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1Departments of Medicine Endocrinology and Cell Biology and 2Department of Ophthalmology, Dean A. McGee Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
Submitted 8 August 2007 ; accepted in final form 28 February 2008
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ABSTRACT |
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 TOP ABSTRACT RESEARCH DESIGN AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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, and VEGF compared with age-matched nondiabetic controls. Ad-PEDF effectively suppressed the overexpression of these proinflammatory factors in diabetic kidneys. In cultured primary human renal mesangial cells (HMC), the high-glucose medium-induced upregulation of VEGF and MCP-1 was largely blocked by PEDF. Furthermore, PEDF inhibited high glucose-induced activation of NF-
B, a key transcription factor mediating inflammatory responses, and hypoxia-inducible factor-1, a major activator of VEGF expression in HMC. These results suggest that the renoprotective effect of PEDF against diabetic nephropathy may be partially through its anti-inflammatory activity, likely by blocking the NF-B and HIF-1 pathways. diabetic complications; gene therapy; angiogenic inhibitor
In recent years, accumulating evidence demonstrates that the pathogenesis of diabetic nephropathy is associated with low-grade inflammation. Increased levels of some proinflammatory factors such as intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) have been found in both type 1 and type 2 diabetic patients with nephropathy (32, 36, 40). In human biopsy samples, it has been shown that increased macrophage infiltration is involved in the pathogenesis of diabetic nephropathy (12). The causative role of inflammation in diabetic nephropathy has been confirmed using the ICAM-1 gene knockout mouse. Knockout of the ICAM-1 gene significantly reduced macrophage infiltration into the kidney after induction of diabetes. Moreover, ICAM-1 deficiency also ameliorated renal structural and functional abnormalities in diabetic mice (32). These results support the notion that inflammation plays an important role in the development and progression of diabetic nephropathy.
Pigment epithelium-derived factor (PEDF) is a neurotrophic factor and a potent angiogenic inhibitor (8). Decreased PEDF levels have been shown to be pathogenic in diabetic retinopathy (DR) (13, 39). Systemic or local delivery of recombinant PEDF protein or viral vector-mediated PEDF gene therapy successfully inhibited retinal neovascularization and reduced retinal vascular permeability in diabetic animals. In addition, PEDF has also been recognized as an important endogenous anti-inflammatory factor (47). Loss or decreased levels of PEDF in the retina may contribute to the pathogenesis of DR by augmentation of retinal inflammation (47).
Our recent studies have demonstrated that decreased PEDF levels in the kidney are implicated in diabetic nephropathy (45). Moreover, systemic administration of an adenovirus expressing the PEDF gene has shown a salutary role on prevention of nephropathy, i.e., drastically reducing albuminuria and amelioration of glomerular hypertrophy in a rat model of type 1 diabetes (46). Moreover, PEDF inhibited the expression of fibronectin in the diabetic kidney. However, the mechanisms for the protective effect of PEDF in the diabetic kidney are undefined. Based on the causative role of inflammation in diabetic nephropathy and the anti-inflammatory activity of PEDF, it is logical to hypothesize that PEDF protects the renal structure and function from diabetic injury via its anti-inflammatory activity. We have tested this hypothesis in the present study.
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RESEARCH DESIGN AND METHODS |
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TOPABSTRACTRESEARCH DESIGN AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Induction of experimental diabetes. Diabetes was induced by an intraperitoneal injection of STZ (50 mg/kg in 10 mmol/l of citrate buffer, pH 4.5) into Brown Norway rats (8 wk of age) after an overnight fast. Age-matched control rats received an injection of citrate buffer alone. Blood glucose levels were measured 2 days after the STZ injection and monitored weekly thereafter. Only the animals with glucose levels higher than 300 mg/dl were considered diabetic. The 24-h urine was collected from each rat in an individual metabolic cage and stored in –80°C after centrifugation at 2,000 g for 5 min.
Cell culture. Primary human glomerular mesangial cells (HMC) were purchased from Cambrex Bio Science (Walkersville, MD). The HMC were cultured in the mesangial cell basal medium (Cambrex) with 0.1% GA-1000 (Cambex) and 5% fetal bovine serum at 37°C in a humidified 5% CO2 atmosphere. Cells from passages 8–10 were used in the experiments. For immunocytochemical study, the cells were grown onto the eight-well chambered slides (Nalge Nunc International, Naperville, IL). After reaching 80% confluence, cells were exposed to a medium with 0.5% serum for 12 h and treated with desired agents.
