KS-WNK1 may stimulate ENaC following aldosterone stimulation (98), although the significance of that action is not clear.Knockout of KS-WNK1 in mice resulted in upregulation of NCC and NKCC2 and downregulation of ENaC, as well as decreased K ϩ secretion due to downregulation of ROMK.The mouse phenotype manifested in mild water and Na ϩ retention with decreased aldosterone secretion, which resulted in increased blood pressure with a high-salt diet (16,53,83).KS-WNK1 expression is increased in PHAII patients exhibiting deletions in intron 1 of the WNK1 gene (23), but the impact on signaling might be masked by the significant simultaneous upregulation of WNK1, which may result in more significant changes to counteract any effects caused by KS-WNK1.Mutations in WNK3 have not been reported in PHAII.The role of WNK4 in the kidney and PHAII has recently been extensively reviewed (51,62,147).

WNKs in the Vasculature
Contractility of resistance vessels is a major determinant of systemic vascular resistance and thus of blood pressure.The contribution of WNKs to vascular control of blood pressure is poorly studied, even though abundant evidence indicates that WNKs play important roles in blood vessel homeostasis and development.Expression analysis of endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) by quantitative RT-PCR shows that WNK1 and WNK3 are the major isoforms expressed in both cell types, while WNK2 and WNK4 are not detected at mRNA or protein levels; both WNK1 and WNK3 are expressed approximately equally in VSMCs, while significantly less WNK3 is expressed compared with WNK1 in ECs (21, 186) (Fig. 2).However, WNK4 has been detected in the specialized endothelium of the blood-brain barrier, but not in the endothelium of other tissues, suggesting possible unique functions for WNK4 within that endothelial setting (69).WNK3 has important roles in VSMCs to control vascular contraction and tone.Accordingly, WNK3 Ϫ/Ϫ mice display decreased blood pressure due to decreased vasoconstriction (105).WNK3 regulates angiotensin II-dependent vascular contraction via the SPAK-NKCC1 pathway, with KLHL2 levels and activity also being downregulated to increase WNK3 amounts following angiotensin II stimulation (186,187).WNK1-null mice die during embryonic development due to angiogenesis defects, which can be rescued by endothelialspecific expression of WNK1 or constitutively active OSR1 (172,173).WNK1 ϩ/Ϫ mice are viable and display hypotension due, at least in part, to decreased vascular contraction of VSMCs ( 9), but WNK1 had no role in angiotensin-II induced SPAK and NKCC1 activation in VSMCs (186).The requirement for an activated WNK pathway in VSMC contraction is highlighted by the fact that SPAK ϩ/Ϫ and SPAK Ϫ/Ϫ mice also display hypotension from decreased contractility of VSMCs (182).WNK1 and WNK3, via OSR1 or SPAK, can activate NKCC1 (119,121,186).NKCC1 is essential for vasoconstriction, as evidenced by the phenotype of NKCC1 Ϫ/Ϫ mice, which are hypotensive due to decreased VSMC contractility (2,42,109).Similar effects are seen with loop diuretics, such as bumetanide, that target NKCC1 and lead to a hypotensive phenotype due to effects on VSMCs and vascular tone (2,42,91,109).However, it is important to note that, due to its pharmacokinetic properties, bumetanide has a major effect on NKCC2 in the kidney rather than vascular NKCC1, as NKCC2 is inhibited at lower concentrations than those required for NKCC1 inhibition (46,50,54,78,86,101). Interestingly, vasoconstrictors, vasodilators, and changes in blood pressure can also regulate NKCC1 activity (2,3,66), suggesting a feedback mechanism that is yet to be defined but could involve modulation of WNK amounts and activity.VSMCs also express ENaC, where it can act as a mechanosensor to mediate stretch-induced VSMC contraction, which is sensitive to amiloride (29,47,64,65).The membrane localization of ENaC in VSMCs is essential for activity, which is controlled in part by WNK-SGK1-Nedd4-2 (176), and its importance in VSMCs points to further potential impact of WNKs on vascular tone and blood pressure control (167).VSMCs also express tran-Fig.2. WNKs in the vasculature.Both endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in blood vessels predominantly express WNK1 and WNK3, with WNK1 being the major isoform in ECs while both isoforms are expressed to similar levels in VSMCs but play different roles (187).Both WNK1 and WNK3 can regulate multiple ion cotransporters and channels, as well as various signaling pathways that control vascular contractility, which contributes to blood pressure.The pathways presented here include defined WNK functions in the vasculature, as well as hypothesized pathways based on the expression of the ion cotransporters and channels in the ECs and VSMCs, coupled with previously characterized WNK interactions and signaling and known functions of these ion cotransporters and channels in the vasculature.See the text for additional definitions.sient receptor potential channels TRPC3, TRPC6, and TRPM4, which are permeable to Ca 2ϩ and to a lesser extent Na ϩ .Knockdown of these channels in isolated arteries results in loss of pressure-induced depolarization and constriction (30,31,168,183).The exact effects of WNK1 or WNK3 on these channels have not been studied, but WNK1 and WNK4 have been shown to regulate surface expression of other TRP channels (36).Furthermore, WNK4, which is not considered a major isoform expressed in VSMCs, has been shown to regulate vascular tone via negative regulation of TRPC3, and this regulation is compromised by WNK4 mutations identified in PHAII patients (111), supporting extrarenal effects of WNK mutations on blood pressure.VSMCs also express KCCs, possibly KCC1 and/or KCC2, and their activation contributes to vasodilation (1).WNK1 and WNK3 can inhibit KCC function (27,122), consistent with the evidence that WNK activity is required for VSMC-mediated vascular constriction.
In the endothelium, WNK1 is the main isoform expressed (Fig. 2).WNK1 was shown to be expressed in the cardiovascular system using a transgenic mouse bearing a WNK1 bacterial artificial chromosome (24).WNK1 knockout results in severe developmental defects in the cardiovascular system with marked angiogenic defects displayed by ECs, which can be partially rescued in vivo and in cell culture models by activation of the WNK-SPAK/OSR1 signaling pathway (21,172,173).The requirement of endothelial WNK1 for angiogenesis is evidenced by findings that both global and endothelial-specific WNK1 deletion exhibit similar phenotypes of embryonic lethality from angiogenesis defects (76,172,173).WNK1-SPAK signaling is required for endothelial proliferation (21).In the endothelium, NKCC1 is activated following growth factor stimulation by a Cl Ϫ -dependent mechanism (67,107), and blocking NKCC1 function using bumetanide or furosemide inhibits EC proliferation (110).However, the impact of NKCC1 in ECs on vascular contractility is not clear, as endothelium-denuded vessels still display vasodilation in response to loop diuretics (109).ECs also express ENaC (48,164), which can cause swelling of ECs in response to aldosterone and stiffening in response to high extracellular Na ϩ (63,102,103).Furthermore, supporting a role for this channel in mediating shear stress responses in ECs, laminar flow increases ENaC activity in ECs (48,164).This shear-mediated activation of ENaC results in endothelial stiffening and leads to decreased endothelial nitric oxide synthase (NOS) activity, which ultimately results in increased VSMC contraction (48,103,114).It has been suggested that hypertension in Liddle syndrome due to global gain-of-function mutation of ENaC could be partly due to increased endothelial ENaC activity resulting in EC stiffening and increased vascular contractility, rather than simply from increased ENaC-mediated Na ϩ retention (63).A recent study showed that increased endothelial ENaC activity and Na ϩ influx inhibits the transport of L-arginine and subsequently NO production, in a hemoxygenase-dependent manner (48).The mechanism by which ENaC regulation controls NOS is also suggested to be phosphoinositide 3-kinase (PI3K) dependent and might be due to direct phosphorylation of NOS by SGK1 (58,114).This suggests that Akt-mediated WNK phosphorylation, which leads to SGK1 activation and decreased ENaC endocytosis, could also inhibit NO production in the endothelium, thereby indirectly regulating vascular contractility and blood pressure through two simultaneous mechanisms.However, WNK1 deficiency in a WNK1 ϩ/Ϫ mouse results in hypocontractility which is independent of NO and not affected by NOS inhibitors (9), and as such the impact of WNK1 on NO levels needs to be further studied and characterized.Other molecules influencing EC-VSMC cross talk to regulate vasoconstriction could also be linked to WNKs.One example is endothelin-1, which is regulated by WNK1 in ECs (21) and can stimulate VSMC contraction ( 89) by an NKCC1-dependent and Cl Ϫ concentration-dependent mechanism (20).ECs also express TRPV4, a Ca 2ϩ and Na ϩ channel, which can be activated by diverse stimuli, including osmotic cell swelling, shear stress, and low intravascular pressure, to induce vasodilatory responses (60).WNK1 decreases TRPV4 at the membrane and thereby decreases its function (36), consistent with the role of WNK1 in inducing vasoconstriction.In contrast, WNK3 has been shown to positively regulate TRPV5 and TRPV6 (188); however, its impact on TRP channels expressed in ECs and VSMCs has not been demonstrated, but this could suggest opposing functions for different WNKs in regulating these channels.ECs also express KCC3 (59), which is inhibited by WNK1 (122).The role of KCC3 in the endothelium has not been studied, but KCC3 mRNA was increased following stimulation of HUVECs with VEGF (59) and IGF1 stimulation of NIH3T3 fibroblasts (138).KCC3 overexpression in fibroblasts enhanced proliferation, an effect which was blocked by inhibition of KCC3 activity (138), although whether KCC3 has a similar role in endothelial cells remains to be explored.
The impact of WNKs on the vasculature is multifaceted, with a current major focus of study on the canonical signaling to OSR1/SPAK and regulation of amount, activity, and localization of ion cotransporters and channels that play a major role in vascular contractility and blood pressure control.However, emerging evidence suggests that WNKs may have vascular roles independently of SPAK/OSR1 signaling, both kinase dependent and independent (21,76,142).These may include effects on angiogenic sprouting and vessel development and organization, with WNKs regulating transcription factors, splicing factors, and other signaling mediators that can affect vessel integrity and functions (21,80).Our understanding of WNKs' roles in the vasculature is limited compared with our broader knowledge of its renal functions, and further studies need to analyze these functions and the full breadth of WNK pathways in ECs and VSMCs.

Conclusions
WNK kinases are central regulators of ion homeostasis and blood pressure.The roles of WNKs have been extensively studied in the kidney, particularly in the regulation of NCC in DCT.Their actions in regulating transporters in other nephron segments require further study.Recently, greater interest in the relevance of WNKs in extrarenal tissues has emerged (23,61,69,112,142), particularly due to the ever-expanding network of WNK-interacting proteins and -regulated signaling networks.This is important as mutations of WNKs affect tissues outside the kidney (74,125).Also, altered WNK functions in the vasculature may conceivably contribute to hypertension in patients with PHAII.While we have highlighted the known contributions of WNK1 and WNK3 in the vasculature, as well as presented potential pathways that WNKs can regulate in ECs and VSMCs based on known interactors, the study of WNKs in vascular biology is still in its infancy.Studies defining contributions of WNKs to vascular function, using primary cells as well as inducible vascular tissue-specific WNK1 or WK3 knockout mice, will provide insight into the roles of WNKs in vascular contractility/relaxation and the mechanisms controlling them, and results could extend to cardiovascular disease and stroke (8,189).Further understanding of WNKs will expand our knowledge of their roles beyond blood pressure control.

Fig. 1 .
Fig. 1.Complexity in regulation of With no Lysine [K] (WNK) levels and activity.The regulation of WNK signaling occurs through several mechanism, with the canonical hyper-or hypotonic stress-mediated activation of OSR1/SPAK and regulation of ion cotransporters (top left) being extensively studied.Recent data have shown other regulatory mechanisms, with KLHL2/3-cullin3 ubiquitinating (ub) WNKs and controlling their levels and degradation (top right), regulation of WNK localization in the cell with an uncharacterized cytoplasmic/vesicular distribution that is altered upon osmotic stress (bottom left), and finally WNK oligomerization (bottom right) controls activity in a positive fashion via reciprocal activation or negative inhibition mediated by the autoinhibitory (AI) domain.