Predicting sex differences in the effects of diuretics in renal epithelial transport during angiotensin II-induced hypertension.

Chronic angiotensin II (ANG II) infusion is an experimental model that induces hypertension in rodents. The natriuresis, diuresis, and blood pressure responses differ between males and females. This is perhaps not unexpected, given the rodent kidney, which plays a key role in blood pressure regulation, exhibits marked sex differences. Under normotensive conditions, compared with males, the female rat nephron exhibits lower Na+/H+ exchanger 3 (NHE3) activity along the proximal tubule but higher Na+ transporter activities along the distal segments. ANG II infusion-induced hypertension induces a pressure natriuretic response that reduces NHE3 activity and shifts Na+ transport capacity downstream. The goals of this study were to apply a computational model of epithelial transport along a rat nephron 1) to understand how a 14-day ANG II infusion impacts segmental electrolyte transport in male and female rat nephrons and 2) to identify and explain any sex differences in the effects of loop diuretics, thiazide diuretics, and K+-sparing diuretics. Model simulations suggest that the NHE3 downregulation in the proximal tubule is a major contributor to natriuresis and diuresis in hypertension, with the effects stronger in males. All three diuretics are predicted to induce stronger natriuretic and diuretic effects under hypertension compared with normotension, with relative increases in sodium excretion higher in hypertensive females than in males. The stronger natriuretic responses can be explained by the downstream shift of Na+ transport load in hypertension and by the larger distal transport load in females, both of which limit the ability of the distal segments to further elevate their Na+ transport.NEW & NOTEWORTHY Sex differences in the prevalence of hypertension are found in human and animal models. The kidney, which regulates blood pressure, exhibits sex differences in morphology, hemodynamics, and membrane transporter distributions. This computational modeling study provides insights into how the sexually dimorphic responses to a 14-day angiotensin II infusion differentially impact segmental electrolyte transport in rats. Simulations of diuretic administration explain how the natriuretic and diuretic effects differ between normotension and hypertension and between the sexes.

Bumetanide induces post-traumatic microglia-interneuron contact to promote neurogenesis and recovery.

Although the Na-K-Cl cotransporter (NKCC1) inhibitor bumetanide has prominent positive effects on the pathophysiology of many neurological disorders, the mechanism of action is obscure. Attention paid to elucidating the role of Nkcc1 has mainly been focused on neurons, but recent single cell mRNA sequencing analysis has demonstrated that the major cellular populations expressing NKCC1 in the cortex are non-neuronal. We used a combination of conditional transgenic animals, in vivo electrophysiology, two-photon imaging, cognitive behavioural tests and flow cytometry to investigate the role of Nkcc1 inhibition by bumetanide in a mouse model of controlled cortical impact (CCI). Here, we found that bumetanide rescues parvalbumin-positive interneurons by increasing interneuron-microglia contacts shortly after injury. The longitudinal phenotypic changes in microglia were significantly modified by bumetanide, including an increase in the expression of microglial-derived BDNF. These effects were accompanied by the prevention of CCI-induced decrease in hippocampal neurogenesis. Treatment with bumetanide during the first week post-CCI resulted in significant recovery of working and episodic memory as well as changes in theta band oscillations 1 month later. These results disclose a novel mechanism for the neuroprotective action of bumetanide mediated by an acceleration of microglial activation dynamics that leads to an increase in parvalbumin interneuron survival following CCI, possibly resulting from increased microglial BDNF expression and contact with interneurons. Salvage of interneurons may normalize ambient GABA, resulting in the preservation of adult neurogenesis processes as well as contributing to bumetanide-mediated improvement of cognitive performance.

Heterogeneous brain distribution of bumetanide following systemic administration in rats.

