Selective Uptake of Macromolecules to the Brain in Microfluidics and Animal Models Using the HAVN1 Peptide as a Blood-Brain Barrier Modulator.

Monoclonal antibodies (mAbs) possess favorable pharmacokinetic properties, high binding specificity and affinity, and minimal off-target effects, making them promising therapeutic agents for central nervous system (CNS) disorders. However, their development as effective therapeutic and diagnostic agents for brain disorders is hindered by their limited ability to efficiently penetrate the blood-brain barrier (BBB). Therefore, it is crucial to develop efficient delivery methods that enhance the penetration of antibodies into the brain. Previous studies have demonstrated the potential of cadherin-derived peptides (i.e., ADTC5, HAVN1 peptides) as BBB modulators (BBBMs) to increase paracellular porosities for penetration of molecules across the BBB. Here, we test the effectiveness of the leading BBBM peptide, HAVN1 (Cyclo(1,6)SHAVSS), in enhancing the permeation of various monoclonal antibodies through the BBB using both in vitro and in vivo systems. In vitro, HAVN1 has been shown to increase the permeability of fluorescently labeled macromolecules, such as a 70 kDa dextran, 50 kDa Fab1, and 150 kDa mAb1, by 4- to 9-fold in a three-dimensional blood-brain barrier (3D-BBB) microfluidics model using a human BBB endothelial cell line (i.e., hCMEC/D3). HAVN1 was selective in modulating the BBB endothelial cell, compared to the pulmonary vascular endothelial (PVE) cell barrier. Co-administration of HAVN1 significantly improved brain depositions of mAb1, mAb2, and Fab1 in C57BL/6 mice after 15 min in the systemic circulation. Furthermore, HAVN1 still significantly enhanced brain deposition of mAb2 when it was administered 24 h after the administration of the mAb. Lastly, we observed that multiple doses of HAVN1 may have a cumulative effect on the brain deposition of mAb2 within a 24-h period. These findings offer promising insights into optimizing HAVN1 and mAb dosing regimens to control or modulate mAb brain deposition for achieving desired mAb dose in the brain to provide its therapeutic effects.

Shear-mediated ALK5 expression regulates endothelial activation.

Calcific aortic valve disease affects the aortic side of the valve, exposed to low magnitude multidirectional ("disturbed) blood flow, more than it affects the ventricular side, exposed to high magnitude uniaxial flow. Overt disease is preceded by endothelial dysfunction and inflammation. Here we investigate the potential role of the transforming growth factor-ß (TGF-ß) receptor ALK5 in this process. Although ECs are always subject to shear stress due to blood flow, and their responses to shear stress are important in healthy valve development and homeostasis, low magnitude multidirectional flow can induce pathophysiological changes. Previous work has shown ALK5 to be an important mechanosensor. ALK5 transduces mechanically sensed signals via the activation of the SMAD2/3 transcriptional modulators. However, it is currently unclear precisely how ALK5-mediated shear stress responses translate into pathological changes under conditions of chronically disturbed flow. Here, we demonstrate that ALK5 mechanosensory signalling influences flow-induced endothelial leukocyte adhesion and paracellular permeability. Low magnitude multidirectional flow resulted in downregulation of the receptor, accompanied by increased SMAD2 phosphorylation, in human umbilical vein endothelial cell (HUVEC) monolayers. These changes correlated with elevated monocyte adhesion and significantly increased transendothelial transport of an albumin-sized tracer. These effects were abolished by inhibition of ALK5 kinase activity. Analysis of ALK5 expression patterns in porcine aortic valve tissue corroborated the findings from cell-based experiments. Together, these results suggest that ALK5 has a role in shear stress-associated cardiovascular disease pathology, emphasising the importance of further mechanistic investigations and supporting it as a potential therapeutic target.

Influence of permeability enhancers on the paracellular permeability of metformin hydrochloride and furosemide across Caco-2 cells.

