The association between therapeutic outcomes and VEGF G-1154A and C-936T gene polymorphisms in patients with glomerulonephritis

Abstract

Background: In this present study, we aimed to investigate the association between therapeutic outcomes and vascular endothelial growth factor (VEGF) G-1154A and C-936T gene polymorphisms in patients with glomerulonephritis. Methods: Thirty-eight patients with glomerulonephritis diagnosed by renal biopsy were included to the study. All patients had proteinuria at least 1 gram (g)/day in urine analysis. At the end of a yearly therapy, patients with proteinuria less than 0.5 g/day were accepted as in complete remission and they were termed as group 1. The patients with proteinuria over 0.5 g/day were accepted as in no remission and they were termed as group 2. Results: The mean age of patients in group 1 and group 2 was 35.88 ± 13.80 years and 37.30 ± 13.89 years, respectively. There were nine (50%) male and nine (50%) female patients in group 1. In group 2, seven (35%) male and 13 (65%) female patients were present. Although VEGF G-1154A (GG) gene polymorphism was found in 55% of group 2 patients, and 22.2% of group 1 patients, but the differences did not reach statistical significance. There were no statistical differences between groups in terms of other gene polymorphisms. Namely, we obtained no statistical differences between therapeutic outcomes and gene polymorphisms. Conclusions: There is a significant difference between groups in terms of VEGF G-1154A (GG) gene polymorphism, but the minority of the patient population has led to not to reach statistical significance. So, this gene polymorphism has to be investigated in larger studies.

• Gene polymorphism
• glomerulonephritis
• proteinuria
• therapeutic outcomes
• vascular endothelial growth factor

Introduction

Glomerulonephritis is a disease that disrupts the structure and function of glomeruli. It is one of the main reasons that lead to end-stage renal failure (ESRF). It ranks third after diabetic nephropathy and hypertension in developing chronic kidney disease in our country.

Vascular endothelial growth factor (VEGF) is a member of platelet-derived growth factor family, and synthesized in various cell types in the body. VEGF has important roles in proliferation, migration, and differentiation of endothelial cells.1–6 VEGF has also a major role in occurring and maintaining the fenestrae in glomerular capillary endothelial cells.7

The fenestrated endothelium in glomeruli, glomerular basement membrane, and podocytes form a physical barrier against transition of macromolecules such as albumin and the other proteins in plasma.8,9 Given the role of VEGF in enhancing microvascular permeability, it is estimated that VEGF may regulate glomerular permeability. There is a close relationship between the distribution of fenestrae and rise in VEGF expression. The signals derived from glomerular basement membrane and podocytes are needed for formation and flattening of the fenestrae. In the absence of VEGF-A, glomerular endothelium does not flatten enough and leads loss of fenestrae. In conclusion, proteinuria will emerge in continuation.

In various studies, it has been shown that the common 936 C/T polymorphism in the 3′ untranslated region of VEGF gene is associated with plasma levels of VEGF, and the plasma level of VEGF was significantly lower in 936T allele positive patients.10 On the other hand, the presence of 1154G allele was associated with higher plasma levels of VEGF.11

In this study, our aim was to investigate the presence of G-1154A and C-936T gene polymorphisms, and their correlation with response to treatment in patients with glomerulonephritis.

Subjects and methods

Sixteen male and 22 female patients were enrolled in the study. After diagnosis of glomerulonephritis, angiotensin converting enzyme inhibitor ± angiotensin receptor blocker ± antilipidemic ± antiaggregant therapy have administered to these patients conservatively. Of the patients, 31 (81.6%) had steroid plus alkylating agents or steroid plus cyclosporine in addition to conservative therapy.

Patients

The study was conducted in a prospective manner in Cumhuriyet University Medical School between January 2007 and August 2011. Thirty-eight patients with glomerulonephritis diagnosed by renal biopsy were included in the study. All patients provided their written consent for the participation in this study. The study was approved by the local Ethics Committee and was in accordance with the Declaration of Helsinki.

Each patient was followed-up during a year at 3 months intervals. All patients had proteinuria at least 1 gram (g)/day in urine analysis. At the end of a yearly therapy, patients with proteinuria less than 0.5 g/day were accepted as in complete remission and they were termed as group 1. The patients with proteinuria over 0.5 g/day were accepted as not in remission and they were termed as group 2.

VEGF G-1154A and C-936T gene polymorphisms and agarose gel electrophoresis

Briefly, VEGF gene polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method with analysis of DNA samples obtained from blood leukocytes. DNA fragments were investigated by using appropriate primers. SNP scanning was performed using MnII (Fermentas, Vilnius, Lithuania) and Hsp92II (Promega, Madison, WI) restriction enzymes for 1154 G/A and 936 C/T polymorphisms, respectively. The amplified polymorphic DNA in a volume of 12.5 µL has been treated with 33 mM Tris-acetate, 10 mM Magnesium acetate, 66 mM Potassium acetate, 0.1 mg/mL BSA (37 °C; pH:7.9) containing RE tamponade, and finally 10 U/µL of tamponade-enzyme mixture for each individual. The end product has been incubated in 37 °C for 14–16 h.

