Figure 6.

C3a–C3aR interaction disrupts actin cytoskeleton in human cultured podocytes. (A and B) Representative images (A) and quantification (B) of cell injury of hiPod exposed to vehicle, C3a (50 nM), or C3a + C3aR-A (50 nM) for 24 h and stained for F-actin and nephrin. (C) Representative images of caspase-3 staining of the same cells pictured above. (D and E) Representative images (D) and quantification (E) of cell injury of amniotic fluid–derived human podocytes exposed to vehicle, C3a (50 nM), or C3a + C3aR-A (50 nM) for 24 h and stained for F-actin and nephrin. *P < 0.05. (F and G) Representative blots and densitometric analysis of Snail (F) and TGF-β (G) expression in hiPod treated with vehicle, C3a, or C3a + C3aR-A for 24 h. Snail was measured in cell lysates; TGF-β was measured in cell supernatants. *P < 0.05; **P < 0.01. (H) Differentially expressed proteins in the supernatants of hiPod exposed for 24 h to vehicle, C3a (50 nM), or C3a + C3aR-A (50 nM; Proteome Profiler Human XL Cytokine Array). The cytokines represented here are the only ones among the 105 analyzed (see Materials and methods) whose expression levels significantly differed in C3a-treated podocytes versus both vehicle- and C3a + C3aR antagonist–treated cells. *P < 0.05 versus vehicle and C3a + C3aR antagonist. (I) Functional network showing the relationship between C3aR, IL-1β, and nephrin in renal tubular cells and in podocytes (https://hb.flatironinstitute.org/gene/; query genes C3aR, IL1B, and NPHS1; tissue, renal tubules or podocytes; maximum number of genes, 15). All experimental data were verified in at least two independent experiments. Scale bars: 20 µm. Error bars are SEM.