1. Volume 81, Issue 3, Pages 256-265 (February 2012)
Uridine adenosine tetraphosphate activation of the purinergic receptor P2Y enhances in vitro vascular calcification 
Mirjam Schuchardt, Markus Tölle, Jasmin Prüfer, Nicole Prüfer, Tao Huang, Vera Jankowski, Joachim Jankowski, Walter Zidek, Markus van der Giet 
Kidney International 
Volume 81, Issue 3, Pages (February 2012)
DOI: /ki
Copyright © 2012 International Society of Nephrology Terms and Conditions

2. Figure 1
Up4A enhances calcification ex vivo. (a) Up4A plasma concentration of healthy controls (n=15) and patients with chronic kidney disease (CKD, n=17) on hemodialysis (HD) treatment. (b) Staining of mineral deposits in aortic rings stimulated for 14 days as indicated (Up4A: 10μmol/l) with Alizarin red or von Kossa staining. (c) Quantification of calcium content with O-cresolphthalein complexone method of decalcified aortic rings incubated for 14 days as indicated (Up4A: 10μmol/l). The calcium concentration is normalized to dry weight of the aortic rings. (a, c) Black bar graphs: control medium±Up4A: 10μmol/l; white bar graphs: CM±Up4A: 10μmol/l; mean±s.e.m.; *P<0.05. CM, calcifying medium; Up4A, uridine adenosine tetraphosphate.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

3. Figure 2
Up4A enhances calcification in vitro. Vascular smooth muscle cells (VSMCs) were cultured for the indicated time points in culture medium (control), CM, or CM+Up4A (10μmol/l). (a) Calcification of VSMCs was visualized with Alizarin red staining. (b) Quantification of calcium content of the cell layers normalized to cellular protein content; mean±s.e.m.; *P<0.05 CM+Up4A versus CM. CM, calcifying medium; Up4A, uridine adenosine tetraphosphate.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

4. Figure 3
Up4A enhances the loss of SMC marker and the increase of OCC marker. Cells were cultured as indicated in control medium, CM, or CM+Up4A (10μmol/l). (a, b) Immunofluorescent staining of the cells with a fluorescein isothiocyanate-conjugated anti-smooth muscle cell-α-actin antibody, (a) detected by fluorescence microscopy and (b) quantified by flow cytometry (n=5; mean±s.e.m.; *P<0.05). (c) ALP enzyme activity was measured photometrically and normalized to cellular protein content; mean±s.e.m.; *P<0.05 CM+Up4A versus CM. ALP, alkaline phosphatase; CM, calcifying medium; OCC, osteochondrogenic cell; SMC, smooth muscle cell; Up4A, uridine adenosine tetraphosphate.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

5. Figure 4
Expression of genes associated with differentiation of VSMCs to OCC. Vascular smooth muscle cells (VSMCs) were stimulated with different concentrations of Up4A (in the absence of CM) for 24h (a–d), 48h (e, g–i), or 12h (f). Expression was measured with real-time PCR using specific primers and normalization to β-actin as a housekeeping gene. For all panels mean±s.e.m.; *P<0.05 Up4A versus control. ALP, alkaline phosphatase; Cbfa1, core binding factor α-1; CM, calcifying medium; OCC, osteochondrogenic cell; OCN, osteocalcin; OPG, osteoprotegerin; OPN, osteopontin; Osx, osterix; Up4A, uridine adenosine tetraphosphate.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

6. Figure 5
Cbfa1 protein content. Vascular smooth muscle cells (VSMCs) were stimulated for 48h with Up4A (1–100μmol/l). Cbfa1 protein was detected by western blot technique in the nuclear protein fraction. (a) Representative western blot. (b) Quantification of band intensity of four independent experiments; mean±s.e.m.; *P<0.05 versus control. Cbfa1, core binding factor α-1; Up4A, uridine adenosine tetraphosphate.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

7. Figure 6
Activation of P2Y. (a) Vascular smooth muscle cells (VSMCs) were stimulated with Up4A (10μmol/l) for 24h±P2Y antagonists; n=10, *P<0.05 Up4A+antagonist versus Up4A. (b) Stimulation with ATPγS in different concentrations or (c) 10μmol/l of each agonist for 24h. (d) VSMCs were stimulated for 21 days in CM, CM+Up4A (10μmol/l), or CM+Up4A (10μmol/l)+antagonists (suramin: 100μmol/l, PPADS: 10μmol/l, RB-2: 10μmol/l, MRS2179: 1μmol/l, MRS2578: 1μmol/l); n=5, *P<0.05 CM+Up4A+antagonists versus CM+Up4A. (e) Aortic rings from P2Y2-/- and control mice were incubated as indicated for 14 days. Calcium content of aortic rings was quantified and normalized to dry weight. ALP, alkaline phosphatase; Cbfa1, core binding factor α-1; CM, calcifying medium; PPADS, pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid; RB-2, reactive blue-2; Up4A, uridine adenosine tetraphosphate.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

8. Figure 7
Intracellular signaling pathway. Measurement of MEK and ERK1/2 phosphorylation via the Luminex technique after (a) time-dependent Up4A (10μmol/l) stimulation (n=3; *P<0.05 Up4A versus control) or after (b) 10min Up4A (10μmol/l). Antagonists were pretreated for 30min (suramin: 100μmol/l, PPADS: 10μmol/l, RB-2: 10μmol/l, MRS2179: 1μmol/l, MRS2578: 1μmol/l, PD98059: 40μmol/l, U0126: 1μmol/l); n=5; *P<0.05 Up4A versus Up4A+antagonist. (c) The influence of PD98059 (40μmol/l) and U0126 (1μmol/l) on Up4A-induced (10μmol/l) Cbfa1 expression measured by real-time PCR. (d) Wnt3a and 7a expression in vascular smooth muscle cells (VSMCs) outgrown from aortic rings and incubated as indicated for 14 days. The medium was changed every 3 days; n=4, *P<0.05. For all panels: mean±s.e.m. (e) The proposed signaling pathway. Cbfa1, core binding factor α-1; OCC, osteochondrogenic cell; OPG, osteoprotegerin; OPN, osteopontin; Osx, osterix; PPADS, pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid; RB-2, reactive blue-2; Up4A, uridine adenosine tetraphosphate.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions