Alpha-1-Anti-Trypsin increased Na+/K+-ATPase and Cx43 expression in an in vitro diabetic retinopathy model

ORTIZ, GUSTAVO A.; POTILINSKI, CONSTANZA; GALLO, JUAN E.

Buenos Aires

Congreso; Reunion Conjunta de Sociedades de Biociencias; 2017

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. Also, Cx43 part of gap junction channels, are abundantly expressed in the retina and play an essential role for maintenance of retinal homeostasis. High glucose or diabetes has been shown to reduce Cx43 expression in retinal cells and inhibit gap junction communication.A1AT may stimulate NKA and Cx43 expression through different cellular mechanisms. For this reason, we aimed at evaluating NKA and Cx43 with A1AT treatment in an in vitro diabetic retinopathy cell model.Methods: 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. Results: Alpha subunit of Na+/K+-ATPase and Cx43 expression were increased in A1AT treated cells. NKA: Diabetic (46.0±4.3)%, Diabetic+A1AT(69.9±17.7)%, P