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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.

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.

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.

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 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.

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.

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.

The goal of the present study was to investigate the functional implications of sexual dimorphism in the pattern of transporters along the rodent nephron as reported by Veiras et al. ( J Am Soc Nephrol 28: 3504–3517, 2017). To do so, we developed sex-specific computational models of water and solute transport along the superficial nephrons from male and female rat kidneys. The models account for the sex differences in the abundance of apical and basolateral transporters, single nephron glomerular filtration rate, and tubular dimensions. Model simulations predict that ~70% and 60% of filtered Na+ is reabsorbed by the proximal tubule of male and female rat kidneys, respectively. The lower fractional Na+ reabsorption in female kidneys is due primarily to their smaller transport area, lower Na+/H+ exchanger activity, and lower claudin-2 abundance, culminating in significantly larger fractional delivery of water and Na+ to the downstream nephron segments in female kidneys. Conversely, the female distal nephron exhibits a higher abundance of key Na+ transporters, including Na+-K+-Cl− cotransporters, Na+-Cl− cotransporters, and epithelial Na+ channels. The higher abundance of transporters accounts for the enhanced water and Na+ transport along the female, relative to male, distal nephron, resulting in similar urine excretion between the sexes. Consequently, in response to a saline load, the Na+ load delivered distally is greater in female rats than male rats, overwhelming transport capacity and resulting in higher natriuresis in female rats.

We studied sex differences in response to high K+(HK) intake on thiazide-sensitive cation (Na+and K+) excretion in wild-type (WT) and ANG II receptor subtype 1a (AT1aR) knockout (KO) mice. Renal clearance experiments were performed to examine Na+-Cl−cotransporter (NCC) activity on mice fed with control and HK (5% KCl, 7 days) diets. Hydrochlorothiazide (HCTZ)-induced changes in urine volume, glomerular filtration rate, absolute Na+and K+excretion, and fractional excretion were compared. HK-induced changes in NCC, Na+/H+exchanger isoform 3 (NHE3), and ENaC expression were examined by Western blot analysis. In WT animals under the control diet, HCTZ-induced cation excretion was greater in female animals, reflecting larger increases in Na+excretion, since there was little sex difference in HCTZ-induced K+excretion. Under the HK diet, the sex difference in HCTZ-induced cation excretion was reduced because of larger increments in K+excretion in male animals. The fraction of K+excretion was 57 ± 5% in male WT animals and 36 ± 4% in female WT animals ( P < 0.05), but this difference was absent in AT1aR KO mice. NCC abundance was higher in female animals than in male animals but decreased by similar fractions on HK diet. NHE3 abundance decreased, whereas cleaved forms of γ-ENaC increased, with HK in all groups; these changes were similar in male and female animals and were not significantly affected by AT1aR ablation. These results indicate that, with the HK diet, male animals display greater distal Na+delivery and greater activation of K+secretion mechanisms, all suggesting a more powerful male adaptation to HK intake.

Abnormally high epithelial Na+ channel (ENaC) activity in the aldosterone-sensitive distal nephron and collecting duct leads to hypertension. Myelin and lymphocyte (Mal) is a lipid raft-associated protein that has been previously shown to regulate Na+-K-2Cl− cotransporter and aquaporin-2 in the kidney, but it is not known whether it regulates renal ENaC. ENaC activity is positively regulated by the anionic phospholipid phosphate phosphatidylinositol 4,5-bisphosphate (PIP2). Members of the myristoylated alanine-rich C-kinase substrate (MARCKS) family increase PIP2 concentrations at the plasma membrane, whereas hydrolysis of PIP2 by phospholipase C (PLC) reduces PIP2 abundance. Our hypothesis was that Mal protein negatively regulates renal ENaC activity by stabilizing PLC protein expression at the luminal plasma membrane. We investigated the association between Mal, MARCKS-like protein, and ENaC. We showed Mal colocalizes with PLC-β3 in lipid rafts and positively regulates its protein expression, thereby reducing PIP2 availability at the plasma membrane. Kidneys of 129Sv mice injected with MAL shRNA lentivirus resulted in increased ENaC open probability in split-open renal tubules. Overexpression of Mal protein in mouse cortical collecting duct (mpkCCD) cells resulted in an increase in PLC-β3 protein expression at the plasma membrane. siRNA-mediated knockdown of MAL in mpkCCD cells resulted in a decrease in PLC-β3 protein expression and an increase in PIP2 abundance. Moreover, kidneys from salt-loaded mice showed less Mal membrane protein expression compared with non-salt-loaded mice. Taken together, Mal protein may play an essential role in the negative feedback of ENaC gating in principal cells of the collecting duct.

