Figure 5

Nrf2 target genes are downregulated after injury but this is improved in Keap1 hypomorphs.

We assessed Keap1, Nrf2, and Nrf2 targets in the IRI mice used for this study. (a) As expected, mRNA levels of Keap1 were consistently decreased at baseline in CTL kidneys in the hypomorphic mice compared to WT mice at all timepoints. At 3 and 10 days post-IRI, there was a decrease in Keap1 expression in all injured kidneys but the hypomorphs were consistently lower than the WT kidneys. Brackets indicate significant differences. (n = 4) (b) Nrf2 mRNA generally showed significant differences between injured and uninjured kidneys, but generally did not show significant differences between similarly treated WT and KEAP kidneys. (n = 4) (c–e) Gene expression levels of GSTM1, GSTP1, and NQO1 were assessed. Brackets indicate groups with significant differences (n = 4–6 for each group). Comparing the uninjured control kidneys (CTL), Keap1 hypomorphs (KEAP1) had higher expression levels compared to wild type (WT) mice. Injury led to suppression of gene expression in all animals. However, this reduction was blunted in the hypomorphs and in general these mice had levels of gene expression that were either closer to, or much higher than, the WT CTL group. Western blots for NQO1 were performed for the wild type mice (f) and hypomorphs (g) over the course of IRI injury. Identical samples (WT CTL) were loaded into lanes 1 and 2 of both gels to facilitate comparison of all the samples over two gels. Densitometry of these bands (n = 3) normalized to GAPDH is shown in (h) and these values were in turn normalized to the samples in lanes 1 and 2. Additional genes were assessed in the 10 day IRI kidneys and revealed increased expression of protective antioxidant genes catalase and the three SODs (i–l). (*P < 0.05 compared to WT group. n = 5–6).