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Qing Zhong, Renu Kowluru; Kelch like-ECH-associated protein 1 (Keap1) and retinal antioxidant defense in the development of diabetic retinopathy. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5564.
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© ARVO (1962-2015); The Authors (2016-present)
In diabetes, retinal superoxide radicals are increased and the intracellular antioxidant GSH becomes subnormal. We have shown that despite increase in the gene expression of the transcription factor, NF-E2-related factor 2 (Nrf2, a master regulator of the antioxidant response), its transcriptional activity is decreased in diabetes. Under normal conditions, Nrf2 is kept in the cytoplasm by Kelch like-ECH-associated protein 1 (Keap1), but in stress conditions it moves to the nucleus to regulate antioxidant genes through binding with the antioxidant-response element (ARE). The aim of this study is to investigate the role of Keap1 in the inhibition of retinal Nrf2 activity in diabetes.
The expressions of Keap1, Nrf2 and the catalytic subunit of glutamylcysteine ligase (GCLC), an enzyme important in GSH synthesis, were analyzed in the retina from streptozotocin-induced diabetic rats. Transcriptional activity of Nrf2 was measured by TransAM Nrf2 ELISA kit, and Nrf2-Keap1 binding was analyzed in the retinal cytosolic fraction (150,000xg supernatant) by co-immunoprecipitation technique. To investigate the effect of inhibition of diabetes-induced increased oxidative stress on Nrf2-Keap1, the retina from rats receiving lipoic acid was analyzed.
Diabetes increased the gene and protein expressions of retinal Keap1 and Nrf2. However, the transcriptional activity of Nrf2 and its downstream gene GCLC were decreased and GSH levels were subnormal. Nrf2-Keap1 binding in the cytosol fraction was increased by 1.5-2 fold compared to the values obtained from age-matched normal rats. Administration of lipoic acid for 10-12 months, in addition to preventing decrease in retinal GSH levels and the development of retinopathy (as evidenced by significant decrease in the number of acellular capillaries in the vasculature), also ameliorated diabetes-induced increase in the gene transcripts of Keap1 and Nrf2, and prevented decrease in Nrf2 activity and GCLC.
Diabetes increases retinal Keap1, and the increased Keap1-Nrf2 binding impedes Nrf2 movement from the cytosol to the nucleus compromising Nrf2-ARE binding. This results in decreased GCLC and subnormal GSH levels. Inhibition of oxidative stress should help maintain the antioxidant defense system in the retina by regulating Keap1-Nrf2, and this will help prevent the development of diabetic retinopathy.
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