Cl Cotransporter Research Articles

The FSGS protein actinin-4 (ACTN4) interacts with NKCC2 to regulate Thick Ascending Limb (TAL) NaCl reabsorption.

In the kidney, the thick Ascending Limb (TAL) of the loop of Henle is crucial for NaCl homeostasis and blood pressure regulation. In animal models of salt-sensitive hypertension, NaCl reabsorption via the apical Na+/K+/2Cl cotransporter (NKCC2) is abnormally increased in the TAL. We showed that NaCl reabsorption is controlled by the presence of NKCC2 at the apical surface of TALs. However, the molecular mechanisms that maintain the steady-state levels of NKCC2 at the apical surface are not clearly understood. Here, we report that NKCC2 interacts with the F-actin cross-linking protein actinin-4 (ACTN4). We find that ACTN4 is expressed in TALs by Western blot and immunofluorescence microscopy. ACTN4 immunoprecipitated with NKCC2 and recombinant GST-ACTN4 pulled down NKCC2 from TAL lysates. ACTN4 is involved in endocytosis in other cells. Therefore, we hypothesized that ACTN4 binds apical NKCC2 and regulates its trafficking. To study this, we silenced ACTN4 in vivo via shRNA or CRISPR/Cas9 system to decrease ACTN4 expression in TALs. We observed that silencing ACTN4 in vivo via shRNA or CRISPR/Cas9 system increased the amount NKCC2 at the apical surface of TALs. Bumetanide-induced diuresis and natriuresis were enhanced by 35% after silencing of ACTN4 in vivo (AV-NKCC2-Cas9: 3841±709 vs AAV-gRNA-ACTN4: 5546±622 µmols Na/8h, n=5, p<0.05). We conclude that ACTN4, binds NKCC2 to regulate its surface expression. Selective depletion of ACTN4 in TALs using shRNA or CRISPR/Cas9 enhances surface NKCC2 and TAL NaCl reabsorption, indicating that regulation of the ACTN4-NKCC2 interaction is important for renal NaCl reabsorption and could be related to hypertension.

#2640 Hyperuricemia in Gitelman syndrome 132 patients

Abstract Background and Aims Thiazide diuretics, the most common prescription for hypertension, were partly restricted by the hyperuricemia induced by the impairment of uric acid clearance serum uric acid. Gitelman syndrome (GS, OMIM263800), caused by mutations of the SLC12A3 gene encodes the thiazide-sensitive human Na–Cl co-transporter (hNCC), might shed light on the precision mechanism of hyperuricemia induced by Thiazide diuretics. Method We retrospectively reviewed 132 GS patients and pertinent articles and analyzed their clinical and genetic characteristics with or without hyperuricemia. Our study also employed Illumina Human Asian Screening Array-24 + v1.0 (ASA) gene chips to screen available samples from 87 patients with Gitelman Syndrome, which included a subgroup of 17 individuals exhibiting elevated uric acid levels characteristic of GS. Results The average uric acid level of 132 genetic-confirmed GS patients was 351.4 ± 95.1 µmol/l, with 21.2% hyperuricemia (mean 482.1 µmol/l) and 2 cases of gout. The GS patients with hyperuricemia had higher BMI (24.3 ± 3.9 vs. 21.6 ± 3.5 kg/m2, P = 0.001), lower FEUA (4.6 ± 1.0 vs. 7.0 ± 2.7%, P &amp;lt; 0.001), urinary calcium/creatinine (0.05 vs. 0.11 mmol/mmol, P = 0.01), and△FECl (0.6 ± 0.4% vs 1.2 ± 1.1%, P = 0.02) of the HCT test, indicating more severe NCC function injury. Fisher's exact test yielded non-significant associations of the ten uric acid-predicting SNPs between the patients with or without hyperuricemia at the conventional P-value cut-off of 0.05. Conclusion The prevalence of hyperuricemia in GS patients exceeds that observed in the general population. GS patients with hyperuricemia might be because of a high body mass index (BMI) and more severe impairment of NCC function, which suggested the crosstalk between NCC and Uric acid transporter.

