Paracellular Pores Research Articles

Role of intestinal claudin-2 in calcium transport and kidney stone disease

Background: Kidney stone disease is characterized by intestinal hyperabsorption of calcium and hypercalciuria, leading to the formation of calcium crystals in the kidney. Claudin-2 is a tight junction protein that forms paracellular cation pores and mutations in its gene are associated with kidney stone disease. We have recently shown that mice deficient in Cldn2 are hypercalciuric due to both decreased renal reabsorption and increased intestinal absorption of calcium and develop medullary mineral deposits reminiscent of kidney stone formers. We, therefore, hypothesized that intestinal claudin-2 is important for calcium secretion, especially in the colon, and that loss of claudin-2 results in increased net intestinal calcium absorption, thereby contributing to kidney stone disease. Method: Villin-Cre (Vil1-Cre/1000Gum) mice were crossed to Cldn2 floxed mice to generate intestine-specific claudin-2 knockout mice. Successful knockout of claudin-2 was confirmed at the mRNA and protein level using qPCR, immunofluorescence staining, and western blot. Male and female littermates were characterized for hypercalciuria from age 3-10 weeks. Circulating levels of parathyroid hormone and 1,25-dihydroxyvitamin D (1,25(OH)2D) were measured by ELISA, and mRNA levels of seven paracellular and transcellular calcium transport genes in the ileum, cecum, distal colon, and nine in the kidney assessed by qPCR. Result: In females, Cldn2 expression was knocked out in the intestine but not in the kidney. However, in males, Cldn2 was knocked out in the intestine and also partially knocked out in the kidneys (where villin is also expressed). Knockout females had normal urine calcium, but knockout males had significantly higher urine calcium at weaning (3 weeks of age) compared to wildtype, which was normalized by 4 weeks of age. At 4 weeks, Cldn19 mRNA was downregulated in knockout mice of both sexes. Male knockout mice had a reduction in intestinal Cldn12, Cldn15, Slc8a1, and increased intestinal Cacnad1, increased renal Cyp27b1 expression, and increased circulating 1,25(OH)2D. Conclusion: Our results suggest that intestine-specific loss of claudin-2, which was only observed in villin-Cre, Cldn2 floxed females, does not cause hypercalciuria. Ongoing whole-body calcium balance and everted sac calcium permeability experiments will determine whether this is because intestinal calcium hyperabsorption is masked by compensatory mechanisms. NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inside Front Cover: Cerebrospinal fluid efflux through dynamic paracellular pores on venules as a missing piece of the brain drainage system (EXP2 2/2024)

Inside Front Cover: Cerebrospinal fluid efflux through dynamic paracellular pores on venules as a missing piece of the brain drainage system (EXP2 2/2024)

Cerebrospinal fluid efflux through dynamic paracellular pores on venules as a missing piece of the brain drainage system

AbstractThe glymphatic system plays a key role in the clearance of waste from the parenchyma, and its dysfunction has been associated with the pathogenesis of Alzheimer's disease (AD). However, questions remain regarding its complete mechanisms. Here, we report that efflux of cerebrospinal fluid (CSF)/interstitial fluid (ISF) solutes occurs through a triphasic process that cannot be explained by the current model, but rather hints at the possibility of other, previously undiscovered routes from paravenous spaces to the blood. Using real‐time, in vivo observation of efflux, a novel drainage pathway was discovered, in which CSF molecules enter the bloodstream directly through dynamically assembled, trumpet‐shaped pores (basolateral ϕ<8 μm; apical ϕ < 2 μm) on the walls of brain venules. As Zn2+ could facilitate the brain clearance of macromolecular ISF solutes, Zn2+‐induced reconstruction of the tight junctions (TJs) in vascular endothelial cells may participate in pore formation. Thus, an updated model for glymphatic clearance of brain metabolites and potential regulation is postulated. In addition, deficient clearance of Aβ through these asymmetric venule pores was observed in AD model mice, supporting the notion that impaired brain drainage function contributes to Aβ accumulation and pathogenic dilation of the perivascular space in AD.

The blood-prostate barrier: an obstacle to drug delivery into the prostate.

