Role of aldose reductase in diabetes-induced retinal microglia activation

Abstract

Diabetes-induced hyperglycemia plays a key pathogenic role in degenerative retinal diseases. In diabetic hyperglycemia, aldose reductase (AR) is elevated and linked to the pathogenesis of diabetic retinopathy (DR) and cataract. Retinal microglia (RMG), the resident immune cells in the retina, are thought to contribute to the proinflammatory phenotype in the diabetic eye. However, we have a limited understanding of the potential role of AR expressed in RMG as a mediator of inflammation in the diabetic retina. Glycated proteins accumulate in diabetes, including Amadori-glycated albumin (AGA) which has been shown to induce a proinflammatory phenotype in various tissues. In this study, we investigated the ability of AGA to stimulate inflammatory changes to RMG and macrophages, and whether AR plays a role in this process. In macrophages, treatment with an AR inhibitor (Sorbinil) or genetic knockdown of AR lowered AGA-induced TNF-α secretion (56% and 40%, respectively) as well as cell migration. In a mouse RMG model, AR inhibition attenuated AGA-induced TNF-α secretion and cell migration (67% and 40%, respectively). To further mimic the diabetic milieu in retina, we cultured RMG under conditions of hypoxia and observed the induction of TNF-α and VEGF protein expression. Downregulation of AR in either a pharmacological or genetic manner prevented hypoxia-induced TNF-α and VEGF expression. In our animal study, increased numbers of RMG observed in streptozotocin (STZ)-induced diabetic retina was substantially lower when diabetes was induced in AR knockout mice. Thus, in vitro and in vivo studies demonstrated that AR is involved in diabetes-induced RMG activation, providing a rationale for targeting AR as a therapeutic strategy for DR.

Keywords: Aldose reductase; Amadori-glycated albumin; Hypoxia; Inflammation; Retinal microglia.

Fig. 1.. Effect of AR inhibition or knockdown on AGA-induced cytokine secretion and migration in RAW264.7.

AGA induces TNF-α secretion in a dose dependent manner (A). Macrophages were treated with vehicle or Sorbinil (10 μM) in the absence or presence of AGA (500 μg/ml) for 24 h for TNF-α detection (B). Macrophages were further transfected with control or AKR1B3 siRNA (siAR) for 72 h followed by treatment with AGA (500 μg/ml) for 24 h for TNF-α detection (C) or 48 h for migration assay (D). The efficiency of AR knockdown was examined by probing with AR antibody (E). Secreted TNF-α was measured by using mouse TNF-α detection ELISA kit. The width of cell-free gap in (D) is 500 μm ± 50 μm. The migration assay was conducted by using wound-healing method. Data shown are means ± SEM (N = 3). *P < 0.05; **P < 0.01.

Fig. 2.. Effect of AR inhibition or knockdown on AGA-induced cytokine secretion and migration in RMG.

RMG cultures were established from C57BL/6 mice and immunostained by microglia marker Iba1 (green) and AR (red) merged with DAPI (blue) to visualize nuclei (A). RMG were treated with vehicle or Sorbinil (10 μM) in the absence or presence of AGA (500 μg/ml) for 24 h for TNF-α detection (B) or migration assay (C). Secreted TNF- α was measured by using mouse TNF-α detection ELISA kit. The migration assay was conducted by using transwell method. Data shown are means ± SEM (N = 3). *P < 0.05.

Fig. 3.. AR inhibition or knockdown prevents hypoxia-induced TNF- α and VEGF secretion.

RMG were treated with 10 μM Sorbinil (A and B) or isolated from ARKO mice (C and D) in normoxia or hypoxia (1% O₂) condition for 24 h. Secreted TNF-α (A and C) and VEGF (B and D) were measured ELISA kit. Data shown are means ± SEM (N = 3). *P, ^#P < 0.05.

Fig. 4.. Effect of AR on RMG activation in diabetic mice

WT-CX3CR1^GFP mice (A and B) and ARKO-CX3CR1^GFP mice (C and D) were injected with STZ (160 mg/kg body weight) for 6 weeks before sacrificed (B and D). Cryosections were obtained from each group and stained with DAPI (blue). RMG were shown in green color by expressing GFP. White arrows indicate migrated RMG in inner or outer nuclear layers. Statistic data was performed graphically (F). Data shown are means ± SEM (N ≥ 5). INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar 100 μm.