[Show abstract][Hide abstract] ABSTRACT: Adherence of human mesangial cells to the surrounding matrix contributes to glomerular homeostasis and is important for the maintenance of glomerular architecture and function in normal adult human kidney. The expression of chemokines and corresponding chemokine receptors on adjacent intrinsic renal cells indicates a novel chemokine/chemokine receptor function on nonimmune cells important for glomerular homeostasis. A constitutive expression of the chemokine SLC/CCL21 on human podocytes and of its corresponding receptor CCR7 on mesangial cells was shown before. SLC/CCL21 has a positive effect on proliferation and migration of mesangial cells and leads to increased cell survival in Fas-induced apoptosis. In leukocytes chemokines mediate integrin-dependent firm adhesion. Therefore, we examined the influence of chemokine receptor CCR7 activation by SLC/CCL21 on adhesive properties of human mesangial cells to matrix molecules.
Adhesion assays, mechanical detachment assays, and evaluation of integrin activation by integrin-linked kinase activity were performed. Changes in the cytoskeletal F-actin were illustrated by phalloidin immunofluorescence staining.
SLC/CCL21 stimulation enhanced adhesiveness to fibronectin in a time- and concentration-dependent manner. SLC/CCL21 also increased the firmness of mesangial cells adhesion as judged by detachment assays. Furthermore activation of integrin-linked kinase occurred with SLC/CCL21 addition to mesangial cells, resulting in increased phosphorylation of glycogen synthase kinase-3 (GSK-3) and protein kinase B (PKB/Akt). Exposure of mesangial cells to SLC/CCL21 also resulted in F-actin rearrangements with membrane ruffling and extensions leading to bridging between mesangial cells.
Activation of CCR7 on mesangial cells by SLC/CCL21 enhances the degree and firmness of cell adhesion and increases cell spreading and the formation of cell-cell contacts. This includes integrin-linked kinase activation and F-actin rearrangements. Thus, local chemokine generation and chemokine receptor expression on mesangial cells may play an important role in the maintenance of glomerular homeostasis and in local remodeling processes.
Full-text · Article · Jan 2005 · Kidney International
[Show abstract][Hide abstract] ABSTRACT: Progressive renal diseases are characterized by an increased synthesis of extracellular matrix (ECM) components. The mechanisms involved in the development of these alterations are not completely known, but a crucial role for TGF-beta 1 has been suggested. Moreover, the ability of the ECM to modulate the phenotypic expression of different cell types has been widely described. In experiments presented here, human mesangial cells (HMC) were grown on collagen type I (COL I) or IV (COL IV). ECM protein and TGF-beta 1 mRNA expression were evaluated by Northern blot analysis, and TGF-beta 1 secretion was evaluated by ELISA. The involvement of tyrosine kinase and serine-threonine kinase pathways was studied by Western blot analysis, immunofluorescence, and in vitro kinase assays. HMC cultured on COL I showed an increased mRNA expression of COL I and COL IV, fibronectin, and TGF-beta 1. Both tyrosine phosphorylation and integrin-linked kinase (ILK) activity increased when HMC were cultured on COL I, and blockade of these pathways inhibited the increased secretion of TGF-beta 1. In conclusion, the present results support a role for extracellular COL I in the regulation of TGF-beta 1 synthesis during progressive renal sclerosis and fibrosis and the subsequent increase in newly synthesized ECM proteins. In addition, ILK, along with the tyrosine kinases, participates in the genesis of this effect.
[Show abstract][Hide abstract] ABSTRACT: Extracellular matrix (ECM) components, through specific peptide motifs such as Arg-Gly-Asp (RGD), interact with integrins and can modify the behavior of cells. Transforming growth factor-beta1 (TGF-beta1) is the main cytokine involved in the synthesis of ECM proteins. We analyzed the effect of a RGD-containing peptide, as Arg-Gly-Asp-Ser (RGDS), on the regulation of TGF-beta1 secretion in cultured human mesangial cells. We found that RGDS increased mRNA expression and secretion of TGF-beta1 by stimulating the TGF-beta1 gene promoter. This effect was dependent on the interaction of RGDS with integrins. We evaluated the signaling pathways implicated in TGF-beta1 production by analyzing the effect of RGDS on kinase-related integrins. RGDS stimulated tyrosine phosphorylation as well as integrin-linked kinase (ILK) activity. However, tyrosine kinase inhibitors did not prevent the RGDS effect. In contrast, the inhibition of ILK by cell transfection with a kinase dead-ILK completely abolished the increased TGF-beta1 secretion and promoter activity in the presence of RGDS. Thus RGDS modulates the secretion of TGF-beta1, probably through increased synthesis by interacting with integrins and activating ILK. This supports a role for ECM components in the regulation of their own secretion.
