Adenosine A2B Receptor-mediated VEGF Induction Promotes Diabetic Glomerulopathy

Angel Cárdenas; Camilo Toledo; Carlos Oyarzún; Angélica Sepúlveda; Claudia Quezada; Elena Guillén-Gómez; Montserrat M Díaz-Encarnación; Marçal Pastor-Anglada; Rody San Martín

Disclosures

Lab Invest. 2013;93(1):135-144. 

In This Article

Results

A2BAR Mediates VEGF Production in Rat Glomeruli

Using isolated glomeruli from rat kidney we determined the role of A2BAR on VEGF production ex vivo. The treatment of glomeruli with the general agonist of ARs induces an increase in the content of VEGF, which is in line with the previously determined increase in mRNA levels.[27] This increase was blocked by MRS1754, the selective antagonist of the A2B and it was not observed when glomeruli were infected with an adenoviral vector expressing a short RNA that interferes with the expression of this AR subtype (Figure 1a). No significant changes in the glomerular content of VEGF have been observed with pharmacological modulators of other ARs.[27] We determined that A2BAR-mediated VEGF production required p42/44 MAPK and PKC signaling molecules activation (Figure 1a). Particularly, we determined that p42/44 MAPK and PKCα isoform were phosphorylated upon A2BAR activation (Figures 1b and c).

Figure 1.

Adenosine A2B receptor signaling mediates vascular endothelial growth factor (VEGF) overexpression in rat glomeruli. (a) The content of VEGF was quantified in glomeruli exposed ex vivo to the general adenosine receptor (ARs) agonist NECA (non-selective P1 receptors agonist) alone or in combination with a selective antagonist of adenosine A2B receptor (A2BAR) (MRS1754), a general inhibitor of PKC (Calphostin C) or the MAPK activation inhibitor (PD98059) for 24 h. Also, the inhibition of the A2BAR expression by siRNA (siRNA A2BAR) was evaluated. When indicated glomeruli were exposed to 25 mM D-glucose (HG) as control of increased VEGF production. The upper images show a representative western blot detection of VEGF content in total protein extracts (50 μg) from treated glomeruli. The graph depicts the ratio between immune signals of VEGF vs β-actin. (b, c) The activation of PKCα and ERK1/2 was quantified by the detection of phosphorylated forms in total protein extracts of glomeruli (50 μg) by western blots. The graphs show the ratio between phosphorylated vs total content of the signaling proteins. (d) Representative western blot analysis of the effect of siRNA-mediated inhibition of A2BAR and scrambled siRNA on VEGF induction by NECA. All graphs represent the means±s.e.m. *P<0.01 vs control without treatment; # P<0.01 vs NECA; ac n=9, d n=4.

Released VEGF was also increased when the general AR agonist NECA was used. This effect was blocked by MRS1754 the selective antagonist of A2BAR (Figure 2a). In contrast, the selective agonist of A1AR decreased the basal release of VEGF from glomeruli. This effect could be blocked by using DPCPX, the selective antagonist of A1AR (Figure 2b).

Figure 2.

Role of A1AR and adenosine A2B receptor (A2BAR) on VEGF release. The release of VEGF from glomeruli was assessed by Enzyme-Linked Immunosorbent Assay. (a) Rat glomeruli were incubated by 6 h in the absence (control) or presence of the general AR agonist (non-selective P1 receptors agonist) alone or in combination with a selective A2BAR antagonist (MRS1754). (b) The role of A1AR on VEGF release was assayed using the selective agonist CPA and the antagonist DPCPX. The graphs show the means±s.e.m. of VEGF content values in the supernatants. *P<0.01 vs control without treatment; # P<0.01 vs NECA or CPA; n=9.

Experimental Diabetes Increases the Expression of A2BAR and VEGF in Kidney Glomeruli

The distribution of A2BAR was assessed in rats' kidney by immunohistochemistry. The expression pattern of A2BAR resembles that of VEGF in the glomeruli of healthy rats (Figure 3). As early as 3 weeks following diabetes induction, we detected a stronger immunosignal and a wide distribution of A2BAR in glomerular cells. Similarly, VEGF content was notably increased (Figure 3). In contrast, the expression of the adenosine A1 receptor subtype appears to decrease in diabetic rats glomeruli (Figure 3).

Figure 3.

Glomerular changes in the expression of adenosine receptors (ARs) induced by experimental diabetes. Following 3 weeks of diabetes induction the expression of ARs in glomeruli were detected by immunohistochemistry. Similarly, the increased glomerular expression of vascular endothelial growth factor (VEGF) at this stage was evidenced in kidney sections. Original magnification × 400. Scale bars 50 μm. The analyses of 50 glomeruli per slide in each condition were performed using the UN-SCANIT 2.0 software. The graphs depict the quantification of intensity of immune signals in glomeruli of rats, normalizing the value of vehicle-treated rats to 1. *P<0.01 vs vehicle, n=6.

Glomerular Adenosine Availability

Owing to the fact that the A2BAR exhibits a low affinity for the ligand,[23] changes in the extracellular adenosine availability should occur to mediate the physiologic effects. We quantified the extracellular concentration of adenine nucleotides and adenosine in glomeruli isolated from healthy and STZ-induced diabetic rats. Following 3 weeks of diabetes induction the contents of ATP, ADP and AMP were not different to the levels seen in normal rats glomeruli (Figure 4a). However, a notably increase in the extracellular levels of adenosine were quantified in glomeruli of STZ-induced diabetic rats (Figure 4a).