Diuretic activity of the hydroalcoholic extracts of rhizomes and leaves of Artemisia abyssinica sch. Bip. Ex A. Rich: In silico and in vivo study

Background/Aim: The majority of communities in developing nations utilise traditional medicine as an alternative or a combination therapy with a clinically approved diuretic regimen. The present study aimed to investigate the in vivo and in silico diuretic properties of the 80 % methanol extracts of the rhizomes and leaves of Artemisia abyssinica, an indigenous traditional diuretic medicinal plant of Ethiopia. Methods: Acute oral toxicity tests of 80 % methanol rhizome and leaf extracts of the plant were conducted in mice. For the diuretic test, six treatment groups were administered 100, 200 and 400 mg/kg doses of rhizome and leaf extracts of the plant. The negative and positive control groups were treated with distilled water (2 mL/100 g) and furosemide (10 mg/kg), respectively. Cumulative urine volume, diuretic action, diuretic activity and saluretic index were then determined. In addition, virtual screening and molecular docking study of the compounds of the genus Artemisia were done. Results: The rhizome and leaf extracts of A abyssinica were found safe at a dose of 2000 mg/kg. Moreover, both extracts showed a significant diuretic action (p &lt; 0.05). However, compared to the standard drug furosemide, the extracts had lower diuretic activity. The rhizome extract in-creased electrolyte excretion at all doses; particularly at the 200 and 400 mg/kg doses, it exhibited a profound natriuretic, chloruretic and kaliuretic effect with the concentration of 109 and 110 mmol/L for Na + , 93 and 106 mmol/L for Cl -and 79 and 86 mmol/L for K + , respectively. These sug-gested inhibition of Na + -K + -2Cl cotransporter as the potential mechanism of action of the extracts. Accordingly, virtual screening and a molecular docking analysis of the compounds of the genus Artemisia revealed that a few of them displayed a strong binding interaction with the cation-chloride cotransporter NKCC1 (PDB: 7S1Y), further indicating the cation-chloride cotransporter as a diuretic target of the constituents of the plant. Conclusion: The current study supports the traditional claim of the plant for diuresis and recommends further isolation of the active constituents.

Osmoregulatory and immunological role of new canceled cells: Mitochondrial rich cells and its future perspective: A concise review.

Mitochondrial-rich cells (MRCs) are one of the most significant canceled type of epithelial cells. Morphologically these cells are totally different from other epithelial cells. These cells primarily implicated in sea-water and fresh-water adaptation, and acid-base regulation. However, in this review paper, we explored some of the most intriguing biological and immune-related functional developmental networks of MRCs. The main pinpoint, MRCs perform a dynamic osmoregulatory and immunological functional role in the gut and male reproductive system. The Na+/K+_ATPase (NKA) and Na+/K+/2Cl cotransporter (NKCC) are key acidifying proteins of MRCs for the ion-transporting function for intestinal homeostasis and maintenance of acidifying the luminal microenvironment in the male reproductive system. Further more importantly, MRCs play a novel immunological role through the exocrine secretion of nano-scale exosomes and multivesicular bodies (MVBs) pathway, which is very essential for sperm maturation, motility, acrosome reaction, and male sex hormones, and these an essential events to produce male gametes with optimal fertilizing ability. This effort is expected to promote the novel immunological role of MRCs, which might be essential for nano-scale exosome secretion.

Endomorphin analog ZH853 shows low reward, tolerance, and affective-motivational signs of withdrawal, while inhibiting opioid withdrawal and seeking

Currently available μ-opioid receptor agonist pharmacotherapies for opioid use disorder possess adverse effects limiting their use and, despite treatment, rates of relapse remain high. We previously showed that endomorphin analog ZH853 had no effect in rodent models that predict abuse liability in humans. Here we extended these findings by examining dependence liability and reinforcing properties in female rats and male rats with previous opioid exposure. The potential use of ZH853 in managing opioid use disorder was evaluated by examining its effect on opioid-seeking behavior and withdrawal. We found that ZH853 did not induce locomotor activation in male and female mice and was not self-administered by female rats. Relative to morphine, ZH853 led to similar somatic signs of withdrawal, but low affective-motivational signs of withdrawal, and absent changes in ventral tegmental area K(+)-Cl(−) co-transporter expression associated with reward dysregulation. The low abuse liability of ZH853 was further supported in oxycodone self-administering male rats, where ZH853 substitution extinguished opioid-seeking behavior. ZH853 priming also did not reinstate morphine conditioned place preference. Lastly, ZH853 inhibited oxycodone-seeking behavior during relapse after forced abstinence and decreased the expression of morphine withdrawal. These findings suggest the potential use of ZH853 as a safer opioid medication for long-term treatment of pain and opioid use disorder.

