Mosaic expression of claudins in thick ascending limbs of Henle results in spatial separation of paracellular Na ⁺ and Mg ²⁺ transport

Abstract

The thick ascending limb (TAL) of Henle's loop drives paracellular Na + , Ca 2+ , and Mg 2+ 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 Mg 2+ , whereas TJs dominated by cldn16 favor Mg 2+ 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 Ca 2+ and Mg 2+ .T he kidney regulates the salt and water balance of the body by filtration and subsequent reabsorption or secretion of ions and water. Thereby it controls blood pressure and maintains acid-base homeostasis. The thick ascending limb (TAL) of Henle's loop drives reabsorption of Na + , Cl − , Ca 2+ , and Mg 2+ from the tubular fluid into the blood. Na + and Cl − are reabsorbed via the transcellular pathway, involving the renal-specific isoform of the Na + /K + /2Cl − cotransporter (NKCC2) in the apical epithelial cell membrane and Na + /K + -ATPase and chloride channel ClC-Kb in the basolateral membrane. K + is circulated via NKCC2 and the renal outer medullary K + channel ROMK1, across the apical cell membrane. These transport processes generate a lumen-positive transepithelial potential that drives additional paracellular reabsorption of Na + as well as the reabsorption of divalent cations, mainly Ca 2+ and Mg 2+ . Paracellular transport is regulated by the tight junction (TJ) in a size-, charge-, and water-selective manner. The main functional constituent of the TJ is the family of claudins with 27 members in mammals. Claudins consist of a four-transmembrane helix bundle, two extracellular segments that expand into the paracellular cleft, and intracellular N and C termini. Claudins interact in cis (within the same plasma membrane) and in trans (with claudins in the plasma membrane of neighbori...

Claudin expression in the rat endolymphatic duct and sac - first insights into regulation of the paracellular barrier by vasopressin

“…In positive control tissue of the rat p4 kidney, an apical immunofluorescence labeling of Claudin 4 was observed in the thin ascending limb as reported in previous studies404142. The expression of Claudin 16 in the positive control kidney was revealed in the cytoplasmic compartment of the thick ascending limb as previously reported4344.…”

“…The confocal microscopic analysis of the subcellular localization of Claudin 6, 7 and 10 in rat p4 kidney control tissue, revealed similar results as were observed in rat p4 and p31 ED and ES and matched with other Claudin expression studies in the kidney21434647. However, in contrast to the adult ES, none of the former studies visualized Claudin 6 by immunostaining in any nephron segment in rat adult kidney4748.…”

Claudins in barrier and transport function—the kidney

“…Milatz and colleagues have recently proposed a new hypothesis to reconcile the renal phenotypes of claudin-10 knockout mice with claudin-16 or -19 knockout mice. Milatz et al found that the TJs in thick ascending limbs possessed a mosaic expression pattern separating claudin-10 from the claudin-16/-19 complex (55). Furthermore, TJs from the medullary thick ascending limb dominated by claudin-10 appear to favor Na + over Mg ++ whereas TJs from the cortical thick ascending limb dominated by claudin-16 favor Mg ++ over Na + (55).…”

“…Milatz et al found that the TJs in thick ascending limbs possessed a mosaic expression pattern separating claudin-10 from the claudin-16/-19 complex (55). Furthermore, TJs from the medullary thick ascending limb dominated by claudin-10 appear to favor Na + over Mg ++ whereas TJs from the cortical thick ascending limb dominated by claudin-16 favor Mg ++ over Na + (55). These results would suggest an axial heterogeneity in thick ascending limb paracellular permeability to various cations.…”

“…Milatz’s seminal discovery of the mosaic expression pattern of claudin-10 and claudin-16 in the thick ascending limb (55) has triggered important new thinking of how tight junction may be regulated through combinatorial claudin expression. Such cellular heterogeneity of claudin expression may exist in other epithelia or endothelia and have important physiologic functions.…”