Potassium-Switch Signaling Pathway Dictates Acute Blood Pressure Response to Dietary Potassium.

BACKGROUND: Potassium (K+)-deficient diets, typical of modern processed foods, increase blood pressure (BP) and NaCl sensitivity. A K+-dependent signaling pathway in the kidney distal convoluted tubule, coined the K+ switch, that couples extracellular K+ sensing to activation of the thiazide-sensitive NaCl cotransporter (NCC) and NaCl retention has been implicated, but causality has not been established. METHODS: To test the hypothesis that small, physiological changes in plasma K+ (PK+) are translated to BP through the switch pathway, a genetic approach was used to activate the downstream switch kinase, SPAK (SPS1-related proline/alanine-rich kinase), within the distal convoluted tubule. The CA-SPAK (constitutively active SPS1-related proline/alanine-rich kinase mice) were compared with control mice over a 4-day PK+ titration (3.8-5.1 mmol) induced by changes in dietary K+. Arterial BP was monitored using radiotelemetry, and renal function measurements, NCC abundance, phosphorylation, and activity were made. RESULTS: As PK+ decreased in control mice, BP progressively increased and became sensitive to dietary NaCl and hydrochlorothiazide, coincident with increased NCC phosphorylation and urinary sodium retention. By contrast, BP in CA-SPAK mice was elevated, resistant to the PK+ titration, and sensitive to hydrochlorothiazide and salt at all PK+ levels, concomitant with sustained and elevated urinary sodium retention and NCC phosphorylation and activity. Thus, genetically locking the switch on drives NaCl sensitivity and prevents the response of BP to potassium. CONCLUSIONS: Low K+, common in modern ultraprocessed diets, presses the K+-switch pathway to turn on NCC activity, increasing sodium retention, BP, and salt sensitivity.

Kinase Scaffold Cab39 Is Necessary for Phospho-Activation of the Thiazide-Sensitive NCC.

BACKGROUND: Potassium regulates the WNK (with no lysine kinase)-SPAK (STE20/SPS1-related proline/alanine-rich kinase) signaling axis, which in turn controls the phosphorylation and activation of the distal convoluted tubule thiazide-sensitive NCC (sodium-chloride cotransporter) for sodium-potassium balance. Although their roles in the kidney have not been investigated, it has been postulated that Cab39 (calcium-binding protein 39) or Cab39l (Cab39-like) is required for SPAK/OSR1 (oxidative stress response 1) activation. This study demonstrates how they control the WNK-SPAK/OSR1-NCC pathway. METHODS: We created a global knockout of Cab39l and a tamoxifen-inducible, NCC-driven, Cab39 knockout. The 2 lines were crossed to generate Cab39-DKO (Cab39 double knockout) animals. Mice were studied under control and low-potassium diet, which activates WNK-SPAK/OSR1-NCC phosphorylation. Western blots were used to assess the expression and phosphorylation of proteins. Blood and urine electrolytes were measured to test for compromised NCC function. Immunofluorescence studies were conducted to localize SPAK and OSR1. RESULTS: Both Cab39l and Cab39 are expressed in distal convoluted tubule, and only the elimination of both leads to a striking absence of NCC phosphorylation. Cab39-DKO mice exhibited a loss-of-NCC function, like in Gitelman syndrome. In contrast to the apical membrane colocalization of SPAK with NCC in wild-type mice, SPAK and OSR1 become confined to intracellular puncta in the Cab39-DKO mice. CONCLUSIONS: In the absence of Cab39 proteins, NCC cannot be phosphorylated, resulting in a Gitelman-like phenotype. Cab39 proteins function to localize SPAK at the apical membrane with NCC, reminiscent of the Cab39 yeast homolog function, translocating kinases during cytokinesis.

Dietary sodium alters aldosterone's effect on renal sodium transporter expression and distal convoluted tubule remodelling.

