Podocyte Integrin-β 3 and Activated Protein C Coordinately Restrict RhoA Signaling and Ameliorate Diabetic Nephropathy

Journal of the American Society of Nephrology, vol. 31, pp. 1762–1780

Abstract

Significance Statement Signaling to integrins is complex and depends on ligands and their binding sites. Signaling-competent integrin ligands that protect podocyte function remain unknown. This study demonstrates that the coagulation protease-activated protein C (aPC) binds via its RGD sequence to podocyte integrin- β 3 . Disruption of the aPC–integrin- β 3 interaction results in excess RhoA activation and podocyte dysfunction. These findings identify the RGD-mediated aPC–integrin- β 3 interaction as a rheostat of RhoA signaling, which is disrupted in diabetic nephropathy. Protease-activated receptor 1 (PAR1) antagonism could ameliorate excess RhoA signaling in the absence of aPC–integrin- β 3 interaction. These data identify a new function of podocyte integrin- β 3 and provide a mechanistic rationale for PAR antagonism as a therapeutic approach for diabetic nephropathy. Background Diabetic nephropathy (dNP), now the leading cause of ESKD, lacks efficient therapies. Coagulation protease–dependent signaling modulates dNP, in part via the G protein–coupled, protease-activated receptors (PARs). Specifically, the cytoprotective protease-activated protein C (aPC) protects from dNP, but the mechanisms are not clear. Methods A combination of in vitro approaches and mouse models evaluated the role of aPC-integrin interaction and related signaling in dNP. Results The zymogen protein C and aPC bind to podocyte integrin- β 3 , a subunit of integrin- α v β 3 . Deficiency of this integrin impairs thrombin-mediated generation of aPC on podocytes. The interaction of aPC with integrin- α v β 3 induces transient binding of integrin- β 3 with G α13 and controls PAR-dependent RhoA signaling in podocytes. Binding of aPC to integrin- β 3 via its RGD sequence is required for the temporal restriction of RhoA signaling in podocytes. In podocytes lacking integrin- β 3 , aPC induces sustained RhoA activation, mimicking the effect of thrombin. In vivo , overexpression of wild-type aPC suppresses pathologic renal RhoA activation and protects against dNP. Disrupting the aPC–integrin- β 3 interaction by specifically deleting podocyte integrin- β 3 or by abolishing aPC’s integrin-binding RGD sequence enhances RhoA signaling in mice with high aPC levels and abolishes aPC’s nephroprotective effect. Pharmacologic inhibition of PAR1, the pivotal thrombin receptor, restricts RhoA activation and nephroprotects RGE-aPC high and wild-type mice. Conclusions aPC–integrin- α v β 3 acts as a rheostat, controlling PAR1-dependent RhoA activation in podocytes in diabetic nephropathy. These results identify integrin- α v β 3 as an essential coreceptor for aPC that is required for nephroprotective aPC-PAR signaling in dNP.

Authors 23

1. Thati Madhusudhan corresponding

   University Medical Center of the Johannes Gutenberg University Mainz · Otto-von-Guericke-Universität Magdeburg

   Affiliation as printed

   Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany

   Institute of Clinical Chemistry and Pathobiochemistry, Otto von Guericke University Magdeburg, Magdeburg, Germany

2. Sanchita Purohit Ghosh

   University Hospital Leipzig · Leipzig University · Otto-von-Guericke-Universität Magdeburg

   Affiliation as printed

   Institute of Clinical Chemistry and Pathobiochemistry, Otto von Guericke University Magdeburg, Magdeburg, Germany

   Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany

3. Wei Dong

   Otto-von-Guericke-Universität Magdeburg

   Affiliation as printed

   Institute of Clinical Chemistry and Pathobiochemistry, Otto von Guericke University Magdeburg, Magdeburg, Germany

4. Dheerendra Gupta

   University Hospital Leipzig · Leipzig University · Otto-von-Guericke-Universität Magdeburg

   Affiliation as printed

   Institute of Clinical Chemistry and Pathobiochemistry, Otto von Guericke University Magdeburg, Magdeburg, Germany

   Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany

5. Ahmed Elwakiel

   University Hospital Leipzig · Leipzig University · Otto-von-Guericke-Universität Magdeburg

   Affiliation as printed

   Institute of Clinical Chemistry and Pathobiochemistry, Otto von Guericke University Magdeburg, Magdeburg, Germany

   Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany

6. Stoyan Stoyanov

   Otto-von-Guericke-Universität Magdeburg

   Affiliation as printed

   German Center for Neurodegenerative Diseases, Otto von Guericke University Magdeburg, Magdeburg, Germany

