Role of KLHL3 and dietary K+ in regulating KS-WNK1 expression.

The physiological role of the shorter isoform of with no lysine kinase (WNK)1 that is exclusively expressed in the kidney (KS-WNK1), with particular abundance in the distal convoluted tubule, remains elusive. KS-WNK1, despite lacking the kinase domain, is nevertheless capable of stimulating the NaCl cotransporter, apparently through activation of WNK4. It has recently been shown that a less severe form of familial hyperkalemic hypertension featuring only hyperkalemia is caused by missense mutations in the WNK1 acidic domain that preferentially affect cullin 3 (CUL3)-Kelch-like protein 3 (KLHL3) E3-induced degradation of KS-WNK1 rather than that of full-length WNK1. Here, we show that full-length WNK1 is indeed less impacted by the CUL3-KLHL3 E3 ligase complex compared with KS-WNK1. We demonstrated that the unique 30-amino acid NH2-terminal fragment of KS-WNK1 is essential for its activating effect on the NaCl cotransporter and recognition by KLHL3. We identified specific amino acid residues in this region critical for the functional effect of KS-WNK1 and KLHL3 sensitivity. To further explore this, we generated KLHL3-R528H knockin mice that mimic human mutations causing familial hyperkalemic hypertension. These mice revealed that the KLHL3 mutation specifically increased expression of KS-WNK1 in the kidney. We also observed that in wild-type mice, the expression of KS-WNK1 was only detectable after exposure to a low-K+ diet. These findings provide new insights into the regulation and function of KS-WNK1 by the CUL3-KLHL3 complex in the distal convoluted tubule and indicate that this pathway is regulated by dietary K+ levels.NEW & NOTEWORTHY In this work, we demonstrated that the kidney-specific isoform of with no lysine kinase 1 (KS-WNK1) in the kidney is modulated by dietary K+ and activity of the ubiquitin ligase protein Kelch-like protein 3. We analyzed the role of different amino acid residues of KS-WNK1 in its activity against the NaCl cotransporter and sensitivity to Kelch-like protein 3.

Metabolic Acidosis, Hyperkalemia, and Renal Unresponsiveness to Aldosterone Syndrome: Response to Treatment with Low-Potassium Diet.

Gordon syndrome involves hyperkalemia, acidosis, and severe hypertension (HTN) with hypercalciuria, low renin and aldosterone levels. It is commonly observed in children and adolescents. Such patients respond successfully to sodium restriction and thiazide diuretics. In this article, we present three cases of metabolic acidosis, hyperkalemia, and renal unresponsiveness to aldosterone (MeHandRU Syndrome). All three patients did not have HTN or hypercalciuria and demonstrated normal renin and aldosterone levels. These patients did not respond to thiazide-type diuretic therapy and salt restriction. Two males (aged 55- and 62-year) and a female patient (aged 68-year) presented to the clinic with unexplained hyperkalemia (5.9 mEq/L, 5.9 mEq/L and 6.2 mEq/L, respectively). On physical examination, blood pressure (BP) was found to be normal (<140/90 mm Hg). Over the counter potassium supplement, nonsteroidal anti-inflammatory drugs, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, potassium sparing diuretic use, as well as hyporeninemic hypoaldosteronism states such as diabetes mellitus were excluded. Plasma renin and aldosterone levels were normal. All three patients had low transtubular potassium gradient, despite high serum potassium levels. None of the patients reported a family history of hyperkalemia or kidney failure. All failed to demonstrate a response to hydrochlorothiazide and salt restriction. After careful consideration, strict low potassium diet (<2 g/day) was initiated in consultation with the dietician. Diuretic therapy was discontinued while BP remained within normal range (<140/90 mm Hg). At eight weeks, all three patients demonstrated normalization of potassium and correction of acidosis. At follow-up of six months, all patients are maintaining a normal potassium level. We suggest that potassium restriction can be successful in patients presenting with MeHandRU syndrome.

Dietary potassium restriction attenuates urinary sodium wasting in the generalized form of pseudohypoaldosteronism type 1.

