BACKGROUND: The inhalational anesthetic isoflurane is commonly utilized in clinical practice, particularly in the field of pediatric anesthesia. Research has demonstrated its capacity to induce neuroinflammation and long-term behavioral disorders; however, the underlying mechanism remains unclear [1]. The cation-chloride cotransporters Na+-K+-2Cl--1 (NKCC1) and K+-2Cl--2 (KCC2) play a pivotal role in regulating neuronal responses to gamma-aminobutyric acid (GABA) [2]. Imbalances in NKCC1/KCC2 can disrupt GABA neurotransmission, potentially leading to neural circuit hyperexcitability and reduced inhibition following neonatal exposure to anesthesia [3]. Therefore, this study postulates that anesthetics have the potential to dysregulate NKCC1 and/or KCC2 during brain development.
METHODS: We administered 1.5% isoflurane anesthesia to neonatal rats for a duration of 4 h at postnatal day 7 (PND7). Anxiety levels were assessed using the open field test at PND28, while cognitive function was evaluated using the Morris water maze test between PND31 and PND34. Protein levels of NKCC1, KCC2, BDNF, and phosphorylated ERK (P-ERK) in the hippocampus were measured through Western blotting analysis. Pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were quantified using ELISA.
RESULTS: We observed a decrease in locomotion trajectories within the central region and a significantly shorter total distance in the ISO group compared to CON pups, indicating that isoflurane induces anxiety-like behavior. In the Morris water maze (MWM) test, rats exposed to isoflurane exhibited prolonged escape latency onto the platform. Additionally, isoflurane administration resulted in reduced time spent crossing in the MWM experiment at PND34, suggesting long-term impairment of memory function. Furthermore, we found that isoflurane triggered activation of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α; downregulated KCC2/BDNF/P-ERK expression; and increased the NKCC1/KCC2 ratio in the hippocampus of PND7 rats. Bumetadine (NKCC1 specific inhibitors) reversed cognitive damage and effective disorder induced by isoflurane in neonatal rats by inhibiting TNF-α activation, normalizing IL-6 and IL-1β levels, restoring KCC2 expression levels as well as BDNF and ERK signaling pathways. Based on these findings, it can be speculated that BDNF, P-ERK, IL-1β, IL-6 and TNF - α may act downstream of the NKCC1/KCC2 pathway.
CONCLUSIONS: Our findings provide evidence that isoflurane administration in neonatal rats leads to persistent cognitive deficits through dysregulation of the Cation-Chloride Cotransporters NKCC1 and KCC2, BDNF, p-ERK proteins, as well as neuroinflammatory processes.

The solute carrier family 12 (SLC12) of cation-chloride cotransporters (CCCs) comprises potassium chloride cotransporters (KCCs, e.g. KCC1, KCC2, KCC3, and KCC4)-mediated Cl- extrusion, and sodium potassium chloride cotransporters (N[K]CCs, NKCC1, NKCC2, and NCC)-mediated Cl- loading. The CCCs play vital roles in cell volume regulation and ion homeostasis. Gain-of-function or loss-of-function of these ion transporters can cause diseases in many tissues. In recent years, there have been considerable advances in our understanding of CCCs\' control mechanisms in cell volume regulations, with many techniques developed in studying the functions and activities of CCCs. Classic approaches to directly measure CCC activity involve assays that measure the transport of potassium substitutes through the CCCs. These techniques include the ammonium pulse technique, radioactive or nonradioactive rubidium ion uptake-assay, and thallium ion-uptake assay. CCCs\' activity can also be indirectly observed by measuring γ-aminobutyric acid (GABA) activity with patch-clamp electrophysiology and intracellular chloride concentration with sensitive microelectrodes, radiotracer 36Cl-, and fluorescent dyes. Other techniques include directly looking at kinase regulatory sites phosphorylation, flame photometry, 22Na+ uptake assay, structural biology, molecular modeling, and high-throughput drug screening. This review summarizes the role of CCCs in genetic disorders and cell volume regulation, current methods applied in studying CCCs biology, and compounds developed that directly or indirectly target the CCCs for disease treatments.

The cation-chloride cotransporters KCC2 and NKCC1 regulate the intracellular Cl- concentration and cell volume of neurons and/or glia. The Cl- extruder KCC2 is expressed at higher levels than the Cl- transporter NKCC1 in mature compared to immature neurons, accounting for the developmental shift from high to low Cl- concentration and from depolarizing to hyperpolarizing currents through GABA-A receptors. Previous studies have shown that KCC2 expression is downregulated following central nervous system injury, returning neurons to a more excitable state, which can be pathological or adaptive. Here, we show that deafferentation of the dendritic segments of granule cells in the outer (oml) and middle (mml) molecular layer of the dentate gyrus via entorhinal denervation in vivo leads to cell-type- and layer-specific changes in the expression of KCC2 and NKCC1. Microarray analysis validated by reverse transcription-quantitative polymerase chain reaction revealed a significant decrease in Kcc2 mRNA in the granule cell layer 7 days post-lesion. In contrast, Nkcc1 mRNA was upregulated in the oml/mml at this time point. Immunostaining revealed a selective reduction in KCC2 protein expression in the denervated dendrites of granule cells and an increase in NKCC1 expression in reactive astrocytes in the oml/mml. The NKCC1 upregulation is likely related to the increased activity of astrocytes and/or microglia in the deafferented region, while the transient KCC2 downregulation in granule cells may be associated with denervation-induced spine loss, potentially also serving a homeostatic role via boosting GABAergic depolarization. Furthermore, the delayed KCC2 recovery might be involved in the subsequent compensatory spinogenesis.

