J Am Soc Nephrol 12: 2300–2309, 2001 Effects of on the TGF-␤ System and Extracellular Matrix in Experimental Glomerulonephritis

CHRISTIAN MORATH,* CLAUDIUS DECHOW,* INGO LEHRKE,* VOLKER HAXSEN,* RUDIGER¨ WALDHERR,* JURGEN¨ FLOEGE,† EBERHARD RITZ,* and JURGEN¨ WAGNER* *Department of Nephrology, University of Heidelberg, Heidelberg, Germany; and †Department of Nephrology, University of Aachen, Aachen, Germany.

Abstract. Transforming growth factor–␤1 (TGF-␤1) overex- compared with untreated animals (P Ͻ 0.025). Cortical expres- pression plays a key role in the glomerular accumulation of sion of TGF receptor II, but not receptor I gene expression, was extracellular matrix proteins in renal disease. Retinoids have significantly lower in animals treated with all-trans retinoic previously been shown to significantly limit glomerular dam- acid or (P Ͻ 0.05). In all-trans –treated age in rat experimental glomerulonephritis. Therefore, the ef- animals with Thy-GN, the increase of glomerular TGF-␤1 fects of all-trans retinoic acid and isotretinoin on the compo- protein (P Ͻ 0.008) and TGF-␤1(P Ͻ 0.025) and TGF nents of the TGF-␤ system and extracellular matrix proteins in receptor II mRNA (P Ͻ 0.015) was significantly less. Immu- anti-Thy1.1-nephritis (Thy-GN) were investigated. Vehicle- nohistochemistry revealed less glomerular staining for TGF-␤1 injected control rats were compared with rats treated with daily and TGF receptor II in the presence of all-trans retinoic acid. subcutaneous injections of 10 mg/kg body wt all-trans retinoic TGF-␤1 immunostaining was not restricted to monocytes and acid or 40 mg/kg body wt isotretinoin (n ϭ 9 per group) either macrophages, as indicated by double-staining. Glomerular with a pretreatment (day Ϫ2 through 8) or posttreatment pro- staining for collagen IV and collagen III was less in animals tocol (day ϩ3 through 8), i.e., starting before or after induction treated with isotretinoin (P Ͻ 0.02 for both) in contrast to of Thy-GN, respectively. Urinary TGF-␤1 excretion was 60% all-trans retinoic acid, whereas fibronectin remained un- lower in all-trans retinoic acid–treated animals with Thy-GN changed. It was concluded that the beneficial effects of retin- (P Ͻ 0.025). The increase of cortical TGF-␤1 gene expression oids on glomerular damage are presumably due to a marked in Thy-GN rats was significantly attenuated with all-trans reduction in renal TGF-␤1 and TGF receptor II expression. retinoic acid and even more with isotretinoin treatment as

Transforming growth factor–␤ (TGF-␤) is a prototype of a The role of retinoids, derivatives of , in embryogen- profibrogenic cytokine (1). TGF-␤ stimulates transcription of esis (including renal development) has been studied in detail many extracellular matrix genes in renal cells (2,3). TGF-␤ (12,13). Little is known, however, about the renal effects of these inhibits extracellular matrix turnover by reducing collagenase compounds. Retinoids exert strong antiproliferative and anti-in- production and by stimulation of tissue inhibitor metallopro- flammatory effects. They act via acid and retinoid X teinase expression (4). In several models of renal disease, receptor subtypes, which belong to the steroid supergene family TGF-␤ has been implicated as a primary mediator of cell (14). These receptors influence gene expression either via retinoid growth and accumulation of extracellular matrix, e.g., diabetic responsive cis-acting elements or via modulation of transcription nephropathy, experimental glomerulonephritis, or unilateral factors such as AP-1, Nf␬B, or GATA (15–19). ureteral obstruction (5,6). Renal TGF-␤1 gene expression is We had demonstrated that retinoids attenuate glomerular markedly elevated in anti–Thy-nephritis or anti-thymocyte se- damage in anti-Thy1.1-nephritis (Thy-GN) (20), as indicated rum nephritis (7–9). Matrix expansion was less when TGF-␤1 by preservation of renal function, less albuminuria, and lower was antagonized by neutralizing antibodies or antisense oligo- capillary occlusion score. It has been suggested that in this nucleotides (10,11). model TGF-␤ is crucial for matrix expansion and fibrosis. We, therefore, decided to examine the effects of retinoids on the renal TGF-␤ system and the expression of collagen III, IV, and Received February 23, 2000. Accepted May 9, 2001. Dr. Richard Glassock served as the Guest Editor and supervised the review and fibronectin in Thy-GN in the rat. We compared non-nephritic final disposition of this manuscript. control rats with Thy-GN animals that were treated with all- Correspondence to Dr. Ju¨rgen Wagner, Department of Nephrology, University trans retinoic acid, isotretinoin, or vehicle. Hospital, Universitiy of Heidelberg, Bergheimerstrasse 56a, D-69115 Heidel- berg, Germany. Phone: ϩ49 6221 91120; Fax: ϩ49 6221 16 24 76. E-mail: [email protected] Materials and Methods 1046-6673/1211-2300 Animal Studies Journal of the American Society of Nephrology Thy-GN was induced by a single injection of OX-7, a monoclonal Copyright © 2001 by the American Society of Nephrology antibody against the Thy1.1-antigen (European Collection of Animal J Am Soc Nephrol 12: 2300–2309, 2001 Effects of Retinoids on TGF in Nephritis 2301

