NEDD4L in essential hypertension

Unravelling the genetic architecture of blood pressure (BP) regulation and hypertension (HTN) is an ongoing process. The progressive sweeps using genome-wide association studies (GWASs) have so far identified 43 single nucleotide polymorphisms (SNPs) to be associated with BP and HTN [1–8]. The effect sizes for each individual SNP are typically 1 mmHg for SBP and 0.5 mmHg for DBP and these SNPs collectively explain only 1–2% of the phenotypic variance of BP. Despite this, when the risk alleles of all the associated SNPs are aggregated into a BP genetic risk score, it is predictive of longitudinal BP and cardiovascular risk [9], although the individual predictive utility of genetic risk scores appears to be limited [10]. Statistical modelling estimates that around 116 common variants with similar effect sizes to the GWAS SNPs will explain underlying BP variation [1]. There is also the possibility of low frequency variants with large effect sizes accounting for some of the missing heritability. A resequencing study of three candidate genes (SLC12A3, SLC12A1, KCNJ1 involved in Bartter's or Gitelman's syndromes) in the Framingham Heart Study population showed that the heterozygous state of 30 distinct rare variants was associated with 6 mmHg lower BP at age 40, and 9.0 mmHg lower BP at age 60, and in aggregate reduces the risk of HTN by 60% at age 60 years [11]. Exome sequencing studies are underway seeking to validate this and identify whether there are other rare variants influencing BP. There is a need for alternate strategies to reliably discover new genetic variants associated with BP that may not be tractable through GWAS. It is in this context that the study by Dahlberg et al.[12] in this issue of the journal needs to be considered. Using a hypothesis-driven approach, they tested whether a salt-sensitive genotype combination of two SNPs in the NEDD4L gene was associated with BP and, after finding an association, they followed this up with evidence that this risk genotype was associated with greater risk of coronary events and cardiovascular mortality in the same population. The amiloride-sensitive epithelial sodium channel (ENaC) is pivotal for the transport of Na+ across the epithelia in the nephron to maintain sodium and fluid volume homeostasis. Here it is primarily expressed in the distal connecting tubules and the cortical collecting tubules of the nephron where it provides the rate-limiting step for Na+ reabsorption into the blood stream. ENaC is regulated largely via physiological mechanisms that control the expression of the transporter at the cell surface of the apical membrane [13,14]. The regulation of Na+ reabsorption is tightly controlled by the actions of the hormones aldosterone and vasopressin. ENaC channel is a heteromultimeric channel consisting of three subunits, α, β, and γ, whereof the α subunit is necessary for appropriate channel function and insertion onto the apical membrane. When active, this channel accounts for electrogenic Na+ reabsorption by active transport powered by the sodium pump. While mediating electrogenic Na+ absorption, transepithelial Cl- absorption is thought to be both paracellular (via tight junctions) and transcellular (across intercalated cells). ENaC mediates Na+ absorption across the apical membrane of principal cells, which provides the driving force for K+ secretion through K+ channels and plays a role in H+ or HCO3- secretion. The lumen-negative potential generated by ENaC also provides a driving force for Cl- absorption across tight junctions and lateral intercellular spaces. ENaC inhibitors, such as amiloride, eliminate the lumen-negative transepithelial voltage, which is thought to reduce Cl- absorption by eliminating the driving force for paracellular Cl- absorption [14]. The rate at which Na+ is transported must be stringently regulated to maintain volume homeostasis in the face of extreme sodium intake. A defect in trafficking of ENaC to the cell surface is responsible for inherited forms of salt-sensitive HTN [15,16]. Liddle's syndrome is a dominant form of monogenic HTN characterized by excessive Na+ reabsorption. This condition is associated with early onset of HTN, hypokalemia alkalosis, suppressed plasma renin activity, and low plasma aldosterone levels leading to elevated ENaC channel expression and activity at the apical membrane [15,16]. A number of reports have identified several deletion/missense mutations that cause Liddle's syndrome, all of which map to the β or γ ENaC subunits [15,17]. These genetic mutations result in either a complete deletion of the C-terminus of the β or γ ENaC subunits, or