Membrane Transport 2023: Recent Research into Ion Channels, Transporters and Epithelial Physiology

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The role of TRPV4 in electrolyte and fluid secretion by choroid plexus epithelia: Implications for hydrocephalus

Hydrocephalus is a devasting disease for which there is no pharmaceutical treatment. Currently the standard of care involves brain surgery – most usually the placement of a shunt to drain excess cerebrospinal fluid (CSF) from the brain to another area of the body. However, shunts routinely fail for a variety of reasons including infection, blockage and equipment malfunction predisposing the patient to multiple brain surgeries. While pediatric hydrocephalus is perhaps the most recognizable form, there are multiple causes of this condition in older children and adults including traumatic brain injury, stroke, infection. Regardless of the precipitating factors, there is an enlargement of the cerebral ventricles and an excess of CSF in the brain. It is our hypothesis that pharmaceuticals that could decrease CSF production on an as-needed basis would be helpful in treating most forms of hydrocephalus. Rational drug design for such potential treatments is based on a more detailed understanding of the production of CSF by the choroid plexus epithelium (CPe). The small tissue comprising the choroid plexus produces approximately 500 ml of CSF per day, the composition of which varies according to physiological, diurnal, and pathophysiological influences.

In a genetic rat model of hydrocephalus, we have shown that antagonists of the transient receptor potential vanilloid 4 (TRPV4) channel ameliorate the development of hydrocephalus, implicating this channel as a key component of CSF production. Preliminary studies indicate that TRPV4 antagonists are also effective in rodent models of post hemorrhagic and post traumatic hydrocephalus. In addition to pre-clinical animal models, we use a human CPe cell model, the HIBCPP (human choroid plexus papilloma) to identify the transepithelial electrolyte and fluid fluxes that occur in response to TRPV4 stimulation. Ussing-style electrophysiology combined with imaging, qRT-PCR and western blotting, have shown that the HIBCPP cells form a moderately tight barrier epithelium consistent with the blood-CSF barrier and express important transporters found in the native epithelium with the correct polarization. Addition of a TRPV4 agonist causes a multicomponent change in transepithelial electrolyte flux as well as a substantial but reversible change in transepithelial permeability. The TRPV4-mediated electrolyte flux appears to result from secondary activation of multiple transport proteins stimulated in response to the TRPV4-mediated influx of Ca²⁺ and Na⁺ and is a complex mixture of movement of both cations and anions. This flux is accompanied by a statistically significant secretory movement of fluid in response to the TRPV4 agonist. Although in vivo the choroid plexus is one of the most secretory epithelia in the body, the degree of fluid secretion by this cell line is unique. The HIBCPP cell line is being used to dissect pathways involved in CSF production. Our results have uncovered unexpected levels of fluid movement in response to TRPV4 stimulation that can inform the role of opposing electrolyte movements and electroneutral transporters that are difficult to measure using electrophysiological techniques.

Nutraceutical targeting of the Farnesoid X Receptor for regulation of CFTR expression in the colonic epithelium

Introduction: The nuclear bile acid receptor, farnesoid X receptor (FXR), is an important regulator of colonic epithelial function and the therapeutic potential for targeting FXR in the treatment of chronic intestinal disease is well documented. We have previously shown that bile acids, acting via FXR, inhibit colonic epithelial Cl^– secretion by downregulating expression of the CFTR Cl^– channel. We have also shown that the pentacyclic triterpene (PCT), hederagenin (HG), a plant-derived bioactive phytochemical, enhances the expression and activity of FXR in colonic epithelial cells and potentiates the effects of FXR on CFTR. Peroxisome proliferator-activated receptors (PPARs), described as nutrient sensors, have recently been shown to modulate FXR expression. Here, we investigated molecular mechanisms underlying FXR regulation of CFTR expression in colonic epithelial cells, and a potential role for PPARs in mediating HG actions on FXR expression and activity.

Methods: Monolayers of T₈₄ colonic epithelial cells were treated with HG, PPARγ, PPARα, or PPARδ agonists, rosiglitazone, WY14643, and GW501516, and antagonists, GW9662, GW6471, and GSK3787, respectively. GW4064 was employed as an FXR agonist and FGF19 used as an index of FXR activation. Levels of FGF19, NF-κB p65, FXR, and ANGPTL4, a PPAR target gene, were measured by qRT-PCR, western blotting, or ELISA. FXR binding to the CFTR promoter was investigated by chromatin immunoprecipitation (ChIP).

Results: FXR activation did not alter expression, phosphorylation, or nuclear translocation of the NF-κB p65 subunit, while preliminary ChIP-qPCR analysis demonstrated enhanced binding of FXR to the CFTR promoter upon GW4064 treatment. Treatment with HG significantly increased mRNA expression of the PPAR target gene, ANGPTL4, to 5.7 ± 1 fold (n = 5; p < 0.05) of untreated cells after 3 hours. The effects of HG on FXR expression were mimicked by rosiglitazone (1 µM), WY14643 (10 µM), and GW501516 (0.5 µM), respectively. The PPARα antagonist, GW6471 (1 µM), reduced HG-induced FXR mRNA and protein expression from 2.8 ± 0.4 to 0.9 ± 0.1 (n = 5; p < 0.05) and 1.8 ± 0.3 to 1 ± 0.1 (n = 3) fold of untreated controls, respectively, whereas the PPARγ and PPARδ antagonists, GW9662 (5 µM) and GSK3787 (1 µM), did not prevent upregulation of FXR by HG.

Conclusion: FXR regulates colonic epithelial CFTR expression and function by a mechanism which appears to involve direct binding of FXR to the CFTR promoter. The bioactive phytochemical, HG, regulates colonic epithelial FXR expression through mechanisms that involve activation of PPARα. By virtue of their ability to upregulate FXR expression, and thereby enhance its antisecretory actions, plant extracts containing HG have excellent potential to be developed as FXR-targeted nutraceuticals for the treatment and prevention of intestinal disease.

Succinate enhances mucociliary clearance in the murine tracheal epithelium by triggering acetylcholine release from chemosensory cells

Introduction: Solitary cholinergic chemosensory cells (SCCC) are rare tracheal epithelial cells, which are considered as a part of the innate immune system sensing invading pathogens within the airways. They express a wide range of GPCR including taste receptors and their signaling machinery (TRPM5, PLCβ2 and ITP3R3) and the acetylcholine (ACh) producing enzyme ChAT. We previously identified bacterial formylated signal peptides as activators of SCCC, which subsequently release ACh and thereby alter ciliary activity. The receptor recognizing these formylated peptides, however, remained unknown. In the intestine it was shown that tuft cells, the counterpart of tracheal SCCC, can be activated by the microbial metabolite succinate. We here investigated if tracheal SCCC are also equipped with the succinate recognizing receptor SucnR1 and if succinate triggers innate defense mechanism through activation of tracheal SCCC.

