Bisindole-based small molecules as transmembrane anion transporters and potential anticancer agents.

Few synthetic ion transporters have been reported incorporating indole as the core moiety. We have developed a novel bisindole-based transporter capable of efficient transmembrane anion antiport. This system induced cytotoxicity in MCF-7 breast cancer cells via chloride ion homeostasis disruption and the associated ROS generation, mitochondrial membrane depolarization, and lysosomal deacidification.

Cryo-EM structure of a fungal mitochondrial calcium uniporter.

The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel localized to the inner mitochondrial membrane. Here, we describe the structure of an MCU orthologue from the fungus Neosartorya fischeri (NfMCU) determined to 3.8 Å resolution by phase-plate cryo-electron microscopy. The channel is a homotetramer with two-fold symmetry in its amino-terminal domain (NTD) that adopts a similar structure to that of human MCU. The NTD assembles as a dimer of dimers to form a tetrameric ring that connects to the transmembrane domain through an elongated coiled-coil domain. The ion-conducting pore domain maintains four-fold symmetry, with the selectivity filter positioned at the start of the pore-forming TM2 helix. The aspartate and glutamate sidechains of the conserved DIME motif are oriented towards the central axis and separated by one helical turn. The structure of NfMCU offers insights into channel assembly, selective calcium permeation, and inhibitor binding.

Cinnamic Acid, Perillic Acid, and Tryptophan Metabolites Differentially Regulate Ion Transport and Serotonin Metabolism and Signaling in the Mouse Ileum In Vitro.

Phytochemicals and tryptophan (Trp) metabolites have been found to modulate gut function and health. However, whether these metabolites modulate gut ion transport and serotonin (5-HT) metabolism and signaling requires further investigation. The aim of this study was to investigate the effects of selected phytochemicals and Trp metabolites on the ion transport and 5-HT metabolism and signaling in the ileum of mice in vitro using the Ussing chamber technique. During the in vitro incubation, vanillylmandelic acid (VMA) reduced (p < 0.05) the short-circuit current, and 100 µM chlorogenic acid (CGA) (p = 0.12) and perillic acid (PA) (p = 0.14) had a tendency to reduce the short-circuit current of the ileum. Compared with the control, PA and N-acetylserotonin treatment upregulated the expression of tryptophan hydroxylase 1 (Tph1), while 100 µM cinnamic acid, indolelactic acid (ILA), and 10 µM CGA or indoleacetaldehyde (IAld) treatments downregulated (p < 0.05) the mRNA levels of Tph1. In addition, 10 µM IAld or 100 µM ILA upregulated (p < 0.05) the expression of monoamine oxidase A (Maoa). However, 10 µM CGA or 100 µM PA downregulated (p < 0.05) Maoa expression. All selected phytochemicals and Trp metabolites upregulated (p < 0.05) the expression of Htr4 and Htr7 compared to that of the control group. VMA and CGA reduced (p < 0.05) the ratios of Htr1a/Htr7 and Htr4/Htr7. These findings may help to elucidate the effects of phytochemicals and Trp metabolites on the regulation of gut ion transport and 5-HT signaling-related gut homeostasis in health and disease.

Structural basis for gating the high-conductance Ca2+-activated K+ channel.

The precise control of an ion channel gate by environmental stimuli is crucial for the fulfilment of its biological role. The gate in Slo1 K+ channels is regulated by two separate stimuli, intracellular Ca2+ concentration and membrane voltage. Slo1 is thus central to understanding the relationship between intracellular Ca2+ and membrane excitability. Here we present the Slo1 structure from Aplysia californica in the absence of Ca2+ and compare it with the Ca2+-bound channel. We show that Ca2+ binding at two unique binding sites per subunit stabilizes an expanded conformation of the Ca2+ sensor gating ring. These conformational changes are propagated from the gating ring to the pore through covalent linkers and through protein interfaces formed between the gating ring and the voltage sensors. The gating ring and the voltage sensors are directly connected through these interfaces, which allow membrane voltage to regulate gating of the pore by influencing the Ca2+ sensors.

