Conditionally Immortal Slc4a11(−/−) Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in Slc4a11(−/−) Mouse Model

PURPOSE: To establish conditionally immortal mouse corneal endothelial cell lines with genetically matched Slc4a11(+/+) and Slc4a11(−/−) mice as a model for investigating pathology and therapies for SLC4A11 associated congenital hereditary endothelial dystrophy (CHED) and Fuchs' endothelial corneal dystrophy. METHODS: We intercrossed H-2Kb-tsA58 mice (Immortomouse) expressing an IFN-γ dependent and temperature-sensitive mutant of the SV40 large T antigen (tsTAg) with Slc4a11(+/+) and Slc4a11(−/−) C57BL/6 mice. The growth characteristics of the cell lines was assessed by doubling time. Ion transport activities (Na(+)/H(+) exchange, bicarbonate, lactate, and Slc4a11 ammonia transport) were analyzed by intracellular pH measurement. The metabolic status of the cell lines was assessed by analyzing TCA cycle intermediates via gas chromatography mass spectrometry (GC-MS). RESULTS: The immortalized Slc4a11(+/+) and Slc4a11(−/−) mouse corneal endothelial cells (MCECs) remained proliferative through passage 49 and maintained similar active ion transport activity. As expected, proliferation was temperature sensitive and IFN-γ dependent. Slc4a11(−/−) MCECs exhibited decreased proliferative capacity, reduced NH(3):H(+) transport, altered expression of glutaminolysis enzymes similar to the Slc4a11(−/−) mouse, and reduced proportion of TCA cycle intermediates derived from glutamine with compensatory increases in glucose flux compared with Slc4a11(+/+) MCECs. CONCLUSIONS: This is the first report of the immortalization of MCECs. Ion transport of the immortalized endothelial cells remains active, except for NH(3):H(+) transporter activity in Slc4a11(−/−) MCECs. Furthermore, Slc4a11(−/−) MCECs recapitulate the glutaminolysis defects observed in Slc4a11(−/−) mouse corneal endothelium, providing an excellent tool to study the pathogenesis of SLC4A11 mutations associated with corneal endothelial dystrophies and to screen potential therapeutic agents.