Preparation and intravenous delivery of an adenovirus expressing human PEDF. A recombinant adenovirus expressing human PEDF (Ad-PEDF) was constructed by cloning a full-length human PEDF cDNA under the control of the CMV promoter (46). The construct sequence was confirmed by DNA sequencing (OMRF, Oklahoma City, OK). A control virus containing a green fluorescent protein (Ad-GFP, Qbiogene, Montreal, QC, Canada) under the same promoter which was obtained from a commercial source (InVivoGen, San Diego, CA). The recombinant virus was amplified, purified, and tittered as described previously (46). One week after the STZ injection, diabetic rats were randomly assigned into three groups. Group 1 received no virus injection (n = 5), and groups 2 and 3 received an intravenous injection of Ad-PEDF (n = 7) and Ad-GFP (n = 5), respectively, at a titer of 4x1010 viral particles/rat.
Measurements of ICAM-1, MCP-1, TNF-, and VEGF by ELISA.
The protein levels of ICAM-1, MCP-1, TNF-, and VEGF in kidney tissue homogenate, in conditioned cell culture media were measured using commercial ELISA kits from R&D Systems (Minneapolis, MN) specific for ICAM-1, MCP-1, TNF-, and VEGF as described previously (47).
Real-time RT-PCR.
Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Primers specific for VEGF (forward, 5'-ctgagctcccggaaaagattg-3'; reverse, 5'-cctgcgaagaacacagcctt-3') and MCP-1 (forward, 5'-gatctcagtgcagaggctcg-3'; reverse, 5'-tgcttgtccaggtggtccat-3') were used for real-time RT-PCR. PCR was performed using a GeneAmp RNA PCR kit and SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) as described (41). The efficiency of real-time PCR was 99.1%. The average CT (threshold cycle) of fluorescence units was used to analyze the mRNA levels. The mRNA levels of target genes were normalized by 18S ribosomal RNA levels. Quantification was calculated as mRNA levels (percent of control) = 2
(CT) with CT = CT, target gene – CT, 18S RNA and (CT) = CT, normal sample(or control cells) – CT, STZ-diabetic sample(or high glucose treated cells).
NF-B activation assay by immunocytochemistry.
NF-B nuclear translocation was evaluated by a commercial assay kit based on an immnofluorescent method (Cellomics, Pittsburgh, PA). Briefly, the cells were fixed with 3.7% formaldehyde for 10 min and permeabilized for 3 min. Then, the cells were incubated with the primary anti-NF-B antibody for 1 h and a secondary FITC-anti-mouse antibody for 1 h and visualized under a fluorescent microscope (Olympus, Hamburg, Germany).
NF-B p65 transcription factor assay.
The nuclear extracts were prepared using a nuclear extract kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions. To quantify NF-B activation in HMC, the translocation of p65, a subunit of NF-B, was assayed by the DNA binding capacity of NF-B using a TransAM NF-B kit (Active Motif). In this assay, an oligonucleotide containing the DNA binding motif of NF-B (5'-GGGACTTTCC-3') is coated onto a 96-well plate. When nuclear extracts are added to the plate, transcriptionally active NF-B binds to the DNA, causing the exposure of an epitope which is recognized by a primary antibody directed against NF-B p65. A HRP-conjugated secondary antibody provides a sensitive colorimetric reaction, which is quantified by spectrophotometry. Briefly, 5 µg of nuclear extracts were applied to the plate and incubated for 1 h at room temperature. After washing, 100 µl of diluted primary antibody was added to all wells and incubated for 1 h, followed by incubation with a secondary antibody for another hour. The plate was read at 450 nm with a reference wave length of 655 nm using a 1420 multilabel counter microplate reader (PerkinElmer, Waltham, MA). TPA+ CI-stimulated Jurkat nuclear extracts were used as positive controls. Specificity of the assay was monitored by competition with a wild-type NF-B consensus oligonucleotide or mutated NF-B consensus oligonucleotide. All the experiments were performed in triplicate. The results were normalized by total protein in the samples and presented as means ± SD.
Hypoxia-inducible factor-1 nuclear translocation assay by immunocytochemistry.
