Susanne Milatz

Claudin-2, a component of the tight junction, forms a paracellular water channel

Whether or not significant amounts of water pass the tight junction (TJ) of leaky epithelia is still unresolved, because it is difficult to separate transcellular water flux from TJ-controlled paracellular water flux. Using an approach without differentiating technically between the transcellular and paracellular route, we measured transepithelial water flux with and without selective molecular perturbation of the TJ to unequivocally attribute changes to the paracellular pathway. To this end, MDCK C7 cells were stably transfected with either claudin-2 or claudin-10b, two paracellular cation-channel-forming TJ proteins that are not endogenously expressed in this cell line. Claudin-2 is typical of leaky, water-transporting epithelia, such as the kidney proximal tubule, whereas claudin-10b is present in numerous epithelia, including water-impermeable segments of the loop of Henle. Neither transfection altered the expression of endogenous claudins or aquaporins. Water flux was induced by an osmotic gradient, a Na+ gradient or both. Under all conditions, water flux in claudin-2-transfected cells was elevated compared with vector controls, indicating claudin-2-mediated paracellular water permeability. Na+-driven water transport in the absence of an osmotic gradient indicates a single-file mechanism. By contrast, claudin-10b transfection did not alter water flux. We conclude that claudin-2, but not claudin-10b, forms a paracellular water channel and thus mediates paracellular water transport in leaky epithelia.

Tricellulin Forms a Barrier to Macromolecules in Tricellular Tight Junctions without Affecting Ion Permeability

Tricellulin is a tight junction protein localized in tricellular tight junctions (tTJs), the meeting points of three cells, but also in bicellular tight junctions (bTJs). To investigate its specific barrier functions in bTJs and tTJs, TRIC-a was expressed in low-level tricellulin-expressing cells, and MDCK II, either in all TJs or only in tTJs. When expressed in all TJs, tricellulin increased paracellular electrical resistance and decreased permeability to ions and larger solutes, which are associated with enhanced ultrastructural integrity of bTJs toward enhanced strand linearity. In tTJs in contrast, ultrastructure was unchanged and tricellulin minimized permeability to macromolecules but not to ions. This paradox is explained by properties of the tTJ central tube which is wide enough for passage of macromolecules, but too rare to contribute significantly to ion permeability. In conclusion, at low tricellulin expression the tTJ central tube forms a pathway for macromolecules. At higher expression, tricellulin forms a barrier in tTJs effective only for macromolecules and in bTJs for solutes of all sizes.

Na+ absorption defends from paracellular back-leakage by claudin-8 upregulation.

In distal colon, the limiting factor for Na(+) absorption is represented by the epithelial sodium channel (ENaC). During absorption, high transepithelial Na(+) gradients are observed. In human colon and in HT-29/B6-GR cells, we investigated whether Na(+) back-leakage is prevented by paracellular sealing. Tissues and cells were incubated with corticosteroids. Barrier properties were analyzed in electrophysiological experiments. Subsequently, analysis of ENaC and tight junction protein expression, localization, and regulation was performed. In colon, nanomolar aldosterone induced sodium absorption via ENaC. Concomitantly, paracellular (22)Na(+) permeability was reduced by half and claudin-8 within the tight junction complex was nearly doubled. Real-time PCR validated an increase of claudin-8 transcripts. Two-path impedance spectroscopy following ENaC induction in HT-29/B6-GR revealed a specific increase of paracellular resistance. These results represent an important physiological implication: Na(+) absorption is paralleled by claudin-8-mediated sealing of the paracellular barrier to prevent Na(+) back-leakage, supporting steep Na(+) gradients in distal colon.

Tight junction proteins as channel formers and barrier builders.

Tight junctions form the paracellular barrier for ions and uncharged solutes not only in “tight” but also in “leaky” epithelia. In the premolecular era of tight junction research, this was believed to be achieved in a perfect or less perfect way, depending mainly on the amount of horizontally oriented tight junction strands. During the past decade it emerged that tight junction molecules, such as claudin‐1 and many others, strengthen the barrier, while a few claudins, such as claudin‐2 or ‐10, weaken it. This report focuses on three claudins: one channel former and two barrier builders. Claudin‐2 represents the prototype of a paracellular, channel‐forming, tight junction protein responsible for specific transfer of solutes across the epithelium without entering the cells. This channel is selective for small cations but nearly impermeable to anions and uncharged solutes of any size. In contrast, claudin‐5, a tight junction protein typical for all endothelia but also found in some epithelia, was characterized as a potent barrier builder. Claudin‐8, another barrier builder, was demonstrated to be regulated by Na+ uptake in surface epithelial cells of human colon. Here, aldosterone enhanced Na+ absorption by dual action: transcellularly by inducing the epithelial sodium channel and paracellularly by preventing back leakage of absorbed Na+ by upregulating claudin‐8.

