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 chloridesensitive 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.ion transport ͉ loop of Henle ͉ protein serine-threonine kinases ͉ hypertension ͉ diuretics

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.angiotensin II receptor ͉ hypertension ͉ distal convoluted tubule ͉ salt reabsorption ͉ thiazide A ldosterone is released from the adrenal glomerulosa in 2 different physiologic conditions: intravascular volume depletion and hyperkalemia. In the former, aldosterone promotes maximal renal Na-Cl reabsorption to preserve and restore intravascular volume, whereas in the latter renal K ϩ secretion is maximized. Classical explanations for these alternative responses have focused on acute changes in solute delivery to the distal nephron. For example, in volume depletion there is enhanced proximal reabsorption of Na ϩ , which reduces the distal electrogenic reabsorption of Na ϩ via the epithelial sodium channel (ENaC) that is required to establish the electrical gradient necessary for K ϩ secretion.The rare autosomal dominant disease pseudohypoaldosteronism type II (PHAII) suggests there must be additional components that regulate the balance between renal salt reabsorption and potassium secretion. Patients with PHAII have chloridedependent hypertension and hyperkalemia despite otherwise normal renal function and normal aldosterone secretion, suggesting that they constitutively reabsorb Na-Cl at the expense of impaired K ϩ secretion. Missense mutations in the serinethreonine kinase WNK4 have been shown to cause PHAII (1). Subsequent studies in X...

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.

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.

San-Cristobal P, Ponce-Coria J, Vázquez N, Bobadilla NA, Gamba G. WNK3 and WNK4 amino-terminal domain defines their effect on the renal Na ϩ -Cl Ϫ cotransporter. Am J Physiol Renal Physiol 295: F1199 -F1206, 2008. First published August 13, 2008 doi:10.1152/ajprenal.90396.2008.-Loss of physiological regulation of the renal thiazide-sensitive Na ϩ -Cl Ϫ 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.distal convoluted tubule; diuretics; hypertension; kinase THE WNKS [with no lysine (K)] are a subfamily of serine/ threonine kinases that lack the conserved lysine (substituted by cysteine), which in all other serine/threonine kinases is located in the ␤-strand 3 of kinase subdomain II of the catalytic domain (35). A total of four genes encoding WNK isoforms are present in the human genome: WNK1, WNK2, WNK3, and WNK4, located in chromosomes 12, 9, X, and 17, respectively. The degree of identity among WNKs is Ͼ80% in the kinase domain and Ͻ17% in the flanking amino-or carboxy-terminal domains. Mutations in WNK1 and WNK4 are the cause of pseudohypoaldosteronism type II (PHAII) (33), an inherited disease that features arterial hypertension, hyperkalemia, and metabolic acidosis. PHAII patients are highly sensitive to thiazide diuretics (18). Clinically, PHAII is the mirror image of Gitelman's disease, an inherited illness characterized by arterial hypotension, hipokalemia, and metabolic alkalosis due to inactivating mutations of the SLC12A3 gene that encodes the thiazide-sensitive Na ϩ -Cl Ϫ cotransporter NCC (28). Thus PHAII seems to be the consequence of overactivity of NCC due to a loss of physiological regulation by the WNKs (15,34,41). This hypothesis is supported by recent studies in Xenopus laevis oocytes (11, 34, 37), culture cells (2), and transgenic mice harboring WNK4 with PHAII-type mutations (15, 41). It is not surprising that the WNKs have emerged as powerful regulators not only of NCC but also ...

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.

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 ␥-adducin interacts with the NCC transporter. ␥-Adducin colocalized with NCC to the distal convoluted tubule.22 Na ϩ uptake experiments in the Xenopus laevis oocyte showed that ␥-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 ␥-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 ␥-adducin on the transporter. ␥-Adducin failed to increase NCC activity when these phosphorylation sites were constitutively inactive or active. In addition, ␥-adducin bound only to the dephosphorylated N terminus of NCC. Taken together, our observations suggest that ␥-adducin dynamically regulates NCC, likely by amending the phosphorylation state, and consequently the activity, of the transporter. These data suggest that ␥-adducin may influence BP homeostasis by modulating renal NaCl transport.

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.