Background

Various growth factors and cytokines have been implicated in different forms of kidney enlargement such as renal growth following induction of diabetes, unilateral nephrectomy, and exposure to high protein diet. Vascular endothelial growth factor (VEGF) is essential for normal renal development and plays a role in diabetes-associated renal and glomerular enlargement.

Methods

To elucidate a possible role for VEGF in high protein-induced renal/glomerular enlargement, we examined the effect of a neutralizing VEGF-antibody (VEGF-ab) on kidney weight and glomerular volume in mice fed a high protein diet for up to seven days.

Results

At day 2 and day 7 of the experimental period, high protein diet induced a significant increase in the mean glomerular volume. This high protein-induced glomerular hypertrophy was completely prevented by treatment with VEGF-ab. Kidney weight was increased significantly only at day 7, and was not influenced by VEGF-ab treatment. High protein diet and/or VEGF-ab treatment had no effect on body weight, food intake, and liver or heart weight.

Conclusions

The administration of a neutralizing VEGF-ab in mice fed a high protein diet for one week completely abolished the glomerular hypertrophy seen in placebo-treated animals on the same diet, without affecting kidney and body weight. These results demonstrate, to our knowledge for the first time, that high protein-induced glomerular hypertrophy is VEGF-dependent.

METHODS

Animals

Adult female NMRI mice (Bomholtgaard, Ry, Denmark) with initial body weights of 28.3 0.2 g were used in the study. The animals were housed 6 to 8 per cage, had free access to water and chow, and were kept at constant temperature (21 1°C), humidity (55 5%) and at a 12-hour light, 12-hour dark cycle (07.00–19.00 light). The study complied with Danish regulations for care and use of laboratory animals.

Study design

The mice were randomly allocated into four groups of 14 to 16 animals. Mice in groups 1 and 2 were fed a custom made synthetic diet containing 20% protein (Altromin, Lage, Germany) (control (C) groups), while mice in groups 3 and 4 were fed an isocaloric, custom made synthetic diet containing 45% protein (Altromin) (high protein (P) groups). Group 1 (control placebo; CP) and group 3 (high protein placebo; PP) were injected with isotype-matched, irrelevant IgG, while mice of groups 2 (control VEGF-ab; CV) and 4 (high protein VEGF-ab; PV) were injected with VEGF-ab. To evaluate the effect of high protein at two time points, one half of the animals in each group was sacrificed at day 2, and the remainder at day 7. Animals sacrificed on day 2 were injected at day 0 with a bolus of 300 g of either the IgG or VEGF-ab. Animals sacrificed on day 7 received the same bolus of either IgG or VEGF-ab at day 0, followed by a dose of 100 g at days 2, 4 and 6. A full characterization of the antibody used has been described elsewhere^6,8. In brief, Balb/c mice were immunized by three repeated booster-injections of recombinant human (rh)-VEGF₁₆₅. The mice with the highest serum titer to rh-VEGF₁₆₅ received an additional injection of rh-VEGF₁₆₅ and later spleen cells were harvested for production of hybridomas to rh-VEGF₁₆₅. Purified IgG was prepared by Protein A chromatography from ascites fluid collected from Balb/c mice that received injections of the cloned hybridomas. The isotype and light chain composition of the VEGF-ab and the characterization of the neutralizing activity were performed as described previously⁸.

The VEGF-ab was dissolved in 0.154 mol/L NaCl and injected intraperitoneally using an injection volume of 0.5 mL. The animals were weighed at day 0, 2 and 7 and their food consumption was determined every day. Before sacrificed, the animals were anesthetized with pentobarbital (50 mg/kg IP) and non-fasting blood samples were collected from the retrobulbar plexus using heparinized capillary tubes. The serum samples were kept at -80°C for later analysis. Liver, heart, left kidney and the poles of the right kidney were removed, weighed and snap frozen in liquid nitrogen. The middle of the right kidney was fixed in 4% paraformaldehyde for later histological preparation. In the PP group sacrificed at day 7, two mice were excluded from the study due to unexplained massive body weight loss in one animal and developmental renal abnormalities in the other.

Serum IGF-I and kidney IGF-I determinations

Serum IGF-I was measured after extraction using acid-ethanol as previously described^9,10. The intra-assay and interassay CV were 5% and 10%, respectively. Tissue extraction of renal IGF-I was performed according to D'Ercole, Stiles and Underwood¹¹ and corrected for the contribution of entrapped serum IGF-I¹².

Measurement of glomerular volume

The middle part of the right kidney was embedded in paraffin for light microscopy examination. Two micron-thick sections were cut on a rotation microtome and stained with periodic acid-Schiff (PAS) and hematoxylin. In each animal, the mean glomerular tuft volume (V_G) was determined from the mean glomerular cross-sectional area (A_G) by light microscopy as previously described^12,13. The areas were estimated with a 2D-version of the nucleator (CAST, Olympus, Denmark)¹⁴ by light microscopy from an average area of 40 to 50 glomerular profiles (that is, the capillary tuft omitting the proximal tubular tissue and the Bowmann capsule). V_G was calculated as:

where = 1.38 is the shape coefficient for spheres (the idealized shape of glomeruli) and k = 1.1 is a size distribution coefficient^15,16.

