Epithelial barrier defects in HT-29/B6 colonic cell monolayers induced by tumor necrosis factor-alpha.

The barrier function of intestinal epithelia relies upon the continuity of the enterocyte monolayer and intact tight junctions. After incubation with tumor necrosis factor-alpha TNF-alpha, however, the number of strands that form the tight junctions decreases, and apoptosis is induced in intestinal epithelial cells. These morphological changes lead to a rise of transepithelial ion permeability, because the paracellular ion permeability increases and leaks associated with sites of apoptosis increase by number and magnitude. Thus apoptosis and degradation of tight junctions contribute to the increased permeability observed after exposure to TNF-alpha. These mechanisms explain clinical manifestations in the inflamed intestinal wall containing cytokine-secreting macrophages--for example, leak flux diarrhea and invasion of bacterial enterotoxins.

Altered tight junction structure contributes to the impaired epithelial barrier function in ulcerative colitis.

BACKGROUND & AIMS: Mechanisms of diarrhea in ulcerative colitis (UC) are still unknown. Functional and structural characterization of epithelial barrier and transport properties in ulcerative colitis (UC) was performed. METHODS: Inflamed sigmoid colon epithelium from UC patients was studied by alternating current impedance analysis to determine the pure epithelial resistance as a measure of intestinal barrier function. Tight junction (TJ) structure was investigated by freeze-fracture electron microscopy. RESULTS: Although total wall resistance was reduced in UC by 50%, impedance analysis uncovered a much more pronounced barrier defect. Epithelial resistance decreased from 95 +/- 5 to 20 +/- 3 omega3. cm2, which in conventional analysis is masked by an increase in subepithelial resistance from 14 +/- 1 to 36 +/- 3 omega3. cm2 caused by inflammation. This was paralleled by a change in epithelial cell TJ structure in UC. Strand count decreased from 6.94 +/- 0.25 to 4.76 +/- 0.47 at the surface and from 7.26 +/- 0.31 to 5.46 +/- 0.37 in the crypts. CONCLUSIONS: The inflamed colonic mucosa in UC has an impaired barrier function that is much more pronounced than previously assumed. An altered TJ structure contributes to this barrier defect which, because of increased back leak, can reduce net ion transport. Thus, a leak-flux mechanism contributes to the diarrhea in UC.

Emergency department hemodialysis in a case of severe ethylene glycol poisoning.

A 36-year-old man with a history of depression presented to the emergency department after ingesting approximately 3,000 mL of ethylene glycol antifreeze in a suicide attempt. The patient's ethylene glycol concentration, 1,889 mg/dL, was higher than any level previously documented in the medical literature. Although his course was complicated by nausea, emesis, lethargy, metabolic acidosis, and kidney failure, the patient survived without persistent kidney failure or other chronic problems. Sustained hemodialysis and ethanol infusion were instituted in the ED, on the basis of the patient's history, before laboratory confirmation of the ingestion was obtained.

Tumor necrosis factor-alpha (TNFalpha) regulates the epithelial barrier in the human intestinal cell line HT-29/B6.

Cytokines are supposed to be mediators in diarrhoeal diseases. The aim of this study is to characterize the effect of tumor necrosis factor-alpha (TNFalpha) on epithelial barrier function in the colonic epithelial cell line HT-29/B6. Active ion transport and barrier function were measured as short-circuit current and transepithelial electrical resistance (Rt), respectively. In parallel, freeze-fracture electron microscopy (EM) of tight junctions (TJ) and immunofluorescence microscopy of the zonula occludens protein-1 (ZO-1) were performed. Serosal addition of TNF(alpha) (100 ng/ml) decreased Rt by 81%. This effect was dose-dependent and could be mimicked by antibodies against the p55 form of the TNF receptor. Cytotoxic effects were excluded by a negative lactate dehydrogenase (LDH) assay. Immunofluorescence localization with anti-ZO-1 antibodies revealed no evidence for disruption of the monolayer after TNFalpha treatment. In freeze-fracture EM, TJ complexity was decreased by TNFalpha, as indicated by a decrease in the number of strands from 4.7 to 3.4. The tyrosine kinase blocker genistein and the protein kinase A inhibitor H-8 reduced the effect of TNFalpha. A combination of TNFalpha with interferon-gamma acted synergistically on the epithelial barrier. In conclusion, TNFalpha impairs epithelial barrier function by altering structure and function of the tight junction, which could be of pathogenic relevance in intestinal inflammation.

