M CAI1,2, E SMITH1,2,N TOUSSAINT1,2, T HEWITSON1,2, S HOLT1,2
1Department of Nephrology, Royal Melbourne Hospital, Parkville, Australia, 2Department of Medicine (RMH), University of Melbourne, Parkville, Australia
Aim: To determine the effects of phosphate and CPP2 on markers of osteoblast differentiation and viability, and to assess the effect of osteoblast differentiation on CPP2 metabolism.
Background: Adynamic bone disease (ABD) occurs in chronic kidney disease (CKD) and is often associated vascular calcification (VC). Phosphate feeding causes ABD in CKD animal models, but mechanisms of action are unclear. Secondary calciprotein particles (CPP2) are calcium phosphate colloidal nanoparticles which circulate in extracellular fluid. Given phosphate promotes CPP2 formation, high phosphate may potentially cause ABD via CPP2. Conversely, ABD may also cause VC via CPP2.
Methods: MC3T3-E1 cells were treated with control (ConM) media, high phosphate media (PiM), CPP2 containing media (CPP2M) or high phosphate and CPP2 containing media (Pi+CPP2M). Osteocalcin expression, alkaline phosphatase (ALP) expression and activity were analysed as markers of osteoblastic differentiation. Cell viability and apoptosis was assessed. To determine the effect of osteoblastic differentiation on CPP2 metabolism, CPP2 uptake and conditioned media CPP quantity and quality was assessed in differentiated and non-differentiated MC3T-E1 cells treated with Osteosense labelled CPP2.
Results: Pi+CPP2M increased osteocalcin expression, but decreased ALP expression and activity. Pi+CPP2M also reduced osteoblast viability and increased apoptosis. MC3T3-E1 cells took up CPP2. This was further enhanced by osteoblastic differentiation. Osteoblastic differentiation affected conditioned media CPP2 characteristics. CPP2 size was significantly increased in non-differentiated cells treated with high phosphate media.
Conclusion: The combination of high phosphate and CPP2 affected clinically relevant markers of ABD in vitro. Reduced osteoblastic differentiation and high phosphate increased CPP2 size, an important contributor to VC. These results suggest that CPP2 plays an important role in the interplay between high phosphate, ABD and VC.
Dr Michael Cai is a nephrologist with Royal Melbourne Hospital. His main research interest is in CKD-MBD, especially on the role of calciprotein particles in mediating the CKD-MBD phenotypes.