Aldosterone is responsible for maintaining volume and potassium homeostasis. Although high salt consumption should suppress aldosterone production, individuals with hyperaldosteronism lose this regulation, leading to a state of high aldosterone despite dietary sodium consumption. The present study examines the effects of elevated aldosterone, with or without high salt consumption, on the expression of key Na+ transporters and remodelling in the distal nephron. Epithelial sodium channel (ENaC) α-subunit expression was increased with aldosterone regardless of Na+ intake. However, ENaC ß- and γ-subunits unexpectedly increased at both a transcript and protein level with aldosterone when high salt was present. Expression of total and phosphorylated Na+ Cl- cotransporter (NCC) significantly increased with aldosterone, in association with decreased blood [K+ ], but the addition of high salt markedly attenuated the aldosterone-dependent NCC increase, despite equally severe hypokalaemia. We hypothesized this was a result of differences in distal convoluted tubule length when salt was given with aldosterone. Imaging and measurement of the entire pNCC-positive tubule revealed that aldosterone alone caused a shortening of this segment, although the tubule had a larger cross-sectional diameter. This was not true when salt was given with aldosterone because the combination was associated with a lengthening of the tubule in addition to increased diameter, suggesting that differences in the pNCC-positive area are not responsible for differences in NCC expression. Together, our results suggest the actions of aldosterone, and the subsequent changes related to hypokalaemia, are altered in the presence of high dietary Na+ . KEY POINTS: Aldosterone regulates volume and potassium homeostasis through effects on transporters in the kidney; its production can be dysregulated, preventing its suppression by high dietary sodium intake. Here, we examined how chronic high sodium consumption affects aldosterone's regulation of sodium transporters in the distal nephron. Our results suggest that high sodium consumption with aldosterone is associated with increased expression of all three epithelial sodium channel subunits, rather than just the alpha subunit. Aldosterone and its associated decrease in blood [K+ ] lead to an increased expression of Na-Cl cotransporter (NCC); the addition of high sodium consumption with aldosterone partially attenuates this NCC expression, despite similarly low blood [K+ ]. Upstream kinase regulators and tubule remodelling do not explain these results.

Dysregulation of the WNK4-SPAK/OSR1 pathway has a minor effect on baseline NKCC2 phosphorylation.

The with-no-lysine kinase 4 (WNK4)-sterile 20/SPS-1-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase 1 (OSR1) pathway mediates activating phosphorylation of the furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC2) and the thiazide-sensitive NaCl cotransporter (NCC). The commonly used pT96/pT101-pNKCC2 antibody cross-reacts with pT53-NCC in mice on the C57BL/6 background due to a five amino acid deletion. We generated a new C57BL/6-specific pNKCC2 antibody (anti-pT96-NKCC2) and tested the hypothesis that the WNK4-SPAK/OSR1 pathway strongly regulates the phosphorylation of NCC but not NKCC2. In C57BL/6 mice, anti-pT96-NKCC2 detected pNKCC2 and did not cross-react with NCC. Abundances of pT96-NKCC2 and pT53-NCC were evaluated in Wnk4-/-, Osr1-/-, Spak-/-, and Osr1-/-/Spak-/- mice and in several models of the disease familial hyperkalemic hypertension (FHHt) in which the CUL3-KLHL3 ubiquitin ligase complex that promotes WNK4 degradation is dysregulated (Cul3+/-/Δ9, Klhl3-/-, and Klhl3R528H/R528H). All mice were on the C57BL/6 background. In Wnk4-/- mice, pT53-NCC was almost absent but pT96-NKCC2 was only slightly lower. pT53-NCC was almost absent in Spak-/- and Osr1-/-/Spak-/- mice, but pT96-NKCC2 abundance did not differ from controls. pT96-NKCC2/total NKCC2 was slightly lower in Osr1-/- and Osr1-/-/Spak-/- mice. WNK4 expression colocalized not only with NCC but also with NKCC2 in Klhl3-/- mice, but pT96-NKCC2 abundance was unchanged. Consistent with this, furosemide-induced urinary Na+ excretion following thiazide treatment was similar between Klhl3-/- and controls. pT96-NKCC2 abundance was also unchanged in the other FHHt mouse models. Our data show that disruption of the WNK4-SPAK/OSR1 pathway only mildly affects NKCC2 phosphorylation, suggesting a role for other kinases in NKCC2 activation. In FHHt models NKCC2 phosphorylation is unchanged despite higher WNK4 abundance, explaining the thiazide sensitivity of FHHt.NEW & NOTEWORTHY The renal cation cotransporters NCC and NKCC2 are activated following phosphorylation mediated by the WNK4-SPAK/OSR1 pathway. While disruption of this pathway strongly affects NCC activity, effects on NKCC2 activity are unclear since the commonly used phospho-NKCC2 antibody was recently reported to cross-react with phospho-NCC in mice on the C57BL/6 background. Using a new phospho-NKCC2 antibody specific for C57BL/6, we show that inhibition or activation of the WNK4-SPAK/OSR1 pathway in mice only mildly affects NKCC2 phosphorylation.