Material and Methods: Expression of the succinate receptor SucnR1 was analyzed in isolated tracheal epithelial cells by RT-PCR and by analyzing existing single cell sequencing data sets. Particle transport speed (PTS) and ciliary beat frequency (CBF) were examined in response to metabolites. Ussing chamber experiments were performed with explanted tracheas to investigate ion transport processes across the tracheal epithelium.

Results: SucnR1 was exclusively expressed by a subset of SCCC within the tracheal epithelium, and there was no epithelial Sucnr1 expression in mice lacking SCCC (Pou2f3^-/-). Succinate, but neither butyrate nor acetate, increased CBF and, consequently, PTS in a dose-dependent manner. This effect required Sucnr1 (lost in SucnR1-deficient mice) and SCCC (lost in Pou2f3- and Trpm5-deficient mice). In mice with SCCC-specific deletion of ChAT (ChAT^fl-Avil^cre) and in the presence of the muscarinic antagonist atropine, the effect of succinate was reduced by 71 and 87%, respectively. In mice lacking muscarinic receptor M3, which is expressed by ciliated cells, the succinate effect was also abrogated by 77%. In Ussing chamber experiments, succinate induced a sharp increase in ion flux across the tracheal epithelium, which was reduced in SucnR1-, Pou2f3- and Trpm5-deficient mice by 81, 82 and 76%, respectively. The succinate-induced increase could be reduced by blocking cholinergic signaling (atropine + mecamylamine, 4-DAMP) and by the general chloride channel inhibitor NPPB (85, 94 and 98%, respectively). Furthermore, the gap junction blocker Gap27 reduced both, the succinate-induced increase in PTS and ion secretion (79 and 74%, respectively).

Conclusion: Succinate activates SCCC by binding to SucnR1, thereby triggering release of ACh. This increases ciliary activity of neighboring ciliated cells most likely via gap junctions and induces secretion of chloride ions into the periciliary fluid. Secretion of chloride ions is required for sufficient production of periciliary fluid. Hence, these effects are synergistic in terms of mucociliary clearance. Interestingly, succinate is produced in high quantities by epithelial cells during infection with Pseudomonas aeruginosa, a common pathogen especially in cystic fibrosis patients. Thus, the present data extend the molecular spectrum surveilled by tracheal SCCC beyond microbial factors to host-derived factors.

Aldosteroneâ€independent ENaC activity: A paradigm shift for renal salt homeostasis

The focus of this presentation will be on emerging evidence for site-specific regulation of the epithelial sodium channel (ENaC) and its role in Na⁺ and K⁺ homeostasis [1]. In most textbooks ENaC function is discussed in the context of the cortical collecting duct (CCD). ENaC is localised in the apical membrane of CCD principal cells and is the rate-limiting step for Na⁺ absorption. It is inhibited by the K⁺ sparing diuretic amiloride and regulated by aldosterone. Aldosterone binds to a cytosolic mineralocortioid receptor (MR) and by complex transcriptional regulation and signaling pathways stimulates ENaC. This is evident in primary hyperaldosteronism or Conn syndrome. In simplified terms, inappropriately increased ENaC activity due to hyperaldosteronism results in enhanced NaCl absorption, expansion of extracellular fluid volume and hypertension. Conn syndrome is mimicked by gain-of-function mutations of ENaC causing Liddle syndrome, a rare genetic form of salt-sensitive hypertension. This highlights the importance of ENaC in Na⁺ homeostasis and long-term control of blood pressure. Hyperaldosteronism and Liddle syndrome are usually associated with hypokalemia due to a tight functional link between ENaC activity and renal K⁺ secretion via the renal outer medullary K⁺ channel ROMK. The classical concept that ENaC is strictly aldosterone-dependent was challenged by our patch-clamp studies exploring the site-specific regulation of ENaC in microdissected mouse tubules with a focus on two clearly distinguishable regions: The transition zone from the late convoluted tubule (CNT) to the CCD referred to as CNT/CCD, and the transition zone from the late distal convoluted tubule (DCT2) to the early connecting tubule referred to as DCT2/CNT. In an aldosterone synthase deficient mouse model we confirmed the aldosterone dependence of ENaC in CNT/CCD, but made the surprising observation that ENaC activity is aldosterone-independent in DCT2/CNT [2]. Using a mouse model with inducible nephron-specific MR deficiency, we demonstrated that aldosterone-dependent ENaC activity in CNT/CCD was completely MR dependent as expected. Interestingly, MR deficiency also largely reduced aldosterone-independent ENaC activity in DCT2/CNT [3], where MR can probably be stimulated by glucocorticoids due to low expression of 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2). Aldosterone-independent but MR-dependent ENaC activity in DCT2/CNT probably explains, why MR antagonists are efficient blood pressure lowering drugs even when aldosterone levels are not elevated. Moreover, in DCT2/CNT baseline apical ROMK activity is high and a low-K⁺ diet reduces ENaC activity, which highlights the important role of this nephron segment in regulating K⁺ secretion in an aldosterone-independent manner [4]. Future studies are needed to explore whether other hormones or local factors also regulate ENaC activity in a site-specific manner. In this context proteolytic channel activation, a unique feature of ENaC, may be of particular interest. To address this, the physiologically relevant endogenous proteases need to be elucidated. Recently, we identified TMPRSS2 as a candidate protease involved in proteolytic ENaC activation [5], but its role in the kidney remains to be determined. Finally, high resolution cryo-EM structures of ENaC are now available. These structural data open up exciting new horizons to study ENaC function and site-specific regulation at the molecular level.

Purinergic P2Y2 receptors are coupled to calcium-dependent mucus and fluid secretion in the human colonic stem cell niche

