Advanced glycation end products regulate extracellular matrix protein and protease expression by human glomerular mesangial cells.

Advanced glycation end products (AGEs) may play a role in the pathogenesis of diabetic nephropathy, by modulating extracellular matrix turnover. AGEs are known to activate specific membrane receptors, including the receptor for AGE (RAGE). In the present study, we analyzed the various receptors for AGEs expressed by human mesangial cells and we studied the effects of glycated albumin and of carboxymethyl lysine on matrix protein and remodelling enzyme synthesis. Membrane RAGE expression was confirmed by FACS analysis. Microarray methods, RT-PCR, and Northern blot analysis were used to detect and confirm specific gene induction. Zymographic analysis and ELISA were used to measure the induction of tPA and PAI-1. We show herein that cultured human mesangial cells express AGE receptor type 1, type 2 and type 3 and RAGE. AGEs (200 microg/ml) induced at least a 2-fold increase in mRNA for 10 genes involved in ECM remodelling, including tPA, PAI-1 and TIMP-3. The increase in tPA synthesis was confirmed by fibrin zymography. The stimulation of PAI-1 synthesis was confirmed by ELISA. AGEs increased PAI-1 mRNA through a signalling pathway involving reactive oxygen species, the MAP kinases ERK-1/ERK-2 and the nuclear transcription factor NF-kappaB, but not AP-1. Carboxymethyl lysine (CML, 5 microM), which is a RAGE ligand, also stimulated PAI-1 synthesis by mesangial cells. In addition, a blocking anti-RAGE antibody partially inhibited the AGE-stimulated gene expression and decreased the PAI-1 accumulation induced by AGEs and by CML. Inhibition of AGE receptors or neutralization of the protease inhibitors TIMP-3 and PAI-1 could represent an important new therapeutic strategy for diabetic nephropathy.

Induction of urokinase receptor expression in nephrotoxic nephritis.

The urokinase receptor (uPAR) is a multifunctional molecule involved in pericellular, fibrinolytic, and proteolytic activities, as well as in cell adhesion and chemotaxis and may play a role in the pathogenesis of tissue remodeling occurring during glomerulonephritis. We analyzed sequentially the expression of uPAR by immunohistochemistry and in situ hybridization in an accelerated model of nephrotoxic nephritis in rats. A strong induction of uPAR mRNA expression was observed in glomeruli as soon as 1 h after nephrotoxic serum injection. The intensity of glomerular uPAR mRNA and antigen expression increased and peaked at 24 h. At that time, numerous glomerular fibrin deposits, monocyte/marcrophage infiltration, and heavy proteinuria were observed. Fibrin deposition was detected at 6 h, peaked at 24 h, and progressively declined over the next 3 weeks, while uPAR antigen expression remained elevated until the end of the study (3 weeks). By double labeling, we showed that the expression of uPAR was mediated by both intrinsic glomerular cells and infiltrating macrophages. Severe podocytic lesions developed within 3 days after antiserum injection, and glomerulosclerosis rapidly progressed within 2-3 weeks. These results show that glomerular uPAR expression is induced in nephrotoxic nephritis and suggest that uPAR may promote local proteolysis and also tissue remodeling, leading to the late development of glomerulosclerosis.

Prognostic value of plasminogen activator inhibitor type 1 mRNA in microdissected glomeruli from transplanted kidneys.

BACKGROUND: Plasminogen activator inhibitor type 1 (PAI-1) exerts antifibrinolytic and profibrotic activities. Inside the glomerulus, PAI-1 is mainly synthesized by mesangial cells. We hypothesized that thrombin, via its receptor protease activated receptor type 1 (PAR-1), present on the membrane of glomerular cells, is an important mediator of PAI-1 synthesis. METHODS: Using the technique of Peten et al., we microdissected the glomeruli of 23 kidney transplanted patients admitted in our department from 1993 to 1997, and we followed-up these patients for up to 5 years, with sometimes iterative renal biopsies. With this technique, we also microdissected the glomeruli of three patients who have had a nephrectomy for cancer (control patients). We investigated mRNA expression of the PAI-1, the thrombin receptor PAR-1, the alpha2 chain of type IV (alpha2 IV) collagen, and of a housekeeping gene (cyclophilin) by reverse transcription-polymerase chain reaction. The results were correlated with the renal function and the histological findings classified into acute rejection (9 biopsies), chronic rejection (22 biopsies), or normal (8 biopsies). RESULTS: A significant up-regulation of PAI-1 and alpha2 IV collagen mRNA was observed in acute rejection (P<0.05) when compared to normal kidneys. A positive correlation exists between alpha2 IV collagen mRNA level and the degree of cellular infiltration. A negative correlation was found between the level of mRNA of PAR-1 and the degree of vascular thrombosis (P=0.005) and glomerulosclerosis (P=0.04). A positive correlation was found between the degradation of renal function and the mRNA level of PAI-1 at the time of the renal biopsy (P<0.05). CONCLUSIONS: These results suggest that glomerular PAI-1 mRNA may be predictive of the long-term renal graft function.