From Fish Physiology to Human Disease: The Discovery of the NCC, NKCC2, and the Cation-Coupled Chloride Cotransporters.

The renal Na-K-2Cl and Na-Cl cotransporters are the major salt reabsorption pathways in the thick ascending limb of Henle loop and the distal convoluted tubule, respectively. These transporters are the target of the loop and thiazide type diuretics extensively used in the world for the treatment of edematous states and arterial hypertension. The diuretics appeared in the market many years before the salt transport systems were discovered. The evolving of the knowledge and the cloning of the genes encoding the Na-K-2Cl and Na-Cl cotransporters were possible thanks to the study of marine species. This work presents the history of how we came to know the mechanisms for the loop and thiazide type diuretics actions, the use of marine species in the cloning process of these cotransporters and therefore in the whole solute carrier cotransproters 12 (SLC12) family of electroneutral cation chloride cotransporters, and the disease associated with each member of the family.

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.

Long-Term Indomethacin Treatment in a Chinese Child with Gitelman Syndrome: Case Report and Literature Review on its Efficacy and Tolerance.

BACKGROUND Gitelman syndrome (GS) is a rare inherited autosomal recessive salt-losing renal tubulopathy. Early-onset GS is difficult to differentiate from Bartter syndrome (BS). It has been reported in some cases that cyclooxygenase (COX) inhibitors, which pharmacologically reduce prostaglandin E2(PGE2) synthesis, are helpful for GS patients, especially in children, but the long-term therapeutic effect has not yet been revealed. CASE REPORT A 4-year-old boy was first brought to our hospital for the chief concern of short stature and growth retardation. Biochemical tests demonstrated severe hypokalemia, hyponatremia, and hypochloremic metabolic alkalosis. The patient's serum magnesium was normal. He was diagnosed with BS and treated with potassium supplementation and indomethacin and achieved stable serum potassium levels and slow catch-up growth. At 11.8 years of age, the patient showed hypomagnesemia and a genetic test confirmed that he had GS with compound heterozygous mutations in the SLC12A3 gene. At the age of 14.8 years, when indomethacin had been taken for nearly 10 years, the boy reported having chronic stomachache, while his renal function remained normal. After proton pump inhibitor and acid inhibitor therapy, the patient's symptoms were ameliorated, and he continued to take a low dose of indomethacin (37.5 mg/d divided tid) with good tolerance. CONCLUSIONS Early-onset GS in childhood can be initially misdiagnosed as BS, and gene detection can confirm the final diagnosis. COX inhibitors, such as indomethacin, might be tolerated by pediatric patients, and long-term therapy can improve the hypokalemia and growth retardation without significant adverse effects.

Dietary potassium stimulates Ppp1Ca-Ppp1r1a dephosphorylation of kidney NaCl cotransporter and reduces blood pressure.

Consumption of low dietary potassium, common with ultraprocessed foods, activates the thiazide-sensitive sodium chloride cotransporter (NCC) via the with no (K) lysine kinase/STE20/SPS1-related proline-alanine-rich protein kinase (WNK/SPAK) pathway to induce salt retention and elevate blood pressure (BP). However, it remains unclear how high-potassium "DASH-like" diets (dietary approaches to stop hypertension) inactivate the cotransporter and whether this decreases BP. A transcriptomics screen identified Ppp1Ca, encoding PP1A, as a potassium-upregulated gene, and its negative regulator Ppp1r1a, as a potassium-suppressed gene in the kidney. PP1A directly binds to and dephosphorylates NCC when extracellular potassium is elevated. Using mice genetically engineered to constitutively activate the NCC-regulatory kinase SPAK and thereby eliminate the effects of the WNK/SPAK kinase cascade, we confirmed that PP1A dephosphorylated NCC directly in a potassium-regulated manner. Prior adaptation to a high-potassium diet was required to maximally dephosphorylate NCC and lower BP in constitutively active SPAK mice, and this was associated with potassium-dependent suppression of Ppp1r1a and dephosphorylation of its cognate protein, inhibitory subunit 1 (I1). In conclusion, potassium-dependent activation of PP1A and inhibition of I1 drove NCC dephosphorylation, providing a mechanism to explain how high dietary K+ lowers BP. Shifting signaling of PP1A in favor of activation of WNK/SPAK may provide an improved therapeutic approach for treating salt-sensitive hypertension.

