Altered Cl- homeostasis hinders forebrain GABAergic interneuron migration in a mouse model of intellectual disability.

Impairments of inhibitory circuits are at the basis of most, if not all, cognitive deficits. The impact of OPHN1, a gene associate with intellectual disability (ID), on inhibitory neurons remains elusive. We addressed this issue by analyzing the postnatal migration of inhibitory interneurons derived from the subventricular zone in a validated mouse model of ID (OPHN1-/y mice). We found that the speed and directionality of migrating neuroblasts were deeply perturbed in OPHN1-/y mice. The significant reduction in speed was due to altered chloride (Cl-) homeostasis, while the overactivation of the OPHN1 downstream signaling pathway, RhoA kinase (ROCK), caused abnormalities in the directionality of the neuroblast progression in mutants. Blocking the cation-Cl- cotransporter KCC2 almost completely rescued the migration speed while proper directionality was restored upon ROCK inhibition. Our data unveil a strong impact of OPHN1 on GABAergic inhibitory interneurons and identify putative targets for successful therapeutic approaches.

Chloride salinity in a chloride-sensitive plant: Focusing on photosynthesis, hormone synthesis and transduction in tobacco.

Chloride (Cl-) is a micronutrient and a beneficial ion for plant growth, but excess Cl- easily leads to Cl- salinity. As a species sensitive to Cl-, tobacco experiences serious damage when encountering excessive Cl-. However, the molecular mechanism by which excess Cl- delays plant growth and development remain largely unknown. In this study, physiological, biochemical and genetic responses were determined in tobacco plants exposed to 12 h, 24 h and 48 h of Cl- salinity. Compared with the control, Cl- salinity increased the content of Cl- and decreased the relative water content (RWC) in tobacco, which severely limited the photosynthetic capacity and reduced photosynthetic products, resulting in decreased levels of auxin (IAA) and gibberellin (GA3). In addition, tobacco increased the content of starch, total phenol and increased phenylalanine ammonia-lyase (PAL) activity in response to Cl- salinity. Multi-omics results revealed that a total of 15,445 genes and 1983 proteins were differentially abundant in response to Cl- salinity. Two metabolic pathways, phenylalanine metabolism and starch and sucrose metabolism, were specifically enriched in the transcriptomic and proteomic data, respectively. In addition, our conjoint analysis of RNA-Seq and proteomics data revealed that 734 differentially abundant genes/proteins were enriched mainly in plant hormone signal transduction, photosynthesis and photosynthesis-antenna protein pathways. Our work presented here not only provides new insights into the molecular response of tobacco to Cl- salinity but also offers important guidance for the improvement of Cl- sensitive crops.

Vasorelaxation elicited by endogenous and exogenous hydrogen sulfide in mouse mesenteric arteries.

H2S causes vasorelaxation however there is considerable heterogeneity in the reported pharmacological mechanism of this effect. This study examines the contribution of endogenously released H2S in the regulation of vascular tone and the mechanism of H2S-induced vasorelaxation in small resistance-like arteries. Mesenteric arteries from C57 and eNOS-/- mice were mounted in myographs to record isometric force. Vasorelaxation responses to NaHS were examined in the presence of various inhibitors of vasorelaxation pathways. Expression and activity of the H2S-producing enzyme, cystathionine-γ-lyase (CSE), were also examined. CSE was expressed in vascular smooth muscle and perivascular adipose cells from mouse mesenteric artery. The substrate for CSE, L-cysteine, caused a modest vasorelaxation (35%) in arteries from C57 mice and poor vasorelaxation (10%) in arteries from eNOS-/- mice that was sensitive to the CSE inhibitor DL-propargylglycine. The fast H2S donor, NaHS, elicited a full and biphasic vasorelaxation response in mesenteric arteries (EC50 (1) 8.7 µM, EC50 (2) 0.6 mM), which was significantly inhibited in eNOS-/- vessels (P < 0.05), unaffected by endothelial removal, or blockers at any point in the NO via soluble guanylate cyclase and cGMP (NO-sGC-cGMP) vasorelaxation pathway. Vasorelaxation to NaHS was significantly inhibited by blocking K+ channels of the KCa and KV subtypes and the Cl-/HCO3- exchanger (P < 0.05). Further experiments showed that NaHS can significantly inhibit voltage-gated Ca2+ channel function (P < 0.05). The vasorelaxant effect of H2S in small resistance-like arteries is complex, involving eNOS, K+ channels, Cl-/HCO3- exchanger, and voltage-gated Ca2+ channels. CSE is present in the smooth muscle and periadventitial adipose tissue of these resistance-like vessels and can be activated to cause modest vasorelaxation under these in vitro conditions.

