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Kolling Institute of Medical Research (S.M.T.), University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia; Department of Diabetic Complications (Z.C., W.C.B., G.B., J.M.F., M.E.C.), Baker Medical Research Institute, Melbourne, Victoria 3004, Australia; and Department of Medicine (S.V.M.), University of Sydney, Sydney 2006, Australia
Address all correspondence and requests for reprints to: Prof. Mark Cooper, Department of Diabetic Complications, Baker Medical Research Institute, Commercial Road, Melbourne, Victoria 3004, Australia. E-mail: mark.cooper{at}baker.edu.au.
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Abstract |
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Introduction |
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Connective tissue growth factor (CTGF) is being increasingly recognized as a possible contributor to diabetic complications (6, 7). CTGF is a potent inducer of extracellular matrix (8) in various cell types, including mesangial cells (9, 10). Elevated extracellular D-glucose increases CTGF mRNA and protein levels in human and rat mesangial cells in vitro (10, 11). Furthermore, CTGF has been reported to be elevated in two different rodent models of diabetic nephropathy (10, 11), and in early (12) and end-stage diabetic nephropathy in humans (13). We recently reported that treatment of human dermal fibroblasts with soluble AGEs induces CTGF mRNA and protein in vitro (14), suggesting a possible link between AGE induction of CTGF and end-organ complications in diabetes. Furthermore, AGE-induced expression of the extracellular matrix protein, fibronectin, has been shown in in vitro studies to be CTGF dependent (15). Fibronectin has been reported to be increased in the diabetic kidney (16, 17), but its link to AGE accumulation and CTGF expression has not been examined previously.
The aims of the current study were, firstly, to determine whether observed increases in CTGF and fibronectin in the diabetic rodent kidney could be prevented by an inhibitor of AGE formation and whether changes in CTGF relate to pathological changes occurring in diabetic nephropathy. This issue was explored in the context of the known actions of aminoguanidine (AG) on the kidney, which include reduction in mesangial expansion (18, 19). AG, an inhibitor of AGE formation, has previously been reported by our group and others to confer renoprotection when administered after the development of diabetes (19, 20). We now report that AG prevents the induction of CTGF and fibronectin in a rodent model of diabetic nephropathy. Secondly, we explored whether there was a direct relationship between AGEs and mesangial CTGF and fibronectin production by performing in vitro studies involving exposure of cultured mesangial cell to carboxymethyl lysine (CML)-containing AGEs.
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Materials and Methods |
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In situ hybridization for CTGF
Gene expression of CTGF was determined by in situ hybridization using a method described previously (24). Antisense riboprobes for rat CTGF were generated from a human CTGF cDNA (25). A 35S-labeled RNA probe for CTGF was prepared with transcription kit (Promega Corp., Madison, WI). Purified riboprobe length was adjusted to approximately 150 bases by alkaline hydrolysis. Sections (4 µm) were cut onto slides precoated with 3-aminopropyltriethoxysilane and incubated overnight at 37 C. Tissue sections were dewaxed and rehydrated in graded ethanol and milliQ water, equilibrated in P buffer [50 mM Tris-HCl (pH 7.5), 5 mM EDTA], and incubated in 125 µg/ml Pronase E in P buffer for 10 min at 37 C. Sections were then washed twice in 0.1 M sodium phosphate buffer (pH 7.2), postfixed in 4% paraformaldehyde for 10 min, washed twice in 0.1 M sodium phosphate buffer, then rinsed in milliQ water, dehydrated in 70% ethanol, and air dried. Hybridization buffer containing 2 x 104 cpm/µl riboprobe in 300 mM NaCl, 10 mM Tris HCl (pH 7.5), 10 mM Na2HPO4, 5 mM EDTA (pH 8.0), 50% deionized formamide, 20 mg/ml yeast RNA, 10% wt/vol dextran sulfate, and 100 mM dithiothreitol was heated to 85 C for 5 min. Twenty-five microliters of this solution were then added to each section and incubated at 60 C overnight in 50% formamide-humidified chambers. As controls for nonspecific signal, sections were incubated with sense riboprobe. Slides were washed in 2x SSC containing 50% formamide prewarmed to 50 C to remove coverslips. Sections were then washed in the above solution for 1 h with shaking at 55 C, rinsed three more times in Rnase buffer [10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8.0), 0.5 M NaCl], and then incubated with Rnase A (150 µg/ml) for 1 h at 37 C. Sections were later washed in 2x SSC for 45 min at 55 C, dehydrated in graded ethanol, air-dried, and exposed to BioMaxMR autoradiographic film for 3–5 d. After in situ hybridization, kidney sections were exposed to x-ray film and then quantitated with the Microcomputer Imaging Device system as previously described. Slides were then dipped in Amersham nuclear emulsion (Ilford, Mobberley, Cheshire, UK), stored in a light-free box with desiccant at 4 C for 21–28 d, brought to room temperature, then immersed in Kodak D19 developer (Eastman Kodak, Rochester, NY) washed in 15% acetic acid, and fixed in Ilford Hypan before staining with hematoxylin and eosin.