Cryo-EM structures of the TMEM16A calcium-activated chloride channel.

Calcium-activated chloride channels (CaCCs) encoded by TMEM16A control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system. To understand how CaCCs mediate and control anion permeation to fulfil these physiological functions, knowledge of the mammalian TMEM16A structure and identification of its pore-lining residues are essential. TMEM16A forms a dimer with two pores. Previous CaCC structural analyses have relied on homology modelling of a homologue (nhTMEM16) from the fungus Nectria haematococca that functions primarily as a lipid scramblase, as well as subnanometre-resolution electron cryo-microscopy. Here we present de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol as determined by single-particle electron cryo-microscopy. These structures reveal the ion permeation pore and represent different functional states. The structure in lauryl maltose neopentyl glycol has one Ca2+ ion resolved within each monomer with a constricted pore; this is likely to correspond to a closed state, because a CaCC with a single Ca2+ occupancy requires membrane depolarization in order to open (C.J.P. et al., manuscript submitted). The structure in nanodiscs has two Ca2+ ions per monomer and its pore is in a closed conformation; this probably reflects channel rundown, which is the gradual loss of channel activity that follows prolonged CaCC activation in 1 mM Ca2+. Our mutagenesis and electrophysiological studies, prompted by analyses of the structures, identified ten residues distributed along the pore that interact with permeant anions and affect anion selectivity, as well as seven pore-lining residues that cluster near pore constrictions and regulate channel gating. Together, these results clarify the basis of CaCC anion conduction.

Activation mechanism of the calcium-activated chloride channel TMEM16A revealed by cryo-EM.

The calcium-activated chloride channel TMEM16A is a ligand-gated anion channel that opens in response to an increase in intracellular Ca2+ concentration. The protein is broadly expressed and contributes to diverse physiological processes, including transepithelial chloride transport and the control of electrical signalling in smooth muscles and certain neurons. As a member of the TMEM16 (or anoctamin) family of membrane proteins, TMEM16A is closely related to paralogues that function as scramblases, which facilitate the bidirectional movement of lipids across membranes. The unusual functional diversity of the TMEM16 family and the relationship between two seemingly incompatible transport mechanisms has been the focus of recent investigations. Previous breakthroughs were obtained from the X-ray structure of the lipid scramblase of the fungus Nectria haematococca (nhTMEM16), and from the cryo-electron microscopy structure of mouse TMEM16A at 6.6 Å (ref. 14). Although the latter structure disclosed the architectural differences that distinguish ion channels from lipid scramblases, its low resolution did not permit a detailed molecular description of the protein or provide any insight into its activation by Ca2+. Here we describe the structures of mouse TMEM16A at high resolution in the presence and absence of Ca2+. These structures reveal the differences between ligand-bound and ligand-free states of a calcium-activated chloride channel, and when combined with functional experiments suggest a mechanism for gating. During activation, the binding of Ca2+ to a site located within the transmembrane domain, in the vicinity of the pore, alters the electrostatic properties of the ion conduction path and triggers a conformational rearrangement of an α-helix that comes into physical contact with the bound ligand, and thereby directly couples ligand binding and pore opening. Our study describes a process that is unique among channel proteins, but one that is presumably general for both functional branches of the TMEM16 family.

Proteolytic activation of the epithelial sodium channel (ENaC) by factor VII activating protease (FSAP) and its relevance for sodium retention in nephrotic mice.