Bumetanide is used widely as a tool and off-label treatment to inhibit the Na-K-2Cl cotransporter NKCC1 in the brain and thereby to normalize intra-neuronal chloride levels in several brain disorders. However, following systemic administration, bumetanide only poorly penetrates into the brain parenchyma and does not reach levels sufficient to inhibit NKCC1. The low brain penetration is a consequence of both the high ionization rate and plasma protein binding, which restrict brain entry by passive diffusion, and of brain efflux transport. In previous studies, bumetanide was determined in the whole brain or a few brain regions, such as the hippocampus. However, the blood-brain barrier and its efflux transporters are heterogeneous across brain regions, so it cannot be excluded that bumetanide reaches sufficiently high brain levels for NKCC1 inhibition in some discrete brain areas. Here, bumetanide was determined in 14 brain regions following i.v. administration of 10 mg/kg in rats. Because bumetanide is much more rapidly eliminated by rats than humans, its metabolism was reduced by pretreatment with piperonyl butoxide. Significant, up to 5-fold differences in regional bumetanide levels were determined with the highest levels in the midbrain and olfactory bulb and the lowest levels in the striatum and amygdala. Brain:plasma ratios ranged between 0.004 (amygdala) and 0.022 (olfactory bulb). Regional brain levels were significantly correlated with local cerebral blood flow. However, regional bumetanide levels were far below the IC50 (2.4 µM) determined previously for rat NKCC1. Thus, these data further substantiate that the reported effects of bumetanide in rodent models of brain disorders are not related to NKCC1 inhibition in the brain.

Influence of administration time and sex on natriuretic, diuretic, and kaliuretic effects of diuretics.

Sex differences in renal function and blood pressure have been widely described across many species. Blood pressure dips during sleep and peaks in the early morning. Similarly, glomerular filtration rate, filtered electrolyte loads, urine volume, and urinary excretion all exhibit notable diurnal rhythms, which reflect, in part, the regulation of renal transporter proteins by circadian clock genes. That regulation is sexually dimorphic; as such, sex and time of day are not two independent regulators of kidney function and blood pressure. The objective of the present study was to assess the effect of sex and administration time on the natriuretic and diuretic effects of loop, thiazide, and K+-sparing diuretics, which are common treatments for hypertension. Loop diuretics inhibit Na+-K+-2Cl- cotransporters on the apical membrane of the thick ascending limb, thiazide diuretics inhibit Na+-Cl- cotransporters on the distal convoluted tubule, and K+-sparing diuretics inhibit epithelial Na+ channels on the connecting tubule and collecting duct. We simulated Na+ transporter inhibition using sex- and time-of-day-specific computational models of mouse kidney function. The simulation results highlighted significant sex and time-of-day differences in the drug response. Loop diuretics induced larger natriuretic and diuretic effects during the active phase. The natriuretic and diuretic effects of thiazide diuretics exhibited sex and time-of-day differences, whereas these effects of K+-sparing diuretics exhibited a significant time-of-day difference in females only. The kaliuretic effect depended on the type of diuretics and time of administration. The present computational models can be a useful tool in chronotherapy, to tailor drug administration time to match the body's diurnal rhythms to optimize the drug effect.NEW & NOTEWORTHY Sex influences cardiovascular disease, and the timing of onset of acute cardiovascular events exhibits circadian rhythms. Kidney function also exhibits sex differences and circadian rhythms. How do the natriuretic and diuretic effects of diuretics, a common treatment for hypertension that targets the kidneys, differ between the sexes? And how do these effects vary during the day? To answer these questions, we conducted computer simulations to assess the effects of loop, thiazide, and K+-sparing diuretics.

Astrocytic NKCC1 inhibits seizures by buffering Cl- and antagonizing neuronal NKCC1 at GABAergic synapses.

