Service de Néphrologie A, Association Claude Bernard, and INSERM U489, Hôpital Tenon, Paris, France
Keywords: kidney; knock-out strategy; PAR1; receptor; thrombin
Introduction
Thrombin is a serine proteinase which cleaves fibrinogen into fibrin and constitutes the key enzyme of blood coagulation. In addition, it is a potent activator of platelet aggregation, and of several cell types including endothelial cells, smooth muscle cells, fibroblasts, neurones, and mononuclear white blood cells. The role of thrombin in renal pathophysiology has been suspected for many years, due to the frequent fibrin deposition observed in the kidney in various vascular or glomerular diseases [1].
During the last 10 years, the cellular receptors mediating the mechanisms of action of thrombin and several other serine proteinases have been discovered, and their roles in vivo have been demonstrated, mainly due to gene knock-out (KO) strategy. It is beyond the scope of this paper to describe all that is known today about these receptors, and excellent recent reviews are available [2,3]. Our objective is to present the relevant results obtained during the last 10 years in the field of renal pathophysiology concerning the thrombin receptors.
The protease-activated receptor family: a subgroup of the 7-transmembrane domain receptors
In 1990, S. Coughlin and co-workers cloned and sequenced the first thrombin receptor [4], a 7-transmembrane domain, G protein-coupled receptor. This group rapidly demonstrated a unique mechanism of activation of this receptor since thrombin, a serine protease, binds to and cleaves the extracellular N-terminal domain of the receptor. A tethered ligand corresponding to the new N-terminus, SFLLRN, is then unmasked, binding to the second extracellular loop of the receptor and activating it. Several intracellular signalling pathways, through Gq and Gi protein activation, protein kinase C (PKC) and MAPK recruitments and gene activation have been demonstrated.
This receptor was then called the protease-activated receptor 1 or PAR1, after another receptor, named PAR2, activated by trypsin but not by thrombin was discovered, cloned and sequenced [5]. Later, two other thrombin receptors, PAR3 and PAR4, were discovered by the group of S. Coughlin. The mechanism of activation of the PARs is similar, i.e. the proteolytic cleavage of the N-terminus of the extracellular domain and significant homology between the sequences suggests that these receptors may be derived from a common ancestral precursor [6]. Human PAR1, PAR2 and PAR3 genes are localized on chromosome 5q13, whereas the PAR4 gene is localized in 19p12 [6]. Invalidation of these genes has been performed in mice, and mild or no phenotype was observed in living KO animals, suggesting a compensatory function of one receptor by others, or a minor effect of these receptors in normal mice.
Interestingly, however, PAR1-deficient embryos appeared to have a 50% intrauterine lethality, which seems to be related to a defect in yolk sack vascular development, as also observed in embryos deficient in tissue factor, factor V or prothrombin [7]. The likely but still unclear role of PAR1 in angiogenesis was the first lesson learnt from the PAR1 KO mouse. The second surprise provided by this animal model was that the platelet aggregation in response to thrombin was normal when the PAR1 gene had been disrupted, while the proliferative response of fibroblasts to thrombin was abolished [8]. This led to the discovery that PAR3 and PAR4 were the main mouse platelet receptors for thrombin, and more recently to the demonstration that mouse PAR3 does not signal, but is a cofactor for thrombin binding and PAR4 activation [9]. In contrast, human platelets have PAR1 and PAR4, but not PAR3, which mediate platelet activation by thrombin with different tempos [10]. PAR1 is expressed by murine and human fibroblasts and is responsible for the proliferative effects of thrombin [11].
The first thrombin receptor: PAR1 is expressed in the kidney
In vitro studies have shown that glomerular epithelial and mesangial cells in culture respond to thrombin, then proliferate and synthesize prostaglandins, nitric oxide, endothelin-1, extracellular matrix components, chemokines such as MCP1, and the serine protease inhibitor plasminogen activator inhibitor type 1 (PAI-1) [12]. We and others have been very interested in the control of PAI-1 synthesis by thrombin in glomerular cells, since an excess in local PAI-1 production may result from the generation of thrombin in several fibrin-associated nephropathies and may contribute to the severity of the renal lesions. Moreover, recent evidence has been provided that PAI-1 promotes the development of tissues fibrosis [13,14]. We showed that human glomerular epithelial and mesangial cells express PAR1, and that all the cellular effects of thrombin can be mimicked by the PAR1 agonist peptide SFLLRN [15,16]. To date there is no evidence that human glomerular epithelial and mesangial cells express PAR3 or PAR4. Similarly, mouse mesangial cells express PAR1, and mesangial cells prepared from PAR1 KO mice lack any response to thrombin (E. Rondeau, unpublished observation).
