Fig. 2

Succinate stimulated macrophage infiltration, activation of profibrotic M2 phenotype, upregulation of profibrotic factors in vivo and in vitro. A F4/80 immunohistochemistry staining indicated succinate markedly increased renal macrophage in the kidney interstitium rather than glomerulus. ***P < 0.001, versus control group, n = 5. B Renal proinflammatory M1 cytokines, including iNOS and IL6 mRNA levels, were reduced by succinate. **P < 0.01, versus the control group, n = 5. C The mRNA levels of anti-inflammatory M2 cytokines (Arg1, Fizz1, Mgl2, and IL-10) in the kidney were markedly increased. **P < 0.01;***P < 0.001, versus control group, n = 5. D Succinate upregulated renal M2 macrophages-related profibrotic factors expression (galectin3, MMP9, MMP12, MMP13, PDGF, and CTGF). ns, not significant; **P < 0.01;***P < 0.001, versus control group, n = 5. RAW 264.7 cells were treated at 500 μM succinate for 24 h, and quantitative PCR analysis and immunoblotting were adopted to detect the effects of succinate on M2 polarization and expression of profibrotic factors in vitro. E Succinate had no effects on the cell viability of RAW 264.7. Succinate downregulated M1 cytokines (iNOS and IL6) mRNA levels (F) while significantly upregulated M2 cytokines (Arg1, Fizz1, Mgl2, and IL-10) (G). ***P < 0.001, versus control group, n = 3, biologically repeated 3 times. Likewise, succinate remarkably increased M2 macrophage-related profibrotic factor expression (MMP9, MMP12, MMP13, PDGF, and CTGF) (H). ns, not significant; ***P < 0.001, versus control group, n = 3, biologically repeated 3 times. The cell supernatant was measured by mouse CTGF ELISA kit according to the manufacturer’s instructions (E-EL-M0340; Elabscience, Bethesda, MD). Similarly, succinate increased CTGF release of macrophages (I)