Transepithelial Leak Research Articles

The multiphasic TNF-α-induced compromise of Calu-3 airway epithelial barrier function

Purpose: Airway epithelial barrier leak and the involvement of proinflammatory cytokines play a key role in a variety of diseases. This study evaluates barrier compromise by the inflammatory mediator Tumor Necrosis Factor-α (TNF-α) in the human airway epithelial Calu-3 model. Methods: We examined the effects of TNF-α on barrier function in Calu-3 cell layers using Transepithelial Electrical Resistance (TER) and transepithelial diffusion of radiolabeled probe molecules. Western immunoblot analyses of tight junctional (TJ) proteins in detergent soluble fractions were performed. Results: TNF-α dramatically reduced TER and increased paracellular permeability of both 14C-D-mannitol and the larger 5 kDa probe, 14C-inulin. A time course of the effects shows two separate actions on barrier function. An initial compromise of barrier function occurs 2-4 hours after TNF-α exposure, followed by complete recovery of barrier function by 24 hrs. Beginning 48 hrs. post-exposure, a second more sustained barrier compromise ensues, in which leakiness persists through 144 hrs. There were no changes in TJ proteins observed at 3 hrs. post exposure, but significant increases in claudins-2, -3, -4, and -5, as well as a decrease in occludin were seen at 72 hrs. post TNF-α exposure. Both the 2-4 hr. and the 72 hr. TNF-α induced leaks are shown to be mediated by the ERK signaling pathway. Conclusion: TNF-α induced a multiphasic transepithelial leak in Calu-3 cell layers that was shown to be ERK mediated, as well as involve changes in the TJ complex. The micronutrients, retinoic acid and calcitriol, were effective at reducing this barrier compromise caused by TNF-α. The significance of these results for airway disease and for COVID-19 specifically are discussed.

Simple graphical approach to investigate differences in transepithelial paracellular leak pathway permeability.

Although many studies have reported differences in epithelial paracellular Leak Pathway permeability following genetic manipulations and treatment with various agents, the basis for these differences remains mostly unclear. Two primary mechanisms which could underlie differences in Leak Pathway permeability are differences in the density of Leak Pathway openings and differences in the opening size. Using a computational approach, we demonstrate that these two possibilities can be readily distinguished graphically by comparing the apparent paracellular permeabilities of a size panel of solutes measured across different cell layers. Using this approach, we demonstrated that depletion of ZO‐1 protein in MDCK Type II renal epithelial cells decreased Leak Pathway opening size and increased opening density. Depletion of ZO‐2 protein either had no effect or minimally decreased opening size and did not markedly change opening density. Comparison of MDCK Type II cells with MDCK Type I cells revealed that Type I cells exhibited a substantially smaller Leak Pathway permeability than did Type II cells. This lower permeability was due to a decrease in opening density with little or no change in opening size. These results demonstrate the utility of this approach to provide insights into the basis for observed differences in epithelial Leak Pathway permeability. This approach has wide applications including analysis of the molecular basis for Leak Pathway permeability, the effects of specific manipulations on Leak Pathway permeability properties, and the effects of permeation enhancers on Leak Pathway permeability properties.

Cobalt chloride compromises transepithelial barrier properties of CaCo-2 BBe human gastrointestinal epithelial cell layers

BackgroundElevation of the transcription factor HIF-1 is a prominent mediator of not only processes that accompany hypoxia, but also the tumor microenvironment and tissue regeneration. This study uses mediators of “chemical hypoxia” to ask the question whether HIF-1α elevation in a healthy epithelial cell layer leads to leakiness in its tight junctional seals.MethodsTransepithelial electrical resistance and transepithelial diffusion of 14C–D-mannitol and other radiolabeled probes are used as indicators of transepithelial barrier function of CaCo-2 BBe human gastrointestinal epithelial cell layers cultured on permeable supports. Western immunoblot analyses of integral tight junctional proteins (occludin and claudins) are used as further indicators of barrier function change.ResultsCobalt, an inhibitor of the prolyl hydroxylase enzymes governing HIF-1α breakdown in the cell, induces transepithelial leakiness in CaCo-2 BBe cell layers in a time and concentration-dependent manner. This increased leakiness is accompanied by significant changes in certain specific integral tight junctional (TJ) proteins such as a decreased level of occludin and increased level of claudin-5. Similar results regarding barrier function compromise also occur with other chemical inhibitors of HIF-1α breakdown, namely ciclopiroxolamine (CPX) and dimethyloxalylglycine (DMOG). The increased leak is manifested by both decreased transepithelial electrical resistance (Rt) and increased paracellular diffusion of D-mannitol (Jm). The induced transepithelial leak shows significant size selectivity, consistent with induced effects on TJ permeability. Less-differentiated cell layers were significantly more affected than well-differentiated cell layers regarding induced transepithelial leak. A genetically modified CaCo-2 variant with reduced levels of HIF-1β, showed reduced transepithelial leak in response to cobalt exposure, further indicating that elevation of HIF-1α levels induced by agents of “chemical hypoxia” is responsible for the compromised barrier function of the CaCo-2 BBe cell layers.ConclusionsExposure to inducers of chemical hypoxia elevated HIF-1α levels and increased transepithelial leak. The degree of epithelial differentiation has significant effects on this action, possibly explaining the varying effects of HIF-1 modulation in epithelial and endothelial barrier function in different physiological and pathophysiological conditions.