Figure 4.

Adenosine contents in streptozotocin-induced diabetic rat. (a) Adenine nucleotides and adenosine levels were quantified by HPLC in glomeruli of control (□) and diabetic rats (▪) following 3 weeks of diabetes induction. The graph shows the means±s.e.m. from individual determinations normalized to 1 μg of total glomerular proteins. *P<0.01, n=7. (b) The adenosine contents in urine of rats were quantified following 4 weeks of diabetes induction. The graph shows individual determinations and horizontal line represents the mean value for control or diabetic group. P=0.038, n=7.

Furthermore, there was a correlation between the altered glomerular adenosine handling and the urinary excretion of the nucleoside. The mean adenosine urinary excretion rate was increased more than twofold in diabetic rats (Figure 4b).

Blockage of Diabetes-induced VEGF Overproduction in MRS1754-Treated Rats

The exposure of ex vivo glomeruli to high glucose concentration induced an overproduction of VEGF (Figure 5a). This result was specific to high glucose levels as using mannitol as osmotic control did not alter the content of VEGF (Figure 5b). We observed that this high glucose effect was blocked when using the A2BAR antagonist, by knocking down the receptor expression or using the PKC and ERK activation inhibitors (Figure 5a).

Figure 5.

Role of adenosine in high glucose-induced vascular endothelial growth factor (VEGF) overproduction. (a) The content of VEGF was quantified in glomeruli exposed ex vivo to D-glucose 25 mM in combination with a selective antagonist of adenosine A2B receptor (A2BAR) (MRS1754), a siRNA interfering with A2BAR expression, a general inhibitor of PKC (Calphostin C) or the MAPK activation inhibitor (PD98059) for 24 h. The upper image shows a representative western blot detection of VEGF content in total protein extracts (50 μg) from treated glomeruli. The graph depicts the ratio between immune signals of VEGF vs β-actin. Control experiments contained D-glucose 5 mM. The graph represents the means±s.e.m. *P<0.01 vs D-glucose 5 mM; # P<0.01 vs D-glucose 25 mM; n=5. (b) The content of VEGF was evaluated in isolated glomeruli exposed to glucose 5 mM, glucose 25 mM and glucose 5 mM supplemented with mannitol 20 mM as osmotic control for 24 h. The figure is a representative western blot analysis showing that increased expression of VEGF was specific to high glucose concentration. The graph shows the means±s.e.m. *P<0.01 vs D-glucose 5 mM; n=4.

In vivo analysis of the A2BAR role on VEGF production were carried out in STZ-induced diabetic rats treated with the selective antagonist MRS1754. Following 4 weeks of diabetes induction, the administration of MRS1754 for 14 days blocked the glomerular increase in the expression of VEGF compared with vehicle-treated diabetic group (Figures 6a and b). The expression of the slit diaphragm protein nephrin was partially recovered following the treatment with the antagonist (Figures 6a and c, and Supplementary Figure 1), and the increase in the urinary excretion of proteins induced by diabetes was blocked by the treatment using MRS1754 (Figure 6e).

Figure 6.

In vivo evaluation of an adenosine A2B receptor (A2BAR) antagonist in experimental diabetic rats. Following 4 weeks of diabetes induction the rats were treated by 14 days with the selective A2BAR antagonist MRS1754 in doses of 0.2 and 1.0 mg/kg of body weight (see Materials and Methods). (a) Glomerulopathy was detected by vascular endothelial growth factor (VEGF), nephrin, α-smooth muscle actin (α-SMA) and Wilms tumor protein (WT-1) immunohistochemistry. Also Periodic acids-Schiff staining (PAS) was included. Original magnification × 400. Scale bars 50 μm. (b, c) The graphs depict the quantification of immune signals intensity in rats glomeruli, normalizing the value of normal rats to 1. # P<0.01 vs diabetes, n=12. (d) The graph shows the percentage of α-SMA-stained glomeruli from total glomeruli on each slide. # P<0.01 vs diabetes, n=12. (e) Total urinary protein contents were quantified in STZ-induced diabetic rats treated with MRS1754. *P<0.01, n=7. (f) Podocyte number was estimated from immunohistochemical staining of nuclear WT-1. The graph depicts the means of positive nuclei in 30 glomeruli of each group. *P<0.01, n=12. (g) The grade of mesangial expansion from normal to severe (1–3) was estimated in PAS-stained glomeruli of each group. The graph shows the means of 30 glomeruli graded in each group.

Furthermore, the glomerular expression of αSMA, a mesangial cells myofibroblast transdifferentiation marker, was also blocked in the kidney of diabetic rats under the treatment with the A2BAR antagonist (Figures 6a and d). The blockade of VEGF induction and mesangial cells activation was in an early stage of glomerular dysfunction as only a decrease in the podocytes number was quantified while mesangial expansion has not yet occurred. A significant difference was detected in the patterns of glomerular WT-1 in all diabetic groups compared with healthy rats (Figures 6a and f). The treatment with MRS1754 did not modify the decreased glomerular WT-1 imnunosignals in diabetic rats. PAS staining was not changed by diabetes or treatment with the antagonist of A2BAR in our experimental conditions (Figures 6a and g).

The treatment with the A2BAR antagonist did not alter the increased levels of plasma glucose in the STZ-induced diabetic rats. However, partial recovery of body weight was observed in rats following treatment with the antagonist (Table 1).

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