PURPOSE OF REVIEW: The apical cotransporter NKCC2 mediates NaCl reabsorption by the thick ascending limb (TAL), contributing to maintenance of blood pressure. Despite effective NKCC2 inhibition by loop diuretics, these agents are not viable for long-term management of blood pressure due to side effects. Novel molecular mechanisms that control NKCC2 activity reveal an increasingly complex picture with interacting layers of NKCC2 regulation. Here we review the latest developments that shine new light on NKCC2-mediated control of blood pressure and potential new long-term therapies to treat hypertension. RECENT FINDINGS: Emerging molecular NKCC2 regulators, often binding partners, reveal a complex overlay of interacting mechanisms aimed at fine tuning NKCC2 activity. Different factors achieve this by shifting the balance between trafficking steps like exocytosis, endocytosis, recycling and protein turnover, or by balancing phosphorylation vs. dephosphorylation. Further molecular details are also emerging on previously known pathways of NKCC2 regulation, and recent in vivo data continues to place NKCC2 regulation at the center of blood pressure control. SUMMARY: Several layers of emerging molecular mechanisms that control NKCC2 activity may operate simultaneously, but they can also be controlled independently. This provides an opportunity to identify new pharmacological targets to fine-tune NKCC2 activity for blood pressure management.

Type 4 Bartter syndrome (BS) is caused by genetic mutations in barttin, which is coded for by BSND. Barttin serves as the β-subunit of the ClC-K chloride (Cl-) channel, which is widely expressed in distal nephrons. Type 4 BS is characterized by severely impaired reabsorption of salt, which may cause polyuria, hypokalemia, and metabolic alkalosis. Calcineurin inhibitors reportedly induce renal salt retention and hyperkalemia by enhancing the phosphorylation of the sodium (Na+)-potassium (K+)-2Cl- cotransporter (NKCC2) and Na+-Cl- cotransporter (NCC). In addition, we have previously reported that tacrolimus, a calcineurin inhibitor, increases the levels of phosphorylated NCC. In this study, we administered tacrolimus to barttin hypomorphic (Bsndneo/neo) mice, a murine model of type 4 BS that exhibits polyuria, hypokalemia, and metabolic alkalosis. Administration of tacrolimus increased the serum K+ level and suppressed urinary K+ excretion. Furthermore, after treatment with tacrolimus, Bsndneo/neo mice increased levels of phosphorylated NCC and NKCC2. We conclude that tacrolimus partially improves clinical phenotypes of Bsndneo/neo mice, and that calcineurin inhibitors might be effective for treating type 4 BS.

We now have an increased understanding of the genetics, cell biology, and physiology of electrolyte transport processes in the mammalian intestine, due to the availability of sophisticated methodologies ranging from genome wide association studies to CRISPR-CAS technology, stem cell-derived organoids, 3D microscopy, electron cryomicroscopy, single cell RNA sequencing, transgenic methodologies, and tools to manipulate cellular processes at a molecular level. This knowledge has simultaneously underscored the complexity of biological systems and the interdependence of multiple regulatory systems. In addition to the plethora of mammalian neurohumoral factors and their cross talk, advances in pyrosequencing and metagenomic analyses have highlighted the relevance of the microbiome to intestinal regulation. This article provides an overview of our current understanding of electrolyte transport processes in the small and large intestine, their regulation in health and how dysregulation at multiple levels can result in disease. Intestinal electrolyte transport is a balance of ion secretory and ion absorptive processes, all exquisitely dependent on the basolateral Na+ /K+ ATPase; when this balance goes awry, it can result in diarrhea or in constipation. The key transporters involved in secretion are the apical membrane Cl- channels and the basolateral Na+ -K+ -2Cl- cotransporter, NKCC1 and K+ channels. Absorption chiefly involves apical membrane Na+ /H+ exchangers and Cl- /HCO3 - exchangers in the small intestine and proximal colon and Na+ channels in the distal colon. Key examples of our current understanding of infectious, inflammatory, and genetic diarrheal diseases and of constipation are provided. © 2019 American Physiological Society. Compr Physiol 9:947-1023, 2019.