The PCR products were digested for 4 h at 37 °C with NLaIII restriction enzyme (Fermentas) at a final concentration of 5 units. After inactivation of the enzyme for 20 min at 65 °C, digested PCR products were separated by electrophoresis using a 3% agarose gel. RFLP band patterns that were visualized by ultraviolet light (Spectroline, New York, NY) are shown in Figures 1 and 2. Positive and negative controls were used to ensure genotyping accuracy.

Figure 1. Genotyping of VEGF gene C-936T polymorphisms. Lane 1 and 4: C/T genotype (208, 122, and 86 bp), Lane 2: T/T genotype (122 and 86 bp), Lane 3 and 5: C/C genotype (208 bp).

Figure 2. Genotyping of vascular endothelial growth factor (VEGF) gene G-1154A polymorphisms. Lane 1 and 2: G/A genotype (184, 150, 34, 22, and 86 bp), Lane 3 and 4: A/A genotype (184 and 22 bp), Lane 5: G/G genotype (150, 34, and 22 bp).

Statistical analysis

All statistical analyses were performed using SPSS version 14.0 (SPSS, Chicago, IL). Variables following normal distribution are presented as the mean ± standard deviation. Mann–Whitney U-test was used to test for differences between independent samples. The Chi square test or Fisher's exact test were used for the comparison of polymorphisms. A p value of <0.05 was considered statistically significant.

Results

The mean age of patients in group 1 and group 2 was 35.88 ± 13.80 years and 37.30 ± 13.89 years, respectively. There were nine (50%) male and nine (50%) female patients in group 1. In group 2, seven (35%) male and 13 (65%) female patients were present. The characteristics of all patients are displayed in Table 1. The histopathological diagnoses of the patients are summarized in Table 2.

Table 1. The baseline characteristics of the patients.

	Group 1 (n = 18)	Group 2 (n = 20)	p Value
Age (years) (mean ± SD)	35.8 ± 14	37.3 ± 14	0.838
Gender (M/F)	9/9	7/13	0.350
Body weight (kg) (mean ± SD)	73.00 ± 11	71.5 ± 12	>0.05
Smoking	4 (22.2%)	2 (10%)	>0.05
Hypertension	5 (27.8%)	6 (30%)	>0.05
Diabetes mellitus	1 (5.6%)	1 (5%)	>0.05

Table 2. The percentage of histopathological diagnosis of the patients.

Diagnosis	n (%)
IgA nephropathy	7 (18.4)
Membranous glomerulonephritis	11 (28.9)
Membranoproliferative glomerulonephritis	6 (15.8)
Focal segmental glomerulosclerosis	4 (10.5)
Lupus nephritis^a	3 (7.9)
Rapidly progressive glomerulonephritis	2 (5.2)
IgG nephropathy	1 (2.6)
IgM mesangial proliferative glomerulonephritis	2 (5.2)
c-ANCA positive glomerulonephritis	2 (5.2)
Total	38 (100)

Note: ^aOf the three patients, two had diffuse proliferative lupus nephritis, and one had focal proliferative lupus nephritis.

VEGF 936 CC gene polymorphism has been detected in 80% of group 2 and 72.2% of group 1. VEGF 936 CT gene polymorphism has been detected in 15% of group 2 and 16.7% of group 1. VEGF 936 TT gene polymorphism has been detected in 5% of group 2 and 11.1% of group 1. These differences were not statistically significant between group 1 and 2 (Table 3).

Table 3. The comparison groups in terms of VEGF C-936T gene polymorphisms.

VEGF C-936T gene polymorphism	Group 1 (n = 18)	Group 2 (n = 20)	p Value
CC	13 (72.2)	16 (80)	0.763
CT	3 (16.7)	3 (15)
TT	2 (11.1)	1 (5)

VEGF 1154 AA gene polymorphism were not seen in group 2; however, it was detected in two patients of group 2 (11.1%). VEGF 1154 GA gene polymorphism was found in 45% of group 2 (n = 9) and 66.7% of group 1 (n = 12). VEGF 1154 GG gene polymorphism was detected in 55% of group 2 (n = 11) and 22.2% of group 1 (n = 4) (Table 4). There were no significant differences between group 1 and 2 in terms of VEGF 1154 gene polymorphisms. In conclusion, the differences between group 1 and 2 were not statistically significant in terms of VEGF C-936T and VEGF G-1154A gene polymorphisms.

Table 4. The comparison groups in terms of VEGF G-1154A gene polymorphisms.