The blood-prostate barrier (BPB), a non-static physical barrier, stands as an obstacle between the prostate stroma and the lumen of the prostate gland tube. The barrier has the ability to dynamically regulate and strictly control the mass exchange between the blood and the prostate, thereby limiting drug penetration into the prostate. The basement membrane, fibrous stromal layer, capillary endothelial cell, prostatic ductal epithelial cell, lipid layer, etc., have been confirmed to be involved in the composition of the barrier structure and altered membrane permeability mainly by regulating the size of paracellular ion pores. Various studies have been conducted to improve the efficiency of drug therapy for prostate diseases by changing the administration approaches, improving barrier permeability and increasing drug penetration. To gain a full understanding of BPB, the composition of BPB, the methodology for evaluating the permeability of BPB and alterations in barrier function under pathological conditions are summarized in this review. To find a shortcut for drug delivery across BPB, the biodistribution of drugs in the prostate and different methods of improving drug penetration across BPB are outlined. This review offers an applied perspective on recent advances in drug delivery across BPB.

The role of claudin-2 in kidney function and dysfunction.

Claudin-2 is a tight junction protein expressed in leaky epithelia where it forms paracellular pores permeable to cations and water. The paracellular pore formed by claudin-2 is important in energy-efficient cation and water transport in the proximal tubules of the kidneys. Mounting evidence now suggests that claudin-2 may modulate cellular processes often altered in disease, including cellular proliferation. Also, dysregulation of claudin-2 expression has been linked to various diseases, including kidney stone disease and renal cell carcinoma. However, the mechanisms linking altered claudin-2 expression and function to disease are poorly understood and require further investigation. The aim of this review is to discuss the current understanding of the role of claudin-2 in kidney function and dysfunction. We provide a general overview of the claudins and their organization in the tight junction, the expression, and function of claudin-2 in the kidney, and the evolving evidence for its role in kidney disease.

#5837 MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF THE PERITONEAL MESOTHELIAL AND ENDOTHELIAL CELL BARRIER

Abstract Background and Aims Solute transport across cellular levels is mediated by paracellular and transcellular pores, channels and carriers. Knowledge on their cell specific expression, regulation and function in peritoneal dialysis (PD) is limited. Method Polarized primary (HPMC) and immortalized human peritoneal mesothelial (MeT-5A), microvascular (HCMEC) and umbilical vein endothelial (HUVEC) cells underwent RNA sequencing and gene enrichment analysis. Key findings were confirmed by western blotting (WB), confocal and single molecule localization microscopy (SMLM). Arteriolar transcriptome and proteome datasets of non-CKD, CKD5 and PD children (n = 6/group) underwent targeted transport pathways analysis. Key transporter proteins were quantified in parietal peritoneum (n = 20–30/group) and related to peritoneal transport rates available in 23 children. Barrier function was studied in vitro (transepithelial electrical resistance, TER, and molecular weight dependent flux), ex vivo and in vivo. Results Junction, transmembrane and transcytotic transporter expression was highly cell type specific on RNA and protein level. Sealing Claudin (CLDN)1 was only expressed in mesothelial cells, sealing CLDN5 in endothelial cells. TER which reflects functional junction status, was 50% lower in HCMEC compared to HUVEC and the two mesothelial cell lines; 4- and 10-kDa dextran permeability was higher in HCMEC. At nanoscale, SMLM yielded highest distance of junction molecules in HCMEC and different spatial organisation, reflecting the low TER. In sheep peritoneum, removal of the mesothelium abolished tissue TER. In mice, short-term LPS exposure to modify mesothelial permeability resulted in faster transperitoneal 4- and 70-kDa dextran transport, suggesting a specific barrier function of the mesothelium. In human parietal peritoneum, total endothelial surface area per section was age dependently 1.5- to 2-fold higher than the respective mesothelial surface area, and further increased with double-chamber PD fluid, due to major hypervascularisation. Tight junction proteins CLDN-1 to -5, and -15, ZO-1, occludin and tricellulin, and transcellular transporter ENaC, PIT1, and SGLT1 were detected in mesothelial and arteriolar endothelial cells. In CKD mesothelial CLDN-1 and arteriolar CLDN-2 and -3 were more abundant than in non-CKD controls, and PD patients had highest mesothelial and arteriolar CLDN-1 and mesothelial CLDN-2, lower mesothelial and arteriolar claudin-3 and lower arteriolar ENaC. D/P creatinine and D/D0 glucose correlated with arteriolar CLDN-2 and with mesothelial CLDN-4 and -15, which are pore forming junctions, and for creatinine with mesothelial PIT-1, a sodium/phosphate co-transporter. Conclusion We provide the first in-depth analysis of peritoneal determinants of solute transport. The molecular expression pattern of the mesothelial and endothelial cell barrier and transporter proteins differs substantially. Disruption of the mesothelial layer increases peritoneal solute absorption rate. In the human peritoneum, peritoneal transporter status is modified by CKD and PD, and pore forming junction proteins are associated with dialytic solute transport function. These represent a promising target for therapeutic intervention.

