Urine release of systemically administered bone morphogenetic protein hybrid molecule.
BACKGROUND
Although it is well known that TGF-beta circulates, the presence and activity of endogenous bone morphogenetic proteins (BMPs) in biological fluids has not been studied. Here we investigated the urine secretion of a systemically administered BMP hybrid molecule.


METHODS
A dimeric recombinant human BMP molecule consisting of the BMP-7 prodomain and the BMP-6 mature domain was constructed and injected into Sprague Dawley rats. The blood was collected from the rats' orbital plexus, and 24-hour urine samples were pooled and purified using a heparin sepharose column. Protein identity was confirmed by Western blot and by liquid chromatography-mass spectrometry (LC-MS) of the resulting peptides. Urine-derived protein from the 35-kDa band was bound to inactive demineralized bone matrix and implanted subcutaneously into rats.


RESULTS
Western blot analysis of sera demonstrated that BMP-7/6 remained intact in the rat plasma and could still be visualized 30 minutes after its systemic administration. Two protein bands at 35 and 39 kDa were detected with anti-BMP antibodies in the urine of rats, corresponding to the mature active BMP-6 dimer and the prodomain of BMP-7, respectively. LC-MS analysis detected only peptides derived from the BMP-7/6 molecule. Histological analysis of implanted pellets revealed formation of a new endochondral bone 14 days following implantation.


CONCLUSIONS
These results show for the first time that systemically administered BMP-7/6 hybrid molecule is secreted into the urine and that its biological activity is preserved, suggesting that analysis of BMP in urine might reflect its presence in serum.
Grgurevic
Lovorka
L
Laboratory of Mineralized Tissues, School of Medicine, University of Zagreb, Zagreb - Croatia.
Macek
Boris
B
Erjavec
Igor
I
Mann
Matthias
M
Vukicevic
Slobodan
S
D000818:Animals; D055418:Bone Morphogenetic Protein 6; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins / Q000008:administration & dosage* / Q000652:urine*; D006801:Humans; D051381:Rats; D017207:Rats, Sprague-Dawley
C525816:BMP6 protein, human; C525819:BMP7 protein, human; C525818:Bmp6 protein, rat; C525821:Bmp7 protein, rat; D055418:Bone Morphogenetic Protein 6; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins