Claudin-10a Deficiency Shifts Proximal Tubular Cl- Permeability to Cation Selectivity via Claudin-2 Redistribution.

BACKGROUND: The tight junction proteins claudin-2 and claudin-10a form paracellular cation and anion channels, respectively, and are expressed in the proximal tubule. However, the physiologic role of claudin-10a in the kidney has been unclear. METHODS: To investigate the physiologic role of claudin-10a, we generated claudin-10a-deficient mice, confirmed successful knockout by Southern blot, Western blot, and immunofluorescence staining, and analyzed urine and serum of knockout and wild-type animals. We also used electrophysiologic studies to investigate the functionality of isolated proximal tubules, and studied compensatory regulation by pharmacologic intervention, RNA sequencing analysis, Western blot, immunofluorescence staining, and respirometry. RESULTS: Mice deficient in claudin-10a were fertile and without overt phenotypes. On knockout, claudin-10a was replaced by claudin-2 in all proximal tubule segments. Electrophysiology showed conversion from paracellular anion preference to cation preference and a loss of paracellular Cl- over HCO3- preference. As a result, there was tubular retention of calcium and magnesium, higher urine pH, and mild hypermagnesemia. A comparison with other urine and serum parameters under control conditions and sequential pharmacologic transport inhibition, and unchanged fractional lithium excretion, suggested compensative measures in proximal and distal tubular segments. Changes in proximal tubular oxygen handling and differential expression of genes regulating fatty acid metabolism indicated proximal tubular adaptation. Western blot and immunofluorescence revealed alterations in distal tubular transport. CONCLUSIONS: Claudin-10a is the major paracellular anion channel in the proximal tubule and its deletion causes calcium and magnesium hyper-reabsorption by claudin-2 redistribution. Transcellular transport in proximal and distal segments and proximal tubular metabolic adaptation compensate for loss of paracellular anion permeability.

Uptake of Tropheryma whipplei by Intestinal Epithelia.

BACKGROUND: Tropheryma whipplei (TW) can cause different pathologies, e.g., Whipple's disease and transient gastroenteritis. The mechanism by which the bacteria pass the intestinal epithelial barrier, and the mechanism of TW-induced gastroenteritis are currently unknown. METHODS: Using ex vivo disease models comprising human duodenal mucosa exposed to TW in Ussing chambers, various intestinal epithelial cell (IEC) cultures exposed to TW and a macrophage/IEC coculture model served to characterize endocytic uptake mechanisms and barrier function. RESULTS: TW exposed ex vivo to human small intestinal mucosae is capable of autonomously entering IECs, thereby invading the mucosa. Using dominant-negative mutants, TW uptake was shown to be dynamin- and caveolin-dependent but independent of clathrin-mediated endocytosis. Complementary inhibitor experiments suggested a role for the activation of the Ras/Rac1 pathway and actin polymerization. TW-invaded IECs underwent apoptosis, thereby causing an epithelial barrier defect, and were subsequently subject to phagocytosis by macrophages. CONCLUSIONS: TW enters epithelia via an actin-, dynamin-, caveolin-, and Ras-Rac1-dependent endocytosis mechanism and consecutively causes IEC apoptosis primarily in IECs invaded by multiple TW bacteria. This results in a barrier leak. Moreover, we propose that TW-packed IECs can be subject to phagocytic uptake by macrophages, thereby opening a potential entry point of TW into intestinal macrophages.

Altered paracellular cation permeability due to a rare CLDN10B variant causes anhidrosis and kidney damage.

Claudins constitute the major component of tight junctions and regulate paracellular permeability of epithelia. Claudin-10 occurs in two major isoforms that form paracellular channels with ion selectivity. We report on two families segregating an autosomal recessive disorder characterized by generalized anhidrosis, severe heat intolerance and mild kidney failure. All affected individuals carry a rare homozygous missense mutation c.144C>G, p.(N48K) specific for the claudin-10b isoform. Immunostaining of sweat glands from patients suggested that the disease is associated with reduced levels of claudin-10b in the plasma membranes and in canaliculi of the secretory portion. Expression of claudin-10b N48K in a 3D cell model of sweat secretion indicated perturbed paracellular Na+ transport. Analysis of paracellular permeability revealed that claudin-10b N48K maintained cation over anion selectivity but with a reduced general ion conductance. Furthermore, freeze fracture electron microscopy showed that claudin-10b N48K was associated with impaired tight junction strand formation and altered cis-oligomer formation. These data suggest that claudin-10b N48K causes anhidrosis and our findings are consistent with a combined effect from perturbed TJ function and increased degradation of claudin-10b N48K in the sweat glands. Furthermore, affected individuals present with Mg2+ retention, secondary hyperparathyroidism and mild kidney failure that suggest a disturbed reabsorption of cations in the kidneys. These renal-derived features recapitulate several phenotypic aspects detected in mice with kidney specific loss of both claudin-10 isoforms. Our study adds to the spectrum of phenotypes caused by tight junction proteins and demonstrates a pivotal role for claudin-10b in maintaining paracellular Na+ permeability for sweat production and kidney function.

