Involvement of aquaporin 5 and Na-K-2Cl cotransporter 1 in the pathogenesis of primary focal hyperhidrosis: evidence from the primary sweat gland cell culture.

People with primary focal hyperhidrosis (PFH) usually have an overactive sympathetic nervous system, which can activate the sweat glands through the chemical messenger of acetylcholine. The role of aquaporin 5 (AQP5) and Na-K-2Cl cotransporter 1 (NKCC1) in PFH is still unknown. The relative mRNA and protein levels of AQP5 and NKCC1 in the sweat gland tissues of three subtypes of patients with PFH (primary palmar hyperhidrosis, PPH; primary axillary hyperhidrosis, PAH; and primary craniofacial hyperhidrosis, PCH) were detected with real-time PCR (qPCR) and Western blot. Primary sweat gland cells from healthy controls (NPFH-SG) were incubated with different concentrations of acetylcholine, and the relative mRNA and protein expression of AQP5 and NKCC1 were also detected. NPFH-SG cells were also transfected with si-AQP5 or shNKCC1, and acetylcholine stimulation-induced calcium transients were assayed with Fluo-3 AM calcium assay. Upregulated AQP5 and NKCC1 expression were observed in sweat gland tissues, and AQP5 demonstrated a positive Pearson correlation with NKCC1 in patients with PPH (r = 0.66, P < 0.001), patients with PAH (r = 0.71, P < 0.001), and patients with PCH (r = 0.62, P < 0.001). Upregulated AQP5 and NKCC1 expression were also detected in primary sweat gland cells derived from three subtypes of patients with PFH when compared with primary sweat gland cells derived from healthy control. Acetylcholine stimulation could induce the upregulated AQP5 and NKCC1 expression in NPFH-SG cells, and AQP5 or NKCC1 inhibitions attenuated the calcium transients induced by acetylcholine stimulation in NPFH-SG cells. The dependence of ACh-stimulated calcium transients on AQP5 and NKCC1 expression may be involved in the development of PFH.NEW & NOTEWORTHY The dependence of ACh-stimulated calcium transients on AQP5 and Na-K-2Cl cotransporter 1 (NKCC1) expression may be involved in the development of primary focal hyperhidrosis (PFH).

Hypernegative GABAA Reversal Potential in Pyramidal Cells Contributes to Medial Prefrontal Cortex Deactivation in a Mouse Model of Neuropathic Pain.

Deactivation of the medial prefrontal cortex (mPFC) has been broadly reported in both neuropathic pain models and human chronic pain patients. Several cellular mechanisms may contribute to the inhibition of mPFC activity, including enhanced GABAergic inhibition. The functional effect of GABAA(γ-aminobutyric acid type A)-receptor activation depends on the concentration of intracellular chloride in the postsynaptic neuron, which is mainly regulated by the activity of Na-K-2Cl cotransporter isoform 1 (NKCC1) and K-Cl cotransporter isoform 2 (KCC2), 2 potassium-chloride cotransporters that import and extrude chloride, respectively. Recent work has shown that the NKCC1-KCC2 ratio is affected in numerous pathological conditions, and we hypothesized that it may contribute to the alteration of mPFC function in neuropathic pain. We used quantitative in situ hybridization to assess the level of expression of NKCC1 and KCC2 in the mPFC of a mouse model of neuropathic pain (spared nerve injury), and we found that KCC2 transcript is increased in the mPFC of spared nerve injury mice while NKCC1 is not affected. Perforated patch recordings further showed that this results in the hypernegative reversal potential of the GABAA current in pyramidal neurons of the mPFC. Computational simulations suggested that this change in GABAA reversal potential is sufficient to significantly reduce the overall activity of the cortical network. Thus, our results identify a novel pathological modulation of GABAA function and a new mechanism by which mPFC function is inhibited in neuropathic pain. Our data also help explain previous findings showing that activation of mPFC interneurons has proalgesic effect in neuropathic, but not in control conditions. PERSPECTIVE: Chronic pain is associated with the presence of depolarizing GABAA current in the spinal cord, suggesting that pharmacological NKCC1 antagonism has analgesic effects. However, our results show that in neuropathic pain, GABAA current is actually hyperinhibitory in the mPFC, where it contributes to the mPFC functional deactivation. This suggests caution in the use of NKCC1 antagonism to treat pain.