Cell Cultures, Salisbury, UK). Male pair-fed Wistar rats (Charles 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C River, Sulzfeld, Germany), weighing 180 to 200 g, were used for all for 1 min carried out 30 times for TGF-␤1, 28 times for TGF receptor studies. In a first experiment (the pretreatment protocol), two groups II, and 32 times for TGF receptor I (subtype R4). In all experiments, of 18 animals each were treated with daily subcutaneous injections of possible contamination with genomic DNA was excluded by PCR 10 mg/kg body wt all-trans retinoic acid (Sigma-Aldrich Chemie amplification in the absence of reverse transcriptase. Amplification GmbH, Steinheim, Germany) dissolved in arachis oil and 5% dimeth- products were separated by agarose gel electrophoresis and then ylsulfoxide or arachis oil and dimethylsulfoxide as vehicle control, digitized by use of a gel documentation system (Intas, Go¨ttingen, respectively. Two days later, animals received by injection into the Germany) and NIH Image (National Institutes of Health, Bethesda, tail vein either 1 mg/kg body wt OX-7 (n ϭ 9) or phosphate-buffered MD). The ratio between the optical density of the endogenous cDNA saline (PBS) as control (n ϭ 9), respectively. The experiment was and the optical density of the mutant DNA was determined. Each terminated 8 d after injection of the antibody. sample was measured in three individual PCR assays for each gene. In a second experiment (the posttreatment protocol), animals were first given OX-7 or PBS and then, 2 d later, 10 mg/kg body wt Enzyme-Linked Immunosorbent Assay for TGF-␤1 all-trans retinoic acid or vehicle. This experiment included two further For enzyme-linked immunosorbent assay (ELISA), glomeruli were experimental groups that were treated with daily subcutaneous injec- isolated and centrifuged in PBS. The pellet was resuspended in 50 ␮l tions of 40 mg/kg body wt isotretinoin (13-cis retinoic acid; Hoffmann of Laemmli buffer 1 (33% 0.5 mM Tris-HCl [pH 6.8], 66% SDS LaRoche, Basel, Switzerland) dissolved in arachis oil and 5% dimeth- 10%). Protein was isolated by three cycles of freeze and thaw. The ylsulfoxide. Each group was composed of nine animals. The experi- supernatant was recovered after centrifugation. The protein concen- ment was terminated 8 d after injection of the antibody. tration was determined according to the method of Bradford (22). BP was determined on day 8 by tail-cuff plethysmography under TGF-␤1 in glomeruli and urine was measured after acid activation light ether anesthesia. For determination of TGF-␤1 in urine, rats were by use of a commercially available inhibitory ELISA kit (Immundi- placed in metabolic cages and urine was collected for 24 h. The agnostik, Bensheim, Germany), according to the manufacturer’s in- experiment was terminated by injection of 5 mg/kg body wt Xylazin structions. Each sample was measured in quadruplicate. intramuscularly (BayerVital, Leverkusen, Germany) and 100 mg/kg body wt Ketamin 10% intramuscularly (WDT, Garbsen, Germany). Kidneys were perfused with normal saline at 37°C that contained 0.5 Immunohistochemistry g/L procaine hydrochloride at a pressure of 110 mm Hg. Glomeruli Saline-perfused 4-␮m slices of renal tissue were fixed in 10% were isolated by a fractionated sieving technique. The yield and the buffered formaline (TGF-␤1, TGF receptor II, and Ki-M2R) or purity of isolated glomeruli at each time point were comparable methyl Carnoy’s solution (fibronectin and collagen IV and III) and between groups (purity Ͼ90%). were processed by a direct or indirect immunoperoxidase technique. Primary antibodies included an affinity-purified IgG fraction of a RNA Isolation and Reverse Transcription polyclonal rabbit anti-rat fibronectin (Chemicon, Temecula, CA), a biotinylated IgG fraction of a polyclonal goat anti-mouse type IV and The Trizol (Life Technologies, Paisley, UK) method was used for type III collagen (Southern Biotech, Birmingham, AL), an IgG frac- RNA isolation, according to the manufacturer’s recommendations. tion of a polyclonal rabbit anti-rat TGF-␤1 and TGF receptor II (Santa Samples were checked for degradation of total RNA on 1% agarose Cruz Biotechnology, Santa Cruz, CA), and a monoclonal mouse gel. RNA concentrations were determined by spectrophotometric anti-rat pan-macrophage antibody Ki-M2R (DPC Biermann, Bad measurements at wavelengths of 260/280 nm. Reverse transcription Nauheim, Germany). was performed as described elsewhere (21). For each sample, reverse Negative controls consisted of substitution of the primary antibody transcription was carried out three times and the resulting cDNA was with equivalent concentrations of an irrelevant murine monoclonal pooled. antibody or normal rabbit IgG. For each kidney, Ͼ30 cross sections of cortical glomeruli with a diameter of at least 100 ␮m were evaluated Quantitative PCR Assay in a blinded fashion by use of a 121-point grid (Leitz, Wetzlar, Quantification of specific mRNA was performed essentially as Germany). Points on stained glomerular areas and all points on described by Wagner et al. (21). For each gene, a DNA deletion glomeruli were counted. The ratio in percent was used to determine mutant was cloned. These had the same sequences as the endogenous the degree of staining. genes, with identical primer binding sites but a deletion of maximally For double-staining of TGF-␤1 and Ki-M2R (macrophages), we 20% that resulted in a shorter amplification product. Reverse-tran- used the Zymed NBA Kit (Zymed, San Francisco, CA) for TGF-␤1 scribed RNA (0.25 ␮g) was used for amplification in the presence of and the DAKO APAAP KIT system (DAKO, Hamburg, Germany) for defined concentrations of DNA deletion mutants as internal standards. Ki-M2R, according to the manufacturer’s recommendations. The concentration of standard DNA was selected to allow comparable degrees of amplification of endogenous and mutant genes. Primers Statistical Analyses ␤ were used for TGF- 1 5'-CACCATCCATGACATGAACC-3' (sense All values are expressed as mean Ϯ SEM. Statistical significance primer) and 5'-TCATGTTGGACAACTGCTCC-3' (antisense), for (defined as P Ͻ 0.05) was evaluated by use of the nonparametric TGF receptor II 5'-CTACAAGGCCAAGCTGAAGC-3' (sense) and Mann-Whitney U test. 5'-AGCCATGGAGTAGACATCCG-3' (antisense), and for TGF re- ceptor I (subtype R4) 5'-ATGGACTCAGCTGTTGAGGC-3' (sense) and 5'-TCAACGGATGGATCAGAAGG-3' (antisense). Results Effects of Retinoids on Systolic BP The PCR reaction mix contained 0.25 mM dNTP, 2.5 mM MgCl2, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 80 nM levels of sense and Pretreatment Protocol. Systolic BP was significantly el- antisense primers, and1UofTaq DNA Polymerase (Life Technol- evated in vehicle-treated rats with Thy-GN on day 8 after ogies). The thermal profile that was used consisted of denaturation at induction of disease, compared with controls (control/vehicle, 2302 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 2300–2309, 2001

