RESUMEN
PURPOSE OF REVIEW: 25âyears after the discovery of claudins as the central constituents of tight junctions, the "hunter-gatherer phase" of claudin research is coming to an end. Deficiency in individual claudins as a cause of rare hereditary diseases is well documented. However, knowledge about the involvement of renal claudins in common kidney diseases and strategies to utilize claudins or their regulators for intervention are still scarce. The present review summarizes novel approaches to address these questions. RECENT FINDINGS: Publicly accessible omics data provide new insights not only into general claudin expression patterns along the nephron, but also into sex-specific differences in claudin expression and into claudin dysregulation in renal injury. Computational association studies identify claudin variants as risk factors for kidney disease such as nephrolithiasis or loss of filtration capacity. The establishment of innovative cell culture and organoid models contributes to a better understanding of junctional and extra-junctional functions of individual claudins. SUMMARY: The current studies lay the foundation for the identification of upstream regulators of renal claudin expression and thus for the development of new concepts for the treatment of kidney disease.
Asunto(s)
Claudinas , Enfermedades Renales , Uniones Estrechas , Claudinas/metabolismo , Claudinas/genética , Humanos , Animales , Enfermedades Renales/metabolismo , Uniones Estrechas/metabolismo , Riñón/metabolismoRESUMEN
Fluorescently labelled compounds are often employed to study the paracellular properties of epithelia. For flux measurements, these compounds are added to the donor compartment and samples collected from the acceptor compartment at regular intervals. However, this method fails to detect rapid changes in permeability. For continuous transepithelial flux measurements in an Ussing chamber setting, a device was developed, consisting of a flow-through chamber with an attached LED, optical filter, and photodiode, all encased in a light-impermeable container. The photodiode output was amplified and recorded. Calibration with defined fluorescein concentration (range of 1 nM to 150 nM) resulted in a linear output. As proof of principle, flux measurements were performed on various cell lines. The results confirmed a linear dependence of the flux on the fluorescein concentration in the donor compartment. Flux depended on paracellular barrier function (expression of specific tight junction proteins, and EGTA application to induce barrier loss), whereas activation of transcellular chloride secretion had no effect on fluorescein flux. Manipulation of the lateral space by osmotic changes in the perfusion solution also affected transepithelial fluorescein flux. In summary, this device allows a continuous recording of transepithelial flux of fluorescent compounds in parallel with the electrical parameters recorded by the Ussing chamber.
Asunto(s)
Proteínas de Uniones Estrechas , Uniones Estrechas , Uniones Estrechas/metabolismo , Epitelio , Línea Celular , Proteínas de Uniones Estrechas/metabolismo , Fluoresceína/metabolismoRESUMEN
In cholinergic urticaria (CholU), small, itchy wheals are induced by exercise or passive warming and reduced sweating has been reported. Despite the described reduced muscarinic receptor expression, sweat duct obstruction, or sweat allergy, the underlying pathomechanisms are not well understood. To gain further insights, we collected skin biopsies before and after pulse-controlled ergometry and sweat after sauna provocation from CholU patients as well as healthy controls. CholU patients displayed partially severely reduced local sweating, yet total sweat volume was unaltered. However, sweat electrolyte composition was altered, with increased K+ concentration in CholU patients. Formalin-fixed, paraffin-embedded biopsies were stained to explore sweat leakage and tight junction protein expression. Dermcidin staining was not found outside the sweat glands. In the secretory coils of sweat glands, the distribution of claudin-3 and -10b as well as occludin was altered, but the zonula occludens-1 location was unchanged. In all, dermcidin and tight junction protein staining suggests an intact barrier with reduced sweat production capability in CholU patients. For future studies, an ex vivo skin model for quantification of sweat secretion was established, in which sweat secretion could be pharmacologically stimulated or blocked. This ex vivo model will be used to further investigate sweat gland function in CholU patients and decipher the underlying pathomechanism(s).
