NKCC1 Inhibitor Research Articles

The roles of sodium-potassium-chloride cotransporter isoform-1 in acute lung injury.

Acute lung injury (ALI) is often characterized by severe lung inflammation and pulmonary edema with poor gas exchange and hypoxemia. Alveolar inflammation and water flooding are, in fact, notable features of ALI pathogenesis. The sodium-potassium-chloride co-transporter isoform 1 (NKCC1), localized at the basolateral surface of the lung epithelium, drives water transport via back transport of Na+ and Cl− to the alveolar air space. NKCC1, therefore, is crucial in regulating alveolar fluid. Increased expression of NKCC1 results in increased alveolar fluid secretion and impaired alveolar fluid clearance. During ALI, the with no lysine kinase (WNK), oxidative stress responsive kinase 1 (OSR1), and STE20/SPS1-related proline/alanine-rich kinase (SPAK) pathways are activated, which upregulates NKCC1 expression. Proinflammatory cytokines also enhance the expression of NKCC1 via c-Jun N-terminal kinase-and p38-dependent pathways. NKCC1 activation also increases the expression of proinflammatory cytokines via cell rupture and activation of macrophages. Increased proinflammatory cytokines, in turn, recruit inflammatory cells to the site of injury and cause further lung damage. Animals with high expression of NKCC1 show more severe lung injury with presentations of more severe pulmonary edema and microvascular permeability, higher expression of proinflammatory cytokines, and greater neutrophilic infiltration. In contrast, animals with low expression of NKCC1 or those treated with NKCC1 inhibitors show less severe lung injury with milder levels of presentations of ALI. These reports collectively highlight a novel role of NKCC1 in ALI pathogenesis. Manipulation of NKCC1 expression levels could, therefore, represent novel modalities for effective ALI treatment.

Design, Synthesis, In Vitro and In Vivo Characterization of Selective NKCC1 Inhibitors for the Treatment of Core Symptoms in Down Syndrome.

Intracellular chloride concentration [Cl–]i is defective in several neurological disorders. In neurons, [Cl–]i is mainly regulated by the action of the Na+–K+–Cl– importer NKCC1 and the K+–Cl– exporter KCC2. Recently, we have reported the discovery of ARN23746 as the lead candidate of a novel class of selective inhibitors of NKCC1. Importantly, ARN23746 is able to rescue core symptoms of Down syndrome (DS) and autism in mouse models. Here, we describe the discovery and extensive characterization of this chemical class of selective NKCC1 inhibitors, with focus on ARN23746 and other promising derivatives. In particular, we present compound 40 (ARN24092) as a backup/follow-up lead with in vivo efficacy in a mouse model of DS. These results further strengthen the potential of this new class of compounds for the treatment of core symptoms of brain disorders characterized by the defective NKCC1/KCC2 expression ratio.

Pathology-selective antiepileptic effects in the focal freeze-lesion rat model of malformation of cortical development

Malformations of cortical development (MCD) represent a group of rare diseases with severe clinical presentation as epileptic and pharmacoresistant encephalopathies. Morphological studies in tissue from MCD patients have revealed reduced GABAergic efficacy and increased intracellular chloride concentration in neuronal cells as important pathophysiological mechanisms in MCD. Also, in various animal models, alterations of GABAergic inhibition have been postulated as a predominant factor contributing to perilesional hyperexcitability. Along with this line, the NKCC1 inhibitor bumetanide has been postulated as a potential drug for treatment of epilepsy, mediating its antiepileptic effect by reduction of the intracellular chloride and increased inhibitory efficacy of GABAergic transmission.In the present study, we focused on the focal freeze-lesion model of MCD to compare antiepileptic drugs with distinct mechanisms of action, including NKCC1 inhibition by bumetanide. For this purpose, we combined electrophysiological and optical methods in slice preparations and assessed the properties of seizure like events (SLE) induced by 4-aminopyridine. In freeze-lesioned but not control slices, SLE onset was confined to the perilesional area, confirming that this region is hyperexcitable and likely triggers pathological activity. Bumetanide selectively reduced epileptic activity in lesion-containing slices but not in slices from sham-treated control rats. Moreover, bumetanide caused a shift in the SLE onset site away from the perilesional area. In contrast, effects of other antiepileptic drugs including carbamazepine, lacosamide, acezatolamide and zonisamide occurred mostly independently of the lesion and did not result in a shift of the onset region.Our work adds evidence for the functional relevance of chloride homeostasis in the pathophysiology of microgyrus formation as represented in the focal freeze-lesion model. Further studies in different MCD models and human tissue will be required to validate the effects across different MCD subtypes and species and to assess the translational value of our findings.