The chloride anion as a signalling effector.

The specific role of the chloride anion (Cl- ) as a signalling effector or second messenger has been increasingly recognized in recent years. It could represent a key factor in the regulation of cellular homeostasis. Changes in intracellular Cl- concentration affect diverse cellular functions such as gene and protein expression and activities, post-translational modifications of proteins, cellular volume, cell cycle, cell proliferation and differentiation, membrane potential, reactive oxygen species levels, and intracellular/extracellular pH. Cl- also modulates functions in different organelles, including endosomes, phagosomes, lysosomes, endoplasmic reticulum, and mitochondria. A better knowledge of Cl- signalling could help in understanding the molecular and metabolic changes seen in pathologies with altered Cl- transport or under physiological conditions. Here we review relevant evidence supporting the role of Cl- as a signalling effector.

Intracellular chloride: a regulator of transepithelial transport in the distal nephron.

PURPOSE OF REVIEW: This review focuses on the role of intracellular chloride in regulating transepithelial ion transport in the distal convoluted tubule (DCT) in response to perturbations in plasma potassium homeostasis. RECENT FINDINGS: Low dietary potassium increases the phosphorylation and activity of the sodium chloride cotransporter (NCC) in the DCT, and vice versa, affecting sodium-dependent potassium secretion in the downstream aldosterone-sensitive distal nephron. In cells, NCC phosphorylation is increased by lowering of intracellular chloride, via activation of the chloride-sensitive with no lysine (WNK)-SPAK/OSR1 (Ste20-related proline/alanine-rich kinase/oxidative stress response) kinase cascade. In-vivo studies have demonstrated pathway activation in the kidney in response to low dietary potassium. A possible mechanism is lowering of DCT intracellular chloride in response to low potassium because of parallel basolateral potassium and chloride channels. Recent studies support a role for these channels in the response of NCC to varying potassium. Studies examining chloride-insensitive WNK mutants, in the Drosophila renal tubule and in the mouse, lend further support to a role for chloride in regulating WNK activity and transepithelial ion transport. Caveats, alternatives, and future directions are also discussed. SUMMARY: Chloride sensing by WNK kinase provides a mechanism to allow coupling of extracellular potassium with NCC phosphorylation and activity to maintain potassium homeostasis.

Pro-contractile role of chloride in arterial smooth muscle: Postnatal decline potentially governed by sympathetic nerves.

NEW FINDINGS: What is the topic of this review? This symposium report discusses the previously unrecognized pro-contractile role of chloride ions in rat arteries at early stages of postnatal development. What advances does it highlight? It highlights the postnatal decline in the contribution of chloride ions to regulation of arterial contractile responses and potential trophic role of sympathetic nerves in these developmental alterations. ABSTRACT: Chloride ions are important for smooth muscle contraction in adult vasculature. Arterial smooth muscle undergoes structural and functional remodelling during early postnatal development, including changes in K+ currents, Ca2+ handling and sensitivity. However, developmental change in the contribution of Cl- to regulation of arterial contraction has not yet been explored. Here, we provide the first evidence that the role of Cl- in α1 -adrenergic arterial contraction prominently decreases during early postnatal ontogenesis. The trophic influence of sympathetic nerves is a potential mechanism for postnatal decline of the contribution of Cl- to the vascular contraction.

Double-edged GABAergic synaptic transmission in seizures: The importance of chloride plasticity.

