Familial Hyperkalemic Hypertension

Familial hyperkalemic hypertension (FHHt) syndrome (1,2), also known as Gordon syndrome (3) or pseudohypoaldosteronism type 2 (4), is a rare inherited form of low-renin hypertension associated with hyperkalemia and hyperchloremic metabolic acidosis in patients with a normal GFR (OMIM no. 145260). This monogenic form of arterial hypertension has excited new interest since the discovery of a new, unsuspected molecular pathway that is responsible for both the biochemical abnormalities and the increase in BP observed. Genetic analysis has led to the identification of mutations in two genes that belong to a new family of kinases, the WNK family. Although the physiologic functions of these kinases and the pathophysiology of FHHt are not completely solved, these results have opened up major new avenues toward understanding the regulation of ion handling in the aldosterone-sensitive nephron. Chasing the Gene Phenotypic and Genetic Heterogeneity Since the first description of the disease by Paver and Pauline in 1964 (1), approximately 50 other cases and families have been reported (5,6). The original case was a 15-yr-old boy with severe hypertension (180/120 mmHg) and very high potassium levels (7.0 to 8.2 mmol/L). Detailed analyses using different diets and pharmacologic stimuli showed that the kidney was probably involved but that the renal tubule reacted normally to an acid load and to carbonic anhydrase inhibitor. Sensitivity to thiazide diuretics was reported a few years later in unrelated, affected individuals (2,7). Gordon’s group reported their first case in 1970 (8) and helped to demonstrate the existence of a unifying syndrome (5). High levels of variability, in terms of age at diagnosis, which may vary from the first few weeks of life (9) until late in adulthood (10), have been reported in sporadic and familial cases. We observed a similarly high level of variability in the first 14 FHHt families studied at our center (6), with the age at diagnosis of index cases ranging from 7 mo to 39 yr. An analysis of the affected and unaffected individuals of our largest kindred showed no relationship between the severity of biochemical abnormalities and age or BP, which seemed to depend primarily on age in affected individuals (11). This phenotypic variability, associated with sensitivity to thiazides—which are widely used in hypertension—and with a low probability of this rare disease’s being recognized by most doctors may have led to an underestimation of its frequency. The mode of inheritance of the disease is consistent with autosomal dominant transmission in most, if not all, of the pedigrees reported. However, we have identified two families in which the parents of the affected individuals were first cousins, suggesting possible autosomal recessive transmission. This would provide further evidence of the genetic heterogeneity of the disease. Indeed, three loci have already been implicated in this disease, and additional loci probably are responsible for the same apparent phenotype. Genomic analysis of kindreds with FHHt revealed no linkage with SLC12A3, encoding the thiazide-sensitive NaCl co-transporter (NCC) (12 and our own data). In 1997, Lifton’s group at Yale University demonstrated locus heterogeneity of the trait, with a multilocus logarithm of odds score of 8.1 for linkage to two loci: a 20- to 33-cM interval on chromosome 1q31-q42 (PHA2A locus) and a 21- to 43-cM linkage interval on chromosome 17p11-q21 (PHA2B locus) (13). Our analysis of a large French pedigree led to the identification of a new locus on chromosome 12p13.3 (14). We were able to exclude linkage with the previously identified loci and with SLC12A3 in three other French kindreds, demonstrating the involvement of at least one other gene in the disease (15). Thus, at least four different genes are responsible for FHHt, suggesting that this syndrome is actually a set of related disorders (Table 1). Two of these genes were identified recently (16). Identification of the WNK1 and WNK4 Genes A fruitful collaboration with Lifton’s group led to the identification of deletions and missense mutations in two genes encoding members of a novel family of serine/threonine kinases. These two genes, WNK1 and WNK4, were shown to be responsible for the disease in six families (16). One large genomic deletion (41 kb) was found in an American family, and a smaller deletion (22 kb) was found in a French family. Both of the deletions that were found in these unrelated kindreds were located in the same part of the first intron of the WNK1 gene. No mutation was identified in the coding sequence of the gene. WNK1 is located at the telomeric part of chromosome 12 (12p12.3). It