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NDT Advance Access published online on November 28, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn659
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Administration of pigment epithelium-derived factor (PEDF) reduces proteinuria by suppressing decreased nephrin and increased VEGF expression in the glomeruli of adriamycin-injected rats

Toshiko Fujimura1, Sho-ichi Yamagishi2, Seiji Ueda1, Kei Fukami1, Ryo Shibata1, Yuriko Matsumoto1, Yusuke Kaida1, Ayako Hayashida1, Kiyomi Koike1, Takanori Matsui2, Kei-ichiro Nakamura3 and Seiya Okuda1

1 Division of Nephrology, Department of Medicine 2 Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications 3 Division of Microscopic & Developmental Anatomy, Department of Anatomy, Kurume University School of Medicine, Kurume, Japan

Correspondence and offprint requests to: Sho-ichi Yamagishi, Division of Cardiovascular Medicine, Department of Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan. Tel: +81-942-31-7580; Fax: +81-942-31-7707; E-mail: shoichi{at}med.kurume-u.ac.jp



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Pigment epithelium-derived factor (PEDF) is a glycoprotein with potent neuronal differentiating activity. We, along with others, have recently found that PEDF inhibits retinal hyperpermeability by counteracting the biological effects of vascular endothelial growth factor (VEGF). However, the protective role of PEDF against nephrotic syndrome (NS), a condition of hyperpermeability in the glomerular capillaries, remains to be elucidated. In this study, we investigated whether and how PEDF reduced proteinuria in rats with adriamycin (ADR)-induced nephropathy (ADN), an experimental model of NS.

Methods. ADN was induced by a single intravenous injection of doxorubicin hydrochloride (n = 12). Half the ADN rats were intravenously administrated human recombinant PEDF; the other half were given vehicle everyday for up to 14 days. Control rats (n = 6) received vehicle only.

Results. In ADN, expression levels of PEDF in isolated glomeruli were significantly decreased, which were associated with a marked proteinuria and increased urinary excretion of nephrin, an index of podocyte damage. Loss of nephrin and decreased podocyte cell number and fusion of foot processes of podocytes with nuclear factor-kappa B (NF-{kappa}B) activation and VEGF overexpression were also observed in the glomeruli of rats with ADN. Intravenous administration of PEDF ameliorated all of these changes in ADN rats.

Conclusion. The present findings suggest that PEDF could reduce proteinuria by suppressing podocyte damage and decreased nephrin as well as increased VEGF expression in the glomeruli of ADN rats. Pharmacological up-regulation or substitution of PEDF may offer a promising therapeutic strategy for the treatment of nephrotic syndrome.

Keywords: hyperpermeability; nephrin; nephrotic syndrome; PEDF; VEGF



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 References
 
Pigment epithelium-derived factor (PEDF) is a glycoprotein that belongs to the superfamily of serine protease inhibitors. It was first purified from the conditioned media of human retinal pigment epithelial cells as a factor that possesses potent neuronal differentiating activity in human retinoblastoma cells [1]. Recently, PEDF has been shown to be a highly effective inhibitor of angiogenesis in cell culture and animal models [2,3]. PEDF inhibits the growth and migration of cultured endothelial cells (ECs), and it potently suppresses ischaemia-induced retinal neovascularization [2,3]. Since PEDF levels in aqueous humour or vitreous fluid are decreased in diabetic patients, especially with proliferative retinopathy [4], the loss of PEDF activity in the eye may contribute to the development and progression of proliferative diabetic retinopathy.

In addition, we, along with others, have recently found that PEDF inhibits retinal hyperpermeability by counteracting the biological effects of vascular endothelial growth factor (VEGF) [5,6], thus suggesting that PEDF may be an important therapeutic adjunct in the treatment of a number of devastating disorders where increased vascular permeability is a pathological mechanism. PEDF is expressed in a broad range of human fetal and adult tissues including kidney [7–10]. Increases in vascular permeability in the kidney have been considered to play a key role in the development of nephrotic syndrome (NS) [11,12]. These findings led us to speculate that PEDF could exert beneficial effects on NS as well by suppressing the effects of VEGF. However, as far as we know, the role of PEDF in NS remains to be elucidated. Therefore, we investigated here whether and how the administration of PEDF could reduce proteinuria in rats with adriamycin (ADR)-induced nephropathy (ADN), an experimental model of NS.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 References
 
Experimental design
The experiments were performed on 8-week-old male Sprague-Dawley (SD) rats weighing ~300 g. ADN was induced in rats (n = 12) by a single intravenous injection of doxorubicin hydrochloride (6.0 mg/kg) diluted in 0.9% saline as previously described [13]. Half the ADN rats were intravenously administrated human recombinant PEDF (30 µg/body diluted in 0.9% saline); the other half were given vehicle everyday for up to 14 days. Control rats (n = 6) received vehicle only. We have previously shown that intravenous administration of 10–30 µg PEDF once a day exerted anti-thrombogenic, anti-permeability and anti-inflammatory effects in 9-week-old SD rats [6,14], whose body weights were nearly the same as those used in this experiment. This is a reason why we chose the treatment condition with intravenous administration of 30 µg PEDF once daily in our model. Rat body weights of each group were nearly equal (~300 g). Therefore, we injected 30 µg PEDF per rat, not per 300 g body weight in this experiment. We also confirmed here that the peak concentration of circulating PEDF level was obtained at 2 h after the 30 µg PEDF injection and its level was increased to about 2-fold of the basal level (Figure 1).


Figure 1
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  		Fig. 1 Serum concentrations of PEDF in SD rats. SD 8-week-old rats were intravenously injected with 30 µg PEDF, and then the serum concentrations of PEDF were measured (each n = 3). *P < 0.05 compared with the basal value at 0 h.

 
BP was measured by a tail-cuff sphygmomanometer using an automated system with a photoelectric sensor (BP-98A; Softron, Tokyo, Japan) on Days –1, 3, 7, 10 and 14. All of the rats were killed on Day 15 and the kidneys were excised for immunohistochemical, morphometric and western blot analysis. All experimental procedures were conducted in accord with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the ethical committee of our institution.

Purification of the PEDF protein
The PEDF protein was prepared and purified as previously described [15]. SDS–PAGE analysis of the purified PEDF protein revealed a single band with a molecular mass of ~50 kDa, which showed positive reactivity with a monoclonal antibody against human PEDF (Transgenic, Kumamoto, Japan).

Measurements of clinical parameters
Plasma and urinary levels of total protein, plasma total cholesterol and albumin were determined with commercially available kits (Wako, Osaka, Japan). Plasma creatinine (Cr) levels were also measured with a commercial kit (Alfresa Pharma Co., Tokyo, Japan). Serum PEDF levels were measured with the ELISA as previously described [16].

Immunofluorescence staining
Frozen kidneys were embedded in a Tissue-Tek (Sakura Finetek, Torrance, CA, USA) 22-oxacalcitriol (OCT) medium and cut on a cryostat microtome into 4 µm sections. Tissue sections were fixed in 2% paraformaldehyde for 5 min at 4°C, and then blocked with 3% normal goat serum (Dako, Glostrup, Denmark). To determine the expression levels of PEDF in the kidney, we performed an indirect immunofluorescence staining with the rabbit anti-PEDF polyclonal antibody (Ab) (1:1000; BioProducts MD, Middletown, MD, USA) as a primary Ab. Further, the sections were double-stained with Abs raised against a podocyte marker, Wilm's tumour-1 (WT-1) (1:100; Dako Cytomation, Glostrup, Denmark), an EC marker, RECA-1 (1:200; AbD serotec, Raleigh, NC, USA) or a mesangial cell marker, Thy1.1 (1:100; Chemicon, Temecula, CA, USA). Goat anti-rabbit IgG Alexa 488 and anti-mouse IgG Alexa 594 (1:2000; Molecular Probes, Eugene, OR, USA) were used as secondary Abs. The samples were analysed under a confocal laser microscope, FV 1000 (OLYMPUS, Tokyo, Japan). Immunofluorescence staining of nephrin was performed using a rabbit polyclonal anti-nephrin Ab (Alpha Diagnostic, San Antonio, TX, USA) and evaluated with a semi-quantitative scoring system (0–4) according to the paper of Gross et al.: score 0, no expression; score 1, mild expression; score 2, moderate expression; score 3, strong expression; score 4, extremely strong expression [17]. All analyses were performed in a blinded manner, i.e. the observer was unaware of the experimental protocol.

Morphometric analysis
The kidneys were fixed in 4% paraformaldehyde and embedded in paraffin wax for sectioning. Three-micrometer paraffin sections were stained with periodic acid-Schiff (PAS) for light microscopic analysis. For electron microscopic analysis, small blocks of the kidneys were fixed with 2.5% glutaraldehyde, post-fixed in 2% osmium tetroxide, dehydrated in graded ethanol, and then embedded in epoxy resin. The ultrathin section (0.1 µm) was stained with uranyl acetate and lead citrate.

Isolation of glomeruli
Glomeruli were isolated by the graded sieving method as previously described [18]. Briefly, rats were deeply anaesthetized and the kidneys were immediately removed. The cortex was excised, cut into fine fragments and homogenized. After being passed through consecutive stainless steel screens of 177-, 125- and 63-µm pore size, the glomeruli were suspended in phosphate-buffered saline and collected by centrifugation at 2000g for 3 min.

Western blot analysis of PEDF, VEGF and P50 subunit of nuclear factor-{kappa}B (NF-{kappa}B) and cAMP response element-binding protein (CREB)
Fifty micrograms of proteins extracted from isolated glomeruli, urine samples or cortical tissues was separated by SDS–PAGE and transferred to nitrocellulose membranes (Biorad, Hercules, CA, USA). Then, the membranes were blotted with anti-PEDF, anti-VEGF or anti-p50 subunit of NF-{kappa}B, and anti-CREB (Santa Cruz Biotechnology, Delaware, CA, USA) Abs. The immune complexes were visualized with an enhanced chemiluminescence detection system (ECL; Amersham Bioscience, Buckinghamshire, UK).

Preparation of nuclear extracts from kidney
Nuclear protein was extracted from cortical lesions of the kidney as previously described [19]. Briefly, 500 mg of cortical tissues was homogenized with 20 even strokes of a glass Teflon homogenizer in 1 mL of ice-cold buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 2 mM MgCl2, 0.1 mM ethylenediaminetetraacetic acid (EDTA), and a cocktail of protease inhibitors (Nakarai Tesque, Kyoto, Japan)]. Then, 325 µL of detergent Nondiet-P40 (2%) was added. The mixture was vortexed 10 times for 1 min each and centrifuged at 13 000g for 5 min. The supernatant was removed, and then the pellet was re-suspended in 300 µL of buffer B [50 mM HEPES, 10% (v/v) glycerol, 300 mM NaCl, 50 mM KCl and the protease inhibitor cocktail]. The samples were centrifuged again and the supernatant (nuclear proteins) was stored in aliquots at –80°C. The protein concentrations were determined by a BCA protein assay kit (Pierce, Rockford, IL, USA).

Measurement of in situ reactive oxygen species (ROS) generation and urinary 8-isoprostane levels
The oxidative fluorescent probe dihydroethidium (DHE) (Invitrogen, Carlsbad, CA, USA) was used to detect in situ levels of ROS in the kidney as previously described [20]. Briefly, the tissue sections were embedded in an OCT medium and cut on a cryostat microtome into 4 µm sections. The sections were stained with 2 µM DHE for 30 min. In situ production of superoxide was visualized as a red fluorescence. We also measured urinary 8-isoprostane levels on Day 14 with an enzyme-linked immunoassay (8-iso-PGF2{alpha} EIA kit; Oxford Biomedical Research, Oxford, MI, USA).

