Pseudohypoaldosteronism Type II Research Articles

Abstract P158: Cullin3 Regulated Endothelial Function by Modulating eNOS Activity

Pseudohypoaldosteronism type II (PHAII) patients expressing dominant negative cullin3 mutations exhibit increased renal NaCl reabsorption and develop hyperkalaemia, metabolic acidosis and hypertension. It is unclear whether loss of cullin3 function in extra-renal tissues contributes to the hypertensive phenotype. In the vasculature, endothelial Nrf2 stability is tightly regulated by cullin3-based E3 ubiquitin ligase via the redox-sensitive adaptor kelch-like ECH-associated protein 1. In the present study, we found that 24-hour treatment with a pan cullin inhibitor MLN4924 (1 μM) caused a 3-fold increase in Nrf2 protein in mouse lung endothelial cells (MLECs), while tert-butyl hydroperoxide (tBHP, 240 μM) had no effect on Nrf2 level. However, both MLN4924 and tBHP triggered time-dependent accumulation of Nrf2 in the nuclei, which peaked at 40 minutes following treatment. As a result, both treatments induced marked upregulation of antioxidant genes including NAD(P)H quinone oxidoreductase 1, heme oxygenase 1, glutamate cysteine ligase (rate-limiting enzyme in glutathione synthesis), and catalase both in MLECs and primary mouse aortic endothelial cells (MAECs). Of note, MLN4924 upregulated Nox4 expression (1.0 ± 0.15 vs 1.7 ± 0.2) and tBHP upregulated Nox1 (1.0 ± 0.2 vs 4.8 ± 1.1), while MLN4924 and tBHP both markedly increased intracellular superoxide as determined by dihydroethidium staining. In addition, intracellular nitric oxide was decreased by half in MLN4924-treated MLECs. This redox imbalance was likely due to impaired eNOS expression and activation as MLN4924 caused a 25% reduction in total eNOS and a 75% reduction in phosphorylated eNOS, while tBHP lead to a 50% reduction in phosphorylated eNOS with no effect on total eNOS. This suggests that decreased eNOS activation contributed to the oxidative stress induced by these agents. These data imply that suppression of cullin3 in arterial endothelial cells may dampen endothelium-dependent vascular relaxation and contribute to the blood pressure elevation observed in PHAII patients with global loss of cullin3 function. Although cullin3 also negatively regulates Nrf2-mediated antioxidant responses in vascular endothelial cells, this likely occurs as a compensatory mechanism.

Abstract 048: Expression of a Hypertension-causing Mutation in Cullin 3 (CUL3Δ9) Specifically in Smooth Muscle Causes Vascular Dysfunction and Hypertension

