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Mutations ofThe trimeric intracellular cation (TRIC) channel TRIC-B is an ER-resident potassium channel that mediates potassium-calcium counterion exchange for inositol trisphosphate-mediated calcium ion release. Using TRIC-B as a tool, we examined the function of ER-localized polycystin-2 and its role in ADPKD pathogenesis in cultured cells, zebrafish, and mouse models.Agonist-induced ER calcium ion release was defective in cells lacking polycystin-2 and reversed by exogenous expression of TRIC-B.Polycystin-2 in the ER appears to be critical for anticystogenesis and likely functions as a potassium ion channel to facilitate potassium-calcium counterion exchange for inositol trisphosphate-mediated calcium release. The results advance the understanding of ADPKD pathogenesis and provides proof of principle for pharmacotherapy by TRIC-B activators.

Large-conductance K(+) (BK) channels expressed in intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) mediate flow-induced K(+) secretion. In the ASDN of mice and rabbits, IC BK channel expression and activity increase with a high-K(+) diet. In cell culture, the long isoform of with-no-lysine kinase 1 (L-WNK1) increases BK channel expression and activity. Apical L-WNK1 expression is selectively enhanced in ICs in the ASDN of rabbits on a high-K(+) diet, suggesting that L-WNK1 contributes to BK channel regulation by dietary K(+). We examined the role of IC L-WNK1 expression in enhancing BK channel activity in response to a high-K(+) diet. Mice with IC-selective deletion of L-WNK1 (IC-L-WNK1-KO) and littermate control mice were placed on a high-K(+) (5% K(+), as KCl) diet for 10 or more days. IC-L-WNK1-KO mice exhibited reduced IC apical + subapical α-subunit expression and BK channel-dependent whole cell currents compared with controls. Six-hour urinary K(+) excretion in response a saline load was similar in IC-L-WNK1-KO mice and controls. The observations that IC-L-WNK1-KO mice on a high-K(+) diet have higher blood K(+) concentration and reduced IC BK channel activity are consistent with impaired urinary K(+) secretion, demonstrating that IC L-WNK1 has a role in the renal adaptation to a high-K(+) diet. NEW & NOTEWORTHY When mice are placed on a high-K(+) diet, genetic disruption of the long form of with no lysine kinase 1 (L-WNK1) in intercalated cells reduced relative apical + subapical localization of the large-conductance K(+) channel, blunted large-conductance K(+) channel currents in intercalated cells, and increased blood K(+) concentration. These data confirm an in vivo role of L-WNK1 in intercalated cells in adaptation to a high-K(+) diet.

The NLRP3 inflammasome mediates the production of proinflammatory cytokines and initiates inflammatory cell death. Although NLRP3 is essential for innate immunity, aberrant NLRP3 inflammasome activation contributes to a wide variety of inflammatory diseases. Understanding the pathways that control NLRP3 activation will help develop strategies to treat these diseases. Here we identify WNK1 as a negative regulator of the NLRP3 inflammasome. Macrophages deficient in WNK1 protein or kinase activity have increased NLRP3 activation and pyroptosis compared with control macrophages. Mice with conditional knockout of WNK1 in macrophages have increased IL-1β production in response to NLRP3 stimulation compared with control mice. Mechanistically, WNK1 tempers NLRP3 activation by balancing intracellular Cl– and K+ concentrations during NLRP3 activation. Collectively, this work shows that the WNK1 pathway has a critical function in suppressing NLRP3 activation and suggests that pharmacological inhibition of this pathway to treat hypertension might have negative clinical implications., The serine/threonine kinase WNK1 is an inhibitor of chloride efflux. Here the authors show that this inhibition is a means of negatively regulating the activation of the NLRP3 inflammasome in macrophages, leading to reduced inflammatory responses.

The appropriate development of myeloid progenitors into macrophages, the body’s professional phagocyte, is essential for organismal development, especially in mammals1. This dependence is exemplified by the observation that loss-of-function mutation in colony stimulating factor 1 receptor (CSF1R) results in multiple tissue abnormalities including osteopetrosis2. Despite this importance, little is known about the molecular and cell biological regulation of macrophage development. Here, we report the surprising finding that the chloride-sensing kinase With-no-lysine 1 (WNK1) is required for embryonic development of tissue-resident macrophages (TRMs). Myeloid-specific deletion of Wnk1 caused a dramatic loss of TRMs and subsequently disrupted organ development, induced systemic neutrophilia, and resulted in mortality between 3 and 4 weeks of age. Specifically, we observed that WNK1 absence stalled macrophage differentiation at the myeloid multipotent progenitor (MPP) stage, instead skewing MPP differentiation towards granulopoiesis. Mechanistically, the cognate CSF1R cytokine, macrophage-colony stimulating factor (M-CSF), triggers macropinocytosis in myeloid progenitors, which in turn induces phosphorylation of WNK1. Importantly, macropinocytosis by myeloid progenitors increases cytosolic chloride, which is directly sensed by WNK1. Perturbing chloride flux during macropinocytosis, inhibiting WNK1 chloride-sensing, and blocking macropinocytosis each skew progenitor differentiation from macrophage lineage to granulocyte lineage. Thus, we have uncovered a novel mechanism that links a cell biological process to a molecular circuit whereby WNK1 chloride-sensing and chloride flux act downstream of M-CSF-induced macropinocytosis by multipotent progenitors to ensure macrophage lineage fidelity.

