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D Rodríguez-Puyol

Publications and source records attributed to D Rodríguez-Puyol.

At least 19 recordsLinked to original sources
PubMed1998

Cyclosporine increases local glomerular synthesis of reactive oxygen species in rats: effect of vitamin E on cyclosporine nephrotoxicity.

BACKGROUND: We report an investigation of the effects of cyclosporine (CsA) on kidney function, the glomerular synthesis of reactive oxygen species, the peroxidation of lipids, and the levels of thromboxane B2 (TXB2). The effect of the simultaneous administration of the antioxidant vitamin E (Vit E) and CsA in rats was also evaluated. METHODS: Adult male Wistar rats were treated for 30 days with CsA (30 mg/kg/day), with Vit E (0.05 mg/ml), with CsA plus Vit E, or with the vehicle used for administration of CsA, namely 12.6% ethanol. RESULTS: CsA induced kidney failure and increased the glomerular synthesis of superoxide anion, H2O2, malonyldialdehyde, and TXB2. Vit E minimized the adverse effects of CsA on kidney function and the glomerular synthesis of these compounds. CONCLUSIONS: Our results suggest that the acute decrease in glomerular filtration rate induced by CsA might be mediated by the synthesis of reactive oxygen species and subsequent peroxidation of lipids, which increases the levels of TXB2. Treatment with Vit E prevented these effects, suggesting a possible role for antioxidants in the prevention of CsA nephrotoxicity.

Animals↗
PubMed1998

Cyclosporin A-induced hydrogen peroxide synthesis by cultured human mesangial cells is blocked by exogenous antioxidants.

Cyclosporin A (CsA) is the immunosupressor most widely used in transplanted patients for preventing organ rejection, but it has some toxic side effects in vascular beds and kidney. The purpose of this work was to study if H2O2, a reactive oxygen species, is involved in the CsA-induced toxic effects on kidney in vitro. Human mesangial cells (HMC) in culture were incubated in presence of CsA (10[-5]-10[-8]M) and H2O2 was measured by flow cytometry. The specificity of the probe used in this method was demonstrated as fluorescence was not detected when superoxide anion generated through a Xanthine-Xanthine oxidase system was present, but fluorescence was noted when H2O2 was present in the incubation medium, both directly and after addition of superoxide dismutase to the medium thus promoting H2O2 synthesis. CsA induced a significant dose and time-response increased H2O2 synthesis by cultured HMC. This increase appeared 5 min after CsA addition, being maximal between 15-45 min at CsA concentration of 10(-7)M. When HMC were preincubated with antioxidants as vitamin E or selenium, the CsA-induced H2O2 production was partially blocked. In addition, selenium also induced an increased activity of glutathion peroxidase in HMC after 24 hours of incubation, suggesting that it exerted its H2O2 scavenging action through the modulation of the activity of this enzyme.

Antioxidants↗
PubMed1998

Role of reactive oxygen species in the signalling cascade of cyclosporine A-mediated up-regulation of eNOS in vascular endothelial cells.

1. Cyclosporine A (CsA) increases eNOS mRNA expression in bovine cultured aortic endothelial cells (BAEC). As some effects of CsA may be mediated by reactive oxygen species (ROS), present experiments were devoted to test the hypothesis that the CsA-induced eNOS up-regulation could be dependent on an increased synthesis of ROS. 2. CsA induced a dose-dependent increase of ROS synthesis, with the two fluorescent probes used, DHR123 (CsA 1 microM: 305+/-7% over control) and H2DCFDA (CsA 1 microM: 178+/-6% over control). 3. Two ROS generating systems, xanthine plus xanthine oxidase (XXO) and glucose oxidase (GO), increased the expression of eNOS mRNA in BAEC, an effect which was maximal after 8 h of incubation (XXO: 168+/-21% of control values. GO: 208+/-18% of control values). The ROS-dependent increased eNOS mRNA expression was followed by an increase in eNOS activity. 4. The effect of CsA on eNOS mRNA expression was abrogated by catalase, and superoxide dismutase (SOD). In contrast, the antioxidant PDTC augmented eNOS mRNA expression, both in basal conditions and in the presence of CsA. 5. The potential participation of the transcription factor AP-1 was explored. Electrophoretic mobility shift assays were consistent with an increase in AP-1 DNA-binding activity in BAEC treated with CsA or glucose oxidase. 6. The present results support a role for ROS, particularly superoxide anion and hydrogen peroxide, as mediators of the CsA-induced eNOS mRNA up-regulation. Furthermore, they situate ROS as potential regulators of gene expression in endothelial cells, both in physiological and pathophysiological situations.