Pseudohypoaldosteronism type II (PHAII) patients with mutations in cullin 3 (CUL3) resulting in exon 9 deletion (CUL3Δ9), exhibit severe early onset hypertension correlated with impaired kidney function. However, the extra-renal mechanisms remain uninvestigated. We hypothesized that expression of CUL3Δ9 protein in smooth muscle in mice impairs endogenous wildtype CUL3 (CUL3-WT) function and causes vascular dysfunction and hypertension. We generated transgenic mice inducibly expressing CUL3Δ9 protein in smooth muscle (S-CUL3Δ9) and measured blood pressure (BP) by radiotelemetry. We assessed vascular responses in the cerebral basilar artery and aorta using a pressurized and a wire myograph, respectively. S-CUL3Δ9 mice exhibited reduced expression of endogenous CUL3WT protein compared to non-transgenic (NT) in aorta. Systolic BP was significantly increased in S-CUL3Δ9 mice (127±2 S-CUL3Δ9 vs 117±1 NT, p=0.02). Basilar artery from S-CUL3Δ9 mice exhibited significantly impaired vasorelaxation to acetylcholine (ACh) (at 100 μM: 15±4% S-CUL3Δ9 vs 65±5% NT, p<0.0001), and to the nitric oxide donor sodium nitroprusside (SNP) (at 100 μM: 59±2% S-CUL3Δ9 vs 90±5% NT, p<0.05). Vasocontraction to angiotensin II (Ang II), phenylephrine (PE) and to endothelin 1 (ET-1) were significantly elevated in S-CUL3Δ9 transgenic mice. Consistent with data from basilar artery, aorta from S-CUL3Δ9 transgenic mice exhibited impaired ACh-mediated relaxation (at 100 μM: 55±2% S-CUL3Δ9 vs 71±7% NT, p<0.0001). Total RhoA protein was significantly elevated in aorta of S-CUL3Δ9 transgenic mice (1.6±0.2 S-CUL3Δ9 vs 1.0±0.1 NT, P<0.05). Serotonin stimulation caused a significant increase in active RhoA in S-CUL3Δ9 aorta (1.83±0.04 S-CUL3Δ9 versus 1.52±0.06 NT, p=0.005). Preincubation with the Rho-kinase inhibitor (Y27632) restored endothelium-dependent relaxation in basilar artery and aorta of S-CUL3Δ9 mice. Ang II infusion via osmotic minipump (200 ng/kg/min) resulted in elevated BP response (Systolic BP: 147 ± 2 S-CUL3Δ9 versus 130 ± 5 NT, p=0.04) and increased aortic stiffening in S-CUL3Δ9 mice. We conclude that CUL3Δ9 acts in a dominant negative manner by interfering with CUL3-WT and contributes at least in part to hypertension via its effects on the vasculature.

With no lysine L-WNK1 isoforms are negative regulators of the K+-Cl- cotransporters.

The K(+)-Cl(-) cotransporters (KCC1-KCC4) encompass a branch of the SLC12 family of electroneutral cation-coupled chloride cotransporters that translocate ions out of the cell to regulate various factors, including cell volume and intracellular chloride concentration, among others. L-WNK1 is an ubiquitously expressed kinase that is activated in response to osmotic stress and intracellular chloride depletion, and it is implicated in two distinct hereditary syndromes: the renal disease pseudohypoaldosteronism type II (PHAII) and the neurological disease hereditary sensory neuropathy 2 (HSN2). The effect of L-WNK1 on KCC activity is unknown. Using Xenopus laevis oocytes and HEK-293 cells, we show that the activation of KCCs by cell swelling was prevented by L-WNK1 coexpression. In contrast, the activity of the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 was remarkably increased with L-WNK1 coexpression. The negative effect of L-WNK1 on the KCCs is kinase dependent. Elimination of the STE20 proline-alanine rich kinase (SPAK)/oxidative stress-responsive kinase (OSR1) binding site or the HQ motif required for the WNK-WNK interaction prevented the effect of L-WNK1 on KCCs, suggesting a required interaction between L-WNK1 molecules and SPAK. Together, our data support that NKCC1 and KCCs are coordinately regulated by L-WNK1 isoforms.

Osmotic stress induces the phosphorylation of WNK4 Ser575 via the p38MAPK-MK pathway.

The With No lysine [K] (WNK)-Ste20-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase 1 (OSR1) pathway has been reported to be a crucial signaling pathway for triggering pseudohypoaldosteronism type II (PHAII), an autosomal dominant hereditary disease that is characterized by hypertension. However, the molecular mechanism(s) by which the WNK-SPAK/OSR1 pathway is regulated remain unclear. In this report, we identified WNK4 as an interacting partner of a recently identified MAP3K, apoptosis signal-regulating kinase 3 (ASK3). We found that WNK4 is phosphorylated in an ASK3 kinase activity-dependent manner. By exploring the ASK3-dependent phosphorylation sites, we identified Ser575 as a novel phosphorylation site in WNK4 by LC-MS/MS analysis. ASK3-dependent WNK4 Ser575 phosphorylation was mediated by the p38MAPK-MAPK-activated protein kinase (MK) pathway. Osmotic stress, as well as hypotonic low-chloride stimulation, increased WNK4 Ser575 phosphorylation via the p38MAPK-MK pathway. ASK3 was required for the p38MAPK activation induced by hypotonic stimulation but was not required for that induced by hypertonic stimulation or hypotonic low-chloride stimulation. Our results suggest that the p38MAPK-MK pathway might regulate WNK4 in an osmotic stress-dependent manner but its upstream regulators might be divergent depending on the types of osmotic stimuli.

