ABSTRACT
While arginine-glycine-aspartic acid-based peptidomimetics have been employed for the treatment of cardiovascular disorders and cancer, their use in other contexts remains to be explored. Arginine-glycine-aspartic acid-serine induces Transforming growth factor-beta1 transcription in human mesangial cells, but the molecular mechanisms involved have not been studied extensively. We explored whether this effect could be due to Activator protein-1 activation and studied the potential pathways involved. Addition of arginine-glycine-aspartic acid-serine promoted Activator protein-1 binding to its cognate sequence within the Transforming growth factor-beta1 promoter as well as c-jun and c-fos protein abundance. Moreover, this effect was suppressed by curcumin, a c-Jun N terminal kinase inhibitor, and was absent when the Activator protein-1 cis-regulatory element was deleted. Activator protein-1 binding was dependent on the activity of integrin linked kinase, as transfection with a dominant negative mutant suppressed both Activator protein-1 binding and c-jun and c-fos protein increment. Integrin linked kinase was, in turn, dependent on Phosphoinositol-3 kinase activity. Arginine-glycine-aspartic acid-serine stimulated Phosphoinositol-3 kinase activity, and Transforming growth factor-beta1 promoter activation was abrogated by the use of Phosphoinositol-3 kinase specific inhibitors. In summary, we propose that arginine-glycine-aspartic acid-serine activates Integrin linked kinase via the Phosphoinositol-3 kinase pathway and this leads to activation of c-jun and c-fos and increased Activator protein-1 binding and Transforming growth factor-beta1 promoter activity. These data may contribute to understand the molecular mechanisms involved in the cellular actions of arginine-glycine-aspartic acid-related peptides and enhance their relevance as these products evolve into clinical therapeutic use.
Subject(s)
Mesangial Cells/metabolism , Peptides, Cyclic/pharmacology , Promoter Regions, Genetic , Signal Transduction/drug effects , Transcription Factor AP-1/metabolism , Transforming Growth Factor beta1/biosynthesis , Up-Regulation/drug effects , Cardiovascular Diseases/drug therapy , Cardiovascular Diseases/metabolism , Cells, Cultured , Enzyme Activation/drug effects , Enzyme Activation/genetics , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Mutation , Neoplasms/drug therapy , Neoplasms/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Signal Transduction/genetics , Transforming Growth Factor beta1/geneticsABSTRACT
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.
Subject(s)
Integrins/metabolism , Oligopeptides/pharmacology , Transforming Growth Factor beta/biosynthesis , Cells, Cultured , Glomerular Mesangium/cytology , Glomerular Mesangium/metabolism , Humans , Models, Biological , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/biosynthesis , Transcriptional Activation , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta1 , Tyrosine/metabolismABSTRACT
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.
Subject(s)
Glomerular Mesangium/drug effects , Somatostatin/pharmacology , Angiotensin II/antagonists & inhibitors , Angiotensin II/pharmacology , Cells, Cultured , Glomerular Filtration Rate/drug effects , Glomerular Mesangium/cytology , Humans , Octreotide/analogs & derivatives , Octreotide/metabolism , Precipitin TestsABSTRACT
Vascular smooth muscle cells (VSMC) exhibit a hypertrophic and contractile response after angiotensin II (Ang II) treatment, and the NADH/NADPH oxidase-dependent synthesis of hydrogen peroxide (H(2)O(2)) seems to play a central role in these responses. Present experiments were designed to analyze the mechanisms responsible for the rapid changes induced by Ang II in the intracellular H(2)O(2) concentration in VSMC. Ang II induced a quick and transient increase of dichlorodihydrofluorescein (DCHF) fluorescence in VSMC, an effect that was completely abolished by catalase and by diethyldithiocarbamate, a cell-permeable superoxide dismutase inhibitor. Losartan and pertussis toxin prevented the stimulatory effect of Ang II. Both diphenylene iodonium (NADH/NADPH oxidase blocker) and 3-(4-octadecylbenzoyl)acrylic acid (phospholipase A2 blocker) inhibited the changes in DCHF fluorescence induced by Ang II, in a dose-dependent fashion, and the effects of both inhibitors were additive. These data demonstrate that Ang II induces a very quick and transient increase of H(2)O(2) in VSMC. This effect depends on the receptor type 1, is linked to a G protein, and involves both NADH/NADPH oxidase and phospholipase A2 activation. The mechanism may be related to the previously proposed role of H(2)O(2) in the genesis of the Ang II-induced cell contraction.
