Fig 1: APOL1 RV expression promotes apoptotic pathways in primary glomerular ECs(A) Cell death in HGECs 24 or 48 h after electroporation, quantified as the percentage GFP cells positive for annexin V staining.(B) Caspase 3/7 and caspase 8 quantification by luminescence 24h after electroporation.(C) TNFR1 protein expression quantification 24h after electroporation.(D) Quantification in live cells of acidic organelles by measuring the spot area of punctate 24h after electroporation. *p < 0.05, **p < 0.01, ***p < 0.001 for one-way ANOVA followed by post hoc test for multiple comparisons. Each dot in the graph represents an independent experiment.
Fig 2: Expression of APOL1 in the kidney and association of APOL1 risk genotypes with EC activation(A) Mean expression and fraction of cells expressing APOL1 in the single-cell transcriptomics dataset from 43 donors (healthy or with AKI or CKD) within the Kidney Precision Medicine Project (KPMP).(B) Volcano plot representing APOL1 differential expression between healthy individuals and those with AKI in peritubular capillary endothelial cells (adjusted p value = 0.05 represented by dotted line, APOL1 in red).(C) Heatmaps showing differential gene expression (log2 fold change) of EC markers from glomerulus (left heatmap) and tubulointerstium (right heatmap) kidney biopsies. Each heatmap shows expression profiles from individuals with nephrotic syndrome and either low-risk or high-risk APOL1 genotypes (see bottom panel).(D–F) Ingenuity pathway analysis (IPA) illustrating the ranking of the most overlapping canonical pathways (D), biofunctions (E), and upstream regulators (F) between the differentially expressed genes in human glomerular biopsies from LR vs. HR individuals (HsLRvsHR; first column), in transgenic mice overexpressing APOL1 under the nephrin promoter (Mm G0vsG1; second column, and Mm G0vsG2; third column). Dark purple represents high enrichment, whereas white represents low enrichment within the dataset. Round dots depict a non-significant enrichment (p > 0.01) of that pathway within the specific dataset.
Fig 3: Changes in cellular complexity in APOL1-expressing ECs(A) Cell morphology quantification by immunofluorescence of ECs.(B) Effect of APOL1 induction with doxycycline (1uM) for 24h in the forward scatter (FSC) and the side scatter (SSC) normalized against untreated control cells, with representative histograms.(C) Nuclear size quantification by immunofluorescence of ECs.(D) Quantification in live cells of acidic organelles by flow cytometry.(E) Mitophagy assessment in cells pre-loaded with a mitophagy dye for 30min followed by APOL1 induction for 24h, and the fluorescence intensity of punctate quantified. *p < 0.05, **p < 0.01, ***p < 0.001 for one-way ANOVA followed by post hoc test for multiple comparisons. Data presented as fold change to non-doxycycline-treated cells. Each dot represents an independent experiment.
Fig 4: APOL1 RV expression promotes primary glomerular EC activation(A) Gene expression analysis of key EC markers 24h after electroporation with green fluorescent protein (GFP) plasmid (C), G0 expressing plasmid with a GFP reporter (G0), or G1 expressing plasmid with a GFP reporter (G1).(B) PECAM1 protein expression quantification 24h after electroporation and representative images.(C) Macrophage adhesion quantification and representative images showing attached macrophages labeled with a cell mask (blue) helping in segmenting the macrophages and allowing quantification (data representative of three independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001 for one-way ANOVA followed by post hoc test for multiple comparisons. Each dot in the graph represents an independent experiment.
Fig 5: In vitro expression of APOL1 iPSC-derived ECs promotes EC activation(A) Schematic representation of the EC differentiation from iPSCs, and representative histograms of the membrane expression of PECAM1 measured by flow cytometry at selected time points.(B) Gene expression analysis of key EC markers after APOL1 induction with 1uM doxycycline for 24h.(C) Membrane protein expression quantification of the adhesion molecule ICAM1 and representative histograms after 24h doxycycline exposure.(D) Membrane protein expression quantification of the endothelial structural protein PECAM1 after 24h exposure to doxycycline (1uM).(E) Macrophage adhesion quantification and representative images showing attached macrophages labeled with a cell mask (blue) helping segment the macrophages and allowing quantification.*p < 0.05, **p < 0.01, ***p < 0.001 for one-way ANOVA followed by post hoc test for multiple comparisons. In B, C, D, and E, the results are presented as fold change expression in doxycycline-treated cells compared to non-doxycycline-treated cells. Each dot represents an independent experiment.
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