Physiological role of hydrogen sulfide in the kidney and its therapeutic implications for kidney diseases.

For over three centuries, hydrogen sulfide (H2S) has been known as a toxic and deadly gas at high concentrations, with a distinctive smell of rotten eggs. However, studies over the past two decades have shown that H2S has risen above its historically notorious label and has now received significant scientific attention as an endogenously produced gaseous signaling molecule that participates in cellular homeostasis and influences a myriad of physiological and pathological processes at low concentrations. Its endogenous production is enzymatically regulated, and when dysregulated, contributes to pathogenesis of renal diseases. In addition, exogenous H2S administration has been reported to exhibit important therapeutic characteristics that target multiple molecular pathways in common renal pathologies in which reduced levels of renal and plasma H2S were observed. This review highlights functional anatomy of the kidney and renal production of H2S. The review also discusses current understanding of H2S in renal physiology and seeks to lay the foundation as a new targeted therapeutic agent for renal pathologies such as hypertensive nephropathy, diabetic kidney disease and water balance disorders.

Antihypertension effect of astragaloside IV during cerebral ischemia reperfusion in rats.

Stroke is one of the leading causes of death from diseases. When the blood supply to the brain tissue is interrupted, neuronal core death occurs due to the lack of glucose and oxygen in min. Blood pressure lowering after ischemic stroke was proven to be an effective strategy to achieve neurovascular protection and reduce the risk of recurrent stroke. Astragaloside IV is a pure small molecular compound isolated from Radix Astragali, and it is well documented that astragaloside IV has neuroprotective effect on cerebral ischemia reperfusion (CIR) injury through many mechanisms, including antioxidant, anti­inflammatory and anti­apoptotic. The present study adopted mean arterial pressure (MAP) monitoring, neurological scoring, 2,3,5­triphenyltetrazolium chloride staining, enzyme­linked immuno­sorbent assay, western blotting and other experimental methods to investigate the effect of astragaloside IV on systemic blood pressure during CIR in a middle cerebral artery occlusion animal model. It was demonstrated that astragaloside IV pretreatment significantly alleviated CIR injury as previously reported. In addition, the elevation of MAP during CIR was significantly inhibited by astragaloside IV administration. Moreover, it was revealed that the expression of Na+­K+­2Cl­ cotransporter isoform 1 in the hypothalamus was inhibited and the subsequent synthesis of vasopressin was reduced by astragaloside IV pretreatment in the CIR animal model. In conclusion, astragaloside IV may alleviate CIR injury partially by lowering systemic blood pressure.

How Staying Negative Is Good for the (Adult) Brain: Maintaining Chloride Homeostasis and the GABA-Shift in Neurological Disorders.

Excitatory-inhibitory (E-I) imbalance has been shown to contribute to the pathogenesis of a wide range of neurodevelopmental disorders including autism spectrum disorders, epilepsy, and schizophrenia. GABA neurotransmission, the principal inhibitory signal in the mature brain, is critically coupled to proper regulation of chloride homeostasis. During brain maturation, changes in the transport of chloride ions across neuronal cell membranes act to gradually change the majority of GABA signaling from excitatory to inhibitory for neuronal activation, and dysregulation of this GABA-shift likely contributes to multiple neurodevelopmental abnormalities that are associated with circuit dysfunction. Whilst traditionally viewed as a phenomenon which occurs during brain development, recent evidence suggests that this GABA-shift may also be involved in neuropsychiatric disorders due to the "dematuration" of affected neurons. In this review, we will discuss the cell signaling and regulatory mechanisms underlying the GABA-shift phenomenon in the context of the latest findings in the field, in particular the role of chloride cotransporters NKCC1 and KCC2, and furthermore how these regulatory processes are altered in neurodevelopmental and neuropsychiatric disorders. We will also explore the interactions between GABAergic interneurons and other cell types in the developing brain that may influence the GABA-shift. Finally, with a greater understanding of how the GABA-shift is altered in pathological conditions, we will briefly outline recent progress on targeting NKCC1 and KCC2 as a therapeutic strategy against neurodevelopmental and neuropsychiatric disorders associated with improper chloride homeostasis and GABA-shift abnormalities.

Tubular-specific CDK12 knockout causes a defect in urine concentration due to premature cleavage of the slc12a1 gene.

