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In cystic fibrosis (CF), reduced HCOsub3/subsup-/supsecretion acidifies the airway surface liquid (ASL), and the acidic pH disrupts host defenses. Thus, understanding the control of ASL pH (pHsubASL/sub) in CF may help identify novel targets and facilitate therapeutic development. In diverse epithelia, the WNK (with-no-lysine [K]) kinases coordinate HCOsub3/subsup-/supand Clsup-/suptransport, but their functions in airway epithelia are poorly understood. Here, we tested the hypothesis that WNK kinases regulate CF pHsubASL/sub. In primary cultures of differentiated human airway epithelia, inhibiting WNK kinases acutely increased both CF and non-CF pHsubASL/sub. This response was HCOsub3/subsup-/supdependent and involved downstream SPAK/OSR1 (Ste20/SPS1-related proline-alanine-rich protein kinase/oxidative stress responsive 1 kinase). Importantly, WNK inhibition enhanced key host defenses otherwise impaired in CF. Human airway epithelia expressed twoiWNK/iisoforms in secretory cells and ionocytes, and knockdown of eitheriWNK1/ioriWNK2/iincreased CF pHsubASL/sub. WNK inhibition decreased Clsup-/supsecretion and the response to bumetanide, an NKCC1 (sodium-potassium-chloride cotransporter 1) inhibitor. Surprisingly, bumetanide alone or basolateral Clsup-/supsubstitution also alkalinized CF pHsubASL/sub. These data suggest that WNK kinases influence the balance between transepithelial Clsup-/supversus HCOsub3/subsup-/supsecretion. Moreover, reducing basolateral Clsup-/supentry may increase HCOsub3/subsup-/supsecretion and raise pHsubASL/sub, thereby improving CF host defenses.

Prenatal dexamethasone has been shown to increase blood pressure in male offspring but the mechanism for the increase in blood pressure is unclear. The present study examined if prenatal programming by maternal injection of dexamethasone on days 15 and 16 of gestation affected the blood pressure comparably in female and male offspring. Our hypothesis was that males would be affected by prenatal dexamethasone to a greater extent than females and that either an increase in renal tubular transporter abundance or an increase in renin or aldosterone system would be associated with hypertension with prenatal programming. Prenatal dexamethasone increased blood pressure at two months and six months of age and resulted in proteinuria and albuminuria at six months in male but not female rat offspring. There was no effect of prenatal dexamethasone on blood pressure and proteinuria at one month in male and in female offspring. While prenatal dexamethasone increased male renal thick ascending limb sodium potassium two chloride cotransporter protein abundance at two months, prenatal dexamethasone on days 15 and 16 of gestation did not affect transporter abundance in males at other ages, nor did it affect proximal tubule sodium/hydrogen exchanger or distal convoluted tubule sodium chloride cotransporter protein abundance at any age. There was no difference in systemic renin or aldosterone in the prenatal dexamethasone group compared to same sex controls. In conclusion, male but not female offspring have an increase in blood pressure and urinary protein excretion with prenatal dexamethasone. The increase in blood pressure with prenatal programming was not associated with a consistent increase in renal tubular transporter protein abundance, nor plasma renin activity and serum aldosterone.

Propionate, a metabolite from the microbial fermentation of carbohydrates, evokes a release of epithelial acetylcholine in rat caecum resulting in an increase of short-circuit current (Isc) in Ussing chamber experiments. The present experiments were performed in order to characterize the ionic mechanisms underlying this response which has been thought to be due to Cl−secretion. As there are regional differences within the caecal epithelium, the experiments were conducted at oral and aboral rat corpus caeci. In both caecal segments, the propionate-inducedIsc(IProp) was inhibited by > 85%, when the experiments were performed either in nominally Cl−- or nominally HCO3−-free buffer. In the case of Cl−, the dependency was restricted to the presence of Cl−in the serosal bath. Bumetanide, a blocker of the Na+-K+-2Cl−-cotransporter, only numerically reducedIPropsuggesting that a large part of this current must be carried by an ion other than Cl−. In the aboral caecum,IPropwas significantly inhibited by mucosally administered stilbene derivatives (SITS, DIDS, DNDS), which block anion exchangers. Serosal Na+-free buffer reduced IPropsignificantly in the oral (and numerically also in aboral) corpus caeci. RT-PCR experiments revealed the expression of several forms of Na+-dependent HCO3−-cotransporters in caecum, which might underlie the observed Na+dependency. These results suggest that propionate sensing in caecum is coupled to HCO3–secretion, which functionally would stabilize luminal pH when the microbial fermentation leads to an increase in the concentration of short-chain fatty acids in the caecal lumen.

Hypertension affects 30% of adults and is the leading risk factor for cardiovascular disease. Kidney sodium reabsorption plays a vital role in the initial stage and development of essential hypertension. It has been extensively reported that the variants of kidney ion handling genes are associated to blood pressure, and clinical features of hypertension. However, the underlying mechanisms by which these variants alter protein function are rarely summarized. In addition, the variation of one single gene is often limited to induce a significant effect on blood pressure. In the past few decades, the influence by genes × genes (G × G) and/or genotype × environment (G × E) interactions on a given trait, for example, blood pressure, have been widely considered, especially in studies on polygenic genetic traits. In the present review, we discuss the progress in genetics studies on kidney ion handling genes, encoding Na+ channels (Na+-Cl− cotransporter [NCC], Na-K-2Cl cotransporter [NKCC2], epithelial Na+ channels [ENaCs]), K+ channel (renal outer medullary potassium channel [ROMK]), and Cl− channels (Pendrin, chloride voltage-gated channel Kb [CLC-Kb]), respectively, and their upstream kinases, WNKs and SGK1. We seek to clarify how these genes are involved in kidney sodium absorption and influence blood pressure, especially emphasizing the underlying mechanisms by which genetic variants alter protein functions and interaction in blood pressure regulation. The present review aims to enhance our understanding of the important role of kidney ion handling genes/channels in blood pressure control.