Significance Rapid regulatory volume increase (RVI) is important for cell survival under hypertonic conditions. RVI is driven by Cl− uptake via the Na–K–Cl cotransporter (NKCC), which is activated by WNK kinases following a reduction in intracellular [Cl−]. However, how intracellular [Cl−] is regulated to modulate the WNK–NKCC axis and engage a protective RVI remains unknown. Our work reveals that LRRC8A-containing chloride channel is a key protective factor against hypertonic shocks. Considering that LRRC8A (SWELL1) is typically activated by low ionic strength under hypotonic stress, our results posed another interesting question: what activates this chloride channel under hypertonic stress? We demonstrated that, upon hyperosmotic activation, the p38-MSK1 pathway gates LRRC8A-containing chloride channel to facilitate activation of WNK–NKCC and an effective RVI., Regulation of cell volume is essential for tissue homeostasis and cell viability. In response to hypertonic stress, cells need rapid electrolyte influx to compensate water loss and to prevent cell death in a process known as regulatory volume increase (RVI). However, the molecular component able to trigger such a process was unknown to date. Using a genome-wide CRISPR/Cas9 screen, we identified LRRC8A, which encodes a chloride channel subunit, as the gene most associated with cell survival under hypertonic conditions. Hypertonicity activates the p38 stress-activated protein kinase pathway and its downstream MSK1 kinase, which phosphorylates and activates LRRC8A. LRRC8A-mediated Cl− efflux facilitates activation of the with-no-lysine (WNK) kinase pathway, which in turn, promotes electrolyte influx via Na+/K+/2Cl− cotransporter (NKCC) and RVI under hypertonic stress. LRRC8A-S217A mutation impairs channel activation by MSK1, resulting in reduced RVI and cell survival. In summary, LRRC8A is key to bidirectional osmotic stress responses and cell survival under hypertonic conditions.

Soluble klotho (sKlotho), the shed ectodomain of α-klotho, protects the heart by down-regulating transient receptor potential canonical isoform 6 (TRPC6)-mediated calcium signaling. Binding to α2-3-sialyllactose moiety of gangliosides in lipid rafts and inhibition of raft-dependent signaling underlies the mechanism. A recent 3-A X-ray structure of sKlotho in complex with fibroblast growth factor receptor (FGFR) and fibroblast growth factor 23 (FGF23) indicates that its β6α6 loop might block access to the proposed binding site for α2-3-sialyllactose. It was concluded that sKlotho only functions in complex with FGFR and FGF23 and that sKlotho's pleiotropic effects all depend on FGF23. Here, we report that sKlotho can inhibit TRPC6 channels expressed in cells lacking endogenous FGFRs. Structural modeling and molecular docking show that a repositioned β6α6 loop allows sKlotho to bind α2-3-sialyllactose. Molecular dynamic simulations further show the α2-3-sialyllactose-bound sKlotho complex to be stable. Domains mimicking sKlotho's sialic acid-recognizing activity inhibit TRPC6. The results strongly support the hypothesis that sKlotho can exert effects independent of FGF23 and FGFR.-Wright, J. D., An, S.-W., Xie, J., Lim, C., Huang, C.-L. Soluble klotho regulates TRPC6 calcium signaling via lipid rafts, independent of the FGFR-FGF23 pathway.

Loss of function of the lipid kinase diacylglycerol kinase ε (DGKε), encoded by the gene DGKE, causes a form of atypical hemolytic uremic syndrome that is not related to abnormalities of the alternative pathway of the complement, by mechanisms that are not understood. By generating a potentially novel endothelial specific Dgke-knockout mouse, we demonstrate that loss of Dgke in the endothelium results in impaired signaling downstream of VEGFR2 due to cellular shortage of phosphatidylinositol 4,5-biphosphate. Mechanistically, we found that, in the absence of DGKε in the endothelium, Akt fails to be activated upon VEGFR2 stimulation, resulting in defective induction of the enzyme cyclooxygenase 2 and production of prostaglandin E2 (PGE2). Treating the endothelial specific Dgke-knockout mice with a stable PGE2 analog was sufficient to reverse the clinical manifestations of thrombotic microangiopathy and proteinuria, possibly by suppressing the expression of matrix metalloproteinase 2 through PGE2-dependent upregulation of the chemokine receptor CXCR4. Our study reveals a complex array of autocrine signaling events downstream of VEGFR2 that are mediated by PGE2, that control endothelial activation and thrombogenic state, and that result in abnormalities of the glomerular filtration barrier.

The distal convoluted tubule (DCT) NaCl cotransporter NCC is activated by phosphorylation, a process that is potassium-regulated and dependent on With-No-Lysine (WNK) kinases. KS-WNK1, a kidney-specific WNK1 isoform lacking the kinase domain, controls WNK signaling pathway localization in the DCT. Its role in NCC regulation, however, is unresolved: while early studies proposed that KS-WNK1 functions as an NCC inhibitor, recent work suggests that it activates NCC. To evaluate the role of KS-WNK1 on potassium-dependent NCC regulation, we studied KS-WNK1-KO mice across a wide range of plasma K+ (2.0-9.0 mmol/L), induced by dietary maneuvers and diuretic challenges. Potassium-deprived KS-WNK1-KO mice exhibited low WNK-dependent NCC phosphorylation compared to littermates, indicating that KS-WNK1 activates NCC during K+ deficiency. In contrast, relative NCC phosphorylation was high in potassium-loaded KS-WNK1-KO mice, consistent with KS-WNK1-mediated NCC inhibition during hyperkalemia. An integrated analysis revealed that KS-WNK1 expands the dynamic range of NCC responsiveness to potassium, steepening the linear inverse relationship between NCC phosphorylation and plasma [K+]. The effect of KS-WNK1 deletion was strongest in potassium-restricted females, as they developed exaggerated hypokalemia and thiazide insensitivity due to low NCC activity. Taken together, these findings indicate that KS-WNK1 is a potassium-responsive signaling amplifier that converts small changes in [K+] into large effects on NCC phosphorylation. This effect predominates in females during potassium deficiency, when high NCC activity is required to maintain salt reabsorption without exacerbating K+ losses. These observations define the role of KS-WNK1 in NCC regulation, and identify a novel mechanism that contributes to sexual dimorphism in the mammalian nephron.