Aldosterone up-regulates basolateral Na+ -K+ -2Cl- cotransporter-1 to support enhanced large-conductance K+ channel-mediated K+ secretion in rat distal colon.

Na+ -K+ -2Cl- cotransporter-1 (NKCC1) facilitates basolateral K+ and Cl- uptake, supporting their efflux across mucosal membranes of colonic epithelial cells. NKCC1 activity has also been shown to be critical for electrogenic K+ secretion induced by aldosterone, which is known to stimulate large-conductance K+ (BK) channel expression in mucosal membranes. This study was aimed to (1) identify whether aldosterone enhances NKCC1 expression specifically to support BK-mediated K+ secretion and (2) to determine whether increased NKCC1 supports electrogenic Cl- secretion in parallel to K+ secretion. Dietary Na+ depletion was used to induce secondary hyperaldosteronism in rats, or aldosterone was administered ex vivo to rat distal colonic mucosae. NKCC1-dependent electrogenic K+ or Cl- secretion was measured as a function of short circuit current (ISC ). qRT-PCR, western blot, and immunofluorescence analyses were performed using standard techniques. Aldosterone enhanced NKCC1 and BKα expression and electrogenic K+ secretion in the distal colon, which was inhibited by either serosal bumetanide (NKCC1 inhibitor) or mucosal iberiotoxin (IbTX; BK channel blocker), but not TRAM-34 (IK channel blocker). Expression of NKCC1 and BKα proteins was enhanced in crypt cells of hyper-aldosterone rats. However, neither NKCC1-dependent Cl- secretion nor CFTR (apical Cl- channel) expression was enhanced by aldosterone. We conclude that aldosterone enhances NKCC1 to support BK-mediated K+ secretion independently of Cl- secretion in the distal colon. The regulation of NKCC1 expression/K+ secretion by aldosterone may be a therapeutic target in treating gastrointestinal disorders associated with alterations in colonic K+ transport, such as colonic pseudo-obstruction, and hyperkalemia associated with renal disease.

Abstract P70: SPAK/OSR1 Signaling as a Novel Target for Post-Stroke Oxidative Stress Brain Injury

Stroke is the second leading cause of death worldwide, and ischemic stroke accounts for the vast majority of stroke cases. Currently, recombinant tissue plasminogen activator and endovascular thrombectomy are the two primary therapy strategies for acute ischemic stroke patients. Reactive oxygen species (ROS)-mediated oxidative stress can cause brain injury during reperfusion. We have shown that the Ste20/SPS1-related proline-alanine-rich protein kinase/oxidative stress-responsive kinase-1 (SPAK/OSR1) are activated in ischemic stroke brains, resulting in worsened outcomes in murine stroke models. SPAK activation induces the production of pro-inflammatory cytokines. Post-stroke administration of a novel SPAK inhibitor ZT-1a attenuates cerebral edema and protects against brain damage in in vivo model of ischemic stroke. However, whether ROS mediated oxidative stress directly activate SPAK/OSR1 pathway and induces SPAK pro-inflammatory cytokine production in ischemic brains remains unknown. In our extended study, we examined activation of SPAK/OSR1 and its substrate Na-K-Cl cotransporter (NKCC1) in cultured mouse primary neurons in response to hydrogen peroxide (H 2 O 2 )-mediated oxidative stress. We found that exposure of neurons to H 2 O 2 for 24 hrs triggered upregulation of protein expression and phosphorylation activation of SPAK/OSR1 and NKCC1 ( p < 0.05), which are accompanied with an increase in intracellular Na + concentration and neuronal death ( p < 0.01). These changes were blocked by an ROS scavenger ebselen. Interestingly, both novel SPAK inhibitor ZT-1a and NKCC1 inhibitor bumetanide are able to block H 2 O 2 -induced neuronal damage. We are in the process to assess effects of SPAK inhibitor ZT-1a in reducing ROS-mediated inflammation and brain injury in in vivo model of ischemic stroke. Together, our study suggests that ROS can activate SPAK/OSR1 complex during reperfusion injury and the therapeutic potentials of SPAK inhibitor ZT-1a for ischemic stroke.

Deletion of the Na-K-2Cl cotransporter NKCC1 results in a more severe epileptic phenotype in the intrahippocampal kainate mouse model of temporal lobe epilepsy

Increased neuronal expression of the Na-K-2Cl cotransporter NKCC1 has been implicated in the generation of seizures and epilepsy. However, conclusions from studies on the NKCC1-specific inhibitor, bumetanide, are equivocal, which is a consequence of the multiple potential cellular targets and poor brain penetration of this drug. Here, we used Nkcc1 knockout (KO) and wildtype (WT) littermate control mice to study the ictogenic and epileptogenic effects of intrahippocampal injection of kainate. Kainate (0.23 μg in 50 nl) induced limbic status epilepticus (SE) in both KO and WT mice with similar incidence, latency to SE onset, and SE duration, but the number of intermittent generalized convulsive seizures during SE was significantly higher in Nkcc1 KO mice, indicating increased SE severity. Following SE, spontaneous recurrent seizures (SRS) were recorded by continuous (24/7) video/EEG monitoring at 0-1, 4-5, and 12-13 weeks after kainate, using depth electrodes in the ipsilateral hippocampus. Latency to onset of electrographic SRS and the incidence of electrographic SRS were similar in WT and KO mice. However, the frequency of electrographic seizures was lower whereas the frequency of electroclinical seizures was higher in Nkcc1 KO mice, indicating a facilitated progression from electrographic to electroclinical seizures during chronic epilepsy, and a more severe epileptic phenotype, in the absence of NKCC1. The present findings suggest that NKCC1 is dispensable for the induction, progression and manifestation of epilepsy, and they do not support the widely held notion that inhibition of NKCC1 in the brain is a useful strategy for preventing or modifying epilepsy.

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.

Effects of the NKCC1 inhibitors bumetanide, azosemide, and torasemide alone or in combination with phenobarbital on seizure threshold in epileptic and nonepileptic mice

The sodium-potassium-chloride (Na–K–Cl) cotransporter NKCC1 is found in the plasma membrane of a wide variety of cell types, including neurons, glia and endothelial cells in the brain. Increased expression of neuronal NKCC1 has been implicated in several brain disorders, including neonatal seizures and epilepsy. The loop diuretic and NKCC inhibitor bumetanide has been evaluated as an antiseizure agent alone or together with approved antiseizure drugs such as phenobarbital (PB) in pre-clinical and clinical studies with varying results. The equivocal efficacy of bumetanide may be a result of its poor brain penetration. We recently reported that the loop diuretic azosemide is more potent to inhibit NKCC1 than bumetanide. In contrast to bumetanide, azosemide is not acidic, which should favor its brain penetration. Thus, azosemide may be a promising alternative to bumetanide for treatment of brain disorders such as epilepsy. In the present study, we determined the effect of azosemide and bumetanide on seizure threshold in adult epileptic mice. A structurally related non-acidic loop diuretic, torasemide, which also blocks NKCC1, was included in the experiments. The drug effects were assessed by determing the maximal electroshock seizure threshold (MEST) in epileptic vs. nonepileptic mice. Epilepsy was induced by pilocarpine, which was shown to produce long-lasting increases in NKCC1 in the hippocampus, whereas MEST did not alter NKCC1 mRNA in this region. None of the three loop diuretics increased MEST or the effect of PB on MEST in nonepileptic mice. In epileptic mice, all three diuretics significantly increased PB's seizure threshold increasing efficacy, but the effect was variable upon repeated MEST determinations and not correlated with the drugs' diuretic potency. These data may indicate that inhibition of NKCC1 by loop diuretics is not an effective means of increasing seizure threshold in adult epilepsy.