Asunto(s)
Urticaria Crónica Inducible , Glándulas Sudoríparas , Sudor , Uniones Estrechas , Sudor/química , Uniones Estrechas/metabolismo , Glándulas Sudoríparas/metabolismo , Ergometría , Proteínas de Uniones Estrechas/metabolismo , Urticaria Crónica Inducible/metabolismo , Urticaria Crónica Inducible/patología , Humanos , Masculino , Femenino , Adulto , Receptor Muscarínico M3/metabolismo , Biopsia con AgujaRESUMEN
The concept of solvent drag, i.e., water and solutes sharing the same pore and their transport being frictionally coupled, was first proposed in the early 1950s. During the following decades, it was applied to transport processes across cell membranes as well as transport along the paracellular pathway. Water-driven solute transport was proposed as the major mechanism for electrolyte and nutrient absorption in the small intestine and for Cl- and HCO3- reabsorption in the renal proximal tubule. With the discovery of aquaporins as transcellular route for water transport and the claudin protein family as the major determinant of paracellular transport properties, new mechanistic insights in transepithelial water and solute transport are emerging and call for a reassessment of the solvent drag concept. Current knowledge does not provide a molecular basis for relevant solvent drag-driven, paracellular nutrient, and inorganic anion (re-)absorption. For inorganic cation transport, in contrast, solvent drag along claudin-2-formed paracellular channels appears feasible.
Asunto(s)
Túbulos Renales Proximales , Agua , Túbulos Renales Proximales/metabolismo , Transporte Biológico , Transporte Iónico , Agua/metabolismo , Solventes/metabolismo , Uniones Estrechas/metabolismoRESUMEN
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.
Asunto(s)
Claudina-2 , Claudinas/metabolismo , Animales , Cationes/metabolismo , Túbulos Renales Proximales/metabolismo , Ratones , Permeabilidad , Uniones Estrechas/fisiologíaRESUMEN
HELIX syndrome, characterized by hypohidrosis, electrolyte imbalance, lacrimal gland dysfunction, ichthyosis, and xerostomia due to claudin-10 (CLDN10) mutations, was recognized in 2017. Here we describe two unrelated Saudi families with this syndrome due to a novel CLDN10 mutation with a unique mechanism of CLDN10 inactivation. The two consanguineous families include 12 affected individuals (three siblings in family 1 and nine members in family 2). They presented with hypokalemia and the above-mentioned features of HELIX syndrome. The underlying mutation was detected by whole exome sequencing, confirmed by Sanger sequencing and functionally indicated by RT-PCR, electrophysiological studies and immunohistochemical staining of transfected HEK293 and MDCK C7 cells, and skin and kidney biopsy tissues. A novel biallelic single nucleotide deletion was identified in exon 5 of CLDN10 (NM_182848.3: c.647delC, p.P216Lfs∗19 for CLDN10a or NM_006984.4: c.653delC, p.P218Lfs∗21 for CLDN10b). The mutation led to frameshift and extension of the original termination codon by nine amino acids with loss of the C-terminus pdz-binding motif. Functional studies showed mRNA degradation and protein retention in intracellular compartments and that the pdz-binding motif is crucial for proper localization of claudin-10 in tight junctions. In the kidney, claudin-10 was replaced by translocation of claudin-2 (proximal tubule) and claudin-19 (thick ascending limb), and in the sweat gland by claudin-3 and occludin. However, these claudins did not functionally compensate for loss of claudin-10. Thus, this novel CLDN10 mutation identified in these two families disrupted the C-terminus pdz-binding motif of claudin-10 causing HELIX syndrome.
Asunto(s)
Anomalías Múltiples/genética , Claudinas , Uniones Estrechas , Claudinas/genética , Consanguinidad , Células HEK293 , Humanos , Aparato Lagrimal/fisiopatología , Mutación , Síndrome , Equilibrio Hidroelectrolítico , Xerostomía/genéticaRESUMEN
BACKGROUND: Expression of the tight junction proteins Cldn1 and 4 is altered in skin diseases such as atopic dermatitis, and Cldn1 deficiency affects skin barrier formation. Impedance spectroscopy (IS) has been proven to allow detection of alterations in the skin barrier but is currently unable to separate effects on viable epidermis (VE) and stratum corneum (SC). METHODS: Effects of siRNA-mediated Cldn1 and 4 knockdown in reconstructed human epidermis (RHE) on VE and SC barrier function were investigated with Ussing chamber-based IS. Barrier components were sequentially altered, employing iron oxide nanoparticles and EGTA, to identify their contribution to the impedance spectrum. Resistance changes due to apically applied hyperosmolar electrolyte were used to identify barrier defects non-invasively. RESULTS: IS of RHE yielded two relaxation frequencies, representing the barrier properties of the SC (~1000 Hz) and VE (~100 Hz). As proof of concept, it was shown that the Cldn1 knockdown-induced resistance drop arises from the impairment of both SC and VE, indicated by a shift of both relaxation frequencies. Hyperosmolar electrolyte penetration allowed non-invasive detection of Cldn1 knockdown via time-dependent frequency shifts. The absence of Cldn4 knockdown-induced changes revealed the weaknesses of transepithelial electrical resistance analysis. CONCLUSION: In conclusion, the present technique allows to separately measure the barrier properties of SC and VE and further evaluate the Cldn1 and 4 knockdown impact on the skin barrier. As the measurement with agarose-embedded electrolyte allowed non-invasive identification of the Cldn1 knockdown, this opens the way to detailed in vivo skin barrier assessment.