Owing to its rarity and severe nature, the treatment for generalized pseudohypoaldosteronism type 1 (PHA1), a genetic disorder in the epithelial sodium channel (ENaC), is exclusively experience-based. In particular, the usefulness of dietary potassium restriction in PHA1 remains unclear with the absence of theoretical background to elucidate its utility. First, we demonstrated the effect of potassium restriction in a 13-month-old patient with ENaC γ-subunit gene mutations via a retrospective chart review; reduction of daily dietary potassium intake from 40 to 20 mEq induced rapid restoration of volume depletion, as evidenced by weight gain, elevation of the serum sodium level from 133 to 141 mEq/L, decreased urinary sodium excretion, and normalized renin activity. The serum potassium level decreased from 5.6 to 4.5 mEq/L. Next, we attempted to elucidate the pathophysiological basis of the usefulness of potassium restriction, leveraged by the increased knowledge regarding the roles of with-no-lysine kinases (WNKs) in the distal nephron. When potassium is restricted, the WNK signal will turn "on" in the distal nephron via reduction in the intracellular chloride level. Consequently, the sodium reabsorption from the Na+Cl- cotransporter (NCC) in the distal convoluted tubule and possibly from pendrin in the ß-intercalated cell will increase. Thus, potassium restriction causes NCC and pendrin to compensate for the non-functional ENaC in the collecting duct. In conclusion, dietary potassium restriction is one of the indispensable treatments for generalized PHA1.

One-month-old girl presenting with pseudohypoaldosteronism leading to the diagnosis of CDK13-related disorder: a case report and review of the literature.

BACKGROUND: It is not uncommon that an infant with a disease of unknown etiology is presented to a physician. Facial dysmorphic features lead to a different diagnosis. It is a challenge to link the presentation to the newfound diagnosis. CASE PRESENTATION: A 37-day-old Yemenite Jewish girl was presented to our institution with a clinical picture of pseudohypoaldosteronism due to abnormal facial features and a psychomotor developmental delay. Further investigation led to the diagnosis of CDK13-related disorder. According to the literature, CDK13 has a key role in the cell cycle, but no interference with the aldosterone signaling pathway or electrolyte balance was described. No mutations in the previously described gene NR3C2 (cytogenetic location 4q31.23), encoding the mineralocorticoid receptor, were found. Although the clinical presentation corresponded to pseudohypoaldosteronism type 1, we could not genetically confirm this. CONCLUSIONS: Probably pseudohypoaldosteronism was a coincidental finding in this girl with a CDK13 mutation, but because only limited information is known about CDK13-related disorders, further investigation could be more informative to clarify this presentation.

Cullin-Ring ubiquitin ligases in kidney health and disease.

PURPOSE OF REVIEW: Members of the Cullin family act as scaffolds in E3 ubiquitin ligases and play a central role in mediating protein degradation. Interactions with many different substrate-binding adaptors permit Cullin-containing E3 ligases to participate in diverse cellular functions. In the kidney, one well established target of Cullin-mediated degradation is the transcription factor Nrf2, a key player in responses to oxidative stress. The goal of this review is to discuss more recent findings revealing broader roles for Cullins in the kidney. RECENT FINDINGS: Cullin 3 acts as the scaffold in the E3 ligase regulating Nrf2 abundance, but was more recently shown to be mutated in the disease familial hyperkalemic hypertension. Studies seeking to elucidate the molecular mechanisms by which Cullin 3 mutations lead to dysregulation of renal sodium transport will be discussed. Disruption of Cullin 3 in mice unexpectedly causes polyuria and fibrotic injury suggesting it has additional roles in the kidney. We will also review recent transcriptomic data suggesting that other Cullins are also likely to play important roles in renal function. SUMMARY: Cullins form a large and diverse family of E3 ubiquitin ligases that are likely to have many important functions in the kidney.

A mouse model of pseudohypoaldosteronism type II reveals a novel mechanism of renal tubular acidosis.

