Does vascular endothelial growth factor (VEGF) play a role in the pathogenesis of minimal change disease?
Geoffrey Boner1,3,6,
Alison J. Cox2,
Darren J. Kelly2,
Ana Tobar4,6,
Joëlle Bernheim5,6,
Robyn G. Langham2,
Mark E. Cooper1 and
Richard E. Gilbert2
1Baker Medical Research Institute, St Kilda Central, Melbourne, 2Department of Medicine, University of Melbourne, St Vincents Hospital, Fitzroy, Australia, 3Institute of Hypertension and Kidney Diseases and 4Department of Pathology, Rabin Medical Center, Beilinson Campus, Petah Tikva, 5Department of Pathology, Sapir Medical Center, Kfar Saba and 6Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
Correspondence and offprints to: G. Boner, 81 Kochav Hayam, Chofit, 40295, Israel. Email: gboner{at}post.tau.ac.il
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Abstract
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Background. Minimal change disease (MCD) is one of the major causes of nephrotic syndrome both in children and adults. The pathogenesis of this condition is not clear and it has been suggested that a plasma permeability factor may play a role. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, has been thought to be one the factors involved. The aim of this study was thus to investigate the role of VEGF in the pathogenesis of MCD.
Methods. The expression of the gene for VEGF and VEGF receptor-2 (VEGFR-2) was estimated using in situ hybridization in renal biopsy specimens taken from patients with nephrotic syndrome and diagnosed histologically as MCD. The results were compared with those obtained in normal renal tissue. Biopsy specimens from eight patients diagnosed as having MCD were randomly selected for the study. The patients were aged 460 years at the time of the biopsy. There were four females and four males. All patients had presented with a nephrotic syndrome, five with recent onset of the disease, two with repeated attacks of the syndrome and one had reduced renal function.
Results. The gene expression for VEGF, measured as the proportional glomerular area occupied by autoradiographic grains, was significantly less in the patients with MCD than in controls (1.9 ± 0.4 vs 4.8 ± 0.6%, P < 0.0025), whereas the gene expression for VEGFR-2 was no different to controls (1.9 ± 0.4 vs 2.0 ± 0.2%).
Conclusions. MCD is associated with a reduction in the expression of the gene for VEGF. As VEGF may play an important role in renal repair and survival, it is postulated that the deficiency, which we have shown, may lead to the dysregulation of the repair process in MCD.
Keywords: in situ hybridization; minimal change disease; VEGF
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Introduction
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Minimal change disease (MCD) is a major cause of the nephrotic syndrome, especially in children. The prevalence of MCD is estimated as being 7090% in children under the age of 10 years, 50% in older children and 1015% in adults with nephrotic syndrome [1]. The pathogenesis of this entity has not been clearly defined, although an abnormality of the T lymphocyte is considered to be one of the major explanations for this condition [1]. Indeed it is postulated that a plasma permeability factor, originating from T cells may induce proteinuria [1]. However, the nature of this substance and the nature of its effect on the filtration barrier are not clear.
Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, plays a role in vascular permeability and endothelial proliferation [2]. This factor has been shown to be produced in the visceral glomerular epithelial cells [3]. Thus, it may be postulated that over-expression of VEGF may play a role in the pathogenesis of proteinuria in MCD and other renal diseases associated with proteinuria. Different groups have examined the expression of VEGF mRNA in the glomerulus and its concentration in plasma and urine in order to investigate whether this factor is involved in the pathogenesis of MCD [2,46]. Their results have been contradictory. Therefore, an alternative approach, using in situ hybridization, was proposed. The gene expression of VEGF and the VEGF receptor-2 (VEGFR-2) was measured in the glomeruli of patients with the nephrotic syndrome and histologic evidence of MCD. The resultant expression was compared with that found in areas of normal renal tissue in nephrectomy specimens, which were taken from patients with renal tumours.
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Subjects and methods
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Patients
Kidney tissue from two groups of patients was studied. The first group consisted of a group of eight patients classified as having MCD who underwent diagnostic biopsy at the Rabin or the Sapir Medical Centers, Israel (Table 1). The diagnosis of MCD was based on characteristic clinical and pathological findings, including immunofluorescence and electron microscopy. In the second group of patients, normal cortical renal tissue was also obtained from kidneys removed for renal malignancies. This group of renal tumour patients included five males and five females with ages ranging from 49 to 71 years. All these control subjects had normal serum creatinine, had no history of hypertension and did not have proteinuria. The control tissue was obtained from kidneys with no evidence of tumour obstruction or vascular invasion, and the samples were removed from areas remote to the malignancy. In all cases the histology of the selected areas was normal. In all patients, kidney tissue was immersion-fixed in 10% neutral-buffered formalin and subsequently embedded in paraffin.