Membranous α-Klotho (mKlotho) interacts with fibroblast growth factor receptor (FGFR) to form coreceptors for fibroblast growth factor 23 (FGF23). The ectodomain of mKlotho is shed (soluble Klotho; sKlotho) and functions as a circulating endocrine or local autocrine/paracrine factor. sKlotho exerts pleiotropic actions. Identification of membrane receptor(s) is essential for designating sKlotho to have “hormonal” function. Lipid rafts are membrane microdomain important in many cellular processes. Here, we review recent studies reporting that sialogangliosides of membrane lipid rafts serve as receptors for sKlotho. The X-ray crystal structure of sKlotho in ternary complex with FGFR and FGF23 has been solved. However, sKlotho binding to lipid rafts does not require FGF23 or FGFR. Computational studies including modeling, docking, and molecular dynamic simulations reveal that slight movement in the KL1 domain of sKlotho allows stable binding of the sialyllactose, headgroup of sialogangliosides. Thus, sKlotho satisfies the criteria to be an FGF23-independent hormone. Binding to lipid rafts may underlie the pleiotropic actions of sKlotho that seem to permeate all cells.

WNK1 (with no lysine [K] kinase 1) is an atypical kinase protein ubiquitously expressed in humans and mice. A mutation in its encoding gene causes hypertension in humans, which is associated with abnormal ion homeostasis. WNK1 is critical for in vitro decidualization in human endometrial stromal cells, thereby demonstrating its importance in female reproduction. Using a mouse model, WNK1 was ablated in the female reproductive tract to define its in vivo role in uterine biology. Loss of WNK1 altered uterine morphology, causing endometrial epithelial hyperplasia, adenomyotic features, and a delay in embryo implantation, ultimately resulting in compromised fertility. Combining transcriptomic, proteomic, and interactomic analyses revealed a potentially novel regulatory pathway whereby WNK1 represses AKT phosphorylation through protein phosphatase 2A (PP2A) in endometrial cells from both humans and mice. We show that WNK1 interacted with PPP2R1A, the alpha isoform of the PP2A scaffold subunit. This maintained the levels of PP2A subunits and stabilized its activity, which then dephosphorylated AKT. Therefore, loss of WNK1 reduced PP2A activity, causing AKT hypersignaling. Using FOXO1 as a readout of AKT activity, we demonstrate that there was escalated FOXO1 phosphorylation and nuclear exclusion, leading to a disruption in the expression of genes that are crucial for embryo implantation., The atypical kinase WNK1 is critical for maintaining uterine homeostasis and mediating proper implantation through the PP2A/AKT signaling pathway.

α-Klotho is a type 1 transmembrane protein that exhibits aging suppression function. The large amino-terminal extracellular domain of α-klotho is shed as soluble klotho (sKlotho) and functions as a circulating cardioprotective hormone. Diacylglycerol (DAG)-activated calcium-permeable TRPC6 channel plays a critical role in stress-induced cardiac remodeling. DAG activates TRPC6 by acting directly on the channel to increase its activity and by stimulation of channel exocytosis. sKlotho protects the heart by inhibiting DAG stimulation of TRPC6 exocytosis. How DAG stimulates TRPC6 exocytosis and thereby inhibition by sKlotho are unknown. Using a compound that directly activates TRPC6 without affecting channel exocytosis, we validate that sKlotho selectively blocks DAG stimulation of channel exocytosis. We further show that DAG stimulates exocytosis of TRPC6-containing vesicles pre-docked to the plasma membrane. Mnuc13 family proteins play important roles in the proper assembly of SNARE proteins and priming the vesicle competent for fusion. We show that DAG stimulates TRPC6 exocytosis by targeting to the C1 domain of Munc13-2. The results provide fresh insights into the molecular mechanism by which DAG regulates vesicle fusion and how sKlotho protects the heart against injury.

Central pacemaker neurons regulate circadian rhythms and undergo diurnal variation in electrical activity in mammals and flies. In mammals, circadian variation in the intracellular chloride concentration of pacemaker neurons has been proposed to influence the response to GABAergic neurotransmission through GABAA receptor chloride channels. However, results have been contradictory, and a recent study demonstrated circadian variation in pacemaker neuron chloride without an effect on GABA response. Therefore, whether and how intracellular chloride regulates circadian rhythms remains controversial. Here, we demonstrate a signaling role for intracellular chloride in the Drosophila ventral lateral (LNv) pacemaker neurons. In control flies, intracellular chloride increases in LNv neurons over the course of the morning. Chloride transport through the sodium-potassium-2-chloride (NKCC) and potassium-chloride (KCC) cotransporters is a major determinant of intracellular chloride concentrations. Drosophila melanogaster with loss-of-function mutations in the NKCC encoded by Ncc69 have abnormally low intracellular chloride six hours after lights on, and a lengthened circadian period. Loss of kcc, which is expected to increase intracellular chloride, suppresses the long-period phenotype of Ncc69 mutant flies. Activation of a chloride-inhibited kinase cascade, consisting of the WNK (With No Lysine (K)) kinase and its downstream substrate, Fray, is necessary and sufficient to prolong period length. Fray activation of an inwardly rectifying potassium channel, Irk1, is also required for the long-period phenotype. These results indicate that the NKCC-dependent rise in intracellular chloride in Drosophila LNv pacemaker neurons restrains WNK-Fray signaling and overactivation of an inwardly rectifying potassium channel to maintain normal circadian period length.

