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R. V. Patil, A. van Hoek, D. Brown, M. B. Wax; Mapping of Aquaporin Isoforms in the Mouse Eye. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3168.
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© ARVO (1962-2015); The Authors (2016-present)
The aquaporins (AQPs) are a family of water transporting proteins expressed in many mammalian epithelia and endothelia. At present 13 mammalian AQPs have been identified and a subset of these aquaporins have been identified in the eye. Recent evidence suggests that aquaporins are involved in corneal and lens transparency, intraocular pressure (IOP) regulation and retinal signal transduction. The purpose of this study was to generate a precise expression map for aquaporins in the various cell types of the eye.
Affinity-purified specific antibodies were generated against rat AQP1 through 9 using 15 amino acids C-terminal peptides. Each peptide was coupled to keyhole limpet hemocyanin and whole serum was affinity purified against the immunizing peptide by using a Pierce affinity purification column. For immunocytochemistry, eyes from mice were removed, injected with and placed in 4% paraformaldehyde, 0.05 M sodium phosphate buffer overnight, sliced in two pieces and washed in PBS. For cryostat sectioning, eyes were infiltrated with 30% sucrose in PBS, placed on a support in OTC compound and frozen. Cryostat sections (5 um) were mounted on microscope slides, dried, rehydrated and incubated with primary and secondary fluorescent-labeled antibodies for one hour and observed with a Nikon FXA epifluorescence microscope. Controls included the incubation of the tissues with the antibodies pre-absorbed with the immunizing peptide.
In the cornea, AQP1 was restricted to keratinocytes of the stroma, and AQP5 localized to the epithelium. In conjunctiva, AQP1 was localized to endothelial and AQP3 to epithelial cells. In the lens, AQP5 was limited to outer lens fibers, AQP7 to the lens capsule, and AQP4 to the lens epithelium. In ciliary body, AQP1 and AQP4 were confined to the non-pigmented epithelium. In retina, we found AQP9 in the retinal ganglion cells (RGC) and outer nuclear layer (ONL) and AQP7 in the outer plexiform layer (OPL) in addition to AQP4 in the outer and inner limiting membrane, OPL, inner plexiform layer (IPL) and RGC layer.
We confirmed the expression of AQP1 in cornea, conjunctiva, lens and ciliary body; AQP3 in conjunctiva; AQP4 in ciliary body and retina; AQP5 in cornea; and AQP9 in RGC layer. In addition, we discovered, for the first time, expression of AQP4, AQP5 and AQP7 in the lens. Another novel observation was the localization of AQP9 in the ONL and AQP7 in the OPL of the retina. The diverse and distinct distribution of aquaporins in the various ocular tissues suggests their important and specific roles in ocular physiology.
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