Abstract 064: Hypokalemia Induced By Loss Of Kir5.1 Initiates WNK-SPAK/OSR1 Signaling In Dahl Salt-sensitive Rats

Background: Hypokalemia promotes Na + reabsorption via the Na + -Cl - (NCC) cotransporter of the distal convoluted tubule (DCT), which reduces the K + losses in downstream nephron segments and increases NaCl retention and blood pressure. In dietary K + depletion, activation of NCC is mediated by the with no lysine (K) kinase (WNK)-Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1) pathway. Our previous work suggested that deletion of Kcnj16 (the gene that encodes K ir 5.1) in Dahl salt-sensitive (SS) rats (SS Kcnj16-/- ) resulted in hypokalemia with a compensatory increase in expression and activity of NCC. Here, we hypothesized that hypokalemia induced by loss of the K ir 5.1 subunit initiates WNK-SPAK/OSR1 signaling. Methods: 8 to 12-week-old male SS WT and SS Kcnj16-/- rats were separated into 3 groups (N=5 per group) and fed different diets for 3 weeks: SS WT (0.4% NaCl); SS Kcnj16-/- (0.4% NaCl); SS Kcnj16-/- (4% NaCl+2% KCl). At the end of the experiment, kidneys were flushed and processed for Western blot, immunofluorescence, and electron microscopy analysis. Results: Electron micrographs exhibited WNK body clusters in SS Kcnj16-/- compared to SS WT rats. Immunofluorescence staining showed dramatically increased expression of both SPAK and phospho-SPAK (pSPAK) in DCTs of SS Kcnj16-/- rats, where pSPAK colocalized with NCC. Similarly, Western blot analysis revealed significantly elevated expression of total SPAK (3.32± 0.18 vs. 1.00 ± 0.19, P &lt;0.0001) and pSPAK (2.09 ± 0.51 vs. 1.00 ± 0.19, P &lt;0.001) in the kidneys of SS Kcnj16/- compared to SS WT rats. Moreover, supplementing the SS Kcnj16-/- rats with high Na + and high K + (4% NaCl+2% KCl) reverses the expression and activity of SPAK. Conclusion: Our results indicate that hypokalemia resulting from loss of K ir 5.1 subunit stimulates WNK-SPAK/OSR1 signaling. Furthermore, the fact that dietary K + supplementation overturned the altered expression of this pathway implies that plasma K + level is responsible for the activation of the WNK-SPAK/OSR1 pathway.

Effect of a C‐terminal Electrostatic Interaction Domain on KCC3 Function

BackgroundThe K:Cl cotransporters (KCCs) are key players to maintain cellular volume, mediate epithelial transport and intracellular chloride concentration, and regulate GABA neurotransmission. Under isotonic conditions, KCCs are normally inhibited due to the phosphorylation of residues located within the cytoplasmic domains. On the contrary, dephosphorylation of those residues in response to hypotonic cell swelling stimulates transport activity. Dysfunction of these proteins and the processes involved in their regulation have been associated with various human diseases by alteration of ionic homeostasis mainly in the kidney and the brain. Based on the cryo‐EM structures recently reported of human KCCs, revealing structural determinants for regulation in both N‐ and C‐terminal domains, in the present study we analyzed the effect of a structural domain located at the C‐ terminal involved in electrostatic interactions. It has been stated that the side chains of residues as Gln (Q), Arg (R) and Lys (K) tend to form hydrogen bonds promoting the stabilization of tertiary and quaternary protein structures. However, whether the phosphorylation status is modified after the electrostatic disruption is unknown.MethodsXenopus laevis oocytes and HEK 293 cells were either microinjected or transfected with KCC3a wild type (WT) or C‐terminal mutants, Q905A, Q905/K934A (2M), Q905/K934/R937A (3M) and Q905/K934/R937/K938A (4M), key residues involved in electrostatic interactions and located within the C‐terminal domain of the cotransporter. The phosphorylation status of KCC3a in response to isotonic and hypotonic conditions was evaluated by immunoblot analysis of three key regulatory phosphorylation sites (pSer96, pThr991 and pThr1048). In parallel, phosphorylation status of SPAK/OSR1 activating regulatory site pSer373/pSer325 was analyzed under the same conditions.ResultsCell swelling‐induced dephosphorylation is not observed on C‐terminal electrostatic interaction domain mutants compared to WT KCC3 in X. laevis oocytes and HEK cells. All three regulatory‐phosphorylation sites in KCC3a mutants remained phosphorylated under hypotonic conditions where dephosphorylation is required for the protein to become active. These results suggest that the disruption of the hydrogen bonds could disrupt the proper folding of the protein and thus interfere with the regulatory dephosphorylation/phosphorylation signaling pathway which is known to activate the transporter after cell swelling.ConclusionsOur results show that the four residues in the electrostatic interaction domain of the C‐termini are involved in KCC3a response to hypotonic conditions affecting the phosphorylation status of the cotransporter. The side chains of these residues seem to be important in maintaining tertiary conformation given their ability to form hydrogen bonds and thus, the disruption of their interactions affects the cotransporter ability to respond to osmotic and cell volume changes.