The Na+-Cl− cotransporter (NCC) in distal convoluted tubule (DCT) plays important roles in renal NaCl reabsorption. The current hypothesis for the mechanism of regulation of NCC focuses on WNK4 and intracellular Cl− concentration ([Cl−]i). WNK kinases bind Cl−, and Cl− binding decreases the catalytic activity. It is believed that hypokalemia under low K+ intake decreases [Cl−]i to activate WNK4, which thereby phosphorylates and stimulates NCC through activation of SPAK. However, increased NCC activity and apical NaCl entry would mitigate the fall in [Cl−]i. Whether [Cl−]i in DCT under low-K+ diet is sufficiently low to activate WNK4 is unknown. Furthermore, increased luminal NaCl delivery also stimulates NCC and causes upregulation of the transporter. Unlike low K+ intake, increased luminal NaCl delivery would tend to increase [Cl−]i. Thus we investigated the role of WNK4 and [Cl−]i in regulating NCC. We generated Wnk4-knockout mice and examined regulation of NCC by low K+ intake and by increased luminal NaCl delivery in knockout (KO) and wild-type mice. Wnk4-KO mice have marked reduction in the abundance, phosphorylation, and functional activity of NCC vs. wild type. Low K+ intake increases NCC phosphorylation and functional activity in wild-type mice, but not in Wnk4-KO mice. Increased luminal NaCl delivery similarly upregulates NCC, which, contrary to low K+ intake, is not abolished in Wnk4-KO mice. The results reveal that modulation of WNK4 activity by [Cl−]i is not the sole mechanism for regulating NCC. Increased luminal NaCl delivery upregulates NCC via yet unknown mechanism(s) that may override inhibition of WNK4 by high [Cl−]i.

The with-no-lysine (K) (WNK) signaling pathway to STE20/SPS1-related proline- and alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1) kinase is an important mediator of cell volume and ion transport. SPAK and OSR1 associate with upstream kinases WNK 1-4, substrates, and other proteins through their C-terminal domains which interact with linear R-F-x-V/I sequence motifs. In this study we find that SPAK and OSR1 also interact with similar affinity with a motif variant, R-x-F-x-V/I. Eight of 16 human inward rectifier K+ channels have an R-x-F-x-V motif. We demonstrate that two of these channels, Kir2.1 and Kir2.3, are activated by OSR1, while Kir4.1, which does not contain the motif, is not sensitive to changes in OSR1 or WNK activity. Mutation of the motif prevents activation of Kir2.3 by OSR1. Both siRNA knockdown of OSR1 and chemical inhibition of WNK activity disrupt NaCl-induced plasma membrane localization of Kir2.3. Our results suggest a mechanism by which WNK-OSR1 enhance Kir2.1 and Kir2.3 channel activity by increasing their plasma membrane localization. Regulation of members of the inward rectifier K+ channel family adds functional and mechanistic insight into the physiological impact of the WNK pathway.

Diuretics are commonly prescribed for treatment in patients with hypertension, edema or heart failure. Studies on hypertensive and salt-losing disorders and on urea transporters have contributed to better understanding of mechanisms of renal salt and water reabsorption and their regulation. Proteins involved in the regulatory pathways are emerging targets for diuretic and aquaretic therapy. Integrative high-throughput screening, protein structure analysis, and chemical modification have identified promising agents for pre-clinical testing in animals. These include WNK-SPAK inhibitors, ClC-K channel antagonists, ROMK channel antagonists, and pendrin and urea transporter inhibitors. We discuss the potential advantages and side effects of these potential diuretics.

Mutations in CLCNKB gene encoding human voltage-gated chloride ClC-Kb (hClC-Kb) channel cause classic Bartter's syndrome (BS). In contrast to antenatal BS, classic BS manifests highly variable phenotypes. The functional severity of mutant channel has been proposed to explain this phenomenon. Due to difficulties in the expression of hClC-Kb in heterologous expression systems, the functional consequences of mutant channels haven't been thoroughly examined, and the genotype-phenotype association hasn't been established. In this study, we found that hClC-Kb, when expressed in human embryonic kidney (HEK) cells, was unstable due to degradation by proteasome. In-frame fusion of green fluorescent protein (GFP) to the C-terminus of the channel may ameliorate proteasome degradation. Co-expression of barttin increased protein abundance and membrane trafficking of hClC-Kb and markedly increased functional chloride current. We then functionally characterized eighteen missense mutations identified in our classic BS cohort and others using HEK cells expressing hClC-Kb-GFP. Most CLCNKB mutations resulted in marked reduction in protein abundance and chloride current, especially those residing at barttin-binding sites, dimer interface, and selectivity filter. We enrolled classic BS patients carrying homozygous missense mutations with well-described functional consequences and clinical presentations for genotype-phenotype analysis. We found significant correlations of mutant chloride current with the age at diagnosis, plasma chloride concentration, and urine calcium excretion rate. In conclusion, hClC-Kb expression in HEK cells is susceptible to proteasome degradation, and fusion of GFP to the C-terminus of hClC-Kb improves the protein expression. The functional severity of CLCNKB mutation is an important determinant of the phenotype in classic BS. This article is protected by copyright. All rights reserved