Campylobacter concisus Impairs Sodium Absorption in Colonic Epithelium via ENaC Dysfunction and Claudin-8 Disruption.

The epithelial sodium channel (ENaC) can increase the colonic absorptive capacity for salt and water. Campylobacter concisus is a common pathogenic epsilonproteobacterium, causing enteritis and diarrhea. It can induce barrier dysfunction in the intestine, but its influence on intestinal transport function is still unknown. Therefore, our study aimed to characterize C. concisus effects on ENaC using the HT-29/B6-GR/MR (epithelial cell line HT-29/B6 transfected with glucocorticoid and mineralocorticoid receptors) cell model and mouse colon. In Ussing chambers, C. concisus infection inhibited ENaC-dependent Na+ transport as indicated by a reduction in amiloride-sensitive short circuit current (-55%, n = 15, p < 0.001). This occurred via down-regulation of ß- and γ-ENaC mRNA expression and ENaC ubiquitination due to extracellular signal-regulated kinase (ERK)1/2 activation, predicted by Ingenuity Pathway Analysis (IPA). In parallel, C. concisus reduced the expression of the sealing tight junction (TJ) protein claudin-8 and induced claudin-8 redistribution off the TJ domain of the enterocytes, which facilitates the back leakage of Na+ ions into the intestinal lumen. In conclusion, C. concisus caused ENaC dysfunction via interleukin-32-regulated ERK1/2, as well as claudin-8-dependent barrier dysfunction-both of which contribute to Na+ malabsorption and diarrhea.

Tilivalline- and Tilimycin-Independent Effects of Klebsiella oxytoca on Tight Junction-Mediated Intestinal Barrier Impairment.

Klebsiella oxytoca causes antibiotic-associated hemorrhagic colitis and diarrhea. This was attributed largely to its secreted cytotoxins tilivalline and tilimycin, inductors of epithelial apoptosis. To study whether Klebsiella oxytoca exerts further barrier effects, T84 monolayers were challenged with bacterial supernatants derived from tilivalline/tilimycin-producing AHC6 or its isogeneic tilivalline/tilimycin-deficient strain Mut-89. Both preparations decreased transepithelial resistance, enhanced fluorescein and FITC-dextran-4kDa permeabilities, and reduced expression of barrier-forming tight junction proteins claudin-5 and -8. Laser scanning microscopy indicated redistribution of both claudins off the tight junction region in T84 monolayers as well as in colon crypts of mice infected with AHC6 or Mut-89, indicating that these effects are tilivalline/tilimycin-independent. Furthermore, claudin-1 was affected, but only in a tilivalline/tilimycin-dependent manner. In conclusion, Klebsiella oxytoca induced intestinal barrier impairment by two mechanisms: the tilivalline/tilimycin-dependent one, acting by increasing cellular apoptosis and a tilivalline/tilimycin-independent one, acting by weakening the paracellular pathway through the tight junction proteins claudin-5 and -8.

Curcumin Mitigates Immune-Induced Epithelial Barrier Dysfunction by Campylobacter jejuni.

Campylobacter jejuni (C. jejuni) is the most common cause of foodborne gastroenteritis worldwide. The bacteria induce diarrhea and inflammation by invading the intestinal epithelium. Curcumin is a natural polyphenol from turmeric rhizome of Curcuma longa, a medical plant, and is commonly used in curry powder. The aim of this study was the investigation of the protective effects of curcumin against immune-induced epithelial barrier dysfunction in C. jejuni infection. The indirect C. jejuni-induced barrier defects and its protection by curcumin were analyzed in co-cultures with HT-29/B6-GR/MR epithelial cells together with differentiated THP-1 immune cells. Electrophysiological measurements revealed a reduction in transepithelial electrical resistance (TER) in infected co-cultures. An increase in fluorescein (332 Da) permeability in co-cultures as well as in the germ-free IL-10-/- mouse model after C. jejuni infection was shown. Curcumin treatment attenuated the C. jejuni-induced increase in fluorescein permeability in both models. Moreover, apoptosis induction, tight junction redistribution, and an increased inflammatory response-represented by TNF-α, IL-1ß, and IL-6 secretion-was observed in co-cultures after infection and reversed by curcumin. In conclusion, curcumin protects against indirect C. jejuni-triggered immune-induced barrier defects and might be a therapeutic and protective agent in patients.

