Abstract

Aim: To study baseline and stimulated tissue factor (TF) production from a normal, albeit immortalised, human kidney proximal tubular cell line (HKC-5), in order to establish a model for investigating the role of inflammatory mediators in the increased urinary TF (uTF) seen in inflammatory and neoplastic disease.

Methods: TF procoagulant activity, expression and secretion in HKC-5 cells were investigated using TF activity and antigen assays, fluorescence confocal microscopy and immunocytochemistry. TF expression in the HKC-5 cells was also studied using reverse transcription (RT)-PCR and its synthesis was suppressed using antisense oligodeoxynucleotide (ODN), directed against human TF mRNA. Cells were stimulated, after serum deprivation, with bacterial lipopolysaccharide (LPS), an agonist known to enhance TF expression in monocytes. They were also subject to serum starvation.

Results: Analysis by RT-PCR showed TF production by stimulated and actively metabolising HKC-5 cells. Antisense ODN treatment resulted in approximately 50% suppression of TF synthesis compared to a mismatch ODN. The amount of TF produced by the HKC-5 cells was time dependent and coincides with a decrease in the intracellular TF levels. LPS up-regulated TF production in HKC-5 cells. Reducing fetal calf serum concentrations in the culture medium decreased TF production and secretion.

Conclusion: Stimulated TF synthesis and secretion in vitro by HKC-5 cells is consistent with the hypothesis that uTF is produced by tubular cells influenced by mediators of disease states and provides a model for further mechanistic investigations.