The Accumulation of VEGFA in the Glomerular Basement Membrane and Its Relationship with Podocyte Injury and Proteinuria in Alport Syndrome

The pathogenesis of proteinuria in Alport syndrome (AS) remains unclear. Vascular endothelial growth factor A (VEGFA) is a key regulator of the glomerular filtration barrier (GFB). This study explored the expression of VEGFA in the glomeruli and its accumulation in the glomerular basement membrane (GBM) and their relationship with podocyte injury and proteinuria in Alport syndrome (AS). Clinical data and renal tissues of control patients (11 cases) and AS patients (25 cases) were included. AS patients were further divided into 2 groups according to the quantities of their urinary protein: mild to moderate proteinuria group (proteinuria <50 mg/kg/d, 15 cases) and heavy proteinuria group (proteinuria ≥50 mg/kg/d, 10 cases). The expression and distribution of VEGFA and VEGF receptor 2 (VEGFR2) in the GFB, the phosphorylation of VEGFR2 (p-VEGFR2) and nephrin (p-nephrin), and the expression of synaptopodin and nephrin in the glomeruli were detected by immune electron microscopy and/or immunofluorescence, and their relationships to proteinuria in AS patients were analyzed. The accumulation of VEGFA in the GBM was increased in AS patients. The expression of VEGFA and the levels of p-VEGFR2 and p-nephrin in glomeruli were increased and were positively correlated with the degree of proteinuria in AS patients. The expression of synaptopodin and nephrin were decreased and were negatively correlated with the degree of proteinuria in AS patients. The over expressed VEGFA in the glomeruli and its accumulation in the GBM may activate the VEGFA-VEGFR2 and nephrin signaling pathways and lead to podocyte injury and occurrence of proteinuria in AS.


Introduction
Alport syndrome (AS) is the most common inherited progressive glomerulonephritis in children and it is caused by defects in type IV collagen (COL IV α3, α4 or α5 chain) in the glomerular basement membrane (GBM) [1,2]. The presence of proteinuria is an important risk factor of disease progression [3]. However, the pathogenesis of proteinuria in AS remains unclear. Dysfunction of the glomerular filtration barrier (GFB) is presumably associated with the development of proteinuria. Vascular endothelial growth factor A (VEGFA) plays an important role in the maintenance of GFB function. Both the over-expression and the down-regulation of VEGFA could cause GFB injuries and the occurrence of proteinuria [4,5]. Vascular endothelial growth factor receptor 2 (VEGFR2) is the main receptor for biological mediation function of VEGFA [6]. It has been reported that VEGFR2 could bind to the intracellular domain of nephrin [7]. The phosphorylation of Tyr1214 within VEGFR-2 triggers the recruitment of Nck and the activation of Fyn, which can mediate the tyrosine phosphorylation of nephrin and initiates signaling events that might dynamically integrate podocyte actin cytoskeleton dynamics and podocyte intercellular junction formation [8,9]. The present study explored the pathogenesis of proteinuria in AS by detecting the expression of VEGFA and VEGFR2 in glomeruli, the accumulation of VEGFA in GBM, the phosphorylation of VEGFR2 and nephrin, and the association of all of these markers with podocyte injury and the development of proteinuria in AS patients.

Materials and Methods Subjects
AS patients who were diagnosed in our hospital between October, 2010 and March, 2014 were included in this study. Diagnosis of AS was conducted in accordance with the criteria reported by Flinter [10], AS can be diagnosed when one of the following conditions is met: (1) abnormal staining of the COL IV α3 or α5-chain in GBM or in skin basement membrane, (2) typical ultrastructural changes visible with an electron microscope, and (3) mutations in COL4 A3-5 genes. The control patients (group 1) were those who underwent nephrectomy, and primary and secondary glomerular nephropathy were excluded. AS patients were further divided into 2 groups according to their degree of proteinuria: group 2, patients with mild and moderate proteinuria (urinary protein <50 mg/kg/d) and group 3, patients with heavy proteinuria (urinary proteinuria 50 mg/kg/d). [11] The clinical and pathological data and renal tissues were collected from AS patients and control patients.
The study was conducted in accordance with the principles outlined in the Declaration of Helsinki with approval from the ethics committee of the First Affiliated Hospital, Sun Yat-Sen University. Written informed consent was obtained from the subjects or their parents.