Animals↗
PubMed1998

CsA and FK506 up-regulate eNOS expression: role of reactive oxygen species and AP-1.

Cyclosporine A (CsA) and FK506 increase endothelial nitric oxide synthase (eNOS) mRNA expression in cultured bovine aortic endothelial cells (BAEC). CsA appears to increase eNOS mRNA levels mainly by increasing the rate of transcription, although a small contribution of mRNA stabilization could not be ruled out. CsA and FK506 induced an increase of ROS synthesis with the fluorescent probe used, DHR123. The ROS generating system glucose oxidase (GO) increased the expression of eNOS mRNA in BAEC. This upregulation of eNOS mRNA by CsA or GO was abrogated by catalase. As AP-1 is a redox-sensitive transcription factor and the bovine eNOS promoter has an AP-1 consensus sequence, a role of this factor in the up-regulation of eNOS mRNA was studied. Electrophoretic mobility shift assays were consistent with an increase in AP-1 DNA-binding activity in BAEC treated with CsA or glucose oxidase. The potential participation of ROS and the transcription factor AP-1 in the regulation of eNOS gene expression is suggested.

Animals↗
PubMed1998

Age-related increase in expression of TGF-beta1 in the rat kidney: relationship to morphologic changes.

In the kidney, aging is characterized by the development of structural changes, including glomerulosclerosis and interstitial fibrosis. Transforming growth factor-beta1 (TGF-beta1) is known to play a critical role in the genesis of these alterations in pathologic conditions. The present experiments were designed to test the hypothesis that TGF-beta1 may be involved in the development of age-related histopathologic changes in rat kidney, and that captopril, an angiotensin-converting enzyme inhibitor, may influence the progression of glomerular and interstitial lesions. In this study, 3-, 18-, 24-, and 30-mo-old rats were examined, and an age-related increase in urinary protein excretion was found; plasma creatinine and systolic BP did not change. Significant structural changes, including glomerular sclerosis and interstitial fibrosis, were found in the group of aged rats (24- and 30-mo-old). Immunostaining for TGF-beta in the renal cortex interstitium was increased in the group of 24-mo-old rats, with a parallel increase in TGF-beta1 mRNA expression, measured with reverse-transcription PCR. Captopril-treated animals showed a statistically significant decrease in urinary protein excretion but no significant changes in BP. Moreover, captopril reduced the extent of interstitial fibrosis, but did not affect the degree of glomerulosclerosis. A significant inhibition of TGF-beta1 mRNA expression was observed in the captopril-treated animals. These findings suggest that TGF-beta1 may act as a fibrogenic growth factor that could be responsible, at least partially, for the renal interstitial fibrosis associated with aging. Treatment with captopril might delay the progression of these lesions.

Aging↗
PubMed1997

Captopril inhibits pp60(c-src) tyrosine phosphorylation in cultured human mesangial cells.

The present experiments were devoted to analyzing the mechanisms involved in the captopril-dependent inhibition of human mesangial cell proliferation. Studies were performed in cultured human mesangial cells incubated with captopril, an angiotensin II-converting enzyme inhibitor with antioxidant properties, lisinopril, a non-antioxidant angiotensin II-converting enzyme inhibitor, and tocopherol, a pure antioxidant. Both angiotensin II-converting enzyme inhibitors significantly inhibited fetal calf serum-induced [3H]thymidine uptake by human mesangial cells, in a dose- and time-dependent manner, an effect which was not observed with tocopherol. The antiproliferative effect of captopril and its ability to block tyrosine phosphorylation of human mesangial cells proteins were significantly greater than those of lisinopril. Moreover, captopril significantly prevented the fetal calf serum-induced tyrosine phosphorylation of pp60(c-src). The present results suggest that the antiproliferative ability of captopril does not completely depend on its angiotensin II-converting enzyme inhibitor properties, pointing to a possible interaction of the drug with the intracellular mechanisms responsible for the transmission of the proliferative signals.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1997

Haemodialyser biocompatibility and erythrocyte structure and function.