Permeability enhancers can affect absorption of paracellularly transported drugs. This study aims to evaluate effects of permeability enhancers (chitosan, methyl-ß -cyclodextrin, sodium caprate, sodium lauryl sulfate, etc.) on the permeability of paracellularly absorbed furosemide and metformin hydrochloride. Methyl thiazole tetrazolium bromide test was carried out to determine the drug concentrations in permeability study. Trans-epithelial electrical resistance (TEER) values determined to assess the integrity of tight junctions. Permeability enhancers were applied at different concentrations alone, in dual/triple combinations. Permeability was determined using human colorectal adenocarcinoma (Caco-2) cells (TEER > 400 Ω·cm2). Permeability enhancers have no significant effect (<2-fold; p > 0.05) on the permeability of furosemide (1.80 × 10-5 ± 4.55 × 10-7 cm/s); however, metformin permeability (1.36 × 10-5 ± 1.25 × 10-6 cm/s) increased significantly (p < 0.05) with 0.3% and 0.5% (w/v) chitosan (2.0- and 2.7-fold, respectively), 1% methyl-ß -cyclodextrin (w/v) (3.5-fold), 10 and 20 µmol/L sodium caprate (2.2- and 2.8-fold, respectively), and 0.012% sodium lauryl sulfate (w/v) (1.9-fold). Furosemide permeability increased significantly (p < 0.05) with chitosan-sodium lauryl sulfate combination (1.7-fold), and all triple combinations (1.4- to 1.9-fold). Chitosan containing dual/triple combinations resulted in significant increase (p < 0.05) in metformin permeability (1.7 to 2.8-fold). All results indicated that absorption of furosemide and metformin can be improved by the combination of permeability enhancers. Therefore, it can be evaluated for the formulation of development strategies containing furosemide and metformin by the pharmaceutical industry.

18ß-Glycyrrhetinic acid altered the intestinal permeability in the human Caco-2 monolayer cell model.

PURPOSE: Glycyrrhizin (GL) and its metabolites 18α-glycyrrhetinic acid (18α-GA) and 18ß-glycyrrhetinic acid (18ß-GA) are used as traditional medicine and food sweeteners. As the major rout of their administration is oral way, therefore their impact on intestinal epithelial cells are investigated. METHODS: The effects of GL and its metabolites on cell viability using MTT assay, on cytotoxicity using LDH release, on integrity of intestinal epithelial cells by measuring the transepithelial electrical resistance (TEER) and Luciferase permeability tests, on the expression of tight junction proteins at mRNA and protein level by qPCR and western blot techniques, and ultimately on the rate of test compounds absorption via Caco-2 cells monolayer were investigated. RESULTS: MTT assay showed a concentration- and time-dependent decrease in metabolic activity of Caco-2 cells induced by GL, 18α-GA, and 18ß-GA, while only 18ß-GA increased the LDH leakage. The monolayer integrity of Caco-2 cells in TEER assay only was affected by 18ß-GA. The permeability of paracellular transport marker was increased by 18α-GA and 18ß-GA and not GL. In transport studies, only metabolites were able to cross from Caco-2 cells monolayer. qPCR analyses revealed that 18ß-GA upregulated the expression of claudin-1 and -4, occludin, junctional adhesion molecules and zonula occludens-1, while 18α-GA upregulated only claudin-4. The expression of claudin-4 at protein level was downregulated non-significantly at 50 µM concentration of 18ß-GA. CONCLUSION: Our results suggest that 18ß-GA may cause cellular damages at higher concentrations on gastrointestinal cells and requires a remarkable attention of the nutraceutical and pharmaceutical industries.

Transport Studies Using Blood-Brain Barrier In Vitro Models: A Critical Review and Guidelines.