OBJECTIVE: A pathological excitatory action of the major inhibitory neurotransmitter γ-aminobutyric acid (GABA) has been observed in epilepsy. Blocking the Cl- importer NKCC1 with bumetanide is expected to reduce the neuronal intracellular Cl- concentration ([Cl- ]i ) and thereby attenuate the excitatory GABA response. Accordingly, several clinical trials of bumetanide for epilepsy were conducted. Although NKCC1 is expressed in both neurons and glial cells, an involvement of glial NKCC1 in seizures has not yet been reported. Astrocytes maintain high [Cl- ]i with NKCC1, and this gradient promotes Cl- efflux via the astrocytic GABAA receptor (GABAA R). This Cl- efflux buffers the synaptic cleft Cl- concentration to maintain the postsynaptic Cl- gradient during intense firing of GABAergic neurons, thereby sustaining its inhibitory action during seizure. In this study, we investigated the function of astrocytic NKCC1 in modulating the postsynaptic action of GABA in acute seizure models. METHODS: We used the astrocyte-specific conditional NKCC1 knockout (AstroNKCC1KO) mice. The seizurelike events (SLEs) in CA1 pyramidal neurons were triggered by tetanic stimulation of stratum radiatum in acute hippocampus slices. The SLE underlying GABAA R-mediated depolarization was evaluated by applying the GABAA R antagonist bicuculline. The pilocarpine-induced seizure in vivo was monitored in adult mice by the Racine scale. The SLE duration and tetanus stimulation intensity threshold and seizure behavior in AstroNKCC1KO mice and wild-type (WT) mice were compared. RESULTS: The AstroNKCC1KO mice were prone to seizures with lower threshold and longer duration of SLEs and larger GABAA R-mediated depolarization underlying the SLEs, accompanied by higher Racine-scored seizures. Bumetanide reduced these indicators of seizure in AstroNKCC1KO mice (which still express neuronal NKCC1), but not in the WT, both in vitro and in vivo. SIGNIFICANCE: Astrocytic NKCC1 inhibits GABA-mediated excitatory action during seizures, whereas neuronal NKCC1 has the converse effect, suggesting opposing actions of bumetanide on these cells.

The loop diuretic torasemide but not azosemide potentiates the anti-seizure and disease-modifying effects of midazolam in a rat model of birth asphyxia.

Loop diuretics such as furosemide and bumetanide, which act by inhibiting the Na-K-2Cl cotransporter NKCC2 at the thick ascending limb of the loop of Henle, have been shown to exert anti-seizure effects. However, the exact mechanism of this effect is not known. For bumetanide, it has been suggested that inhibition of the NKCC isoform NKCC1 in the membrane of brain neurons may be involved; however, NKCC1 is expressed by virtually all cell types in the brain, which makes any specific targeting of neuronal NKCC1 by bumetanide impossible. In addition, bumetanide only poorly penetrates the brain. We have previously shown that loop diuretics azosemide and torasemide also potently inhibit NKCC1. In contrast to bumetanide and furosemide, azosemide and torasemide lack a carboxylic group, which should allow them to better penetrate through biomembranes by passive diffusion. Because of the urgent medical need to develop new treatments for neonatal seizures and their adverse outcome, we evaluated the effects of azosemide and torasemide, administered alone or in combination with phenobarbital or midazolam, in a rat model of birth asphyxia and neonatal seizures. Neither diuretic suppressed the seizures when administered alone but torasemide potentiated the anti-seizure effect of midazolam. Brain levels of torasemide were below those needed to inhibit NKCC1. In addition to suppressing seizures, the combination of torasemide and midazolam, but not midazolam alone, prevented the cognitive impairment of the post-asphyxial rats at 3 months after asphyxia. Furthermore, aberrant mossy fiber sprouting in the hippocampus was more effectively prevented by the combination. We assume that either an effect on NKCC1 at the blood-brain barrier and/or cells in the periphery or the NKCC2-mediated diuretic effect of torasemide are involved in the present findings. Our data suggest that torasemide may be a useful option for improving the treatment of neonatal seizures and their adverse outcome.

Diuretics in States of Volume Overload: Core Curriculum 2022.

Volume overload, defined as excess total body sodium and water with expansion of extracellular fluid volume, characterizes common disorders such as congestive heart failure, end-stage liver disease, chronic kidney disease, and nephrotic syndrome. Diuretics are the cornerstone of therapy for volume overload and comprise several classes whose mechanisms of action, pharmacokinetics, indications, and adverse effects are essential principles of nephrology. Loop diuretics are typically the first-line treatment in the management of hypervolemia, with additional drug classes indicated in cases of diuretic resistance and electrolyte or acid-base disorders. Separately, clinical trials highlight improved outcomes in some states of volume overload, such as loop diuretics and sodium/glucose cotransporter 2 inhibitors in patients with congestive heart failure. Resistance to diuretics is a frequent, multifactorial clinical challenge that requires creative and physiology-based solutions. In this installment of AJKD's Core Curriculum in Nephrology, we discuss the pharmacology and therapeutic use of diuretics in states of volume overload and strategies to overcome diuretic resistance.

