KS-WNK1 is required for the renal response to extreme changes in potassium intake.

Kidney-specific with-no-lysine kinase 1 (KS-WNK1) is an isoform of WNK1 kinase that is predominantly found in the distal convoluted tubule of the kidney. The precise physiological function of KS-WNK1 remains unclear. Some studies have suggested that it could play a role in regulating potassium renal excretion by modulating the activity of the Na+-Cl- cotransporter (NCC). However, changes in the potassium diet from normal to high failed to reveal a role for KS-WNK1, but under a normal-potassium diet, the expression of KS-WNK1 is negligible. It is only detectable when mice are exposed to a low-potassium diet. In this study, we investigated the role of KS-WNK1 in regulating potassium excretion under extreme changes in potassium intake. After following a zero-potassium diet (0KD) for 10 days, KS-WNK1-/- mice had lower plasma levels of K+ and Cl- while exhibiting higher urinary excretion of Na+, Cl-, and K+ compared with KS-WNK1+/+ mice. After 10 days of 0KD or normal-potassium diet (NKD), all mice were challenged with a high-potassium diet (HKD). Plasma K+ levels markedly increased after the HKD challenge only in mice previously fed with 0KD, regardless of genotype. KSWNK1+/+ mice adapt better to HKD challenge than KS-WNK1-/- mice after a potassium-retaining state. The difference in the phosphorylated NCC-to-NCC ratio between KS-WNK1+/+ and KS-WNK1-/- mice after 0KD and HKD indicates a role for KS-WNK1 in both NCC phosphorylation and dephosphorylation. These observations show that KS-WNK1 helps the distal convoluted tubule to respond to extreme changes in potassium intake, such as those occurring in wildlife.NEW & NOTEWORTHY The findings of this study demonstrate that kidney-specific with-no-lysine kinase 1 plays a role in regulating urinary electrolyte excretion during extreme changes in potassium intake, such as those occurring in wildlife. .

Arginine vasopressin regulates the renal Na+-Cl- and Na+-K+-Cl- cotransporters through with-no-lysine kinase 4 and inhibitor 1 phosphorylation.

Vasopressin regulates water homeostasis via the V2 receptor in the kidney at least in part through protein kinase A (PKA) activation. Vasopressin, through an unknown pathway, upregulates the activity and phosphorylation of Na+-Cl- cotransporter (NCC) and Na+-K+-2Cl- cotransporter 2 (NKCC2) by Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1), which are regulated by the with-no-lysine kinase (WNK) family. Phosphorylation of WNK4 at PKA consensus motifs may be involved. Inhibitor 1 (I1), a protein phosphatase 1 (PP1) inhibitor, may also play a role. In human embryonic kidney (HEK)-293 cells, we assessed the phosphorylation of WNK4, SPAK, NCC, or NKCC2 in response to forskolin or desmopressin. WNK4 and cotransporter phosphorylation were studied in desmopressin-infused WNK4-/- mice and in tubule suspensions. In HEK-293 cells, only wild-type WNK4 but not WNK1, WNK3, or a WNK4 mutant lacking PKA phosphorylation motifs could upregulate SPAK or cotransporter phosphorylation in response to forskolin or desmopressin. I1 transfection maximized SPAK phosphorylation in response to forskolin in the presence of WNK4 but not of mutant WNK4 lacking PP1 regulation. We observed direct PP1 regulation of NKCC2 dephosphorylation but not of NCC or SPAK in the absence of WNK4. WNK4-/- mice with desmopressin treatment did not increase SPAK/OSR1, NCC, or NKCC2 phosphorylation. In stimulated tubule suspensions from WNK4-/- mice, upregulation of pNKCC2 was reduced, whereas upregulation of SPAK phosphorylation was absent. These findings suggest that WNK4 is a central node in which kinase and phosphatase signaling converge to connect cAMP signaling to the SPAK/OSR1-NCC/NKCC2 pathway.NEW & NOTEWORTHY With-no-lysine kinases regulate the phosphorylation and activity of the Na+-Cl- and Na+-K+-2Cl- cotransporters. This pathway is modulated by arginine vasopressin (AVP). However, the link between AVP and WNK signaling remains unknown. Here, we show that AVP activates WNK4 through increased phosphorylation at putative protein kinase A-regulated sites and decreases its dephosphorylation by protein phosphatase 1. This work increases our understanding of the signaling pathways mediating AVP actions in the kidney.

