Characterization of Rho-Family of Small GTPases Interactome in Podocytes

Background: Podocyte injury is the major cause of proteinuria, a hallmark of kidney diseases and an important risk factor for progression to kidney failure. Rho GTPases play critical roles in podocyte cytoskeleton regulation and their alteration leads to foot process effacement. However, their regulatory mechanisms and signaling networks are largely unknown. Here, we investigated the interactomes of RhoA, Rac1, and Cdc42 in podocytes. Methods: We defined Rho GTPases interactomes in human podocytes (HP) using the proximity-dependent biotinylation assay (BioID). We generated RhoA guanine nucleotide exchange factors (GEFs) KO lines by CRISPR/Cas9 and KIAA1522 KD zebrafish by morpholinos. We used CRISPR/Cas9 to generate systemic Arhgef6 KO mice then adriamycin and LPS injections to induce FSGS phenotype. Results: Network analysis revealed that ~50% of the interactions are unique to podocytes while enrichment analyses highlighted biological processes related to cell adhesion and cell shape organization. BioID revealed a large panel of potential effectors, including the uncharacterized protein KIAA1522. We found that KIAA1522 translocates to filopodia in response to Cdc42 activation. KIAA1522 KD in Zebrafish induced pericardial effusion, similar to Nephrin KD. BioID also identified 20 GEFs, eleven of which interacted with RhoA. Analyses of public scRNAseq datasets showed that RhoA regulators are highly enriched in primary human podocytes. KO of the majority of identified RhoA-GEFs reduced podocyte migration, cell size and basal RhoA activity, with ARHGEF12 KO having the highest impact on RhoA activity. In addition, BioID detected ARHGEF6 as a Rac1-specific GEF. Interrogation of public transcriptomic datasets showed that Arhgef6 expression is low in healthy kidneys and increases in glomeruli with FSGS. uACR analysis showed that Arhgef6 KO mice are protected against proteinuria 5 weeks post-ADR and 24h post-LPS injections. KO mice were also resistant to ADR-induced weight restriction. Conclusion: We dissected for the first time the upstream regulators and downstream effectors of Rho GTPases in podocytes. We identified KIAA1522 as a novel Cdc42 effector. We identified RhoA as a predominant Rho GTPase in podocytes and uncovered its key regulators. Our findings suggest that increased ARHGEF6 expression might be implicated in FSGS progression. Funding: Government Support – Non-U.S.