WNK1 Research Articles

Effect of heterozygous deletion of WNK1 on the WNK-OSR1/ SPAK-NCC/NKCC1/NKCC2 signal cascade in the kidney and blood vessels.

We found that a mechanism of hypertension in pseudohypoaldosteronism type II (PHAII) caused by a WNK4 missense mutation (D561A) was activation of the WNK-OSR1/SPAK-NCC signal cascade. However, the pathogenic effect of intronic deletions in WNK1 genes also observed in PHAII patients remains unclear. To understand the pathophysiological roles of WNK1 in vivo, WNK1(+/-)mice have been analyzed, because homozygous WNK1 knockout is embryonic lethal. Although WNK1(+/-) mice have been reported to have hypotension, detailed analyses of the WNK signal cascade in the kidney and other organs of WNK1(+/-) mice have not been performed. We assess the effect of heterozygous deletion of WNK1 on the WNK-OSR1/SPAK-NCC/NKCC1/NKCC2 signal cascade in the kidney and blood vessels. Contrary to the previous report, the blood pressure of WNK1(+/-) mice was not decreased, even under a low-salt diet. Under a WNK4(D561A/+) background, the heterozygous deletion of the WNK1 gene did not reduce the high blood pressure either. We then evaluated the phosphorylation status of OSR1, SPAK, NCC, NKCC1, and NKCC2 in the kidney, but no significant decrease in the phosphorylation was observed in WNK1(+/-) mice or WNK1(+/-)WNK4(D561A/+) mice. In contrast, a significant decrease in NKCC1 phosphorylation in the aorta and a decreased pressure-induced myogenic response in the mesenteric arteries were observed in WNK1(+/-) mice. The contribution of WNK1 to total WNK kinase activity in the kidney may be small, but that WNK1 may play a substantial role in the regulation of blood pressure in the arteries.

Pathogenesis of pseudohypoaldosteronism type 2 by WNK1 mutations

Pseudohypoaldosteronism type 2 (PHA2) is a rare autosomal dominant form of human arterial hypertension, associated with hyperkalemia and hyperchloremic metabolic acidosis. WNK1 and WNK4 are two of the genes mutated in PHA2 patients. This review focuses on the mechanisms by which deletions of the first intron of WNK1 found in PHA2 patients trigger the disease. The WNK1 gene gives rise to a ubiquitous kinase (L-WNK1) and to a shorter kinase-defective isoform, KS-WNK1 (for kidney-specific WNK1), expressed only in the distal convoluted tubule (DCT) and connecting tubule. WNK1 first intron deletion leads to overexpression of L-WNK1 in the DCT and ubiquitous ectopic expression of KS-WNK1. The increased expression of L-WNK1 in the DCT results in increased activity of the Na-Cl cotransporter (NCC) and thus hypervolemia and hypertension. Contrarily, the mechanisms underlying the hyperkalemia and metabolic acidosis remain unclear. As particularly small doses of thiazide diuretics, inhibitors of NCC activity, correct both the blood pressure and metabolic disorders in PHA2 patients, it was believed that increased NCC was directly responsible for all PHA2 features. Studies performed in mouse models of KS-WNK1 inactivation or WNK4-related PHA2, however, have revealed that the situation is much more complex.