With-no-lysine (WNK) kinases regulate renal sodium-chloride cotransporter (NCC) to maintain body sodium and potassium homeostasis. Gain-of-function mutations of WNK1 and WNK4 in humans lead to a Mendelian hypertensive and hyperkalemic disease pseudohypoaldosteronism type II (PHAII). X-ray crystal structure and in vitro studies reveal chloride ion (Cl(−)) binds to a hydrophobic pocket within the kinase domain of WNKs to inhibit its activity. The mechanism is thought to be important for physiological regulation of NCC by extracellular potassium. To test the hypothesis that WNK4 senses the intracellular concentration of Cl(−) physiologically, we generated knockin mice carrying Cl(−)-insensitive mutant WNK4. These mice displayed hypertension, hyperkalemia, hyperactive NCC, and other features fully recapitulating human and mouse models of PHAII caused by gain-of-function WNK4. Lowering plasma potassium levels by dietary potassium restriction increased NCC activity in wild-type, but not in knockin, mice. NCC activity in knockin mice can be further enhanced by the administration of norepinephrine, a known activator of NCC. Raising plasma potassium by oral gavage of potassium inactivated NCC within 1 hour in wild-type mice, but had no effect in knockin mice. The results provide compelling support for the notion that WNK4 is a bona fide physiological intracellular Cl(−) sensor and that Cl(−) regulation of WNK4 underlies the mechanism of regulation of NCC by extracellular potassium.

Adhesion and migration of T cells are controlled by chemokines and by adhesion molecules, especially integrins, and have critical roles in the normal physiological function of T lymphocytes. Using an RNA-mediated interference screen, we identified the WNK1 kinase as a regulator of both integrin-mediated adhesion and T cell migration. We found that WNK1 is a negative regulator of integrin-mediated adhesion, whereas it acts as a positive regulator of migration via the kinases OXSR1 and STK39 and the ion co-transporter SLC12A2. WNK1-deficient T cells home less efficiently to lymphoid organs and migrate more slowly through them. Our results reveal that a pathway previously known only to regulate salt homeostasis in the kidney functions to balance T cell adhesion and migration.

With-no-lysine (K)-1 (WNK1) is the founding member of family of four protein kinases with atypical placement of catalytic lysine that play important roles in regulating epithelial ion transport. Gain-of-function mutations of WNK1 and WNK4 cause a mendelian hypertension and hyperkalemic disease. WNK1 is ubiquitously expressed and essential for embryonic angiogenesis in mice. Increasing evidence indicates the role of WNK kinases in tumorigenesis at least partly by stimulating tumor cell proliferation. Here, we show that human hepatoma cells xenotransplanted into zebrafish produced high levels of vascular endothelial growth factor (VEGF) and WNK1, and induced expression of zebrafish wnk1. Knockdown of wnk1 in zebrafish decreased tumor-induced ectopic vessel formation and inhibited tumor proliferation. Inhibition of WNK1 or its downstream kinases OSR1 (oxidative stress responsive kinase 1)/SPAK (Ste20-related proline alanine rich kinase) using chemical inhibitors decreased ectopic vessel formation as well as proliferation of xenotransplanted hepatoma cells. The effect of WNK and OSR1 inhibitors is greater than that achieved by inhibitor of VEGF signaling cascade. These inhibitors also effectively inhibited tumorigenesis in two separate transgenic zebrafish models of intestinal and hepatocellular carcinomas. Endothelial-specific overexpression of wnk1 enhanced tumorigenesis in transgenic carcinogenic fish, supporting endothelial cell-autonomous effect of WNK1 in tumor promotion. Thus, WNK1 can promote tumorigenesis by multiple effects that include stimulating tumor angiogenesis. Inhibition of WNK1 may be a potent anti-cancer therapy.

Hyperchloremic metabolic acidosis along with hypertension and hyperkalemia are features of pseudohypoaldosteronism type II (PHAII). Increased activity of sodium chloride cotransporter (NCC) is believed to be an important mechanism of these phenotypic features (1). Gain-of-function mutations of WNK4 in PHAII activate NCC in the distal convoluted tubule, which leads to enhanced sodium and chloride reabsorption causing hypertension and diminished sodium delivery to the downstream cortical collecting duct, resulting in hyperkalemia. Our recent report (2) that chloride-insensitive WNK4 knockin mouse exhibits increased WNK4 kinase activity and fully recapitulates PHAII phenotype supports the notion. Still, as mentioned by Farfel et al. (3), there remain debates on the … [↵][1]1To whom correspondence may be addressed. Email: laurence1234kimo{at}yahoo.com.tw. [1]: #xref-corresp-1-1