Permeation is one of the most evaluated parameters using preclinical in vitro blood-brain barrier models, as it has long been considered to be one of the major factors influencing central nervous system drug delivery. Blood-brain barrier permeability can be defined as the speed at which a compound crosses the brain endothelial cell barrier and is employed to assess barrier tightness, which is a crucial feature of brain capillaries in vivo. In addition, it is used to assess brain drug penetration. We review traditionally used methods to assess blood-brain barrier permeability in vitro and summarize often neglected in vivo (e.g., plasma protein and brain tissue binding) or in vitro (e.g., culture insert materials or methodology) factors that influence this property. These factors are crucial to consider when performing BBB permeability assessments, and especially when comparing permeability data obtained from different models, since model diversification significantly complicates inter-study comparisons. Finally, measuring transendothelial electrical resistance can be used to describe blood-brain barrier tightness; however, several parameters should be considered while comparing these measurements to the blood-brain barrier permeability to paracellular markers.

Endothelial cell barriers: Transport of molecules between blood and tissues.

An endothelial cell monolayer separates interstitia from blood and lymph, and determines the bidirectional transfer of solutes and macromolecules across these biological spaces. We review advances in transport modalities across these endothelial barriers. Glucose is a major fuel for the brain and peripheral tissues, and insulin acts on both central and peripheral tissues to promote whole-body metabolic signalling and anabolic activity. Blood-brain barrier endothelial cells display stringent tight junctions and lack pinocytic activity. Delivery of blood glucose and insulin to the brain occurs through their respective carrier (Glucose transporter 1) and receptor (insulin receptor), enacting bona fide transcytosis. At supraphysiological concentrations, insulin is also likely transferred by fluid phase cellular uptake and paracellular transport, especially in peripheral microvascular endothelia. The lymphatic microvasculature also transports insulin but in this case from tissues to lymph and therefrom to blood. This serves to end the hormone's action and to absorb highly concentrated subcutaneously injected insulin in diabetic individuals. The former function may involve receptor-mediated transcytosis into lymphatic endothelial cells, the latter fluid phase uptake and paracellular transport. Lymphatic capillaries also mediate carrier-dependent transport of other nutrients and macromolecules. These findings challenge the notion that lymphatic capillaries only transport macromolecules through intercellular flaps.

Transcytosis within PVN capillaries: a mechanism determining both hypertension-induced blood-brain barrier dysfunction and exercise-induced correction.

Although hypertension disrupts the blood-brain barrier (BBB) integrity within the paraventricular nucleus of hypothalamus (PVN) and increases the leakage into the brain parenchyma, exercise training (T) was shown to correct it. Since there is scarce and contradictory information on the mechanism(s) determining hypertension-induced BBB deficit and nothing is known about T-induced improvement, we sought to evaluate the paracellular and transcellular transport across the BBB within the PVN in both conditions. Spontaneously hypertensive rats (SHR) and WKY submitted to 4-wk aerobic T or sedentary (S) protocol were chronically catheterized for hemodynamic recordings at rest and intra-arterial administration of dyes (Rhodamine-dextran 70 kDa + FITC-dextran 10 kDa). Brains were harvesting for FITC leakage examination, qPCR evaluation of different BBB constituents and protein expression of caveolin-1 and claudin-5, the main markers of transcytosis and paracellular transport, respectively. Hypertension was characterized by increased arterial pressure and heart rate, augmented sympathetic modulation of heart and vessels, and reduced cardiac parasympathetic control, marked FITC extravasation into the PVN which was accompanied by increased caveolin-1 gene and protein expression, without changes in claudin-5 and others tight junctions' components. SHR-T vs. SHR-S showed a partial pressure reduction, resting bradycardia, improvement of autonomic control of the circulation simultaneously with correction of both FITC leakage and caveolin-1 expression; there was a significant increase in claudin-5 expression. Caveolin-1 content was strongly correlated with improved autonomic control after exercise. Data indicated that within the PVN the transcytosis is the main mechanism governing both hypertension-induced BBB leakage, as well as the exercise-induced correction.