Statistical analysis
All data were expressed as mean ± SD. One-way ANOVA followed by the Scheffe F-test was performed for statistical comparisons. P < 0.05 was considered statistically significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 References
 
Immunofluorescence staining for PEDF-positive cells in normal rat kidney
PEDF is expressed in a broad range of human fetal and adult tissues including kidney [7–10]. Further, its levels are higher in renal cortex than those in the medulla [10,21]. However, the precise localization of PEDF-positive cells in the kidney remains to be determined. So, we first examined which types of cells were positive for PEDF staining in the kidney. PEDF was expressed in glomerular areas, arteries, veins and capillaries in normal rat kidneys (Figure 2A a). Negative control staining with 3% goat serum/PBS is shown in Figure 2A b. Double staining with anti-RECA-1 (Figure 2B a–d), anti-WT-1 (Figure 2B e–h) or anti-Thy1.1 Abs (Figure 2B i–l) revealed that PEDF was expressed in podocytes, ECs and mesangial cells. DAPI staining confirmed that PEDF was expressed within the cells (Figure 2C a, b).


Figure 2
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  		Fig. 2 Immunofluorescence staining for PEDF in kidney. (A) Immunofluorescence staining of PEDF. The sections were incubated with (a) or without (b) (negative staining) PEDF antibody (Ab). (c) is a phase contrast for (b). Magnification x200. Arrow and arrowhead indicate the location of glomerulus and blood vessel, respectively. (B) Double staining of the glomeruli with PEDF Ab (a, e, i), anti-RECA-1 Ab (b), anti-WT-1 Ab (f) or anti-Thy1.1 Ab (j). PEDF-positive podocytes are indicated as arrows. Magnification: (a–c, e–g, i–k), x400; (d, h, l), x800. (C) PEDF immunofluoresscence with (a) or without (b) DAPI staining. Magnification x600.

 
PEDF expression levels in glomeruli of rats with ADN and PEDF-treated ADN
We next evaluated the expression levels of PEDF in the glomeruli with two different methods, immunofluorescence staining (Figure 3A) and western blot analysis (Figure 3B). As shown in Figure 3A, PEDF-positive immunofluorescence in the glomeruli of rats with ADN was significantly decreased, compared with that in control rats; the glomerular PEDF levels were reduced to ~4/5 of those of control rats (Figure 3B), which was prevented by the treatments with intravenous administration of PEDF (Figure 3A and B).


Figure 3
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  		Fig. 3 PEDF expression in the glomeruli of control, ADN and PEDF-treated ADN rats. (A) Immunofluorescence staining for PEDF in control (a), ADN (b) and PEDF-treated ADN rats (c). Magnification x600. (B) Western blot analysis of PEDF in the glomeruli isolated from control, ADN and PEDF-treated ADN rats. Left panel shows the representative results of western blots. Right panel shows the quantitative data. Data were normalized by the intensity of β-actin bands (n = 6, respectively). *P < 0.05 compared with the controls, #P < 0.05 compared with the ADN rats.

 
Effects of PEDF administration on proteinuria in ADN rats
In order to investigate the pathophysiological relevance of PEDF in ADN, we next examined the effects of PEDF on urinary protein excretion in ADN rats. As shown in Table 1, intravenous administration of PEDF significantly reduced proteinuria and prevented the decrease in serum albumin as well as the increase in total cholesterol levels in ADN rats. Body weight and creatinine clearance (Ccr) were not different among three groups (Table 1). There was no significant difference in systolic blood pressure (SBP) among each group (Table 2).


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  		Table 1 Clinical variables on Day 14

 

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  		Table 2 Effects of PEDF administration on SBP in ADN rats

 
Effects of PEDF administration on renal histopathology in ADN rats
Light microscopic examination showed subtle histopathological changes in glomerular and tubulointerstitial areas of the ADN rats (Figure 4). However, electron microscopic examination revealed that foot processes of podocytes were fused in the ADN rats, which was significantly improved by the treatment of PEDF (Figure 5).


