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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 1, 2003 as doi:10.1096/fj.03-0084fje. |
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,2
* Institut National de la Santé et de la Recherche Médicale U489,
Association Claude Bernard,
Department of Pathology and of
Nephrology A, Hopital Tenon, 75020 Paris, France; and
¶ Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium
2Correspondence: Inserm U489, 4, rue de la Chine, 75020 Paris, France. E-mail: eric.rondeau{at}tnn.ap-hop-paris.fr
SPECIFIC AIM
Type 1 plasminogen activator inhibitor (PAI-1) is the primary inhibitor of tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. Whereas PAI-1 is not expressed in normal kidneys, it is strongly induced in glomerular diseases, and thus could promote the local accumulation of fibrin. To address this issue, passive anti-glomerular basement membrane (GBM) glomerulonephritis (GN) was induced in PAI-1 gene-deficient mice and in wild-type mice of the same genetic background.
PRINCIPAL FINDINGS
1. PAI-1 is induced in the kidneys during the passive model of crescentic glomerulonephritis
Northern blot analysis of RNAs extracted from total kidneys disclosed a clear induction of PAI-1 mRNA synthesis in PAI-1 +/+ mice on day 7 after injection of nephrotoxic serum. This induction was not observed in PAI-1 -/- mice. By contrast, we found the same level of expression of 
2-antiplasmin (the main inhibitor of plasmin) mRNA in control mice and in mice with GN in both genotypes.
2. PAI-1 deficiency aggravates histological lesions, renal function impairment, and lethality
We observed a significant difference in histological features of GN between both genotypes. While both PAI-1 -/- and PAI-1 +/+ mice developed fibrinous and crescentic GN, disease was surprisingly more severe in PAI-1 -/- mice, which showed a significantly higher incidence of fibrin-positive crescents (cf. Fig. 1
). By immunofluorescence studies, glomeruli from both genotypes disclosed leukocyte accumulation in the vicinity of injured glomeruli with CD4+ T cell and macrophage infiltrates. Infiltration by CD4+ T cells was, however, markedly increased in PAI-1 -/- mice compared with PAI-1 +/+ mice, suggesting the possibility that PAI-1 deficiency leads to enhanced glomerular lesions because of a facilitated CD4+ T cell response. Baseline values of proteinuria were comparable in PAI-1 +/+ and PAI-1 -/- mice. Injection of nephrotoxic serum induced a sharp increase in daily proteinuria in both strains, but to a higher extent in PAI-1 -/- mice, on days 7, 15, and 28 (Fig. 2
). Similarly, both genotypes developed renal failure, but serum creatinine at day 28 was significantly higher in PAI-1 -/- mice. In addition, when a cohort of 10 mice in each group was prospectively followed on a long-term period, PAI-1 -/- mice showed a dramatic increase in mortality compared with PAI-1 +/+ mice.
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3. Thrombin is increased in the kidneys from PAI-1 gene-deficient mice
To reconcile the apparent paradox that PAI-1-deficient animals have a poorer outcome and more fibrin deposition despite a theoretically enhanced fibrinolytic activity, we explored the possibility that the rate of fibrin formation could exceed its clearance in the context of PAI-1 deficiency. Since thrombin is critically involved in crescentic GN through its procoagulant and proinflammatory properties mediated by its specific receptor, PAR1, we hypothesized that PAI-1-deficient mice presented a more severe GN because of enhanced thrombin production. In agreement, thrombin-antithrombin III (TAT) complex concentrations were found to be higher in PAI-1 -/- mice than in PAI-1 +/+ mice during anti-GBM glomerulonephritis (1.71±0.4 µg/mg prot in PAI-1 -/- mice vs. 0.66±0.15 µg/mg prot in PAI-1 +/+ mice, P<0.05). TAT complexes were undetectable in renal tissue in mice from both genotypes without GN.
4. Diseased kidneys from PAI-1 knockout mice exhibit an enhancement of plasminogen activators but not of plasmin activity
The activity of PAs was then explored by fibrin zymography. Lack of PAI-1 resulted in a frank increase of uPA and tPA-related fibrinolysis. However, intrarenal plasmin activity was found to be similar during GN in both genotypes. To further determine whether PAI-1 deficiency exacerbates glomerular injury through plasmin-dependent or PA-dependent effects, we used tranexamic acid, a potent inhibitor of plasmin proteolysis and of plasmin generation. This treatment induced an early increase in the mortality and considerably enhanced the glomerular deposition of fibrin in both strains.
