VEGF –460 genotype plays an important role in progression to chronic kidney disease stage 5

Angela M. Summers, Beatrice M. Coupes, Mary Frances Brennan, Shirley A. Ralph, Colin D. Short and Paul E. C. Brenchley

Manchester Institute of Nephrology and Transplantation, Manchester Royal Infirmary, Manchester M13 9WL, UK

Correspondence and offprint requests to: Dr Angela M. Summers, Department of Renal Research, Manchester Institute of Nephrology and Transplantation, Manchester Royal Infirmary, Manchester M13 9WL, UK. Email: Angela.Summers{at}cmmc.nhs.uk



   Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Changes in renal vasculature, with vascular and interstitial fibrosis, are hallmarks of progression to chronic kidney disease (CKD) stage 5. Vascular endothelial growth factor (VEGF) is a potent angiogenic and vascular permeability factor. Transforming growth factor-ß1 (TGF-ß1) plays a critical role in promoting extracellular matrix (ECM) deposition and fibrosis. This study investigates whether genetic polymorphisms of VEGF or TGF-ß1 are associated with (i) progressive decline in renal function in patients with glomerular disorders (cohort 1) and (ii) predisposition to CKD stage 5 in a separate group of renal transplant recipients with various primary diseases (cohort 2).

Methods. Two patient groups were studied. Cohort 1 comprised 91 patients with biopsy-proven glomerular disease who were followed-up for 5 years before categorization as either non-progressors (with stable serum creatinine or ≤30% increase over 5 years, n = 39) or progressors (requiring dialysis, transplantation or whose serum creatinine increased by >30% over 5 years, n = 52). Cohort 2 comprised 107 patients with various primary renal diseases, who had reached CKD stage 5 and undergone renal transplantation at the time of study. All patients were genotyped for the VEGF polymorphisms at positions –460 (C/T) and +405 (G/C). Linkage disequilibrium (LD) was established using EHplus. SNPHAP was used to estimate haplotype frequency and to infer haplotypes to all patients. Cohort 1 patients were genotyped for the TGF-ß1 polymorphisms at positions –800, –509, codons 10 and 25. Genotyping was performed by polymerase chain reaction-restriction length polymorphism (PCR-RFLP).

Results. In cohort 1, there was a significant increase in frequency of the –460 VEGF CC genotype 30.8 vs 5.1%, P = 0.008; odds ratio (OR), CC vs TT 10.67, 95% confidence interval (CI), 1.94–58.72 and C allele 56.7 vs 37.2%, P = 0.009; OR 2.22, 95% CI, 1.21–4.04, in the progressor patients when compared with the non-progressors. In cohort 2, there was a significant increase in the VEGF –460 CC genotype when compared with healthy volunteers 37 vs 20.8%, P = 0.011; OR CC vs TT 1.59, 95% CI, 0.72–3.51. The –460 and +405 polymorphisms were in LD P<0.00007. There were significant differences in diplotype (haplotype pair) frequencies in cohort 1 and 2, P = 0.018, which confirmed the importance of the –460C allele. There were no associations between the VEGF +405 or TGF-ß1 polymorphisms and progressive renal disease.

Conclusion. In this study, we have demonstrated an association between the VEGF –460 polymorphism and progression to CKD stage 5. The function of this polymorphism remains unclear although previous evidence suggests that promoter constructs containing this single nucleotide polymorphism (SNP) have been associated with increased activity. Clearly there is a role for TGF-ß1 in chronic kidney disease. However, this study found no associations with four TGF-ß1 polymorphisms in this cohort.

Keywords: chronic kidney disease stage 5; haplotypes; polymorphisms; progressive renal disease; transforming growth factor ß-1; vascular endothelial growth factor



   Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The prognosis of patients at diagnosis of glomerular disease is highly variable, ranging from spontaneous or therapy-induced resolution to the development of end-stage renal failure (ESRF; CKD stage 5), either rapidly or slowly. Risk factors for progression include hypertension, persistent proteinuria and histological evidence of glomerular and interstitial fibrosis [1]. In many renal disorders, once a significant degree of renal impairment is reached, a progressive fall in creatinine almost inevitably ensues, irrespective of the primary disease and whether or not the original insult has been successfully eradicated. It is probable that under these circumstances this inexorable decline to CKD stage 5 is mediated via a common pathway involving many factors. Vascular endothelial growth factor (VEGF) and transforming growth factor-ß (TGF-ß1) have both been implicated in the pathogenesis of renal dysfunction.