Upregulation of Spinal ASIC1 and NKCC1 Expression Contributes to Chronic Visceral Pain in Rats.

Aims: To determine whether acid-sensing ion channel 1 (ASIC1)–sodium-potassium-chloride cotransporter 1 (NKCC1) signaling pathway participates in chronic visceral pain of adult rats with neonatal maternal deprivation (NMD).Methods: Chronic visceral pain was detected by colorectal distension (CRD). Western blotting and Immunofluorescence were performed to detect the expression and location of ASIC1 and NKCC1. Whole-cell patch-clamp recordings were performed to record spinal synaptic transmission.Results: The excitatory synaptic transmission was enhanced and the inhibitory synaptic transmission was weakened in the spinal dorsal horn of NMD rats. ASIC1 and NKCC1 protein expression in the spinal dorsal horn was significantly up-regulated in NMD rats. Incubation of Amiloride reduced the amplitude of mEPSCs. Incubation of Bumetanide (BMT) increased the amplitude of mIPSCs. Intrathecal injection of ASIC1 or NKCC1 inhibitors reversed the threshold of CRD in NMD rats. Also, Amiloride treatment significantly reversed the expression of NKCC1 in the spinal dorsal horn of NMD rats.Conclusion: Our data suggest that the ASIC1-NKCC1 signaling pathway is involved in chronic visceral pain in NMD rats.

The search for brain-permeant NKCC1 inhibitors for the treatment of seizures: Pharmacokinetic-pharmacodynamic modelling of NKCC1 inhibition by azosemide, torasemide, and bumetanide in mouse brain

Because of its potent inhibitory effect on the Na+-K+-2Cl- symporter isotype 1 (NKCC1) in brain neurons, bumetanide has been tested with varying results for treatment of seizures that potentially evolve as a consequence of abnormal NKCC1 activity. However, because of its physicochemical properties, bumetanide only poorly penetrates into the brain. We previously demonstrated that NKCC1 can be also inhibited by azosemide and torasemide, which lack the carboxyl group of bumetanide and thus should be better brain-permeable. Here we studied the brain distribution kinetics of azosemide and torasemide in comparison with bumetanide in mice and used pharmacokinetic-pharmacodynamic modelling to determine whether the drugs reach NKCC1-inhibitory brain concentrations. All three drugs hardly distributed into the brain, which seemed to be the result of probenecid-sensitive efflux transport at the blood–brain barrier. When fractions unbound in plasma and brain were determined by equilibrium dialysis, only about 6–17% of the brain drug concentration were freely available. With the systemic doses (10 mg/kg i.v.) used, free brain concentrations of bumetanide and torasemide were in the NKCC1-inhibitory concentration range, while levels of azosemide were slightly below this range. However, all three drugs exhibited free plasma levels that would be sufficient to block NKCC1 at the apical membrane of brain capillary endothelial cells. These data suggest that azosemide and torasemide are interesting alternatives to bumetanide for treatment of seizures involving abnormal NKCC1 functionality, particularly because of their longer duration of action and their lower diuretic potency, which is an advantage in patients with seizures.

Phenobarbital and midazolam suppress neonatal seizures in a noninvasive rat model of birth asphyxia, whereas bumetanide is ineffective.

