Journal: PLoS ONE

Article Title: Rapamycin Ameliorates Kidney Fibrosis by Inhibiting the Activation of mTOR Signaling in Interstitial Macrophages and Myofibroblasts

doi: 10.1371/journal.pone.0033626

Figure Lengend Snippet: Kidney tissues derived from wide type C57BL/6J mice, moderate IRI and UUO mouse models were used for analysis. Animals were treated as described in . A(A′)–C(C′): Costaining of pS6K (red) and LTL (green) in renal sections from post-natal day 1(P1, A: low-power and A′: high-power), post-natal day 7 (P7, B: low-power and B′: high-power), 6-week (6w, C) and 16-week mice (16w, C′). D–F: Representative costaining images of pS6K (red) and LTL (green) in kidney sections derived from moderate IRI mice, including 48-hour (IR-48 hr, D), 7-day (IR-7d, E) and 2-week (IR-2w, F) after operation. G–I: Representative costaining images of pS6K (red) and LTL (green) in kidney sections derived from UUO models, including 1-day (U1d, G), 3-day (U3d, H) and 7-day (U7d, I) post-obstruction. J: Western blot and quantitative analysis of p-mTOR and pS6K in developing kidneys (right panel), moderate IRI model (middle panel) and UUO model (left panel). n = 5 animals in each group. *P<0.05, **P<0.01, NS, no significance. Error bars represent S.E.

Article Snippet: In brief, cryosections were air-dried for 15 min, then primary antibodies against the following proteins were used: pS6K (rabbit, 1∶100, Cell signaling, USA), F4/80 (rat, 1∶100, Abcam, USA), CD3 (rabbit 1∶100, Vectorlab, USA), CD4 (mouse, 1∶100, Abcam, USA), anti-neutrophil (rat, 1∶50, Santa Cruz, USA), αSMA (mouse, 1∶200, Abcam, USA), collagen-I (rabbit, 1∶500, Abcam, USA), Kim-1 (goat, 1∶100, R&D, USA), Lotus tetragonolobus lectin (LTL, 1∶1000, Vectorlab, USA).

Techniques: Derivative Assay, Western Blot

Journal: PLoS ONE

Article Title: Rapamycin Ameliorates Kidney Fibrosis by Inhibiting the Activation of mTOR Signaling in Interstitial Macrophages and Myofibroblasts

doi: 10.1371/journal.pone.0033626

Figure Lengend Snippet: UUO mice received daily i.p. injections of rapamycin (2 mg/kg of body weight) or vehicle respectively, starting 1 day prior to operation and continuing until termination of the experiment. A–H: Representative co-staining images of pS6K (red) and LTL (green) in UUO kideny sections with (E–H) or without (A–D) rapamycin treatment, from day 0 (A, E), day 3 (B, F), day 7(C, G) and day 14(D, H). Nuclei were labeled with DAPI (blue). Scale bar = 50 µm. I: Representative western blot (panel left) and quantitative analysis (panel right) of pS6K expression in renal sections from vehicle-treated or rapamycin-treated UUO mice. *P<0.05, **P<0.01 vs. vehicle treated groups. Error bars represent S.E.

Article Snippet: In brief, cryosections were air-dried for 15 min, then primary antibodies against the following proteins were used: pS6K (rabbit, 1∶100, Cell signaling, USA), F4/80 (rat, 1∶100, Abcam, USA), CD3 (rabbit 1∶100, Vectorlab, USA), CD4 (mouse, 1∶100, Abcam, USA), anti-neutrophil (rat, 1∶50, Santa Cruz, USA), αSMA (mouse, 1∶200, Abcam, USA), collagen-I (rabbit, 1∶500, Abcam, USA), Kim-1 (goat, 1∶100, R&D, USA), Lotus tetragonolobus lectin (LTL, 1∶1000, Vectorlab, USA).

