My game closes on start up or gives me an error message that I'm missing a file. Remember to extract the RAR file with an unzipping program like WinRAR,. I know about the joyful route and the rodriguez route, but that's pretty much all. To unlock one ending, respond to Lanks by picking all of the TOP (first).
9-cRA inhibited foetal calf serum-induced CHMC proliferation. It also prevented CHMC death induced by the inflammatory mediator H 2O 2. This preventive effect was not due to any increase in H 2O 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. Introduction Glomerular diseases remain the most common cause of progressive renal failure that may require dialysis or kidney transplantation.
Remarkable recent advances in our understanding of the pathogenesis of various forms of glomerulonephritis have made it obvious that glomerular cells and especially mesangial cells play an active part in the inflammatory response to glomerular injury. For instance, these cells contribute to phagocyte infiltration of the glomerulus during glomerular inflammation since they express cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (;, ) or secrete osteopontin (OP) , a glycoprotein with cell adhesive properties which plays a role in macrophage infiltration (reviewed by; ), and fibronectin (FN) , a major glomerular matrix component which has been shown to mediate monocyte accumulation in the damaged kidney. Furthermore, the mesangial cells constitute a highly specialized pericapillary tissue and they have evolved three prominent proinflammatory features as a result of the cross-communication with invading immune cells : (1) increased inflammatory mediator production; (2) increased matrix production by mesangial cells; and (3) increased mesangial cell proliferation. Interestingly, mesangial cells may also contribute to the repair process of experimental proliferative glomerulonephritis through a reduction of hypercellularity by means of the process of apoptosis. We have recently shown that all-trans-retinoic acid (tRA) inhibits OP mRNA expression in cultured human mesangial cells (CHMC) and prevents the cytotoxicity of H 2O 2–one of the mediators implicated in the pathophysiology of inflammatory renal diseases –in CHMC, in parallel to an increase in catalase activity and reduced glutathione content (GSH). There is now strong evidence that two families of nuclear retinoic acid receptors, RAR-α, -β, -γ and RXR-α, -β, -γ mediate the effects of retinoids (reviewed by ).
TRA only activates RARs whereas 9-cis retinoic acid (9-cRA) is a pan-agonist and it activates both RARs and RXRs (; ). Thus 9-cRA may lead to a broader or a different spectrum of anti-inflammatory activity than tRA. Studies were therefore conducted to explore the ability of 9-cRA: (1) to modulate mesangial cell number through antiproliferative mechanisms; (2) to inhibit monocyte adhesion to CHMC; (3) to inhibit mesangial expression of molecules involved in monocyte adhesion to CHMC; and (4) to prevent the toxic effect of H 2O 2 on CHMC, which may be secreted by activated macrophages. To gain insight on the role of RARs and RXRs in the observed effects, we also studied the modulatory effect of 9-cRA on mRNA expression of these receptors. Finally, we studied retinoid toxicity on CHMC.
Cell culture Human mesangial cells were obtained from adult specimens, as we have previously described. The identity of the cells was confirmed by morphologic and functional criteria. Under phase-contrast microscopy all the cells appeared large and stellate and no cells with epithelial or endothelial morphologic characteristics were seen. Mesangial cells showed histochemical evidence of containing actomyosin fibres, and they did not stain for factor VIII, unlike endothelial cells.
In addition all the cells examined contracted after incubation with platelet activating factor, angiotensin II and arginine-vasopressin. Cultured medium was made of RPMI 1640 supplemented with 10% foetal calf serum (FCS), 200 m M L-glutamine and antibiotics (penicillin 100 u ml −1, streptomycin 100 μg ml −1 and amphotericin B 0.25 μg ml −1). Confluent cells between 12th and 15th passages were used and they were made quiescent when appropriate by 48 h incubation with medium supplemented with 0.5% FCS. Experimental design 9-cRA was dissolved in ethanol and appropriate controls consisting of either vehicle (final concentration 0.09%) or medium were always included (when necessary, retinoid effects were first tested in dose-response and time-response experiments (in some experiments tRA effects were also tested)).
Studies performed were: (1) Modulation of cell number. We assessed the inhibitory effect of 9-cRA on FCS-stimulated mesangial cell proliferation by three different methods: direct cell count3H-thymidine incorporation and reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). We also studied retinoid toxicity.
These later studies were mainly performed with tRA (since it was a stronger inducer of apoptosis than 9-cRA) and they consisted of assessment of the loss of viable cells, identification of apoptotic death and changes in antioxidant defence systems such as catalase activity and reduced glutathione content; (2) Prevention of H 2O 2-induced cell death. Quiescent CHMC in 96-well plates (typically 20,000 cells per well) were preincubated either with 9-cRA or tRA for 24 h and then incubated with H 2O 2 for 24 h.
H 2O 2-induced cytotoxicity was quantified, as previously described , by measuring LDH efflux from the cytosol of the damaged cells. Since tRA stimulates antioxidant defences , additional experiments were performed to compare the relative potency of tRA and 9-cRA in terms of maximal stimulation of the GSH system and to confirm if prevention was really due to increased H 2O 2 catabolism (by measuring the rate of comsuption of H 2O 2 exogenously added to CHMC preincubated for 24 h with the either 9-cRA or tRA); (3) Inhibition of monocyte adhesion to CHMC. Experiments to study the inhibition by 9-cRA of monocyte adhesion to CHMC stimulated with 20% FCS were performed as follows: confluent CHMC were incubated for 24 h in 20% FCS containing 9-cRA or vehicle. Cells were then washed and human monocytes, isolated from blood from healthy donors, were added to CHMC. After coincubation for 2 h, the adhesion of monocytes to CHMC was quantified as described below; (4) Inhibition of the expression of FN, OP, ICAM-1 and VCAM-1. Experiments similar to those described above were performed. At definite time intervals along the incubation period with 9-cRA, total RNA or protein was extracted and the expression of the mentioned molecules was assessed by Northern and Western blot techniques; (5) Expression of mRNA for RARs and RXRs in mesangial cells and its modulation by 9-cRA.
Retinoid effects were tested in time-response experiments, using a concentration of 1 μ M to ensure a maximal response. At definite time intervals along the incubation period, total RNA was extracted and the mRNA expression of the mentioned molecules was assessed by Northern blot techniques.
Measurement of cell proliferation Cells at ≈80% confluence in 96-well plates (MTT assay) or in 24-well plates ( 3H-thymidine incorporation) or in 6-well plates (direct cell count) were incubated in RPMI 1640 medium +0.5% FCS for 48 h to induce quiescence. Cells were then preincubated for 2 h with the retinoids, and then with 20% FCS for 24 h.
Monolayers were then tested for the ability to reduce exogenous 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan or to incorporate 3H-thymidine. Each experimental condition was assayed in quadruplicate. The MTT assay was performed in cells incubated for 6 h with MTT (0.15 μg ml −1). The blue formazan product was solubilized with 0.04 M HCl in isopropanol and the plates were then read on a microELISA reader using a test wavelength of 595 nm.
Alternatively, treated cells were incubated for 6 h with 3H-thymidine (1 μCi well −1, 10 Ci mmol −1, American Radiolabeled Chemicals, St Louis, MO, U.S.A.) and, after removing medium, cells were washed three times with ice-cold phosphate-buffered saline. Then ice-cold 10% trichloroacetic acid was added to remove acid-soluble materials and, 5 min later, it was removed. Cells were then solubilized overnight in 0.1 M NaOH and subjected to liquid scintillation counting. Finally, direct cell counting was performed on trypsinized cells from 6-well plates, non-viable cells (Trypan blue analysis) were excluded from the count.
Apoptosis assays (1) Microscopic analyses: Morphologic examination was performed by light and fluorescent microscopy. Cells for light microscopy were fixed using 10% buffered formaline and stained for 60 min with Giemsa reagent. Cells were also examined under fluorescent microscopy, after staining with acridine orange (4 μg ml −1) for 10 min. Apoptosis was identified using morphological criteria including pycnotic nuclei, marked chromatin condensation, nuclear fragmentation into spherical structures, and apoptotic bodies. We used as a positive control, mesangial cells cultured without foetal calf serum for 6 days, and as a negative control mesangial cells cultured with 15% of foetal calf serum. (2) In situ detection of apoptosis: Mesangial cells were washed and fixed during 20 min at room temperature with 4% formaldehyde containing phosphate-buffered saline. Then, in situ end labelling (TUNEL) was performed by using the In situ cell death detection kit (Boehringer Mannheim) according to the manufacturer's instructions.
