Epithelial Transport Research Articles

A Review of Epithelial Ion Transporters and Their Roles in Equine Infectious Colitis.

Equine colitis is a devastating disease with a high mortality rate. Infectious pathogens associated with colitis in the adult horse include Clostridioides difficile, Clostridium perfringens, Salmonella spp., Neorickettsia risticii/findlaynesis, and equine coronavirus. Antimicrobial-associated colitis can be associated with the presence of infectious pathogens. Colitis can also be due to non-infectious causes, including non-steroidal anti-inflammatory drug administration, sand ingestion, and infiltrative bowel disease. Current treatments focus on symptomatic treatment (restoring fluid and electrolyte balance, preventing laminitis and sepsis). Intestinal epithelial ion channels are key regulators of electrolyte (especially sodium and chloride) and water movement into the lumen. Dysfunctional ion channels play a key role in the development of diarrhea. Infectious pathogens, including Salmonella spp. and C. difficile, have been shown to regulate ion channels in a variety of ways. In other species, there has been an increased interest in ion channel manipulation as an anti-diarrheal treatment. While targeting ion channels also represents a promising way to manage diarrhea associated with equine colitis, ion channels have not been well studied in the equine colon. This review provides an overview of what is known about colonic ion channels and their known or putative role in specific types of equine colitis due to various pathogens.

Generation of heterozygous KCNA2 knockout induced pluripotent stem cell line

The KCNA2 gene is the voltage-gated ion channel from both functional and structural perspectives. KCNA2 is involved in diverse functions including the regulation of neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction, and cell volume. To investigate the relevant pathophysiological mechanisms, we generated heterozygous KCNA2 knockout human induced pluripotent stem cell (iPSC) line via CRISPR/Cas9 gene editing. The generated iPSCs had a normal karyotype, were free of genetically integrated epitaxial plasmids, expressed pluripotency markers, and maintained trilineage differentiation potential.

Bee pollen from bracatinga (Mimosa scabrella): Effects of gastrointestinal digestion and epithelial transport in vitro on phenolic profile and bioactivities

The main objective of the present work was to assess the phenolic profile of bracatinga (Mimosa scabrella) bee pollen, and its antioxidant and anti-inflammatory activities after gastrointestinal digestion in vitro and epithelial transport in a Caco-2 cell monolayer model. The botanical origin of bee pollen was confirmed by optical microscopy and scanning electron microscopy. As major results, 34 phenolic compounds (13 phenylamides, 14 flavonols, and 7 flavanones) were tentatively identified in the extract of bracatinga bee pollen by HPLC-ESI-QTOF-MS. The aglycone forms of quercetin and p-coumaric acid were identified only after digestion, indicating the breakage of flavonols and phenylamides, respectively. These compounds may have contributed to the decrease in NF-κΒ activation up to 54% and in the release of TNF-α and CXCL2/MIP-2 by 26% and 21%, respectively, in raw 264.7 murine macrophages activated with microbial lipopolysaccharide and treated with the digested fraction. Among all tentatively identified phenolic compounds, five of them were found in the basolateral fraction. These compounds, represented by four aglycone flavonoids (quercetin, kaempferol, naringenin, and herbacetin methyl ether) and a phenolic acid (p-coumaric acid) may be responsible for its outstanding antioxidant activity in Caco-2 cells, as well as for its remaining capacity in mitigating CXCL2/MIP-2 release after transport through the Caco-2 cell monolayer, as an intestinal barrier model. Therefore, our work sheds light on the phenolic profile and bioactivities of an interesting functional food produced by bees throughout a simulated gastrointestinal system.

Oxaluria in inflammatory bowel diseases (review)

AIM: to analyse and synthesize Russian and foreign literature, to get acquainted with the concept of oxaluria, its types, transport mechanisms of oxalate transport in the intestine and the relationship between hyperoxaluria and inflammatory bowel diseases in order to identify possible options for therapeutic action on the mechanisms of development of these pathologies. MATERIALS AND METHODS: the literature review was based on the Internet data, including bibliographic directories, books, journals, and original articles. The literature sources used for the article reflect the essence of the described problem to the fullest extent possible and can be useful for both practicing physicians and students of medical universities. RESULTS: the gastrointestinal tract through epithelial transport of oxalate plays an exclusive role in oxalate homeostasis and hyperoxaluria. Metabolism of dietary oxalate and the formation of endogenous oxalate, its secretion, absorption, transport and biodegradation by intestinal microflora may influence the excretion of this compound by the kidneys. Knowledge of the interrelated relationships of the gut-kidney axis, mechanisms of transport, transport and biodegradation of oxalate, especially in inflammatory bowel disease, is of great importance for understanding the pathophysiology of hyperoxaluria as a risk factor for urinary stone formation with a point of pharmacological action in the gut. This literature review introduces the concept and forms of oxaluria, shows the classification of oxaluria, describes each form, and broadly explains the metabolism and mechanisms of oxalate transport in the human body. Special attention is given to intestinal hyperoxaluria and anion exchangers belonging to the large multifunctional SLC26 gene family, most of which are expressed throughout the gastrointestinal tract. The authors emphasise their current role in intestinal oxalate transport, as well as methods of possible drug action on the mechanisms of hyperoxaluria. CONCLUSION: a multidisciplinary approach is needed to address the problems of intestinal hyperoxaluria and, consequently, the treatment of urolithiasis. The role of newly identified intestinal and renal anion exchangers is not fully understood, hence the targets and mechanisms of action on these types of exchangers with the possibility of preventing the development of urolithiasis are not fully understood. Further randomised studies on the problem under investigation are needed.

Tea Polyphenols Inhibit Methanogenesis and Improve Rumen Epithelial Transport in Dairy Cows.

This study systematically investigated the effects of tea polyphenols on methane (CH4) production and the rumen epithelial cell transport capability in cattle using both in vitro and animal experiments, employing multi-omics techniques. The in vitro results demonstrated that, compared to the control group, tea polyphenols significantly reduced CH4 production and the acetate/propionate ratio (p < 0.05). Tea polyphenols reduced CH4 production by inhibiting the relative abundance of unclassified_d_Archaea methanogens and the protozoa Pseudoentodinium and g__Balantioides. The animal experiments showed that tea polyphenols significantly increased the concentrations of T-AOC and GSH-PX in bovine blood (p < 0.05). In addition, microbial groups such as Rikenellaceae_RC9_gut_group, Ruminococcaceae_NK4A214_group, and Butyrivibrio_2 were significantly enriched in the ruminal fluid of the tea polyphenol group (p < 0.05). The proteomic results indicated significant upregulation of proteins such as COIII, S100A8, FABP1, SLC2A8, and SLC29A1 (p < 0.05) and downregulation of proteins including HBB, RAB4A, RBP4, LOC107131172, HBA, and ZFYVE19 (p < 0.05), with FABP1 showing a positive correlation with propionate concentration, and RAB4A had a negative correlation (p < 0.05). Overall, tea polyphenols modulate the microbial composition within the rumen, inhibiting CH4 production and enhancing the host's rumen epithelial cell transport capacity for volatile fatty acids.

With No Lysine (K) Kinases and Sodium Transporter Function in Solute Exchange with Implications for BP Regulation as Elucidated through Drosophila.

Like other multicellular organisms, the fruit fly Drosophila melanogaster must maintain homeostasis of the internal milieu, including the maintenance of constant ion and water concentrations. In mammals, the with no lysine (K) (WNK)-Ste20-proline/alanine rich kinase/oxidative stress response 1 kinase cascade is an important regulator of epithelial ion transport in the kidney. This pathway regulates SLC12 family cotransporters, including sodium-potassium-2-chloride, sodium chloride, and potassium chloride cotransporters. The WNK-Ste20-proline/alanine rich kinase/oxidative stress response 1 kinase cascade also regulates epithelial ion transport via regulation of the Drosophila sodium-potassium-2-chloride cotransporter in the Malpighian tubule, the renal epithelium of the fly. Studies in Drosophila have contributed to the understanding of multiple regulators of WNK pathway signaling, including intracellular chloride and potassium, the scaffold protein Mo25, hypertonic stress, hydrostatic pressure, and macromolecular crowding. These will be discussed together, with implications for mammalian kidney function and BP control.