Cyclin-dependent kinase 12 (CDK12) plays a critical role in regulating gene transcription. CDK12 inhibition is a potential anticancer therapeutic strategy. However, several clinical trials have shown that CDK inhibitors might cause renal dysfunction and electrolyte disorders. CDK12 is abundant in renal tubular epithelial cells (RTECs), but the exact role of CDK12 in renal physiology remains unclear. Genetic knockout of CDK12 in mouse RTECs causes polydipsia, polyuria, and hydronephrosis. This phenotype is caused by defects in water reabsorption that are the result of reduced Na-K-2Cl cotransporter 2 (NKCC2) levels in the kidney. In addition, CKD12 knockout causes an increase in Slc12a1 (which encodes NKCC2) intronic polyadenylation events, which results in Slc12a1 truncated transcript production and NKCC2 downregulation. These findings provide novel insight into CDK12 being necessary for maintaining renal homeostasis by regulating NKCC2 transcription, which explains the critical water and electrolyte disturbance that occurs during the application of CDK12 inhibitors for cancer treatment. Therefore, there are safety concerns about the clinical use of these new anticancer drugs.

The Acute Effects of Furosemide on Na-K-Cl Cotransporter-1, Fetuin-A and Pigment Epithelium-Derived Factor in the Guinea Pig Cochlea.

Background: Furosemide is a loop diuretic used to treat edema; however, it also targets the Na-K-Cl cotransporter-1 (NKCC1) in the inner ear. In very high doses, furosemide abolishes the endocochlear potential (EP). The aim of the study was to gain a deeper understanding of the temporal course of the acute effects of furosemide in the inner ear, including the protein localization of Fetuin-A and PEDF in guinea pig cochleae. Material and Method: Adult guinea pigs were given an intravenous injection of furosemide in a dose of 100 mg per kg of body weight. The cochleae were studied using immunohistochemistry in controls and at four intervals: 3 min, 30 min, 60 min and 120 min. Also, cochleae of untreated guinea pigs were tested for Fetuin-A and PEDF mRNA using RNAscope® technology. Results: At 3 min, NKCC1 staining was abolished in the type II fibrocytes in the spiral ligament, followed by a recovery period of up to 120 min. In the stria vascularis, the lowest staining intensity of NKCC1 presented after 30 min. The spiral ganglion showed a stable staining intensity for the full 120 min. Fetuin-A protein and mRNA were detected in the spiral ganglion type I neurons, inner and outer hair cells, pillar cells, Deiters cells and the stria vascularis. Furosemide induced an increased staining intensity of Fetuin-A at 120 min. PEDF protein and mRNA were found in the spiral ganglia type I neurons, the stria vascularis, and in type I and type II fibrocytes of the spiral ligament. PEDF protein staining intensity was high in the pillar cells in the organ of Corti. Furosemide induced an increased staining intensity of PEDF in type I neurons and pillar cells after 120 min. Conclusion: The results indicate rapid furosemide-induced changes of NKCC1 in the type II fibrocytes. This could be part of the mechanism that causes reduction of the EP within minutes after high dose furosemide injection. Fetuin-A and PEDF are present in many cells of the cochlea and probably increase after furosemide exposure, possibly as an otoprotective response.

Glial Chloride Homeostasis Under Transient Ischemic Stress.

High water permeabilities permit rapid adjustments of glial volume upon changes in external and internal osmolarity, and pathologically altered intracellular chloride concentrations ([Cl-]int) and glial cell swelling are often assumed to represent early events in ischemia, infections, or traumatic brain injury. Experimental data for glial [Cl-]int are lacking for most brain regions, under normal as well as under pathological conditions. We measured [Cl-]int in hippocampal and neocortical astrocytes and in hippocampal radial glia-like (RGL) cells in acute murine brain slices using fluorescence lifetime imaging microscopy with the chloride-sensitive dye MQAE at room temperature. We observed substantial heterogeneity in baseline [Cl-]int, ranging from 14.0 ± 2.0 mM in neocortical astrocytes to 28.4 ± 3.0 mM in dentate gyrus astrocytes. Chloride accumulation by the Na+-K+-2Cl- cotransporter (NKCC1) and chloride outward transport (efflux) through K+-Cl- cotransporters (KCC1 and KCC3) or excitatory amino acid transporter (EAAT) anion channels control [Cl-]int to variable extent in distinct brain regions. In hippocampal astrocytes, blocking NKCC1 decreased [Cl-]int, whereas KCC or EAAT anion channel inhibition had little effect. In contrast, neocortical astrocytic or RGL [Cl-]int was very sensitive to block of chloride outward transport, but not to NKCC1 inhibition. Mathematical modeling demonstrated that higher numbers of NKCC1 and KCC transporters can account for lower [Cl-]int in neocortical than in hippocampal astrocytes. Energy depletion mimicking ischemia for up to 10 min did not result in pronounced changes in [Cl-]int in any of the tested glial cell types. However, [Cl-]int changes occurred under ischemic conditions after blocking selected anion transporters. We conclude that stimulated chloride accumulation and chloride efflux compensate for each other and prevent glial swelling under transient energy deprivation.

