1D11 prevents fibrosis in the NEP25 podocyte ablation model.

<p>(A and B) Representative images of NEP25 mouse kidney samples with silver- (A) and PAS staining (B). Disease was induced by injection of LMB2, and 1D11 or 13C4 (control IgG) was administered on day -1 and day 0, followed by further injections every other day until sacrifice at day 28. Scale bar = 100 μm. 40x objective. (C) Glomerular mRNA content was analyzed with laser-captured glomeruli from cryosectioned kidney samples. † <i>P</i> < 0.05 compared to healthy controls by t-test with Welch correction.</p

1D11 administration after the onset of proteinuria prevents podocyte detachment.

<p>Left upper panel: healthy control, right upper panel: LMB2-induced disease mouse. Lower panels: LMB2-induced disease in mice that received 1D11 after detection of proteinuria on day 3. Representative images are shown.</p

1D11 ameliorates fibrosis when administered after the onset of proteinuria prevents glomerular fibrosis.

<p>(A) Three days after disease induction, spot urine was tested for proteinuria and mice that were positive of proteinuria were randomly allocated to either 1D11 or 13C4 group and histology was evaluated day 28. Each dot represents the median of the glomerular scar score, which was evaluated by a blinded observer in approximately 80–100 glomeruli of a PAS-stained section. † <i>P</i> < 0.05 compared to healthy controls by Dunn’s post-hoc test compared to no disease controls, following Kruskal-Walis one-way ANOVA. * <i>P</i> < 0.05 compared to LMB2 only group by Mann Whitney test. (B) Glomerular mRNA content was measured with laser-captured glomeruli from cryosectioned kidney samples. † <i>P</i> < 0.05 compared to healthy controls by Bonferroni’s post-hoc test compared to no disease controls, following one-way ANOVA. * <i>P</i> < 0.05 compared to LMB2 only group by unpaired, two-tailed, <i>t</i>-test. (C and D) Representative PAS staining images (C) and immunohistochemical evaluation of type I collagen (D) from the experiment analyzed in A and B are shown. Scale bar = 20 μm. 20x objective. (E) mRNA expression of three TGF-β isoforms in whole kidney of NEP25 mice was analyzed by qPCR. Relative changes compared to day 0 are shown. *<i>P</i> < 0.05 compared to day 0. (F) Mice were injected with 1.0 mg/kg BW of adriamycin via tail vain, and 1D11 administration was started at day 3, followed by every other day, and mice were sacrificed at day 10 for evaluation. Glomerular scar score was evaluated as described for Fig 5A. † <i>P</i> < 0.05 compared to healthy controls. * <i>P</i> < 0.05 compared to LMB2 only group by Mann Whitney test.</p

1D11 does not affect proteinuria in NEP25 model.

<p>Urine albumin, and urine and serum creatinine concentations were analyzed by ELISA kits. Urine albumin to creatinine ratio (left) and serum creatinine concentration (right) were shown after logarithmic transformation. † <i>P</i> < 0.05 compared to healthy controls by Bonferroni’s post-hoc test compared to no disease controls, following one-way ANOVA.</p

1D11 suppresses TGF-β signaling <i>in vitro</i>.

<p>(A) Formalin-fixed, paraffin-embedded kidney sections were stained for pSmad3 (brown nuclei) followed by counterstaining with hematoxylin (blue nuclei). Scale bar = 100 μm. 40x objective with oil. (B) mRNA expression of TGF-β isoforms was evaluated with whole kidney lysates. † <i>P</i> < 0.01, compared to healthy control, * <i>P</i> < 0.01 compared to disease control (3^rd column from the left), † <i>P</i> < 0.01, compared to healthy control by Bonferroni’s post-hoc test following one-way ANOVA.</p

Anti-TGF-β Antibody, 1D11, Ameliorates Glomerular Fibrosis in Mouse Models after the Onset of Proteinuria - Fig 6

<p>1D11 prevents podocyte loss (A) WT-1 positive cells numbers per 10⁻³ mm² were counted in ~100 glomeruli in a PAS-stained kidney section using the TissueGnostics system. Each dot represents the median of WT-1 positivity in a mouse, corrected for a healthy control as 1 from 3 independent experiments. (B) WT-1 positivity was also analyzed with mice that received 1D11 after the onset of proteinuria positive of proteinuria or relevant control mice. Each dot represents median of WT-1-positive cell numbers per 10⁻³ mm² in glomeruli. † <i>P</i> < 0.05 compared to healthy controls by Dunn’s post-hoc test compared to no disease controls, following Kruskal-Walis one-way ANOVA. Representative images of glomeruli are shown in the bottom panel. WT1: DAB, Nuclei: Hematoxylin. Scale bar = 20 μm. 40x objective. (C) Percentage of pSmad3 positive nuclei over total nucleus numbers per glomerulus was evaluated in ~100 glomeruli per section using the TissueGnositics system. † <i>P</i> < 0.05 by Bonferroni’s post-hoc test compared to healthy controls, following one-way ANOVA. Images taken by Nuance system were remixed for color separation and composite images are shown. Representative images of glomeruli are shown in the bottom panel. pSmad3: DAB, Nuclei: Hematoxylin. Scale bar = 20μm. 40x objective.</p

1D11 reversed pre-existing hepatic fibrosis.

