Volume 81, Issue 3, Pages 280-292 (February 2012) Altered expression of microRNA miR-146a correlates with the development of chronic renal inflammation  Osamu Ichii, Saori Otsuka, Nobuya Sasaki, Yuka Namiki, Yoshiharu Hashimoto, Yasuhiro Kon  Kidney International  Volume 81, Issue 3, Pages 280-292 (February 2012) DOI: 10.1038/ki.2011.345 Copyright © 2012 International Society of Nephrology Terms and Conditions

Figure 1 miR-146a expression in kidneys. (a) Comparison of miR-146a expression between 12-month-old C57BL/6 and B6.MRLc1 mice (n=4). (b) Comparison of primary-miRNA expression between 12-month-old C57BL/6 and B6.MRLc1 mice (n=4). (c) Time-course expression in B6.MRLc1 mice (n≥4 at each age). Expression values are expressed as a fold increase to those of C57BL/6 mice at 12 months. Values=means±s.e. *Mann–Whitney U-test (P<0.05). Kidney International 2012 81, 280-292DOI: (10.1038/ki.2011.345) Copyright © 2012 International Society of Nephrology Terms and Conditions

Figure 2 Correlations between renal histopathology and miR-146a expression. The vertically arranged figures are derived from the same individuals. Panels g–r are light micrographs of immunofluorescence. (a, d, g, j, m, and p) Renal cortices from B6.MRLc1 mice with miR-146a expression values=0.4. Histopathological changes are not clear, and F4/80-, CD3- Gr-1-, and IL-1F6-positive cells are scarcely observed (m, arrow). (b, e, h, k, n, and q) Renal cortices from B6.MRLc1 mice with miR-146a expression values=1.7. Glomerular lesions, e.g., glomerular hypertrophy, expansion of the mesangial matrix, and increased number of glomerular cells, and mild tubule interstitial lesions (arrowheads), e.g., mononuclear cell infiltration, are observed (b and e). F4/80-positive cells are observed in interstitial lesions (h, arrowheads). CD3-positive mononuclear cells are observed in the glomerulus and interstitium (k, arrows). Gr-1-positive cells are mainly observed in the glomerulus (n, arrow). IL-1F6-positive reactions are observed in the cytoplasm and nucleus of tubular cells (q). (c, f, i, l, o, and r) Renal cortices from B6.MRLc1 mice with miR-146a expression values=3.1. Glomerular lesions and tubulointerstitial lesions are the most severe (compare a–c). Fibrotic areas are increased in the interstitial lesions (blue regions, f). The number or area of positive reactions of F4/80, CD3, and IL-1F6, except for Gr-1, are remarkably increased compared with the other two mice. AS, aniline blue stain; CD3, T-cell marker; F4/80, macrophage marker; Gr-1, granulocyte marker; IL-1F6, tubular damage marker; PAS, periodic acid-Schiff stain. Bars=50μm. Kidney International 2012 81, 280-292DOI: (10.1038/ki.2011.345) Copyright © 2012 International Society of Nephrology Terms and Conditions

Figure 3 Histopathological parameters and miR-146a expression in kidneys. (a, d, g, j, m, and p) Comparison between 12-month-old C57BL/6 and B6.MRLc1 mice (n=4). Values=means±s.e. *Mann–Whitney U-test (P<0.05). (b, e, h, k, n, and q) Time-course analysis in B6.MRLc1 mice (n≥4 at each age). Values=means±s.e. (c, f, i, l, o, and r) Correlation between the histopathological scores and miR-146a expression (n=24). Values=means±s.e. Spearman's correlation test (P<0.001) was used to analyze the correlation between two parameters. CD3, T-cell marker; F4/80, macrophage marker; GL, glomerular lesion; Gr-1, granulocyte marker; IL, interstitial lesion; IL-1F6, tubular damage marker; ND, not detected. Kidney International 2012 81, 280-292DOI: (10.1038/ki.2011.345) Copyright © 2012 International Society of Nephrology Terms and Conditions

Figure 4 mRNA expression of inflammatory cell-specific markers and miR-146a expression in kidneys. (a, d, g and j) Comparison between 12-month-old C57BL/6 and B6.MRLc1 mice (n=4). Values=means±s.e. *Mann–Whitney U-test (P<0.05). (b, e, h, and k) Time-course analysis in B6.MRLc1 mice (n≥4 at each age). Values=means±s.e. (c, f, i and l) Correlation between the histopathological scores and miR-146a expression (n=24). Values=means±s.e. Spearman's correlation test (P<0.001) was used to analyze the correlation between two parameters. Expression values are expressed as a fold increase to those of C57BL/6 mice at 12 months. Kidney International 2012 81, 280-292DOI: (10.1038/ki.2011.345) Copyright © 2012 International Society of Nephrology Terms and Conditions