Protein-protein interactions at the tight junctions interface.

The epithelial and endothelial tissues are the physiological barriers against chemical and biological insurgents by constituting the tight junctions. Tight junctions are specialized adhesion constructs formed by the assembly of claudin family proteins. Membrane-embedded claudin proteins are the functional determinants of the tight junctions. In the paracellular space between two adjacent cells, claudins form size and charge selective pores that impede the free diffusion of these solutes. The vital role of tight junctions in various tissue barriers such as the blood-brain barrier, renal barrier, and gut barrier are currently active research fields, yet a complete molecular understanding of the tight junctions is elusive. Here we have employed multiscale molecular dynamics simulations to gain insight into the underpinnings of claudin assembly at tight junctions. From these simulations, a clear picture of how the membrane milieu influences the tight junction's assembly is evident. Both cholesterol and ceramide lipids limit claudin-claudin interaction in the lipid bilayer due to reduced fluidity. Further, we demonstrated that claudins preferentially assemble into distinct dimers that are the primary building blocks of the paracellular pores. The molecular-level insights gained from this work are critical in unraveling the physiological roles of tight junctions.

The impact of pathogenic and artificial mutations on Claudin-5 selectivity from molecular dynamics simulations

Tight-junctions (TJs) are multi-protein complexes between adjacent endothelial or epithelial cells. In the blood-brain-barrier (BBB), they seal the paracellular space and the Claudin-5 (Cldn5) protein forms their backbone. Despite the fundamental role in brain homeostasis, little is known on Cldn5-based TJ assemblies. Different structural models were suggested, with Cldn5 protomers generating paracellular pores that restrict the passage of ions and small molecules. Recently, the first Cldn5 pathogenic mutation, G60R, was identified and shown to induce Cl−-selective channels and Na+ barriers in BBB TJs, providing an excellent opportunity to validate the structural models. Here, we used molecular dynamics to study the permeation of ions and water through two distinct G60R-Cldn5 paracellular architectures. Only the so-called Pore I reproduces the functional modification observed in experiments, displaying a free energy (FE) minimum for Cl− and a barrier for Na+ consistent with anionic selectivity. We also studied the artificial Q57D and Q63D mutations in the constriction region, Q57 being conserved in Cldns except for cation permeable homologs. In both cases, we obtain FE profiles consistent with facilitated passage of cations. Our calculations provide the first in-silico description of a Cldn5 pathogenic mutation, further assessing the TJ Pore I model and yielding new insight on BBB’s paracellular selectivity.

Computational Assessment of Different Structural Models for Claudin-5 Complexes in Blood-Brain Barrier Tight Junctions.

The blood-brain barrier (BBB) strictly regulates the exchange of ions and molecules between the blood and the central nervous system. Tight junctions (TJs) are multimeric structures that control the transport through the paracellular spaces between the adjacent brain endothelial cells of the BBB. Claudin-5 (Cldn5) proteins are essential for TJ formation and assemble into multiprotein complexes via cis-interactions within the same cell membrane and trans-interactions across two contiguous cells. Despite the relevant biological function of Cldn5 proteins and their role as targets of brain drug delivery strategies, the molecular details of their assembly within TJs are still unclear. Two different structural models have been recently introduced, in which Cldn5 dimers belonging to opposite cells join to generate paracellular pores. However, a comparison of these models in terms of ionic transport features is still lacking. In this work, we used molecular dynamics simulations and free energy (FE) calculations to assess the two Cldn5 pore models and investigate the thermodynamic properties of water and physiological ions permeating through them. Despite different FE profiles, both structures present single/multiple FE barriers to ionic permeation, while being permissive to water flux. These results reveal that both models are compatible with the physiological role of Cldn5 TJ strands. By identifying the protein-protein surface at the core of TJ Cldn5 assemblies, our computational investigation provides a basis for the rational design of synthetic peptides and other molecules capable of opening paracellular pores in the BBB.