Subject(s)
Angiotensin II/pharmacology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Reactive Oxygen Species/metabolism , Acrylates/pharmacology , Angiotensin II/metabolism , Animals , Benzoates , Catalase/metabolism , Catalase/pharmacology , Cells, Cultured , Ditiocarb/pharmacology , Enzyme Inhibitors/pharmacology , Fluoresceins/chemistry , Fluoresceins/metabolism , Fluorescence , Hydrogen Peroxide/metabolism , Indomethacin/pharmacology , Losartan/pharmacology , Muscle, Smooth, Vascular/cytology , NADH, NADPH Oxidoreductases/antagonists & inhibitors , NADH, NADPH Oxidoreductases/metabolism , Onium Compounds/pharmacology , Pertussis Toxin/pharmacology , Phospholipases A/antagonists & inhibitors , Phospholipases A/metabolism , Phospholipases A2 , Rats , Rats, WistarABSTRACT
In experimental models and in humans, somatostatin (SRIF) is able to contract certain vascular structures. The present experiments were designed to assess the capacity of SRIF to contract cultured rat aortic vascular smooth muscle cells (VSMC), and to analyze the possible mechanisms involved. Cells incubated with SRIF showed a significant reduction in planar cell surface area, in a time- and dose-dependent manner. This effect was partially blocked by preincubating the cells with a combination of calcium antagonists (10 microM verapamil, plus 10 microM 3,4,5-Trimethoxybenzoic acid 3-(diethylanino) octyl ester TMB)-8). SRIF was also able to stimulate myosin light-chain phosphorylation in VSMC. A small but significant increase of intracellular calcium concentration, and decreased levels of cAMP, without changes in cGMP, were detected in VSMC incubated with SRIF. A search for the known SRIF receptors present in these cells, by reverse transcription-polymerase chain reaction, only SRIF receptor-4 was found to be present. These results demonstrate the ability of SRIF to contract cultured rat VSMC. The contraction observed in these cells appears to be due to a mixed mechanism, that involves [Ca2+]i and cAMP as second messengers, and is likely mediated via SRIF receptor-4.
Subject(s)
Muscle, Smooth, Vascular/drug effects , Somatostatin/pharmacology , Animals , Base Sequence , Cells, Cultured , DNA Primers , Muscle Contraction/drug effects , Muscle, Smooth, Vascular/physiology , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Platelets seem to be involved in the pathogenesis of some kidney diseases, but the exact relationships between platelets and the changes in renal function are incompletely known. Mesangial cells (MC) were incubated with platelet-supernatants (PS) and cellular surface area (CSA) and myosin light-chain phosphorylation (MLCP) were measured. CSA of PS-incubated MC (PS-MC) significantly diminished, as compared to control MC (70 +/- 6% vs. 100 +/- 5%). PS induced a significant increase in MLCP with respect to control cells (150 +/- 23% vs. 100 +/- 18%). When platelets were pretreated with indomethacin, the PS-dependent contraction was abolished. Pretreatment with sulotroban (SU) or BN-52021 (BN), a thromboxane A2 (TXA2) and a platelet-activating factor (PAF) receptor blocker respectively, also completely blocked the PS effects. In other experiments, platelets were activated with thrombin (T), adding the so obtained PS to MC. Moreover, cells were also preincubated with T and then added PS. No changes in CSA were observed in either case. It may be concluded that PS contracted cultured MC, and these changes could be related to the decreased glomerular filtration rate (GFR) observed in some diseases in which platelets seem to be involved. TXA2 and PAF may be responsible for this effect. In contrast, T incubation inhibited the effect of PS, perhaps through a direct relaxing effect of T in MC.
Subject(s)
Blood Platelets/physiology , Glomerular Mesangium/cytology , Glomerular Mesangium/physiology , Animals , Cells, Cultured , Glomerular Mesangium/drug effects , Glomerular Mesangium/ultrastructure , Myosins/metabolism , Phosphorylation , Platelet Activating Factor/pharmacology , Rats , Rats, Wistar , Thrombin/pharmacologyABSTRACT
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.