Involvement of selective autophagy mediated by p62/SQSTM1in KLHL3-dependent WNK4 degradation.

We reported that kelch-like protein 3 (KLHL3)-Cullin3 E3 ligase ubiquitinates with-no-lysine kinase 4 (WNK4) and that impaired WNK4 ubiquitination causes pseudohypoaldosteronism typeII, a hereditary hypertensive disease. However, we also found that KLHL3-induced WNK4 degradation could not be inhibited completely by a proteasome inhibitor. Rather, on exposure, for 24h, of HEK293T cells expressing WNK4 and KLHL3 to a proteasome inhibitor, epoxomicin, the WNK4 protein level was further decreased. As proteasome inhibition is known to activate p62-mediated selective autophagy, we investigated whether WNK4 degradation induced by KLHL3 is also mediated by such an autophagic mechanism. 3-Methyladenine, an autophagy inhibitor, blocked the epoxomicin-induced decrease in WNK4. Co-immunoprecipitation assays revealed that KLHL3 formed a complex not only with WNK4 but also with p62 via its kelch repeat domain. Under proteasome inhibition, p62 overexpression decreased KLHL3 and WNK4 protein levels, and p62 knockdown dramatically increased KLHL3 and WNK4 protein levels. Based on immunofluorescent staining, transiently overexpressed WNK4 showed punctate localization in the cytoplasm where it co-localized with KLHL3, p62 and light chain 3, a marker of autophagosomes. Thus, WNK4 was degraded not only by proteasomes but also by p62-KLHL3-mediated selective autophagy, which may be involved in WNK regulation under certain pathophysiological conditions.

Kelch-like 3/Cullin 3 ubiquitin ligase complex and WNK signaling in salt-sensitive hypertension and electrolyte disorder.

Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by salt-sensitive hypertension, hyperkalemia and thiazide sensitivity. Mutations in with-no-lysine kinase 1 (WNK1) and WNK4 genes are reported to cause PHAII. Rigorous studies have demonstrated that WNK kinases constitute a signaling cascade with oxidative stress-responsive gene 1 (OSR1), Ste20-related proline-alanine-rich kinase (SPAK) and the solute carrier family 12a (SLC12a) transporter, including thiazide-sensitive NaCl cotransporter. The WNK-OSR1/SPAK-SLC12a signaling cascade is present in the kidneys and vascular smooth muscle cells (VSMCs) and regulates salt sensitivity physiologically, i.e. urinary sodium excretion and arterial tone by various hormonal and dietary factors. However, although it was clear that the abnormal activation of this signaling cascade is the molecular basis of PHAII, the molecular mechanisms responsible for the physiological regulation of WNK signaling and the effect of WNK4 mutations on PHAII pathogenesis are poorly understood. Two additional genes responsible for PHAII, Kelch-like 3 (KLHL3) and Cullin 3 (CUL3), were identified in 2012. WNK1 and WNK4 have been shown to be substrates of KLHL3-CUL3 E3 ubiquitin ligase both in vitro and in vivo In PHAII, the loss of interaction between KLHL3 and WNK4 induces increased levels of WNK kinases due to impaired ubiquitination. These results indicate that WNK signaling is physiologically regulated by KLHL3/CUL3-mediated ubiquitination. Here, we review recent studies investigating the pathophysiological roles of the WNK signaling cascade in the kidneys and VSMCs and recently discovered mechanisms underlying the regulation of WNK signaling by KLHL3 and CUL3.