Wnt Signaling in Inner Blood-Retinal Barrier Maintenance.

The retina is a light-sensing ocular tissue that sends information to the brain to enable vision. The blood-retinal barrier (BRB) contributes to maintaining homeostasis in the retinal microenvironment by selectively regulating flux of molecules between systemic circulation and the retina. Maintaining such physiological balance is fundamental to visual function by facilitating the delivery of nutrients and oxygen and for protection from blood-borne toxins. The inner BRB (iBRB), composed mostly of inner retinal vasculature, controls substance exchange mainly via transportation processes between (paracellular) and through (transcellular) the retinal microvascular endothelium. Disruption of iBRB, characterized by retinal edema, is observed in many eye diseases and disturbs the physiological quiescence in the retina's extracellular space, resulting in vision loss. Consequently, understanding the mechanisms of iBRB formation, maintenance, and breakdown is pivotal to discovering potential targets to restore function to compromised physiological barriers. These unraveled targets can also inform potential drug delivery strategies across the BRB and the blood-brain barrier into retinas and brain tissues, respectively. This review summarizes mechanistic insights into the development and maintenance of iBRB in health and disease, with a specific focus on the Wnt signaling pathway and its regulatory role in both paracellular and transcellular transport across the retinal vascular endothelium.

Combinatorial expression of claudins in the proximal renal tubule and its functional consequences.

The proximal renal tubule (PT) is characterized by a highly conductive paracellular pathway, which contributes to a significant amount of solute and water reabsorption by the kidney. Claudins are tight junction proteins that, in part, determine the paracellular permeability of epithelia. In the present study, we determined the expression pattern of the major PT claudins. We found that claudin-2 and claudin-10 are coexpressed throughout the PT, whereas claudin-3 is coexpressed with claudin-2 predominantly in the proximal straight tubule. Additionally, claudin-2 and claudin-3 are expressed separately within mutually exclusive populations of descending thin limbs. We developed a novel double-inducible Madin-Darby canine kidney I cell model to characterize in vitro the functional effect of coexpression of PT claudins. In keeping with previous studies, we found that claudin-2 alone primarily increased cation (Na+ and Ca2+) permeability, whereas claudin-10a alone increased anion (Cl-) permeability. Coexpression of claudin-2 and claudin-10a together led to a weak physical interaction between the isoforms and the formation of a monolayer with high conductance but neutral charge selectivity. Claudin-3 expression had a negligible effect on all measures of cell permeability, whether expressed alone or together with claudin-2. In cells coexpressing a claudin-2 mutant, S68C, together with claudin-10a, inhibition of cation permeability through the claudin-2 pore with a thiol-reactive pore blocker did not block anion permeation through claudin-10a. We conclude that claudin-2 and claudin-10a form independent paracellular cation- and anion-selective channels that function in parallel.

The role of SLC34A2 in intestinal phosphate absorption and phosphate homeostasis.

There has recently been significant interest in the concept of directly targeting intestinal phosphate transport to control hyperphosphatemia in patients with chronic kidney disease. However, we do not have a complete understanding of the cellular mechanisms that govern dietary phosphate absorption. Studies in the 1970s documented both active and passive pathways for intestinal phosphate absorption. However, following the cloning of the intestinal SLC34 cotransporter, NaPi-IIb, much of the research focused on the role of this protein in active transcellular phosphate absorption and the factors involved in its regulation. Generation of a conditional NaPi-IIb knockout mouse has demonstrated that this protein is critical for the maintenance of skeletal integrity during periods of phosphate restriction and that under normal physiological conditions, the passive sodium-independent pathway is likely be the more dominant pathway for intestinal phosphate absorption. The review aims to summarise the most recent developments in our understanding of the role of the intestine in phosphate homeostasis, including the acute and chronic renal adaptations that occur in response to dietary phosphate intake. Evidence regarding the overall contribution of the transcellular and paracellular pathways for phosphate absorption will be discussed, together with the clinical benefit of inhibiting these pathways for the treatment of hyperphosphatemia in chronic kidney disease.

