In this study, we test the hypothesis that diabetes promotes a greater than normal cytosolic calcium level in rod cells that activates a Ca-sensitive protease, calpain, resulting in oxidative stress and inflammation, two pathogenic factors of early diabetic retinopathy (DR). Nondiabetic and 2-months diabetic C57Bl/6J and calpain1 knockout (Capn1) mice were studied; subgroups were treated with a calpain inhibitor (CI). Ca content was measured in photoreceptors using Fura-2. Retinal calpain expression was studied by qTR-PCR and immunohistochemistry. Superoxide and expression of inflammatory proteins were measured using published methods. Proteomic analysis was conducted on photoreceptors isolated from diabetic mice untreated or treated daily with CI for 2-months. Cytosolic Ca content was increased two-fold in photoreceptors of diabetic mice as compared to nondiabetic mice. Capn1 expression increased 5-fold in photoreceptor outer segments of diabetic mice. Pharmacologic inhibition or genetic deletion of Capn1 significantly suppressed diabetes-induced oxidative stress and expression of pro-inflammatory proteins in retina. Proteomics identified a protein (WWOX; WW domain-containing oxidoreductase) whose expression was significantly increased in photoreceptors from mice diabetic for 2-months and was inhibited with CI. Knockdown of Wwox using specific siRNA in vitro inhibited increase in superoxide caused by the high glucose. These results suggest that reducing Ca accumulation, suppressing calpain activation, and / or reducing Wwox upregulation are novel targets for treating early DR.Copyright © 2021. Published by Elsevier Inc.
Photoreceptor cell calcium dysregulation and calpain activation promote pathogenic photoreceptor oxidative stress and inflammation in prodromal diabetic retinopathy.