Familial Hyperkalemic Hypertension: A New Early-onset Pediatric Case

The Familial hyperkalemic hypertension (FHHt) syndrome (OMIM #145260), first described in 1964, and also known as Gordon’s syndrome or pseudohypoaldosteronism type II (PHA II), is a rare autosomal dominant disease characterized by hypertension, hyperkalemia, hyperchloremic metabolic acidosis, and normal renal and adrenal function, with suppressed plasma rennin activity. Thiazides correct the metabolic abnormalities and hypertension (1,2,3). FHHt syndrome may be due to mutations in the WNK1 and WNK4 genes, encoding the with-no-lysine kinases 1 and 4 respectively. WNK4 mutants fail to inhibit the thiazide-sensitive Na-Cl cotransporter (NCC), increasing reabsorption of chloride in the distal collecting tubule and causing volume expansion and hypertension, and fail to inhibit the ROMK1 channel, causing hyperkalemia. WNK1 mutants also cause an increase in chloride adsorption both through the activation of EnaC and reduced inhibition of NCC through its influence on the WNK4 activity (4,5,6). Three loci associated with FHHt have been identified (12p13.3 and 17p11-q21 and 1q31-q42), but other loci, not yet identified, are probably involved, demonstrating the genetic heterogeneity of FHH. The syndrome has been described primarily in adolescents and young adults, and in most cases, hypertension has been the presenting symptom; however, the investigations performed in the relatives of patients with the FHHt syndrome, showed that the metabolic disorders preceded hypertension, and no relationship between the severity of biochemical abnormalities and severity of hypertension was observed (7, 8). Short stature, hypercalciuria with urolithiasis and dental abnormalities have also been reported. The long-term prognosis remains uncertain because follow-up data are still limited.

A minor role of WNK3 in regulating phosphorylation of renal NKCC2 and NCC co-transporters in vivo

SummaryMutations in WNK1 and WNK4 kinase genes have been shown to cause a human hereditary hypertensive disease, pseudohypoaldosteronism type II (PHAII). We previously discovered that WNK kinases phosphorylate and activate OSR1/SPAK kinases that regulate renal SLC12A family transporters such as NKCC2 and NCC, and clarified that the constitutive activation of this cascade causes PHAII. WNK3, another member of the WNK kinase family, was reported to be a strong activator of NCC/NKCC2 when assayed in Xenopus oocytes, suggesting that WNK3 also plays a major role in regulating blood pressure and sodium reabsorption in the kidney. However, it remains to be determined whether WNK3 is in fact involved in the regulation of these transporters in vivo. To clarify this issue, we generated and analyzed WNK3 knockout mice. Surprisingly, phosphorylation and expression of OSR1, SPAK, NKCC2 and NCC did not decrease in knockout mouse kidney under normal and low-salt diets. Similarly, expression of epithelial Na channel and Na/H exchanger 3 were not affected in knockout mice. Na+ and K+ excretion in urine in WNK3 knockout mice was not affected under different salt diets. Blood pressure in WNK3 knockout mice was not lower under normal diet. However, lower blood pressure was observed in WNK3 knockout mice fed low-salt diet. WNK4 and WNK1 expression was slightly elevated in the knockout mice under low-salt diet, suggesting compensation for WNK3 knockout by these WNKs. Thus, WNK3 may have some role in the WNK-OSR1/SPAK-NCC/NKCC2 signal cascade in the kidney, but its contribution to total WNK kinase activity may be minimal.

Common variation in WNK1 and blood pressure responses to dietary sodium or potassium interventions

Objectives: Blood pressure response to dietary sodium and potassium intake varies considerably among individuals. WNK1 is a member of the WNK family of serine/threonine kinases with no lysine (K), and these kinases have been implicated as important modulators of sodium and potassium homeostasis in the kidney. Aims of the study were to investigate whether WNK1 gene polymorphism are associated with blood pressure variations to dietary salt or potassium intervention. Methods: In Northern China, probands with SBP between 130 and 160mmHg and/or DBP between 85 and 100mmHg and their siblings, spouses, and offspring were recruited for the dietary intervention study.

Pseudohypoaldosteronism type 2 presenting with hypertension and hyperkalaemia due to a novel mutation in the WNK4 gene

