Figure 4.

The binding of HuR to Nox4 mRNA is increased in kidneys from type 1 diabetic animals. (A) HuR and Nox4 protein levels were increased in kidneys from STZ-induced mice. Kidneys from control and STZ-induced mice were isolated and total protein was subjected to western blot, and GAPDH was used as a loading control. Representative images were shown here for two mice in each group. (B) HuR staining in kidneys was increased in STZ-induced mice. Paraffin-embedded sections of kidneys from controls or STZ-induced mice were prepared and subjected to standard immuno-histochemistry staining for HuR (scale bar, 60 mm). The intensities of HuR were quantified by Image J software, and 3 mice were quantified for each group. (C) HuR protein levels were increased in diabetic OVE26 mice as compared to control FVB mice. The kidney cortex from 3-month old OVE26 and control FVB mice were prepared and the expression of HuR was assessed by western blotting assay from these cortical lysates. (D) Nox4 RNA levels in the HuR IP complex were increased in diabetic mice, as compared to the ones in control animals. Kidney cortex lysates were prepared from control and STZ-induced mice, and immunoprecipitated by the HuR antibody. Then the presence of Nox4 mRNA in these IP complexes was assessed by RT-PCR (upper panel). Histogram showed the quantification against the trace amount presented in the total IgG complex (bottom panel). (E) Representative images showed HuR and Nox4 staining in paraffin embedded sections from healthy human and DN patients (scale bar, 60 μm). (F) The intensities of HuR and Nox4 staining were quantified by Image J and normalized to the lowest expression level sample. The plot showed the positive correlation between HuR (x-axis) and Nox4 staining (y-axis). All data are presented as the mean ± S.E of three repeats. ∗, p < 0.05; ∗∗, p < 0.01 in comparison with control or as indicated. The Student's t-test was used to compare two groups; and one-way ANOVA was used for comparison between multiple groups.