90 Ϯ 3 mmHg; control/all-trans retinoic acid, 91 Ϯ 4 mmHg; Thy-GN/vehicle, 112 Ϯ 5 mmHg, P Ͻ 0.005 versus control/ vehicle; Thy-GN/all-trans retinoic acid, 88 Ϯ 3 mmHg, P Ͻ 0.001 versus Thy-GN/vehicle). Pretreatment with all-trans reti- noic acid had no effect on systolic BP in non-nephritic control rats but completely abrogated the BP increase in Thy-GN rats. BP levels were similar in the posttreatment protocol (data not shown).

Retinoids Reduce Urinary TGF-␤1 Excretion Pretreatment Protocol. Figure 1 shows a 20-fold increase in urinary TGF-␤1 protein in vehicle-treated rats with Thy-GN 8 d after injection of OX-7, compared with controls (P Ͻ 0.0004). In Thy-GN rats treated with all-trans retinoic acid, urinary TGF-␤1 protein increased only 8-fold (P Ͻ 0.025 versus Thy-GN ϩ vehicle). In non-nephritic control rats, all- trans retinoic acid had no effect on urinary TGF-␤1 excretion.

Less Cortical Expression of the TGF-␤1 Gene in the Presence of Retinoids Pretreatment Protocol. Figure 2A shows that treatment with all-trans retinoic acid before injection of OX-7 antibody had no effect on cortical TGF-␤1 mRNA expression in non- nephritic animals. Expression of the TGF-␤1 gene was mark- edly higher in the renal cortex of vehicle-treated Thy-GN rats. In Thy-GN animals pretreated with all-trans retinoic acid, TGF-␤1 gene expression was significantly less (P Ͻ 0.025). Post-treatment Protocol. Cortical TGF-␤1 gene expres- sion was also determined in animals that were treated with all-trans retinoic acid or isotretinoin after induction of Thy-GN (Figure 2B). Similar to the pretreatment study, cortical TGF-␤1 gene expression was low in Thy-GN rats with all-trans retinoic acid treatment. In comparison, TGF-␤1 mRNA was signifi- cantly less in isotretinoin than in all-trans retinoic acid–treated Figure 2. Effect of retinoids on TGF-␤1 gene expression in the renal cortex. (A) Pretreatment with Ra suppresses cortical TGF-␤1 overex- pression in rats with Thy-GN. (B) Reduction of cortical TGF-␤1 expression by Ra or isotretinoin (Iso) in the posttreatment protocol. No change of TGF-␤1 expression is observed in control rats either with the pretreatment or posttreatment protocols.

Thy-GN rats (P Ͻ 0.003). Neither all-trans retinoic acid nor isotretinoin had an effect on cortical TGF-␤1 mRNA in non- nephritic control rats.

Effects of All-Trans Retinoic Acid and Isotretinoin on Cortical Expression of the TGF Receptor Genes Pretreatment Protocol. In vehicle-treated Thy-GN rats, cortical expression of TGF receptor II was significantly higher (Figure 3A) than in non-nephritic control rats (P Ͻ 0.002) but was low after pretreatment with all-trans retinoic acid (P Ͻ Figure 1. Retinoids reduce urinary transforming growth factor–␤1 0.004). No significant difference was observed between all- (TGF-␤1) excretion in Thy-GN rats. Significant reduction of urinary trans retinoic acid–treated and vehicle-treated non-nephritic TGF-␤1 excretion by all-trans retinoic acid (Ra) in rats with Thy-GN. control rats (Figure 3A). In contrast to TGF receptor II, no No change of urinary TGF-␤1 excretion is observed in control rats significant difference in cortical expression of TGF receptor I treated with Ra. was observed between vehicle-treated and all-trans retinoic J Am Soc Nephrol 12: 2300–2309, 2001 Effects of Retinoids on TGF in Nephritis 2303

Figure 3. Expression of the TGF receptors in the renal cortex. (A and B) Cortical TGF receptor II expression is less in Ra-treated or Iso-treated Thy-GN rats than in vehicle-treated nephritic rats in the pretreatment (A) and posttreatment (B) protocols. (C and D) No effect of Ra or Iso on cortical TGF receptor I (subtype R4) expression was found in rats with Thy-GN in the pretreatment (C) and posttreatment (D) studies.

