780 research outputs found

    T cell deletional tolerance restricts AQP4 but not MOG CNS autoimmunity

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    Aquaporin-4 (AQP4)-specific Th17 cells are thought to have a central role in neuromyelitis optica (NMO) pathogenesis. When modeling NMO, only AQP4-reactive Th17 cells from AQP4-deficient (AQP4-/-), but not wild-type (WT) mice, caused CNS autoimmunity in recipient WT mice, indicating that a tightly regulated mechanism normally ensures tolerance to AQP4. Here, we found that pathogenic AQP4 T cell epitopes bind MHC II with exceptionally high affinity. Examination of T cell receptor (TCR) α/ÎČ usage revealed that AQP4-specific T cells from AQP4-/- mice employed a distinct TCR repertoire and exhibited clonal expansion. Selective thymic AQP4 deficiency did not fully restore AQP4-reactive T cells, demonstrating that thymic negative selection alone did not account for AQP4-specific tolerance in WT mice. Indeed, AQP4-specific Th17 cells caused paralysis in recipient WT or B cell-deficient mice, which was followed by complete recovery that was associated with apoptosis of donor T cells. However, donor AQP4-reactive T cells survived and caused persistent paralysis in recipient mice deficient in both T and B cells or mice lacking T cells only. Thus, AQP4 CNS autoimmunity was limited by T cell-dependent deletion of AQP4-reactive T cells. In contrast, myelin oligodendrocyte glycoprotein (MOG)-specific T cells survived and caused sustained disease in WT mice. These findings underscore the importance of peripheral T cell deletional tolerance to AQP4, which may be relevant to understanding the balance of AQP4-reactive T cells in health and in NMO. T cell tolerance to AQP4, expressed in multiple tissues, is distinct from tolerance to MOG, an autoantigen restricted in its expression

    SLC26A6-selective inhibitor identified in a small-molecule screen blocks fluid absorption in small intestine

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    SLC26A6 (also known as putative anion transporter 1 [PAT1]) is a Cl-/HCO3- exchanger expressed at the luminal membrane of enterocytes where it facilitates intestinal Cl- and fluid absorption. Here, high-throughput screening of 50,000 synthetic small molecules in cells expressing PAT1 and a halide-sensing fluorescent protein identified several classes of inhibitors. The most potent compound, the pyrazolo-pyrido-pyrimidinone PAT1inh-B01, fully inhibited PAT1-mediated anion exchange (IC50 ~350 nM), without inhibition of the related intestinal transporter SLC26A3 (also known as DRA). In closed midjejunal loops in mice, PAT1inh-B01 inhibited fluid absorption by 50%, which increased to >90% when coadministered with DRA inhibitor DRAinh-A270. In ileal loops, PAT1inh-B01 blocked fluid absorption by >80%, whereas DRAinh-A270 was without effect. In colonic loops, PAT1inh-B01 was without effect, whereas DRAinh-A270 completely blocked fluid absorption. In a loperamide constipation model, coadministration of PAT1inh-B01 with DRAinh-A270 increased stool output compared with DRAinh-A270 alone. These results provide functional evidence for complementary and region-specific roles of PAT1 and DRA in intestinal fluid absorption, with PAT1 as the predominant anion exchanger in mouse ileum. We believe that PAT1inh-B01 is a novel tool to study intestinal ion and fluid transport and perhaps a drug candidate for small intestinal hyposecretory disorders such as cystic fibrosis-related meconium ileus and distal intestinal obstruction syndrome

    Chloride channel accessory 1 integrates chloride channel activity and mTORC1 in aging‐related kidney injury

