The bovine retinal pigment epithelium (BRPE) drives solute-coupled water transport from the retinal side of the retinal pigment epithelium (RPE) towards the choroidal vasculature through Bruch?s membrane which was measured with high accuracy by a refined capacitance probe method. With recent advances in such measurement techniques employed in our laboratory this research study aim to measure the hydraulic conductivity of the BRPE and to directly measure the flows generated by small gradients. The RPE transports water from the retinal side of the RPE to the choroidal vasculature via Bruch?s membrane. As the Bruch?s membrane-choroid complex is in series with the RPE, individual hydraulic conductivities have been difficult to resolve, as isolating the RPE is often unavoidable without compromising the integrity of the epithelium. We have been able to resolve individual hydraulic conductivities of the series components using a novel method and our study is the first establish the hydraulic conductivity for the Bruch?s membrane in an intact epithelium. Retina-to-choroid hydrostatic pressure steps of 1-5 cmH2O failed to generate measurable transepithelial flows. Osmotic gradients of approximately 70 mOsm (sucrose) applied across the RPE in both retina-to-choroid and choroid-to-retina directions generated sizeable and reversible alterations in water flows spontaneously generated by the epithelium. The osmotic hydraulic conductivity thus measured, approximately 35-fold lower than previously reported, is sufficient for an intraepithelial gradient of 20 mOsm to drive physiologically observed absorption rates of approximately 5 microliter/cm2/hr. Choroid-to-retina hydrostatic steps reversibly disrupt RPE tight junctions and drive very high water flows through the Bruch?s membrane as the primary barrier. Thus, Bruch?s membrane is not a barrier in healthy eyes and a pressure of less than 1 cmH2O allows the absorbate to drain into the choriocapillaries. These measurements will allow a deeper understanding of mechanisms which underlie ocular pathophysiology and thus provide an enhanced assessment of abnormal fluid accumulation in and around the subretinal space. This study establishes the RPE as the major series barrier to water absorption, rather than the previously thought Bruch?s membrane. Therefore, we establish that the healthy Bruch?s membrane does not present a significant barrier to absorption of water and with a pressure of less than 1cmH2O water, water absorbed by and facilitated across the RPE can easily cross the Bruch?s membrane. The implications of this study include a novel method for measuring the hydraulic conductivity of Bruch?s membrane introduced here which will permit accurate assessment of progressive reduction of hydraulic conductivity with aging. Future directions include a better understanding of the control signals for regulation of various membrane transporters that are responsible for generating water transport which need further clarification. Additionally, the structural parameters of the cell membranes (membrane area, integral membrane proteins e.g. aquaporins, ultrastructural morphometry) of the RPE involved in fluid transport need to be further identified and measured.