A 41-year-old man was found to have high blood pressure of 220/100 mmHg during a routine health check-up for medical insurance. He was asymptomatic, and physical examination was unremarkable apart from a soft ejection systolic murmur heard loudest at the left sternal edge. Initial investigations showed serum sodium 144 mmol/l (reference range 132–144), potassium 6.0 mmol/l (3.5–5.5) and creatinine 100 μmol/l (45–120). There was no evidence of left ventricular hypertrophy on an electrocardiogram, and an echocardiography showed no abnormality apart from trivial tricuspid regurgitation. Renal ultrasound, renal perfusion scan and chest radiograph were also normal. The patient was given lifestyle advice, and was commenced on atenolol 100 mg once a day. Over the next 7 years, further antihypertensive agents—including amlodipine 10 mg, doxazosin 16 mg and lisinopril 5 mg—were gradually added. During this period, following an episode of renal colic, he was found to have a radio-opaque renal stone and was treated with lithotripsy. Despite multiple antihypertensive agents, his blood pressure was still consistently above 150/90 mmHg. In addition, his serum potassium levels, measured on several occasions, remained high between 5.8 and 6.6 mmol/l. Therefore, the patient was referred to the Endocrine outpatient clinic for further review. In the Endocrine clinic, it was noted that his mother and his only sister also suffered from hypertension and hyperkalaemia. We excluded pseudohyperkalaemia in our patient by analysing serum potassium level immediately after venesection, and carried out further tests to investigate possible …

Novel polymorphic AluYb8 insertion in the WNK1 gene is associated with blood pressure variation in Europeans

Mutations in WNK1 and WNK4 cause familial hypertension, the Gordon syndrome. WNK1 and WNK4 conserved noncoding regions were targeted to polymorphism screening using DHPLC and DGGE. The scan identified an undescribed polymorphic AluYb8 insertion in WNK1 intron 10. Screening in primates revealed that this Alu-insertion has probably occurred in human lineage. Genotyping in 18 populations from Europe, Asia, and Africa (n = 854) indicated an expansion of the WNK1 AluYb8 bearing chromosomes out of Africa. The allele frequency in Sub-Saharan Africa was ∼3.3 times lower than in other populations (4.8 vs. 15.8%; P = 9.7 × 10−9). Meta-analysis across three European sample sets (n = 3,494; HYPEST, Estonians; BRIGHT, the British; CADCZ, Czech) detected significant association of the WNK1 AluYb8 insertion with blood pressure (BP; systolic BP, P = 4.03 × 10−3, effect 1.12; diastolic BP, P = 1.21 × 10−2, effect 0.67). Gender-stratified analysis revealed that this effect might be female-specific (n = 2,088; SBP, P = 1.99 × 10−3, effect 1.59; DBP P = 3.64 × 10−4, effect 1.23; resistant to Bonferroni correction), whereas no statistical support was identified for the association with male BP (n = 1,406). In leucocytes, the expressional proportions of the full-length WNK1 transcript and the splice-form skipping exon 11 were significantly shifted in AluYb8 carriers compared to noncarriers. The WNK1 AluYb8 insertion might affect human BP via altering the profile of alternatively spliced transcripts. Hum Mutat 32:1–9, 2011. © 2011 Wiley-Liss, Inc.

Association of WNK1 exon 1 polymorphisms with essential hypertension in Hani and Yi minorities of China

The association of polymorphisms in exon 1 of the WNK1 gene with essential hypertension in the minority groups of Hani and Yi of China was investigated in the case-control study. The sequence of 1257bp containing the WNK1 gene exon 1 was determined in 1307 individuals (649 essential hypertension subjects and 658 controls) to identify SNPs in Hani and Yi minority groups. Four of eleven previously known SNPs (rs3168640, rs11885, rs11554421 and rs34880640) were identified. The SNP analysis indicated that SNPs rs11885 and rs11554421 were significantly associated with hypertension in both Hani and Yi populations, and rs34880640 was significantly associated with hypertension in Hani but not in Yi population, adjusted for covariates. Haplotype analysis indicated that the haplotype H1 significantly decreased the risk of hypertension in both populations. These results suggested that WNK1 polymorphisms were involved in the predisposition of essential hypertension in Hani and Yi populations and its effects showed a clear population specificity. This finding supported the importance of population specificity in determining the genetic factors associated with diseases and thus disease treatment.

