The influence of ghrelin on the thyroid-stimulating hormone (TSH)-induced increase in thyroglobulin (Tg) and cAMP production.

<p>The influence of ghrelin on the TSH-induced increase in Tg and cAMP production at three different concentrations of TSH (0.1 IU/L, 0.5 IU/L and 1 IU/L). The basal levels, i.e. the values in the absence of TSH, were subtracted, before the groups were compared. Grey = vehicle, pattern = ghrelin (100 nM). Means (+SEM). *P < 0.05 compared to the control (vehicle). <b>A)</b> Ghrelin inhibited the TSH-induced increase in Tg production measured by enzyme-linked immunosorbent assay (ELISA) in primary cultures of human thyroid cells for the TSH concentration of 0.1 IU/L. n = 8 (0.1 IU/L) and n = 6 (0.5 and 1 IU/L) in triplets. Two patient samples were excluded due to lack of basal TSH-induced Tg production. <b>B)</b> No influence of ghrelin on the TSH-induced increase in cAMP production at three different concentrations of TSH (0.1 IU/L, 0.5 IU/L and 1 IU/L) measured by a competitive protein binding method in primary cultures of human thyroid cells. n = 8 (0.1 IU/L and 1 IU/L) and n = 6 (0.5 IU/L) in triplets.</p

Ghrelin receptor (GhrR) mRNA expression level.

<p>GhrR mRNA expression level in relation to the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression level in human brain, thyroid tissue and cell cultures measured by real-time quantitative polymerase chain reaction (RT-qPCR). n = 2.</p

Primer sequences for RT-qPCR.

<p>Primer sequences for RT-qPCR.</p

The influence of ghrelin on the thyroid-stimulating hormone (TSH)-induced (0.1 IU/L) mRNA expression of four thyroid components.

<p>The expression of the TSH receptor (TSH-R), thyroperoxidase (TPO), thyroglobulin (Tg) and sodium iodide symporter (NIS) measured by real-time quantitative polymerase chain reaction (RT-qPCR) in a primary culture of human thyroid cells in presence and absence of ghrelin. Indicated as fold change of mRNA expression compared to basal level (dashed line). IL-6 was used as a negative control. Grey = vehicle, pattern = ghrelin (100 nM). Means (±SEM), n = 6. *P < 0.05 compared to the control (vehicle). Two patients were excluded due to unknown sample material.</p

Effect of BMP7 on bone morphology and bone mineral density.

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

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

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