Kelch-Like Protein 2 Mediates Angiotensin II-With No Lysine 3 Signaling in the Regulation of Vascular Tonus.

Recently, the kelch-like protein 3 (KLHL3)-Cullin3 complex was identified as an E3 ubiquitin ligase for with no lysine (WNK) kinases, and the impaired ubiquitination of WNK4 causes pseudohypoaldosteronism type II (PHAII), a hereditary hypertensive disease. However, the involvement of WNK kinase regulation by ubiquitination in situations other than PHAII has not been identified. Previously, we identified the WNK3-STE20/SPS1-related proline/alanine-rich kinase-Na/K/Cl cotransporter isoform 1 phosphorylation cascade in vascular smooth muscle cells and found that it constitutes an important mechanism of vascular constriction by angiotensin II (AngII). In this study, we investigated the involvement of KLHL proteins in AngII-induced WNK3 activation of vascular smooth muscle cells. In the mouse aorta and mouse vascular smooth muscle (MOVAS) cells, KLHL3 was not expressed, but KLHL2, the closest homolog of KLHL3, was expressed. Salt depletion and acute infusion of AngII decreased KLHL2 and increased WNK3 levels in the mouse aorta. Notably, the AngII-induced changes in KLHL2 and WNK3 expression occurred within minutes in MOVAS cells. Results of KLHL2 overexpression and knockdown experiments in MOVAS cells confirmed that KLHL2 is the major regulator of WNK3 protein abundance. The AngII-induced decrease in KLHL2 was not caused by decreased transcription but increased autophagy-mediated degradation. Furthermore, knockdown of sequestosome 1/p62 prevented the decrease in KLHL2, suggesting that the mechanism of KLHL2 autophagy could be selective autophagy mediated by sequestosome 1/p62. Thus, we identified a novel component of signal transduction in AngII-induced vascular contraction that could be a promising drug target.

Actions of the protein kinase WNK1 on endothelial cells are differentially mediated by its substrate kinases OSR1 and SPAK.

The with no lysine (K) (WNK) family of enzymes is best known for control of blood pressure through regulation of the function and membrane localization of ion cotransporters. In mice, global as well as endothelial-specific WNK1 gene disruption results in embryonic lethality due to angiogenic and cardiovascular defects. WNK1(-/-) embryos can be rescued by endothelial-specific expression of a constitutively active form of the WNK1 substrate protein kinase OSR1 (oxidative stress responsive 1). Using human umbilical vein endothelial cells (HUVECs), we explored mechanisms underlying the requirement of WNK1-OSR1 signaling for vascular development. WNK1 is required for cord formation in HUVECs, but the actions of the two major WNK1 effectors, OSR1 and its close relative SPAK (STE20/SPS1-related proline-, alanine-rich kinase), are distinct. SPAK is important for endothelial cell proliferation, whereas OSR1 is required for HUVEC chemotaxis and invasion. We also identified the zinc-finger transcription factor Slug in WNK1-mediated control of endothelial functions. Our study identifies a separation of functions for the WNK1-activated protein kinases OSR1 and SPAK in mediating proliferation, invasion, and gene expression in endothelial cells and an unanticipated link between WNK1 and Slug that is important for angiogenesis.

Impaired degradation of WNK1 and WNK4 kinases causes PHAII in mutant KLHL3 knock-in mice.

Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by salt-sensitive hypertension, hyperkalemia and metabolic acidosis, and genes encoding with-no-lysine kinase 1 (WNK1) and WNK4 kinases are known to be responsible. Recently, Kelch-like 3 (KLHL3) and Cullin3, components of KLHL3-Cullin3 E3 ligase, were newly identified as responsible for PHAII. We have reported that WNK4 is the substrate of KLHL3-Cullin3 E3 ligase-mediated ubiquitination. However, WNK1 and Na-Cl cotransporter (NCC) were also reported to be a substrate of KLHL3-Cullin3 E3 ligase by other groups. Therefore, it remains unclear which molecule is the target(s) of KLHL3. To investigate the pathogenesis of PHAII caused by KLHL3 mutation, we generated and analyzed KLHL3(R528H/+) knock-in mice. KLHL3(R528H/+) knock-in mice exhibited salt-sensitive hypertension, hyperkalemia and metabolic acidosis. Moreover, the phosphorylation of NCC was increased in the KLHL3(R528H/+) mouse kidney, indicating that the KLHL3(R528H/+) knock-in mouse is an ideal mouse model of PHAII. Interestingly, the protein expression of both WNK1 and WNK4 was significantly increased in the KLHL3(R528H/+) mouse kidney, confirming that increases in these WNK kinases activated the WNK-OSR1/SPAK-NCC phosphorylation cascade in KLHL3(R528H/+) knock-in mice. To examine whether mutant KLHL3 R528H can interact with WNK kinases, we measured the binding of TAMRA-labeled WNK1 and WNK4 peptides to full-length KLHL3 using fluorescence correlation spectroscopy, and found that neither WNK1 nor WNK4 bound to mutant KLHL3 R528H. Thus, we found that increased protein expression levels of WNK1 and WNK4 kinases cause PHAII by KLHL3 R528H mutation due to impaired KLHL3-Cullin3-mediated ubiquitination.

WNK4 is the major WNK positively regulating NCC in the mouse kidney

By analysing the pathogenesis of a hereditary hypertensive disease, PHAII (pseudohypoaldosteronism type II), we previously discovered that WNK (with-no-lysine kinase)–OSR1/SPAK (oxidative stress-responsive 1/Ste20-like proline/alanine-rich kinase) cascade regulates NCC (Na–Cl co-transporter) in the DCT (distal convoluted tubules) of the kidney. However, the role of WNK4 in the regulation of NCC remains controversial. To address this, we generated and analysed WNK4−/− mice. Although a moderate decrease in SPAK phosphorylation and a marked increase in WNK1 expression were evident in the kidneys of WNK4−/− mice, the amount of phosphorylated and total NCC decreased to almost undetectable levels, indicating that WNK4 is the major WNK positively regulating NCC, and that WNK1 cannot compensate for WNK4 deficiency in the DCT. Insulin- and low-potassium diet-induced NCC phosphorylation were abolished in WNK4−/− mice, establishing that both signals to NCC were mediated by WNK4. As shown previously, a high-salt diet decreases phosphorylated and total NCC in WNK4+/+ mice via AngII (angiotensin II) and aldosterone suppression. This was not ameliorated by WNK4 knock out, excluding the negative regulation of WNK4 on NCC postulated to be active in the absence of AngII stimulation. Thus, WNK4 is the major positive regulator of NCC in the kidneys.

Regulation of with‐no‐lysine kinase signaling by Kelch‐like proteins

In 2001, with-no-lysine (WNK) kinases were identified as the genes responsible for the human hereditary hypertensive disease pseudohypoaldosteronism type II (PHAII). It took a further 6 years to clarify that WNK kinases participate in a signaling cascade with oxidative stress-responsive gene 1 (OSR1), Ste20-related proline-alanine-rich kinase (SPAK), and thiazide-sensitive NaCl cotransporter (NCC) in the kidney and the constitutive activation of this signaling cascade is the molecular basis of PHAII. Since this discovery, the WNK–OSR1/SPAK–NCC signaling cascade has been shown to be involved not only in PHAII but also in the regulation of blood pressure under normal and pathogenic conditions, such as hyperinsulinemia. However, the molecular mechanisms of WNK kinase regulation by dietary and hormonal factors and by PHAII-causing mutations remain poorly understood. In 2012, two additional genes responsible for PHAII, Kelch-like 3 (KLHL3) and Cullin3, were identified. At the time of their discovery, the molecular mechanisms underlying the interaction between these genes and their involvement in PHAII were unknown. Here we review the pathophysiological roles of the WNK signaling cascade clarified to date and introduce a new mechanism of WNK kinase regulation by KLHL3 and Cullin3, which provides insight on previously unknown mechanisms of WNK kinase regulation.