Glucose/Fructose Delivery to the Distal Nephron Activates the Sodium-Chloride Cotransporter via the Calcium-Sensing Receptor.

BACKGROUND: The calcium-sensing receptor (CaSR) in the distal convoluted tubule (DCT) activates the NaCl cotransporter (NCC). Glucose acts as a positive allosteric modulator of the CaSR. Under physiologic conditions, no glucose is delivered to the DCT, and fructose delivery depends on consumption. We hypothesized that glucose/fructose delivery to the DCT modulates the CaSR in a positive allosteric way, activating the WNK4-SPAK-NCC pathway and thus increasing salt retention. METHODS: We evaluated the effect of glucose/fructose arrival to the distal nephron on the CaSR-WNK4-SPAK-NCC pathway using HEK-293 cells, C57BL/6 and WNK4-knockout mice, ex vivo perfused kidneys, and healthy humans. RESULTS: HEK-293 cells exposed to glucose/fructose increased SPAK phosphorylation in a WNK4- and CaSR-dependent manner. C57BL/6 mice exposed to fructose or a single dose of dapagliflozin to induce transient glycosuria showed increased activity of the WNK4-SPAK-NCC pathway. The calcilytic NPS2143 ameliorated this effect, which was not observed in WNK4-KO mice. C57BL/6 mice treated with fructose or dapagliflozin showed markedly increased natriuresis after thiazide challenge. Ex vivo rat kidney perfused with glucose above the physiologic threshold levels for proximal reabsorption showed increased NCC and SPAK phosphorylation. NPS2143 prevented this effect. In healthy volunteers, cinacalcet administration, fructose intake, or a single dose of dapagliflozin increased SPAK and NCC phosphorylation in urinary extracellular vesicles. CONCLUSIONS: Glycosuria or fructosuria was associated with increased NCC, SPAK, and WNK4 phosphorylation in a CaSR-dependent manner.

Blood pressure effects of sodium transport along the distal nephron.

The mammalian distal nephron is a target of highly effective antihypertensive drugs. Genetic variants that alter its transport activity are also inherited causes of high or low blood pressure, clearly establishing its central role in human blood pressure regulation. Much has been learned during the past 25 years about salt transport along this nephron segment, spurred by the cloning of major transport proteins and the discovery of disease-causing genetic variants. Recognition is increasing that substantial cellular and segmental heterogeneity is present along this segment, with electroneutral sodium transport dominating more proximal segments and electrogenic sodium transport dominating more distal segments. Coupled with recent insights into factors that modulate transport along these segments, we now understand one important mechanism by which dietary potassium intake influences sodium excretion and blood pressure. This finding has solved the aldosterone paradox, by demonstrating how aldosterone can be both kaliuretic, when plasma potassium is elevated, and anti-natriuretic, when extracellular fluid volume is low. However, what also has become clear is that aldosterone itself only stimulates a portion of the mineralocorticoid receptors along this segment, with the others being activated by glucocorticoid hormones instead. These recent insights provide an increasingly clear picture of how this short nephron segment contributes to blood pressure homeostasis and have important implications for hypertension prevention and treatment.

Molecular mechanisms for the modulation of blood pressure and potassium homeostasis by the distal convoluted tubule.

Epidemiological and clinical observations have shown that potassium ingestion is inversely correlated with arterial hypertension prevalence and cardiovascular mortality. The higher the dietary potassium, the lower the blood pressure and mortality. This phenomenon is explained, at least in part, by the interaction between salt reabsorption in the distal convoluted tubule (DCT) and potassium secretion in the connecting tubule/collecting duct of the mammalian nephron: In order to achieve adequate K+ secretion levels under certain conditions, salt reabsorption in the DCT must be reduced. Because salt handling by the kidney constitutes the basis for the long-term regulation of blood pressure, losing salt prevents hypertension. Here, we discuss how the study of inherited diseases in which salt reabsorption in the DCT is affected has revealed the molecular players, including membrane transporters and channels, kinases, and ubiquitin ligases that form the potassium sensing mechanism of the DCT and the processes through which the consequent adjustments in salt reabsorption are achieved.

