1Department of Nephrology, University of Heidelberg, 69115 Heidelberg; and 2Department of Dermatology, University Medical Center Benjamin Franklin, The Free University of Berlin, 14195 Berlin, Germany
Submitted 5 May 2003 ; accepted in final form 26 October 2003
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ABSTRACT |
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retinoid receptors; retinol dehydrogenase; retinal dehydrogenase; isotretinoin; acute anti-Thy1.1-glomerulonephritis; rat
We examined the glomerular expression of RAR,
,
, and RXR
subtypes, the expression of the enzymes RoDH 1 and 2 as well as RalDH 1 and 2. RoDH 1 and 2 are mainly expressed in the rat kidney and lung but also in the liver, testis, and brain (3). RalDH 2 is involved in the differentiation of renal epithelium; it is highly expressed in the embryonic kidney. Additionally, it is a regulator of local RA concentrations (39).
RA possesses autoregulatory potential by inducing or repressing key enzymes of its own metabolism. Excess RA activates the cytochrome P-450 system, which transforms RA into inactive metabolites (31). Alternatively, RA may enhance the storage pathway by activating the enzyme lecithin retinol acyltransferase, resulting in the formation of retinyl esters (19, 34). Also, RA may reduce the activity of RalDH 2 (25) and induce the expression of retinol and RA-binding proteins (24). Despite the variety of these findings, RA metabolism still remains to be understood in its complexity.
We previously demonstrated that retinoids effectively reduce renal injury in the models of acute and chronic mesangioproliferative glomerulonephritis in the rat (32, 43). Retinoids exhibited antiproliferative and anti-inflammatory effects by preserving renal function, significantly reducing albuminuria, and reducing glomerular or tubular damage (8, 16). In turn, inflammatory mediators activate retinoid pathways (9).
For this reason, we examined the regulation of the renal retinoid system in response to inflammatory renal injury in the course of acute anti-Thy1.1-glomerulonephritis (THY-GN). Furthermore, we wanted to know whether retinoid supplementation causes changes in the endogenous retinoid system. Therefore, in a second set of experiments we investigated the influence of isotretinoin supplementation on the components of the renal retinoid system.
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MATERIALS AND METHODS |
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We chose male Wistar rats (Charles River, Sulzfeld, Germany), weighing 150-160 g, as the object of our investigations. All animal experimentation was performed according to the "Deutsches Tierschutzgesetz" (German Animal Protection Law). The experimental groups represented nephritic groups. We induced acute anti-Thy1.1-mesangioproliferative glomerulonephritis by administering 500 µg of Moab 1-22-3 dissolved in PBS (15) as a single-shot injection into the tail vein of the rats on day 0. Moab 1-22-3 is a monoclonal antibody directed against the Thy1.1-like antigen on the surface of rat mesangial cells (14). In contrast to the monoclonal antibody OX-7, Moab 1-22-3 induces more severe mesangial cell injury, resulting in earlier mesangiolytic changes, matrix expansion, and higher proteinuria (35).
Between groups, pair-feeding of the rats was used to ensure that all animals received identical amounts of nutrients.
At the end of the experiments, the rats were given intramuscular injections of 5 mg/kg body wt xylazine (Bayer Vital, Leverkusen, Germany) and 100 mg/kg body wt ketamine 10% (WDT, Garbsen, Germany). The rats were then perfused with saline solution containing 0.5 g/l procaine hydrochloride via a cannula retrogradely inserted into the abdominal aorta (45). To drain blood, the inferior vena cava was incised. The perfusion pressure was adjusted to the individual systolic blood pressure (SBP). Glomeruli were isolated by a fractional sieving technique as described elsewhere (37). The kidneys of each rat were sieved individually. We used three grids with a final mesh size of 53 µm for glomeruli. The yield and purity of isolated glomeruli were comparable between groups at any time (purity >90%).
