Serum P and endothelial function Shuto et al
Supplemental Appendix
Supplemental Appendix
Supplementary Materials and Methods
Chemicals and antibodies
Diphenyl iodonium chloride (DPI; NAD(P)H oxidase inhibitor), oxypurinol (xanthine oxidase inhibitor), rotenone (mitochondrial respiratory chain system inhibitor), 2-phenyl-1,2-benzisoselenazol-3[2H]-one (ebselen; antioxidant), phosphonoformic acid (PFA; sodium-dependent phosphate transporter inhibitor), 2,2,6,6-tetramethyl-4-hydroxy-piperidine-N-oxyl (Tempol; antioxidant), bradykinin, acetylcholine, phenylephrine, Hank’s balanced salt solution (HBSS), sodium nitroprusside (SNP), calcium ionophore, and ATP were purchased from Sigma-Aldrich Japan (Tokyo, Japan). Gö6976 (conventional protein kinase C (PKC) inhibitor), H-89 (protein kinase A (PKA) inhibitor), wortmannin (Phosphatidylinositol 3-kinase (PI3K) inhibitor) were purchased from Calbiochem (San Diego, CA, USA). Nitro blue tetrazolium chloride (NBT) was purchased from Invitrogen (Grand Island, NY). Aminophenyl fluorescein (APF) and diacetyl-diaminofluorescein-2 (DAF-2DA) were purchased from Daiichi Chemical (Tokyo, Japan). Anti-eNOS monoclonal antibody (1:2500), anti-caveolin polyclonal antibody (1:5000) and anti-phospho-eNOS Thr495 monoclonal antibody (1:1000), anti-PKC monoclonal antibody (1:1000) were purchased from BD Bioscience Pharmingen (San Diego, CA, USA). These antibodies were used as the primary antibodies for the western blot analyses. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (1:5000) were purchased from Bio-Rad Japan (Tokyo, Japan) and were used as secondary antibodies.
Measurement of intracellular Ca2+ change
Fluo-4 and Fura-red were used for the measurement of intracellular Ca2+ changes. BAECs were incubated in the presence of 5 µM Fluo-4AM, 5 µM Fura Red-AM, and 0.05 % Puronic F127 (Molecular Probes, Eugene, OR) for 30 min in serum-free Medium 199. Cells were then washed twice with Medium 199 and postincubated in the presence of Medium 199 plus 20 % FBS for 20 min. Cells loaded with these dyes were washed three times with HBSS supplemented with 1.8 mM Mg2+. After washing, the cells were finally placed in HBSS supplement with 2 mM P or without, and were mounted on the inverted stage of the confocal microscope. Cells were examined at an excitation wavelength of 488 nm and emission at 500-540 nm (Fluo-4) and 600-680 nm (Fura-Red) were acquired simultaneously. During acquisition, the cells were treated with 0.5 µM ATP, 2 µM ATP, and 10 µM calcium ionophore (final concentrations) to increase intracellular Ca2+ concentration. Regions of interest (ROI) were traced on the monitor and the ratio value of Fluo-4/Fura-Red for each ROI was calculated using a built-in accessory program in the confocal system. The processing of ratio images was done off-line using the ratio imaging module of OpenLab software (Improvision Inc).
Reverse transcription-PCR
Total RNA was extracted from the BAECs with Trizol reagent (Invitrogen) according to the manufacture’s instruction. Complementary DNA was synthesized from 2 µg total RNA using a cDNA synthesis kit (Invitrogen) with random primers. Reverse transcription-PCR analysis was performed using Go Taq Green Master Mix (Promega, Osaka, Japan) with Thermal CyclerTM (ASTEC, Fukuoka, Japan). The primer sequences used in PCR are shown in Supplemental Table 1. The PCR reactions were conducted under the following conditions: precycling at 95 C for 1 min followed with 35 cycles consisting of denaturation at 95 C for 1 min, annealing at 58 C for 1 min, and polymerization at 72 C for 1 min. The size of the PCR product was first verified on a 2.0 % agarose gel. Amplified products were subjected to sequence analysis and confirmed.