Proteolytic activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases is thought to contribute to renal sodium retention in nephrotic syndrome. However, the identity of the responsible proteases remains elusive. This study evaluated factor VII activating protease (FSAP) as a candidate in this context. We analyzed FSAP in the urine of patients with nephrotic syndrome and nephrotic mice and investigated its ability to activate human ENaC expressed in Xenopus laevis oocytes. Moreover, we studied sodium retention in FSAP-deficient mice (Habp2-/-) with experimental nephrotic syndrome induced by doxorubicin. In urine samples from nephrotic humans, high concentrations of FSAP were detected both as zymogen and in its active state. Recombinant serine protease domain of FSAP stimulated ENaC-mediated whole-cell currents in a time- and concentration-dependent manner. Mutating the putative prostasin cleavage site in γ-ENaC (γRKRK178AAAA) prevented channel stimulation by the serine protease domain of FSAP. In a mouse model for nephrotic syndrome, active FSAP was present in nephrotic urine of Habp2+/+ but not of Habp2-/- mice. However, Habp2-/- mice were not protected from sodium retention compared to nephrotic Habp2+/+ mice. Western blot analysis revealed that in nephrotic Habp2-/- mice, proteolytic cleavage of α- and γ-ENaC was similar to that in nephrotic Habp2+/+ animals. In conclusion, active FSAP is excreted in the urine of nephrotic patients and mice and activates ENaC in vitro involving the putative prostasin cleavage site of γ-ENaC. However, endogenous FSAP is not essential for sodium retention in nephrotic mice.

Thymol improves salinity tolerance of tobacco by increasing the sodium ion efflux and enhancing the content of nitric oxide and glutathione.

BACKGROUND AND OBJECTIVE: Salt stress is one of the most important abiotic stresses affecting the yield and quality of tobacco (Nicotiana tabacum). Thymol (a natural medicine) has been widely used in medical research because of its antibacterial and anti-inflammatory activities. However, the influence of thymol on the root growth of tobacco is not fully elucidated. In this study, the regulatory effects of different concentrations of thymol were investigated. METHODOLOGY: Here, histochemical staining and biochemical methods, non-invasive micro-test technology (NMT), and qPCR assay were performed to investigate the effect of thymol and mechanism of it improving salinity tolerance in tobacco seedlings. RESULTS: In this study, our results showed that thymol rescued root growth from salt stress by ameliorating ROS accumulation, lipid peroxidation, and cell death. Furthermore, thymol enhanced contents of NO and GSH to repress ROS accumulation, further protecting the stability of the cell membrane. And, thymol improved Na+ efflux and the expression of SOS1, HKT1, and NHX1, thus protecting the stability of Na+ and K+. CONCLUSION: Our study confirmed the protecting effect of thymol in tobacco under salt stress, and we also identified the mechanism of it, involving dynamic regulation of antioxidant system and the maintenance of Na+ homeostasis. It can be a new method to improve salinity tolerance in plants.

Transport, functions, and interaction of calcium and manganese in plant organellar compartments.

Calcium (Ca2+) and manganese (Mn2+) are essential elements for plants and have similar ionic radii and binding coordination. They are assigned specific functions within organelles, but share many transport mechanisms to cross organellar membranes. Despite their points of interaction, those elements are usually investigated and reviewed separately. This review takes them out of this isolation. It highlights our current mechanistic understanding and points to open questions of their functions, their transport, and their interplay in the endoplasmic reticulum (ER), vesicular compartments (Golgi apparatus, trans-Golgi network, pre-vacuolar compartment), vacuoles, chloroplasts, mitochondria, and peroxisomes. Complex processes demanding these cations, such as Mn2+-dependent glycosylation or systemic Ca2+ signaling, are covered in some detail if they have not been reviewed recently or if recent findings add to current models. The function of Ca2+ as signaling agent released from organelles into the cytosol and within the organelles themselves is a recurrent theme of this review, again keeping the interference by Mn2+ in mind. The involvement of organellar channels [e.g. glutamate receptor-likes (GLR), cyclic nucleotide-gated channels (CNGC), mitochondrial conductivity units (MCU), and two-pore channel1 (TPC1)], transporters (e.g. natural resistance-associated macrophage proteins (NRAMP), Ca2+ exchangers (CAX), metal tolerance proteins (MTP), and bivalent cation transporters (BICAT)], and pumps [autoinhibited Ca2+-ATPases (ACA) and ER Ca2+-ATPases (ECA)] in the import and export of organellar Ca2+ and Mn2+ is scrutinized, whereby current controversial issues are pointed out. Mechanisms in animals and yeast are taken into account where they may provide a blueprint for processes in plants, in particular, with respect to tunable molecular mechanisms of Ca2+ versus Mn2+ selectivity.