In situ hybridization
In situ hybridization was performed as described previously [7]. In brief, riboprobes were synthesized from cDNA encoded for VEGF and VEGFR-2 (gifts of Dr Steven Stacker, Ludwig Institute for Cancer Research, Melbourne, Australia) [8]. The cDNAs were cloned into pGEM 4Z (Promega, Madison, WI) and linearized with HindIII to produce antisense probes using T7 RNA polymerase. Four-micron thick sections cut from formalin-fixed paraffin-embedded kidney tissue were placed onto slides pre-coated with 3-aminopropyltriethoxysilane and baked overnight at 37°C. Tissue sections were dewaxed, rehydrated in graded ethanol and milliQ water, equilibrated in P buffer (50 mM TrisHCl, 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 in 0.1 M sodium phosphate buffer (pH 7.2), briefly re-fixed in 4% paraformaldehyde for 10 min, rinsed in milliQ water, dehydrated in 70% ethanol and air dried. Hybridization buffer containing 2 x 104 c.p.m./µl riboprobe in 300 mM NaCl, 10 mM TrisHCl (pH 7.5), 10 mM Na2HPO4, 5 mM EDTA (pH 8.0), 1x Denhardts solution, 50% formamide, 17 mg/ml yeast RNA, 10% weight/volume dextran sulfate was heated to 85°C for 5 min and 25 µl of this solution was then added to each section. Hybridization was performed overnight at 60°C in 50% formamide-humidified chambers. Sections hybridized with sense probes for VEGF and VEGFR-2 were used as controls for non-specific binding. After hybridization, slides were washed in 2x SSC containing 50% formamide pre-warmed to 50°C to remove coverslips. Sections were then washed in the above solution for 1 h at 55°C, rinsed three more times in RNAse buffer (10 mM TrisHCl, pH 7.5, 1 mM EDTA, pH 8.0, 0.5 M NaCl) and 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 Kodak X-Omat autoradiographic film for 13 days. Slides were subsequently dipped in Ilford K5 nuclear emulsion (Ilford, Mobberley, Cheshire, UK), stored in a light-free box with desiccant at room temperature for 23 weeks, immersed in Kodak D19 developer, fixed in Ilford Hypam and stained with haematoxylin and eosin.
Quantification of in situ hybridization
All tissue sections were processed in an identical fashion and were hybridized in the same batch. Glomerular gene expression was assessed in emulsion-coated sections using computer-assisted image analysis to quantify autoradiographic grains, as described previously [9,10]. In brief, light microscopic images viewed through a 20x objective lens were captured and digitized using a Fujix HC-2000 digital camera (Fuji, Tokyo, Japan). The outline of each glomerulus (210/biopsy), as defined by interactive tracing, was used to assess glomerular gene expression in each kidney section. Glomerular gene expression was then assessed by quantifying the proportion of the area of each glomerulus occupied by autoradiographic grains [8,9], using computerized image analysis (Analytical Imaging Station, Imaging Research Inc., St Catherines, Ontario, Canada). Two to ten representative glomeruli were examined in each specimen. All sections were hybridized to their respective probes in the same experiment and analysed in duplicate with the observer masked to the underlying diagnosis.
Statistics
The results were expressed as mean ± SEM, unless otherwise specified. The results of the two groups were compared using the MannWhitney non-parametric test.
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Results
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Clinical data
Clinical details of the eight patients are summarized in Table 1. The age of the patients at biopsy ranged from 4 to 60 years. There were four females and four males. They were all diagnosed histologically as having MCD. Five of them (numbers 2, 4, 6, 7 and 8) had presented with a recent onset nephrotic syndrome. Two patients, numbers 3 and 5 had had repeated attacks of nephrotic syndrome over a period of
20 years, and were biopsied on more than one occasion. Both these patients had responded to corticosteroid treatment in the past and had normal renal function at the time of the present biopsy. A previous biopsy, performed 17 years prior to the present one in patient number 5, showed some focal sclerosis, but the present biopsy was diagnostic of MCD. The long duration of disease in this patient and completely normal renal function (serum creatinine 0.6 mg/100 ml), support the diagnosis of MCD and not focal sclerosing glomerulosclerosis. Patient number 1 had a clinical and histologic picture typical of MCD, with reduced renal function. At the time of biopsy none of the patients were receiving an ACE inhibitor. Patient number 1 had received corticosteroids prior to the biopsy. Patient number 5 was in the recovery phase of an exacerbation of the nephrotic syndrome and was tapering down her dosage of prednisone. Patient number 7 was receiving prednisone 35 mg every second day and cyclophosphamide 2.5 mg/day, while patient number 8 was receiving prednisone 1 mg/day. The remaining patients (numbers 2, 3, 4 and 6) all had a full-blown nephrotic syndrome at the time of biopsy and had not received steroid therapy. Renal biopsies from both MCD and nephrectomy patients appeared normal when examined by light microscopy.