Techniques: Staining, Labeling, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Rapamycin Ameliorates Kidney Fibrosis by Inhibiting the Activation of mTOR Signaling in Interstitial Macrophages and Myofibroblasts

doi: 10.1371/journal.pone.0033626

Figure Lengend Snippet: Kidney sections derived from either UUO or IRI models were analyzed by immunohistochemistry using antibodies against pS6K, F4/80, CD4 or anti-neutrophil. A–A′″ and B–B′″: Expression of pS6K (red) is immediately induced in F4/80+ macrophages (green) after kidney injury. Large portions of interstitial macrophages in obstructed kidney (A–A′″) and IRI kidney (B–B″) are co-stained with F4/80 and pS6K (indicated by arrwos). C–E: Co-staining of CD4 (green) and pS6K in obstructed kidneys. F: Costaining of anti-neutrophil and pS6K in obstructed kidneys. Scale bar: 20 µm.

Article Snippet: In brief, cryosections were air-dried for 15 min, then primary antibodies against the following proteins were used: pS6K (rabbit, 1∶100, Cell signaling, USA), F4/80 (rat, 1∶100, Abcam, USA), CD3 (rabbit 1∶100, Vectorlab, USA), CD4 (mouse, 1∶100, Abcam, USA), anti-neutrophil (rat, 1∶50, Santa Cruz, USA), αSMA (mouse, 1∶200, Abcam, USA), collagen-I (rabbit, 1∶500, Abcam, USA), Kim-1 (goat, 1∶100, R&D, USA), Lotus tetragonolobus lectin (LTL, 1∶1000, Vectorlab, USA).

Techniques: Derivative Assay, Immunohistochemistry, Expressing, Staining

Journal: PLoS ONE

Article Title: Rapamycin Ameliorates Kidney Fibrosis by Inhibiting the Activation of mTOR Signaling in Interstitial Macrophages and Myofibroblasts

doi: 10.1371/journal.pone.0033626

Figure Lengend Snippet: A–B: Co-immunostainging images of kidney sections from day-1 post-obstruction with administration of rapamycin (A–A′″) or vehicle (B–B′″), using anti-pS6K (red), anti-F4/80 (green) and DAPI (blue) for immunofluorescent staining. Representative areas in A″ and B″ (indicated by white square) are magnified in A′″ and B′″, respectively. Scale bar: 50 µm. C. Assessment of inflammatory activity of isolated macrophages from obstructed kidney on day-1 post-operation. mRNA level of proinflammatory chemokines, including TNF-α, IL-1β and MCP-1, were determined by realtime-PCR. *P<0.05, **P<0.01, vs control group (sham operation); # P<0.05, ## P<0.01 vs vehicle-treated group. n = 5 animals in each group. Ctrl: control group, U1d-Ve: 1-day post UUO operation with administration of vehicle, U1d-Rp: 1-day post UUO operation with administration of rapamycin. Error bars represent S.E.

Article Snippet: In brief, cryosections were air-dried for 15 min, then primary antibodies against the following proteins were used: pS6K (rabbit, 1∶100, Cell signaling, USA), F4/80 (rat, 1∶100, Abcam, USA), CD3 (rabbit 1∶100, Vectorlab, USA), CD4 (mouse, 1∶100, Abcam, USA), anti-neutrophil (rat, 1∶50, Santa Cruz, USA), αSMA (mouse, 1∶200, Abcam, USA), collagen-I (rabbit, 1∶500, Abcam, USA), Kim-1 (goat, 1∶100, R&D, USA), Lotus tetragonolobus lectin (LTL, 1∶1000, Vectorlab, USA).