Development of enzyme-linked immunosorbent assays for urinary thiazide-sensitive Na-Cl cotransporter measurement

The Na-Cl cotransporter (NCC) in the distal convoluted tubules in kidney is known to be excreted in urine. However, its clinical significance has not been established because of the lack of quantitative data on urinary NCC. We developed highly sensitive enzyme-linked immunosorbent assays (ELISAs) for urinary total NCC (tNCC) and its active form, phosphorylated NCC (pNCC). We first measured the excretion of tNCC and pT55-NCC in urinary exosomes in pseudohypoaldosteronism type II (PHAII) patients since PHAII is caused by NCC activation. Highly increased excretion of tNCC and pNCC was observed in PHAII patients. In contrast, the levels of tNCC and pNCC in the urine of patients with Gitelman's syndrome were not detectable or very low, indicating that both assays could specifically detect the changes in urinary NCC excretion caused by the changes of NCC activity in the kidney. Then, to test whether these assays could be feasible for a more general patient population, we measured tNCC and pNCC in the urine of outpatients with different clinical backgrounds. Although urinary protein levels >30 mg/dl interfered with our ELISA, we could measure urinary pNCC in all patients without proteinuria. Thus we established highly sensitive and quantitative assays for urinary NCC, which could be valuable tools for estimating NCC activity in vivo.

Chemical library screening for WNK signalling inhibitors using fluorescence correlation spectroscopy

WNKs (with-no-lysine kinases) are the causative genes of a hereditary hypertensive disease, PHAII (pseudohypoaldosteronism typeII), and form a signal cascade with OSR1 (oxidative stress-responsive 1)/SPAK (STE20/SPS1-related proline/alanine-rich protein kinase) and Slc12a (solute carrier family 12) transporters. We have shown that this signal cascade regulates blood pressure by controlling vascular tone as well as renal NaCl excretion. Therefore agents that inhibit this signal cascade could be a new class of antihypertensive drugs. Since the binding of WNK to OSR1/SPAK kinases was postulated to be important for signal transduction, we sought to discover inhibitors of WNK/SPAK binding by screening chemical compounds that disrupt the binding. For this purpose, we developed a high-throughput screening method using fluorescent correlation spectroscopy. As a result of screening 17000 compounds, we discovered two novel compounds that reproducibly disrupted the binding of WNK to SPAK. Both compounds mediated dose-dependent inhibition of hypotonicity-induced activation of WNK, namely the phosphorylation of SPAK and its downstream transporters NKCC1 (Na/K/Cl cotransporter 1) and NCC (NaCl cotransporter) in cultured cell lines. The two compounds could be the promising seeds of new typesof antihypertensive drugs, and the method that we developed could be applied as a general screening method to identify compounds that disrupt the binding of two molecules.

A molecular update on pseudohypoaldosteronism type II

The DCT (distal convoluted tubule) is the site of microregulation of water reabsorption and ion handling in the kidneys, which is mainly under the control of aldosterone. Aldosterone binds to and activates mineralocorticoid receptors, which ultimately lead to increased sodium reabsorption in the distal part of the nephron. Impairment of mineralocorticoid signal transduction results in resistance to aldosterone and mineralocorticoids, and, therefore, causes disturbances in electrolyte balance. Pseudohypoaldosteronism type II (PHAII) or familial hyperkalemic hypertension (FHHt) is a rare, autosomal dominant syndrome characterized by hypertension, hyperkalemia, metabolic acidosis, elevated or low aldosterone levels, and decreased plasma renin activity. PHAII is caused by mutations in the WNK isoforms (with no lysine kinase), which regulate the Na-Cl and Na-K-Cl cotransporters (NCC and NKCC2, respectively) and the renal outer medullary potassium (ROMK) channel in the DCT. This review focuses on new candidate genes such as KLHL3 and Cullin3, which are instrumental to unraveling novel signal transductions pathways involving NCC, to better understand the cause of PHAII along with the molecular mechanisms governing the pathophysiology of PHAII and its clinical manifestations.