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    The mechanism of kidney injury in aging are not well understood. In order to identify hitherto unknown pathways of aging‐related kidney injury, we performed RNA‐Seq on kidney extracts of young and aged mice. Expression of chloride (Cl) channel accessory 1 (CLCA1) mRNA and protein was increased in the kidneys of aged mice. Immunostaining showed a marked increase in CLCLA1 expression in the proximal tubules of the kidney from aged mice. Increased kidney CLCA1 gene expression also correlated with aging in marmosets and in a human cohort. In aging mice, increased renal cortical CLCA1 content was associated with hydrogen sulfide (H2S) deficiency, which was ameliorated by administering sodium hydrosulfide (NaHS), a source of H2S. In order to study whether increased CLCA1 expression leads to injury phenotype and the mechanisms involved, stable transfection of proximal tubule epithelial cells overexpressing human CLCA1 (hCLCA1) was performed. Overexpression of hCLCA1 augmented Cl− current via the Ca++‐dependent Cl− channel TMEM16A (anoctamin‐1) by patch‐clamp studies. hCLCA1 overexpression also increased the expression of fibronectin, a matrix protein, and induced the senescence‐associated secretory phenotype (SASP). Mechanistic studies underlying these changes showed that hCLCA1 overexpression leads to inhibition of AMPK activity and stimulation of mTORC1 as cellular signaling determinants of injury. Both TMEM16A inhibitor and NaHS reversed these signaling events and prevented changes in fibronectin and SASP. We conclude that CLCA1‐TMEM16A‐Cl− current pathway is a novel mediator of kidney injury in aging that is regulated by endogenous H2S.Chloride channel accessory 1 expression is increased in the tubular epithelial cells of aged kidneys. In vitro experiments show that CLCA1 augments the activity of TMEM16A, a Ca++‐dependent Chloride Channel, activates mTORC1, augments the synthesis of matrix proteins, and induces SASP, whcih contribute to aging associated kidney injury.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/168458/1/acel13407_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/168458/2/acel13407-sup-0001-Supinfo.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/168458/3/acel13407.pd

    A small molecule inhibitor of the chloride channel TMEM16A blocks vascular smooth muscle contraction and lowers blood pressure in spontaneously hypertensive rats

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    Hypertension is a major cause of cardiovascular morbidity and mortality, despite the availability of antihypertensive drugs with different targets and mechanisms of action. Here, we provide evidence that pharmacological inhibition of TMEM16A (ANO1), a calcium-activated chloride channel expressed in vascular smooth muscle cells, blocks calcium-activated chloride currents and contraction in vascular smooth muscle in vitro and decreases blood pressure in spontaneously hypertensive rats. The acylaminocycloalkylthiophene TMinh-23 fully inhibited calcium-activated TMEM16A chloride current with nanomolar potency in Fischer rat thyroid cells expressing TMEM16A, and in primary cultures of rat vascular smooth muscle cells. TMinh-23 reduced vasoconstriction caused by the thromboxane mimetic U46619 in mesenteric resistance arteries of wild-type and spontaneously hypertensive rats, with a greater inhibition in spontaneously hypertensive rats. Blood pressure measurements by tail-cuff and telemetry showed up to a 45-mmHg reduction in systolic blood pressure lasting for four-six hours in spontaneously hypertensive rats after a single dose of TMinh-23. A minimal effect on blood pressure was seen in wild-type rats or mice treated with TMinh-23. Five-day twice daily treatment of spontaneously hypertensive rats with TMinh-23 produced sustained reductions of 20-25 mmHg in daily mean systolic and diastolic blood pressure. TMinh-23 action was reversible, with blood pressure returning to baseline in spontaneously hypertensive rats by three days after treatment discontinuation. Thus, our studies provide validation for TMEM16A as a target for antihypertensive therapy and demonstrate the efficacy of TMinh-23 as an antihypertensive with a novel mechanism of action

    CFTR modulator therapy for cystic fibrosis caused by the rare c.3700A>G mutation

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    BackgroundThe c.3700A>G mutation, a rare cystic fibrosis (CF)-causing CFTR mutation found mainly in the Middle East, produces full-length transcript encoding a missense mutation (I1234V-CFTR), and a cryptic splice site that deletes 6 amino acids in nucleotide binding domain 2 (I1234del-CFTR).MethodsFRT cell models expressing I1234V-CFTR and I1234del-CFTR were generated. We also studied an I1234del-CFTR-expressing gene-edited human bronchial (16HBE14o-) cell model, and primary cultures of nasal epithelial cells from a c.3700A>G homozygous subject. To identify improved mutation-specific CFTR modulators, high-throughput screening was done using I1234del-CFTR-expressing FRT cells. Motivated by the in vitro findings, Trikafta was tested in two c.3700A>G homozygous CF subjects.ResultsFRT cells expressing full-length I1234V-CFTR had similar function to that of wildtype CFTR. I1234del-CFTR showed reduced activity, with modest activation seen with potentiators VX-770 and GLPG1837, correctors VX-809, VX-661 and VX-445, and low-temperature incubation. Screening identified novel arylsulfonyl-piperazine and spiropiperidine-quinazolinone correctors, which when used in combination with VX-445 increased current ~2-fold compared with the VX-661/VX-445 combination. The combination of VX-770 with arylsulfonamide-pyrrolopyridine, piperidine-pyridoindole or pyrazolo-quinoline potentiators gave 2-4-fold greater current than VX-770 alone. Combination potentiator (co-potentiator) efficacy was also seen in gene-edited I1234del-CFTR-expressing human bronchial epithelial cells. In two CF subjects homozygous for the c.3700A>G mutation, one subject had a 27 mmol/L decrease in sweat chloride and symptomatic improvement on Trikafta, and a second subject showed a small improvement in lung function.ConclusionsThese results support the potential benefit of CFTR modulators, including co-potentiators, for CF caused by the c.3700A>G mutation