GABAergic synaptic inhibition, which is a critical regulator of neuronal excitability, is closely involved in epilepsy. Interestingly, fast GABAergic transmission mediated by Cl- permeable GABAA receptors can bi-directionally exert both seizure-suppressing and seizure-promoting actions. Accumulating evidence suggests that chloride plasticity, the driving force of GABAA receptor-mediated synaptic transmission, contributes to the double-edged role of GABAergic synapses in seizures. Large amounts of Cl- influx can overwhelm Cl- extrusion during seizures not only in healthy tissue in a short-term "activity-dependent" manner, but also in chronic epilepsy in a long-term, irreversible "pathology-dependent" manner related to the dysfunction of two chloride transporters: the chloride importer NKCC1 and the chloride exporter KCC2. In this review, we address the importance of chloride plasticity for the "activity-dependent" and "pathology-dependent" mechanisms underlying epileptic events and provide possible directions for further research, which may be clinically important for the design of GABAergic synapse-targeted precise therapeutic interventions for epilepsy.

Extracellular Cl- regulates electrical slow waves and setting of smooth muscle membrane potential by interstitial cells of Cajal in mouse jejunum.

NEW FINDINGS: What is the central question of this study? The aim was to investigate the roles of extracellular chloride in electrical slow waves and resting membrane potential of mouse jejunal smooth muscle by replacing chloride with the impermeant anions gluconate and isethionate. What is the main finding and its importance? The main finding was that in smooth muscle cells, the resting Cl- conductance is low, whereas transmembrane Cl- movement in interstitial cells of Cajal (ICCs) is a major contributor to the shape of electrical slow waves. Furthermore, the data confirm that ICCs set the smooth muscle membrane potential and that altering Cl- homeostasis in ICCs can alter the smooth muscle membrane potential. Intracellular Cl- homeostasis is regulated by anion-permeable channels and transporters and contributes to excitability of many cell types, including smooth muscle and interstitial cells of Cajal (ICCs). Our aims were to investigate the effects on electrical activity in mouse jejunal muscle strips of replacing extracellular Cl- (Cl-o ) with the impermeant anions gluconate and isethionate. On reducing Cl-o , effects were observed on electrical slow waves, with small effects on smooth muscle membrane voltage (Em ). Restoration of Cl- hyperpolarized smooth muscle Em proportional to the change in Cl-o concentration. Replacement of 90% of Cl-o with gluconate reversibly abolished slow waves in five of nine preparations. Slow waves were maintained in isethionate. Gluconate and isethionate substitution had similar concentration-dependent effects on peak amplitude, frequency, width at half peak amplitude, rise time and decay time of residual slow waves. Gluconate reduced free ionized Ca2+ in Krebs solutions to 0.13 mm. In Krebs solutions containing normal Cl- and 0.13 mm free Ca2+ , slow wave frequency was lower, width at half peak amplitude was smaller, and decay time was faster. The transient hyperpolarization following restoration of Cl-o was not observed in W/Wv mice, which lack pacemaker ICCs in the small intestine. We conclude that in smooth muscle cells, the resting Cl- conductance is low, whereas transmembrane Cl- movement in ICCs plays a major role in generation or propagation of slow waves. Furthermore, these data support a role for ICCs in setting smooth muscle Em and that altering Cl- homeostasis in ICCs can alter smooth muscle Em .

Lack of Impact of Hyperchloremia in Brain-Dead Organ Donors on the Onset of Kidney Allograft Function in the Recipients.