The neurogliovascular unit in hepatic encephalopathy.

Hepatic encephalopathy (HE) is a neurological complication of hepatic dysfunction and portosystemic shunting. It is highly prevalent in patients with cirrhosis and is associated with poor outcomes. New insights into the role of peripheral origins in HE have led to the development of innovative treatment strategies like faecal microbiota transplantation. However, this broadening of view has not been applied fully to perturbations in the central nervous system. The old paradigm that HE is the clinical manifestation of ammonia-induced astrocyte dysfunction and its secondary neuronal consequences requires updating. In this review, we will use the holistic concept of the neurogliovascular unit to describe central nervous system disturbances in HE, an approach that has proven instrumental in other neurological disorders. We will describe HE as a global dysfunction of the neurogliovascular unit, where blood flow and nutrient supply to the brain, as well as the function of the blood-brain barrier, are impaired. This leads to an accumulation of neurotoxic substances, chief among them ammonia and inflammatory mediators, causing dysfunction of astrocytes and microglia. Finally, glymphatic dysfunction impairs the clearance of these neurotoxins, further aggravating their effect on the brain. Taking a broader view of central nervous system alterations in liver disease could serve as the basis for further research into the specific brain pathophysiology of HE, as well as the development of therapeutic strategies specifically aimed at counteracting the often irreversible central nervous system damage seen in these patients.

Advances in the development of novel compounds targeting cation-chloride cotransporter physiology.

For about half a century, the pharmacology of electroneutral cation-chloride cotransporters has been dominated by a few drugs that are widely used in clinical medicine. Because these diuretic drugs are so good at what they do, there has been little incentive in expanding their pharmacology. The increasing realization that cation-chloride cotransporters are involved in many other key physiological processes and the knowledge that different tissues express homologous proteins with matching transport functions have rekindled interest in drug discovery. This review summarizes the methods available to assess the function of these transporters and describe the multiple efforts that have made to identify new compounds. We describe multiple screens targeting KCC2 function and one screen designed to find compounds that discriminate between NKCC1 and NKCC2. Two of the KCC2 screens identified new inhibitors that are 3-4 orders of magnitude more potent than furosemide. Additional screens identified compounds that purportedly increase cell surface expression of the cotransporter, as well as several FDA-approved drugs that increase KCC2 transcription and expression. The technical details of each screen biased them toward specific processes in the life cycle of the transporter, making these efforts independent and complementary. In addition, each drug discovery effort contributes to our understanding of the biology of the cotransporters.

Na+-K+-2Cl- cotransporter 2 located in the human and murine gastric mucosa is involved in secretagogue-induced gastric acid secretion and is downregulated in lipopolysaccharide-treated mice.