Molecular basis of inhibition of the amino acid transporter B0AT1 (SLC6A19)

The epithelial neutral amino acid transporter B0AT1 (SLC6A19) is the major transporter for the absorption of neutral amino acids in the intestine and their reabsorption in the kidney. Mouse models have demonstrated that lack of B0AT1 can normalize elevated plasma amino acids in rare disorders of amino acid metabolism such as phenylketonuria and urea-cycle disorders, implying a pharmacological approach for their treatment. Here we employ a medicinal chemistry approach to generate B0AT1 inhibitors with IC50-values of 31-90 nM. High-resolution cryo-EM structures of B0AT1 in the presence of two compounds from this series identified an allosteric binding site in the vestibule of the transporter. Mechanistically, binding of these inhibitors prevents a movement of TM1 and TM6 that is required for the transporter to make a conformational change from an outward open state to the occluded state.

Phytase super-dosing modulates bone parameters and the concentration of the calcium epithelial carrier calbindin-D28k in quails (Coturnix coturnix japonica) under thermal stress

Context Thermal stress causes significant changes in the bioavailability of minerals, especially calcium, in laying birds, resulting in reduced egg production, and compromising egg quality. Therefore, it is necessary to use additives that provide better nutrient availability and minimise the negative effects caused by high temperatures. Aims This study aimed to evaluate the effects of super-dosing the diet of laying quails submitted to different temperatures with phytase on their bone parameters and on the epithelial calcium transporter calbindin-D28K positivity. Methods Seven hundred and twenty (720) Japanese quails in the production phase were distributed in a completely randomised design, in a 5 × 3 factorial design, with five supplementation levels of phytase (0, 500, 1000, 1500, and 3000 FTU/kg), and three temperatures (24°C, 30°C and 36°C), totalling 15 treatments, and six replications of eight birds each. The study began at the 8th week of age and continued for two cycles of 21 days, totalling 42 days of experiment. Bone parameters of the tibias and calbindin-D28K positivity were measured by immunohistochemistry. The data were submitted to variance, Tukey, and regression analyses. Key results At 24°C, birds had higher averages for proximal, medial, and distal diaphyses than did birds at 30°C and 36°C, and a greater breaking strength of the tibias than did those at 36°C. There was an increasing linear effect on the medial diaphysis and Seedor index in the tibias with an increasing phytase supplementation level. Calcium (Ca) in the tibias was higher in birds kept at 30°C at a supplementation level of up to 1000 FTU of phytase, than in the birds at 24°C and 36°C, equalling at 1500 FTU, and lower at 3000 FTU. There was a quadratic effect for phosphorus (P) and the Ca:P ratio in the tibias of quails submitted to increasing concentrations of phytase. Quails kept at 30°C showed greater anti-calbindin-D28K positivity in the duodenum and jejunum, when compared to animals subjected to temperatures of 24°C and 36°C. Phytase doses of 500 and 1000 FTU increased calbindin-D28K positivity in the duodenum of quails kept at 30°C. At a supplementation level of 1500 FTU, phytase provided greater eggshell thickness and greater calbindin-D28K positivity in the uterus of quails kept at 36°C. Conclusions Therefore, it is recommended to use 1500 FTU of phytase for Japanese quail exposed to high temperatures. Implications These findings suggest that dietary phytase super-dosing can help alleviate thermal stress in Japanese quail.

Cryo-EM Structures of Clostridium perfringens Enterotoxin Bound to its Human Receptor, Claudin-4.

Pathogenic strains of Clostridium perfringens secrete an enterotoxin (CpE) that causes prevalent, severe, and sometimes deadly gastrointestinal disorders in humans and domesticated animals. CpE binds selectively to membrane protein receptors called claudins on the apical surfaces of small intestinal epithelium. Claudins normally construct tight junctions that regulate epithelial paracellular transport but are hijacked from doing so by CpE and are instead led to form claudin/CpE small complexes. Small complexes are building blocks for assembling oligomeric β-barrel pores that penetrate the plasma membrane and induce gut cytotoxicity. Here we present structures of CpE in complexes with its native claudin receptor in humans, claudin-4, at 4.0 and 2.8 Å using cryogenic electron microscopy. The structures reveal the overall architecture of the small complex, that the small complex can be kinetically trapped, and resolve its key features; like the residues used in claudin/CpE complex binding, the orientation of CpE relative to the membrane, and CpE-induced structural changes to claudin-4. Further, the structures allude to the biophysical procession from small complex to cytotoxic β-barrel pore used by CpE during pathogenesis and the role of trypsin in this process. In full, this work elucidates the structure and mechanism of claudin-bound CpE pore assembly and provides strategies to obstruct its formation to treat CpE-induced gastrointestinal diseases.

