Correlation of TGF-β and GREM1 expression in transgenic line A homozygous mice.

<p>TGF-β gene expression was measured in FA-injected GREM1 transgenic mice. We observed a strongly positive correlation between TGF-β and GREM1 expression (p≤0.029, R  =  0.67; 4-9 mice per group).</p

FA injection induces interstitial cell infiltration in transgenic line A homozygous mice.

<p>The inflammatory cell infiltration was characterized by immunohistochemistry with anti-F4/80 (monocytes/macrophages) and anti-CD3 (T cells) antibodies. (A and B). Representative immunostaining of one mouse from each group (x400 magnification). (C) Quantification of positive IHC signals were quantified for (a) F4/80, (b) CD3 and (c) PCNA using KS300 image analyzer software. All parameters were significantly increased in transgenic mice. Data are shown as the mean ± SEM of 14–18 mice per group * p < 0.05; ** p < 0.01 vs WT-FA.</p

Generation and validation of transgenic mice with specific tubular GREM1 overexpression.

<p>(a) Illustration of the pKAP GREM1-c-myc-IRES-eGFP plasmid. Restriction sites used for transgene isolation are indicated with EV (<i>Eco</i>RV) and A (<i>Ase</i>I). (b) <b>eGFP and c-myc detection in renal tubular epithelial cells of transgenic mice.</b> Immunofluorescence against eGFP and immunohistochemistry for c-myc (peroxidase immunostaining) to detect these proteins in the kidney tissue of transgenic males from line A and WT mice (400x). (c) Kidneys were dissected from WT and transgenic male mice of lines A, B, C, D and E, and isolated proteins were subjected to western blotting using an antibody against c-myc (1∶1000); anti β-actin (1∶2500) was used as a loading control. GREM1 expression was determined by densitometric analysis of the c-myc/β-actin ratio and normalized to transgenic line C expression.</p

Molecular characterization of transgenic mice.

<p>Qualitative and quantitative data from the transgenic lines are shown. Copy number and GREM1 expression levels were determined using real-time PCR. The eGFP signal was qualitatively analyzed. c-myc levels were quantified by densitometric analysis in each line and normalized to line C. Ectopic expression is indicated as the number of the eight analyzed extrarenal organs that demonstrated increased for GREM1 expression compared with wild-type expression. M, male; F, female; n.d., no data. *** p < 0.001.</p

Effect of FA administration on GREM1 expression in transgenic line A homozygous mice.

<p>Gremlin expression in transgenic mice was examined 7 and 14 days after treatment with FA. The GREM1 relative expression in homozygous mice of transgenic line A was increased at 7 days after injection with FA and remained increased at 14 days. Data are shown as the mean ± SEM of 4-9 mice per group * p≤0.0049 TG-FA vs TG-Veh, used as control because no WT littermates of the transgenic homozygotes mice were available.</p

In vitro validation of the GREM1 plasmid.

<p>(a) EBNA293 cells were transfected with pCDNA3-GREM1-c-myc-IRES-eGFP, as described in the methods. Immunofluorescence shows that eGFP (green) and c-myc (red) are expressed in the same transfected EBNA293 cell. Nuclei were stained with DAPI (blue) (1000x). (b) In HK-2 cells transfected with pCDNA3-GREM1-c-myc-IRES-eGFP, GREM-1 expression was evaluated by immunocytochemistry using an antibody against GREM-1, followed by a secondary TRICT antibody (red staining). The figure shows eGFP and GREM-1 expressed in the same cell (800x). (c) Confocal immunofluorescence of HK-2 transfected cells, showing the loss of E-cadherin and induction of vimentin in eGFP-positive GREM-1-expressing cells (1600X). E-cadherin and vimentin immunostaining was detected with secondary anti-FITC antibodies (green).</p