The research on the biological pacemaker has been very active in recent years. And turning nonautomatic ventricular cells into pacemaking cells is believed to hold the key to making a biological pacemaker. In the study, the inward-rectifier K+ current (IK1) is depressed to induce the automaticity of the ventricular myocyte, and then, the effects of the other membrane ion currents on the automaticity are analyzed. It is discovered that the L-type calcium current (ICaL) plays a major part in the rapid depolarization of the action potential (AP). A small enough ICaL would lead to the failure of the automaticity of the ventricular myocyte. Meanwhile, the background sodium current (IbNa), the background calcium current (IbCa), and the Na+/Ca2+ exchanger current (INaCa) contribute significantly to the slow depolarization, indicating that these currents are the main supplementary power of the pacing induced by depressing IK1, while in the 2D simulation, we find that the weak electrical coupling plays a more important role in the driving of a biological pacemaker.

Mummichogs (Fundulus heteroclitus) can tolerate abrupt changes in environmental salinity because of their ability to rapidly adjust the activities of ionocytes in branchial and opercular epithelia. In turn, the concerted expression of sub-cellular effectors of ion transport underlies adaptive responses to fluctuating salinities. Exposure to seawater (SW) stimulates the expression of Na+/K+/2Cl− cotransporter 1 (nkcc1) and cystic fibrosis transmembrane regulator (cftr) mRNAs in support of ion extrusion by SW-type ionocytes. Given the incomplete understanding of how freshwater (FW)-type ionocytes actually operate in mummichogs, the transcriptional responses essential for ion absorption in FW environments remain unresolved. In a subset of species, a ‘fish-specific’ Na+/Cl− cotransporter denoted Ncc2 (Slc12a10) is responsible for the uptake of Na+ and Cl− across the apical surface of FW-type ionocytes. In the current study, we identified an ncc2 transcript that is highly expressed in gill filaments and opercular epithelium of FW-acclimated mummichogs. Within 1 day of transfer from SW to FW, ncc2 levels in both tissues increased in parallel with reductions in nkcc1 and cftr. Conversely, mummichogs transferred from FW to SW exhibited marked reductions in ncc2 concurrent with increases in nkcc1 and cftr. Immunohistochemical analyses employing a homologous antibody revealed apical Ncc2-immunoreactivity in Na+/K+-ATPase-immunoreactive ionocytes of FW-acclimated animals. Our combined observations suggest that Ncc2/ncc2-expressing ionocytes support the capacity of mummichogs to inhabit FW environments.

Spreading depolarizations (SDs) indicate injury progression and predict worse clinical outcome in acute brain injury. We demonstrate in rodents that acute brain swelling upon cerebral ischemia impairs astroglial glutamate clearance and increases the tissue area invaded by SD. The cytotoxic extracellular glutamate accumulation (>15 µM) predisposes an extensive bulk of tissue (4–5 mm2) for a yet undescribed simultaneous depolarization (SiD). We confirm in rat brain slices exposed to osmotic stress that SiD is the pathological expansion of prior punctual SD foci (0.5–1 mm2), is associated with astrocyte swelling, and triggers oncotic neuron death. The blockade of astrocytic aquaporin-4 channels and Na+/K+/Cl− co-transporters, or volume-regulated anion channels mitigated slice edema, extracellular glutamate accumulation (

Apoptotic cell clearance (efferocytosis) elicits an anti-inflammatory response by phagocytes, but the mechanisms that underlie this response are still being defined. Here, we uncover a chloride-sensing signalling pathway that controls both the phagocyte 'appetite' and its anti-inflammatory response. Efferocytosis transcriptionally altered the genes that encode the solute carrier (SLC) proteins SLC12A2 and SLC12A4. Interfering with SLC12A2 expression or function resulted in a significant increase in apoptotic corpse uptake per phagocyte, whereas the loss of SLC12A4 inhibited corpse uptake. In SLC12A2-deficient phagocytes, the canonical anti-inflammatory program was replaced by pro-inflammatory and oxidative-stress-associated gene programs. This 'switch' to pro-inflammatory sensing of apoptotic cells resulted from the disruption of the chloride-sensing pathway (and not due to corpse overload or poor degradation), including the chloride-sensing kinases WNK1, OSR1 and SPAK-which function upstream of SLC12A2-had a similar effect on efferocytosis. Collectively, the WNK1-OSR1-SPAK-SLC12A2/SLC12A4 chloride-sensing pathway and chloride flux in phagocytes are key modifiers of the manner in which phagocytes interpret the engulfed apoptotic corpse.

