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K Kuang, M Yiming, Y Li, M Li, P Iserovich, AS Verkman, J Fischbarg; Water Transport Across Cultured Layers of Corneal Endothelium from Aquaporin-1 (AQP1) Knockout Mice . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3249.
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Purpose: We have reported that bovine corneal endothelium express AQP1. In this study, we investigated the role of AQP1 in water movement across the cultured normal mouse (strain CD1) corneal endothelial cells (MCE) and cultured AQP1 knockout mice corneal endothelial cells (KMCE). Methods: 1). Cultured mice corneal endothelial cells: eyeballs were removed from mice. The cornea was excised and transferred to a 35 mm dish containing media. Descemet's membrane was gently removed with Jeweler's forceps and placed into a 4-well culture plate in DMEM containing 10% fetal calf serum, FGF (5 ng/ml), EGF (10ng/ml), insulin (5 mg/ml) and penicillin/streptomycin. 2). RT-PCR analysis of AQP1 transcript expression: After extraction of total RNA from MCE, mouse kidney, and KMCE with the ULTRASPECTM RNA reagent method (Biotecx), RT-PCR reaction was performed with the S UPERSCRIPTTM Preamplification System (GIBCOBRL). Two specific primers used in PCR were based on the sequence of mice AQP1. 3). Fluid transport measurement: MCE and KMCE layers were grown to confluence on 24 mm permeable membrane inserts (Transwell" Costar" #3450). Inserts were mounted between two chambers filled with solutions (T=37 C). Fluid flow was measured by nanoinjector-driven volume clamp of the bottom chamber. Results: The transcript for AQP1 was detected in MCE and kidney but not in KMCE. The knockout mice had grossly transparent corneas, just as normal mice. The spontaneous rate of fluid transport was 4.29 ± 0.64 µl × h-1 × cm-2 (n=9) for MCE and 3.70 ± 0.66 µl × h-1 × cm-2 (n=9) for KMCE, both from the basolateral to the apical side. 300 mM HgCl2, a blocker of water channels, markedly reduced the MCE osmotic water flow induced by a 100 mOsm sucrose gradient across the layer (from 8.04 ± 0.87 to 3.09 ± 0.56 µl × h-1 × cm-2, n=5). However, HgCl2 had no significant effect on the fluid transport by these MCE cells (from 4.38 ± 0.98 to 4.20 ± 0.99 µl × h-1 × cm-2, n=5). Conclusions: Deletion of AQP1 in mice affected osmotic permeability but did not significantly affect spontaneous transendothelial fluid transport function by the corneal endothelium. As no aquaporins other than AQP1 have been detected so far in corneal endothelium, the precise role of AQP1 in spontaneous fluid transport requires further investigation. Cr: N Supported by NIH Grant EY06178 (JF), DK35124 (ASV) and RPB, Inc.
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