Claudins 6, 9, and 13 are developmentally expressed renal tight junction proteins

Abstract

The adult proximal tubule is a low-resistance epithelium where there are high rates of both active transcellular and passive paracellular NaCl transport. We have previously demonstrated that the neonatal rabbit and rat proximal tubule have substantively different passive paracellular transport properties than the adult proximal tubule, which results in a maturational change in the paracellular passive flux of ions. Neonatal proximal tubules have a higher P(Na)/P(Cl) ratio and lower chloride and bicarbonate permeabilities than adult proximal tubules. Claudins are a large family of proteins which are the gate keepers of the paracellular pathway, and claudin isoform expression determines the permeability characteristics of the paracellular pathway. Previous studies have shown that claudins 1, 2, 3, 4, 5, 7, 8, 10, 11, 12, 15, and 16 are expressed in the adult mouse kidney. To determine whether there are developmental claudin isoforms, we compared the claudin isoforms present in the neonatal and adult kidney using RT-PCR to detect mRNA of claudin isoforms. Claudin 6, claudin 9, and claudin 13 were either not expressed or barely detectable in the adult mouse kidney using traditional PCR, but were expressed in the neonatal mouse kidney. Using real-time RT-PCR, we were able to detect a low level of claudin 6 mRNA expression in the adult kidney compared with the neonate, but claudin 9 and claudin 13 were only detected in the neonatal kidney. There was the same maturational decrease in these claudin proteins with Western blot analysis. Immunohistochemistry showed high levels of expression of claudin 6 in neonatal proximal tubules, thick ascending limb, distal convoluted tubules, and collecting ducts in a paracellular distribution but there was no expression of claudin 6 in the adult kidney. Using real-time RT-PCR claudin 6 and 9 mRNA were present in 1-day-old proximal convoluted tubules and were virtually undetectable in proximal convoluted tubules from adults. Claudin 13 was not detectable in neonatal or adult proximal convoluted tubules. In summary, we have identified developmentally expressed claudin isoforms, claudin 6, claudin 9, and claudin 13. These paracellular proteins may play a role in the maturational changes in paracellular permeability.

Fig. 1

P_Na/PCl and P_HCO3/P_Cl in neonatal (~10 days) and adult mouse proximal tubules where P is permeability. P_Na/PCl and P_HCO3/P_Cl were measured using dilution potentials in the absence of active transport. There is a maturational change in both consistent with a maturational difference in the paracellular pathway during development in the mouse proximal tubule.

Fig. 2

Gel showing PCR product for claudin 1–16 in 1-day-old neonates and adults. Arrows denote the presence of claudin 6, claudin 9, and claudin 13 in the neonate. Note that there is no detectable claudin 6, claudin 9, and claudin 13 band in the adult samples. Top band is GAPDH.

Fig. 3

PCR profile for claudin 6, 9, 13 in 1-day-old, 2-wk-old, and adult mice kidney using quantitative real-time RT-PCR. To the right of each profile is a summary of experiments showing a developmental decrease in mRNA expression. This difference in these claudin isoforms was significant showing a maturational change during postnatal development.

Fig. 4

Immunoblots showing claudin claudin 6, 9, 13 expression in 1-day-old, 2-wk-old, and adult kidney. β-Actin is below showing equal loading. Also shown is the result of densitometry showing that there is a developmental decrease in expression of these claudin isoforms.

Fig. 5

Expression of claudin 6 in the kidneys of neonatal, 2-wk-old, and adult mice. Kidney sections were costained with anti-claudin 6 antibody (red) and markers of nephron segments (green). Tubular markers used were LTA for proximal tubules (pt), NKCC2 for thick ascending limb (tal), TSC for distal tubules (dt), and DBA for collecting duct (cd). Note the expression of claudin 6 at the apical aspect of the intercellular junction (arrows) in all the above nephron segments in neonatal kidneys (a, d, g, and j). In 2-wk-old mouse kidneys, claudin 6 was detected in proximal tubules (b) and in thick ascending limbs (e), but not in distal tubules (h) and collecting duct (k). In adult kidneys, claudin 6 expression was not detected by immunostaining (c, f, i, and l). Nuclei were counterstained with DAPI and images were merged. Scale bar = 20 μm.

Fig. 6

Gel showing PCR product for claudin 1–16 in 1-day-old neonatal and adult-dissected proximal convoluted tubules. Arrow denotes the presence of claudin 6 in the neonate. Note that there is no detectable claudin 6 in the adult. Claudin 5 in the neonate is likely contaminant from glomerular vessels (33). A faint band for claudin 9 was sometimes found. Claudin 10b splice variant was not detected, but the claudin 10a splice variant was detected in adult and neonatal proximal tubules. The band for claudin 10a was run on a separate gel, and the image was merged on this figure.

Fig. 7

PCR profile for claudin 1, 2, 6, 9, 10a, 12 and occludin in 1-day-old and adult mice proximal convoluted tubules (PCT) using quantitative real-time RT-PCR. Shown is a summary of 6 experiments with ~20 mm of PCT demonstrating comparable claudin 1, 2, 10a, 12 and occludin mRNA expression in 1-day-old and adult PCT. Claudin 6 and claudin 9 were detectable in 1-day-old tubules but virtually undetectable in adult PCT. This difference in these claudin isoforms was significant showing a maturational change during postnatal development. Claudin 13 was not detected in 1-day-old or adults.