VEGF is a key mediator of normal and abnormal angiogenesis and is important in maintaining the integrity of the renal vasculature [2]. In the kidney, VEGF is primarily localized to the glomerular podocyte, the tubular cells, and the outer medulla and medullary rays. VEGF expression is upregulated in mesangial proliferative disease, minimal change disease, chronic tubulointerstitial injury, membranous nephropathy and diabetic nephropathy [3].

We have previously described polymorphisms in the VEGF gene [4]. Two polymorphisms were chosen in this study because they occur at a relatively high minor allele frequency in the general population. These single nucleotide polymorphisms (SNPs) also alter VEGF expression [4–6]. The polymorphism at +405(G -> C) has been shown to regulate VEGF expression. Higher VEGF production by peripheral blood mononuclear cells (PBMCs) in response to lipopolysaccharide (LPS) has been associated with the +405 G allele [4]. However, internal ribosome entry site B (IRES-B) activity is higher in promoter constructs containing the VEGF +405 C allele during normoxia and hypoxia, and production of L-VEGF is impaired by the +405 G in the same conditions [5]. Haplotypes containing the common polymorphisms at –460C in the promoter and +405G have a 71% higher basal promoter activity when compared with the wild-type sequence [6]. Elevated serum VEGF levels have previously been associated with the +405 C allele, and this polymorphism was associated with retinopathy in type 2 diabetes [7]. A second study in type 1 and 2 diabetics demonstrated an association of the –460C allele with retinopathy [8].

TGF-ß1 plays a critical role in renal pathophysiology by promoting extracellular matrix deposition and fibrosis, leading to loss of functional renal tissue. In renal fibrosis, TGF-ß1 is upregulated in the glomerulus and is thought to derive from mesangial cells, platelets and infiltrating cells [9]. Upregulation of TGF-ß1 in the glomerulus is found in IgA nephropathy (IgAN) and mesangial proliferative glomerulonephritis [10]. In the latter, the degree of immunostaining for TGF-ß1 correlates with the amount of mesangial matrix. Epithelial to mesenchymal transition (EMT) is a major contributor to the pathogenesis of renal fibrosis as it leads to a substantial increase in the number of myofibroblasts, leading to tubular atrophy. TGF-ß1 is a well-established inducer of EMT involving renal tubular cells [11].

Seven SNPs have been described in the TGF-ß1 gene in the coding region and throughout the promoter [12]. This study examines four of these SNPs at positions –800 and –509 from the first transcribed nucleotide, and Leu10 -> Pro and Arg25 -> Pro in the signal peptide sequence. These polymorphisms influence TGF-ß1 production [13].

We postulate that these polymorphic variants in the VEGF and/or the TGF-ß1 genes could predispose certain individuals with renal disease to the progression to CKD stage 5. The aims of this study are therefore 2-fold: first, to determine whether polymorphisms in the VEGF and TGF-ß1 genes influence the progression of renal impairment, in patients with glomerular disease, and secondly, to establish whether any association can be identified in a population of renal transplant recipients with a variety of primary diseases who, by definition, had reached CKD stage 5 as a consequence of their primary renal disease.



   Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study groups
Cohort 1
Ninety-one patients with biopsy-proven glomerular renal disease, including IgAN, membranous nephropathy (IMN), focal and segmental glomerular sclerosis (FSGS), idiopathic glomerulonephritis and mesangio capillary glomerulonephritis (MCGN) were monitored over a 5 year follow-up period and, on the basis of their renal function, divided into two groups (Table 1): (i) non-progressors, i.e. patients with renal disease who had stable renal function with serum creatinine levels which did not rise by >30% over the 5 years; and (ii) progressors, i.e. patients with progressive renal disease, defined as a rise in serum creatinine of >30%, or development of CKD stage 5, requiring dialysis or transplantation. All patients with progressive disease had rapidly rising creatinine levels (doubling over the 5 year period).


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Table 1. Demographics of patient cohort 1

 
Cohort 2
This group comprised 107 patients who had received renal allografts; the patient demographics, ethnicity and cause of primary renal disease are shown in Table 2.


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Table 2. Demographics of patient cohort 2

 
Healthy volunteer group
This group comprised 102 healthy volunteers with no history of renal disease who were recruited from a similar geographical region to that of the patients.

VEGF and TGF-ß1 genotyping
Venous blood was collected in EDTA-coated tubes. DNA was prepared from whole blood using a standard salting out process.

The VEGF and TGF-ß1 polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) as previously described [4,13].

VEGF haplotype and diplotype analysis
EHplus was used to determine the pattern of observed and expected frequencies, and hence predict whether the two SNPs were in linkage disequilibrium (LD). The expectation–maximization algorithm was used to assign haplotypes to individuals with >94% certainty (http://www.gene.cimr.cam.ac.uk/clayton/software/SNPHAP.txt). From this, individuals were grouped according to their diplotypes (haplotype pair).