Seizure incidence, severity, and antiseizure medication (ASM) efficacy varies between males and females. Differences in sex-dependent signaling pathways that determine network excitability may be responsible. The identification and validation of sex-dependent molecular mechanisms that influence seizure susceptibility is an emerging focus of neuroscience research. The electroneutral cation-chloride cotransporters (CCCs) of the SLC12A gene family utilize Na+-K+-ATPase generated electrochemical gradients to transport chloride into or out of neurons. CCCs regulate neuronal chloride gradients, cell volume, and have a strong influence over the electrical response to the inhibitory neurotransmitter GABA. Acquired or genetic causes of CCCs dysfunction have been linked to seizures during early postnatal development, epileptogenesis, and refractoriness to ASMs. A growing number of studies suggest that the developmental expression of CCCs, such as KCC2, is sex-dependent. This review will summarize the reports of sexual dimorphism in epileptology while focusing on the role of chloride cotransporters and their associated modulators that can influence seizure susceptibility.

In the early 80s, renal microperfusion studies led to the identification of a basolateral K(+)‐Cl(−) cotransport mechanism in the proximal tubule, thick ascending limb of Henle and collecting duct. More than ten years later, this mechanism was found to be accounted for by three different K(+)‐Cl(−) cotransporters (KCC1, KCC3 and KCC4) that are differentially distributed along the renal epithelium. Two of these isoforms (KCC1 and KCC3) were also found to be expressed in arterial walls, the myocardium and a variety of neurons. Subsequently, valuable insights have been gained into the molecular and physiological properties of the KCCs in both the mammalian kidney and cardiovascular system. There is now robust evidence indicating that KCC4 sustains distal renal acidification and that KCC3 regulates myogenic tone in resistance vessels. However, progress in understanding the functional significance of these transporters has been slow, probably because each of the KCC isoforms is not identically distributed among species and some of them share common subcellular localizations with other KCC isoforms or sizeable conductive Cl(−) pathways. In addition, the mechanisms underlying the process of K(+)‐Cl(−) cotransport are still ill defined. The present review focuses on the knowledge gained regarding the roles and properties of KCCs in renal and cardiovascular tissues.

Background Alterations of bioelectrical properties of cells and tissues are known to function as wide-ranging signals during development, regeneration and wound-healing in several species. The Drosophila follicle-cell epithelium provides an appropriate model system for studying the potential role of electrochemical signals, like intracellular pH (pHi) and membrane potential (Vmem), during development. Therefore, we analysed stage-specific gradients of pHi and Vmem as well as their dependence on specific ion-transport mechanisms. Results Using fluorescent indicators, we found distinct alterations of pHi- and Vmem-patterns during stages 8 to 12 of oogenesis. To determine the roles of relevant ion-transport mechanisms in regulating pHi and Vmem and in establishing stage-specific antero-posterior and dorso-ventral gradients, we used inhibitors of Na+/H+-exchangers and Na+-channels (amiloride), V-ATPases (bafilomycin), ATP-sensitive K+-channels (glibenclamide), voltage-dependent L-type Ca2+-channels (verapamil), Cl−-channels (9-anthroic acid) and Na+/K+/2Cl−-cotransporters (furosemide). Either pHi or Vmem or both parameters were affected by each tested inhibitor. While the inhibition of Na+/H+-exchangers (NHE) and amiloride-sensitive Na+-channels or of V-ATPases resulted in relative acidification, inhibiting the other ion-transport mechanisms led to relative alkalisation. The most prominent effects on pHi were obtained by inhibiting Na+/K+/2Cl−-cotransporters or ATP-sensitive K+-channels. Vmem was most efficiently hyperpolarised by inhibiting voltage-dependent L-type Ca2+-channels or ATP-sensitive K+-channels, whereas the impact of the other ion-transport mechanisms was smaller. In case of very prominent effects of inhibitors on pHi and/or Vmem, we also found strong influences on the antero-posterior and dorso-ventral pHi- and/or Vmem-gradients. For example, inhibiting ATP-sensitive K+-channels strongly enhanced both pHi-gradients (increasing alkalisation) and reduced both Vmem-gradients (increasing hyperpolarisation). Similarly, inhibiting Na+/K+/2Cl−-cotransporters strongly enhanced both pHi-gradients and reduced the antero-posterior Vmem-gradient. To minor extents, both pHi-gradients were enhanced and both Vmem-gradients were reduced by inhibiting voltage-dependent L-type Ca2+-channels, whereas only both pHi-gradients were reduced (increasing acidification) by inhibiting V-ATPases or NHE and Na+-channels. Conclusions Our data show that in the Drosophila follicle-cell epithelium stage-specific pHi- and Vmem-gradients develop which result from the activity of several ion-transport mechanisms. These gradients are supposed to represent important bioelectrical cues during oogenesis, e.g., by serving as electrochemical prepatterns in modifying cell polarity and cytoskeletal organisation. Electronic supplementary material The online version of this article (10.1186/s12861-019-0192-x) contains supplementary material, which is available to authorized users.