Intracellular phosphate sensing and regulation of phosphate transport systems in plants.

Recent research on the regulation of cellular phosphate (Pi) homeostasis in eukaryotes has collectively made substantial advances in elucidating inositol pyrophosphates (PP-InsP) as Pi signaling molecules that are perceived by the SPX (Syg1, Pho81, and Xpr1) domains residing in multiple proteins involved in Pi transport and signaling. The PP-InsP-SPX signaling module is evolutionarily conserved across eukaryotes and has been elaborately adopted in plant Pi transport and signaling systems. In this review, we have integrated these advances with prior established knowledge of Pi and PP-InsP metabolism, intracellular Pi sensing, and transcriptional responses according to the dynamics of cellular Pi status in plants. Anticipated challenges and pending questions as well as prospects are also discussed.

The emerging role of GABA as a transport regulator and physiological signal.

While the proposal that γ-aminobutyric acid (GABA) acts a signal in plants is decades old, a signaling mode of action for plant GABA has been unveiled only relatively recently. Here, we review the recent research that demonstrates how GABA regulates anion transport through aluminum-activated malate transporters (ALMTs) and speculation that GABA also targets other proteins. The ALMT family of anion channels modulates multiple physiological processes in plants, with many members still to be characterized, opening up the possibility that GABA has broad regulatory roles in plants. We focus on the role of GABA in regulating pollen tube growth and stomatal pore aperture, and we speculate on its role in long-distance signaling and how it might be involved in cross talk with hormonal signals. We show that in barley (Hordeum vulgare), guard cell opening is regulated by GABA, as it is in Arabidopsis (Arabidopsis thaliana), to regulate water use efficiency, which impacts drought tolerance. We also discuss the links between glutamate and GABA in generating signals in plants, particularly related to pollen tube growth, wounding, and long-distance electrical signaling, and explore potential interactions of GABA signals with hormones, such as abscisic acid, jasmonic acid, and ethylene. We conclude by postulating that GABA encodes a signal that links plant primary metabolism to physiological status to fine tune plant responses to the environment.

Structure, inhibition and regulation of two-pore channel TPC1 from Arabidopsis thaliana.

Two-pore channels (TPCs) comprise a subfamily (TPC1-3) of eukaryotic voltage- and ligand-gated cation channels with two non-equivalent tandem pore-forming subunits that dimerize to form quasi-tetramers. Found in vacuolar or endolysosomal membranes, they regulate the conductance of sodium and calcium ions, intravesicular pH, trafficking and excitability. TPCs are activated by a decrease in transmembrane potential and an increase in cytosolic calcium concentrations, are inhibited by low luminal pH and calcium, and are regulated by phosphorylation. Here we report the crystal structure of TPC1 from Arabidopsis thaliana at 2.87 Å resolution as a basis for understanding ion permeation, channel activation, the location of voltage-sensing domains and regulatory ion-binding sites. We determined sites of phosphorylation in the amino-terminal and carboxy-terminal domains that are positioned to allosterically modulate cytoplasmic Ca(2+) activation. One of the two voltage-sensing domains (VSD2) encodes voltage sensitivity and inhibition by luminal Ca(2+) and adopts a conformation distinct from the activated state observed in structures of other voltage-gated ion channels. The structure shows that potent pharmacophore trans-Ned-19 (ref. 17) acts allosterically by clamping the pore domains to VSD2. In animals, Ned-19 prevents infection by Ebola virus and other filoviruses, presumably by altering their fusion with the endolysosome and delivery of their contents into the cytoplasm.