    Repurposing calcium-sensing receptor agonist cinacalcet for treatment of CFTR-mediated secretory diarrheas

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    Diarrhea is a major cause of global mortality, and outbreaks of secretory diarrhea such as cholera remain an important problem in the developing world. Current treatment of secretory diarrhea primarily involves supportive measures, such as fluid replacement. The calcium-sensing receptor (CaSR) regulates multiple biological activities in response to changes in extracellular Ca2+. The FDA-approved drug cinacalcet is an allosteric activator of CaSR used for treatment of hyperparathyroidism. Here, we found by short-circuit current measurements in human colonic T84 cells that CaSR activation by cinacalcet reduced forskolin-induced Cl– secretion by greater than 80%. Cinacalcet also reduced Cl– secretion induced by cholera toxin, heat-stable E. coli enterotoxin, and vasoactive intestinal peptide (VIP). The cinacalcet effect primarily involved indirect inhibition of cystic fibrosis transmembrane conductance regulator–mediated (CFTR-mediated) Cl– secretion following activation of CaSR and downstream phospholipase C and phosphodiesterases. In mice, cinacalcet reduced fluid accumulation by more than 60% in intestinal closed loop models of cholera and traveler’s diarrhea. The cinacalcet effect involved both inhibition of CFTR-mediated secretion and stimulation of sodium-hydrogen exchanger 3–mediated absorption. These findings support the therapeutic utility of the safe and commonly used drug cinacalcet in CFTR-dependent secretory diarrheas, including cholera, traveler’s diarrhea, and VIPoma

    Therapeutic Cleavage of Anti-Aquaporin-4 Autoantibody in Neuromyelitis Optica by an IgG-Selective Proteinase

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    ABSTRACT Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system caused by binding of pathogenic IgG autoantibodies (NMO-IgG) to astrocyte water channel aquaporin-4 (AQP4). Astrocyte damage and downstream inflammation require NMO-IgG effector function to initiate complement-dependent cytotoxicity (CDC) and antibodydependent cell-mediated cytotoxicity (ADCC). Here, we evaluated the potential therapeutic utility of the bacterial enzyme IdeS (IgG-degrading enzyme of Streptococcus pyogenes), which selectively cleaves IgG antibodies to yield Fc and F(ab9) 2 fragments. In AQP4-expressing cell cultures, IdeS treatment of monoclonal NMO-IgGs and NMO patient sera abolished CDC and ADCC, even when IdeS was added after NMO-IgG was bound to AQP4. Binding of NMO-IgG to AQP4 was similar to that of the NMO-F(ab9) 2 generated by IdeS cleavage. NMO-F(ab9) 2 competitively displaced pathogenic NMO-IgG, preventing cytotoxicity, and the Fc fragments generated by IdeS cleavage reduced CDC and ADCC. IdeS efficiently cleaved NMO-IgG in mice in vivo, and greatly reduced NMO lesions in mice administered NMO-IgG and human complement. IgGselective cleavage by IdeS thus neutralizes NMO-IgG pathogenicity, and yields therapeutic F(ab9) 2 and Fc fragments. IdeS treatment, by therapeutic apheresis or direct administration, may be beneficial in NMO

    TITLE PAGE Small-Molecule Vasopressin-2 Receptor Antagonist Identified by a G- Protein Coupled Receptor 'Pathway' Screen MOL #34496 2 RUNNING TITLE PAGE Running title: Small-molecule V 2 R antagonist Corresponding author: Number of text pages: 31 Number o

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    Number of references: 40 Number of words in Abstract: 200 Number of words in Introduction: 496 Number of words in Discussion: 1,102 List of abbreviations: GPCR, G-protein coupled receptor; cAMP, cyclic adenosine monophospate; CFTR

    Accelerated cataract formation and reduced lens epithelial water permeability in aquaporin-1-deficient mice