Neonatal seizures are the most frequent type of neurological emergency in newborn infants, often being a consequence of prolonged perinatal asphyxia. Phenobarbital is currently the most widely used antiseizure drug for treatment of neonatal seizures, but fails to stop them in ~50% of cases. In a neonatal hypoxia-only model based on 11-day-old (P11) rats, the NKCC1 inhibitor bumetanide was reported to potentiate the antiseizure activity of phenobarbital, whereas it was ineffective in a human trial in neonates. The aim of this study was to evaluate the effect of clinically relevant doses of bumetanide as add-on to phenobarbital on neonatal seizures in a noninvasive model of birth asphyxia in P11 rats, designed for better translation to the human term neonate. Intermittent asphyxia was induced for 30minutes by exposing the rat pups to three 7+3-minute cycles of 9% and 5% O2 at constant 20% CO2 . Drug treatments were administered intraperitoneally either before or immediately after asphyxia. All untreated rat pups had seizures within 10minutes after termination of asphyxia. Phenobarbital significantly blocked seizures when applied before asphyxia at 30mg/kg but not 15mg/kg. Administration of phenobarbital after asphyxia was ineffective, whereas midazolam (0.3 or 1mg/kg) exerted significant antiseizure effects when administered before or after asphyxia. In general, focal seizures were more resistant to treatment than generalized convulsive seizures. Bumetanide (0.3mg/kg) alone or in combination with phenobarbital (15 or 30mg/kg) exerted no significant effect on seizure occurrence. The data demonstrate that bumetanide does not increase the efficacy of phenobarbital in a model of birth asphyxia, which is consistent with the negative data of the recent human trial. The translational data obtained with the novel rat model of birth asphyxia indicate that it is a useful tool to evaluate novel treatments for neonatal seizures.

NKCC-1 mediated Cl\u2212 uptake in immature CA3 pyramidal neurons is sufficient to compensate phasic GABAergic inputs

Activation of GABAA receptors causes in immature neurons a functionally relevant decrease in the intracellular Cl− concentration ([Cl−]i), a process termed ionic plasticity. Amount and duration of ionic plasticity depends on kinetic properties of [Cl−]i homeostasis. In order to characterize the capacity of Cl− accumulation and to quantify the effect of persistent GABAergic activity on [Cl−]i, we performed gramicidin-perforated patch-clamp recordings from CA3 pyramidal neurons of immature (postnatal day 4–7) rat hippocampal slices. These experiments revealed that inhibition of NKCC1 decreased [Cl−]i toward passive distribution with a time constant of 381 s. In contrast, active Cl− accumulation occurred with a time constant of 155 s, corresponding to a rate of 15.4 µM/s. Inhibition of phasic GABAergic activity had no significant effect on steady state [Cl−]i. Inhibition of tonic GABAergic currents induced a significant [Cl−]i increase by 1.6 mM, while activation of tonic extrasynaptic GABAA receptors with THIP significantly reduced [Cl−]i.. Simulations of neuronal [Cl−]i homeostasis supported the observation, that basal levels of synaptic GABAergic activation do not affect [Cl−]i. In summary, these results indicate that active Cl−-uptake in immature hippocampal neurons is sufficient to maintain stable [Cl−]i at basal levels of phasic and to some extent also to compensate tonic GABAergic activity.

Aldosterone up-regulates voltage-gated potassium currents and NKCC1 protein membrane fractions

Na+–K+–2Cl− Cotransporter (NKCC1) is a protein that aids in the active transport of sodium, potassium, and chloride ions across cell membranes. It has been shown that long-term systemic treatment with aldosterone (ALD) can enhance NKCC1 protein expression and activity in the aging cochlea resulting in improved hearing. In the present work, we used a cell line with confirmed NKCC1 expression to demonstrate that in vitro application of ALD increased outward voltage-gated potassium currents significantly, and simultaneously upregulated whole lysate and membrane portion NKCC1 protein expression. These ALD-induced changes were blocked by applying the mineralocorticoid receptor antagonist eplerenone. However, application of the NKCC1 inhibitor bumetanide or the potassium channel antagonist Tetraethyl ammonium had no effect. In addition, NKKC1 mRNA levels remained stable, indicating that ALD modulates NKCC1 protein expression via the activation of mineralocorticoid receptors and post-transcriptional modifications. Further, in vitro electrophysiology experiments, with ALD in the presence of NKCC1, K+ channel and mineralocorticoid receptor inhibitors, revealed interactions between NKCC1 and outward K+ channels, mediated by a mineralocorticoid receptor-ALD complex. These results provide evidence of the therapeutic potential of ALD for the prevention/treatment of inner ear disorders such as age-related hearing loss.