Structure of the voltage-gated two-pore channel TPC1 from Arabidopsis thaliana.

Two-pore channels (TPCs) contain two copies of a Shaker-like six-transmembrane (6-TM) domain in each subunit and are ubiquitously expressed in both animals and plants as organellar cation channels. Here we present the crystal structure of a vacuolar two-pore channel from Arabidopsis thaliana, AtTPC1, which functions as a homodimer. AtTPC1 activation requires both voltage and cytosolic Ca(2+). Ca(2+) binding to the cytosolic EF-hand domain triggers conformational changes coupled to the pair of pore-lining inner helices from the first 6-TM domains, whereas membrane potential only activates the second voltage-sensing domain, the conformational changes of which are coupled to the pair of inner helices from the second 6-TM domains. Luminal Ca(2+) or Ba(2+) can modulate voltage activation by stabilizing the second voltage-sensing domain in the resting state and shift voltage activation towards more positive potentials. Our Ba(2+)-bound AtTPC1 structure reveals a voltage sensor in the resting state, providing hitherto unseen structural insight into the general voltage-gating mechanism among voltage-gated channels.

Esc peptides as novel potentiators of defective cystic fibrosis transmembrane conductance regulator: an unprecedented property of antimicrobial peptides.

Mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein lead to persistent lung bacterial infections, mainly due to Pseudomonas aeruginosa, causing loss of respiratory function and finally death of people affected by CF. Unfortunately, even in the era of CFTR modulation therapies, management of pulmonary infections in CF remains highly challenging especially for patients with advanced stages of lung disease. Recently, we identified antimicrobial peptides (AMPs), namely Esc peptides, with potent antipseudomonal activity. In this study, by means of electrophysiological techniques and computational studies we discovered their ability to increase the CFTR-controlled ion currents, by direct interaction with the F508del-CFTR mutant. Remarkably, this property was not explored previously with any AMPs or peptides in general. More interestingly, in contrast with clinically used CFTR modulators, Esc peptides would give particular benefit to CF patients by combining their capability to eradicate lung infections and to act as promoters of airway wound repair with their ability to ameliorate the activity of the channel with conductance defects. Overall, our findings not only highlighted Esc peptides as the first characterized AMPs with a novel property, that is the potentiator activity of CFTR, but also paved the avenue to investigate the functions of AMPs and/or other peptide molecules, for a new up-and-coming pharmacological approach to address CF lung disease.

Antisense oligonucleotide-mediated correction of CFTR splicing improves chloride secretion in cystic fibrosis patient-derived bronchial epithelial cells.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, encoding an anion channel that conducts chloride and bicarbonate across epithelial membranes. Mutations that disrupt pre-mRNA splicing occur in >15% of CF cases. One common CFTR splicing mutation is CFTR c.3718-2477C>T (3849+10 kb C>T), which creates a new 5' splice site, resulting in splicing to a cryptic exon with a premature termination codon. Splice-switching antisense oligonucleotides (ASOs) have emerged as an effective therapeutic strategy to block aberrant splicing. We test an ASO targeting the CFTR c.3718-2477C>T mutation and show that it effectively blocks aberrant splicing in primary bronchial epithelial (hBE) cells from CF patients with the mutation. ASO treatment results in long-term improvement in CFTR activity in hBE cells, as demonstrated by a recovery of chloride secretion and apical membrane conductance. We also show that the ASO is more effective at recovering chloride secretion in our assay than ivacaftor, the potentiator treatment currently available to these patients. Our findings demonstrate the utility of ASOs in correcting CFTR expression and channel activity in a manner expected to be therapeutic in patients.

Piezo1 and BKCa channels in human atrial fibroblasts: Interplay and remodelling in atrial fibrillation.