Sieving characteristics of cytokine- and peroxide-induced epithelial barrier leak: Inhibition by berberine.

To study whether the inflammatory bowel disease (IBD) colon which exhibits varying severity and cytokine levels across its mucosa create varying types of transepithelial leak. We examined the effects of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1-β (IL1β) and hydrogen peroxide (H2O2) - singly and in combinations - on barrier function of CACO-2 cell layers. Our focus was on the type (not simply the magnitude) of transepithelial leak generated by these agents as measured by transepithelial electrical resistance (TER) and transepithelial flux of (14)C-D-mannitol, (3)H-Lactulose and (14)C-Polyethylene glycol as radiolabeled probe molecules. The isoquinoline alkaloid, berberine, was then examined for its ability to reduce specific types of transepithelial leak. Exposure to TNF-α alone (200 ng/mL; 48 h) induced a 50% decrease in TER, i.e., increased leak of Na(+) and Cl(-) - with only a marginal but statistically significant increase in transepithelial leak of (14)C-mannitol (Jm). Exposure to TNF-α + IFN-γ (200 ng/mL; 48 h) + IL1β (50 ng/mL; 48 h) did not increase the TER change (from TNF-α alone), but there was now a 100% increase in Jm. There however was no increase in transepithelial leak of two larger probe molecules, (3)H-lactulose and (14)C-polyethylene glycol (PEG). However, exposure to TNF-α + IFN-γ + IL1β followed by a 5 h exposure to 2 mmol/L H2O2 resulted in a 500% increase in (14)C-PEG leak as well as leak to the luminal mitogen, epidermal growth factor. This model of graded transepithelial leak is useful in evaluating therapeutic agents reducing IBD morbidity by reducing barrier leak to various luminal substances.

Transepithelial leak in Barrett's esophagus patients: The role of proton pump inhibitors

To determine if the observed paracellular sucrose leak in Barrett's esophagus patients is due to their proton pump inhibitor (PPI) use. The in vivo sucrose permeability test was administered to healthy controls, to Barrett's patients and to non-Barrett's patients on continuous PPI therapy. Degree of leak was tested for correlation with presence of Barrett's, use of PPIs, and length of Barrett's segment and duration of PPI use. Barrett's patients manifested a near 3-fold greater, upper gastrointestinal sucrose leak than healthy controls. A decrease of sucrose leak was observed in Barrett's patients who ceased PPI use for 7 d. Although initial introduction of PPI use (in a PPI-naïve population) results in dramatic increase in sucrose leak, long-term, continuous PPI use manifested a slow spontaneous decline in leak. The sucrose leak observed in Barrett's patients showed no correlation to the amount of Barrett's tissue present in the esophagus. Although future research is needed to determine the degree of paracellular leak in actual Barrett's mucosa, the relatively high degree of leak observed with in vivo sucrose permeability measurement of Barrett's patients reflects their PPI use and not their Barrett's tissue per se.