7. Moh’d Mohanad Al‐Dabet

   American University of Madaba · University Hospital Leipzig · Leipzig University · Otto-von-Guericke-Universität Magdeburg

   Affiliation as printed

   Department of Medical Laboratories, Faculty of Health Sciences, American University of Madaba, Amman, Jordan

   Institute of Clinical Chemistry and Pathobiochemistry, Otto von Guericke University Magdeburg, Magdeburg, Germany

   Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany

8. Shruthi Krishnan

   University Hospital Leipzig · Leipzig University · Otto-von-Guericke-Universität Magdeburg

   Affiliation as printed

   Institute of Clinical Chemistry and Pathobiochemistry, Otto von Guericke University Magdeburg, Magdeburg, Germany

   Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany

9. Ronald Biemann

   University Hospital Leipzig · Leipzig University · Otto-von-Guericke-Universität Magdeburg

   Affiliation as printed

   Institute of Clinical Chemistry and Pathobiochemistry, Otto von Guericke University Magdeburg, Magdeburg, Germany

   Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany

10. Sumra Nazir

    Otto-von-Guericke-Universität Magdeburg

    Affiliation as printed

    Institute of Clinical Chemistry and Pathobiochemistry, Otto von Guericke University Magdeburg, Magdeburg, Germany

11. Silke Zimmermann

    University Hospital Leipzig · Leipzig University · Otto-von-Guericke-Universität Magdeburg

    Affiliation as printed

    Institute of Clinical Chemistry and Pathobiochemistry, Otto von Guericke University Magdeburg, Magdeburg, Germany

    Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany

12. Akash Mathew

    University Hospital Leipzig · Leipzig University · Otto-von-Guericke-Universität Magdeburg

    Affiliation as printed

    Institute of Clinical Chemistry and Pathobiochemistry, Otto von Guericke University Magdeburg, Magdeburg, Germany

    Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany

13. Ihsan Gadi

    University Hospital Leipzig · Leipzig University · Otto-von-Guericke-Universität Magdeburg

    Affiliation as printed

    Institute of Clinical Chemistry and Pathobiochemistry, Otto von Guericke University Magdeburg, Magdeburg, Germany

    Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany

14. Rajiv Rana

    University Hospital Leipzig · Leipzig University · Otto-von-Guericke-Universität Magdeburg

    Affiliation as printed

    Institute of Clinical Chemistry and Pathobiochemistry, Otto von Guericke University Magdeburg, Magdeburg, Germany

    Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany

15. Jinyang Zeng-Brouwers

    Goethe University Frankfurt · University Hospital Frankfurt

    Affiliation as printed

    Institute of Pharmacology, University Hospital and Goethe University, Frankfurt, Germany

16. Marcus Johannes Moeller Aachen Division of Nephrology and Immunology

    Universitätsklinikum Aachen · RWTH Aachen University

    Affiliation as printed

    Division of Nephrology and Immunology, University Hospital of the Rheinisch-Westfälische Technische Hochschule, Aachen University of Technology, Aachen, Germany

17. Liliana Schaefer

    Goethe University Frankfurt · University Hospital Frankfurt

    Affiliation as printed

    Institute of Pharmacology, University Hospital and Goethe University, Frankfurt, Germany

18. Charles T. Esmon

    Oklahoma Medical Research Foundation · University of Oklahoma Health Sciences Center

    Affiliation as printed

    Coagulation Biology Laboratory, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma

19. Shrey Kohli

    University Hospital Leipzig · Leipzig University · Otto-von-Guericke-Universität Magdeburg

    Affiliation as printed

    Institute of Clinical Chemistry and Pathobiochemistry, Otto von Guericke University Magdeburg, Magdeburg, Germany

    Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany

20. Jochen Reiser

    Rush University Medical Center

    Affiliation as printed

    Department of Medicine, Rush University Medical Center, Chicago, Illinois

21. Alireza R. Rezaie

    Oklahoma Medical Research Foundation · University of Oklahoma Health Sciences Center

    Affiliation as printed

    Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma

22. Wolfram Ruf

    Scripps Research Institute · University Medical Center of the Johannes Gutenberg University Mainz

    Affiliation as printed

    Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany

    Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, California

23. Berend Isermann corresponding

    University Hospital Leipzig · Leipzig University · Otto-von-Guericke-Universität Magdeburg

    Affiliation as printed

    Institute of Clinical Chemistry and Pathobiochemistry, Otto von Guericke University Magdeburg, Magdeburg, Germany

    Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany