Tuesday, May 13, 2025
5/13/2025
Mechanisms of Kidney Diseases Associated With APOL1 Variation - ABSTRACT Severe, progressive kidney diseases (CKD) are more common among Black patients than in other populations. The excess risk for non-diabetic CKD in this population is explained, in part, by the presence of genetic variants in the APOL1 gene, which are unique to some African ancestral populations. The APOL1 high risk (HR) genotype, defined as the presence of two risk alleles (G1 or G2), is strongly associated with some idiopathic proteinuric podocytopathies, primarily FSGS. The pathogenic mechanisms responsible for the genetic association remain poorly understood, although over-expression systems suggest kidney diseases result from HR variant-dependent cytotoxicity. However published data suggest that both loss-of protection and gain of toxic function pathways may mediate APOL-associated kidney diseases. To develop hypotheses about mechanisms, we characterized the G0 and G2 spatial proteomes using peroxidase-catalyzed proximity labelling and mass spectrometry to identify candidate APOL1-regulated pathways in HeLa cells with regulated APOL1 expression. APOL1 induction did not cause cell death as assayed by a method agnostic to death mechanism or by an apoptosis-specific assay. The compositions of the G0 and G2 spatial proteomes are markedly enriched in secretory pathway membrane and luminal proteins but their protein neighborhoods diverge in the Golgi where G0 and HR-APOL1s are loaded into a subset of RAB6+ vesicles. The G0 and G2 spatial proteomes suggest that cargos of these vesicles are APOL1-isoform specific, with G0 but not G2 being in proximity of cell surface proteins. Using a N-hydroxysuccinimide biotin ester enrichment method, we demonstrated G2 expression altered cell surface proteins with functional consequences. Interference reflectance microscopy suggests G2 expressing but not G0 expressing cells fail to maintain adhesion. Importantly, these phenotypes are recapitulated in isogenic IPSC-derived podocytes with G0, G1 and G2 genotypes. Based on these data, we hypothesize that APOL1 regulates podocyte surfaceome to maintain podocyte adhesion and differentiated functions. G2 fails to do this resulting in podocyte depletion and progressive podocytopathy. We propose the following experiments to better understand APOL1 reference and kidney risk variant function: Aim 1: Use stable isotope labelling by amino acids in cell culture (SILAC) and mass spectrometry to characterize the surface proteome of podocytes expressing APOL1-G0, G1 and G2 variants in the presence and absence of Interferon-γ. Aim 2: Characterize the RAB6A+ post-Golgi vesicular compartment using biochemical and microscopic methods to define the differential trafficking of RAB6A+ vesicles and the associated cargo that is dependent upon APOL1 genotype. Aim 3: Prove that the APOL1 isoform-specific cargos in RAB6+ vesicles regulate the podocyte surfaceome and define the APOL1–dependent mechanisms for the podocyte adhesion defect.
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