Paracellular transport of phosphate along the intestine.

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.

Hypokalemia Associated With a Claudin 10 Mutation: A Case Report.

Hypokalemia of renal origin can arise from genetic abnormalities in a variety of transporters or channel proteins that mediate tubular handling of potassium. Recently, mutations in claudin 10 have been documented in patients with hypokalemia in association with a range of other electrolyte abnormalities and skin and sweat gland manifestations. We report a 12-year-old Hispanic boy who presented with anhydrosis, aptyalism, alacrima, hypokalemia, and hypocalciuria, in whom we detected a homozygous mutation in the claudin 10 gene. During the 4-year follow-up period, he developed hypermagnesemia and a decline in estimated glomerular filtration rate to 59mL/min/1.73m2. His unaffected parents and siblings were heterozygous for the mutation. We summarize the clinical phenotype encountered in patients with claudin 10 mutations. It is characterized by significant heterogeneity in electrolyte and extrarenal abnormalities and is associated with a risk for progressive loss of kidney function in up to 33% of cases. Awareness of this association between claudin 10 mutations and electrolyte abnormalities, namely hypokalemia and hypermagnesemia, sheds new light on the physiology of potassium and magnesium handling along the nephron and increases the likelihood of identifying the underlying tubular mechanism in patients with newly diagnosed hypokalemia with or without concomitant hypermagnesemia.

HIV-1 Tat and opioids act independently to limit antiretroviral brain concentrations and reduce blood-brain barrier integrity.

Poor antiretroviral penetration may contribute to human immunodeficiency virus (HIV) persistence within the brain and to neurocognitive deficits in opiate abusers. To investigate this problem, HIV-1 Tat protein and morphine effects on blood-brain barrier (BBB) permeability and drug brain penetration were explored using a conditional HIV-1 Tat transgenic mouse model. Tat and morphine effects on the leakage of fluorescently labeled dextrans (10-, 40-, and 70-kDa) into the brain were assessed. To evaluate effects on antiretroviral brain penetration, Tat+ and Tat- mice received three antiretroviral drugs (dolutegravir, abacavir, and lamivudine) with or without concurrent morphine exposure. Antiretroviral and morphine brain and plasma concentrations were determined by LC-MS/MS. Morphine exposure, and, to a lesser extent, Tat, significantly increased tracer leakage from the vasculature into the brain. Despite enhanced BBB breakdown evidenced by increased tracer leakiness, morphine exposure led to significantly lower abacavir concentrations within the striatum and significantly less dolutegravir within the hippocampus and striatum (normalized to plasma). P-glycoprotein, an efflux transporter for which these drugs are substrates, expression and function were significantly increased in the brains of morphine-exposed mice compared to mice not exposed to morphine. These findings were consistent with lower antiretroviral concentrations in brain tissues examined. Lamivudine concentrations were unaffected by Tat or morphine exposure. Collectively, our investigations indicate that Tat and morphine differentially alter BBB integrity. Morphine decreased brain concentrations of specific antiretroviral drugs, perhaps via increased expression of the drug efflux transporter, P-glycoprotein.

Urate Transport via Paracellular Route across Epithelial Cells.

