Ankyrin-3 is a novel binding partner of the voltage-gated potassium channel Kv1.1 implicated in renal magnesium handling.

The voltage-gated potassium channel, Kv1.1, was recently identified as a causative gene in isolated dominant hypomagnesemia. The channel is situated in the distal convoluted tubule, where it participates in maintaining a favorable electrical gradient for driving magnesium ion into the cell through the transient receptor potential melastatin 6 channel. Pull-down experiments coupled to mass spectrometry using the carboxy-terminal domain of Kv1.1 as bait were used in mouse kidney lysates. Ankyrin-3 (ANK3) was identified as a binding partner of Kv1.1 and was enriched in isolated distal convoluted tubules as compared to whole kidney. Electrophysiology studies performed in HEK293 cells expressing Kv1.1 showed that ANK3 significantly inhibited Kv1.1-mediated currents (267 compared to 125 pA/pF) for control and ANK3, respectively. Finally, to evaluate a potential role of ANK3 in magnesium handling, the intrarenal abundance of ANK3 was measured in mice fed a low-, normal-, or high-magnesium diet for 10 days. Mice maintained on high dietary magnesium significantly doubled their fractional urinary excretion of magnesium, which coincided with a 1.8-fold increase in the renal expression of ANK3 compared to mice on a normal- or low-magnesium diet. Thus, our observations demonstrate a novel role for ANK3 in modulating the biophysical properties of Kv1.1. Such regulation appears to be particularly important in conditions of high dietary magnesium.

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.

The impact of dietary magnesium restriction on magnesiotropic and calciotropic genes.

BACKGROUND: Magnesium (Mg(2+)) is an essential electrolyte with important physiological functions. Consequently, hypomagnesaemia, an electrolyte disorder frequently diagnosed in critically ill patients, can have life-threatening consequences. The kidney plays a central role in the regulation of the Mg(2+) balance. The present study investigated the molecular consequences of dietary Mg(2+) restriction on renal Mg(2+) transporters. METHODS: Two groups of 10 mice were fed a Mg(2+)-deficient diet or a Mg(2+)-enriched diet for 2 weeks. Serum and urine electrolyte concentrations were assayed. Next, renal mRNA expression levels of Mg(2+)-related genes were measured to determine their sensitivity to the dietary Mg(2+) content. Subsequently, parvalbumin (PV) and the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), both co-expressed in the distal convoluted tubule (DCT) with TRPM6, were further analysed at the protein level using immunoblotting and immunohistochemistry. RESULTS: Serum and urine electrolyte measurements revealed that dietary Mg(2+) restriction resulted in significant reduction of serum Mg(2+) and Ca(2+) levels, and that the urinary excretion of these ions was also markedly reduced, while phosphate (Pi) excretion was significantly increased. In addition, the serum FGF23 level was markedly increased, whereas Pi was not significantly changed in the Mg(2+)-restricted mouse group. The renal abundance of hepatocyte nuclear factor 1 homeobox B (HNF1B) and the epithelial Mg(2+) channel TRPM6 were increased in response to dietary Mg(2+) restriction, whereas other magnesiotropic transporters were not affected. PV abundance was upregulated, while NCC was significantly downregulated. Furthermore, the expression levels of the epithelial Ca(2+) channel TRPV5 and calbindin-D28K were markedly reduced in the low Mg(2+) group. CONCLUSIONS: Our data indicate an essential adaptive role for DCT during hypomagnesaemia since TRPM6, HNF1B, PV and NCC expression levels were adjusted. Moreover, hypomagnesaemia resulted in severe changes in Ca(2+) and Pi reabsorption and expression levels of calciotropic proteins.

A primary culture of distal convoluted tubules expressing functional thiazide-sensitive NaCl transport.