Asunto(s)
Dermatitis Atópica , Espectroscopía Dieléctrica , Células Epidérmicas , Epidermis , Humanos , Piel , Uniones EstrechasRESUMEN
Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. This review summarizes our current knowledge of this large protein family and discusses recent advances in our understanding of their structure and physiological functions.
Asunto(s)
Claudinas/genética , Claudinas/metabolismo , Transducción de Señal/fisiología , Uniones Estrechas/metabolismo , Animales , Claudinas/química , Epitelio/metabolismo , Regulación de la Expresión Génica/fisiología , Humanos , PermeabilidadRESUMEN
Strong communication and interaction between the retinal pigment epithelium (RPE) and the photoreceptor (PR) cells is essential for vision. RPE cells are essential for supporting and maintaining PR cells by transporting nutrients, waste products and ions, and phagocytosing photoreceptor outer segments (POS). POS phagocytosis follows a circadian pattern, taking place in the morning in human, mice and other organisms. However, it remains unknown whether other RPE processes follow a daily rhythm. To study the daily rhythm of RPE cells, we isolated murine RPE cells at six different time points during a 24 h period, after which RNA was isolated and sequenced. Murine RPE flatmounts were isolated at four different time points to study daily rhythm in protein abundance and localisation. EnrichR pathway analysis resulted in 13 significantly-enriched KEGG pathways (p < 0.01) of which seven showed a large number of overlapping genes. Several genes were involved in intracellular trafficking, possibly playing a role in nutrient transport, POS phagocytosis or membrane protein trafficking, with different expression patterns during the day-night cycle. Other genes were involved in actin cytoskeleton building, remodelling and crosslinking and showed a high expression in the morning, suggesting actin cytoskeleton remodelling at this time point. Finally, tight junction proteins Cldn2 and Cldn4 showed a difference in RNA and protein expression and tight junction localisation over time. Our study suggests that several important processes in the RPE follow a day-night rhythm, including intracellular trafficking, and processes involving the actin cytoskeleton and tight junctions. The differential protein localisation of Cldn2 in the RPE during the day-night cycle suggest that Cldn2 may facilitate paracellular water and sodium transport during the day.
Asunto(s)
Ritmo Circadiano/fisiología , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Proteínas de Uniones Estrechas/genética , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Epitelio Pigmentado de la Retina/citología , Proteínas de Uniones Estrechas/biosíntesisRESUMEN
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.
Asunto(s)
Claudinas/genética , Riñón/metabolismo , Isoformas de Proteínas/genética , Insuficiencia Renal/genética , Animales , Transporte Biológico/genética , Cationes/metabolismo , Claudinas/metabolismo , Células Epiteliales/metabolismo , Humanos , Hipohidrosis , Riñón/patología , Ratones , Microscopía Electrónica , Mutación Missense , Permeabilidad , Isoformas de Proteínas/metabolismo , Insuficiencia Renal/metabolismo , Insuficiencia Renal/patología , Uniones EstrechasRESUMEN
The thick ascending limb (TAL) of Henle's loop drives paracellular Na+, Ca2+, and Mg2+ reabsorption via the tight junction (TJ). The TJ is composed of claudins that consist of four transmembrane segments, two extracellular segments (ECS1 and -2), and one intracellular loop. Claudins interact within the same (cis) and opposing (trans) plasma membranes. The claudins Cldn10b, -16, and -19 facilitate cation reabsorption in the TAL, and their absence leads to a severe disturbance of renal ion homeostasis. We combined electrophysiological measurements on microperfused mouse TAL segments with subsequent analysis of claudin expression by immunostaining and confocal microscopy. Claudin interaction properties were examined using heterologous expression in the TJ-free cell line HEK 293, live-cell imaging, and Förster/FRET. To reveal determinants of interaction properties, a set of TAL claudin protein chimeras was created and analyzed. Our main findings are that (i) TAL TJs show a mosaic expression pattern of either cldn10b or cldn3/cldn16/cldn19 in a complex; (ii) TJs dominated by cldn10b prefer Na+ over Mg2+, whereas TJs dominated by cldn16 favor Mg2+ over Na+; (iii) cldn10b does not interact with other TAL claudins, whereas cldn3 and cldn16 can interact with cldn19 to form joint strands; and (iv) further claudin segments in addition to ECS2 are crucial for trans interaction. We suggest the existence of at least two spatially distinct types of paracellular channels in TAL: a cldn10b-based channel for monovalent cations such as Na+ and a spatially distinct site for reabsorption of divalent cations such as Ca2+ and Mg2.