A genome-wide association study-derived candidate gene seeks replication: STK39

A genome-wide association study-derived candidate gene seeks replication: STK39

A WNK4 gene variant relates to osteoporosis and not to hypertension in the Portuguese population

Germline mutations in the WNK4 gene originate Gordon syndrome or pseudohypoaldosteronism type II, a familial form of hypertension with hyperkalemia and hypercalciuria. In order to elucidate the contribution of WNK4 genetic variants to hypertension and/or osteoporosis, we analyzed 271 control individuals and a cohort of 448 hypertensive and 372 osteoporosis patients from the Portuguese population. Ten genetic variants were detected in 4.3% of the population under study, none of which revealed any significant association to the hypertension phenotype. In contrast, a rare missense alteration within exon 17 in a highly conserved arginine residue showed a possible tendency for association to the osteoporosis group. Our data suggest that WNK4 polymorphism rs56116165 is a rare allelic variant in a candidate gene with a biological function in renal calcium homeostasis that may contribute to a genetic predisposition to osteoporosis.

Association of the C1155547T polymorphism in WNK4 gene with essential hypertension in Xinjiang Kazakhs

To investigate whether the WNK lysine deficient protein kinase 4 (WNK4) gene C1155547T polymorphism is associated with essential hypertension (EH) in Xinjiang Kazakhs and to assess the effect of the interaction between this polymorphism and environment factors on EH. The study covered 556 hypertension patients and 341 normotensive controls. The C1155547T was determined by Taqman probe real-time PCR method. Some biochemical index such as glucose, triglyceride and total cholesterol were also measured. All of these results were analyzed with Logistic regression analysis. Additive model was applied to assess the effect of interaction between the WNK4 gene C1155547T polymorphism and environment factors on hypertension. The C1155547T polymorphism was consistent with Hardy-Weinberg equilibrium in both the case and control groups. There was significant difference in the genotype frequencies (P=0.003). The T allele frequency was significantly higher in the patient group (P=0.002). Logistic regression analysis revealed that the age, body mass index (BMI), total cholesterol as well as the CT+TT genotype frequency conferred increased risks for EH. Positive interaction between the C1155547T polymorphism and gender, BMI, glucose was observed. The ORs were 3.85 (95%CI:1.23-12.04), 5.91 (95%CI:1.99-17.57) and 8.77 (95%CI:1.04-73.93), respectively. The result suggested that the exon 7 C1155547T polymorphism in WNK4 gene might be associated with EH in Xinjiang Kazakhs, the T allele might be the risk factor of essential hypertension. There were interactive effects between the WNK4 gene C1155547T polymorphism and gender, BMI and glucose.

The soybean root-specific protein kinase GmWNK1 regulates stress-responsive ABA signaling on the root system architecture

In humans, members of the WNK protein kinase family are osmosensitive regulators of cell volume homeostasis and epithelial ion transport, and mutation of these proteins causes a rare inherited form of hypertension due to increased renal NaCl re-absorption. A related class of kinases was recently discovered in plants, but their functions are largely unknown. We have identified a root-specific WNK kinase homolog, GmWNK1, in soybean (Glycine max). GmWNK1 expression was detected in the root, specifically in root cells associated with lateral root formation, and was down-regulated by abscisic acid (ABA), as well as by mannitol, sucrose, polyethylene glycol and NaCl. In vitro and in vivo experiments showed that GmWNK1 interacts with another soybean protein, GmCYP707A1, which is a key ABA 8'-hydroxylase that functions in ABA catabolism. Furthermore, 35S-GmWNK1 transgenic soybean plants had reduced lateral root number and length compared with wild-type, suggesting a role of GmWNK1 in the regulation of root system architecture. We propose that GmWNK1 functions to fine-tune ABA-dependent ABA homeostasis, thereby mediating the regulation of the root system architecture by ABA and osmotic signals. The study has revealed a new function of a plant WNK1 gene from the important staple crop soybean, and has identified a new component of a regulatory pathway that is involved not only in ABA signaling, but also in the repression of lateral root formation by an ABA-dependent mechanism distinct from known ABA signaling pathways.