In the first investigation, three experimental (THY; n = 8) and three control groups (CON; n = 6) were established. Day 3, 7, or 14 after the injection of the antibody represented the experimental end point when SBP was determined by tail-cuff plethysmography (tail plethysmograph built by the University of Heidelberg) under light ether anesthesia. SBP was also taken on day 0. The SBP for each rat was calculated as the average of three separate measurements at each session.
In the second investigation, we established two experimental and two control groups. One experimental (THY; n = 8) and one control (CON; n = 6) group was pretreated with 10 mg/kg body wt isotretinoin (F. Hoffmann-La Roche, Basel, Switzerland) orally for 3 days (day -3 to day -1) before Moab 1-22-3 or PBS administration. Isotretinoin-chow was prepared according to Schaier et al. (32). The other groups received a placebo (vehicle) chow. On day 7 the experiment was terminated. SBP was measured on days -3, 0, and 7.
Renal morphology. Tissues for light microscopy were fixed in 10% buffered formalin and embedded in paraffin. Sections (4 µm) were stained with the periodic acid-Schiff reagent and counterstained with hematoxylin. For each kidney, total glomerular counts of cell nuclei were determined in at least 30 cortical glomeruli with diameters of at least 100 µm. The investigator was blinded for the treatment protocol.
Solid-phase extraction and HPLC analysis. Before HPLC, individually isolated glomeruli and serum of each rat were prepared under dimmed yellow light by solid-phase extraction following the protocol of Collins et al. (6). HPLC analysis was performed on 10-µl samples of the eluate as described elsewhere (6, 41). 13-Cis RA and all-trans RA were determined by simultaneously measuring light absorption at 340 and 356 nm using a Shimadzu SPD-10AV detector. Retinoid levels were quantified by comparing peak areas and authentic standards (Sigma-Aldrich, Dreisenhofen, Germany).
RNA isolation and reverse transcription. RNA of shock-frozen glomeruli and liver tissue of every rat was isolated using TRIzol following the manufacturer's protocol (Invitrogen, Paisley, UK). Individual samples were checked for degradation of total RNA on 1% agarose gels. First, concentrations were spectrophotometrically determined at 260/280 nm and then adjusted to a final value of 0.2 µg/µl. For each sample, reverse transcription, as described elsewhere (44), was performed three times. The resulting cDNA was pooled.
Quantitative PCR assay. Protocols by Paul et al. (30) and Wagner et al. (44), adjusted to our needs, were used to quantify mRNA. For each gene, a DNA deletion mutant was cloned (2), having the same sequence as the wild-type gene and identical primer binding sites but a deletion of 20% at most. This resulted in a shorter amplification product. For amplification, 0.1 µg of reversely transcribed RNA was used. Defined concentrations of DNA deletion mutants served as internal standards. (Primer sequences and hybridization positions are given in Table 1.) The PCR mixture contained 0.25 mmol/l deoxynucleoside triphosphate (dNTP; Promega, Madison, WI), 2.5 mmol/l MgCl2, 20 mmol/l Tris·HCl (pH = 8.4), 50 mmol/l KCl, 80 nmol/l sense and antisense primers (Invitrogen), and 1 U of Taq DNA polymerase (Invitrogen). The thermal profile we used consisted of denaturation at 94°C for 30 s, annealing for 30 s as described in Table 1, and extension at 72°C for 30 s. Possible contamination with genomic DNA was prevented by PCR amplification in the absence of RT. Amplification products of the individual samples were separated by agarose gel electrophoresis and then digitized by using a gel documentation system (Intas, Göttingen, Germany) and Scion Image software (National Institutes of Health, Bethesda, MD). The ratio of the optical density (OD) of endogenous cDNA to the OD of mutant DNA was determined. Each sample was measured in two individual PCR assays for every investigated gene.