Full-text · Article · Sep 2003 · The FASEB Journal
[Show abstract][Hide abstract] ABSTRACT: The effects of reactive oxygen species (ROS) on different cellular types are variable. In some conditions they can be harmful metabolites, but they can also act as intracellular messengers that are able to activate different transcription factors. Based on previous reports in which ROS were shown to stimulate the proliferation of mesenchymal cells, this study was carried out to assess this effect on bovine aortic endothelial cells (BAECs). When cells were incubated with glucose oxidase (GO), an enzyme that generates H2O2 continuously, a significant increase in BAEC proliferation was detected. BAEC proliferation was measured by the incorporation of [3H]-thymidine in the DNA of BAECs, and also by an increase in the number of cells. The effect observed with GO was maximal at 8-24 h. Catalase abolishes proliferation. We also tested the ability of GO to phosphorylate tyrosine residues in endothelial cell proteins. A significant increase in tyrosine phosphorylation was found, which might constitute the molecular basis for proliferative effect of GO. In conclusion, these results demonstrate the ability of H2O2 to stimulate BAEC proliferation at least under certain experimental conditions. We suggest a general activation of the cascade of tyrosine phosphorylation as one of the possible cellular mechanisms responsible for GO-induced BAEC proliferation.
No preview · Article · Feb 2000 · Journal of Cardiovascular Pharmacology
[Show abstract][Hide abstract] ABSTRACT: The mechanisms responsible for age-related glomerular sclerosis (GS) have not been clearly identified. The present experiments were aimed at assessing the importance of the oxidant/antioxidant balance in the early stages of this process. For this purpose, the renal function (biochemical and clearance studies), some characteristics of isolated glomeruli, and reactive oxygen production (superoxide anion, hydrogen peroxide) as well as the antioxidant ability (superoxide dismutase, catalase, glutathione peroxidase) of glomeruli and cultured mesangial cells were studied in 3- and 18-month-old Fischer 344 rats (YOUNG and OLD rats, respectively). OLD animals show a normal renal function, increased urine protein excretion, and augmented protein glomerular content, an indirect index of GS. Isolated glomeruli from these rats produced increased amounts of superoxide anion and hydrogen peroxide, and catalase activity was increased. The glomerular thiobarbituric acid-reactive substances (TBARS) content was higher in OLD than in YOUNG animals. Similar results were obtained in cultured mesangial cells. In summary, the present results demonstrate, at an early stage of rat GS development, an association between the functional and structural changes of this process and an increased TBARS content (likely indicative of lipid oxidative damage) at the glomerular structures as well as in cultured mesangial cells. More extensive studies are needed to confirm the nature of this association.
No preview · Article · Feb 1997 · Free Radical Biology and Medicine
[Show abstract][Hide abstract] ABSTRACT: Oxidative stress induces tyrosine phosphorylation of PDGF α- and β-receptors and pp60c-src in mesangial cells. Reactive oxygen species are autocrine and paracrine modulators of cell behavior. Hydrogen peroxide, a cellular oxidant, has been shown to stimulate mesangial cell proliferation. In the present study we analyzed the H2O2-induced early signaling events. Immunofluorescence analysis revealed a H2O2 induced dose-dependent increase in tyrosine phosphorylation. Short treatment (2 or 5 min) with 5 mM H2O2 induced a mitogenic response and a significant (P < 0.01) increase in the number of cells compared to non-treated controls. Proteins extracted from H2O2 (0.1 to 10 mM) treated cells were separated on SDS-PAGE and subjected to immunoblot analysis with anti-phosphotyrosine. A dose-dependent induction of tyrosine phosphorylation of 180 kDa, 120 kDa and 60 kDa proteins was observed within 1 to 10 minutes. By sequentially using immunoprecipitation and immunoblotting the 180 kDa tyrosine phosphorylated band was shown to represent both PDGF α-and β-receptors. The tyrosine phosphorylated 60 kDa protein was identified as the cytoplasmic protein tyrosine kinase pp60c-src. The c-src phosphorylation was associated with an inhibition of c-src kinase activity, suggesting phosphorylation of tyrosine 527 in the c-src regulatory domain. Pretreatment with catalase completely abrogated the H2O2-induced PDGF receptor and c-src tyrosine phosphorylation. These data support the notion that the activation of a signaling pathway involving the PDGF receptors and c-src contributes to the mitogenic effects of reactive oxygen species.