There is controversy as to the clinical importance of providing haemodialysis (HD) with biocompatible versus non-biocompatible membranes. The effects of both acute and chronic dialysis with a biocompatible membrane (polyacrylonitrile, PAN) and a non-biocompatible membrane (cuprophane, CU) on the structural and functional properties of human erythrocytes have been examined. All 27 studied HD patients had increased erythrocyte osmotic fragility (OF) compared to controls; a single CU HD decreased mean OF (% lysis) by 13% without altering cell cholesterol. A single PAN HD decreased OF by a significantly greater amount (24%) and was associated with a 20% reduction in cell cholesterol. Chronic PAN HD for 6 months was associated with a sustained reduction in osmotic fragility compared to chronic CU HD (mean lysis 16% vs 45%) with no differences in mean pre-HD cell cholesterol. A single CU HD was associated with increased mean erythrocyte malonyldialdehyde (MDA) and reduced membrane content of spectrin and band 3 and this was significantly different from the effects of PAN. A single CU or PAN HD had no significant action on reduced glutathione (GSH), ankyrin, actin or sodium pump activity. Chronic HD was associated with increased GSH, and decreased ankyrin and band 3 protein compared with controls but the results for CU and PAN were not different. There was a non-significant tendency for higher MDA levels after chronic CU HD compared to PAN. These results indicate that the structural integrity of erythrocytes is improved by PAN HD with respect to CU but this difference cannot easily be ascribed to gross changes in structural proteins, ionic homeostasis or oxidation status.

Acrylic Resins↗
PubMed1997

Oxidant/antioxidant balance in isolated glomeruli and cultured mesangial cells.

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.

Aging↗
PubMed1997

Involvement of phospholipase A2 in gentamicin-induced rat mesangial cell activation.

The role of the phospholipase A2 (PLA2) activation in the gentamicin (Gent)-induced rat mesangial cell activation has been studied. Gent (10(-5) M) induced a time-dependent mesangial planar cell surface area reduction that was significantly inhibited by the PLA2 inhibitors aristolochic acid (AA) and quinacrine, by the platelet-activating factor (PAF) blocker BN-52021 (BN), and by verapamil. These substances also inhibited Gent-induced [3H]thymidine incorporation into DNA (AA, 504 +/- 20; quinacrine, 555 +/- 66; BN, 1,126 +/- 120; and verapamil, 896 +/- 109; vs. 1,818 +/- 35 cpm/well in cells treated with Gent alone) and the Gent-induced increase in cell number (AA, 20,116 +/- 2,696; quinacrine, 24,687 +/- 1,481; BN, 26,122 +/- 1,016; and verapamil, 27,566 +/- 1,214; vs. 47,486 +/- 1,124 cells/well in cells treated with Gent alone). Gent induced a twofold increase in [3H]acetate incorporation into PAF (27 +/- 3 vs. 12 +/- 2 cpm/microgram protein in control conditions) that was completely blocked by AA, BN, or verapamil. Gent increased thromboxane B2 and prostaglandin E2 (PGE2) production, with both increases inhibited by either AA or verapamil. BN only inhibited the Gent-induced mesangial PGE2 production. In addition, Gent increased PLA2 activity (measured as [3H]arachiodonic acid release, 29,849 +/- 2,151 vs. 20,104 +/- 2,308 cpm/well in basal conditions), an effect that was blocked by AA (11,804 +/- 684 cpm/well). These data suggest a major role for PLA2 activation in Gent-induced mesangial cell contraction, proliferation and prostanoid secretion.

Acetates↗
PubMed1997

Prevention by tretinoin (all-trans-retinoic acid) of age-related renal changes.