Silencing of Ago-2 Interacting Protein SERBP1 Relieves KCC2 Repression by miR-92 in Neurons.

RNA-binding proteins (RBPs) play important roles in modulating miRNA-mediated mRNA target repression. Argonaute2 (Ago2) is an essential component of the RNA-induced silencing complex (RISC) that plays a central role in silencing mechanisms via small non-coding RNA molecules known as siRNAs and miRNAs. Small RNAs loaded into Argonaute proteins catalyze endoribonucleolytic cleavage of target RNAs or recruit factors responsible for translational silencing and mRNA target destabilization. In previous studies we have shown that KCC2, a neuronal Cl (−) extruding K (+) Cl (−) co-transporter 2, is regulated by miR-92 in neuronal cells. Searching for Ago2 partners by immunoprecipitation and LC-MS/MS analysis, we isolated among other proteins the Serpine mRNA binding protein 1 (SERBP1) from SH-SY5Y neuroblastoma cells. Exploring the role of SERBP1 in miRNA-mediated gene silencing in SH-SY5Y cells and primary hippocampal neurons, we demonstrated that SERBP1 silencing regulates KCC2 expression through the 3′ untranslated region (UTR). In addition, we found that SERBP1 as well as Ago2/miR-92 complex bind to KCC2 3′UTR. Finally, we demonstrated the attenuation of miR-92-mediated repression of KCC2 3′UTR by SERBP1 silencing. These findings advance our knowledge regarding the miR-92-mediated modulation of KCC2 translation in neuronal cells and highlight SERBP1 as a key component of this gene regulation.

Abstract 135: A case of acquired Gitelman syndrome presenting as hypokalemic paralysis and AKI

Introduction: Gitelman syndrome is usually an inherited disorder, it is induced by defect in the thiazide sensitive Na –Cl co-transporter in distal tubules. Case reports on hypokalemia, AKI secondary to GS has been discussed in present case report.Clinical Case: A 18 year old female referred from department of General Medicine as a case of AKI having recurrent hypokalemia. She gave history of hypokalemic paralysis. There was no potential cause of dehydration such as diarrhoea, vomiting, nephrotoxic agent intake or contrast exposure. Family history was negative. During current hospitalization, she was normotensive with normal renal function test. Other electrolyte levels including calcium and routine laboratory parameters were determined to be normal. Kidney dimensions and echogenicity were determined to be normal without hydronephrosis in the urinary system as oer USG finding. Further workup for hypokalemia revealed urine potassium creatinine ratio (17 mmol/g), ABG (pH 7.44, bicarbonate 26.5 mmol/l), urine chloride (111.3 mmol/l) and urine calcium creatinine ratio (0.05 mmol/mmol) respectively with serum Mg (1.7 mg/dl) on lower range. Based on above findings, a diagnosis of Gitelman syndrome has been made. Autoimmune etiology were ruled out. These finding suggested that patient had AKI secondary to hypokalemia. She was started on oral potassium supplementation and magnesium supplementation.Conclusion: GS is a rare tubulopathy transmitted as an autosomal recessive trait. It is caused by mutation in thiazide sensitive Na Cl co-transporter coding gene, SLC12A3. Patients may be referred with hypokalemia, hypomagnesemia and metabolic alkalosis associated muscular weakness and cramping. By contrast, worsening hypokalemia and profound volume depletion as the predisposing factors for ischemic AKI appears to coexist.

