Regenerative and proinflammatory effects of thrombin on human proximal tubular cells.
Interstitial fibrin deposition is a common histologic feature of tubulointerstitial diseases, which suggests that the coagulation system is activated. Thrombin, generated during the activation of the coagulation cascade, is a powerful activating factor for different cell types. Although proximal tubular cells are potential targets for this coagulation factor, no information is available on the effect of thrombin on these cells. Thus, the expression of protease-activated receptor-1 (PAR-1), the main thrombin receptor, was investigated in human proximal tubular cells (hPTC) in vivo and in vitro. A diffuse expression of PAR-1 was observed by immunohistochemistry along the basolateral membrane of PTC in normal human kidney. This observation was confirmed in vitro in cultured hPTC. Because tubular damage and monocyte infiltration are two hallmarks of tubulointerstitial injury, the effect of thrombin on DNA synthesis and monocyte chemotactic peptide-1 (MCP-1) gene and protein expression was evaluated in cultured hPTC. Thrombin induced a significant and dose-dependent increase in thymidine uptake and a striking upregulation of MCP-1 mRNA expression and protein release into the supernatant. Although PAR-1 is a G protein-coupled receptor, its activation in hPTC, as in other cell systems, resulted in a transient increase in cellular levels of tyrosine-phosphorylated proteins. An increased level of tyrosine-phosphorylated c-src suggested the activation of this cytoplasmic tyrosine kinase in response to thrombin and its potential role in thrombin-induced protein-tyrosine phosphorylation. Interestingly, thrombin-induced DNA synthesis and MCP-1 gene expression were completely blocked by genistein, a specific tyrosine kinase inhibitor, but not by its inactive analogue daidzein, demonstrating a central role for tyrosine kinase activation in the thrombin effects on hPTC. Moreover, the specific src inhibitor PP1 abolished the thrombin effect on DNA synthesis. In conclusion, thrombin might represent a powerful regenerative and proinflammatory stimulus for hPTC in acute and chronic tubulointerstitial diseases.
Grandaliano
Giuseppe
G
Division of Nephrology/Department of Emergency and Transplantation, University of Bari, Italy.
Monno
Raffaella
R
Division of Nephrology/Department of Emergency and Transplantation, University of Bari, Italy.
Ranieri
Elena
E
Division of Nephrology/Department of Emergency and Transplantation, University of Bari, Italy.
Gesualdo
Loreto
L
Division of Nephrology/Department of Emergency and Transplantation, University of Bari, Italy.
Schena
Francesco P
FP
Division of Nephrology/Department of Emergency and Transplantation, University of Bari, Italy.
Martino
Carmela
C
Division of Nephrology/Department of Emergency and Transplantation, University of Bari, Italy.
Ursi
Michele
M
Division of Nephrology/Department of Emergency and Transplantation, University of Bari, Italy.
D000704:Analysis of Variance; D000911:Antibodies, Monoclonal; D002460:Cell Line; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006801:Humans; D007120:Immunochemistry; D007249:Inflammation / Q000503:physiopathology; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000378:metabolism / Q000503:physiopathology*; D015336:Molecular Probe Techniques; D010766:Phosphorylation; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000378:metabolism*; D012038:Regeneration / Q000502:physiology; D013917:Thrombin / Q000494:pharmacology / Q000502:physiology*; D015854:Up-Regulation
D000911:Antibodies, Monoclonal; D018932:Chemokine CCL2; D012333:RNA, Messenger; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D012313:RNA; D004247:DNA; D013917:Thrombin