Nsets, 12006. One-way ANOVA, n = 5, data are means 6 SD. ***: vs NS day 28, P,0.001. doi:10.1371/journal.pone.0055027.gwere probed with goat ��-Sitosterol ��-D-glucoside anti-rabbit or goat anti-rat with Alexa Fluor 555 conjugate (1:2000; Molecular Probes, Eugene, OR). Sections were counterstained with 4, 6-diamidino-2 phenylindole (DAPI) to visualize nuclei and mounted with Fluorescence Mounting Medium (Dako Cytomation). Sections were analyzed with an Olympus Fluoview 1000 confocal microscope (Olympus, Tokyo, Japan), FV10-ASW software (version 1.3c; Olympus), oil UPLFL 60x objective (NA1.25; Olympus) at x2 or x3 digital zoom. Contrast and brightness of the images were adjusted further in ImageJ.TUNEL AssayApoptotic assays were performed by TdT mediated X-dUTP nicked labeling (TUNEL) reaction using ApopTagH Fluorescein In Situ Apoptosis Detection Kit (Merck Millipore, Kilsyth, Vic, Australia). Apoptotic endothelial cells and podocytes were identified by double labelling using TUNEL and anti-CD31 or anti-synaptopodin. Goat anti-rat Alexa Fluor 555 conjugate (1:2000) and goat anti-rabbit Alexa Fluor 555 conjugate (1:2000) were used. Sections were counterstained with DAPI.SDS-PAGE gel before transferring to a PVDF membrane. After blocking for 30 minutes at 4uC in blocking buffer (5 skim milk powder in PBS with 0.1 Tween 20), the membrane was incubated overnight with rabbit anti-synaptopodin (1:8000) or rabbit anti-eNOS (1:4000) (Santa Cruz Biotechnology, Inc). The membrane was washed and incubated for 30 minutes 
at room temperature with a goat anti-rabbit antibody conjugated with HRP. After further washing, the membrane was detected with ECL kit (Amersham Pharmacia Biotech, Arlington, IL, USA). atubulin and GAPDH were used as internal controls and detected by mouse anti-a-tubulin antibody conjugated with HRP and mouse anti-GAPDH antibody conjugated with HRP. Western blotting images were captured by Kodak 4000 mm and density of the bands was quantitated by using ImageJ (http://rsb.info.nih. gov/ij/).Statistical AnalysesData are mean 6 SD with statistical analyses performed using one way or two-way ANOVA from GraphPad Prism 5.0 (GraphPad Software, San Diego, CA) and post test Tukey analysis when appropriate. P,0.05 was considered statistically significant.Western blottingKidney tissues and cell culture samples were sonicated and lysed in 0.4 ml RIPA lysis buffer. The tissue and cell extracts were centrifuged at 3000 rpm and 4uC for 30 minutes to remove cell debris. The SC 1 web protein concentrations were measured by modified Lowry protein assay using BSA as a 11967625 protein standard (DC protein assay kit, Biorad). Proteins were electrophoresed through a 10Results Characteristics of ADR-Induced Nephropathy in C57BL/6 mice with eNOS deficiencyIn normal saline (NS)-treated wild type and eNOS-deficient C57BL/6 groups, glomeruli and tubulointerstitium histology wereGlomerular Endothelial Cell InjuryFigure 6. Glomerular endothelial cell and podocyte injury in ADR-induced nephropathy in Balb/c mice. (A) Western blotting detected expression of CD31 and synaptopodin in NS-treated and ADR-treated kidneys in Balb/c mice. (B) Quantification of CD31/a-Tubulin and synaptopodin/ a-Tubulin in Western blotting. Immunostaining of CD31+ (glomerular endothelial cells) (D) and synaptopodin+ (podocytes) (F) in ADR-induced nephropathy. NS-treated kidneys were used as normal controls (C E). Quantification of CD31 (G) and synaptopodin (H) staining in NS-treated and ADR-treated kidneys. One-way