The human colonic stem cell niche provides a safe harbour for long-lived stem cells. Intestinal stem cells are responsible for driving tissue renewal and cellular differentiation that underpins intestinal barrier function. Neuronal and non-non-neuronal (i.e. crypt autonomous) neurotransmitters are emerging as key regulators of physiological function in the stem cell niche. Similarly, downstream calcium signals are emerging as central integrators of signals that regulate the physiology of the stem cell niche. The aim of this study was to determine the mechanism of purinergic coupled calcium signals in the human colonic stem cell niche and investigate their physiological consequences. Methods: Human colonic crypts were isolated from colorectal tissue samples obtained at surgical resection (NREC approval) and cultured as crypts in the short term (or propagated as crypt-like organoids over the long-term (5 years). The spatio-temporal characteristics of intracellular calcium was monitored by Fura-2/Fluo-4/Calbryte-630 imaging and the mechanism of receptor-mediated calcium mobilisation was characterised by pharmacological and knockdown gene approaches. Calcium signalling toolkit expression was investigated by bulk RNAseq and visualised by fluorescence immunolabelling and super-resolution imaging. Mucus secretion was visualised by Muc2 immunofluorescence depletion assays and real-time imaging of fluorescently tagged mucin-2, using MUC2::mNEON crypt-like organoids generated by CRISPR-HOT. An organoid swelling assay was used as a proxy for fluid secretion. Results: Bulk RNA seq demonstrated mRNA expression of metabotropic P2Y1 (P2RY1), P2Y2 (P2RY2), P2Y11 (P2RY11) and P2Y13 (P2RY13) in native human colonic mucosa, isolated crypts, and cultured organoids. A focus on P2Y2 immunolocalisation revealed expression on basolateral membranes lining the colonic crypt-axis. P2Y2 co-labelled with LGR5+ stem cells along their basolateral membranes at the crypt-base, in addition to labelling MUC2-positive goblet cells, and sparse numbers of chromogranin-positive enteroendocrine cells. Both ATP and UTP, a P2Y2-selective ligand, stimulated intracellular calcium levels in the absence of extracellular calcium, and was inhibited by the InsP3R receptor antagonists 2-APB (100 mM), but not by inhibitors of two pore channels, NED19 (250 μM or tetrandrine (20 μM). UTP induced a reduction of intracellular MUC2 immunofluorescence intensity in colonic crypt-bases and crypt-like organoids. Similarly, UTP stimulated a decrease in intracellular MUC2-mNEON fluorescence and a concomitant increase in luminal MUC2-mNEON fluorescence. UTP also promoted an increase in the cross-sectional area of closed organoids. UTP effects on intracellular calcium, intracellular and luminal MUC2 levels and organoid cross sectional area were abolished by the P2Y2 antagonist, AR-C118925XX (5 μM). These functional observations of fluid secretion are supported by basal expression of NKCC1 and apical expression of ANO1. Conclusions: Puringeric input stimulates P2Y2 receptor mediated calcium mobilisation from intracellular stores, mucus secretion and fluid secretion. These effects suggest that purinergic input regulates the microenvironment of the human colonic stem cell niche.

Multiplatform analysis of kidney tubule-specific mechanisms during low dietary potassium intake

Western diets are typically high in sodium, widely regarded to increase blood pressure (BP). They are also characterised by a relatively low potassium (K) intake, also linked to higher BP. Previous studies have shown that the ability of low dietary K to increase BP is almost fully accounted for by an increase in the abundance and activity of the thiazide-sensitive sodium-chloride transporter (NCC) in the kidney distal convoluted tubule (DCT). However, there is a gap in knowledge for how NCC is increased, but also in the mechanisms underlying an accompanying hypertrophy of the DCT. This study aims to increase our understanding of the specific effects of low K intake on the molecular landscape of the DCT.

Methods: Due to the relatively low abundance of DCT cells vs. whole kidney, many DCT-specific changes are undetectable when analysing whole kidney or cortex samples. To uncover these mechanisms, Parvalbumin-GFP+ mice (GFP expressed only in DCT) were fed control (1% K) or a low K (0.2% K, n=7) diet for 4 days. Kidneys were collected on day 5, a single cell suspension prepared and GFP^– and GFP⁺ (DCT) cells separated using FACS. Cells were prepared for both bulk RNAseq and protein mass spectrometry. In parallel, kidney samples were embedded in paraffin, tubules were isolated using a laser microdissection system (LMD) and a protocol optimised to analyse these samples with mass spectrometry.

Results: Compared to control diets, mice on 0.2% K intake had increased NCC (+44%) and phosphorylated (active) NCC (+51%), and decreased expression of α-ENaC (-20%) and cleaved γ-ENaC (-10%). Mice on 0.2% K also had higher BP by tail cuff plethysmography (SBP 121.4 ± 2.1 vs. 110.8 ± 1.1 mmHg), reduced urine volume and urinary K excretion (5.5-fold). Plasma K was slightly decreased compared to control diets (4.06 vs. 4.35mmol/l). FACS of living GFP⁺ cells was optimised to 95 ± 2% purity and enrichment of DCT cells confirmed at the protein and RNA level for various DCT specific genes of interest. Mass spectrometry returned 1426 identified proteins and 75 proteins with TTest<0.05 (Control vs. LK). LK diet increases NCC as expected. Kir5.1 and Parvalbumin also increase, while V-ATPase subunits C and E decrease. Other potentially interesting proteins are involved in zinc transport, glycolysis and tubular structure. More generally, results suggest a remodelling of the DCT with DCT1 expanding and DCT2/CNT shrinking.

LMD sample analysis is ongoing. LMD studies are addressing the specific proteomes of proximal tubule cells and collecting duct cells subsequent to 0.2% K intake.

Perspectives: Understanding the mechanisms of K handling and their effects on BP may offer new targets for prevention or treatment of hypertension, both through drug and dietary intervention. We have developed a multiplatform approach to study DCT cells by proteomics, transcriptomics and coupled it to functional studies. We have also developed a protocol for processing laser microdissection samples that allows for a good recovery of proteins from a low number of tubules.

Conserved hydrophobic interactions play a critical role in inhibiting the epithelial sodium channel (ENaC) by Î±- and Î³-inhibitory peptides

ENaC is a heterotrimeric ion channel typically formed by α-, β-, and γ-subunits [1, 2]. Channel activation requires proteolytic release of inhibitory tracts from the extracellular domains of α- and γ-ENaC [1-3]. In good agreement with this, applying synthetic inhibitory peptides corresponding to key inhibitory sequences within the α- or γ-inhibitory tracts was shown to reduce ENaC activity [4, 5]. Recently published structural data of ENaC (PDB-ID: 6WTH; [2]) provided information regarding the putative binding pockets of the inhibitory peptides. However, the functional importance of individual residues belonging to these binding pockets is still incompletely understood. We performed atomistic molecular dynamic (MD) simulations to predict critical interactions between the inhibitory peptides and their binding sites in α- and γ-ENaC. Computer predictions were verified by combining site-directed mutagenesis with two-electrode voltage clamp current measurements of human wild-type or mutant αβγ-ENaC heterologously expressed in Xenopus laevis oocytes. ENaC function was assessed by measuring amiloride-sensitive currents (ΔI_ami). Values are presented as mean ± SEM. One-way ANOVA with Bonferroni posthoc test was used for statistical analysis. MD simulations demonstrated that α- and γ-inhibitory peptides form stable interactions with a central hydrophobic patch in their respective binding site. Interestingly, these hydrophobic patches are formed by four amino acid residues, which are conserved in α- (α_F226, α_W251, α_H255, α_Y447) and γ-ENaC (γ_F204, γ_W229, γ_H233, γ_Y425). To assess the functional importance of these residues, we individually replaced them by an alanine and compared the effects of synthetic α or γ inhibitory peptides on channels with corresponding mutations in α or γ ENaC with those on wild-type ENaC. Except for α_F226Aβγ- and αβγ_Y425A-ENaC, which for unknown reasons could not be functionally expressed, all other ENaC mutants produced measurable ΔI_ami in oocytes. Compared to its inhibitory effect on wild-type ENaC (−76±2 %, n=41) the inhibitory effect of the synthetic α-peptide (30 µM) was strongly reduced in α_W251Aβγ-ENaC (−28±4 %, n=14, p<0.001) and to lesser extents in α_H255Aβγ-ENaC (−61±4 %, n=12, p<0.001) and α_Y447Aβγ-ENaC (−47±4 %, n=14, p<0.001). To investigate the effect of the γ-inhibitory peptide on ENaC, oocytes were pre-treated with chymotrypsin to remove the endogenous γ-inhibitory tract, which otherwise would have occupied its binding site in γ-ENaC. Compared to its inhibitory effect on wild-type ENaC (−58±2 %, n=44), the inhibitory effects of the synthetic γ-peptide (10 µM) on αβγ_F204A-ENaC (−15±1 %, n=13, p<0.001), αβγ_W229A– (−17±6 %, n=12, p<0.001) and αβγ_H233A-ENaC (−24±1 %, n=17, p<0.001) were significantly reduced. Consistent with these findings, we found that hydrophobic amino acid residues belonging to the endogenous γ-ENaC inhibitory tract are critical for its inhibitory effect on the channel. The leucine residue γ_L160, which is expected to interact with γ_W229, appeared to be particularly important. Its substitution by an alanine increased baseline currents by ~2-fold, mimicking the stimulatory effect of chymotrypsin on the channel. These findings highlight the functional importance of conserved hydrophobic interactions of synthetic inhibitory peptides or endogenous inhibitory tracts with their corresponding binding pockets in α- and γ-ENaC. This adds to our molecular understanding of proteolytic ENaC activation.

