Reconstitution of functional tight junctions with individual claudin subtypes in epithelial cells.

The claudin family of membrane proteins is responsible for the backbone structure and function of tight junctions (TJs), which regulate the paracellular permeability of epithelia. It is thought that each claudin subtype has its own unique function and the combination of expressed subtypes determines the permeability property of each epithelium. However, many issues remain unsolved in regard to claudin functions, including the detailed functional differences between claudin subtypes and the effect of the combinations of specific claudin subtypes on the structure and function of TJs. To address these issues, it would be useful to have a way of reconstituting TJs containing only the claudin subtype(s) of interest in epithelial cells. In this study, we attempted to reconstitute TJs of individual claudin subtypes in TJ-deficient MDCK cells, designated as claudin quinKO cells, which were previously established from MDCK II cells by deleting the genes of claudin-1, -2, -3, -4, and -7. Exogenous expression of each of claudin-1, -2, -3, -4, and -7 in claudin quinKO cells resulted in the reconstitution of functional TJs. These TJs did not contain claudin-12 and -16, which are endogenously expressed in claudin quinKO cells. Furthermore, overexpression of neither claudin-12 nor claudin-16 resulted in the reconstitution of TJs, demonstrating the existence of claudin subtypes lacking TJ-forming activity in epithelial cells. Exogenous expression of the channel-forming claudin-2, -10a, -10b, and -15 reconstituted TJs with reported paracellular channel properties, demonstrating that these claudin subtypes form paracellular channels by themselves without interaction with other subtypes. Thus, the reconstitution of TJs in claudin quinKO cells is advantageous for further investigation of claudin functions.Key words: tight junction, claudin, paracellular permeability, epithelial barrier.

Na+-dependent intestinal glucose absorption mechanisms and its luminal Na+ homeostasis across metamorphosis from tadpoles to frogs.

The abrupt morphological changes of the intestine during metamorphosis have been detailed in frogs. The features of intestinal metamorphosis are shortening of the intestine and remodeling of the intestinal epithelium. It is believed that the purpose of the morphological changes of the intestine is adaptation from aquatic herbivorous to carnivorous life. However, little is known about the physiological importance of these morphological changes. To elucidate the functional changes during metamorphosis, we measured luminal Na+ concentrations and Na+-dependent glucose uptake in tadpoles and adult African clawed frogs Xenopus laevis. The small intestine was isolated and divided into four segments in length, the luminal contents collected for analysis of ion concentration by ion chromatography. Phlorizin-sensitive glucose-induced short-circuit current (ΔIsc) was measured in intestinal preparations mounted in Ussing chambers. Although dietary sodium intake was extremely low in tadpoles, luminal Na+ concentration gradually increased along the proximal to the middle part of the intestine (>70 mM), and this Na+ concentration was comparable with that of carnivorous adult frogs. The increment of glucose-induced ΔIsc was observed in tadpole intestine. We also measured the ΔIsc induced by acetic acid, which is the major short-chain fatty acid produced by fermentation. The expression levels of mRNA for Na+-dependent glucose transporter 1 and tight junction protein claudin-15 in each intestinal segment was measured. These results suggest that luminal Na+ homeostasis is important and luminal Na+ is kept at a high concentration for Na+-dependent nutrient absorption mechanisms.

Increase in Paracellular Leakage of Amino Acids Mediated by Aging-Induced Reduction of Claudin-4 Expression.