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Immunoblotting. Proteins were isolated from frozen glomeruli according to the manufacturer's instructions provided together with an immunoblotting kit (Santa Cruz Biotechnology, Santa Cruz, CA). After fractionating by SDS-PAGE in a 10-slot gel chamber, proteins were electrotransferred onto nitrocellulose filters that were blocked with 5% milk-0.05% Tween 20. Membranes were incubated with RAR antibody (1:250) for 1 h and then with 1:5,000 dilution of goat anti-rabbit IgG-horseradish peroxidase (both antibodies: Santa Cruz Biotechnology) and finally detected by chemoluminescence (11).
Statistical analyses. All values are expressed as means ± SE. Data were analyzed by using the nonparametric Mann-Whitney test or multivariate ANOVA and Bonferroni's posttest, as indicated. Statistical significance was accepted at P < 0.05.
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RESULTS |
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Isotretinoin treatment reduced the steepness of the rise in SBP in THY-GN rats but did not have any effect in nonnephritic CON on day 7 (P < 0.01; Fig. 1B).
Compared with CON rats, the glomerular count of cell nuclei was significantly lower in THY-GN rats on day 3 (P < 0.01) but doubled in value on day 7 and thus was significantly higher than in the CON groups on days 7 and 14 (P < 0.001; Fig. 1C). Again, isotretinoin treatment of THY-GN rats reduced the increase in glomerular numbers of cell nuclei, whereas elevated values in vehicle-treated THY-GN rats were retained (P < 0.01; Fig. 1D).
Serum and tissue retinoid concentrations. Serum 13-cis RA and all-trans RA levels were higher in isotretinoin-treated than in vehicle-treated groups (Fig. 2, A and B).
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13-Cis RA and all-trans RA levels in isolated glomeruli of vehicle-treated groups were close to or below the detection limit of the assay but made a shift to detectable values in the case of isotretinoin treatment (Fig. 2, C and D).
Time-dependent changes in glomerular gene expressions of retinoid receptors in the course of acute THY-GN. On day 3, levels of glomerular expression of the retinoid receptors RXR, RAR
, and RAR
significantly fell below control values in THY-GN rats (P < 0.01), RAR
being the only receptor expressed at control levels. On day 7, all receptors experienced expression peaks with values three times the control value (P < 0.001; RAR
: 3.58x, RAR
: 2.75x, RAR
: 3.22x, RXR
: 2.80x). On day 14, expression levels for all receptors were normalizing. RAR
and RAR
mRNA returned to the level of controls. The normalization of RAR
and RXR
expression was less compared with the other receptors. It remained minimally elevated in THY-GN vs. CON (P < 0.001; Fig. 3, A-D).
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Hepatic mRNA expression of RAR and RXR
in THY-GN. No difference in the hepatic expression of RAR
and RXR
could be observed between nephritic and nonnephritic groups on day 7.
Time-dependent changes in glomerular gene expressions of retinoid-metabolizing enzymes in the course of acute THY-GN. The glomerular expression pattern of all retinoid-metabolizing enzymes (RalDH 1 and 2, RoDH 1 and 2) showed obvious similarities to the expression pattern of retinoid receptors: a 80% decline in relation to control values on day 3 (P < 0.0001; RoDH 1: 83.04%, RoDH 2: 77.53%, RalDH 1: 75.71%, RalDH 2: 82.0%), a surge to values about four times the control values on day 7 (RoDH 1: 5.56x, RoDH 2: 8.3x, RalDH 1: 2.79x, RalDH 2: 1.65x), and the return to or below control levels on day 14 (Fig. 4, A-D).
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Effect of isotretinoin treatment on the glomerular expression of retinoid receptors and retinoid-metabolizing enzymes in the course of acute THY-GN. Oral supplementation with isotretinoin did not influence glomerular gene expression of retinoid receptors and of retinoid-synthesizing enzymes in nonnephritic rats. Isotretinoin did not influence the threefold elevation of the expression of these genes in THY-GN rats on day 7 (Table 2).