In Colon Epithelia, Clostridium perfringens Enterotoxin Causes Focal Leaks by Targeting Claudins Which are Apically Accessible Due to Tight Junction Derangement.

Clostridium perfringens enterotoxin (CPE) causes food poisoning and antibiotic-associated diarrhea. It uses some claudin tight junction proteins (eg, claudin-4) as receptors to form Ca2+-permeable pores in the membrane, damaging epithelial cells in small intestine and colon. We demonstrate that only a subpopulation of colonic enterocytes which are characterized by apical dislocation of claudins are CPE-susceptible. CPE-mediated damage was enhanced if paracellular barrier was impaired by Ca2+ depletion, proinflammatory cytokine tumor necrosis factor α, or dedifferentiation. Microscopy, Ca2+ monitoring, and electrophysiological data showed that CPE-mediated cytotoxicity and barrier disruption was limited by extent of CPE-binding. The latter was restricted by accessibility of non-junctional claudin molecules such as claudin-4 at apical membranes. Focal-leaks detected in HT-29/B6 colonic monolayers were verified for native tissue using colon biopsies. These mechanistic findings indicate how CPE-mediated effects may turn from self-limiting diarrhea into severe clinical manifestation such as colonic necrosis-if intestinal barrier dysfunction, eg, during inflammation facilitates claudin accessibility.

Yersinia enterocolitica Affects Intestinal Barrier Function in the Colon.

Infection with Yersinia enterocolitica causes acute diarrhea in early childhood. A mouse infection model presents new findings on pathological mechanisms in the colon. Symptoms involve diarrhea with watery feces and weight loss that have their functional correlates in decreased transepithelial electrical resistance and increased fluorescein permeability. Y. enterocolitica was present within the murine mucosa of both ileum and colon. Here, the bacterial insult was of focal nature and led to changes in tight junction protein expression and architecture. These findings are in concordance with observations from former cell culture studies and suggest a leak flux mechanism of diarrhea.

Interleukin-13 affects the epithelial sodium channel in the intestine by coordinated modulation of STAT6 and p38 MAPK activity.

KEY POINTS: Interleukin-13 (IL-13) causes intestinal epithelial barrier dysfunction, and is implicated in the pathogenesis of Th2-driven intestinal inflammation (e.g. ulcerative colitis). However, it is unclear whether the epithelial sodium channel (ENaC) - the main limiting factor for sodium absorption in the distal colon - is also influenced by IL-13 and if so, by what mechanism(s). We demonstrate in an intestinal cell model as well as in mouse distal colon that IL-13 causes reduced ENaC activity. We show that IL-13 impairs ENaC-dependent sodium transport by activating the JAK1/2-STAT6 signalling pathway. These results improve our understanding of the mechanisms through which IL-13 functions as a key effector cytokine in ulcerative colitis, thereby contributing to the distinct pathology of this disease. ABSTRACT: Interleukin-13 (IL-13) has been strongly implicated in the pathogenesis of ulcerative colitis, possibly by disrupting epithelial integrity. In the distal colon, the epithelial sodium channel (ENaC) is an important factor in the regulation of sodium absorption, and therefore plays a critical role in minimizing intestinal sodium and water losses. In the present study, we investigated whether IL-13 also acts as a potent modulator of epithelial sodium transport via ENaC, and the signalling components involved. The effect of IL-13 on ENaC was examined in HT-29/B6-GR/MR human colon cells, as well as in mouse distal colon, by measuring amiloride-sensitive short-circuit current (ISC ) in Ussing chambers. The expression levels of ENaC subunits and the cellular components that contribute to ENaC activity were analysed by qRT-PCR and promoter gene assay. We show that IL-13, in both the cell model and in native intestinal tissue, impaired epithelial sodium absorption via ENaC (JNa ) as a result of decreased transcription levels of ß- and γ-ENaC subunits and SGK1, a post-translational regulator of ENaC activity, due to impaired promoter activity. The reduction in JNa was prevented by inhibition of JAK1/2-STAT6 signalling. This inhibition also affected the IL-13-induced decrease in p38 MAPK phosphorylation. The contribution of STAT6 to IL-13-mediated ENaC inactivation was confirmed in a STAT6(-/-) mouse model. In conclusion, these results indicate that IL-13, the levels of which are elevated in ulcerative colitis, contributes to impaired ENaC activity via modulation of the STAT6/p38 MAPK pathways.