Statistical analysis

Data were examined for normal distribution and analyzed using the Student unpaired t test if appropriate. For data not following a normal distribution the Mann-Whitney rank sum test was used. All data are expressed as mean SEM, with N indicating the number of mice studied. Statistics were performed by the statistical package SPSS for Windows. P < 0.05 was considered statistically significant in a two-tailed test.

RESULTS

Body weight and food consumption

Body weight and food intake are given in Table 1. At day 2 and day 7, the mean body weights of the different groups were not significantly changed by high protein diet and/or VEGF-ab treatment Table 1. Food consumption over 24 hours was measured during the study period on a group basis. At day 0 mean food intake per mouse was 3.4 0.2 g/24 h with no significant differences between the groups. At day 2 and day 7, food consumption, presented as the mean value of day 0 to 2 and day 3 to 7, respectively, did not differ significantly in any of the groups.

Table 1 - Mean body weight and food consumption at day 2 and day 7 in control and high protein fed mice treated with placebo or VEGF-ab.

Full table

Kidney weight and glomerular volume

At the end of the experimental periods, right kidney weight was determined. The mean right kidney weights are given in Figure 1. Control groups had a similar mean kidney weight at day 2 (CP 154 10 mg, CV 147 7 mg) and day 7 (CP 146 5 mg, CV 149 2 mg). At day 2, kidney weight was not significantly increased in the high protein groups (PP 169 5 mg, PV 164 2 mg) when compared to controls Figure 1. However, at day 7, there was a 22% increase in kidney weight in the PP group (179 6 mg vs. 146 5 mg, P = 0.001) when compared to CP and a 16% increase in the PV group (173 6 mg vs. 149 2 mg, P = 0.005) when compared to CV. VEGF-ab treatment had no influence on kidney weight in the control mice nor in the high protein fed mice.

Figure 1.

Mean right kidney weights at day 2 and day 7 in control mice treated with placebo (CP) or vascular endothelial growth factor-antibody (VEGF-ab; group CV) and high protein fed mice treated with placebo (PP) or VEGF-ab (PV). Values are means SEM, N = 6 to 8 in each group. *P < 0.01 vs. control groups (CP and CV).

Full figure and legend (14K)

Mean glomerular volume is given in Figure 2. At day 2 and day 7, mean glomerular volume in the PP group was significantly increased with 19% and 12%, respectively, when compared to the CP groups (2.10 0.06 10⁵m³ and 2.08 0.05 10⁵m³; P < 0.05). VEGF-ab treatment prevented the high protein-induced increase in mean glomerular volume both at day 2 and day 7.

Figure 2.

Mean glomerular volume in the right kidney at day 2 and day 7 in control mice treated with placebo (CP) or VEGF-ab (CV) and high protein fed mice treated with placebo (PP) or VEGF-ab (PV). Values are means SEM, N = 6 to 8 in each group. *P < 0.05 vs. CP day 2 and day 7.

Full figure and legend (15K)

Mean glomerular volume at day 2 (CP 1.77 0.07 10⁵m³ and CV 1.80 0.04 10⁵m³) and day 7 (CP 1.87 0.06 10⁵m³ and CV 1.88 0.04 10⁵m³) was not statistically different between control groups.

Liver and heart weight

High protein diet and/or VEGF-ab treatment for 2 or 7 days had no significant influence on the weight of liver and heart Table 2.

Table 2 - Mean liver and heart weight at day 2 and day 7 in control and high protein fed mice treated with placebo or VEGF-ab.

Full table

Kidney and serum IGF-I

Kidney and serum IGF-I levels are given in Table 3. After two days of high protein diet, kidney IGF-I was almost doubled (increase of 97%) both in PP and PV when compared to the control groups CP and CV, respectively. Serum IGF-I was increased to a lesser extent with 18% (PP vs. CP) and 28% (PV vs. CV). After 7 days of high protein diet, kidney IGF-I was still elevated in the high protein fed groups (45% PP vs. CP and 55% PV vs. CV) compared to control groups, whereas serum IGF-I was not different among the four groups. VEGF-ab treatment had no influence on kidney or serum IGF-I levels when compared with the respective control groups.

Table 3 - Mean kidney and serum IGF-I levels at day 2 and day 7 in control and high protein fed mice treated with placebo or VEGF-ab.

Full table

DISCUSSION

The major new finding of the present study is the specific inhibitory effect of VEGF-ab treatment on high protein-induced glomerular hypertrophy in mice. Treatment with VEGF-ab completely normalized the glomerular enlargement induced by high protein diet without affecting body weight, food consumption, liver, heart, or kidney weight. This study indicates that VEGF plays a major role in high protein-induced glomerular growth.