mutate a proline or tyrosine within a short sequence called the PY motif (Pro Pro X Tyr) [15,18]. This PY motif or the extended PY motif (PPXYXXL) are highly conserved in the C-terminus of all ENaC subunits and serve as binding sites to the NEDD4 (neuronal precursor cell-expressed developmentally downregulated protein) family of ubiquitin ligases [19,20]. The PY motif of the ENaC subunits interacts specifically with NEDD4-1 and NEDD4-2 (also referred to as NEDD4L) proteins as a first step in the ubiquitination and clearance of ENaC. NEDD4L binds with higher affinity to ENaC than NEDD4-1. NEDD4 family members are E3 ubiquitin ligages comprising a C2 domain responsible for membrane targeting, three to four WW domains that bind the PY motif of ENaC, and the ubiquitin ligase domain HECT (homologous to the E6-AP carboxyl terminus) [20]. Studies have shown that NEDD4L suppresses ENaC, during times of high salt intake, via ubiquitylation [21]. Two mechanisms have been proposed to explain the increased surface expression levels of ENaC. First, ENaC is controlled by a sequence conserved in the C-terminus of the α, β, and γ subunits (PPPXYXXL). Missense mutation of this sequence leads to increased ENaC surface expression, and excess sodium reabsorption [15,18,21]. Second, the PPPXYXXL sequence fits the consensus of the PY motif to bind NEDD4L. The NEDD4-related E3 ubiquitin-protein ligases contain three to four WW domains that bind to PY motifs in all ENaC subunits [19,22]. As NEDD4L targets ENaC for degradation, it plays a key role in minimizing Na+ transport during times of high salt intake; conversely, ENaC surface availability must increase to facilitate enhanced Na+ reabsorption in times of sodium depletion. In-vitro experiments in Xenopus oocytes have demonstrated that mutations in the PY motif of the β or γ subunits of ENaC found in Liddle's syndrome severely attenuate the degradation of ENaC by NEED4L, leading to reduced clearance of ENaC from the tubule membrane, augmented retention of mutant ENaC at the apical cell surface resulting in enhanced activity and expression of the channel, thus increasing Na+ reabsorption [23]. A conditional knockout rodent model of Liddle's syndrome (deletion of the PY motif on the β ENaC subunit) developed HTN when fed a high salt diet [24]. As ENaC is positively regulated in the kidney by the hormone aldosterone, studies have focused on serum and glucocorticoid-induced kinase (SGK), an aldosterone target, to further elucidate ENaC function in disease. SGK a Ser/Thr kinase was shown to elevate ENaC expression and activity via NEDD4L regulation. NEDD4L contains phosphorylation sites for SGK and upon phosphorylation, it is prevented from downregulating ENaC [25]. Reports this far show that the ENaC-NEDD4L proteasome system plays a key role in BP regulation and sodium homeostasis; however, to further improve understanding of this system and to provide insight into the pathogenesis and genetic epidemiology of essential HTN, the study on effect of common variations in the gene encoding NEDD4L is required. Dunn et al.[26] demonstrated that a frameshift mutation (a C2 encoding exon) of NEDD4L, rs4149601 (G/A), results in the premature truncation of the NEDD4L protein, and was associated with ambulatory BP and progression of BP over time. A functional study by Dahlberg et al. [27] in 2007 demonstrated that individuals carrying the G allele of rs4149601 have shown significant association with the phenotypes of HTN inconsistent with reports from Araki et al.[28] who suggest that NEDD4L isoform I, which is generated by rs4149601 A allele leads to abnormal Na+ reabsorption. Manunta et al.[29] observed that BP responses to diuretic treatment were greater in hypertensive individuals who carry a combination of the common alleles of NEDD4L. In a Chinese study, Luo et al.[30] showed that the A allele of rs4149601 was associated with HTN, but protective against orthostatic hypotension in hypertensive individuals. They went on to show that the anithypertenisve response to hydrochlorothiazide is more sensitive in A allele carriers. The study by Dahlberg et al.[12] in this journal provides an incremental advance on the pathophysiological role of NEDD4L in essential HTN. One weakness of the study is that the outcome study was performed on the same population as the discovery study; however, this does not minimize the value of the results and should encourage other investigators to validate or refute this risk variant. Nevertheless, NEDD4L remains an attractive target for further molecular mechanistic and population studies. ACKNOWLEDGEMENTS Conflicts of interest There are no conflicts of interest.