The role of HCO3- in propionate-induced anion secretion across rat caecal epithelium.

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-induced Isc (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 reduced IProp suggesting that a large part of this current must be carried by an ion other than Cl-. In the aboral caecum, IProp was significantly inhibited by mucosally administered stilbene derivatives (SITS, DIDS, DNDS), which block anion exchangers. Serosal Na+-free buffer reduced IProp significantly 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.

Vascular control of kidney epithelial transporters.

A major pathway in hypertension pathogenesis involves direct activation of ANG II type 1 (AT1) receptors in the kidney, stimulating Na+ reabsorption. AT1 receptors in tubular epithelia control expression and stimulation of Na+ transporters and channels. Recently, we found reduced blood pressure and enhanced natriuresis in mice with cell-specific deletion of AT1 receptors in smooth muscle (SMKO mice). Although impaired vasoconstriction and preserved renal blood flow might contribute to exaggerated urinary Na+ excretion in SMKO mice, we considered whether alterations in Na+ transporter expression might also play a role; therefore, we carried out proteomic analysis of key Na+ transporters and associated proteins. Here, we show that levels of Na+-K+-2Cl- cotransporter isoform 2 (NKCC2) and Na+/H+ exchanger isoform 3 (NHE3) are reduced at baseline in SMKO mice, accompanied by attenuated natriuretic and diuretic responses to furosemide. During ANG II hypertension, we found widespread remodeling of transporter expression in wild-type mice with significant increases in the levels of total NaCl cotransporter, phosphorylated NaCl cotransporter (Ser71), and phosphorylated NKCC2, along with the cleaved, activated forms of the α- and γ-epithelial Na+ channel. However, the increases in α- and γ-epithelial Na+ channel with ANG II were substantially attenuated in SMKO mice. This was accompanied by a reduced natriuretic response to amiloride. Thus, enhanced urinary Na+ excretion observed after cell-specific deletion of AT1 receptors from smooth muscle cells is associated with altered Na+ transporter abundance across epithelia in multiple nephron segments. These findings suggest a system of vascular-epithelial in the kidney, modulating the expression of Na+ transporters and contributing to the regulation of pressure natriuresis.NEW & NOTEWORTHY The use of drugs to block the renin-angiotensin system to reduce blood pressure is common. However, the precise mechanism for how these medications control blood pressure is incompletely understood. Here, we show that mice lacking angiotensin receptors specifically in smooth muscle cells lead to alternation in tubular transporter amount and function. Thus, demonstrating the importance of vascular-tubular cross talk in the control of blood pressure.

CATION-CHLORIDE CO-TRANSPORTER 1 (CCC1) Mediates Plant Resistance against Pseudomonas syringae.

Plasma membrane (PM) depolarization functions as an initial step in plant defense signaling pathways. However, only a few ion channels/transporters have been characterized in the context of plant immunity. Here, we show that the Arabidopsis (Arabidopsis thaliana) Na+:K+:2Cl- (NKCC) cotransporter CCC1 has a dual function in plant immunity. CCC1 functions independently of PM depolarization and negatively regulates pathogen-associated molecular pattern-triggered immunity. However, CCC1 positively regulates plant basal and effector-triggered resistance to Pseudomonas syringae pv. tomato (Pst) DC3000. In line with the compromised immunity to Pst DC3000, ccc1 mutants show reduced expression of genes encoding enzymes involved in the biosynthesis of antimicrobial peptides, camalexin, and 4-OH-ICN, as well as pathogenesis-related proteins. Moreover, genes involved in cell wall and cuticle biosynthesis are constitutively down-regulated in ccc1 mutants, and the cell walls of these mutants exhibit major changes in monosaccharide composition. The role of CCC1 ion transporter activity in the regulation of plant immunity is corroborated by experiments using the specific NKCC inhibitor bumetanide. These results reveal a function for ion transporters in immunity-related cell wall fortification and antimicrobial biosynthesis.