Statistical analysis
Genotype frequencies were compared using a 3 x 2 {chi}2 analysis, where statistical significance was taken as P<0.05. Allele frequencies were compared using a 2 x 2 {chi}2 or Fishers exact test. Odds ratios (ORs) of the most common genotype vs other genotypes were calculated with a 95% confidence interval (CI). Diplotype frequencies in cohorts 1 and 2 were compared using {chi}2 analyses.



   Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study groups
In cohort 1, the mean ages, gender ratios, ethnicity and distribution of primary renal diseases were not significantly different between the non-progressors and the progressors (Table 1). All patients in this group had primary idiopathic glomerular disease.

The demographics of cohort 2 are shown in Table 2. The primary diseases leading to CKD stage 5 in this group were not all glomerular in origin. The inclusion of other renal pathologies allowed investigation of the genetic polymorphisms with the development of CKD stage 5 regardless of primary disease. The healthy volunteer group was predominantly Caucasian (99.1%). This group differed significantly from cohort 2 in age (38.6±9.2 vs 42.9±12.6 years, respectively, P = 0.01) and gender distribution (26% male vs 62%, respectively, P = 0.0001).

VEGF and TGF-ß1 genotypes in cohort 1
The distribution of VEGF genotypes is illustrated in Table 3. There was an increased frequency of the VEGF –460 CC genotype in the progressors when compared with the non-progressors; P = 0.008; OR CC vs TT 10.7, 95% CI, 1.94–58.72. This was also significant at the allelic level; P = 0.009; OR 2.22, 95% CI, 1.2–4.04.


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Table 3. VEGF genotypes in cohort 1

 
There was no significant difference in the VEGF +405 or the TGF-ß1 genotype distribution between the two groups (data not shown for TGF-ß1).

In addition, there was no significant difference in distribution for any of the genotypes studied between cohort 1 and the healthy volunteer group; P = 0.86; OR CC vs TT 1.2, 95% CI, 0.51–2.95 (data not shown).

VEGF genotypes and cohort 2
The distribution of VEGF genotypes is illustrated in Table 4. There was a significant increase in the VEGF –460CC genotype frequency in cohort 2 when compared with the healthy volunteer group; P = 0.012; OR CC vs TT 1.59, 95% CI, 0.72–3.51.


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Table 4. VEGF genotypes in cohort 2 compared with healthy volunteers

 
There were no significant differences between the VEGF +405 genotype frequencies.

Linkage disequilibrium
Evidence for very strong LD between the VEGF –460 and the +405 was obtained through EHplus (P<0.0007).

Haplotype and diplotype analysis
SNPHAP was used to assign nine haplotype pairs (diplotypes) with >94% certainty. The results were expressed as, for example, TG/CG; –460T, +405G/–460C, +405G (Tables 5 and 6).


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Table 5. Diplotype comparison cohort 1

 

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Table 6. Diplotype comparison cohort 2

 
VEGF diplotypes and cohort 1
The distribution of VEGF diplotypes is illustrated in Table 5. A {chi}2 analysis shows the frequencies to be significantly different between the two groups, P = 0.018. Notably, the frequency of the CG/CG diplotypes was increased in the progressors when compared with the non-progressors. In contrast, the frequency of TG/CG was reduced in the progressors vs the non-progressors. The only point at which this diplotype differs is at the –460 polymorphism. Also of note is an increase in the frequency of the TC/CG diplotype which is heterozygous for both polymorphisms.

VEGF diplotypes and cohort 2
The distribution of VEGF diplotypes is illustrated in Table 6. The difference in diplotype frequencies approaches significance between the two groups, P = 0.05 with a {chi}2 test for trend giving a P-value of 0.0061. Like the progressive group, the frequency of the CG/CG diplotype was increased in cohort 2 when compared with healthy controls and that of the TG/CG diplotype was reduced. However, in contrast to the progressive group, there was a decrease in the TC/CG diplotype in the transplant group.



   Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study has demonstrated a significant increase in the VEGF –460CC genotype in patients with progressive glomerular disease, when compared with those with stable renal function (cohort 1). The increase in the VEGF –460CC genotype was also found in a separate cohort of renal allograft recipients with various primary renal disorders (cohort 2). These findings suggest that this polymorphism influences susceptibility to progression to CKD stage 5. VEGF genotype does not appear to increase susceptibility to renal disease per se, since the genotype distribution of cohort 1 did not differ significantly from the healthy volunteer group. However, this study was not powered to assess susceptibility to individual primary renal diseases.