VEGF gene expression
By both quantitative and semi-quantitative methods gene expression for VEGF was reduced in glomeruli from patients with MCD when compared with controls. Mean VEGF gene expression in MCD patients was 40% that of control subjects (1.9 ± 0.4 vs 4.8 ± 0.6%, P < 0.01) (Figure 1). All the MCD patients had low values for the VEGF gene, except patient number 1, with reduced renal function, whose result was 5% (value with asterisk). The three patients receiving corticosteroids had the lowest levels for VEGF (see solid triangles in Figure 1). The major site of renal VEGF gene expression was in the glomeruli (Figure 2). Furthermore, as shown in both the high-power light and dark field photomicrographs (Figure 2), VEGF mRNA localized to the glomerular epithelial cells. In MCD patients there was minimal VEGF gene expression in the glomeruli (Figure 2).

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Fig. 1. Gene expression of VEGF in glomeruli from control subjects and patients with MCD. The gene expression (y-axis) is expressed as a percentage of the glomerular area occupied by the autoradiographic grains. Individual data and the median are shown. The solid diamonds are the results of those patients receiving corticosteroids at the time of biopsy and the asterisk denotes the patient with reduced renal function.
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Fig. 2. Photomicrographs of glomeruli labelled in situ with anti-sense riboprobes to VEGF. Upper left section (magnification x340) shows a representative glomerulus from a control subject demonstrating expression of VEGF in the glomerulus and especially in epithelial cells. The upper right darkfield photomicrograph (magnification x260) from a control subject, is punctate and represents localization to the glomerular epithelial cells. The lower left photomicrograph (magnification x1030) shows the clear localization of the silver granules to the epithelial cells from a renal biopsy specimen of a control subject. The lower right photomicrograph (magnification x340), taken from an MCD subject shows minimal renal expression of VEGF.
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VEGFR-2 gene expression
No difference in mRNA for VEGFR-2 was noted in glomeruli from patients with MCD when compared with controls (1.9 ± 0.4 vs 2.0 ± 0.2%, individual data not shown). Similarly, there was no difference in the pattern of distribution, with VEGFR-2 mRNA being localized to glomerular capillary endothelial cells.
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Discussion
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The present study has documented reduced VEGF gene expression in MCD. This would suggest that VEGF is not the vascular permeability factor that has been thought to be one of the possible factors in the pathogenesis of the nephrotic syndrome in MCD in both children and adults. In this study, VEGF was localized to visceral epithelial cells, as has been demonstrated previously using both in situ hybridization and immunohistochemistry [3]. The patients in this study all had histologic features of MCD. The major finding was a marked reduction in VEGF gene expression in most MCD patients. Indeed, in only one patient was VEGF expression not decreased and this was in the context of decreased renal function. Of interest, those subjects receiving corticosteroids had even lower VEGF expression, although one must be cautious in detailed interpretation of these findings, based on a relatively small number of patients. It has been shown in a model of mesangial glomerulonephritis in rats that i.p. dexamethasone resulted in increased proteinuria, associated with an increase in VEGF release, but in an insignificant decrease in glomerlular mRNA levels [11]. In spite of the fact that the VEGF expression was reduced in this group of MCD patients, there was no difference in expression of the VEGF-2 receptor between the controls and MCD patients. Thus, the results in this group of patients with a histologic diagnosis of MCD documents specifically reduced VEGF gene expression.
The demonstration of renal VEGF expression in the human kidney was subsequently followed by studies performed by the group of Shulman, where they explored the expression of this growth factor in a range of kidney diseases [4]. Expression of VEGF was noted in glomeruli from subjects with MCD although no direct comparison was performed with kidneys from control subjects [4]. In other renal disorders, there was, in general, reduced VEGF expression in sclerotic areas, areas of amyloidosis and at sites of glomerular collapse secondary to crescent formation. Another group has used non-radioisotopic in situ hybridization to examine VEGF gene expression in proteinuric renal disease [2]. These investigators measured the number of VEGF mRNA positive cells in histologic sections from MCD subjects as well as in those with diabetic and membranous nephropathies. Using this approach the investigators reported increased, decreased and no change in VEGF positive cells in MCD, diabetic and membranous nephropathies, respectively. Based on these preliminary studies, the authors postulated a role for VEGF in MCD.