(3) Trypan blue analysis: Cells were trypsinized, stained with trypan blue dye and total cell count was performed in a Neubauer chamber. Assays were repeated four times. (4) Measurement of antioxidant defences: (i) Catalase activity : Sample was diluted in phosphate buffer (KH 2PO 4 50 m M, pH 7.0) containing 0.2% Triton-X-100 and 2 ml were added to 1 ml of 30 m M H 2O 2. Changes in absorbance at 240 nm were measured for 30 s. The rate constant of a first-order reaction (k) was used as a unit by the equation.
Where t 2− t 1 is the measured interval in seconds and A 1 and A 2 are the absorbances at initial and final measurement points, respectively. Results were corrected to cellular protein content : (ii) Reduced glutathione content: Cells were lysed in a cold room and proteins were precipitated with 0.9 ml perchloric acid. After neutralization with 0.3 ml 1 M KOH/KHCO 3 and centrifugation, 0.15 ml of supernatant were collected on plastic tubes, o-phtal-dialdehyde was added (0.15 ml of a 7.46 μ M solution) and tubes were incubated at room temperature for 15 min. GSH was measured in a scanning fluorescence spectrophotometer (Perkin-Elmer model LS-5B) at 420 nm with excitation wavelength of 350 nm, using a standard curve. Results were corrected for cellular protein. H 2O 2 assay Consumption of H 2O 2 was measured by the horseradish peroxidase-mediated oxidation of phenol red , as described for renal tissue : H 2O 2 (100 μ M final concentration) was added to CHMC and, at the appropriate times, 40 μl of medium were removed and added to 1.12 ml of the phenol red solution (0.1 g phenol red l −1), 140 m M NaCl, 10 m M potassium phosphate buffer and 5.5 m M dextrose) with 40 μl of horseradish peroxidase (1.5 g/l). The cuvettes were incubated for 5 min at room temperature and brought to pH 12.5 by the addition of 72 μl of 1 N NaOH.
Absorbance was read at 610 nm against an H 2O 2-standard curve. Human monocyte isolation and adhesion assays Human monocytes were isolated from 25 ml heparinized blood from healthy donors. In brief, mononuclear cells were prepared by Ficoll-Hypaque density gradient centrifugation (400× g, 30 min), washed and seeded on plastic culture plates (Falcon, U.S.A.) containing RPMI1640 medium and 20% FCS in 5% CO 2/95% air atmosphere in order to enrich the sample in monocytes by 1 h adherence to culture plates.
This procedure gave a purity 90% as determined by flow cytometry using CD14 antibodies as an specific monocyte-macrophage marker , and a viability 95% as detected by trypan blue exclusion. Monocyte adhesion to CHMC was tested by a previously described method to study monocyte/endothelial interactions, with several modifications.
Enriched monocytes (800,000 ml −1) in complete RPMI 1640 medium were layered onto confluent monolayers of CHMC in 35 mm Petri plates (total volume: 400 μl per plate) and cells were co-incubated at 37°C for 2 h (this time was chosen on the basis of preliminary time-course experiments). The suspension was then aspirated, and non-adherent monocytes were dislodged by gently rinsing the monolayers three times with sterile phosphate-buffered saline (pH 7.3). Cells were fixed with methanol/ethanol 1: 1 (v v −1) for 30 min on ice and then stained with Giemsa during 3 h. These experiments were performed in triplicate plates for every experimental condition. Under light microscopy (×100), CHMC grew in monolayers and showed a characteristic stellate appearance with smooth peripheral borders, whereas monocytes exhibited a round profile, with little presence of cytoplasm and a pycnotic nucleus, and a cell size around 3–5 times smaller than CHMC. Identification of these cells as monocytes was confirmed by staining for nonspecific esterase. Counts of adherent monocytes was performed by three independent observers in a double-blind fashion (every observer selected in a random way 10 microscopic fields in every plate).
Northern blots mRNA levels were measured in total RNA extracts isolated from CHMC. Total RNA was electrophoresed in a denaturing 1% agarose gel, transferred to a nylon membrane and probed with 32P-labeled-probes. The filters were washed at room temperature twice with 2× standard saline citrate (1× standard saline citrate: 0.15 M NaCl, 0.015 M Na 3-citrate, pH 7.0), at 65°C twice with 1× standard saline citrate containing 1% sodium dodecylsulfate and at 65°C twice with 0.1× standard saline citrate containing 0.1% sodium dodecyl sulphate. As a loading control, blots were also probed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. The blots were exposed to film (Kodak, XOMAT) or visualized in a Phosphorimager (Molecular Dynamics, Sunnyvale, CA, U.S.A.).