Nonspecific lipid-transfer proteins trigger TLR2 and NOD2 signaling and undergo ligand-dependent endocytosis in epithelial cells

BackgroundAllergens can cross the epithelial barrier to enter the body but how this cellular passage affects protein structures and the downstream interactions with the immune system are still open questions. ObjectiveWe show the molecular details and the effects of three non-specific lipid transfer proteins (nsLTPs; Mal d 3, Cor a 8 and Pru p 3) upon epithelial cell uptake and transport. MethodsWe used fluorescent imaging, flow cytometry, proteomic and lipidomic screenings to identify the mechanism involved in nsLTP cellular uptake and signaling on selected epithelial and transgenic cell lines. ResultsNsLTPs are transported across the epithelium without affecting cell membrane stability or viability and allergen uptake was largely impaired by inhibition of clathrin-mediated endocytosis (CME). Analysis of the lipidome associated with nsLTPs showed a wide variety of lipid ligands predicted to bind inside the allergen hydrophobic cavity. Importantly, the internalization of nsLTPs was contingent upon these ligands in the protein complex.nsLTPs were found to initiate cellular signaling via TLR2 but not the CD1d receptor, despite neither being essential for nsLTP endocytosis. We also provide evidence that the three allergens induced intracellular stress signaling through activation of the NOD2 pathway. ConclusionsOur work consolidates the current model on nsLTP-epithelial cell interplay and adds molecular details about cell transport and signaling. Additionally, we have developed a versatile toolbox to extend these investigations to other allergens and cell types.

Sodium-Glucose Cotransporter-2 (SGLT-2) Inhibitors: Antidiabetics Medications for Treating Diabetes

Type 2 diabetes (T2D) is a progressive disease, and most patients need to treat with monotherapy and combinations of oral hypoglycaemic drugs. At present about 500 million people worldwide are suffering from T2D. Sodium-glucose cotransporter-2 (SGLT-2) inhibitors are a novel antidiabetic drug class that mediates epithelial glucose transport at the renal proximal tubules, inhibiting glucose absorption that result in glycosuria, modest weight loss and improve glycaemic control with a low risk of hypoglycaemia. These are used by T2D patients together with diet and exercise, either alone or in combination with other diabetes medicines. The main side-effects of SGLT-2 inhibitors are urinary tract and genital infections, as well as euglycemic diabetic ketoacidosis. Some other probable adverse effects are lower limb amputations, risk of bone fractures, Fournier gangrene, male bladder cancer, female breast cancer, orthostatic hypotension, and acute kidney injury. This mini review explains aspects of SGLT-2 inhibitors with treatment procedures and side-effects.

Effect of bile salts on intestinal epithelial function in gilthead seabream (Sparus aurata)

In the context of modern aquaculture, the effort to reduce the reliance on fishmeal/marine ingredients in fish diets has led to the exploration of plant-based protein sources as potential substitutes, a dietary shift that disrupts the bile acid profile in fish. Therefore, bile salts are being sought as additives. However, artificially increased intestinal levels of bile acids may significantly impact mucosal function. Therefore, here, we explored the regulatory role in the intestine of gilthead sea bream (Sparus aurata) of (i) chenodeoxycholic acid (CDC), (ii) a mixture formed by two bile acids, 3% cholic acid and 97% deoxycholic acid (MIX), and (iii) a conjugated bile salt sodium taurocholate (TC) in Ussing chambers with the epithelial voltage clamp technique. We tested the bile salts in a 50–500 μg/ml concentration range, and all of them promoted ion absorption. Yet, clear concentration-dependent and more pronounced effects on the ion transport were observed in the posterior intestine. On the other hand, bile salts had no or minor effects on tissue resistance. However, there are indications that the MIX could have adverse effects at high concentrations (500 μg/ml), promoting a threefold increase in tissue permeability measured using FITC-dextran (4 kD) regardless of the intestinal region, thus suggesting an alteration in intestinal permeability at high bile salt concentrations. The findings from our study emphasize the importance of considering intestinal function when contemplating the possible use of a particular bile salt as a dietary supplement. It appears that bile salts, whether acting individually or in combination, play a pivotal role in orchestrating nutrient absorption by influencing the function of epithelial ion transport. However further research is needed to fully grasp the region-dependent nuances of bile salt effects on ion transport and the ultimate consequences for nutrient absorption in the context of fish aquaculture.

