Kidney Week

Abstract: SA-PO086

Identification of Small Molecule Inducers of Heme Oxygenase-1 (HO-1) in Renal Epithelial Cells

Session Information

• AKI: Mechanisms - Primary Injury and Repair - II
  November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

• 103 AKI: Mechanisms

Authors

• Traylor, Amie, University of Alabama at Birmingham, Mountain Brk, Alabama, United States

Amie Traylor,

• Mathew, Bini, Southern Research, Birmingham, Alabama, United States

Bini Mathew,

• Bostwick, James Robert, Southern Research, Birmingham, Alabama, United States

James Robert Bostwick,

• Nebane-Ngwa, Ntsang Miranda, Southern Research, Birmingham, Alabama, United States

Ntsang Miranda Nebane-Ngwa,

• George, James F., University of Alabama at Birmingham, Mountain Brk, Alabama, United States

James F. George,

• Esman, Stephanie, University of Alabama at Birmingham, Mountain Brk, Alabama, United States

Stephanie Esman,

• Zhai, Ling, Southern Research, Birmingham, Alabama, United States

Ling Zhai,

• Suto, Mark J., Southern Research, Birmingham, Alabama, United States

Mark J. Suto,

• Agarwal, Anupam, University of Alabama at Birmingham, Mountain Brk, Alabama, United States

Anupam Agarwal,

Background

Acute kidney injury (AKI) is a major public health concern that is associated with increased morbidity and mortality in hospitalized patients. Furthermore, the rising cost of care for these patients presents an increasing economic burden. While several promising biomarkers are emerging to aid in the early identification of AKI, no new therapies have succeeded in clinical trials. Currently, the only FDA approved treatment for AKI is dialysis. Recent studies from our laboratory and others have demonstrated the beneficial effects of HO-1, an enzyme that catalyzes heme breakdown into biliverdin, carbon monoxide and iron, in animal models of AKI.

Methods

We designed an assay suitable for high throughput screening to assist in the identification of novel small molecule targets that both induce HO-1 and have desirable pharmacologic characteristics. We created a stable HEK293 cell line that contains portions of the human HO-1 promoter and a unique 220 bp enhancer sequence in a luciferase reporter vector to screen a library of >150,000 compounds.

Results

We identified 2240 candidate compounds in the initial screen. Based on chemical structure, we pared these down to 800. Compounds exhibiting E_max ≥ 70% of 5uM hemin and EC₅₀< 10mM were assayed for endogenous HO-1 expression in HEK293 cells. The screen was repeated on a library of >4,000 FDA approved compounds and several additional candidates were identified, including broxaldine, an antiprotozoal drug. At low micromolar concentrations (1-5uM), broxaldine induced markedly high expression of HO-1 mRNA, protein and enzyme activity. Using RNA seq, the transcription factor, Nrf2 was identified as a target for broxaldine induced HO-1 expression, which was confirmed using siRNA experiments. We further assessed the potential for broxaldine to inhibit cisplatin-induced cytotoxicity in HEK293 cells. Pretreatment with broxaldine preserved cell viability and reduced cleaved caspase-3 expression.

Conclusion

These studies provide a methodology for the identification of compounds that are clinically relevant and efficacious in inducing HO-1, and therefore confer protection against a variety of pathologies, including AKI.

Funding

• NIDDK Support