Increased protein intake is known to cause renal hypertrophy and an increase in kidney function^17,18,19,20. Kidney size increases linearly with protein intake rate both in male and female mice¹⁸. A similar, but less pronounced effect of dietary protein level on glomerular filtration rate (GFR) has been shown, however, this effect was significant only in male mice¹⁸. In rats, an increase in GFR in response to high protein diet has been shown to occur within 24 hours with no further change after 48 hours²¹. Consistently, an increase in nephron filtration rate has been found after two days of high protein intake²².

Several mechanisms have been suggested to be players in the renal effects of high protein diets. Nitric oxide has been demonstrated to mediate the renal hemodynamic effects of a high dietary protein intake (hyperfiltration and renal vasodilation) in rats²³. Prostaglandin production was enhanced by dietary protein intake in rats, both in glomeruli²⁴ and tubuli²⁵, and prostaglandins are known to regulate GFR and renal plasma flow. Sodium load has been suggested to be a major factor triggering tubular growth after high protein intake, while an increase in nephron filtration rate after high protein intake is thought to induce glomerular growth²². Ornithine decarboxylase (ODC) is the rate-controlling enzyme for the biosynthesis of growth regulatory polyamines²⁶. A significant increase in renal ODC activity is observed in rats fed a high protein diet and may be linked to the increase in GFR²¹. Finally, a role for IGF-I in the induction of protein-induced renal growth is strongly supported by a study in rats that found a positive correlation between renal IGF-I gene expression and dietary protein content³.

Our results support a role for VEGF in high protein-induced glomerular growth. The VEGF system consists of a group of five different isoforms and two VEGF receptors (VEGFR-1 and VEGFR-2)²⁷. VEGF and VEGF receptors (VEGFRs) are essential for normal embryonic development and for normal postnatal growth and development⁴, particularly of glomeruli⁵, as shown in knockout animals^28,29 and by postnatal VEGF inactivation, respectively. Both VEGF and VEGFRs are expressed in the glomeruli and tubules of normal rat kidney³⁰. In the glomerulus, VEGF mRNA and protein are found in the visceral epithelial cells (podocytes), whereas VEGFR-2 mRNA and protein are detected in the glomerular endothelial cells³⁰. Increased renal expression of VEGF and VEGFRs has been demonstrated in experimental diabetes³⁰, and administration of a neutralizing VEGF-ab in a rat model of type 1 diabetes reduced the diabetes-associated increase in GFR and urinary albumin excretion⁶. These findings suggest a role for VEGF in the pathogenesis of diabetic renal changes^2,6,30. Interestingly, VEGF-ab administration in diabetic rats abolished glomerular hypertrophy without affecting the diabetes-associated renal enlargement⁶. This observation is comparable to the effect of VEGF-ab administration obtained in high protein fed mice in the present study. Overall, these results support the contention that VEGF, besides being essential for normal glomerulogenesis, is an important growth factor for glomerular changes in pathophysiological conditions.

A relationship has been suggested to exist between IGF-I and VEGF. In vitro, IGF-I stimulated VEGF mRNA and protein expression in human SaOS-2 osteoblast-like cells and murine osteoblasts³¹. In a mouse model of proliferative retinopathy with well-characterized VEGF-dependence, administration of JB3, an IGF-I receptor antagonist, suppressed retinal neovascularization in vivo⁷. JB3 did not reduce retinal protein/mRNA levels of VEGF and VEGFRs nor VEGFR-2 phosphorylation, but reduced the VEGF activation of the p44/42 mitogen-activated protein kinase (MAPK) pathway; MAPK being essential for VEGF-stimulated retinal endothelial cell growth⁷. As mentioned above, IGF-I has been implicated in high protein-induced renal growth³. In our study renal IGF-I protein levels were up-regulated at day 2 and day 7 in high protein fed mice. VEGF-ab administration affected neither serum nor kidney IGF-I levels. Accordingly, our results are in concert with the hypothesis of an interrelation between VEGF and IGF-I with VEGF being a downstream mediator of IGF-I. However, the assumption that IGF-I induces glomerular hypertrophy through VEGF remains to be proven.

In conclusion, administration of a neutralizing VEGF-ab in mice fed a high protein diet for one week completely prevented the glomerular hypertrophy seen in placebo-treated animals on the same diet, without affecting kidney or body weight. The present study, to our knowledge for the first time, supports a specific role for VEGF in high protein-induced glomerular growth.

References

References

Acknowledgments

The work was supported by the Danish Medical Research Council (Grant # 9700592), the Eva and Henry Frænkels Memorial Foundation, the Danish Kidney Foundation, the Ruth König Petersen Foundation, the Danish Diabetes Association, the Novo Foundation, the Nordic Insulin Foundation, the Johanne and Aage Louis Petersen Memorial Foundation, the Institute of Experimental Clinical Research, University of Aarhus, Denmark and the Aarhus University-Novo Nordisk Centre for Research in Growth and Regeneration (Danish Medical Research Council Grant # 9600822). B.F. Schrijvers is supported by a grant from the Institute for the Promotion of Innovation by Science and Technology in Flanders.