Children with repeated exposures to anesthesia at an early age are at an increased risk of cognitive impairment. Data in the literature link increased developmental depolarizing γ-aminobutyric acid (GABA) type A receptor (GABAAR) at younger age to neurodevelopmental disorders. Here we investigated the involvement of GABAergic signaling during development in mediating the adverse effects of repeated sevoflurane exposures.Sprague-Dawley male rats received repeated exposures to 3 % sevoflurane for 2 h daily for 3 consecutive days on postnatal days (P) 4, 5, and 6; maternally separated and unseparated rats served as controls. A subgroup of rats received three injections of the Na(+)-K(+)-2Cl(-) cotransporter inhibitor, bumetanide (1.82 mg/kg, intraperitoneally) 15 min prior to initiation of each sevoflurane exposure.The results showed that repeated neonatal sevoflurane exposures contribute to learning and memory impairment in the Morris water maze (MWM) at P60. The corticosterone level was significantly increased immediately after repeated neonatal sevoflurane exposures. Repeated neonatal sevoflurane exposures heightened the secretion of corticosterone in response to stress in P7 and P60 rats. Pretreatment of male rats prior to each sevoflurane exposure with bumetanide attenuated the corticosterone level immediately after repeated neonatal sevoflurane exposures, normalized endocrine response to stress at P7 and P60, and attenuated the sevoflurane-induced learning and memory impairment in the MWM.These data suggested that the heightened stress response and cognitive impairment after repeated neonatal sevoflurane exposures might be linked to excessive GABAAR-mediated depolarization.

The solute carrier family 12, as numbered according to Human Genome Organisation (HUGO) nomenclature, encodes the electroneutral cation-coupled chloride cotransporters that are expressed in many cells and tissues; they play key roles in important physiological events, such as cell volume regulation, modulation of the intracellular chloride concentration, and transepithelial ion transport. Most of these family members are expressed in specific regions of the nephron. The Na-K-2Cl cotransporter NKCC2, which is located in the thick ascending limb, and the Na-Cl cotransporter, which is located in the distal convoluted tubule, play important roles in salt reabsorption and serve as the receptors for loop and thiazide diuretics, respectively (Thiazide diuretics are among the most commonly prescribed drugs in the world.). The activity of these transporters correlates with blood pressure levels; thus, their regulation has been a subject of intense research for more than a decade. The K-Cl cotransporters KCC1, KCC3, and KCC4 are expressed in several nephron segments, and their role in renal physiology is less understood but nevertheless important. Evidence suggests that they are involved in modulating proximal tubule glucose reabsorption, thick ascending limb salt reabsorption and collecting duct proton secretion. In this work, we present an overview of the physiological roles of these transporters in the kidney, with particular emphasis on the knowledge gained in the past few years.

scl is a spontaneous medaka mutant deficient in P450c17I, which is required for production of sex steroids, but not of cortisol, the major role of which is osmoregulation in teleost fish. The scl mutant provides a new model to study the functions of these hormones. We first found that fish homozygous for this mutation have plasma cortisol constitutively at a high physiological level (1000 nM). Since we previously showed that this level reversed the seawater-type differentiation of the medaka gastrointestinal tract, hypoosmoregulation of the scl mutant was analyzed. Muscle water contents in freshwater were normal in scl homozygotes, but the contents were lower than those of the wild type (WT) after seawater transfer. There were no differences in gill mRNA levels of corticosteroid receptors or ion transporters between scl homozygotes and WT. In the intestine, expression of glucocorticoid receptors and Na(+)/K(+)/2Cl(-) cotransporter were induced in WT during seawater acclimation, but not in scl homozygotes. The high plasma cortisol may prevent hypoosmoregulation by inhibition of increased intestinal water absorption, essentially by the Na(+)/K(+)/2Cl(-) cotransporter, in seawater.

