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Kidney Week

Abstract: SA-PO173

Impact of HIF Stabilizer ICA on Nrf2/Keap1 Pathway in Macrophages

Session Information

  • AKI: Mechanisms - III
    November 04, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
    Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • Kurzhagen, Johanna T., Friedrich-Alexander-Universitat Erlangen-Nurnberg, Erlangen, Bayern, Germany
  • Schauer, Max, Friedrich-Alexander-Universitat Erlangen-Nurnberg, Erlangen, Bayern, Germany
  • Noel, Sanjeev, Johns Hopkins University, Baltimore, Maryland, United States
  • Rabb, Hamid, Johns Hopkins University, Baltimore, Maryland, United States
  • Willam, Carsten, Friedrich-Alexander-Universitat Erlangen-Nurnberg, Erlangen, Bayern, Germany
Background

Mice treated with HIF-stabilizer 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA) were protected from ischemia-reperfusion induced AKI, as were mice after treatment with Nrf2-inducer 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im). However, the potential interactions of HIF and Nrf2/Keap1 pathways have not been studied in this context.

Methods

Bone marrow was isolated from wildtype (WT) and HIF1α-KO mice and differentiated into bone marrow-derived macrophages (BMDM). Cells were cultured for 5 days before treatment with vehicle, CDDO-Im (50nM), ICA (250µM) or CDDO-Im+ICA (50nM+250µM) for 24 hours. Cells were harvested and mRNA expression of Nrf2- (Nqo1) and HIF1α-target genes (Hmox1, Glut1, Ldha) using quantitative real time PCR was analyzed. Protein expression of Keap1, Nqo1 and HIF1α was studied by Western blotting. Statistical analyses were performed using one-way ANOVA and Dunnett post hoc test.

Results

In BMDM from WT mice, treatment with CDDO-Im, ICA or the combination of both (CDDO-Im+ICA) resulted in an upregulation of Nqo1 mRNA expression (Vehicle: 1.3±0.2 vs. CDDO-Im: 4.4±1.3 (p<0.05); vs. ICA: 4.1±1.4 (p>0.05); vs. CDDO-Im+ICA: 4.4±1.5 (p<0.05)) and Hmox1 (Vehicle: 1.3±0.2 vs. CDDO-Im: 1.7±0.4 (p>0.05); vs. ICA: 5.0±1.3 (p>0.001); vs. CDDO-Im+ICA: 4.1±0.6 (p<0.001)). This effect was not reversed in HIF1α-KO cells for Nqo1 (Vehicle: 1.4±0.3 vs. CDDO-Im: 5.4±1.5 (p<0.001); vs. ICA: 3.9±0.1 (p<0.05); vs. CDDO-Im+ICA: 4.8±0.6 (p<0.01)). However, HIF1α-target genes Hmox1, Glut1 and Ldha were significantly upregulated by ICA treatment in WT mice, but not by CDDO-Im or vehicle treatment. Western blotting confirmed these results at the protein level with a decrease of Keap1 expression (Vehicle: 1 vs. ICA: 0.7±0.5) and an increase of Nqo1 expression (Vehicle: 1 vs. ICA: 1.3±0.4) in WT and HIF1α-KO mice.

Conclusion

These in vitro studies demonstrate an upregulation of the Nrf2-target gene Nqo1 following HIF-stabilizer, ICA, treatment. However, this effect was Hif1α independent. These results indicate a potential overlap of the two pathways HIF and Nrf2/Keap1 playing important roles in ischemic AKI. In vivo studies in AKI-models are warranted to assess the impact on these dual effects.

Funding

  • Government Support – Non-U.S.