BACKGROUND: Claudins (CLDNs), major components of tight junctions, control paracellular permeabilities of mineral ions and wastes. The absorption of nutrients including glucose and amino acids (AAs) is regulated by intestinal epithelial cells. However, the role of CLDNs is not fully understood. OBJECTIVES: The purpose of this study was to clarify the effect of AA deprivation on the expression of AA transporters and CLDNs, as well as the role of CLDNs in the regulation of paracellular AA fluxes. METHODS: The messenger RNA and protein expression of various CLDNs were examined by real-time quantitative polymerase chain reaction and Western blot analyses, respectively. The AA selectivity of CLDNs was estimated using liquid chromatography-tandem mass spectrometry (LC-MS) analysis. RESULTS: The expression levels of some AA transporters, CLDN4, and CLDN15 were increased by AA deprivation in normal mouse colon-derived MCE301 cells. The expression of AA transporters and CLDN15 in the mouse colon was positively correlated with aging but the expression of CLDN4 was not. The AA deprivation-induced elevation of CLDN4 expression was inhibited by MHY1485, a mammalian target of rapamycin (mTOR) activator. Furthermore, CLDN4 expression was increased by rapamycin, an mTOR inhibitor. mTOR may be involved in the transcriptional activation of CLDN4. The fluxes of AAs from the basal to apical compartments were decreased and increased by CLDN4 overexpression and silencing, respectively. LC-MS analysis showed that the fluxes of all AAs, especially Lys, His, and Arg, were enhanced by CLDN4 silencing. CONCLUSIONS: CLDN4 is suggested to form a paracellular barrier to AAs, especially alkaline AAs, which is attenuated with aging.

Na+-Coupled Nutrient Cotransport Induced Luminal Negative Potential and Claudin-15 Play an Important Role in Paracellular Na+ Recycling in Mouse Small Intestine.

Many nutrients are absorbed via Na+ cotransport systems, and therefore it is predicted that nutrient absorption mechanisms require a large amount of luminal Na+. It is thought that Na+ diffuses back into the lumen via paracellular pathways to support Na+ cotransport absorption. However, direct experimental evidence in support of this mechanism has not been shown. To elucidate this, we took advantage of claudin-15 deficient (cldn15-/-) mice, which have been shown to have decreased paracellular Na+ permeability. We measured glucose-induced currents (ΔIsc) under open- and short-circuit conditions and simultaneously measured changes in unidirectional 22Na+ fluxes (ΔJNa) in Ussing chambers. Under short-circuit conditions, application of glucose resulted in an increase in ΔIsc and unidirectional mucosal to serosal 22Na+ (∆JNaMS) flux in both wild-type and cldn15-/- mice. However, under open-circuit conditions, ΔIsc was observed but ∆JNaMS was strongly inhibited in wild-type but not in cldn15-/- mice. In addition, in the duodenum of mice treated with cholera toxin, paracellular Na+ conductance was decreased and glucose-induced ∆JNaMS increment was observed under open-circuit conditions. We concluded that the Na+ which is absorbed by Na+-dependent glucose cotransport is recycled back into the lumen via paracellular Na+ conductance through claudin-15, which is driven by Na+ cotransport induced luminal negativity.

Upregulation of Claudin-7 Expression by Angiotensin II in Colonic Epithelial Cells of Mice Fed with NaCl-Depleted Diets.

Dietary NaCl depletion increases Na+ and Cl- absorption in the colon, but the mechanisms are not fully understood. So far, we reported that the expression of claudin-7 (CLDN7), a tight junction (TJ) protein, was upregulated in the mice fed with NaCl-depleted diets, but the regulatory mechanism has not been clarified. Here, we found that angiotensin II (ANGII) increases the mRNA level of CLDN7, which was inhibited by losartan, a type 1 ANGII (AT1) receptor antagonist. Immunofluorescence measurement showed that CLDN7 is colocalized with zonula occludens-1 at the TJ in untreated and ANGII-treated cells. ANGII decreased transepithelial electrical resistance (TER) and increased permeability to C1- without affecting permeability to lucifer yellow, a paracellular flux marker. In contrast, TER was increased by CLDN7 knockdown in the absence and presence of ANGII. ANGII increased the nuclear distribution of phosphorylated p65 subunit of NF-κB, which was inhibited by losartan. The ANGII-induced elevation of CLDN7 expression was blocked by BAY 11-7082 (BAY), an NF-κB inhibitor. Luciferase reporter assay showed that ANGII increases promoter activity of CLDN7, which was inhibited by the treatment with losartan or BAY, and introduction of mutations in κB-binding motifs in the promoter. The binding of p65 on the promoter region of CLDN7 was increased by ANGII, which was inhibited by losartan and BAY in chromatin immunoprecipitation assay. Our data suggest that ANGII acts on AT1 receptor and increases paracellular permeability to Cl- mediated by the elevation of CLDN7 expression in the colon.