Claudin-17 forms tight junction channels with distinct anion selectivity.

Barrier properties of tight junctions are determined by the claudin protein family. Many claudins seal this barrier, but others form paracellular channels. Among these, no claudins with general and clear-cut anion selectivity have yet been described, while for claudin-10a and claudin-4, only circumstantial or small anion selectivities have been shown. A claudin with unknown function and tissue distribution is claudin-17. We characterized claudin-17 by overexpression and knock-down in two renal cell lines. Overexpression in MDCK C7 cell layers caused a threefold increase in paracellular anion permeability and switched these cells from cation- to anion-selective. Knockdown in LLC-PK(1) cells indorsed the finding of claudin-17-based anion channels. Mutagenesis revealed that claudin-17 anion selectivity critically depends on a positive charge at position 65. Claudin-17 expression was found in two organs: marginal in brain but abundant in kidney, where expression was intense in proximal tubules and gradually decreased towards distal segments. As claudin-17 is predominantly expressed in proximal nephrons, which exhibit substantial, though molecularly not defined, paracellular chloride reabsorption, we suggest that claudin-17 has a unique physiological function in this process. In conclusion, claudin-17 forms channels within tight junctions with distinct anion preference.

Cell polarity-determining proteins Par-3 and PP-1 are involved in epithelial tight junction defects in coeliac disease.

BACKGROUND: Epithelial barrier defects are well known in coeliac disease, but the mechanisms are only poorly defined. It is unclear, whether barrier disturbance reflects upregulated epithelial transcytosis or paracellular leakage. OBJECTIVE: To characterise the molecular structure and function of the epithelial tight junction (TJ) and mechanisms of its dysregulation. METHODS: Molecular analysis of proteins involved in TJ assembly and their regulation was performed by western blotting and confocal microscopy correlated to electrophysiology. RESULTS: A complex alteration of the composition of epithelial TJ proteins (with more pore-forming claudins like claudin-2 and a reduction in tightening claudins like claudin-3, -5 and -7) was found for protein expression and subcellular localisation, responsible for an increase in paracellular biotin-NHS uptake. In contrast, epithelial apoptosis was only moderately elevated (accounting for a minor portion of barrier defects) and epithelial gross lesions--for example, at cell extrusion zones, were absent. This TJ alteration was linked to an altered localisation/expression of proteins regulating TJ assembly, the polarity complex protein Par-3 and the serine-/threonine phosphatase PP-1. CONCLUSIONS: Changes in cell polarity proteins Par-3 and PP-1 are associated with altered expression and assembly of TJ proteins claudin-2, -3, -5 and -7 and ZO-1, causing paracellular leakage in active coeliac disease.

TNFalpha-induced and berberine-antagonized tight junction barrier impairment via tyrosine kinase, Akt and NFkappaB signaling.

TNFα-mediated tight junction defects contribute to diarrhea in inflammatory bowel diseases (IBDs). In our study, the signaling pathways of the TNFα effect on barrier- or pore-forming claudins were analyzed in HT-29/B6 human colon monolayers. Berberine, a herbal therapeutic agent that has been recently established as a therapy for diabetes and hypercholesterinemia, was able to completely antagonize the TNFα-mediated barrier defects in the cell model and in rat colon. Ussing chamber experiments and two-path impedance spectroscopy revealed a decrease of paracellular resistance after TNFα to 11±4%, whereas transcellular resistance was unchanged. The permeability of the paracellular marker fluorescein was increased fourfold. Berberine alone had no effect while it fully prevented the TNFα-induced barrier defects. This effect on resistance was confirmed in rat colon. TNFα removed claudin-1 from the tight junction and increased claudin-2 expression. Berberine prevented TNFα-induced claudin-1 disassembly and upregulation of claudin-2. The effects of berberine were mimicked by genistein plus BAY11-7082, indicating that they are mediated via tyrosine kinase, pAkt and NFκB pathways. In conclusion, the anti-diarrheal effect of berberine is explained by a novel mechanism, suggesting a therapeutic approach against barrier breakdown in intestinal inflammation.