The developmental changes in intrinsic and synaptic properties of prefrontal neurons enhance local network activity from the second to the third postnatal weeks in mice

The prefrontal cortex (PFC) is characterized by protracted maturation. The cellular mechanisms controlling the early development of prefrontal circuits are still largely unknown. Our study delineates the developmental cellular processes in the mouse medial PFC (mPFC) during the second and the third postnatal weeks and characterizes their contribution to the changes in network activity. We show that spontaneous inhibitory postsynaptic currents (sIPSC) are increased, whereas spontaneous excitatory postsynaptic currents (sEPSC) are reduced from the second to the third postnatal week. Drug application suggested that the increased sEPSC frequency in mPFC at postnatal day 10 (P10) is due to depolarizing γ-aminobutyric acid (GABA) type A receptor function. To further validate this, perforated patch-clamp recordings were obtained and the expression levels of K-Cl cotransporter 2 (KCC2) protein were examined. The reversal potential of IPSCs in response to current stimulation was significantly more depolarized at P10 than P20 while KCC2 expression is decreased. Moreover, the number of parvalbumin-expressing GABAergic interneurons increases and their intrinsic electrophysiological properties significantly mature in the mPFC from P10 to P20. Using computational modeling, we show that the developmental changes in synaptic and intrinsic properties of mPFC neurons contribute to the enhanced network activity in the juvenile compared with neonatal mPFC.

Frequent SLC12A3 mutations in Chinese Gitelman syndrome patients: structure and function disorder

PurposesThis study was conducted to identify the frequent mutations from reported Chinese Gitelman syndrome (GS) patients, to predict the three-dimensional structure change of human Na–Cl co-transporter (hNCC), and to test the activity of these mutations and some novel mutations in vitro and in vivo.MethodsSLC12A3 gene mutations in Chinese GS patients previously reported in the PubMed, China National Knowledge Infrastructure, and Wanfang database were summarized. Predicted configurations of wild type (WT) and mutant proteins were achieved using the I-TASSER workplace. Six missense mutations (T60M, L215F, D486N, N534K, Q617R, and R928C) were generated by site-directed mutagenesis. 22Na+ uptake experiment was carried out in the Xenopus laevisoocyte expression system. In the study, 35 GS patients and 20 healthy volunteers underwent the thiazide test.ResultsT60M, T163M, D486N, R913Q, R928C, and R959frameshift were frequent SLC12A3 gene mutations (mutated frequency >3%) in 310 Chinese GS families. The protein’s three-dimensional structure was predicted to be altered in all mutations. Compared with WT hNCC, the thiazide-sensitive 22Na+ uptake was significantly diminished for all six mutations: T60M 22 ± 9.2%, R928C 29 ± 12%, L215F 38 ± 14%, N534K 41 ± 15.5%, Q617R 63 ± 22.1%, and D486N 77 ± 20.4%. In thiazide test, the net increase in chloride fractional excretion in 20 healthy controls was significantly higher than GS patients with or without T60M or D486N mutations.ConclusionsFrequent mutations (T60M, D486N, and R928C) and novel mutations (L215F, N534K, and Q617R) lead to protein structure alternation and protein dysfunction verified by 22Na+ uptake experiment in vitro and thiazide test on the patients.

All-Atom Simulations Uncover the Molecular Terms of the NKCC1 Transport Mechanism.

The secondary-active Na-K-Cl cotransporter 1 (NKCC1), member of the cation-chloride cotransporter (CCC) family, ensures the electroneutral movement of Cl-, Na+, and K+ ions across cellular membranes. NKCC1 regulates Cl- homeostasis and cell volume, handling a pivotal role in transepithelial water transport and neuronal excitability. Aberrant NKCC1 transport is hence implicated in a variety of human diseases (hypertension, renal disorders, neuropathies, and cancer). Building on the newly resolved NKCC1 cryo-EM structure, all-atom enhanced sampling simulations unprecedentedly unlock the mechanism of NKCC1-mediated ion transport, assessing the order and the molecular basis of its interdependent ion translocation. Our outcomes strikingly advance the understanding of the physiological mechanism of CCCs and disclose a key role of CCC-conserved asparagine residues, whose side-chain promiscuity ensures the transport of both negatively and positively charged ions along the same translocation route. This study sets a conceptual basis to devise NKCC-selective inhibitors to treat diseases linked to Cl- dishomeostasis.

