Characterization of a plasminogen activator and its inhibitor in human mesangial cells

Characterization of a plasminogen activator and its inhibitor in human mesangial cells. In the course of some pathological and experimental nephropathies, intraglomerular fibrin deposits develop, possibly as a consequence of inefficient fibrinolysis. In vitro human glomeruli exhibit fibrinolytic activity due to the synthesis of plasminogen activators (PAs) such as, tissue-type PA (t-PA) and urokinase-type PA (u-PA). Immunofluorescence studies have previously shown that t-PA is localized in the capillary tufts and u-PA in the visceral epithelial cells. We have now investigated the fibrinolytic activity of cultured human mesangial cells. Inhibitory activity towards u-PA or t-PA but not plasmin was found in both conditioned medium and cellular extracts. Analysis of the conditioned medium by zymography revealed a single band of PA-activity (Mr: 110 to 120 kDa). Immunoneutralization with anti-t-PA and antiplasminogen activator inhibitor (PAI-1) IgG but not anti-u-PA or anti-PAI-2 removed this band. Reverse fibrin autography demonstrated the presence of PAI-1 in both cellular extracts and in conditioned medium. Western Blot analysis showed that two bands (50 kD and 120 kD) were recognized by the anti-PAI-1 antibody. By ELISA t-PA and PAI-1 antigens were found to increase progressively with time in the culture medium but not in cellular extracts. Both t-PA and PAI-1, but not u-PA and PAI-2, were also detected by immunofluorescence studies. Thus human glomerular mesangial cells synthesize and secrete t-PA and PAI-1 in vitro. PAI-1 is produced in excess, therefore t-PA is only found in the form of a complex with PAI-1

Regulation of growth-hormone-receptor gene expression by growth hormone and pegvisomant in human mesangial cells

Regulation of growth-hormone-receptor gene expression by growth hormone and pegvisomant in human mesangial cells.BackgroundMice transgenic for growth hormone develop mesangial proliferation, glomerular hypertrophy, and progressive glomerular sclerosis suggesting that the growth hormone–insulin-like growth factor I (IGF-I) pathway plays an important role. Therefore, we studied the impact of variable concentrations of 22 kD, 20 kD growth hormone, as well as of the growth hormone receptor antagonist pegvisomant (B2036-PEG), on both the growth hormone receptor (GHR/GHBP) gene expression and growth hormone binding protein (GHBP) formation in a human glomerular mesangial cell line. Further, the impact on collagen, IGF-I and IGF binding protein-1 (IGFBP-1) formation was studied.MethodsIn order to assess transcription, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used.ResultsPhysiologic doses of 22 kD or 20 kD growth hormone caused a dose-dependent and significant (P < 0.01) up-regulation of GHR/GHBP gene transcription, whereas supraphysiologic doses (50 and 500ng/mL) resulted in down-regulation (P < 0.001). Whenever pegvisomant was used, there was no increase in GHR/GHBP expression. These data were confirmed using run-on experiments. Further, the assessment of GHBP presented a constant, dose-dependent increase, which was completely abolished in the experiments where pegvisomant was used.ConclusionWe present data showing that growth hormone has a direct impact on GHR/GHPB gene transcription and that pegvisomant is a potent growth hormone receptor antagonist in human mesangial cells. In addition, although the GHR/GHBP gene transcription is down-regulated by supraphysiologic growth hormone concentrations, this effect was not found when GHBP levels were measured. This finding may reflect a self-inhibitory effect of growth hormone on the level of GHR/GHBP gene transcription, which does not involve the regulation of the shedding of GHBP and may, therefore, be of physiologic interest

Stable cell line of T-SV40 immortalized human glomerular visceral epithelial cells

Stable cell line of T-SV40 immortalized human glomerular visceral epithelial cells. Human subcultures (third passage) of glomerular visceral epithelial cells (VEC) isolated from one month old kidney were successfully transfected by two recombinant plasmids containing the cloned oncogenes from the simian virus 40 large T antigen and H-ras gene. One postcrisis cell clone (56/10 A1) was selected, propagated and characterized. One hundred percent of the 56/10 A1 cells (current passage > 100th; doubling time 30 hrs) expressed the nuclear T-SV40 antigen assayed by IF; the cells failed to express H-ras (RNA blot analysis). Immortalized cells were morphologically and phenotypically compared to parental cell type (third passage). Phenotypic characterization of the 56/10 Al cells was achieved using indirect immunofluorescence (IF) and immunogold silver staining coupled to bright field and epipolarization microscopy. Both parental and 56/10 A1 cells displayed positivity for cytokeratin, CALLA and PHM5, whereas von Willebrand factor was not detected in the two cell types. Since we have previously shown that human glomerular epithelial cells in culture synthetize plasminogen activator (PA) related compounds, we investigated the secretion pattern of these products in parental and transfected cells. Zymographic analysis of secreted PA related compounds revealed production of free urokinase (u-PA) and type 1 plasminogen activator inhibitor (PAI-1) complexed to tissular plasminogen activator (t-PA). Finally, in the transfected cells, increased cGMP generation under atrial natriuretic factor (ANF) stimulation agreed with previous work performed on nontransfected human VEC. In conclusion, the establishment of a human permanent cell line which retains most of the phenotypic features of parental glomerular visceral epithelial cells should represent a new tool to study human glomerular cell functions