Proton Pump Inhibitor-Induced Transepithelial Leak in Gastric Mucosa: Decrease of Leak with Long-Term PPI Use

Purpose: In previous patient-based studies our group has shown that proton pump inhibitors (PPIs) induce a transepithelial leak in gastric mucosa (Mullin, et al. 2008). This leak seemingly occurs without the onset of tissue inflammation or damage as assessed endoscopically. Using rat gastric corpus mucosa in Ussing chamber studies, we observed that PPI-induced leak is bidirectional and has a size limit of 10 kDa for solutes than can permeate through it (Murray, et al. 2009). We also observed that whereas the leak allows for charged as well as uncharged molecules to permeate, it exhibits a surprising degree of structural specificity in the types of molecules that can pass (Gabello, et al. 2009). Two small molecule drugs, digoxin and phenytoin, showed surprisingly different patterns, with digoxin able to permeate but phenytoin unable to permeate. We likewise observed that the small peptide bradykinin, can permeate, but the small peptide, oxytocin, cannot (Gabello, et al. 2010). Concerning kinetics, a (standard) 40 mg dose of esomeprazole is required for five days before a statistically significant leak is observed clinically. However, in rat gastric corpus Ussing chamber studies, exposure to 25 μM omeprazole results in instantaneous leak. The current study aimed to assess the long term effect of PPI use on transepithelial gastric leak. Methods: Subjects were enrolled with up to 20 years duration of PPI usage. To quantify transepithelial leak, subjects drank a concentrated sucrose solution followed by overnight collection of urine with subsequent measurement of sucrose concentration. Results: We observed that the PPI-induced leak effect decreases spontaneously in the first two years of PPI usage. Most of the decrease in leak is occurring in the first 10 months of PPI use. The phenomenon therefore exhibits tachyphyllaxis, self-correcting in the continued presence of the PPI. A linear regression of the sucrose leak in mg of sucrose vs. months on PPIs for 41 patients shows a declining slope (m=-0.8) with a y intercept of 220 mg and an r2 of 0.07. If the data are grouped as PPI use less than 3 months (n=11) and greater than 3 months (n=30) the mean values for leak are 330 mg and 130 mg respectively (P < 0.0003, Student's t). Conclusion: While PPI-induced trans-epithelial sucrose leak is present, it appears to decrease as a function of time. Most of this decrease occurs in the first 10 months of usage. Statistical significance of this decrease in leak was demonstrated based on whether patients had been taking PPIs for less than 3 months or greater than 3 months.

M1889 Omeprazole-Induced Transepithelial Leak in Gastric Mucosa: Implications for Small Molecule Drugs and Peptides

M1889 Omeprazole-Induced Transepithelial Leak in Gastric Mucosa: Implications for Small Molecule Drugs and Peptides

Proton pump inhibitors: actions and reactions

Proton pump inhibitors are the second most commonly prescribed drug class in the United States. The increased utilization of PPIs parallels the rising incidence of reflux disease. Owing to their clinical efficacy and relative lack of tachyphylaxis, PPIs have largely displaced H-2 receptor antagonists in the treatment of acid peptic disorders. The elevation of intragastric pH and subsequent alterations of gastric physiology induced by PPIs may yield undesired effects within the upper GI tract. The ubiquity of the various types of H(+), K(+)-ATPase could also contribute to non-gastric effects. PPIs may influence physiology in other ways, such as inducing transepithelial leak.

Omeprazole Induces a Transepithelial Leak in Gastric Mucosa

Purpose: Having observed a paracellular leak to oral sucrose in esophagitis patients, we tested if PPI therapy would decrease (paracellular) sucrose leak by allowing for mucosal healing. Methods: 23 study subjects presenting to their primary care physician with symptoms of GERD were placed on an 8 week course of esomeprazole (40 mg daily). Subjects were PPI and H-2 blocker naive. All subjects reported symptom alleviation by 4 weeks. Before beginning esomeprazole therapy and at the end of therapy, subjects consumed at bedtime a solution of the disacharide, sucrose (100 gms in 200 cc tap water), and collected an overnight urine specimen. Sucrose, as a disacharide, can enter the bloodstream only by leaking paracellularly out of the upper GI lumen. Once in the blood, sucrose is filtered quantitatively into urine. Sucrose concentration in urine was measured by enzymatic/spectrophotometric means. The sucrose concentration × the total urine volume yields the total amount of sucrose in the urine, which equals the the amount of sucrose which leaked paracellularly from the upper GI lumen. Results: We observed that 17 of 23 patients exhibited a marked increase in sucrose leak by 8 weeks of omeprazole therapy (667% increase). Similar sucrose leak is observed after only 8 days of esomeprazole therapy. Considering all 23 patients, the Wilcoxon Signed Rank Test indicated a significant increase in sucrose leak (P= 0.007). In rat gastric mucosa, 200 μM omeprazole induces an immediate paracellular leak to D-mannitol, with a simultaneous decrease in transepithelial electrical resistance. Both phenomena indicate that omeprazole is likely producing a tight junction leak in gastric mucosa. Conclusion: PPI therapy may be associated with a paracellular leak in the acid secreting region of the stomach. Future studies will examine the characteristics of this leak and the mechanism by which omeprazole induces the leak.

