Activation of the Signal Transducer and Activator of Transcription Signaling Pathway in Renal Proximal Tubular Cells by Albumin

Figure 2. Activation of Jak2 in mouse renal proximal tubular (mProx24) cells treated with albumin mediates the activation of Stat1 and Stat5. (A) Phosphorylated Jak2 was detected 15 min after addition of albumin, demonstrating that albumin induced JAK signaling pathways. Jak1 and Tyk2 showed no significant changes. Western blot results for Jak2, Jak1, and Tyk2 are shown as controls. (B) mProx24 cells were transferred to a medium containing albumin (30 mg/ml medium) in the presence of AG490 (20 μM). AG490 inhibited the activation of Jak2 (only control and 30-min data shown) and prevented the activation of Stat1 and Stat5 compared with the control, indicating that albumin-induced activation of Stat1 and Stat5 was mediated mostly by Jak2. Western blot analyses for Stat1 and Stat5 are shown as controls. Three independent experiments were performed, and results are shown for one representative experiment.

Figure 3. Effects of cycloheximide or anti–IFN-γ antibody on albumin-induced Stat1 and Stat5 activation. Mouse renal proximal tubular (mProx24) cells were transferred to a medium containing albumin (30 mg/ml medium) in the presence of cycloheximide (2 μg/ml) or anti–IFN-γ antibody (0.05 ng/ml). (A) Cycloheximide could not inhibit albumin-induced activation of Stat1 and Stat5. (B) Anti–IFN-γ antibody could not inhibit albumin-induced activation of Stat1. Three independent experiments were performed, and results are shown for one representative experiment.

Figure 4. Western blot analyses of activated Stat1 and Stat5 in mouse renal proximal tubular (mProx24) cells treated with addition of apoTf. Phosphorylated Stat1 and Stat5 were detected 15 min after addition of apoTf, indicating that apoTf induced signal transducer and activator of transcription (STAT) signaling pathways. Western blot analyses for Stat1 and Stat5 are shown as controls. Three independent experiments were performed, and results are shown for one representative experiment.

Figure 5. Assessment of reactive oxygen species (ROS) by fluorescence-activated cell sorter analysis. After CM-H₂DCFDA incubation, mouse renal proximal tubular (mProx24) cells were treated with albumin for 15 min. Accumulation of DCF was measured with a flow cytometer by monitoring fluorescence at 526 nm. The increase was assumed to be proportional to the concentration of superoxide anions and hydrogen peroxide in the mProx24 cells. (A) Results are shown for one representative experiment. ROS generation was observed even in the control mProx24 cells (black line). The ROS generation was upregulated within 15 min after albumin (yellow line; 3 mg/ml, blue line; 30 mg/ml) overloading, but NAC (green line), a precursor of glutathione (GSH) and a ROS scavenger, blocked ROS generation. (B) Intracellular ROS formation was expressed as a ratio of the mean fluorescence intensity of control cells incubated in an albumin-free medium. Results are the means ± SD of triplicate experiments. $P < 0.01 versus control cells. *P < 0.005 versus control cells. **P < 0.001 versus control cells.

Figure 6. Effects of N-acetyl-l-cysteine (NAC) on albumin-induced Stat1 and Stat5 activation. Mouse renal proximal tubular (mProx24) cells were transferred to a medium containing albumin (30 mg/ml medium) in the presence of NAC (20 mM). (A) Western blot analyses showed that activation of Stat1 and Stat5 was inhibited by NAC, indicating that the activation of Stat1 and Stat5 is mediated by reactive oxygen species (ROS). Western blot analyses for Stat1 and Stat5 are shown as controls. (B) Electrophoretic mobility shift assay (EMSA) also confirmed the inhibition of Stat1 and Stat5 by NAC. Three independent experiments were performed, and results are shown for one representative experiment.

Figure 7. Effects of rotenone or diphenylene iodonium chloride (DPI) on albumin-induced Stat1 and Stat5 activation. Mouse renal proximal tubular (mProx24) cells were transferred to a medium containing albumin (30 mg/ml medium) in the presence of rotenone (10 μM) or DPI (10 μM). Western blot analyses showed that activation of Stat1 and Stat5 was inhibited by either rotenone (B) or DPI (C), compared with the positive control (A), indicating that the activation of Stat1 and Stat5 was mediated by reactive oxygen species (ROS). Western blot analyses for Stat1 and Stat5 are shown as controls. Three independent experiments were performed, and results are shown for one representative experiment.