BACKGROUND: Hyperchloremia produces renal vasoconstriction and fall in glomerular filtration rate. In 90% of brain-dead organ donors, diabetes insipidus develops, characterized by inappropriate diuresis, hyperosmolality, and hyperchloremia. The aim of this study was to determine the relationship between the serum concentration of chlorides of the donor and the onset of the function of the kidney allograft in the recipient. METHODS: We retrospectively studied 213 donors and kidney allograft recipients. Serum creatinine concentrations and glomerular filtration rates on the 1st, 7th, and 30th days after transplantation of the recipients from hyperchloremic donors were compared with the recipients from normochloremic donors, as well as the incidences of acute tubular necrosis and delayed graft function. RESULTS: On the 1st day, serum creatinine concentrations of the recipients from hyperchloremic and normochloremic donors, respectively, were 448.2 ± 212.1 µmol/L and 502.2 ± 197.8 µmol/L (P = .1), on the 7th day, 168.6 ± 102.6 µmol/L and 196.9 ± 120.6 µmol/L (P = .13), and on the 30th day, 129.4 ± 43.3 µmol/L and 131.8 ± 43.6 µmol/L (P = .73). The differences were statistically significant. The groups also did not differ significantly in glomerular filtration rates and incidences of acute tubular necrosis and delayed graft function. CONCLUSIONS: In this study, no significant correlation between serum chloride concentrations of the organ donors and the onset of the function of kidney allografts in the recipients was found.

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins.

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable - ideally monochromatic - light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

Effects of taurine on plasma glucose concentration and active glucose transport in the small intestine.

Taurine lowers blood glucose levels and improves hyperglycemia. However, its effects on glucose transport in the small intestine have not been investigated. Here, we elucidated the effect of taurine on glucose absorption in the small intestine. In the oral glucose tolerance test, addition of 10 mmol/L taurine suppressed the increase in hepatic portal glucose concentrations. To investigate whether the suppressive effect of taurine occurs via down-regulation of active glucose transport in the small intestine, we performed an assay using the everted sac of the rat jejunum. Addition of taurine to the mucosal side of the jejunum suppressed active glucose transport via sodium-glucose cotransporter 1 (SGLT1). After elimination of chloride ions from the mucosal solution, taurine did not show suppressive effects on active glucose transport. These results suggest that taurine suppressed the increase in hepatic portal glucose concentrations via suppression of SGLT1 activity in the rat jejunum, depending on chloride ions.

Endogenous and exogenous control of gastrointestinal epithelial function: building on the legacy of Bayliss and Starling.

Transport of fluid and electrolytes in the intestine allows for appropriate adjustments in luminal fluidity while reclaiming water used in digesting and absorbing a meal, and is closely regulated. This article discusses various endogenous and exogenous mechanisms whereby transport is controlled in the gut, placing these in the context of the ideas about the neurohumoral control of alimentary physiology that were promulgated by William Bayliss and Ernest Starling. The article considers three themes. First, mechanisms that intrinsically regulate chloride secretion, centred on the epidermal growth factor receptor (EGFr), are discussed. These may be important in ensuring that excessive chloride secretion, with the accompanying loss of fluid, is not normally stimulated by intestinal distension as the meal passes through the gastrointestinal tract. Second, mechanisms whereby probiotic microorganisms can impart beneficial effects on the gut are described, with a focus on targets at the level of the epithelium. These findings imply that the commensal microbiota exert important influences on the epithelium in health and disease. Finally, mechanisms that lead to diarrhoea in patients infected with an invasive pathogen, Salmonella, are considered, based on recent studies in a novel mouse model. Diarrhoea is most likely attributable to reduced expression of absorptive transporters and may not require the influx of neutrophils that accompanies infection. Overall, the goal of the article is to highlight the many ways in which critical functions of the intestinal epithelium are regulated under physiological and pathophysiological conditions, and to suggest possible targets for new therapies for digestive disease states.

MDCK cells are capable of water secretion and reabsorption in response to changes in the ionic environment.