Luminal Na+ homeostasis has an important role in intestinal peptide absorption in vivo.

Intestinal cell line studies indicated luminal Na+ homeostasis is essential for proton-coupled peptide absorption, because the driving force of PepT1 activity is supported by the apical Na+/H+ exchanger NHE3. However, there is no direct evidence demonstrating the importance of in vivo luminal Na+ for peptide absorption in animal experiments. To investigate the relationship between luminal Na+ homeostasis and peptide absorption, we took advantage of claudin 15-deficient (cldn15-/-) mice, whereby Na+ homeostasis is disrupted. We quantitatively assessed the intestinal segment responsible for peptide absorption using radiolabeled nonhydrolyzable dipeptide (glycylsarcosine, Gly-Sar) and nonabsorbable fluid phase marker polyethylene glycol (PEG) 4000 in vivo. In wild-type (WT) mice, the concentration ratio of Gly-Sar to PEG 4000 decreased in the upper jejunum, suggesting the upper jejunum is responsible for peptide absorption. Gly-Sar absorption was decreased in the jejunum of cldn15-/- mice. To elucidate the mechanism underlining these impairments, a Gly-Sar-induced short-circuit ( Isc) current was measured. In WT mice, increments of Gly-Sar-induced Isc were inhibited by the luminal application of a NHE3-specific inhibitor S3226 in a dose-dependent fashion. In contrast to in vivo experiments, robust Gly-Sar-induced Isc increments were observed in the jejunal mucosa of cldn15-/- mice. Gly-Sar-induced Isc was inhibited by S3226 or a reduction of luminal Na+ concentration, which mimics low luminal Na+ concentrations in vivo . Our study demonstrates that luminal Na+ homeostasis is important for peptide absorption in native epithelia and that there is a cooperative functional relationship between PepT1 and NHE3. NEW & NOTEWORTHY Our study is the first to demonstrate that luminal Na+ homeostasis is important for proton-coupled peptide absorption in in vivo animal experiments.

Tight junctional localization of claudin-16 is regulated by syntaxin 8 in renal tubular epithelial cells.

Claudin-16 (CLDN16) regulates the paracellular reabsorption of Mg(2+) in the thick ascending limb of Henle's loop. However, the mechanism regulating the tight junctional localization of CLDN16 remains unknown. In yeast two-hybrid systems, we found that CLDN16 bound to syntaxin 8 (STX8), a target soluble N-ethylmaleimide-sensitive factor attachment protein receptor. We have examined the effect of STX8 on the localization and function of CLDN16 using Madin-Darby canine kidney cells expressing FLAG-tagged CLDN16. A pulldown assay showed that the carboxyl cytoplasmic region of human CLDN16 bound to STX8. CLDN16 was localized in the thick ascending limb, whereas STX8 was widely distributed throughout the rat kidney. An association between CLDN16 and STX8 was observed in rat renal homogenates and Madin-Darby canine kidney cells. STX8 siRNA decreased the cell surface localization of CLDN16 and transepithelial electrical resistance and permeability to Mg(2+) but increased the co-localization of CLDN16 with early endosome and lysosome markers. Dephosphorylation of CLDN16 by protein kinase A inhibitors and S217A mutant, a dephosphorylated form, decreased the association with STX8 and the cell surface localization of CLDN16. Recycling assays indicated that STX8 siRNA decreased the trafficking of CLDN16 to the plasma membrane without affecting endocytosis. Dominant negative Rab11 and recycling inhibitor primaquine decreased the cell surface localization of CLDN16, which was similar to that in STX8 siRNA-transfected cells. These results suggest that STX8 mediates the recycling of CLDN16 and constitutes an important component of the CLDN16 trafficking machinery in the kidney.