Asunto(s)
Claudinas/metabolismo , Asa de la Nefrona/metabolismo , Magnesio/metabolismo , Sodio/metabolismo , Animales , Claudinas/genética , Células HEK293 , Humanos , Asa de la Nefrona/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas Sprague-Dawley , Uniones Estrechas/metabolismoRESUMEN
Inorganic phosphate (Pi) is crucial for many biological functions, such as energy metabolism, signal transduction, and pH buffering. Efficient systems must exist to ensure sufficient supply for the body of Pi from diet. Previous experiments in humans and rodents suggest that two pathways for the absorption of Pi exist, an active transcellular Pi transport and a second paracellular pathway. Whereas the identity, role, and regulation of active Pi transport have been extensively studied, much less is known about the properties of the paracellular pathway. In Ussing chamber experiments, we characterized paracellular intestinal Pi permeabilities and fluxes. Dilution potential measurements in intestinal cell culture models demonstrated that the tight junction is permeable to Pi, with monovalent Pi having a higher permeability than divalent Pi. These findings were confirmed in rat and mouse intestinal segments by use of Ussing chambers and a combination of dilution potential measurements and fluxes of radiolabeled 32Pi. Both techniques yielded very similar results, showing that paracellular Pi fluxes were bidirectional and that Pi permeability was ~50% of the permeability for Na+ or Cl-. Pi fluxes were a function of the concentration gradient and Pi species (mono- vs. divalent Pi). In mice lacking the active transcellular Pi transport component sodium-dependent Pi transporter NaPi-IIb, the paracellular pathway was not upregulated. In summary, the small and large intestines have a very high paracellular Pi permeability, which may favor monovalent Pi fluxes and allow efficient uptake of Pi even in the absence of active transcellular Pi uptake.NEW & NOTEWORTHY The paracellular permeability for phosphate is high along the entire axis of the small and large intestine. There is a slight preference for monovalent phosphate. Paracellular phosphate fluxes do not increase when transcellular phosphate transport is genetically abolished. Paracellular phosphate transport may be an important target for therapies aiming to reduce intestinal phosphate absorption.
Asunto(s)
Espacio Extracelular/fisiología , Mucosa Intestinal/metabolismo , Transporte Iónico/fisiología , Fosfatos , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb/metabolismo , Uniones Estrechas/fisiología , Animales , Células Cultivadas , Absorción Intestinal , Ratones , Permeabilidad , Fosfatos/química , Fosfatos/metabolismo , RatasRESUMEN
The tight junction proteins claudin-10 and -16 are crucial for the paracellular reabsorption of cations along the thick ascending limb of Henle's loop in the kidney. In patients, mutations in CLDN16 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis, while mutations in CLDN10 impair kidney function. Mice lacking claudin-16 display magnesium and calcium wasting, whereas absence of claudin-10 results in hypermagnesemia and interstitial nephrocalcinosis. In order to study the functional interdependence of claudin-10 and -16 we generated double-deficient mice. These mice had normal serum magnesium and urinary excretion of magnesium and calcium and showed polyuria and sodium retention at the expense of increased renal potassium excretion, but no nephrocalcinosis. Isolated thick ascending limb tubules of double mutants displayed a complete loss of paracellular cation selectivity and functionality. Mice lacking both claudin-10 and -16 in the thick ascending limb recruited downstream compensatory mechanisms and showed hypertrophic distal convoluted tubules with changes in gene expression and phosphorylation of ion transporters in this segment, presumably triggered by the mild decrease in serum potassium. Thus, severe individual phenotypes in claudin-10 and claudin-16 knockout mice are corrected by the additional deletion of the other claudin.
