Up-regulation of tissue factor activity on human proximal tubular epithelial cells in response to Shiga toxin.
BACKGROUND
The pathophysiology of hemolytic uremic syndrome (HUS) is incompletely established. Based on clinical studies demonstrating the presence of prothrombotic plasma markers in patients with HUS, we hypothesized that Shiga toxin might cause activation of the coagulation pathway by augmenting tissue factor, the major initiator of coagulation.


METHODS
Human proximal tubular epithelial cells (PTECs) [human kidney-2 (HK-2 cells)] were exposed to Shiga toxin-1, and expression of tissue factor, cell detachment, protein synthesis, caspase-3 activity, and Shiga toxin-1 binding were examined. Results. HK-2 cells expressed constitutive surface tissue factor activity and increased their tissue factor expression upon exposure to Shiga toxin-1. Shiga toxin-1 bound to HK-2 cells and inhibited protein synthesis. The up-regulation of tissue factor was dose- and time-dependent and strongly correlated with cell detachment and increase in caspase-3 activity caused by Shiga toxin-1 exposure. A general caspase inhibitor simultaneously inhibited HK-2 cell detachment and tissue factor up-regulation while mutant Shiga toxin-1 neither caused cell detachment, protein synthesis inhibition, nor increase in tissue factor activity. Tissue factor activity elicited by Shiga toxin-1 was abrogated by a monoclonal antitissue factor antibody. Calphostin C, a protein kinase C (PKC) inhibitor, partially blocked tissue factor up-regulation, indicating possible involvement of PKC-dependent mechanism.


CONCLUSIONS
These data, taken together, suggest a strong link between Shiga toxin-induced up-regulation of tissue factor activity, cytotoxicity, and apoptosis in HK-2 cells. The proximal tubule is a target of Shiga toxin in HUS, and it seems plausible that injured proximal tubular cells trigger the activation of the coagulation system, the formation of intrarenal platelet-fibrin thrombi, and the development of acute renal failure in HUS.
Nestoridi
Eirini
E
Pediatric Nephrology Laboratory, MassGeneral Hospital for Children at Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
Kushak
Rafail I
RI
Duguerre
Dayana
D
Grabowski
Eric F
EF
Ingelfinger
Julie R
JR
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000378:metabolism; D008074:Lipoproteins / Q000502:physiology; D014176:Protein Biosynthesis / Q000187:drug effects; D011493:Protein Kinase C / Q000502:physiology; D022622:Shiga Toxin 1 / Q000378:metabolism / Q000633:toxicity*; D013925:Thromboplastin / Q000032:analysis / Q000096:biosynthesis*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015854:Up-Regulation
D000590:Amino Acid Chloromethyl Ketones; D008074:Lipoproteins; D022622:Shiga Toxin 1; D014409:Tumor Necrosis Factor-alpha; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C051928:lipoprotein-associated coagulation inhibitor; D013925:Thromboplastin; D011493:Protein Kinase C; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases