Thrombin stimulates proinflammatory and proliferative responses in primary cultures of human proximal tubule cells

Abstract

Fibrin deposition is frequently observed within the tubulointerstitium in various forms of chronic renal disease. This suggests the presence of active components of the coagulation pathway, which may contribute to the progressive deterioration in renal function. The aim of this study was to investigate the proinflammatory and fibroproliferative effects of the coagulation protease thrombin on human proximal tubular cells (PTC) in culture. Primary cultures of PTC were established from normal kidney tissue and grown under serum-free conditions with or without thrombin or the protease-activated receptor (PAR) activating peptides TFLLRN-NH(2), SLIGKV-NH(2), and SFLLRN-NH(2) (100 to 400 micromol/L). DNA synthesis (thymidine incorporation), intracellular Ca(2+) mobilization (fura-2 fluorimetry), fibronectin secretion [enzyme-linked immunosorbent assay (ELISA), immunoblotting], monocyte chemoattractant protein-1 (MCP-1) secretion (ELISA), and transforming growth factor-beta1 (TGF-beta1) secretion (ELISA) were measured. Reverse transcription-polymerase chain reaction (RT-PCR) was used to assess PAR mRNA expression in these cells. Thrombin enhanced DNA synthesis, fibronectin secretion, MCP-1 secretion, and TGF-beta1 secretion in a concentration-dependent manner. Cell injury [lactate dehydrogenase (LDH) release] and cellular protein levels were unaffected. RT-PCR showed that cultures of PTC expressed mRNA transcripts for the thrombin receptors PAR-1 and PAR-3, but not PAR-4. Thrombin and each of the PAR activating peptides enhanced intracellular calcium mobilization. However, the other effects of thrombin were only fully reproduced by the PAR-2-specific peptide, SLIGKV-NH(2), only partially by SFLLRN-NH(2), (a PAR-1 peptide that can activate PAR-2), and not at all by the PAR-1-specific peptide, TFLLRN-NH(2). Thrombin-induced DNA synthesis, fibronectin, and MCP-1 secretion were unaffected by a TGF-beta neutralizing antibody, the matrix metalloproteinase (MMP) inhibitor, GM6001 and the epidermal growth factor (EGF) receptor kinase inhibitor AG1478. Thrombin initiates both proinflammatory and fibroproliferative responses in human PTC. These responses which are dependent on its protease activity appear not to be mediated by PAR-1 activation, the autocrine action of thrombin-induced TGF-beta1 secretion, MMP activation, or EGF receptor transactivation. The proinflammatory and fibroproliferative actions of thrombin on human PTC may help explain the extent of tubulointerstitial fibrosis observed in kidney diseases where fibrin deposition is evident.