acid–treated Thy-GN rats. There was also no difference be- Glomerular TGF-␤1 and TGF Receptor II Gene tween vehicle-treated control and Thy-GN rats (Figure 3C). Expression in Thy-GN Rats Treated with All-Trans Posttreatment Protocol. Cortical TGF receptor II and I Retinoic Acid gene expression was also determined in animals that were Pretreatment Protocol. As in the cortex, TGF-␤1 gene treated with all-trans retinoic acid or isotretinoin after induc- expression was markedly higher in glomeruli of Thy-GN rats. tion of Thy-GN (Figure 3, B and D). The results for both Expression of this gene was significantly less in animals that receptors were similar to those obtained in the pretreatment were treated with all-trans retinoic acid (P Ͻ 0.025; Figure study. The effects of all-trans retinoic acid and isotretinoin on 4A). In contrast to TGF-␤1 mRNA expression in whole-cortex cortical TGF receptor II and I gene expression in Thy-GN rats preparations, gene expression was significantly lower in iso- were not significantly different. Neither all-trans retinoic acid lated glomeruli of all-trans retinoic acid–treated non-nephritic nor isotretinoin had an effect on cortical TGF receptor II and I control rats when compared with vehicle-treated rats (P Ͻ mRNA in non-nephritic control rats. 0.025; Figure 4A). Similarly, Figure 4B shows lower TGF 2304 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 2300–2309, 2001

Figure 5. Ra lowers glomerular TGF-␤1 protein content. Glomerular TGF-␤1 protein is markedly higher in vehicle-treated Thy-GN rats. Ra treatment normalizes glomerular TGF-␤1 concentration. No effect of Ra is observed in control rats.

different from that of non-nephritic controls. All-trans retinoic acid did not alter glomerular TGF-␤1 protein in non-nephritic control rats (Figure 5).

Glomerular Staining for TGF-␤1 and TGF Receptor II and Double-Staining for TGF-␤1 and Ki-M2R Figure 6 shows representative examples of glomerular stain- ing for TGF-␤1 (Figure 6, A through C) and TGF receptor II (Figure 6, D through F) in rats with Thy-GN treated with vehicle (Figure 6, B and E) or all-trans retinoic acid (Figure 6, C and F). Figure 6, A and D shows glomerular staining for TGF-␤1 and TGF receptor II in non-nephritic control rats. No Figure 4. Ra limits glomerular TGF-␤1 and TGF receptor II expres- significant expression of TGF-␤1 protein was found in non- sion in Thy-GN. (A) Glomerular TGF-␤1 gene expression is enhanced nephritic vehicle-treated glomeruli (Figure 6A). TGF receptor in vehicle-treated Thy-GN rats, compared with controls. Glomerular II immunostaining was found in tubular cells but was almost TGF-␤1 mRNA is significantly less in the presence of Ra. Glomerular absent in non-nephritic glomeruli (Figure 6D). Staining was TGF-␤1 mRNA is significantly less in Ra-treated control rats versus not influenced in presence of all-trans retinoic acid in non- vehicle-treated control rats. (B) In vehicle-treated Thy-GN rats, glo- nephritic kidneys (data not shown). merular TGF receptor II expression is markedly higher than in control In Thy-GN rats, prominent staining for TGF-␤1 (Figure 6B) groups but is significantly less when rats were treated with Ra. and TGF receptor II (Figure 6E) was noted within the Thy-GN Glomerular TGF receptor II mRNA is significantly less in Ra-treated control rats versus vehicle-treated control rats. glomeruli, whereas staining was much less in all-trans retinoic acid–treated Thy-GN glomeruli (Figure 6, C and F). Staining of serial sections for TGF-␤1 and monocytes/mac- receptor II gene expression in glomeruli of all-trans retinoic rophages (Ki-M2R; Figure 6G) as well as double-staining acid–treated Thy-GN rats than in vehicle-treated Thy-GN an- procedures (Figure 6H) showed that TGF-␤1 expression in imals (P Ͻ 0.015). Glomerular TGF receptor II gene expres- all-trans retinoic acid–treated Thy-GN glomeruli was not ex- sion in non-nephritic rats with all-trans retinoic acid treatment clusively related to monocytes/macrophages but was also was lower than that in vehicle-treated control rats (P Ͻ 0.002). found in glomerular areas where no monocyte/macrophage accumulation was evident. No Increase in Glomerular TGF-␤1 Protein in Thy-GN Rats Treated with All-Trans Retinoic Acid Retinoids Modify the Renal Expression of Extracellular Pretreatment Protocol. Figure 5 shows lower glomerular Matrix Proteins TGF-␤1 protein in glomeruli of Thy-GN rats pretreated with Posttreatment Protocol. Figure 7 depicts representative all-trans retinoic acid, compared with vehicle-treated Thy-GN examples of glomerular staining for collagen IV in rats with rats (P Ͻ 0.008). TGF-␤1 protein content was not significantly Thy-GN treated with vehicle (Figure 7B), isotretinoin (Figure J Am Soc Nephrol 12: 2300–2309, 2001 Effects of Retinoids on TGF in Nephritis 2305

Figure 6. Representative examples of immunohistochemical staining for TGF-␤1 (A through C), TGF receptor II (D through F), staining for monocytes/macrophages (Ki-M2R) (G), and double-staining for TGF-␤1 and Ki-M2R (H). Staining in glomeruli of non-nephritic rats is shown in vehicle-treated control rats (A and D) and in Thy-GN rats treated either with vehicle (B and E) or Ra (C and F). (G) Staining for monocytes/macrophages in a glomerulus of an Ra-treated Thy-GN rat. (H) Double-staining for monocytes/macrophages (red) and TGF-␤1 (brown) in the same group.