The Antiedematous Effect of Exogenous Lactate Therapy in Traumatic Brain Injury: A Physiological and Mechanistic Approach

BackgroundSodium lactate (SL) has been described as an efficient therapy in treating raised intracranial pressure (ICP). However, the precise mechanism by which SL reduces intracranial hypertension is not well defined. An antiedematous effect has been proposed but never demonstrated. In this context, the involvement of chloride channels, aquaporins, or K–Cl cotransporters has also been suggested, but these mechanisms have never been assessed when using SL.MethodsIn a rat model of traumatic brain injury (TBI), we compared the effect of SL versus mannitol 20% on ICP, cerebral tissue oxygen pressure, and brain water content. We attempted to clarify the involvement of chloride channels in the antiedematous effects associated with lactate therapy in TBI.ResultsAn equimolar single bolus of SL and mannitol significantly reduced brain water content and ICP and improved cerebral tissue oxygen pressure 4 h after severe TBI. The effect of SL on brain water content was much longer than that of mannitol and persisted at 24 h post TBI. Western blot and immunofluorescence staining analyses performed 24 h after TBI revealed that SL infusion is associated with an upregulation of aquaporin 4 and K–Cl cotransporter 2.ConclusionsSL is an effective therapy for treating brain edema after TBI. This study suggests, for the first time, the potential role of chloride channels in the antiedematous effect induced by exogenous SL.

Downregulation of Cullin 3 Ligase Signaling Pathways Contributes to Hypertension in Preeclampsia.

Background: Preeclampsia (PE) is a leading cause of maternal and perinatal morbidity and mortality; however, its etiology and pathophysiology remain obscure. PE is initiated by inadequate spiral artery remodeling and subsequent placental ischemia/hypoxia, which stimulates release of bioactive factors into maternal circulation, leading to hypertension and renal damage.Methods and Results: Abundance of key components of cullin 3-ring ubiquitin ligase (CRL3), including cullin 3 (CUL3) and its neddylated modification, and adaptors including Kelch-like 2 (KLHL2) and Rho-related BTB domain containing protein 1 was all decreased in spiral arteries and placentas of PE patients. Similar changes were found in aortic tissues and renal distal tubules of pregnant mice treated with Nω-nitro-l-arginine methyl ester hydrochloride. The downregulation of CRL3 function led to accumulation of with-no-lysine kinases, phosphodiesterase 5, and RhoA in vessels and renal distal tubules, which promoted vasoconstriction and Na–Cl cotransporter activation in the distal convoluted tubule (DCT), as well as vascular and DCT structure remodeling. Proton pump inhibitor esomeprazole partially restored CRL3 function. In vitro studies have shown that increased abundance of JAB1, a component of the COP9 signalosome, inhibited CUL3 neddylation and promoted the expression of hypoxia-inducible factor 1α, which downregulated peroxisome proliferator–activated receptor γ and further promoted CUL3 inactivation. KLHL3/2 was degraded by increased autophagy.Conclusion: These findings support that the downregulation of CRL3 function disrupts the balance of vasoconstriction and vasodilation and aggravates excess reabsorption of sodium in PE.

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.

Severe impairment of salivation in Na+/K+/2Cl- cotransporter (NKCC1)-deficient mice.

The salivary fluid secretory mechanism is thought to require Na(+)/K(+)/2Cl(-) cotransporter-mediated Cl(-) uptake. To directly test this possibility we studied the in vivo and in vitro functioning of acinar cells from the parotid glands of mice with targeted disruption of Na(+)/K(+)/2Cl(-) cotransporter isoform 1 (Nkcc1), the gene encoding the salivary Na(+)/K(+)/2Cl(-) cotransporter. In wild-type mice NKCC1 was localized to the basolateral membranes of parotid acinar cells, whereas expression was not detected in duct cells. The lack of functional NKCC1 resulted in a dramatic reduction (>60%) in the volume of saliva secreted in response to a muscarinic agonist, the primary in situ salivation signal. Consistent with defective Cl(-) uptake, a loss of bumetanide-sensitive Cl(-) influx was observed in parotid acinar cells from mice lacking NKCC1. Cl(-)/ HCO(3)(-) exchanger activity was increased in parotid acinar cells isolated from knockout mice suggesting that the residual saliva secreted by mice lacking NKCC1 is associated with anion exchanger-dependent Cl(-) uptake. Indeed, expression of the Cl(-)/ HCO(3)(-) exchanger AE2 was enhanced suggesting that this transporter compensates for the loss of functional Na(+)/K(+)/2Cl(-) cotransporter. Furthermore, the ability of the parotid gland to conserve NaCl was abolished in NKCC1-deficient mice. This deficit was not associated with changes in the morphology of the ducts, but transcript levels for the alpha-, beta-, and gamma-subunits of the epithelial Na(+) channel were reduced. These data directly demonstrate that NKCC1 is the major Cl(-) uptake mechanism across the basolateral membrane of acinar cells and is critical for driving saliva secretion in vivo.

