SRGAP1 Controls Small Rho GTPases To Regulate Podocyte Foot Process Maintenance

Manuel Rogg; Jasmin I. Maier; Robert Dotzauer; Nadine Artelt; Oliver Kretz; Martin Helmstädter; Ahmed Abed; Alena Sammarco; August Sigle; Dominik Sellung; Patrick Dinse; Karoline Reiche; Mako Yasuda-Yamahara; Martin L. Biniossek; Gerd Walz; Martin Werner; Nicole Endlich; Oliver Schilling; Tobias B. Huber; Christoph Schell

doi:10.1681/ASN.2020081126

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Visual Abstract

Significance Statement

Although the role of the podocyte cytoskeleton in the integrity of glomerular filtration is well established, the specific contribution of an upstream regulatory network comprising the Rho family of small GTPases—modulators of the actin cytoskeleton—is poorly characterized. The authors provide a comprehensive map of the podocyte Rho GTPase affinity interactome and identify a small GTPase-activating protein, SRGAP1, as a podocyte-specific RhoGAP. Through in vivo models of experimental FSGS, they demonstrate that SRGAP1 prevents podocyte foot-process effacement. They also show that SRGAP1 mediates spatial restriction of the activity of the Rho GTPase RAC1, thereby maintaining morphologic plasticity in disease conditions. These findings indicate that a comprehensive understanding of the regulatory networks of small Rho GTPases is needed for precisely targeted therapeutic interventions in glomerular diseases.

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. Srgap1^fl/fl*Six2Cre but not Srgap1^fl/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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