Diverse ultrastructural landscape of atherosclerotic endothelium

Background and aimsThe endothelium plays a major role in atherosclerosis, yet the endothelial plaque surface is a largely uncharted territory. Here we hypothesize that atherosclerosis-driven remodeling of the endothelium is a dynamic process, involving both damaging and regenerative mechanisms. MethodsUsing scanning electron microscopy (SEM) and immuno-SEM, we studied endothelial junction ultrastructure, endothelial openings and immune cell-endothelium interactions in eight apoe−/− mice and two human carotid plaques. ResultsThe surface of early mouse plaques (n = 11) displayed a broad range of morphological alterations, including junctional disruptions and large transcellular endothelial pores with the average diameter between 0.6 and 3 μm. The shoulder region of advanced atherosclerotic lesions (n = 7) had a more aggravated morphology with 8 μm-size paracellular openings at two-fold higher density. In contrast, the central apical surface of advanced plaques, i.e., the plaque body (n = 7), displayed endothelial normalization, as shown by a significantly higher frequency of intact endothelial junctions and a lower incidence of paracellular pores. This normalized endothelial phenotype correlated with low immune cell density (only 5 cells/mm2).The human carotid plaque surface (n = 2) displayed both well-organized and disrupted endothelium with similar features as described above. In addition, they were accompanied by extensive thrombotic areas. ConclusionsOur study unveils the spectrum of endothelial abnormalities associated with the development of atherosclerosis. These were highly abundant in early lesions and in the shoulder region of advanced plaques, while normalized at the advanced plaque's body. Similar endothelial features were observed in human atherosclerotic plaques, underlining the versatility of endothelial transformations in atherosclerosis.

Increased intestinal phosphate absorption, an often-overlooked effect of vitamin D.

Increased intestinal phosphate absorption, an often-overlooked effect of vitamin D.

SiRNA targeting Schlemm’s canal endothelial tight junctions enhances outflow facility and reduces IOP in a steroid-induced OHT rodent model

Systemic or localized application of glucocorticoids (GCs) can lead to iatrogenic ocular hypertension, which is a leading cause of secondary open-angle glaucoma and visual impairment. Previous work has shown that dexamethasone increases zonula occludens-1 (ZO-1) protein expression in trabecular meshwork (TM) cells, and that an antisense oligonucleotide inhibitor of ZO-1 can abolish the dexamethasone-induced increase in trans-endothelial flow resistance in cultured Schlemm’s canal (SC) endothelial and TM cells. We have previously shown that intracameral inoculation of small interfering RNA (siRNA) targeting SC endothelial cell tight junction components, ZO-1 and tricellulin, increases aqueous humor outflow facility ex vivo in normotensive mice by reversibly opening SC endothelial paracellular pores. In this study, we show that targeted siRNA downregulation of these SC endothelial tight junctions reduces intraocular pressure (IOP) in vivo, with a concomitant increase in conventional outflow facility in a well-characterized chronic steroid-induced mouse model of ocular hypertension, thus representing a potential focused clinical application for this therapy in a sight-threatening scenario.

In vivo and ex vivo assessment of bladder hyper-permeability and using molecular targeted magnetic resonance imaging to detect claudin-2 in a mouse model for interstitial cystitis.