HAPLOTYPES IN THE WNK1 AND SLC12A3 GENES ARE ASSOCIATED TO HYPERTENSION IN A CHINESE POPULATION: 6B.02

Objective: Deletions in with-no-lysine-kinase 1 gene (WNK1) cause Gordon's syndrome, a rare Mendelian form of hypertension. The mechanism might partially be WNK1 activates thiazide-sensitive Na-Cl cotransporter(NCC, encoded by SLC12A3), leading to increased salt reabsorption in distal nephron. Our study aimed to evaluate the role of genetic variants at WNK1 and a possible interaction with SLC12A3 on BP and hypertension in a Chinese population. Methods: 13 polymorphisms were selected at the WNK1 and SLC12A3 genes and 550 hypertensives (HT1), a more severely affected group HT2 (n = 227) and 538 normotensive controls (NT) were collected. All SNPs were genotyped using Taqman assay. Multi-dimensional reduction (MDR) and R package HaploStats were used for data analysis. Results: Genotype and allele frequencies distribution of rs2286028 were different between cases and controls (p = 0.05 and 0.03 for allele for HT1, HT2, p = 0.006 for genotype for HT2) even after multiple correction the latter association remained the significance (p = 0.04). We found one common WNK1 haplotype significantly associated with higher risk for hypertension (P = 0.03 and P = 0.007 for HT1 and HT2) and BP (P = 0.005 and P = 0.001 for SBP and DBP) after adjustment for confounding factors and multiple testing. Another common haplotype was associated with potassium excretion (P = 0.04). One rare WNK1 haplotype was strongly associated with hypertension (p = 0.003and 0.005 for HT1 and HT2), BP (p = 0.03 and 0.005 for SBP and DBP) and potassium excretion (P = 0.04). Using MDR we generated a five-locus model in the two genes for gene interaction analysis. One common allelic combination showed significant association with SBP (p = 0.0009) and hypertension in HT1 (P = 0.01). Combinations with lower frequencies showed more striking associations with hypertension and BP, one of which presented a high risk factor for hypertension with highest OR = 31.5 (P = 0.00005). Conclusion: Our results strongly support the findings that WNK1 gene contributes to hypertension and BP, also in the Chinese population. A synergistic genetic interaction between WNK1 and SLC12A3 could play a significant role in hypertension and BP variation.

Pathophysiological roles of WNK kinases in the kidney

Since the discovery of mutations in the WNK1 and WNK4 genes in pseudohypoaldosteronism type II (PHAII), the pathophysiological role of WNK kinases in hypertension and renal ion transport has been a hot topic for investigation. Analyses from a mouse model carrying the same mutation as seen in PHAII patients, reveal a new signal cascade in the kidney that regulates NaCl and K balance in the body. WNK kinases phosphorylate and activate oxidative stress responsive kinase 1 (OSR1) and STE20-like proline and alanine-rich kinase (SPAK), and OSR1 and SPAK phosphorylate and activate the thiazide-sensitive Na-Cl cotransporter (NCC). Furthermore, this cascade is regulated by aldosterone, indicating that WNK-OSR1/SPAK-NCC cooperates with this system including the epithelial Na channel (ENaC) to conserve NaCl. With regard to K excretion, however, both systems work in opposite directions whereby PHAII and Liddle syndrome show hyperkalemia and hypokalemia, respectively. Thus, the identification of such aldosterone effecters other than ENaC, will reveal a novel regulatory mechanism of K excretion in the distal nephron, and also provides basic evidence for the therapeutic use of thiazide in various clinical situations.

Study on the association between genetic polymorphism on WNK4 genes and essential hypertension among Kazakhs ethnic population, in Xinjiang