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    PURPOSE. To investigate the involvement of aquaporin (AQP)-1 in lens epithelial cell water permeability and maintenance of lens transparency in experimental models of cataract formation. METHODS. Comparative studies were performed on wild-type versus AQP1-null mice. Osmotic water permeability was measured in calcein-stained epithelial cells in intact lenses from fluorescence changes in response to osmotic gradients. Lens water content was measured by gravimetry using kerosenebromobenzene density gradients, and from wet/dry weight measurements. Lens transparency was measured by contrast analysis of transmitted grid images. Cataract formation was induced in vitro by incubation in high-glucose solutions and in vivo by acetaminophen toxicity. RESULTS. Immunofluorescence showed AQP1 expression in wild-type mice in epithelial cells covering the anterior surface of the lens. AQP1 deletion did not alter baseline lens morphology or transparency, though basal water content was Ïł3% greater (P Ïœ 0.001). AQP1 deficiency reduced plasma membrane water permeability in lens epithelium by 2.8 Ïź 0.3-fold (P Ïœ 0.0001). Loss of lens transparency was accelerated by more than 50-fold in AQP1-null lenses bathed in a 55-mM glucose solution for 18 hours. At 4 hours after acetaminophen administration in 3-methylcholantrene-treated mice, lens opacification was seen in none of the six wild-type mice and in six of six AQP1-null mice. 1 Lens opacification is a multifactorial process, in which posttranslational modification of lens structural proteins enhances their aggregation, fragmentation, and precipitation, leading to cataract formation. 3,4 An epithelial cell monolayer extends from the anterior pole of the lens to its equatorial surface, surrounding the elongated lens fibers, which are arranged in a stratified manner with the oldest fibers in the lens interior. Nourishment to the lens is provided by diffusion from the aqueous and vitreous humors. However, it is thought that simple diffusion cannot sustain the metabolic needs of the lens interior. 5 A circulatory system has been proposed, in which an asymmetric distribution of ion pumps, transporters, channels, and cell junctions drive ion-coupled fluid absorption, facilitating the entry of nutrients and metabolites into the inner lens across the polar regions and their exit through the lens equator. The role of AQP1 in lens epithelium is unknown. We tested the hypothesis that AQP1 is involved in lens water permeability and maintenance of lens transparency. The motivations for this work included (1) the consistent expression of AQP1 in lens epithelium across multiple mammalian species; (2) the involvement of corneal endothelial AQP1 in the maintenance of corneal transparency 10 ; and (3) our observations on lens opacification in AQP1-null mice during eye surgery. In this study, we demonstrated for the first time AQP1 expression and water channel function in mouse lens epithelium. Although AQP1 deficiency was not associated with demonstrable abnormalities in baseline lens structure or transparency, we found remarkably accelerated cataractogenesis in both in vitro and in vivo models of cataract formation. Our results provide evidence for a novel role of AQP1 in ocular physiology. MATERIALS AND METHODS Mice Wild-type and AQP1-deficient mice in a CD1 genetic background matched by age (8 -10 weeks) were used, with littermates used in most experiments. Transgenic mice deficient in AQP1 were generated b

    Cystic fibrosis transmembrane conductance regulator dysfunction in platelets drives lung hyperinflammation.

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    Cystic fibrosis (CF) lung disease is characterized by an inflammatory response that can lead to terminal respiratory failure. The cystic fibrosis transmembrane conductance regulator (CFTR) is mutated in CF, and we hypothesized that dysfunctional CFTR in platelets, which are key participants in immune responses, is a central determinant of CF inflammation. We found that deletion of CFTR in platelets produced exaggerated acute lung inflammation and platelet activation after intratracheal LPS or Pseudomonas aeruginosa challenge. CFTR loss of function in mouse or human platelets resulted in agonist-induced hyperactivation and increased calcium entry into platelets. Inhibition of the transient receptor potential cation channel 6 (TRPC6) reduced platelet activation and calcium flux, and reduced lung injury in CF mice after intratracheal LPS or Pseudomonas aeruginosa challenge. CF subjects receiving CFTR modulator therapy showed partial restoration of CFTR function in platelets, which may be a convenient approach to monitoring biological responses to CFTR modulators. We conclude that CFTR dysfunction in platelets produces aberrant TRPC6-dependent platelet activation, which is a major driver of CF lung inflammation and impaired bacterial clearance. Platelets and TRPC6 are what we believe to be novel therapeutic targets in the treatment of CF lung disease
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