5. Lack of PAI-1 induces the overactivation of transforming growth factor ß
Based on the observation that CD4+ T cell infiltration is greater in the kidneys of PAI-1 -/- mice, combined with an increased thrombin production and an early enhancement of proteinuria compared with wild-type mice, we sought to determine the mechanisms by which PAI-1 deficiency could potentiate T cell response to heterologous anti-GBM serum. Among their biological substrates, the PA/plasmin system is known to activate the latent form of transforming growth factor ß, a multifunctional cytokine shown to be involved in the development of crescentic anti-GBM glomerulonephritis. Whereas both genotypes had comparable kidney levels of total TGF-ß1, active TGF-ß1 level was dramatically higher in PAI-1 -/- mice during GN (199.2±71.9 pg/mg protein in PAI-1 -/- mice vs. 10.9 ± 5.9 pg/mg protein in PAI-1 +/+ mice, P=0.01).
6. Plasminogen activators activate latent TGF-ß in vitro
By in vitro experiments, we found a dose-dependent activation of recombinant human latent TGF-ß by both PAs in the absence of plasmin. This activation was suppressed by aprotinin, a potent inhibitor of serine proteases, but not by tranexamic acid.
7. Neutralization of TGF-ß serves a protective role during GN in PAI-1-deficient mice
In vivo neutralization of TGF-ß by specific antibodies in PAI-1 -/- mice induced a significant reduction of proteinuria compared with PAI-1 -/- animals injected with rabbit control IgGs on days 14 and 28 after induction. Histological analysis showed a significant reduction in the proportion of fibrin-positive crescents (18.4±4.2% in mice treated with anti-TGF-ß antibodies vs. 36.4 ± 6.5% in mice treated with control IgGs, P<0.05). By immunofluorescence study, a significant reduction of CD4+ T cells was also observed (1.23±0.16 cell/gl in the group treated with control rabbit IgG vs. 0.54±0.20 cell/gl in the group treated with anti-TGF-ß antibodies, P<0.05). This protective effect was not observed in PAI-1 +/+ mice.
CONCLUSIONS
Our findings show that PAI-1 deficiency drastically exacerbates the course of passive, immune-mediated crescentic glomerulonephritis. Absence of PAI-1 resulted in a greater histological and functional damage and in a higher intrarenal concentration of thrombin, a key mediator of the disease. PAI-1 -/- mice were also found to have superior induction of the synthesis and of activation of TGF-ß, most likely related to enhanced plasminogen activator activity. Moreover, systemic neutralization of TGF-ß significantly attenuated the severity of their disease.
Several arguments indicate that all these findings were related to an increased PA activity rather than to an increased plasmin activity. We found that 1) the intrarenal activity of plasmin was not enhanced in the absence of PAI-1 during glomerulonephritis; 2) there is substantial evidence in the literature that plasmin has a protective effect during anti-GBM glomerulonephritis, as attested by the enhanced severity of disease in plasminogen-deficient mice; 3) accordingly, we found in both genotypes that tranexamic acid aggravates the course of the disease; and finally, 4) 2-antiplasmin, but not PAI-1, seems to be the main regulator of fibrinolysis in the kidneys after endotoxin injection. In our model the persistent expression of 2-antiplasmin seems sufficient to blunt the enhanced generation of plasmin resulting from the increased PAs activity in PAI-1-deficient mice. Taken together, this suggests that the increased activity of PAs we observed in the absence of PAI-1 worsens the course of glomerulonephritis independent of a role in the generation of plasmin, but because of proper enzymatic action.
The PA/plasmin system has biological substrates other than fibrin and matrix proteins, including growth factors such as TGF-ß. We demonstrated in our model that the absence of PAI-1 leads to a much higher activation of TGF-ß than in wild-type mice, probably due to the higher enzymatic activity of PAs independent from plasmin, and that in vivo neutralization of TGF-ß induces a significant decrease in proteinuria and crescentic lesions in PAI-1 -/- mice. It is likely that an enhanced activation of TGF-ß is deleterious during crescentic anti-GBM glomerulonephritis. Kanamaru and co-workers generated transgenic mice expressing inhibitory Smad7, an intracellular antagonist of TGF-ß/Smad signaling, under the control of a distal lck promoter that directed high expression in peripheral T cells. When inducing antibody-mediated glomerulonephritis, they observed that glomerulonephritis was suppressed in these mice, which had a marked reduction in the infiltration of CD4+ T cell into glomeruli.
In summary, our results bring evidence that, converse to our initial hypothesis, PAI-1 deficiency dramatically worsens the course of experimental crescentic GN. Our findings suggest that PAI-1 deficiency induces a PA-related overactivation of TGF-ß that amplifies T cell-mediated immune glomerular injury in the anti-GBM model. These results demonstrate a new immunomodulatory role for PAI-1.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0084fje; doi: 10.1096/fj.03-0084fje 
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