Figure 5 mRNA expression of inflammatory cytokines/chemokines and miR-146a expression in kidneys. (a, d, g, j, and m) Comparison between 12-month-old C57BL/6 and B6.MRLc1 mice (n=4). Values=means±s.e. *Mann–Whitney U-test (P<0.05). (b, e, h, k, and n) Time-course analysis in B6.MRLc1 mice (n≥4 at each age). Values=means±s.e. (c, f, i, l, and o) Correlation between the examined mRNA and miR-146a expression (n=24). Values=means±s.e. Spearman's correlation test (P<0.05) was used to analyze the correlation between two parameters. Expression values are expressed as a fold increase to those of C57BL/6 mice at 12 months. Kidney International 2012 81, 280-292DOI: (10.1038/ki.2011.345) Copyright © 2012 International Society of Nephrology Terms and Conditions

Figure 6 Localization of miR-146a-expressing cells. Panels a–c, e, f, h, and i are light micrographs of in situ hybridizations. Panels d and g are periodic acid-Schiff–stained sections. (a–c) Positive signals for miR-146a are observed in interstitial lesions of B6.MRLc1 (b, arrows). These reactions are not observed in the sections from C57BL/6 or negative control (a and c). G, glomerulus; T, renal tubule. Panels b and c are serial sections. (d–f) Positive signals for miR-146a are observed in perivascular lesions containing mononuclear cell infiltrations (d and e, arrows). These reactions are not observed in the serial negative control sections (f). A, arteriole; G, glomerulus; V, venule. (g–i) Strong positive signals for miR-146a are observed in an enlarged mediastinal lymph node of B6.MRLc1 as a positive control. These reactions are not observed in the negative control sections (i). *Same regions in serial sections. Bars=50μm. Kidney International 2012 81, 280-292DOI: (10.1038/ki.2011.345) Copyright © 2012 International Society of Nephrology Terms and Conditions

Figure 7 Local expression of miR-146a. (a–d) Glomerulus (GL) and interstitial lesions (IL) containing mononuclear cell infiltrations are isolated by laser as shown in a–d. (e) RT-PCR analysis of cDNA synthesized from total RNA extracted from the laser-cut sections. Cd68, macrophage marker; Il1f6, tubulointerstitial lesion marker; M, molecular markers; Nfkb, genes of nuclear factor-kappa B (NF-κB). (f) Comparison between GLs and ILs. Expression values are expressed as a fold increase to those of GLs at 12 months. Three different regions were cut and collected from three animals. Values=means±s.e. Bars=50μm. Kidney International 2012 81, 280-292DOI: (10.1038/ki.2011.345) Copyright © 2012 International Society of Nephrology Terms and Conditions

Figure 8 Silencing of miR-146a/b in human monocytes. (a) Confirmation of the transfection efficiency for the anti-miRNA inhibitor by the detection of the carboxyfluorescein-labeled anti-miRNA negative control in THP-1 human monocyte cells. (b and c) Comparison of morphology between THP-1 cells transfected with the negative control (b) and the anti-miRNA inhibitor (c). Although the size of the latter is relatively small compared with the control, no obvious morphological changes are observed. (d) miR-146a and miR-146b-5p expression in transfected THP-1 cells. (e) mRNA expression of nuclear factor-kappa B (NF-κB) molecules, IRAK1, and TRAF6, and in transfected THP-1 cells. (f) IRAK1 and TRAF6 protein levels in transfected THP-1 cells. Upper panel, immunoblotting. Lower graph, quantification by band intensity. (g) mRNA expression of inflammatory mediators in transfected THP-1 cells. n=3. Values=means±s.e. *Mann–Whitney U-test (vs. control, P<0.05). Kidney International 2012 81, 280-292DOI: (10.1038/ki.2011.345) Copyright © 2012 International Society of Nephrology Terms and Conditions

Figure 9 miR-146a levels in urine. (a) Comparison between supernatant and sediment from B6.MRLc1 bladder urine at 12–14 months of age. n=4. Values=means±s.e. (b) Comparison of bladder urine sediment from 12-month-old C57BL/6 (n=6) and B6.MRLc1 (n=19) mice. Values=means±s.e. Kidney International 2012 81, 280-292DOI: (10.1038/ki.2011.345) Copyright © 2012 International Society of Nephrology Terms and Conditions