Studying the molecular regulation of the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is important for understanding how the kidney contributes to blood pressure regulation. Until now, a native mammalian cell model to investigate this transporter remained unknown. Our aim here is to establish, for the first time, a primary distal convoluted tubule (DCT) cell culture exhibiting transcellular thiazide-sensitive Na(+) transport. Because parvalbumin (PV) is primarily expressed in the DCT, where it colocalizes with NCC, kidneys from mice expressing enhanced green-fluorescent protein (eGFP) under the PV gene promoter (PV-eGFP-mice) were employed. The Complex Object Parametric Analyzer and Sorter (COPAS) was used to sort fluorescent PV-positive tubules from these kidneys, which were then seeded onto permeable supports. After 6 days, DCT cell monolayers developed transepithelial resistance values of 630 ± 33 Ω·cm(2). The monolayers also established opposing transcellular concentration gradients of Na(+) and K(+). Radioactive (22)Na(+) flux experiments showed a net apical-to-basolateral thiazide-sensitive Na(+) transport across the monolayers. Both hypotonic low-chloride medium and 1 µM angiotensin II increased this (22)Na(+) transport significantly by four times, which could be totally blocked by 100 µM hydrochlorothiazide. Angiotensin II-stimulated (22)Na(+) transport was also inhibited by 1 µM losartan. Furthermore, NCC present in the DCT monolayers was detected by immunoblot and immunocytochemistry studies. In conclusion, a murine primary DCT culture was established which expresses functional thiazide-sensitive Na(+)-Cl(-) transport.

Novel NCC mutants and functional analysis in a new cohort of patients with Gitelman syndrome.

Gitelman syndrome (GS) is an autosomal recessive disorder characterized by hypokalemic metabolic alkalosis in conjunction with significant hypomagnesemia and hypocalciuria. The GS phenotype is caused by mutations in the solute carrier family 12, member 3 (SLC12A3) gene that encodes the thiazide-sensitive NaCl cotransporter (NCC). We analyzed DNA samples of 163 patients with a clinical suspicion of GS by direct sequencing of all 26 exons of the SLC12A3 gene. In total, 114 different mutations were identified, 31 of which have not been reported before. These novel variants include 3 deletions, 18 missense, 6 splice site and 4 nonsense mutations. We selected seven missense mutations to investigate their effect on NCC activity and plasma membrane localization by using the Xenopus laevis oocyte expression system. The Thr392Ile mutant did not display transport activity (probably class 2 mutation), while the Asn442Ser and Gln1030Arg NCC mutants showed decreased plasma membrane localization and consequently function, likely due to impaired trafficking (class 3 mutation). Even though the NaCl uptake was hampered for NCC mutants Glu121Asp, Pro751Leu, Ser475Cys and Tyr489His, the transporters reached the plasma membrane (class 4 mutation), suggesting an effect on NCC regulation or ion affinity. The present study shows the identification of 38 novel mutations in the SLC12A3 gene and provides insight into the mechanisms that regulate NCC.

γ-Adducin stimulates the thiazide-sensitive NaCl cotransporter.

The thiazide-sensitive NaCl cotransporter (NCC) plays a key role in renal salt reabsorption and the determination of systemic BP, but the molecular mechanisms governing the regulation of NCC are not completely understood. Here, through pull-down experiments coupled to mass spectrometry, we found that γ-adducin interacts with the NCC transporter. γ-Adducin colocalized with NCC to the distal convoluted tubule. (22)Na(+) uptake experiments in the Xenopus laevis oocyte showed that γ-adducin stimulated NCC activity in a dose-dependent manner, an effect that occurred upstream from With No Lysine (WNK) 4 kinase. The binding site of γ-adducin mapped to the N terminus of NCC and encompassed three previously reported phosphorylation sites. Supporting this site of interaction, competition with the N-terminal domain of NCC abolished the stimulatory effect of γ-adducin on the transporter. γ-Adducin failed to increase NCC activity when these phosphorylation sites were constitutively inactive or active. In addition, γ-adducin bound only to the dephosphorylated N terminus of NCC. Taken together, our observations suggest that γ-adducin dynamically regulates NCC, likely by amending the phosphorylation state, and consequently the activity, of the transporter. These data suggest that γ-adducin may influence BP homeostasis by modulating renal NaCl transport.

Novel molecular pathways in renal Mg2+ transport: a guided tour along the nephron.