Immunohistochemistry for CTGF
Immunohistochemical staining was performed as previously described, according to a modified method (26). The CTGF polyclonal antibody used (27) was raised in a New Zealand white rabbit, against full-length purified recombinant human CTGF protein (28). In brief, 20-µm frozen kidney sections were cut on a cryostat at -20 C. Frozen sections were fixed with cold acetone, and endogenous peroxidase was inactivated using 0.1% hydrogen peroxide in PBS. The sections were incubated with protein-blocking agent, and endogenous nonspecific binding for biotin/avidin was blocked using a Biotin/Avidin Blocking Kit (Vector Laboratories, Inc., Burlingame, CA). The kidney sections were incubated for 1 h at room temperature with CTGF antibody at a dilution of 1:200. Biotinylated horse antimouse Ig (Vector Laboratories, Inc.) was used as a second antibody, followed by horseradish peroxidase-conjugated streptavidin. Peroxidase activity was identified by reaction with 3,3'-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) substrate.
Immunohistochemistry for AGEs and fibronectin
Immunohistochemical staining for AGEs and fibronectin was performed as previously described (29). In brief, formalin-fixed, paraffin-embedded sections (4 µm) of kidney were rehydrated and treated with 1% H2O2/methanol followed by incubation in Protein Blocking Agent (Lipshaw-Immunon, Pittsburgh, PA) for 20 min at room temperature. Sections were then sequentially incubated with an anti-AGE (30) or anti-fibronectin (DAKO Corp., Carpinteria, CA) antibody for 30 min at room temperature, washed in PBS, and incubated with biotinylated goat antirabbit Ig (DAKO Corp.). This was followed by another PBS wash and incubation with peroxidase-conjugated streptavidin (DAKO Corp.). Peroxidase localization was revealed using diaminobenzidine tetrahydrochloride as the chromogen. The AGE antibody used in this protocol has been described in detail previously (30). This antibody detects advanced glycated proteins including advanced glycated BSA and RNase. Studies to characterize the epitope of this antibody indicate that it detects CML-containing proteins but does not detect native BSA, native Rnase, or the AGE, pentosidine (31).
Histomorphometry
Quantification of immunostaining for CTGF, fibronectin, and AGEs were performed by calculation of the proportion of area occupied by the brown staining in the glomeruli using the Imaging Analysis System (AIS, Imaging Research, Inc. St. Catherines, Ontario, Canada) as described previously (26).
Study protocol 2
Human fetal mesangial cells were isolated after serial sieving from kidneys obtained from termination of pregnancies in the second trimester and were cultured as outgrowths from glomerular isolates. Their identity and purity as mesangial cells was established using previously published criteria (32). Cells were grown in RPMI 1640 medium containing 5 mM glucose (Cytosystems, New South Wales, Australia), and in 10% fetal calf serum in an atmosphere of 5% CO2 and air. After an overnight incubation in serum-free RPMI medium, near-confluent cells grown in six-well plates between the second and fourth passage were treated with reagents in fresh serum-free medium.