AIMS: Atrial Fibrillation (AF) is an arrhythmia of increasing prevalence in the aging populations of developed countries. One of the important indicators of AF is sustained atrial dilatation, highlighting the importance of mechanical overload in the pathophysiology of AF. The mechanisms by which atrial cells, including fibroblasts, sense and react to changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in SAC- presence and activity associated with AF. METHODS AND RESULTS: Using primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or sustained AF, we combine electrophysiological, molecular and pharmacological tools to identify SAC. Two electrophysiological SAC- signatures were detected, indicative of cation-nonselective and potassium-selective channels. Using siRNA-mediated knockdown, we identified the cation-nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel, its sensitivity to calcium, paxilline or iberiotoxin (blockers), and NS11021 (activator), indicated presence of calcium-dependent 'big potassium channels' (BKCa). In cells from AF patients, Piezo1 activity and mRNA expression levels were higher than in cells from sinus rhythm patients, while BKCa activity (but not expression) was downregulated. Both Piezo1-knockdown and removal of extracellular calcium from the patch pipette resulted in a significant reduction of BKCa current during stretch. No co-immunoprecipitation of Piezo1 and BKCa was detected. CONCLUSIONS: Human atrial fibroblasts contain at least two types of ion channels that are activated during stretch: Piezo1 and BKCa. While Piezo1 is directly stretch-activated, the increase in BKCa activity during mechanical stimulation appears to be mainly secondary to calcium influx via SAC such as Piezo1. During sustained AF, Piezo1 is increased, while BKCa activity is reduced, highlighting differential regulation of both channels. Our data support the presence and interplay of Piezo1 and BKCa in human atrial fibroblasts in the absence of physical links between the two channel proteins.

Novel Correctors and Potentiators Enhance Translational Readthrough in CFTR Nonsense Mutations.

Premature-termination codons (PTCs) in CFTR (cystic fibrosis [CF] transmembrane conductance regulator) result in nonfunctional CFTR protein and are the proximate cause of ∼11% of CF-causing alleles, for which no treatments exist. The CFTR corrector lumacaftor and the potentiator ivacaftor improve CFTR function with terminal PTC mutations and enhance the effect of readthrough agents. Novel correctors GLPG2222 (corrector 1 [C1]), GLPG3221 (corrector 2 [C2]), and potentiator GLPG1837 compare favorably with lumacaftor and ivacaftor in vitro. Here, we evaluated the effect of correctors C1a and C2a (derivatives of C1 and C2) and GLPG1837 alone or in combination with the readthrough compound G418 on CFTR function using heterologous Fischer rat thyroid (FRT) cells, the genetically engineered human bronchial epithelial (HBE) 16HBE14o- cell lines, and primary human cells with PTC mutations. In FRT lines pretreated with G418, GLPG1837 elicited dose-dependent increases in CFTR activity that exceeded those from ivacaftor in FRT-W1282X and FRT-R1162X cells. A three-mechanism strategy consisting of G418, GLPG1837, and two correctors (C1a + C2a) yielded the greatest functional improvements in FRT and 16HBE14o- PTC variants, noting that correction and potentiation without readthrough was sufficient to stimulate CFTR activity for W1282X cells. GLPG1837 + C1a + C2a restored substantial function in G542X/F508del HBE cells and restored even more function for W1282X/F508del cells, largely because of the corrector/potentiator effect, with no additional benefit from G418. In G542X/R553X or R1162X/R1162X organoids, enhanced forskolin-induced swelling was observed with G418 + GLPG1837 + C1a + C2a, although GLPG1837 + C1a + C2a alone was sufficient to improve forskolin-induced swelling in W1282X/W1282X organoids. Combination of CFTR correctors, potentiators, and readthrough compounds augments the functional repair of CFTR nonsense mutations, indicating the potential for novel correctors and potentiators to restore function to truncated W1282X CFTR.

Bile acid composition regulates the manganese transporter Slc30a10 in intestine.

