Paracellular Permeability Properties Research Articles

C-type natriuretic peptide regulates the molecular components of the rainbow trout gill epithelium tight junction complex

Freshwater (FW) fish experience passive paracellular loss of ions into the surrounding environment across water-exposed epithelia such as the gill. The mitigation of paracellular ion loss is thought to be regulated by proteins of the tight junction (TJ) complex and in particular, the large superfamily of claudin (cldn) TJ proteins plays an important role. Transcript and protein levels of TJ proteins in teleosts are known to be under endocrine control of several important osmoregulatory hormones and the current study was aimed at determining whether the osmoregulatory hormone, C-type natriuretic peptide (CNP), can alter paracellular permeability and TJ protein abundance in a primary cultured gill epithelium derived from rainbow trout. Natriuretic peptide receptors were detected in the cultured trout gill epithelium. It was found that (i) developing cultured gill epithelia "grown" in the presence of 10 nM CNP, and (ii) mature cultured gill epithelia exposed to 10 nM CNP for 48 h, exhibited augmented barrier properties. This occurred in association with reduced flux rates of a paracellular permeability marker (polyethylene glycol, molecular mass 400; PEG-400) and, reduced ion efflux (i.e. ion loss) when preparations were exposed to apical FW. Exposure to CNP altered mRNA abundance of cldn-3a, -5a, -6, - 8c, -20a, -25b, -28a, -32a and cgn, but differences in the transcriptional response were observed between chronic and acute CNP exposure. In contrast, chronic and acute exposure to CNP resulted in reduced cldn-10e/Cldn-10e abundance. Data suggest that CNP may play a role in regulating the molecular physiology of the TJ complex in the fish gill epithelium and contribute to the regulation of salt and water balance by influencing the paracellular permeability properties of this tissue.

Coexpression of proximal tubule claudins‐2 and ‐10a increases conductance and decreases permselectivity in a renal epithelial cell line

The mammalian nephron is composed of a series of tubule segments with distinct patterns of tight junction morphology, ion selectivity, and protein expression. Paracellular permeability of epithelial tissues is determined in part by claudins, a family of tight junction membrane proteins. The most abundantly expressed claudins in the proximal tubule (PT) are claudin‐2 and ‐10a. In general, claudin‐2 is expressed in leaky epithelia and functions as a cation‐permeable paracellular channel, whereas claudin‐10a has been shown to form anion‐selective pores. We hypothesized that coexpression of these PT claudins leads to distinct paracellular permeability properties, and contribute to ion transport in this segment.We established a double‐inducible, titratable gene expression system using Madin‐Darby canine kidney cells (MDCK I). Utilizing tetracycline‐controlled transcriptional activation (Tet‐Off), we developed stable cell lines which expressed claudin‐2 in the absence of tetracycline antibiotics. From this parent line, we generated stable transfectants expressing claudin‐10a under the control of a cumate‐inducible switch (SBI). Induction of claudin‐2, ‐10a, or both isoforms, all reduced TER compared to cells with no claudin overexpression (p<0.0001 using 2‐way ANOVA). We found a marked increase in PCl in cells induced to express claudin‐10a alone (17.4 ± 1.7 * 106 cm/s vs. 0.55 ± 0.06 * 106 cm/s with no induction), and a marked increase in PNa in cells induced to express claudin‐2 alone (12.2 ± 0.6 * 106 cm/s vs. 0.59 ± 0.07 * 106 cm/s with no induction). When both claudin‐2 and ‐10a were expressed, PCl and PNa were both increased (to 9.5 ± 1.5 * 106 cm/s and 6.2 ± 0.4 * 106 cm/s, respectively) and PNa/PCl was unchanged (0.76 ± 0.10 vs. 1.07 ± 0.05 with no induction). Calcium transport is believed to occur via a paracellular route in the PT, and so we measured the effect of PT claudin overexpression on unidirectional 45calcium radiotracer flux. Claudin‐2 expression increased calcium flux by over 7‐fold (p<0.0001), while claudin‐10a had no effect compared with no expression (p=0.99). No difference was detected in calcium flux between claudin‐2 expression and claudin‐2 and ‐10a in combination (p=0.89). These results further support claudin‐2 as the mediator of calcium reabsorption in the PT, and suggest that an interaction between PT claudin isoforms reduces the permselectivity of the PT to sodium and chloride.Support or Funding InformationSupported in part by NIH grant F30DK109605This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enhanced paracellular transport of insulin can be achieved via transient induction of myosin light chain phosphorylation

