Kinase-dependent, retinoic acid receptor-independent up-regulation of cyclooxygenase-2 by all-trans retinoic acid in human mesangial cells.

BACKGROUND AND PURPOSE: Preliminary results in human mesangial cells (MC) suggested that all-trans retinoic acid (ATRA) increased the expression of COX-2 and the production of prostaglandin E2 (PGE2), a PG with anti-inflammatory effects in MC. The aim of this work is to confirm that ATRA increases the expression of COX-2 in MC and to examine the mechanisms involved. EXPERIMENTAL APPROACH: Cultured MC were treated with ATRA. COX expression and kinase activity were analyzed by Western blot. Transcriptional mechanisms were analyzed by Northern blot, RT-PCR and promoter assays. KEY RESULTS: COX-2 and COX-1 expression and PGE2 production were increased by ATRA. COX-2 played a role in PGE2 production as production was only partially inhibited by COX-1 inhibitor SC-560. COX-2 up-regulation by ATRA was due to transcriptional mechanisms as pre-incubation with actinomycin D abolished it and ATRA increased the expression of COX-2 mRNA and the activity of a human COX-2 promoter construct, whereas post-transcriptional mechanisms were not found. Retinoic acid receptors (RAR) were not involved in the up-regulation of COX-2 by ATRA since it was not inhibited by RAR-pan-antagonists and the RAR-pan-agonist TTNPB did not up-regulate COX-2. Instead ATRA might act through a sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) since up-regulation of COX-2 was prevented by inhibition of the activation of ERK1/2 with PD098059. Also ERK1/2, as well as downstream signalling proteins from ERK1/2, remained phosphorylated when COX-2 increased 24 h later. CONCLUSIONS AND IMPLICATIONS: These results highlight the relevance of RAR-independent mechanisms to the biological effects of ATRA.

Retinoids as a potential treatment for experimental puromycin-induced nephrosis.

1 Puromycin aminonucleoside (PAN)-induced nephrosis is a model of human minimal change disease. In rats, PAN induces nephrotic-range proteinuria, renal epithelial cell (podocyte) damage, infiltration of mononuclear leukocytes, and apoptosis of several renal cell types. 2 Retinoic acid (RA) modulates a wide range of biological processes, such as inflammation and apoptosis. Since renal damage by PAN is characterized by inflammatory infiltration and epithelial cell death, the effect of treatment with all-trans RA (tRA) was examined in the PAN nephrosis model and in the cultured differentiated podocyte. 3 Treatment with tRA 4 days after PAN injection did not inhibit the proteinuria peak but reversed it significantly. However, treatment with tRA both before and 2 days after the injection of PAN protected the glomerular epithelial cells, diminishing the cellular edema and diffuseness of the foot process effacement. Preservation of the podocyte architecture correlated with the inhibition of proteinuria. The anti-inflammatory effect of tRA was evidenced by the inhibition of PAN-induced interstitial mononuclear cell infiltration and the decreased renal expression of two molecules involved in monocyte infiltration: fibronectin and monocyte chemoattractant protein-1. TUNEL assays showed that tRA inhibited the PAN-induced apoptosis of cultured differentiated mouse podocytes. 4 We conclude that tRA treatment may prevent proteinuria by protecting the podocytes from injury and diminishing the interstitial mononuclear infiltrate in the model of PAN nephrosis. Retinoids are a potential new treatment for kidney diseases characterized by proteinuria and mononuclear cell infiltration.

Phenotypic modifications of human mesangial cells by extracellular matrix: the importance of matrix in the contractile response to reactive oxygen species.

The progression of chronic renal diseases is characterized by the accumulation of extracellular matrix proteins in the glomerulus. The present experiments were designed to analyze the effect of hydrogen peroxide on the contractile and proliferative phenotypes of human mesangial cells grown on different culture substrates: plastic, collagen type I, and collagen type IV. Contraction was analyzed by measuring planar cell surface area and myosin light chain phosphorylation, whereas proliferation was studied by [(3)H]thymidine incorporation. No changes were detected in the proliferation rate of human mesangial cells grown on different culture substrates, neither under basal conditions nor in the presence of fetal calf serum or H(2)O(2). Cells grown on plastic or collagen did not contract in the presence of H(2)O(2), but cells grown on collagen I elicited a significant contraction with H(2)O(2). Platelet-activating factor induced contraction of human mesangial cells on the three culture substrates. The different contractile responses observed were not due to different degradation rates of H(2)O(2). The present experiments support the importance of extracellular matrix in the response to exogenous stimuli and point to the possibility that patients with changes in the mesangial matrix as a result of chronic renal diseases may have an increased susceptibility to the pathological actions of reactive oxygen species.

Human renal mesangial cells are a target for the anti-inflammatory action of 9-cis retinoic acid.