Decrease of WNK4 ubiquitination by disease-causing mutations of KLHL3 through different molecular mechanisms

Recently, we demonstrated that WNK4 is a substrate for KLHL3–Cullin3 (CUL3) E3 ubiquitin ligase complexes and that impaired WNK4 ubiquitination is a common mechanism for pseudohypoaldosteronism type II (PHAII) caused by WNK4, KLHL3, and CUL3 mutations. Among the various KLHL3 mutations that cause PHAII, we demonstrated that the R528H mutation in the Kelch domain decreased the binding to WNK4, thereby causing less ubiquitination and increased intracellular levels of WNK4. However, the pathogenic mechanisms of PHAII caused by other KLHL3 mutants remain to be determined. In this study, we examined the pathogenic effects of three PHAII-causing mutations in different KLHL3 domains; the protein levels of these mutants significantly differed when they were transiently expressed in HEK293T cells. In particular, S410L expression was low even with increased plasmid expression. The cycloheximide chase assay revealed that an S410L mutation in the Kelch domain significantly decreased the intracellular stability. Mutations in E85A in the BTB domain and C164F in the BACK domain decreased the binding to CUL3, and S410L as well as R528H demonstrated less binding to WNK4. In vitro and in vivo assays revealed that these mutants decreased the ubiquitination and increased the intracellular levels of WNK4 compared with wild-type KLHL3. Therefore, the KLHL3 mutants causing PHAII investigated in this study exhibited less ability to ubiquitinate WNK4 because of KLHL3’s low stability and/or decreased binding to CUL3 or WNK4.

KLHL2 interacts with and ubiquitinates WNK kinases

Mutations in the WNK1 and WNK4 genes result in an inherited hypertensive disease, pseudohypoaldosteronism type II (PHAII). Recently, the KLHL3 and Cullin3 genes were also identified as responsible genes for PHAII. Although we have reported that WNK4 is a substrate for the KLHL3–Cullin3 E3 ligase complex, it is not clear whether all of the WNK isoforms are regulated only by KLHL3. To explore the interaction of WNKs and other Kelch-like proteins, we focused on KLHL2 (Mayven), a human homolog of Drosophila Kelch that shares the highest similarity with KLHL3. We found that KLHL2, as well as KLHL3, was co-immunoprecipitated with all four WNK isoforms. The direct interaction of KLHL2 with WNKs was confirmed on fluorescence correlation spectroscopy. Co-expression of KLHL2 and Cullin3 decreased the abundance of WNK1, WNK3 and WNK4 within HEK293T cells, and a significant increase of WNK4 ubiquitination by KLHL2 and Cullin3 was observed both in HEK293T cells and in an in vitro ubiquitination assay. These results suggest that KLHL2–Cullin3 also functions as an E3-ligase for WNK isoforms within the body.

CUL3 gene analysis enables early intervention for pediatric pseudohypoaldosteronism type II in infancy

Four genes responsible for pseudohypoaldosteronism type II (PHA-II) have been identified, thereby facilitating molecular diagnostic testing. A 1-year-old boy with prolonged hyperkalemia, metabolic acidosis, hyperchloremia, growth delay, and mild hypertension was diagnosed with PHA-II based on the detection of exon 9 skipping in CUL3 mRNA. The impaired splicing was the result of a de novo, previously unreported single nucleotide substitution in the splice acceptor site of CUL3 intron 8. Among the four genes reported to be involved in PHA-II, CUL3 was the primary suspect in our patient because in patients with the CUL3 mutation, the onset of disease is often early in infancy and the phenotypes of PHA-II are more severe. Our patient was treated with trichlormethiazide, which inhibits the function of the sodium-chloride co-transporter (NCC), and the outcome was favorable, with correction of body fluids and blood electrolyte homeostasis. Since chronic acidosis and hypertension associated with PHA-II can result in delayed growth and development in pediatric patients, genetic analysis to detect the CUL3 mutation and to enable intervention early in the disease course would be beneficial for infants with suspected PHA-II.