Inhibition of cell proliferation We asked if 9-cRA would inhibit CHMC proliferation. We therefore added 20% FCS to quiescent CHMC preincubated or not with 9-cRA. FCS added alone stimulated a 15–18% increase in MTT reduction and cell count increased from 335,209±18,204 cells per well to 381,204±23,326 cells per well ( n=3).
9-cRA caused dose-dependent inhibition of MTT reduction within a narrow dose range: threshold inhibition was found at 10 n M 9-cRA and maximal inhibition of FCS-stimulated MTT reduction occurred at 100 n M 9-cRA (cell count was 346,207±21,409 cells per well). In a similar way, maximal inhibition of FCS-stimulated 3H-thymidine incorporation occurred at 100 n M 9-cRA (the per cent of inhibition was 61±5, n=3) Maximal inhibition by tRA (the per cent of inhibition was 69±8, n=3) was of similar magnitude to that found with 9-cRA but the inhibitory threshold was found to be 5 μ M, that is to say 500 fold higher than the threshold for 9-cRA antiproliferative effect.
9-cRA at 1 n M to 25 μ M neither reduced mesangial cell viability as judged by trypan blue dye exclusion nor induced any apoptosis in the range within the range of concentrations tested for the proliferation studies (results are not shown). Inhibition by 9-cRA of FCS-stimulated mesangial cell proliferation (measured as MTT reduction). Quiescent mesangial cells were incubated for 2 h with 9-cRA. FCS (20%) or an equivalent amount of medium (to obtain the basal values of every experimental condition) was then added and cells were incubated for 24 h. Cell proliferation was quantified by measuring the ability of monolayers to reduce exogenous MTT to formazan after 6 h incubation (as described in Methods). In control cells, the difference between the absorbances corresponding to 20% FCS-stimulated cells versus 0.5% FCS-quiescent cells was considered as 100%.
In 9-cRA treated cells, the difference between absorbances found in 20% FCS-stimulated cells versus 0.5% FCS cells was obtained and then results were expressed as per cent of change with respect the 100% established for control cells. There were no statistically significant differences between the absorbances found in 0.5% FCS cells in the various experimental conditions tested. Results are the mean±s.d of three separate experiments in quadruplicate. ANOVA,. P.
Effects of high concentrations of retinoids on the morphology and viability of mesangial cells We first performed microscopic examination of whether occurrence of apoptosis after treatment with retinoids occurred in CHMC or not. Because of the absence of definitive molecular markers, morphologic examination is the only way to be sure that cells are undergoing apoptosis.
Before treatment, the mesangial cells grew in monolayers until confluence was reached. After exposure to 25 μ M tRA or 50 μ M 9-cRA for 24 to 72 h, however, significant numbers of cells showed the typical morphology of apoptosis including pycnotic nuclei, marked chromatin condensation, nuclear fragmentation into spherical structures, and apoptotic bodies (, left side, upper panels). Such cells also exhibited condensed chromatin acridine orange dye in contrast to the control (, left side, lower panels). We also assessed retinoid-induced apoptotic cell death in mesangial cells by TUNEL determination of DNA fragmentation. The number of TUNEL-positive cells – this indicating the occurrence of DNA fragmentation – was significantly increased by incubation of cells for 24 to 72 h either in the presence of 50 μ M 9-cRA or in the absence of FCS.
Again 50 μ M 9-cRA produced similar effects than 25 μ M tRA (results not shown). Few positive cells were observed when cells were cultured in the presence of 20% FCS, and this was a consistent background level of apoptosis. Given that tRA was a stronger apoptosis inductor than 9-cRA, most of the remaining studies were performed with tRA. To quantify the effect of tRA on mesangial cell death, we incubated cells for various intervals of time with 25 μ M tRA and then measured both total cell count and cell viability: the incubation of mesangial cells with tRA resulted in loss of viable cells (, right side). Retinoid-induced apoptosis in CHMC. (A) Effect of high concentrations of tRA on the morphology and viability of CHMC. The left panel shows light microphotographs (upper panels) where apoptotic mesangial cells exhibits nuclear fragmentation into spherical structures and condensed cytoplasm, but they are still surrounded by a cell membrane, and fluorescent microphotographs (lower panels) where several nuclei with condensed chromatin are observed.
