Figure 3
AGE-BSA induces the AT1-dependent signaling cascade in rat kidney epithelial cells. (a) Immunoblot analysis of phosphorylation of ERK in response to 100 µg/mL BSA, Ang II or AGE-BSA for 5, 15,30, or 60 min with or without 1 µM olmesartan (AT1 blocker, ARB) in NRK52E cells. The graph indicates the intensity ratio of phosphorylated ERK (P-ERK) /total ERK(T-ERK) normalized to that with vehicle control (number(n) = 1, Ang II, n = 4, the other groups). *p < 0.05 determined by Student’s t-test. (b) Confirmation of knockdown efficiency of siRNA by quantitative real-time PCR in NRK-52E. (n = 5, each) The expression level of each gene was normalized using GAPDH mRNA as an internal control. The differences were determined by one-way ANOVA with Bonferroni correction. *p < 0.01 vs scramble siRNA; ♰p < 0.05 vs scramble siRNA. (c) Immunoblot analysis of phosphorylation of ERK in response to 100 µg/mL BSA or AGE-BSA for 30 min in combination with siRNA of scramble, RAGE or AT1 in NRK52E cells. The graph indicates the intensity ratio of P-ERK /T-ERK normalized to that with vehicle control (n = 4, each). The differences were determined by one-way ANOVA with Bonferroni correction. *p < 0.01 vs. scramble siRNA treated with BSA. (d) NF-κB luciferase reporter assay performed employing a treatment regimen of 50 μg/mL BSA or AGE-BSA for 2 h on treatment with 1 μM olmesartan (ARB) or 1 μM FPS-ZM1 (RAGE inhibitor) (n = 4, each). The differences were determined by one-way ANOVA with Bonferroni correction. *p < 0.01 vs. AGE-BSA treatment; ♰p < 0.05 vs. AGE-BSA treatment. (e) cAMP concentration in response to vehicle ,0.3 µg/mL HMGB1, 100 µg/mL AGE-BSA, or 10–10 M angiotensin II after the treatment with the indicated siRNA in NRK52E (n = 10, each). The concentration in vehicle-treated wells in cells with siVehicle was normalized to 100%. The differences were determined by one-way ANOVA with Bonferroni correction. *p < 0.01 vs. vehicle treatment; ♰p < 0.05 vs. vehicle treatment. (f, g) Immunoblot analysis of α-smooth muscle actin (SMA) as a measure of epithelial–mesenchymal transition after stimulation with (f) 100 μg/mL BSA, 100 μg/mL AGE-BSA, (g) vehicle, 3 µg/mL HMGB1, or 20 ng/mL TGFβ for 72 h. (n = 4, each). Treatment with 1 μM losartan (ARB), or irbesartan (ARB) was carried out 24 h before stimulation. The graph indicates the intensity ratio of α-SMA /control α-tubulin normalized to that with BSA (f) or vehicle (g). The differences were determined by one-way ANOVA with Bonferroni correction. *p < 0.05 vs. the other treatments; ♰p < 0.05 vs. vehicle treatment. Full-length blots are presented in Supplementary Fig. 2.
