Nephropathy in Hypertensive Animals Is Linked to M2 Macrophages and Increased Expression of the YM1/Chi3l3 Protein

Figures

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  Figure 1. The macrophages of hypertensive animals have a predisposition toward the M2 phenotype. Four mice for each group, 6 wells per animal. (a) CD86 gene expression. Values were expressed as the mean ± SEM. ∗p < 0.05 compared to M0 groups and #p < 0.05 relative to IL-4+IL-13 groups, with ANOVA followed by Bonferroni correction for multiple comparisons. (b) CD206 gene expression. Values expressed as the mean ± SEM. ∗p < 0.05 compared to M0 groups, #p < 0.05 relative to LPS+IFN-γ groups, and §§p < 0.05 compared to IL-4+IL-13 from control mice, with ANOVA followed by Bonferroni correction for multiple comparisons. (c) Ratio M2/M1. Values are expressed as the mean ± SEM. ∗p < 0.05 compared to the control group with the t-test. The M2/M1 ratio reached 288%.

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  Figure 2. High levels of collagen were found in the kidneys of the hypertensive group. (a) Picro sirius red stained renal paraffin sections (renal cortex and medulla) of nonhypertensive control (C) and of rAOGEN transgenic hypertensive (H) mice at the age of 12 weeks. Light microscopic images were taken using a Keyence microscope (BZ-9000). Yellow arrows represent the perivascular fibrosis and black arrows the interstitial fibrosis. The scale bar = 300 μm. (b) Analysis and quantification of total fibrosis (interstitial and perivascular) were performed on digital renal images using a Keyence BZII analyzer. Data were presented as the area of fibrosis in % of whole renal section (4 sections/animal/groups). Histology with four mice of each group.

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  Figure 3. F4/80 gene expression indicates the presence of macrophages in the kidneys of hypertensive mice. Ang II levels are elevated, but Ang II is probably not binding to AT1aR. (a) F4/80 gene presented a significant difference when compared to the respective controls. Values are expressed as the mean ± SEM. ∗p < 0.05 compared to the control group with the t-test. (b) AT1aR gene presented no significant difference when compared to the respective controls. Values are expressed as the mean ± SEM. ∗p < 0.05 compared to the control group with the t-test. Five mice for each group.

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  Figure 4. Three markers of the M1 phenotype showed an increase, but the changes were smaller than twofold. Values are expressed as the mean ± SEM. ∗p < 0.05 compared to the control group with the t-test. The genes iNOS, TNFα, and IL-1β presented a significant difference when compared to the respective controls. Five mice for each group.

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  Figure 5. There was no increase in levels of the M1 marker proteins TNFα and IL-1β. (a) Renal levels of TNFα by ELISA. (b) Renal levels of IL-1β by ELISA. Values are expressed as the mean ± SEM. ∗p < 0.05 compared to the control group with the t-test. Five mice for each group.

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  Figure 6. There was no increase in levels of M1 marker proteins TNFα, IL-6, IFN-γ, and MCP-1: (a) renal levels of TNFα by CBA; (b) renal levels of IL-6 by CBA; (c) renal levels of IFN-γ by CBA; (d) renal levels of MCP-1 by CBA. Values were expressed as the mean ± SEM. ∗p < 0.05 compared to the control group with the t-test. Five mice for each group.

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  Figure 7. High levels of F4/80 in the hypertensive group indicate the presence of macrophages, and the YM1 marker shows the predominance for the M2 phenotype. There was no increase in iNOS, an M1 phenotype marker: (a) renal levels of F4/80 by IF; (b) renal levels of YM1 by IF; (c) renal levels of iNOS by IF. Values were expressed as the mean ± SEM. ∗p < 0.05 compared to the control group with the t-test. Seven sections/animal/groups. Four mice for each group.

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  Figure 8. Increase in markers related to the M2 phenotype in the kidneys. The genes YM1, TGF-β1, KIM-1, fibronectin, type I collagen, and type III collagen presented a significant difference when compared to the respective controls, and these results were higher than twofold. Values were expressed as the mean ± SEM. ∗p < 0.05 compared to the control group with the t-test. Five mice for each group.

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  Figure 9. IL-10 do not represent a form of protection against renal fibrosis in the hypertensive animals. Values were expressed as the mean ± SEM. Five mice for each group.

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  Figure 10. Levels of M2 marker proteins. M2 macrophages produced high levels of YM1 protein (91,89%): (a) renal levels of YM1/YM2/β-actin by WB; (b) renal levels of TGF-β1/β-actin by WB. Values were expressed as the mean ± SEM. ∗p < 0.05 compared to the control group with the t-test. Five mice for each group.

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  Figure 11. Immunofluorescence images of renal tissue showed colocalization between the F4/80, a macrophage marker, and YM1, suggesting that high levels of the YM1 protein were secreted by M2 macrophages. On the other hand, there was no increase in iNOS, an M1 marker: (a) DAPI, iNOS, and F4/80 by IF; (b) 3D overlapping images of DAPI, iNOS, and F4/80 by IF; (c) panel of DAPI, YM1, and F4/80 by IF; (d) 3D overlapping images of DAPI, YM1, and F4/80 by IF. The scale bar = 5 μm.

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  Figure 12. Macrophage polarization in the kidneys of 10- to 12-week-old hypertensive mice. The hypertensive stimuli from RAAS, as high levels of AOGEN contributed to macrophage polarization in the kidneys of hypertensive mice with a clear predominance of the M2 phenotype. The high levels of TGF-β1 may be involved in the development of fibrosis. Basal levels of IL-10 do not represent a form of protection against renal fibrosis in the hypertensive animals. In addition, at this stage of hypertension, our data do not support the polarization of macrophages to an M1 phenotype. It seems that the AT1aR in the kidneys from the hypertensive animals were not activated. High levels of AOGEN plus the hypertensive environment contribute to the M2 macrophage polarization. M2 macrophages produced high levels of YM1/Chi3l3 protein (91,89%).