The right panel shows the decrease in viable cells (as assessed by direct count under light microscopy of cells excluding trypan blue dye) after 24 h incubation with 25 μ M tRA (Student's t-test. P. Time course profile of catalase activity and reduced glutathione content during tRA-induced apoptosis We have previously found that non-apoptotic concentrations of tRA stimulate catalase activity and increase reduced glutathione content in a concentration-dependent, time-dependent manner.
Here we found that when tRA is added at doses high enough to induce apoptosis, there is also an increase in the antioxidant defences coinciding with the development of the apoptotic cell death. We then studied whether there was a cause-effect relationship by exploring two opposite possibilities: first, that the increase in the antioxidant defences is a defensive cellular response to oxidative stress, which is relevant since oxygen free radicals are involved in several models of apoptosis.
However, preincubation with the antioxidant dl-α-tocopherol, or with catalase, which specifically detoxifies H 2O 2, or with with the cell-permeable O 2 − scavenger tiron , did not inhibit tRA-induced CHMC apoptotic death. Secondly we studied the possibility that catalase and/or GSH were directly involved in the apoptotic signalling. Again, tRA-triggered apoptosis was not inhibited by aminotriazol, an irreversible inhibitor of catalase activity , or by L-buthionine-(S-R)-sulphoximine (BSO), an irreversible inhibitor of γ-glutamylcysteine synthetase, the rate-limiting step of glutathione synthesis.
Prevention of H 2O 2-induced cell death H 2O 2 induces concentration-dependent damage in mesangial cells, characterized by apoptosis followed by secondary necrosis with plasma membrane damage resulting in loss of cytosolic LDH. H 2O 2-induced LDH release is clearly abolished by tRA, within a narrow concentration range: 10 μ M tRA has the maximal protective action against H 2O 2 and 1 μ M tRA has no effect at all. Shows the results obtained when using retinoids at concentrations high enough to reach maximal prevention against H 2O 2. It is clear that maximal prevention with 9-cRA requires a 1000 fold lower concentration than that required with tRA.
Since H 2O 2-induced cell death is apoptotic , we also confirmed by morphological examination with acridine orange-ethidium bromide staining that H 2O 2-induced apoptosis was suppressed by pretreatment with retinoids (results are not shown). (A) Prevention by retinoids of H 2O 2-induced cytotoxicity. Confluent, quiescent CHMC cultured in 96-well microtiter plates, were incubated for 24 h with either 10 n M 9-cRA or 10 μ M tRA. Cells were then washed and incubated for 24 h with either 25 or 100 μ M H 2O 2.
Control cells were incubated in parallel but without adding retinoids and H 2O 2. Cytotoxicity was quantified by measuring LDH efflux from the cytosol of the damaged cells to the supernatant. Results were expressed as the per cent of the control value (LDH released by control cells incubated with 2% Triton-X-100 was considered as 100% LDH efflux) and are mean±s.d. Of three separate experiments in quadruplicate. As shown, 10 n M 9-cRA and 10 μ M tRA prevented in a similar extension the H 2O 2-induced loss of cell viability (. P0.05).
Additional experiments were performed to assess if this protective effect was due to retinoid-induced stimulation of antioxidant defences. This possibility was raised from previous results showing that catalase activity and GSH content are increased by 2.5 fold in tRA-incubated CHMC. We studied first the relative potency of tRA and 9-cRA in terms of maximal stimulation of the GSH system. Time-course experiments have previously shown that a 48 h incubation with 10 μ M tRA is needed to reach a maximal increase in the intracellular GSH content from CHMC. We have studied here the relative potency of the two retinoids and found in four separate experiments in triplicate that, after a 48 h incubation with 10 μ M of either 9-cRA or tRA, the respective intracellular GSH contents (nmol mg −1 protein) were 68+5 and 53+4 (results are mean±s.d.; Student's t-test: P. Inhibition of monocyte adhesion to CHMC The co-culture of monocytes with the FCS-stimulated CHMC resulted in time-dependent adhesion of monocytes to the underlying mesangial monolayer.
Adhesion was substantially inhibited when CHMC were pretreated with 9-cRA but within a narrow concentration range: maximal inhibition was found at 1 μ M and the threshold inhibition appeared at 10 n M (results are not shown). Although we do not show the results, we found that tRA inhibited adhesion to a similar extension. Inhibition by retinoids of the adhesion of monocytes to cultured human mesangial cells.
