Abstract
Purpose :
Diabetic retinopathy (DR) is associated with persistent inflammation with damage to the vascular bed. Consequently, water accumulation in the retina leads to retinal edema. The retinal pigment epithelium (RPE) forms the epithelial component of the blood-retinal barrier where aquaporins (AQPs) are involved in water transportation. Altered expression and/or impaired function of AQPs facilitate edema development through water transport dysregulation.
The ophthalmic therapy for this retinal pathology is focused on the severe stages of the disease. Previous results obtained by our group show that Alpha-1 Antitrypsin (A1AT) acts as an anti-inflammatory agent that could play a role in DR treatment. However, it is important to know the effect of A1AT on AQPs that are relevant to retinal water homeostasis. There are many members of the AQPs family, but one of the most studied and described in ARPE-19 line is Aquaporin 4 (AQP4).
Therefore, we studied the expression of AQP4 on the retinal epithelial ARPE-19 cell line exposed to high glucose and/or A1AT treatment.
Methods :
ARPE-19 cells (ATCC® CRL-2302TM, USA) were maintained in DMEM/F12 (Invitrogen, USA) containing 2μM L-glutamine, 100U/ml penicillin, 100μg/ml streptomycin, and 10% fetal bovine serum. ARPE-19 cells (passages 9-12) were incubated for 16h with DMEM 5,5mM glucose (Control), DMEM 5,5mM glucose + 4.5mg/ml A1AT (Control + A1AT), DMEM 30mM glucose (High glucose), DMEM 30mM glucose + 4.5mg/ml A1AT (High glucose + A1AT). Cells were harvested with RIPA for Western blot or fixed for immunohistochemistry. Immunoblot quantifications were performed with FIJI and relativized to Actin. Measurements were expressed as mean ± SD and statistically analyzed with ANOVA and Tukey multiple comparison test. A p-value <0.05 was considered to be statistically significant.
Results :
We observed an impaired expression of AQP4 in cells exposed to high glucose (0.57±0.02 Control vs. 0.36±0.03 High glucose; p>0.01), (0.78±0.07 Control + A1AT vs. 0.36±0.03 High glucose; p>0.0001) and an increment on cells exposed to A1AT treatment (0.57±0.02 Control vs. 0.78±0.07 Control + A1AT; p>0.01), (0.36±0.03 High glucose vs. 0,71±0.06 High glucose + A1AT; p>0.001).
Conclusions :
In conclusion, A1AT could restore Aquaporin4 disrupted protein levels helping to reestablish altered water homeostasis in ARPE-19 cells with high glucose.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.