Western Blot analysis
The proteins in the samples (20 µg protein) were separated by electrophoresis on 11 % polyacrylamide gels containing 0.1% sodium dodecyl sulfate (SDS). The separated proteins were subsequently transferred onto PVDF membranes (Immobilon-P; Millipore Japan, Tokyo, Japan) by electroblotting. The membrane was blocked with 5 % skim milk/TBS-0.1% Tween 20 for 1 hour at room temperature and then they were incubated with appropriate primary antibody in 5% skim milk/TBS-0.1% Tween 20 for 1 hour at room temperature, or overnight at 4 C. After washing out the unbound antibodies with TBS-0.1% Tween 20, the membrane was incubated with either horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG for 1 hour at room temperature. After washing out the unbound antibodies, the immunoreactive bands were visualized using enhanced chemiluminescence (ECL plus kit; GE Healthcare, Buckinghamshire, UK) and analyzed with LAS-3000 Lumino-image analyzer (Fujifilm, Tokyo, Japan).
PKC activity assay
Whole cell lysates were prepared from BAECs using lysis buffer attached to the TruLight PKC Assay Kit (Calbiochem, Tokyo, Japan). The PKC activity was measured following the protocol of the kit and detected by reading the fluorescence using an excitation wavelength of 450 nm and an emission wavelength of 490 nm in micro-plate reader (Infinit TM200, TECAN, Osaka, Japan).
Evaluation of vasodilation and vasoconstriction using rat thoracic aorta rings.
Male, 12-wk–old Sprague Dawley (SD) rats (Japan SLC Inc), were individually caged in a climate-controlled room (23 ± 2 °C with humidity 70–75%; specific pathogen-free) with a 12-h light:12-h dark cycle. Prior to the initiation of our study, the rats were allowed free access to standard rodent powder diet (MF; Oriental Yeast) and water.S1 The Institutional Animal Care and Oversight Committee approved the experimental protocols of the study. The experiments were carried out according to the guidelines and principles for the care and use of animals at the University of Tokushima.
The thoracic aorta was excised from the rats, and immediately placed in Krebs–Henseleit buffer (KHB) of the following composition: NaCl 118.1 mM, NaHCO3 24.8 mM, glucose 5.6 mM, KCl 4.9 mM, CaCl2 2.5 mM, MgSO4･7H2O 1.2 mM, KH2PO41.2 mM, pH7.4. The aortic rings were cut to 3 mm slices, and then connected to an isometric force transducer. For ex vivo experiments, the aortic rings were incubated with KHB buffer containing 1.2 mM P or 2.4 mM P for 1 hour at 37°C, otherwise all other experiments using aortic rings were done with KHB buffer containing 1.2 mM P. To confirm the endothelium-dependent vasodilation, the endothelium was removed from the aorta with a cotton thread as previously described.S2, S3 The aorta samples were equilibrated under a resting tension of 1.0 g for 60 min, and the solution was changed at 20 min intervals. All vessels were preconstricted with phenylephrine (100 nM), and then were treated with acetylcholine (1 to 100 nM) for the measurement of endothelium-dependent dilation or sodium nitroprusside (SNP, 1 to 100 nM) for endothelium-independent dilation. Relaxation responses to acetylcholine and SNP were expressed as percentage of relaxation relative to a submaximal phenylephrine-induced constriction (100 nM).
Diets and P-supplementations for human study
As described in a previous report,S4 the test meals were follows: P400 meal consisted of steamed rice, boiled egg, ham, and milk (690 kcal, 110g carbohydrate, 23g protein, 16g fat, 400 mg P and 200 mg Ca) with placebo supplement (containing 1022 mg Na in NaCl dissolved in 22ml of water to keep the intake of Na constant between test meals); P1200 meal consisted of steamed rice, boiled egg, ham, and milk (690 kcal, 110g carbohydrate, 23g protein, 16g fat, 400 mg P and 200 mg Ca) with 800 mg P supplement (a neutral sodium phosphate (mixture of Na2HPO4 and NaH2PO4) dissolved in 22 ml of water). The subjects drank either P supplement or placebo supplement in the same time during a meal. All of the meals were consumed over 7-14min. A standard dinner (650 kcal, 100g carbohydrate, 23g protein, 16g fat, 480mg P, 250mg Ca) was served on the day before each study day.
Endothelial function testing: Flow-mediated dilation (FMD) measurements
Endothelial function was evaluated by means of FMD, as previously published guidelines.S5 Volunteers were required to lie at rest for at least 15 min, FMD was assessed in each their right arm in a supine position in a quiet temperature controlled room (27 °C) by using high-resolution ultrasound (UNEXEF18G, UNEX Corporation, Nagoya, Japan). The brachial artery was scanned laterally and its diameter at end-diastole (i.e, from the inner border line of adventitia to adventitia) was measured.
The cuff was obtained 5 cm proximal to the antecubital fossa, fitted at 8 cm distal to the brachial artery, near the wrist. The transmit focus zone was set at the depth of the anterior wall. Anatomical landmarks and snapshot images were used to assess FMD in the exact same vessel section on each study day and at each time point. A view of a 5 cm transversal section of the brachial artery was recorded for periods of 30 s at baseline and during peak (up to 2 min after cuff release) reactive hyperemia (after deflation of the blood pressure cuff previously inflated to 50 mm Hg above the volunteer's systolic blood pressure around the forearm for 5 min). Vessel diameter was automatically estimated with built-in software. FMD was calculated as the percentage change in diameter from the baseline value before cuff release to the peak value after cuff release. Ultrasound studies and image analysis were separately performed by an independent investigator in an observer-blinded fashion to minimize the effect of inter-observer variability. The intra-observer variability in measuring brachial artery diameter (mean±SEM of the absolute difference) was 0.08±0.02 mm (average coefficient of variation=1.37%). This value is an acceptable reproducibility as recommended in current guidelines for FMD.S5
References
S1. Segawa H, Yamanaka S, Ito M, Kuwahata M, Shono M, Yamamoto T, Miyamoto K: Internalization of renal type IIc Na-Pi cotransporter in response to a high-phosphorus diet. Am J Physiol Renal Physiol 288: F587-F596, 2005
S2. Nakamura A, Harada N, Takahashi A, MAwatari K, Nakano M, Tsutsumi K, Nakaya Y: NO-1886, a lipoprotein lipase activator, attenuates vascular smooth muscle contraction in rat aorta. Eur J Pharmacol. 554: 183-190, 2007
S3. Harada N, Sakamoto S, Niwa Y, Nakaya Y. Involvement of adenosine in vascular contractile preconditioning. Am J Physiol Heart Circ Physiol. 280: H2911-H2919, 2001
S4. Nishida Y, Taketani Y, Yamanaka-Okumura H, Imamura F, Taniguchi A, Sato T, Shuto E, Nashiki K, Arai H, Yamamoto H, Takeda E: Acute effect of oral phosphate loading on serum fibroblast growth factor 23 levels in healthy men. Kidney Int 70: 2141-2147, 2006
S5. Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbounneau F, Creager MA, Deanfield J, Drexler H, Gerhard-Herman M, Herrington D, Vallance P, Vita J, Vogal R: Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: A report of the international brachial artery reactivity task force. J Am Coll Cardiol 39:257-265, 2002
Supplemental Table1. Primer alignments.
NaPi-IIa sense 5’-gaagctgtccagtgctttcc-3’ (1395-1414)
anti-sense 5’-aaccagcgatacttggctgt-3’ (1522-1541)
NaPi-IIb sense 5’-tgaatgatgagtcggtccaa-3’ (860-879)
anti-sense 5’-tggtccaggtgtacaaacca-3’ (990-1009)
Pit-1 sense 5’-gggcttaattgacgtggaga-3’ (273-292)
anti-sense 5’-gttgcaccgacgatacaatg-3’ (403-422)
Pit-2 sense 5’-gtggatgcggaggaagataa-3’ (708-727)
anti-sense 5’-acagtgctgtcatcgtgagc-3’ (835-854)
Positions are counted from translation initiation codon “A” as +1.
1
S