Effects of sodium chloride intake on urea-N recycling and renal urea-N kinetics in lactating Holstein cows

The effects of high (2.5% of DM) versus normal dietary sodium chloride (NaCl) intake on renal urea-N kinetics and urea-N metabolism were investigated in 9 rumen-cannulated and multi-catheterized lactating dairy cows in a crossover design with 21-d periods. It was hypothesized that urinary urea-N excretion would be greater, and blood urea-N concentration lower in response to greater diuresis induced by high NaCl intake. Also, urea-N transport across ruminal and portal drained viscera (PDV) tissues was hypothesized to be affected by dietary sodium intake. A second experiment was conducted using 8 lactating cows in a crossover design with 14-d periods to test high NaCl (2.5% of DM) versus high KCl (3.2% of DM) intake on milk yield and milk urea-N concentrations. Experiment 1 showed that despite greater diuresis there was no effect of high NaCl intake on urinary urea-N excretion or blood urea-N concentration. The high NaCl intake did not affect rumen ammonia concentrations, total rumen VFA concentrations, ruminal venous - arterial concentration differences for ammonia, or ammonia absorption indicating that high NaCl did not adversely affect ruminal fermentation and microbial protein synthesis. High NaCl intake did not affect the total amount of urea-N transport from blood to gut, but ruminal venous - arterial concentration differences for urea-N were lower with high NaCl and ruminal extraction of arterial urea-N was numerically smaller, indicating that the ruminal epithelial urea-N transport was lower with high NaCl. Energy corrected milk yield was greater with high NaCl (3.2 ± 1.5 kg/d); however, milk urea-N concentrations were not affected by treatment. In experiment 2, ECM was greater with NaCl (1.4 ± 0.31 kg/d) compared with KCl (30.2 and 28.8 ± 0.91 kg ECM / d, respectively). Milk urea-N concentration was lower with KCl, suggesting a urea-N lowering effect in milk not evident with high NaCl intake. In conclusion, the present data show that dietary Na intake of 12-13 g/kg DM was followed by greater diuresis but did not impact urea-N excretion or blood urea-N concentration. High NaCl intake did not affect the total amount of urea-N transfer across PDV tissues. Energy corrected milk yield was greater with high NaCl compared with both control and feeding KCl, however, with KCl milk urea-N decreased.

In vitro digestion and culture in Caco‐2 cells to assess the bioavailability of fatty acids: A case study in meat matrix enriched with ω‐3 microcapsules

AbstractThis work aimed to evaluate the Caco‐2 cells as a model to study the epithelial transport of intestinal lipid extracts subjected to in vitro digestion, to establish a standard protocol for the determination of bioaccessibility and bioavailability of fatty acids in meat matrix, especially in those enriched with ω‐3 (eicosapentaenoic (EPA) and docosahexaenoic acids (DHA)). Samples were first subjected to in vitro digestion, and then, the intestinal extract was incubated with Caco‐2 cells. A first trial was conducted to select the most influencing variables on the fatty acid transport during Caco‐2 cell incubation: fat quantity on the intestinal extract, incubation time, and shaking. Then, a second experiment was carried out to determine the influence of these variables, being the fat quantity and the incubation time the most influencing factors on the transport and bioavailability of fatty acids. The effect of shaking was not so marked but seemed to improve the bioavailability of saturated fatty acids. This study also allows establishing the most suitable conditions: intestinal extracts with 30 mg of fat, longer incubation times (8 h), and shaking, achieving active and passive fatty acid transport without compromising the integrity of the Caco‐2 cell monolayer. The accurate results obtained for major and minor fatty acids, especially EPA and DHA are remarkable, due to the interest in these bioactive compounds. Thus, this study provides a combined protocol based on static in vitro digestion followed by Caco‐2 cell incubation to assess the bioaccessibility and bioavailability of fatty acids in meat samples.

Intestinal Epithelial Creatine Transporter SLC6A8 Dysregulation in Inflammation and in Response to Adherent Invasive E. coli Infection.

