Roles Of Aquaporins In The Cornea

Aquaporin 1 in Corneal Endothelium - its Role in Corneal Transparency

Maintenance of corneal transparency requires precise regulation of stromal water content (41, 42). Aquaporin 1 is expressed in corneal endothelial cells and AQPs 3 and 5 in epithelial cells. To test the possible involvement of AQPs in the maintenance of corneal volume, transparency and thickness, water permeability and response to experimental swelling was measured in wild-type versus AQP1-null mice (43). Compared to wild-type mice, which have a corneal thickness of 123 ^m, corneal thickness was reduced in AQP1-null mice (101 ^m). Thickness measurements were made in fixed eyes (Fig. 3A), as well as

Fig. 3. Involvement of aquaporin 1 in the maintenance of corneal transparency. (A) Reduced thickness in AQP1-deficient adult mouse corneas in paraffin-embedded central corneal sections. Bar = 50 |im. (B) Osmotic water-transport across the corneal endothelium. Top: schematic showing micropipette placement for anterior chamber perfusion. Bottom: time course of corneal thickness following corneal endothelial exposure to hypotonic saline in wild-type (open circles) and AQP1-null (filled circles) mice. (C) Restoration of corneal thickness after osmotic swelling. Top: transient exposure of the corneal surface to hypotonic saline to induce corneal swelling and to follow recovery of thickness. Bottom: time course of corneal thickness following exposure of the corneal surface to hypotonic saline. Corneal thickness measured in vivo by z-scanning confocal microscopy. Adapted from (43).

Fig. 3. Involvement of aquaporin 1 in the maintenance of corneal transparency. (A) Reduced thickness in AQP1-deficient adult mouse corneas in paraffin-embedded central corneal sections. Bar = 50 |im. (B) Osmotic water-transport across the corneal endothelium. Top: schematic showing micropipette placement for anterior chamber perfusion. Bottom: time course of corneal thickness following corneal endothelial exposure to hypotonic saline in wild-type (open circles) and AQP1-null (filled circles) mice. (C) Restoration of corneal thickness after osmotic swelling. Top: transient exposure of the corneal surface to hypotonic saline to induce corneal swelling and to follow recovery of thickness. Bottom: time course of corneal thickness following exposure of the corneal surface to hypotonic saline. Corneal thickness measured in vivo by z-scanning confocal microscopy. Adapted from (43).

in vivo by brightfield scanning confocal microscopy. Aquaporin 1 water-transport function in corneal endothelium in vivo was demonstrated by slowed corneal swelling upon hypotonic challenge at the endothelial surface, utilizing an anterior chamber microperfusion method (Fig. 3B). An important role for AQP1 in the maintenance of corneal transparency was demonstrated in an experimental model of corneal edema produced by transient exposure of the corneal surface to hypotonic solution, in which AQP1 deficiency was associated with remarkably impaired recovery of corneal transparency and thickness. Although baseline corneal transparency was not impaired by AQP1 deletion, the return of corneal transparency and thickness after hypotonic swelling (10 minutes exposure of corneal surface to distilled water) was remarkably delayed in AQP1-null mice, with approximately 75% recovery at seven minutes in wild-type mice compared to 5% recovery in AQP1-null mice (Fig. 3C). The impaired recovery of corneal transparency in AQP1-null mice provides evidence for the involvement of AQP1 in active extrusion of fluid from the corneal stroma across the corneal endothelium.

The mechanisms by which AQP1 deletion impairs maintenance of corneal transparency remain unclear. In primary corneal endothelial cell cultures, AQP1 deficiency reduced osmotically driven cell membrane water permeability, but did not impair active near-isosmolar transcellular fluid transport (44). The generally assumed mechanism of transcellular, AQP-facilitated fluid transport has been questioned in relation to the corneal endothelium, with Fischbarg and colleagues proposing a central role for electro-osmotic coupling of fluid transport to recirculating currents in intercellular junctions (45). This model, which is described more fully in another chapter in this volume, posits that AQP1 contributes primarily to cell volume regulation, a role that remains difficult to reconcile with the dramatic corneal swelling phenotype of AQP1-null mice and with the substantially slower rate of cell volume regulation versus osmotic equilibration.

Aquaporins and Ocular Surface Fluid Secretion

The ocular surface is lined by stratified corneal and conjunctival epithelia, which lie in contact with the tear film. The water permeability of the ocular surface, together with the rates of evaporative water-loss and tear-fluid production and drainage, determine tear-film volume and osmolality, as well as corneal stromal water content. Active Cl-secretion and Na+ absorption drive net water secretion into tears across both corneal and conjunctival epithelia (reviewed in ref. 46). The ocular surface, and the conjuncti-val epithelium in particular (covering 17 times more area than the cornea in humans), contributes to active tear-fluid secretion under basal conditions, and even more so upon stimulation. A computational model of tear-film balance was developed to investigate the theoretical importance of ocular surface water permeability on tear-film osmolality. The model demonstrated the sensitivity of tear film osmolarity to both excessive tear evaporation and inadequate tear-fluid secretion, the two principal causes of dry-eye syndrome (Fig. 4; ref. 47). In this model, tear fluid generated by osmotic flux (Jv) and isosmolar active secretion (Js) is removed by evaporation (Je) and isotonic drainage (Jd), such that in the steady state, Js + Jv = Je + Jd. Predicted tear-film osmolarity depended strongly on both passive water permeation and active fluid secretion at the ocular surface.

A series of measurements indicated the involvement of AQPs in osmotically-driven water transport at the ocular surface (47). Mice lacking AQP5 have increased corneal thickness (144 versus 123 ^m, from ref. 43). Plasma membrane osmotic water permeability of corneal epithelial cells was measured in mice utilizing an ocular surface perfusion method (Fig. 5A) involving microfluorimetric measurement of calcein quenching in surface cells. Water permeability was high (0.045 cm/s), andreducedapproximatelytwofoldin AQP5defi-ciency (Fig. 5B). Membrane water permeability was AQP3 dependent in conjunctiva when measured similarly, with approximately fourfold slowing of osmotic equilibration in AQP3 deficiency. Water permeability across the intact cornea and conjunctiva (Pf), the relevant parameters describing water movement into the hyperosmolar tear film in vivo, was measured by a dye-dilution method from the fluorescence of Texas red dextran in an anisosmolar solution in a microchamber at the ocular surface. The Pf of whole cornea was 0.0017 cm/s, and

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