The intestinal epithelium functions to effectively restrict the causal uptake of luminal contents but has been demonstrated to transiently increase paracellular permeability properties to provide an additional entry route for dietary macromolecules. We have examined a method to emulate this endogenous mechanism as a means of enhancing the oral uptake of insulin. Two sets of stable Permeant Inhibitor of Phosphatase (PIP) peptides were rationally designed to stimulate phosphorylation of intracellular epithelial myosin light chain (MLC) and screened using Caco-2 monolayers in vitro. Apical application of PIP peptide 640, designed to disrupt protein–protein interactions between protein phosphatase 1 (PP1) and its regulator CPI-17, resulted in a reversible and non-toxic transient reduction in Caco-2 monolayer trans-epithelial electric resistance (TEER) and opening of the paracellular route to 4kDa fluorescent dextran but not 70kDa dextran in vitro. Apical application of PIP peptide 250, designed to impede MYPT1-mediated regulation of PP1, also decreased TEER in a reversible and non-toxic manner but transiently opened the paracellular route to both 4 and 70kDa fluorescent dextrans. Direct injection of PIP peptides 640 or 250 with human insulin into the lumen of rat jejunum caused a decrease in blood glucose levels that was PIP peptide and insulin dose-dependent and correlated with increased pMLC levels. Systemic levels of insulin suggested approximately 3–4% of the dose injected into the intestinal lumen was absorbed, relative to a subcutaneous injection. Measurement of insulin levels in the portal vein showed a time window of absorption that was consistent with systemic concentration-time profiles and approximately 50% first-pass clearance by the liver. Monitoring the uptake of a fluorescent form of insulin suggested its uptake occurred via the paracellular route. Together, these studies add validation to the presence of an endogenous mechanism used by the intestinal epithelium to dynamically regulate its paracellular permeability properties and better define the potential to enhance the oral delivery of biopharmaceuticals via a transient regulation of an endogenous mechanism controlling the intestinal paracellular barrier.

Significant water absorption goes paracellular in kidney proximal tubules

it had been a fundamental dispute for several decades, whether water passes epithelia such as kidney proximal tubule or small intestine along the transcellular, paracellular, or both pathways. By discovery of the transmembranal aquaporin water channels (AQP), the molecular basis of the

Claudin‐5a in developing zebrafish brain barriers: another brick in the wall

Claudins serve essential roles in regulating paracellular permeability properties within occluding junctions. Recent studies have begun to elucidate developmental roles of claudins within immature tissues. This work has uncovered an involvement of several claudins in determining tight junction properties that have an effect on embryonic morphogenesis and physiology. During zebrafish brain morphogenesis, Claudin-5a determines the paracellular permeability of tight junctions within a transient neuroepithelial-ventricular barrier that maintains the hydrostatic fluid pressure required for brain ventricular lumen expansion. However, the roles of Claudins in development may well extend beyond being mere junctional components. Several post-translational modifications of Claudins have been characterized that indicate a direct regulation by developmental signals. This review focuses on the involvement of Claudin-5a in cerebral barrier formation in the zebrafish embryo and includes some speculations about possible modes of regulation.

Claudin-2–deficient mice are defective in the leaky and cation-selective paracellular permeability properties of renal proximal tubules

Claudin-2 is highly expressed in tight junctions of mouse renal proximal tubules, which possess a leaky epithelium whose unique permeability properties underlie their high rate of NaCl reabsorption. To investigate the role of claudin-2 in paracellular NaCl transport in this nephron segment, we generated knockout mice lacking claudin-2 (Cldn2(-/-)). The Cldn2(-/-) mice displayed normal appearance, activity, growth, and behavior. Light microscopy revealed no gross histological abnormalities in the Cldn2(-/-) kidney. Ultrathin section and freeze-fracture replica electron microscopy revealed that, similar to those of wild types, the proximal tubules of Cldn2(-/-) mice were characterized by poorly developed tight junctions with one or two continuous tight junction strands. In contrast, studies in isolated, perfused S2 segments of proximal tubules showed that net transepithelial reabsorption of Na(+), Cl(-), and water was significantly decreased in Cldn2(-/-) mice and that there was an increase in paracellular shunt resistance without affecting the apical or basolateral membrane resistances. Moreover, deletion of claudin-2 caused a loss of cation (Na(+)) selectivity and therefore relative anion (Cl(-)) selectivity in the proximal tubule paracellular pathway. With free access to water and food, fractional Na(+) and Cl(-) excretions in Cldn2(-/-) mice were similar to those in wild types, but both were greater in Cldn2(-/-) mice after i.v. administration of 2% NaCl. We conclude that claudin-2 constitutes leaky and cation (Na(+))-selective paracellular channels within tight junctions of mouse proximal tubules.

