Figure 4. Promoting HIF-1 function attenuates renal ROS excess and mitochondrial respiration in mouse models of diabetes.

Kidneys were harvested from wild-type (WT) and Lepr^db/db diabetic mice (db/db) that were treated with placebo (vehicle) or DMOG (A–B, E, G), and from non-diabetic control (Ctrl) or diabetic (Db) wild-type (WT) and Egln1^+/- mice (C–D, F, H). (A and C) HIF-1α (green), pimonidazole (red, hypoxia marker) and DAPI (blue, nuclear staining) signals were detected by fluorescent immunohistochemistry, and relative HIF-1α expression levels were quantified (A, n = 4–5; C, n = 4–6). Scale bar: 100 μm. (B and D) Renal ROS levels were detected using the OxiSelect HNE adduct competitive ELISA kit (B, n = 7–10; D, n = 5–8). (E and F) Mitochondrial respiratory function was evaluated using high resolution respirometry (E, n = 4–9; F, n = 11–17). (G and H) PDK1 gene expression in kidneys (G, n = 4–9; H, n = 4–6). (I and J) mIMCD-3 cells were transfected with plasmids encoding GFP or GFP-PDK1,and exposed to hypoxia and 30 mM glucose (H30) for 24 hr. (I) Expression of GFP and GFP-HIF-1α (green) and nuclear DAPI staining (blue) were detected using confocal microscopy. Scale bar: 50 μm. (J) Mitochondrial ROS levels are shown (n = 4). Data are shown as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 using one-way ANOVA (A, B, E, G) and two-way ANOVA (C, D, F, H) followed by multi-comparison post hoc tests, and unpaired two-sided Student t-test (J). This figure has three figure supplements. Source data are shown in Figure 4—source data 1.

Figure 4—figure supplement 1. Kidney in diabetes is more hypoxic.

Pimonidazole (60 mg/kg body weight) was i.p. administered to mice 90 min prior to tissue harvest from wild-type (WT) and Lepr^db/db diabetic mice (db/db) that were treated with placebo (vehicle) or DMOG (A), and from non-diabetic control (Ctrl) or diabetic (Db) wild-type (WT) and Egln1^+/- mice (B). Pimonidazole adducts were detected on kidney sections using fluorescent immunohistochemistry and fold induction of pimonidazole signal is shown. *, p < 0.05 using one-way ANOVA (A) and two-way ANOVA (B) followed by multi-comparison post hoc tests. Source data are shown in Figure 4—figure supplement 1—source data 1.

Figure 4—figure supplement 2. DMOG increases HIF-1 target gene expression in Lepr^db/db mice without affecting blood glucose levels.

Lepr^db/db diabetic mice (db/db) were treated with placebo (vehicle) or DMOG (50 mg / kg) for 4 weeks. (A) There was no difference in blood glucose in Lepr^db/db mice treated with placebo or DMOG (n = 7). (B) QPCR results demonstrate that DMOG increased the gene expression of HIF-1 target genes (n = 4–5). Data are shown as mean ± SEM. ns = not significant. **, p < 0.01; ****, p < 0.0001 analysed using unpaired two-sided Student’s t-test. Source data are shown in Figure 4—figure supplement 2—source data 1.

Figure 4—figure supplement 3. Egln1 haplodeficiency increases HIF-1 target gene expression in diabetic mice without affecting blood glucose levels.

Egln1 haplodeficient (Egln1^+/-, HZ) and corresponding Wild-type (WT) mice were induced diabetes using STZ. HbA1c (A) and gene expression (B) of Egln1 and HIF-1 target gene GLUT3 were assessed in non-diabetic control and diabetic WT and Egln1^+/- mice (n = 4–8). Data are shown as mean ± SEM, and were analyzed using unpaired two-sided Student’s t-test (A) and two-way ANOVA followed by Bonferroni’s post hoc test (B). ns = not significant; *, p < 0.05; ****, p < 0.0001. Source data are shown in Figure 4—figure supplement 3—source data 1.