KCC2 Chloride Transport Contributes to the Termination of Ictal Epileptiform Activity.

Recurrent seizures intensely activate GABAA receptors (GABAA-Rs), which induces transient neuronal chloride ([Cl-]i) elevations and depolarizing GABA responses that contribute to the failure of inhibition that engenders further seizures and anticonvulsant resistance. The K+-Cl- cotransporter KCC2 is responsible for Cl- extrusion and restoration of [Cl-]i equilibrium (ECl) after synaptic activity, but at the cost of increased extracellular potassium which may retard K+-Cl- extrusion, depolarize neurons, and potentiate seizures. Thus, KCC2 may either diminish or facilitate seizure activity, and both proconvulsant and anticonvulsant effects of KCC2 inhibition have been reported. It is now necessary to identify the loci of these divergent responses by assaying both the electrographic effects and the ionic effects of KCC2 manipulation. We therefore determined the net effects of KCC2 transport activity on cytoplasmic chloride elevation and Cl- extrusion rates during spontaneous recurrent ictal-like epileptiform discharges (ILDs) in organotypic hippocampal slices in vitro, as well as the correlation between ionic and electrographic effects. We found that the KCC2 antagonist VU0463271 reduced Cl- extrusion rates, increased ictal [Cl-]i elevation, increased ILD duration, and induced status epilepticus (SE). In contrast, the putative KCC2 upregulator CLP257 improved chloride homeostasis and reduced the duration and frequency of ILDs in a concentration-dependent manner. Our results demonstrate that measuring both the ionic and electrographic effects of KCC2 transport clarify the impact of KCC2 modulation in specific models of epileptiform activity. Anticonvulsant effects predominate when KCC2-mediated chloride transport rather than potassium buffering is the rate-limiting step in restoring ECl and the efficacy of GABAergic inhibition during recurrent ILDs.

The role of KCC2 in hyperexcitability of the neonatal brain

BackgroundThe hyperpolarizing activity of γ-aminobutyric acid A (GABAA) receptors depends on the intracellular chloride gradient that is developmentally regulated by the activity of the chloride extruder potassium (K) chloride (Cl) cotransporter 2 (KCC2). In humans and rodents, KCC2 expression can be detected at birth. In rodents, KCC2 expression progressively increases and reaches adult-like levels by the second postnatal week of life. Several studies report changes in KCC2 expression levels in response to early-life injuries. However, the functional contribution of KCC2 in maintaining the excitation-inhibition balance in the neonatal brain is not clear. In the current study, we examined the effect of KCC2 antagonism on the neonatal brain activity under hyperexcitable conditions ex vivo and in vivo. MethodsEx vivo electrophysiology experiments were performed on hippocampal slices prepared from 7 to 9 days-old (P7-P9) male rats. Excitability of CA1 pyramidal neurons bathed in zero-Mg2+ buffer was measured using single-unit extracellular (loose) or cell-attach protocol before and after application of VU0463271, a specific antagonist of KCC2. To examine the functional role of KCC2 in vivo, the effect of VU0463271 on hypoxia-ischemia (HI)-induced ictal (seizures and brief runs of epileptiform discharges - BREDs), and inter-ictal spike and sharp-wave activity was measured in P7 male rats. A highly sensitive LC–MS/MS method was used to determine the distribution and the concentration of VU0463271 in the brain. ResultsEx vivo blockade of KCC2 by VU0463271 significantly increased the frequency of zero-Mg2+-triggered spiking in CA1 pyramidal neurons. Similarly, in vivo administration of VU0463271 significantly increased the number of ictal events, BREDs duration, and spike and sharp-wave activity in HI rats. LC–MS/MS data revealed that following systemic administration, VU0463271 rapidly reached brain tissues and distributed well among different brain regions. ConclusionThe results suggest that KCC2 plays a critical functional role in maintaining the balance of excitation-inhibition in the neonatal brain, and thus it can be used as a therapeutic target to ameliorate injury associated with hyperexcitability in newborns.