Creatine transporter (CrT1) mediates cellular uptake of creatine (Cr), a nutrient pivotal in maintaining energy homeostasis in various tissues including intestinal epithelial cells (IECs). The impact of CrT1 deficiency on the pathogenesis of various psychiatric and neurological disorders has been extensively investigated. However, there are no studies on its regulation in IECs in health and disease. Current studies have determined differential expression of CrT1 along the length of the mammalian intestine and its dysregulation in inflammatory bowel disease (IBD)-associated inflammation and Adherent Invasive E. coli (AIEC) infection. CrT1 mRNA and protein levels in normal intestines and their alterations in inflammation and following AIEC infection were determined in vitro in model IECs (Caco-2/IEC-6) and in vivo in SAMP1/YitFc mice, a model of spontaneous ileitis resembling human IBD. CrT1 is differentially expressed in different regions of mammalian intestines with its highest expression in jejunum. In vitro, CrT1 function (Na+-dependent 14C-Cr uptake), expression and promoter activity significantly decreased following TNFα/IL1β treatments and AIEC infection. SAMP1 mice and ileal organoids generated from SAMP1 mice also showed decreased CrT1 mRNA and protein compared to AKR controls. Our studies suggest that Cr deficiency in IECs secondary to CrT1 dysregulation could be a key factor contributing to IBD pathogenesis.

EPS9.08 A modelling framework for epithelial airway fluid and ion transport with multiple cell types: implications for success or failure in gene therapies for cystic fibrosis

EPS9.08 A modelling framework for epithelial airway fluid and ion transport with multiple cell types: implications for success or failure in gene therapies for cystic fibrosis

Voltage-gated ion channels as novel regulators of epithelial ion transport in the osmoregulatory organs of insects.

Voltage-gated ion channels (VGICs) respond to changes in membrane potential (Vm) and typically exhibit fast kinetic properties. They play an important role in signal detection and propagation in excitable tissues. In contrast, the role of VGICs in non-excitable tissues like epithelia is less studied and less clear. Studies in epithelia of vertebrates and invertebrates demonstrate wide expression of VGICs in epithelia of animals. Recently, VGICs have emerged as regulators of ion transport in the Malpighian tubules (MTs) and other osmoregulatory organs of insects. This mini-review aims to concisely summarize which VGICs have been implicated in the regulation of ion transport in the osmoregulatory epithelia of insects to date, and highlight select groups for further study. We have also speculated on the roles VGICs may potentially play in regulating processes connected directly to ion transport in insects (e.g., acid-base balance, desiccation, thermal tolerance). This review is not meant to be exhaustive but should rather serve as a thought-provoking collection of select existing highlights on VGICs, and to emphasize how understudied this mechanism of ion transport regulation is in insect epithelia.

Modulation of the rat intestinal microbiota in the course of Anisakis pegreffii infection.

Anisakis are globally distributed, marine parasitic nematodes that can cause human health problems, including symptoms such as vomiting, acute diarrhea, and allergic reactions. As parasitic nematodes that primarily affect the patient's digestive tract, intestinal helminths can interact directly with the host microbiota through physical contact, chemicals, or nutrient competition. It is widely accepted that the host microbiota plays a crucial role in the regulation of immunity. Nematodes collected from the abdominal cavity of marine fish were identified by molecular biology and live worms were artificially infected in rats. Infection was determined by indirect ELISA based on rat serum and worm extraction. Feces were collected for 16S rDNA-based analysis of microbiota diversity. Molecular biology identification based on ITS sequences identified the collected nematodes as A. pegreffii. The success of the artificial infection was determined by indirect ELISA based on serum and worm extraction from artificially infected rats. Microbiota diversity analysis showed that a total of 773 ASVs were generated, and PCoA showed that the infected group was differentiated from the control group. The control group contained five characterized genera (Prevotellaceae NK3B31 group, Turicibacter, Clostridium sensu stricto 1, Candidatus Stoquefichus, Lachnospira) and the infected group contained nine characterized genera (Rodentibacter, Christensenella, Dubosiella, Streptococcus, Anaeroplasma, Lactococcus, Papillibacter, Desulfovibrio, Roseburia). Based on the Wilcoxon test, four processes were found to be significant: bacterial secretion system, bacterial invasion of epithelial cells, bacterial chemotaxis, and ABC transporters. This study is the first to analyze the diversity of the intestinal microbiota of rats infected with A. pegreffii and to determine the damage and regulation of metabolism and immunity caused by the infection in the rat gut. The findings provide a basis for further research on host-helminth-microbe correlationships.