Members of With-no-lysine (WNK) family of serine-threonine kinase are key regulators of chloride ion transport in diverse cell types, controlling the activity and the surface expression of cation-chloride (Na + /K + -Cl − ) co-transporters. Mutations in WNK1 and WNK4 are linked to a hereditary form of hypertension, and WNKs have been extensively investigated pertaining to their roles in renal epithelial ion homeostasis. However, some members of the WNK family and their splice isoforms are also expressed in the mammalian brain, and have been implicated in aspects of hereditary neuropathy as well as neuronal and glial survival. WNK2, which is exclusively enriched in neurons, is well known as an anti-proliferative tumor suppressor. WNK3, on the other hand, appears to promote cell survival as its inhibition enhances neuronal apoptosis. However, loss of WNK3 has been recently shown to reduce ischemia-associated brain damage. In this review, I surveyed the potentially context-dependent roles of WNKs in neurological disorders and neuronal survival.

The furosemide-sensitive Na+-K+-2Cl(-)-cotransporter (NKCC2) is crucial for NaCl reabsorption in kidney thick ascending limb (TAL) and drives the urine concentrating mechanism. NKCC2 activity is modulated by N-terminal phosphorylation and dephosphorylation. Serine-threonine kinases that activate NKCC2 have been identified, but less is known about phosphatases that deactivate NKCC2. Inhibition of calcineurin phosphatase has been shown to stimulate transport in the TAL and the distal convoluted tubule. Here, we identified NKCC2 as a target of the calcineurin A beta isoform. Short-term cyclosporine administration in mice augmented the abundance of phospho-NKCC2, and treatment of isolated TAL with cyclosporine increased the chloride affinity and transport activity of NKCC2. Because sorting-related receptor with A-type repeats (SORLA) may affect NKCC2 phosphoregulation, we used SORLA-knockout mice to test whether SORLA is involved in calcineurin-dependent modulation of NKCC2. SORLA-deficient mice showed more calcineurin A beta in the apical region of TAL cells and less NKCC2 phosphorylation and activity compared with littermate controls. In contrast, overexpression of SORLA in cultured cells reduced the abundance of endogenous calcineurin A beta. Cyclosporine administration rapidly normalized the abundance of phospho-NKCC2 in SORLA-deficient mice, and a functional interaction between calcineurin A beta and SORLA was further corroborated by binding assays in rat kidney extracts. In summary, we have shown that calcineurin A beta and SORLA are key components in the phosphoregulation of NKCC2. These results may have clinical implications for imnnunosuppressive therapy using calcineurin inhibitors.

Background Dietary sodium intake regulates the thiazide-sensitive Na-Cl cotransporter (NCC) in the distal convoluted tubule (DCT). Whether the basolateral, inwardly rectifying potassium channel Kir4.1/Kir5.1 (a heterotetramer of Kir4.1/Kir5.1) in the DCT is essential for mediating the effect of dietary sodium intake on NCC activity is unknown. Methods We used electrophysiology, renal clearance techniques, and immunoblotting to examine effects of Kir4.1/Kir5.1 in the DCT and NCC in wild-type and kidney-specific Kir4.1 knockout mice. Results Low sodium intake stimulated basolateral Kir4.1/Kir5.1 activity, increased basolateral K + conductance, and hyperpolarized the membrane. Conversely, high sodium intake inhibited the potassium channel, decreased basolateral K + currents, and depolarized the membrane. Low sodium intake increased total and phosphorylated NCC expression and augmented hydrochlorothiazide-induced natriuresis; high sodium intake had opposite effects. Thus, elevated NCC activity induced by low sodium intake was associated with upregulation of Kir4.1/Kir5.1 activity in the DCT, whereas inhibition of NCC activity by high sodium intake was associated with diminished Kir4.1/Kir5.1 activity. In contrast, dietary sodium intake did not affect NCC activity in knockout mice. Further, Kir4.1 deletion not only abolished basolateral K + conductance and depolarized the DCT membrane, but also abrogated the stimulating effects induced by low sodium intake on basolateral K + conductance and hyperpolarization. Finally, dietary sodium intake did not alter urinary potassium excretion rate in hypokalemic knockout and wild-type mice. Conclusions Stimulation of Kir4.1/Kir5.1 by low intake of dietary sodium is essential for NCC upregulation, and inhibition of Kir4.1/Kir5.1 induced by high sodium intake is a key step for downregulation of NCC.