To determine if the URO-MCP-1 mouse model for bladder IC/BPS is associated with in vivo bladder hyper-permeability, as measured by contrast-enhanced MRI (CE-MRI), and assess whether molecular-targeted MRI (mt-MRI) can visualize in vivo claudin-2 expression as a result of bladder hyper-permeability. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic, painful condition of the bladder that affects primarily women. It is known that permeability plays a substantial role in IC/BPS. Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. Claudin-2 is a molecular marker that is associated with increased hyperpermeability in the urothelium. CE-MRI was used to measure bladder hyper-permeability in the URO-MCP-1 mice. A claudin-2-specific mt-MRI probe was used to assess in vivo levels of claudin-2. The mt-MRI probe consists of an antibody against claudin-2 conjugated to albumin that had Gd-DTPA (gadolinium diethylenetriamine pentaacetate) and biotin attached. Verification of the presence of the mt-MRI probe was done by targeting the biotin moiety for the probe with streptavidin-horse radish peroxidase (SA-HRP). Trans-epithelial electrical resistance (TEER) was also used to assess bladder permeability. The URO-MCP-1 mouse model for IC/BPS was found to have a significant increase in bladder permeability, following liposaccharide (LPS) exposure, compared to saline-treated controls. mt-MRI- and histologically-detectable levels of the claudin-2 probe were found to increase with LPS -induced bladder urothelial hyper-permeability in the URO-MCP-1 IC mouse model. Levels of protein expression for claudin-2 were confirmed with immunohistochemistry and immunofluorescence imaging. Claudin-2 was also found to highly co-localize with zonula occlidens-1 (ZO-1), a tight junction protein. The combination of CE-MRI and TEER approaches were able to demonstrate hyper-permeability, a known feature associated with some IC/BPS patients, in the LPS-exposed URO-MCP-1 mouse model. This MRI approach could be clinically translated to establish which IC/BPS patients have bladder hyper-permeability and help determine therapeutic options. In addition, the in vivo molecular-targeted imaging approach can provide invaluable information to enhance our understanding associated with bladder urothelium hyper-permeability in IC/BPS patients, and perhaps be used to assist in developing further therapeutic strategies.

Interclaudin Antagonism: A New Mechanism Of Tight Junction Dependent Barrier Regulation

Tight junctions form selectively‐permeable barriers that regulate paracellular flux. Individual members of the claudin protein family have been categorized as having either pore‐forming or sealing functions. For example, claudin‐2 forms paracellular cation‐selective pores. Conversely, overexpression of claudin‐4 can enhance the paracellular barrier, i.e., reduce permeability, in vitro; claudin‐4 has therefore been classified as a sealing claudin. Although specific residues that form paracellular pores, e.g., within claudin‐2, have been defined, the molecular mechanisms by which sealing claudins, e.g., claudin‐4, regulate the barrier have not. The goal of these studies was to determine the means by which the representative sealing protein claudin‐4 reduces paracellular permeability.METHODSClaudin‐4 was knocked out in Madin‐Darby Canine Kidney I (MDCK I) epithelial cells, which do not express claudin‐2 or other pore‐forming claudins, e.g., claudin‐15. These Cldn4‐KO MDCK I cells were stably transfected to inducibly express mCherry‐claudin‐4 with or without constitutive expression of EGFP‐claudin‐2. Transepithelial resistance (TER) and bi‐ionic potential measurements were used to assess barrier function. Protein anchoring and exchange were determined by Fluorescence Recovery After Photobleaching (FRAP). Widefield, confocal, and super‐resolution microscopy were used for live‐cell imaging of fluorescent claudin proteins in MDCK I and undifferentiated, fibroblast‐like cells (U2OS).RESULTSSurprisingly, claudin‐4 knockout had no effect on the paracellular barrier. Inducible mCherry‐claudin‐4 expression was also insufficient to modify TER. In contrast, EGFP‐claudin‐2 expression reduced TER 10‐fold (p<0.0001). Induction of mCherry‐claudin‐4 expression in EGFP‐claudin‐2‐expressing, but not claudin‐2‐deficient, MDCK I increased TER by 92±18% (p<0.001). FRAP analysis showed that mCherry‐claudin‐4 doubled the mobile fraction of tight junction‐associated EGFP‐claudin‐2 from 16±3% to 32±4% (p<0.001). In U2OS cells, mCherry‐claudin‐4 alone was incapable of forming tight junction‐like strands whereas EGFP‐claudin‐2 assembled into polymeric strands. Induction of mCherry‐claudin‐4 expression disrupted strands formed by EGFP‐claudin‐2, which was subsequently internalized and trafficked to LAMP2‐positive late endosomes. Although MitMAB (dynamin inhibitor) prevented EGFP‐claudin‐2 removal from the junction after mCherry‐claudin‐4 induction, monolayer TER was still increased by mCherry‐claudin‐4 expression.CONCLUSIONSThese data refute the widely‐held view that claudin‐4 forms polymers that seal the paracellular pathway by demonstrating that i) neither knockout nor overexpression of claudin‐4 affect barrier function; and ii) claudin‐4 does not assemble into tight junction‐like strands (polymers). Instead, the results support an alternative model where claudin‐4 enhances barrier function by destabilizing and inhibiting flux via claudin‐2 pores. This challenges existing dogma regarding claudin function and reveals interclaudin antagonism as an unsuspected mechanism of barrier regulation.Support or Funding InformationNIH 5R01DK06 (JRT), CCF RFA 622459 (NS)

Predicting selectivity of paracellular pores for biomimetic applications

Systematic approach to predicting selectivity of paracellular pores for biomimetic applications.