Cardiovascular diseases, the leading life-threatening conditions, involve cardiac arrhythmia, coronary artery disease, myocardial infarction, heart failure, cardiomyopathy, and heart valve disease that are associated with the altered functioning of cation-chloride cotransporters. The decreased number of cation-chloride cotransporters leads to reduced reactivity to adrenergic stimulation. The KCC family is crucial for numerous physiological processes including cell proliferation and invasion, regulation of membrane trafficking, maintaining ionic and osmotic homeostasis, erythrocyte swelling, dendritic spine formation, maturation of postsynaptic GABAergic inhibition, and inhibitory/excitatory signaling in neural tracts. KCC2 maintains intracellular chlorine homeostasis and opposes β-adrenergic stimulation-induced Cl- influx to prevent arrhythmogenesis. KCC3-inactivated cardiac tissue shows increased vascular resistance, aortic distensibility, heart size and weight (i.e. hypertrophic cardiomyopathy). Due to KCC4\'s high affinity for K+, it plays a vital role in cardiac ischemia with increased extracellular K+. The NKCC and NCC families play a vital role in the regulation of saliva volume, establishing the potassium-rich endolymph in the cochlea, sodium uptake in astrocytes, inhibiting myogenic response in microcirculatory beds, regulation of smooth muscle tone in resistance vessels, and blood pressure. NKCC1 regulates chlorine homeostasis and knocking it out impairs cardiomyocyte depolarization and cardiac contractility as well as impairs depolarization and contractility of vascular smooth muscle rings in the aorta. The activation of NCC in vascular cells promotes the formation of the abdominal aortic aneurysm. This narrative review provides a deep insight into the structure and function of KCCs, NKCCs, and NCC in human physiology and cardiac pathobiology. Also, it provides cell-specific (21 cell types) and region-specific (6 regions) expression of KCC1, KCC2, KCC3, KCC4, NKCC1, NKCC2, and NCC in heart.

Loop and thiazide diuretics have been cornerstones of clinical management of hypertension and fluid overload conditions for more than five decades. The hunt for their molecular targets led to the discovery of cation-chloride cotransporters (CCCs) that catalyze electroneutral movement of Cl- together with Na+ and/or K+. CCCs consist of two 1 Na+-1 K+-2 Cl- (NKCC1-2), one 1 Na+-1 Cl- (NCC), and four 1 K+-1 Cl- (KCC1-4) transporters in human. CCCs are fundamental in trans-epithelia ion secretion and absorption, homeostasis of intracellular Cl- concentration and cell volume, and regulation of neuronal excitability. Malfunction of NKCC2 and NCC leads to abnormal salt and water retention in the kidney and, consequently, imbalance in electrolytes and blood pressure. Mutations in KCC2 and KCC3 are associated with brain disorders due to impairments in regulation of excitability and possibly cell volume of neurons. A recent surge of structures of CCCs have defined their dimeric architecture, their ion binding sites, their conformational changes associated with ion translocation, and the mechanisms of action of loop diuretics and small molecule inhibitors. These breakthroughs now set the stage to expand CCC pharmacology beyond loop and thiazide diuretics, developing the next generation of diuretics with improved potency and specificity. Beyond drugging renal-specific CCCs, brain-penetrable therapeutics are sorely needed to target CCCs in the nervous system for the treatment of neurological disorders and psychiatric conditions.

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.

Epilepsies represent one of the most common neurological diseases worldwide. They are characterized by recurrent spontaneous seizures with severe impact on a patient\'s life. An imbalance in excitatory and inhibitory signalling is considered the main underlying pathophysiological mechanism. Therefore, GABA-mimetic drugs, strengthening the main inhibitory signalling system in the CNS, are frequently used as antiepileptic or anticonvulsant drugs. However, the therapeutic effect of such treatment depends on the chloride gradient along the plasma membrane. Impairment of chloride homeostasis, caused by alterations in the functional balance of chloride transporters, was implicated in the pathophysiology of epilepsy and numerous other diseases. Breakdown or even inversion of the chloride gradient may result in ineffective or in worst cases proconvulsant effects of GABA-mimetics. Unfortunately, such situations are reported in considerable number. Consequently, bumetanide, an inhibitor of Na-K-Cl cotransporters gained interest as potential add-on therapy re-establishing the chloride gradient and thereby the hyperpolarizing effects of GABA-mimetic drugs. Indeed, preclinical studies yielded encouraging results, especially when applied in combination with GABA-mimetics in epilepsy models. However, bumetanide induces a strong diuretic effect and displays poor penetration across the blood-brain barrier, two adverse features for chronic antiepileptic treatment. Therefore, new compounds overcoming these limitations are under development. This review focuses on alterations in chloride homeostasis and its underlying molecular mechanisms in epilepsy, on the potential impact of impaired chloride homeostasis on the treatment of epilepsy and on concepts to overcome this problem including recent development of bumetanide derivatives with improved pharmacological profile.