7C), or all-trans retinoic acid (Figure 7D). Figure 7A shows found in vehicle-treated Thy-GN rats. Isotretinoin had no ef- glomerular staining for collagen IV in control rats. In Thy-GN fect on fibronectin content and was not different from controls. rats, glomerular staining for collagen IV was markedly higher In all-trans retinoic acid–treated Thy-GN rats, there was a than in non-nephritic control rats but was significantly less in significant increase of fibronectin compared with both the isotretinoin-treated Thy-GN rats (P Ͻ 0.02). There was no isotretinoin-treated and vehicle-treated groups (P Ͻ 0.008 for difference in glomerular collagen IV staining between vehicle- both). treated and all-trans retinoic acid–treated Thy-GN rats (Figure 8A). Discussion Figure 8B shows no glomerular expression of collagen III in The data document that retinoic acids lower gene and protein control rats. Collagen III was markedly increased in vehicle- expression of the components of the TGF-␤ system in Thy-GN treated Thy-GN rats. Glomerular expression of collagen III rats and modulate the expression of extracellular matrix pro- was significantly less in presence of isotretinoin than in the teins in the renal cortex. vehicle-treated group (P Ͻ 0.02). In contrast, all-trans retinoic The relationship between retinoids and TGF-␤ is complex. acid had no effect on collagen III expression or even tended to Much information has been provided by in vitro studies, but increase it without reaching statistical significance. the results on the effects of retinoids on the expression of Figure 8C shows the result of the quantitative analysis of TGF-␤1 have been conflicting; depending on cell type, culture expression of fibronectin protein. No increase of basal fi- conditions, and type of retinoid used, both increases and de- bronectin expression was seen in control rats in the different creases have been reported (23,24). Jakowlew et al. (25) sug- treatment groups. Only a slight increase of fibronectin was gest that the effect of retinoids depends on the basal levels of 2306 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 2300–2309, 2001

Figure 7. Representative example of immunohistochemical staining for collagen IV in glomeruli of Thy-GN rats. Staining for collagen IV in the glomeruli of non-nephritic rats is shown in vehicle-treated control rats (A) and in Thy-GN rats treated either with vehicle (B), Iso (C), or Ra (D).

TGF-␤ expression; TGF-␤ was lowered in cells with high basal system and extracellular matrix proteins because we had pre- expression and increased in cells with low natural abundance viously demonstrated that retinoids strongly reduce glomerular of TGF-␤. The effects of retinoids on cellular differentiation damage in this model (20); retinoids clearly reduce TGF-␤1 partly depend on the presence of TGF-␤ (24). In Hep 2G gene and protein expression on different levels. Urinary hepatocarcinoma cells stimulated by 12-O-tetradecanoyl phor- TGF-␤1 protein is markedly reduced in nephritic rats after bol-13-acetate in vitro, retinoids down-regulate TGF-␤ via exposure to retinoids. Because, however, the pattern of urinary AP-1 binding sites on the TGF-␤1 promoter (17). TGF-␤1 excretion closely follows the degree of albuminuria, In vivo data on the effects of retinoids are scarce, although reduction in urinary TGF-␤1 may reflect increased tubular some information is available concerning wound healing and reabsorption of TGF-␤1 in Thy-GN rats treated with retinoids inflammation (26,27). Alteration of body retinoid status re- (20). In whole-cortex preparations and in isolated glomeruli, sulted in increased TGF-␤ content in some organs and a both TGF-␤1 gene expression and TGF-␤1 protein were lower decrease in others (28,29). in retinoid-treated Thy-GN animals. Immunohistochemistry Many data support a role of TGF-␤1 in accumulation of for TGF-␤1 revealed positive staining in mesangial cells and extracellular matrix in Thy-GN (10,11,30,31). A causative role glomerular epithelial cells and segmentally in highly damaged of TGF-␤ in this model is suggested by studies of Isaka et al. glomerular capillaries. Simultaneously, the glomerular and cor- (31), who used a gene transfer system to introduce TGF-␤ tical expression of TGF receptor II was reduced in retinoic expression vectors into the glomeruli. This maneuver aggra- acid–treated Thy-GN rats. We cannot decide whether the re- vated renal damage. Conversely, blockade of local TGF-␤1 duction in TGF receptor II expression is due to the lower synthesis by antisense oligonucleotides or neutralizing anti- number of glomerular cells in all-trans retinoic acid–treated bodies abrogated matrix expansion (10,11). Isaka et al. (30) nephritic rats or whether expression per cell is reduced. The reported that a TGF receptor II IgG-Fc fusion protein inhibited latter is probable, given that TGF receptor I mRNA is not glomerular damage in this model. altered. Immunohistochemistry also indicated that TGF recep- We examined the effects of retinoids on the renal TGF-␤ tor II staining was less expressed on a per-cell basis. Glomer- J Am Soc Nephrol 12: 2300–2309, 2001 Effects of Retinoids on TGF in Nephritis 2307

Figure 8. Glomerular staining score of extracellular matrix proteins. Significantly lower collagen IV (A) and collagen III (B) staining was found in glomeruli of rats with Thy-GN after posttreatment with Iso. The staining for fibronectin is higher in Thy-GN rats than in controls (C). In Thy-GN rats treated with Iso, staining for fibronectin was not different from that of control animals. Treatment with Ra had no discernible effect on staining for collagen IV (A) and collagen III (B) but led to high staining for fibronectin in Thy-GN rats (C).