Confluent CHMC were incubated for 24 h in 20% FCS containing 1 μ M 9-cRA or vehicle. Cells were then washed and human monocytes, isolated from blood from healthy donors as described in Methods, were added to CHMC. After coincubation for 2 h, the adhesion of monocytes to CHMC was quantified as described in Methods. Data are mean±s.d. Of three different experiments in triplicate.
The actual value for 100% adhesion was 71±8 monocytes per well. P.
![Best rar extractor windows Best rar extractor windows](/uploads/1/2/5/3/125352749/787245610.png)
Changes in the CHCM expression of FN, OP, ICAM-1 and VCAM-1 mRNA expression We also studied the effect of 9-cRA on the mRNA and protein expression of four molecules which are involved in the adhesion of monocytes to mesangial cells: FN, OP, ICAM-1 and VCAM-1. These experiments were performed using identical experimental conditions to those used in the study of monocyte adhesion to CHMC, excepting that cells were stimulated with 10% FCS instead of 20% FCS. Results obtained may be classified into two categories: those related to molecules secreted by CHMC (i.e., FN and OP) and those involving cell adhesion molecules present in the cell surface of mesangial cells (i.e., ICAM-1 and VCAM-1). 9-cRA inhibited OP and FN mRNA and protein expression ( and ) in FCS-stimulated mesangial cells. On the contrary, 9-cRA had no effect on the ICAM-1 mRNA expression (data not shown) nor on the ICAM-1 protein expression. Finally, 9-cRA stimulated VCAM-1 mRNA expression (data not shown) but this effect did not result in increased VCAM-1 protein expression , this being probably due to the fact that post-transcriptional mechanisms may be relevant determinants of VCAM-1 protein expression (; ). Although we do not show the results, tRA had similar effects on the expression of the four adhesion molecules.
Effect of 9-cRA on osteopontin and fibronectin mRNA expression in human mesangial cells. Confluent human mesangial cells were incubated up to 24 h in 10% FCS with 1 μ M 9-cRA and total RNA was extracted. Northern blotting for OP (A) and FN (B) was carried out as described in Methods. The blots were stripped and rehybridized with a cDNA probe for the housekeeping gene GAPDH to adjust for small variations in RNA loading and transfer to the filter. The autoradiograms shown are representative of three separate experiments. Effect of 9-cRA on the protein expression of osteopontin, fibronectin, VCAM-1 and ICAM-1 in human mesangial cells. Confluent human mesangial cells were incubated up to 24 h in 10% FCS with 1 μ M 9-cRA and total protein was extracted.
Twenty μg of every protein extract was electrophoresed and transferred onto nitrocellulose. Membranes were probed with primary antibodies against OP, FN, VCAM and ICAM-1 and blots were developed using secondary antibodies conjugated with horseradish peroxidase for viewing enhanced chemiluminescence as described in Methods.
Expression of mRNA for RARs and RXRs in mesangial cells and its modulation by 9-cRA We characterized the steady state mRNA levels of all six retinoid receptors as well as their regulation by 9-cRA. As shown in all major RARs isotypes were expressed in CHMC regardless of the presence or absence of 9-cRA. Transcripts to RAR-α have been previously found both in normal human renal tissue and in 12 human renal cell carcinoma lines. In these lines 13-cis retinoic acid failed to increase RAR-α expression this result being in contrast with the 9-cRA-triggered induction of this receptor in CHMC. RAR-γ mRNA expression was also present in CHMC and it was not modified by 9-cRA. Although previous studies also showed that the renal expression of this receptor may be modulated by nuclear hormones such as androgens , the retinoid 13-cis retinoic acid did not modify the RAR-γ expression in renal cell carcinoma lines.
Low but reproducibly detectable transcripts to RAR-β were also present in CHMC and the addition of 9-cRA increased their levels. Increased expression of RAR-β mRNA has also been documented in several cell lines after 9-cRA treatment (; ) and it has been related to the antiproliferative effects of 13-cis-retinoic acid in human renal cell carcinomas and to the response of renal cancer patients to 13-cis retinoic acid. The identification of a retinoic acid response element located on the promoter of the RAR-β gene suggests that the RAR-β gene might amplify the response to retinoids in target tissues by raising its own receptor expression level. Expression of mRNA for RARs and RXRs and its modulation by 9-cRA. Quiescent, confluent human mesangial cells were incubated up to 8 h with 1 μ M 9-cRA and total RNA was extracted. Northern blot for the subtypes α, β and γ of either RAR or RXR was carried out as described in Methods. The blots were stripped and rehybridized with a cDNA probe for the housekeeping gene GAPDH to adjust for small variations in RNA loading and transfer to the filter.