Aim: The phosphorylation level of the furosemide-sensitive Na +-K +-2Cl − cotransporter (NKCC2) in the thick ascending limb (TAL) is used as a surrogate marker for NKCC2 activation and TAL function. However, in mice, analyses of NKCC2 phosphorylation with antibodies against phosphorylated threonines 96 and 101 (anti-pT96/pT101) give inconsistent results. We aimed (a) to elucidate these inconsistencies and (b) to develop a phosphoform-specific antibody that ensures reliable detection of NKCC2 phosphorylation in mice. Methods: Genetic information, molecular biology, biochemical techniques and mouse phenotyping was used to study NKCC2 and kidney function in two commonly used mouse strains (ie 129Sv and in C57BL/6 mice). Moreover, a new phosphoform-specific mouse NKCC2 antibody was developed and characterized. Results: Amino acids sequence alignment revealed that C57BL/6 mice have a strain-specific five amino acids deletion (ΔF97-T101) in NKCC2 that diminishes the detection of NKCC2 phosphorylation with previously developed pT96/pT101 NKCC2 antibodies. Instead, the antibodies cross-react with the phosphorylated thiazide-sensitive NaCl cotransporter (NCC), which can obscure interpretation of results. Interestingly, the deletion in NKCC2 does not impact on kidney function and/or expression of renal ion transport proteins as indicated by the analysis of the F2 generation of crossbred 129Sv and C57BL/6 mice. A newly developed pT96 NKCC2 antibody detects pNKCC2 in both mouse strains and shows no cross-reactivity with phosphorylated NCC. Conclusion: Our work reveals a hitherto unappreciated, but essential, strain difference in the amino acids sequence of mouse NKCC2 that needs to be considered when analysing NKCC2 phosphorylation in mice. The new pNKCC2 antibody circumvents this technical caveat.

The solute carrier family has been reported to play critical roles in the progression of several cancers; however, the relationship between solute carrier family 12 member 8 (SLC12A8) and bladder cancer (BC) has not been clearly confirmed. This study explores the prognostic value of SLC12A8 for BC and its correlation with immune cell infiltration. We found that the expression of SLC12A8 mRNA was significantly overexpressed in BC tissues compared with noncancerous tissues in multiple public databases, and the result was validated using real-time PCR and immunohistochemistry (IHC). The Kaplan-Meier method and Cox proportional hazards models were used to evaluate the prognostic value of SLC12A8 for BC. The high expression of SLC12A8 led to a shorter overall survival time and was an unfavorable prognostic biomarker for BC. The mechanisms of SLC12A8 promoting tumorigenesis were investigated by Gene Set Enrichment Analysis (GSEA). Moreover, the correlations of SLC12A8 expression with the tumor-infiltrating immune cells (TICs) in BC were explored using TIMER 2.0 and CIBERSORT. SLC12A8 was associated with CD4+ T cells, dendritic cells, neutrophils, and macrophages infiltration. The expression of SLC12A8 was positively correlated with crucial immune checkpoint molecules. In conclusion, SLC12A8 might be an unfavorable prognostic biomarker in BC related to tumor immune cell infiltration.

Two orthologues of the gene encoding the Na+-Cl− cotransporter (NCC), termed ncca and nccb, were found in the sea lamprey genome. No gene encoding the Na+-K+-2Cl− cotransporter 2 (nkcc2) was identified. In a phylogenetic comparison among other vertebrate NCC and NKCC sequences, the sea lamprey NCCs occupied basal positions within the NCC clades. In freshwater, ncca mRNA was found only in the gill and nccb only in the intestine, whereas both were found in the kidney. Intestinal nccb mRNA levels increased during late metamorphosis coincident with salinity tolerance. Acclimation to seawater increased nccb mRNA levels in the intestine and kidney. Electrophysiological analysis of intestinal tissue ex vivo showed this tissue was anion absorptive. After seawater acclimation, the proximal intestine became less anion absorptive, whereas the distal intestine remained unchanged. Luminal application of indapamide (an NCC inhibitor) resulted in 73% and 30% inhibition of short-circuit current (Isc) in the proximal and distal intestine, respectively. Luminal application of bumetanide (an NKCC inhibitor) did not affect intestinal Isc. Indapamide also inhibited intestinal water absorption. Our results indicate that NCCb is likely the key ion cotransport protein for ion uptake by the lamprey intestine that facilitates water absorption in seawater. As such, the preparatory increases in intestinal nccb mRNA levels during metamorphosis of sea lamprey are likely critical to development of whole animal salinity tolerance.

In this review, we discuss the role of adrenoceptors in the regulation of the rheological functions of erythrocytes. β-adrenoceptors play an important role in the regulation of erythrocytes functions and metabolism. They participate in the modification of transport membrane proteins (Na+/K+-ATPase, Ca+2-ATPase, Na+/K+/2Cl-cotransporter, Na+/H+-antiporter, CAT-1, Ca2+-dependent K+ channels (Gard channels), the activity of adenylate cyclase and cAMP, AMP-dependent activation of the L-arginine/NOS system and erythrocyte NOS) and by this way modulate the cells volume, rheological properties (deformability, aggregability), intensity of NO synthesis and ATP reliase. These properties of erythrocytes determine, that, in addition to the transport of gases, they play the oxygen sensors role and can participate in the mechanisms of vasorelaxation and maintenance of a normal level of microcirculation.

