Blood and urine parameters in healthy free ranging lynx, healthy captive lynx and captive lynx with renal disease.

<p>Blood and urine parameters in healthy free ranging lynx, healthy captive lynx and captive lynx with renal disease.</p

Ultrasonographic and radiologic images of the kidneys.

<p>(A, B) healthy lynx, (C, D) lynx with renal disease. Diseased kidneys are smaller, have irregular contour, show loss of normal architecture and mineralization.</p

Von Kossa stained sections of the kidney, stomach, lung, heart and aorta from Iberian lynx.

<p>(A-E) Healthy controls that died in car accidents; (F-J) Animals that died as a consequence of vitamin D toxicosis and renal failure. Foci of calcification (arrows) are stained brown. Magnification = x200.</p

Dkk-1 inhibits the high phosphate-induced osteogenic-like characteristics in rat mesenchymal stem cells.

<p>A) Rat mesenchymal cells treated with high phosphate and Dkk-1 were stained for β-catenin immunofluorescence (green) and counterstained with DAPI (blue) to determine β-catenin subcellular localization. Merged images of β-catenin immunofluorescence and DAPI staining are shown. Dkk-1 administration reduced nuclear translocation of β-catenin. Original magnification: 40x. B) BMP-2 mRNA expression in rat mesenchymal stem cells treated with high phosphate and Dkk-1 was determined by RT-PCR (a p<0.001 vs high phosphate treated cells). C) Calcium content and alkaline phosphatase activity (Units/mg protein) in rat mesenchymal stem cells treated with high phosphate and Dkk-1 (a p<0.001 vs high phosphate alone). Image is representative of three experiments.</p

TGF-β administration prevents osteogenic effects induced by high phosphate.

<p>A) High phosphate (P) increased the expression of BMP-2 while TGF-β or the combination of TGF-β plus high phosphate decreased significantly the expression of this osteogenic marker (a p<0.001 vs. all groups). Results are expressed as fold change vs. Control cells. B) High phosphate (P) decreased significantly SM22α and myocardin expression with respect to Control cells (b p<0.01 for SM22α and a p<0.001 for myocardin) and TGF-β group (c p<0.001). The combination of TGF- β and high phosphate (TGF-β + P) decreased the expression of SM22α and Myocardin although less than high phosphate alone (c p <0.001vs. TGF-β group). C) TGF-β alone did not change significantly the alkaline phosphatase activity. This activity increased after high phosphate treatment (a p<0.001 vs. all others groups). The combination of TGF-β and high phosphate for 14 days significantly decreased this activity when compared with high phosphate group. D) Calcium content was significantly increased after high phosphate treatment (a p<0.001 vs other groups). The combination of TGF-β and high phosphate prevented this increase of calcium induced by high phosphate alone.</p

Wnt/β-catenin pathway activation enhances the high phosphate-induced osteogenic-like characteristics in rat mesenchymal stem cells.

<p>A) Rat mesenchymal cells treated with high phosphate and CHIR98014 (0.4 µM) or lithium chloride (5 mM) were stained for β-catenin immunofluorescence (green) and counterstained with DAPI (blue) to determine β-catenin subcellular localization. Merged images of β-catenin immunofluorescence and DAPI staining are shown. Both Wnt activators (CHIR98014 and lithium chloride) increased nuclear translocation of β-catenin. Original magnification: 40x. B) BMP-2 protein and C) mRNA expression in rat mesenchymal stem cells treated with high phosphate and CHIR98014 or lithium chloride was determined by western blot and RT-PCR respectively (a p < 0.001 vs high phosphate alone). D) Calcium content and E) alkaline phosphatase activity in rat mesenchymal stem cells treated with high phosphate and CHIR98014or lithium chloride (a p<0.001 vs. high phosphate alone). Image is representative of three experiments.</p

High phosphate activates Wnt/β-catenin pathway.

<p>A) Rat mesenchymal cells treated with TGF-β and/or high phosphate were stained for β-catenin immunofluorescence (green) and counterstained with DAPI (blue) to determine β-catenin subcellular localization. Merged images of β-catenin immunofluorescence and DAPI staining are shown. High phosphate induced nuclear translocation of β-catenin while the addition of TGF-β inhibited this translocation. Original magnification: 40x. Image is representative of three experiments. B) Quantification of β-catenin confocal immunofluorescence was performed with Image J software (a p<0.001 vs. all groups). C) With respect to control cells high phosphate decreased the expression of Dkk1 (b p<0.001) and Gsk3β (b p<0.001) while increased the expression of Lrp5 with respect to other groups (d p<0.001). These differences were also significant respect to TGF-β treated groups (c p<0.001 vs. TGF groups). TGF-β alone increased the expression of Dkk1 and Gsk3β (a p<0.001).</p

TGF-β induces vascular smooth muscle cells differentiation of mesenchymal stem cells through nuclear translocation of Smad3.

<p>A) Rat mesenchymal cells treated with TGF-β for 14 days were stained for phospho-Smad3 immunofluorescence (red) and counterstained with DAPI (blue) to determine phospho-Smad3 subcellular localization. In TGF-β treated cells, positive phospho-Smad3 immunofluorescence was localized into the nucleus. Original magnification: 40x. B) Vascular smooth muscle actin (VSM-actin, green) was stained and the nuclei were counter-stained with DAPI showing cytoskeleton organization in Control cells and TGF-β treated cells. Original magnification: 20x.C) After 7 and 14 days, TGF-β induced the expression of vascular smooth muscle cells markers such as VSM-actin, SM22α, Myocardin and Myosin heavy chain with respect to control cells (a p<0.001 vs. control cells). Images are representative of three experiments.</p

TGF-β addition inhibits nuclear translocation of Smad 1/5/8 induced by high Phosphate.

<p>A) Rat mesenchymal stem cells treated with high phosphate showed nuclear localization of phospho-Smad1/5/8 (Red) (a p<0.001 vs. all groups). Cells treated with TGF-β (alone or plus high phosphate) were negative for phospho-Smad1/5/8. Merged images of phospho-Smad1/5/8 immunofluorescence and DAPI staining are shown. Original magnification: 40x. Image is representative of three experiments. Colocalization Finder plugging from Image J software was carried out to analyse nuclear localization of Smad 1/5/8 showing a submask with white areas specific to nuclear colocalization with DAPI. Original magnification: 40x. B) Quantification of confocal immunofluorescence was performed with Image J software.</p

BMP2 inhibition prevents the osteogenic effects of high phosphate.

<p>A and B) Noggin administration (200 ng/ml) prevented the expression of osteogenic markers such as Osterix and Runx2 (a p<0.001 vs. high phosphate treated cells) and reduced calcium deposition. C) Alkaline phosphatase activity was not modified after Noggin administration. The figures are representative of at least three experiments.</p