Background: Calcineurin inhibitors (CNIs), such as cyclosporine A (CsA), tacrolimus (TAC), and voclosporin (VCS), have been used to regulate the immune response in a number of conditions, including kidney transplantation, lupus nephritis and other autoimmune disorders. Whereas the immunosuppressive functions of CNIs are regulated by the calcium-calmodulin pathway, the mechanisms by which they control glomerular cell biology remain elusive. We previously showed that CsA treatment in LPS-induced proteinuria was associated with cytoskeletal stabilization of podocytes leading to preserved glomerular integrity. Methods: We combine network, enrichment and pathway analyses of transcriptomics, isobaric-tagged quantitative phospho- and total proteomics, atomic force microscope elastography and high content image analytics with classical cell biological assays to understand the role of CNIs in podocyte biology and biomechanics. Results: We discovered that podocyte physiology is significantly modulated by all CNIs, and that this regulation converged on the cytoskeletal architecture. Divergent phosphoproteomic signatures highlighted the differential impact of each drug in microtubule organization, post-translational modifications, and histone methylation. Downstream gene transcription showed a significant upregulation of focal adhesion molecules, GEF/GAPs and cytoskeletal-adhesion organizers. Our phoshoproteomic observations recapitulated several previously known targets, such as synaptopodin, and detected a number of novel cytoskeletal posttranslational modifications. Interestingly, we observed that at maximal clinically relevant concentrations lower than CsA and TAC, VCS affected these signaling pathways for enhancing cytoskeletal stability, which is confirmed via focal adhesion morphometrics, spreading area and cellular elasticity. Conclusion: Based on these findings, our study contributes significantly to the understanding of the role of CNIs in podocyte function, independently from other cell biological impacts, and it represents an integrative cellular and molecular atlas to guide the discovery of novel drug-induced modulators of glomerulopathies and evaluate the effect of new CNIs on podocyte biophysical and biomechanical features.

Integrative Systems Analyses Reveal Calcineurin Inhibitor-Mediated Control of Human Podocyte Biophysics and Physiology

Santini, Maria Paola
2024-01-01

Abstract

Background: Calcineurin inhibitors (CNIs), such as cyclosporine A (CsA), tacrolimus (TAC), and voclosporin (VCS), have been used to regulate the immune response in a number of conditions, including kidney transplantation, lupus nephritis and other autoimmune disorders. Whereas the immunosuppressive functions of CNIs are regulated by the calcium-calmodulin pathway, the mechanisms by which they control glomerular cell biology remain elusive. We previously showed that CsA treatment in LPS-induced proteinuria was associated with cytoskeletal stabilization of podocytes leading to preserved glomerular integrity. Methods: We combine network, enrichment and pathway analyses of transcriptomics, isobaric-tagged quantitative phospho- and total proteomics, atomic force microscope elastography and high content image analytics with classical cell biological assays to understand the role of CNIs in podocyte biology and biomechanics. Results: We discovered that podocyte physiology is significantly modulated by all CNIs, and that this regulation converged on the cytoskeletal architecture. Divergent phosphoproteomic signatures highlighted the differential impact of each drug in microtubule organization, post-translational modifications, and histone methylation. Downstream gene transcription showed a significant upregulation of focal adhesion molecules, GEF/GAPs and cytoskeletal-adhesion organizers. Our phoshoproteomic observations recapitulated several previously known targets, such as synaptopodin, and detected a number of novel cytoskeletal posttranslational modifications. Interestingly, we observed that at maximal clinically relevant concentrations lower than CsA and TAC, VCS affected these signaling pathways for enhancing cytoskeletal stability, which is confirmed via focal adhesion morphometrics, spreading area and cellular elasticity. Conclusion: Based on these findings, our study contributes significantly to the understanding of the role of CNIs in podocyte function, independently from other cell biological impacts, and it represents an integrative cellular and molecular atlas to guide the discovery of novel drug-induced modulators of glomerulopathies and evaluate the effect of new CNIs on podocyte biophysical and biomechanical features.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11571/1517017
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