contains 28 exons in a 156-kb segment. This segment is particularly large as a result, in part, of the large size (60 kb) of intron 1 (NM_018979.1). Several WNK1 isoforms have been identified (see below). An analysis of WNK1 transcript levels in leukocytes from affected and unaffected members of one of these FHHt families revealed that the intronic deletion was associated with five times higher levels of its expression (16). The identification of mutations in WNK1 and knowledge of the existence of a putative PHA2 locus on chromosomes 1 and 17 led to the identification of WNK4, located on 17q21-q22. This gene contains 18 exons in a 16-kb segment (NM_032387). WNK1 and WNK4 have similar sequences and intron-exon organizations, but the WNK4 mutations that were found in several FHHt kindreds differ considerably from the WNK1 mutations identified. The WNK4 mutations are missense mutations that affect short (approximately 10 amino acids) sequences that are highly conserved in the WNK family, immediately downstream from the first and second coiled-coil domains (Figure 1). They result in the substitution of a charged residue in these negatively and positively charged sequences (16,17). As coiled-coil domains are generally thought to be involved in protein–protein interactions, the mutations may affect the interaction of WNK4 with its partners. WNK Kinase Structure WNK (with no lysine [K] [18]) proteins form a small family of serine/threonine kinases that were identified by Xu et al. (19). They lack a conserved lysine that usually is found in subdomain II of the catalytic domain that is critical for ATP binding to the catalytic site and strictly conserved in all other serine/threonine kinases that have been identified to date. In WNK, this lysine is replaced by a cysteine, and the catalytic lysine is located in subdomain I. This new subfamily of protein kinases has been found only in multicellular organisms. Four members of the family have been identified in humans: WNK1, WNK2, WNK3, and WNK4, located on 12p13.3, 9q22.31, Xp11.22, and 17p11-q21, respectively (16,20,21). New insight recently has been provided into the structure and function of these kinases. The structure of the kinase domain of WNK1 has been resolved at a resolution of 1.8 A (22). This structure has confirmed that the lysine residue that is responsible for kinase activity is located in strand β2 rather than in β3 as in other protein kinases and the precise conformation of the activation loop and has identified residues that contribute to substrate specificity. These findings should facilitate the design of WNK1 inhibitors. In addition to the catalytic domain located near the N-terminus, WNK1 and WNK4 contain an autoinhibitory domain, two predicted coiled-coil domains, and three proline-rich regions that may interact with the SH3 domains of other proteins, all of which are strongly conserved in WNK (23). Several in vitro studies have shown that WNK oligomerize and that WNK1 may act as a tetramer (24). WNK1 phosphorylates itself and a generic substrate (19,25). WNK1 activation requires the autophosphorylation of at least one serine residue, Ser-382, within the WNK1 activation loop, and is affected by extracellular ion concentration (19). Changes in NaCl concentration and other osmotic challenges activate the kinase in kidney epithelial cells and in a variety of cell lines, suggesting that WNK1 acts as an osmotic sensor (26). The autoinhibitory sequence is 55 residues long and is located just after the kinase domain. It regulates the kinase activity of WNK1 and probably also its interactions with substrates and/or partners. The WNK1 autoinhibitory domain was shown recently to inhibit the autophosphorylation of WNK4, suggesting that these two kinases may belong to the same cascade (26). WNK1 probably has many functions. It has been shown to be involved in the extracellular signal-regulated kinase 5 mitogen-activated protein kinase pathway, suggesting that it may be regulated by the growth factors and stress stimuli that control this cascade (26). WNK1 has also been demonstrated to be one of the numerous substrates of Akt/protein kinase B, a kinase involved in the metabolic and mitogenic functions of insulin (27). WNK1 interacts with and phosphorylates synaptotagmin 2 (Syt2) (28), whereas WNK4 does not. Synaptotagmins are involved in the regulation of membrane trafficking and vesicle fusion via a calcium-sensing mechanism (29). Both binding and phosphorylation are enhanced by Ca2+ binding to Syt2. As the phosphorylation of Syt2 inhibits the interaction of this protein with phospholipid vesicles, the binding of Syt2 to the membrane requires higher Ca2+ concentrations after phosphorylation by WNK1. Thus, WNK1 is