The Role of Schlemm's Canal Endothelium Cellular Connectivity in Giant Vacuole Formation: A 3D Electron Microscopy Study.

PurposeWe investigated whether cellular connectivity between Schlemm's canal (SC) inner wall (IW) endothelium, and juxtacanalicular connective tissue (JCT), and between IW endothelial cells, plays a role in giant vacuole (GV) and pore formation by comparing perfusion- and immersion-fixed eyes.MethodsNormal human donor eyes (n = 4) were either immersion-fixed (0 mm Hg) or perfusion-fixed (15 mm Hg). Trabecular meshwork near SC was imaged using serial block-face scanning electron microscopy. A total of 12 IW cells from each group were 3D-reconstructed from ∼7040 electron micrographs and compared. In each cell, connections between IW cells and JCT cells/matrix were quantified; IW/IW connectivity was measured by cell border overlap length. GV volume, density, shape, and intracellular and paracellular pores were analyzed.ResultsThe mean number of IW/JCT cell-cell connections per cell significantly decreased (P < 0.01) while the summed GV volume per cell significantly increased (P < 0.01) in perfusion-fixed eyes compared to immersion-fixed eyes. Intracellular pores were observed in 14.6% of GVs in perfusion-fixed eyes and not observed in immersion-fixed eyes. The mean IW/IW overlap length per cell decreased (P < 0.01), and paracellular pores were found only in regions where IW/IW connectivity was minimal (overlap length = 0 μm) in perfusion-fixed eyes and not observed in immersion-fixed eyes.ConclusionsOur data suggest that changes in IW/JCT connectivity may be an important factor in the formation of larger GVs, and decreased IW/IW connectivity may promote paracellular pore formation. Targeting the IW/JCT and IW/IW connectivity may therefore be a potential strategy to regulate outflow resistance and IOP.

Urinary K+ promotes irritative voiding symptoms and pain in the face of urothelial barrier dysfunction

The internal surface of the bladder is lined by the urothelium, a stratified epithelium that forms an impermeable barrier to water and urine constituents. Abnormalities in the urothelial barrier have been described in certain forms of cystitis and were hypothesized to contribute to irritative voiding symptoms and pain by allowing the permeation of urinary K+ into suburothelial tissues, which then alters afferent signaling and smooth muscle function. Here, we examined the mechanisms underlying organ hyperactivity and pain in a model of cystitis caused by adenoviral-mediated expression of claudin-2 (Cldn2), a tight junction protein that forms paracellular pores and increases urothelial permeability. We found that in the presence of a leaky urothelium, intravesical K+ sensitizes bladder afferents and enhances their response to distension. Notably, dietary K+ restriction, a maneuver that reduces urinary K+, prevented the development of pelvic allodynia and inflammation seen in rats expressing Cldn2. Most importantly, intravesical K+ causes and is required to maintain bladder hyperactivity in rats with increased urothelial permeability. Our study demonstrates that in the face of a leaky urothelium, urinary K+ is the main determinant of afferent hyperexcitability, organ hyperactivity and pain. These findings support the notion that voiding symptoms and pain seen in forms of cystitis that coexist with urothelial barrier dysfunction could be alleviated by cutting urinary K+ levels.

