Increased fluid secretion after adenoviral-mediated transfer of the aquaporin-1 cDNA to irradiated rat salivary glands

Abstract

A replication-deficient, recombinant adenovirus encoding human aquaporin-1 (hAQP1), the archetypal water channel, was constructed. This virus, AdhAQP1, directed hAQP1 expression in several epithelial cell lines in vitro. In polarized MDCK cell monolayers, hAQP1 was localized in the apical and basolateral plasma membranes. Fluid movement across monolayers infected by AdhAQP1 in response to an osmotic gradient was approximately 4-fold that seen with uninfected monolayers or monolayers infected by a control virus. When AdhAQP1 was administered to rat submandibular glands by retrograde ductal instillation, significant hAQP1 expression was observed by Western blot analysis in crude plasma membranes and by immunohistochemical staining in both acinar and ductal cells. Three or four months after exposure to a single radiation dose (17.5 or 21 Gy, respectively), AdhAQP1 administration to rat submandibular glands led to a two- to threefold increase in salivary secretion compared with secretion from glands administered a control virus. These results suggest that hAQP1 gene transfer may have potential as an unique approach for the treatment of postradiation salivary hypofunction.

Figures

Figure 1

hAQP1 expression in 293 cells and other epithelial cells. (A) Northern blot analysis was performed using 10 μg of total RNA, and 293 cells were infected at a MOI of 100 with either Adα1AT or AdhAQP1 and hybridized with a 813-bp radiolabeled probe of hAQP1 as described in Methods. (B) Ten micrograms of crude membranes from 293 cells infected at a MOI of 100 with either Adα1AT or AdhAQP1 were analyzed by Western blot as described in Methods. (C) Ten micrograms of crude membranes from MDCK and SMIE cells were analyzed by Western blot as above. The cells were infected at a MOI of 100 with either Adα1AT or AdhAQP1.

Figure 2

Localization of hAQP1 in polarized MDCK cells. Confluent MDCK cells, grown on filters, were infected for 24 h at the basolateral side at a MOI of 100 with either Adα1AT (A and B) or AdhAQP1 (C and D). hAQP1 expression was determined as described in Methods. Micrographs of horizontal (xy; A and C), and vertical (xz; B and D) optical sections through MDCK cells are shown.

Figure 3

Net fluid secretion rate of MDCK cells. Net fluid secretion rate of control, Adα1AT-(MOI of 100)- and AdhAQP1-(MOI of 100)-infected MDCK cells was measured as described in Methods. The results are expressed as water flow in microliters of fluid secreted per cm2 per h and are the mean ± SEM of three separate experiments, each performed in triplicate. The results were analyzed using an ANOVA. ∗, P < 0.05 compared with control and Adα1AT-infected cells.

Figure 4

Expression and function of the recombinant hAQP1 in vivo. (A) Ten micrograms of crude membranes from rat submandibular glands, which were cannulated and infected for 24 h with 5 × 109 pfu of either Addl312 or AdhAQP1, were used for Western blot analysis as in Fig. 1. (B) Submandibular glands of sham-irradiated (Non-IR) or irradiated (IR) rats were cannulated and retrograde instilled with 5 × 109 pfu of either Addl312 (hashed bar) or AdhAQP1 (solid bar), and then saliva was collected after 3 days as described in Methods. The results are expressed as saliva secretion in microliters per 100 g of rat body weight per 15 min and are the mean ± SEM. The results were analyzed using an ANOVA. ∗, P < 0.05 between the two groups with different instilled viruses.

Figure 5

Localization of hAQP1 in rat submandibular glands infected with AdhAQP1. hAQP1 localization was determined by immunohistochemistry (see Methods) on paraffin sections from sham-irradiated rat submandibular glands infected with 5 × 109 pfu of either Addl312 (A) or AdhAQP1 (B). Arrows indicate endothelial cells in capillaries and venules. a, acinar cells; d, ductal cells. Similar results were found with irradiated glands (not shown).