Epithelial tight junction structure in the jejunum of children with acute and treated celiac sprue.

Tight junction morphology was analyzed in freeze fracture electron micrographs from biopsies at two locations along the surface-crypt axis in the jejunum of children with treated and untreated sprue and in control subjects. In control jejunum, strand number, meshwork depth, and total depth of the tight junction decreased from surface to crypt, consistent with the concept of the crypt being more permeable than the surface epithelium. In acute sprue, strand number was reduced in all regions along the surface-crypt axis, from 5.5+/-0.2 to 3.4+/-0.3 (surface) and from 4.7+/-0.2 to 3.6+/-0.1 (crypt). Meshwork depth was also reduced at all regions along the surface-crypt axis. Strand discontinuities were more frequent in acute sprue. Aberrant strands appeared below the main meshwork of crypt tight junctions in acute sprue. In asymptomatic children treated with the gluten-free diet, jejunal tight junctional structure only partially recovered. Strand number was restored to normal at the surface, but was still decreased in the crypts, from 4.7+/-0.2 to 3.9+/-0.3. We conclude that the epithelial barrier function of the small intestine is seriously disturbed by structural modifications of the tight junction in acute symptomatic celiac disease, thereby accounting for increased ionic permeability noted in a parallel study on identical specimens. This epithelial barrier defect may contribute to diarrhea in celiac disease by a "leak flux mechanism." In children with sprue treated with a gluten-free diet, barrier dysfunction was only partly recovered, suggesting a level of "minimal damage."

Clinical models of intestinal adaptation.

Mucosal adaptation of the small intestine is morphologically restricted to only three different patterns, namely, atrophy, hyperplasia, and hyperregeneration. The hyperplastic mucosa in the experimental short bowel syndrome exhibits unchanged epithelial barrier properties and a differential functional adaptation with a 150% increase in Na-glucose cotransport but no change in electroneutral NaCl cotransport. In the hyperregeneratively transformed mucosa of the self-filling blind loop of rat jejunum, absorption is seriously impaired, as indicated by the 80% decrease in Na-glucose cotransport. To compensate for this, epithelial barrier function is upregulated by an increase in tight junction complexity to prevent leak flux of ions and substrates. In contrast, the hyperregeneratively transformed mucosa in celiac sprue shows reduced tight junction complexity. Possible candidates responsible for the heterogeneity of tight junction adaptation in these conditions could be cytokines, because tumor necrosis factor-alpha can specifically downregulate the tight junction, as indicated in the intestinal HT-29/B6 cell model.

Osmotic water flow pathways across Necturus gallbladder: role of the tight junction.

To explore the quantitative significance of passive water flow through tight junctions of leaky epithelia, transepithelial water flow rates were measured in Necturus gallbladder mounted in chambers. Osmotic flows generated by raffinose gradients were asymmetrical with the greater flow in the mucosal-to-serosal direction. In tissue fixed in situ, intercellular spaces were dilated during mucosal-to-serosal flow and closed during serosal-to-mucosal flow. Tight junctions were focally separated (blistered), which correlated with the magnitude of mucosal-to-serosal flow. Blisters were not observed during serosal-to-mucosal flow or in nontransporting gallbladders. In freeze-fracture replicas, blisters appeared as pockets between intramembranous strands. Protamine, which decreases electrical conductance and increases depth and complexity of the tight junction, reduced osmotic water flow by approximately 30% in the mucosal-to-serosal direction (100 mosmol/kg gradient) without altering serosal-to-mucosal flow. We suggest that in the steady state, at least 30% of osmotically driven water passes transjunctionally in the mucosal-to-serosal direction, but flow is transcellular in the serosal-to-mucosal direction. Directionally divergent pathways may account for flow asymmetry.