thought to regulate the function of Syt2, depending on cellular Ca2+ concentration. Like Syt2, WNK1 is present in the cerebellum (C. Delaloy et al., unpublished observations) and in neuroendocrine cells, in which WNK1 and Syt2 co-localize with secretory granules (28). Synaptotagmins are involved in exocytosis, endocytosis, and regulating the membrane insertion of transporters and channels. WNK1 phosphorylation therefore may affect these processes. Much less is known about the determinants of WNK4 kinase activity. Comparisons of the WNK1 and WNK4 kinase domains showed differences in structure and function. Min et al. (22) looked at differences that might account for substrate specificity. They generated a homology-based structural model of WNK4, using the WNK1 coordinates. They identified two residues, at positions 318 and 448, that differed between the two enzymes and that seemed to mediate stable binding between WNK1 and Syt2. This may account for the poor phosphorylation of synaptotagmin 2 by WNK4 in their experiments. Wang et al. (25), using GST fusion constructs, confirmed the capacity of WNK1 constructs to autophosphorylate and to phosphorylate the generic substrate histone. Conversely, the WNK4 kinase domain displayed no kinase activity, either in vitro or in HEK 293 cells, suggesting that factors are for WNK4 kinase In these the WNK4 autoinhibitory domain WNK1 kinase activity, consistent with the interaction demonstrated in with et al. demonstrated the of and WNK4 in HEK 293 cells, but it is not known this interaction is or The of WNK4 therefore to be identified. of WNK1 and WNK4 WNK1 and WNK4 are in many different analysis has shown that WNK1 is in the and in and in and has shown that WNK1 is present in the kidney and in WNK1 expression is either or to the depending on the WNK4 is in the it is present in the and of the tubule and (16). WNK4 and protein are also present in several epithelial particularly in one of WNK4 has been whereas isoforms are from the WNK1 gene as a result of the existence of three two and three exons (Figure The first two and are located from and within and isoforms that contain the kinase domain. A is located in intron and the of a with from a This most of the kinase domain and is in large in the and tubule The of the identified intronic deletions on the of of the WNK1 isoforms are is if we are to the precise mechanism of the disease. It is that all that WNK1 and WNK4 are involved in In the of the of the co-transporter and of the are by WNK4 in cells, WNK4 by but does not the of No was observed in a with an The and the pharmacologic of these that were to the et al. showed that WNK4 phosphorylates 1 to These proteins are the major membrane proteins involved in regulating ion As no on structure was observed on these findings that WNK4 is involved in regulating the that in the studies in showed that the of WNK4 is not to it to the regulation of a of in the (Figure The of WNK4 into membrane expression of the co-transporter and of the renal and that of the renal epithelial WNK4 to regulate and membrane expression in a In is by and is of WNK4 kinase activity. However, two have studied the of and have results the for the WNK4 kinase domain. et al. showed that an mutation in the kinase domain the observed in et al. showed that the part of WNK4 was for this whereas the kinase domain was not. These results the of WNK4 in and NaCl and in the The regulation of WNK4 in individuals with would activity Conversely, in individuals with WNK4 regulation would inhibit The differences in the of WNK4 in of hyperkalemia and probably other One of these factors may be the WNK1 which has been shown to be by in vitro and may regulate the interaction between WNK1 and WNK4 (see below). the regulation of by WNK4 may it possible to regulate the of and An relationship has been found between the of of these two in the This might account for the observed in FHHt, as a result of WNK4 mutations (see It might also the increase in and that was observed recently in a large family with FHHt The physiologic functions of WNK1 are less Several studies in vitro have shown that it may act as an osmotic sensor in cells (24). In it has been to a of the functions of WNK1 of expression of the long WNK1 and expression of the short in the In a cell that a physiologic concentrations of have been shown to the expression of the WNK1 but not that of the long isoforms of this short the cellular of the et al. that might affect the or activity of the epithelial but not its The possible of WNK1 on was very recently confirmed by in vitro studies from WNK1 was shown to and activate the and protein kinase by its In WNK1 in via the These new findings that WNK1 regulate in the late by the of