Transepithelial Leak in Barrett’s Esophagus

Using orally administered sucrose as a probe of gastrointestinal permeability, this study focused on determining whether Barrett's metaplasia exhibits a paracellular transepithelial leak to small nonelectrolytes. Subjects in five separate classes (nonendoscoped, asymptomatic controls; endoscoped, asymptomatic controls; gastroesophageal reflux disease without mucosal complications; grossly visible esophagitis; and Barrett's esophagus) consumed a sucrose solution at bedtime and collected all overnight urine. Urine volume was measured and sucrose concentration was determined by high-performance liquid chromatography. Patients with Barrett's were observed to exhibit a transepithelial leak to sucrose whose mean value was threefold greater than that seen in healthy control subjects or patients with reflux but without any mucosal defect. A parallel study of claudin tight junction proteins in endoscopy biopsy samples showed that whereas Barrett's metaplasia contains dramatically more claudin-2 and claudin-3 than is found in normal esophageal mucosa, it is markedly lower in claudins 1 and 5, indicating very different tight junction barriers.

Detection of Barrett's Esophagus by Transepithelial Leak of Sucrose

An inexpensive test for Barrett's esophagus in the general population and among patients with gastroesophageal reflux disease is described. Patients swallow a concentrated solution of sucrose (100 gms in 200 cc of water) at bedtime, and collect any urine voided for 7 hrs. Urine sucrose is determined by HPLC. The amount of sucrose voided (urine volume x sucrose concentration) equals the amount of sucrose that leaked transepithelially out of the upper gastrointestinal (GI) tract. Unlike, glucose or fructose, the disaccharide sucrose cannot transit through cells, since it lacks a membrane transport protein. However any paracellular leak in the upper GI tract will allow sucrose to leave the luminal compartment and enter the vasculature. Once in the bloodstream, without a reabsorptive pathway in the kidney, all sucrose will enter the urine. This test has been well described for diagnosis of gastric ulcer disease (Meddings et al., 1993). A test that could cheaply identify: 1) GERD patients most in need of upper endoscopy to diagnose Barrett's, and 2) Barrett's patients who have undetected dysplasia and are in the need of the most frequent repeat endoscopic surveillance, would be clinically valuable. Our group has shown that precancerous epithelium in colon is characterized by transepithelial leaks in tight junctions (Soler et al., 1998) and that Barrett's metaplasia contains different tight junction proteins than in adjacent squamous esophageal mucosa (Rendon Huerta et al., 2003). Claudin-1 is lower in Barrett's epithelium. Claudin-2, nondetectable in normal esophagus, is apparent in Barrett's epithelium. Occludin is not different in the two epithelia on a per mg protein basis. We hypothesized that the esophageal mucosa of Barrett's patients would be leakier than that of healthy controls or GERD patients. Our results indicate that controls and GERD patients manifest the same degree of sucrose leak (66 mg in urine of controls [n = 15, + 39 std dev] vs 62 mg excreted by GERD patients [n = 9, + 39 std dev]). Barrett's patients exhibit greater sucrose leak (177 mg + 102 std. dev., n = 14) which is significantly different from both prior groups (P<0.0002, Wilcoxon). All enrolled patients were on PPI therapy prior to taking the sucrose. Neither the Barrett's nor the GERD patients had clinically detectable esophagitis. The mechanistic significance for promotion of tumorigenesis, of high permeability across the Barrett's epithelium, is discussed. An inexpensive test for Barrett's esophagus in the general population and among patients with gastroesophageal reflux disease is described. Patients swallow a concentrated solution of sucrose (100 gms in 200 cc of water) at bedtime, and collect any urine voided for 7 hrs. Urine sucrose is determined by HPLC. The amount of sucrose voided (urine volume x sucrose concentration) equals the amount of sucrose that leaked transepithelially out of the upper gastrointestinal (GI) tract. Unlike, glucose or fructose, the disaccharide sucrose cannot transit through cells, since it lacks a membrane transport protein. However any paracellular leak in the upper GI tract will allow sucrose to leave the luminal compartment and enter the vasculature. Once in the bloodstream, without a reabsorptive pathway in the kidney, all sucrose will enter the urine. This test has been well described for diagnosis of gastric ulcer disease (Meddings et al., 1993). A test that could cheaply identify: 1) GERD patients most in need of upper endoscopy to diagnose Barrett's, and 2) Barrett's patients who have undetected dysplasia and are in the need of the most frequent repeat endoscopic surveillance, would be clinically valuable. Our group has shown that precancerous epithelium in colon is characterized by transepithelial leaks in tight junctions (Soler et al., 1998) and that Barrett's metaplasia contains different tight junction proteins than in adjacent squamous esophageal mucosa (Rendon Huerta et al., 2003). Claudin-1 is lower in Barrett's epithelium. Claudin-2, nondetectable in normal esophagus, is apparent in Barrett's epithelium. Occludin is not different in the two epithelia on a per mg protein basis. We hypothesized that the esophageal mucosa of Barrett's patients would be leakier than that of healthy controls or GERD patients. Our results indicate that controls and GERD patients manifest the same degree of sucrose leak (66 mg in urine of controls [n = 15, + 39 std dev] vs 62 mg excreted by GERD patients [n = 9, + 39 std dev]). Barrett's patients exhibit greater sucrose leak (177 mg + 102 std. dev., n = 14) which is significantly different from both prior groups (P<0.0002, Wilcoxon). All enrolled patients were on PPI therapy prior to taking the sucrose. Neither the Barrett's nor the GERD patients had clinically detectable esophagitis. The mechanistic significance for promotion of tumorigenesis, of high permeability across the Barrett's epithelium, is discussed.