ular TGF receptor II immunostaining was found in mesangial than all-trans retinoic acid. Because we had selected high doses cells, glomerular epithelial cells, and podocytes. Gene expres- of both compounds, according to the recommendations by Dr. sion and immunostaining of TGF receptor II (and also of Klaus (Hoffmann-LaRoche), these findings support the idea TGF-␤1) is enhanced in tubuli of Thy-GN rats. This increase in that retinoids differ in their potency to modulate expression of Thy-GN rats may not reflect a tubular damage in Thy-GN but this cytokine. rather the activation of tubular cells by high protein load, The mechanism by which retinoids inhibit TGF-␤1 expres- which was also described by others (32,33). sion cannot be determined from these studies. One has to In contrast to the extensive studies on the interaction of consider the possibility that lower renal expression of TGF-␤1 retinoids and TGF-␤1, little is known about the interaction of is only the consequence of reduced glomerular damage in this retinoids with TGF receptors. Mercier et al. (34) have de- model. Macrophages and monocytes are well-known sources scribed down-regulation of TGF receptor II by retinoids. The of TGF-␤1, i.e., in acute puromycin aminonucleoside nephro- TGF receptor promoter contains two AP-1 binding sites. Reti- sis (40). Our data indicate fewer glomerular ED-1(ϩ) cells in noids inhibit AP-1–dependent pathways (35). This suggests, isotretinoin-treated rats (20). However, double-staining for but does not prove, that TGF receptor II expression is reduced TGF-␤1 and monocytes/macrophages (Ki2MR) demonstrated by retinoids via an AP-1–dependent mechanism. that TGF-␤1 is also expressed in glomerular cells other than The interaction of TGF receptors I and II has not yet been monocytes/macrophages. This indicates that reduction in glo- fully understood. There is only one TGF receptor II, but there merular monocyte/macrophage count is not the only explana- are different TGF receptor I subtypes, of which involvement tion for the reduced TGF-␤1 staining (Figure 6, G and H). with TGF-␤ signaling has only been demonstrated for subtype All-trans retinoic acid completely abrogated the BP increase in R4 (36,37). TGF receptor II binds TGF-␤1 and acts as a this model. Because shear stress is known to induce TGF-␤1 constitutively active kinase. The complex of TGF receptor II expression (41), the beneficial effects of retinoids might be, at and TGF-␤1 is thought to recruit and phosphorylate TGF least in part, the consequence of normalized BP. The finding of receptor I, which then initiates the downstream cascade lower glomerular TGF-␤1 gene expression in non-nephritic (38,39). rats treated with all-trans retinoic acid suggests, however, a The simultaneous decrease of TGF-␤1 and its receptor sug- direct effect of retinoids on glomerular TGF-␤1 independent gests very effective lowering of TGF-␤1 action after treatment from BP and shear stress. with retinoids. This observation supports the notion that the Retinoids exert strong anti–AP-1 activity. TGF-␤1 expres- sensitivity of glomerular cells to TGF-␤1 is reduced after sion depends at least in part on this transcription factor. The treatment with retinoids. The effects of retinoids on TGF-␤1 TGF-␤1 promotor contains three AP-1 consensus sites that were comparable in the pretreatment and the posttreatment mediate retinoid-dependent suppression of TPA-stimulated protocols, which indicates that the time point of initiation of TGF-␤1 gene activation (17). Haxsen et al. (42) demonstrated retinoid treatment does not influence its effect on TGF-␤1 that retinoids lower basal TGF-␤1 gene expression and inhibit expression. Interestingly, however, comparison of the effects angiotensin II induction of TGF-␤1 expression in vascular of isotretinoin and all-trans retinoic acid shows that isotretinoin smooth muscle cells. Morishita et al. (43) demonstrated that reduced the level of TGF-␤1 expression significantly more angiotensin II induction of TGF-␤1 gene expression depends 2308 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 2300–2309, 2001 on AP-1 activation. These findings suggest that retinoids may 5. Kaneto H, Morrissey J, Klahr S: Increased expression of TGF- also have a direct effect on TGF-␤1 via these pathways. beta 1 mRNA in the obstructed kidney of rats with unilateral Retinoids also influence the expression of glomerular matrix ureteral ligation. Kidney Int 44: 313–321, 1993 proteins (44–47). Retinoids are capable of either increasing or 6. Schneider A, Thaiss F, Rau HP, Wolf G, Zahner G, Jocks T, decreasing expression of extracellular matrix proteins. Some Helmchen U, Stahl RA: Prostaglandin E1 inhibits collagen ex- pression in anti-thymocyte antibody-induced glomerulonephritis: matrix proteins, e.g., laminin, contain retinoic acid–responsive Possible role of TGF beta. Kidney Int 50: 190–199, 1996 elements on their promotor (46,47), whereas others do not, i.e., 7. Johnson RJ, Lombardi D, Eng E, Gordon K, Alpers CE, Pritzl P, fibronectin and collagen III. Scita et al. (44) demonstrated that Floege J, Young B, Pippin J, Couser WG, Gabbiani G: Modu- retinoic acid reduced fibronectin levels in NIH-3T3 cells. Taub lation of experimental mesangial proliferative nephritis by inter- et al. (45) demonstrated a significant reduction of laminin, feron-gamma. Kidney Int 47: 62–69, 1995 collagen IV, and heparan sulfate in canine kidney cells after 8. Okuda S, Languino LR, Ruoslahti E, Border WA: Elevated