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Abstract: SA-PO010

Integrative Systems Analysis of Calcineurin Inhibitor Action on Podocytes and Proximal Tubular Epithelial Cells

Session Information

Category: Bioengineering

  • 400 Bioengineering

Authors

  • Mendoza, Anthony, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Santini, Maria Paola, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Wong, Jenny, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Rehaume, Linda M., Aurinia Pharmaceuticals Inc, Victoria, British Columbia, Canada
  • Viel, John, Aurinia Pharmaceuticals Inc, Victoria, British Columbia, Canada
  • Campbell, Kirk N., Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Azeloglu, Evren U., Icahn School of Medicine at Mount Sinai, New York, New York, United States
Background

Calcineurin inhibitors (CNI), including Cyclosporin A (CSA), Tacrolimus (TAC) and Voclosporin (VCS) are used to treat proteinuric kidney disease. The mechanisms driving differential podocyte and tubular cell responses upon clinical exposure are poorly understood. Here, we tested the in vitro cellular response of these three most commonly used CNIs using a combination of integrated systems biology methods.

Methods

We used high content image analysis to assess the changes in cytoskeletal and focal adhesion architecture; isobaric-tagged LC-MS/MS to assess altered proteomic response; image-based and colorimetric assays to assess viability and metabolic response. For all above assays, immortalized human podocytes and proximal tubular epithelial cells were treated with increasing doses of CSA, TAC and VCS. Respective drug dose responses were mapped back to the translational plasma concentrations as measured in the associated clinical trial.

Results

All CNIs were well tolerated by immortalized human podocytes with minimal change in viability. Immortalized human tubular epithelial cells also showed little change in cell viability with high levels of cell death only in the highest doses of TAC. Morphometric changes were similar in CNIs, with increased focal adhesion area and aspect ratio. Levels of remodeling for all CNIs mirrored their respective calcineurin IC50 values with VCS showing a more robust dose response for both altered focal complex signaling and cell spreading. Quantitative proteomics at the physiologically relevant drug doses recapitulated the altered focal adhesion complex signatures for all CNIs. While network analyses of the differentially abundant podocyte adhesome showed a core CNI proteomic signature, this was supplemented by differing peripheral drug-specific networks that showed different neighborhoods for each CNI.

Conclusion

While integrated viability, proteomic and high-content morphometric analyses show a common CNI-induced signature within human proximal tubular epithelial cells and podocytes, differences were observed among CSA, TAC and VCS for both differentially abundant proteins and drug dose responses. Consistent adhesome and cytoskeletal changes may point to a conserved biophysical role of calcineurin in podocytes with differences in off-target effects.

Funding

  • Commercial Support – Aurinia