Activation of focal adhesion kinase via M1 muscarinic acetylcholine receptor is required in restitution of intestinal barrier function after epithelial injury.

Impairment of epithelial barrier is observed in various intestinal disorders including inflammatory bowel diseases (IBD). Numerous factors may cause temporary damage of the intestinal epithelium. A complex network of highly divergent factors regulates healing of the epithelium to prevent inflammatory response. However, the exact repair mechanisms involved in maintaining homeostatic intestinal barrier integrity remain to be clarified. In this study, we demonstrate that activation of M1 muscarinic acetylcholine receptor (mAChR) augments the restitution of epithelial barrier function in T84 cell monolayers after ethanol-induced epithelial injury, via ERK-dependent phosphorylation of focal adhesion kinase (FAK). We have shown that ethanol injury decreased the transepithelial electrical resistance (TER) along with the reduction of ERK and FAK phosphorylation. Carbachol (CCh) increased ERK and FAK phosphorylation with enhanced TER recovery, which was completely blocked by either MT-7 (M1 antagonist) or atropine. The CCh-induced enhancement of TER recovery was also blocked by either U0126 (ERK pathway inhibitor) or PF-228 (FAK inhibitor). Treatment of T84 cell monolayers with interferon-γ (IFN-γ) impaired the barrier function with the reduction of FAK phosphorylation. The CCh-induced ERK and FAK phosphorylation were also attenuated by the IFN-γ treatment. Immunological and binding experiments exhibited a significant reduction of M1 mAChR after IFN-γ treatment. The reduction of M1 mAChR in inflammatory area was also observed in surgical specimens from IBD patients, using immunohistochemical analysis. These findings provide important clues regarding mechanisms by which M1 mAChR participates in the maintenance of intestinal barrier function under not only physiological but also pathological conditions.

Hyperosmolarity-Induced Down-Regulation of Claudin-2 Mediated by Decrease in PKCß-Dependent GATA-2 in MDCK Cells.

Hyperosmolarity decreases claudin-2 expression in renal tubular epithelial cells, but the molecular mechanism remains undefined. Here, we found that the hyperosmolarity-induced decrease in claudin-2 expression is inhibited by Go6983, a non-selective protein kinase C (PKC) inhibitor, and PKCß specific inhibitor in Madin-Darby canine kidney II cells. Hyperosmolarity increased intracellular free Ca(2+) concentration and phosphorylated PKCß level, which were inhibited by RN-1734, an antagonist of transient receptor potential vanilloid 4 channel. Phorbol 12-myristate 13-acetate, a PKC activator, decreased claudin-2 expression. These results indicate hyperosmolarity decreases claudin-2 expression mediated by the activation of RN-1734-sensitive channel and PKCß. Hyperosmolarity decreased promoter activity of claudin-2, which was inhibited by Go6983 and PKCß inhibitor similar to those in real-time PCR and Western blotting. The effect of hyperosmolarity on promoter activity was not observed in the construct of -469/-6, a deletion mutant. Claudin-2 has hyperosmolarity-sensitive region in its promoter, which includes GATA binding site. Hyperosmolarity decreased the nuclear level of GATA-2, which was inhibited by Go6983 and PKCß inhibitor. Mutation of GATA binding site decreased the basal promoter activity and inhibited the effect of hyperosmolarity. In contrast, the hyperosmolarity-induced decrease in reporter activity and claudin-2 expression were rescued by over-expression of wild type GATA-2. Chromatin immunoprecipitation assay showed that GATA-2 bound to promoter region of claudin-2. These results suggest that hyperosmolarity decreases the expression level of claudin-2 via a decrease in PKCß-dependent GATA-2 transcriptional activity in renal tubular epithelial cells.

Muscarinic cholinoceptor-mediated activation of JNK negatively regulates intestinal secretion in mice.

Regulation of intestinal secretion is important for body fluid homeostasis. We investigated the role of three MAP kinases (MAPKs) as negative regulators in muscarinic cholinoceptor (mAChR)-mediated intestinal secretion in mice. Electrophysiological analyses revealed that mAChR stimulation enhanced intestinal chloride secretion, which was further augmented by the inhibition of JNK but not by that of ERK or p38 with specific inhibitors SP600125, U0126 or SB203580, respectively. Immunoblot analyses in colonic mucosa showed that mAChR stimulation increased MAPKs phosphorylation that was suppressed by the specific inhibitor for each MAPK. This suggests that JNK is a major negative regulator in mAChR-induced intestinal secretion.

Hyperosmolarity-induced up-regulation of claudin-4 mediated by NADPH oxidase-dependent H2O2 production and Sp1/c-Jun cooperation.

Claudin-4 is exclusively localized in the tight collecting ducts in the renal tubule. We examined what molecular mechanism is involved in the regulation of claudin-4 expression. In Madin-Darby canine kidney cells, hyperosmolarity increased the expression level of claudin-4 and the production of reactive oxygen species, which were inhibited by diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, and manganese (III) tetrakis (4-benzoic acid)porphyrin (MnTBAP), a scavenger of H2O2. Both hyperosmolarity and H2O2 increased p-ERK1/2 and p-JNK, which were inhibited by U0126, a MEK inhibitor, and SP600125, a JNK inhibitor, respectively. Immunoprecipitation assay showed that hyperosmolarity increased the association of nuclear Sp1 with c-Jun, which was inhibited by U0126 and SP600125. In mouse inner medullary collecting duct cells and rat kidney slices, hyperosmolarity increased the expression level of claudin-4, which was inhibited by DPI, MnTBAP, U0126, and SP600125. Hyperosmolarity increased luciferase reporter activity of claudin-4, which was inhibited by U0126, SP600125, Sp1 siRNA, and c-Jun siRNA. The activity was inhibited by the mutation in the Sp1 binding site. Chromatin immunoprecipitation assay and avidin-biotin conjugated DNA assay showed that Sp1 and c-Jun are associated with the Sp1 binding site. These results suggest that hyperosmolarity increases nuclear Sp1/c-Jun complex and the association of the complex with the Sp1 binding site, resulting in the segment-specific expression of claudin-4 in the kidney.

Roseiterribacter gracilis gen. nov., sp. nov., a novel filterable alphaproteobacterium isolated from soil using a gel-filled microwell array device.

Our previous studies indicate the abundant and diverse presence of yet-to-be-cultured microorganisms in the micropore-filtered fractions of various environmental samples. Here, we isolated a novel bacterium (designated as strain TMPK1T) from a 0.45-µm-filtered soil suspension by using a gel-filled microwell array device comprising 900 microwells and characterized its phylogenetic and physiological features. This strain showed low 16S rRNA gene sequence identities (<91%) and low average nucleotide identity values (<70%) to the closest validly described species, and belonged to a novel-family-level lineage within the order Rhodospirillales of Alphaproteobacteria. Strain TMPK1T exhibited small cell sizes (0.08-0.23 µm3) and had a high cyclopropane fatty acid content (>13%), and these characteristics were differentiated from other Rhodospirillales bacteria. A comprehensive habitability search using amplicon datasets suggested that TMPK1T and its close relatives are mainly distributed in soil and plant-associated environments. Based on these results, we propose that strain TMPK1T represents a novel genus and species named Roseiterribacter gracilis gen. nov., sp. nov. (JCM 34627T = KCTC 82790T). We also propose Roseiterribacteraceae fam. nov. to accommodate the genus Roseiterribacter.

Temporal and fertilizer-dependent dynamics of soil bacterial communities in buckwheat fields under long-term management.

