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.

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.

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.

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.

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.

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.

Phospholipid effects on SGLT1-mediated glucose transport in rabbit ileum brush border membrane vesicles.

In small intestine, sodium-glucose cotransporter SGLT1 provides the main mechanism for sugar uptake. We investigated the effect of membrane phospholipids (PL) on this transport in rabbit ileal brush border membrane vesicles (BBMV). For this, PL of different charge, length, and saturation were incorporated into BBMV. Transport was measured related to (i) membrane surface charge (membrane-bound MC540 fluorescence), (ii) membrane thickness (PL incorporation of different acyl chain length), and (iii) membrane fluidity (r12AS, fluorescence anisotropy of 12-AS). Compared to phosphatidylcholine (PC) carrying a neutral head group, inhibition of SGLT1 increased considerably with the acidic phosphatidic acid (PA) and phosphatidylinositol (PI) that increase membrane negative surface charge. The order of PL potency was PI>PA > PE = PS > PC. Inhibition by acidic PA-oleate was 5-times more effective than with neutral PE (phosphatidylethanolamine)-oleate. Lineweaver-Burk plot indicated uncompetitive inhibition of SGLT1 by PA. When membrane thickness was increased by neutral PC of varying acyl chain length, transport was increasingly inhibited by 16:1 PC to 22:1 PC. Even more pronounced inhibition was observed with mono-unsaturated instead of saturated acyl chains which increased membrane fluidity (indicated by decreased r12AS). In conclusion, sodium-dependent glucose transport of rabbit ileal BBMV is modulated by (i) altered membrane surface charge, (ii) length of acyl chains via membrane thickness, and (iii) saturation of PL acyl chains altering membrane fluidity. Transport was attenuated by charged PL with longer and unsaturated acyl residues. Alterations of PL may provide a principle for attenuating dietary glucose uptake.

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.

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 fetus impairs barrier function in HT-29/B6 cells through focal tight junction alterations and leaks.

Infections by Campylobacter species are the most common foodborne zoonotic disease worldwide. Campylobacter jejuni and C. coli are isolated most frequently from human stool samples, but severe infections by C. fetus (Cf), which can cause gastroenteritis, septicemia, and abortion, are also found. This study aims at the characterization of pathological changes in Cf infection using an intestinal epithelial cell model. The Cf-induced epithelial barrier defects appeared earlier than those of avian Campylobacter species like C. jejuni/C. coli. Two-path impedance spectroscopy (2PI) distinguished transcellular and paracellular resistance contributions to the overall epithelial barrier impairment. Both transcellular and paracellular resistance of Cf-infected HT-29/B6 monolayers were reduced. The latter was attributed to activation of active anion secretion. Western blot analysis showed no decrease in tight junction (TJ) protein expression (claudin-1, -2, -3, and -4) but showed redistribution of claudin-1 off the TJ domain. In addition, Cf induced epithelial cell death, cell detachment, and lesions (focal leaks), as the result of which macromolecule flux (10-kDa dextran) was increased in Cf-invaded cell monolayers. In conclusion, barrier dysfunction from Cf infection was due to TJ protein redistribution, cell death induction, and leak formation, resulting in bacterial translocation, ion leak flux, and antigen uptake (leaky gut).

Tight junction strand formation by claudin-10 isoforms and claudin-10a/-10b chimeras.

Claudins are integral components of tight junctions (TJs) in epithelia and endothelia. When expressed in cell lines devoid of TJs, claudins are able to form TJ-like strands at contacts between adjacent cells. According to a current model of TJ strand formation, claudin protomers assemble in an antiparallel double row within the plasma membrane of each cell (cis-interaction) while binding to corresponding double rows from the neighboring cells (trans-interaction). Cis-interaction was proposed to involve two interfaces of the protomers' first extracellular segment (extracellular loop (ECL)1). In the current study, three naturally occurring claudin-10 isoforms and two claudin-10 chimeras were used to investigate strand formation. All constructs were able to interact in cis (Förster/fluorescence resonance energy transfer (FRET)), to integrate into TJs of MDCK-C7 cells (confocal laser scanning microscopy), and to form TJ-like strands in HEK293 cells (freeze-fracture electron microscopy). Strand formation occurred despite the fact that isoform claudin-10a_i1 lacks both structural ECL1 elements reported to be crucial for cis-interaction. Furthermore, results from FRET experiments on claudin-10 chimeras indicated that identity of the first transmembrane region rather than ECL1 is decisive for claudin-10 cis-interaction. Therefore, in addition to the interaction interfaces suggested in the current model for TJ strand assembly, alternative interfaces must exist.

Epithelial barrier dysfunction in lymphocytic colitis through cytokine-dependent internalization of claudin-5 and -8.