Role of NCC in the pathophysiology of hypertension in primary aldosteronism.

PURPOSE OF REVIEW: An increasing amount of evidence points out to a role for the thiazide-sensitive Na+:Cl- cotransporter, NCC, in the blood pressure alterations observed in conditions of pathologically high or pathologically low aldosterone. Here, we briefly review this evidence that is changing our perception of the pathophysiology of primary aldosteronism. RECENT FINDINGS: Although initially NCC was thought to be a direct target of aldosterone, more recent evidence suggests that NCC is only indirectly regulated by aldosterone, at least in a chronic setting. Aldosterone-induced changes in plasma K+ concentration that are prompted by the modulation of K+ secretion in principal cells of the connecting tubule and collecting duct are actually responsible for the modulation of NCC in conditions of altered aldosterone levels. A mounting amount of evidence suggests that this indirect effect of aldosterone on NCC may be key to produce the blood pressure alterations observed in aldosterone excess or aldosterone deficit. Finally, recent insights into the molecular pathways involved in NCC modulation by K+ are briefly reviewed. SUMMARY: The evidence reviewed here suggests that correction of K+ alterations in patients with hyper or hypoaldosteronism may substantially affect blood pressure levels. Mechanistically, this may be related to the K+-mediated modulation of NCC.

Regulation of the WNK4-SPAK-NCC pathway by the calcium-sensing receptor.

PURPOSE OF REVIEW: Regulation of the sodium chloride cotransporter (NCC) in the distal convoluted tubule (DCT) plays a crucial role in renal salt handling. The calcium-sensing receptor (CaSR) has been shown to activate NCC through the WNK4-SPAK pathway, which is independent of the Renin-Angiotensin-Aldosterone system. In this review, we examine new information about the mechanism of how the CaSR regulates NCC through the WNK4-SPAK pathway and its physiological and therapeutic implications. RECENT FINDINGS: The activation of CaSR in TALH cells during hypercalcemia inhibits NKCC2 and ROMK activity, reducing paracellular Ca2+ reabsorption but decreasing salt reabsorption. This pathway enables NaCl reabsorption in the DCT while promoting Ca2+ excretion. CaSR activation in the apical DCT stimulates a signaling pathway involving PKC, WNK4, and SPAK, which increases NCC activation to recover the NaCl not reabsorbed in TAHL. Glucose or fructose acting as calcimimetics enhance apical CaSR sensitivity, increasing NCC activity, which contribute to the mechanism of hypertension prevalence in diabetic patients or in those with high fructose consumption. SUMMARY: These findings reveal the importance of the CaSR-mediated activation of the WNK4-SPAK pathway in regulating salt and calcium homeostasis and its potential as a therapeutic target for hypertension and related diseases.

Thirty years of the NaCl cotransporter: from cloning to physiology and structure.

The primary structure of the thiazide-sensitive NaCl cotransporter (NCC) was resolved 30 years ago by the molecular identification of the cDNA encoding this cotransporter, from the winter's flounder urinary bladder, following a functional expression strategy. This review outlines some aspects of how the knowledge about thiazide diuretics and NCC evolved, the history of the cloning process, and the expansion of the SLC12 family of electroneutral cotransporters. The diseases associated with activation or inactivation of NCC are discussed, as well as the molecular model by which the activity of NCC is regulated. The controversies in the field are discussed as well as recent publication of the three-dimensional model of NCC obtained by cryo-electron microscopy, revealing not only the amino acid residues critical for Na+ and Cl- translocation but also the residues critical for polythiazide binding to the transporter, opening the possibility for a new era in thiazide diuretic therapy.

NGAL is a Novel Target in Hypertension by Modulating the NCC-Mediated Renal Na Balance.