AGE reagents were synthesized by previously described methods, using BSA as the protein substrate (15). BSA (Sigma; RIA grade, fraction V) at 10 mM was coincubated in sterile PBS with 0.5 M D-ribose for 10 d, under aerobic conditions at 37 C. To generate control BSA for comparison with AGE treatments, tubes were prepared with simultaneous incubations under the same conditions without the addition of the D-ribose. Additionally, in parallel preparations, AG at 100 mM, as an inhibitor of formation of products of nonenzymatic glycosylation, was added to the BSA and ribose, and this reagent is termed AG-BSA. In addition the AGE reagent termed N-
carboxymethyl lysine-BSA (CML-BSA) was generated as previously reported (33, 34). In brief, 176 mg BSA was dissolved in sodium phosphate buffer to which 14.3 mg (0.155 M) glyoxylic acid (Sigma) was added, together with 19.8 mg (0.315 M) of the reducing agent, sodium cyanoborohydride (Sigma), all in a total reaction volume of 1 ml, followed by an incubation time of 24 h at 37 C. To generate control BSA for comparison, tubes were prepared with simultaneous incubations under the same conditions without the addition of the glyoxylic acid. All preparations were extensively dialyzed in PBS, using a low molecular weight cut-off membrane (Spectrapor 1, 6–8 kDa, Spectrum Industries, Los Angeles, CA), to remove free AG, reducing agent, and unincorporated sugars.
The AGE content in the preparations was assessed using fluorescence and ELISA. The fluorescence content, measured with a fluorescence spectrometer at 390-nm emission after a 450-nm excitation, in relative fluorescence units per milligram of BSA, was 50.5 ± 2.5 (mean ± SD) for control BSA, 187.9 ± 4.6 for AGE-BSA, and 55.7 ± 5.2 for AG-BSA. By competitive ELISA performed by Alteon Inc. (Ramsey, NJ), using a synthetic N- CML analog as the standard (15), the CML content of the CML-based preparations (picomoles of CML per milligram of BSA ± 95% confidence interval) was 13 ± 1.4 for the CML-BSA, and it was undetectable (<1) for control BSA.
Cells were treated with soluble AGE-BSA, AG-BSA, or control BSA (each at 100 µg/ml BSA) for 72 h. In other experiments, cells were treated for 72 h with 0–600 µg/ml of CML-BSA or control-BSA. After cell treatments, total RNA was isolated and reverse transcribed, and CTGF mRNA and fibronectin mRNA were then measured by quantitative real-time RT-PCR, using specific probes and primers, all as previously described (15). The 
- method was used to calculate relative changes in mRNA levels of the same transcript, corrected for the housekeeping gene, human ß-actin (35).
Statistics
Logarithmic transformation of urinary albumin data was performed to yield a normal distribution for this parameter. The specific contributions of diabetes or treatment in the animal and cell culture studies were assessed by ANOVA using the Statview II Program (Brainpower, Calabasas, CA) on a Macintosh G3 personal computer (Apple, Cupertino CA). Comparisons among groups were performed by Fisher’s least significant difference method. A P value less than 0.05 was viewed as statistically significant. All data are shown as mean ± SEM unless otherwise specified.
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Results |
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). No difference in any of these parameters was observed in diabetic rats treated with AG when compared with untreated diabetic animals. Systolic blood pressure was modestly elevated in diabetic rats, with no significant effect of AG on this parameter (Table 1).
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). These increases were absent in diabetic rats treated with AG (Table 1). These results indicate that the AG used in the study blocked AGE formation in the renal glomerulus of treated diabetic rats.
CTGF expression.
Assessment of CTGF gene expression was initially performed by quantitation of autoradiographs of whole kidney cross-sections after in situ hybridization with a radiolabeled CTGF riboprobe. Representative cross-sections in Fig. 1 demonstrate that the CTGF mRNA signal in diabetic rats (Fig. 1B) was more intense and frequent than in nondiabetic controls (Fig. 1A). The signal in diabetic rats was confined predominantly to punctate areas in the renal cortex. The diabetic animals treated with AG (Fig. 1C) had lower CTGF mRNA signal compared with diabetic animals not treated with AG, at a level similar to that seen in the nondiabetic controls. Hybridization using a negative control sense riboprobe for CTGF did not show any specific signal (data not shown).
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, CTGF mRNA levels in diabetic rats were higher than in nondiabetic controls. The diabetic animals treated with AG had reduced CTGF gene expression when compared with diabetic animals not treated with AG. This level of CTGF gene expression was not significantly different from that seen in control kidney sections.
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), CTGF gene expression in untreated diabetic animals was increased mainly in glomeruli (Fig. 3B). The cell types expressing CTGF were spindle shaped, characteristic of mesangial cells. AG treatment of diabetic rats (Fig. 3C) prevented the induction of CTGF gene expression when compared with untreated diabetic rats.