In this study, we have also shown that the VEGF –460 and +405 SNPs are in LD. The finding that the CG/CG diplotypes are increased in the progressors whereas TG/CG is decreased supports our original finding that the C allele at –460 may be important, as these two diplotypes only differ at one allele at the –460 locus. The influence of the +405 allele is more complex as there are two kinds of distribution in cohorts 1 and 2. In cohort 1, the TC/CG diplotype is increased in the progressors. In cohort 2, there is a decrease in the TC/CG and an increase in the CG/CG diplotype, meaning that heterozygosity at both loci may influence progression to ESRF. The area of the VEGF gene containing the VEGF +405 polymorphism does influence the post-transcriptional activity of VEGF [14]. IRES-B activity is higher in promoter constructs containing the VEGF +405C allele during normoxia and hypoxia, with impaired production of L-VEGF by the +405G allele under the same conditions [5]. This apparent reduction in VEGF activity in constructs containing the VEGF +405G allele conflicts with our finding of increased activity for constructs containing the same allele. We previously showed, using a promoter readout assay to analyse the impact of the common VEGF –460/+405 polymorphisms in response to oestrogen and phorbol ester, that a complex haplotype, which contains the VEGF –460C and +405G alleles, is associated with increased VEGF promoter activity when compared with wild type. This study describes two promoter haplotypes containing the VEGF –460C and +405G alleles, which differ in activity. The promoter constructs differ at the –160 C/T (allele frequency 0.017) and the –116 G/A (allele frequency 0.3) polymorphisms. This suggests that one or both of these SNPs are functionally important in a haplotype which contains the VEGF –406C and +405 G alleles [6]. Since the promoter region contains several other SNPs, it is possible that these may be influential in VEGF production. Lambrechts et al. [5] used constructs ranging from –947 to –484 (relative to the translation start site) for the IRES-B experiments and the entire 5'-untranslated region for the L-VEGF data. We used larger constructs which incorporated many other SNPs including the VEGF –460 SNP, allowing speculation as to the possible interaction between SNPs. In addition, we did not study promoter activity during hypoxia, although under normoxic conditions there was no difference in activity.

Our previous report of increased PBMC VEGF production associated with the G allele may reflect linkage to a more complicated diplotype: this was a small study (n = 32) which did not have the power to exclude the role of the VEGF –460 polymorphism and VEGF production [4]. Elevated serum levels of VEGF were associated with the +405C allele, but it is arguable that serum is the correct medium to measure VEGF, as platelets represent a large source of stored VEGF [7,15].

This new diplotypic study implies that it is the C allele at –460 which may form part of a complex haplotype associated with progressive glomerular disease. This haplotype may affect protein production through altered mRNA transcription or translational processing (IRES-B) activity, although the evidence for this is inconclusive. One possible mechanism in progressive renal disease is that high levels of VEGF may initially increase blood flow and raise the permeability of glomerular endothelial cells. The chemotactic effect of VEGF would amplify the inflammatory process and promote atherosclerosis. Activation of macrophages and monocytes may cause localized immune injury affecting epithelial cells, podocytes and mesangial cells, triggering further VEGF release and exacerbating the process of epithelial cell destruction. Expression of VEGF and its receptors is significantly increased in the PHN and PAN rat models of proteinuria, suggesting a role for VEGF in the disease process [16]. Chronic tubulointerstitial injury (CTI) including tubular atrophy and interstitial fibrosis represents one major determinant for the progression of chronic renal disease regardless of cause. In CTI, an interesting pattern of increased VEGF expression by renal tubules, especially morphologically intact or hypertrophic ones, has been demonstrated [17]. VEGF is thought to be an important mediator in diabetic nephropathy where it may have a role in mediating glomerular hypertrophy and proteinuria. In contrast, in some studies of progressive renal disease (e.g. the remnant kidney model), a loss of VEGF is associated with a loss of peritubular and glomerular capillaries and enhanced progression, and providing exogenous VEGF can slow the renal disease [18].

The evidence to support a role for TGF-ß1 polymorphisms in progression of renal disease is limited and conflicting. Previous studies in patients with both type 1 and type 2 diabetic nephropathy implicate the codon 10 polymorphism [19]. However, this has not been confirmed in a recent study [20]. We can speculate that differences in genotype frequencies in ethnic groups may explain these discrepancies. In the present study, there is no evidence to suggest that TGF-ß1 polymorphisms influence progression to CKD stage 5 in this predominantly Caucasian cohort. The multi-layered control mechanisms governing the expression and activation of TGF-ß1 may mask any genetic component. We cannot disregard a role for TGF-ß1 in the pathogenesis of CKD stage 5.

This genotypic study has added to the emerging evidence that VEGF plays an important role in progression to CKD stage 5.

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 10. 6.05
Accepted in revised form: 24. 6.05