BACKGROUND: The expression of NGAL/lcn2 (neutrophil gelatinase-associated lipocalin) is directly modulated by mineralocorticoid receptor activation but its role in blood pressure control is unclear. METHODS: a potential relationship between NGAL plasma levels, systolic blood pressure and urinary Na excretion was assessed in the STANISLAS cohort. The specific role of NGAL/lcn2 in salt-sensitive hypertension was studied using lcn2-knockout mice (lcn2 KO) fed with low-Na diet (0Na). RESULTS: we show that NGAL plasma levels positively correlate with systolic blood pressure, whereas they negatively correlate with urinary Na excretion in subjects of the STANISLAS cohort. Prolonged feeding of lcn2 KO mice with a 0Na diet induced lower systolic blood pressure than that of the control group (wildtype), suggesting a role for NGAL/lcn2 in Na-balance homeostasis. Short-term or prolonged 0Na increased Na-Cl cotransporter (NCC) phosphorylation in the cortex of wildtype mice, which was prevented in lcn2 KO mice. Recombinant mouse lcn2 injections in lcn2 KO mice induced NCC phosphorylation in the kidney cortex, associated with decreased urinary Na excretion. Ex vivo experiments using kidney slices from lcn2 KO mice showed increased NCC phosphorylation by recombinant murine lcn2. In addition, recombinant murine lcn2 induced activation of CamK2ß (calcium/calmodulin-dependent protein kinase II ß subunit) phosphorylation in lcn2 KO mice and in kidney slices, providing an underlying mechanism involved in lcn2-induced NCC phosphorylation. Indeed, the inhibition of CamK2ß prevented NCC phosphorylation induced by recombinant lcn2 in kidney slices. CONCLUSIONS: we highlight a novel role of NGAL/lcn2 as a modulator of the activity of the renal sodium transporter NCC affecting salt-sensitive blood pressure.

Kir4.1 deletion prevents salt-sensitive hypertension in early streptozotocin-induced diabetic mice via Na + -Cl - cotransporter in the distal convoluted tubule.

OBJECTIVES: Functional impairment of renal sodium handling and blood pressure (BP) homeostasis is an early characteristic manifestation of type 1 diabetes. However, the underlying mechanisms remain unclear. METHODS: Metabolic cages, radio-telemetry, immunoblotting, and electrophysiology were utilized to examine effects of high salt (8% NaCl, HS) intake on Na + /K + balance, BP, Na + -Cl - cotransporter (NCC) function, and basolateral K + channel activity in the distal convoluted tubule (DCT) under diabetic conditions. RESULTS: Improper Na + balance, hypernatremia, and a mild but significant increase in BP were found in streptozotocin (STZ)-induced diabetic mice in response to HS intake for 7 days. Compared to the vehicle, STZ mice showed increased Kir4.1 expression and activity in the DCT, a more negative membrane potential, higher NCC abundance, and enhanced hydrochlorothiazide-induced natriuretic effect. However, HS had no significant effect on basolateral Kir4.1 expression/activity and DCT membrane potential, or NCC activity under diabetic conditions, despite a downregulation in phosphorylated NCC abundance. In contrast, HS significantly downregulated the expression of Na + -H + exchanger 3 (NHE3) and cleaved epithelial sodium channel-γ in STZ mice, despite an increase in NHE3 abundance after STZ treatment. Kir4.1 deletion largely abolished STZ-induced upregulation of NCC expression and prevented BP elevation during HS intake. Interestingly, HS causes severe hypokalemia in STZ-treated kidney-specific Kir4.1 knockout (Ks-Kir4.1 KO) mice and lead to death within a few days, which could be attributed to a higher circulating aldosterone level. CONCLUSIONS: We concluded that Kir4.1 is required for upregulating NCC activity and may be essential for developing salt-sensitive hypertension in early STZ-induced diabetes.

Upregulation of SLC12A3 and SLC12A9 Mediated by the HCP5/miR-140-5p Axis Confers Aggressiveness and Unfavorable Prognosis in Uveal Melanoma.