Figure 4
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  		Fig. 4 Effects of PEDF administration on renal histopathology in ADN rats. Light microscopic examination showed subtle histopathological changes in glomerular and tubulointerstitial areas of the normal (a, d), ADN (b, e) and PEDF-treated ADN (c, f) rats. Magnification x200 (a, b, c), x600 (d, e, f)

 

Figure 5
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  		Fig. 5 Effects of PEDF administration on electron microscopic findings of the podocytes in ADN rats. Electron microscopic findings of the glomeruli in normal control (a, d), ADN (b, e) and PEDF-treated ADN rats (c, f). Magnification x6000 (a–c), x10 000 (d–f). The scale bar shows 0.5 µm. The arrows show the fusions of podocyte foot processes in ADN rats.

 
Effects of PEDF administration on podocyte loss and expression levels of nephrin in ADN rats
We next examined the number of podocyte and the expression levels of nephrin, which localizes to the slit pore of podocytes [22–24], in the glomeruli of ADN rats. Immunofluorescence staining revealed that the numbers of WT-1 positive (Figure 6A) or nephrin-positive cells (Figure 6B) were decreased in the glomeruli of ADN rats, compared with those of the control rats, both of which were partially restored by the treatment of PEDF.


Figure 6
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  		Fig. 6 Effects of PEDF administration on podocyte and expression levels of nephrin in ADN rats. (A) Immunofluorescence staining for WT-1, podocyte marker in normal control (a), ADN (b) and PEDF-treated ADN (c) rats. Right panel shows the number of WT-1 positive cells per glomerulus in these three groups (n = 6). **P < 0.0001 compared with control, *P < 0.05 compared with control, #P < 0.05 compared with ADN. (B) Immunofluorescence staining for nephrin in control (a), ADN (b) and PEDF-treated ADN rats (c). Magnification x400. Right panel shows the glomerular nephrin staining scores (n = 6, respectively). *P < 0.05 compared with the controls, #P < 0.05 compared with the ADN rats. (C) Effects of PEDF administration on urinary excretion levels of nephrin on ADN rats. Left panel shows the representative results of western blots. Right panel shows the quantitative data (n = 6, respectively). *P < 0.05 compared with the controls, #P < 0.05 compared with the values of ADN rats.

 
Nephrin is excreted into urine in the early stages of experimental NS [24,25]. So, we next investigated whether PEDF administration reduced urinary excretion levels of nephrin in ADN rats. As shown in Figure 6C, nephrin was not detected in urine samples of the control rats. Urinary excretion levels of nephrin were increased in ADN rats, which were prevented by the treatment of PEDF.

Effects of PEDF administration on VEGF expression in the glomeruli of ADN rats
We next investigated the effects of PEDF administration on VEGF, a potent vasopermeability factor, expression in the glomeruli of ADN rats. VEGF expression was up-regulated in the glomeruli of ADN rats, which was significantly decreased by PEDF treatment (Figure 7).


Figure 7
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  		Fig. 7 Effects of PEDF administration on VEGF expression in the glomeruli of ADN rats. Left panel shows representative results of western blots. Right panel shows the quantitative data. Data were normalized by the intensity of β-actin bands (n = 6, respectively). #P < 0.05 compared with the values of ADN rats.

 
Effects of PEDF on NF-{kappa}B activation in ADN rats
NF-{kappa}B is also known as one of the key mediators in experimental NS [19,26,27]. Therefore, in order to further investigate the molecular mechanism by which PEDF prevented the decrease in nephrin as well as the increase in VEGF expression in ADN rats, we investigated the effects of PEDF administration on NF-{kappa}B activation in the glomeruli of ADN rats. The p50 protein expression levels were densitometrically measured and normalized to the CREB expression because CREB is a nuclear marker protein [28]. As shown in Figure 8A, nucleus levels of p50 subunit of NF-{kappa}B were increased in the glomeruli of ADN rats, which were blocked by the treatment of PEDF. Since glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is not a specific cytoplasmic marker protein and also exists in the nucleus [29], we were not able to confirm the purity of the nuclear extracts by western blots raised against GAPDH. We show a Comassie blue staining as a loading control in Figure 8B.