Ion transport in the experimental short bowel syndrome of the rat.

The adaptational changes of epithelial ion transport in the short bowel syndrome were studied. Ileal remnants of rats were investigated 8 weeks after 70% proximal small intestinal resection. Pure epithelial resistance measured by impedance analysis decreased from 27 +/- 1 to 21 +/- 1 omega.cm2, and polyethylene glycol 4000 fluxes increased from 2.5 +/- 0.3 to 3.6 +/- 0.3 nmol.h-1.cm-2, indicating increased permeability of the short bowel. Unidirectional flux measurements in control ileum showed absorptive net fluxes of Na+ and Cl- that were assigned to electroneutral NaCl absorption and a short-circuit current that was accounted for by the residual flux (HCO3- secretion). Neither NaCl absorption nor HCO3- secretion were altered in the short bowel. Also, electrogenic Cl- secretion, defined after maximal stimulation by theophylline and prostaglandin E1 was not changed in the short bowel. In contrast, electrogenic Na+/glucose cotransport increased in Vmax from 2.0 +/- 0.3 in controls to 5.0 +/- 1.0 mumol.h-1.cm-2 in the short bowel. Tight junction structure was studied by freeze-fracture electron microscopy. The number of horizontal strands was unchanged, whereas tight junction depth was slightly increased in the short bowel. Microvillus area of short bowels was increased by 20% in villus regions. Under the light microscope, villus height was increased by 30%. In conclusion, the short bowel mucosa undergoes adaptive responses to reduced overall absorptive area by increasing glucose-dependent electrogenic Na+ absorption to 250%, which is partly caused by increased villus and microvillus surface area. Electrogenic Cl- and HCO3- secretion and electroneutral NaCl absorption remained unchanged. The decreased epithelial resistance is caused by mucosal surface amplification.

pH dependence of protamine action on apical membrane permeability in Necturus gallbladder epithelium.

Protamine reversibly decreases cation permeability and alters the structure of Necturus gallbladder tight junctions. Conflicting results, however, have been published whether or not it also affects apical cell membrane permeability. We investigated this issue more systematically by measuring voltage (psi mc) and fractional resistance (fRa) of the apical membrane at varying concentrations of protamine, K+, and H+ in the bathing solution. At pH 7.6 and [K+] 2.5 mM, (Poler, M.S. and Reuss, L. (1987) Am. J. Physiol. 253, C662) 6 microM protamine caused psi mc to depolarize from -58 to -51 mV and fRa to decrease from 0.74 to 0.67. If we increased pH to 8.1 these effects were even more pronounced. At [K+] 2.5 mM, but not 4.5 mM, psi mc transiently hyperpolarized for about 5 min after adding protamine. Most importantly, if [K+] was 4.5 mM and pH was adjusted to 7.1 (Bentzel et al. (1987) J. Membr. Biol. 95, 9) no significant changes of psi mc and fRa occurred. In any case, at a supramaximal concentration of 200 microM, protamine did not further increase the paracellular response but produced decreasing psi mc and fRa. We conclude that 6 microM protamine decreases K+ conductance of the apical membrane, if it is already tuned high by high pH. At low control K+ conductance as observed at lower pH, protamine action is restricted to the paracellular pathway. Thus, conflicting results were due to different experimental conditions. At a solution pH of 7.1, 6 microM protamine fulfills criteria of a selective tool for reversibly altering structure and function of the tight junction in Necturus gallbladder.

Adaptation of epithelial ion transport in the short bowel syndrome.