<p><b>A</b>. Representative photomicrographs of picrosirius red stained liver sections following treatment with TAA for 8 weeks (“baseline”; TAA-8W), or an additional 8 weeks with 13C4 (13C4–16W) or 1D11 (1D11–16W) after cessation of TAA administration. A normal rat liver is also shown (Normal). Livers of rats dosed with TAA for 8 weeks (TAA-8W) had substantial lesions with widely spread fibrous bands (septa), originating from portal areas and extending into the parenchyma. 8 weeks after stopping TAA dosing, fibrotic areas were further expanded, with extensive architectural disorganization and more fibrosis covering a greater percentage of the livers (13C4–16W). Treatment with 1D11 for 8 weeks significantly ameliorated the TAA-mediated histopathological lesions, as shown by an overall improvement in hepatic morphology (1D11–16W). Liver collagen deposition in rats treated with 1D11 was noticeably much reduced than the pre-established fibrosis seen at baseline (TAA-W8), shown in rats that were examined at baseline. Magnification: 40x. <b>B</b>. Morphometric analysis of picrosirius red stained sections. Significantly less collagen was deposited in livers treated with 1D11 (▴) for 4 weeks or 8 weeks, when compared to the PBS (•, * p<0.01) or 13C4 (▪, * p<0.01) treatment groups. Moreover, comparison of 1D11 treated livers to those at week 8 before therapeutic treatment started, showed significantly less collagen deposition (treatment for 4 weeks: 9.61%; 8 weeks: 7.53%, * p<0.05) than that harvested at baseline (week 8∶12.7%). Morphometric quantification further supports the histology of a reversal of pre-existing hepatic fibrosis when TGF-β was neutralized by 1D11. Values represent the percentage of collagen deposition in the total liver section and are expressed as mean ± SE (n = 8, except n = 4 for normal controls at week 0).</p

Increased PAI-1 protein in fibrotic liver was reduced in rats dosed with 1D11.

<p><b>A</b>. A representative Western blot of pooled samples (n = 3) showed marked increase in PAI-1 expression before the start of therapeutic dosing at week 8. Levels were maintained in both PBS and 13C4 groups at the end of study at week 16. Levels of PAI-1 were normalized in livers treated with 1D11. <b>B and C</b>. Hepatic PAI-1 protein was also analyzed by ELISA. At the end of TAA administration, total (B) and active PAI-1 (C) were markedly elevated in fibrotic livers, with a further increase by week 16 in rats treated with PBS or 13C4. After 8 weeks of 1D11 dosing, total PAI-1 and active PAI-1 had returned to normal levels, confirming the immunoblotting data. Values are expressed as mean ± SE, n = 8; * p<0.01 vs. normal controls at week 8; ** p<0.01 vs. PBS or 13C4 group at week 16.</p

Experimental protocol of TAA induced liver fibrosis.

<p>Fischer rats were dosed with TAA intraperitoneally for 8 weeks (300 mg/kg for 6 weeks, three times weekly, then two times weekly for 2 weeks). A cohort of rats received PBS as normal controls. At the end of 8 weeks, TAA was withdrawn and rats were divided into three cohorts that were given PBS, 13C4 or 1D11 for 8 weeks. 13C4 and 1D11 were dosed at a concentration of 5 mg/kg, intraperitoneally, three times per week.</p

1D11 reduced cholangiocarcinomas induced by TAA.

<p><b>A</b>. TAA induced profound neoplastic changes in biliary ductules (cholangiocarcinoma) at week 16. As visualized by CK staining, these neoplastic cells extended into the parenchyma, as fibrosis evolved (arrow heads). The neoplasia displayed a typical “intestinal metaplasia”-like appearance (arrows). The number of neoplastic biliary ductules or cells was much lower with 1D11 treatment, compared to the PBS or 13C4 groups. For comparison, a normal liver stained for CK is shown. Magnification: x100 <b>B</b>. CK staining was quantitated. The TAA-induced neoplasia was significantly reduced in rats treated with 1D11 for 8 weeks. In contrast, the neoplasia showed no reduction in rats dosed with PBS or 13C4. Values are expressed as mean ± SE of percentage of CK staining of the entire area of liver section. n = 8; * p<0.01 vs. normal controls; ** p<0.01 vs. PBS, 13C4 and normal control groups. <b>C</b>. To quantify the areas of cholangiocarcinoma, liver sections from the 16 time point were scanned by ChromaVision Imaging Analysis System. The area of the cholangiocarcinomas defined as neoplastic biliary epithelial cells plus stromal fibrotic tissue and infiltrated cells, was measured and expressed as a percentage of the total area of the liver section. Data revealed a striking reduction with 1D11 treatment, as compared to the PBS and 13C4 groups, reflecting a diminishment of cholangiocarcinoma by TGF-β neutralization. n = 8, * p<0.01 vs. normal controls; ** p<0.01 vs. PBS, 13C4 and normal control groups. Values represent mean ± SE.</p