Coupled transport Cation-chloride cotransporters move chloride and cations across the cell membrane and are important in regulating cell volume and setting the chloride concentration inside the cell. Mutations lead to serious diseases, such as epilepsy. Liu et al. present the structure of the human potassium-chloride cotransporter KCC1, as determined by cryo–electron microscopy. Based on the structure, functional studies, and molecular dynamics simulations, they propose an ion transport model. The structure provides a framework for interpreting disease-related mutations in potassium-chloride cotransporters. Science , this issue p. 505

Introduction Potassium (K+) intake is intrinsically linked to blood pressure. High-K+ intake decreases hypertension and associated lower mortality. On the other hand, hyperkalemia causes sudden death with fatal cardiac arrhythmia and is also related to higher mortality. Renal sodium (Na+)–chloride (Cl‒) cotransporter (NCC), expressed in the distal convoluted tubule, is a key molecule in regulating urinary K+ excretion. K+ intake affects the activity of the NCC, which is related to salt-sensitive hypertension. A K+-restrictive diet activates NCC, and K+ loading suppresses NCC. Hyperpolarization caused by decreased extracellular K+ concentration ([K+]ex) increases K+ and Cl‒ efflux, leading to the activation of Cl‒-sensitive with-no-lysine (WNK) kinases and their downstream molecules, including STE20/SPS1-related proline/alanine-rich kinase (SPAK) and NCC. Results We investigated the role of the ClC-K2 Cl‒ channel and its β-subunit, barttin, using barttin hypomorphic (Bsndneo/neo) mice and found that these mice did not show low-K+-induced NCC activation and salt-sensitive hypertension. Additionally, we discovered that the suppression of NCC by K+ loading was regulated by another mechanism, whereby tacrolimus (a calcineurin [CaN] inhibitor) inhibited high-K+-induced NCC dephosphorylation and urinary K+ excretion. The K+ loading and the tacrolimus treatment did not alter the expression of WNK4 and SPAK. The depolarization induced by increased [K+]ex activated CaN, which dephosphorylates NCC. Conclusions We concluded that there were two independent molecular mechanisms controlling NCC activation and K+ excretion. This review summarizes the clinical importance of K+ intake and explains how NCC phosphorylation is regulated by different molecular mechanisms between the low- and the high-K+ condition. Electronic supplementary material The online version of this article (10.1007/s10157-019-01766-x) contains supplementary material, which is available to authorized users.

Quercetin has multiple potential to control various cell function keeping our body condition healthy. In this review article, we describe the molecular mechanism on how quercetin exerts its action on blood pressure, neurite elongation and epithelial ion transport based from a viewpoint of cytosolic Cl- environments, which is recently recognized as an important signaling factor in various types of cells. Recent studies show various roles of cytosolic Cl- in regulation of blood pressure and neurite elongation, and prevention from bacterial and viral infection. We have found the stimulatory action of quercetin on Cl- transporter, Na+-K+-2Cl- cotransporter 1 (NKCC1; an isoform of NKCC), which has been recognized as one of the most interesting, fundamental actions of quercetin. In this review article, based on this stimulatory action of quercetin on NKCC1, we introduce the molecular mechanism of quercetin on: 1) blood pressure, 2) neurite elongation, and 3) epithelial Cl- secretion including tight junction forming in epithelial tissues. 1) Quercetin induces elevation of the cytosolic Cl- concentration via activation of NKCC1, leading to anti-hypertensive action by diminishing expression of epithelial Na+ channel (ENaC), a key ion channel involved in renal Na+ reabsorption, while quercetin has no effects on the blood pressure with normal salt intake. 2) Quercetin also has stimulatory effects on neurite elongation by elevating the cytosolic Cl- concentration via activation of NKCC1 due to tubulin polymerization facilitated through Cl--induced inhibition of GTPase. 3) Further, in lung airway epithelia quercetin stimulates Cl- secretion by increasing the driving force for Cl- secretion via elevation of the cytosolic Cl- concentration: this leads to water secretion, participating in prevention of our body from bacterial and viral infection. In addition to transcellular ion transport, quercetin regulates tight junction function via enhancement of tight junction integrity by modulating expression and assembling tight junction-forming proteins. Based on these observations, it is concluded that quercetin is a useful medicinal compound keeping our body to be in healthy condition.

Hyperglycemia exacerbates edema formation and worsens neurological outcome in ischemic stroke. Edema formation in the early hours of stroke involves transport of ions and water across an intact blood-brain barrier (BBB), and swelling of astrocytes. We showed previously that high glucose (HG) exposures of 24 hours to 7 days increase abundance and activity of BBB Na(+)-K(+)-2Cl(−) cotransport (NKCC) and Na(+)/H(+) exchange 1 (NHE1). Further, bumetanide and HOE-642 inhibition of these transporters significantly reduces edema and infarct following middle cerebral artery occlusion in hyperglycemic rats, suggesting that NKCC and NHE1 are effective therapeutic targets for reducing edema in hyperglycemic stroke. The mechanisms underlying hyperglycemia effects on BBB NKCC and NHE1 are not known. In the present study we investigated whether serum-glucocorticoid regulated kinase 1 (SGK1) and protein kinase C beta II (PKCβII) are involved in HG effects on BBB NKCC and NHE1. We found transient increases in phosphorylated SGK1 and PKCβII within the first hour of HG exposure, after 5-60 min for SGK1 and 5 min for PKCβII. However, no changes were observed in cerebral microvascular endothelial cell SGK1 or PKCβII abundance or phosphorylation (activity) after 24 or 48 h HG exposures. Further, we found that HG-induced increases in NKCC and NHE1 abundance were abolished by inhibition of SGK1 but not PKCβII, whereas the increases in NKCC and NHE activity were abolished by inhibition of either kinase. Finally, we found evidence that STE20/SPS1-related proline/alanine-rich kinase and oxidative stress-responsive kinase-1 (SPAK/OSR1) participate in the HG-induced effects on BBB NKCC.