Targeting the WNK-SPAK/OSR1 Pathway and Cation-Chloride Cotransporters for the Therapy of Stroke.

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.

Renal lymphatic vessel dynamics.

Similar to other organs, renal lymphatics remove excess fluid, solutes, and macromolecules from the renal interstitium. Given the kidney's unique role in maintaining body fluid homeostasis, renal lymphatics may be critical in this process. However, little is known regarding the pathways involved in renal lymphatic vessel function, and there are no studies on the effects of drugs targeting impaired interstitial clearance, such as diuretics. Using pressure myography, we showed that renal lymphatic collecting vessels are sensitive to changes in transmural pressure and have an optimal range of effective pumping. In addition, they are responsive to vasoactive factors known to regulate tone in other lymphatic vessels including prostaglandin E2 and nitric oxide, and their spontaneous contractility requires Ca2+ and Cl-. We also demonstrated that Na+-K+-2Cl- cotransporter Nkcc1, but not Nkcc2, is expressed in extrarenal lymphatic vessels. Furosemide, a loop diuretic that inhibits Na+-K+-2Cl- cotransporters, induced a dose-dependent dilation in lymphatic vessels and decreased the magnitude and frequency of spontaneous contractions, thereby reducing the ability of these vessels to propel lymph. Ethacrynic acid, another loop diuretic, had no effect on vessel tone. These data represent a significant step forward in our understanding of the mechanisms underlying renal lymphatic vessel function and highlight potential off-target effects of furosemide that may exacerbate fluid accumulation in edema-forming conditions.

Functional characterization and osmoregulatory role of the Na+-K+-2Cl- cotransporter in the gill of sea lamprey (Petromyzon marinus), a basal vertebrate.

The present study provides molecular and functional characterization of Na+-K+-2Cl- cotransporter (NKCC1/Slc12a2) in the gills of sea lamprey (Petromyzon marinus), the most basal extant vertebrate with an osmoregulatory strategy. We report the full-length peptide sequence for the lamprey Na-K-Cl cotransporter 1 (NKCC1), which we show groups strongly with and occupies a basal position among other vertebrate NKCC1 sequences. In postmetamorphic juvenile lamprey, nkcc1 mRNA was present in many tissues but was fivefold higher in the gill than any other examined tissue, and NKCC1 protein was only detected in the gill. Gill mRNA and protein abundances of NKCC1 and Na+-K+-ATPase (NKA/Atp1a1) were significantly upregulated (20- to 200-fold) during late metamorphosis in fresh water, coinciding with the development of salinity tolerance, and were upregulated an additional twofold after acclimation to seawater (SW). Immunohistochemistry revealed that NKCC1 in the gill is found in filamental ionocytes coexpressing NKA, which develop during metamorphosis in preparation for SW entry. Lamprey treated with bumetanide, a widely used pharmacological inhibitor of NKCC1, exhibited higher plasma Cl- and osmolality as well as reduced muscle water content after 24 h in SW; there were no effects of bumetanide in freshwater-acclimated lamprey. This work provides the first functional characterization of NKCC1 as a mechanism for branchial salt secretion in lampreys, providing evidence that this mode of Cl- secretion has been present among vertebrates for ~550 million years.

Do Vitamin D Level and Dietary Calcium Intake Modify the Association Between Loop Diuretics and Bone Health?

