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

Abstract

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.

Keywords: FRET; claudin interaction; microperfusion; paracellular ion transport; tight junction.

Fig. 1.

Mosaic TJ expression of claudins in single segments of cortex/OSOM TAL. (A) Kidney sections stained for occludin (ocln) and claudins (cldn). Cldn3 and cldn19 are expressed in the intracellular compartment of all cortex/OSOM TAL cells. A certain portion of cells lacks cldn10 expression (arrowhead). Cldn16 is not detected intracellularly but is strictly localized to the TJ (arrow). All claudins are expressed within the TJ in only a certain number of cells. (B) Single isolated TAL tubule. In contrast to sections, intracellular cldn expression is not distinguishable from background. Claudins localize to TJs (arrowheads). Cldn16 (magenta) and cldn19 (green) are colocalized within joint TJs (the congruence of magenta and green is shown as white in the merged image), but cldn10 (yellow) never forms joint strands with any of the other claudins. (C) A single isolated TAL tubule. Cldn3 (red) and cldn19 (green) are colocalized within joint TJs (congruence is shown in yellow). Thus, TJs were equipped with either cldn10b alone or with cldn3/16/19 together. (D) 3D projection of the TAL tubule shown in B. Each cell–cell contact expresses a certain claudin setting with tricellular junctions (arrows) as “switchover points.” DAPI staining is shown in blue. (Scale bars, 10 µm.)

Fig. S1.

Mosaic TJ expression of claudins in a single rat TAL segment. Shown are parts of an isolated single TAL segment ranging from medulla (arrow) to cortex (arrowhead). In the cortex, cells have extensive lateral interdigitations and express the claudin TJ mosaic of cldn10b and cldn3/16/19. Toward the medulla, cells have less complex interdigitations. (Scale bar, 10 µm.)

Fig. 2.

Claudin expression and paracellular permeabilities. (A) Single TAL tubules were microperfused, and diffusion potentials were measured. (B) Subsequently, tubules were transferred to object slides. The arrow indicates the perfusion site. (C) Tubules were costained for cldn10b and cldn16, and z stacks were taken. The TJ length positive for cldn10b or cldn16 was measured for ∼100 µm from the perfusion site. The arrowhead depicts cell–cell borders retraced with the overlay tool of Zeiss software. The percentages of the tubule length positive for cldn10b and cldn16 relative to the total tubule TJ length were calculated. (D–G) P_Na/P_Cl, P_Mg/P_Na, P_Na, and P_Mg are plotted against the cldn16 percentage in single TAL tubules. ISOM TJs contain cldn10b almost exclusively, without expression of cldn16. OSOM and cortex TJs express varying amounts of cldn10b and cldn16. Solid lines show the linear regression without the ISOM of the TAL; dashed lines depict the linear regression including the ISOM of the TAL. R² values are provided for regressions including only cortex and OSOM. (D) P_Na/P_Cl as a measure for cation selectivity is highest in cldn10b-expressing TJs and declines as cldn10b decreases. R² = 0.29. (E) Cldn10b-dominated TAL prefer Na⁺ over Mg²⁺ (P_Na > P_Mg > P_Cl); cldn16-dominated TAL favor Mg²⁺ over Na⁺ (P_Mg > P_Na > P_Cl). R² = 0.25. (F) P_Na decreases with cldn10b reduction. R² = 0.25. (G) P_Mg is not correlated with cldn10b or cldn16 expression. R² = 0.03.

Fig. 3.

Claudin mosaic expression in cldn10-deficient or cldn16-deficient TAL. (A) Cldn10 deficiency in the TAL of mice results in a complete abrogation of the TJ mosaic pattern with broad expansion of cldn3/16/19 over all TAL TJs, shown here for cldn19 (congruence with the TJ marker occludin). (B) Cldn16 deficiency does not lead to an altered distribution of cldn10 (there is no expansion into all occludin-stained TJs), probably because the TJs are still occupied by cldn3/cldn19. (Scale bars, 10 µm.)