The Calcium-Sensing Receptor Increases Activity of the Renal NCC through the WNK4-SPAK Pathway.

Background Hypercalciuria can result from activation of the basolateral calcium-sensing receptor (CaSR), which in the thick ascending limb of Henle's loop controls Ca2+ excretion and NaCl reabsorption in response to extracellular Ca2+ However, the function of CaSR in the regulation of NaCl reabsorption in the distal convoluted tubule (DCT) is unknown. We hypothesized that CaSR in this location is involved in activating the thiazide-sensitive NaCl cotransporter (NCC) to prevent NaCl loss.Methods We used a combination of in vitro and in vivo models to examine the effects of CaSR on NCC activity. Because the KLHL3-WNK4-SPAK pathway is involved in regulating NaCl reabsorption in the DCT, we assessed the involvement of this pathway as well.Results Thiazide-sensitive 22Na+ uptake assays in Xenopus laevis oocytes revealed that NCC activity increased in a WNK4-dependent manner upon activation of CaSR with Gd3+ In HEK293 cells, treatment with the calcimimetic R-568 stimulated SPAK phosphorylation only in the presence of WNK4. The WNK4 inhibitor WNK463 also prevented this effect. Furthermore, CaSR activation in HEK293 cells led to phosphorylation of KLHL3 and WNK4 and increased WNK4 abundance and activity. Finally, acute oral administration of R-568 in mice led to the phosphorylation of NCC.Conclusions Activation of CaSR can increase NCC activity via the WNK4-SPAK pathway. It is possible that activation of CaSR by Ca2+ in the apical membrane of the DCT increases NaCl reabsorption by NCC, with the consequent, well known decrease of Ca2+ reabsorption, further promoting hypercalciuria.

C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity.

WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overactivation of the renal Na+/Cl- cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4-/- mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4's C terminus at several positions increased its kinase activity toward Ste20-related proline/alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-binding site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn2+-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate.

Pathophysiology of functional mutations of the thiazide-sensitive Na-Cl cotransporter in Gitelman disease.

Most of the missense mutations that have been described in the human SLC12A3 gene encoding the thiazide-sensitive Na(+)-Cl(-) cotransporter (TSC, NCC, or NCCT), as the cause of Gitelman disease, block TSC function by interfering with normal protein processing and glycosylation. However, some mutations exhibit considerable activity. To investigate the pathogenesis of Gitelman disease mediated by such mutations and to gain insights into structure-function relationships on the cotransporter, five functional disease mutations were introduced into mouse TSC cDNA, and their expression was determined in Xenopus laevis oocytes. Western blot analysis revealed immunoreactive bands in all mutant TSCs that were undistinguishable from wild-type TSC. The activity profile was: wild-type TSC (100%) > G627V (66%) > R935Q (36%) = V995M (32%) > G610S (12%) > A585V (6%). Ion transport kinetics in all mutant clones were similar to wild-type TSC, except in G627V, in which a small but significant increase in affinity for extracellular Cl(-) was observed. In addition, G627V and G610S exhibited a small increase in metolazone affinity. The surface expression of wild-type and mutant TSCs was performed by laser-scanning confocal microscopy. All mutants exhibited a significant reduction in surface expression compared with wild-type TSC, with a profile similar to that observed in functional expression analysis. Our data show that biochemical and functional properties of the mutant TSCs are similar to wild-type TSC but that the surface expression is reduced, suggesting that these mutations impair the insertion of a functional protein into the plasma membrane. The small increase in Cl(-) and thiazide affinity in G610S and G627V suggests that the beginning of the COOH-terminal domain could be implicated in defining kinetic properties.