PAR1 expression is regulated in vitro. After the addition of thrombin or SFLLRN to cultured glomerular cells, activated PAR1 is rapidly internalized to be degraded in the lysosomes, as described in many other cell types. After a transient exposure of the cell to thrombin, intracellular stores of PAR1 are mobilized to the membrane so the cell once more becomes sensitive to thrombin. In contrast, prolonged stimulation of mesangial cells by thrombin induces a decrease in both the membrane and the intracellular pool of PAR1 [16]. PAR1 gene transcription is stimulated in response to PAR1 activation [17] through a pathway involving Gi, src, P13-kinase, ras and MAPK. The homologous internalization of PAR1 is a PKC-independent process, in contrast to the heterologous internalization that can be induced by the PKC activator, phorbol myristate acetate. Endothelin-1, which also activates PKC in glomerular epithelial cells, can induce an heterologous PAR1 internalization [18].
Similarly, in vivo, by northern blot, PAR1 mRNA has been demonstrated in the mouse and human kidney, whereas no PAR3 [19] and traces of PAR4 mRNA are detectable [20]. Using a monoclonal antibody directed against the SFLLRN sequence of PAR1 (thus this antibody recognizes both the intact and cleaved PAR1) and a specific cDNA probe, PAR1 expression can be demonstrated in the normal human kidney: it is found in the endothelial cells of glomerular and interstitial capillaries and arterioles, and in podocytes and mesangial cells. Smooth muscle cells of diseased arterioles and activated interstitial myofibroblasts may also express PAR1 [21]. Tubular epithelial cells do not usually express PAR1 spontaneously. Recently, Grandaliano et al. reported that PAR1 may be present at the basolateral membrane of proximal tubular cells, but this immunohistochemical finding was not controlled by in situ hybridization [22]. In fibrin-associated kidney diseases, such as crescentic glomerulonephritis and thrombotic microangiopathy, we found a reduced expression of the PAR1 protein in glomerular lesions while there was no change in mRNA expression, suggesting a thrombin-induced internalization and degradation of PAR1 in these diseases [21]. The question then arose of how to demonstrate that PAR1 plays a role in the pathogenesis of glomerular lesions. The answer was given by the PAR1 KO mouse.
Experimental nephropathies in PAR1-/- mice
The first kidney disease model studied in PAR1-deficient mice was the accelerated anti-GBM glomerulonephritis model [23]. Interestingly, we were able to demonstrate that, when compared to wild-type controls of similar genetic background, PAR1-/- had less severe renal failure, a lower percentage of crescent formation, and less glomerular fibrin deposition and macrophage infiltration. In addition, twice daily infusion of the agonist peptide SFLLRN worsens the severity of glomerular lesions in wild-type, but not in PAR1-/- mice, further demonstrating the role of PAR1 in these lesions. Finally, in the same model, hirudin infusion, which strongly inhibits thrombin activity and thus PAR1 activation, almost completely prevents the development of glomerular fibrin deposition and crescent formation. Clearly, PAR1 is involved in the pathogenesis of crescentic glomerulonephritis and is a new target for therapeutic intervention.
In contrast, in the endotoxin model of renal fibrin deposition, the role of PAR1 seems less pronounced since no difference in uPA gene repression, tPA and PAI1 gene induction and fibrin deposition in the kidneys was found between wild-type and PAR1-/- mice (E. Rondeau, unpublished data). These results indicate that during severe sepsis, PAI1 is induced in the kidney independently of PAR1 activation by thrombin, which, on the other hand, promotes fibrin deposition.
Conclusions and perspectives
The last 10 years have yielded much information for the elucidation of the mechanisms of cell activation by thrombin, i.e. how thrombin talks to cells [2], and for the role of PAR1 in platelet aggregation in humans. KO technology has been extremely useful and powerful in demonstrating the role of PAR1 in the pathogenesis of crescent formation during extracapillary glomerulonephritis. Similarly, PAR1 has been shown to play a role in arterial wall thickening after vascular injury [24], and in motor neurone degeneration and death [25]. In contrast, although anticipated, no defect in cutaneous wound healing has been found in PAR1-/- mice. The role of PAR1 in atherosclerosis and in tumoural angiogenesis, `the big killers, is also anticipated but remains to be demonstrated. In renal diseases, we would like to know which cellular expression of PAR1 on infiltrating inflammatory cells, on resident glomerular cells or on both is required for crescent formation. Targeted disruption of the PAR1 gene would be helpful to answer this question. In addition, we hypothesise that PAR1 may contribute to the progression of chronic renal failure since glomerular microthrombosis has been demonstrated in experimental models of glomerulosclerosis after subtotal nephrectomy and thrombin has been shown recently to induce the synthesis of connective tissue growth factor through PAR1 activation [26].
Notes
Correspondence and offprint requests to: Professeur Eric Rondeau, Service de Néphrologie A Hôpital Tenon 4 rue de la Chine F-75970 Paris cedex 20, France.
References