This study integrated bacterial community and soil chemicals to characterize the soil ecosystem in an open upland field managed by six controlled fertilizer programs using the minimum amount of pesticides. Amplicon sequencing the 16S rRNA gene revealed that inorganic nitrogen fertilizer and compost altered the diversity and structure of the soil bacterial community throughout buckwheat (Fagopyrum esculentum Moench 'Hitachiakisoba') cultivation. The bacterial community comprised three clusters that contained bacteria that are prevalent in soils fertilized with nitrogen (cluster 1, 340 taxa), without nitrogen and compost (cluster 2, 234 taxa), and with compost-fertilized (cluster 3, 296 taxa). Cluster 2 contained more taxa in Actinobacteriota and less in Acidobacteriota, and cluster 3 contained more taxa in Gemmatimonadota compared with the other clusters. The most frequent taxa in cluster 1 were within the Chloroflexi phylum. The bacterial community structure correlated with soil chemical properties including pH, total organic carbon, SO42-, soluble Ca2+. A co-occurrence network of bacterial taxa and chemicals identified key bacterial groups comprising the center of a community network that determined topology and dynamics of the network. Temporal dynamics of the bacterial community structure indicated that Burkholderiales were associated with buckwheat ripening, indicating plant-bacteria interaction in the ecosystem.

The effect of claudin-15 deletion on cationic selectivity and transport in paracellular pathways of the cecum and large intestine.

The large intestine plays a pivotal role in water and electrolyte balance. Paracellular transport may play a role in ion transport mechanisms in the cecum and large intestine; however, these molecular mechanisms and their physiological roles have not been fully studied. Claudin-15 forms a cation channel in tight junctions in the small intestine, but its role in the cecum and large intestine has not been investigated. This study aimed to explore the physiological role of claudin-15 in the cecum and large intestine using claudin-15 (Cldn15) KO mice. Electrical conductance, short-circuit current, Na+ flux, and dilution potential were assessed in isolated tissue preparations mounted in Ussing chambers. The induced short-circuit current of short-chain fatty acids, which are fermentative products in the intestinal tract, was also measured. Compared to wild type mice, the electrical conductance and paracellular Na+ flux was decreased in the cecum, but not the middle large intestine, while in both the cecum and the middle large intestine, paracellular Na+ permeability was decreased in Cldn15 KO mice. These results suggest that claudin-15 is responsible for Na+ permeability in the tight junctions of the cecum and large intestine and decreased Na+ permeability in the cecum may cause impaired absorption function.

Regulatory mechanisms of glucose absorption in the mouse proximal small intestine during fasting and feeding.

Fasting is known to alter the function of various organs and the mechanisms of glucose metabolism, which affect health outcomes and slow aging. However, it remains unclear how fasting and feeding affects glucose absorption function in the small intestine. We studied the effects of the fasting and feeding on glucose-induced short-circuit current (Isc) in vitro using an Ussing chamber technique. Glucose-induced Isc by SGLT1 was observed in the ileum, but little or no Isc was observed in the jejunum in ad libitum-fed mice. However, in mice fasted for 24-48 h, in addition to the ileum, robust glucose-induced Isc was observed over time in the jejunum. The expression of SGLT1 in the brush border membranes was significantly decreased in the jejunum under fed conditions compared to 48 h fasting, as analyzed by western blotting. Additionally, when mice were fed a 60% high glucose diet for 3 days, the increase in glucose-induced Isc was observed only in the ileum, and totally suppressed in the jejunum. An increase in Na+ permeability between epithelial cells was concomitantly observed in the jejunum of fasted mice. Transepithelial glucose flux was assessed using a non-metabolizable glucose analog, 14C-methyl α-D-glucopyranoside glucose (MGP). Regardless of whether fed or fasted, no glucose diffusion mechanism was observed. Fasting increased the SGLT1-mediated MGP flux in the jejunum. In conclusion, segment-dependent up- and down-regulation mechanisms during fasting and feeding are important for efficient glucose absorption once the fast is broken. Additionally, these mechanisms may play a crucial role in the small intestine's ability to autoregulate glucose absorption, preventing acute hyperglycemia when large amounts of glucose are ingested.

