Journal: Journal of the American Society of Nephrology : JASN

Article Title: Specific Macrophage Subtypes Influence the Progression of Rhabdomyolysis-Induced Kidney Injury

doi: 10.1681/ASN.2014040320

Figure Lengend Snippet: The kidney macrophage subtype evolution mirrors kidney function. (A) The elevated BUN levels observed at day 2 (Figure 1F) decreased by day 8, indicating ongoing kidney repair (n=6–11). (B–D) Analysis of macrophages obtained from kidney cell suspensions. Three regions were discriminated according to the expression levels of CD11b and F4/80 as follows: F4/80-CD11b+ (R0), F4/80lowCD11bhigh (R1), and F4/80highCD11b+ (R2). CD11b corresponds to R0+R1+R2 sum. Macrophage distribution among the three regions was affected by glycerol injection at day 2 and day 8. (B) Representative dotplot gated on 30,000 live CD45+ cells. (C) R0, R1, R2, and total CD11b+ cell counts in kidney samples (n=3–5; **P<0.01 and ***P<0.001 compared with day 0; #P<0.05 and ####P<0.0001 compared with day 2). (D) Distribution of CD11b+ cells in the three regions (n=3–5). Macrophage subpopulations were characterized by expression of surface markers using flow cytometry. Mean fluorescence intensity (MFI) is reported on the yaxis for the following markers: (E) CD206 (M2 marker) and (F) CD36 (M2 marker). (n=3–9; *A star above a line displays P<0.05; #displays significant difference between R2 at day 2 or day 8 compared with R2 at day 0 [p<0.05]; $displays significant difference between R1 at day 8 compared with R1 at day 2 [p<0.05]. Lines display means±SEM for individual data).

Article Snippet: Specific primary antibodies were incubated on mouse tissue sections for the detection of F4/80 (rat anti-mouse, 1/100, clone BM8, MF48000; Invitrogen, Saint Aubin, France), collagen III (rabbit anti-mouse, BP8014; Acris Antibodies GmbH, Herford, Germany) or fibronectin (rabbit anti-human/mouse, F3648; Sigma-Aldrich, Saint-Quentin Fallavier, France).

Techniques: Expressing, Injection, Flow Cytometry, Fluorescence, Marker

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Specific Macrophage Subtypes Influence the Progression of Rhabdomyolysis-Induced Kidney Injury

doi: 10.1681/ASN.2014040320

Figure Lengend Snippet: CL-mediated macrophage depletion attenuates kidney lesions 2 days after glycerol injection. Mice received EL or lCL in the saline (NaCl) or the glycerol (Gly) condition according to the protocol shown in Figure 6E. (A and B) CL reduced glycerol-induced fibronectin (Original magnification, ×400 in A) and collagen III (Original magnification, ×200 in B) accumulation at the protein level (left panel; n=3–9) and at the mRNA level in the whole kidney (right panel; n=5–6). (C) Hmox1 was upregulated by glycerol treatment and not modified by CL. CL reduced glycerol-induced Ccl2 and Ccl7 mRNA expression (n=5–6). (Versus NaCl EL: ***P<0.001 and ****P<0.0001. Versus glycerol EL: ##P<0.01; ###P<0.001; ####P<0.0001.) (D) CL reduced glycerol-induced lesions on periodic acid-Schiff staining (Original magnification, ×200 in representative examples). (E) CL did not affect rhabdomyolysis intensity (n=4–8).

Article Snippet: Specific primary antibodies were incubated on mouse tissue sections for the detection of F4/80 (rat anti-mouse, 1/100, clone BM8, MF48000; Invitrogen, Saint Aubin, France), collagen III (rabbit anti-mouse, BP8014; Acris Antibodies GmbH, Herford, Germany) or fibronectin (rabbit anti-human/mouse, F3648; Sigma-Aldrich, Saint-Quentin Fallavier, France).

Techniques: Injection, Saline, Modification, Expressing, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Specific Macrophage Subtypes Influence the Progression of Rhabdomyolysis-Induced Kidney Injury

doi: 10.1681/ASN.2014040320

Figure Lengend Snippet: CL-mediated macrophage depletion attenuates kidney fibrosis. (A–D) One month after rhabdomyolysis. CTL, age-matched control group; Gly, glycerol-treated group; Gly CL, CL-treated mice according to the pre, post, and glycerol CL protocol depicted in Figure ​Figure6C.6C. (A) BUN levels (n=2–6). (B) CD206+ expression was significantly increased in the glycerol CL condition among CD11b+ cells (R0, R1, and R2) (n=5–8). (C) Collagen III and Masson trichrome staining (representative examples are shown). (D) Collagen III deposit quantification (n=5–7). (Versus control: **P<0.01 and ****P<0.0001. Versus Gly: #P<0.05 and ##P<0.01.) (E–H) Seven months after rhabdomyolysis. NaCl EL, glycerol EL, and pre-glycerol early CL refer to mice treated according to the protocol depicted in Figure 6A. (E) BUN levels (n=6–14). (F) Kidney mass indicated global atrophy after glycerol injection. (n=6–14) (G) Collagen III and Masson trichrome staining (representative examples are shown). (H) Collagen III deposits were still significantly increased in the glycerol condition, which was partially prevented by CL (n=6–14). (Versus NaCl EL: *P<0.05 and ****P<0.0001. Versus glycerol: #P<0.05, ##P<0.01, ####P<0.0001.)

Article Snippet: Specific primary antibodies were incubated on mouse tissue sections for the detection of F4/80 (rat anti-mouse, 1/100, clone BM8, MF48000; Invitrogen, Saint Aubin, France), collagen III (rabbit anti-mouse, BP8014; Acris Antibodies GmbH, Herford, Germany) or fibronectin (rabbit anti-human/mouse, F3648; Sigma-Aldrich, Saint-Quentin Fallavier, France).

Techniques: Control, Expressing, Staining, Injection

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Specific Macrophage Subtypes Influence the Progression of Rhabdomyolysis-Induced Kidney Injury

doi: 10.1681/ASN.2014040320

Figure Lengend Snippet: Proposed early mechanisms involved in rhabdomyolysis-induced kidney injury. (1) In response to myoglobin, tubular cells secrete macrophage chemoattractants (Ccl2, Ccl7). (2) In response to these chemoattractants blood monocytes migrate to the renal interstitium. (3) In addition to its effect on tubular cells, myoglobin polarizes these macrophages toward a proinflammatory phenotype. (4) CD11bhighF4/80lowLy6-Bhigh subtype macrophages enhance renal injury by secreting extracellular matrix compounds (fibronectin, collagen III), proinflammatory cytokines (Il1b, Il12p40) and by increasing recruitment of newly generated macrophages (secretion of Ccl2 and Ccl7).

Article Snippet: Specific primary antibodies were incubated on mouse tissue sections for the detection of F4/80 (rat anti-mouse, 1/100, clone BM8, MF48000; Invitrogen, Saint Aubin, France), collagen III (rabbit anti-mouse, BP8014; Acris Antibodies GmbH, Herford, Germany) or fibronectin (rabbit anti-human/mouse, F3648; Sigma-Aldrich, Saint-Quentin Fallavier, France).

Techniques: Generated