Asunto(s)
Claudinas/deficiencia , Hipercalciuria/prevención & control , Túbulos Renales Distales/metabolismo , Asa de la Nefrona/metabolismo , Deficiencia de Magnesio/prevención & control , Animales , Calcio/metabolismo , Claudinas/genética , Modelos Animales de Enfermedad , Eliminación de Gen , Predisposición Genética a la Enfermedad , Hipercalciuria/genética , Hipercalciuria/metabolismo , Hipercalciuria/fisiopatología , Túbulos Renales Distales/patología , Túbulos Renales Distales/fisiopatología , Asa de la Nefrona/patología , Asa de la Nefrona/fisiopatología , Magnesio/metabolismo , Deficiencia de Magnesio/genética , Deficiencia de Magnesio/metabolismo , Deficiencia de Magnesio/fisiopatología , Ratones Endogámicos C57BL , Ratones Noqueados , Nefrocalcinosis/genética , Nefrocalcinosis/metabolismo , Nefrocalcinosis/fisiopatología , Nefrocalcinosis/prevención & control , Fenotipo , Sodio/metabolismoRESUMEN
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.
Asunto(s)
Claudinas/antagonistas & inhibidores , Infecciones por Clostridium/patología , Clostridium perfringens/patogenicidad , Colon/patología , Enterotoxinas/toxicidad , Enfermedades Transmitidas por los Alimentos/patología , Uniones Estrechas/patología , Línea Celular , Enterocitos/patología , Humanos , Mucosa Intestinal/patología , PermeabilidadRESUMEN
Tight junction (TJ) strands between epithelial or endothelial cells are formed by claudins, a protein family comprising up to 27 members in mammals. Although many more proteins are involved in the formation of TJ complexes, claudins are the only TJ proteins that are able to form TJ-like strands when overexpressed in cells that are normally devoid of TJs (e.g., fibroblasts). Within the paracellular cleft, the extracellular domains of claudins provide the matrix that seals the paracellular pathway. However, within this matrix, some claudins act as channels that specifically allow certain ions to cross this barrier. Barrier-forming claudins predominate in epithelia that enclose compartments containing harmful ion concentrations (e.g., H+ in the stomach, K+ in the inner ear endolymph) or high pressures (e.g., in blastocoel or brain ventricle formation during development). Here, even seemingly minor alterations in TJ composition may be detrimental to the organism. In contrast, in many transporting epithelia, channel-forming claudins are essential for transcellular and paracellular transport coupling. Mutation or knockout of channel-forming claudins in these tissues brings both transcellular and paracellular transports to a standstill. The present review will present examples to illustrate the importance of single members of the claudin family in general epithelial transport physiology.
Asunto(s)
Claudinas/metabolismo , Epitelio/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Humanos , Uniones Estrechas/metabolismoRESUMEN
The renal proximal tubule achieves the majority of renal water and solute reabsorption with the help of paracellular channels which lead through the tight junction. The proteins forming such channels in the proximal tubule are claudin-2, claudin-10a, and possibly claudin-17. Claudin-2 forms paracellular channels selective for small cations like Na+ and K+. Independently of each other, claudin-10a and claudin-17 form anion-selective channels. The claudins form the paracellular "pore pathway" and are integrated, together with purely sealing claudins and other tight junction proteins, in the belt of tight junction strands surrounding the tubular epithelial cells. In most species, the proximal tubular tight junction consists of only 1-2 (pars convoluta) to 3-5 (pars recta) horizontal strands. Even so, they seal the tubule very effectively against leak passage of nutrients and larger molecules. Remarkably, claudin-2 channels are also permeable to water so that 20-25% of proximal water absorption may occur paracellularly. Although the exact structure of the claudin-2 channel is still unknown, it is clear that Na+ and water share the same pore. Already solved claudin crystal structures reveal a characteristic ß-sheet, comprising ß-strands from both extracellular loops, which is anchored to a left-handed four-transmembrane helix bundle. This allowed homology modeling of channel-forming claudins present in the proximal tubule. The surface of cation- and anion-selective claudins differ in electrostatic potentials in the area of the proposed ion channel, resulting in the opposite charge selectivity of these claudins. Presently, while models of the molecular structure of the claudin-based oligomeric channels have been proposed, its full understanding has only started.