Bile acids (BAs) comprise heterogenous amphipathic cholesterol-derived molecules that carry out physicochemical and signaling functions. A major site of BA action is the terminal ileum, where enterocytes actively reuptake BAs and express high levels of BA-sensitive nuclear receptors. BA pool size and composition are affected by changes in metabolic health, and vice versa. One of several factors that differentiate BAs is the presence of a hydroxyl group on C12 of the steroid ring. 12α-Hydroxylated BAs (12HBAs) are altered in multiple disease settings, but the consequences of 12HBA abundance are incompletely understood. We employed mouse primary ileum organoids to investigate the transcriptional effects of varying 12HBA abundance in BA pools. We identified Slc30a10 as one of the top genes differentially induced by BA pools with varying 12HBA abundance. SLC30A10 is a manganese efflux transporter critical for whole-body manganese excretion. We found that BA pools, especially those low in 12HBAs, induce cellular manganese efflux and that Slc30a10 induction by BA pools is driven primarily by lithocholic acid signaling via the vitamin D receptor. Administration of lithocholic acid or a vitamin D receptor agonist resulted in increased Slc30a10 expression in mouse ileum epithelia. These data demonstrate a previously unknown role for BAs in intestinal control of manganese homeostasis.

Sex difference in kidney electrolyte transport III: Impact of low K intake on thiazide-sensitive cation excretion in male and female mice.

We compared the regulation of the NaCl cotransporter (NCC) in adaptation to a low-K (LK) diet in male and female mice. We measured hydrochlorothiazide (HCTZ)-induced changes in urine volume (UV), glomerular filtration rate (GFR), absolute (ENa, EK), and fractional (FENa, FEK) excretion in male and female mice on control-K (CK, 1% KCl) and LK (0.1% KCl) diets for 7 days. With CK, NCC-dependent ENa and FENa were larger in females than males as observed previously. However, with LK, HCTZ-induced ENa and FENa increased in males but not in females, abolishing the sex differences in NCC function as observed in CK group. Despite large diuretic and natriuretic responses to HCTZ, EK was only slightly increased in response to the drug when animals were on LK. This suggests that the K-secretory apparatus in the distal nephron is strongly suppressed under these conditions. We also examined LK-induced changes in Na transport protein expression by Western blotting. Under CK conditions females expressed more NCC protein, as previously reported. LK doubled both total (tNCC) and phosphorylated NCC (pNCC) abundance in males but had more modest effects in females. The larger effect in males abolished the sex-dependence of NCC expression, consistent with the measurements of function by renal clearance. LK intake did not change NHE3, NHE2, or NKCC2 expression, but reduced the amount of the cleaved (presumably active) form of γENaC. LK reduced plasma K to lower levels in females than males. These results indicated that males had a stronger NCC-mediated adaptation to LK intake than females.

Ivacaftor partially corrects airway inflammation in a humanized G551D rat.

Animal models have been highly informative for understanding the pathogenesis and progression of cystic fibrosis (CF) lung disease. In particular, the CF rat models recently developed have addressed mechanistic causes of the airway mucus defect characteristic of CF, and how these may change when cystic fibrosis transmembrane conductance regulator (CFTR) activity is restored using new modulator therapies. We hypothesized that inflammatory changes to the airway would develop spontaneously and progressively, and that these changes would be resolved with modulator therapy. To test this, we used a humanized-CFTR rat expressing the G551D variant that responds to the CFTR modulator ivacaftor. Markers typically found in the CF lung were assessed, including neutrophil influx, small airway histopathology, and inflammatory cytokine concentration. Young hG551D rats did not express inflammatory cytokines at baseline but did upregulate these in response to inflammatory trigger. As the hG551D rats aged, histopathology worsened, accompanied by neutrophil influx into the airway and increasing concentrations of TNF-α, IL-1α, and IL-6 in the airways. Ivacaftor administration reduced concentrations of these cytokines when administered to the rats at baseline but was less effective in the rats that had also received inflammatory stimulus. Therefore, we conclude that administration of ivacaftor resulted in an incomplete resolution of inflammation when rats received an external trigger, suggesting that CFTR activation may not be enough to resolve inflammation in the lungs of patients with CF.