VEGF G-1154A gene polymorphism	Group 1 (n = 18)	Group 2 (n = 20)	p Value
AA	2 (11.1)	0 (0)	0.061
GA	12 (66.7)	9 (45)
GG	4 (22.2)	11 (55)

Discussion

Glomerulonephritis may show variable clinical courses. Some may display sudden onset and then spontaneous recovery or treatment induced complete remissions may evolve. In contrast, some leads to rapidly progressive glomerulonephritis in a short time or chronic kidney disease in years. This process may be affected by age, gender, presence of crescent, tubulointerstitial involvement, degree of proteinuria, and ACE gene polymorphism, however, this situation is still not clear.

VEGF is synthesized by glomerular podocytes, and VEGF receptors are found on glomerular capillary endothelial cells.1,7,12 Vascular endothelial growth factor receptor-1 (VEGFR1) and vascular endothelial growth factor receptor-2 (VEGFR2) are expressed in preglomerular, glomerular, and peritubular endothelial cells.13–16 VEGF mainly activates VEGFR2. VEGF is essential for growth and proliferation of glomerular and peritubular endothelial cells.1,17 VEGF has an important role in occurring and maintaining the fenestrae in glomerular capillary endothelial cells.7

In general, VEGF is expressed from hypoxic cells.1,18 The presence of hypoxy leads VEGF production by tubular cells.1 The kidneys are quite sensitive to ischemia, and VEGF plays a vital role in ischemic situations. In ischemic damage, VEGF stimulates angiogenesis which is a protective mechanism for kidneys.19 VEGF is needed for tubular hypertrophy and proliferation. It has been shown that glomerulosclerosis and tubulointerstitial fibrosis is associated with a loss of VEGF.1 In rat studies, the pharmacological inhibition of VEGF release from podocytes has led to loss of fenestrae, endotheliosis, loss of podocytes, and proteinuria.7,20

Khakoo et al.21 have stated that proteinuria leads to an increase in VEGF expression. Ayerder et al.22 have shown that VEGF expression is diminished significantly when blood pressure was normalized by angiotensin converting enzyme inhibitors. Honkanen et al.23 have stated that urinary VEGF level may be used as a marker in activation of membranous glomerulonephritis. In another study, urinary VEGF levels were compared between patients with nephrotic syndrome and healthy subjects, and it has been found that VEGF levels are increased in patients with nephrotic syndrome and are correlated with proteinuria.24–28 Shimizu et al.29 demonstrated that VEGF 165 therapy resulted in an improvement of crescentic and necrotizing lesions, endothelial cell proliferation, capillary reparation, and loss of proteinuria in rats with anti-glomerular basement membrane glomerulonephritis.

In the literature, multiple polymorphisms of VEGF gene were identified. The first is determined by Watson et al.3 Some of these polymorphisms are found correlated with differences in VEGF expression.30 Recently, 15 different polymorphisms containing 460 C/T, 405 G/C, and 141 A/C in VEGF gene were determined. The most seen haplotypes in normal population are 460C/405G and 460T/405C. The VEGF 405 production is highest in GG genotype, is moderate in GC genotype, and finally is lowest in CC genotype has been shown in in vitro and in vivo studies.3,31 And also, the combination of genotype 405 and the other polymorphisms result in much more VEGF promotor activity.32 A deletion/insertion polymorphism in VEGF promotor region at 2549 has been found related with an enhanced transcriptional activity.33

Renner et al.10 have found an association between VEGF plasma levels and VEGF 936 C/T gene polymorphism, and stated that patients with 936 T allele have significantly lower VEGF plasma levels.34 Shahbazi et al.11 have found that patients with coronary heart disease, who have VEGF 1154 G allele, have higher VEGF plasma levels. Chow et al.30 have performed a study on patients with IgA nephropathy [119 female (61%) and 76 male (39%)]. They stated in their study that VEGF 2578 promoter CC genotype is associated with an increase in renal insufficiency.

In the literature, we could not encounter with a study investigating the relationship between VEGF C-936T and VEGF G-1154A polymorphisms and the remission rates in patients with glomerulonephritis. So, we aimed to investigate this relationship in our prospective study. However, we found no statistical significance between therapeutic outcomes and VEGF C-936T and VEGF G-1154A polymorphisms.

The results of this study are subjected to some limitations. First, this is a single-center study with a relatively small sample size. Second, we could not study serum VEGF levels and renal mRNA expression due to absence of commercial kits. However, it must be stated that measuring serum VEGF levels are not reliable in patients withglomerulonephritis under angiotensin converting enzyme inhibitor and corticosteroid therapy. In this setting, we suggest that serum VEGF levels might be measured before treatment.

In conclusion, the literature data suggest that VEGF and VEGF polymorphisms may be a marker in determining the progression in glomerular diseases.29,30,35,36 Although we could not demonstrate a statistical significance between therapeutic outcomes and VEGF C-936T and VEGF G-1154A polymorphisms, nonetheless more specifically designed prospective and observational studies in a larger cohort are needed to make certain conviction.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.