Cerebral edema is exacerbated in diabetic ischemic stroke through poorly understood mechanisms. We showed previously that blood–brain barrier (BBB) Na–K–Cl cotransport (NKCC) and Na/H exchange (NHE) are major contributors to edema formation in normoglycemic ischemic stroke. Here, we investigated whether hyperglycemia-exacerbated edema involves changes in BBB NKCC and NHE expression and/or activity and whether inhibition of NKCC or NHE effectively reduces edema and injury in a type I diabetic model of hyperglycemic stroke. Cerebral microvascular endothelial cell (CMEC) NKCC and NHE abundances and activities were determined by Western blot, radioisotopic flux and microspectrofluorometric methods. Cerebral edema and Na in rats subjected to middle cerebral artery occlusion (MCAO) were assessed by nuclear magnetic resonance methods. Hyperglycemia exposures of 1-7d significantly increased CMEC NKCC and NHE abundance and activity. Subsequent exposure to ischemic factors caused more robust increases in NKCC and NHE activities than in normoglycemic CMEC. MCAO-induced edema and brain Na uptake were greater in hyperglycemic rats. Intravenous bumetanide and HOE-642 significantly attenuated edema, brain Na uptake and ischemic injury. Our findings provide evidence that BBB NKCC and NHE contribute to increased edema in hyperglycemic stroke, suggesting that these Na transporters are promising therapeutic targets for reducing damage in diabetic stroke.

The molecular mechanisms of melamine-induced renal toxicity have not been fully understood. The purpose of the study aimed to investigate whether melamine and cyanuric acid induced NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation in the kidney, which may contribute to abnormal water and sodium handling in a rat model. Wistar rats received melamine (Mel; 200 mg·kg body wt−1·day−1), cyanuric acid (CA; 200 mg·kg body wt−1·day−1), or Mel plus CA (Mel + CA; 100 mg·kg body wt−1·day−1, each) for 2 wk. Mel + CA caused damaged tubular epithelial structure and organelles, dilated tubular lumen, and inflammatory responses. Crystals were observed in urine and serum specimen, also in the lumen of dilated distal renal tubules. The combined ingestion of Mel and CA in rats caused a markedly impaired urinary concentration, which was associated with reduced protein expression of aquaporin (AQP)1, 2, and 3 in inner medulla and α-Na-K-ATPase and Na-K-2Cl transporters in cortex and outer medulla. Mel + CA treatment was associated with increased protein expression of CD3 and mRNA levels of CD68 and F4/80 as well as phosphorylation of NF-κB in the kidney. Mel + CA treatment increased protein and mRNA expression of NLRP3 inflammasome components apoptosis-associated speck-like protein containing a caspase recruitment domain, caspase-1, and IL-1β in the inner medulla of rats. NF-κB inhibitor Bay 11-7082 reduced IL-1β expression induced by Mel + CA and prevented downregulation of AQP2 in inner medullary collecting duct cell suspensions. In conclusion, Mel + CA treatment caused urinary-concentrating defects and reduced expression of renal AQPs and key sodium transporters, which is likely due to the inflammatory responses and activation of NLRP3 inflammasome induced by crystals formed in the kidney.

Postcapillary venules (PCVs) play a critical role in regulating capillary hydrostatic pressure, but their contractile mechanisms are not well understood. We examined the properties of spontaneous vasomotion and corresponding Ca(2+) transients in gastric PCV. In the rat gastric submucosa, changes in PCV diameter and intracellular Ca(2+) dynamics were visualised by video tracking system and fluorescent Ca(2+) imaging, respectively, while PCV morphology was examined by immunohistochemistry. Stellate-shaped PCV mural cells expressing α-smooth muscle actin exhibited synchronised spontaneous Ca(2+) transients to develop vasomotion which was abolished by nifedipine (1 μM), cyclopiazonic acid (10 μM), or Ca(2+)-activated Cl(-) channel inhibitors (100 μM niflumic acid, 1 μM T16Ainh-A01). A gap junction blocker (3 μM carbenoxolone) disrupted the synchrony of spontaneous Ca(2+) transients amongst PCV mural cells and attenuated spontaneous vasomotion. Low chloride solution ([Cl(-)]0 = 12.4 mM) also disrupted the synchrony of spontaneous Ca(2+) transients and abolished vasomotion. Na(+)-K(+)-Cl(-) co-transporter inhibitors (10 μM bumetanide, 30 μM furosemide) suppressed spontaneous Ca(2+) transients and vasoconstrictions. A phosphodiesterase type 5 (PDE5) inhibitor (1 μM tadalafil) disrupted the spontaneous Ca(2+) transient synchrony and abolished vasomotion in a nitric oxide (NO)-dependent manner. Thus, gastric PCVs exhibit spontaneous vasomotion, resulting from synchronised spontaneous Ca(2+) transients within a network of stellate-shaped PCV mural cells. An active Cl(-) accumulation partly via Na(+)-K(+)-Cl(-) co-transport appears to be fundamental in maintaining depolarisation upon the opening of Ca(2+)-activated Cl(-) channels that triggers Ca(2+) influx via voltage-dependent L-type Ca(2+) channels. Basal PDE5 activity may continuously counteract vaso-relaxing effects of endothelial NO to maintain spontaneous vasomotion.