The autoradiograms shown are representative of three separate experiments. A second RXRγ autoradiogram with lower exposition is also shown (centre right) in order to improve the observation of the two bands between 28S and 18S rRNA. On the other hand, all major RXRs isotypes were also expressed in CHMC regardless of the presence or absence of 9-cRA.
Regarding RXR-β and RXR-γ, these findings are in good agreement with previous reports showing that rat glomeruli express RXR-β transcripts and RXR-γ protein. However, these studies failed to find transcripts to RXR-α and RXR-α protein in glomeruli and this contrasts with our results since we have found mRNA for RXR-α in glomerular CHMC. Interestingly, RXR-α transcripts have also been found in cultured rat mesangial cells (Q. Kitamura, unpublished observation). 9-cRA increased the expression of RXR-α and inhibited the expression of RXR-γ. Regulation of RXRs may be particularly relevant since they have been shown to function as coregulators that enhance the binding of other nuclear hormone receptors (including RARs, thyroid hormone receptor, vitamin D receptor and peroxisome proliferator-activated receptors) to their cognate response elements (reviewed by ).
Discussion Glomerular cells and especially mesangial cells play an active part in the inflammatory response to glomerular injury. This work is mainly a prospective study on the possibility that 9-cRA may act as an anti-inflammatory agent at the mesangial cell level. The approach involved the study of three relevant effects: inhibition of CHMC proliferation, prevention of H 2O 2-induced mesangial cell death, and inhibition by retinoids of monocyte adhesion to CHMC.
Glomerular mesangial cell proliferation plays an important role in the pathogenesis of many types of progressive glomerular disease and its inhibition has, therefore, a potential therapeutic interest. 9-cRA had antiproliferative action on CHMC , a well known effect of retinoids on many normal and transformed cells. TRA also inhibited CHMC proliferation–although the concentration required was 500 fold higher–and this effect has been attributted to the inhibition of activating protein-1 activity in CHMC. Ligand-activated RARs, which are able to inhibit activating-protein-1 activity , have been traditionally linked to the antiproliferative effect of retinoids.
Therefore this could be one of the mechanisms through which 9-cRA, an RAR ligand , exerts its antiproliferative effect on CHMC. However, a contribution of RXRs can not be ruled out: recent work suggest that activation of RXRs by selective retinoids can also mediate growth inhibitory effects and 9-cRA activates both RARs and RXRs (; ).
The potent growth-inhibitory properties of 9-cRA on serum-stimulated CHMC are not associated with cytotoxicity. However, we asked if higher concentrations are toxic for CHMC and we found that concentrations of 9-cRA higher than 50 μ M induced apoptosis.
This concentration is much greater than the concentrations required for the effects with a potential anti-inflammatory value (see Results). In contrast, apoptosis in CHMC was found with 25 μ M tRA (that is to say, a concentration slightly higher than that needed to obtain anti-inflammatory effects), although the onset of tRA-induced apoptosis was associated with a strong increase in both catalase activity and GSH. Further studies using several modulators (see the Results section) did not confirm our effect suggesting that the oxidation/antioxidation balance might be involved in the mechanism of retinoid-induced apoptosis. RARs are, on the contrary, likely to play a role in the mechanism of retinoid-induced CHMC apoptosis since TTNPB, another RAR pan-agonist like tRA , induced apoptosis at concentrations as low as 10 μ M (F.J. Lucio, unpublished data).
The results obtained for the prevention by retinoids of H 2O 2-induced mesangial cell death are exactly equivalent to those found in the experiments of inhibition of CHMC proliferation: maximal prevention with 9-cRA requires a 1000 fold lower concentration than that required with tRA. Recently we have found that the apoptotic signal of H 2O 2 on CHMC is blocked by tRA inhibiting both c-Jun NH 2-terminal kinase and activating protein-1 expression. This is the most likely mechanism involved in the preventive effect of retinoids against H 2O 2-induced cell death since our results (see and Results section) exclude any role of antioxidant defences.