Nitric oxide attenuates and xanthine oxidase exaggerates lung damage-induced gut injury.

Aspirated gastric contents can evoke multiorgan failure. We hypothesized that secondary intestinal epithelial dysfunction after lung damage would be mediated by xanthine oxidase (XO) and antagonized by endogenous gut nitric oxide (NO). Isosmotic saline or HCl solutions were instilled intratracheally in anesthetized rats, and intestinal injury was assessed 190 min later by measuring the blood-to-lumen clearance of 51Cr-labeled EDTA (51Cr-EDTA clearance) and gut wall neutrophil population density. Intratracheal HCl increased 51Cr-EDTA clearance, and this transepithelial leak was attenuated by either systemic L-arginine or intraluminal NO and by chronic dietary pretreatment with allopurinol or sodium tungstate. Conversely, lung damage-induced gut leak was exaggerated by NO synthase inhibition or intravenous XO administration. Intratracheal HCl also increased intestinal wall neutrophil density and myeloperoxide activity. We conclude that two enzymatic systems involved in remote gut barrier dysfunction after endobronchial acidification are XO as mediator and NO synthase as antagonist.

Polarity and kinetics of Na(+)-H+ exchange in cultured opossum kidney cells

Opossum kidney (OK) cells (an established cell line) were loaded with 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF; a fluorescent dye with a pH-sensitive spectrum), and intracellular pH (pHi) was examined by microfluorometry. Single cells, within a confluent monolayer and grown on a permeant support, were examined for the mechanism of recovery from an acid load as imposed by exposure to ammonium chloride (NH4 prepulse). The Na(+)-dependent recovery of pHi from an acid load (Na(+)-H+ exchange) is examined in terms of the Na+ activation kinetics of the recovery and the polarity of the response. In 80% of the cells examined (33/41), both apical and basolateral Na+ cause recovery from an acid load. The response of cells to apical Na+ is well fit by Michaelis-Menten kinetics [Kt(Na) = 35 mM], but the response to basolateral Na+ is not. The response to basolateral Na+ addition is modeled in terms of variable transepithelial leak of Na+ and variable amounts of basolateral Na(+)-H+ exchange. Despite an average response to basolateral (145 mM) Na+ that is 34% of the response to apical Na+, modeling suggests that basolateral Na(+)-H+ exchange must be less than 10% of the cellular total to fit the basolateral Na+ activation kinetics. The model, and experiments using ordered addition of Na+ from the apical vs. basolateral medium, also suggest that transepithelial leak (of basolateral Na+ to the apical compartment) is required to explain the pHi recovery observed due to addition of basolateral Na+. Direct estimation of (basolateral to apical) transepithelial leak demonstrates that the response due to basolateral Na+ addition is explained by transepithelial leak and a Na(+)-H+ exchange that is expressed solely in the apical membrane.