Aerolysin from Aeromonas hydrophila perturbs tight junction integrity and cell lesion repair in intestinal epithelial HT-29/B6 cells.

BACKGROUND: Aeromonads cause a variety of infections, including gastroenteritis, sepsis, and wound necrosis. Pathogenesis of Aeromonas hydrophila and its hemolysin has been characterized, but the mechanism of the epithelial barrier dysfunction is currently poorly understood. METHODS: Human colon epithelial monolayers HT-29/B6 were apically inoculated with clinical isolates of A. hydrophila or with the secreted pore-forming toxin aerolysin. Epithelial resistance and permeability for several markers were determined in Ussing chambers, using 2-path impedance spectroscopy. The subcellular distribution of tight junction (TJ) and cytoskeleton proteins was analyzed by Western blotting and confocal laser-scanning microscopy. RESULTS: A. hydrophila infection induces chloride secretion with a small decrease in transcellular resistance. However, the major effect of A. hydrophila, mediated by its toxin aerolysin, was a sustained reduction of paracellular resistance by retracting sealing TJ proteins from the TJ strands. Aerolysin-treated monolayers showed increased paracellular permeability to FITC-dextran-4000 (0.104 ± 0.014 vs 0.047 ± 0.001 10(-6) cm/s in control; P < .05). Moreover, restitution of epithelial lesions was impaired. The effects were myosin light chain kinase (MLCK) dependent and mediated by intracellular Ca(2+) signaling. CONCLUSIONS: During Aeromonas infection, pore formation by aerolysin impairs epithelial integrity by promoting TJ protein redistribution and consequently affecting wound closure. Thus, Aeromonas-induced diarrhea is mediated by 2 mechanisms, transcellular secretion and paracellular leak flux.

Tight junction channels claudin-10b and claudin-15: Functional mapping of pore-lining residues.

Although functional and structural models for paracellular channels formed by claudins have been reported, mechanisms regulating charge and size selectivity of these channels are unknown in detail. Here, claudin-15 and claudin-10b cation channels showing high-sequence similarity but differing channel properties were analyzed. Mutants of pore-lining residues were expressed in MDCK-C7 cells. In claudin-15, proposed ion interaction sites (D55 and E64) conserved between both claudins were neutralized. D55N and E64Q substitutions decreased ion permeabilities, and D55N/E64Q had partly additive effects. D55N increased cation dehydration capability and decreased pore diameter. Additionally, residues differing between claudin-15 and -10b close to pore center were analyzed. Claudin-10b-mimicking W63K affected neither assembly nor function of claudin-15 channels. In contrast, in claudin-10b, corresponding (claudin-15b-mimicking) K64W and K64M substitutions disturbed integration into tight junction and slightly altered relative permeabilities for differently sized monovalent cations. Removal of claudin-10b-specific negative charge (D36A substitution) was without effect. The data suggest that a common tetra-aspartate ring (D55/D56) in pore center of claudin-15/-10b channels directly attracts cations, while E64/D65 may be at least partly shielded by W63/K64. Charge at position W63/K64 affects assembly and properties for claudin-10b but not for claudin-15 channels. Our findings add to the mechanistic understanding of the determinants of paracellular cation permeability.

Yersinia enterocolitica induces epithelial barrier dysfunction through regional tight junction changes in colonic HT-29/B6 cell monolayers.

