Figure 3.

Temporal, dose-dependent, and injury-induced increase in vascular smooth muscle cell (vSMC) tissue factor (TF) with uremic solutes. A, Uric acid (UA)–induced upregulation of TF occurs within 4 hours of treatment. Lysates from vSMCs treated with NaOH and UA were harvested at different time periods and immunoblotted with TF and pancadherin antibodies. Representative blot from 3 experiments is shown. Densitometry analysis was performed. Average of 3 experiments is shown. P<0.01 for 4- and 24-hour UA and P<0.02 for 48-hour UA compared with NaOH. Error bars=SEM. B, Doubling of TF with concentrations of UA observed in patients with chronic renal failure. vSMCs treated with UA with increasing concentrations for 24 hours are shown. Representative blot from 3 experiments is shown. Densitometry analysis was performed. Average of 3 experiments is shown. P<0.02 for 80 μg/mL and P<0.001 for 100 μg/mL UA compared with NaOH. Error bars=SEM. C, Indole-3-acetic acid (IA) and indoxyl sulfate (IS) induce TF expression within 4 hours of treatment. vSMCs treated with IA and IS were harvested at different time intervals, and lysates were immunoblotted. Cells treated with human serum albumin served as control. Representative blot from 3 experiments is shown. Densitometry analysis was performed. Average of 3 experiments is shown. P<0.01 for 4, P<0.001 for 24, and P<0.01 for 48 hours for IA compared with human serum albumin; and P<0.01 for 4, 24, and 48 hours for IS compared with human serum albumin. Error bars=SEM. D, Increase in TF expression with increasing dose of IA and IS. vSMCs treated with IA and IS with increasing concentrations for 24 hours were harvested for immunoblotting. Cells treated with human serum albumin served as control. Representative blot from 3 experiments is shown. Densitometry analysis was performed. Average of 3 experiments is shown. P<0.05 for 1.75 μg/mL and P<0.01 for 3.25 μg/mL IA compared with human serum albumin; P<0.01 for 12.5 and 25 μg/mL IS compared with human serum albumin. Error bars=SEM. E, Injury further augments upregulation of TF. vSMCs 1·10⁶ treated with uremic solutes for 24 hours were injured with a cell scraper. The cells were washed and incubated with fresh medium without uremic solutes and harvested after 2 hours. Lysates were probed with the use of TF and pancadherin antibodies. Representative blot from 4 experiments is shown. Densitometry analysis was performed. Average of 4 experiments is shown. All P values refer to TF increase without vs with scratch injury. P<0.02 for control, P<0.01 for IA and IS, and P<0.05 for uric acid. Error bars=SEM. WB indicates westernblot.