Interaction between transcellular and paracellular water transport pathways through Aquaporin 5 and the tight junction complex

Abstract

To investigate potential physiological interactions between the transcellular and paracellular pathways of water transport, we asked whether targeted deletion of Aquaporin 5 (AQP5), the major transcellular water transporter in salivary acinar cells, affected paracellular transport of 4-kDa FITC-labeled dextran (FITC-D), which is transported through the paracellular but not the transcellular route. After i.v. injection of FITC-D into either AQP5 wild-type or AQP5-/- mice and saliva collection for fixed time intervals, we show that the relative amount of FITC-D transported in the saliva of AQP5-/- mice is half that in matched AQP5+/+ mice, indicating a 2-fold decrease in permeability of the paracellular barrier in mice lacking AQP5. We also found a significant difference in the proportion of transcellular vs. paracellular transport between male and female mice. Freeze-fracture electron microscopy revealed an increase in the number of tight junction strands of both AQP5+/+ and AQP5-/- male mice after pilocarpine stimulation but no change in strand number in female mice. Average acinar cell volume was increased by approximately 1.4-fold in glands from AQP5-/- mice, suggesting an alteration in the volume-sensing machinery of the cell. Western blots revealed that expression of Claudin-7, Claudin-3, and Occludin, critical proteins that regulate the permeability of the tight junction barrier, were significantly decreased in AQP5-/- compared with AQP5+/+ salivary glands. These findings reveal the existence of a gender-influenced molecular mechanism involving AQP5 that allows transcellular and paracellular routes of water transport to act in conjunction.

Fig. 1.

Comparison between AQP5+/+ and AQP5−/− mice of each gender (n = 7 AQP5+/+ males; n = 6 AQP5−/− males; n = 6 AQP5+/+ females; n = 5 AQP5−/− females). (a) FITC-D clearance by salivary glands (amount of FITC-D transported in the saliva relative to its concentration in plasma in 15 min) corrected for BW of the mice. (b) Total saliva volume secreted in 15 min.

Fig. 2.

Measurement of transport. (a) Comparison of concentration of FITC-D in saliva collected in 15 min between AQP5+/+ and AQP5−/− mice of each gender (n = 7 +/+ males; n = 6 −/− males; n = 6 +/+ females; n = 5 −/− females). (b) Plasma concentration of FITC-D in plasma. Blood samples were collected from the tail vein at 0-, 5-, 10-, 15-, 20-, and 35-min time points after the start of saliva collection.

Fig. 3.

Histology of parotid glands. Absence of AQP5 is associated with increased acinar volume of parotid salivary glands in AQP5−/− mice. Sections from AQP5+/+ and AQP5−/− parotid glands of each gender were stained with H&E. (a) AQP5+/+ females (b) AQP5−/− females (d) AQP5+/+ males and (e) AQP5−/− males. Quantitation of acinar area in AQP5+/+ and AQP5−/− mice was done by using Zeiss Axiovision software (c) females and (f) males. A minimum of four animals were studied in each group.

Fig. 4.

Freeze-fracture electron microscopy of parotid acinar cell tight junctions of male mice indicates an increase in strand number after pilocarpine stimulation. (a) AQP5+/+. (b) AQP5+/+, pilocarpine stimulated. (c) AQP5−/−. (d) AQP5−/−, pilocarpine stimulated. Black arrowheads indicate P-face junctional strands; white arrowheads indicate E-face grooves. Lu, lumen. (Scale bar = 0.25 μm.)

Fig. 5.

Western blots. AQP5 deletion alters the expression of molecular components of the tight junction barrier. (a) Western blot analysis of tight junction proteins from total protein extracts of female parotid salivary gland. (b) Quantitation of the Western blots by using β-actin as the loading control. (c) Western blot analysis of tight junction proteins from total protein extracts of male parotid salivary gland. (d) Quantitation of Western blots by using β-actin as the loading control.

Fig. 6.

Proposed scenarios for evaluating relative changes to paracellular FITC-D transport. (a) Transport of transcellular and paracellular fluid in AQP5+/+ mice. Water transport from transcellular pathway dilutes FITC-D concentration in saliva to yield a hypothetical baseline concentration = X ng/μl. (b) Both transcellular and paracellular water transport is decreased equally resulting in FITC-D concentration = X ng/μl. (c) Larger decrease in transcellular compared with paracellular transport resulting in FITC-D concentration > X ng/μl. (d) Larger decrease in paracellular transport compared with decrease in transcellular transport resulting in FITC-D concentration < X ng/μl.