Subject(s)
Acrylic Resins/pharmacology , Biocompatible Materials/pharmacology , Cellulose/analogs & derivatives , Erythrocytes/cytology , Erythrocytes/physiology , Membranes, Artificial , Renal Dialysis/instrumentation , Adult , Cellulose/pharmacology , Cholesterol/blood , Erythrocyte Membrane/metabolism , Erythrocytes/metabolism , Female , Glutathione/blood , Humans , Male , Malondialdehyde/blood , Membrane Proteins/blood , Middle Aged , Osmotic Fragility , Ouabain/pharmacology , Sensitivity and Specificity , Sodium-Potassium-Exchanging ATPase/bloodABSTRACT
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.
Subject(s)
Blood Platelets/physiology , Glomerular Mesangium/cytology , Platelet Activating Factor/metabolism , Thromboxane A2/metabolism , Animals , Cell Communication , Cells, Cultured , Glomerular Mesangium/physiology , Platelet Activating Factor/biosynthesis , Rats , Rats, Wistar , Thromboxane A2/biosynthesis , Thromboxane B2/biosynthesis , Thromboxane B2/metabolismABSTRACT
Rats receiving daily IP injections of dl-alpha-tocopheryl acetate (50 mg/kg) for 4 days showed levels of plasma alpha-tocopherol slightly increased and normal platelet count but platelet aggregation (induced by ADP or adrenaline) was significantly decreased when compared with the control animals. In addition, both platelet-rich plasma and platelet-poor plasma-thromboelastograms from treated rats showed thromboplastine-formation times significantly higher than those from control animals. Serum biochemical parameters showed several minor modifications in treated rats with respect the control ones. It suggests that vitamin E could be considered as a possible useful tool for the therapy of several hyperaggregable or hypercoagulable states.
Subject(s)
Blood Coagulation/drug effects , Platelet Aggregation Inhibitors/pharmacology , Vitamin E/analogs & derivatives , alpha-Tocopherol/analogs & derivatives , Adenosine Diphosphate/pharmacology , Alkaline Phosphatase/blood , Animals , Blood Proteins/metabolism , Creatinine/blood , Epinephrine/pharmacology , Male , Platelet Aggregation/drug effects , Rats , Rats, Inbred Strains , Tocopherols , Vitamin E/pharmacologyABSTRACT
It has been studied the in vitro direct action of alpha-tocopherol on osmotic fragility of red blood cells (RBCs) as well as its protective role in presence of two oxidizing drugs: phenylhydrazine hydrochloride or hydrogen peroxide. Incubation of RBCs with dl-alpha-tocopheryl acetate, at doses near to the plasma concentration in rat (6 micrograms/ml) did not modify the osmotic fragility of RBCs but increasing doses of tocopherol induces changes in osmotic fragility which directly parallelled the vitamin E concentrations. In addition, tocopherol did not protect RBCs from the hemolytic effect of the oxidizing drugs tested. Data from osmotic fragility after preincubation with dl-alpha-tocopheryl acetate and later incubation with these oxidizing drugs point to a possible toxic effect of higher dose vitamin E supplementation rather to a protective role on membrane stability.
Subject(s)
Erythrocytes/drug effects , Vitamin E/pharmacology , Animals , Erythrocytes/physiology , Hydrogen Peroxide/pharmacology , In Vitro Techniques , Male , Osmotic Fragility/drug effects , Phenylhydrazines/pharmacology , Rats , Rats, Inbred StrainsSubject(s)
Blood Coagulation Disorders/physiopathology , Phenylhydrazines/toxicity , Plasma/physiology , Animals , Blood Coagulation Disorders/chemically induced , Blood Platelets/physiology , Erythrocytes/physiology , Free Radicals , Male , Oxidation-Reduction , Platelet Aggregation , Rats , Rats, Inbred Strains , ThrombelastographyABSTRACT
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.