Loop diuretics (LD) may affect bone health by inhibiting renal calcium reuptake. However, whether vitamin D status and dietary calcium intake modify the association between LD and bone outcome is unclear. Therefore, this study aimed to evaluate whether vitamin D level or calcium intake modify the association between LD and various indices of bone health including bone mineral density (BMD) and Trabecular Bone Score (TBS). From The Rotterdam Study, a prospective population-based cohort study, we used data from 6990 participants aged > 45 year with a DXA scan (2002-2008), 6908 participants with femoral neck (FN)-BMD, 6677 participants with lumbar spine (LS)-BMD and 6476 participants with LS-TBS measurements. Use of LD was available from pharmacy dispensing records. Vitamin D (25(OH)D) level was measured in serum, and dietary calcium intake was measured with a validated food frequency questionnaire. Almost eight percent of the participants used LD. The association between LD (past-users compared to never-users) and LS-TBS was significantly different by 25(OH)D concentrations (P for interaction = 0.04). A significantly lower LS-TBS among LD past-users was observed for 25(OH)D ≥ 50 nmol/l compared to ≤ 20 and 20-50 nmol/l (ß = - 0.036, 95% CI - 0.060; - 0.013 vs. ß = - 0.012, 95% CI - 0.036; 0.013 and ß = - 0.031, 95% CI - 0.096; 0.034, respectively). However, no other significant effect modification by 25(OH)D and dietary calcium intake was found in the associations between LD use and bone health outcomes (P-interaction > 0.13). This study suggests that the association between LD use and indices of bone health is not consistently modified by vitamin D or dietary calcium intake.

Impaired glucose tolerance, glucagon, and insulin responses in mice lacking the loop diuretic-sensitive Nkcc2a transporter.

The Na+K+2Cl- cotransporter-2 (Nkcc2, Slc12a1) is abundantly expressed in the kidney and its inhibition with the loop-diuretics bumetanide and furosemide has been linked to transient or permanent hyperglycemia in mice and humans. Notably, Slc12a1 is expressed at low levels in hypothalamic neurons and in insulin-secreting ß-cells of the endocrine pancreas. The present study was designed to determine if global elimination of one of the Slc12a1 products, i.e., Nkcc2 variant a (Nkcc2a), the main splice version of Nkcc2 found in insulin-secreting ß-cells, has an impact on the insulin and glucagon secretory responses and fuel homeostasis in vivo. We have used dynamic tests of glucose homeostasis in wild-type mice and mice lacking both alleles of Nkcc2a (Nkcc2aKO) and assessed their islet secretory responses in vitro. Under basal conditions, Nkcc2aKO mice have impaired glucose homeostasis characterized by increased blood glucose, intolerance to the sugar, delayed/blunted in vivo insulin and glucagon responses to glucose, and increased glycemic responses to the gluconeogenic substrate alanine. Further, we provide evidence of conserved quantitative secretory responses of Nkcc2aKO islets within a context of increased islet size related to hyperplastic/hypertrophic glucagon- and insulin-positive cells (α-cells and ß-cells, respectively), normal total islet Cl- content, and reduced ß-cell expression of the Cl- extruder Kcc2.

Genetic and pharmacological inactivation of apical Na+-K+-2Cl- cotransporter 1 in choroid plexus epithelial cells reveals the physiological function of the cotransporter.

Choroid plexus epithelial cells (CPECs) secrete cerebrospinal fluid (CSF). They express Na+-K+-ATPase and Na+-K+-2Cl- cotransporter 1 (NKCC1) on their apical membrane, deviating from typical basolateral membrane location in secretory epithelia. Given this peculiarity, the direction of basal net ion fluxes mediated by NKCC1 in CPECs is controversial, and cotransporter function is unclear. Determining the direction of basal NKCC1-mediated fluxes is critical to understanding the function of apical NKCC1. If NKCC1 works in the net efflux mode, it may be directly involved in CSF secretion. Conversely, if NKCC1 works in the net influx mode, it would have an absorptive function, contributing to intracellular Cl- concentration ([Cl-]i) and cell water volume (CWV) maintenance needed for CSF secretion. We resolve this long-standing debate by electron microscopy (EM), live-cell-imaging microscopy (LCIM), and intracellular Na+ and Cl- measurements in single CPECs of NKCC1+/+ and NKCC1-/- mouse. NKCC1-mediated ion and associated water fluxes are tightly linked, thus their direction is inferred by measuring CWV changes. Genetic or pharmacological NKCC1 inactivation produces CPEC shrinkage. EM of NKCC1-/- CPECs in situ shows they are shrunken, forming large dilations of their basolateral extracellular spaces, yet remaining attached by tight junctions. Normarski LCIM shows in vitro CPECs from NKCC1-/- are ~17% smaller than NKCC1+/+. CWV measurements in calcein-loaded CPECs show that bumetanide (10 µM) produces ~16% decrease in CWV in NKCC1+/+ but not in NKCC1-/- CPECs. Our findings suggest that under basal conditions apical NKCC1 is continuously active and works in the net inward flux mode maintaining [Cl-]i and CWV needed for CSF secretion.