Yersinia enterocolitica is a common cause of acute gastroenteritis. This study aimed to clarify the mechanisms leading to barrier dysfunction and diarrhea. Exposure of human colonic HT-29/B6 cells to Y. enterocolitica resulted in a decrease in transepithelial resistance from 404±23 to 163±21 Ω cm² (P<0.001) in parallel with an increase in mannitol (182 Da) and fluorescein (332 Da) permeability, whereas short circuit current did not change. This effect was time dependent, required the presence of living bacteria, could not be triggered by bacterial supernatants and was not due to Yersinia outer proteins. Concomitantly, Y. enterocolitica induced necrosis as indicated by an increase in lactate dehydrogenase-release, whereas epithelial apoptosis was not upregulated. Local changes in conductivity were detected by conductance scanning, indicating 'leaky regions' within the epithelium that were visualized by biotinylation and confocal microscopy. In these regions, claudin-3 and -4 and, especially claudin-8, were redistributed off the tight junction (TJ) into the cytoplasm. In addition, the expression of claudin-2, -3, -8, -10 and ZO-1 was diminished as quantified by immunoblotting. Moreover, we found claudin-8 to be regulated by the c-Jun N-terminal kinase, the inhibition of which attenuated the Y. enterocolitica-induced decrease in transepithelial resistance and restored claudin-8 protein level. In conclusion, barrier dysfunction in Y. enterocolitica infection is due to circumscribed epithelial TJ protein changes and necrotic cell loss, as a consequence of which leak flux diarrhea and antigen-uptake provoking extraintestinal arthritis may be triggered.

Transforming growth factor-ß, a whey protein component, strengthens the intestinal barrier by upregulating claudin-4 in HT-29/B6 cells.

TGFß (isoforms 1-3) has barrier-protective effects in the intestine. The mechanisms involved in regulating tight junction protein expression are poorly understood. The aim of this study was to elucidate TGFß-dependent protective effects with special attention to promoter regulation of tight junction proteins using the HT-29/B6 cell model. In addition, the effects of whey protein concentrate 1 (WPC1), a natural source of TGFß in human nutrition, were examined. For this purpose, the claudin-4 promoter was cloned and tested for its activity. It exhibited transactivation in response to TGFß1, which was intensified when Smad-4 was cotransfected, indicating a Smad-4-dependent regulatory component. Shortening and mutation of the promoter altered and attenuated this effect. WPC1 induced an increase in the claudin-4 protein level and resistance of HT-29/B6 cell monolayers. Anti-TGFß(1-3) antibodies blocked these whey protein effects, suggesting that a main part of this function was mediated through TGFß. This effect was observed on intact monolayers as well as when barrier function was impaired by preexposure to IFNγ. In conclusion, TGFß1 affects claudin-4 gene expression via Smad-4-dependent and -independent transcriptional regulation, resulting in barrier protection, a cytokine effect that is also found in whey protein concentrates used in enteral nutrition.

Escherichia coli Alpha-Hemolysin HlyA Induces Host Cell Polarity Changes, Epithelial Barrier Dysfunction and Cell Detachment in Human Colon Carcinoma Caco-2 Cell Model via PTEN-Dependent Dysregulation of Cell Junctions.

Escherichia coli (E. coli) of the B2 phylotype reside in human and animal intestines. The bacteria possess pathogenicity factors such as α-hemolysin (HlyA) that can induce intestinal epithelial leaks. We addressed the questions which host cell processes were dysregulated by E. coli HlyA that can potentiate intestinal diseases. The colon carcinoma cell line Caco-2 was infected by HlyA+ E. coli. Cell polarity regulation was analyzed by live cell imaging for the phosphatidylinositol-4,5-bisphosphate (PIP2) abundance. In Caco-2 monolayers, transepithelial electrical resistance was measured for characterization of barrier function. Cell proliferation and separation were assessed microscopically. Epithelial regulation and cell signaling were analyzed by RNA-Seq and Ingenuity Pathway Analysis (IPA). Our main findings from E. coli HlyA toxinogenicity in the colon carcinoma cell line are that (i) PIP2 at the membrane decrease, (ii) PTEN (phosphatase and tensin homolog) inhibition leads to cell polarity changes, (iii) epithelial leakiness follows these polarity changes by disruption of cell junctions and (iv) epithelial cell detachment increases. HlyA affected pathways, e.g., the PTEN and metastasis signaling, were identified by RNA-Seq bioinformatics calculations in IPA. In conclusion, HlyA affects cell polarity, thereby inducing epithelial barrier dysfunction due to defective tight junctions and focal leak induction as an exemplary mechanism for leaky gut.

Glucocorticoids and tumor necrosis factor-alpha synergize to induce absorption by the epithelial sodium channel in the colon.