exposure to retinoic acid. expression of transforming growth factor-beta and proteoglycan Renal induction of collagen III and IV and fibronectin in production in experimental glomerulonephritis. Possible role in Thy-GN rats in our experiments is in accordance with other expansion of the mesangial extracellular matrix [published erra- data obtained in this model (48,49). Parallel to the decrease in tum appears in J Clin Invest 86: 2175, 1990]. J Clin Invest 86: renal TGF-␤1 and TGF receptor II in Thy-GN rats, glomerular 453–462, 1990 9. Peters H, Border WA, Noble NA: Targeting TGF-beta overex- collagen III and IV were decreased after isotretinoin treatment, pression in renal disease: Maximizing the antifibrotic action of whereas fibronectin was not influenced. This indicates that angiotensin II blockade. Kidney Int 54: 1570–1580, 1998 isotretinoin has an overall antifibrotic effect in this model. This 10. Akagi Y, Isaka Y, Arai M, Kaneko T, Takenaka M, Moriyama T, is in accordance with studies of Shigematsu and Tajima (50), Kaneda Y, Ando A, Orita Y, Kamada T, Ueda N, Imai E: who found that the antifibrotic action of isotretinoin was stron- Inhibition of TGF-beta 1 expression by antisense oligonucleo- ger than that of retinoic acid. In contrast, although all-trans tides suppressed extracellular matrix accumulation in experimen- retinoic acid reduced glomerular TGF-␤1, it did not reduce tal glomerulonephritis. Kidney Int 50: 148–155, 1996 collagen III or IV. It evoked a slight but significant increase in 11. Border WA, Okuda S, Languino LR, Sporn MB, Ruoslahti E: fibronectin. From these data, it cannot be safely concluded that Suppression of experimental glomerulonephritis by antiserum all-trans retinoic acid and isotretinoin have differential effects against transforming growth factor beta 1. Nature 346: 371–374, on extracellular matrix proteins. Differences in retinoid metab- 1990 olism or activation or shifts in dose-response 12. Merlet-Benichou C, Vilar J, Lelievre-Pegorier M, Gilbert T: Role of retinoids in renal development: Pathophysiological implica- curves may explain such different effects. Studies on vascular tion. Curr Opin Nephrol Hypertens 8: 39–43, 1999 smooth muscle cells, however, have indicated that, at the same 13. Morriss-Kay G: Retinoic acid receptors in normal growth and concentration, all-trans retinoic acid and isotretinoin, indeed, development. Cancer Surv 14: 181–193, 1992 have different effects on TGF-␤1 and procollagen IV expres- 14. Chambon P: A decade of molecular biology of retinoic acid sion (preliminary unpublished observations), as has already receptors. Faseb J 10: 940–954, 1996 been proposed by Shigematsu and Tajima (50). 15. Datta PK, Lianos EA: Retinoic acids inhibit inducible nitric In future experiments, it will be necessary to evaluate oxide synthase expression in mesangial cells. Kidney Int 56: whether the various retinoids, i.e., specific retinoid receptor 486–493, 1999 agonists, have different effects and to establish their dose- 16. Miano JM, Topouzis S, Majesky MW, Olson EN: Retinoid response relationships. In conclusion, the above data indicate receptor expression and all-trans retinoic acid-mediated growth that the beneficial effects of retinoids on glomerular damage in inhibition in vascular smooth muscle cells. Circulation 93: 1886–1895, 1996 Thy-GN are in part due to reduction of renal TGF-␤1 gene 17. Salbert G, Fanjul A, Piedrafita FJ, Lu XP, Kim SJ, Tran P, Pfahl expression. Our findings clearly warrant further studies to M: Retinoic acid receptors and retinoid X receptor-alpha down- assess the potential therapeutic effects of retinoids in renal regulate the transforming growth factor-beta 1 promoter by an- disease, to delineate the mechanisms of retinoid action, and to tagonizing AP-1 activity. Mol Endocrinol 7: 1347–1356, 1993 identify retinoid receptor–specific pathways in the kidney. 18. Simonson MS: Anti-AP-1 activity of all-trans retinoic acid in glomerular mesangial cells. Am J Physiol 267: F805–F815, 1994 References 19. Zhou XF, Shen XQ, Shemshedini L: Ligand-activated retinoic 1. Border WA, Noble NA: Transforming growth factor beta in acid receptor inhibits AP-1 transactivation by disrupting c-Jun/ tissue fibrosis. N Engl J Med 331: 1286–1292, 1994 c-Fos dimerization. Mol Endocrinol 13: 276–285, 1999 2. Kasinath BS: Glomerular endothelial cell proteoglycans–regula- 20. Wagner J, Dechow C, Morath C, Lehrke I, Amann K, Waldherr tion by TGF-beta 1. Arch Biochem Biophys 305: 370–377, 1993 R, Floege J, Ritz E: Retinoic acid reduces glomerular injury in a 3. Nakamura T, Miller D, Ruoslahti E, Border WA: Production of rat model of glomerular damage. J Am Soc Nephrol 11: 1479– extracellular matrix by glomerular epithelial cells is regulated by 1487, 2000 transforming growth factor-beta 1. Kidney Int 41: 1213–1221, 21. Wagner J, Gehlen F, Ciechanowicz A, Ritz E: Angiotensin II 1992 receptor type 1 gene expression in human glomerulonephritis and 4. Edwards DR, Murphy G, Reynolds JJ, Whitham SE, Docherty diabetes mellitus. J Am Soc Nephrol 10: 545–551, 1999 AJ, Angel P, Heath JK: Transforming growth factor beta mod- 22. Bradford MM: A rapid and sensitive method for the quantitation ulates the expression of collagenase and metalloproteinase inhib- of microgram quantities of protein utilizing the principle of itor. Embo J 6: 1899–1904, 1987 protein-dye binding. Anal Biochem 72: 248–254, 1976 J Am Soc Nephrol 12: 2300–2309, 2001 Effects of Retinoids on TGF in Nephritis 2309