Role of the third intracellular loop and of the cytoplasmic tail in the mitogenic signaling of the protease-activated receptor 1.

The activation of the protease-activated receptor 1 (PAR-1) by thrombin has been shown to induce an activation of the MAP kinase cascade and to stimulate cell proliferation. To examine the mechanisms of signal transduction by PAR-1, we constructed several PAR-1 mutants which were stably expressed in CHO cells. When compared to wild-type PAR-1, mutation of Ser306-->Ala (S306A) in the third intracellular loop of PAR-1 inhibited MAP kinase activation and cell proliferation stimulated by thrombin. The thrombin activation of MAP kinase was inhibited by pertussis toxin, suggesting a role for a Gi-like protein. As shown by calcium signaling and inosotol trisphosphate generation, the Ser306-mutated PAR-1 induced a strong activation of phospholipase C after thrombin addition. Deletion of the cytoplasmic tail of PAR-1 also inhibited thrombin-induced DNA synthesis but the MAP kinase pathway was activated as with wild-type PAR-1. In contrast, the deletion of the C-tail of PAR-1 prevented almost completely the activation of the phospholipase C pathway. Taken together these results suggest that the C-tail of PAR-1 is a critical site for PAR-1 coupling to phospholipase C activation, while the third intracellular loop of PAR-1 is implicated in PAR-1 coupling to Gi and MAP kinase activation. In addition, these results also show that MAP kinase activation is necessary but not sufficient for thrombin to induce cell proliferation.

Internalisation of the protease-activated receptor 1: role of the third intracellular loop and of the cytoplasmic tail.

To analyse the mechanisms of PAR-1 internalisation, we constructed several PAR-1 mutants and stably expressed them in CHO cells. Our study shows that the Ser(306)-->Ala mutation (S306A), which eliminates a potential site of phosphorylation by PKC in the third intracellular loop of PAR-1, did not change the rate of phosphorylation but reduced the rate of thrombin-induced internalisation of the PAR-1 mutant (58 versus 78% of membrane PAR-1 in 15 min, p<0.005). Deletion of the last 43 amino acid residues of the PAR-1 cytoplasmic tail completely suppressed the thrombin phosphorylation of the mutated receptor and significantly reduced its internalisation upon activation. This deletion also inhibited the PMA-induced and the agonist-independent internalisation of the receptor. The Tyr(371)--> Ala mutation (Y371A), in a NPXXY motif of the seventh transmembrane domain of the receptor had no effect on the receptor behaviour. Our results indicate that both the C-tail and the third intracellular loop are involved in PAR-1 internalisation induced by thrombin while only the C-tail plays a role in the PMA-induced and in the agonist-independent PAR-1 internalisation.

[Plasminogen activator inhibitor type 1: physiology and role in renal physiopathology]. / L'inhibiteur de type 1 des activateurs du plasminogène: physiologie et rôle en physiopathologie rénale.

Plasminogen activator inhibitor type 1 plays a prominent part in the regulation of extra and intra-vascular fibrinolysis through the inhibition of plasmin formation. In addition to its role in the resolution of blood clots, PAI-1 is involved in a variety of other biological processes including extracellular remodeling, cellular mobility, embryo implantation, development and tumoral proliferation. Moreover, PAI-1 is also implicated in various pathological processes such as thromboembolic diseases, atherosclerosis and fibrosis formation, particularly in the kidney and the lung. Inhibition of PAI-1 activity or of PAI-1 synthesis by specific antibodies, peptidic antagonists, antisens oligonucleotides or decoy oligonucleotides has been obtained in vitro but need to be evaluated in vivo. All these findings may have new therapeutical implications, explaining the importance of studies on PAI-1 production and regulation.

[Descriptive epidemiological study of acne on scholar pupils in France during autumn 1996]. / Epidémiologie descriptive de l'acné dans la population scolarisée en France métropolitaine pendant l'automne 1996.

