Polymorphisms in the WNK1 gene are asociated with blood pressure variation and urinary potassium excretion

WNK1 - a serine/threonine kinase involved in electrolyte homeostasis and blood pressure (BP) control - is an excellent candidate gene for essential hypertension (EH). We and others have previously reported association between WNK1 and BP variation. Using tag SNPs (tSNPs) that capture 100% of common WNK1 variation in HapMap, we aimed to replicate our findings with BP and to test for association with phenotypes relating to WNK1 function in the British Genetics of Hypertension (BRIGHT) study case-control resource (1700 hypertensive cases and 1700 normotensive controls). We found multiple variants to be associated with systolic blood pressure, SBP (7/28 tSNPs min-p = 0.0005), diastolic blood pressure, DBP (7/28 tSNPs min-p = 0.002) and 24 hour urinary potassium excretion (10/28 tSNPs min-p = 0.0004). Associations with SBP and urine potassium remained significant after correction for multiple testing (p = 0.02 and p = 0.01 respectively). The major allele (A) of rs765250, located in intron 1, demonstrated the strongest evidence for association with SBP, effect size 3.14 mmHg (95%CI:1.23–4.9), DBP 1.9 mmHg (95%CI:0.7–3.2) and hypertension, odds ratio (OR: 1.3 [95%CI: 1.0–1.7]).We genotyped this variant in six independent populations (n = 14,451) and replicated the association between rs765250 and SBP in a meta-analysis (p = 7×10−3, combined with BRIGHT data-set p = 2×10−4, n = 17,851). The associations of WNK1 with DBP and EH were not confirmed. Haplotype analysis revealed striking associations with hypertension and BP variation (global permutation p10 mmHg reduction) and risk for hypertension (OR<0.60). Our data indicates that multiple rare and common WNK1 variants contribute to BP variation and hypertension, and provide compelling evidence to initiate further genetic and functional studies to explore the role of WNK1 in BP regulation and EH

Characterisation of the Cullin-3 mutation that causes a severe form of familial hypertension and hyperkalaemia

Deletion of exon 9 from Cullin‐3 (CUL3, residues 403–459: CUL3Δ403–459) causes pseudohypoaldosteronism type IIE (PHA2E), a severe form of familial hyperkalaemia and hypertension (FHHt). CUL3 binds the RING protein RBX1 and various substrate adaptors to form Cullin‐RING‐ubiquitin‐ligase complexes. Bound to KLHL3, CUL3‐RBX1 ubiquitylates WNK kinases, promoting their ubiquitin‐mediated proteasomal degradation. Since WNK kinases activate Na/Cl co‐transporters to promote salt retention, CUL3 regulates blood pressure. Mutations in both KLHL3 and WNK kinases cause PHA2 by disrupting Cullin‐RING‐ligase formation. We report here that the PHA2E mutant, CUL3Δ403–459, is severely compromised in its ability to ubiquitylate WNKs, possibly due to altered structural flexibility. Instead, CUL3Δ403–459 auto‐ubiquitylates and loses interaction with two important Cullin regulators: the COP9‐signalosome and CAND1. A novel knock‐in mouse model of CUL3WT/Δ403–459 closely recapitulates the human PHA2E phenotype. These mice also show changes in the arterial pulse waveform, suggesting a vascular contribution to their hypertension not reported in previous FHHt models. These findings may explain the severity of the FHHt phenotype caused by CUL3 mutations compared to those reported in KLHL3 or WNK kinases

Mother-child histocompatibility and risk of rheumatoid arthritis and systemic lupus erythematosus among mothers.

The study objective was to test the hypothesis that having histocompatible children increases the risk of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), possibly by contributing to the persistence of fetal cells acquired during pregnancy. We conducted a case control study using data from the UC San Francisco Mother Child Immunogenetic Study and studies at the Inova Translational Medicine Institute. We imputed human leukocyte antigen (HLA) alleles and minor histocompatibility antigens (mHags). We created a variable of exposure to histocompatible children. We estimated an average sequence similarity matching (SSM) score for each mother based on discordant mother-child alleles as a measure of histocompatibility. We used logistic regression models to estimate odds ratios (ORs) and 95% confidence intervals. A total of 138 RA, 117 SLE, and 913 control mothers were analyzed. Increased risk of RA was associated with having any child compatible at HLA-B (OR 1.9; 1.2-3.1), DPB1 (OR 1.8; 1.2-2.6) or DQB1 (OR 1.8; 1.2-2.7). Compatibility at mHag ZAPHIR was associated with reduced risk of SLE among mothers carrying the HLA-restriction allele B*07:02 (n = 262; OR 0.4; 0.2-0.8). Our findings support the hypothesis that mother-child histocompatibility is associated with risk of RA and SLE

