Carl J. Bentzel

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.

Antagonism of the effects of furosemide by in domethacin in normal and hypertensive man

Furosemide and the prostaglandin synthetase inhibitor, indomethacin, were administered singly and in combination to four normal subjects and six patients with essential hypertension in order to determine whether the antihypertensive, natriuretic and other effects of furosemide could be altered by inhibition of prostaglandin synthesis. In all subjects indomethacin treatment alone resulted in a significant elevation of blood pressure and a fall in plasma renin without any change in sodium excretion. Furosemide alone resulted in a significant blood pressure fall with a rise in plasma renin and urinary aldosterone with a marked increase in urinary sodium loss. These effects were either obviated or blunted by addition of indomethacin. The results are compatible with hypothesis that the antihypertensive and natriuretic effects of furosemide might be mediated at least in part by prostaglandin synthesis. In addition, the effects of indomethacin should be considered when using this drug in hypertensive patients and in subjects requiring diuretic therapy.

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.”

Uric acid excretion and renal function in the acute hyperuricemia of leukemia: Pathogenesis and therapy of uric acid nephropathy

Abstract Uric acid excretion and discrete renal functions were measured in forty-three studies performed on twenty-six patients with active leukemia. Patients with hyperuricemia had an increased urinary excretion rate and urinary concentration of uric acid. The increase in uric acid excretion associated with acute endogenous hyperuricemia promotes uric acid precipitation in the acid, concentrated urine of the distal tubules and collecting ducts, with resultant obstruction of nephrons. In hyperuricemic patients, there was a diminished clearance of C 14 -labeled inulin (C inulin ) usually with minimal depression of para-aminohippurate clearance (C PAH ). The rapidly reversible nature of uric acid nephropathy suggests that the lesion is primarily obstructive. Successful prophylaxis as well as therapy of uric acid nephropathy depends on effective urinary alkalinization and a brisk urine flow, in order to attain maximal solubility of uric acid. A regimen to accomplish the foregoing, through administration of acetazolamide, sodium bicarbonate and intravenous hydration, is outlined.

Epithelial Barrier Defects in HT-29/B6 Colonic Cell Monolayers Induced by Tumor Necrosis Factor-α

Abstract: The barrier function of intestinal epithelia relies upon the continuity of the enterocyte monolayer and intact tight junctions. After incubation with tumor necrosis factor‐α TNF‐α, 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‐α. These mechanisms explain clinical manifestations in the inflamed intestinal wall containing cytokinesecreting macrophages‐for example, leak flux diarrhea and invasion of bacterial enterotoxins.

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.

Protamine reversibly decreases paracellular cation permeability inNecturus gallbladder

SummaryProtamine, a naturally occurring arginine-rich polycationic protein (pI 9.7 to 12), was tested inNecturus gallbladder using a transepithelial AC-impedance technique. Protamine sulfate or hydrochloride (100 μg/ml=20 μm), dissolved in the mucosal bath, increased transepithelial resistance by 89% without affecting the resistance of subepithelial layers. At the same time, transepithelial voltage (ψms) turned from slightly mucosapositive values to mucosa-negative values of approximately +1 to −5 mV. The effect of protamine on transepithelial resistance was minimal at concentrations below 5 μg/ml but a maximum response was achieved between 10 and 20 μg/ml. Resistance started to increase within 1 min and was maximal after 10 min. These effects were not inhibited by serosal ouabain (5×10−4m) but could be readily reversed by mucosal heparin. The sequence of protamine effect and heparin reversal could be repeated several times in the same gallbladder. Mucosal heparin, a strong negatively charged mucopolysaccharide, or serosal protamine were without effect. Mucosal protamine reversibly decreased the partial ionic conductance of K and Na by a factor of 3, but did not affect Cl conductance. Net water transport from mucosa to serosa was reversibly increased by 60% by protamine. We conclude that protamine reversibly decreases the conductance of the cation-selective pathway through the tight junction. Although this effect is similar to that reported for 2,4,6-triamino-pyrimidinium (TAP), the mechanism of action may differ. We propose that protamine binds to the apical cell membrane and induces a series of intracellular events which leads to a conformational alteration of the tight junction structure resulting in decreased cationic permeability.

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

SummaryProtamine 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 μm), induces about a twofold increase in transepithelial resistance inNecturus gallbladder within 10 min. Conductance decreased concomitantly with cation selectivity.In this leaky epithelium, where >90% of an applied current passes between cells, an increment in resistance of this magnitude suggests a paracellular actiona 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 50mm) 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 fixedin 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.

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 patients 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 patients history, before laboratory confirmation of the ingestion was obtained.

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.