23. Danielpour D, Kim KY, Winokur TS, Sporn MB: Differential and its effect on TGF-beta binding to the type II receptor. Science regulation of the expression of transforming growth factor-betas 260: 1344–1348, 1993 1 and 2 by retinoic acid, epidermal growth factor, and dexameth- 38. Kretzschmar M, Massague J: SMADs: Mediators and regulators asone in NRK-49F and A549 cells. J Cell Physiol 148: 235–244, of TGF-beta signaling. Curr Opin Genet Dev 8: 103–111, 1998 1991 39. Wrana JL, Attisano L, Wieser R, Ventura F, Massague J: Mech- 24. Roberts AB, Sporn MB: Mechanistic interrelationships between anism of activation of the TGF-beta receptor. Nature 370: 341– two superfamilies: The steroid/retinoid receptors and transform- 347, 1994 ing growth factor-beta. Cancer Surv 14: 205–220, 1992 40. Ding G, Pesek-Diamond I, Diamond JR: Cholesterol, macro- 25. Jakowlew SB, Cubert J, Danielpour D, Sporn MB, Roberts AB: phages, and gene expression of TGF-beta 1 and fibronectin Differential regulation of the expression of transforming growth during nephrosis. Am J Physiol 264: F577–F584, 1993 factor-beta mRNAs by growth factors and retinoic acid in 41. Cucina A, Sterpetti AV, Borrelli V, Pagliei S, Cavallaro A, chicken embryo chondrocytes, myocytes, and fibroblasts. J Cell D’Angelo LS: Shear stress induces transforming growth factor- Physiol 150: 377–385, 1992 beta 1 release by arterial endothelial cells. Surgery 123: 212–217, 26. Anstead GM: Steroids, retinoids, and wound healing. Adv Wound 1998 Care 11: 277–285, 1998 42. Haxsen V, Adam-Stitah S, Ritz E, Wagner J: Retinoids inhibit 27. Bennett RT, Mazzaccaro RJ, Chopra N, Melman A, Franco I: Suppression of renal inflammation with vitamins A and E in the actions of angiotensin II on vascular smooth muscle cells. ascending pyelonephritis in rats. J Urol 161: 1681–1684, 1999 Circ Res 88: 637–644, 2001 28. Glick AB, McCune BK, Abdulkarem N, Flanders KC, Lumadue 43. Morishita R, Gibbons GH, Horiuchi M, Kaneda Y, Ogihara T, JA, Smith JM, Sporn MB: Complex regulation of TGF beta Dzau VJ: Role of AP-1 complex in angiotensin II-mediated expression by retinoic acid in the vitamin A-deficient rat. Devel- transforming growth factor-beta expression and growth of opment 111: 1081–1086, 1991 smooth muscle cells: Using decoy approach against AP-1 29. Mahmood R, Flanders KC, Morriss-Kay GM: Interactions be- binding site. Biochem Biophys Res Commun 243: 361–367, tween retinoids and TGF betas in mouse morphogenesis. Devel- 1998 opment 115: 67–74, 1992 44. Scita G, Darwiche N, Greenwald E, Rosenberg M, Politi K, De 30. Isaka Y, Akagi Y, Ando Y, Tsujie M, Sudo T, Ohno N, Border Luca LM: Retinoic acid down-regulation of fibronectin and WA, Noble NA, Kaneda Y, Hori M, Imai E: Gene therapy by retinoic acid receptor alpha proteins in NIH-3T3 cells. Blocks of transforming growth factor-beta receptor-IgG Fc chimera sup- this response by ras transformation. J Biol Chem 271: 6502– pressed extracellular matrix accumulation in experimental glo- 6508, 1996 merulonephritis [see comments]. Kidney Int 55: 465–475, 1999 45. Taub M: Retinoic acid inhibits basement membrane protein 31. Isaka Y, Fujiwara Y, Ueda N, Kaneda Y, Kamada T, Imai E: biosynthesis while stimulating dome formation by Madin Darby Glomerulosclerosis induced by in vivo transfection of transform- canine kidney cells in hormonally defined serum-free medium. ing growth factor-beta or platelet-derived growth factor gene into J Cell Physiol 148: 211–219, 1991 the rat kidney. J Clin Invest 92: 2597–2601, 1993 46. Vasios G, Mader S, Gold JD, Leid M, Lutz Y, Gaub MP, 32. Eddy AA, Giachelli CM: Renal expression of genes that promote Chambon P, Gudas L: The late retinoic acid induction of laminin interstitial inflammation and fibrosis in rats with protein-over- B1 gene transcription involves RAR binding to the responsive load proteinuria. Kidney Int 47: 1546–1557, 1995 element. Embo J 10: 1149–1158, 1991 33. Mezzano SA, Droguett MA, Burgos ME, Ardiles LG, Aros CA, 47. Vasios GW, Gold JD, Petkovich M, Chambon P, Gudas LJ: A Caorsi I, Egido J: Overexpression of chemokines, fibrogenic retinoic acid-responsive element is present in the 5' flanking cytokines, and myofibroblasts in human membranous nephrop- region of the laminin B1 gene. Proc Natl Acad Sci USA 86: athy. Kidney Int 57: 147–158, 2000 9099–9103, 1989 34. Mercier T, Gaillard-Sanchez I, Martel P, Heberden C: TGF-beta receptors are diminished after retinoid exposure in rat liver 48. Floege J, Eng E, Young BA, Couser WG, Johnson RJ: Heparin epithelial cells. J Cell Biochem 61: 230–237, 1996 suppresses mesangial cell proliferation and matrix expansion in 35. Schule R, Rangarajan P, Yang N, Kliewer S, Ransone LJ, Bolado J, experimental mesangioproliferative glomerulonephritis. Kidney Verma IM, Evans RM: Retinoic acid is a negative regulator of AP-1- Int 43: 369–380, 1993 responsive genes. Proc Natl Acad Sci USA 88: 6092–6096, 1991 49. Floege J, Johnson RJ, Gordon K, Iida H, Pritzl P, Yoshimura A, 36. Bassing CH, Yingling JM, Howe DJ, Wang T, He WW, Campbell C, Alpers CE, Couser WG: Increased synthesis of Gustafson ML, Shah P, Donahoe PK, Wang XF: A transforming extracellular matrix in mesangial proliferative nephritis. Kidney growth factor beta type I receptor that signals to activate gene Int 40: 477–488, 1991 expression. Science 263: 87–89, 1994 50. Shigematsu T, Tajima S: Modulation of collagen synthesis and 37. Ebner R, Chen RH, Shum L, Lawler S, Zioncheck TF, Lee A, cell proliferation by retinoids in human skin fibroblasts. J Der- Lopez AR, Derynck R: Cloning of a type I TGF-beta receptor matol Sci 9: 142–145, 1995