Journal: Frontiers in Immunology
Article Title: Angiotensinogen and C3 compete for renin-induced complement activation
doi: 10.3389/fimmu.2025.1563868
Figure Lengend Snippet: Renin cleavage of C3 is inhibited by aliskiren. (a) Renin-mediated C3 cleavage to C3b using the Iowa protocol without magnesium (Mg, lane 1) and with Mg as per the reference (lane 2). C3 alone, as the negative control, using the Iowa protocol with Mg (lane 3). Total C3 cleavage to C3b using the Lund protocol without Mg (lane 4) and with Mg (lane 5). C3 alone as per the Lund protocol (lane 6). All incubations were performed for 24h at 37°C. The figure shows one representative experiment, similar results were obtained in five separate experiments. (b) C3 incubated with renin (Lund protocol) for shorter incubation times of 30 min to 3h. (c) C3 alone showed no cleavage (lane 1). C3 incubated with renin showed cleavage to C3b (lane 2). C3 incubated with renin and aliskiren showed no cleavage (lane 3). Renin was preincubated with aliskiren for 2h and after addition of C3 an additional 18h at 37°C. For comparison, C3 convertase-mediated cleavage of C3 is shown in lane 4. (d) C3 alone showed no cleavage (lane 1), C3 incubated with trypsin (0.5 µg/ml) showed cleavage to C3b (lane 2), and C3 incubated with trypsin and aliskiren showed cleavage to C3b and lack of inhibition (lane 3). Trypsin was preincubated with aliskiren for 30 min and with C3 an additional 15 min at 37°C. All lanes were run on the same gel as shown and C3 detected by immunoblotting. Full blot images corresponding to panels a–d are presented in the supplement. (e) C3a generation when recombinant renin was incubated with C3 for up to 90 min. The two left columns show background C3a present in the sample before and after incubation for 90 min in the absence of renin. Statistical analysis was performed by Kruskal–Wallis test followed by Dunn’s multiple comparisons test. * P < 0.05, ** P < 0.01.
Article Snippet: Renin cleavage (final concentration 100 μg/ml) by C3 (100 μg/ml) was also performed in the presence of the renin inhibitor aliskiren hemifumarate (final concentration 50 mM, Tocris Bioscience, Bristol, UK) diluted in 10× PBS.
Techniques: Negative Control, Incubation, Comparison, Inhibition, Western Blot, Recombinant