A prerequisite for tissue electrolyte homeostasis is highly regulated ion and water transport through kidney or intestinal epithelia. In the present work, we monitored changes in the cell and luminal volumes of type II Madin-Darby canine kidney (MDCK) cells grown in a 3D environment in response to drugs, or to changes in the composition of the basal extracellular fluid. Using fluorescent markers and high-resolution spinning disc confocal microscopy, we could show that lack of sodium and potassium ions in the basal fluid (tetramethylammonium chloride (TMACl) buffer) induces a rapid increase in the cell and luminal volumes. This transepithelial water flow could be regulated by inhibitors and agonists of chloride channels. Hence, the driving force for the transepithelial water flow is chloride secretion, stimulated by hyperpolarization. Chloride ion depletion of the basal fluid (using sodium gluconate buffer) induces a strong reduction in the lumen size, indicating reabsorption of water from the lumen to the basal side. Lumen size also decreased following depolarization of the cell interior by rendering the membrane permeable to potassium. Hence, MDCK cells are capable of both absorption and secretion of chloride ions and water; negative potential within the lumen supports secretion, while depolarizing conditions promote reabsorption.

Mapping the Nephron Exercise Incorporates Multiple Learning Strategies.

Introduction: Understanding the location and action of nephron transporters and channels is important to the understanding of renal function. As each region of the nephron is unique in its inclusion of specific transporters and channels, mapping of the nephron is an effective first step in understanding overall nephron processing. We describe a small-group, active-learning exercise that facilitates students' ability to understand renal processing within each region of the nephron. Methods: Following an overview lecture on renal transporters and channels, small groups of students worked cooperatively to map the nephron. This 2-hour, collaborative exercise was developed to reinforce key concepts in renal processing of ions and nutrients and, at the same time, utilize effective learning strategies. Learning strategies incorporated in this exercise include small-group collaboration, peer teaching, retrieval practice using an audience response system, and elaboration through discussion. Results: Written examination was used to assess student understanding. Students demonstrated higher performance on a subset of questions related to this learning activity compared to the overall exam. Highly positive feedback was provided by a convenience sample of students completing an anonymous survey. Discussion: This nephron-mapping exercise was an effective means to promote synthesis and analysis of lecture content and engage students in methods that enhance learning.

Regulation of neuronal chloride homeostasis by neuromodulators.

KCC2 is the central regulator of neuronal Cl(-) homeostasis, and is critical for enabling strong hyperpolarizing synaptic inhibition in the mature brain. KCC2 hypofunction results in decreased inhibition and increased network hyperexcitability that underlies numerous disease states including epilepsy, neuropathic pain and neuropsychiatric disorders. The current holy grail of KCC2 biology is to identify how we can rescue KCC2 hypofunction in order to restore physiological levels of synaptic inhibition and neuronal network activity. It is becoming increasingly clear that diverse cellular signals regulate KCC2 surface expression and function including neurotransmitters and neuromodulators. In the present review we explore the existing evidence that G-protein-coupled receptor (GPCR) signalling can regulate KCC2 activity in numerous regions of the nervous system including the hypothalamus, hippocampus and spinal cord. We present key evidence from the literature suggesting that GPCR signalling is a conserved mechanism for regulating chloride homeostasis. This evidence includes: (1) the activation of group 1 metabotropic glutamate receptors and metabotropic Zn(2+) receptors strengthens GABAergic inhibition in CA3 pyramidal neurons through a regulation of KCC2; (2) activation of the 5-hydroxytryptamine type 2A serotonin receptors upregulates KCC2 cell surface expression and function, restores endogenous inhibition in motoneurons, and reduces spasticity in rats; and (3) activation of A3A-type adenosine receptors rescues KCC2 dysfunction and reverses allodynia in a model of neuropathic pain. We propose that GPCR-signals are novel endogenous Cl(-) extrusion enhancers that may regulate KCC2 function.

Cold acclimation allows regulation of chloride secretion in a eurythermic teleost fish Fundulus heteroclitus.