Asunto(s)
Claudinas/metabolismo , Túbulos Renales Proximales/metabolismo , Uniones Estrechas/metabolismo , Animales , Claudinas/química , Humanos , Túbulos Renales Proximales/fisiología , Túbulos Renales Proximales/ultraestructura , Uniones Estrechas/ultraestructuraRESUMEN
During the postweaning period, piglets are prone to gastrointestinal infections. The resulting impairment of intestinal barrier function may cause diarrhea associated with growth retardation or even death of piglets. Orally applied Zn is commonly used to prevent and treat diarrhea, but its mode of action still needs to be elucidated. To analyze the molecular mechanism whereby Zn acts on porcine intestinal barrier function, ex vivo studies on piglet jejunum and accompanying in vitro studies on a porcine jejunal epithelial cell line, IPEC-J2/PS, were performed with electrophysiological tools. Feeding pharmacological Zn doses exerted no significant electrophysiologically ascertainable short- and long-term effects on jejunal barrier function ex vivo. However, in IPEC-J2/PS, basolateral Zn was cytotoxic since its application caused a release of lactate dehydrogenase and an irreversible breakdown of the epithelial barrier. In contrast, apical Zn application caused an immediate increase in paracellular resistance and a decrease in permeability to the paracellular marker fluorescein, reflecting overall barrier strengthening in vitro. Apical effects were fully reversible upon washout. This indicates that Zn supplemented to feed was completely washed out during ex vivo jejunum preparation. We conclude that there is no evidence for long-term barrier effects through prophylactic Zn supplementation and that extracellular Zn acts acutely and reversibly from the apical side via tightening the paracellular route, thus counteracting leak-flux diarrhea.NEW & NOTEWORTHY Therapeutically administered Zn successfully treats diarrhea in veterinary and human medicine. Here we present data that Zn strengthens the porcine jejunal epithelial barrier by reversibly tightening the paracellular route for inorganic ions and small solutes. Acute or long-lasting Zn effects on transcellular transport (Cl- secretion) were not detected. We therefore conclude that Zn is useful for acutely treating leak-flux diarrhea rather than secretory diarrhea. Suitability as prophylactic feed supplement, however, is questionable.
Asunto(s)
Polaridad Celular , Suplementos Dietéticos , Células Epiteliales/efectos de los fármacos , Uniones Intercelulares/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Yeyuno/efectos de los fármacos , Acetato de Zinc/farmacología , Administración Oral , Aminoácidos/farmacología , Animales , Animales Recién Nacidos , Bicarbonatos/farmacología , Línea Celular , Claudinas/metabolismo , Conductividad Eléctrica , Impedancia Eléctrica , Células Epiteliales/metabolismo , Uniones Intercelulares/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Yeyuno/citología , Yeyuno/metabolismo , Permeabilidad , Sus scrofa , Factores de Tiempo , Acetato de Zinc/administración & dosificaciónRESUMEN
Claudin-17 is a paracellular channel-forming tight junction protein. Unlike the cation channels claudin-2 and -15, claudin-17 forms a distinct anion-selective channel. Aim of this study was to determine the molecular basis of channel formation and charge selectivity of this protein. To achieve this, residues located in the extracellular loops (ECL) 1 and 2 of claudin-17 were substituted, preferably those whose charges differed in claudin-17 and in claudin-2 or -15. The respective mutants were stably expressed in MDCK C7 cells and their ability to form charge-selective channels was analyzed by measuring ion permeabilities and transepithelial electrical resistance. The functional data were combined with homology modeling of the claudin-17 protomer using the structure of claudin-15 as template. In ECL1, K65, R31, E48, and E44 were found to be stronger involved in Cldn17 channel function than the clustered R45, R56, R59, and R61. For K65, not only charge but also stereochemical properties were crucial for formation of the anion-selective channel. In ECL2, both Y149 and H154 were found to contribute to constitution of the anion channel in a distinct manner. In conclusion, we provide insight into the molecular mechanism of the formation of charge- and size-selective paracellular ion channels. In detail, we propose a hydrophilic furrow in the claudin-17 protomer spanning from a gap between the ends of TM2 and TM3 along R31, E48, and Y67 to a gap between K65 and S68 lining the anion channel.