Pathways in the Pathophysiology of Coronavirus 19 Lung Disease Accessible to Prevention and Treatment.

Background: In COVID 19 related lung disease, which is a leading cause of death from this disease, cytokines like tumor necrosis factor-alpha (TNF alpha) may be pivotal in the pathogenesis. TNF alpha reduces fluid absorption due to impairment of sodium and chloride transport required for building an osmotic gradient across epithelial cells, which in the airways maintains airway surface liquid helping to keep airways open and enabling bacterial clearance and aids water absorption from the alveolar spaces. TNF alpha can, through Rho-kinase, disintegrate the endothelial and epithelial cytoskeleton, and thus break up intercellular tight junctional proteins, breaching the intercellular barrier, which prevents flooding of the interstitial and alveolar spaces with fluid.Hypotheses: (1) Preservation and restoration of airway and alveolar epithelial sodium and chloride transport and the cytoskeleton dependent integrity of the cell barriers within the lung can prevent and treat COVID 19 lung disease. (2) TNF alpha is the key mediator of pulmonary edema in COVID 19 lung disease.Confirmation of hypothesis and implications: The role of a reduction in the function of epithelial sodium and chloride transport could with regards to chloride transport be tested by analysis of chloride levels in exhaled breath condensate and levels correlated with TNF alpha concentrations. Reduced levels would indicate a reduction of the function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and a correlation with TNF alpha levels indicative of its involvement. Anti-TNF alpha treatment with antibodies is already available and needs to be tested in randomized controlled trials of COVID 19 lung disease. TNF alpha levels could also be reduced by statins, aspirin, and curcumin. Chloride transport could be facilitated by CFTR activators, including curcumin and phosphodiesterase-5 inhibitors. Sodium and chloride transport could be further regulated to prevent accumulation of alveolar fluid by use of Na(+)/K(+)/2Cl(−) cotransporter type 1 inhibitors, which have been associated with improved outcome in adults ventilated for acute respiratory distress syndrome (ARDS) in randomized controlled trials. Primary prevention of coronavirus infection and TNF alpha release in response to it could be improved by induction of antimicrobial peptides LL-37 and human beta defensin-2 and reduction of TNF alpha production by vitamin D prophylaxis for the population as a whole.

Involvement of activin a receptor type 1 (ACVR1) in the pathogenesis of primary focal hyperhidrosis

The pathogenesis of primary focal hyperhidrosis (PFH) is still not clear. PFH is thought to be a genetic disease. Whether activin A receptor type 1 (ACVR1) is involved in the pathogenesis of PFH is unknown. In this study, the expression of ACVR1 in sweat glands of patients with PAH was detected by western blot and immunofluorescence. The primary sweat gland cells obtained from primary axillary hyperhidrosis (PAH) patients were transfected with acvr1 vector. Cell proliferation, apoptosis and cell cycling of gland cells were measured after transfection with acvr1 vector. The mRNA and protein expression of aquaporin 5 (AQP5) and Na:K:2Cl Cotransporter 1 (NKCC1/SLC12A2) were detected. Our data showed that ACVR1 expression in axillary sweat gland tissue of PAH patients was significantly higher than that of normal control group. The function of ACVR1 was further investigated in the gland cells obtained from PAH patients. Compared with NC group, ACVR1 overexpression significantly promoted the proliferation of sweat gland cells and inhibited the apoptosis of sweat gland cells. Meanwhile, ACVR1 overexpression significantly reduced the percentage of cells in G0/G1 and G2/M phases, and increased the percentage of cells in S phase. In addition, ACVR1 overexpression significantly promoted the expression of AQP5 and NKCC1 at both mRNA and protein levels. Together, ACVR1 expression is related to PFH and ACVR1 overexpression can promote the proliferation of sweat gland cells and inhibit apoptosis by promoting the expression of AQP5 and NKCC1.