Fundulus heteroclitus (mummichog or common killifish) is an ideal model for ion transport regulation in chloride cells of the opercular epithelium (OE) and the response to thermal challenge. Mummichogs were acclimated to warm (20 °C) and cold (5 °C) seawater and opercular epithelia dissected and mounted in isolated Ussing-style epithelia chambers. The α2 adrenergic agonist clonidine inhibited the Cl(-) secretion (measured as short-circuit current, Isc), while the ß-adrenergic agonist isoproterenol and 1.0mM dibutyryl cyclic adenosine monophosphate (db-cAMP) plus 0.1mM isobutyl methylxanthine (IBMX) stimulated Isc in OE from warm and cold acclimated fish, measured at 20 °C. In contrast, rapid cooling partially inhibited Isc, but totally blocked the inhibition by clonidine and stimulation by isoproterenol and db-cAMP+IBMX in OE from warm-acclimated fish, while OE from cold-acclimated animals responded normally at 5 °C. Warming epithelia from 5 °C to 20 °C restored Isc and stimulation by db-cAMP+IBMX markedly increased Isc to levels similar to warm acclimated epithelia, while isoproterenol was much less effective. The isoproterenol insensitivity suggests a downregulation of ß-adrenergic receptors in the cold. We infer from present results and previous work (Buhariwalla et al. 2012) that cold shock of plasma membranes induces a phase shift from liquid to gel state that impaired plasma membrane protein mobility of necessary hormone regulatory functions, while cold acclimation preserved ion transport regulation via homeoviscous adaptation of plasma membrane lipids.

Exercise modulates chloride homeostasis after spinal cord injury.

Activity-based therapies are routinely integrated in spinal cord injury (SCI) rehabilitation programs because they result in a reduction of hyperreflexia and spasticity. However, the mechanisms by which exercise regulates activity in spinal pathways to reduce spasticity and improve functional recovery are poorly understood. Persisting alterations in the action of GABA on postsynaptic targets is a signature of CNS injuries, including SCI. The action of GABA depends on the intracellular chloride concentration, which is determined largely by the expression of two cation-chloride cotransporters (CCCs), KCC2 and NKCC1, which serve as chloride exporters and importers, respectively. We hypothesized that the reduction in hyperreflexia with exercise after SCI relies on a return to chloride homeostasis. Sprague Dawley rats received a spinal cord transection at T12 and were assigned to SCI-7d, SCI-14d, SCI-14d+exercise, SCI-28d, SCI-28d+exercise, or SCI-56d groups. During a terminal experiment, H-reflexes were recorded from interosseus muscles after stimulation of the tibial nerve and the low-frequency-dependent depression (FDD) was assessed. We provide evidence that exercise returns spinal excitability and levels of KCC2 and NKCC1 toward normal levels in the lumbar spinal cord. Acutely altering chloride extrusion using the KCC2 blocker DIOA masked the effect of exercise on FDD, whereas blocking NKCC1 with bumetanide returned FDD toward intact levels after SCI. Our results indicate that exercise contributes to reflex recovery and restoration of endogenous inhibition through a return to chloride homeostasis after SCI. This lends support for CCCs as part of a pathway that could be manipulated to improve functional recovery when combined with rehabilitation programs.

Revisiting CFTR inhibition: a comparative study of CFTRinh -172 and GlyH-101 inhibitors.

BACKGROUND AND PURPOSE: For decades, inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have been used as tools to investigate the role and function of CFTR conductance in cystic fibrosis research. In the early 2000s, two new and potent inhibitors of CFTR, CFTRinh -172 and GlyH-101, were described and are now widely used to inhibit specifically CFTR. However, despite some evidence, the effects of both drugs on other types of Cl(-) -conductance have been overlooked. In this context, we explore the specificity and the cellular toxicity of both inhibitors in CFTR-expressing and non-CFTR-expressing cells. EXPERIMENTAL APPROACH: Using patch-clamp technique, we tested the effects of CFTRinh -172 and GlyH-101 inhibitors on three distinct types of Cl(-) currents: the CFTR-like conductance, the volume-sensitive outwardly rectifying Cl(-) conductance (VSORC) and finally the Ca(2+) -dependent Cl(-) conductance (CaCC). We also explored the effect of both inhibitors on cell viability using live/dead and cell proliferation assays in two different cell lines. KEY RESULTS: We confirmed that these two compounds were potent inhibitors of the CFTR-mediated Cl(-) conductance. However,GlyH-101 also inhibited the VSORC conductance and the CaCC at concentrations used to inhibit CFTR. The CFTRinh -172 did not affect the CaCC but did inhibit the VSORC, at concentrations higher than 5 µM. Neither inhibitor (20 µM; 24 h exposure) affected cell viability, but both were cytotoxic at higher concentrations. CONCLUSIONS AND IMPLICATIONS: Both inhibitors affected Cl(-) conductances apart from CFTR. Our results provided insights into their use in mouse models.