Urate is the final oxidation product of purine metabolism in humans. We have recently reported that the paracellular route is the major urate transport pathway across the blood-placental barrier. In this study, the mechanism of urate paracellular transport was investigated in several epithelial cell lines including Madin-Darby canine kidney (MDCK) type I, Lilly Laboratories cell-porcine kidney 1 (LLC-PK1) and Caco-2 cells. Very little urate passed through MDCK and LLC-PK1 cell layers. In contrast, one of the Caco-2 cell lines was found to be urate-permeable. This urate paracellular movement across Caco-2 cell layer was not inhibited by the urate transporter inhibitor benzbromarone but was partially inhibited by 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS), which inhibits chloride transport. Detection and quantification of claudin proteins that are important for paracellular transport of ions were performed by LC/MS. Claudins 1, 3, 4, 6, 7 and 12 were detected in urate-permeable cell lines, BeWo cells and Caco-2 cells. We compared claudin expression patterns in urate-permeable and urate-non-permeable Caco-2 cells by LC/MS and found that claudin 12 had a higher expression level in urate-permeable Caco-2 cells. Overexpression of these claudins in MDCK cells did not increase urate paracellular transport. Although there were differences in claudin expression pattern between urate-permeable and non-permeable cells, increased expression of single claudin alone did not explain paracellular permeability of urate.

Temporal Effects of Quercetin on Tight Junction Barrier Properties and Claudin Expression and Localization in MDCK II Cells.

: Kidney stones affect 10% of the population. Yet, there is relatively little known about how they form or how to prevent and treat them. The claudin family of tight junction proteins has been linked to the formation of kidney stones. The flavonoid quercetin has been shown to prevent kidney stone formation and to modify claudin expression in different models. Here we investigate the effect of quercetin on claudin expression and localization in MDCK II cells, a cation-selective cell line, derived from the proximal tubule. For this study, we focused our analyses on claudin family members that confer different tight junction properties: barrier-sealing (Cldn1, -3, and -7), cation-selective (Cldn2) or anion-selective (Cldn4). Our data revealed that quercetin's effects on the expression and localization of different claudins over time corresponded with changes in transepithelial resistance, which was measured continuously throughout the treatment. In addition, these effects appear to be independent of PI3K/AKT signaling, one of the pathways that is known to act downstream of quercetin. In conclusion, our data suggest that quercetin's effects on claudins result in a tighter epithelial barrier, which may reduce the reabsorption of sodium, calcium and water, thereby preventing the formation of a kidney stone.

Phosphatidylcholine Passes by Paracellular Transport to the Apical Side of the Polarized Biliary Tumor Cell Line Mz-ChA-1.

Phosphatidylcholine (PC) translocation into mucus of the intestine was shown to occur via a paracellular transport across the apical/lateral tight junction (TJ) barrier. In case this could also be operative in biliary epithelial cells, this may have implication for the pathogenesis of primary sclerosing cholangitis (PSC). We here evaluated the transport of PC across polarized cholangiocytes. Therefore, the biliary tumor cell line Mz-ChA-1 was grown to confluency. In transwell culture systems the translocation of PC to the apical compartment was analyzed. After 21 days in culture, polarized Mz-ChA-1 cells revealed a predominant apical translocation of choline containing phospholipids including PC with minimal intracellular accumulation. Transport was suppressed by TJ destruction employing chemical inhibitors and pretreatment with siRNA to TJ forming proteins as well as the apical transmembrane mucin 3 as PC acceptor. Apical translocation was dependent on a negative apical electrical potential created by the cystic fibrosis transmembrane conductance regulator (CFTR) and the anion exchange protein 2 (AE2). It was stimulated by apical application of secretory mucins. The results indicated the existence of a paracellular PC passage across apical/lateral TJ of the polarized biliary epithelial tumor cell line Mz-ChA-1. This has implication for the generation of a protective mucus barrier in the biliary tree.

Effect of Npt2b deletion on intestinal and renal inorganic phosphate (Pi) handling.

