Fig 1.
PEGylated FUD retains the ability of FUD to inhibit plasma and cellular fibronectin fibrillogenesis by human fibroblasts and proximal tubular epithelial cells in vitro.
Microtiter assays measuring fluorescence associated with incorporation of fibronectin into ECM (left panels) and corresponding cell viability (right panels) following doses of FUD and PEG-FUD up to 500 nM. A) Human fibroblast incorporation of A488-plasma fibronectin; B) human fibroblast and C) human proximal tubular epithelial cell incorporation of cellular fibronectin recognized with A488-anti-EDA mAb. Data were collected in triplicate per dose and is depicted as mean +/- SD.
Fig 2.
Control peptide, PEG-mFUD, does not inhibit plasma fibronectin fibrillogenesis by human fibroblasts in vitro.
Microtiter assay measuring fluorescence associated with A488-plasma fibronectin into ECM (left panel) and corresponding cell viability (right panel) following doses of FUD, PEG-FUD and PEG-mFUD up to 20 μM, approximating the dose used in vivo. Data was collected in triplicate per dose and is depicted as mean +/- SD.
Fig 3.
Subcutaneously administered PEG-FUD is found intact in UUO kidneys, recognized by anti-FUD polyclonal antibody.
Immunoblotting of 10 μg/lane ECM fractions of UUO kidney extracts from mice subcutaneously administered with saline, PEG, FUD or PEG-FUD. The first two lanes contain purified FUD (1000 ng) and PEG-FUD (0.005 ng). Blot was reacted with 0.7 μg/ml rabbit polyclonal antibody generated to KLH-C-terminal half of FUD. Loading control for ECM fractions was achieved with rabbit-anti Histone3 (1:10000). Molecular weight markers are depicted to the left of the blot. Primary antibodies were followed by a 1:10000 dilution of anti-rabbit-HRP-IgG and chemiluminescence. Mouse ID numbers are depicted above corresponding lane. Note the polyclonal anti-FUD 1) recognizes PEGylated FUD much better than unconjugated FUD both in purified form and tissue extracts; and 2) is highly specific for the two bands associated with PEG-FUD without spurious reactivity towards other proteins in kidney tissues; 3) no PEG-FUD fragmentation was detected.
Fig 4.
IHC shows the increased level of fibronectin found in the interstitium of UUO compared to contralateral kidneys was significantly reduced with PEG-FUD treatment.
Representative images from the central cortex of 4 μm kidney sections stained with rabbit polyclonal antibody to fibronectin IgG (RamFN) at 0.5 μg/ml. UUO and contralateral kidneys were stained simultaneously. Except for the contralateral kidney of saline treated mice shown in top left panel, all other images are from UUO kidneys for comparison of treatment with saline, PEG, FUD, PEG-FUD and PEG-mFUD. Bar = 200 μm. Quantitation of staining was performed using Image J and the mean of six images per treatment per cohort +/- SD is graphed. Significance is denoted as *** p<0.001; **p<0.01, *p<0.05.
Fig 5.
Fibronectin is enriched in the ECM fraction of UUO kidneys from mice treated with saline.
Distribution of fibronectin into different tissue compartments was analyzed by fractionation of UUO and contralateral kidneys from three different mice treated with saline (mouse IDs above lanes) into DOC-insoluble (pellets) corresponding largely to ECM and DOC-soluble (lysates) corresponding to cytosolic and membrane proteins (10 μg/lane). A) Western blotting was carried out incubating the blot with RamFN IgG at 2 ng/ml followed by anti-rabbit-HRP IgG at 1:10000. Loading controls were GAPDH for lysate fractions and histone 3 (H3) for DOC-insoluble fractions. Molecular weight markers are depicted to the left of the blot. B) Quantitation of bands was carried out using Image J. Mouse ID numbers are depicted above corresponding lane. The means of fibronectin (FN) bands normalized to loading control bands from three mice +/- SD are presented. Significance is denoted as ** p< 0.01, as per Student t-Test analysis.
Fig 6.
Immunoblotting shows PEG-FUD significantly decreased fibronectin without degradation in ECM fractions of UUO kidneys.
DOC-insoluble fractions from UUO kidneys (10 μg/lane) from mice treated with saline, PEG, FUD or PEG-FUD were immunoblotted using RamFN IgG at 2 ng/ml and anti-histone3 at 1:10000, followed by anti-rabbit-HRP IgG at 1:10000. Quantitation of fibronectin and histone 3 band intensities was carried out using Image J. The means of fibronectin (FN) bands normalized to H3 from 3 or 5 mice (as indicated) +/- SD are presented. Mouse ID numbers are depicted above corresponding lane. Significance is denoted as * p < 0.05; ** p < 0.01, as per Student t-Test analysis.
Fig 7.
Collagens increased in the interstitium of UUO kidneys is significantly reduced by FUD and PEG-FUD.
Representative images from the central cortex of 4 μm kidney sections stained with picrosirius red, which stains primarily collagens I and III. Birefringence elicited from exposure to polarized light was imaged. UUO and contralateral kidneys were stained simultaneously. Except for the contralateral kidney of saline treated mice, all other images are from UUO kidneys for comparison of treatment with saline, PEG, FUD, PEG-FUD and PEG-mFUD. Bar = 200 μm. Quantitation of staining was performed using Image J and the mean of six images per treatment per cohort +/- SD was graphed. Significance is denoted as * p < 0.05; ** p<0.01, as per Student t-Test analysis.
Fig 8.
IHC shows the increased level of CD45+ leukocytes found primarily in the interstitium of UUO kidneys was significantly reduced with FUD and PEG-FUD treatment.
Representative images from the central cortex of 4 μm kidney sections stained with rat anti-mouse CD45 at 2.5 μg/ml. UUO and contralateral kidneys were stained simultaneously. Except for the contralateral kidney of saline treated mice, all other images are from UUO kidneys for comparison of treatment with saline, PEG, FUD, PEG-FUD and PEG-mFUD. Bar = 200 μm. Quantitation of staining was performed using Image J and the mean of six images per treatment per cohort +/- SD is graphed. Significance is denoted as * p< 0.05; ** p<0.001.
Fig 9.
Hematoxylin-eosin staining of UUO kidneys shows improved histology with PEG-FUD treatment.
Representative images from the central cortex of 4 μm kidney sections stained with H&E. UUO and contralateral kidneys were stained simultaneously. Except for the contralateral kidney of saline treated mice, all other images are from UUO kidneys for comparison of treatment with saline, PEG, FUD, PEG-FUD and PEG-mFUD. Eosin staining was more prominent in the saline treated contralateral kidney, was associated with intact proximal tubules (arrowheads) and with defined glomeruli containing red blood cells (double asterisks). Hematoxilin staining associated with infiltrating cells, interstitial cell proliferation (arrows) and less defined glomeruli lacking red blood cells (single asterisk) were more prominent in the saline, PEG or PEG-mFUD-treated UUO kidneys. Diminished hematoxylin, increased eosin staining associated with tubular structures, and vascularized glomeruli similar to the contralateral kidney, were more apparent in the FUD and, to a greater extent, the PEG-FUD-treated kidneys, suggesting improvement in tubular and glomerular health. Bar = 100 μm.