PURPOSE OF REVIEW: This review highlights recent advances in renal magnesium (Mg) handling. The understanding of the molecular processes of epithelial Mg transport has expanded considerably due to the identification of novel genes involved in hypomagnesemic disorders. RECENT FINDINGS: Mg deficiency remains one of the most common electrolyte disorders. Detailed genetic analysis of families with inherited forms of hypomagnesemia has led to the identification of new genes involved in Mg homeostasis. As such, familial hypomagnesemia has been linked to mutations in the claudin-16/19 complex located in the thick ascending limb. Moreover, the pro-epidermal growth factor, the potassium channels Kv1.1 and Kir4.1, and the hepatocyte nuclear factor 1B have recently been identified as causative factors in syndromes of hereditary hypomagnesemia. These proteins play key roles in regulating electrolyte balance within the distal convoluted tubule, either by directly affecting the epithelial Mg channel, transient receptor potential channel melastatin member 6, or by altering the driving force for Mg influx via the channel. SUMMARY: Recent genetic and molecular studies have further elucidated the processes that govern renal Mg transport and hence systemic Mg balance. This has provided us with new tools to understand the molecular pathology behind hypomagnesemia.

Angiotensin II signaling increases activity of the renal Na-Cl cotransporter through a WNK4-SPAK-dependent pathway.

Mutations in the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a syndrome featuring hypertension and high serum K(+) levels (hyperkalemia). WNK4 has distinct functional states that regulate the balance between renal salt reabsorption and K(+) secretion by modulating the activities of renal transporters and channels, including the Na-Cl cotransporter NCC and the K(+) channel ROMK. WNK4's functions could enable differential responses to intravascular volume depletion (hypovolemia) and hyperkalemia. Because hypovolemia is uniquely associated with high angiotensin II (AngII) levels, AngII signaling might modulate WNK4 activity. We show that AngII signaling in Xenopus oocytes increases NCC activity by abrogating WNK4's inhibition of NCC but does not alter WNK4's inhibition of ROMK. This effect requires AngII, its receptor AT1R, and WNK4, and is prevented by the AT1R inhibitor losartan. NCC activity is also increased by WNK4 harboring mutations found in PHAII, and this activity cannot be further augmented by AngII signaling, consistent with PHAII mutations providing constitutive activation of the signaling pathway between AT1R and NCC. AngII's effect on NCC is also dependent on the kinase SPAK because dominant-negative SPAK or elimination of the SPAK binding motif in NCC prevent activation of NCC by AngII signaling. These effects extend to mammalian cells. AngII increases phosphorylation of specific sites on SPAK and NCC that are necessary for activation of each in mpkDCT cells. These findings place WNK4 in the signaling pathway between AngII and NCC, and provide a mechanism by which hypovolemia maximizes renal salt reabsoprtion without concomitantly increasing K(+) secretion.

WNK3 and WNK4 amino-terminal domain defines their effect on the renal Na+-Cl- cotransporter.

Loss of physiological regulation of the renal thiazide-sensitive Na+-Cl- cotransporter (NCC) by mutant WNK1 or WNK4 results in pseudohypoaldosteronism type II (PHAII) characterized by arterial hypertension and hyperkalemia. WNK4 normally inhibits NCC, but this effect is lost by eliminating WNK4 catalytic activity or through PHAII-type mutations. In contrast, another member of the WNK family, WNK3, activates NCC. The positive effect of WNK3 on NCC also requires its catalytic activity. Because the opposite effects of WNK3 and WNK4 on NCC were observed in the same expression system, sequences within the WNKs should endow these kinases with their activating or inhibiting properties. To gain insight into the structure-function relationships between the WNKs and NCC, we used a chimera approach between WNK3 and WNK4 to elucidate the domain of the WNKs responsible for the effects on NCC. Chimeras were constructed by swapping the amino or carboxyl terminus domains, which flank the central kinase domain, between WNK3 and WNK4. Our results show that the effect of chimeras toward NCC follows the amino-terminal domain. Thus the amino terminus of the WNKs contains the sequences that are required for their activating or inhibiting properties on NCC.

Regulation of NKCC2 by a chloride-sensing mechanism involving the WNK3 and SPAK kinases.