Mesangial cells play an active role in the inflammatory response to glomerular injury. We have studied in cultured human mesangial cells (CHMC) several effects of 9-cis retinoic acid (9-cRA), an activator of both retinoic acid receptors (RARs) and retinoid X receptors (RXRs). 9-cRA inhibited foetal calf serum-induced CHMC proliferation. It also prevented CHMC death induced by the inflammatory mediator H(2)O(2). This preventive effect was not due to any increase in H(2)O(2) catabolism and it persisted even when both catalase and glutathione synthesis were inhibited. Finally, 9-cRA diminished monocyte adhesion to FCS-stimulated CHMC. Interestingly, the retinoid also inhibited in FCS-stimulated cells the protein expression of two mesangial adhesion molecules, fibronectin and osteopontin, but it did not modify the protein expression of intercellular adhesion molecule-1 and vascular adhesion molecule-1. All major RARs and RXRs isotypes were expressed in CHMC regardless of the presence or absence of 9-cRA. Transcripts to RAR-alpha, RAR-beta and RXR-alpha increased after incubation with 9-cRA whereas RXR-gamma was inhibited, suggesting a major role for RARs and RXRs in 9-cRA-anti-inflammatory effects. 9-cRA was toxic only at 50 microM (a concentration 50 - 5000 times higher than required for the effects above). Cell death occurred by apoptosis, whose onset was associated with a pronounced increase in catalase activity and reduced glutathione content, being more effectively induced by all-trans retinoic acid. Modulation of the oxidant/antioxidant balance failed to inhibit apoptosis. We conclude that mesangial cells might be a target for the treatment of inflammatory glomerulopathies with 9-cRA.

Tretinoin prevents age-related renal changes and stimulates antioxidant defenses in cultured renal mesangial cells.

Age-related progressive glomerular sclerosis in the rat is associated with increased expression of tumor necrosis factor-beta1 and increased protein content in the renal cortex, enhanced production of H2O2, in both renal glomeruli and mesangial cells (MCs) cultured from them, as well as augmented glomerular oxidative damage. We have previously shown that tretinoin-treated old male Fischer 344 rats have 30% lower protein content in the renal cortex than control old rats. Here, we report that this effect may depend on the inhibition of the expression of tumor necrosis factor-beta1, a matrigenic cytokine, and osteopontin, a protein with cell adhesive and chemotactic properties. In addition, we show that tretinoin prevents the cytotoxicity of H2O2 in cultured human MCs by increasing both the catalase activity and the reduced glutathione content, which are dose- and time-dependent changes. These increases were not dependent on each other: when these effects were previously inhibited with 3-amino-1,2,4-atriazole or L-buthionine-(S, R)-sulfoximine, respectively, tretinoin still induced the increase of the other noninhibited antioxidant defense. An enhanced gene transcription is the most likely mechanism involved in the tretinoin-induced stimulation of MC antioxidant defense systems because 1) preincubation of MCs with actinomycin D or cycloheximide fully abolished it; 2) tretinoin-incubated MCs showed increased levels of catalase mRNA and gamma-glutamyl-cysteine synthetase (catalytic subunit) mRNA, the latter being the rate-limiting step in de novo reduced glutathione synthesis; and 3) the stability of both mRNA was unchanged by tretinoin. These results show one strategy of protecting renal cells from H2O2-mediated injury based on increasing their antioxidant defenses.

Reactive oxygen species and platelet-activating factor synthesis in age-related glomerulosclerosis.

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.

Modulation of Na-H antiporter activity in human lymphoblasts by altered membrane cholesterol.

The effects of changes in membrane cholesterol on Na-H antiporter activity in culture human lymphoblasts are described. Lymphoblast cholesterol alteration was achieved with liposomes of phosphatidylcholine (cholesterol depletion) or phosphatidylcholine plus cholesterol (cholesterol enrichment). Lymphoblast intracellular pH (pHi) was examined by fluorimetry using cells loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)5(6)-carboxyfluorescein, and the Na-dependent proton efflux rate at a pHi of 6.0 was taken as the maximum velocity of the Na-H antiporter. Lymphoblast membrane cholesterol depletion activated the Na-H antiporter, and enrichment of membrane cholesterol caused inhibition of the antiporter activity. This study demonstrates that in situ modification of membrane cholesterol can modulate the activity of the Na-H antiporter.

In vitro effect of plasma during phenylhydrazine-induced erythrocyte oxidative damage.

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.

Effects of dietary hyperlipidemia-hypercholesterolemia on rat erythrocytes.

We report in the present work that membrane fluidity in erythrocytes from rats under alimentary hyperlipidemia-hypercholesterolemia is slightly diminished despite the fact that in these cells the cholesterol/phospholipid molar ratio is nearly 45% higher than in erythrocytes from control animals. In addition, the erythrocyte count in the rats given the high-fat, high-cholesterol diet was normal. Only a slight reticulocytosis was observed in these animals although their erythrocytes showed a marked decrease in their ability to extrude sodium actively. A tentative explanation for these results is proposed on the basis of other findings suggesting cellular compensatory mechanisms.

dl-alpha-Tocopheryl acetate induces hypocoagulability and platelet hypoaggregability in rats.

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.

In vitro response of erythrocytes to alpha-tocopherol exposure.

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.