Stroke is one of the major culprits responsible for morbidity and mortality worldwide, and the currently available pharmacological strategies to combat this global disease are scanty. Cation-chloride cotransporters (CCCs) are expressed in several tissues (including neurons) and extensively contribute to the maintenance of numerous physiological functions including chloride homeostasis. Previous studies have implicated two CCCs, the Na+-K+-Cl- and K+-Cl- cotransporters (NKCCs and KCCs) in stroke episodes along with their upstream regulators, the with-no-lysine kinase (WNKs) family and STE20/SPS1-related proline/alanine rich kinase (SPAK) or oxidative stress response kinase (OSR1) via a signaling pathway. As the WNK-SPAK/OSR1 pathway reciprocally regulates NKCC and KCC, a growing body of evidence implicates over-activation and altered expression of NKCC1 in stroke pathology whilst stimulation of KCC3 during and even after a stroke event is neuroprotective. Both inhibition of NKCC1 and activation of KCC3 exert neuroprotection through reduction in intracellular chloride levels and thus could be a novel therapeutic strategy. Hence, this review summarizes the current understanding of functional regulations of the CCCs implicated in stroke with particular focus on NKCC1, KCC3, and WNK-SPAK/OSR1 signaling and discusses the current and potential pharmacological treatments for stroke.

Objectives Clinical and epidemiological studies have suggested a correlation between vitamin D deficiency (VDD) and high blood pressure (BP). This study aimed to test the hypothesis that high BP induced by VDD is associated with altered expression and covalent modification of apical sodium transporters along the nephron. The contributions of the intrarenal renin-angiotensin system (RAS) and oxidative stress were also investigated. Methods Male Wistar rats were fed a vitamin D-free (n = 26) or standard diet (n = 25) for 30 days. BP was recorded using noninvasive and invasive procedures. The expression levels of total and phosphorylated apical sodium transporters in rat renal cortex and medulla were evaluated by immunoblotting. Intrarenal RAS components were assessed by immunoblotting and ELISA. Renal oxidative stress was analyzed by measuring the concentrations of thiobarbituric acid reactive substances and reduced glutathione. Results Higher BP levels in VDD rats than controls were accompanied by overexpression and hyperphosphorylation of renal cortical and medullary Na+-K+-2Cl- cotransporter type 2, enhanced levels of phosphorylated Na+/H+ exchanger type 3, and reduced expression levels of total and phosphorylated Na+/Cl- cotransporter. Changes in intrarenal RAS induced by VDD vs. controls included the marked elevation of medullary renin expression, higher expression of cortical angiotensinogen, higher urinary angiotensinogen excretion, and higher cortical and medullary angiotensin II content. VDD rats displayed higher thiobarbituric acid reactive substances/glutathione ratios in the renal cortex and medulla than controls. Conclusion These results suggest that the molecular mechanisms underlying the effects of VDD on BP may include the upregulation of Na+-K+-2Cl- cotransporter type 2 and activation of intrarenal RAS and oxidative stress.

Abnormal activity of red cell KCl cotransport (KCC) is involved in pathogenesis of sickle cell anaemia (SCA). KCC-mediated solute loss causes shrinkage, concentrates HbS, and promotes HbS polymerisation. Red cell KCC also responds to various stimuli including pH, volume, urea, and oxygen tension, and regulation involves protein phosphorylation. The main aim of this study was to investigate the role of the WNK/SPAK/OSR1 pathway in sickle cells. The pan WNK inhibitor WNK463 stimulated KCC with an EC50 of 10.9 ± 1.1 nM and 7.9 ± 1.2 nM in sickle and normal red cells, respectively. SPAK/OSR1 inhibitors had little effect. The action of WNK463 was not additive with other kinase inhibitors (staurosporine and N-ethylmaleimide). Its effects were largely abrogated by pre-treatment with the phosphatase inhibitor calyculin A. WNK463 also reduced the effects of physiological KCC stimuli (pH, volume, urea) and abolished any response of KCC to changes in oxygen tension. Finally, although protein kinases have been implicated in regulation of phosphatidylserine exposure, WNK463 had no effect. Findings indicate a predominant role for WNKs in control of KCC in sickle cells but an apparent absence of downstream involvement of SPAK/OSR1. A more complete understanding of the mechanisms will inform pathogenesis whilst manipulation of WNK activity represents a potential therapeutic approach.