SPAK [STE20 (sterile 20)/SPS1-related proline/alanine-rich kinase] kinase consists of a full-length (FL-) and an alternatively spliced kidney-specific (KS-) isoform. SPAK regulates the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). The relative abundance and role of FL- vs. KS-SPAK in regulating Na+-K+-2Cl− cotransporter (NKCC2) in thick ascending limb (TAL) are not completely understood. Here, we report that FL-SPAK mRNA was the most abundant in medullary TAL (mTAL), followed by cortical TAL (cTAL) and DCT. KS-SPAK mRNA abundance was relatively lower than FL-SPAK. The ratios of FL-SPAK to KS-SPAK in mTAL, cTAL, and DCT were 12.3, 12.5, and 10.2, respectively. To examine the role of SPAK in the regulation of sodium transport in TAL, we used in vitro microperfusion of mTAL and cTAL isolated from wild-type (WT) and SPAK knockout mice (SPAK-KO) that lack both FL- and KS-SPAK. The rates of sodium absorption in cTAL and mTAL of SPAK-KO mice were 34.5 and 12.5% of WT tubules, respectively. The mRNA levels of related OSR1 kinase and SPAK protease Dnpep in SPAK-KO tubules were not significantly different from WT tubules. We next examined the role of SPAK in the regulation of sodium reabsorption by vasopressin in TAL. Vasopressin increased sodium reabsorption by ∼80% in both mTAL and cTAL from WT mice. While baseline sodium reabsorption was lower in SPAK-KO tubules, vasopressin increased sodium reabsorption over twofold. In conclusion, the combined net effect of SPAK isoforms on sodium reabsorption in TAL is stimulatory. SPAK is not essential for vasopressin stimulation of sodium reabsorption in TAL.

With-no-lysine (WNK) kinases coordinate volume and potassium homeostasis by regulating renal tubular electrolyte transport. In the distal convoluted tubule (DCT), potassium imbalance causes WNK signaling complexes to concentrate into large discrete foci, which we call “WNK bodies.” Although these structures have been reported previously, the mechanisms that drive their assembly remain obscure. Here, we show that kidney-specific WNK1 (KS-WNK1), a truncated kinase-defective WNK1 isoform that is highly expressed in the DCT, is critical for WNK body formation. While morphologically distinct WNK bodies were evident in the distal tubules of mice subjected to dietary potassium loading and restriction, KS-WNK1 knockout mice were deficient in these structures under identical conditions. Combining in vivo observations in kidney with reconstitution studies in cell culture, we found that WNK bodies are dynamic membraneless foci that are distinct from conventional organelles, colocalize with the ribosomal protein L22, and cluster the WNK signaling pathway. The formation of WNK bodies requires an evolutionarily conserved cysteine-rich hydrophobic motif harbored within a unique N-terminal exon of KS-WNK1. We propose that WNK bodies are not pathological aggregates, but rather are KS-WNK1–dependent microdomains of the DCT cytosol that modulate WNK signaling during physiological shifts in potassium balance.

Soluble Klotho (sKlotho) is the shed ectodomain of antiaging membrane Klotho that contains 2 extracellular domains KL1 and KL2, each of which shares sequence homology to glycosyl hydrolases. sKlotho elicits pleiotropic cellular responses with a poorly understood mechanism of action. Notably, in injury settings, sKlotho confers cardiac and renal protection by down-regulating calcium-permeable transient receptor potential canonical type isoform 6 (TRPC6) channels in cardiomyocytes and glomerular podocytes. Inhibition of PI3K-dependent exocytosis of TRPC6 is thought to be the underlying mechanism, and recent studies showed that sKlotho interacts with α2-3-sialyllactose-containing gangliosides enriched in lipid rafts to inhibit raft-dependent PI3K signaling. However, the structural basis for binding and recognition of α2-3-sialyllactose by sKlotho is unknown. Using homology modeling followed by docking, we identified key protein residues in the KL1 domain that are likely involved in binding sialyllactose. Functional experiments based on the ability of Klotho to down-regulate TRPC6 channel activity confirm the importance of these residues. Furthermore, KL1 domain binds α2-3-sialyllactose, down-regulates TRPC6 channels, and exerts protection against stress-induced cardiac hypertrophy in mice. Our results support the notion that sialogangliosides and lipid rafts are membrane receptors for sKlotho and that the KL1 domain is sufficient for the tested biologic activities. These findings can help guide the design of a simpler Klotho mimetic.-Wright, J. D., An, S.-W., Xie, J., Yoon, J., Nischan, N., Kohler, J. J., Oliver, N., Lim, C., Huang, C.-L. Modeled structural basis for the recognition of α2-3-sialyllactose by soluble Klotho.

Fil: Dalton, George. University of Texas Southwestern Medical Center. Department of Medicine; Estados Unidos Fil: An, Sung-Wan. University of Texas Southwestern Medical Center. Department of Medicine; Estados Unidos Fil: Al-Juboori, Saif I. University of Colorado Denver. Department of Electrical Engineering; Estados Unidos Fil: Nischan, Nicole. University of Texas Southwestern Medical Center. Department of Biochemistry; Estados Unidos Fil: Yoon, Joonho. University of Texas Southwestern Medical Center. Department of Medicine; Estados Unidos Fil: Dobrinskikh, Evgenia. University of Colorado Denver. Department of Medicine; Estados Unidos Fil: Hilgemann, Donald W. University of Texas Southwestern Medical Center. Department of Physiology; Estados Unidos Fil: Xie, Jian. University of Texas Southwestern Medical Center. Department of Medicine; Estados Unidos Fil: Luby-Phelps, Kate. University of Texas Southwestern Medical Center. Department of Cell Biology; Estados Unidos Fil: Luby-Phelps, Kate. University of Texas Southwestern Medical Center. Live Cell Imaging Core Facility; Estados Unidos Fil: Kohler, Jennifer J. University of Texas Southwestern Medical Center. Department of Biochemistry; Estados Unidos Fil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina Fil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences. Neurobiology Laboratory; Estados Unidos Fil: Huang, Chou-Long. University of Texas Southwestern Medical Center. Department of Medicine; Estados Unidos Abstract: Soluble klotho, the shed ectodomain of the antiaging membrane protein α-klotho, is a pleiotropic endocrine/paracrine factor with no known receptors and poorly understood mechanism of action. Soluble klotho down-regulates growth factor-driven PI3K signaling, contributing to extension of lifespan, cardioprotection, and tumor inhibition. Here we show that soluble klotho binds membrane lipid rafts. Klotho binding to rafts alters lipid organization, decreases membrane's propensity to form large ordered domains for endocytosis, and down-regulates raft-dependent PI3K/Akt signaling. We identify α2-3-sialyllactose present in the glycan of monosialogangliosides as targets of soluble klotho. α2-3-Sialyllactose is a common motif of glycans. To explain why klotho preferentially targets lipid rafts we show that clustering of gangliosides in lipid rafts is important. In vivo, raft-dependent PI3K signaling is up-regulated in klotho-deficient mouse hearts vs. wild-type hearts. Our results identify ganglioside-enriched lipid rafts to be receptors that mediate soluble klotho regulation of PI3K signaling. Targeting sialic acids may be a general mechanism for pleiotropic actions of soluble klotho.