Ileal remnants 8 weeks after 70% proximal small intestinal resection were used as a model for the short bowel syndrome in man. For comparing active ion transport between control ileum and short bowel with the Ussing technique, the relative contribution of the subepithelial resistance has to be considered. Epithelial/subepithelial voltage divider ratios were determined in the Ussing chamber by positioning the tip of a microelectrode just below the epithelium. In control ileum, the ratio of total to epithelial voltage deflection was 1:0.56 +/- 0.03 (n = 48) and decreased to 1:0.42 +/- 0.01 (n = 67; p less than 0.001) under the short bowel condition. Thus, the factors by which a measured short-circuit current (Isc) underestimates the true electrogenic transport was 1.78 +/- 0.09 (n = 48) in control ileum and 2.36 +/- 0.08 (n = 67; p less than 0.001) in the short bowel. Glucose-dependent electrogenic Na absorption was defined using bathing media containing 48 mM 3-o-methyl-glucose as the decrease in Isc (delta Isc) after addition of 0.5 mM phlorizin. After correction for the respective contributions of the subepithelial resistance, delta Isc was -1.4 +/- 0.2 microEq.h-1.cm-2 (n = 13) in control ileum and -3.2 +/- 0.7 microEq.h-1.cm-2 (n = 11; p less than 0.01) in the short bowel. We conclude that the mucosa in the short bowel syndrome is characterized by an increase in glucose-dependent electrogenic Na absorption, probably as an adaptive response to the reduced overall absorptive area of the remaining intestine.

Tight junction regulation during impaired ion transport in blind loops of rat jejunum.

Epithelial cell tight junction structure in self-filling blind loops of rat jejunum, a model for blind loop syndrome in humans, was analyzed morphometrically along the crypt-villus axis. In control jejunum, the number of strands and junctional depth, including meshwork depth, decreased from crypt to villus tip. In the blind loop, aberrant strands appeared below the meshwork, particularly in crypt cells. Consequently, total junctional depth was greater than in controls. Furthermore, strand number and junctional meshwork depth were increased in blind loops at the villus tip. It is that site along the crypt-villus axis which showed the most shallow junction in control jejunum. This structural change is paralleled by a three-fold increase in epithelial resistance as previously measured by alternating current impedance analysis. Relative Na over Cl permeability (PNa:Cl) was obtained from dilution potential measurements. PNa:Cl was 1.50:1 in control jejunum and 1.35:1 in the blind loop (n.s.). Considering the cation selectivity of the tight junction, the increase in epithelial resistance in blind loops cannot be attributed to a collapse of the lateral intercellular space but is due to changes in tight junctional permeability resulting from structural alteration. The blind loop syndrome represents a further example of diminished epithelial ion transport and concomitant decrease in tight junction permeability, thus supporting the general concept of regulation of the tight junction in response to active transport activity.

Electrophysiologic study of rabbit proximal tubular cell monolayers in primary culture.

Primary cultures of renal cortical cells prepared by selective sieves have been found to display some characteristics of renal proximal tubular epithelium but their site of origin has not been confirmed by electrophysiologic studies. Cells were cultured in a defined medium on collagen gels. Confluency was approached after 7-10 days but gels were found to have zero transepithelial resistance unless they were allowed to contract spontaneously. With the appearance of a nonzero resistance, there was a change in morphology to a more columnar cell with better developed microvilli. These structural features were particularly prominent in clusters of proliferating cells observed on and around remnants of original tubules embedded in the gel. In noncontracted cultures there was no focal cell clustering and cells were squamous-like with rudimentary microvilli, similar in appearance to cells grown on plastic culture dishes. Measurements made in contracted monolayers yielded an average transepithelial resistance of 6.5 omega cm2, a spontaneous transepithelial potential difference of +0.9 mV, measured with respect to the serosa, and an apical membrane potential of -75 mV when cells were bathed in 0.4 mM K and -49 mV when cells were bathed in 4 mM K media. Mucosal protamine (50 micrograms/ml) increased transepithelial resistance by 22%, suggesting that the epithelial cell tight junctions were responsive to external stimuli. Monolayers were anion selective, giving a dilution potential (lumen-directed NaCl gradient) of -2.6 mV with respect to the serosa. These experiments show that primary culture of rabbit renal cortical cells separated by differential sieves displays electrophysiologic and morphologic characteristics of a proximal renal tubular epithelium. Confluency and attainment of differentiated morphology and function are promoted when monolayer cells are not bound to an unyielding substrate.

