Figure 4.

Upregulation of the Nrf2-Mediated Antioxidant Response Pathway in FH-Deficient Cells and Tumors

(A) Heat map comparing the patterns of expression for 14 Nrf2 pathway genes for control, Fh1^−/−Hif-1α^−/−, and Fh1^−/− mouse kidneys (age = 15 weeks, n = 4 per group). Red and green indicate up- or downregulation, respectively. The Heatmap was generated using R (Ihaka and Gentleman, 1996) with differentially regulated genes specific to the Nrf2 pathway.

(B) Q-PCR validation of a subset of genes in control, Fh1^−/−, and Fh1^−/−Hif-1α^−/− mouse kidneys using the same template mRNA as for microarray analysis and compared with renal tissue from Fh1^−/−Hif-2α^−/− mice confirms significant reduction of mRNA for Fh1 and Hif-1α as expected and upregulation of the Nrf2 target genes Gsta1, Hmox1, and Nqo1 in Fh1^−/−, Fh1^−/−Hif-1α^−/−, and Fh1^−/−Hif-2α^−/− mouse kidneys. Error bars indicate ± 1 SD calculated from three biological replicates, each assayed in duplicate; p < 0.02 (students t test). Increased expression of the Hif-1α target gene Pdk1 in Fh1^−/− kidneys is ameliorated by Hif-1α deletion and to a lesser extent by Hif-2α deletion.

(C) IHC for Fh1, Hif-1α, Nrf2, and Nqo1 was performed on kidney sections from 17-week-old control, Fh1^−/−, and Fh1^−/− Hif-1α^−/− mice. In contrast to the ubiquitous expression of Fh1 in the controls, Fh1 is deleted in cysts of both Fh1^−/− and Fh1^−/−Hif-1α^−/− mice, but is retained in the interstitium and in a proportion of the renal tubules. Hif-1α is stabilized in the nuclei of cells lining the cysts in Fh1^−/− kidneys, while this staining is absent in the control tissue and Fh1^−/−Hif-1α^−/− kidneys. Renal cysts from both these groups show increased nuclear expression of Nrf2 and Nqo1 compared with the interstitium and most non-cystic tubules and with the control; scale bar = 100 μm.

(D) H&E staining and IHC for NRF2 and NQO1 in pRCC shows strong staining for both in the tumor cells exclusively and not the stroma; scale bar = 100 μm.

See also Tables S1 and S2.