CrossTalk proposal: Apical NKCC1 of choroid plexus epithelial cells works in the net inward flux mode under basal conditions, maintaining intracellular Cl- and cell volume.

CrossTalk proposal: Apical NKCC1 of choroid plexus epithelial cells works in the net inward flux mode under basal conditions, maintaining intracellular Cl- and cell volume.

Role of NKCC1 Activity in Glioma K+ Homeostasis and Cell Growth: New Insights With the Bumetanide-Derivative STS66.

Introduction: Na+-K+-2Cl− cotransporter isoform 1 (NKCC1) is important in regulating intracellular K+ and Cl− homeostasis and cell volume. In this study, we investigated a role of NKCC1 in regulating glioma K+ influx and proliferation in response to apoptosis inducing chemotherapeutic drug temozolomide (TMZ). The efficacy of a new bumetanide (BMT)-derivative NKCC1 inhibitor STS66 [3-(butylamino)-2-phenoxy-5-[(2, 2, 2-trifluoroethylamino) methyl] benzenesulfonamide] in blocking NKCC1 activity was compared with well-established NKCC1 inhibitor BMT. Methods: NKCC1 activity in cultured mouse GL26 and SB28-GFP glioma cells was measured by Rb+ (K+) influx. The WNK1-SPAK/OSR1-NKCC1 signaling and AKT/ERK-mTOR signaling protein expression and activation were assessed by immunoblotting. Cell growth was determined by bromodeoxyuridine (BrdU) incorporation assay, MTT proliferation assay, and cell cycle analysis. Impact of STS66 and BMT on cell Rb+ influx and growth was measured in glioma cells treated with or without TMZ. Results: Rb+ influx assay showed that 10 μM BMT markedly decreased the total Rb+ influx and no additional inhibition detected at >10 μM BMT. In contrast, the maximum effects of STS66 on Rb+ influx inhibition were at 40–60 μM. Both BMT and STS66 reduced TMZ-mediated NKCC1 activation and protein upregulation. Glioma cell growth can be reduced by STS66. The most robust inhibition of glioma growth, cell cycle, and AKT/ERK signaling was achieved by the TMZ + STS66 treatment. Conclusion: The new BMT-derivative NKCC1 inhibitor STS66 is more effective than BMT in reducing glioma cell growth in part by inhibiting NKCC1-mediated K+ influx. TMZ + STS66 combination treatment reduces glioma cell growth via inhibiting cell cycle and AKT-ERK signaling.

Discovery of a Small Molecule Drug Candidate for Selective NKCC1 Inhibition in Brain Disorders

SummaryAberrant expression ratio of Cl− transporters, NKCC1 and KCC2, is implicated in several brain conditions. NKCC1 inhibition by the FDA-approved diuretic drug, bumetanide, rescues core symptoms in rodent models and/or clinical trials with patients. However, bumetanide has a strong diuretic effect due to inhibition of the kidney Cl− transporter NKCC2, creating critical drug compliance issues and health concerns. Here, we report the discovery of a new chemical class of selective NKCC1 inhibitors and the lead drug candidate ARN23746. ARN23746 restores the physiological intracellular Cl− in murine Down syndrome neuronal cultures, has excellent solubility and metabolic stability, and displays no issues with off-target activity in vitro. ARN23746 recovers core symptoms in mouse models of Down syndrome and autism, with no diuretic effect, nor overt toxicity upon chronic treatment in adulthood. ARN23746 is ready for advanced preclinical/manufacturing studies toward the first sustainable therapeutics for the neurological conditions characterized by impaired Cl− homeostasis.

Acute Hyperglycemia Aggravates Lung Injury via Activation of the SGK1-NKCC1 Pathway.

Acute lung injury (ALI) is characterized by severe hypoxemia and has significantly high mortality rates. Acute hyperglycemia occurs in patients with conditions such as sepsis or trauma, among others, and it results in aggravated inflammation and induces damage in patients with ALI. Regulation of alveolar fluid is essential for the development and resolution of pulmonary edema in lung injury. Pulmonary sodium-potassium-chloride co-transporter 1 (NKCC1) regulates the net influx of ions and water into alveolar cells. The activation of with-no-lysine kinase 4 (WNK4), STE20/SPS1-related proline/alanine rich kinase (SPAK) and the NKCC1 pathway lead to an increase in the expression of NKCC1 and aggravation of ALI. Moreover, hyperglycemia is known to induce NKCC1 expression via the activation of the serum-glucocorticoid kinase 1 (SGK1)–NKCC1 pathway. We aim to evaluate the influence of acute hyperglycemia on the SGK1–NKCC1 pathway in ALI. ALI was induced using a high tidal volume for four hours in a rat model. Acute hyperglycemia was induced by injection with 0.5 mL of 40% glucose solution followed by continuous infusion at 2 mL/h. The animals were divided into sham, sham+ hyperglycemia, ALI, ALI + hyperglycemia, ALI + inhaled bumetanide (NKCC1 inhibitor) pretreatment, ALI + hyperglycemia + inhalational bumetanide pretreatment, and ALI + hyperglycemia + post-ALI inhalational bumetanide groups. Severe lung injury along with pulmonary edema, alveolar protein leakage, and lung inflammation was observed in ALI with hyperglycemia than in ALI without hyperglycemia. This was concurrent with the higher expression of pro-inflammatory cytokines, infiltration of neutrophils and alveolar macrophages (AM) 1, and NKCC1 expression. Inhalational NKCC1 inhibitor significantly inhibited the SGK1–NKCC1, and WNK4–SPAK–NKCC1 pathways. Additionally, it reduced pulmonary edema, inflammation, levels of pro-inflammatory cytokines, neutrophils and AM1 and increased AM2. Therefore, acute hyperglycemia aggravates lung injury via the further activation of the SGK1–NKCC1 pathway. The NKCC1 inhibitor can effectively attenuate lung injury aggravated by acute hyperglycemia.

Blockade of Cell Volume Regulatory Protein NKCC1 Increases TMZ-Induced Glioma Apoptosis and Reduces Astrogliosis.

Glioma is one of the most common primary malignant tumors of the central nervous system accounting for approximately 40% of all intracranial tumors. Temozolomide is a conventional chemotherapy drug for adjuvant treatment of patients with high-risk gliomas, including grade II to grade IV. Our bioinformatic analysis of The Cancer Genome Atlas and Chinese Glioma Genome Atlas datasets and immunoblotting assay show that SLC12A2 gene and its encoded Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) protein are abundantly expressed in grade II-IV gliomas. NKCC1 regulates cell volume and intracellular Cl- concentration, which promotes glioma cell migration, resistance to temozolomide, and tumor-related epilepsy in experimental glioma models. Using mouse syngeneic glioma models with intracranial transplantation of two different glioma cell lines (GL26 and SB28), we show that NKCC1 protein in glioma tumor cells as well as in tumor-associated reactive astrocytes was significantly upregulated in response to temozolomide monotherapy. Combination therapy of temozolomide with the potent NKCC1 inhibitor bumetanide reduced tumor proliferation, potentiated the cytotoxic effects of temozolomide, decreased tumor-associated reactive astrogliosis, and restored astrocytic GLT-1 and GLAST glutamate transporter expression. The combinatorial therapy also led to suppressed tumor growth and prolonged survival of mice bearing GL26 glioma cells. Taken together, these results demonstrate that NKCC1 protein plays multifaceted roles in the pathogenesis of glioma tumors and presents as a therapeutic target for reducing temozolomide-mediated resistance and tumor-associated astrogliosis.

Androgenic Modulation of the Chloride Transporter NKCC1 Contributes to Age-dependent Isoflurane Neurotoxicity in Male Rats.