Chronic neuropathic pain is a debilitating condition that remains difficult to treat. Diminished synaptic inhibition by GABA and glycine and increased NMDA receptor (NMDAR) activity in the spinal dorsal horn are key mechanisms underlying neuropathic pain. However, the reciprocal relationship between synaptic inhibition and excitation in neuropathic pain is unclear. Here, we show that intrathecal delivery of K(+)-Cl(-) cotransporter-2 (KCC2) using lentiviral vectors produces a complete and long-lasting reversal of pain hypersensitivity induced by nerve injury. KCC2 gene transfer restores Cl(-) homeostasis disrupted by nerve injury in both spinal dorsal horn and primary sensory neurons. Remarkably, restoring Cl(-) homeostasis normalizes both presynaptic and postsynaptic NMDAR activity increased by nerve injury in the spinal dorsal horn. Our findings indicate that nerve injury recruits NMDAR-mediated signaling pathways through the disruption of Cl(-) homeostasis in spinal dorsal horn and primary sensory neurons. Lentiviral vector-mediated KCC2 expression is a promising gene therapy for the treatment of neuropathic pain.

The Na(+)-K(+)-Cl(-) cotransporter NKCC1 plays a major role in the regulation of intraneuronal Cl(-) concentration. Abnormal functionality of NKCC1 has been implicated in several brain disorders, including epilepsy. Bumetanide is the only available selective NKCC1 inhibitor, but also inhibits NKCC2, which can cause severe adverse effects during treatment of brain disorders. A NKCC1-selective bumetanide derivative would therefore be a desirable option. In the present study, we used the Xenopus oocyte heterologous expression system to compare the effects of bumetanide and several derivatives on the two major human splice variants of NKCCs, hNKCC1A and hNKCC2A. The derivatives were selected from a series of ~5000 3-amino-5-sulfamoylbenzoic acid derivatives, covering a wide range of structural modifications and diuretic potencies. To our knowledge, such structure-function relationships have not been performed before for NKCC1. Half maximal inhibitory concentrations (IC50s) of bumetanide were 0.68 (hNKCC1A) and 4.0μM (hNKCC2A), respectively, indicating that this drug is 6-times more potent to inhibit hNKCC1A than hNKCC2A. Side chain substitutions in the bumetanide molecule variably affected the potency to inhibit hNKCC1A. This allowed defining the minimal structural requirements necessary for ligand interaction. Unexpectedly, only a few of the bumetanide derivatives examined were more potent than bumetanide to inhibit hNKCC1A, and most of them also inhibited hNKCC2A, with a highly significant correlation between IC50s for the two NKCC isoforms. These data indicate that the structural requirements for inhibition of NKCC1 and NKCC2 are similar, which complicates development of bumetanide-related compounds with high selectivity for NKCC1.

Perturbations of γ-aminobutyric acid (GABA) neurotransmission in the human prefrontal cortex have been implicated in the pathogenesis of schizophrenia (SCZ), but the mechanisms are unclear. NKCC1 (SLC12A2) is a Cl(-)-importing cation-Cl(-) cotransporter that contributes to the maintenance of depolarizing GABA activity in immature neurons, and variation in SLC12A2 has been shown to increase the risk for schizophrenia via alterations of NKCC1 mRNA expression. However, no disease-causing mutations or functional variants in NKCC1 have been identified in human patients with SCZ. Here, by sequencing three large French-Canadian (FC) patient cohorts of SCZ, autism spectrum disorders (ASD), and intellectual disability (ID), we identified a novel heterozygous NKCC1 missense variant (p.Y199C) in SCZ. This variant is located in an evolutionarily conserved residue in the critical N-terminal regulatory domain and exhibits high predicted pathogenicity. No NKCC1 variants were detected in ASD or ID, and no KCC3 variants were identified in any of the three neurodevelopmental disorder cohorts. Functional experiments show Y199C is a gain-of-function variant, increasing Cl(-)-dependent and bumetanide-sensitive NKCC1 activity even in conditions in which the transporter is normally functionally silent (hypotonicity). These data are the first to describe a functional missense variant in SLC12A2 in human SCZ, and suggest that genetically encoded dysregulation of NKCC1 may be a risk factor for, or contribute to the pathogenesis of, human SCZ.