Figure 8
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  		Fig. 8 Effects of PEDF administration on nuclear translocation of NF-{kappa}B p50 subunit in the glomeruli of ADN rats. (A) Left panel shows representative results of western blots of p50 and CREB. Right panel shows the quantitative data. The expression levels of p50 were normalized by the intensity of CREB bands (n = 6, respectively). *P < 0.05 compared with the controls. #P < 0.05 compared with the values of ADN rats. (B) After electrophoresis, proteins were visualized with Coomassie blue staining.

 
Effects of PEDF administration on ROS generation in ADN rats
Since NF-{kappa}B is a redox-sensitive transcriptional factor [30], we further investigated the effects of PEDF on ROS generation in ADN rats. As shown in Figure 9, superoxide generation in the glomeruli and urinary excretion levels of oxidative stress marker, 8-isoprostane [31] were increased in ADN rats, both of which were not affected by the treatment of PEDF.


Figure 9
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  		Fig. 9 Effect of PEDF administration on ROS generation in ADN rats. (A) Representative photographs of DHE staining of the glomeruli in control (a, b), ADN (c, d) and PEDF-treated ADN rats (e, f). Magnification x100 in (a, c, e) and x400 in (b, d, f). (B) Urinary excretion levels of 8-isoprostane/Cr. *P < 0.05 compared with the controls.

 


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
References
 
We, along with others, have recently found that PEDF inhibits retinal hyperpermeability by counteracting the biological effects of VEGF [5,6], thus suggesting that PEDF may be a novel therapeutic strategy for the treatment of various disorders in which vasopermeability is involved. However, there are a few studies to show the implication of PEDF in the pathogenesis of proteinuria, a condition of barrier dysfunction of the glomerular capillary wall [21,32]. Indeed, Wang et al. only reported that PEDF levels were decreased in kidneys of streptozotocin-induced diabetic rats and that injection of adenovirus PEDF alleviated microalbuminuria and suppressed overexpression of two major fibrogenic factors, transforming growth factor-β and connective tissue growth factor in the diabetic kidneys [21,32]. In the present study, we have extended the previous findings that PEDF exerted salutary effects on proteinuria. We demonstrated here for the first time that administration of PEDF could reduce proteinuria by inhibiting podocytes loss as well as increased VEGF expression via blockade of NF-{kappa}B activation in the glomeruli of ADN rats, an experimental model of NS on the basis of the following evidence: (1) endogenous levels of glomerular PEDF expression levels were significantly decreased in ADN rats, compared with control rats; (2) administration of PEDF to ADN rats significantly ameliorated the decrease in glomerular PEDF levels and subsequently reduced proteinuria and prevented fusion of foot processes of podocytes and loss of podocytes in the glomeruli; (3) PEDF treatment also decreased urinary excretion levels of nephrin, an index of podocyte damage in ADN rats; and (4) NF-{kappa}B activation and VEGF up-regulation in the glomeruli of ADN rats were significantly blocked by the treatment of PEDF.

Nephrin is a key component of the slit diaphragm, the main site of control of glomerular permeability [22–24]. Indeed, nephrin mRNA or protein levels were reported to decrease in the glomeruli of various experimental models of hypertension, diabetes and membranous nephropathy, thereby being involved in proteinuria in these models [24,33–36]. In addition, nephrin is excreted into urine in the early stages of experimental NS and human diabetic nephropathy [24,25,35,36]. These observations suggest that the loss of nephrin may play a role in the pathogenesis of NS and urinary nephrin excretion could be a biomarker of podocyte damage in NS [24,25]. In the present study, we found that PEDF administration ameliorated loss of podocyte and nephrin in the glomeruli as well as the increase in its urinary excretion in ADN rats. Further, loss of podocytes and fusion of podocyte foot processes in ADN rats were prevented by the treatment of PEDF. Taken together, the present findings suggest that PEDF may have a protective role against podocyte damage and subsequently reduce proteinuria in ADN rats.

