Manuel Rogg1,2, Jasmin I Maier1, Robert Dotzauer2, Nadine Artelt3, Oliver Kretz4, Martin Helmstädter2, Ahmed Abed2, Alena Sammarco1, August Sigle2,5, Dominik Sellung2,6, Patrick Dinse2, Karoline Reiche1, Mako Yasuda-Yamahara2,7, Martin L Biniossek8, Gerd Walz2, Martin Werner1, Nicole Endlich3, Oliver Schilling1, Tobias B Huber9, Christoph Schell10,11. 1. Institute of Surgical Pathology, Faculty of Medicine, Medical Center - University of Freiburg, Freiburg, Germany. 2. Department of Medicine IV, Faculty of Medicine, Medical Center - University of Freiburg, Freiburg, Germany. 3. Department of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany. 4. III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. 5. Department of Urology, Faculty of Medicine, Medical Center - University of Freiburg, Freiburg, Germany. 6. Department of Neurology, Heimer Institute for Muscle Research, University Hospital Bergmannsheil, Ruhr-University Bochum, Bochum, Germany. 7. Department of Medicine, Shiga University of Medical Science, Otsu, Japan. 8. Institute of Molecular Medicine and Cell Research, Faculty of Medicine, Medical Center - University of Freiburg, Freiburg, Germany. 9. III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany christoph.schell@uniklinik-freiburg.de t.huber@uke.de. 10. Institute of Surgical Pathology, Faculty of Medicine, Medical Center - University of Freiburg, Freiburg, Germany christoph.schell@uniklinik-freiburg.de t.huber@uke.de. 11. Berta-Ottenstein Program, Medical Faculty, Medical Center - University of Freiburg, Freiburg, Germany.
Abstract
BACKGROUND: Previous research demonstrated that small Rho GTPases, modulators of the actin cytoskeleton, are drivers of podocyte foot-process effacement in glomerular diseases, such as FSGS. However, a comprehensive understanding of the regulatory networks of small Rho GTPases in podocytes is lacking. METHODS: We conducted an analysis of podocyte transcriptome and proteome datasets for Rho GTPases; mapped in vivo, podocyte-specific Rho GTPase affinity networks; and examined conditional knockout mice and murine disease models targeting Srgap1. To evaluate podocyte foot-process morphology, we used super-resolution microscopy and electron microscopy; in situ proximity ligation assays were used to determine the subcellular localization of the small GTPase-activating protein SRGAP1. We performed functional analysis of CRISPR/Cas9-generated SRGAP1 knockout podocytes in two-dimensional and three-dimensional cultures and quantitative interaction proteomics. RESULTS: We demonstrated SRGAP1 localization to podocyte foot processes in vivo and to cellular protrusions in vitro. Srgap1fl/fl*Six2Cre but not Srgap1fl/fl*hNPHS2Cre knockout mice developed an FSGS-like phenotype at adulthood. Podocyte-specific deletion of Srgap1 by hNPHS2Cre resulted in increased susceptibility to doxorubicin-induced nephropathy. Detailed analysis demonstrated significant effacement of podocyte foot processes. Furthermore, SRGAP1-knockout podocytes showed excessive protrusion formation and disinhibition of the small Rho GTPase machinery in vitro. Evaluation of a SRGAP1-dependent interactome revealed the involvement of SRGAP1 with protrusive and contractile actin networks. Analysis of glomerular biopsy specimens translated these findings toward human disease by displaying a pronounced redistribution of SRGAP1 in FSGS. CONCLUSIONS: SRGAP1, a podocyte-specific RhoGAP, controls podocyte foot-process architecture by limiting the activity of protrusive, branched actin networks. Therefore, elucidating the complex regulatory small Rho GTPase affinity network points to novel targets for potentially precise intervention in glomerular diseases.
BACKGROUND: Previous research demonstrated that small Rho GTPases, modulators of the actin cytoskeleton, are drivers of podocyte foot-process effacement in glomerular diseases, such as FSGS. However, a comprehensive understanding of the regulatory networks of small Rho GTPases in podocytes is lacking. METHODS: We conducted an analysis of podocyte transcriptome and proteome datasets for Rho GTPases; mapped in vivo, podocyte-specific Rho GTPase affinity networks; and examined conditional knockout mice and murine disease models targeting Srgap1. To evaluate podocyte foot-process morphology, we used super-resolution microscopy and electron microscopy; in situ proximity ligation assays were used to determine the subcellular localization of the small GTPase-activating protein SRGAP1. We performed functional analysis of CRISPR/Cas9-generated SRGAP1 knockout podocytes in two-dimensional and three-dimensional cultures and quantitative interaction proteomics. RESULTS: We demonstrated SRGAP1 localization to podocyte foot processes in vivo and to cellular protrusions in vitro. Srgap1fl/fl*Six2Cre but not Srgap1fl/fl*hNPHS2Cre knockout mice developed an FSGS-like phenotype at adulthood. Podocyte-specific deletion of Srgap1 by hNPHS2Cre resulted in increased susceptibility to doxorubicin-induced nephropathy. Detailed analysis demonstrated significant effacement of podocyte foot processes. Furthermore, SRGAP1-knockout podocytes showed excessive protrusion formation and disinhibition of the small Rho GTPase machinery in vitro. Evaluation of a SRGAP1-dependent interactome revealed the involvement of SRGAP1 with protrusive and contractile actin networks. Analysis of glomerular biopsy specimens translated these findings toward human disease by displaying a pronounced redistribution of SRGAP1 in FSGS. CONCLUSIONS: SRGAP1, a podocyte-specific RhoGAP, controls podocyte foot-process architecture by limiting the activity of protrusive, branched actin networks. Therefore, elucidating the complex regulatory small Rho GTPase affinity network points to novel targets for potentially precise intervention in glomerular diseases.
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