The hormone erythropoietin (EPO), which is synthesized in the kidney or liver of adult mammals, controls erythrocyte production and is regulated by the stress-responsive transcription factor hypoxia-inducible factor-2 (HIF-2). We previously reported that the lysine acetyltransferase CREB-binding protein (CBP) is required for HIF-2α acetylation and efficient HIF-2-dependent EPO induction during hypoxia. We now show that these processes require acetate-dependent acetyl CoA synthetase 2 (ACSS2). In human Hep3B hepatoma cells and in EPO-generating organs of hypoxic or acutely anemic mice, acetate levels rise and ACSS2 is required for HIF-2α acetylation, CBP-HIF-2α complex formation, CBP-HIF-2α recruitment to the EPO enhancer and efficient induction of EPO gene expression. In acutely anemic mice, acetate supplementation augments stress erythropoiesis in an ACSS2-dependent manner. Moreover, in acquired and inherited chronic anemia mouse models, acetate supplementation increases EPO expression and the resting hematocrit. Thus, a mammalian stress-responsive acetate switch controls HIF-2 signaling and EPO induction during pathophysiological states marked by tissue hypoxia.

We present a case of an 82-year-old woman with elevated parathyroid hormone (PTH) levels, hypocalciuria, hypercalcemia, and stage 3 chronic kidney disease. Hypocalciuria initially was attributed to chronic kidney disease, and hypercalcemia was attributed to primary hyperparathyroidism. Subsequent laboratory studies showed autoantibodies in the patient's serum directed against the calcium-sensing receptor (CaSR). Functional testing in a CaSR-transfected human embryonic kidney-293 cell line showed that the patient's antibodies inhibited CaSR-mediated intracellular signaling that ordinarily would have been stimulated by extracellular calcium ions. Her serum calcium and PTH levels were normalized by treatment with the calcimimetic cinacalcet. We advise consideration of the presence of inhibitory autoantibodies directed at the CaSR in patients with hypercalcemic hyperparathyroidism and unexplained hypocalciuria or with confounding conditions affecting interpretation of urinary calcium measurement. A calcimimetic is an effective treatment for the hypercalcemia and elevated PTH levels in acquired hypocalciuric hypercalcemia caused by inhibitory anti-CaSR autoantibodies.

TRPV5 and TRPV6 channels are expressed in distal renal tubules and play important roles in the transcellular Ca 2 + reabsorption in kidney. They are regulated by multiple intracellular factors including protein kinases A and C, membrane phospholipid PIP 2 , protons, and divalent ions Ca 2 + and Mg 2 + . Here, we report that fluid flow that generates shear force within the physiological range of distal tubular fluid flow activated TRPV5 and TRPV6 channels expressed in HEK cells. Flow-induced activation of channel activity was reversible and did not desensitize over 2 min. Fluid flow stimulated TRPV5 and 6-mediated Ca 2 + entry and increased intracellular Ca 2 + concentration. N-glycosylation-deficient TRPV5 channel was relatively insensitive to fluid flow. In cells coexpressing TRPV5 (or TRPV6) and Slo1 -encoded maxi-K channels, fluid flow induced membrane hyperpolarization, which could be prevented by the maxi-K blocker iberiotoxin or TRPV5 and 6 blocker La 3 + . In contrast, fluid flow did not cause membrane hyperpolarization in cells coexpressing ROMK1 and TRPV5 or 6 channel. These results reveal a new mechanism for the regulation of TRPV5 and TRPV6 channels. Activation of TRPV5 and TRPV6 by fluid flow may play a role in the regulation of flow-stimulated K + secretion via maxi-K channels in distal renal tubules and in the mechanism of pathogenesis of thiazide-induced hypocalciuria.

Renal microangiopathies and membranoproliferative GN (MPGN) can manifest similar clinical presentations and histology, suggesting the possibility of a common underlying mechanism in some cases. Here, we performed homozygosity mapping and whole exome sequencing in a Turkish consanguineous family and identified DGKE gene variants as the cause of a membranoproliferative-like glomerular microangiopathy. Furthermore, we identified two additional DGKE variants in a cohort of 142 unrelated patients diagnosed with membranoproliferative GN. This gene encodes the diacylglycerol kinase DGK epsilon, which is an intracellular lipid kinase that phosphorylates diacylglycerol to phosphatidic acid. Immunofluorescence confocal microscopy demonstrated that mouse and rat Dgk epsilon colocalizes with the podocyte marker WT1 but not with the endothelial marker CD31. Patch-clamp experiments in human embryonic kidney (HEK293) cells showed that DGK epsilon variants affect the intracellular concentration of diacylglycerol. Taken together, these results not only identify a genetic cause of a glomerular microangiopathy but also suggest that the phosphatidylinositol cycle, which requires DGKE, is critical to the normal function of podocytes. J Am Soc Nephrol 24: 377-384, 2013. doi: 10.1681/ASN.2012090903