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) and in the interstitium of the renal cortex. In comparison with the controls, immunostaining for CTGF was more intense in diabetic rat kidney (9.9 ± 1.3 vs. 4.1 ± 0.7% in control). CTGF immunoreactivity was greater in both the glomerulus and the renal interstitium, including areas adjacent to proximal tubules. These patterns of staining were not seen when the same titer of control normal rabbit serum was used in place of the CTGF antiserum. The diabetic rats treated with AG (Fig. 4C) had reduced levels of CTGF protein expression (5.9 ± 1.0%) compared with untreated diabetic animals and a similar level to that observed in control animals (4.1 ± 0.7%).
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), with minimal expression in the tubulointerstitium in the control kidney. Glomerular fibronectin protein expression was increased in diabetic rats (12.0 ± 0.9% vs. control, 8.5 ± 1.0%). Treatment with AG prevented the diabetes-related increase in glomerular fibronectin expression (8.3 ± 1.9%).
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). Because the AGE adduct CML is increased in the rodent diabetic kidney in vivo and is prevented by AG treatment of these diabetic animals (Table 1), we then tested whether the specific AGE reagent CML-BSA regulated CTGF and fibronectin in the mesangial cells. Treatment of these cells with CML-BSA caused a dose-dependent induction of CTGF mRNA and fibronectin mRNA (Fig. 6B). These data show that AGEs induce CTGF mRNA and fibronectin mRNA in human mesangial cells in vitro, and they support the in vivo data implicating AGEs in these observed effects.
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Discussion |
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There is strong circumstantial evidence to implicate CTGF in contributing to diabetic renal complications. CTGF is known to be induced in renal mesangial cells in vitro by high glucose (10, 11), and renal CTGF is increased in vivo in experimental (10, 11) and human diabetes (12, 13). A recent study in humans showed that increases in CTGF mRNA levels in diabetic glomeruli correlate with early increases in glomerular collagen type IV expression and with microalbuminuria (12). Moreover, factors implicated in diabetic complications, such as oxidative stress (37), cyclic mechanical strain (10), TGF-ß1 (9), and VEGF (38) induce CTGF in a variety of cell types. Of particular relevance to this study is our previous in vitro finding demonstrating that AGEs induce CTGF expression, albeit in a nonrenal context (14). We have now presented novel data in mesangial cells showing that both CTGF and fibronectin mRNA are induced by AGE treatment of cells.
The in vivo studies involved an immunohistochemical approach to assess renal AGE accumulation using an antibody that predominantly detects CML-containing proteins. Therefore, to further assess whether these AGEs per se are linked to CTGF and fibronectin expression, in vitro studies were performed that showed that CML-containing AGEs induce both CTGF and fibronectin. These complementary in vitro and in vivo studies emphasize the likelihood that CTGF plays a central role in diabetes and AGE-associated renal extracellular matrix accumulation, which ultimately results in mesangial expansion. Further evidence linking CTGF expression to renal disease is suggested by a range of studies performed using various potentially renoprotective treatments. ACE inhibitors, angiotensin II antagonists, and statins have been reported to reduce CTGF expression either in vitro or in experimental studies, albeit not in a diabetic milieu (39, 40). Clearly, specific methods to inhibit CTGF induction and/or bioactivity in vivo are required before causality for CTGF in diabetic renal disease can be definitively determined.
The glomerulus, specifically the mesangial cell, is the main source of CTGF expression in the diabetic kidney (41). Our study showed that the increases in renal CTGF gene expression in diabetes are predominantly but not exclusively localized to the mesangial cells in the glomerulus, and this is consistent with previous reports in diabetes, in which glomeruli were the main renal source of CTGF expression (10). As previously reported (10), CTGF gene expression in nondiabetic animals was very low throughout the kidney in the present study. CTGF produced in mesangial cells may have an autocrine and/or paracrine role. In human renal mesangial cells in vitro, soluble recombinant human CTGF potently induced collagens and fibronectin (11), potentially contributing to mesangial matrix expansion. Although CTGF may exert its bioactivity through binding classes of integrin (42, 43) and/or through MAPK pathway activation (44), its exact cellular mechanism of action in mesangial cells is yet to be defined.