N-linked glycosylation contributes to the absence of protease-sensitivity in epithelial sodium channels containing the Î´-subunit

Introduction
There are four epithelial sodium channel (ENaC) subunits (α, β, γ, δ) which form heterotrimeric αβγ- or δβγâ€‘ENaCs that are vital in the salt and water homeostasis in vertebrates. As they mature through the secretory pathway, the ENaC subunits undergo N-linked glycosylation. αβγ-ENaC open probability is tightly coupled to the processing of its α- and γ-ENaC subunits by both intracellular and extracellular proteases. We have previously shown that guinea pig or Xenopus laevis αβγ-ENaC is potently activated by chymotrypsin, whereas δβγâ€‘ENaC is not (Gettings et al., 2021; Wichmann et al., 2018). Since ENaC activation by chymotrypsin requires cleavage of the γ-ENaC subunit, it is unknown how the presence of the δ-ENaC subunit prevents ENaC activation by the protease. Glycosylation of ENaC subunits has been shown to impact the channel’s activation by proteases (Kashlan et al., 2018). As such, we aimed to investigate whether glycans in the δ-ENaC subunit contribute to the observed differences in the activation of ENaC isoforms by chymotrypsin.

Methods
N-linked glycosylation sites were predicted on guinea pig δâ€‘ENaC using the NetNGlyc 1.0 server. With site-directed mutagenesis, the glycosylated asparagines (N) in the δâ€‘ENaC subunit extracellular domain were substituted with alanine (A). We heterologously expressed wild type αβγ-ENaC, wild type δβγ-ENaC or δβγâ€‘ENaC containing the N/A substituted δ-ENaC subunits in Xenopus laevis oocytes. ENaC activity was measured using the two-electrode voltage-clamp technique at – 60 mV.

Results
Extracellular chymotrypsin (2 µg/ml) significantly stimulated αβγ-ENaC currents by 2.8 ± 0.34-fold (n=11) in comparison with control experiments without chymotrypsin (0.82 ± 0.07-fold, n=12; p<0.0001, unpaired t-test). There was no difference in the fold-activation of δβγ-ENaC currents without (0.97 ± 0.02-fold, n=10) and with chymotrypsin (0.95 ± 0.03-fold, n=13, p=0.6, unpaired t-test). The NetNGlyc 1.0 server predicted three glycosylated asparagines in the extracellular loop of the guinea pig δâ€‘ENaC subunit: N129, N174 and N344. When these asparagines were substituted one at a time by alanine, none of the channels were activated by chymotrypsin (δ_N129Aβγ-ENaC:with chymotrypsin 0.92 ± 0.03-fold, n=12, without chymotrypsin 0.84 ± 0.06, n=8; p=0.2080, unpaired t-test; δ_N174Aβγ-ENaC: with chymotrypsin 0.97 ± 0.05-fold, n=9, without chymotrypsin 0.87 ± 0.04, n=8; p=0.1482, unpaired t-test; and δ_N344Aβγ-ENaC: with chymotrypsin 0.91 ± 0.04-fold, n=8, without chymotrypsin 0.89 ± 0.05, n=9; p=0.8438, unpaired t-test). When all three asparagines were substituted, extracellular chymotrypsin significantly stimulated the mutant δ_{N129,174,344A}βγ-ENaC by 1.72 ± 0.14-fold (n=15) in comparison with the control experiments without chymotrypsin (0.9 ± 0.03-fold, n=7; p=0.0007, unpaired t-test).

Conclusion
Taken together, these data indicate that removal of glycans in the δ-ENaC subunit renders guinea pig δβγâ€‘ENaC sensitive to activation by chymotrypsin. N-glycans in the δ-ENaC subunit might alter the accessibility of the γâ€‘ENaC subunit to proteases.

CFTR structure and function: insight from chimeric channels

A powerful approach to investigate ion channel structure-function relationships is to exploit functional differences between orthologues from divergent species. Here, this approach is used to study the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Mouse CFTR has 79% shared amino acid identity with human CFTR. Its function differs from human CFTR in several important respects: altered gating pattern distinguished by prolonged residence in a sub-conductance state, reduced single-channel conductance and strong inward rectification (1,2). Transfer of the regulatory (R) domain of mouse CFTR to human CFTR was without effect, whereas transfer of both nucleotide-binding domains (NBDs) endowed human CFTR with greatly prolonged channel openings without conferring human CFTR with the sub-conductance state, single-channel conductance and inward rectification of mouse CFTR (3). To test the idea that the membrane-spanning domains (MSDs) govern the conduction properties of human and murine CFTR, the human-murine CFTR (hmCFTR) chimeras hmTM1-6, hmTM7-12 and hmTM1-6:TM7-12 containing MSD1, MSD2 and MSD1+MSD2 of mouse CFTR on a human CFTR background were synthesized. To study the single-channel behaviour of these hmCFTR chimeras, a large Cl^– concentration gradient ([Cl^–]_int, 147 mM; [Cl^–]_ext, 10 mM) was imposed across inside-out membrane patches excised from transiently transfected CHO cells and voltage clamped at -50 mV, whereas to analyse macroscopic currents, membrane patches were bathed in symmetrical 147 mM Cl^– solutions and voltage clamped at 0 mV. All intracellular solutions contained protein kinase A (PKA; 75 nM) and ATP (1 mM) to activate and sustain CFTR channel activity; temperature was 37 °C. The three hmCFTR chimeras possessed a gating pattern intermediate between those of human and mouse CFTR. Like human CFTR, hmTM7-12 rarely transitioned to sub-conductance states, whereas hmTM1-6 sojourned to multiple sub-conductance states and hmTM1-6:TM7-12 resided in a tiny sub-conductance state resembling that of mouse CFTR. Consistent with hmTM1-6:TM7-12 possessing human, not mouse, NBDs, this hmCFTR chimera resided in the sub-conductance state for noticeably shorter periods than mouse CFTR. Upon quantification, both the single-channel conductance and open probability of the full open-state of hmCFTR chimeras decreased in the rank order: human CFTR > hmTM7-12 > hmTM1-6 ≥ hmTM1-6:TM7-12 (n = 5 – 10). Using 2-s voltage ramps from -100 to +100 mV to acquire current-voltage relationships, human CFTR Cl^– currents weakly inwardly rectified, whereas those of mouse CFTR exhibited strong inward rectification. Transfer of both MSDs of mouse CFTR to human CFTR in the chimera hmTM1-6:TM7-12 reproduced the strong inward rectification of mouse CFTR (n = 4). Interestingly, the inward rectification of hmTM1-6 closely resembled that of mouse CFTR, whereas that of hmTM7-12 was intermediate between human and mouse CFTR (n = 6 – 9). Taken together, these data demonstrate that sequences from both MSDs specify the gating, conduction and rectification properties of CFTR with sequences from MSD1 playing a dominant role in determining the different rectification behaviour of human and mouse CFTR.

