Figure 2.
Complexity of uremic solutes to increase tissue factor (TF) levels and activity in vascular smooth muscle cells (vSMCs). A, Uric acid increases TF expression. vSMCs were treated with water-soluble and -insoluble uremic solutes at concentrations observed in patients with chronic renal failure for 24 hours. Cell lysates were probed for TF and pancadherin. Cells treated with water or NaOH served as control. Representative blot from 3 experiments is shown. Densitometry analysis was performed on TF bands with the use of pancadherin to normalize the signal. Average of 3 experiments is shown. P<0.001 for uric acid compared with NaOH. Error bars=SEM. Crea indicates creatinine; and Oxal, oxalic acid. B, Immunofluorescence of TF. vSMCs treated with uremic solutes for 24 hours were fixed and stained with TF. DAPI was used to stain nuclei. C, Indole-3-acetic acid (IA) and indoxyl sulfate (IS) increase TF expression in vSMCs. Cell lysates of vSMCs treated with protein-bound uremic solutes were probed for TF and pancadherin antibodies. Cells treated with media containing human serum albumin served as control. Representative blot from 3 experiments is shown. Densitometry analysis was performed as above. Average of 3 experiments is shown. P<0.02 for IA and P<0.01 for IS compared with human serum albumin (HSA) (control). Error bars=SEM. D, Immunofluorescence of TF. vSMCs treated with protein-bound uremic solutes were fixed and stained with TF. DAPI was used to stain nuclei. E, Increase in TF activity in vSMCs treated with IA, IS, and uric acid. vSMCs 1·105 seeded in 6-well plates and exposed to exposed to uremic solutes serum are shown. Cell lysates prepared after 24 hours were subjected to TF activity measurements. TF activity was normalized to number of vSMCs. Mean of 3 experiments performed in duplicates is shown. Error bars=SEM. DMSO indicates dimethyl sulfoxide; and Hom, homocysteine.