Subject(s)
Angiotensin II/antagonists & inhibitors , Glomerular Mesangium/drug effects , Somatostatin/pharmacology , Adenylate Cyclase Toxin , Angiotensin II/pharmacology , Angiotensin II/physiology , Animals , Cells, Cultured , Glomerular Filtration Rate/drug effects , Glomerular Mesangium/cytology , Glomerular Mesangium/metabolism , Myosins/metabolism , Pertussis Toxin , Phosphorylation , Rats , Rats, Wistar , Somatostatin/physiology , Virulence Factors, Bordetella/pharmacologyABSTRACT
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.
Subject(s)
Glomerular Mesangium/drug effects , Glomerular Mesangium/enzymology , Guanylate Cyclase/metabolism , Somatostatin/pharmacology , 3',5'-Cyclic-GMP Phosphodiesterases/antagonists & inhibitors , Animals , Cell Division/drug effects , Cells, Cultured , Cyclic GMP/metabolism , Enzyme Activation/drug effects , Glomerular Mesangium/metabolism , Purinones/pharmacology , Rats , Second Messenger Systems , Somatostatin/metabolism , Vasodilation/drug effectsABSTRACT
Although the cGMP-dependent relaxation of contractile cells seems to depend on the ability of the cyclic nucleotide to interfere with intracellular calcium, this does not appear to be the only mechanism involved. The present experiments were designed to analyse alternative mechanisms, trying to test the hypothesis that cGMP could relax rat mesangial cells by activating myosin light-chain phosphatase (MLC-PP), with the subsequent dephosphorylation of myosin light chain (MLC). The effect of a cGMP analogue, dibutyryl cGMP (dbcGMP), on angiotensin II-(AII) and PMA-induced MLC phosphorylation (MLCP) was tested, in the presence of calyculin A (CA), an inhibitor of MLC-PP. MLCP was measured, after cell labelling with (32)P, by immunoprecipitation. dbcGMP prevented the increased MLCP induced by AII or PMA, and this inhibition was blocked by CA. dbcGMP also increased the MLC dephosphorylation observed in cells incubated with AII and in which MLC kinase and protein kinase C activities were blocked. The AII-elicited increased intracellular calcium concentration was only partially inhibited by dbcGMP. These results suggest that the cGMP-induced mesangial-cell relaxation could be due, at least partially, to the stimulation of MLC-PP.
Subject(s)
Dibutyryl Cyclic GMP/pharmacology , Glomerular Mesangium/drug effects , Glomerular Mesangium/physiology , Phosphoprotein Phosphatases/metabolism , Angiotensin II/antagonists & inhibitors , Angiotensin II/pharmacology , Animals , Azepines/pharmacology , Calcium/metabolism , Calcium Signaling/drug effects , Cell Size/drug effects , Cells, Cultured , Dibutyryl Cyclic GMP/antagonists & inhibitors , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Glomerular Mesangium/cytology , Glomerular Mesangium/enzymology , Marine Toxins , Myosin Light Chains/metabolism , Myosin-Light-Chain Kinase/antagonists & inhibitors , Myosin-Light-Chain Kinase/metabolism , Myosin-Light-Chain Phosphatase , Oxazoles/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphorylation/drug effects , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Rats , Rats, Wistar , Staurosporine/pharmacology , Tetradecanoylphorbol Acetate/antagonists & inhibitors , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
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.
Subject(s)
Collagen Type I/metabolism , Extracellular Matrix/genetics , Fibroblasts/metabolism , Glomerular Mesangium/metabolism , Transforming Growth Factor beta/metabolism , Cells, Cultured , Collagen Type I/pharmacology , Collagen Type IV/metabolism , Collagen Type IV/pharmacology , Extracellular Matrix Proteins/biosynthesis , Extracellular Matrix Proteins/genetics , Fibroblasts/drug effects , Glomerular Mesangium/cytology , Glomerular Mesangium/drug effects , Humans , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Transforming Growth Factor beta/biosynthesis , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta1 , Tyrosine/metabolism , Up-Regulation/drug effects , Up-Regulation/geneticsABSTRACT
The present study was designed to evaluate the influence of two haemodialysis membranes of different biocompatibility on red blood cell function. Twelve patients were studied in two consecutive dialyses, with cuprophan and cellulose acetate. Blood was extracted at 0, 20 and 180 min after the beginning of the haemodialysis session and general haematological parameters, osmotic fragility, deformability, methaemoglobin concentration and malonyldialdehyde (MDA) red blood cell content were determined. Osmotic fragility improved with both membranes, but this improvement was more marked with cellulose acetate. MDA red blood cell content showed a tendency to increase after 3 h with cuprophan (114 +/- 11% of the basal value), whereas it tended to decrease with cellulose acetate (92 +/- 12%), the differences between the two groups being statistically significant. These results suggest that red cell function may improve by changing the characteristics of haemodialysis membranes. This phenomenon could be related to a better biocompatibility.
