Abstract: TH-PO361

Differential Effects of Two Nrf2 Inducers on Renal Tubule Cells

Session Information

Category: Cell Biology

  • 201 Cell Signaling, Oxidative Stress

Authors

  • King, Austin, University of Louisville, Louisville, Kentucky, United States
  • Wadhwa, Shruti, University of Louisville, Louisville, Kentucky, United States
  • Ghayoumi, Kayvon, University of Louisville, Louisville, Kentucky, United States
  • Isaacs, Susan M., University of Louisville, Louisville, Kentucky, United States
  • Merchant, Michael, University of Louisville Medicine, Louisville, Kentucky, United States
  • Rane, Madhavi J., University of Louisville, Louisville, Kentucky, United States
  • Barati, Michelle T., University of Louisville, Louisville, Kentucky, United States
Background

A variety of compounds inducing the cytoprotective transcription factor Nrf2 have shown promise to reduce renal injury in experimental murine models. However, comparison of mechanisms of action of these agents on renal cell biology is limited. This study examined effects two Nrf2 inducing compounds on renal tubule cells: Dimethyl fumarate (DMF), an FDA approved and clinically used drug, and Protandim, a dietary supplement with no previously reported findings in renal cells.

Methods

Human proximal tubule cells (HK11 cells) were treated with DMF (10-80μM) or Protandim (5-80μg/ml; comprised of Milk Thistle, Bacopa, Ashwagandha, turmeric, and green tea extracts) for various time points. Cell viability was analyzed by reduction of MTT and cell counting. Immunoblotting used to analyze Nrf2 expression/localization, phosphorylation of kinases known to regulate Nrf2 activation (Akt, GSK3β, ERK and p38 MAPK), and expression of Nrf2 transcriptional targets (NQO1 and SOD-1). Apoptosis was analyzed by immunostaining for cleaved caspase 3.

Results

MTT reduction and number of adherent cells was decreased with ≧40μM DMF and ≧40μg/ml Protandim, and DMF caused cells to round up while Protandim caused cell shrinkage, at these concentrations. The lowest concentration of Protandim (5μg/ml) and DMF (10μM) ) increased nuclear localization of Nrf2, indicative of Nrf2 activation. Both inducers increased expression of NQO1 at multiple concentrations whereas expression of SOD1 was induced more by DMF. Protandim caused a concentration-dependent increase in p38 phosphorylation. Neither compound altered Akt phosphorylation whereas phospho-ERK and GSK3β-Ser9 phosphorylation (inactive GSK3β) were increased by Protandim. High concentrations of Protandim (80μg/ml) increased caspase3 cleavage and nuclear condensation, indicating apoptosis, while 80μM DMF caused nuclear swelling.

Conclusion

Activation of p38 by Protandim may activate Nrf2 through direct phosphorylation and inhibition of GSK3β, a cellular Nrf2 inhibitor. Alternatively, p38 may play a role in high concentration Protandim-induced cell death. The results suggest that differential effects on tubule cell morphology, kinase signaling, and induction of Nrf2 transcriptional targets by DMF and Protandim may result in different tubule cell responses during stress stimuli or kidney injury.

Funding

  • NIDDK Support