Structural insights into the gating dynamics of bacterial mechanosensitive channels

Ion channels control solute flux by undergoing conformational changes which regulate their function in response to stimuli. Bacterial mechanosensitive channels sense changes in lateral tension generated in the membrane, acting as pressure-safety valves to prevent cell lysis (1). However, some of these channels also have unique features that suggest additional physiological roles. Bacterial mechanosensitive channels can be divided into two structural families: MscL and the MscS-like superfamily (1). The large-conductance (MscL) and small-conductance (MscS) mechanosensitive channels are well characterized structurally and functionally, but an understanding of the link between structure and function is missing. Lipid availability within pockets has been linked with mechanosensation in these channels (2), but the gating mechanisms and discrete subconducting states have not been fully defined. Studies on other members of the MscS-like family, such as YbiO, are limited. Several of these mechanosensitive channels have been implicated in bacterial pathogenicity and virulence, so understanding their mechanisms could provide avenues for the development of antimicrobials (3).

Previously, it was shown that lipid removal from transmembrane pockets through a point mutation (L89W) leads to the stabilization of an expanded subconducting state (2). However, whether there is a correlation between the tension-mediated state and the state derived by pocket delipidation in the absence of tension remained unknown. To investigate the physiological relevance of this subconducting state we probed solvent accessibility using a combination of electron paramagnetic resonance (EPR) spectroscopy and hydrogen-deuterium exchange (HDX) mass spectrometry (4). This was coupled with membrane stretching molecular dynamics (MD) which allowed the stabilization of a state under tension. This allowed us to investigate the structural changes associated with distinctively derived states. We found that pocket delipidation by either membrane tension or molecular modification causes MscL to occupy a similar structurally defined subconducting state (5). These findings revealed the mutation stabilized state is an intermediate in the channel activation pathway. They also provide support for models that implicate lipid availability in the regulation of channel gating in mechanosensitive channels. Ongoing structural work aims to investigate whether lipid availability within transmembrane pockets is also a regulatory mechanism of channel gating within larger members of the MscS-like superfamily.

Structural Insight to Human Urea Transporter Function and Implications for Medicines

Introduction: Urea is a key molecule in several physiological processes including urea cycle and urine volume regulation (1). These functions require urea transport across the plasma membrane, which in humans is facilitated by two urea transporters, UT-A (also called UT-2 or SLC14A2) and UT-B (UT-1, SLC14A1) (1). UT-A isoforms are expressed in various compartments of the loops of Henle to reabsorb urea from glomerular filtrate to tubular cells and eventually to blood (1), thereby regulating urine volume via the osmotic gradient. In contrast, UT-B is widely expressed in human tissues and so is involved in a wider range of physiological processes (2). Additionally, UT-B has a unique clinical implication as the primary antigen for the Kidd blood group system (3), where its Asp/Asn amino acid variation at residue 280 determines its Jk blood group phenotype. These make UT-A and UT-B important targets not only from drug discovery perspective but also for clinical applications.

Aims: This study aimed to determine high-resolution structures of human UT-A and UT-B to understand structural aspects of their function, and to enable the development of therapeutics targeting these urea transporters.

Methods: Human urea transporters were expressed in HEK293 cells and purified in detergents for structural studies. Atomic-resolution (2.3 – 2.9 Å) structures of apo UT-A and UT-B, and inhibitor-bound UT-B, were determined by X-ray crystallography and cryo-electron microscopy. Computational tools such as APBS electrostatics, ICM-Pro and MOLE were used to analyse the proteins’ surface charges, inhibitor binding, and channel pore radius, respectively.

Results: In this talk, I present insights into the molecular mechanisms of human UT-A and UT-B function from our high-resolution structures of the proteins in apo and inhibitor-bound states. These structures reveal UT-A’s extracellular surface to be more negatively charged than UT-B, and that its surface charge can significantly vary within the pH ranges of 5.5 and 7.5 observed in the tubular lumen. This suggests that the function of UT-A may be regulated by the pH of the convoluted tubules, in contrast to UT-B. The divergent surface properties between the two transporters also establish the basis for structure-guided drug discovery programs which target each protein individually. Lastly, the position of UT-B’s antigenic surface raises interesting questions about the epitope targeted by anti-Jk antibodies.

Conclusion: The urea transporter structures further our physiological understanding of urea transport and advance efforts for therapeutic development programs targeting them.

Using scRNAseq datasets to explore glucose transporters in airway epithelia

Introduction: The recent development of new technologies such as single cell RNA sequencing (scRNAseq) has enabled identification of the mRNA transcripts expressed by single cells of many tissues including the airway. We analysed recent scRNAseq studies to investigate the expression of facilitative glucose transporters (GLUTs) and sodium coupled glucose transporters (SGLTs) in the epithelial cells of the airway epithelium from trachea to alveolus.

Methods: We used six studies that reported single cell data for the human airway for interrogation based on the basis of the data availability, the lung cell types included and the range glucose transporters reported (even if not detected) (1-6). The number of cells in a cluster in which RNA transcripts were detected (frequency, normally expressed as %) and the number of RNA transcripts recorded per cell (transcripts per million) were obtained.

Results: Not all studies reported the full range of transporters. Glucose transporter mRNA transcripts were expressed at lower levels than other epithelial marker genes and there were differences between cells freshly isolated from the airways and those grown in vitro. Nevertheless, these studies highlighted that there were differences in cellular expression of GLUTs and SGLTs. GLUT1 was the most abundant of the broadly expressed transporters that included GLUT8, 10 and 13. GLUT9 transcripts were more common in basal cells and GLUT12 in ionocytes and ciliated cells. SGLT1 transcripts were present in alveolar and secretory cells. GLUT3 mRNA transcripts were expressed in a cell cluster that expressed monocarboxylate (MCT2) transporters.