Na+-K+-2Cl- cotransporter (NKCC) is expressed at exceptionally high levels in gastric parietal cells. Bumetanide, a potent loop diuretic that blocks NKCC, usually causes a decrease in gastric acid secretion. Endotoxaemia causes hypochlorhydria in vivo, in which lipopolysaccharide (LPS) plays an important role. This study aimed to investigate the effect of NKCC2 on gastric acid secretion and its alteration in LPS-treated mice. The scanning ion-selective electrode technique and real-time pH titration combined with RNA interference were used to determine the effects of bumetanide on gastric acid secretion. Immunochemistry and Western blotting were performed to investigate the changes in NKCC2 expression in LPS-treated mice. Immunoreactivity of NKCC1 and NKCC2 was mainly observed near the basolateral and apical membranes of parietal cells, respectively. Pretreatment with bumetanide reduced the histamine-stimulated H+ flux in the mouse gastric mucosa. The apical, but not the basolateral, addition of bumetanide inhibited forskolin- or histamine/3-isobutyl-1-methylxanthine(IBMX)-induced gastric acid secretion. In vivo treatment with NKCC2 siRNA inhibited forskolin-induced acid secretion. Upon histamine stimulation, the majority of NKCC2 was targeted to the apical membrane in the gastric mucosa and in primary cultured parietal cells. The expression of NKCC2 and vesicle-associated membrane protein-2 (VAMP2), but not that of H+/K+-ATPase, was decreased in the gastric mucosa of LPS-treated mice. Blocking apical NKCC2, but not basolateral NKCC1, by bumetanide inhibited secretagogue-induced gastric acid secretion, during which the membrane trafficking of NKCC2 may be necessary. The downregulation of NKCC2 and VAMP2 may be related to the reduced gastric acid secretion induced by LPS.

Asthmatic Airway Vagal Hypertonia Involves Chloride Dyshomeostasis of Preganglionic Neurons in Rats.

Airway vagal hypertonia is closely related to the severity of asthma; however, the mechanisms of its genesis are unclear. This study aims to prove that asthmatic airway vagal hypertonia involves neuronal Cl- dyshomeostasis. The experimental airway allergy model was prepared with ovalbumin in male adult Sprague-Dawley rats. Plethysmography was used to evaluate airway vagal response to intracisternally injected γ-aminobutyric acid (GABA). Immunofluorescent staining and Western-blot assay were used to examine the expression of microglia-specific proteins, Na+-K+-2Cl- co-transporter 1 (NKCC1), K+-Cl- co-transporter 2 (KCC2) and brain-derived nerve growth factor (BDNF) in airway vagal centers. Pulmonary inflammatory changes were examined with hematoxylin and eosin staining of lung sections and ELISA assay of ovalbumin-specific IgE in bronchoalveolar lavage fluid (BALF). The results showed that histochemically, experimental airway allergy activated microglia, upregulated NKCC1, downregulated KCC2, and increased the content of BDNF in airway vagal centers. Functionally, experimental airway allergy augmented the excitatory airway vagal response to intracisternally injected GABA, which was attenuated by intracisternally pre-injected NKCC1 inhibitor bumetanide. All of the changes induced by experimental airway allergy were prevented or mitigated by chronic intracerebroventricular or intraperitoneal injection of minocycline, an inhibitor of microglia activation. These results demonstrate that experimental airway allergy augments the excitatory response of airway vagal centers to GABA, which might be the result of neuronal Cl- dyshomeostasis subsequent to microglia activation, increased BDNF release and altered expression of Cl- transporters. Cl- dyshomeostasis in airway vagal centers might contribute to the genesis of airway vagal hypertonia in asthma.

Sodium Transporters in Human Health and Disease.

Sodium (Na+) electrochemical gradients established by Na+/K+ ATPase activity drives the transport of ions, minerals, and sugars in both excitable and non-excitable cells. Na+-dependent transporters can move these solutes in the same direction (cotransport) or in opposite directions (exchanger) across both the apical and basolateral plasma membranes of polarized epithelia. In addition to maintaining physiological homeostasis of these solutes, increases and decreases in sodium may also initiate, directly or indirectly, signaling cascades that regulate a variety of intracellular post-translational events. In this review, we will describe how the Na+/K+ ATPase maintains a Na+ gradient utilized by multiple sodium-dependent transport mechanisms to regulate glucose uptake, excitatory neurotransmitters, calcium signaling, acid-base balance, salt-wasting disorders, fluid volume, and magnesium transport. We will discuss how several Na+-dependent cotransporters and Na+-dependent exchangers have significant roles in human health and disease. Finally, we will discuss how each of these Na+-dependent transport mechanisms have either been shown or have the potential to use Na+ in a secondary role as a signaling molecule.

Cryo-EM structures of DrNKCC1 and hKCC1: a new milestone in the physiology of cation-chloride cotransporters.