The Na(+):K(+):2Cl(-) cotransporter (NKCC2) is the target of loop diuretics and is mutated in Bartter's syndrome, a heterogeneous autosomal recessive disease that impairs salt reabsorption in the kidney's thick ascending limb (TAL). Despite the importance of this cation/chloride cotransporter (CCC), the mechanisms that underlie its regulation are largely unknown. Here, we show that intracellular chloride depletion in Xenopus laevis oocytes, achieved by either coexpression of the K-Cl cotransporter KCC2 or low-chloride hypotonic stress, activates NKCC2 by promoting the phosphorylation of three highly conserved threonines (96, 101, and 111) in the amino terminus. Elimination of these residues renders NKCC2 unresponsive to reductions of [Cl(-)](i). The chloride-sensitive activation of NKCC2 requires the interaction of two serine-threonine kinases, WNK3 (related to WNK1 and WNK4, genes mutated in a Mendelian form of hypertension) and SPAK (a Ste20-type kinase known to interact with and phosphorylate other CCCs). WNK3 is positioned upstream of SPAK and appears to be the chloride-sensitive kinase. Elimination of WNK3's unique SPAK-binding motif prevents its activation of NKCC2, as does the mutation of threonines 96, 101, and 111. A catalytically inactive WNK3 mutant also completely prevents NKCC2 activation by intracellular chloride depletion. Together these data reveal a chloride-sensing mechanism that regulates NKCC2 and provide insight into how increases in the level of intracellular chloride in TAL cells, as seen in certain pathological states, could drastically impair renal salt reabsorption.

WNK kinases, renal ion transport and hypertension.

Two members of a recently discovered family of protein kinases are the cause of an inherited disease known as pseudohypoaldosteronism type II (PHAII). These patients exhibit arterial hypertension together with hyperkalemia and metabolic acidosis. This is a mirror image of Gitelman disease that is due to inactivating mutations of the SLC12A3 gene that encodes the thiazide-sensitive Na(+):Cl(-) cotransporter. The uncovered genes causing PHAII encode for serine/threonine kinases known as WNK1 and WNK4. Physiological and biochemical studies have revealed that WNK1 and WNK4 modulate the activity of several transport pathways of the aldosterone-sensitive distal nephron, thus increasing our understanding of how diverse renal ion transport proteins are coordinated to regulate normal blood pressure levels. Observations discussed in the present work place WNK1 and WNK4 as genes involved in the genesis of essential hypertension and as potential targets for the development of antihypertensive drugs.

WNK3 bypasses the tonicity requirement for K-Cl cotransporter activation via a phosphatase-dependent pathway.

SLC12A cation/Cl- cotransporters are mutated in human disease, are targets of diuretics, and are collectively involved in the regulation of cell volume, neuronal excitability, and blood pressure. This gene family has two major branches with different physiological functions and inverse regulation: K-Cl cotransporters (KCC1-KCC4) mediate cellular Cl- efflux, are inhibited by phosphorylation, and are activated by dephosphorylation; Na-(K)-Cl cotransporters (NCC and NKCC1/2) mediate cellular Cl- influx and are activated by phosphorylation. A single kinase/phosphatase pathway is thought to coordinate the activities of these cotransporters in a given cell; however, the mechanisms involved are as yet unknown. We previously demonstrated that WNK3, a paralog of serine-threonine kinases mutated in hereditary hypertension, is coexpressed with several cation/Cl- cotransporters and regulates their activity. Here, we show that WNK3 completely prevents the cell swelling-induced activation of KCC1-KCC4 in Xenopus oocytes. In contrast, catalytically inactive WNK3 abolishes the cell shrinkage-induced inhibition of KCC1-KCC4, resulting in a >100-fold stimulation of K-Cl cotransport during conditions in which transport is normally inactive. This activation is completely abolished by calyculin A and cyclosporine A, inhibitors of protein phosphatase 1 and 2B, respectively. Wild-type WNK3 activates Na-(K)-Cl cotransporters by increasing their phosphorylation, and catalytically inactive kinase inhibits Na-(K)-Cl cotransporters by decreasing their phosphorylation, such that our data suggest that WNK3 is a crucial component of the kinase/phosphatase signaling pathway that coordinately regulates the Cl- influx and efflux branches of the SLC12A cotransporter family.