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Genome-wide scans performed in affected sib pairs have revealed small and often inconsistent clues to the loci responsible for the inherited components of hypertension. Since blood pressure is a quantitative trait regulated by many loci, two siblings at opposite extremes of the blood pressure distribution are more likely to have inherited different alleles at any given locus. Hence, we investigated an extreme discordant sib pair strategy to analyse markers from two previous loci of interest: (1) the Gordons syndrome locus that includes the WNK4 gene and (2) the ROMK locus identified in our first genome-wide scan. For this study, 24 sib pairs with strong family histories of essential hypertension were selected from the top and bottom 10% of the blood pressure distribution and genotyped for highly polymorphic microsatellite markers on chromosomes 11 and 17. The mean age of the population was 39.8 ± 7.8 years. A significant inverse correlation was found between the squared difference in pulse pressure and the number of alleles shared by IBD between the siblings for the DS11925 marker (r = −0.44, p = 0.031), systolic pressure and chromosome 17 markers (D17S250: r = −0.42, p = 0.040; D17S799 (r = −0.51, p = 0.011), and this relationship persisted after correcting for age and gender. Markers on chromosome 17 (D17S250, D17S928 and D17S1301) and 11 (D11S1999) also correlated with diastolic pressure. These results illustrate the successful use of discordant sib pair analysis to detect linkage within relatively small numbers of pedigrees with hypertension. Further analysis of this cohort may be valuable in complementing findings from the large genome wide scans in affected sib pairs. [ABSTRACT FROM AUTHOR]

Thiazide diuretics are among the most widely used treatments for hypertension, but thiazide-induced hyponatremia (TIH), a clinically significant adverse effect, is poorly understood. Here, we have studied the phenotypic and genetic characteristics of patients hospitalized with TIH. In a cohort of 109 TIH patients, those with severe TIH displayed an extended phenotype of intravascular volume expansion, increased free water reabsorption, urinary prostaglandin [E.sub.2] excretion, and reduced excretion of serum chloride, magnesium, zinc, and antidiuretic hormone. GWAS in a separate cohort of 48 TIH patients and 2,922 controls from the 1958 British birth cohort identified an additional 14 regions associated with TIH. We identified a suggestive association with a variant in SLCO2A1, which encodes a prostaglandin transporter in the distal nephron. Resequencing of SLCO2A1 revealed a nonsynonymous variant, rs34550074 (p.A396T), and association with this SNP was replicated in a second cohort of TIH cases. TIH patients with the p.A396T variant demonstrated increased urinary excretion of prostaglandin [E.sub.2] and metabolites. Moreover, the SLCO2A1 phospho-mimic p.A396E showed loss of transporter function in vitro. These findings indicate that the phenotype of TIH involves a more extensive metabolic derangement than previously recognized. We propose one mechanism underlying TIH development in a subgroup of patients in which SLCO2A1 regulation is altered., Introduction Cardiovascular disease is the leading cause of mortality worldwide (1, 2) and hypertension one of its most important modifiable causes. Thiazide diuretics inhibit the thiazide-sensitive sodium chloride cotransporter NCC [...]

Gordon Syndrome (GS) is a rare familial hypertension syndrome with a characteristic hyperkalaemia which distinguishes it from other syndromic forms of hypertension that typically cause hypokalaemia. Patients with GS respond to aggressive salt-restriction or relatively small doses of thiazide diuretics, which suggests that activation of the thiazide-sensitive Na/Cl cotransporter (NCC) in the distal nephron is to blame. However, the mechanism has proved to be complex. In 2001, mutations in genes encoding two serine/threonine kinases, WNK1 and WNK4, were identified as causing GS. However, it took several years to appreciate that these kinases operated in a cascade with downstream serine/threonine kinases (SPAK and OSR1) actually phosphorylating and activating NCC and the closely related cotransporters NKCC1 and NKCC2. The hyperkalaemia in GS arises from an independent action of WNK1/WNK4 to reduce cell-surface expression of ROMK, the secretory K-channel in the collecting ducts. However, mutations in WNK1/4 are present in a small minority of GS families, and further genes have emerged (CUL3 and KLHL3) that code for Cullin-3 (a scaffold protein in an ubiquitin-E3 ligase) and an adaptor protein, Kelch3, respectively. These new players regulate the ubiquitination and proteasomal degradation of WNK kinases, thereby adding to the complex picture we now have of NCC regulation in the distal nephron., Author(s): Kevin M. O'Shaughnessy[sup.1] Author Affiliations: (1) Division of Experimental Medicine and Immunotherapeutics, Department of Medicine, Addenbrooke's Hospital-University of Cambridge, Cambridge, CB2 2QQ UK Introduction The first report of a [...]