Osmoregulatory Challenges Faced by Mosquitoes and Techniques for Studying the Transporters Involved in Their Adaptations.

Mosquitoes are the most important disease vector in the world, and gaining knowledge of their physiology to develop novel population control strategies has been a focus of research for some time. Both aquatic larvae and terrestrial adults face harsh environmental factors that severely challenge their salt and water balance, which are regulated by the function of epithelia of various organs. The regulated passage of water and solutes across epithelia occurs, in part, through transporters expressed in epithelial cell membranes. Identifying these transporters and their localization is necessary to understand how mosquitoes regulate salt and water balance. Here, we review environmental challenges faced by mosquitoes and how they cope with them, in addition to introducing techniques used to identify organ epithelial transporters.

Role of Protein Phosphatase 1 (PP1) Upon KCCs Regulation by WNK3 Kinase During Cell Volume Regulation and Ion Transport

Background: The K-Cl cotransporters (KCCs) are SLC12A cation/chloride cotransporters (CCCs) that mediate epithelial transport, maintain cellular volume, and regulate GABA neurotransmission. These mechanisms are complex, and contain multiple sensors, transducers, and effectors, often operating in parallel pathways. It has been shown that KCCs activity is affected by direct phosphorylation and dephosphorylation. In particular dephosphorylation of important regulatory sites on KCCs is necessary to become fully activated, whereas phosphorylation by SPAK/OSR1 kinases will result in KCCs inhibition. Protein phosphatases (PP) inhibitors reduce KCCs function and so, it is generally accepted the presence of phosphatases on the KCCs signaling pathway. Several studies have proposed protein phosphatase 1 (PP1) as the major PP responsible for KCCs dephosphorylation. On the other hand, WNK3 kinases have been shown to directly regulate KCCs activity bypassing their osmotic regulation. That is wild type (WT) WNK3 phosphorylates KCCs under hypotonic conditions where they are normally active, whereas kinase dead WNK3 (DA) activates KCCs function under isotonic conditions where they are normally inhibited. On the present study we analyze the role of protein phosphatase 1 (PP1) upon KCCs regulation by WNK3 kinase during cell volume regulation and ion transport. Methods: HEK 293 cells and Xenopus laevis oocytes were either transfected or microinjected with KCC3 or KCC4 and one of the following PP1 plasmids: WT, H248K (inactive) or T320A (constitutive active), and/or WT WNK3 /DA WNK3. KCCs dephosphorylation in response to cell swelling, hypotonic conditions, was evaluated by Western blot analysis of KCCs regulatory phosphorylation site (pThr1048 in KCC3a). Phosphatase/kinase interaction and the possible formation of a complex was analyzed by transfecting cells or microinjecting oocytes with WNK3 PP1 putative binding sites mutants (WNK3 PP1a y PP1b). Phosphorylation of SPAK/OSR1 activating regulatory site Ser373 was analyzed in parallel under the same conditions. Results: WT or constitutive active mutant PP1 T320A induced KCCs Thr1048 dephosphorylation in HEK cells and X. laevis oocytes even under isotonic conditions, where KCCs are normally phosphorylated. WT WNK3 prevented KCCs T1048 dephosphorylation under hypotonic conditions, ablating their response to cell swelling. Nevertheless, when co-expressed with PP1 T320A, KCCs became dephosphorylated in both osmotic conditions eliminating WT WNK3 inhibitory effect. PP1 inactivating mutation H248K prevented KCCs Thr1048 dephosphorylation in response to hypotonic conditions. However, when co-expressed with DA WNK3, this KCCs regulatory site, became dephosphorylated under hypotonic conditions. SPAK Ser373 regulation was unaffected in any off the studied conditions. Conclusions: Our results show a direct role for PP1 on KCCs function. PP1WT and PP1T320A regulate KCCs activity by its dephosphorylation. In presence of WT WNK3, PP1 activity predominates over WNK3 surpassing the inhibitory phosphorylation of the regulatory Thr residue. KCCs are not dephosphorylated by PP1H248K under hypotonic conditions. Coexpression of kinase dead WNK3 overcomes this effect, supporting the hypothesis of a phosphatase/kinase complex regulating KCCs cell volume regulatory response. AM CF-2023-I-532, INCar 22-1345 PdlH Papiit IN228123. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.