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Glomerular protein expression of RAR on day 7. The glomerular expression of protein RAR
was analyzed in vehicle- and isotretinoin-treated CON and THY-GN rats. RAR
was found in glomeruli, where it was more abundant in THY-GN than in nonnephritic rats. Isotretinoin treatment did not alter RAR
expression levels (P < 0.05; Fig. 5, A and B).
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DISCUSSION |
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The results of this study complement previous findings, in which there had been shown that isotretinoin reduces the blood pressure increase in acute THY-GN (43). The fact that the number of resident glomerular cells is reduced on day 3, reflecting early mesangiolysis in response to antibody-mediated injury, is characteristic of the model (14). In contrast, on day 7 the number of mesangial cells is increased again as a result of mesangial cell proliferation (12). This course of THY-GN was confirmed in our model.
In agreement with our earlier work, treatment with isotretinoin lowered blood pressure, proliferation of mesangial cells, and glomerular damage (8, 22, 43). The mechanism of the antihypertensive effect of retinoids has not yet been clarified. The retinoid-mediated decrease in SBP is not specific for isotretinoin, because synthetic retinoids also lowered blood pressure in the THY-GN model (16). It may reflect alleviation of renal damage by retinoids, allowing the kidney to normalize SBP. Additionally, however, retinoids lower the expression of the angiotensin receptor in vitro and in vivo. They block the effects of angiotensin II (8, 11). This suggests another possible mechanism of blood pressure-lowering action by retinoids.
Antiproliferative effects of retinoids were demonstrated in different cell types, e.g., mesangial, vascular smooth muscle, endothelial, and tubular cells (11, 28, 36). The mechanisms of the antiproliferative action of retinoids have not been completely elucidated. One pathway is the interference with AP-1 by protein-protein interaction of retinoid receptors or by downregulation of its units c-Jun and c-Fos (33). Thus proliferation by AP-1-dependent genes, e.g., angiotensin II, PDGF, or endothelins, is lowered by retinoids (11, 20, 46).
Changes in the endogenous retinoid system in response to acute glomerular injury were observed at the level of retinoid receptor expression, serum and glomerular retinoid levels, and glomerular expression of retinoid-metabolizing enzymes.
Regulation of the different subtypes of retinoid receptors under different circumstances has been described in the past (4, 5, 17). On day 3, we observed a uniform decrease in glomerular retinoid receptor gene expression, with the exception of RAR. Because receptor expression was determined in isolated glomeruli and expressed per microgram RNA, it is obvious that this decrease does not reflect merely the effects of mesangiolysis but indicates either a relative reduction in retinoid receptor-expressing cells within the glomeruli or a reduced expression of the receptor number per cell.
Mesangial cells are known to express retinoid receptors (11), but so do endothelial and inflammatory cells, i.e., monocytes/macrophages. In contrast to the other receptors, RAR was not affected. Because this receptor is supposed to be ubiquitously expressed, its expression might not have been influenced by a shift in the relative distribution of glomerular cells, although it cannot be excluded that these are receptor subtype-specific differences in receptor regulation.
Similar to the expression of glomerular retinoid receptors on day 3, the expression of retinoid-metabolizing enzymes was reduced as well. The cellular origin of expression of these enzymes in the glomerulus has not yet been determined. The reduction of gene expression of these enzymes may suggest a decrease in the local production of retinoids in the state of early glomerular damage in THY-GN. Whether these alterations locally influence the level of retinoids in the glomeruli is unknown.
On day 7, a completely different expression pattern of the retinoid receptors and retinoid-metabolizing enzymes was observed: In contrast to day 3, the expression of all retinoid receptors and retinoid-metabolizing enzymes was significantly higher in nephritic compared with nonnephritic glomeruli. On the mRNA level, retinoid receptor and RalDH expression was increased about threefold, and RoDHs expression about five-fold. On day 7, both mRNA expression and protein expression of RAR in the glomeruli were increased.
