Journal: PLoS ONE

Article Title: TLR4 Activation Promotes Podocyte Injury and Interstitial Fibrosis in Diabetic Nephropathy

doi: 10.1371/journal.pone.0097985

Figure Lengend Snippet: mRNA expression of TLR4 and its ligands in kidney was upregulated at 10 weeks after diabetes induction (A & B). The data are presented as means ± SD; * p <0.05; ** p <0.01; *** p <0.001. n = 7 per group. (C) Representative sections of kidney were stained with a rabbit anti-mouse-TLR4 polyclonal antibody in the top panels and a goat anti-mouse-TLR4 polyclonal antibody in the bottom panels.

Article Snippet: Sections were then incubated with 10% normal horse serum followed by 60-minute incubation with primary antibodies: a rat anti-mouse CD68 antibody (ABD Serotec Inc., Oxford, UK), a rat anti-mouse F4/80 (ABD Serotec Inc.), rabbit anti-mouse-TLR4 antibody (Invitrogen, Catalogue No. 48-2300) for glomerular , goat anti-mouse-TLR4 antibody (Santa Cruz Biotechnology, Catalogue No sc-12511) for tubular , rabbit anti-WT1 antibody (Abcam, Cambridge, UK), or concentration-matched IgG as an isotype negative control.

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: TLR4 Activation Promotes Podocyte Injury and Interstitial Fibrosis in Diabetic Nephropathy

doi: 10.1371/journal.pone.0097985

Figure Lengend Snippet: WT diabetic mice showed significant glomerular injury including increased kidney to body weight ratio (A), glomerular volume (B), glomerular cellularity (C, week 24), glomerular mesangial matrix (D & E), decreased podocin staining (F) and WT1 + podocyte numbers (H & I) compared to non-diabetic WT controls. These were attenuated in TLR4 −/− diabetic mice. (E) Photomicrographs of representative sections of glomerular changes at 24 weeks on PAS staining (×400 magnification). (G) Representative sections of glomeruli were stained for podocin at week 24, with similar staining intensity evident in non-diabetic WT and TLR4 −/− mice but reduced staining in diabetic mice with a substantially greater reduction seen in WT versus TLR4 −/− diabetic mice. (I) Representative sections were stained for WT1 at week 24. The data are presented as the means ± SEM; * p <0.05; *** p <0.001; *** p <0.0001. The number of animals per group was described in .

Article Snippet: Sections were then incubated with 10% normal horse serum followed by 60-minute incubation with primary antibodies: a rat anti-mouse CD68 antibody (ABD Serotec Inc., Oxford, UK), a rat anti-mouse F4/80 (ABD Serotec Inc.), rabbit anti-mouse-TLR4 antibody (Invitrogen, Catalogue No. 48-2300) for glomerular , goat anti-mouse-TLR4 antibody (Santa Cruz Biotechnology, Catalogue No sc-12511) for tubular , rabbit anti-WT1 antibody (Abcam, Cambridge, UK), or concentration-matched IgG as an isotype negative control.

Techniques: Staining

Journal: PLoS ONE

Article Title: TLR4 Activation Promotes Podocyte Injury and Interstitial Fibrosis in Diabetic Nephropathy

doi: 10.1371/journal.pone.0097985

Figure Lengend Snippet: (A) Significant interstitial collagen accumulation was evident in WT but not TLR4 −/− mice with diabetes at 12 and 24 weeks. (B) Illustration of representative sections of the histological changes on PSR staining for collagen. (C) Immunohistochemical staining showed upregulation of α-SMA in WT, but not TLR4 −/− mice, with diabetes as compared to non-diabetic controls at week 24. (D) Significant upregulation of KIM-1 was apparent in WT mice with diabetes, but attenuated in TLR4 −/− diabetic kidneys. The data are present as the means ± SEM; * p <0.05; ** p <0.01; *** p <0.001. The number of animals per group was described in .

Article Snippet: Sections were then incubated with 10% normal horse serum followed by 60-minute incubation with primary antibodies: a rat anti-mouse CD68 antibody (ABD Serotec Inc., Oxford, UK), a rat anti-mouse F4/80 (ABD Serotec Inc.), rabbit anti-mouse-TLR4 antibody (Invitrogen, Catalogue No. 48-2300) for glomerular , goat anti-mouse-TLR4 antibody (Santa Cruz Biotechnology, Catalogue No sc-12511) for tubular , rabbit anti-WT1 antibody (Abcam, Cambridge, UK), or concentration-matched IgG as an isotype negative control.

Techniques: Staining, Immunohistochemical staining

Journal: PLoS ONE

Article Title: TLR4 Activation Promotes Podocyte Injury and Interstitial Fibrosis in Diabetic Nephropathy

doi: 10.1371/journal.pone.0097985

Figure Lengend Snippet: Macrophage (CD68 + or F8/40 + ) accumulation was evident in WT mice with diabetes, but prevented by TLR4 deficiency in both the interstitial (A) and glomerular (B & C) compartments. Data are present as means ± SEM; * p <0.05; *** p <0.001.