Elevation of extracellular osmolarity improves signs of myotonia congenita in vitro: a preclinical animal study.

KEY POINTS: During myotonia congenita, reduced chloride (Cl- ) conductance results in impaired muscle relaxation and increased muscle stiffness after forceful voluntary contraction. Repetitive contraction of myotonic muscle decreases or even abolishes myotonic muscle stiffness, a phenomenon called 'warm up'. Pharmacological inhibition of low Cl- channels by anthracene-9-carboxylic acid in muscle from mice and ADR ('arrested development of righting response') muscle from mice showed a relaxation deficit under physiological conditions compared to wild-type muscle. At increased osmolarity up to 400 mosmol L-1 , the relaxation deficit of myotonic muscle almost reached that of control muscle. These effects were mediated by the cation and anion cotransporter, NKCC1, and anti-myotonic effects of hypertonicity were at least partly antagonized by the application of bumetanide. ABSTRACT: Low chloride-conductance myotonia is caused by mutations in the skeletal muscle chloride (Cl- ) channel gene type 1 (CLCN1). Reduced Cl- conductance of the mutated channels results in impaired muscle relaxation and increased muscle stiffness after forceful voluntary contraction. Exercise decreases muscle stiffness, a phenomena called 'warm up'. To gain further insight into the patho-mechanism of impaired muscle stiffness and the warm-up phenomenon, we characterized the effects of increased osmolarity on myotonic function. Functional force and membrane potential measurements were performed on muscle specimens of ADR ('arrested development of righting response') mice (an animal model for low gCl- conductance myotonia) and pharmacologically-induced myotonia. Specimens were exposed to solutions of increasing osmolarity at the same time as force and membrane potentials were monitored. In the second set of experiments, ADR muscle and pharmacologically-induced myotonic muscle were exposed to an antagonist of NKCC1. Upon osmotic stress, ADR muscle was depolarized to a lesser extent than control wild-type muscle. High osmolarity diminished myotonia and facilitated the warm-up phenomenon as depicted by a faster muscle relaxation time (T90/10 ). Osmotic stress primarily resulted in the activation of the NKCC1. The inhibition of NKCC1 with bumetanide prevented the depolarization and reversed the anti-myotonic effect of high osmolarity. Increased osmolarity decreased signs of myotonia and facilitated the warm-up phenomenon in different in vitro models of myotonia. Activation of NKCC1 activity promotes warm-up and reduces the number of contractions required to achieve normal relaxation kinetics.

A Novel Na+-K+-Cl- Cotransporter 1 Inhibitor STS66* Reduces Brain Damage in Mice After Ischemic Stroke.

Background and Purpose- Inhibition of brain NKCC1 (Na+-K+-Cl- cotransporter 1) with bumetanide (BMT) is of interest in ischemic stroke therapy. However, its poor brain penetration limits the application. In this study, we investigated the efficacy of 2 novel NKCC1 inhibitors, a lipophilic BMT prodrug STS5 (2-(Dimethylamino)ethyl 3-(butylamino)-4-phenoxy-5-sulfamoyl-benzoate;hydrochloride) and a novel NKCC1 inhibitor STS66 (3-(Butylamino)-2-phenoxy-5-[(2,2,2-trifluoroethylamino)methyl]benzenesulfonamide), on reducing ischemic brain injury. Methods- Large-vessel transient ischemic stroke in normotensive C57BL/6J mice was induced with 50-min occlusion of the middle cerebral artery and reperfusion. Focal, permanent ischemic stroke in angiotensin II (Ang II)-induced hypertensive C57BL/6J mice was induced by permanent occlusion of distal branches of middle cerebral artery. A total of 206 mice were randomly assigned to receive vehicle DMSO, BMT, STS5, or STS66. Results- Poststroke BMT, STS5, or STS66 treatment significantly decreased infarct volume and cerebral swelling by ≈40% to 50% in normotensive mice after transient middle cerebral artery occlusion, but STS66-treated mice displayed better survival and sensorimotor functional recovery. STS5 treatment increased the mortality. Ang II-induced hypertensive mice exhibited increased phosphorylatory activation of SPAK (Ste20-related proline alanine-rich kinase) and NKCC1, as well as worsened infarct and neurological deficit after permanent distal middle cerebral artery occlusion. Conclusions- The novel NKCC1 inhibitor STS66 is superior to BMT and STS5 in reducing ischemic infarction, swelling, and neurological deficits in large-vessel transient ischemic stroke, as well as in permanent focal ischemic stroke with hypertension comorbidity.