BACKGROUND: Acne is the most common symptom prompting patients to consult a dermatologist. No previous study has been conducted in France to determine the prevalence of acne and describe the main epidemiological features. SUBJECTS AND METHODS: A cross sectional study was conducted in November 1996 and included 913 school children aged 11 to 18 years. This sample was statistically representative of the entire secondary school population in France during the 1996-1997 school year. The subjects were stratified by 5 criteria: age, sex, rural or urban residence, sun exposure, type of school. RESULTS: Taking the clinical diagnosis made by the dermatologist investigator as the main criteria, the overall prevalence of acne was 72 p. 100. It was 76.1 p. 100 using the new ECLA grading system previously described. The prevalence of acne was sex and age dependent: highest scores were found for girls aged 14-16 years and for boys aged 16-17 years. Genetic factors were very important for the outcome of acne. Finally, 41 p. 100 of the acneic subjects were following a treatment, prescribed by a dermatologist in two-third of the cases. DISCUSSION: These results are in agreement with those previously published in the literature although some differences were disclosed. It would appear important to distinguish between minimal acne with a few retentional pimples occuring during adolescence and severe acne (more than 20 pimples on the face) requiring early medical care to avoid scarring.

[ECLA grading: a system of acne classification for every day dermatological practice]. / La grille ECLA: un système de cotation de l'acné pour la pratique quotidienne du dermatologue.

BACKGROUND: Different acne gradings have been proposed: global grading, semi-global grading, quantitative grading and photographic grading. They are mainly used in clinical studies for the evaluation of acne treatment. However, it would be important for physicians to have an acne grade which would be useful for assessing acne lesions prior to treatment and following treatment efficacy. METHODS: Six French dermatologists developed an acne grading scaled called "ECLA" which only takes 2 minutes to fill out and specifically designed for use by dermatologist practitioners. In addition, the intra- and inter-observer reliability of the grading scale was assessed. RESULTS: This analysis demonstrated the excellent reliability of the ECLA grading scale both in terms of intra- and inter-observer variability except for the retentional factor. The cross physician reliability decreased with time specificity for the retention factor, indicating that previous training prior to a multicentric clinical study would be necessary. CONCLUSION: ECLA grading appears to be an interesting and useful tool in dermatology for the follow-up of acne patients.

Tissue-type plasminogen activator activity in HIV-associated HUS.

BACKGROUND: In children, haemolytic uraemic syndrome (HUS) is associated with high plasma plasminogen activator inhibitor type 1 (PAI-1), which may contribute to the persistence of renal glomerular and arteriolar thrombi. HUS has been described in HIV-infected patients, but the pathophysiology of HUS in these patients is poorly understood. The aim of the study was to investigate plasma fibrinolytic activity in 18 patients with HIV-associated HUS. METHODS: We measured tissue type plasminogen activator (t-PA) and PAI-1 activities in the plasma of 18 HIV-infected patients with biopsy-proven HUS (HIV+/HUS+) and 48 HIV-infected patients without HUS (HIV+/HUS-). RESULTS: Patients with HUS had a significantly higher serum creatinine, a lower platelet count and an increased incidence of cytomegalovirus (CMV) infection (72% of patients HIV+/HUS+, vs 25% of patients HIV+/HUS-). Unexpectedly, plasma PAI-1 activity was similar in both groups. However, t-PA activity was significantly higher in HUS cases (11.5 vs 4.5 U/ml, P=0.001). Patients with CMV infection, with or without HUS, had significantly increased t-PA level (P=0.01). Multivariate analysis identified high t-PA (RR=9.21) and CMV infection (RR=3.36) as risk factors for HUS. CONCLUSION: This study provides evidence that HIV-infected patients with HUS have high plasma t-PA activity. PAI-1 plasma activity is not significantly increased, as opposed to non-HIV-associated HUS. Thus, in the setting of HIV infection, HUS cannot be attributed to decreased fibrinolytic activity.

Specific receptor binding of renin on human mesangial cells in culture increases plasminogen activator inhibitor-1 antigen.