To further explore the role of VEGF in MCD, plasma and urinary VEGF levels and peripheral blood mononuclear cell VEGF gene expression were assessed in children with relapsing yet steroid-sensitive nephrotic syndrome. These three parameters were also assessed in healthy children and in children in remission from MCD and the results were shown to be similar in the healthy subjects to those in patients with active MCD [5]. In a recent study Matsumoto et al. [6] found elevated VEGF in the urine of patients with MCD. They found that the levels of VEGF in the urine correlated with the degree of proteinuria. They were of the opinion that this VEGF was derived from the plasma and was thus nothing more than a marker of proteinuria and with no bearing on the pathophysiology of MCD. This would imply that VEGF is not elevated in MCD.
The role of VEGF in mediating proteinuria in various renal diseases remains speculative. For example, administration of VEGF to rats induces a decrease in blood pressure but has no effect on proteinuria [5]. In contrast, in a study of a rat model of nephrosis induced by i.p. injection of BSA there was up-regulation of VEGF and its receptors [12]. These findings do not provide evidence that VEGF is responsible for the proteinuria and indeed it has been suggested that the up-regulation of VEGF may be secondary to the proteinuria [12]. To further explore the link between VEGF and proteinuria, a range of more direct approaches have been considered. For example, Ostendorf et al. [13] have generated aptamers to VEGF-165, which act as antagonists. These compounds failed to reduce proteinuria. In contrast, in the model of streptozotocin diabetes, administration of an antibody to VEGF reduced albuminuria [14]. A more recent study of a rat model of nephrosis (puromycin aminonucleoside-induced nephrosis) showed down-regulation of both VEGF and its receptors [15]. A number of other roles for VEGF in the kidney have been described. This includes proliferative effects on glomerular endothelial cellular proliferation as demonstrated in a model of glomerulonephritis [13]. In fact, VEGF may be involved in the repair process, this action being of particular significance in the haemolyticuraemic syndrome [16].
In the present study, VEGFR-2 was localized to glomerular endothelial cells, as has been reported previously by several groups using a range of complementary techniques, including radioligand binding, in vitro and in vivo autoradiography, immunohistochemistry and various molecular techniques to assess gene expression [1719].
The pathogenesis of MCD is still unclear with a role for VEGF remaining speculative. A factor, which is heat labile, vasoactive and has a molecular weight of
100 kDa has been reported to be found in plasma taken from subjects with MCD, and recent studies have provided evidence that this may be related to the human glycoprotein hemopexin [20,21]. Indeed, in additional studies involving infusion of human hemopexin into the rat has resulted in proteinuria and renal alterations resembling human MCD [22]. Other studies have implicated various interleukins, some of which including IL-10 and IL-13 inhibit vascular permeability, whereas others such as IL-12 and IL-15 increase vascular permeability [2325]. These investigators were unable to provide clear evidence of an interaction between these interleukins and VEGF. In a more recent study, they showed an increase in concavalin-A-induced VEGF from peripheral blood T-cells taken from MCD patients [6]. The increased release was inhibited by TGF-ß1, suggesting a possible role for TGF-ß1 in the regulation of VEGF in MCD.
In this study we used as controls, tissue taken from patients with renal tumours. It may be postulated that by using tissue taken from patients with renal tumours as controls would result in a spuriously high level for VEGF mRNA and thus affect the results of our study. Several groups have investigated the expression of VEGF in human renal tissues and have found increased VEGF only in the area of the tumour and in surrounding vascular tissue, but not in the area of normal renal tissue [2628]. Although a relatively small number of patients were included in this study, the uniformity of the results, decreased expression of VEGF and normal expression of VEGFR-2, support the hypothesis that in patients with MCD there is a specific down-regulation of VEGF gene expression. Corticosteroid treatment may further decrease this expression. Most previous investigators have demonstrated increased VEGF in MCD, also in a relatively small number of subjects [2,4]. We are unable to explain the difference between their findings and ours. However, as it is now thought that VEGF is an important inducer of angiogenesis and possibly fibrosis [29], it is to be expected that in MCD with no signs of proliferative renal disease or glomerulosclerosis, there would not be an up-regulation of the VEGF gene.
In summary, renal VEGF expression was reduced in patients with MCD. In view of reports emphasizing the potential role of VEGF in renal repair and survival [12,30], it is postulated that deficiency of VEGF in this disease, possibly secondary to the underlying cause of this disease, leads to dysregulation of the renal repair process, which is involved in the maintenance of normal structure and function of the glomerular capillary wall. This does not rule out the possibility that other plasmatic factors may play a role in inducing the disease or in suppression of VEGF.
Conflict of interest statement. None declared.
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Received for publication: 29. 8.01
Accepted in revised form: 21. 5.03