Subject(s)
Cellulose/analogs & derivatives , Erythrocytes/physiology , Kidneys, Artificial , Adult , Biocompatible Materials , Erythrocyte Deformability/drug effects , Female , Hematocrit , Hemoglobins/analysis , Hemolysis/drug effects , Humans , Male , Malondialdehyde/blood , Membranes, Artificial , Methemoglobin/metabolism , Middle Aged , Osmotic FragilityABSTRACT
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.
Subject(s)
Kidney/physiopathology , Pancreatitis/physiopathology , Somatostatin/therapeutic use , Acute Disease , Animals , Bile Acids and Salts/administration & dosage , Disease Models, Animal , Kidney/drug effects , Male , Pancreatitis/blood , Pancreatitis/drug therapy , Rats , Rats, Wistar , Renal CirculationABSTRACT
The effects of reactive oxygen species (ROS) on cultured rat mesangial cells were studied by measuring planar cell surface area (PCSA) after incubation with xanthine plus xanthine oxidase (XXO), in the presence of superoxide dismutase (SOD; 5 micrograms/ml) or catalase (CAT; 20 micrograms/ml), or after incubation with H2O2. Myosin light chain (MLC) phosphorylation was assessed in cells prelabeled with o-[32P]phosphoric acid and incubated with H2O2, after protein separation with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A possible intermediate role for platelet-activating factor (PAF) was analyzed by preincubation of the cells with a PAF antagonist BN 52021 (BN, 5 x 10(-5) M) and by measuring PAF-specific [3H]acetate incorporation and immunoassayable PAF. XXO significantly decreased PCSA (14%), an effect abolished by CAT but not by SOD. H2O2 induced a similar effect, in a dose-dependent and time-dependent manner. MLC phosphorylation increased by 81 +/- 15% after H2O2 incubation, and this effect was blocked by BN. BN also completely blocked the effect of H2O2 on PCSA. PAF-specific [3H]acetate incorporation increased in the presence of H2O2 (from 6,886 +/- 2,030 to 58,703 +/- 16,063 counts.min-1.mg-1) as well as the immunoassayable PAF production by cells (from 0.90 +/- 0.19 to 6.71 +/- 2.27 ng/mg). These results suggest that ROS, particularly H2O2, could modulate the surface area of mesangial cells, modifying the ultrafiltration coefficient, thus explaining the decrease in glomerular filtration rate in those pathological situations characterized by an increased ROS synthesis. PAF could be involved in the genesis of these effects.
Subject(s)
Glomerular Mesangium/drug effects , Kidney Glomerulus/drug effects , Reactive Oxygen Species/pharmacology , Animals , Cells, Cultured , Glomerular Mesangium/cytology , In Vitro Techniques , Myosins/metabolism , Phosphorylation , Platelet Activating Factor/physiology , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Dinoprostone/biosynthesis , Hydrogen Peroxide/pharmacology , Kidney Glomerulus/metabolism , Platelet Activating Factor/metabolism , Thromboxane B2/biosynthesis , Animals , Cells, Cultured , Rats , Rats, WistarABSTRACT
Erythrocytes were incubated for 60 min either in plasma or phosphate-buffered saline containing 10 mM phenylhydrazine hydrochloride. Plasma prevented the decrease in membrane fluidity observed in saline-phenylhydrazine incubated erythrocytes but these cells showed decreases in both filterability and active extrusion of Na+ that were nearly 100% lower than those found in erythrocytes incubated in plasma-phenyl-hydrazine. Also erythrocyte lipid peroxidation, as measured as thiobarbituric acid reactive products, was 100% higher in the presence of plasma. These results suggest that plasma could play "in vivo" an active role during oxidant-induced erythrocyte damage, contributing significantly to the hemolytic effects of oxidation.