New milestones have been reached in the field of cation-Cl- cotransporters with the recently released cryo-electron microscopy (EM) structures of the Danio rerio (zebrafish) Na+-K+-2Cl- cotransporter (DrNKCC1) and the human K+-Cl- cotransporter (hKCC1). In this review we provide a brief timeline that identifies the multiple breakthroughs in the field of solute carrier 12 transporters that led to the structure resolution of two of its key members. While cation-Cl- cotransporters share the overall architecture of carriers belonging to the amino acid-polyamine-organocation (APC) superfamily and some of their substrate binding sites, several new insights are gained from the two individual structures. A first major feature relates to the largest extracellular domain between transmembrane domain (TMD) 5 and TMD6 of KCC1, which stabilizes the dimer and forms a cap that likely participates in extracellular gating. A second feature is the conservation of the K+ and Cl- binding sites in both structures and evidence of an unexpected second Cl- coordination site in the KCC1 structure. Structural data are discussed in the context of previously published studies that examined the basic and kinetics properties of these cotransport mechanisms. A third characteristic is the evidence of an extracellular gate formed by conserved salt bridges between charged residues located toward the end of TMD3 and TMD4 in both transporters and the existence of an additional neighboring bridge in the hKCC1 structure. A fourth feature of these newly solved structures relates to the multiple points of contacts between the monomer forming the cotransporter homodimer units. These involve the TMDs, the COOH-terminal domains, and the large extracellular loop for hKCC1.

Seasonal exploration of ultrastructure and Na+/K+-ATPase, Na+/K+/2Cl- cotransporter of mitochondria-rich cells in the small intestine of turtles.

Despite the exploration of mitochondria-rich cells (MRCs) in different animal classes, very limited information has been documented about MRCs in reptiles. The present study was designed to investigate the effect of seasonal variation on the cell ultrastructure and ion transport protein activity of MRCs during hibernation and non-hibernation of Chinese soft-shelled turtle's intestine. Transmission electron microscopy revealed that, during hibernation the high-density cytoplasm of MRCs occupied large cross-sectional area and showed heterogeneous abundance of mitochondria and an expanded extensive tubular system as compared to non-hibernation. During hibernation the cytoplasm of MRCs exhibited more mitochondrial vacuolization, autophagosomes, phagophore formation and well-structured endoplasmic reticulum. During hibernation, MRCs connected with absorptive cells through wide interdigitation, and created tight junction and more desmosomes as compared to non-hibernation. Immunohistochemistry and immunofluorescence showed, the strong immunopositive reactions and immunosignaling of Na+/K+-ATPase (NKA) and Na+/K+/2Cl- cotransporter (NKCC) at basolateral region of mucosal surface of intestine during hibernation. However, weak immunopositive reactions and immunosignaling of NKA and NKCC during non-hibernation. The statistical analysis showed that the number and size of MRCs with NKA-associated immunoreactivity were significantly increased during hibernation. NKA and NKCC mRNA expression was significantly increased during hibernation via qPCR. Further confirmed, the intensity of NKA and NKCC proteins was more elevated during hibernation than non-hibernation shown by immunobloting. However, the concentrations of the plasma ions Na+ and Cl- were significantly higher during hibernation; conversely, K+ concentration was significantly higher during non-hibernation. The findings suggest that the potential role of MRCs is affected by seasonal fluctuations, during which intestinal homeostasis and hydromineral balance are essential for turtles.

Superoxide increases surface NKCC2 in the rat thick ascending limbs via PKC.

The apical Na+-K+-2Cl- cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb (TAL). The free radical superoxide ( O2- ) stimulates TAL NaCl absorption by enhancing NKCC2 activity. In contrast, nitric oxide (NO) scavenges O2- and inhibits NKCC2. NKCC2 activity depends on the number of NKCC2 transporters in the TAL apical membrane and its phosphorylation. We hypothesized that O2- stimulates NKCC2 activity by enhancing apical surface NKCC2 expression. We measured surface NKCC2 expression in rat TALs by surface biotinylation and Western blot analysis. Treatment of TALs with O2- produced by exogenous xanthine oxidase (1 mU/ml) and hypoxanthine (500 µM) stimulated surface NKCC2 expression by ~18 ± 5% (P < 0.05). O2- -stimulated surface NKCC2 expression was blocked by the O2- scavenger tempol (50 µM). Scavenging H2O2 with 100 U/ml catalase did not block the stimulatory effect of xanthine oxidase-hypoxanthine (22 ± 8% increase from control, P < 0.05). Inhibition of endogenous NO production with Nω-nitro-l-arginine methyl ester enhanced surface NKCC2 expression by 21 ± 6% and, when added together with xanthine oxidase-hypoxanthine, increased surface NKCC2 by 41 ± 10% (P < 0.05). Scavenging O2- with superoxide dismutase (300 U/ml) decreased this stimulatory effect by 60% (39 ± 4% to 15 ± 10%, P < 0.05). Protein kinase C inhibition with Gö-6976 (100 nM) blocked O2- -stimulated surface NKCC2 expression (P < 0.05). O2- did not affect NKCC2 phosphorylation at Thr96/101 or its upstream kinases STE20/SPS1-related proline/alanine-rich kinase-oxidative stress-responsive kinase 1. We conclude that O2- increases surface NKCC2 expression by stimulating protein kinase C and that this effect is blunted by endogenous NO. O2- -stimulated apical trafficking of NKCC2 may be involved in the enhanced surface NKCC2 expression observed in Dahl salt-sensitive rats.