Conclusions: The differences in cellular transporter expression potentially underlie cell specific metabolic requirements to support proliferation, ion transport, mucous secretion, environment sensing and airway glucose homeostasis. These studies also highlighted the role of glucose transporters in the movement of dehydroascorbic acid/vitamin C, myoinositol and urate which are factors important to the innate immune properties of the airways. Discrepancies in the reported detection of mRNAs, protein and function of glucose transporters in the lungs remains. Nevertheless, understanding which glucose transporters are present in the lung, their cellular location and how they change in respiratory disease will provide further insights into the function of airway cells and their contribution to cancer progression, the regulation of ASL glucose homeostasis and innate defence against infection.

Cryptic calcium signals for intestinal membrane transport â€“ utility of ex vivo 3D culture systems

The main functions of the human intestinal epithelium are to form a vital selective barrier between the systemic circulation and the luminal contents of the gut, whilst facilitating digestion and absorption of nutrients. Preservation of barrier function is underpinned by rapid stem cell-driven tissue renewal, and by secretion of a protective mucus layer. These physiological processes are regulated through the bilateral convergence of systemic and luminal signals which enable the gut epithelium to adapt to changes in the gut microenvironment. Not surprisingly, loss of gut epithelial tissue homeostasis precedes the onset of major intestinal conditions such as inflammatory bowel disease and cancer. Until relatively recently, investigation of the molecular and cellular processes that regulate and enact the physiological functions of the of the human gut epithelium in health and disease has been hampered by the lack of appropriate 3D ex vivo model systems.

The human colonic epithelial monolayer invaginates millions of times to form a carpet of crypts that line the gut lumen. At the base of each crypt, a population of 5-10 active LGR5-positive stem cells divides on a daily basis and gives rise to progenitor cells that migrate along the crypt-axis and differentiate into absorptive colonocytes or secretory goblet cells, enteroendocrine cells and tuft cells. Tissue renewal is completed by shedding of differentiated cells from the surface epithelium into the gut lumen. Significantly, the cellular hierarchy of tissue renewal aligns with the topology of the crypt-axis, as do the processes of fluid secretion and absorption.

Secretion of fluid from stem/progenitor cells located in the lower half of the crypt is coupled to the secretion of mucus from crypt-base-goblet-cells which combine to periodically flush the microenvironment of the stem cell niche and the macroscopic crypt lumen. Regulation and coordination of mucus and fluid secretion from collaborating goblet and stem/progenitor cells is achieved in part via the polarised expression of the associated molecular machinery across these gut epithelial cell types.

Recent advances in 3D ‘near-native’ and organoid model systems have made significant progress in recapitulating the topology and the polarity of the molecular machinery that orchestrates intestinal epithelial membrane transport along the crypt-axis. I will showcase the utility of cultured colonic crypts and organoids, in conjunction with fixed clinical specimens, for the study of calcium-dependent excitation-mucus/fluid secretion coupling in the human colon.

Identification of novel TMEM175 modulators using high throughput automated patch clamp and solid supported membrane- (SSM-) based electrophysiology platforms

TMEM175 is a novel, constitutively active ion channel involved in regulating lysosomal pH and autophagy. Mutations in this gene impair normal lysosomal and mitochondrial function, thereby increasing aggregation of insoluble proteins such as phosphorylated α-synuclein, leading to symptoms typical of Parkinson’s Disease (PD). Consequently, TMEM175 demonstrates significant potential as a key player in the treatment of PD. The lack of specific pharmacological tools has hampered further investigation into the exact role of TMEM175 in normal lysosomal function and pathological processes.

Advancements in high-throughput screening technologies have allowed rapid assessment of large numbers of compounds against ion channel drug targets using automated patch-clamp. We have successfully developed recombinant cell lines expressing wild-type (WT) TMEM175, a gain of function (Q65P), and loss of function (M393T) mutants using stably transfected HEK cells. Here, we report the characterization of the WT TMEM175 cell line performed using high-throughput automated patch-clamp electrophysiology and show reproducible concentration-response curves with the potassium channel inhibitor 4-aminopyridine (4-AP). A rapid and robust, automated high-throughput electrophysiology screening assay was subsequently developed to enable the identification of both activators and inhibitors of TMEM175.

Additionally, we have used solid-supported membrane- (SSM-) based electrophysiology (SSM-E) for TMEM175 recordings from lysosomal membranes purified and prepared from the WT TMEM175 cell line. Dose-dependent signal enhancement and inhibition of TMEM175 currents were successfully characterized. The SSM-E approach allows for stable and robust recordings from proteins residing in organellar membranes with a throughput of up to 10,000 data points per day.

In summary, the TMEM175 stable cell line was characterized using an automated patch-clamp and SSM-based electrophysiology. We developed and executed robust, high throughput, and high content direct electrophysiological intracellular screening assays, with unusually high success rates. The successful development of TMEM175 electrophysiology assays capable of identifying novel pharmacological tools will enable investigation of the role of this exciting target in normal physiology and in disease.

Flotillins, nicotinic acetylcholine receptors, endothelial NO synthase, and the P2X7 receptor interact to control interleukin-1Î² release

Objective: The pro-inflammatory cytokine interleukin (IL)-1β contributes to the pathogenesis of diverse inflammatory diseases. An example of high clinical relevance is life-threatening systemic inflammation caused by accidental trauma or major surgery that is inter alia induced by extracellular ATP originating from damaged cells. Extracellular ATP activates the P2X7 receptor (P2X7R) and thereby triggers the maturation and release of IL-1β. Therefore, mechanisms controlling ATP-mediated IL-1β release are of substantial clinical interest. We identified a cholinergic mechanism that inhibits the damage-mediated release of IL-1β by human monocytes via flux-independent signaling of nicotinic acetylcholine receptors (nAChRs) containing subunits α7, α9 and/or α10 [1]. Stimulation of nAChRs activates endothelial NO synthase and inhibits the P2X7R by cysteine nitrosylation. Here, we investigate if the lipid raft-associated flotillins are involved in this pathway.

Methods: Human monocytic THP-1 cells, THP-1 cell-derived macrophages, murine peripheral blood mononuclear cells (PBMCs), and murine bone marrow-derived macrophages (BMDMs) isolated from wild-type and flotillin-1 (Flot1) and/or -2 (Flot2) gene-deficient mice were primed with lipopolysaccharide. The ATP-induced release of IL-1β was studied in the presence and the absence of classical (e.g. acetylcholine) and unconventional (e.g. phosphocholine) nAChR agonists. To test for the involvement of nAChRs, the conopeptides [V11L;V16D]ArIB (specific for subunit α7) and RgIA4 (specific for subunits α9/α10) were used. Animals received humane care according to NIH “Guide for the Care and Use of Laboratory Animals”.