A putative role for AHNAK1 in maintaining gastric epithelial permeability barrier properties

The organization and integrity of the plasma membrane of gastric epithelial cells is crucial for providing a protective barrier for the stomach. AHNAK1 is a scaffolding protein that has been described as a protein marker of cells with barrier properties and is implicated in the regulation and organization of the cytoskeleton and the plasma membrane of epithelial cells. Previously, we reported AHNAK1 protein is expressed in different regions of the stomach of healthy human organ donors, and in cultured non‐transformed rat gastric mucosal (RGM1) polarized epithelial cells. We showed AHNAK1 protein is highly expressed in the cytoplasm and at the plasma membrane of RGM1 cells. Here, we investigated a possible function for AHNAK1 in mediating the selective paracellular permeability properties of gastric epithelial cells. An siRNA mediated knockdown of AHNAK1 protein in RGM1 cells by approximately 70% was determined by Western blot and densitometric analysis. Immunofluorescence studies revealed AHNAK1 protein colocalizes with the tight junction associated protein ZO2. In vitro cell permeability assays showed an increase in permeation of fluorescein isothiocyanate (FITC)‐dextran across RGM1 monolayers after transfecting cells with a smartpool of siRNAs targeting AHNAK1 compared to transfecting cells with nontargeting siRNAs or treating cells with EDTA. Taken together, our results suggest a role for AHNAK1 in maintaining the integrity of the permeability barrier properties of gastric mucosal cells. This research was supported by an American Heart Association Predoctoral Fellowship and a Veterans Affairs Merit grant.

Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

BackgroundTight junctions are an intercellular adhesion complex of epithelial and endothelial cells, and form a paracellular barrier that restricts the diffusion of solutes on the basis of size and charge. Tight junctions are formed by multiprotein complexes containing cytosolic and transmembrane proteins. How these components work together to form functional tight junctions is still not well understood and will require a complete understanding of the molecular composition of the junction.ResultsHere we identify a new transmembrane component of tight junctions: MarvelD3, a four-span transmembrane protein. Its predicted transmembrane helices form a Marvel (MAL and related proteins for vesicle traffic and membrane link) domain, a structural motif originally discovered in proteins involved in membrane apposition and fusion events, such as the tight junction proteins occludin and tricellulin. In mammals, MarvelD3 is expressed as two alternatively spliced isoforms. Both isoforms exhibit a broad tissue distribution and are expressed by different types of epithelial as well as endothelial cells. MarvelD3 co-localises with occludin at tight junctions in intestinal and corneal epithelial cells. RNA interference experiments in Caco-2 cells indicate that normal MarvelD3 expression is not required for the formation of functional tight junctions but depletion results in monolayers with increased transepithelial electrical resistance.ConclusionsOur data indicate that MarvelD3 is a third member of the tight junction-associated occludin family of transmembrane proteins. Similar to occludin, normal expression of MarvelD3 is not essential for the formation of functional tight junctions. However, MarvelD3 functions as a determinant of epithelial paracellular permeability properties.

Claudins at the gate: determinants of renal epithelial tight junction paracellular permeability

The epithelial tight junction (TJ) is responsible for the control of paracellular transport between epithelial cells (gate function) and the maintenance of apical/basolateral polarity by preventing the diffusion of membrane lipids and/or proteins from one surface domain to another (fence function). Renal tubule epithelia in the mammalian nephron have TJs that determine paracellular transport characteristics. Paracellular transport across renal tubular epithelial TJs (gate function) varies in different segments of the nephron. A large family of recently identified TJ-associated transmembrane proteins named claudins appear to determine the paracellular permeability properties of the TJ. A combination of inherited human diseases, renal epithelial cell culture models, and nephron expression patterns of claudins is providing important clues about how claudin molecules determine the TJ gate function of renal epithelia in different segments of the nephron.

Extracellular Signal-regulated Kinases 1/2 Control Claudin-2 Expression in Madin-Darby Canine Kidney Strain I and II Cells

The tight junction of the epithelial cell determines the characteristics of paracellular permeability across epithelium. Recent work points toward the claudin family of tight junction proteins as leading candidates for the molecular components that regulate paracellular permeability properties in epithelial tissues. Madin-Darby canine kidney (MDCK) strain I and II cells are models for the study of tight junctions and based on transepithelial electrical resistance (TER) contain "tight" and "leaky" tight junctions, respectively. Overexpression studies suggest that tight junction leakiness in these two strains of MDCK cells is conferred by expression of the tight junction protein claudin-2. Extracellular signal-regulated kinase (ERK) 1/2 activation by hepatocyte growth factor treatment of MDCK strain II cells inhibited claudin-2 expression and transiently increased TER. This process was blocked by the ERK 1/2 inhibitor U0126. Transfection of constitutively active mitogen-activated protein kinase/extracellular signal-regulated kinase kinase into MDCK strain II cells also inhibited claudin-2 expression and increased TER. MDCK strain I cells have higher levels of active ERK 1/2 than do MDCK strain II cells. U0126 treatment of MDCK strain I cells decreased active ERK 1/2 levels, induced expression of claudin-2 protein, and decreased TER by approximately 20-fold. U0126 treatment also induced claudin-2 expression and decreased TER in a high resistance mouse cortical collecting duct cell line (94D). These data show for the first time that the ERK 1/2 signaling pathway negatively controls claudin-2 expression in mammalian renal epithelial cells and provide evidence for regulation of tight junction paracellular transport by alterations in claudin composition within tight junction complexes.