Full-text · Article · Jul 1996 · Kidney International
[Show abstract][Hide abstract] ABSTRACT: Reactive oxygen species are autocrine and paracrine modulators of cell behavior. Hydrogen peroxide, a cellular oxidant, has been shown to stimulate mesangial cell proliferation. In the present study we analyzed the H2O2-induced early signaling events. Immunofluorescence analysis revealed a H2O2 induced dose-dependent increase in tyrosine phosphorylation. Short treatment (2 or 5 min) with 5 mM H2O2 induced a mitogenic response and a significant (P < 0.01) increase in the number of cells compared to non-treated controls. Proteins extracted from H2O2 (0.1 to 10 mM) treated cells were separated on SDS-PAGE and subjected to immunoblot analysis with anti-phosphotyrosine. A dose-dependent induction of tyrosine phosphorylation of 180 kDa, 120 kDa and 60 kDa proteins was observed within 1 to 10 minutes. By sequentially using immunoprecipitation and immunoblotting the 180 kDa tyrosine phosphorylated band was shown to represent both PDGF alpha- and beta-receptors. The tyrosine phosphorylated 60 kDa protein was identified as the cytoplasmic protein tyrosine kinase pp60c-src. The c-src phosphorylation was associated with an inhibition of c-src kinase activity, suggesting phosphorylation of tyrosine 527 in the c-src regulatory domain. Pretreatment with catalase completely abrogated the H2O2-induced PDGF receptor and c-src tyrosine phosphorylation. These data support the notion that the activation of a signaling pathway involving the PDGF receptors and c-src contributes to the mitogenic effects of reactive oxygen species.
Full-text · Article · Jul 1996 · Kidney International
[Show abstract][Hide abstract] ABSTRACT: This work deals with the hypothesis that an increased synthesis of reactive oxygen species (ROS) or platelet-activating factor (PAF) (or both) may be implied in the genesis of age-related glomerulosclerosis. Plasma concentration and urinary excretion of both thiobarbituric acid-reactive substances (TBARS) and PAF were measured in young and old human beings and rats. Moreover, these same parameters as well as H2O2 synthesis and reduced glutathione (GSH) content were measured in isolated glomeruli of young (3 months) and old (18 months) Wistar rats. H2O2 synthesis and GSH content were also measured in cultured rat mesangial cells from young and old animals. Both human beings and rats showed a decreased glomerular filtration rate and an increased urinary protein excretion with respect to young individuals. Isolated glomeruli from old animals showed a higher protein content and a lower number of cell nuclei than those from young rats. No changes were detected in plasma concentration and urinary excretion of TBARS and PAF in either human beings or rats. Glomeruli from 18-month-old rats exhibited a higher content of TBARS and GSH and an increased synthesis of H2O2 and PAF than did those from 3-month-old rats. GSH content and H2O2 synthesis were higher in cultured cells from old rats than in those from young rats. These results point to the possibility that ROS or PAF could mediate some of the changes that characterize age-related glomerulosclerosis.
No preview · Article · Nov 1994 · Journal of Laboratory and Clinical Medicine
[Show abstract][Hide abstract] ABSTRACT: The present work deals with the hypothesis that probucol, through its antioxidant properties, may be a useful tool in the prevention of age-related glomerular sclerosis. Fifteen month-old male Sprague-Dawley rats were fedad libitum during three months with standard chow containing or not 10 g probucol/kg diet. Rats eating chow with probucol exhibited 25% lower content of glomerular protein than control rats, whereas no differences were found in the number of cells per glomerulus, since both the number of cellular nuclei/unit of glomerular area and the DNA/glomerulus ratio were similar in the two groups of rats. Glomeruli from probucol-treated animals also exhibited a 90% lower production of superoxide anion than those of control rats, while the glomerular activities of catalase and superoxide dismutase were similar in the two groups. These results suggest that probucol, through its antioxidant properties, may be a useful agent in the prevention of age-related glomerular changes.