The present work shows that dietary tretinoin (all-trans-retinoic acid) supplementation may be a useful manoeuvre for the prevention of age-related renal changes. 18-month-old male Fischer 344 rats fed during three months with standard chow plus tretinoin (1 mg/kg/day) did not exhibit any adverse effect in terms of bodyweight, urinary volume, renal handling of sodium and both hematological and blood chemistry parameters. Although the diet did not reduce age-related proteinuria nor renal lipid peroxidation, glomerular filtration rate and renal cortex protein content were, respectively, 30% higher and 30% lower than in age-matched control rats. These results suggest that dietary tretinoin supplementation may be a useful manoeuvre to slow the progression of age-related renal changes. Since glomerular H2O2 production increases during renal aging in rats, we studied the effect of tretinoin on the biology of cultured glomerular rat mesangial cells exposed to H2O2. Preincubation with tretinoin abolished cell proliferation or cell death induced, respectively, by low and high concentrations of H2O2. These results suggest that the modulation of the cellular actions of H2O2 may be relevant in the mechanisms through which tretinoin prevents age-related renal changes.

Aging↗
PubMed1996

Oxidative stress induces tyrosine phosphorylation of PDGF alpha-and beta-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 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.

Animals↗
PubMed1996

Regulation of endothelial NO synthase expression by cyclosporin A in bovine aortic endothelial cells.

The introduction of cyclosporin A (CsA) is considered a cornerstone advance in immunosuppression. However, serious side effects such as hypertension have fostered an important body of research regarding their pathophysiology. Although the participation of several vasoactive factors in the hypertensive response has been described, recent attention has focused on endothelium-derived vasoactive factors, and several reports describe an overproduction of endothelin-1 and a deficient endothelium-dependent vasodilation. In the case of the latter, no definitive clues for the precise molecular mechanisms have been provided. We demonstrate that endothelial cells in culture synthesize more NO in the presence of CsA for 24 h in a concentration-response manner. This augmentation is correlated with a threefold increase in the endothelial constitutive NO synthase (ecNOS) transcript, which is time dependent and maximal at 24 h. The CsA-induced increase in ecNOS mRNA expression was blocked by actinomycin D but unaltered by cycloheximide. Levels of ecNOS protein were also enhanced by CsA after 24 h. These data establish that NO synthesis is moderately enhanced in endothelial cells exposed to CsA for long periods of time and describe a new mode of regulating ecNOS gene expression.

Animals↗
PubMed1995

Thromboxane A2 and platelet-activating factor decrease in the platelet-mesangial cell interactions.

To analyze the metabolisms of platelet-activating factor (PAF) and Thromboxane A2 (TxA2) when platelets and mesangial cells (MC) interact, immunoreactive thromboxane B2 (TxB2) and PAF were measured after incubation of cultured rat MC with platelets (P) and with platelet supernatants (PS). In both cases, TxB2 significantly decreased with respect to the P synthesis and to the PS content, suggesting an increased degradation of this metabolite or even the existence of a specific effect of MC upon platelet TxB2. When immunoreactive PAF was measured, results were comparable to those observed for TxB2. Moreover, when intrinsic mesangial cell synthesis of PAF was assessed by analyzing the [3H]-acetate incorporation by prelabeled MC in the HPLC fraction coeluting with cold PAF standards, it was possible to demonstrate that P or PS did not modify PAF synthesis in these cells. In summary, present results support the existence of a specific effect of mesangial cells upon platelet TxA2 and PAF.

Animals↗
PubMed1995

Effects of somatostatin on cultured human mesangial cells.