Primary HMC were cultured on four-chamber slides (Nalge Nunc International) to reach 80% confluence. After exposure to 30 mM glucose or mannitol for 48 h, PEDF was added to the culture medium to 160 nM and incubated with the cells for another 24 h. After fixation with 4% paraformaldehyde for 10 min, cells were incubated with an anti-hypoxia-inducible factor-1 (HIF-1) antibody (1:200, Santa Cruz Biotechnology) for 2 h and then incubated with an FITC-conjugated donkey anti-rabbit IgG antibody for 1 h. The slide was examined and analyzed under a fluorescent microscope (Olympus).
HIF-1 DNA-binding assay.
The nuclear extracts were prepared using a nuclear extract kit (Active Motif) according to the manufacturer's instructions. HIF-1 binding to the hypoxia response element was assessed using a Trans-AM HIF-1 transcription factor assay kit (Active Motif). In this assay, an oligonucleotide containing the HIF-1 binding site (5'-TACGTGCT-3') is attached to a 96-well plate. The active form of HIF-1 contained in nuclear extracts specifically binds to this oligonucleotide and can be revealed by incubation with antibodies using enzyme-linked immunosorbent assay. Ten micrograms of nuclear extract were applied to the precoated plate and incubated for 1 h. After extensive washes, a primary antibody detecting HIF-1 was added and incubated for 1 h at room temperature followed by incubation with a secondary antibody for another 1 h. The plate was read at 450 nm. CoCl2-treated COS-7 nuclear extract was used as a positive control. Specificity of the assay was monitored by competition with a wild-type HIF-1 consensus oligonucleotide or mutated HIF-1 consensus oligonucleotide. All the experiments were performed in triplicate. The results were normalized by total protein in the samples and are presented as means ± SD.
Statistical analysis. Data were calculated and are expressed as means ± SD. Statistical analyses were performed using Student's t-test, ANOVA, and Bonferroni's multiple comparison test. Statistical difference was considered significant at a P value of <0.05.
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RESULTS |
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TOPABSTRACTRESEARCH DESIGN AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Ad-PEDF decreases renal levels of proinflammatory factors ICAM-1, MCP-1, and TNF- in diabetic rats.
To explore the mechanisms underlying the salutary effect of PEDF on the reduction of albuminuria, we determined levels of ICAM-1, MCP-1, and TNF- in the kidney of diabetic rats after Ad-PEDF treatment, with the same titer of Ad-GFP as control. Three weeks after the injection of Ad-PEDF, renal levels of ICAM-1, MCP-1, and TNF- were significantly increased in diabetic rats treated with the control virus compared with age-matched nondiabetic rats (Fig. 1). An injection of Ad-PEDF significantly decreased renal levels of ICAM-1 (Fig. 1A), MCP-1 (Fig. 1B), and TNF- (Fig. 1C) in diabetic rats compared with Ad-GFP, suggesting an anti-inflammatory effect of PEDF in the diabetic kidney.
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. The Ad-PEDF treatment significantly reduced the protein levels of VEGF in the kidney of diabetic rats compared with that in Ad-GFP-treated rats (P < 0.05, n = 5, Fig. 2B). The overexpression of the VEGF mRNA was also significantly blocked by Ad-PEDF (Fig. 2A).
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B activation in cultured renal mesangial cells (15). In the present study, we examined the effects of PEDF on MCP-1 secretion induced by a high-glucose medium. After incubation with a high-glucose medium for 48 h, the MCP-1 secretion was increased by 1.7-fold over that of the low-glucose control (Fig. 3A). PEDF at 10 nM significantly blocked the increase in MCP-1 induced by high glucose. The expression of MCP-1 at the mRNA level was also significantly inhibited by PEDF (Fig. 3B).
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B nuclear translocation in HMC cultured in a high-glucose medium.
As NF-B is the major transcription factor regulating a variety of inflammatory factors, including MCP-1 and ICAM-1 (6, 20, 29, 36, 42), we further investigated whether the inhibitory effects of PEDF on MCP-1 and ICAM-1 expression are through the inhibition of NF-B activation. HMC were exposed to 30 mM glucose in the culture medium or the same concentration of mannitol as osmolarity control for 48 h. PEDF was added into the medium (160 nM) and incubated with the cells for 24 h. The cells were fixed and stained with an antibody specific for NF-B. The results showed that under normal conditions, NF-B was diffusely distributed in the cytoplasm (Fig. 4A-a). Exposure to high glucose (Fig. 4A-c), but not mannitol (Fig. 4A-b), induced significant NF-B translocation from the cytoplasm to the nuclei, indicating that NF-B was activated. PEDF almost completely blocked the activation of NF-B induced by high glucose (Fig. 4A-d).