Perturbation of solute carriers (SLCs) has been implicated in metabolic disorders and cancer, highlighting the potential for drug discovery and therapeutic opportunities. However, there is relatively little exploration of the clinical relevance and potential molecular mechanisms underlying the role of the SLC12 family in uveal melanoma (UVM). Here, we performed an integrative multiomics analysis of the SLC12 family in multicenter UVM datasets and found that high expression of SLC12A3 and SLC12A9 was associated with unfavorable prognosis. Moreover, SLC12A3 and SLC12A9 were highly expressed in UVM in vivo. We experimentally characterized the roles of these proteins in tumorigenesis in vitro and explored their association with the prognosis of UVM. Lastly, we identified the HCP5-miR-140-5p axis as a potential noncoding RNA pathway upstream of SLC12A3 and SLC12A9, which was associated with immunomodulation and may represent a novel predictor for clinical prognosis and responsiveness to checkpoint blockade immunotherapy. These findings may facilitate a better understanding of the SLCome and guide future rationalized development of SLC-targeted therapy and drug discovery for UVM.

Calcineurin inhibitors stimulate Kir4.1/Kir5.1 of the distal convoluted tubule to increase NaCl cotransporter.

We examine whether calcineurin or protein phosphatase 2B (PP2B) regulates the basolateral inwardly rectifying potassium channel Kir4.1/Kir5.1 in the distal convoluted tubule (DCT). Application of tacrolimus (FK506) or cyclosporine A (CsA) increased whole-cell Kir4.1/Kir5.1-mediated K+ currents and hyperpolarized the DCT membrane. Moreover, FK506-induced stimulation of Kir4.1/Kir5.1 was absent in kidney tubule-specific 12 kDa FK506-binding protein-knockout mice (Ks-FKBP-12-KO). In contrast, CsA stimulated Kir4.1/Kir5.1 of the DCT in Ks-FKBP-12-KO mice, suggesting that FK506-induced stimulation of Kir4.1/Kir5.1 was due to inhibiting PP2B. Single-channel patch-clamp experiments demonstrated that FK506 or CsA stimulated the basolateral Kir4.1/Kir5.1 activity of the DCT, defined by NPo (a product of channel number and open probability). However, this effect was absent in the DCT treated with Src family protein tyrosine kinase (SFK) inhibitor or hydroxyl peroxide. Fluorescence imaging demonstrated that CsA treatment increased membrane staining intensity of Kir4.1 in the DCT of Kcnj10fl/fl mice. Moreover, CsA treatment had no obvious effect on phosphorylated NaCl cotransporter (pNCC) expression in Ks-Kir4.1-KO mice. Immunoblotting showed acute FK506 treatment increased pNCC expression in Kcnj10fl/fl mice, but this effect was attenuated in Ks-Kir4.1-KO mice. In vivo measurement of thiazide-induced renal Na+ excretion demonstrated that FK506 enhanced thiazide-induced natriuresis. This effect was absent in Ks-FKBP-12-KO mice and blunted in Ks-Kir4.1-KO mice. We conclude that inhibition of PP2B stimulates Kir4.1/Kir5.1 of the DCT and NCC and that PP2B inhibition-induced stimulation of NCC is partially achieved by stimulation of the basolateral Kir4.1/Kir5.1.

Control of sodium and potassium homeostasis by renal distal convoluted tubules.

Distal convoluted tubules (DCT), which contain the Na-Cl cotransporter (NCC) inhibited by thiazide diuretics, undergo complex modulation to preserve Na+ and K+ homeostasis. The lysine kinases 1 and 4 (WNK1 and WNK4), identified as hyperactive in the hereditary disease pseudohypoaldosteronism type 2, are responsible for activation of NCC and consequent hypokalemia and hypertension. WNK4, highly expressed in DCT, activates the SPAK/OSR1 kinases, which phosphorylate NCC and other regulatory proteins and transporters in the distal nephron. WNK4 works as a chloride sensor through a Cl- binding site, which acts as an on/off switch at this kinase in response to changes of basolateral membrane electrical potential, the driving force of cellular Cl- efflux. High intracellular Cl- in hyperkalemia decreases NCC phosphorylation and low intracellular Cl- in hypokalemia increases NCC phosphorylation and activity, which makes plasma K+ concentration a central modulator of NCC and of K+ secretion. The WNK4 phosphorylation by cSrc or SGK1, activated by angiotensin II or aldosterone, respectively, is another relevant mechanism of NCC, ENaC, and ROMK modulation in states such as volume reduction, hyperkalemia, and hypokalemia. Loss of NCC function induces upregulation of electroneutral NaCl reabsorption by type B intercalated cells through the combined activity of pendrin and NDCBE, as demonstrated in double knockout mice (KO) animal models, Ncc/pendrin or Ncc/NDCBE. The analysis of ks-Nedd-4-2 KO animal models introduced the modulation of NEDD4-2 by intracellular Mg2+ activity as an important regulator of NCC, explaining the thiazide-induced persistent hypokalemia.