The Na

Extracellular fluid (ECF) potassium concentration ([K(+)]) is maintained by adaptations of kidney and skeletal muscle, responses heretofore studied separately. We aimed to determine how these organ systems work in concert to preserve ECF [K(+)] in male C57BL/6J mice fed a K(+)-deficient diet (0K) versus 1% K(+) diet (1K) for 10 days (n = 5–6/group). During 0K feeding, plasma [K(+)] fell from 4.5 to 2 mM; hindlimb muscle (gastrocnemius and soleus) lost 28 mM K(+) (from 115 ± 2 to 87 ± 2 mM) and gained 27 mM Na(+) (from 27 ± 0.4 to 54 ± 2 mM). Doubling of muscle tissue [Na(+)] was not associated with inflammation, cytokine production or hypertension as reported by others. Muscle transporter adaptations in 0K- versus 1K-fed mice, assessed by immunoblot, included decreased sodium pump α2-β2 subunits, decreased K(+)-Cl(−) cotransporter isoform 3, and increased phosphorylated (p) Na(+),K(+),2Cl(−) cotransporter isoform 1 (NKCC1p), Ste20/SPS-1-related proline-alanine rich kinase (SPAKp), and oxidative stress-responsive kinase 1 (OSR1p) consistent with intracellular fluid (ICF) K(+) loss and Na(+) gain. Renal transporters’ adaptations, effecting a 98% reduction in K(+) excretion, included two- to threefold increased phosphorylated Na(+)-Cl(−) cotransporter (NCCp), SPAKp, and OSR1p abundance, limiting Na(+) delivery to epithelial Na(+) channels where Na(+) reabsorption drives K(+) secretion; and renal K sensor Kir 4.1 abundance fell 25%. Mass balance estimations indicate that over 10 days of 0K feeding, mice lose ~48 μmol K(+) into the urine and muscle shifts ~47 μmol K(+) from ICF to ECF, illustrating the importance of the concerted responses during K(+) deficiency.

The SLC12 family of cation-chloride-cotransporters (CCCs) is comprised of potassium chloride cotransporters (KCCs), which mediate Cl− extrusion and sodium-potassium chloride cotransporters (N[K]CCs), which mediate Cl− loading. The CCCs play vital roles in cell volume regulation and ion homeostasis. The functions of CCCs influence a variety of physiological processes, many of which overlap with the pathophysiology of cardiovascular disease. Although not all of the cotransporters have been linked to Mendelian genetic disorders, recent studies have provided new insights into their functional role in vascular and renal cells in addition to their contribution to cardiovascular diseases. Particularly, an imbalance in potassium levels promotes the pathogenesis of atherosclerosis and disturbances in sodium homeostasis are one of the causes of hypertension. Recent findings suggest hypothalamic signaling as a key signaling pathway in the pathophysiology of hypertension. In this review, we summarize and discuss the role of CCCs in cardiovascular disease with particular emphasis on knowledge gained in recent years on NKCCs and KCCs.

Each day, ~1.7 kg of NaCl and 180 liters of water are reabsorbed by nephron segments in humans, with urinary excretion fine tuned to meet homeostatic requirements. These tasks are coordinated by a spectrum of renal Na(+) transporters and channels. The goal of the present study was to investigate the extent to which inhibitors of transepithelial Na(+) transport (T(Na)) along the nephron alter urinary solute excretion and how those effects may vary between male and female subjects. To accomplish that goal, we developed sex-specific multinephron models that represent detailed transcellular and paracellular transport processes along the nephrons of male and female rat kidneys. We simulated inhibition of Na(+)/H(+) exchanger 3 (NHE3), bumetanide-sensitive Na(+)-K(+)-2Cl(−) cotransporter (NKCC2), Na(+)-Cl(−) cotransporter (NCC), and amiloride-sensitive epithelial Na(+) channel (ENaC). NHE3 inhibition simulations predicted a substantially reduced proximal tubule T(Na), and NKCC2 inhibition substantially reduced thick ascending limb T(Na). Both gave rise to diuresis, natriuresis, and kaliuresis, with those effects stronger in female rats. While NCC inhibition was predicted to have only minor impact on renal T(Na), it nonetheless had a notable effect of enhancing excretion of Na(+), K(+), and Cl(−), particularly in female rats. Inhibition of ENaC was predicted to have opposite effects on the excretion of Na(+) (increased) and K(+) (decreased) and to have only a minor impact on whole kidney T(Na). Unlike inhibition of other transporters, ENaC inhibition induced stronger natriuresis and diuresis in male rats than female rats. Overall, model predictions agreed well with measured changes in Na(+) and K(+) excretion in response to diuretics and Na(+) transporter mutations.

Background Arginine vasopressin dependent antidiuresis plays a key role in water‐sodium retention in heart failure. In recent years, the role of glucocorticoids in the control of body fluid homeostasis has been extensively investigated. Glucocorticoid deficiency can activate V2R (vasopressin receptor 2), increase aquaporins expression, and result in hyponatremia, all of which can be reversed by glucocorticoid supplement. Methods and Results Heart failure was induced by coronary artery ligation for 8 weeks. A total of 32 rats were randomly assigned to 4 groups (n=8/group): sham surgery group, congestive heart failure group, dexamethasone group, and dexamethasone in combination with glucocorticoid receptor antagonist RU486 group. An acute water loading test was administered 6 hours after drug administration. Left ventricular function was measured by a pressure‐volume catheter. Protein expressions were determined by immunohistochemistry and immunoblotting. The pressure‐volume loop analysis showed that dexamethasone improves cardiac function in rats with heart failure. Western blotting confirmed that dexamethasone remarkably reduces the expressions of V2R, aquaporin 2, and aquaporin 3 in the renal‐collecting ducts. As a result of V2R downregulation, the expressions of glucocorticoid regulated kinase 1, apical epithelial sodium channels, and the furosemide‐sensitive Na‐K‐2Cl cotransporter were also downregulated. These favorable effects induced by dexamethasone were mostly abolished by the glucocorticoid receptor inhibitor RU486, indicating that the aforementioned effects are glucocorticoid receptor mediated. Conclusions Glucocorticoids can reverse diluted hyponatremia via inhibiting the vasopressin receptor pathway in rats with heart failure.