Results: Classical and unconventional nAChR agonists efficiently inhibited the ATP-mediated release of IL-1β by monocytic cells and exert similar functions in THP-1 cell-derived macrophages and BMDMs (n ≥ 5, P ≤ 0.05, Friedman followed by Wilcoxon signed-rank test). This inhibitory effect was reversed by the specific conopeptides indicating an involvement of nAChR subunits α7, α9 and/or α10 in mononuclear phagocytes. When investigating the ATP-induced IL-1β release by murine PBMCs and BMDMs, classical and unconventional nAChR agonists efficiently inhibited the ATP-induced IL-1β release by cells from wild-type mice (n ≥ 5, P ≤ 0.05, Friedman followed by Wilcoxon signed-rank test). This inhibitory effect was completely reversed in in mice with a double-deï¬ciency in Flot1 and Flot2 and partially reversed in cells from either Flot1 or Flot2 gene-deficient mice (n ≥ 5, P ≤ 0.05, Kruskal Wallis followed by Mann Whitney rank sum test).

Conclusion: We provide first evidence that the lipid raft-related proteins flotillin-1 and -2 are required for the cholinergic control of ATP-mediated IL-1β release by mononuclear phagocytes. Flotillins have been shown to be associated with several signal transduction pathways. Their exact physiological function is, however, at best partially understood. In the light of the high clinical relevance of the control of ATP-mediated IL-1β release, the exact molecular mechanisms of the interaction of nAChRs, the endothelial NO synthase, the P2X7R, and flotillins deserve further investigation.

Mechanisms of disturbed fluid and bicarbonate transport in the intestine in Cystic Fibrosis and In-flammatory Bowel Disease, and potential treatment options

Maldigestion and –absorption, constipation and intestinal obstructive episodes (DIOS) are major health problems in cystic fibrosis patients. The defective Cl^– channel results in reduced fluidity and low pH of the pancreatic ducts and the gut lumen, which leads to ductal obstruction, maldigestion, mucus hyperviscosity, dysbiosis, mucosal inflammation, and slow intestinal transit.

In a strategic research center funded by the Cystic Fibrosis Trust the ability of three FDA-approved drugs against constipation-prone irritable bowel syndrome, i.e. the guanylyl cyclase and cGMP-linked agonist Linaclotide, and the prostaglandin E1 analogue Lubiprostone, and the NHE3-selective inhibitor Tenapanor, was tested for their ability to restore luminal fluidity and pH in CF mouse models. In parallel, the effect of these compounds on the activity of the NHE3 (SLC9A3) Na/H exchanger, the major intestinal sodium absorptive transporter and regulator of fluid absorption, was measured in stem cell-derived human intestinal organoids generated from ileal biopsies donated by homozygous F508del CF patients. Tenapanor was chosen for an intervention trial in CFTR null mice. Following retraction of their daily osmotic laxative, mice were gavaged twice daily with 30µg tenapanor or vehicle for 21 days, and carefully monitored for the occurrence of an intestinal obstruction. After the end of the intervention, or if a suspicion of an intestinal obstruction arose, the mice were sacrificed, the intestine excised, the microbial composition of the luminal contents and the mucosa-adherent mucus layer was analysed, and the mucosa was studied histologically. The results suggested that an intestine-specific inhibition of NHE3 results not only in an increase in gut fluidity and pH, but also reduces mucus hypersecretion and prevents intestinal obstruction.

A hallmark of inflammatory bowel disease (IBD) is chronic or recurrent “inflammatory diarrhea”. Reduced epithelial Na⁺ and fluid absorption is a prominent feature. Fluid absorption in the ileocolonic region is predominantly mediated by the Na⁺/H⁺ exchanger NHE3, the H⁺-short chain fatty acid (SCFA) cotransporter MCT1, the Cl^–/HCO₃-exchanger SLC26A3 (DRA), and the epithelial Na⁺ channel (ENaC). We asked the question whether the downregulation of specific absorptive ion transporters or a general defect in enterocyte differentiation correlates with the degree of diarrhea and inflammation. mRNA expression and/or protein abundance was systematically studied of a panel of genes that determine or correlate with the differentiation state of the colonic epithelium, including the transport genes named above, in the different segments of the ileocolon of healthy individuals and IBD patients with mildly, moderate-severely, or noninflamed colonic mucosa. Clinical scores for disease activity and diarrhea were obtained. The results from a comparison of the IBD and healthy control biopsies suggested that inflammation results in a dysregulation of differentiation, with an aberrant proliferative response, an elongation of the proliferative zone, and an incomplete differentiation of the surface enterocytes. This results in a dramatically reduced expression of the colonic absorptive transporters and an increased expression of the poreforming “leaky” claudin2, resulting in diarrhea. This dysregulation is reversible, and noninflamed IBD colon has the same differentiation pattern as that of healthy controls. Thus, anti-inflammatory treatment appears as the only option to improve inflammatory diarrhea.

Impaired Renal Bicarbonate Excretion in Cystic Fibrosis: Its Physiology and Clinical Implications

Intro: Cystic fibrosis (CF) is a lethal inherited multi-organ disease caused by loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Respiratory insufficiency is the primary cause of mortality in CF, but additional complications such as alkalosis and a Bartter-like phenotype have been observed, potentially contributing to end-stage hypercapnic respiratory failure. The mechanism behind the development of alkalosis in CF has been unclear.

Methods: This study utilized various molecular techniques (immunoblotting, immunostainings, in vitro cell studies, etc.), ex vivo renal tubule perfusion, in vivo whole animal studies, and clinical investigations involving individuals with CF and healthy controls.

Results: The function of renal β-intercalated cells, which are responsible for physiological regulation of renal HCO₃^– excretion, is reliant on CFTR. Loss of CFTR function leads to dysfunctional pendrin regulation and impaired renal HCO₃^– excretion, resulting in exacerbated metabolic alkalosis and hypoventilation during acute base loading. Individuals with CF exhibit a significantly reduced ability to excrete an oral HCO₃^– load. The level of challenged urine HCO₃^– excretion correlates with important disease features such as lung function, pancreatic insufficiency, mutation type, and risk of persistent pulmonary infections. Treatment with the CFTR modulator drug Elexacaftor/Tezacaftor/Ivacaftor significantly improves urine HCO₃^– excretion. Furthermore, CFTR modulator therapy may restore pendrin function, reducing the risk of electrolyte disorders and metabolic alkalosis. This is highlighted by the correction of the salt-wasting phenotype in persons with CF following CFTR modulator treatment.