ANOVA, n = 6,.Nsets, 12006. One-way ANOVA, n = 5, data are means 6 SD. ***: vs NS day 28, P,0.001. doi:10.1371/journal.pone.0055027.gwere probed with goat anti-rabbit or goat anti-rat with Alexa Fluor 555 conjugate (1:2000; Molecular Probes, Eugene, OR). Sections were counterstained with 4, 6-diamidino-2 phenylindole (DAPI) to visualize nuclei and mounted with Fluorescence Mounting Medium (Dako Cytomation). Sections were analyzed with an Olympus Fluoview 1000 confocal microscope (Olympus, Tokyo, Japan), FV10-ASW software (version 1.3c; Olympus), oil UPLFL 60x objective (NA1.25; Olympus) at x2 or x3 digital zoom. Contrast and brightness of the images were adjusted further in ImageJ.TUNEL AssayApoptotic assays were performed by TdT mediated X-dUTP nicked labeling (TUNEL) reaction using ApopTagH Fluorescein In Situ Apoptosis Detection Kit (Merck Millipore, Kilsyth, Vic, Australia). Apoptotic endothelial cells and podocytes were identified by double labelling using TUNEL and anti-CD31 or anti-synaptopodin. Goat anti-rat Alexa Fluor 555 conjugate (1:2000) and goat anti-rabbit Alexa Fluor 555 conjugate (1:2000) were used. Sections were counterstained with DAPI.SDS-PAGE gel before transferring to a PVDF membrane. After blocking for 30 minutes at 4uC in blocking buffer (5 skim milk powder in PBS with 0.1 Tween 20), the membrane was incubated overnight with rabbit anti-synaptopodin (1:8000) or rabbit anti-eNOS (1:4000) (Santa Cruz Biotechnology, Inc). The membrane was washed and incubated for 30 minutes at room temperature with a goat anti-rabbit antibody conjugated with HRP. After further washing, the membrane was detected with ECL kit (Amersham Pharmacia Biotech, Arlington, IL, USA). atubulin and GAPDH were used as internal controls and detected by mouse anti-a-tubulin antibody conjugated with HRP and mouse anti-GAPDH antibody conjugated with HRP. Western blotting images were captured by Kodak 4000 mm and density of the bands was quantitated by using ImageJ (http://rsb.info.nih. gov/ij/).Statistical AnalysesData are mean 6 SD with statistical analyses performed using one way or two-way ANOVA from GraphPad Prism 
5.0 (GraphPad Software, San Diego, CA) and post test Tukey analysis when appropriate. P,0.05 was considered statistically significant.Western blottingKidney tissues and cell culture samples were sonicated and lysed in 0.4 ml RIPA lysis buffer. The tissue and cell extracts were centrifuged at 3000 rpm and 4uC for 30 minutes to remove cell debris. The protein concentrations were measured by modified Lowry protein assay using BSA as a 11967625 protein standard (DC protein assay kit, Biorad). Proteins were electrophoresed through a 10Results Characteristics of ADR-Induced Nephropathy in C57BL/6 mice with eNOS deficiencyIn normal saline (NS)-treated wild type and eNOS-deficient C57BL/6 groups, glomeruli and tubulointerstitium histology wereGlomerular Endothelial Cell InjuryFigure 6. Glomerular endothelial cell and podocyte injury in ADR-induced nephropathy in Balb/c mice. (A) Western blotting detected expression of CD31 and synaptopodin in NS-treated and ADR-treated kidneys in Balb/c mice. (B) Quantification of CD31/a-Tubulin and synaptopodin/ a-Tubulin in Western blotting. Immunostaining of CD31+ (glomerular endothelial cells) (D) and synaptopodin+ (podocytes) (F) in ADR-induced nephropathy. NS-treated kidneys were used as normal controls (C E). Quantification of CD31 (G) and synaptopodin (H) staining in NS-treated and ADR-treated kidneys. One-way ANOVA, n = 6,.