Activation of Kir4.1/Kir5.1 contributes to the cyclosporin A-induced stimulation of the renal NaCl cotransporter and hyperkalemic hypertension.

AIM: Cyclosporin A (CsA) is a widely used immunosuppressive drug that causes hypertension and hyperkalemia. Moreover, CsA-induced stimulation of the thiazide-sensitive NaCl cotransporter (NCC) in the kidney has been shown to be responsible for the development of hyperkalemic hypertension. In this study, we tested whether CsA induces the activation of NCC by stimulating the basolateral Kir4.1/Kir5.1 channel in the distal convoluted tubule (DCT). METHODS: Electrophysiology, immunoblotting, metabolic cages, and radio-telemetry methods were used to examine the effects of CsA on Kir4.1/Kir5.1 activity in the DCT, NCC function, and blood pressure in wild-type (WT) and kidney-specific Kir4.1 knockout (KS-Kir4.1 KO) mice. RESULTS: The single-channel patch clamp experiment demonstrated that CsA stimulated the basolateral 40 pS K+ channel in the DCT. Whole-cell recording showed that short-term CsA administration (2 h) not only increased DCT K+ currents but also shifted the K+ current (IK ) reversal potential to the negative range (hyperpolarization). Furthermore, CsA administration increased phosphorylated NCC (pNCC) levels and inhibited renal Na+ and K+ excretions in WT mice but not in KS-Kir4.1 KO mice, suggesting that Kir4.1 is required to mediate CsA effects on NCC function. Finally, long-term CsA infusion (14 days) increased blood pressure, plasma K+ concentration, and total NCC or pNCC abundance in WT mice, but these effects were blunted in KS-Kir4.1 KO mice. CONCLUSION: We conclude that CsA stimulates basolateral K+ channel activity in the DCT and that Kir4.1 is essential for CsA-induced NCC activation and hyperkalemic hypertension.

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.

Differential IL18 signaling via IL18 receptor and Na-Cl co-transporter discriminating thermogenesis and glucose metabolism regulation.

White adipose tissue (WAT) plays a role in storing energy, while brown adipose tissue (BAT) is instrumental in the re-distribution of stored energy when dietary sources are unavailable. Interleukin-18 (IL18) is a cytokine playing a role in T-cell polarization, but also for regulating energy homeostasis via the dimeric IL18 receptor (IL18r) and Na-Cl co-transporter (NCC) on adipocytes. Here we show that IL18 signaling in metabolism is regulated at the level of receptor utilization, with preferential role for NCC in brown adipose tissue (BAT) and dominantly via IL18r in WAT. In Il18r-/-Ncc-/- mice, high-fat diet (HFD) causes more prominent body weight gain and insulin resistance than in wild-type mice. The WAT insulin resistance phenotype of the double-knockout mice is recapitulated in HFD-fed Il18r-/- mice, whereas decreased thermogenesis in BAT upon HFD is dependent on NCC deletion. BAT-selective depletion of either NCC or IL18 reduces thermogenesis and increases BAT and WAT inflammation. IL18r deletion in WAT reduces insulin signaling and increases WAT inflammation. In summary, our study contributes to the mechanistic understanding of IL18 regulation of energy metabolism and shows clearly discernible roles for its two receptors in brown and white adipose tissues.

Chloride-sensitive signaling turns the potassium switch on.

The potassium switch refers to plasma potassium regulation of the sodium-chloride cotransporter (NCC), which controls distal sodium delivery and therefore potassium secretion. Low extracellular potassium activates NCC by relieving chloride inhibition of With-No-Lysine 4 (WNK4). A new mouse model carrying a chloride-insensitive WNK4 mutant still shows NCC activation on low potassium diet. These effects are mediated by WNK4 activation and kelch-like 3 (KLHL3) inhibition and reveal additional chloride-sensitive pathways for NCC activation.