Cognitive deficits after perinatal anesthetic exposure are well established outcomes in animal models. This vulnerability is sex-dependent and associated with expression levels of the chloride transporters NKCC1 and KCC2. The hypothesis was that androgen signaling, NKCC1 function, and the age of isoflurane exposure are critical for the manifestation of anesthetic neurotoxicity in male rats. Flutamide, an androgen receptor antagonist, was administered to male rats on postnatal days 2, 4, and 6 before 6 h of isoflurane on postnatal day 7 (ntotal = 26). Spatial and recognition memory were subsequently tested in adulthood. NKCC1 and KCC2 protein levels were measured from cortical lysates by Western blot on postnatal day 7 (ntotal = 20). Bumetanide, an NKCC1 antagonist, was injected immediately before isoflurane exposure (postnatal day 7) to study the effect of NKCC1 inhibition (ntotal = 48). To determine whether male rats remain vulnerable to anesthetic neurotoxicity as juveniles, postnatal day 14 animals were exposed to isoflurane and assessed as adults (ntotal = 30). Flutamide-treated male rats exposed to isoflurane successfully navigated the spatial (Barnes maze probe trial F[1, 151] = 78; P < 0.001; mean goal exploration ± SD, 6.4 ± 3.9 s) and recognition memory tasks (mean discrimination index ± SD, 0.09 ± 0.14; P = 0.003), unlike isoflurane-exposed controls. Flutamide changed expression patterns of NKCC1 (mean density ± SD: control, 1.49 ± 0.69; flutamide, 0.47 ± 0.11; P < 0.001) and KCC2 (median density [25th percentile, 75th percentile]: control, 0.23 [0.13, 0.49]; flutamide, 1.47 [1.18,1.62]; P < 0.001). Inhibiting NKCC1 with bumetanide was protective for spatial memory (probe trial F[1, 162] = 6.6; P = 0.011; mean goal time, 4.6 [7.4] s). Delaying isoflurane exposure until postnatal day 14 in males preserved spatial memory (probe trial F[1, 140] = 28; P < 0.001; mean goal time, 6.1 [7.0] s). Vulnerability to isoflurane neurotoxicity is abolished by blocking the androgen receptor, disrupting the function of NKCC1, or delaying the time of exposure to at least 2 weeks of age in male rats. These results support a dynamic role for androgens and chloride transporter proteins in perinatal anesthetic neurotoxicity.

TRPV1 activation stimulates NKCC1 and increases hydrostatic pressure in the mouse lens.

The porcine lens response to a hyperosmotic stimulus involves an increase in the activity of an ion cotransporter sodium-potassium/two-chloride cotransporter 1 (NKCC1). Recent studies with agonists and antagonists pointed to a mechanism that appears to depend on activation of transient receptor potential vanilloid 1 (TRPV1) ion channels. Here, we compare responses in lenses and cultured lens epithelium obtained from TRPV1-/- and wild type (WT) mice. Hydrostatic pressure (HP) in lens surface cells was determined using a manometer-coupled microelectrode approach. The TRPV1 agonist capsaicin (100 nM) caused a transient HP increase in WT lenses that peaked after ∼30 min and then returned toward baseline. Capsaicin did not cause a detectable change of HP in TRPV1-/- lenses. The NKCC inhibitor bumetanide prevented the HP response to capsaicin in WT lenses. Potassium transport was examined by measuring Rb+ uptake. Capsaicin increased Rb+ uptake in cultured WT lens epithelial cells but not in TRPV1-/- cells. Bumetanide, A889425, and the Akt inhibitor Akti prevented the Rb+ uptake response to capsaicin. The bumetanide-sensitive (NKCC-dependent) component of Rb+ uptake more than doubled in response to capsaicin. Capsaicin also elicited rapid (<2 min) NKCC1 phosphorylation in WT but not TRPV1-/- cells. HP recovery was shown to be absent in TRPV1-/- lenses exposed to hyperosmotic solution. Bumetanide and Akti prevented HP recovery in WT lenses exposed to hyperosmotic solution. Taken together, responses to capsaicin and hyperosmotic solution point to a functional role for TRPV1 channels in mouse lens. Lack of NKCC1 phosphorylation and Rb+ uptake responses in TRPV1-/- mouse epithelium reinforces the notion that a hyperosmotic challenge causes TRPV1-dependent NKCC1 activation. The results are consistent with a role for the TRPV1-activated signaling pathway leading to NKCC1 stimulation in lens osmotic homeostasis.