High-precision plant height measurement by drone with RTK-GNSS and single camera for real-time processing.

Conventional crop height measurements performed using aerial drone images require 3D reconstruction results of several aerial images obtained through structure from motion. Therefore, they require extensive computation time and their measurement accuracy is not high; if the 3D reconstruction result fails, several aerial photos must be captured again. To overcome these challenges, this study proposes a high-precision measurement method that uses a drone equipped with a monocular camera and real-time kinematic global navigation satellite system (RTK-GNSS) for real-time processing. This method performs high-precision stereo matching based on long-baseline lengths (approximately 1 m) during the flight by linking the RTK-GNSS and aerial image capture points. As the baseline length of a typical stereo camera is fixed, once the camera is calibrated on the ground, it does not need to be calibrated again during the flight. However, the proposed system requires quick calibration in flight because the baseline length is not fixed. A new calibration method that is based on zero-mean normalized cross-correlation and two stages least square method, is proposed to further improve the accuracy and stereo matching speed. The proposed method was compared with two conventional methods in natural world environments. It was observed that error rates reduced by 62.2% and 69.4%, for flight altitudes between 10 and 20 m respectively. Moreover, a depth resolution of 1.6 mm and reduction of 44.4% and 63.0% in the error rates were achieved at an altitude of 4.1 m, and the execution time was 88 ms for images with a size of 5472 × 3468 pixels, which is sufficiently fast for real-time measurement.

Effects of EOS789, a novel pan-phosphate transporter inhibitor, on phosphate metabolism : Comparison with a conventional phosphate binder.

BACKGROUND: Inorganic phosphate (Pi) binders are the only pharmacologic treatment approved for hyperphosphatemia. However, Pi binders induce the expression of intestinal Pi transporters and have limited effects on the inhibition of Pi transport. EOS789, a novel pan-Pi transporter inhibitor, reportedly has potent efficacy in treating hyperphosphatemia. We investigated the properties of EOS789 with comparison to a conventional Pi binder. METHODS: Protein and mRNA expression levels of Pi transporters were measured in intestinal and kidney tissues from male Wistar rats fed diets supplemented with EOS789 or lanthanum carbonate (LC). 32Pi permeability was measured in intestinal tissues from normal rats using a chamber. RESULTS: Increased protein levels of NaPi-2b, an intestinal Pi transporter, and luminal Pi removal were observed in rats treated with LC but not in rats treated with EOS789. EOS789 but not LC suppressed intestinal protein levels of the Pi transporter Pit-1 and sodium/hydrogen exchanger isoform 3. 32Pi flux experiments using small intestine tissues from rats demonstrated that EOS789 may affect transcellular Pi transport in addition to paracellular Pi transport. CONCLUSION: EOS789 has differing regulatory effects on Pi metabolism compared to LC. The properties of EOS789 may compensate for the limitations of LC therapy. The combined or selective use of EOS789 and conventional Pi binders may allow tighter control of hyperphosphatemia. J. Med. Invest. 70 : 260-270, February, 2023.

Role of N-glycosylation in cell surface expression and protection against proteolysis of the intestinal anion exchanger SLC26A3.