BACKGROUND: Watery diarrhea is the cardinal symptom of lymphocytic colitis (LC). We have previously shown that colonic Na malabsorption is one of the major pathologic alterations of LC and found evidence for an epithelial barrier defect. On these grounds, this study aimed to identify the inherent mechanisms of this epithelial barrier dysfunction and its regulatory features. METHODS: Epithelial resistance (R epi) was determined by one-path impedance spectroscopy and 3H-mannitol fluxes were performed on biopsies from sigmoid colon in miniaturized Ussing chambers. Tight junction proteins were analyzed by Western blot and confocal microscopy. Inflammatory signaling was characterized in HT-29/B6 cells. Apoptosis and mucosal surface parameters were quantified morphologically. RESULTS: R epi was reduced to 53% and 3H-mannitol fluxes increased 1.7-fold in LC due to lower expression of claudin-4, -5, and -8 and altered subcellular claudin-5 and -8 distributions off the tight junction. TNFα and IFNγ could mimic subcellular redistribution in HT-29/B6 cells, a process which was independent on MLCK activation. Epithelial apoptosis did not contribute to barrier dysfunction in LC and mucosal surface area was unchanged. CONCLUSIONS: Epithelial barrier dysfunction in LC occurs through downregulation of claudin-4, -5, and -8, and redistribution of claudin-5 and -8 off the tight junction, which contributes to diarrhea by a leak-flux mechanism. The key effector cytokines TNFα and IFNγ turned out to be the trigger for redistribution of claudin-5 and -8. Thus, alongside sodium malabsorption, leak-flux is yet another important diarrheal mechanism in LC.

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.

ENaC Dysregulation Through Activation of MEK1/2 Contributes to Impaired Na+ Absorption in Lymphocytic Colitis.

BACKGROUND: Lymphocytic colitis (LC) causes watery diarrhea. We aimed to identify mechanisms of altered Na absorption and regulatory inputs in patients with LC by examining the epithelial Na channel (ENaC) function as the predominant Na transport system in human distal colon. METHODS: Epithelial Na channel function and regulation was analyzed in biopsies from sigmoid colon of patients with LC and in rat distal colon in Ussing chambers. ENaC-subunit expression was measured by real-time PCR and RNA sequencing. Correction factors for subepithelial resistance contributions were determined by impedance spectroscopy. Upstream regulators in LC were determined by RNA sequencing. RESULTS: Epithelial Na channel-mediated electrogenic Na transport was inhibited despite aldosterone stimulation in human sigmoid colon of patients with LC. The increase in γ-ENaC mRNA expression in response to aldosterone was MEK1/2-dependently reduced in LC, since it could be restored toward normal by MEK1/2 inhibition through U0126. Parallel experiments for identification of signaling in rat distal colon established MEK1/2 to be activated by a cytokine cocktail of TNFα, IFNγ, and IL-15, which were identified as the most important regulators in the upstream regulator analysis in LC. CONCLUSIONS: In the sigmoid colon of patients with LC, the key effector cytokines TNFα, IFNγ, and IL-15 inhibited γ-ENaC upregulation in response to aldosterone through a MEK1/2-mediated pathway. This prevents ENaC to reach its maximum transport capacity and results in Na malabsorption which contributes to 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.

The plant-derived glucocorticoid receptor agonist Endiandrin A acts as co-stimulator of colonic epithelial sodium channels (ENaC) via SGK-1 and MAPKs.

In a search for secondary plant compounds that bind to the glucocorticoid receptor (GR), the cyclobutane lignan endiandrin A was discovered from the rainforest tree Endiandra anthropophagorum Domin. Our present study aims to characterize the effect of endiandrin A on GR-dependent induction of colonic sodium transport. The effect of endiandrin A was analyzed in GR-expressing colonic HT-29/B6 cells (HT-29/B6-GR). GR transactivation and subcellular localization were investigated by reporter gene assay and immunofluorescence. Epithelial sodium channel (ENaC) was analyzed by qRT-PCR and by measuring amiloride-sensitive short-circuit current (I(sc)) in Ussing chambers. Endiandrin A (End A) has been identified as GR receptor binder. However, it did not cause significant GR transactivation as pGRE-luciferase activity was only 7% of that of the maximum effect of dexamethasone. Interestingly, endiandrin A had a significant impact on dexamethasone-dependent sodium absorption in cells co-exposed to tumor necrosis factor (TNF)-α. This was in part due to up-regulation of ß- and γ-ENaC subunit expression. Endiandrin A potentiated GR-mediated transcription by increasing GR protein expression and phosphorylation. It inhibited c-Jun N-terminal kinase (JNK) activation induced by dexamethasone and/or TNF-α and increased levels of GR localized to the nucleus. Additionally, endiandrin A increased the serum- and glucocorticoid-induced kinase (sgk)-1 via activation of p38. Finally, the regulation of ENaC function by endiandrin A was confirmed in rat native colon. In conclusion, endiandrin A potentiates glucocorticoid-driven activation of colonic epithelial sodium channels via JNK inhibition and p38 activation due to transcriptional up-regulation of ß- and γ-ENaC-subunits along with induction of sgk-1.

Ion transport and barrier function are disturbed in microscopic colitis.

In this paper, we identify mechanisms of watery diarrhea in microscopic colitis (MC). Biopsies from the sigmoid colon of patients with collagenous colitis and treated lymphocytic colitis were analyzed in miniaturized Ussing chambers for electrogenic sodium transport and barrier function with one-path impedance spectroscopy. Cytometric bead arrays (CBA) served to analyze cytokine profiles. In active MC, electrogenic sodium transport was diminished and epithelial resistance decreased. CBA revealed a Th1 cytokine profile featuring increased IFN-γ, TNF-α, and IL-1ß levels. After four weeks of steroid treatment with budesonide, electrogenic sodium transport recovered while epithelial barrier defects remained. Diarrhea in MC results at least in part from a combination of impaired electrogenic sodium transport and barrier defects. From a therapeutic perspective it can be postulated that the functional importance of loss of ions may be higher than that caused by barrier impairment.