9-cRA also inhibited monocyte adhesion to FCS-stimulated CHMC. Western blot analysis of adhesion molecules revealed that 9-cRA inhibited the expression of FN and OP but not VCAM-1 and ICAM-1 expression. The inhibitory effect of the retinoid on the mRNA expression of FN and OP is an effect likely responsible for the inhibition of the expression of the respective proteins. Although VCAM-1, ICAM-1, FN and OP are involved in monocyte adhesion to mesangial cells (;;; ) it is worthy of remark that increased monocyte adhesion does not necessarily correlate to increased VCAM-1 or ICAM-1 expression. For instance, in the model of low-density lipoprotein-stimulated monocyte adhesion to cultured human mesangial cells–characterized by increased expression of VCAM-1, ICAM-1 and FN–monocyte adhesion is inhibited by treatment of CHMC with the antibody anti-fibronectin but not with antibodies anti-VCAM-1 or anti-ICAM-1. In the same way, monocyte entry in human atherosclerotic lesions does not correlate with the endothelial areas of increased VCAM-1 expression but with increased expression of connecting segment-1 of FN. Therefore, it may be speculated that the inhibition of the expression of FN and OP might be sufficient to explain the retinoid-induced inhibition of monocyte adhesion to CHMC.
However, specific studies must be undertaken to confirm this point. Retinoids are known to exert their biological effects by binding to specific nuclear receptors. Each subtype of nuclear retinoid receptors is thought to regulate the expression of distinct genes because the subtypes exhibit specific patterns of expression during embryonal development and different distribution in adult tissues. Therefore we characterized the RAR/RXR mRNA expression pattern in CHMC. Several interesting observations evolved from this characterization.
First, the presence of all three RARs and all three RXRs, implies that mesangial cells can respond to exogenously administered retinoids in a receptor-mediated manner. Taking into account that endogenous ligand activity of 9-cRA has been detected in kidney , the presence of RARs and RXRs in mesangial cells suggests that these receptors are likely to play a significant role in controlling mesangial-specific gene expression and mesangial cell phenotype in vivo. This notion is supported by our data showing that CHMC are highly responsive to 9-cRA in terms of modulation of RARs and RXRs: 9-cRA treatment stimulated expression of RAR-α, RAR-β, and RXR-α genes, and it inhibited the expression of RXR-γ. These changes may be critical for the effects of 9-cRA.
Upon binding to RARs and RXRs, retinoids regulate cellular processes by directly modulating the expression of responsive genes. Therefore the changes induced by 9-cRA on mRNA expression of the RARs and RXRs may be critical for the effects of this retinoid on CHMC. On the other hand, the changes in RXRs could trigger a broad spectrum of phenotypic modifications given that the regulation of the expression of target genes by nuclear hormone receptors, including RARs, VDR (vitamin D receptors), TR (thyroid receptors) and PPARs (peroxisome proliferator activated receptors), usually requires prior ligand-activated nuclear hormone receptor heterodimerization with RXRs.
It is important to emphasize, however, that our studies have not assigned a retinoid receptor-mediated process to any action of 9-cRA in CHMC. Such studies will require dominant negative retinoid receptors, synthetic retinoids that specifically activate one given receptor or that bind specific receptors without activating them, or the cultivation of glomerular mesangial cells from knock-out mice deficient in specific retinoid receptors. The glomerular capillary network is unique in that circulating phagocytes may potentially adhere to both endothelial and mesangial cells, as the latter may be found interposed between endothelial cells. The fact that 9-cRA inhibited monocyte adhesion to FCS-stimulated CHMC is particularly important (FCS resembles an inflammatory situation, given that it contains a high number of active factors present during inflammation) taking into account that monocytes can be considered as the real effectors of the immune system in the pathogenesis of glomerular diseases. The same applies to the prevention of mesangial cell death by H 2O 2 – that may be released from activated monocytes/macrophages –and to the inhibition of FCS-induced mesangial cell proliferation. The potential of 9-cRA for the treatment of inflammatory glomerular diseases is even more evident if we take into account the fact that the concentration of 9-cRA used in our studies is comparable to the blood levels found in cancer patients receiving this retinoid.
However, the relevance of the findings presented here needs to be confirmed by in vivo studies in animal models of glomerulonephritis.