Evaluation of behavior in transgenic mouse models to understand human congenital pain conditions

BACKGROUND: Containing a brain for signal processing and decision making, and a peripheral component for sensation and response, the nervous system provides higher organisms a powerful method of interacting with their environment. The specific neurons involved in pain sensation are known as nociceptors and are the source of normal nociceptive pain signaling to prompt appropriate responses. Though acute hypersensitization can be advantageous by encouraging an organism to allow an injured area to heal, chronic pain conditions can be pathological and can markedly reduce quality of life. While a variety of genes have been associated with congenital pain conditions, two rare cases examined in this study have not had their mutated genes identified. Potassium voltage-gated channel subfamily H member 8, or KCNH8, is involved in regulating action potential production and propagation, and has not been linked with pain processing of any kind to date. Here, a male patient evaluated at Boston Children’s Hospital contains a novel single-base KCNH8 mutation and possesses an extremely low sensitivity to cold temperatures and mechanical pain, but a higher sensitivity to warmer temperatures. A separate protein, intersectin-2, or ITSN2, normally functions in clathrin-mediated endocytosis and exocytosis. A second patient at Boston Children’s Hospital expresses a previously-unseen point mutation in ITSN2 and experiences erythromelalgia, characterized by episodes of intense pain and red, swollen limbs during ambient warm temperatures. Through the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing, this study will produce these specific genetic mutations in mouse lines to explore their effects on mammalian behavior. OBJECTIVES: This project employs two transgenic mouse models to study the behavioral phenotypes associated with rare potentially damaging mutations in KCNH8 and ITSN2 exhibited in the human patients. Through these experiments, a greater understanding of neural pain signaling and sensitivity changes can occur. METHODS: The differences in temperature preference of KCNH8 and ITSN2 mutant mice compared to wild type mice lacking these mutations was studied using thermal plates under cold and warm conditions. Direct application of acetone and von Frey filaments to mouse paws was used to study cold and mechanical sensitivity. Further testing of stamina, anxiety, coordination, and strength were also evaluated. RESULTS: A marked decrease in sensitivity to von Frey stimulation (p<0.01) and acetone administration (p<0.05) was observed in KCNH8 mutant mice. Thermal preference testing demonstrated a decreased preference for warmer temperatures as compared to wild type mice. In addition, anxiety levels were also observed to be slightly higher in these mutant KCNH8 mice (p<0.05). The mutant ITSN2 mice spent less time at cooler temperatures, though surprisingly they significantly preferred warmer conditions as compared to their wild type littermates. A full and partial reversal of these temperature preferences was demonstrated in cold and heat thermal conditions respectively after intraperitoneal gabapentin injection, which normalized the mice toward wild type behavior. CONCLUSIONS: Data from the KCNH8 mutant mouse model indicates an aversion to warmer temperatures and a decreased ability to detect cold or mechanical pressure, much like the human patient. The mutant ITSN2 mice were less likely to spend time at cooler temperatures, indicating heightened sensory sensitivity, but their preference for warmer temperatures suggests a possible desensitization of the affected nociceptors. These results often mirror the patient’s phenotype, but the preference for ambient warmer environments appears opposite to the patient. As the ITSN2 mice feel discomfort at cooler temperatures, a proposed desensitization at warmer temperatures would result in a more comfortable environment and could explain the observed preference. The trends toward normal neural firing rates achieved through gabapentin injection suggest that the aberrant responses in mutant ITSN2 mice is due to altered sensitization, but additional examination under these conditions with a larger group of mice is necessary to further unravel these signaling pathways. However, these extremely encouraging data introduce two new molecular targets for acute pain control

Semiparametric Regression Estimation in Null Recurrent Nonlinear Time Series

Estimation theory in a nonstationary environment has been very popular in recent years. Existing studies focus on nonstationarity in parametric linear, parametric nonlinear and nonparametric nonlinear models. In this paper, we consider a partially linear model and propose to estimate both alpha and g semiparametrically. We then show that the proposed estimator of alpha is still asymptotically normal with the same rate as for the case of stationary time series. We also establish the asymptotic normality for the nonparametric estimator of the function g and the uniform consistency of the nonparametric estimator. The simulated example is given to show that our theory and method work well in practice.asymptotic normality; beta-null recurrent Markov chain; consistency; kernel estimator; partially linear model

Mechanisms of CFTR Functional Variants That Impair Regulated Bicarbonate Permeation and Increase Risk for Pancreatitis but Not for Cystic Fibrosis

CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR bicarbonate permeability are altered by CFTRBD variants through multiple mechanisms. CFTRBD variants are associated with clinically significant disorders of the pancreas, sinuses, and male reproductive system.Fil: LaRusch, Jessica. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Jung, Jinsei. Yonsei University College of Medicine; Corea del SurFil: General, Ignacio. University of Pittsburgh; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lewis, Michele D.. Mayo Clinic. Division of Gastroenterology and Hepatology; Estados UnidosFil: Park, Hyun Woo. Yonsei University College of Medicine; Corea del SurFil: Brand, Randall E.. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Gelrud, Andres. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Anderson, Michelle A.. University of Michigan; Estados UnidosFil: Banks, Peter A.. Brigham and Women’s Hospital. Division of Gastroenterology; Estados UnidosFil: Conwell, Darwin. Brigham and Women’s Hospital. Division of Gastroenterology; Estados UnidosFil: Lawrence, Christopher. Medical University of South Carolina; Estados UnidosFil: Romagnuolo, Joseph. Medical University of South Carolina; Estados UnidosFil: Baillie, John. University of Duke; Estados UnidosFil: Alkaade, Samer. St. Louis University. School of Medicine; Estados UnidosFil: Cote, Gregory. Indiana University; Estados UnidosFil: Gardner, Timothy B.. Dartmouth-Hitchcock Medical Center; Estados UnidosFil: Amann, Stephen T.. North Mississippi Medical Center; Estados UnidosFil: Slivka, Adam. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Sandhu, Bimaljit. Virginia Commonwealth University Medical Center; Estados UnidosFil: Aloe, Amy. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Kienholz, Michelle L.. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Yadav, Dhiraj. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Barmada, M. Michael. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Bahar, Ivet. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Lee, Min Goo. Yonsei University College of Medicine; Corea del SurFil: Whitcomb, David C.. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: North American Pancreatitis Study Group. No especifica

Contribution of the sympathetic nervous system to the pathogenesis of salt-sensitive hypertension

Dysregulation of the sodium-chloride cotransporter (NCC) is believed to significantly impact blood pressure. Recent studies have implicated overactivity of the sympathetic nervous system as a mechanism driving renal NCC dysregulation to evoke the development of salt-sensitive hypertension. It is proposed that the sympathetic nervous system accomplishes this by norepinephrine (NE)-mediated over-activation of the beta2-adrenergic receptors located in the distal tubules of the kidney. Beta2-adrenoreceptor activation is hypothesized to stimulate the protein kinases SPAK and OxSR1 to phosphorylate and thus activate NCC. This beta2-receptor-SPAK/OxSR1-NCC pathway was elucidated in studies that challenged salt-resistant mice with high-salt diets, bilateral adrenalectomies, and NE infusion. To expand the scope of these studies, we investigated the effects of elevated circulating NE on blood pressure, NCC activity, and expression of NCC proteins, SPAK, and OxSR1 in a different salt-resistant animal species (the Sprague-Dawley rat). In this study we implanted male Sprague-Dawley rats with osmotic minipumps delivering a subcutaneous infusion of either saline, NE, a 50:50 solution of DMSO/isotonic saline, a combination of NE and the NCC antagonist hydrochlorothiazide, or a combination of NE and the beta-adrenoreceptor antagonist propranolol. Following implantation of the pumps the rats were randomly assigned to either a standard diet (0.4% NaCl) or a high-salt diet (8% NaCl) for two weeks. After fourteen days all animals underwent acute femoral artery, vein, and bladder cannulation in order to monitor heart rate and blood pressure, administer drugs intravenously, and track renal function, respectively. Following surgical recovery, blood pressure and heart rate were measured continuously, and urine was collected in ten-minute intervals in order to assess peak natriuretic responses to amiloride and hydrochlorothiazide. Following this protocol the rats received an intravenous bolus of hexamethonium (30 mg/kg), and their peak drops in blood pressure were recorded. Afterwards both kidneys were harvested and frozen at -80 °C for measurement of NCC proteins, SPAK, and OxSR1 expression. This study demonstrates that increased circulating NE induces salt-sensitive hypertension in the naturally salt-resistant Sprague-Dawley rat. Chronic infusion of NE raised the blood pressure of the rats, and a high-salt diet exacerbated this effect. Furthermore, NE prevented salt-evoked suppression of NCC activity and NCC, SPAK, and OxSR1 protein expression. Co-infusion of hydrochlorothiazide with NE attenuated NE-mediated hypertension and caused no variance in the blood pressures between the standard salt and high-salt groups. This indicates that chronically antagonizing NCC eliminated the salt-sensitive component of NE-mediated hypertension. Beta-receptor antagonism combined with NE infusion completely eliminated the hypertensive influence of NE and downregulated the expression of NCC proteins, SPAK, and OxSR1. However, NCC activity still remained at a level comparable with that observed in the NE-infused rats, demonstrating dissociation between protein expression and function. These data, the first report in a rat model of an interaction of NE and a high salt intake that impairs NCC function, demonstrate that increased levels of NE in combination with a high dietary salt intake result in NCC dysregulation and the development of NE-mediated salt-sensitive hypertension. To an extent the data also support the proposition that NE activates the beta-adrenergic receptors to influence the activity of NCC and the expression of NCC proteins, SPAK, and OxSR1. Beta-antagonism combined with NE infusion attenuated the effects of NE on blood pressure and the expression of NCC proteins, SPAK, and OxSR1. However, the NE-mediated elevation of NCC activity still remained high. We propose that the beta-receptors are not the only adrenergic receptors that can influence NCC activity. The presence of alpha-adrenergic receptors in the distal tubules suggests that they may be able to keep NCC activity elevated through a pathway independent of the beta-receptors, SPAK, and OxSR1