Kidney-specific with-no-lysine kinase 1 (KS-WNK1) is a kinase-deficient variant of WNK1 that is expressed exclusively in the kidney. It is abundantly expressed in the distal convoluted tubule (DCT) and to a lesser extent in the cortical thick ascending limb (cTAL), connecting tubule, and cortical collecting duct (CCD). KS-WNK1 inhibits Na+-K+-2Cl−- and sodium chloride cotransporter-mediated Na+reabsorption in cTAL and DCT, respectively. Here, we investigated the role of KS-WNK1 in regulating Na+and K+transport in CCD using in vitro microperfusion of tubules isolated from KS-WNK1 knockout mice and control wild-type littermates. Because baseline K+secretion and Na+reabsorption were negligible in mouse CCD, we studied tubules isolated from mice fed a high-K+diet for 2 wk. Compared with that in wild-type tubules, K+secretion was reduced in KS-WNK1 knockout CCD perfused at a low luminal fluid rate of ∼1.5 nl/min. Na+reabsorption and the lumen-negative transepithelial potential difference were also lower in the KS-WNK1 knockout CCD compared with control CCD. Increasing the perfusion rate to ∼5.5 nl/min stimulated K+secretion in the wild-type as well as knockout CCD. The magnitudes of flow-stimulated increase in K+secretion were similar in wild-type and knockout CCD. Maxi-K+channel inhibitor iberiotoxin had no effect on K+secretion when tubules were perfused at ∼1.5 nl/min, but completely abrogated the flow-dependent increase in K+secretion at ∼5.5 nl/min. These findings support the notion that KS-WNK1 stimulates ROMK-mediated K+secretion, but not flow-dependent K+secretion mediated by maxi-K+channels in CCD. In addition, KS-WNK1 plays a role in regulating Na+transport in the CCD.

An increasing number of studies have linked high dietary phosphate (Pi) intake to hypertension. It is well established that the rise in sympathetic nerve activity (SNA) and blood pressure (BP) during physical exertion is exaggerated in many forms of hypertension, which are primarily mediated by an overactive skeletal muscle exercise pressor reflex (EPR). However, it remains unknown whether high dietary Pi intake potentiates the EPR-mediated SNA and BP response to exercise. Accordingly, we measured renal SNA (RSNA) and mean BP (MBP) in normotensive Sprague-Dawley rats fed a normal Pi diet (0.6%, n = 13) or high Pi diet (1.2%, n = 13) for 3 mo. As previously reported, we found that resting BP was significantly increased by 1.2% Pi diet in both conscious and anesthetized animals. Activation of the EPR by electrically induced hindlimb contraction triggered greater increases in ΔRSNA and ΔMBP in the 1.2% compared with 0.6% Pi group (126 ± 25 vs. 42 ± 9%; 44 ± 5 vs. 14 ± 2 mmHg, respectively, P < 0.01). Activation of the muscle mechanoreflex, a component of the EPR, by passively stretching hindlimb muscle also evoked greater increases in ΔRSNA and ΔMBP in the 1.2% compared with 0.6% Pi group (109 ± 27 vs. 24 ± 7%, 38 ± 7 vs. 8 ± 2 mmHg, respectively, P < 0.01). A similar response was produced by hindlimb intra-arterial capsaicin administration to stimulate the metaboreflex arm of the EPR. Thus, our data demonstrate a novel action of dietary Pi loading in augmenting EPR function through overactivation of both the muscle mechanoreflex and metaboreflex.

Klotho is a type-1 membrane protein predominantly produced in the kidney, the extracellular domain of which is secreted into the systemic circulation. Membranous and secreted Klotho protect organs, including the kidney, but whether and how Klotho directly protects the glomerular filter is unknown. Here, we report that secreted Klotho suppressed transient receptor potential channel 6 (TRPC6)-mediated Ca2+ influx in cultured mouse podocytes by inhibiting phosphoinositide 3-kinase-dependent exocytosis of the channel. Furthermore, soluble Klotho reduced ATP-stimulated actin cytoskeletal remodeling and transepithelial albumin leakage in these cells. Overexpression of TRPC6 by gene delivery in mice induced albuminuria, and exogenous administration of Klotho ameliorated the albuminuria. Notably, immunofluorescence and in situ hybridization revealed Klotho expression in podocytes of mouse and human kidney. Heterozygous Klotho-deficient CKD mice had aggravated albuminuria compared with that in wild-type CKD mice with a similar degree of hypertension and reduced clearance function. Finally, disrupting the integrity of glomerular filter by saline infusion-mediated extracellular fluid volume expansion increased urinary Klotho excretion. These results reveal a potential novel function of Klotho in protecting the glomerular filter, and may offer a new therapeutic strategy for treatment of proteinuria.

The With no Lysine [K] (WNK) family of enzymes are central in the regulation of blood pressure. WNKs have been implicated in hereditary hypertension disorders, mainly through control of the activity and levels of ion cotransporters and channels. Actions of WNKs in the kidney have been heavily investigated, and recent studies have provided insight into not only the regulation of these enzymes but also how mutations in WNKs and their interacting partners contribute to hypertensive disorders. Defining the roles of WNKs in the cardiovascular system will provide clues about additional mechanisms by which WNKs can regulate blood pressure. This review summarizes recent developments in the regulation of the WNK signaling cascade and its role in regulation of blood pressure.