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B translocation in the nuclear extracts from HMC using an NF-B DNA-binding activity assay. The results showed that activated NF-B was significantly increased by 1.8- and 1.7-fold in the HMC treated with high glucose compared with untreated and mannitol controls, respectively (Fig. 4B, n = 3, P < 0.01). This increase was almost completley blocked by PEDF at 160 nM (Fig. 4B, n = 3, P < 0.05). These results suggested that the anti-inflammatory effect of PEDF in diabetic kidneys might be, at least partially, through the inhibition of the NF-B activation. PEDF inhibits high glucose-induced overexpression of VEGF in HMC. To further determine the direct effect of PEDF on VEGF expression in the kidney, we have measured the VEGF secretion from HMC exposed to a high-glucose medium with or without PEDF treatment. After incubation of HMC with a high-glucose (30 mM) medium for 72 h, secreted VEGF levels were significantly increased over the control (Fig. 5). PEDF decreased high glucose-induced VEGF secretion to the level of the control. This inhibitory effect of PEDF on VEGF secretion was also observed at 96 h of the high-glucose culture.
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activation in HMC exposed to high glucose.
HIF-1 is the major transcription factor regulating VEGF expression (17). To explore the mechanism underlying the inhibition of VEGF expression by PEDF, we examined the effect of PEDF on HIF-1 activation in HMC exposed to the high-glucose medium. In the cells exposed to the normal glucose medium, HIF-1 was predominantly distributed in the cytoplasm (Fig. 6, A–C). Incubation with mannitol did not change the distribution pattern of HIF-1 in HMC (Fig. 6, G–I). Exposure to the high-glucose medium for 24 h resulted in a translocation of HIF-1 from the cytoplasm to the nuclei, indicating an activation of HIF-1 induced by high glucose (Fig. 6, D–F). PEDF at 160 nM dramatically inhibited the high glucose-induced HIF-1 nuclear translocation (Fig. 6, J–L), indicating that PEDF inhibits HIF-1 activation induced by high glucose.
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translocation was further quantified by an HIF-1 DNA-binding activity assay in the nuclear extracts from HMC. The results showed that activated HIF-1 was significantly increased by 1.5-fold in the HMC treated with high glucose compared with both of the untreated and mannitol controls (Fig. 6M, n = 3, P < 0.05). This increase was almost completely blocked by PEDF at 160 nM (Fig. 6M, n = 3, P < 0.05). |
DISCUSSION |
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TOPABSTRACTRESEARCH DESIGN AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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, MCP-1, ICAM-1, and VEGF, in diabetic kidneys, correlating with the renal-protective effects of PEDF in diabetic kidneys. Our experiments showed that these effects are likely via inhibition of NF-B and HIF-1 activation.
The roles of inflammation in diabetic nephropathy have been intensively studied (10, 12). It has been shown that TNF- and IFN-
are overexpressed in the kidneys of rats with long-term STZ-induced diabetes (16). In the same diabetic rat model, macrophages were recruited into the glomeruli at the very early phase of hyperglycemia in the glomeruli of rats with diabetes, and immunohistochemistry detected ED1 (monocyte/macrophage marker) in the glomeruli at day 8 of diabetes (16, 28, 33). In a kidney biopsy study conducted in diabetic patients, the number of macrophages and common leukocyte marker-positive cells increased significantly in the glomeruli at the moderate stage of glomerulosclerosis compared with the mild or advanced stage (12). Macrophages may transiently infiltrate during the moderate stage of diffuse diabetic glomerulosclerosis, contributing to irreversible structural damage (12). In ICAM-1-deficient (ICAM-1–/–) mice with experimental diabetes, the infiltration of macrophages was markedly suppressed compared with that in ICAM-1+/+ mice with diabetes, suggesting that ICAM-1 plays an important role in inflammation in the diabetic kidney (32). Furthermore, urinary albumin excretion, glomerular hypertrophy, and mesangial matrix expansion were significantly lower in diabetic ICAM-1–/– mice than in diabetic ICAM-1+/+ mice (32). These studies suggest that inflammation is critically involved in the pathogenesis of diabetic nephropathy.
