Exogenous BMP7 in aortae of rats with chronic uremia ameliorates expression of profibrotic genes, but does not reverse established vascular calcification

<div><p>Hyperphosphatemia and vascular calcification are frequent complications of chronic renal failure and bone morphogenetic protein 7 (BMP7) has been shown to protect against development of vascular calcification in uremia. The present investigation examined the potential reversibility of established uremic vascular calcification by treatment of uremic rats with BMP7. A control model of isogenic transplantation of a calcified aorta from uremic rats into healthy littermates examined whether normalization of the uremic environment reversed vascular calcification. Uremia and vascular calcification were induced in rats by 5/6 nephrectomy, high phosphate diet and alfacalcidol treatment. After 14 weeks severe vascular calcification was present and rats were allocated to BMP7, vehicle or aorta transplantation. BMP7 treatment caused a significant decrease of plasma phosphate to 1.56 ± 0.17 mmol/L vs 2.06 ± 0.34 mmol/L in the vehicle group even in the setting of uremia and high phosphate diet. Uremia and alfacalcidol resulted in an increase in aortic expression of genes related to fibrosis, osteogenic transformation and extracellular matrix calcification, and the BMP7 treatment resulted in a decrease in the expression of profibrotic genes. The total Ca-content of the aorta was however unchanged both in the abdominal aorta: 1.9 ± 0.6 μg/mg tissue in the vehicle group vs 2.2 ± 0.6 μg/mg tissue in the BMP7 group and in the thoracic aorta: 71 ± 27 μg/mg tissue in the vehicle group vs 54 ± 18 μg/mg tissue in the BMP7 group. Likewise, normalization of the uremic environment by aorta transplantation had no effect on the Ca-content of the calcified aorta: 16.3 ± 0.6 μg/mg tissue pre-transplantation vs 15.9 ± 2.3 μg/mg tissue post-transplantation. Aortic expression of genes directly linked to extracellular matrix calcification was not affected by BMP7 treatment, which hypothetically might explain persistent high Ca-content in established vascular calcification. The present results highlight the importance of preventing the development of vascular calcification in chronic kidney disease. Once established, vascular calcification persists even in the setting when hyperphosphatemia or the uremic milieu is abolished.</p></div

Effect of BMP7 on bone morphology and bone mineral density.

<p>Effect of BMP7 on bone morphology and bone mineral density.</p

BMP7 had a plasma phosphate-lowering effect in uremic rats.

<p>The duration of uremia was 22 weeks. To induce severe vascular calcification the rats were treated with alfacalcidol from week 8 to 14. Then the CRF rats were treated with BMP7 (250 μg/kg/week) or vehicle for 8 weeks. (<b>A</b>) CRF rats had increased plasma phosphate (P) levels at 8 weeks, and alfacalcidol treatment resulted in a further increase at 14 weeks. At 22 weeks plasma P decreased in the vehicle treated rats to the level observed before alfacalcidol treatment. In the CRF/BMP7 group a significant further decrease in plasma P was observed, resulting in near normal plasma P, despite uremia and high P diet. (<b>B</b>) At 14 weeks plasma PTH was completely suppressed in CRF animals and increased to very high levels at 22 weeks similar in BMP7- and vehicle-treated animals. (<b>C-D</b>) Plasma total calcium (Tca) and Ca²⁺ were increased with a slight difference in plasma Ca²⁺ between the two uremic groups at 14 weeks. At 22 weeks plasma TCa and Ca²⁺ were normalized. Plasma P, TCa and Ca²⁺ is presented as mean ± SD and PTH as median and IQR, n = 6–10. *P<0.05 vs 8 weeks and ** P<0.05 vs 14 weeks by two-tailed paired t-test. ^#P<0.05 vs control and ^#P<0.05 vs vehicle by two-tailed unpaired t-test. ¹P<0.005 vs control by Mann Whitney U-test. ²P<0.05 vs 14 weeks by Wilcoxon matched-pairs signed rank test.</p

The effect of BMP7 treatment and normalisation of the uremic milieu on aortic Ca-content.

<p>Uremic alfalcalcidol-treated rats had significant calcification of both the distal abdominal, proximal abdominal and proximal thoracic aorta. (<b>A-B</b>) The Ca-content of the proximal thoracic aorta was 20–30 fold higher compared to the distal abdominal aorta. No differences were seen between BMP7- and vehicle-treated rats in the Ca-content in the established vascular calcification of either the abdominal or thoracic aorta. (<b>C</b>) Complete normalization of the uremic environment by aorta transplantation (ATx) did not change the Ca-content of the calcified aorta from CRF rats (CRF/ATx) and the surgery did not induce calcifications of the normal aorta from healthy animals (Ctrl/ATx). Mean ± SD, n = 6–15; *P<0.0001 vs control by two-tailed unpaired t-test.</p

BMP7 treatment and kidney gene expression.

<p>(<b>A</b>) Uremia significantly increased the expression of genes related to fibrosis and EMT: Postn, Fn1, Tgfb1, Vim and Inhba. Postn (periostin) and Inhba (Activin A) were not expressed (NE) in kidneys from control rats, but significantly induced in uremic rats. No differences between CRF/BMP7 and CRF/Veh were seen in the expression levels of the examined genes. (<b>B</b>) No differences were seen between controls and CRF/Veh and CRF/BMP7 in the expression of the phosphate transporters Napi2a, Napi2c and Pit2 or in the FGF23 co-receptor, Klotho. In the CRF/Veh group there was a small increase in the expression of Fgfr1 and this increase was not seen in the CRF/BMP7 group. Mean ± SD, n = 6–10. *P<0.01; **P = 0.0007 vs control by two-tailed t-test.</p

Effect of BMP7 on bone morphology visualized by microCT imaging.

<p>Representative pictures of distal femur from control rats, uremic vehicle treated rats (CRF/Veh) and uremic BMP7 treated rats (CRF/BMP7). (<b>A</b>) Cortical cross-sectional area midshaft. In comparison to control rats both uremic groups had thicker cortical bone with porosity, indicated by arrow. (<b>B</b>) Trabecular bone in region of interest (ROI). Uremic rats had increased trabecular thickness, greater trabecular spacing and less trabecular number. (<b>C</b>) Sagittal image of distal femur. No effect of BMP7 treatment was seen on bone microstructure.</p

BMP7 ameliorated disturbed gene expression in the calcified aorta.

<p>The two figures depict expression levels of genes related to the calcification process. (<b>A</b>) In uremic rats the expression of genes involved in fibrosis and epithelial-to-mesenchymal transition (EMT) was strongly increased. The BMP7-treated group had significantly lower expression levels of these genes compared to vehicle. (<b>B</b>) There was a strong induction of genes related to extracellular matrix calcification and osteogenic transformation in the uremic rat aorta, but no significant differences were seen in the expression levels of these genes between the BMP7- and vehicle-treated uremic groups. Mean ± SD, n = 6–10. *P<0.05 vs control; **P<0.001; ***P<0.0001 vs control; ^#P<0.05 vas vehicle; ^##P = 0.006 vs vehicle by two-tailed unpaired t-test.</p