SLC26A3 is a Cl(-)/HCO(3)(-) exchanger that plays a major role in Cl(-) absorption from the intestine. Its mutation causes congenital chloride-losing diarrhea. It has been shown that SLC26A3 are glycosylated, with the attached carbohydrate being extracellular and perhaps modulating function. However, the role of glycosylation has yet to be clearly determined. We used the approaches of biochemical modification and site-directed mutagenesis to prevent glycosylation. Deglycosylation experiments with glycosidases indicated that the mature glycosylated form of SLC26A3 exists at the plasma membrane, and a putative large second extracellular loop contains all of the N-linked carbohydrates. Deglycosylation of SLC26A3 causes depression of transport activity compared with wild-type, although robust intracellular pH changes were still observed, suggesting that N-glycosylation is not absolutely necessary for transport activity. To localize glycosylation sites, we mutated the five consensus sites by replacing asparagine (N) with glutamine. Immnoblotting suggests that SLC26A3 is glycosylated at N153, N161, and N165. Deglycosylation of SLC26A3 causes a defect in cell surface processing with decreased cell surface expression. We also assessed whether SLC26A3 is protected from tryptic digestion. While the mature glycosylated SLC26A3 showed little breakdown after treatment with trypsin, deglycosylated SLC26A3 exhibited increased susceptibility to trypsin, suggesting that the oligosaccharides protect SLC26A3 from tryptic digestion. In conclusion, our data indicate that N-glycosylation of SLC26A3 is important for cell surface expression and for protection from proteolytic degradation that may contribute to the understanding of pathogenesis of congenital disorders of glycosylation.

Enhancement of cell-cell contact by claudin-4 in renal epithelial Madin-Darby canine kidney cells.

Claudin-4 regulates ion permeability via a paracellular pathway in renal epithelial cells, but its other physiological functions have not been examined. We found that hyperosmotic stress increases claudin-4 expression in Madin-Darby canine kidney cells. Here, we examined whether claudin-4 affects cell motility, cell association, and the intracellular distribution of endogenous junctional proteins. Doxycycline-inducible expression of claudin-4 did not change endogenous levels of claudin-1, claudin-2, claudin-3, occludin, E-cadherin, and ZO-1. Claudin-4 overexpression increased cell association and decreased cell migration without affecting cell proliferation. Doxycycline did not change cell junctional protein levels, cell association or cell migration in mock-transfected cells. The insolubility of claudin-1 and -3 in Triton X-100 was increased by claudin-4 overexpression, but that of claudin-2, occludin, ZO-1, and E-cadherin was unchanged. Immunocytochemistry showed that claudin-4 overexpression increases the accumulation of claudin-1 and -3 in tight junctions (TJs). Furthermore, claudin-4 overexpression increased the association of claudin-4 with claudin-1 and -3. These results suggest that claudin-4 accumulates claudin-1 and -3 in TJs to enhance cell-cell contact in renal tubular epithelial cells.

Loss of claudin-15, but not claudin-2, causes Na+ deficiency and glucose malabsorption in mouse small intestine.

BACKGROUND & AIMS: In the small intestine, the paracellular transport of Na(+) is thought to be critical for luminal Na(+)-homeostasis and the transcellular absorption of nutrients by Na(+)-driven transporters. Na(+) is supplied to the intestinal lumen from the submucosa and serum through tight junctions, which form a paracellular barrier between the cells of epithelial sheets. However, the molecular basis for this paracellular transport of Na(+) is not well understood. Here, we examined this mechanism by performing loss-of-function studies of claudin-2 and claudin-15, two tight-junctional membrane proteins that are specifically and age-dependently expressed in the villi and/or crypts of small intestinal epithelia. METHODS: Knockout mice for claudin-2 or claudin-15 were subjected to histologic, cell biologic, electrophysiologic, and physiologic analyses. RESULTS: Examination of the knockout mice revealed that both claudin-2 and claudin-15 play crucial roles in the transepithelial paracellular channel-like permselectivity for extracellular monovalent cations, particularly Na(+), in infants and adults. Especially in Cldn15(-/-) adults, the luminal Na(+) concentration in the small intestine measured directly in vivo was abnormally low, and glucose absorption was impaired, as assessed by the oral glucose tolerance test and estimation of unabsorbed glucose. CONCLUSIONS: We propose that the "Na(+)-leaky" claudin-15 is indispensable in vivo for the paracellular Na(+) permeability, luminal Na(+)-homeostasis, and efficient glucose absorption in the small intestine, but claudin-2 is indispensable for only the first of these functions. Claudin-15 knockout leads to Na(+) deficiency and glucose malabsorption in the mouse adult small intestine.