TGF-ß1 is a potent inducer of CTGF in mesangial cells (9), and the rat and human CTGF promoters contain a unique TGF-ß response element, conserved across species (45). In in vitro studies in rodent mesangial cells, CTGF was shown to mediate the TGF-ß1-induced increase in fibronectin (9), and, in other cell types, CTGF mediates TGF-ß1 induced increases in fibronectin (46) and type 1 collagen (47). High glucose induces CTGF in vitro in mesangial cells at least partially through increases in TGF-ß1 bioactivity (10, 11). Because AGEs can induce TGF-ß1 in rodent kidney in both in vitro studies as well as in vivo studies (48), it is possible that the induction of CTGF in the current study is at least partially occurring through induction of TGF-ß1. If this is the case, then the protection from diabetic nephropathy produced by treatment of diabetic mice with neutralizing TGF-ß antibodies (49) may be due to prevention of induction of CTGF by TGF-ß1. In a recent preliminary report in diabetic mice specifically overexpressing in the myocardium the protein kinase C isoform, ß2, up-regulation of CTGF in the heart in association with the development of fibrosis was apparent before an increase in TGF-ß was observed (50). These findings provide further evidence that fibrosis, as part of the pathology of diabetic complications, may be partly related to CTGF rather than to TGF-ß alone. In contrast to TGF-ß1, CTGF is not thought to have an important role in immune modulation (47), and therefore it is potentially a more specific and downstream target for preventing diabetic complications (6).
AG has previously been reported to influence a range of other growth factors, including VEGF, TGF-ß, and IGFs (4, 5, 51). However, in contrast to TGF-ß, IGF-I has not been shown to directly induce CTGF in vitro or in vivo. IGF-I potentiates the induction of type IV collagen by CTGF in normal rat kidney cells (52), although the mechanism of this cellular effect has not been defined, and it is not clear whether the observed weak in vitro binding of CTGF to IGF-I (28) is responsible. Whether the increased IGF-I and CTGF synergize in vivo in their pathological effects in diabetic nephropathy remains to be determined.
AGEs have recently been reported in vitro to stimulate fibronectin production, which could be attenuated by coadministration of a neutralizing antibody to CTGF (15). The present study provides evidence that this phenomenon occurs in vivo. Indeed, AG prevented the increase in glomerular fibronectin expression observed in the diabetic kidney. Fibronectin has previously been shown to be up-regulated in the diabetic kidney and has been reported to be reduced by renoprotective regimens such as protein kinase C inhibition (53). This reduction in extracellular matrix accumulation is associated with previous quantitative histomorphometric studies that demonstrated reduced mesangial expansion in response to inhibition of renal AGE accumulation with AG (19, 30).
AG is the most well characterized and widely studied inhibitor of AGE formation. That we observed inhibition of CTGF overexpression in parallel with inhibition of AGE accumulation by AG in the diabetic rats suggests that AG is working through inhibition of formation of AGEs to prevent CTGF up-regulation. This is consistent with our in vitro finding of induction of CTGF expression by AGEs (14). Furthermore, the novel data presented in the current work showing induction of CTGF mRNA and fibronectin mRNA by AGEs in human mesangial cells is supportive of this concept. AG is also known to have other actions, which include inhibition of nitric oxide synthase (54). We have previously observed in the same rodent model of diabetes that administration of more specific inhibitors of nitric oxide synthase does not reproduce the renal effects of AG (30). Those results and the current in vivo study, supported by the in vitro data, suggest that AGEs cause renal pathology in this model of diabetes, at least partly through the up-regulation of renal CTGF. Studies using specific CTGF inhibitors are now warranted to determine to what extent CTGF mediates renal pathology caused by AGEs, and by other agents, in diabetes.
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Acknowledgments |
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Footnotes |
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Z.C. is an advanced Post-Doctoral Fellow supported by the Juvenile Diabetes Research Foundation.
Abbreviations: AG, Aminoguanidine; AGE, advanced glycation end-product; CML, carboxymethyl lysine; CTGF, connective tissue growth factor; VEGF, vascular endothelial growth factor.
Received June 13, 2002.
Accepted for publication September 3, 2002.
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(IIb)ß(3). J Biol Chem 274:24321–24327vß3, promotes endothelial cell survival, and induces angiogenesis in vivo. Mol Cell Biol 19:2958–2966This article has been cited by other articles:
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