To investigate whether G1155942T polymorphism in WNK4 gene is associated with essential hypertension in a population with Kazakhs ethnicity, in Xinjiang. This study covered 563 hypertension patients and 346 normotensive controls. The variant of G1155942T was determined by the TaqMan probe real-time PCR method. Some biochemical indices such as glucose (GLU), triglyceride (TG) and total cholesterol (TC) were also measured. All of these results were under logistic regression analysis. Addictive model was applied to assess the interactive effects between WNK4 gene G1155942T mutation and environmental factors on hypertension. The G1155942T polymorphism was consistent with Hardy-Weinberg expectations in both case and control groups. Genotype and allele frequencies of G1155942T were observed (P = 0.004, P = 0.003). Data through logistic regression analysis showed that factors as age, BMI, total cholesterol as well as the GT + TT genotype frequencies of Exon 8 G1155942T polymorphism in WNK4 were responsible for the increased risks for hypertension. Positive interactions between G1155942T mutation and gender, BMI, GLU, the OR were 3.75 (95%CI: 1.19 - 11.80), 5.77 (95%CI: 1.93 - 17.21) and 8.67 (95%CI: 1.03 - 72.99), respectively. Our result suggested that the Exon 8 G1155942T polymorphism in WNK4 gene was associated with hypertension in the studied Kazakhs ethnic group in Xinjiang and the T allele might be the risk factor for essential hypertension. There were interactive effects between WNK4 gene G1155942T mutation, gender, BMI, and GLU.

Targeted disruption of the Wnk4 gene decreases phosphorylation of Na-Cl cotransporter, increases Na excretion and lowers blood pressure

We recently generated Wnk4(D561A/+) knockin mice and found that a major pathogenesis of pseudohypoaldosteronism type II was the activation of the OSR1/SPAK kinase-NaCl cotransporter (NCC) phosphorylation cascade by the mutant WNK4. However, the physiological roles of wild-type WNK4 on the regulation of Na excretion and blood pressure, and whether wild-type WNK4 functions positively or negatively in this cascade, remained to be determined. In the present study, we generated WNK4 hypomorphic mice by deleting exon 7 of the Wnk4 gene. These mice did not show hypokalemia and metabolic alkalosis, but they did exhibit low blood pressure and increased Na and K excretion under low-salt diet. Phosphorylation of OSR1/SPAK and NCC was significantly reduced in the mutant mice as compared with their wild-type littermates. Protein levels of ROMK and Maxi K were not changed, but epithelial Na channel appeared to be activated as a compensatory mechanism for the reduced NCC function. Thus, wild-type WNK4 is a positive regulator for the WNK-OSR1/SPAK-NCC cascade, and WNK4 is a potential target of anti-hypertensive drugs.

LINGO-1 Interacts with WNK1 to Regulate Nogo-induced Inhibition of Neurite Extension

LINGO-1 is a component of the tripartite receptor complexes, which act as a convergent mediator of the intracellular signaling in response to myelin-associated inhibitors and lead to collapse of growth cone and inhibition of neurite extension. Although the function of LINGO-1 has been intensively studied, its downstream signaling remains elusive. In the present study, a novel interaction between LINGO-1 and a serine-threonine kinase WNK1 was identified by yeast two-hybrid screen. The interaction was further validated by fluorescence resonance energy transfer and co-immunoprecipitation, and this interaction was intensified by Nogo66 treatment. Morphological evidences showed that WNK1 and LINGO-1 were co-localized in cortical neurons. Furthermore, either suppressing WNK1 expression by RNA interference or overexpression of WNK1-(123-510) attenuated Nogo66-induced inhibition of neurite extension and inhibited the activation of RhoA. Moreover, WNK1 was identified to interact with Rho-GDI1, and this interaction was attenuated by Nogo66 treatment, further indicating its regulatory effect on RhoA activation. Taken together, our results suggest that WNK1 is a novel signaling molecule involved in regulation of LINGO-1 mediated inhibition of neurite extension.

Genetic variants in angiogenesis pathway associated with clinical outcome in NSCLC patients (pts) treated with bevacizumab in combination with carboplatin and paclitaxel: Subset pharmacogenetic analysis of ECOG 4599