in the membrane (Figure The second mechanism of of WNK1 in the to be its interaction with Indeed, WNK4, WNK1 was found to have no on activity in the expression However, if WNK4 and WNK1 are WNK1 the of This requires an interaction between the kinase domain of WNK1 and WNK4 and to be by the part of WNK4 These results therefore that a WNK1 protein that the kinase domain, as the would not be able to regulate the of activity. The precise mechanism of interaction between the two kinases is as results have been It is also to that is no evidence that WNK1 activity itself is regulated by (24). Thus, we not the or activity of WNK1 is affected by other than the of in NaCl the physiologic of WNK1 on BP by the et al. generated a of WNK1 by gene in that were for the mutation in the first of whereas showed a in BP than that in However, no in concentrations were in that were a suggesting that the in BP was not to in the These that WNK1 is for and is a of BP as the of a of the gene led to a in of FHHt The mechanism of FHHt has been for many The high sensitivity of both hypertension and metabolic disorders to thiazide diuretics and the low levels have been as via the thiazide-sensitive co-transporter in the This would the of to the and the the in potassium et al. an suggesting that the syndrome may result from high levels of This would via the potassium In both low-renin hypertension and hyperkalemic metabolic acidosis result primarily from with potassium These have to be to the knowledge of mutations at the WNK4 and WNK1 FHHt by WNK4 In WNK4 expression and expression at the membrane (Figure No are observed in the same with a WNK4 the mutation results were recently with a novel mutation that cell expression of but expression of the of these it has been that the mutations that are responsible for FHHt may be the physiologic of by WNK4, in high levels of the thiazide-sensitive However, other mutations and have been shown to have as an on activity as WNK4 This that the mechanism of the disease may not be the same in all FHHt kindreds, the existence of phenotypic However, this as all of these missense mutations in a highly conserved sequence and affect the of this negatively charged segment in a similar The of the and mutations for the of in the membrane have also been studied in the Both mutations further the of and expression of the and mutations a on than WNK4 It was further demonstrated that WNK4 phosphorylation of the proteins 1 to and that WNK4 further phosphorylation These that mutations in WNK4 would as mutations for but mutations for and WNK4 to have of of which are kinase activity and of which are kinase WNK4 probably by the physiologic of by WNK4 or should to potassium by the pathway, WNK4 further concentration. These three major involved in in the which a in and potassium They are also consistent with the and observed in patients with high BP, and with high sensitivity to thiazide Although these similar lines, it should be that the levels of provided by the studies are not The in epithelial renal cell lines, by two that all FHHt mutations studied increase the pathway is consistent with the of WNK4 with the protein in the and with the by than However, the from the model should not be that not all of the FHHt mutations to levels at the is a of also that the of WNK4 in renal epithelial cells the of However, this not differ between and Our understanding therefore and further studies of the WNK pathway are In that results in are of Lifton’s group recently findings that that were for WNK4 a higher thiazide-sensitive co-transporter protein and a at the in FHHt by WNK1 As previously two large genomic deletions (41 and in the first intron of WNK1 have been identified as being responsible for FHHt in two unrelated kindreds, with no mutation in the coding sequence (16). An analysis of WNK1 transcript levels in leukocytes from affected and unaffected members of one of these families revealed that the intronic deletion was associated with a increase in the levels of a WNK1 the of which has to be does the of WNK1 have in the renal WNK4 and WNK1 are in WNK1 completely the of et al. recently confirmed that WNK1 the of WNK4 on activity but that a WNK1 the kinase domain the of Thus, the might not be able to inhibit WNK4, as it the kinase domain WNK1 intronic mutations may increase of the WNK1 that is normally in only small This in would the of by WNK4 and increase activity NaCl This requires in The second major mechanism by which in WNK1 expression might affect to its activity on as shown by Xu et al. and Thus, the of WNK1 mutations in the kidney would not be their on WNK4 but also a further increase in activity (Figure an an expression of the long WNK1 would contribute to and hypertension