Keith Siew

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.

Critical role of the SPAK protein kinase CCT domain in controlling blood pressure

The STE20/SPS1-related proline/alanine-rich kinase (SPAK) controls blood pressure (BP) by phosphorylating and stimulating the Na-Cl (NCC) and Na-K-2Cl (NKCC2) co-transporters, which regulate salt reabsorption in the kidney. SPAK possesses a conserved carboxy-terminal (CCT) domain, which recognises RFXV/I motifs present in its upstream activator [isoforms of the With-No-lysine (K) kinases (WNKs)] as well as its substrates (NCC and NKCC2). To define the physiological importance of the CCT domain, we generated knock-in mice in which the critical CCT domain Leu502 residue required for high affinity recognition of the RFXI/V motif was mutated to Alanine. The SPAK CCT domain defective knock-in animals are viable, and the Leu502Ala mutation abolished co-immunoprecipitation of SPAK with WNK1, NCC and NKCC2. The CCT domain defective animals displayed markedly reduced SPAK activity and phosphorylation of NCC and NKCC2 co-transporters at the residues phosphorylated by SPAK. This was also accompanied by a reduction in the expression of NCC and NKCC2 protein without changes in mRNA levels. The SPAK CCT domain knock-in mice showed typical features of Gitelman Syndrome with mild hypokalaemia, hypomagnesaemia, hypocalciuria and displayed salt wasting on switching to a low-Na diet. These observations establish that the CCT domain plays a crucial role in controlling SPAK activity and BP. Our results indicate that CCT domain inhibitors would be effective at reducing BP by lowering phosphorylation as well as expression of NCC and NKCC2.

Extrarenal roles of the with-no-lysine[K] kinases (WNKs).

Identified over a decade ago, the with‐no‐lysine[K] kinases (WNKs) have been the subsequent focus of intense research into the renal handling of Na+, Cl− and K+ and several rare monogenetic diseases. However, the potential extrarenal roles for WNKs have been less well explored. Thiazides and Gordon syndrome are known to have effects on bone mineral density, Ca2+ and PO43− homeostasis, which were originally assumed to be an indirect effect through the kidney. However, current data suggest a complex and direct role for WNKs in the physiology of bone. The WNKs also modulate systemic blood pressure at several levels, including the vascular resistance vessels, where they cause vasoconstriction by altering the abundance of the transient receptor potential canonical channel 3 and/or phosphorylation of the Na+−K+−2Cl− cotransporter 1 in vascular smooth muscle cells. The WNKs and many of the cation‐coupled Cl− cotransporters they regulate are highly expressed in the central nervous system and recent work suggests that WNK dysfunction may have a role in the development of autism, schizophrenia and hereditary sensory and autonomic neuropathy Type 2. Finally, the WNK–sterile 20 kinase signalling axis represents an evolutionarily ancient mechanism for maintaining osmotic homeostasis, but a rapidly expanding body of evidence also shows a role in immunity and cellular regulation.

Phenotypic and pharmacogenetic evaluation of patients with thiazide-induced hyponatremia

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 E2 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 E2 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.

Characterization of [125I]GLP-1(9-36), a novel radiolabeled analog of the major metabolite of glucagon-like peptide 1 to a receptor distinct from GLP1-R and function of the peptide in murine aorta

AIMS Glucagon-like peptide 1 (GLP-1) is an insulin secretagogue, released in response to meal ingestion and efficiently lowers blood glucose in Type 2 diabetic patients. GLP-1(7-36) is rapidly metabolized by dipeptidyl peptidase IV to the major metabolite GLP-1(9-36)-amide, often thought to be inactive. Inhibitors of this enzyme are widely used to treat diabetes. Our aim was to characterize the binding of GLP-1(9-36) to native mouse tissues and to cells expressing GLP1-R as well as to measure functional responses in the mouse aorta compared with GLP-1(7-36). MAIN METHODS The affinity of [(125)I]GLP-1(7-36) and [(125)I]GLP-1(9-36) was measured in mouse tissues by saturation binding and autoradiography used to determine receptor distribution. The affinity of both peptides was compared in binding to recombinant GLP-1 receptors using cAMP and scintillation proximity assays. Vasoactivity was determined in mouse aortae in vitro. KEY FINDINGS In cells expressing GLP-1 receptors, GLP-1(7-36) bound with the expected high affinities (0.1 nM) and an EC50 of 0.07 nM in cAMP assays but GLP-1(9-36) bound with 70,000 and 100,000 fold lower affinities respectively. In contrast, in mouse brain, both labeled peptides bound with a single high affinity, with Hill slopes close to unity, although receptor density was an order of magnitude lower for [(125)I]GLP-1(9-36). In functional experiments both peptides had similar potencies, GLP-1(7-36), pD2=7.40 ± 0.24 and GLP-1(9-36), pD2=7.57 ± 0.64. SIGNIFICANCE These results suggest that GLP-1(9-36) binds and has functional activity in the vasculature but these actions may be via a pathway that is distinct from the classical GLP-1 receptor and insulin secretagogue actions.

The matrix proteins aggrecan and fibulin-1 play a key role in determining aortic stiffness

Stiffening of the aorta is an important independent risk factor for myocardial infarction and stroke. Yet its genetics is complex and little is known about its molecular drivers. We have identified for the first time, tagSNPs in the genes for extracellular matrix proteins, aggrecan and fibulin-1, that modulate stiffness in young healthy adults. We confirmed SNP associations with ex vivo stiffness measurements and expression studies in human donor aortic tissues. Both aggrecan and fibulin-1 were found in the aortic wall, but with marked differences in the distribution and glycosylation of aggrecan reflecting loss of chondroitin-sulphate binding domains. These differences were age-dependent but the striking finding was the acceleration of this process in stiff versus elastic young aortas. These findings suggest that aggrecan and fibulin-1 have critical roles in determining the biomechanics of the aorta and their modification with age could underpin age-related aortic stiffening.

Functional characterization of common BCL11B gene desert variants suggests a lymphocyte-mediated association of BCL11B with aortic stiffness.

The recent genome-wide analysis of carotid–femoral pulse wave velocity (PWV) identified a significant locus within the 14q32.2 gene desert. Gene regulatory elements for the transcriptional regulator B-cell CLL/lymphoma 11B (BCL11B) are within this locus and an attractive target for the gene association. We investigated the functional impact of these gene desert SNPs on BCL11B transcript in human aorta to characterize further its role in aortic stiffness. To do this, we used a large repository of aortic tissues (n = 185) from an organ transplant program and assessed ex vivo stiffness of the aortic rings. We tested association of three lead SNPs from the GWAS meta-analysis with ex vivo aortic stiffness and BCL11B aortic mRNA expression: rs1381289 and rs10782490 SNPs associated significantly with PWV and showed allele-specific differences in BCL11B mRNA. The risk alleles associated with lower BCL11B expression, suggesting a protective role for BCL11B. Despite strong association, we could not detect BCL11B protein in the human aorta. However, qPCR for CD markers showed that BCL11B transcript correlated strongly with markers for activated lymphocytes. Our data confirm the significance of the 14q32.2 region as a risk locus for aortic stiffness and an upstream regulator of BCL11B. The BCL11B transcript detected in the human aorta may reflect lymphocyte infiltration, suggesting that immune mechanisms contribute to the observed association of BCL11B with aortic stiffness.