Pseudohypoaldosteronism type II (PHAII) is a genetic disease characterized by association of hyperkalemia, hyperchloremic metabolic acidosis, hypertension, low renin, and high sensitivity to thiazide diuretics. It is caused by mutations in the WNK1, WNK4, KLHL3 or CUL3 gene. There is strong evidence that excessive sodium chloride reabsorption by the sodium chloride cotransporter NCC in the distal convoluted tubule is involved. WNK4 is expressed not only in distal convoluted tubule cells but also in ß-intercalated cells of the cortical collecting duct. These latter cells exchange intracellular bicarbonate for external chloride through pendrin, and therefore, account for renal base excretion. However, these cells can also mediate thiazide-sensitive sodium chloride absorption when the pendrin-dependent apical chloride influx is coupled to apical sodium influx by the sodium-driven chloride/bicarbonate exchanger. Here we determine whether this system is involved in the pathogenesis of PHAII. Renal pendrin activity was markedly increased in a mouse model carrying a WNK4 missense mutation (Q562E) previously identified in patients with PHAII. The upregulation of pendrin led to an increase in thiazide-sensitive sodium chloride absorption by the cortical collecting duct, and it caused metabolic acidosis. The function of apical potassium channels was altered in this model, and hyperkalemia was fully corrected by pendrin genetic ablation. Thus, we demonstrate an important contribution of pendrin in renal regulation of sodium chloride, potassium and acid-base homeostasis and in the pathophysiology of PHAII. Furthermore, we identify renal distal bicarbonate secretion as a novel mechanism of renal tubular acidosis.

Consequences of SPAK inactivation on Hyperkalemic Hypertension caused by WNK1 mutations: evidence for differential roles of WNK1 and WNK4.

Mutations of the gene encoding WNK1 [With No lysine (K) kinase 1] or WNK4 cause Familial Hyperkalemic Hypertension (FHHt). Previous studies have shown that the activation of SPAK (Ste20-related Proline/Alanine-rich Kinase) plays a dominant role in the development of FHHt caused by WNK4 mutations. The implication of SPAK in FHHt caused by WNK1 mutation has never been investigated. To clarify this issue, we crossed WNK1+/FHHt mice with SPAK knock-in mice in which the T-loop Thr243 residue was mutated to alanine to prevent activation by WNK kinases. We show that WNK1+/FHHT:SPAK 243A/243A mice display an intermediate phenotype, between that of control and SPAK 243A/243A mice, with normal blood pressure but hypochloremic metabolic alkalosis. NCC abundance and phosphorylation levels also decrease below the wild-type level in the double-mutant mice but remain higher than in SPAK 243A/243A mice. This is different from what was observed in WNK4-FHHt mice in which SPAK inactivation completely restored the phenotype and NCC expression to wild-type levels. Although these results confirm that FHHt caused by WNK1 mutations is dependent on the activation of SPAK, they suggest that WNK1 and WNK4 play different roles in the distal nephron.

Mechanisms and controversies in mutant Cul3-mediated familial hyperkalemic hypertension.

Autosomal dominant mutations in cullin-3 ( Cul3) cause the most severe form of familial hyperkalemic hypertension (FHHt). Cul3 mutations cause skipping of exon 9, which results in an internal deletion of 57 amino acids from the CUL3 protein (CUL3-∆9). The precise mechanism by which this altered form of CUL3 causes FHHt is controversial. CUL3 is a member of the cullin-RING ubiquitin ligase family that mediates ubiquitination and thus degradation of cellular proteins, including with-no-lysine [K] kinases (WNKs). In CUL3-∆9-mediated FHHt, proteasomal degradation of WNKs is abrogated, leading to overactivation of the WNK targets sterile 20/SPS-1 related proline/alanine-rich kinase and oxidative stress-response kinase-1, which directly phosphorylate and activate the thiazide-sensitive Na+-Cl- cotransporter. Several groups have suggested different mechanisms by which CUL3-∆9 causes FHHt. The majority of these are derived from in vitro data, but recently the Kurz group (Schumacher FR, Siew K, Zhang J, Johnson C, Wood N, Cleary SE, Al Maskari RS, Ferryman JT, Hardege I, Figg NL, Enchev R, Knebel A, O'Shaughnessy KM, Kurz T. EMBO Mol Med 7: 1285-1306, 2015) described the first mouse model of CUL3-∆9-mediated FHHt. Analysis of this model suggested that CUL3-∆9 is degraded in vivo, and thus Cul3 mutations cause FHHt by inducing haploinsufficiency. We recently directly tested this model but found that other dominant effects of CUL3-∆9 must contribute to the development of FHHt. In this review, we focus on our current knowledge of CUL3-∆9 action gained from in vitro and in vivo models that may help unravel this complex problem.

Mutant Cullin 3 causes familial hyperkalemic hypertension via dominant effects.