The present experiments were devoted to analyzing the hypothesis that somatostatin (SS) could modulate glomerular filtration rate by interacting with mesangial cells. Studies were performed in cultured human mesangial cells, passages 3-5. Radioligand experiments demonstrated the presence in the cells of two kinds of receptors, with high (dissociation constant 14 pM. Number of sites: 426 fmol/mg) and low (dissociation constant 56 pM. Number of sites: 20, 111 fmol/mg) affinity. SS prevented in a dose-dependent manner the reduction in planar cell surface area induced by 100 nM Angiotensin II (AII). This effect was not inhibited by the blockade of the vasorelaxing prostaglandins (indomethacin, 10 microM), nitric oxide (L-N-methyl-arginine, 0.2 mM), adenylate cyclase (2,5'-dideoxyadenosine, 0.1 mM), or guanylate cyclase (Methylene blue, 30 microM; LY-83583, 10 microM), but it was potentiated by zaprinast, an inhibitor of the cyclic GMP (cGMP)-specific phosphodiesterase. SS also blocked the increase in myosin light chain phosphorylation induced by AII. SS increased cGMP synthesis by cultured human mesangial cells, an effect that seemed to be dependent on the stimulation of a particulate guanylate cyclase. Preincubation of the cells with pertussis toxin (0.5 microgram/ml) inhibited the effect of SS on the AII-dependent changes in planar cell surface area, as well as the SS-dependent cGMP stimulation. In summary, these results demonstrate the ability of SS to relax cultured human mesangial cells, thus supporting a role for this peptide in the regulation of the glomerular filtration rate. The SS-dependent mesangial cell relaxation may be due to changes in the intracellular concentrations of cGMP, as a consequence of the activation of a particulate guanylate cyclase.

Angiotensin II↗
PubMed1995

A possible role for platelet-activating factor in the hydrogen peroxide-induced TXB2 and PGE2 glomerular synthesis.

Hydrogen peroxide stimulates both prostanoid and platelet-activating factor (PAF) biosynthesis in cultured rat mesangial cells and isolated rat glomeruli. The present experiments were designed to try to establish some relationship between prostanoid and PAF synthesis in these renal structures, in the presence of hydrogen peroxide. Cells and glomeruli were incubated with hydrogen peroxide under different experimental conditions, and thromboxane B2 (TXB2), the stable metabolite of thromboxane A2 (TXA2), and prostaglandin E2 (PGE2) concentrations were measured in the supernatants of the cells or glomeruli. Moreover, H2O2-dependent PAF synthesis was measured by high performance liquid chromatography (HPLC) ([3H]acetate incorporation) and radioimmunoassay. H2O2 induced increased TXB2 and PGE2 production in cultured rat mesangial cells and isolated rat glomeruli. This effect was blocked by incubation in the presence of a PAF-receptor antagonist, BN-52021. This antagonist has no intrinsic effect either in basal prostanoid synthesis or in arachidonic acid-stimulated glomerular TXB2 synthesis. Alprazolam, another PAF antagonist, nonchemically related to BN-52021, also completely blocked the H2O2-induced production of TXB2 by isolated rat glomeruli. Moreover, H2O2 was also able to induce an increased [3H]acetate incorporation into a fraction comigrating with a PAF standard in HPLC in isolated glomeruli, and this effect was dependent on the H2O2 concentration tested. Moreover, H2O2 was also able to induce an increased [3H]acetate incorporation and increased synthesis of radioimmunoassayable PAF in cultured mesangial cells. These results suggest that the increased synthesis of PGE2 and TXB2 induced by H2O2 could be dependent on platelet-activating factor production.

Animals↗
PubMed1994

Somatostatin activates particulate guanylate cyclase in cultured rat mesangial cells.

Although the ability of somatostatin (ST) to relax cultured rat mesangial cells has recently been described, the intimate cellular mechanisms responsible for this effect have not been adequately clarified. The present experiments were designed to test the hypothesis that cyclic GMP (cGMP) could be involved in the genesis of this relaxation. ST increased cGMP synthesis by cultured rat mesangial cells, in basal conditions and in the presence of isobutylmethylxanthine or zaprinast. This effect was dose-dependent, with a threshold value of about 1 nM and a maximal response at ST concentrations between 0.1 and 1 microM. This increased cGMP synthesis was dependent on the stimulation by ST of a particulate guanylate cyclase, as the synthesis of cGMP by a particulate membrane fraction obtained from the cells increased in the presence of ST. When the cGMP-specific phosphodiesterase of mesangial cells was blocked with zaprinast, the ST-dependent relaxation, assessed both by morphological and biochemical criteria, significantly increased with respect to the experiments performed without zaprinast. These results support a role for cGMP in the ST-dependent relaxation of cultured rat mesangial cells. The increased cGMP synthesis appears to be the consequence of the activation of some form of particulate guanylate cyclase.

3',5'-Cyclic-GMP Phosphodiesterases↗