The cellular mechanisms of calcification in sea urchin larvae are still not well understood. Primary mesenchyme cells within the larval body cavity form a syncytium to secrete CaCO3 spicules from intracellular amorphous CaCO3 (ACC) stores. We studied the role of Na(+)K(+)2Cl(-) cotransporter (NKCC) in intracellular ACC accumulation and larval spicule formation of Strongylocentrotus droebachiensis. First, we incubated growing larvae with three different loop diuretics (azosemide, bumetanide, and furosemide) and established concentration-response curves. All loop diuretics were able to inhibit calcification already at concentrations that specifically inhibit NKCC. Calcification was most effectively inhibited by azosemide (IC50=6.5 μM), while larval mortality and swimming ability were not negatively impacted by the treatment. The inhibition by bumetanide (IC50=26.4 μM) and furosemide (IC50=315.4 μM) resembled the pharmacological fingerprint of the mammalian NKCC1 isoform. We further examined the effect of azosemide on the maintenance of cytoplasmic cords and on the occurrence of calcification vesicles using fluorescent dyes (calcein, FM1-43). Fifty micromolars of azosemide inhibited the maintenance of cytoplasmic cords and resulted in increased calcein fluorescence within calcification vesicles. The expression of NKCC in S. droebachiensis was verified by PCR and Western blot with a specific NKCC antibody. In summary, the pharmacological profile of loop diuretics and their specific effects on calcification in sea urchin larvae suggest that they act by inhibition of NKCC via repression of cytoplasmic cord formation and maintenance.

Cyst enlargement in autosomal dominant polycystic kidney disease (ADPKD) requires the transepithelial secretion of fluid into the cyst lumen. We previously showed that physiological amounts of ouabain enhance cAMP-dependent fluid secretion and cyst growth of human ADPKD cyst epithelial cells in culture and formation of cyst-like dilations in metanephric kidneys from Pkd1 mutant mice. Here, we investigated the mechanisms by which ouabain promotes cAMP-dependent fluid secretion and cystogenesis. Ouabain (3 nM) enhanced cAMP-induced cyst-like dilations in embryonic kidneys from Pkd1 m1Bei mice, but had no effect on metanephroi from Pkd1 m1Bei mice that lack expression of the cystic fibrosis transmembrane conductance regulator (CFTR). Similarly, ouabain stimulation of cAMP-induced fluid secretion and in vitro cyst growth of ADPKD cells were abrogated by CFTR inhibition, showing that CFTR is required for ouabain effects on ADPKD fluid secretion. Moreover, ouabain directly enhanced the cAMP-dependent Cl− efflux mediated by CFTR in ADPKD monolayers. Ouabain increased the trafficking of CFTR to the plasma membrane and up-regulated the expression of the CFTR activator PDZK1. Finally, ouabain decreased plasma membrane expression and activity of the Na,K-ATPase in ADPKD cells. Altogether, these results show that ouabain enhances net fluid secretion and cyst formation by activating apical anion secretion via CFTR and decreasing basolateral Na+ transport via Na,K-ATPase. These results provide new information on the mechanisms by which ouabain affects ADPKD cells and further highlight the importance of ouabain as a non-genomic stimulator of cystogenesis in ADPKD.

Eccrine sweat glands help to maintain homoeostasis, primarily by stabilizing body temperature. Derived from embryonic ectoderm, millions of eccrine glands are distributed across human skin and secrete litres of sweat per day. Their easy accessibility has facilitated the start of analyses of their development and function. Mouse genetic models find sweat gland development regulated sequentially by Wnt, Eda and Shh pathways, although precise subpathways and additional regulators require further elucidation. Mature glands have two secretory cell types, clear and dark cells, whose comparative development and functional interactions remain largely unknown. Clear cells have long been known as the major secretory cells, but recent studies suggest that dark cells are also indispensable for sweat secretion. Dark cell-specific Foxa1 expression was shown to regulate a Ca(2+) -dependent Best2 anion channel that is the candidate driver for the required ion currents. Overall, it was shown that cholinergic impulses trigger sweat secretion in mature glands through second messengers - for example InsP3 and Ca(2+) - and downstream ion channels/transporters in the framework of a Na(+) -K(+) -Cl(-) cotransporter model. Notably, the microenvironment surrounding secretory cells, including acid-base balance, was implicated to be important for proper sweat secretion, which requires further clarification. Furthermore, multiple ion channels have been shown to be expressed in clear and dark cells, but the degree to which various ion channels function redundantly or indispensably also remains to be determined.