Claudin-2 is selectively expressed in proximal nephron in mouse kidney.

The proximal nephron possesses a leaky epithelium with unique paracellular permeability properties that underlie its high rate of passive NaCl and water reabsorption, but the molecular basis is unknown. The claudins are a large family of transmembrane proteins that are part of the tight junction complex and likely form structural components of a paracellular pore. To localize claudin-2 in the mouse kidney, we performed in situ hybridization using an isoform-specific riboprobe and immunohistochemistry using a polyclonal antibody directed against a COOH-terminal peptide. Claudin-2 mRNA and protein were found throughout the proximal tubule and in the contiguous early segment of the thin descending limb of long-looped nephrons. The level of expression demonstrated an axial increase from proximal to distal segments. In confocal images, the subcellular localization of claudin-2 protein coincided with that of the tight junction protein ZO-1. Our findings suggest that claudin-2 is a component of the paracellular pathway of the most proximal segments of the nephron and that it may be responsible for their uniquely leaky permeability properties.

Claudin-2 is selectively expressed in proximal nephron in mouse kidney

First published August 15, 2001; 10.1152/ajprenal.00021.2001.—The proximal nephron possesses a leaky epithelium with unique paracellular permeability properties that underlie its high rate of passive NaCl and water reabsorption, but the molecular basis is unknown. The claudins are a large family of transmembrane proteins that are part of the tight junction complex and likely form structural components of a paracellular pore. To localize claudin-2 in the mouse kidney, we performed in situ hybridization using an isoform-specific riboprobe and immunohistochemistry using a polyclonal antibody directed against a COOH-terminal peptide. Claudin-2 mRNA and protein were found throughout the proximal tubule and in the contiguous early segment of the thin descending limb of long-looped nephrons. The level of expression demonstrated an axial increase from proximal to distal segments. In confocal images, the subcellular localization of claudin-2 protein coincided with that of the tight junction protein ZO-1. Our findings suggest that claudin-2 is a component of the paracellular pathway of the most proximal segments of the nephron and that it may be responsible for their uniquely leaky permeability properties.

Heterogeneity in expression and subcellular localization of claudins 2, 3, 4, and 5 in the rat liver, pancreas, and gut

Background & Aims: Paracellular transport varies widely among epithelia of the gastrointestinal tract. We determined whether members of the claudin family of tight junction proteins are differentially expressed consistent with a potential role in creating these variable properties. Methods: Rabbit polyclonal antibodies were produced against peptides from claudins 2 through 5. The distribution of individual claudins was detected by immunoblotting, and their cell type and subcellular localization were determined by immunofluorescence on cryosections of rat liver, pancreas, stomach, and small and large intestine. Results: All antibodies detected single bands of the expected size on immunoblots and were monospecific based on peptide competition studies. Immunoblotting detected strong differences among tissues in the expression level of each claudin. Immunolocalization confirmed these differences and revealed striking variations in expression patterns. In the liver, claudin 2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, claudin 3 is uniformly expressed, claudin 4 is absent, and claudin 5 is only expressed in endothelial junctions. In the pancreas, claudin 2 is only detected in junctions of the duct epithelia, claudin 5 only in junctions of acinar cells, whereas claudin 3 and 4 are in both. Among differences in the gut are a crypt-to-villus decrease in claudin 2, a highly restricted expression of claudin 4 to colonic surface cells, and the finding that some claudins can be junctional, lateral, or show a gradient in junctional vs. lateral localization along the crypt-to-villus surface axis. Conclusions: Claudins have very different expression patterns among and within gastrointestinal tissues. We propose these patterns underlie differences in paracellular permeability properties, such as electrical resistance and ion selectivity that would complement known differences in transcellular transport.GASTROENTEROLOGY 2001;120:411-422

Endothelial cell junctions in the ciliary body microvasculature

Endothelial cells of the microvasculature (arterioles, fenestrated capillaries, and small venules) of the rabbit ciliary body have been examined in freeze-fracture replicas in ultrahigh vacuum and at very low temperatures, with special regard to their junctional boundaries. The junctional complexes of fenestrated capillaries and venular endothelium are discontinuous and do not seal the interendothelial clefts. They cannot be compared with zonulae occludentes or tight junctions, while the junctions in the arteriolar endothelium are continuous and well organized, suggesting a very low degree of paracellular permeability. It would be of interest to know if these structural differences at the junctional level in each segment of the microvasculature correspond to different paracellular permeability properties.