Prostaglandin and thromboxane synthesis by highly enriched rabbit proximal tubular cells in culture.

Prostaglandin synthetic profiles were studied in monolayers of highly enriched rabbit renal proximal tubular cells cultured in serum-free, hormone-supplemented, defined media. The cultures were initiated from glomeruli-free cortical suspensions. Cells in culture demonstrated morphologic and functional characteristics highly suggestive of proximal tubular cells. The basal and stimulated synthesis of immunoassayable prostaglandin (PG) E2, PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane (Tx) B2 in response to various agonists, as well as the effect of two cyclooxygenase inhibitors, was assessed. Under both basal and stimulated conditions, PGE2 was the major product synthesized. PGF2 alpha and 6-keto-PGF1 alpha were synthesized to a lesser extent, and TxB2 was undetectable. The basal synthesis of PGE2 and PGF2 alpha in cultured cells was found to be higher than in isolated proximal tubular fragments by sevenfold and fivefold, respectively. Exogenous arachidonate, angiotensin II, and the divalent cation ionophore A23187 stimulated all three immunoassayable prostaglandins in a dose-dependent manner. Arginine vasopressin (10(-5) mol/L) had no stimulatory effect. In Ca++-free media or in the presence of 10(-5) mol/L Ca++ channel blocker, verapamil, the stimulatory effects of angiotensin II and A23187 were ameliorated. The stimulatory effect of angiotensin II was inhibited by saralasin (10(-5) mol/L), indicating that receptor binding could mediate PGE2 synthesis. Both indomethacin and sulindac sulfide (10(-5) mol/L) reversibly inhibited PGE2 synthesis.

Protamine alters structure and conductance of Necturus gallbladder tight junctions without major electrical effects on the apical cell membrane.

Protamine is a naturally occurring basic protein (pI; 9.7 to 12.0). We have recently reported that protamine dissolved in the mucosal bath (2 to 20 microM), induces about a twofold increase in transepithelial resistance in Necturus gallbladder within 10 min. Conductance decreased concomitantly with cation selectivity. In this leaky epithelium, where greater than 90% of an applied current passes between cells, an increment in resistance of this magnitude suggests a paracellular action a priori. To confirm this, ionic conductance across the apical cell membrane was studied with microelectrodes. Protamine increased transepithelial resistance without changing apical cell membrane voltage or fractional membrane resistance. Variation in extracellular K concentration (6 to 50 mM) caused changes in apical membrane voltage not different from control. To determine if protamine-induced resistance changes were associated with structural alteration of tight junctions, gallbladders were fixed in situ at peak response and analyzed by freeze-fracture electron microscopy. According to a morphometrical analysis, the tight junctional intramembranous domain expands vertically due to incorporation of new strands (fibrils) into the main compact fibrillar meshwork. Since morphologic changes are complete within 10 min, strands are probably recycled into and out of the tight junctional membrane domain possibly by the cytoskeleton either from cytoplasmic vesicles or from intramembranous precursors. Regulation of tight junctional permeability by protamine and other perturbations may constitute a common mechanism by which leaky epithelia regulate transport, and protamine, in concentrations employed in this study, seems reasonably specific for the tight junction.

Adaptation of the jejunal mucosa in the experimental blind loop syndrome: changes in paracellular conductance and tight junction structure.