Cation transport in the distal mammalian nephron relies on the SLC12 family of membrane cotransporters that include the thiazide-sensitive [Na.sup.+]-[Cl.sup.-] cotransporter (NCC). NCC is regulated through a scaffold of interacting proteins, including the WNK kinases, WNK 1 and WNK 4, which are mutated in the hypertensive Gordon's syndrome. Dynamic regulation of NCC function by kinases must involve dephosphorylation by phosphatases, as illustrated by the role of PP1 and PP2B in the regulation of KCC members of the SLC12 family. There are 2 phosphorylation-controlled regulatory pathways for NCC: type 1, mediated by WNK4 and affecting trafficking to the surface membrane, and type 2, affecting intrinsic transporter kinetics by phosphorylation of conserved N-terminal S/T amino acids. Using the Xenopus oocyte expression system, we show that PP4 inhibits NCC activity--but not trafficking to the surface membrane--by a mechanism that requires phosphatase activity and a conserved N-terminal amino acid of NCC, threonine 58. This action is distinct from WNK4 regulation of membrane trafficking. In the mouse kidney, PP4 is selectively expressed in the distal nephron, including cells of the distal convoluted tubule cells, suggesting that PP4 may have a physiological role in regulating NCC and hence NaCl reabsorption in vivo. Key words: SLC12 cotransporter regulation, protein phosphorylation, kidney tubular function, distal nephron. Dans la partie distale du nephron mammifere, le transport de cations est assure entre autres par la famille de co-transporteurs SLC12. Le cotransporteur de sodium et chlore sensible au thiazide (NCC) est un membre de cette famille. La regulation de NCC implique un ensemble de proteines dont font partie les kinases WNK, WNK1 et WNK4, qui sont mutees dans le syndrome de Gordon. La dynamique de regulation de la fonction de NCC par les kinases devrait impliquer une dephosphorylation par des phosphatases comme l'illustre le role de PP1 et PP2B dans la regulation des KCC, autres membres de la famille SLC12. Pour NCC il existe deux voies de regulation controkee par phosphorylation : celle de type 1, impliquant WNK4 et affectant le trafic vers la membrane plasmique, et celle de type 2, affectant la cinetique intrinseque des transporteurs par phosphorylation des acides amines S/T conserves en N-terminal. A l'aide du systeme d'expression des ovocytes de xenope, nous montrons que PP4 inhibe l'activite de NCC--mais pas le trafic vers la membrane plasmique--par un mecanisme requerant une activite phosphatase et la presence d'un acide amine; conserve; en N-terminal de NCC, la threonine 58. Cette action est distincte de la regulation du trafic vers la membrane plasmique par WNK4. Dans le rein de souris, PP4 est exprime selectivement dans le neephron distal et en particulier dans les cellules du tubule contourne distal, suggerant que PP4 pourrait avoir un role physiologique dans la regulation de NCC et donc dans la reabsorption du NaCl in vivo. Mots-cles: regulation des cotransporteurs SLC12, phosphorylation des proteeines, fonction tubulaire distale, nephron distal., Introduction The cation-coupled chloride cotransporter family of membrane proteins (SLC12) is important for electrolyte transport in many secretary epithelia, including the kidney (Kahle et al. 2004). The thiazide-sensitive [Na.sup.+]-[Cl.sup.-] cotransporter [...]

The rate of aldosterone synthesis by adrenal glomerulosa cells relies on the selective permeability of the glomerulosa cell to [K.sup.+] ions. In rodent and bovine adrenal glomerulosa cells, this background potassium current is provided by a two-pore loop potassium (K2P) channel: largely TASK-3 in the rat and TREK-1 in the cow. The nature of the K2P channel in the human adrenal cortex is not known, and we have addressed this issue here using the H295R human adrenal cell line. We show that these cells express mRNA and protein for both TASK-3 and TREK-1 K2P channels. Using a potentiometric dye (FMP), we also show that TASK-3 and TREK-1 channel modulators can affect the membrane potential of H295R cells. Transfecting H295R cells with TASK-3 or TREK-1 dominant-negative mutants (TASK-3 G95E or TREK-1 G144E) produced depolarization of H295R cells and altered K-stimulated aldosterone secretion. Finally, transfection of a constitutively active mutant of G[[alpha].sub.q] into H295R cells (GTPase-deficient G[[alpha].sub.q]-QL) depolarized them and increased basal aldosterone secretion. Taken together, our data support both TASK-3 and TREK-1 as being functionally operational in the H295R cell line. This suggests that human adrenal glomerulosa cells may utilize both of these K2P channels for their background potassium current. glomerulosa cells; adrenal cortex; voltage-sensitive dyes; angiotensin II

The novel serine/threonine kinases (with no lysine kinases or WNKs), WNK1 and WNK4, are encoded by the disease genes for Gordon syndrome (PRKWNK1 and PRKWNK4), a rare monogenic syndrome of hypertension and hyperkalemia. These proteins alter the expression of the thiazide-sensitive Na/Cl cotransporter (NCCT) in Xenopus laevis oocytes, although the details are controversial. We describe here our own experience and confirm that kinase-dead WNK4 (318D>A) is unable to affect [Na.sup.+] fluxes through the thiazide-sensitive Na/Cl transporter (NCCT) or its membrane expression as an ECFP-NCCT fusion protein. However, the kinase domain is not sufficient for a functional WNK4 since deletion of the acidic motif (a motif unique to WNK family members) completely abolishes functional activity. Indeed, the N[H.sup.2] terminal of WNK4 (1-620) containing the kinase domain and acidic motif retains full activity, but does not interact directly with NCCT in pull-down assays. Coexpression of WNK1 antagonizes the action of WNK4, and kinase-dead WNK1 (368D>A) or WNK1 carrying a WNK4 disease mutation (565Q>E) behaves in the same way as wild-type WNK1. This suggests kinase activity and charge conservation within the acidic motif are not essential for the WNK1-WNK4 interaction. We also report that WNK4 probably reduces surface expression largely through an effect on forward trafficking. Hence, the effect of WNK4 on NCCT expression is mimicked by dynamin, but the dominant-negative K44A dynamin mutant does not block the action of WNK4 itself. These results further highlight important differences in the mechanism by which WNK kinases affect expression of NCCT vs. other membrane proteins such as ROMK. kidney transporters; WNK kinases; regulation of membrane expression