BACKGROUND: Hyperphosphatemia is common in chronic kidney disease and is associated with morbidity and mortality. The intestinal Na+-dependent phosphate transporter Npt2b is thought to be an important molecular target for the prevention of hyperphosphatemia. The role of Npt2b in the net absorption of inorganic phosphate (Pi), however, is controversial. METHODS: In the present study, we made tamoxifen-inducible Npt2b conditional knockout (CKO) mice to analyze systemic Pi metabolism, including intestinal Pi absorption. RESULTS: Although the Na+-dependent Pi transport in brush-border membrane vesicle uptake levels was significantly decreased in the distal intestine of Npt2b CKO mice compared with control mice, plasma Pi and fecal Pi excretion levels were not significantly different. Data obtained using the intestinal loop technique showed that Pi uptake in Npt2b CKO mice was not affected at a Pi concentration of 4 mM, which is considered the typical luminal Pi concentration after meals in mice. Claudin, which may be involved in paracellular pathways, as well as claudin-2, 12, and 15 protein levels were significantly decreased in the Npt2b CKO mice. Thus, Npt2b deficiency did not affect Pi absorption within the range of Pi concentrations that normally occurs after meals. CONCLUSION: These findings indicate that abnormal Pi metabolism may also be involved in tight junction molecules such as Cldns that are affected by Npt2b deficiency.

Transepithelial transport of milk-derived angiotensin I-converting enzyme inhibitory peptide with the RLSFNP sequence.

BACKGROUND: To exert an antihypertensive effect after oral administration, angiotensin I-converting enzyme (ACE)-inhibitory peptides must remain active after intestinal transport. The purpose of this article is to elucidate the transport permeability and route of ACE-inhibitory peptide Arg-Leu-Ser-Phe-Asn-Pro (RLSFNP) across the intestinal epithelium using Caco-2 cell monolayers. RESULTS: Intact RLSFNP and RLSFNP breakdown fragments F, FNP, SFNP and RLSF were found in RLSFNP transport solution across Caco-2 cell monolayers using ultra-performance liquid chromatography-tandem mass spectrometry. RLSFNP fragments FNP, SFNP and RLSF also contributed to ACE inhibitory effects. Protease inhibitors (bacitracin and leupeptin) and absorption enhancers (sodium glycocholate hydrate, sodium deoxycholate and Na2 EDTA) improved the transport flux of RLSFNP. A transport inhibitor experiment showed that intact RLSFNP may be transported via the paracellular route. CONCLUSION: Intact RLSFNP can be transported across the Caco-2 cell monolayers via the paracellular route. Extensive hydrolysis was the chief reason for the low permeability of RLSFNP. © 2017 Society of Chemical Industry.

One gene, two paracellular ion channels-claudin-10 in the kidney.

Claudins are tight junction membrane proteins and regulate the paracellular passage of ions and water. They can seal the paracellular cleft against solute passage but also form paracellular channels. They are tetraspan proteins with two extracellular segments. Claudin-10 exists in at least two functional isoforms, claudin-10a and claudin-10b, that differ in their first transmembrane segment and first extracellular segment. Both isoforms act as selective paracellular ion channels, either for anions (claudin-10a) or for cations (claudin-10b). Their diverse functions are reflected in completely different expression patterns in the body, especially in the kidney. Their structural and functional similarities and differences make them ideal subjects to study determinants of claudin charge selectivity and pore formation. This review aims to summarise research on permeability properties of the claudin-10 channels and their role in physiology and pathophysiology of the kidney.

Physiological roles of claudins in kidney tubule paracellular transport.

The paracellular pathways in renal tubular epithelia such as the proximal tubules, which reabsorb the largest fraction of filtered solutes and water and are leaky epithelia, are important routes for transepithelial transport of solutes and water. Movement occurs passively via an extracellular route through the tight junction between cells. The characteristics of paracellular transport vary among different nephron segments with leaky or tighter epithelia. Claudins expressed at tight junctions form pores and barriers for paracellular transport. Claudins are from a multigene family, comprising at least 27 members in mammals. Multiple claudins are expressed at tight junctions of individual nephron segments in a nephron segment-specific manner. Over the last decade, there have been advances in our understanding of the structure and functions of claudins. This paper is a review of our current knowledge of claudins, with special emphasis on their physiological roles in proximal tubule paracellular solute and water transport.