Asunto(s)
Aniones/metabolismo , Claudinas/química , Claudinas/metabolismo , Secuencia de Aminoácidos , Animales , Claudinas/genética , Perros , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas , Conformación Proteica , Alineación de SecuenciaRESUMEN
The aim of the present study was to elucidate the effects of the probiotic feed additive Enterococcus faecium NCIMB 10415 (E. faecium) on porcine jejunal epithelial cells (IPEC-J2) during an in vitro challenge with enterotoxigenic Escherichia coli (ETEC). Cells were incubated with E. faecium, ETEC, or both, and the effects on barrier function and structure and intra- and intercellular signaling were determined. Coincubation with E. faecium abolished the ETEC-induced decrease in transepithelial resistance (Rt) (p ≤ 0.05). No differences were seen in the expression levels of the intercellular connecting tight junction proteins examined. However, for the first time, a reorganization of the monolayer was observed in ETEC-infected cells but not in coincubated cells. ETEC induced an increase in cytotoxicity that was prevented by coincubation (p ≤ 0.05), whereas apoptosis rates were not affected by bacterial treatment. ETEC increased the mRNA expression and release of proinflammatory cytokines TNF-α, IL-1α, and IL-6 which could be prevented by coincubation for TNF-α mRNA expression and IL-6 protein (p ≤ 0.05). Likewise, cAMP concentrations elevated by ETEC were reduced in coincubated cells (p ≤ 0.05). These findings indicate a protective effect of the probiotic E. faecium on inflammatory responses during infection with ETEC.
Asunto(s)
Enterococcus faecium/patogenicidad , Escherichia coli Enterotoxigénica/patogenicidad , Células Epiteliales/microbiología , Animales , Apoptosis/fisiología , Línea Celular , Citocinas , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/citología , Interleucina-1alfa/metabolismo , Interleucina-6/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , PorcinosRESUMEN
Subacute ruminal acidosis is induced by high concentrations of short-chain fatty acids (SCFA, mainly acetate, propionate, and butyrate) that release protons to decrease the pH of the ruminal digesta. This low pH, in turn, is thought to damage epithelial barrier function. The present study applied a model of simulated ruminal acidosis ex vivo to investigate if SCFA directly contribute to epithelial barrier failure beyond their role as proton donors. Epithelial tissues from the rumen of slaughtered sheep were mounted in Ussing chambers and incubated under 3 different conditions. Two groups were incubated in the absence of SCFA at mucosal pH 6.1 (control) and pH 5.1, respectively, for 7 h. A third group was first incubated in a mucosal solution containing 100 mM SCFA at pH 5.1 for 2 h and, thereafter, in a mucosal solution without SCFA at pH 6.1 for the remaining 5 h. Transepithelial conductance (Gt), short-circuit current (Isc), and fluorescein fluxes were determined. After 7 h of incubation, the expression levels of claudin-1, claudin-4, claudin-7, and occludin were measured by quantitative reverse-transcription PCR and Western blot. Furthermore, the local distribution of these tight junction (TJ) proteins was examined by confocal laser scanning microscopy. A 7-h incubation at pH 5.1 in the absence of SCFA did not influence either Gt or fluorescein flux rates of ruminal tissues ex vivo compared with the control. In contrast, incubation at pH 5.1 with SCFA for only 2 h induced increases in Gt and fluorescein flux rates that continued even after tissues were returned back to pH 6.1. Expression analysis showed that pH 5.1 without SCFA for 7 h induced no changes in mRNA expression of claudin-1, claudin-4, claudin-7, and occludin and a selective decrease in protein expression of only claudin-4 compared with the control. However, a 2-h incubation at pH 5.1 in the presence of SCFA decreased the mRNA-expression of claudin-7, as well as the protein expression of claudin-4, claudin-7, and occludin. The decreased expression of these TJ proteins in the group incubated with SCFA was also evident in immunohistochemistry. Immunohistochemistry additionally evidenced a considerable retraction of all tested TJ proteins out of the TJ in that group. We conclude that a low mucosal pH of 5.1 is tolerated well by ruminal epithelia for several hours. However, a low pH in combination with SCFA induces damage to the TJ and disturbs barrier function, which is not immediately reversible upon the removal of the acidotic insult.