Although PEDF has been shown to be a potent inhibitor of angiogenesis, it was recently found to have an anti-inflammatory function as well. Recent studies (47) by our group showed that retinal and plasma PEDF levels were drastically decreased in rats with endotoxin-induced uveitis. Intravitreal injection of PEDF significantly reduced vascular hyperpermeability in rat models of diabetes and oxygen-induced retinopathy, correlating with the decreased levels of retinal inflammatory factors, including VEGF, VEGF receptor-2, MCP-1, TNF-, and ICAM-1 (47). In cultured retinal capillary endothelial cells, PEDF significantly decreased TNF- and ICAM-1 expression under hypoxia. Moreover, downregulation of PEDF expression by siRNA resulted in significant increases of VEGF and TNF- secretion in retinal Müller cells (47). These findings suggest that PEDF is an endogenous anti-inflammatory factor in the eye. The present study provides further evidence showing that PEDF is also an endogenous anti-inflammatory factor in the kidney, and the decrease of renal PEDF levels in diabetic rat models may contribute to inflammation in the kidney.
Overexpression of intercellular adhesion molecules ICAM-1 (41), E-selectin and P-selectin, (37) and MCP-1 (3, 21) has been found to be involved in pathogenesis of diabetic nephropathy in diabetic patients and in animal models. TNF-, which is produced mainly by monocytes and macrophages and also glomerular mesangial cells (4, 18), has been found to be increased in the kidney of diabetic animal models (16, 31). Although the mechanisms of upregulation for these molecules have not been defined, high ambient glucose levels have been suggested to be a major causative factor. Our present study showed that MCP-1, ICAM-1, and TNF- expression in the kidneys of STZ rats was significantly increased compared with that in nondiabetic control rats. Treatment with PEDF has been shown to suppress renal MCP-1, ICAM-1, and TNF- expression and to attenuate renal injury without altering glycemic control in diabetic rats. Taken together, these results suggest that PEDF has anti-inflammatory activities in the diabetic rat kidney, which may be responsible for the salutary effect of PEDF in diabetic nephropathy.
NF-B is one of the most important transcription factors regulating ICAM-1 expression (14). On activation, NF-B upregulates transcription of a number of target genes, including cytokines, adhesion molecules, nitric oxide synthases, and a variety of other inflammatory proteins involved in the pathogenesis of diabetic nephropathy (14). Our observation that PEDF can prevent nuclear translocation of NF-B suggests a potential mechanism for the anti-inflammatory effect and renoprotective action of PEDF.
VEGF is strongly implicated in the pathogenesis of diabetic microvascular complications, as it increases vascular permeability to macromolecules and stimulates monocyte chemotaxis and tissue factor production, all of which can contribute to microvascular complications (9, 30). Although the role of VEGF in regulating glomerular permeability has not yet been defined, a growing body of clinical and experimental evidence suggests that VEGF be responsible for proteinuria in diabetic nephropathy (22, 23, 38). In the present study, Ad-PEDF treatment of rats with STZ-induced diabetes inhibited renal VEGF overexpression, which may contribute to the reduction of albuminuria after the PEDF treatment. This finding suggests that the effect of PEDF on albuminuria may be through blockade of VEGF expression in the diabetic kidney.
HIF-1 is a transcription factor that is increased in conditions of reduced cellular O2 (11, 34). The binding site of HIF-1 is present in the promoters of several genes, such as VEGF, that are sensitive to changes in PO2 (27, 35). A recent report (7) suggested that HIF-1 could also be induced during inflammation. Inhibiting HIF-1 translocation by PEDF could be another important mechanism for the anti-inflammatory activities of PEDF in diabetic nephropathy. It is not clear whether the inhibitory effect of PEDF on HIF-1 activation is related to or independent of the inhibition of NF-B.
In summary, our data suggest that PEDF is an endogenous anti-inflammatory factor in the kidney, and decreased PEDF levels in diabetic kidney may contribute to the inflammation in the kidney, subsequently leading to diabetic nephropathy. The salutary effect of PEDF on diabetic nephropathy may be mediated, at least in part, by its anti-inflammatory activity. Therefore, PEDF treatment may become an anti-inflammatory therapy in diabetic nephropathy.
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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.
* J. J. Wang and S. X. Zhang contributed equally to this study. 
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