Cloning of the Murine Na+-K+-2Cl-Cotransporter Gene Promoter and the Effect of 20-HETE on Its Transcriptional Activity / 中国医科大学学报

Objective To clone the murine Na+-K+-2Cl-cotransporter (Nkcc2) gene promoter and analyze 20-HETE regulation of the murine Nkcc2 gene transcriptional activity. Methods A fragment of the murine Nkcc2 gene promoter was analyzed using bioinformatics software AliBaba and TRANSFAC TESS. The murine Nkcc2 gene promoter fragment (-1 462 bp-+40 bp) was amplified by PCR using murine genomic DNA as a template and then cloned into a pGL3-Basic vector to generate a luciferase reporter construct. The recombinant reporter construct was transiently transfected into HEK293 T cells using Lipofectamine 2000 for 24 h. The transfected HEK293 T cells were treated with 20-HETE for 2 h followed by measurement of the luciferase activity using the Dual-Luciferase Reporter Assay system. Results A luciferase reporter construct containing the murine Nkcc2 gene promoter was successfully generated. The results showed that 20-HETE significantly reduced the transcriptional activity of the construct. Conclusion 20-HETE may reduce the expression of the murine Nkcc2 gene through transcriptional regulation.

Inhibition of NKCC1 Modulates Alveolar Fluid Clearance and Inflammation in Ischemia-Reperfusion Lung Injury via TRAF6-Mediated Pathways.

Background: The expression of Na-K-2Cl cotransporter 1 (NKCC1) in the alveolar epithelium is responsible for fluid homeostasis in acute lung injury (ALI). Increasing evidence suggests that NKCC1 is associated with inflammation in ALI. We hypothesized that inhibiting NKCC1 would attenuate ALI after ischemia-reperfusion (IR) by modulating pathways that are mediated by tumor necrosis-associated factor 6 (TRAF6). Methods: IR-ALI was induced by producing 30 min of ischemia followed by 90 min of reperfusion in situ in an isolated and perfused rat lung model. The rats were randomly allotted into four groups comprising two control groups and two IR groups with and without bumetanide. Alveolar fluid clearance (AFC) was measured for each group. Mouse alveolar MLE-12 cells were cultured in control and hypoxia-reoxygenation (HR) conditions with or without bumetanide. Flow cytometry and transwell monolayer permeability assay were carried out for each group. Results: Bumetanide attenuated the activation of p-NKCC1 and lung edema after IR. In the HR model, bumetanide decreased the cellular volume and increased the transwell permeability. In contrast, bumetanide increased the expression of epithelial sodium channel (ENaC) via p38 mitogen-activated protein kinase (p38 MAPK), which attenuated the reduction of AFC after IR. Bumetanide also modulated lung inflammation via nuclear factor-κB (NF-κB). TRAF6, which is upstream of p38 MAPK and NF-κB, was attenuated by bumetanide after IR and HR. Conclusions: Inhibition of NKCC1 by bumetanide reciprocally modulated epithelial p38 MAPK and NF-κB via TRAF6 in IR-ALI. This interaction attenuated the reduction of AFC via upregulating ENaC expression and reduced lung inflammation.

Activation of TRPV1 channels leads to stimulation of NKCC1 cotransport in the lens.