In the retina, ON- and OFF-type bipolar cells are classified by subtype-specific center responses, which are attributed to differences in glutamate receptor subtypes. However, the mechanisms by which ON- and OFF-type bipolar cells generate subtype-specific surround responses remain unclear. One hypothesis for surround responses is that intracellular Cl concentrations ([Cl-]i) are set at different levels to achieve opposite polarities for GABA responses in ON- and OFF-type bipolar cells. Although this hypothesis is supported by previous findings obtained from rod (ON-) type bipolar cells, there is currently no information on OFF-type bipolar cells. In the present study, we examined the distribution and function of the Cl transporters, the Na-K-Cl co-transporter (NKCC1) and K-Cl co-transporter (KCC2), in rod (ON-) and OFF-type bipolar cells using immunohistochemical, in situ hybridization, and electrophysiological methods. Rod (ON-) and OFF-type bipolar cells both expressed NKCC1 and KCC2. However, the functional contribution of NKCC1 and KCC2 to the regulation of [Cl-]i differed between rod (ON-) and OFF-type bipolar cells. Strong NKCC1 activity increased [Cl-]i in rod (ON-) type bipolar cells, while that of KCC2 decreased [Cl-]i in OFF-type bipolar cells. We also confirmed the presence of a [Cl-]i gradient between dendrites and axon terminals in rod (ON-type) bipolar cells. Thus, the subtype-specific control of [Cl-]i is achieved by the activity of NKCC1 relative to that of KCC2 and appears to influence the polarity of surround responses.

PAPPA2 is a metalloproteinase which cleaves insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5, and its role in pregnancy and postnatal growth is primarily studied. Using exclusion mapping, we reported a subcongenic (26-P) rat where a 0.71-Mbp region containing the pregnancy-associated plasma protein a2 ( Pappa2) allele of salt-insensitive Brown Norway (BN) was introgressed into Dahl saltsensitive (SS) genetic background, resulting in the reduction of salt sensitivity. Pappa2 was differentially expressed in the adult kidney of 26-P and SS rats. Here, the expression and cellular localization of Pappa2 in embryonic and postnatal kidneys of 26-P and SS rats were examined. Pappa2 mRNA expression was 5-fold higher in the embryonic kidney (day 20.5) of the 26-P rat compared with the SS rat. Pappa2 mRNA expression progressively increased with the development of kidney, reaching a peak at postnatal day 5 before trending downward in subsequent stages of development in both strains. At all tested time points, Pappa2 remained higher in the 26-P compared with the SS rat kidney. Immunohistochemistry studies localized PAPPA2 in the ureteric bud (UB) and distal part of S-shaped body. PAPPA2 was colocalized with IGFBP-5 in the UB and Na+/K+/2Cl−cotransporter–stained tubules, respectively. Future studies are needed to determine the role of Pappa2 in kidney development and mechanistic pathways involved in this process.

With no lysine kinase 4 (WNK4) is essential to activate the thiazide-sensitive NaCl cotransporter (NCC) along the distal convoluted tubule, an effect central to the phenotype of familial hyperkalemic hypertension. Although effects on potassium and sodium channels along the connecting and collecting tubules have also been documented, WNK4 is typically believed to have little role in modulating sodium chloride reabsorption along the thick ascending limb of the loop of Henle. Yet wnk4−/−mice (knockout mice lacking WNK4) do not demonstrate the hypocalciuria typical of pure distal convoluted tubule dysfunction. Here, we tested the hypothesis that WNK4 also modulates bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) function along the thick ascending limb. We confirmed that w nk4−/−mice are hypokalemic and waste sodium chloride, but are also normocalciuric. Results from Western blots suggested that the phosphorylated forms of both NCC and NKCC2 were in lower abundance in wnk4−/−mice than in controls. This finding was confirmed by immunofluorescence microscopy. Although the initial response to furosemide was similar in wnk4−/−mice and controls, the response was lower in the knockout mice when reabsorption along the distal convoluted tubule was inhibited. Using HEK293 cells, we showed that WNK4 increases the abundance of phosphorylated NKCC2. More supporting evidence that WNK4 may modulate NKCC2 emerges from a mouse model of WNK4-mediated familial hyperkalemic hypertension in which more phosphorylated NKCC2 is present than in controls. These data indicate that WNK4, in addition to modulating NCC, also modulates NKCC2, contributing to its physiological function in vivo.

The thalamus is important for sensory integration with the ventrobasal thalamus (VB) as relay controlled by GABAergic projections from the nucleus reticularis thalami (NRT). Depending on the [Cl−]i primarily set by cation-chloride-cotransporters, GABA is inhibitory or excitatory. There is evidence that VB and NRT differ in terms of GABA action, with classical hyperpolarization in VB due to the expression of the Cl− extruder KCC2 and depolarizing/excitatory GABA action in the NRT, where KCC2 expression is low and Cl− accumulation by the Cl− inward transporter NKCC1 has been postulated. However, data on NKCC1 expression and functional analysis of both transporters are missing. We show that KCC2-mediated Cl− extrusion set the [Cl−]i in VB, while NKCC1 did not contribute substantially to Cl− accumulation and depolarizing GABA action in the NRT. The finding that NKCC1 did not play a major role in NRT neurons is of high relevance for ongoing studies on the therapeutic use of NKCC1 inhibitors trying to compensate for a disease-induced up-regulation of NKCC1 that has been described for various brain regions and disease states like epilepsy and chronic pain. These data suggest that NKCC1 inhibitors might have no major effect on healthy NRT neurons due to limited NKCC1 function.