Exposure to low pH induces molecular level changes in the marine worm, Platynereis dumerilii

© 2015 Elsevier Inc. Fossil fuel emissions and changes in net land use lead to an increase in atmospheric CO 2 concentration and a subsequent decrease of ocean pH. Noticeable effects on organisms' calcification rate, shell structure and energy metabolism have been reported in the literature. To date, little is known about the molecular mechanisms altered under low pH exposure, especially in non-calcifying organisms. We used a suppression subtractive hybridisation (SSH) approach to characterise differentially expressed genes isolated from Platynereis dumerilii, a non-calcifying marine polychaeta species, kept at normal and low pH conditions. Several gene sequences have been identified as differentially regulated. These are involved in processes previously considered as indicators of environment change, such as energy metabolism (NADH dehydrogenase, 2-oxoglutarate dehydrogenase, cytochrome c oxidase and ATP synthase subunit F), while others are involved in cytoskeleton function (paramyosin and calponin) and immune defence (fucolectin-1 and paneth cell-specific alpha-defensin) processes. This is the first study of differential gene expression in a non-calcifying, marine polychaete exposed to low pH seawater conditions and suggests that mechanisms of impact may include additional pathways not previously identified as impacted by low pH in other species

Les WNK kinases et les effets de WNK3 sur l'activité du canal ENaC

Les WNK kinases sont une famille de sérine/thréonine protéines kinases, des enzymes capables de phosphoryler le résidu OH de sérine ou thréonine. Quatre membres (WNK 1-4) ont été identifiés, largement distribués dans les cellules et tissus des mammifères et dont les fonctions sont de réguler des canaux ioniques et cotransporteurs. Leur particularité se situe au niveau de leur structure puisque une lysine a été substituée par une cystéine dans le domaine catalytique, d'où leur nom « with-no-lysine kinase » (McCormick and Ellison, 2011). Leur rôle dans le bon fonctionnement du rein et plus particulièrement dans le contrôle et maintien du bilan hydro-sodé n'est plus à vérifier puisque des mutations dans WNK 1 et WNK 4 sont connues pour causer la maladie de l'hypertension familiale ou syndrome de pseudohypoaldostéronisme de type 2, aussi appelé syndrome de Gordon (ou Fhht = Familial hyperkalaemic hypertension) (Furgeson and Linas, 2010). Le syndrome de Gordon est une maladie génétique autosomale dominante caractérisée par une hypertension, une hyperkaliémie et une acidose métabolique. Les WNKs contrôlent de nombreux canaux et transporteurs dans le rein, devenant des acteurs importants dans la régulation du bilan sodique et potassique. Leurs effets sont nombreux et variables et les canaux jouant un rôle clé dans cette régulation sont ENaC, NCC et ROMK (Kahle et al., 2008). Dans ce travail, nous commencerons par une partie théorique faisant le point sur l'organisation des néphrons et la régulation du bilan sodique et les différents mécanismes entrant en jeu. Nous intégrerons des informations générales concernant les WNKs ainsi qu'une revue plus détaillée de leurs effets respectifs dans le néphron. Pour terminer, nous aborderons les résultats d'une expérience qui a été réalisée en laboratoire sur des oocytes de Xenopus laevis. L'implication de WNK1 et WNK4 dans le contrôle du sodium ayant déjà été prouvée à de nombreuses reprises, nous nous sommes intéressés ici aux effets de WNK3 sur le canal ENaC ainsi que l'implication de Nedd4-2 dans ce procédé