Experimental Protocols
The expression levels of synaptopodin, nephrin, VEGFA and VEGFR2 and the levels of p-VEGFR2 and p-nephrin within the glomeruli were detected by immunofluorescence. The expression and distribution of VEGFA and VEGFR2 were detected by immune electron microscopy. Vehicle control samples were set by adding tris buffered saline (TBS) without either primary or secondary antibodies during the detections. The protocols were as follows.

Statistical Analysis
Statistical analyses were performed with SPSS 13.0 software. The indexes of age and immunofluorescence in each group were expressed as the median (maximum, minimum). The indexes of data with normal distribution were expressed as mean ± standard deviation (SD). Data were analyzed using the Kruskal-Wallis test within 3 groups, and the Bonferroni test was used in between-group comparison (statistical significance was considered to be p<0.05/3 = 0.017). The Spearman test was used for the correlation analysis, and statistical significance was considered to be p<0.05.

Clinical data
Twenty five AS patients (19 male, 4 female) were included in the present study (Table 1). Fifteen patients (12 male, 3 female) had mild to moderate proteinuria (group 2, urinary protein was 18.45±6.04 mg/kg/d) and their median age at renal biopsy was 3.5 years (1.9 years, 11.0 years). Ten patients (9 male, 1 female) had heavy proteinuria (group 3, urinary protein was 108.45±45.43 mg/kg/d) and their median age at renal biopsy was 10.5 years (5.9 years, 17.0 years). Hearing impairment was observed in 5 of the 25 AS patients, one in group 2 and four in group 3. Optical injury was not observed in any patient in both groups.
The levels of Scr (65.8±32.3 μmol/l) and blood urine nitrogen (BUN, 9.5±4.05 mmol/l) of patients in group 3 were higher than those (Scr 29.6±8.03 μmol/l and BUN 4.5±1.15 mmol/l) of patients in group 2 (p values were <0.001 both, Table 2). Both Scr and BUN were positively correlated with the levels of proteinuria (both of p values were 0.001). The mean glomerular filtration rate (GFR, which was calculated by the Schwartz formula) was 135.3±42.93 ml/min/ 1.73 m 2 in group 2 and 109.9±62.08 in group 3. The differences in GFR between two groups and the relationships between GFR and proteinuria were both of no significance (Table 2).
Eleven patients were included in the control group (2 male and 9 female: Wilm's tumor 5 cases, renal carcinoma 3 cases, duplex kidney 2 cases and pyelo carcinoma 1 case); the median age at nephrectomy was 6.7 years (0.2 years, 42 years). There was no significant age difference between control patients and AS patients. Two of the AS specimens with heavy proteinuria exhibited focal segmental glomerulosclerosis, the remaining specimens showed minimal change disease or mild-to-moderate mesangial proliferative glomerulonephritis.

Experimental Data
The expression and distribution of VEGFA and VEGFR2, the phosphorylation of VEGFR2 in glomeruli, and their association with proteinuria, Scr, BUN and GFR in AS patients. Immune electron microscopy revealed that VEGFA was detected in the three layers of the GFB (podocytes, GBM and endothelial cells); the accumulation of VEGFA within the GBM was increased in AS patients and was apparent in patients with heavy proteinuria (Fig 1a-1c). VEGFR2 expression was observed both in endothelial cells and in podocytes (Fig 1e-1g).
Immunofluorescence revealed that the VEGFA and VEGFR2 expression levels were low in control patients and were slightly increased in AS patients with mild proteinuria. The VEGFA and VEGFR2 expression levels were significantly upregulated in patients with heavy proteinuria and were observed in mass distribution (Table 3, Fig 2a-2h). The expression levels of VEGFA and VEGFR2 were positively correlated with the degree of proteinuria in AS patients, respectively ( Table 4). The VEGFA expression levels were positively correlated with the levels of Scr and BUN. However, the VEGFA and VEGFR2 expression levels were not correlated with GFR (Table 4).
The fluorescence of p-VEGFR2 was faint or undetectable in control patients and in some patients with mild proteinuria (5 cases); the fluorescence of p-VEGFR2 was slightly increased in patients with moderate proteinuria and became bright in patients with heavy proteinuria ( Table 3, Fig 2i-2l). The level of p-VEGFR2 was positively correlated with the degree of proteinuria in AS patients ( Table 4). The levels of p-VEGFR2 were positively correlated with the levels of Scr and BUN, but they showed no relationship with levels of GFR ( Table 4).
The expression of synaptopodin and nephrin and the phosphorylation of p-nephrin in glomeruli and their association with proteinuria, Scr, BUN and GFR in AS patients. Immunofluorescence revealed that the expression levels of synaptopodin and nephrin in the glomeruli were high and regular and appeared linear or reticular in control patients. The expression levels of synaptopodin and nephrin were slightly decreased in patients with mild and moderate proteinuria, and their expression levels were significantly down-regulated in patients with heavy preoteiuria (Table 3, Fig 3a-3h). The expression levels of synaptopodin and nephrin were negatively correlated with the degree of proteinuria and the levels of Scr and BUN in AS patients, but were not correlated with the levels of GFR (Table 4).
The fluorescence of p-nephrin was faint or undetectable in control patients and in some patients with mild proteinuria (3 cases). The fluorescence of p-nephrin was significantly increased in patients with heavy proteinuria ( Table 3, Fig 3i-3l). The level of p-nephrin was positively correlated with the degree of proteinuria and the levels of Scr and BUN in AS patients, but they were not correlated with GFR ( Table 4).
The association between VEGFA, VEGFR2, p-VEGFR2, synaptopodin, nephrin and pnephrin ( Table 5). The VEGFA and VEGFR2 expression levels and the level of p-VEGFR2 were negatively correlated with the expression levels of synaptopodin and nephrin, but they were positively correlated with the level of p-nephrin in glomeruli. The VEGFA expression level was positively correlated with the expression level of VEGFR2 and the level of p-VEGFR2.

Discussion
AS is a progressive inherited glomerulonephritis [1,2]. Hematuria is the initial presentation at early stage [13]. Proteinuria emerges with disease progression [3]. AS could be evidenced by the lack of COL IV α3 chain or α5 chain in GBM. In progressed AS patients, electron microscopy revealed distinct structural changes of the GBM; the GBM became thinner, thicker, layered or  splitting [14]. In the present study, all patients were diagnosed by their lacking of COL IV α5 and/or α3 chain in GBM, and/or typical GBM changes of AS under EM. Both the levels of Scr and BUN were correlated with the levels of proteinuria in the present study, which indicated that proteinuria was associated with the deterioration of renal function. Although the GFR levels were not correlated with proteinuria which may attribute to the small number of patients.  VEGFA is a key factor for the development and maturation of the GFB and for the maintenance of GFB function [15]. VEGFA in GFB mainly comes from podocytes [16,17]. Either over-expression or down-regulation of VEGFA would lead to damage of the GFB and the development of proteinruia. The up regulation of VEGFA in the podocytes of mice could cause proteinuria, fusion of the foot precess, effacement of the diaphragm and down-regulation of nephrin [18]. Increased expression of VEGFA in glomeruli was also detected in minimal change of disease, membranous nephropathy, mesangial proliferative glomerulonephritis, lupus nephritis, and diabetic nephropathy [19]. In rats with adriamycin nephropathy or Table 4. Correlation analyses between the indexes of renal impairments (degrees of proteinuria, Scr, BUN and GFR) and the expression levels of VEGFA, VEGFR2, synaptopodin and nephrin as well as the levels of p-nephrin and p-VEGFR2 in AS patients.  puromycin progressive nephritis, decreased expression of VEGFA in the glomeruli was observed and was associated with the development of proteinuia [5]. Clinically, the use of anti-VEGFA antibodies in patients with neoplastic diseases could cause proteinuria [19].
In the present study, the expression of VEGFA in glomeruli was up-regulated in AS patients, especially in patients with heavy proteinuria. The expression level of VEGFA was positively correlated with the degree of proteinuria and the levels of Scr and BUN in AS patients. Immune electron microscopy revealed that increased accumulation of VEGFA was observed within the three layers of the GFB (podocytes, GBM and glomerular endothelial cells) in AS patients. The accumulation of VEGFA in the GBM was prominent in patients with heavy proteinuria. These results indicated that VEGFA was over-expressed in glomeruli and was associated with the development of proteinuria in AS patients. However, the mechanism remains unclear.
AS is characterized by defect in COL IVα3 or α5in GBM [1,2,19]. At the early stage, AS patients manifest with microhematuria and the GBM appears as normal under electron microscopy; in advanced patients, proteinuria develops and the GBM shows distinct structural changes; such as attenuation, thicken, layering or splitting [14]. These changes may affect the function of the GBM as the support of the glomerular capillary as well as the signal crass-talk platform between the glomerular endothelial cells and podocytes [20]. Whether these changes would affect the expression of VEGFA and its distribution in GBM deserves more studies.
VEGFR2 is the main receptor of VEGFA [21], and it plays a role in regulating angiogenesis, mitogenic signaling and permeability-enhancing effects [6]. VEGFR2 is expressed both in the podocytes and endothelial cells of glomeruli, and VEGFA plays its role by autocrine and paracrine pathways after being secreted from podocytes [22,23]. However, controversy exists regarding whether VEGFR2 is expressed in podocytes in vivo [24]. In the present study, we evidenced by immune electron microscopy that VEGFR2 was expressed in podocytes both in control and AS patients. Immunofluorescence showed that the expression of VEGFR2 and the level of p-VEGFR2 were increased in AS patients, especially in patients with heavy proteinuria. These results indicated that the VEGFA-VEGFR2 signaling pathway was activated in AS patients. Moreover, we found that the expression of VEGFR2 in glomeruli was positively correlated with the expression of VEGFA in AS patients. It has been reported that increased expression of VEGFA could induce up-regulation of VEGFR2 in podocytes in vitro [24]. In AS patients, whether the up-regulation of VEGFR2 is induced by the up-regulation of VEGFA deserves further study.
VEGFR2 can bind to the intracellular domains of nephrin [7]. Nephrin is the skeleton of the foot process diaphragm of the podocytes as well as a key signal transductor [25]. After binding with VEGFA, VEGFR2 could be phosphorylated, alter the binding status with nephrin and recruit Nck to format VEGFR2-nephrin-Nck complex, which plays a role in regulating the reorganization and polymerization of actin and lead to changes in podocyte modality [7]. Podocyte injury has been proven to be associated with the development of proteinuria in AS. Ding et al [11] has reported that the width of the foot process was positively correlated with the degree of proteinuria in AS patients, and the expression levels of synaptopodin and nephrin were negatively correlated with the degree of proteinuria and the levels of Scr and BUN. Previously, we also found that the development of proteinuria was associated with podocyte injury; the degree of foot process fusion was more severe in patients with persistent proteinuria than in patients with mild proteinuria [26]. Synaptopodin is an important structural protein of podocytes and it plays a significant role in maintaining the stability of podocyte skeleton [27].
In the present study, we also found that the expression of synaptopodin and nephrin were down-regulated and negatively correlated with the degree of proteinuria, and the distribution of synaptopodin and nephrin was disturbed in AS patients with heavy proteinuria. Furthermore, we found that the level of p-nephrin was low in the control group and AS patients with mild proteinuria, but it was prominently increased in patients with heavy proteinuria and it was also positively correlated with the degree of proteinuria. These results indicated that the activation of the nephrin signaling pathway is associated with podocyte injury and the development of proteinuria in AS.
It has been reported that over-expression of VEGFA in podocytes could lead to phosphorylation of VEGFR2, down-regulation of nephrin and proteinuria in mice [4,28]. Phosphorylation of VEGFR2 (Y1214) could recruit Nck, activate the Fyn signaling pathway, and enhance the phosphorylation of nephrin [29]. In the present study, we also found that the expression of VEGFA and the level of p-VEGFR2 were positively correlated with the level of p-nephrin, but they were negatively correlated with the expression of synaptopodin and nephrin, indicating that up-regulated VEGFA expression in glomeruli may activate the VEGFA-VEGFR2 signaling pathway, which would then activate the nephrin signaling pathway and cause podocyte injury and the development of proteinuria in AS patients.

Conclusion
In conclusion, the expression of VEGFA in glomeruli and its accumulation within GBM were increased as were the levels of p-VEGFR2 and p-nephrin, all of which were positively correlated with the degree of proteinuria in AS patients. The expression levels of synaptopodin and nephrin were down-regulated and negatively correlated with degree of proteinuria. These results indicated that the defect of the GBM in AS may lead to over-expression of VEGFA in the glomeruli and its accumulation in GBM, which could occur through the activation of the VEGFA-VEGFR2 and nephrin signaling pathways, leading to podocyte injury and the development of proteinuria.