Claudin Function in the Thick Ascending Limb of Henle's Loop

During the past decade, claudins have been established as major determinants of paracellular permeablilty in epithelia. In the kidney, each nephron segment expresses a distinct pattern of claudins. Cells of the thick ascending limb of Henles loop (TAL), which is characterized by high paracellular cation permeability, co‐express an unusually large number of different claudins: claudin‐10, ‐16, and ‐19 and, depending on the species, also claudin‐3, ‐4, ‐8, and/or ‐11. The function of most of these claudins has been investigated in vitro. We present a summary of their function with special emphasis on claudin‐16 and ‐19. Mutations in the corresponding human genes lead to severely impaired renal Ca2+ and Mg2+ handling. To date, 42 different claudin‐16 mutations and three claudin‐19 mutations have been reported. These mutations prevent the claudins from reaching the surface membrane, decrease membrane residence time, or render them functionless. In spite of the clear clinical symptoms such as hypomagnesemia, hypercalciuria, nephrocalcinosis, and renal insufficiency, mechanisms that link claudin‐16 and ‐19 to these symptoms are still unknown. Depending on the cell type used in overexpression studies, claudin‐16 appears to cause a mild increase in paracellular Mg2+‐permeability or a pronounced increase in Na+ permeability. Claudin‐19 selectively decreases Cl− permeability, thus synergistically increasing relative cation permeability, or indiscriminately decreases paracellular permeability. In the light of these results it is hypothesized that the renal Mg2+/Ca2+ waste may not be solely due to reduced resorption in the TAL but at least in part to paracellular back‐leak of Mg2+/Ca2+ into the tubular lumen of the distal convoluted tubule.

Mosaic expression of claudins in thick ascending limbs of Henle results in spatial separation of paracellular Na+ and Mg2+ transport

Significance The thick ascending limb (TAL) of Henle’s loop is a nephron segment that reabsorbs Na+, Ca2+, and Mg2+ via the paracellular pathway, the tight junction (TJ). TJ permeability is regulated by claudin proteins. We show that the TAL expresses claudins cldn3, cldn10b, cldn16, and cldn19 in a TJ mosaic pattern with cldn3/cldn16/cldn19 in a complex and cldn10b alone. This mutual exclusiveness is facilitated by different claudin interaction properties. TJs with cldn10b favor Na+ over Mg2+, whereas TJs with cldn3/cldn16/cldn19 prefer Mg2+ over Na+. Hence we conclude that mono- and divalent cations in the TAL take different paracellular routes, and their reabsorption can be regulated independently. This spatial separation is important for renal ion homeostasis and its discovery improves our understanding of paracellular transport organization. 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+.

Probing the cis-arrangement of prototype tight junction proteins claudin-1 and claudin-3.

Claudins form a large family of TJ (tight junction) proteins featuring four transmembrane segments (TM1-TM4), two extracellular loops, one intracellular loop and intracellular N- and C-termini. They form continuous and branched TJ strands by homo- or heterophilic interaction within the same membrane (cis-interaction) and with claudins of the opposing lateral cell membrane (trans-interaction). In order to clarify the molecular organization of TJ strand formation, we investigated the cis-interaction of two abundant prototypic claudins. Human claudin-1 and claudin-3, fused to ECFP or EYFP at the N- or C-terminus, were expressed in the TJ-free cell line HEK (human embryonic kidney)-293. Using FRET analysis, the proximity of claudin N- and C-termini integrated in homopolymeric strands composed of claudin-3 or of heteropolymeric strands composed of claudin-1 and claudin-3 were determined. The main results are that (i) within homo- and heteropolymers, the average distance between the cytoplasmic ends of the TM1s of cis-interacting claudin molecules is shorter than the average distance between their TM4s, and (ii) TM1 segments of neighbouring claudins are oriented towards each other as the cytoplasmic end of TM1 is in close proximity to more other TM1 segments than TM4 is to other TM4 segments. The results indicate at least two different cis-interaction interfaces within claudin-3 homopolymers as well as within claudin-1/claudin-3 heteropolymers. The data provide novel insight into the molecular TJ architecture consistent with a model with an antiparallel double-row cis-arrangement of classic claudin protomers within strands.

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.

Altered paracellular cation permeability due to a rare CLDN10B variant causes anhidrosis and kidney damage

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.

A Novel Hypokalemic-Alkalotic Salt-Losing Tubulopathy in Patients with CLDN10 Mutations.

Mice lacking distal tubular expression of CLDN10, the gene encoding the tight junction protein Claudin-10, show enhanced paracellular magnesium and calcium permeability and reduced sodium permeability in the thick ascending limb (TAL), leading to a urine concentrating defect. However, the function of renal Claudin-10 in humans remains undetermined. We identified and characterized CLDN10 mutations in two patients with a hypokalemic-alkalotic salt-losing nephropathy. The first patient was diagnosed with Bartter syndrome (BS) >30 years ago. At re-evaluation, we observed hypocalciuria and hypercalcemia, suggesting Gitelman syndrome (GS). However, serum magnesium was in the upper normal to hypermagnesemic range, thiazide responsiveness was not blunted, and genetic analyses did not show mutations in genes associated with GS or BS. Whole-exome sequencing revealed compound heterozygous CLDN10 sequence variants [c.446C>G (p.Pro149Arg) and c.465-1G>A (p.Glu157_Tyr192del)]. The patient had reduced urinary concentrating ability, with a preserved aquaporin-2 response to desmopressin and an intact response to furosemide. These findings were not in line with any other known salt-losing nephropathy. Subsequently, we identified a second unrelated patient showing a similar phenotype, in whom we detected compound heterozygous CLDN10 sequence variants [c.446C>G (p.(Pro149Arg) and c.217G>A (p.Asp73Asn)]. Cell surface biotinylation and immunofluorescence experiments in cells expressing the encoded mutants showed that only one mutation caused significant differences in Claudin-10 membrane localization and tight junction strand formation, indicating that these alterations do not fully explain the phenotype. These data suggest that pathogenic CLDN10 mutations affect TAL paracellular ion transport and cause a novel tight junction disease characterized by a non-BS, non-GS autosomal recessive hypokalemic-alkalotic salt-losing phenotype.