Iron Overload Accelerates the Progression of Diabetic Retinopathy in Association with Increased Retinal Renin Expression

Figure 1. Iron accumulation in diabetic retina. (A) Quantification of ferritin levels in the (a) serum and (b) eye tissues of 12-week type 1 (STZ) and type 2 (db/db) diabetic mice by ELISA. (B) Heavy chain H-ferritin and light chain L-ferritin levels in the retinas of type 1 (STZ) and type 2 (db/db) diabetic mice models by western blot. Blots cropped from different parts of the same gel or from different gels are separated by white space. (C) Representative images of retinal sections from control vs type 1 (STZ) and type 2 (db/db) diabetic mice immunostained for (a) heavy chain H-ferritin and (b) light chain L-ferritin. (D) Labile iron (Fe2+) in retinal tissues of control vs type 1 (STZ) and type 2 (db/db) 12-week diabetic mice were detected using FeRhoNox-1 fluorescent imaging probe. (E) Representative images of retinal sections from normal and diabetic human patients (Diabetic retinopathy, DR) stained for labile iron (Fe2+) using FeRhoNox-1 fluorescent imaging probe. GCL-Ganglion Cell Layer, INL-Inner Nuclear Layer, ONL-Outer Nuclear Layer, RPE-Retinal Pigment Epithelium. *p < 0.05; **p < 0.001; ***p < 0.0001.

Figure 2. Ferritin expression is upregulated in the retina of diabetic HFE KO mice model of iron overload. (A) Western blot for Heavy chain (H-) and Light chain (L-) Ferritin in the retina of non-diabetic control and 16-week diabetic HFE WT and KO mice. β-actin was used as a loading control. Blots cropped from different parts of the same gel or from different gels are separated by white space. (B) ELISA for ferritin in the retina of non-diabetic and diabetic HFE WT and KO mice. *p < 0.05; **p < 0.001.

Figure 3. Altered outer and inner retinal barrier function in diabetic HFE KO mice. (A) Tight junction protein Zonula occludin-1 (ZO-1) immunofluorescence in retinal pigment epithelium (RPE) flat mounts prepared from non-diabetic control and 16-week post-diabetic HFE WT and KO mice. (B) Western blot analysis of ZO-1 expression in protein extracted from the RPE of non-diabetic and diabetic HFE WT and KO mice. β-actin was used as a loading control. Blots cropped from different parts of the same gel or from different gels are separated by white space. (C) FITC-Dextran (40-kD) trans epithelial permeability assay performed on confluent monolayers of primary RPE cells established from HFE WT and HFE KO mouse eyes grown in normal glucose (5.5 mM) or high glucose (25 mM). Data are presented as mean ± S.E. *p < 0.01. (D) Fluorescein angiography and fundoscopic imaging of non-diabetic control vs diabetic HFE WT and KO mice.

Figure 4. Accelerated neuronal cell loss and morphological changes in retina of diabetic HFE KO mice. (A) TUNEL assay in the eye sections of diabetic HFE WT and KO mice. Green signals indicate apoptotic cells. (B) Hematoxylin and eosin stained retinal sections from non-diabetic HFE WT and KO mice (top panel) compared to diabetic HFE WT (middle panel) and diabetic HFE KO mice (bottom panel) (C) Quantification of number of cells in ganglion cell layer in the central and peripheral regions of retinal sections from non-diabetic and diabetic HFE WT and KO mice. *p < 0.01.

Figure 5. Increased oxidative stress and NLRP3 inflammasome activation in retina of diabetic HFE KO mouse model of iron overload. (A) 4-Hydroxynonenal staining in non-diabetic control and diabetic HFE WT and KO mouse retina. (B) Expression levels of NLRP3 and IL-1ß mRNA in non-diabetic control and diabetic HFE WT and KO mouse retina by RT-PCR (C) Active caspase-1 levels detected using the FAM-FLICA Assay Kit (green). Data are presented as mean ± S.E. *p < 0.01, **p < 0.001.

Figure 6. Iron overload during diabetic retinopathy upregulates retinal renin expression in a GPR91 dependent mechanism. (A) Pro/renin mRNA quantified in retina of non-diabetic and diabetic HFE WT and HFE KO mice by RT PCR (B) Renin protein levels quantified in retina of non-diabetic and diabetic HFE WT and HFE KO mice by western blot. Blots cropped from different parts of the same gel or from different gels are separated by white space. (C) Lentivirus-mediated knockdown of GPR91 expression in ARPE-19 cells treated with two different GPR91-specific shRNAs34. GPR91 knockdown was quantified by RT-PCR with 18S RNA as an internal control. Control ARPE-19 cells (vector only) and GPR91 shRNA-expressing ARPE-19 cells were treated with 100 μg/mL FAC for 72 hours, and during the last 16 hours of this 72-hour FAC treatment, cells were incubated with or without 2 mM succinate. (D) Renin mRNA levels were analyzed using RT-PCR in RNA from control ARPE-19 cells (vector only) and GPR91 shRNA-expressing ARPE-19 cells with or without the treatment of FAC and succinate. 18S RNA was used as an internal control. Data presented as mean ± SE of three experiments. *p < 0.05; **p < 0.001; ***p < 0.0001.

Figure 7. Illustration of the pathways linking iron and renin angiotensin system in diabetic retina. A schematic of the signaling pathways involved in the induction of retinal renin angiotensin system (RAS) during diabetes mediated by iron overload, GPR91 and succinate.