Fig 1.
Macrophages are activated by high Pi concentration.
Unpolarized M0φs were washed three times and incubated with the indicated concentration of Pi for 3 days (A) or incubated with 2.5 mmol/L Pi for the indicated time (B). Statistical significance was determined by one-way ANOVA analysis of variance followed by Tukey´s multicomparison test. (C) Flow cytometry studies showing activation of M0φs after exposure to high [Pi] (2.5 mmol/L) for 7 days. (D) Quantification of CD11b and F4/80, revealing significantly higher levels of macrophage-specific surface markers on MPiφs than on M0φs. Statistical significance was determined by Student’s t-test. Results are presented as mean ± SE of 3 independent experiments with 3 mice per experiment.*, P<0.05; **,P<0.01; ***,P<0.001.
Fig 2.
MPiφs degrade arginine via Arg1.
(A) RNAseq data summarizing fold mRNA upregulation in MPiφs of collagen types and enzymes involved in the urea cycle. (B) Arg1 and iNOS mRNA levels. (C) Arginase activity. Statistical significance was determined by Student’s t-test. Results are presented as mean ± SE of 4 independent experiments with 3 mice per experiment. ***, P<0.001.
Fig 3.
MPiφs have an enhanced energetic profile.
(A) RNAseq data summarizing fold mRNA downregulation or upregulation in MPiφs of enzymes in the main pathways involved in glucose and fatty acid catabolism. (B) mRNA levels for HIF-1α and PGC-1β mRNA levels. (C) Intracellular ATP and ADP/ATP ratio. Statistical significance was determined by Student’s t-test. Results are presented as mean ± SE of 4 independent experiments with 3 mice per experiment. **, P<0.01; ***, P<0.001.
Fig 4.
MPiφs show elevated antioxidant synthesis.
(A) RNAseq data summarizing fold mRNA downregulation or upregulation in MPiφs of enzymes involved in glutathione metabolism. (B) Total antioxidant capacity. (C) GSH/GSSG ratio. (D) Total GSH. Statistical significance was determined Student’s t-test. Results are presented as mean ± SE of 4 independent experiments with 3 mice per experiment. *, P<0.05; **, P<0.01.
Fig 5.
MPiφs prevent calcium-phosphate deposition by increasing extracellular ATP and PPi.
Fixed VSMCs were incubated in pro-calcifying medium over 5 days with 0.4 μm polycarbonate transwells containing unpolarized M0φsor Pi-activated MPiφs. (A, B) Extracellular ATP and PPi released by the indicated macrophage types. (C) PPi/ATP ratio. Statistical significance was determined by unpaired Student’s t-test. Results are presented as mean ± SE of 3 independent experiments with 3 mice per experiment. (D) Calcium deposition on fixed VSMCs in absence (Control) or presence of M0φs or MPiφs. Experiments were performed in the presence or absence of exogenous alkaline phosphatase (-AP and +AP). Statistical significance was determined by one-way ANOVA analysis of variance followed by Tukey’s multicomparison test. Results are presented as mean ± SE of 4 independent experiments with 3 mice per experiment. *, P<0.05; **, P<0.01; ***, P<0.001.
Fig 6.
Extracellular PPi metabolism in MPiφs.
(A) mRNA levels for the indicated ectoenzymes in unpolarized M0φs and Pi-activated MPiφs. (B) Immunoblot analysis of the indicated ectoenzymes. Relative levels were quantified by densitometry and were normalized to α–tubulin. (C) eNTPD activity, quantified as PPi produced from hydrolysis of 1 μmol/L ATP in 1h. (D) Alkaline phosphatase activity. Statistical significance was determined by unpaired Student’s t-test. Results are presented as mean ± SE of 4 independent experiments with 3 mice per experiment. ATP, adenosine triphosphate; AMP, adenosine monophosphate; PPi, pyrophosphate; Pi, inorganic phosphate; eNPP1, ectoenzyme nucleotide pyrophosphatase/phosphodiesterase-1; eNTPD1, ectonucleoside triphosphate diphosphohydrolase 1; TNAP, tissue non-specific alkaline phosphatase. **, P<0.01; ***, P<0,001.
Fig 7.
Increased release of ATP and PPi to the extracellular environment by Pi-activated macrophages protects tissues against Pi-induced calcification.
Unpolarized M0φs exposed to high Pi concentrations (hyperphosphatemia) are polarized to Pi-activated macrophages (MPiφs), which release ATP and pyrophosphate (PPi), known endogenous inhibitors of calcium-phosphate crystal formation.