Abstract 5215: Vitamin D receptor regulates intestinal barriers in colon cancer

Abstract Background Vitamin D and Vitamin D receptor (VDR) play important roles in the development of colon cancer. Tight junction structures are essential in intestinal barrier integrity, inflammation, and cancer. The disruption of tight junctions is a common manifestation of various diseases including cancers. Claudin-5 is a tight junction protein that is expressed in epithelia and endothelia and form paracellular barriers and pores that determine permeability. It is downregulated in colon cancer. Although VDR and Claudin-5 are all involved in colon cancer, it remains unclear whether they are closely related or function independently. Methods In the current study, we investigate the novel role of VDR in regulating Claudin-5, using whole body VDR knockout mice (VDR-/-) and intestinal epithelial VDR knockout mice (VDRΔIEC). In vitro, VDR-/- embryonic fibroblasts (MEF) cells, cultured human intestinal epithelial cells, organoids, and human colon cancer samples were used to determine the molecular mechanism. Results In intestinal samples of colon cancer patients, VDR expression is low, whereas the expression of Claudin-5 is also decreased. In the colon of VDR-/- mice and VDRΔIEC mice, deletion of VDR led to lower claudin-5 at the protein and mRNA levels. This observation was also confirmed by immunostaining of Claudin-5. In VDR-/- MEF cells, both Western blot and real time PCR revealed that VDR deletion led to lower protein and mRNA levels of claudin-5 in cells without any treatment. Vitamin D3 is known to increase VDR expression and activate VDR signaling. Protein and mRNA levels of Claudin-5 were significantly elevated in mice, mouse organoids, and human intestinal epithelial SKCO15 cells treated with 1, 25 vitamin D3. In contrast, knockdown of Claudin-5 using siRNA did not change the expression level of VDR protein in human colonic epithelial cells. Our data suggest that the Claudin-5 gene is a downstream target of the VDR signaling pathway. Conclusion Taken together, we show that VDR is important for the maintenance of physiological level of Claudin-5. This study also reveals a complex role for vitamin D/VDR regulation of intestinal barrier functions in colon cancer. Citation Format: Yong-guo Zhang, Rong Lu, Danika Bakke, Yinglin Xia, Jun Sun. Vitamin D receptor regulates intestinal barriers in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5215.

Claudin Loss-of-Function Disrupts Tight Junctions and Impairs Amelogenesis.

Claudins are a family of proteins that forms paracellular barriers and pores determining tight junctions (TJ) permeability. Claudin-16 and -19 are pore forming TJ proteins allowing calcium and magnesium reabsorption in the thick ascending limb of Henle's loop (TAL). Loss-of-function mutations in the encoding genes, initially identified to cause Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis (FHHNC), were recently shown to be also involved in Amelogenesis Imperfecta (AI). In addition, both claudins were expressed in the murine tooth germ and Claudin-16 knockout (KO) mice displayed abnormal enamel formation. Claudin-3, an ubiquitous claudin expressed in epithelia including kidney, acts as a barrier-forming tight junction protein. We determined that, similarly to claudin-16 and claudin-19, claudin-3 was expressed in the tooth germ, more precisely in the TJ located at the apical end of secretory ameloblasts. The observation of Claudin-3 KO teeth revealed enamel defects associated to impaired TJ structure at the secretory ends of ameloblasts and accumulation of matrix proteins in the forming enamel. Thus, claudin-3 protein loss-of-function disturbs amelogenesis similarly to claudin-16 loss-of-function, highlighting the importance of claudin proteins for the TJ structure. These findings unravel that loss-of-function of either pore or barrier-forming TJ proteins leads to enamel defects. Hence, the major structural function of claudin proteins appears essential for amelogenesis.

Application of a Human Intestinal Epithelial Cell Monolayer to the Prediction of Oral Drug Absorption in Humans as a Superior Alternative to the Caco-2 Cell Monolayer

A human small intestinal epithelial cell (HIEC) monolayer was recently established in our laboratories as a novel system to evaluate the Papp (apparent permeability coefficient) of compounds during their absorption in humans. An effusion-based analysis using polyethylene glycol oligomers with molecular weights ranging from 194-898 indicated that HIEC and Caco-2 cell monolayers both had paracellular pores with 2 distinct radiuses (∼5 and 9-14 Å), whereas the porosity of large pores was 11-fold higher in the HIEC monolayer (44 × 10−8) than in the Caco-2 cells (4 × 10−8). A comparison between the fraction-absorbed (Fa) values observed in humans and those predicted from Papp values in both monolayers indicated that the HIEC monolayer had markedly higher precision to predict Fa values with root mean square error of 9.40 than the Caco-2 cells (root mean square error = 16.90) for 10 paracellularly absorbed compounds. Furthermore, the accuracy of the HIEC monolayer to classify the absorption of 23 test drugs with diverse absorption properties, including different pathways in the presence or absence of susceptibility to efflux transporters, was higher than that of the Caco-2 cell monolayer. In conclusion, the HIEC monolayer exhibited advantages over Caco-2 cells in the ranking and prediction of absorption of compounds in humans.