Article Snippet: Sections were then incubated with 10% normal horse serum followed by 60-minute incubation with primary antibodies: a rat anti-mouse CD68 antibody (ABD Serotec Inc., Oxford, UK), a rat anti-mouse F4/80 (ABD Serotec Inc.), rabbit anti-mouse-TLR4 antibody (Invitrogen, Catalogue No. 48-2300) for glomerular , goat anti-mouse-TLR4 antibody (Santa Cruz Biotechnology, Catalogue No sc-12511) for tubular , rabbit anti-WT1 antibody (Abcam, Cambridge, UK), or concentration-matched IgG as an isotype negative control.

Techniques:

Journal: PLoS ONE

Article Title: TLR4 Activation Promotes Podocyte Injury and Interstitial Fibrosis in Diabetic Nephropathy

doi: 10.1371/journal.pone.0097985

Figure Lengend Snippet: The expression of endogenous TLR ligands was upregulated in WT-DN kidneys and TLR4 −/− diabetic kidneys at week 6 compared with WT non-diabetic controls. (A). Gene expression of IL-6 (A), TNF-α (B), CCL2 (C), CXCL10 (D), TGF-β (E) and fibronectin (F) were upregulated in WT mice with diabetes, but suppressed in TLR4 −/− mice with diabetes, as measured by RT-PCR. Data are presented as means ± SD; ** p <0.01; *** p <0.001. The number of animals per group was described in .

Article Snippet: Sections were then incubated with 10% normal horse serum followed by 60-minute incubation with primary antibodies: a rat anti-mouse CD68 antibody (ABD Serotec Inc., Oxford, UK), a rat anti-mouse F4/80 (ABD Serotec Inc.), rabbit anti-mouse-TLR4 antibody (Invitrogen, Catalogue No. 48-2300) for glomerular , goat anti-mouse-TLR4 antibody (Santa Cruz Biotechnology, Catalogue No sc-12511) for tubular , rabbit anti-WT1 antibody (Abcam, Cambridge, UK), or concentration-matched IgG as an isotype negative control.

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: TLR4 Activation Promotes Podocyte Injury and Interstitial Fibrosis in Diabetic Nephropathy

doi: 10.1371/journal.pone.0097985

Figure Lengend Snippet: Primary podocyte cultures were defined by positive staining for the podocyte markers podocin and nephrin (A). Primary cultures of podocytes harvested from WT and TLR4 −/− mice, were cultured in media containing physiological concentration glucose (5.5 mM glucose+24.5 mM mannitol) as an osmotic control or high-concentration glucose (5.5 mM glucose+24.5 mM glucose) for 12 hours. High glucose induced upregulation of TLR4 (B), its ligands (C) and downstream molecules (D) in WT podocytes compared to osmotic controls. Upregulation of downstream molecules induced by high glucose was not seen in TLR4 −/− podocytes(D). NF-κB DNA binding activity was increased by 2 fold in WT podocytes by high glucose stimulation, but not in TLR4 −/− podocytes (E & F). Data are presented as means ± SEM; * p <0.05; ** p <0.01; *** p <0.001.

Article Snippet: Sections were then incubated with 10% normal horse serum followed by 60-minute incubation with primary antibodies: a rat anti-mouse CD68 antibody (ABD Serotec Inc., Oxford, UK), a rat anti-mouse F4/80 (ABD Serotec Inc.), rabbit anti-mouse-TLR4 antibody (Invitrogen, Catalogue No. 48-2300) for glomerular , goat anti-mouse-TLR4 antibody (Santa Cruz Biotechnology, Catalogue No sc-12511) for tubular , rabbit anti-WT1 antibody (Abcam, Cambridge, UK), or concentration-matched IgG as an isotype negative control.

Techniques: Staining, Cell Culture, Concentration Assay, Control, Binding Assay, Activity Assay

Journal: PLoS ONE

Article Title: TLR4 Activation Promotes Podocyte Injury and Interstitial Fibrosis in Diabetic Nephropathy

doi: 10.1371/journal.pone.0097985

Figure Lengend Snippet: Primary cultures of TECs, harvested from WT and TLR4 −/− mice, were cultured in media containing physiological concentration glucose (5.5 mM glucose+24.5 mM mannitol) as an osmotic control or high-concentration glucose (5.5 mM glucose+24.5 mM glucose) for 12 hours. High glucose induced upregulation of TLR4 (A), its ligands (B) and downstream molecules(C) in WT TECs compared to osmotic controls. Upregulation of downstream molecules induced by high-glucose was not seen in TLR4 −/− TECs (C). Data are presented as means ± SEM; * p <0.05; ** p <0.01; *** p <0.001.

Article Snippet: Sections were then incubated with 10% normal horse serum followed by 60-minute incubation with primary antibodies: a rat anti-mouse CD68 antibody (ABD Serotec Inc., Oxford, UK), a rat anti-mouse F4/80 (ABD Serotec Inc.), rabbit anti-mouse-TLR4 antibody (Invitrogen, Catalogue No. 48-2300) for glomerular , goat anti-mouse-TLR4 antibody (Santa Cruz Biotechnology, Catalogue No sc-12511) for tubular , rabbit anti-WT1 antibody (Abcam, Cambridge, UK), or concentration-matched IgG as an isotype negative control.

Techniques: Cell Culture, Concentration Assay, Control