Hypoxia-inducible factor-1α causes renal cyst expansion through calcium-activated chloride secretion.

Polycystic kidney diseases are characterized by numerous bilateral renal cysts that continuously enlarge and, through compression of intact nephrons, lead to a decline in kidney function over time. We previously showed that cyst enlargement is accompanied by regional hypoxia, which results in the stabilization of hypoxia-inducible transcription factor-1α (HIF-1α) in the cyst epithelium. Here we demonstrate a correlation between cyst size and the expression of the HIF-1α-target gene, glucose transporter 1, and report that HIF-1α promotes renal cyst growth in two in vitro cyst models-principal-like MDCK cells (plMDCKs) within a collagen matrix and cultured embryonic mouse kidneys stimulated with forskolin. In both models, augmenting HIF-1α levels with the prolyl hydroxylase inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate enhanced cyst growth. In addition, inhibition of HIF-1α degradation through tubule-specific knockdown of the von Hippel-Lindau tumor suppressor increased cyst size in the embryonic kidney cyst model. In contrast, inhibition of HIF-1α by chetomin and knockdown of HIF-1α both decreased cyst growth in these models. Consistent with previous reports, plMDCK cyst enlargement was driven largely by transepithelial chloride secretion, which consists, in part, of a calcium-activated chloride conductance. plMDCKs deficient for HIF-1α almost completely lacked calcium-activated chloride secretion. We conclude that regional hypoxia in renal cysts contributes to cyst growth, primarily due to HIF-1α-dependent calcium-activated chloride secretion. These findings identify the HIF system as a novel target for inhibition of cyst growth.

Cl⁻ homeodynamics in gap junction-coupled astrocytic networks on activation of GABAergic synapses.

The electrophysiological properties and functional role of GABAergic signal transmission from neurons to the gap junction-coupled astrocytic network are still unclear. GABA-induced astrocytic Cl⁻ flux has been hypothesized to affect the driving force for GABAergic transmission by modulating [Cl⁻]o. Thus, revealing the properties of GABA-mediated astrocytic responses will deepen our understanding of GABAergic signal transmission. Here, we analysed the Cl⁻ dynamics of neurons and astrocytes in CA1 hippocampal GABAergic tripartite synapses, using Cl⁻ imaging during GABA application, and whole cell recordings from interneuron-astrocyte pairs in the stratum lacunosum-moleculare. Astrocytic [Cl⁻]i was adjusted to physiological conditions (40 mm). Although GABA application evoked bidirectional Cl⁻ flux via GABAA receptors and mouse GABA transporter 4 (mGAT4) in CA1 astrocytes, a train of interneuron firing induced only GABAA receptor-mediated inward currents in an adjacent astrocyte. A GAT1 inhibitor increased the interneuron firing-induced currents and induced bicuculline-insensitive, mGAT4 inhibitor-sensitive currents, suggesting that synaptic spillover of GABA predominantly induced the astrocytic Cl⁻ efflux because GABAA receptors are localized near the synaptic clefts. This GABA-induced Cl⁻ efflux was accompanied by Cl⁻ siphoning via the gap junctions of the astrocytic network because gap junction inhibitors significantly reduced the interneuron firing-induced currents. Thus, Cl⁻ efflux from astrocytes is homeostatically maintained within astrocytic networks. A gap junction inhibitor enhanced the activity-dependent depolarizing shifts of reversal potential of neuronal IPSCs evoked by repetitive stimulation to GABAergic synapses. These results suggest that Cl⁻ conductance within the astrocytic network may contribute to maintaining GABAergic synaptic transmission by regulating [Cl⁻]o.