Alpha-1-Anti-Trypsin increased Na+/K+-ATPase expression in an in vitro Müller cells diabetic retinopathy model

ORTIZ, G; GALLO, JE; POTILINSKI, MC

Baltimore

Congreso; 2017 ARVO Annual Meeting; 2017

ASSOCIATION FOR RESEARCH IN VISION AND OPHTHALMOLOGY

Purpose : The ophthalmic therapy for diabetic retinopathy is focused on severe stages of the disease. Previous results obtained in our group show that Alpha-1-Anti-Trypsin (A1AT) acts like an anti-inflammatory agent that could play a role on diabetic retinopathy treatment. It is important to evaluate A1AT impact on cellular components that are essential to retina function like Na+/K+-ATPase (NKA). This protein is responsible of Na+ and K+ gradients in cells and it is involved in synaptic activity and action potentials in this tissue. It is known that NKA activity and expression is diminished in diabetic retinopathy. A1AT may stimulate NKA expression through different cellular mechanisms. For this reason we aimed at evaluating NKA with A1AT treatment in an in vitro diabetic retinopathy cell model.Methods : Eight mouse retinas were obtained from freshly enucleated eyes incubated with collagenase I and Trypsin. Retinas were desegregated and incubated with DMEM for 5 days to allow the enrichment of Müller cells population. Müller cells obtained, were incubated 24h with DMEM 30mM glucose (Control), DMEM 30mM glucose + 4.5mg/ml A1AT (Control + A1AT), DMEM 100mM glucose (Diabetic), DMEM 100mM glucose + 4.5mg/ml A1AT (Diabetic + A1AT). Cells were harvested with RIPA buffer for Western Blot Assay or Fixed for Immunohistochemistry. Western blot and Immunohistochemistry were performed with using specific primary antibodies for total α-Na+/K+-ATP-ase (H-300, Santa Cruz Biotechnology, California, USA).Results : Alpha subunit of Na+/K+-ATPase expression was increased in A1AT treated cells.Conclusions : Results support the hypothesis that A1AT promotes Na+/K+-ATPase expression. This is a novel aspect about NKA expression modulation. Although molecular mechanisms involved remained unknown, A1AT might play a new role in diabetic retinopathy treatment.