Lens ion homeostasis is crucial in maintaining water content and, in turn, refractive index and transparency of the multicellular syncytium-like structure. New information is emerging on the regulation of ion transport in the lens by mechanisms that rely on transient receptor potential vanilloid (TRPV) ion channels. We found recently that TRPV1 activation leads to Ca2+/PKC-dependent ERK1/2 signaling. Here, we show that the TRPV1 agonist capsaicin (100 nM) and hyperosmotic solution (350 vs. 300 mosM) each caused an increase of bumetanide-inhibitable Rb uptake by intact porcine lenses and Na-K-2Cl cotransporter 1 (NKCC1) phosphorylation in the lens epithelium. The TRPV1 antagonist A889425 (1 µM) abolished the increases of Rb uptake and NKCC1 phosphorylation in response to hyperosmotic solution. Exposing lenses to hyperosmotic solution in the presence of MEK/ERK inhibitor U0126 (10 µM) or the with-no-lysine kinase (WNK) inhibitor WNK463 (1 µM) also prevented NKCC1 phosphorylation and the Rb uptake responses to hyperosmotic solution. WNK463 did not prevent the increase in ERK1/2 phosphorylation that occurs in response to capsaicin or hyperosmotic solution, suggesting that ERK1/2 activation occurs before WNK activation in the sequence of signaling events. Taken together, the evidence indicates that activation of TRPV1 is a critical early step in a signaling mechanism that responds to a hyperosmotic stimulus, possibly lens shrinkage. By activating ERK1/2 and WNK, TRPV1 activation leads to NKCC1 phosphorylation and stimulation of NKCC1-mediated ion transport.

Molecular Basis and Differentiation-Associated Alterations of Anion Secretion in Human Duodenal Enteroid Monolayers.

BACKGROUND & AIMS: Human enteroids present a novel tool to study human intestinal ion transport physiology and pathophysiology. The present study describes the contributions of Cl- and HCO3- secretion to total cyclic adenosine monophosphate (cAMP)-stimulated electrogenic anion secretion in human duodenal enteroid monolayers and the relevant changes after differentiation. METHODS: Human duodenal enteroids derived from 4 donors were grown as monolayers and differentiated by a protocol that includes the removal of Wnt3A, R-spondin1, and SB202190 for 5 days. The messenger RNA level and protein expression of selected ion transporters and carbonic anhydrase isoforms were determined by quantitative real-time polymerase chain reaction and immunoblotting, respectively. Undifferentiated and differentiated enteroid monolayers were mounted in the Ussing chamber/voltage-current clamp apparatus, using solutions that contained as well as lacked Cl- and HCO3-/CO2, to determine the magnitude of forskolin-induced short-circuit current change and its sensitivity to specific inhibitors that target selected ion transporters and carbonic anhydrase(s). RESULTS: Differentiation resulted in a significant reduction in the messenger RNA level and protein expression of cystic fibrosis transmembrane conductance regulator, (CFTR) Na+/K+/2Cl- co-transporter 1 (NKCC1), and potassium channel, voltage gated, subfamily E, regulatory subunit 3 (KCNE3); and, conversely, increase of down-regulated-in-adenoma (DRA), electrogenic Na+/HCO3- co-transporter 1 (NBCe1), carbonic anhydrase 2 (CA2), and carbonic anhydrase 4 (CA4). Both undifferentiated and differentiated enteroids showed active cAMP-stimulated anion secretion that included both Cl- and HCO3- secretion as the magnitude of total active anion secretion was reduced after the removal of extracellular Cl- or HCO3-/CO2. The magnitude of total anion secretion in differentiated enteroids was approximately 33% of that in undifferentiated enteroids, primarily owing to the reduction in Cl- secretion with no significant change in HCO3- secretion. Anion secretion was consistently lower but detectable in differentiated enteroids compared with undifferentiated enteroids in the absence of extracellular Cl- or HCO3-/CO2. Inhibiting CFTR, NKCC1, carbonic anhydrase(s), cAMP-activated K+ channel(s), and Na+/K+-adenosine triphosphatase reduced cAMP-stimulated anion secretion in both undifferentiated and differentiated enteroids. CONCLUSIONS: Human enteroids recapitulate anion secretion physiology of small intestinal epithelium. Enteroid differentiation is associated with significant alterations in the expression of several ion transporters and carbonic anhydrase isoforms, leading to a reduced but preserved anion secretory phenotype owing to markedly reduced Cl- secretion but no significant change in HCO3- secretion.