in An expression of the would to hypertension of It to be in vitro and in the WNK1 intronic deletions to or in WNK1 We also to our understanding of the expression of the WNK1 in the and that or WNK genes are being by and should to the of WNK physiologic functions and FHHt The identification of WNK1 and WNK4 has it possible to a few large FHHt and observed that affected individuals in an family the missense mutation displayed in addition to hyperkalemic metabolic This was associated with a in In in our WNK1 affected patients hyperkalemic metabolic acidosis of similar severity to that observed in the family but similar to unaffected (11). The of an increase in and an increase in in the would not be the relationship between and However, this does not for WNK1 The between the two families may be related to a interaction between WNK4 and the However, et al. found no between WNK4 and Thus, these findings is not the phenotypic differences may be to WNK1 expression in and to and/or Thus, the two FHHt probably the pathophysiology of the two is probably not strictly the It is that in both metabolic disorders In the French hypertension not until patients were in their The patients of the 17 the mutation were but displayed metabolic abnormalities of similar severity to of the were reported for the pedigree In this family, the between of hyperkalemia and of hypertension was yr. In both affected patients concentrations than unaffected but the of the was similar in and concentration that to is to the disease, but that individuals until that is less than in other of monogenic as type 2 or and the to It is that hypertension resolved two in previously of the whereas hyperkalemia and of the functions of WNK1 and WNK4 in the regulation of ion handling has opened up new and of that may our understanding of However, the precise FHHt for both WNK1 and The on and to be in as the possible of on WNK1 expression and activity. if WNK1 does result in the activation of and WNK4 missense mutations to via by both of hypertension a mechanism with The of affected kindreds also the of metabolic disorders and patients with FHHt until adulthood may provide insight into the the of which with In terms of one of the findings of the few years is the that several loci are responsible for Two genes have already been and the involvement of locus is with a locus as (15). In addition to the identification of substrates and/or of WNK1 and WNK4 should to the genes it to the regulation of ion in the nephron. that is after the identification of genes that are responsible for a the that mutations might be responsible for of the disease. of the genes that are responsible for monogenic of hypertension have results in analyses of However, several of evidence WNK genes in individuals are to thiazide which are particularly in patients with The locus that contains WNK4 within the largest genetic linkage for BP observed in and in the This has also been to hypertension in a but has not been reported in other in large analysis of the coding sequence of WNK4 revealed no mutation in the with to the control In a of genetic have been identified in of and No has been reported between WNK4 and but by of and In that a analysis of WNK1 in families recently showed a but between one located from the WNK1 and the severity of hypertension with the that expression of WNK1 might contribute to to Structure of the WNK4 gene and description of the familial hyperkalemic hypertension (FHHt) missense The WNK4 gene is long and contains The kinase domain is by exons 1 to the autoinhibitory domain is by exons to 7 and 17 the first and second coiled-coil domains, and the small the FHHt missense mutations were These 10 and amino acid are conserved the WNK Structure of the WNK1 gene. The two the renal and the two and are exons and kidney The large intronic deletions found in the American and French kindreds are by Structure and expression of WNK1 The and located of and in to long isoforms that contain the kinase domain. isoforms are with a expression in the and the as shown in the isoforms are The to a the major part of the kinase domain and and strongly in the as shown in the with the to this WNK4 and in experiments. a and with the thiazide-sensitive NaCl co-transporter (NCC) in WNK4 inhibits by the of present at the A WNK4 has no on in the all FHHt mutations only the mutation is to in the with a and with the potassium WNK4 inhibits by of the This is kinase and is by a In to is with the and mutations to an of of the further of WNK1 on in the tubule of the nephron. Two major are to a long and In the is than The possible of these two isoforms on the are (see for Genetic heterogeneity of familial hyperkalemic was by from the French of and et