Conclusions: This research provides insights into the role of CFTR in renal HCO₃^– excretion and the development of alkalosis in CF. Mechanistically, Loss of CFTR causes dysfunctional pendrin regulation and CFTR is necessary for alkalosis-induced pendrin activation. Pendrin is the HCO₃^–-secretory pathway in the distal renal tubular system and is furthermore important for renal fluid and NaCl conservation. CFTR modulator therapy shows promise in improving urine HCO₃^– excretion and correcting electrolyte imbalances. Acute renal HCO₃^– excretion may serve as a useful measure of CFTR function and as a prognostic marker in persons with CF. The precise mechanism by which CFTR regulates pendrin function remains unresolved.

The Effect of Microgravity and Galactic Cosmic Radiation on Renal Structure and Function

Background: Missions into Deep Space (DS) are planned for the next two decades; a permanent orbital lunar station by end of the 2020s and first manned trip to Mars by late 2030s. The biology of kidney function in spaceflight has been largely overlooked. We suspect that kidney physiology may be disturbed in spaceflight, either by microgravity (MG) and/or Galactic Cosmic Radiation (GCR) potentially leading to kidney injury and stone formation.

MG exposed astronauts have unusually high rates of kidney stones. Indeed, MG associated changes in urinary biochemistry favour stone formation, and current thinking suggests this is secondary to bone demineralisation due to unloading. However, the compartmental fluid shifts associated with MG cause changes in differential perfusion pressures in tissue beds and changes in baroreceptor activation. We predicted this would cause physiological changes in renal sodium and calcium transporter expression (which tend to be physiologically linked), resulting in elevated urinary calcium loss, kidney stone formation and decreased bone mineral density as kidney-driven primary events.

The kidney is an exquisitely radiation sensitive organ; it is the dose limiting organ in abdominal radiotherapy. Chronic kidney dysfunction can occur with acute low linear energy transfer (LET) radiation doses as low as <0.5 Gy; the LET dose expected on a Mars Mission. However, GCR is comprised of low-LET x- and γ-rays, protons and high energy ions of heavier elements 'HZE' (e.g. iron). We hypothesise that GCR may cause renal damage within the timeframe and dose expected for a mission to Mars.

Methods: We performed biomolecular (Epigenomic, bulk and spatial mRNA and miRNA transcriptomic, proteomic, phosphoproteomic, metabolomic, metagenomic), clinical chemistry (electrolytes, endocrine and biochemistry biomarkers) and imaging (histology, immunofluorescence, miRNA FISH, 3D morphometry) analyses using samples and datasets available from 11 spaceflight-exposed mouse (13-75 days on ISS/shuttle) and 5 human (3-180 days on ISS/Shuttle) collections of missions, and 4 simulated GCR-exposed mouse experiments (0.5-0.75Gy ≈ 1.5-2.5-year dose / necropsy 1-180 days post-exposure). In total the study encompassed approximately ~100 humans / ~155 mice (n per group). All animal and human studies had local ethical committee approval and licenses where appropriate.

Results: Historical human data showed hypercalciuria, hyperphosphaturia and reduced urine volume during spaceflight. Multiomic pathway enrichment analysis indicated changes in kidney gene products associated with nephrolithiasis. MG-exposed animals had significant dephosphorylation of thiazide-sensitive SLC12A3 and furosemide-sensitive SLC12A1 canonical transporters known to impact nephrolithiasis and bone mineral density, as well as dysbiosis of faecal microbial genera known to influence nephrolithiasis.

Pathway analysis of human and mouse plasma and kidney samples showed enrichment of gene products associated with renal fibrosis, inflammation, glomerular and tubular injury (Fig.1). GCR-exposed animals had decreased mitochondrial protein expression, and elevation in pathogenic miRNA species. Spatial transcriptomics revealed immune cell infiltration of the cortex, biochemistry revealed elevated urinary protein loss and histopathological investigation revealed incidences of thrombotic microangiopathy.

Conclusion: Our data provide strong evidence that prolonged exposure to DS will induce significant harm to renal health and function that may prove mission critical. Development of mitigation strategies will be essential for long-term missions to the moon, mars and beyond.

Registration opens	03 April 2023
Abstract submission opens	03 April 2023
Abstract submission closes	30 April 2023
Abstract decisions issued	25 May 2023
Conference Attendance Award deadline	31 May 2023
Early bird registration deadline	06 July 2023
Registration deadline	10 August 2023
Meeting dates	24 – 25 August 2023

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Thursday 24 August - Friday 25 August 2023

Membrane Transport 2023: Recent Research into Ion Channels, Transporters and Epithelial Physiology

Date

Location

09:30 SA01 The role of TRPV4 in electrolyte and fluid secretion by choroid plexus epithelia: Implications for hydrocephalus

10:15 SA02 Nutraceutical targeting of Farnesoid X Receptor for regulation of CFTR expression in the colonic epithelium

10:30 C01 Succinate enhances mucociliary clearance in the murine tracheal epithelium by triggering acetylcholine release from chemosensory cells

11:15 SA03 Aldosterone‐independent ENaC activity: A paradigm shift for renal salt homeostasis

12:00 C02 Purinergic P2Y2 receptors are coupled to calcium-dependent mucus and fluid secretion in the human colonic stem cell niche

12:15 C03 Multiplatform analysis of kidney tubule-specific mechanisms during low dietary potassium intake

13:30 SA04 Structural insights into the trimeric assembly of epithelial sodium channels

14:15 C04 Conserved hydrophobic interactions play a critical role in inhibiting the epithelial sodium channel (ENaC) by α- and γ-inhibitory peptides

14:30 C05 N-linked glycosylation contributes to the absence of protease-sensitivity in epithelial sodium channels containing the δ-subunit

15:15 SA05 CFTR structure and function: Insight from chimeric channels

16:00 SA06 Structural insights into the dynamics of bacterial mechanosensitive channels

16:15 C06 Structural insight to human urea transporter function and implications for medicines

09:30 SA07 Ion channel engineering

10:00 SA08 Using scRNAseq datasets to explore glucose transporters in airway epithelia

11:00 SA09 Metabolic communication in SGLT2 inhibition

11:30 SA10 Cryptic calcium signals for intestinal membrane transport: Utility of ex vivo 3D culture systems

12:00 SA11 Identification of novel TMEM175 modulators using high throughput automated patch clamp and solid supported membrane- (SSM-) based electrophysiology platforms

13:30 SA12 The role of a newly identified Ca2+-permeable channel in cardiac dysfunction

14:15 SA13 Circadian regulation and dysregulation of the thiazide-sensitive sodium chloride co-transporter (NCC)

14:30 C07 Flotillins, nicotinic acetylcholine receptors, endothelial NO synthase, and the P2X7 receptor interact to control interleukin-1b release

15:15 SA14 Mechanisms of disturbed fluid and bicarbonate transport in the intestine in Cystic Fibrosis and Inflammatory Bowel Disease, and potential treatment options

16:00 SA15 Impaired renal bicarbonate excretion in Cystic Fibrosis: Its physiology and clinical implications

16:15 C08 The effect of microgravity and galactic cosmic radiation on renal structure and function

17:00 SA16 The kidneys’ inner workings and needs: Lessons from inhibiting a glucose transporter

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