Because at that time the number of mesangial cells was elevated as a result of the ongoing repair processes, it cannot be decided whether the enhanced receptor and enzyme expression was due to an increased glomerular number of retinoid receptor-expressing cells or an enhanced expression per cell. The findings on day 14, however, answer this question, because at that point the expression of retinoid system components and glomerular cell number did not go in parallel. On day 14, the expression of the different retinoid receptors had almost returned to normal, whereas the number of glomerular cells was still elevated. Similar findings were obtained with respect to the retinoid-metabolizing enzymes.
These changes, therefore, cannot be explained on the basis of the number of retinoid receptor-expressing cells but strongly suggest a regulated cellular expression of these receptors. This interpretation is also supported by the fact that hepatic expression of RAR and RXR
was not altered on day 7, when their expression in the glomeruli was significantly elevated, suggesting time-specific regulation. Because the expression of these two receptors did not change in the liver on the day of maximal change in the kidney, we took this as further evidence that the changes in the retinoid system are induced locally in the kidney due to renal disease.
A regulated response of the retinoid system to glomerular damage is further supported by the findings of a decreased amount of 13-cis RA and all-trans RA in the serum on day 7 in vehicle-treated nephritic rats. This may indicate "consumption" of the retinoids. A local reduction in available retinoids at the site of inflammation may trigger increased expression of retinoid receptors and of metabolizing enzymes. In the glomeruli, the endogenous retinoid levels were low or at the detection limit of these assays, precluding further analysis.
We therefore applied isotretinoin to overcome a potential local reduction in retinoids. As a result of this maneuver, the elevated concentrations of 13-cis RA and its isomer all-trans RA were meshed in the serum of nonnephritic and nephritic rats as well as in the glomeruli. In the past, isotretinoin had been shown to reduce the number of glomerular cells and the level of SBP as surrogate markers of glomerular damage (22, 32, 42, 43). The fact that isotretinoin influenced neither the glomerular expression of retinoid receptors nor that of metabolizing enzymes is puzzling, because isotretinoin is metabolized to all-trans RA in the glomeruli. 13-Cis RA binds neither CRABP nor retinoid receptors (48), but it has been shown to act as a prodrug for all-trans RA in the skin (40). These findings indicate that retinoid supplementation is not the trigger for retinoid receptor expression in this model. Our results are in conflict with the results of other investigators, who had demonstrated that the levels of vitamin A or of retinoids can influence retinoid receptor expression (13). On the other hand, induction of retinoid receptors on day 7 may render renal tissue more sensitive to the action of isotretinoin, further supporting its anti-inflammatory and antiproliferative effects.
The results of our experiments indicate an active and specific response of the renal retinoid system in the early repair phase of acute mesangioproliferative glomerulonephritis.
Retinoids have long been associated with wound healing. In dermatology, for instance, retinoids are used for the treatment of psoriasis, acne, and seborrhoea (26, 27). Previous work has indicated that retinoids play a role in the control of inflammation and wound healing, because they exert strong anti-inflammatory and antiproliferative effects (7, 11, 21). Therefore, a local reduction in available retinoids at the site of inflammation may trigger increased expression of retinoid receptors and of metabolizing enzymes.
Conversely, a deficiency of vitamin A retards tissue repair. Retinoids reverse the inhibitory effects of glucocorticoids on wound healing by promoting epithelization and synthesis of collagen and ground-substance (1). Paquette et al. (29) have recently shown that topical all-trans RA treatment of patients with chronic leg ulcerations stimulates formation of granulation tissue, angiogenesis, and synthesis of new collagen.
Above all, our findings suggest that renal tissue responds to exogenous retinoids and that the endogenous retinoid system is altered in response to renal injury. Whether these changes reflect the activation of this system or the response to local deprivation of retinoids after renal injury remains to be elucidated. Clearly, the changes in the endogenous system suggest an active role of retinoids in glomerular damage repair.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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