Some proteases possess a membrane receptor that focalizes their proteolytic activity on the cell surface and may mediate a proliferative effect, such as urokinase on glomerular epithelial cells. Since some hypertensive states are associated with high concentrations of renin and proliferation of arteriolar smooth muscle cells, we asked whether renin, an aspartyl-protease, would bind to mesangial cells that are smooth-muscle derived cells, which would induce their proliferation. The binding of 125I labeled recombinant human renin (125I-R) was studied on human primary mesangial cells and mesangial cells immortalized by transfection with SV40-T antigen. At 37 degrees C, the binding of 125I-R was time dependent and reached a plateau after two hours. 125I-R was found to bind in a saturable and specific manner with a Kd = 0.4 nM and 1 nM and 8,000 and 2,000 binding sites/cell, for primary and immortalized cells, respectively. When binding experiments were performed in the presence RO 42-5892, a synthetic inhibitor of renin, RO 42-5892 could inhibit the specific binding of labeled renin only at concentrations 1,000 times superior to the IC 50, indicating that the renin-mesangial receptor interaction did not depend on the active site of renin. Analysis by SDS-PAGE and autoradiography of cross-linking experiments of 125I-R bound to a membrane preparation showed a band of approximately 110 to 120 kDa, suggesting a Mr of 70 to 80 kDa for the renin receptor. Incubation of mesangial cells with 100 nM renin for 24 hours provoked a 100% increase of 3H thymidine incorporation that was not accompanied by an increase of the cell number, even after a seven day period of incubation. However, the incubation of mesangial cells with renin for 24 hours induced a significant increase (170% of control, P = 0.04) of plasminogen activator inhibitor-1 (PAI1) antigen in the conditioned medium. In conclusion, we have shown that human mesangial cells in culture express a specific receptor for renin, and that the binding of renin increases 3H thymidine incorporation independently of renin enzymatic activity. The absence of cell proliferation, the increase of 3H thymidine incorporation and the increase of PAI1 antigen suggest that the binding of renin can induce mesangial cell activation, which is reflected by a change in the fibrinolytic capacity of the cells. The role of this receptor remains to be determined in nephropathies and hypertensive states associated with high plasma/tissue renin concentrations, hypertrophy of mesangial or smooth muscle cells and extracellular matrix remodeling.

Endothelin-1 induces rapid and long lasting internalization of the thrombin receptor in human glomerular epithelial cells.

Endothelin-1 acts as a potent autocrine and paracrine mediator in the kidney. Glomeruli and renal arterioles also express functional thrombin receptors which mediate the cellular effects of thrombin. Using the binding on glomerular epithelial cells of 125I-labeled ATAP2, a monoclonal antibody raised against the functional thrombin receptor, we demonstrate here that endothelin-1 induces thrombin receptor internalization in a dose- and a time-dependent manner. The maximal effect is observed at 10(-7) M endothelin-1 corresponding to internalization of approximately 25% of thrombin receptors. It is maximal at 20 min and persists for at least 24 h. This effect is mediated by the endothelin receptor subtype A (ETA), and involves a pertussis toxin-sensitive G-protein and protein kinase C activation. Endothelin-1 does not induce thrombin receptor degradation nor change in thrombin receptor mRNA level after 2 h and 24 h of incubation. These results indicate that partial heterologous internalization of the functional thrombin receptor is induced by endothelin-1.

[Altitude and pregnancy. Apropos of an experimental study on the pregnant rat]. / Altitude et grossesse. A propos d'un travail expérimental sur la rate gestante.

Pregnancies that occur at high altitude are bedevilled by many complications and particularly those due to pressure disorders. Although there are many maternal and placental mechanisms that are brought to bear to "blunt" the effects of hypoxia in the fetus, these pregnancies are characterised by low birth weights. This work has been carried out to find out the effects of hypoxia during part only of the pregnancy. The study was carried out on three groups of ten pregnant Wistar Rats: a control group with normal oxygenation (N); a group exposed to hypobaric hypoxia during the 4th to the 12th day of pregnancy (480 mmHg, equivalent to altitude of 5200 m) (HP); and a group exposed to the same levels of hypoxia between the 12th and 20th days of pregnancy (HT). The litters were the same size in each group. Mortality of the little rats respectively were 0%, 8.1% and 30.6% in groups N, HP and HT (p < 0.001). With hypoxia the rats eat less at whichever stage of pregnancy. Weight gain in pregnancy overall was less in the groups exposed to hypoxia. The weight gain in the compression chamber was particularly low in the early exposed group (HP). The mean weight of the small rats was lowered most in group HT (p 0.001). In conclusion, exposure to hypoxia is particularly damaging if it occurs in the second half of pregnancy but if hypoxia occurs only in the first half of pregnancy there are disturbances. Exposure to hypoxia goes up mainly as an increase in neonatal mortality with low birth weights.(ABSTRACT TRUNCATED AT 250 WORDS)