Upon activation by with-no-lysine kinases, STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) phosphorylates and activates SLC12A transporters such as the Na(+)-Cl(-) cotransporter (NCC) and Na(+)-K(+)-2Cl(-) cotransporter type 1 (NKCC1) and type 2 (NKCC2); these transporters have important roles in regulating BP through NaCl reabsorption and vasoconstriction. SPAK knockout mice are viable and display hypotension with decreased activity (phosphorylation) of NCC and NKCC1 in the kidneys and aorta, respectively. Therefore, agents that inhibit SPAK activity could be a new class of antihypertensive drugs with dual actions (i.e., NaCl diuresis and vasodilation). In this study, we developed a new ELISA-based screening system to find novel SPAK inhibitors and screened >20,000 small-molecule compounds. Furthermore, we used a drug repositioning strategy to identify existing drugs that inhibit SPAK activity. As a result, we discovered one small-molecule compound (Stock 1S-14279) and an antiparasitic agent (Closantel) that inhibited SPAK-regulated phosphorylation and activation of NCC and NKCC1 in vitro and in mice. Notably, these compounds had structural similarity and inhibited SPAK in an ATP-insensitive manner. We propose that the two compounds found in this study may have great potential as novel antihypertensive drugs.

Patients with advanced cirrhosis have abnormal sodium homoeostasis. The study was undertaken to quantify the sodium transport across the plasma membrane of red blood cells (RBC) in patients with cirrhosis. RBC efflux and influx of sodium were studied in vitro with tracer (22) Na(+) according to linear kinetics in 24 patients with cirrhosis and 14 healthy controls. The sodium efflux was modified by ouabain (O), furosemide (F) and a combination of O and F (O + F). RBC sodium was significantly decreased (4·6 versus control 6·3 mmol l(-1) , P

Intracellular chloride ([Cl-](i)) andpH(pH(i)) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl-](i) and pH(i), but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl-and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl-](i) and pH(i) at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl-](i) and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic two-photon excitation protocol to simultaneously determine the changes of pHi and [Cl-](i) in response to hypercapnia and seizure activity.

BACKGROUND Liver cirrhosis is associated with enhanced renal tubular sodium retention, the mechanism of which is still debated. We hypothesized that liver cirrhosis is associated with increased expression of renal epithelial sodium transporter(s). METHODS Liver cirrhosis was induced by bile duct ligation (BDL) in rats. Steady state mRNA of ENaC subunits alpha, beta, gamma serum and glucocorticoid inducible kinase (Sgk1) were measured by TaqMan PCR in kidney homogenates at week 1, 2, 3 and 4 after BDL. Renal protein content of ENaC subunits, ubiquitin-protein-ligase Nedd4-2 and NaCl cotransporter (NCC) were assessed by western blot. Subcellular localization of ENaC subunits and NCC were analysed by immunohistochemistry. RESULTS Steady state mRNA of ENaC alpha, beta and gamma were unchanged during the 4 weeks investigated, while ENaC protein decreased most prominently at week 2 (control vs BDL; alpha, -46%; beta, -81%; and gamma, -63%; n = 6). Subcellular localization of ENaC subunits was not altered at week 2. Sgk1 mRNA did not change, whereas Nedd4-2 protein was reduced by >50% 2-4 weeks after BDL. NCC protein significantly increased at week 1 (control vs BDL: +66%, n = 6, P

BACKGROUND: Little information is available on a long-term follow-up in Bartter syndrome type I and II. METHODS: Clinical presentation, treatment and long-term follow-up (5.0-21, median 11 years) were evaluated in 15 Italian patients with homozygous (n = 7) or compound heterozygous (n = 8) mutations in the SLC12A1 (n = 10) or KCNJ1 (n = 5) genes. RESULTS: Thirteen new mutations were identified. The 15 children were born pre-term with a normal for gestational age body weight. Medical treatment at the last follow-up control included supplementation with potassium in 13, non-steroidal anti-inflammatory agents in 12 and gastroprotective drugs in five patients. At last follow-up, body weight and height were within normal ranges in the patients. Glomerular filtration rate was