Mutations in the ubiquitin ligase scaffold protein Cullin 3 (CUL3) cause the disease familial hyperkalemic hypertension (FHHt). In the kidney, mutant CUL3 (CUL3-Δ9) increases abundance of With-No-Lysine [K] Kinase 4 (WNK4), with excessive activation of the downstream Sterile 20 (STE20)/SPS-1-related proline/alanine-rich kinase (SPAK) increasing phosphorylation of the Na+-Cl- cotransporter (NCC). CUL3-Δ9 promotes its own degradation via autoubiquitination, leading to the hypothesis that Cul3 haploinsufficiency causes FHHt. To directly test this, we generated Cul3 heterozygous mice (CUL3-Het), and Cul3 heterozygotes also expressing CUL3-Δ9 (CUL3-Het/Δ9), using an inducible renal epithelial-specific system. Endogenous CUL3 was reduced to 50% in both models, and consistent with autoubiquitination, CUL3-Δ9 protein was undetectable in CUL3-Het/Δ9 kidneys unless primary renal epithelia cells were cultured. Abundances of WNK4 and phosphorylated NCC did not differ between control and CUL3-Het mice, but they were elevated in CUL3-Het/Δ9 mice, which also displayed higher plasma [K+] and blood pressure. Abundance of phosphorylated Na+-K+-2Cl- cotransporter (NKCC2) was also increased, which may contribute to the severity of CUL3-Δ9-mediated FHHt. WNK4 and SPAK localized to puncta in NCC-positive segments but not in NKCC2-positive segments, suggesting differential effects of CUL3-Δ9. These results indicate that Cul3 haploinsufficiency does not cause FHHt, but dominant effects of CUL3-Δ9 are required.

Pseudohypoaldosteronism types I and II: little more than a name in common.

Pseudohypoaldosteronism (PHA) comprises a diverse group of rare diseases characterized by sodium and potassium imbalances incorrectly attributed to a defect in aldosterone production. Two different forms of PHA have been described, type I (PHAI) and type II (PHAII). PHAI has been subclassified into renal and systemic. Given the rarity and heterogeneity of this group of disorders we report three patients who carry PHA and a brief revision of current literature focused on the comparative analysis of PHAI and PHAII. Cases 1 and 2 presented with hyponatremia, hyperkalemia, metabolic acidosis and elevated plasma aldosterone and plasma renin activity in the neonatal period. Sequence analysis of the NRC2 gene demonstrated a novel heterozygous c.403C>T mutation in case 1 and a complete deletion in case 2, confirming the diagnosis of renal PHAI. Case 3 was a 4-year-old with hypertension, hyperkalemia, metabolic acidosis, normal plasma aldosterone and decreased plasma renin activity. Sequence analysis of the CUL3 gene demonstrated a previously unreported heterozygous c.1377+2T>3 mutation, confirming the diagnosis of PHAII-E. We highlight the importance of the determination of plasma aldosterone and plasma renin activity in the context of persistent sodium and potassium imbalances in children.

KLHL3 Knockout Mice Reveal the Physiological Role of KLHL3 and the Pathophysiology of Pseudohypoaldosteronism Type II Caused by Mutant KLHL3.

Mutations in the with-no-lysine kinase 1 (WNK1), WNK4, kelch-like 3 (KLHL3), and cullin3 (CUL3) genes are known to cause the hereditary disease pseudohypoaldosteronism type II (PHAII). It was recently demonstrated that this results from the defective degradation of WNK1 and WNK4 by the KLHL3/CUL3 ubiquitin ligase complex. However, the other physiological in vivo roles of KLHL3 remain unclear. Therefore, here we generated KLHL3-/- mice that expressed ß-galactosidase (ß-Gal) under the control of the endogenous KLHL3 promoter. Immunoblots of ß-Gal and LacZ staining revealed that KLHL3 was expressed in some organs, such as brain. However, the expression levels of WNK kinases were not increased in any of these organs other than the kidney, where WNK1 and WNK4 increased in KLHL3-/- mice but not in KLHL3+/- mice. KLHL3-/- mice also showed PHAII-like phenotypes, whereas KLHL3+/- mice did not. This clearly demonstrates that the heterozygous deletion of KLHL3 was not sufficient to cause PHAII, indicating that autosomal dominant type PHAII is caused by the dominant negative effect of mutant KLHL3. We further demonstrated that the dimerization of KLHL3 can explain this dominant negative effect. These findings could help us to further understand the physiological roles of KLHL3 and the pathophysiology of PHAII caused by mutant KLHL3.

