Tissue factor expression by a human kidney proximal tubular cell line in vitro: a model relevant to urinary tissue factor secretion in disease?
OBJECTIVE
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.


CONCLUSIONS
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.
Lwaleed
Bashir A
BA
Department of Urology, Southampton University Hospitals, Southampton, UK. bashir@soton.ac.uk
Vayro
Steven
S
Racusen
Lorraine C
LC
Cooper
Alan J
AJ
D002472:Cell Transformation, Viral; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D006801:Humans; D007124:Immunoenzyme Techniques; D007674:Kidney Diseases / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D018613:Microscopy, Confocal; D008954:Models, Biological; D016376:Oligonucleotides, Antisense / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D044967:Serum; D013925:Thromboplastin / Q000096:biosynthesis* / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects
D003470:Culture Media; D008070:Lipopolysaccharides; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D013925:Thromboplastin