Techniques: Staining, Activity Assay, Isolation, Control

Journal: PLoS ONE

Article Title: Rapamycin Ameliorates Kidney Fibrosis by Inhibiting the Activation of mTOR Signaling in Interstitial Macrophages and Myofibroblasts

doi: 10.1371/journal.pone.0033626

Figure Lengend Snippet: Immunofluorescent staining of kidney sections derived from UUO models on day-1 post-obstruction was performed, using anti-αSMA (green, A–D) and anti-pS6K (red, A′–D′). Images were merged (A″–D″) and co-labeled with DAPI (A′″–D′″). Representative areas in B–B′″ (white square) were magnified in C–C′″. Arrow heads (A–A′″) and asterisks (C–C′″) indicate arterioles in the kidneys Arrows indicate representative myofibroblasts expressing pS6K (C–C′″). Scale bar: 50 µm. NIH3T3 cells were cultured for 24 hours in the absence (E–E″) or presence of 20 ng/ml recombinant human TGF-β1 (F–F′″, G–G′″), with administration of vehicle (F–F′″) or rapamycin (G–G′″). The cells were stained for αSMA (green) and pS6K (red). DAPI was used to stain the nuclei. Magnification was 400×. (H–I): Representative Western blot and quantitative assessment for expression of αSMA and pS6K in NIH3T3 cells. β-actin was used in this experiment to control for equal protein loading. *P<0.05, **P<0.01 vs. control groups. # P<0.05, ## P<0.01 vs. TGF-β+vehicle groups. Error bars represent S.E.

Article Snippet: In brief, cryosections were air-dried for 15 min, then primary antibodies against the following proteins were used: pS6K (rabbit, 1∶100, Cell signaling, USA), F4/80 (rat, 1∶100, Abcam, USA), CD3 (rabbit 1∶100, Vectorlab, USA), CD4 (mouse, 1∶100, Abcam, USA), anti-neutrophil (rat, 1∶50, Santa Cruz, USA), αSMA (mouse, 1∶200, Abcam, USA), collagen-I (rabbit, 1∶500, Abcam, USA), Kim-1 (goat, 1∶100, R&D, USA), Lotus tetragonolobus lectin (LTL, 1∶1000, Vectorlab, USA).

Techniques: Staining, Derivative Assay, Labeling, Expressing, Cell Culture, Recombinant, Western Blot, Control

Journal: PLoS ONE

Article Title: Rapamycin Ameliorates Kidney Fibrosis by Inhibiting the Activation of mTOR Signaling in Interstitial Macrophages and Myofibroblasts

doi: 10.1371/journal.pone.0033626

Figure Lengend Snippet: HK2 cells or Animals were treated as described in . A–C. Representative immunofluorescent costaining images of kidney sections derived from UUO mice or IRI mice, using anti-Kim-1 and anti-αSMA (A), anti-Kim-1 and anti-CD11b (B), anti-pS6K and anti-Kim-1 (C). Nuclei were labeled with DAPI (blue). Scale bar: 50 µm. D. HK2 cells were cultured for 48 hours in the absence or presence of aristolochic acid (AA), with or without administration of rapamycin. The cells were stained for Ki67 or pS6K. DAPI was used to stain the nuclei. E. Representative western blot and densitometric analyses for expression of CTGF, Collagen-I, and pS6K in cultured HK2 cells. β-actin was used in this experiment for equal protein loading control. Data were presented as mean ± SE. ** P<0.01, NS no significance. F. Assessment of proinflammatory and profibrogenic gene expression in culture HK2 cells. mRNA level of MCP-1, TGF-β, CTGF and Collagen-I were determined by realtime-PCR. ** P<0.01, NS no significance. Error bars represent S.E.

Article Snippet: In brief, cryosections were air-dried for 15 min, then primary antibodies against the following proteins were used: pS6K (rabbit, 1∶100, Cell signaling, USA), F4/80 (rat, 1∶100, Abcam, USA), CD3 (rabbit 1∶100, Vectorlab, USA), CD4 (mouse, 1∶100, Abcam, USA), anti-neutrophil (rat, 1∶50, Santa Cruz, USA), αSMA (mouse, 1∶200, Abcam, USA), collagen-I (rabbit, 1∶500, Abcam, USA), Kim-1 (goat, 1∶100, R&D, USA), Lotus tetragonolobus lectin (LTL, 1∶1000, Vectorlab, USA).

Techniques: Derivative Assay, Labeling, Cell Culture, Staining, Western Blot, Expressing, Control, Gene Expression