No preview · Article · Sep 1994 · Geriatric Nephrology and Urology
[Show abstract][Hide abstract] ABSTRACT: 1. The present study was designed to determine the changes in renal function in two models of experimental pancreatitis in rats, in an attempt to assess the possible pathogenic role of reactive oxygen species and to elucidate a possible therapeutic role for somatostatin. 2. Mild pancreatitis was induced by low blockade of the biliary duct and severe pancreatitis was evoked by retrograde infusion of bile salts. Renal function was studied by clearance techniques in rats with pancreatitis, treated or not treated with somatostatin. Plasma and glomerular malonyldialdehyde levels were measured by the thiobarbituric acid method. 3. Renal function did not change in rats with low blockade of the biliary duct, but animals receiving a retrograde infusion of bile salts showed a significant decrease in glomerular filtration rate and renal plasma flow with respect to sham-operated animals. 4. Plasma malonyldialdehyde levels increased significantly in rats treated with bile salts with respect to control animals, whereas no changes were detected in glomerular malonyldialdehyde levels. Thus, the renal dysfunction does not seem to be related to an increased production of reactive oxygen metabolites at the glomerular level. 5. Somatostatin infusion significantly improved renal function in rats with severe pancreatitis (retrograde infusion of bile salts) by increasing glomerular filtration rate, renal plasma flow and filtration fraction. These results support a possible therapeutic role for somatostatin in the renal dysfunction associated with the severe forms of pancreatitis.
No preview · Article · Dec 1993 · Clinical Science
[Show abstract][Hide abstract] ABSTRACT: The present experiments were designed to analyze the ability of somatostatin to modulate the proliferation of cultured rat mesangial cells. In the absence of fetal calf serum, somatostatin stimulated cell proliferation in a dose-dependent manner. In contrast, in proliferating cells, somatostatin inhibited cell proliferation, also in a dose-dependent fashion. Zaprinast, a rather specific cyclic GMP phosphodiesterase blocker, inhibited the somatostatin-dependent proliferation in the absence of growth factors. However, it potentiated the inhibitory effect on proliferating cells. These results support a dual role for somatostatin in the regulation of mesangial cell proliferation. The inhibitory effect of the peptide may be mediated by cyclic GMP.
No preview · Article · Oct 1993 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: The effects of somatostatin (ST) on the regulation of the glomerular filtration rate have not been extensively studied. The present experiments were designed to analyze this possible relationship. ST alone did not modify the planar cell surface area (PCSA) of cultured rat mesangial cells (CRMC), but it prevented and reversed the reduction in PCSA induced by 10 nM angiotensin II (Ang II) in a dose- and time-dependent manner. ST (1 microM) completely prevented and reversed the increase in the myosin light chain phosphorylation induced by 10 nM Ang II. Incubation with pertussis toxin (PT, 0.5 micrograms/ml) inhibited the effect of ST on the Ang II-dependent changes in PCSA, but this effect was not inhibited by the blockade of the vasodilatory prostaglandins (indomethacin, 10 microM) or nitric oxide (L-N-methyl-arginine, 0.2 mM) synthesis. 2',5'-dideoxyadenosine (DDA, 0.1 mM), an adenylate cyclase blocker, and methylene blue (MB, 30 microM), a soluble guanylate cyclase blocker, did not interfere with the ST inhibitory effect on the Ang II-dependent reduction in PCSA of rat mesangial cells. ST also blocked the reduction in PCSA induced by phorbol myristate acetate (PMA, 300 nM). ST was also able to prevent and revert the Ang II dependent reduction in glomerular cross-sectional area of isolated rat glomeruli, also in a dose- and time-dependent fashion. Finally, intravenous administration of ST (200 ng/kg body wt as a bolus plus a continuous injection of 25 ng/min/kg body wt) partially blocked the reduction in GFR (measured as CIn) and RPF (measured as CPAH) and the increase in filtration fraction induced by the intravenous administration of Ang II (1.7 micrograms/min/kg body wt) in anesthetized rats. In summary, these results suggest that ST could antagonize the renal actions of Ang II, increasing the GFR and RPF decreased by Ang II, and this effect could be dependent, at least partially, on a direct relaxing effect of ST on mesangial cells.
No preview · Article · Mar 1993 · Kidney International