Control of electrolyte balance through ubiquitination

The specific causes of elevated blood pressure cannot be determined in the vast majority of patients with hypertension. However, identification of genes causing rare Mendelian forms of hypertension has provided penetrating insights into the basic mechanisms of human hypertension, highlighting the key role of altered sodium handling by the kidney as a final common pathway in hypertension pathogenesis (1). Pseudohypoaldosteronism type II (PHAII) is one of these Mendelian syndromes, characterized by the unusual combination of hypertension and high levels of potassium in the blood (hyperkalemia). The Lifton laboratory had previously found mutations in four distinct genes that can cause PHAII (2, 3). In PNAS, Shibata et al. show that impaired ubiquitination of With no lysine kinases (WNKs), key molecular switches for regulating electrolyte flux in the distal nephron, explain clinical features of PHAII (4).

Kelch-like 3 and Cullin 3 regulate electrolyte homeostasis via ubiquitination and degradation of WNK4

Pseudohypoaldosteronism type II (PHAII) is a rare Mendelian syndrome featuring hypertension and hyperkalemia resulting from constitutive renal salt reabsorption and impaired K(+) secretion. Recently, mutations in Kelch-like 3 (KLHL3) and Cullin 3 (CUL3), components of an E3 ubiquitin ligase complex, were found to cause PHAII, suggesting that loss of this complex's ability to target specific substrates for ubiquitination leads to PHAII. By MS and coimmunoprecipitation, we show that KLHL3 normally binds to WNK1 and WNK4, members of WNK (with no lysine) kinase family that have previously been found mutated in PHAII. We show that this binding leads to ubiquitination, including polyubiquitination, of at least 15 specific sites in WNK4, resulting in reduced WNK4 levels. Dominant disease-causing mutations in KLHL3 and WNK4 both impair WNK4 binding, ubiquitination, and degradation. WNK4 normally induces clearance of the renal outer medullary K(+) channel (ROMK) from the cell surface. We show that WT but not mutant KLHL3 inhibits WNK4-induced reduction of ROMK level. We show that PHAII-causing mutations in WNK4 lead to a marked increase in WNK4 protein levels in the kidney in vivo. These findings demonstrate that CUL3-RING (really interesting new gene) ligases that contain KLHL3 target ubiquitination of WNK4 and thereby regulate WNK4 levels, which in turn regulate levels of ROMK. These findings reveal a specific role of CUL3 and KLHL3 in electrolyte homeostasis and provide a molecular explanation for the effects of disease-causing mutations in both KLHL3 and WNK4.

Impaired KLHL3-Mediated Ubiquitination of WNK4 Causes Human Hypertension

Mutations in WNK kinases cause the human hypertensive disease pseudohypoaldosteronism type II (PHAII), but the regulatory mechanisms of the WNK kinases are not well understood. Mutations in kelch-like 3 (KLHL3) and Cullin3 were also recently identified as causing PHAII. Therefore, new insights into the mechanisms of human hypertension can be gained by determining how these components interact and how they are involved in the pathogenesis of PHAII. Here, we found that KLHL3 interacted with Cullin3 and WNK4, induced WNK4 ubiquitination, and reduced the WNK4 protein level. The reduced interaction of KLHL3 and WNK4 by PHAII-causing mutations in either protein reduced the ubiquitination of WNK4, resulting in an increased level of WNK4 protein. Transgenic mice overexpressing WNK4 showed PHAII phenotypes, and WNK4 protein was indeed increased in Wnk4(D561A/+) PHAII model mice. Thus, WNK4 is a target for KLHL3-mediated ubiquitination, and the impaired ubiquitination of WNK4 is a common mechanism of human hereditary hypertension.