VEGF is expressed in foot processes of podocytes [37,38]. There is a growing body of evidence that podocyte-derived VEGF may play a central role in the maintenance of glomerular capillary permeability as well as the regulation of proteinuria in various types of NS [39–42]. In this study, VEGF expression was up-regulated in the glomeruli of ADN rats, which was prevented by the treatment of PEDF. These observations suggest that VEGF-elicited vascular permeability in the kidney may be counterbalanced by glomerular PEDF and that PEDF administration could ameliorate proteinuria, by not only restoring the loss of podocytes and decreased nephrin expression but also suppressing VEGF levels in the glomeruli of ADN rats. Since VEGF is a potent vasodilator, it would be possible that the inhibition of VEGF (the proposed mechanism of PEDF) may reduce tissue perfusion, flow and area, thereby decreasing proteinuria in ADN rats [43]. However, as we have reported in the previous paper [6], PEDF significantly inhibits the decrease in transendothelial electrical resistance, an indicator of EC barrier function in advanced glycation end-product-exposed ECs, by suppressing VEGF expression. Therefore, PEDF could improve barrier function in ECs, independent of vessel area or flow. Furthermore, we found here that PEDF treatment did not affect systemic blood pressure (Table 2) or Ccr in ADN rats (Table 1). These findings suggest that PEDF treatment may mainly decrease proteinuria in ADN rats through its anti-permeability effect, but not its haemodynamic one, via suppression of VEGF.

In this study, we found that expression levels of NF-{kappa}B p50 subunit in nuclei of the glomeruli were increased in ADN rats, which was blocked by the treatment of PEDF. Suppression of the NF-{kappa}B activation may be a central mechanism by which PEDF inhibited the ADR-induced decrease in podocyte number and nephrin expression as well as the increase in VEGF generation in the glomeruli on the basis of the following evidence: (1) NF-{kappa}B activation was associated with the loss of nephrin expression and proteinuria in anti-glomerular basement membrane nephritis [27]; (2) podocyte NF-{kappa}B overactivation was observed in patients with non-proliferative glomerulopathy and its levels were positively correlated with the severity of proteinuria in those patients [44] and (3) NF-{kappa}B binding site exists in the promoter region of VEGF and that NF-{kappa}B activation is indispensable for VEGF gene induction in various types of disorders [45–47]. Although we did not know the molecular mechanisms underlying the ADR-induced NF-{kappa}B activation, PEDF may suppress the NF-{kappa}B activation in the glomeruli of ADN rats in an ROS-independent manner because we found here that PEDF did not affect ROS generation in ADN rats by two distinct methods: one is DHE staining that can detect in situ superoxide production, and the other is the measurement of urinary 8-isoprostane excretion.

There are some limitations of the present study. Due to technical reasons, we were not able to study the potential benefit of a more continuous administration of PEDF in ADN and to include an additional group of PEDF-treated healthy rats. However, we already confirmed in another study (unpublished data) that intravenous PEDF administration to normal rats for 2 weeks increased serum PEDF levels to 150 ng/mL, which had no effect on urinary albumin excretion levels (control rats versus PEDF-injected rats; 0.03 ± 0.01 versus 0.04 ± 0.02 mg albumin/g creatinine).

In conclusion, our present study suggests that pharmacological up-regulation or substitution of PEDF may offer a promising therapeutic strategy for the treatment of NS.



   Acknowledgments
 
This work was supported in part by Grants of Venture Research and Development Centers from the Ministry of Education, Culture, Sports, Science and Technology, Japan, to S.Y.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 7. 2.08
Accepted in revised form: 3.11.08


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