NRF2 plays a protective role in diabetic retinopathy in mice

Abstract

Aims/hypothesis: Although much is known about the pathophysiological processes contributing to diabetic retinopathy (DR), the role of protective pathways has received less attention. The transcription factor nuclear factor erythroid-2-related factor 2 (also known as NFE2L2 or NRF2) is an important regulator of oxidative stress and also has anti-inflammatory effects. The objective of this study was to explore the potential role of NRF2 as a protective mechanism in DR.

Methods: Retinal expression of NRF2 was investigated in human donor and mouse eyes by immunohistochemistry. The effect of NRF2 modulation on oxidative stress was studied in the human Müller cell line MIO-M1. Non-diabetic and streptozotocin-induced diabetic wild-type and Nrf2 knockout mice were evaluated for multiple DR endpoints.

Results: NRF2 was expressed prominently in Müller glial cells and astrocytes in both human and mouse retinas. In cultured MIO-M1 cells, NRF2 inhibition significantly decreased antioxidant gene expression and exacerbated tert-butyl hydroperoxide- and hydrogen peroxide-induced oxidative stress. NRF2 activation strongly increased NRF2 target gene expression and suppressed oxidant-induced reactive oxygen species. Diabetic mice exhibited retinal NRF2 activation, indicated by nuclear translocation. Superoxide levels were significantly increased by diabetes in Nrf2 knockout mice as compared with wild-type mice. Diabetic Nrf2 knockout mice exhibited a reduction in retinal glutathione and an increase in TNF-α protein compared with wild-type mice. Nrf2 knockout mice exhibited early onset of blood-retina barrier dysfunction and exacerbation of neuronal dysfunction in diabetes.

Conclusions/interpretation: These results indicate that NRF2 is an important protective factor regulating the progression of DR and suggest enhancement of the NRF2 pathway as a potential therapeutic strategy.

Fig. 1

Expression of NRF2 in human and mouse retinas. Paraffin-embedded human retinal sections were immunostained with NRF2 antibody (blue) and counter-stained with nuclear fast red. (a) Retina from donors showed NRF2 immunoreactivity in Müller cells spanning the retina. NRF2 was also expressed in the GCL. (b) Negative immunohistochemical staining in human retina with rabbit control IgG. Arrows indicate Müller cell processes and asterisks indicate the nerve fibre layer. The images are representative of eight donors and were taken at 20× objective. (c) Mouse retinal cryosections were analysed by immunofluorescence for NRF2 (green) and Vimentin (red). (d) Mouse retinal cryosections were analysed by immunofluorescence for NRF2 (green) and GFAP (red). (e) Immunofluorescence for control IgG (green). DAPI (blue) was used to stain nuclei in all retinal sections

Fig. 2

NRF2 protects against oxidant-induced ROS increase in human Müller cells. (a) Relative expression of NRF2-responsive antioxidant genes in MIO-M1 cells after 48 h of NRF2 siRNA and KEAP1 siRNA transfection. White bars, control siRNA; grey bars, NRF2 siRNA; black bars, KEAP1 siRNA. n=4; *p<0.05, **p<0.01. (b) Western blot analysis of NRF2, NQO1 and HO-1 expression after 48 h of NRF2 siRNA and KEAP1 siRNA transfection. (c) The effect of knocking down NRF2 or KEAP1 on different oxidant-induced ROS increase in MIO-M1 cells. Forty-eight hours after siRNA transfection, cells were challenged by different doses of TBH or H₂O₂ for 1 h. ROS levels were determined by DCF assay. White bars, control siRNA; grey bars, NRF2 siRNA; black bars, KEAP1 siRNA. n=4; *p<0.05 and **p<0.01 vs control siRNA with PBS treatment; ^†p<0.05, ^‡p<0.05 and ^‡‡p<0.01 vs control siRNA with respective treatment

Fig. 3

Nuclear translocation of NRF2 in diabetic mouse retinas. (a) Western blot analysis of NRF2 with retina nuclear extract from wild-type mice after 8 weeks of diabetes. Lamin B was used as a loading control. (b). Quantitative analysis of nuclear NRF2 protein level. The intensity of NRF2 is presented relative to Lamin B in the same sample and normalised to non-diabetic control retinas. n=6 for each group; **p<0.01

Fig. 4

NRF2-deficient mice exhibit increased oxidative stress in early diabetes. Fresh retinas were isolated from wild-type mice and Nrf2^−/− mice after 5 weeks of diabetes. The level of retinal superoxide was determined by lucigenin assay. White bars, non-diabetic control mice; black bars, diabetic mice. Non-diabetic Nrf2^+/+ mice, n = 6; diabetic Nrf2^+/+ mice, n = 7; non-diabetic Nrf2^−/− mice, n = 6; diabetic Nrf2^−/− mice, n = 5; *p<0.05, **p<0.01

Fig. 5

NRF2-deficient diabetic mice exhibit lower retinal GSH levels compared with wild-type diabetic mice. GSH levels were measured in retinal protein lysates from wild-type and Nrf2^−/− mice after 8 weeks of diabetes using a GSH assay kit. White bars, non-diabetic control mice; black bars, diabetic mice. n=7 for each group; *p<0.05

Fig. 6

NRF2-deficient mice exhibit increased retinal TNF-α protein levels in diabetes. After 8 weeks of diabetes, retinas were isolated and processed for TNF-α ELISA assay. White bars, non-diabetic control mice; black bars, diabetic mice. Non-diabetic Nrf2^+/+ mice, n=10; diabetic Nrf2^+/+ mice, n=14; non-diabetic Nrf2^−/− mice, n=10, diabetic Nrf2^−/− mice, n=8; *p<0.05; **p<0.01

Fig. 7

NRF2-deficient mice exhibit earlier onset of BRB dysfunction in diabetes. BRB dysfunction was determined in Nrf2^+/+ and Nrf2^−/− mice after 8 weeks of diabetes. Radioactivity per mg of retina was measured 1 h after i.p. injection of [³H]mannitol. White bars, non-diabetic control mice; black bars, diabetic mice. Non-diabetic Nrf2^+/+ mice, n=4; diabetic Nrf2^+/+ mice, n=5; non-diabetic Nrf2^−/− mice, n=4; diabetic Nrf2^−/− mice, n=4; *p<0.05; **p<0.01

Fig. 8

NRF2-deficient mice have increased visual dysfunction induced by diabetes. After 8 weeks of diabetes, the SF (a) and CS (b) thresholds were obtained in wild-type and Nrf2^−/− mice using Optomotry optokinetic testing. White bars, non-diabetic control mice; black bars, diabetic mice. Non-diabetic Nrf2^+/+ mice, n=6; diabetic Nrf2^+/+ mice, n=6; non-diabetic Nrf2^−/− mice, n=6; diabetic Nrf2^−/− mice, n=4; *p<0.05 and **p<0.01