Ciliary beating amplitude controlled by intracellular Cl- and a high rate of CO2 production in ciliated human nasal epithelial cells.

The ciliary transport is controlled by two parameters of the ciliary beating, frequency (CBF) and amplitude. In this study, we developed a novel method to measure both CBF and ciliary bend distance (CBD, an index of ciliary beating amplitude) in ciliated human nasal epithelial cells (cHNECs) in primary culture, which are prepared from patients contracting allergic rhinitis and chronic sinusitis. An application of Cl--free NO3- solution or bumetanide (an inhibitor of Na+/K+/2Cl- cotransport), which decreases intracellular Cl- concentration ([Cl-]i), increased CBD, not CBF, at 37 °C; however, it increased both CBD and CBF at 25 °C. Conversely, addition of Cl- channel blockers (5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and 4-[[4-Oxo-2-thioxo-3-[3-trifluoromethyl]phenyl]-5-thiazolidinylidene]methyl] benzoic acid (CFTR(inh)-172)), which increase [Cl-]i, decreased both CBD and CBF, suggesting that CFTR plays a crucial role for maintaining [Cl-]i in these cells. We speculate that Cl- modulates activities of the molecular motors regulating both CBD and CBF in cHNECs. Moreover, application of the CO2/HCO3--free solution did not change intracellular pH (pHi), and addition of an inhibitor of carbonic anhydrase (acetazolamide) sustained pHi increase induced by the NH4+ pulse, which transiently increased pHi in the absence of acetazolamide. These results indicate that the cHNEC produces a large amount of CO2, which maintains a constant pHi even under the CO2/HCO3--free condition.

Furosemide reduces BK-αß4-mediated K+ secretion in mice on an alkaline high-K+ diet.

Special high-K diets have cardioprotective effects and are often warranted in conjunction with diuretics such as furosemide for treating hypertension. However, it is not understood how a high-K diet (HK) influences the actions of diuretics on renal K+ handling. Furosemide acidifies the urine by increasing acid secretion via the Na+-H+ exchanger 3 (NHE3) in TAL and vacuolar H+-ATPase (V-ATPase) in the distal nephron. We previously found that an alkaline urine is required for large conductance Ca2+-activated K+ (BK)-αß4-mediated K+ secretion in mice on HK. We therefore hypothesized that furosemide could reduce BK-αß4-mediated K+ secretion by acidifying the urine. Treating with furosemide (drinking water) for 11 days led to decreased urine pH in both wild-type (WT) and BK-ß4-knockout mice (BK-ß4-KO) with increased V-ATPase expression and elevated plasma aldosterone levels. However, furosemide decreased renal K+ clearance and elevated plasma [K+] in WT but not BK-ß4-KO. Western blotting and immunofluorescence staining showed that furosemide treatment decreased cortical expression of BK-ß4 and reduced apical localization of BK-α in connecting tubules. Addition of the carbonic anhydrase inhibitor, acetazolamide, to furosemide water restored urine pH along with renal K+ clearance and plasma [K+] to control levels. Acetazolamide plus furosemide also restored the cortical expression of BK-ß4 and BK-α in connecting tubules. These results indicate that in mice adapted to HK, furosemide reduces BK-αß4-mediated K+ secretion by acidifying the urine.