8032 Background: E4599 was a randomized phase III study which demonstrated a survival advantage in advanced NSCLC pts treated with bevacizumab (bev) + carboplatin/paclitaxel (BPC) versus carboplatin/paclitaxel alone (PC). Recent studies have shown certain germline polymorphisms (SNPs) are associated with clinical outcome in advanced breast cancer pts treated with paclitaxel plus bev and in advanced colorectal cancer pts treated with first line 5-FU or capecitabine in combination with oxaliplatin and bev. A study also found 2 SNPs in WNK1 gene were associated with bev-induced hypertension. We tested the hypothesis that SNPs involved in angiogenesis pathway (VEGF, EGF, EGFR, IL-8, KDR, ICAM1, FGFR4), DNA repair pathway (ERCC1, XPD, XRCC1, GSTP1) and WNK1 may predict clinical outcome in a subset of pts enrolled on E4599. Methods: Of 878 pts enrolled, samples from 146 pts were available for the current pharmacogenetic study and only 133 of the samples (67 PC, 66 BPC) came from eligible pts. PCR-RFLP assays were performed on genomic DNA extracted from sera of pts. The Kaplan-Meier method was used to estimate time-to-event distributions. Multivariable Cox models adjusted for gender, PS, stage, adrenal, liver and bone mets were separately fitted for each SNP to obtain estimates of hazard ratios. Results: Median OS for the 133 patients was 10.3 mos (8.2–15.6) for PC and 13.0 mos (10.2–16.6) for BPC. Median PFS was 4.6 mos (3.6–5.6) for PC & 6.5 mos (5.4–8.3) for BPC. Pts with mutant homozygote CC genotype for ICAM1 T469C had significantly higher tumor response rate (39%) than heterozygote CT (13%) and homozygote TT (20%) genotype (Fisher's test, p=0.04). Tests for whether treatment effect differs by genotype via interaction terms in the Cox models were statistically significant (p<0.05) for VEGF G-634C, ICAM1 T469C and WNK1-rs11064560 for OS, and for ICAM1 T469C, EGF A-61G and CXCR2 C785T for PFS. Conclusions: Although exploratory, our preliminary results suggest germline SNPs in angiogenesis pathway may predict response, PFS and OS in NSCLC pts treated with BPC. Prospective trials based on these correlative studies are warranted. [Table: see text]

Polymorphisms in the WNK1 Gene Are Associated 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 p<10−7). We identified several common haplotypes to be associated with increased BP and multiple low frequency haplotypes significantly associated with lower BP (>10 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.

J026 A role for L-WNK1 in cardiovascular development and vasoconstriction

Mutations at the WNK1 gene cause Gordon syndrome, a rare inherited form of arterial hypertension. The gene encodes a short kidney-specific isoform and a complete long isoform, L-WNK1, expressed ubiquitously and notably within the vascular system. The aim of our study was to investigate the vascular role of L-WNK1 in mice. We used mice bearing a constitutive homozygous inactivation of L-WNK1 (L-WNK1-/-) which die in utero before day 13 of gestation as well as adult healthy heterozygous (L-WNK1+/-) and wild-type (L-WNK1+/+) littermates. We isolated vessels from L-WNK1+/- and L-WNK1+/+ to perform in vitro pharmacological studies in a wire-myograph system and an arteriograph system. We first showed that L-WNK1-/- embryos present growth retardation and severe oedema. An abnormal vascular remodeling was observed at day 10.5 within the primary vascular network of L-WNK1-/- embryos as well as in the yolk sac, suggesting an important role of L-WNK1 in cardiovascular development. We next showed that vascular diameters of pressurized arteries as well as arterial blood flow velocities measured by echo-Doppler were comparable between L-WNK1+/- and L-WNK1+/+ mice. Endothelium-dependent dilatations induced either by acetylcholine or by flow were also comparable between both groups of mice. In contrast, phenylephrine-induced vasoconstrictions were significantly reduced in L-WNK1+/- mice compared to L-WNK1+/+ mice in thoracic aorta as well as in mesenteric arteries (27.45 % (P=0.04) and 39.6 % (P=0.0015) decrease at maximal concentration, respectively) whereas potassium chloride contractions remained comparable. Furthermore, myogenic tone in L-WNK1+/- mesenteric arteries was also significantly blunted when compared to L-WNK1+/+ mice (P<0.0001). Our results evidenced for the first time a vascular role for L-WNK1 in pressure- and agonist- induced contractions.