The regulation of Na+Cl- cotransporter by with-no-lysine kinase 4.

PURPOSE OF REVIEW: Abundant evidence supports that the NaCl cotransporter (NCC) activity is tightly regulated by the with-no-lysine (WNK) kinases. Here, we summarize the data regarding NCC regulation by WNKs, with a particular emphasis on WNK4. RECENT FINDINGS: Several studies involving in-vivo and in-vitro models have provided paradoxical data regarding WNK4 regulation of the NCC. Although some studies show that WNK4 can activate the NCC, other equally compelling studies show that WNK4 inhibits the NCC. Recent studies have shown that WNK4 is regulated by the intracellular chloride concentration ([Cl]i), which could account for these paradoxical results. In conditions of high [Cl]i, WNK4 could act as an inhibitor via heterodimer formation with other WNKs. In contrast, when [Cl]i is low, WNK4 can activate Ste20-related, proline-alanine-rich kinase (SPAK)/oxidative stress responsive kinase 1 (OSR1) and thus the NCC. Modulation of WNK4 by [Cl]i has been shown to account for the potassium-sensing properties of the distal convoluted tubule. Other regulators of WNK4 include hormones and ubiquitination. SUMMARY: Modulation of WNK4 activity by [Cl]i can account for its dual role on the NCC, and this has important physiological implications regarding the regulation of extracellular potassium concentration. Defective regulation of WNKs by ubiquitination explains most cases of familial hyperkalemic hypertension.

Pseudohipoaldosteronismo y aparente exceso de mineralocorticoides: cara y ceca en dos pacientes pediátricos / Pseudohypoaldosteronism and apparent mineralocorticoid excess: two faces, two pediatric patients

Los síndromes endocrinológicos con hipofunción o hiperfunción con niveles paradójicos de dosajes hormonales han sido bien caracterizados en los últimos años del siglo XX, a partir del desarrollo de técnicas genéticas y moleculares. Presentamos dos pacientes con pseudohipoaldosteronismo y aparente exceso de mineralocorticoides como síndromes en espejo, con la intención de alertar al médico clínico respecto de su consideración como entidad diagnóstica en niños con alteraciones hidroelectrolíticas. (AU)

Endocrinological syndromes with underactive or overactive hormonal levels with paradoxical dosages have been well characterized over the years of the twentieth century, from the development of genetic and molecular techniques. We present two patients with pseudohypoaldosteronism and apparent mineralocorticoid excess as mirror syndromes, with the aim to alert the clinician regarding their consideration as a diagnostic entity in children with fluid and electrolyte disturbances. (AU)

Regulation of Renal Electrolyte Transport by WNK and SPAK-OSR1 Kinases.

The discovery of four genes responsible for pseudohypoaldosteronism type II, or familial hyperkalemic hypertension, which features arterial hypertension with hyperkalemia and metabolic acidosis, unmasked a complex multiprotein system that regulates electrolyte transport in the distal nephron. Two of these genes encode the serine-threonine kinases WNK1 and WNK4. The other two genes [kelch-like 3 (KLHL3) and cullin 3 (CUL3)] form a RING-type E3-ubiquitin ligase complex that modulates WNK1 and WNK4 abundance. WNKs regulate the activity of the Na(+):Cl(-) cotransporter (NCC), the epithelial sodium channel (ENaC), the renal outer medullary potassium channel (ROMK), and other transport pathways. Interestingly, the modulation of NCC occurs via the phosphorylation by WNKs of other serine-threonine kinases known as SPAK-OSR1. In contrast, the process of regulating the channels is independent of SPAK-OSR1. We present a review of the remarkable advances in this area in the past 10 years.

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: CUL3(Δ403-459)) 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, CUL3(Δ403-459), is severely compromised in its ability to ubiquitylate WNKs, possibly due to altered structural flexibility. Instead, CUL3(Δ403-459) auto-ubiquitylates and loses interaction with two important Cullin regulators: the COP9-signalosome and CAND1. A novel knock-in mouse model of CUL3(WT) (/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.