We recently reported the case of a young patient with multisystem failure carrying a de novo mutation in SLC12A2, the gene encoding the Na-K-2Cl cotransporter-1 (NKCC1). Heterologous expression studies in nonepithelial cells failed to demonstrate dominant-negative effects. In this study, we examined expression of the mutant cotransporter in epithelial cells. Using Madin-Darby canine kidney (MDCK) cells grown on glass coverslips, permeabilized support, and Matrigel, we show that the fluorescently tagged mutant cotransporter is expressed in cytoplasm and at the apical membrane and affects epithelium integrity. Expression of the mutant transporter at the apical membrane also results in the mislocalization of some of the wild-type transporter to the apical membrane. This mistargeting is specific to NKCC1 as the Na+-K+-ATPase remains localized on the basolateral membrane. To assess transporter localization in vivo, we created a mouse model using CRISPR/cas9 that reproduces the 11 bp deletion in exon 22 of Slc12a2. Although the mice do not display an overt phenotype, we show that the colon and salivary gland expresses wild-type NKCC1 abundantly at the apical pole, confirming the data obtained in cultured epithelial cells. Enough cotransporter must remain, however, on the basolateral membrane to participate in saliva secretion, as no significant decrease in saliva production was observed in the mutant mice.

Neuronal chloride regulation is a determinant factor for the dynamic tuning of GABAergic inhibition during and beyond brain development. This regulation is mainly dependent on the two co-transporters K+/Cl- co-transporter KCC2 and Na+/K+/Cl- co-transporter NKCC1, whose activity can decrease or increase neuronal chloride concentrations respectively. Altered expression and/or activity of either of these co-transporters has been associated with a wide variety of brain disorders including developmental disorders, epilepsy, schizophrenia and stroke. Here, we review current knowledge on chloride transporter expression and activity regulation and highlight the intriguing potential for existing and future interventions to support chloride homeostasis across a wide range of mental disorders and neurological conditions.

Purpose of review The kidney Na-K-2Cl cotransporter (NKCC2) is essential for urinary concentration and renal electrolyte handling. Loss of function mutations in the NKCC2 gene cause urinary salt and potassium wasting, whereas excessive NKCC2 function has been linked to high blood pressure. Loop diuretics, targeting the transporter, are instrumental for relieving edema or hypertension. This review focuses on intrinsic mechanisms regulating NKCC2 activity at the posttranslational level, namely its trafficking and phosphorylation. Recent findings Protein networks mediating cellular turnover of NKCC2 have recently received major attention. Several key components of its apical trafficking were identified, including respective chaperones, SNARE protein family members and raft-associated proteins. NKCC2 internalization has been characterized qualitatively and quantitatively. Kinase and phosphatase pathways regulating NKCC2 activity have been clarified and links between NKCC2 phosphorylation and trafficking proposed. Constitutive and inducible NKCC2 trafficking and phosphorylation mechanisms have been specified with focus on endocrine control of thick ascending limb (TAL) function by vasopressin. Summary Proper NKCC2 trafficking and phosphorylation are critical to the TAL function in the physiological context of urinary concentration and extracellular volume regulation. Clarification of the underlying mechanisms and respective protein networks may open new therapeutic perspectives for better management of renal electrolyte disorders and blood pressure control.

Antenatal Bartter syndrome (aBS) is a heterogenous disease resulting from defective ion transport in the thick ascending limb of the loop of Henle. Novel insights into the pathophysiology, as well as the recent identification of a novel genetic cause of aBS, merit an update on this topic.In aBS, severe salt losing is further aggravated by defective salt sensing in the macula densa, where a reduced tubular salt concentration is perceived and glomerular filtration is increased instead of decreased. As patients with aBS come of age, there is an increased incidence of proteinuria and impaired renal function.Moreover, we recently reported a new form of aBS. Indeed, we described a series of nine families in whom pregnancies with male fetuses where complicated by acute polyhydramnios, preterm delivery and with severe but transient polyuria. We identified mutations in melanoma-associated antigen D2 in all study participants and showed, in vivo and in vitro, reduced expression of the furosemide and thiazide sensitive transporters sodium-potassium-2-chloride cotransporter and sodium chloride cotransporter, respectively.Genetic studies revealed the complexity of ion transport in the thick ascending limb of the loop of Henle and will help to clarify the pathophysiology, which is essential to design new therapies.

Vasopressin (AVP) induces antidiuresis, thus playing an essential role in body water and electrolyte homeostasis. Its antidiuretic effects are mediated chiefly by V2 vasopressin receptors (V2R) expressed along the distal nephron and collecting duct epithelia. NaCl reabsorption in the distal nephron, which includes the thick ascending limb (TAL) and distal convoluted tubule (DCT), largely depends on the activity of two structurally related Na-(K)-Cl cotransporters, NKCC2 in TAL and NCC in DCT. AVP-induced activation of these transporters contributes to urine concentration and renal electrolyte reabsorption. Previous work has specified molecular pathways mediating the effects of V2R activation in TAL and DCT, and protein networks involved in intracellular trafficking and phosphoregulation of the two transporters have been identified. This review summarizes recent progress in understanding AVP signalling mechanisms that are responsible for the activation of NKCC2 and NCC. Implications in the pathophysiology of diseases such as nephrogenic diabetes insipidus, diabetes mellitus and salt-sensitive hypertension are discussed in this context.