Receptor binding and degradation of urokinase-type plasminogen activator by human mesangial cells

Receptor binding and degradation of urokinase-type plasminogen activator by human mesangial cells. The binding of [125I] labeled urokinase-type plasminogen activator (u-PA) was studied on human mesangial cells (MC) in culture. The binding of active [125I]u-PA at 37°C reached a plateau after 30 minutes of incubation and remained stable for at least four hours. When the supernatant was analyzed with trichloracetic acid (TCA), TCA soluble radioactive material could be detected after a lag phase of 30 minutes, and then increased linearly for four hours. Analysis by electrophoresis on SDS PAGE and autoradiography of the cell associated radioactivity and of the intracellular content showed that active u-PA and u-PA complexed to plasminogen activator inhibitor type-1 (PAI-1) were bound to the cell surface, but only u-PA/PAI-1 complexes were internalized and degraded. Therefore, the Kd and the number of binding sites were determined by competitive inhibition curves at 4°C using diisopropyl-fluorophosphate (DFP) u-PA. Scatchard plots showed a Kd = 400 ± 30 pM, and Bmax = 240,000 ± 25,000 sites/cell. Excess of the amino terminal fragment of u-PA (ATF) completely blocked the specific binding of [125I]u-PA, confirming that the binding of u-PA was independent of the presence of the active site and/or of the formation of complexes with PAI-1. 3H thymidine incorporation by mesangial cells after stimulation with 100nM active u-PA showed that u-PA had a moderate but significant mitogenic effect, in contrast to inactive u-PA and ATF. However, this mitogenic effect was not accompanied by a proliferative effect. Pretreatment of mesangial cell with a phosphoinositol-specific phospholipase C decreased the binding of [125I]u-PA by 60%, indicating that the majority of the u-PA receptor is anchored in the membrane by a phosphatidylinositol group. These results, together with a positive labeling of MC with monoclonal antibodies to the receptor of U937 cells, and the positive RNA hybridization with the cDNA probe for the human receptor cloned from U937 cells, indicate that the u-PA receptor on mesangial cells is identical to the one of U937 cells. In conclusion, human mesangial cells in culture express a specific receptor for u-PA, which could play a major role in the regulation of u-PA activity by degrading u-PA complexed to PAI-1

Introduction. La sclérose rénale : un problème de Santé Publique

L’insuffisance rénale (IR) terminale touche 45 000 patients dont 7 000 nouveaux par an. Leur traitement substitutif par la dialyse et/ou la transplantation représente plus de 1 % du budget de l’assurance maladie, malgré le faible nombre de patients.L’IR peut avoir une évolution ralentie par des traitements symptomatiques anti-hypertenseur, anti-diabétique et/ou anti-hyperlipemian.La progression de l’IR chronique vers l’IR terminale est secondaire à la déposition de collagènes dans les différentes structures du rein. Afin de ralentir la progression de l’insuffisance rénale, il faut donc connaître les différents collagènes déposés, les systèmes protéases/anti-protéases intervenant sur le remodelage de la matrice extracellulaire ainsi que l’effet de certaines protéases/anti-protéases sur des médiateurs activant la synthèse des collagènes.Une fois acquis ces connaissances, de nouvelles thérapeutiques, pharmacologiques, géniques et/ou cellulaires pourront limiter, voire faire disparaître les dépôts de collagènes

Protéases et antiprotéases dans la progression des lésions de l’insuffisance rénale chronique

Le rôle des protéases et des antiprotéases dans la progression de l’insuffisance rénale chronique a été bien documenté. La plupart des études ont concerné la plasmine et les activateurs du plasminogène de la famille des sérine-protéases, et les métalloprotéases matricielles. Récemment, l’attention s’est portée sur la rénine, une aspartyl-protéase. La rénine est une enzyme essentielle du système rénine angiotensine et le clivage de l’angiotensinogène en angiotensine I est l’étape limitante de la génération d’angiotensine II (Ang II), puissant peptide vaso-actif également impliqué dans les processus de fibrose rénale. Notre groupe vient de cloner un récepteur fonctionnel de la rénine. La fixation de la (pro)rénine est associée à deux événements majeurs : d’une part, l’augmentation de l’activité catalytique de la (pro)rénine pour son substrat l’angiotensinogène, d’autre part, l’activation des MAP kinases ERK1/2 (Extracellular Regulated Kinases) dont on connaît le rôle dans les processus de différenciation, d’hypertrophie et de prolifération cellulaires. L’analyse immunohistochimique de coupes de cœur et de rein humains normaux montre que le récepteur de la rénine est trouvé dans les artères coronaires et rénales, ainsi que dans les glomérules, associé aux cellules musculaires lisses vasculaires et aux cellules mésangiales glomérulaires. Dans les cellules musculaires lisses, la conversion de l’angiotensine I en Ang II serait plus efficace du fait de la proximité de l’enzyme de conversion membranaire et les effets biologiques seraient optimisés, l’Ang II étant générée au voisinage des ses récepteurs. L’analyse par Northern blot indique que l’ARN messager du récepteur est très abondamment exprimé dans le cœur, le cerveau et le placenta, et à un moindre degré dans le rein, le foie et le pancréas