Role of Claudins in Renal Branching Morphogenesis.

Claudins are a family of tight junction proteins that are expressed during mouse kidney development. They regulate paracellular transport of solutes along the nephron and contribute to the final composition of the urinary filtrate. To understand their roles during development, we used a protein reagent, a truncated version of the Clostridium perfringens enterotoxin (C-CPE), to specifically remove a subset of claudin family members from mouse embryonic kidney explants at embryonic day 12. We observed that treatment with C-CPE decreased the number and the complexity of ureteric bud tips that formed: there were more single and less bifid ureteric bud tips when compared to control-treated explants. In addition, C-CPE-treated explants exhibited ureteric bud tips with larger lumens when compared to control explants (p < .05). Immunofluorescent analysis revealed decreased expression and localization of Claudin-3, -4, -6, and -8 to tight junctions of ureteric bud tips following treatment with C-CPE. Interestingly, Claudin-7 showed higher expression in the basolateral membrane of the ureteric bud lineage and poor localization to the tight junctions of the ureteric bud lineage both in controls and in C-CPE-treated explants. Taken together, it appears that claudin proteins may play a role in ureteric bud branching morphogenesis through changes in lumen formation that may affect the efficiency by which ureteric buds emerge and branch.

Claudin-7, -16, and -19 during mouse kidney development.

Members of the claudin family of tight junction proteins are critical for establishing epithelial barriers and for the regulation of paracellular transport. To understand their roles during kidney development, we first performed RT-PCR analyses and determined that 23 claudin family members were expressed in embryonic day (E) 13.5 mouse kidneys. Based on their developmental expression and phenotypes in mouse models, we hypothesized that 3 claudin members could affect nephron formation during kidney development. Using whole mount in situ hybridization and immunohistochemistry, we demonstrated that Claudin-7 (Cldn7) was expressed in the nephric duct, the emerging ureteric bud, and in tubules derived from ureteric bud branching morphogenesis. In contrast, Claudin-16 (Cldn16) and Claudin-19 (Cldn19) were expressed at later stages of kidney development in immature renal tubules that become the Loop of Henle. To determine if a loss of these claudins would perturb kidney development, we examined newborn kidneys from mutant mouse models lacking Cldn7 or Cldn16. In both models, we noted no evidence for any congenital renal malformation and quantification of nephron number did not reveal a decrease in nephron number when compared to wildtype littermates. In summary, Cldn7, Cldn16, and Cldn19 are expressed in different epithelial lineages during kidney development. Mice lacking Cldn7 or Cldn16 do not have defects in de novo nephron formation, and this suggests that these claudins primarily function to regulate paracellular transport in the mature nephron.

The tight junction protein claudin-3 shows conserved expression in the nephric duct and ureteric bud and promotes tubulogenesis in vitro.

The claudin family of proteins is required for the formation of tight junctions that are contact points between epithelial cells. Although little is known of the cellular events by which epithelial cells of the ureteric bud form tubules and branch, tubule formation is critical for kidney development. We hypothesize that if claudin-3 (Cldn3) is expressed within tight junctions of the ureteric bud, this will affect ureteric bud cell shape and tubule formation. Using transmission electron microscopy, we identified tight junctions within epithelial cells of the ureteric bud. Whole mount in situ hybridization and immunoassays were performed in the mouse and chick and demonstrated that Cldn3 transcript and protein were expressed in the nephric duct, the ureteric bud, and its derivatives at critical time points during tubule formation and branching. Mouse inner medullary collecting duct cells (mIMCD-3) form tubules when seeded in a type I collagen matrix and were found to coexpress CLDN3 and the tight junction marker zonula occludens-1 in the cell membrane. When these cells were stably transfected with Cldn3 fused to the enhanced green fluorescent protein reporter, multiple clones showed a significant increase in tubule formation compared with controls (P < 0.05) due in part to an increase in cell proliferation (P < 0.01). Cldn3 may therefore promote tubule formation and expansion of the ureteric bud epithelium.