Self-filling blind loops of rat jejunum exhibit hyperregenerative transformation of the mucosa. We used this experimental model to characterise mechanisms, which may occur under similar conditions in man (stagnant loop syndrome). Epithelial and subepithelial resistance were measured in the Ussing-chamber by voltage divider ratio measurements after positioning a microelectrode between epithelium and subepithelial tissue layers. In the blind loop, epithelial resistance increased from 8 +/- 1 to 23 +/- 1 omega cm2 and subepithelial resistance from 39 +/- 4 to 86 +/- 8 omega cm2 as compared with control jejunum. The increase in the subepithelial resistance was paralleled anatomically by an increase in the thickness of the subepithelial tissue layers from 63 +/- 4 microns to 177 +/- 19 microns. Ultrastructural analysis of the tight junction area by freeze fracture electron microscopy revealed an increase in the total junctional 'depth' in the crypts from 243 +/- 9 nm in control jejunum to 396 +/- 17 nm in the blind loop, while the number of horizontally oriented 'strands' remained unchanged. Villus tight junctions did not differ between blind loop and control. We interpret the alterations in the self-filling blind loop as an adaptive response of the epithelium which reduces backleakage of already absorbed electrolytes across the tight junction into the intestinal lumen. This mechanism is suitable to support the intestine in maintaining body electrolyte and water contents during cellular electrolyte malabsorption.

Reduction of proteinuria by indomethacin in patients with nephrotic syndrome.

Immediate and longer-term (five-day) effects of indomethacin on proteinuria and renal function were examined in a group of nephrotic subjects with glomerular filtration rates (GFR) that ranged from near normal to moderately impaired. The modifying role of the patients' sodium/volume (S/V) status on renal prostaglandin inhibition was systematically evaluated by renal clearance and balance studies. After patients were S/V-depleted for five days, indomethacin (75 mg/d) decreased protein excretion by 45%. The decrement in proteinuria was greater than 2 times greater than the fall in creatinine clearance and was unrelated to baseline clearance. In acute clearance studies, 75 mg indomethacin administered orally immediately reduced protein excretion, effective renal plasma flow (CPAH), GFR (C inulin), Na, K, and free water excretion. Indomethacin responsiveness (reduced proteinuria) correlated with the change in PGE2 excretion. The effect of indomethacin on protein excretion and renal hemodynamics was apparent, but blunted, when dietary Na intake was increased to 200 mEq/d. Mean BP increased during indomethacin therapy only when patients were S/V-expanded.

Nodular glomerulosclerosis associated with multiple myeloma. Role of light chain isoelectric point.

A 68-year-old female patient with multiple myeloma exhibited advanced nodular glomerulosclerosis. Immunofluorescence of the kidney showed kappa light chain deposition in the mesangium and in glomerular and tubular basement membrane. Isoelectric focusing and immunofixation of urinary proteins revealed an isolated kappa light chain with an unusually high isoelectric point of 8.4. Most light chain proteins have isoelectric points in the 4.6 to 6.7 range. Since loss of fixed negative charges may precede experimental glomerulosclerosis, it is proposed that this cationic circulating kappa chain may have interacted with glomerular polyanion, thereby inducing a nodular sclerotic reaction leading to irreversible renal damage.

Peritoneal permeability in the rat: modulation by microfilament-active agents.

A model of peritoneal dialysis in the rat was used to determine the effects of cytochalasins on ultrastructure and peritoneal permeability to molecules of varying molecular weight. The permeability to urea, inulin, and plasma albumin were determined after intraperitoneal administration of cytochalasin B (2 to 10 X 10(-6) M) and cytochalasins D and E (2 X 10(-6) M). Cytochalasin B (20 X 10(-6) M) increased the permeability to inulin, urea, and albumin by 30, 60, and 150%, respectively. These effects were, to a large degree, reversible. Cytochalasins D and E produced greater increments in permeability for all molecules; this increase was only partially reversible. Ultrastructure analysis by scanning electron microscopy revealed extensive development of membrane protuberances (zeiotic knobs) on mesothelial cells exposed to cytochalasin B. A return to a normal apical cell surface was apparent although incomplete at 24 hr. Tight junctions were not grossly altered and major changes in intramembranous junctional strands were not observed. The major effect of cytochalasins on the cell surface may be responsible for the increased permeability to urea, predominately a transcellular probe. Inulin, which follows a paracellular route, was less affected. Altered protein permeability may be due to the action of cytochalasin on the exposed capillary endothelium in subdiaphragmatic areas where the mesothelium is discontinuous.