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Abstract: PO0944

Can Nrf2 Inducers Cause Renal Proximal Tubule Epithelial Cell De-Differentiation?

Session Information

Category: Diabetic Kidney Disease

  • 601 Diabetic Kidney Disease: Basic

Authors

  • Eskridge, Jessica, University of Louisville, Louisville, Kentucky, United States
  • Isaacs, Susan M., University of Louisville, 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

Nrf2 is a transcription factor serving as a master regulator of cytoprotective responses and Nrf2-inducing agents have been shown to attenuate renal injury in animal models of kidney disease. However, use of these agents in the clinical setting are limited and defining/comparing mechanisms of action of different agents remains to be defined. Previous lab studies showed two Nrf2 inducers (Protandim [nutritional supplement] and DMF [Dimethyl fumarate]) caused differing effects on proximal tubule cell shape, actin and tubulin cytoskeleton, junction proteins, and extracellular matrix (ECM) secretion. Some of these responses were similar to tubule cell changes during epithelial to mesenchymal transition (EMT) and diabetes. This study tested the hypothesis that the combined effect of high glucose and DMF or Protandim may cause tubule cell de-differentiation.

Methods

Human proximal tubule cells (HK-11 cells) were cultured in high glucose (HG; 25mM) or normal glucose concentrations (NG; 5mM) for 24h, followed by additional treatment with 5µg/ml Protandim or 10µM DMF for another 24h. To test markers of EMT and cytoskeleton, cells were immuno-stained for vimentin and fibronectin, and actin filaments stained with FITC-phalloidin. Cell extracts were immunoblotted for E-cadherin and vimentin.

Results

Protandim and DMF increased vimentin filaments by image analysis of immunostained cells, and HG+DMF conditions led to a further increase. Treatment with Protandim following culture in both HG and NG caused collapse of actin filaments (as previously observed) and vimentin encapsulated and co-localized with the collapsed actin. The cell-cell junction protein E-cadherin was downregulated by DMF and culture in HG prior to DMF was not additive to this effect. Protandim did not alter E-Cadherin expression. Extracellularly deposited fibronectin increased with HG and this effect was augmented by additional treatment with DMF.

Conclusion

Protandim and DMF distinctively regulate the cell cytoskeleton. Increased vimentin filament formation with Protandim may be a compensatory mechanism due to collapse of actin filaments. DMF decreases E-Cadherin and increases vimentin and ECM deposition, suggestive of tubule cell de-differentiation. The disparate effects of these two Nrf2 inducers may lead to varying outcomes if used for treatment of diabetic or other types of kidney disease.

Funding

  • NIDDK Support