Fig. 4.

Mosaic expression of cldn10 and cldn16 in kidney sections from animals subjected to different dietary Ca²⁺ conditions. The expression and localization of cldn10 or cldn16 are not altered at high dietary Ca²⁺ (B) compared with control conditions (A). The number of cells without intracellular expression of cldn10 (arrowheads) is unaltered in the two groups.

Fig. 5.

The capability for cis interaction of TAL claudins determined by FRET analysis within TJs. CFP– or YFP–claudin fusion proteins were expressed in TJ-free HEK 293 cells. FRET occurs when tagged claudins are in close proximity (within a maximum range of 8 nm) and indicates cis interaction. All TAL claudins can interact with themselves and form homomers. Because cldn16 lacks trans interaction capability, its E_F^max is lower than that of other homomers. Heteromer formation occurs in only two combinations; other combinations are incompatible with heteromer formation.

Fig. 6.

The capability for trans interaction of TAL claudins. CFP– or YFP–claudin fusion proteins were expressed in TJ-free HEK 293 cells (and were cocultured when two different claudins were tested). Enrichment of fluorescence intensity at cell–cell contacts (arrows) compared with cell membranes without contact to a transfected cell (arrowheads) indicates trans interaction and strand formation. (A and B) Cldn3, -10b, and -19 are capable of trans interactions (A), but cldn16 is not (B). (C) No heterotypic interaction is seen between TAL claudins, as shown for cldn16/cldn19 (contact indicated by dotted arrow). (D) When cldn16 and cldn19 are coexpressed within the same cells, both claudins are integrated into strands.

Fig. 7.

Summary of claudin cis (Upper Right) and trans (Lower Left) interactions. Cldn19 can form heteromers with both cldn3 and cldn16 and facilitate insertion into joint cldn3/16/19 complexes. Cldn10b is the only TAL claudin not capable of any interaction other than with itself and thus must form its own strands.

Fig. S2.

Interactions of cldn11. (A) Cldn11 is capable of homomer formation and shows slight cis interaction with cldn16. (B) Exemplary trans interactions. Cldn11 cannot interact in trans with cldn16 but can interact in trans with cldn19. Arrowheads depict cell–cell contacts with (Lower) or without (Upper) contact enrichment, respectively.

Fig. 8.

Summary of homophilic interaction properties of cldn10b/cldn16 chimeras compared with wild types. Cldn16’s lack of trans interaction cannot be overcome by replacing cldn16 ECS2 with cldn10b ECS2. The inverse exchange destroys cldn10b’s trans-interaction capability, indicating that ECS2 has a role in trans interactions. Chimeras without membrane localization in HEK 293 cells were excluded from data interpretation.

Fig. S3.

Homomer formation of cldn10b/cldn16 chimeras. The E_F^max of chimeras are similar to that of wild-type cldn16. Homomeric cis-interaction capability and membrane localization are considered indicators of correct protein folding.

Fig. 9.

Scheme of claudin expression pattern and interaction capabilities in cortex/OSOM TAL. All cells express cldn3, cldn19, and potentially cldn16, but only 77% also express cldn10b. Those cells have the fundamental claudin setting to establish both TJ composition variants but form cldn10b TJs. Cldn16 can be integrated into the TJ only when cldn19 is present. Cldn19 forms heteromers with cldn3 and cldn16 and allows the formation of joint strands. Cldn10b forms channels with a preference for monovalent cations and thus mainly conducts Na⁺. TJs dominated by the cldn3/16/19 complex prefer Mg²⁺ over Na⁺. The molecular identity of the channel for divalent cations remains elusive.

Fig. S4.

TJ colocalization of cldn3 and cldn10b in HEK 293 cells. Although cdln3 and cldn10b cannot interact in cis or in trans, they colocalize within the cell membrane (arrowheads) and show no mutual exclusiveness as seen in TAL epithelium. The same is true for cldn16/cldn10b and cldn19/cldn10b.