BACKGROUND & AIMS: The epithelial sodium channel (ENaC) mediates electrogenic sodium absorption in distal colon. In patients with inflammatory bowel disease (IBD), ENaC induction is impaired, mainly through transcriptional suppression by proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha. Glucocorticoid therapy rapidly increases sodium absorption; we investigated the molecular mechanisms underlying the interaction among TNF-alpha, glucocorticoids, and ENaC induction. METHODS: ENaC-mediated sodium transport in glucocorticoid receptor (GR)-expressing HT-29/B6 cells and rat distal colon, under the influence of the synthetic glucocorticoid dexamethasone and TNF-alpha, was quantified in Ussing chambers. ENaC messenger RNA (mRNA) levels were monitored by real-time polymerase chain reaction. GR transactivation and expression were investigated by gene reporter, immunoblot, and confocal immunofluorescence microscopy analyses. The GR mRNA half-life was determined. Signaling pathways were characterized using mitogen-activated protein kinase inhibitors. RESULTS: Dexamethasone not only prevented TNF-alpha-mediated ENaC suppression but caused synergistic induction of ENaC-dependent sodium absorption in HT-29/B6-GR cells and rat distal colon. This synergy resulted from TNF-alpha-mediated increases in GR protein levels because of GR mRNA stabilization and subsequent GR transactivation by dexamethasone. As a consequence, transcription of the ENaC beta- and gamma-subunits was up-regulated, increasing ENaC-dependent sodium absorption. p38 Mitogen-activated protein kinase is required for this synergistic effect: p38 inhibition blocked the increase in GR protein expression and ENaC-dependent sodium absorption. CONCLUSIONS: TNF-alpha and dexamethasone induce ENaC, explaining the rapid and intense proabsorptive effect of glucocorticoid therapies.

Cnksr3 is a direct mineralocorticoid receptor target gene and plays a key role in the regulation of the epithelial sodium channel.

Aldosterone is the principal hormonal regulator of sodium homeostasis in vertebrates. It exerts its actions through the mineralocorticoid receptor (MR) that regulates the transcription of specific target genes. In recent years, a number of MR target genes have been identified that are involved in the regulation of the epithelial sodium channel (ENaC), a key modulator of renal sodium absorption. Here we report the identification of cnksr3 as a direct MR target gene that is up-regulated in response to physiological concentrations of aldosterone. The cnksr3 promoter exhibits two functional aldosterone-responsive regions, which were bound by the MR as assessed by chromatin immunoprecipitation (ChIP). In vivo, CNKSR3 was highly expressed in the renal cortical collecting duct (CCD), the prime target segment of aldosterone-regulated sodium retention in the kidney. CCD cell lines stably overexpressing or silencing CNKSR3 were electrophysiologically analyzed and show that CNKSR3 expression correlated with and is required for ENaC-mediated transepithelial sodium transport. In parallel, CNKSR3 expression led to decreased MEK phosphorylation. We conclude that CNKSR3, a homologue of scaffold proteins involved in MAPK pathway regulation, is a direct target of MR and is required for the maintenance of transepithelial sodium transport in the kidney.

Zinc prevents intestinal epithelial barrier dysfunction induced by alpha-hemolysin-producing Escherichia coli 536 infection in porcine colon.

Zinc treatment is beneficial for infectious diarrhea or colitis. This study aims to characterize the pathomechanisms of the epithelial barrier dysfunction caused by alpha-hemolysin (HlyA)-expressing Escherichia coli in the colon mucosa and the mitigating effects of zinc ions. We performed Ussing chamber experiments on porcine colon epithelium and infected the tissues with HlyA-producing E. coli. Colon mucosa from piglets was obtained from a feeding trial with defined normal or high dose zinc feeding (pre-conditioning). Additional to the zinc feeding, zinc was added to the luminal compartment of the Ussing chamber. Transepithelial electrical resistance (TER) was measured during the infection of the living tissue and subsequently the tissues were immuno-stained for confocal microscopy. Zinc applied to the luminal compartment was effective in preventing from E. coli-induced epithelial barrier dysfunction in Ussing chamber experiments. In contrast, zinc pre-conditioning of colon mucosae when zinc ions were missing subsequently in the luminal compartment was not sufficient to prevent epithelial barrier impairment during E. coli infection. The pathological changes caused by E. coli HlyA were alterations of tight junction proteins claudin-4 and claudin-5, focal leak formation, and cell exfoliation which reflected the paracellular barrier defect measured by a reduced TER. In microscopic analysis of luminal zinc-treated mucosae these changes were absent. In conclusion, continuous presence of unbound zinc ions in the luminal compartment is essential for the protective action of zinc against E. coli HlyA. This suggests the usage of zinc as therapeutic regimen, while prophylactic intervention by high dietary zinc loads may be less useful.