1. Volume 59, Issue 2, Pages 579-592 (February 2001)
TGF-β1 induces proliferation in human renal fibroblasts via induction of basic fibroblast growth factor (FGF-2) 
Frank Strutz, Michael Zeisberg, Anja Renziehausen, Birgit Raschke, Volker Becker, Cees Van Kooten, Gerhard A. Müller 
Kidney International 
Volume 59, Issue 2, Pages (February 2001)
DOI: /j x
Copyright © 2001 International Society of Nephrology Terms and Conditions

2. Figure 1
Characterization of primary cortical fibroblasts by immunofluorescence. Primary cortical fibroblast line Tk 455 stained positively for vimentin (A), collagen type I (C), collagen type III (E), and α-smooth muscle actin (F). No staining was observed for cytokeratin (B) and factor VIII (D). Costainings with the Hoechst dye H33258 identifying cell nuclei were performed.
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

3. Figure 2
FGF-2 mRNA levels in Tk 173 fibroblasts after stimulation with TGF-β1 (1 ng/mL) for 0, 3, 6, 9, 12, and 24 hours. (A) One representative Northern blot with a main band at 7.0 kb 18S-RNA was used as internal control. (B) The densitometric analyses after five different stimulations () and mRNA changes after the addition of cyclohexamide (▪). *P < 0.05; **P < 0.01 vs. control (= 0 hour).
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

4. Figure 3
Detection of FGF-2 protein in the supernatant by immunoblot (A) and ELISA (B). Cells were stimulated with 1 ng/mL TGF-β1 for 0, 3, 6, 12, 24, and 48 hours. Protein of supernatant was corrected for cell number. (A) A characteristic immunoblot illustrating the robust increase in FGF-2 protein in the supernatant peaking after 24 hours. The de novo detection of the protein after only three hours of stimulation points to a role of TGF-β1 in release of preformed FGF-2. (B) A summary of ELISAs for FGF-2 in the supernatant confirming the immunoblot data. Protein values were corrected for cell number. *P < 0.05; **P < 0.01 vs. control (= 0 hour).
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

5. Figure 4
TGF-β1–induced proliferation as determined by bromodeoxyuridine incorporation in human kidney fibroblasts. (A) The time dependency of TGF-β1–induced proliferation in Tk 173 fibroblasts after stimulation with TGF-β1 at 1 ng/mL. Cells grown in Iscove's medium without additives were used as controls (= 100%). (B) The time dependency in Tk 455 cells. The addition of a neutralizing antibody to TGF-β (gray columns) and to FGF-2 (dark gray columns) prevented the TGF-β1–induced proliferation, whereas a neutralizing antibody to EGF (light gray columns) had no effect. *P < 0.05 vs. control; **P < 0.01 vs. control.
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

6. Figure 5
Partial dose dependency of TGF-β1–induced proliferation in Tk 173 (A) and Tk 455 (B) fibroblasts, indicating a maximal promitogenic effect of 1 ng/mL. Cells were originally plated at a density of 4000 per well and stimulated with 0.1, 1.0 or 10 ng/mL TGF-β1 and counted after 72 hours. Again, neutralizing antibodies to TGF-β and FGF-2 inhibited TGF-β1–induced proliferation, whereas an irrelevant IgG had no effect. *P < 0.05 vs. control; **P < 0.01 vs. control (Iscove's medium = 100%).
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

7. Figure 6
Effects of two tyrphostines on TGF-β–induced proliferation in kidney fibroblasts. Cells were plated at a density of 103 per well and counted after 72 hours of stimulation with TGF-β1 (1 ng/mL). Whereas the FGF-receptor tyrosine kinase inhibitor tyrphostin AG1296 inhibited TGF-β1–induced proliferation in Tk 173 (A) and Tk 455 (B) kidney fibroblasts, no effect was seen with tyrphostin B42, which blocks EGF receptor signal transduction. Cells grown in Iscove's medium alone served again as controls. Neutralizing antibodies to TGF-β1 and FGF-2 demonstrated the specificity of induced proliferation. **P < 0.01 vs. control.
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

8. Figure 7
Effects of a neutralizing antibody to PDGF on TGF-β1–induced proliferation in primary kidney and skin fibroblasts. Primary cortical fibroblast lines Tk 458 (▪) and Tk 467 (gray boxes) as well as primary skin fibroblast lines Sk 448 () and Sk 449 () were subjected to stimulation with TGF-β1 (1 ng/mL). In addition, neutralizing antibodies to TGF-β1, FGF-2, PDGF, or EGF were added. After 72 hours, bromodeoxyuridine incorporation was determined. Unstimulated cells served as controls (= 100%). Whereas the neutralizing antibody to FGF-2 inhibited TGF-β1–induced proliferation in cortical fibroblasts, only partial inhibition was observed in skin fibroblasts. Conversely, the neutralizing antibody to PDGF prevented TGF-β1–induced proliferation in skin fibroblasts but had only minor effects in kidney fibroblasts. *P < 0.05 vs. control; **P < 0.01 vs. control.
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

9. Figure 8
Immunoblots for cdk 2 (A) and p27kip1 (B) in Tk 173 and Tk 455 human kidney fibroblasts. Cells were stimulated with 1 ng/mL TGF-β1 for 0, 24, 48, and 72 hours. Both cell lines displayed robust up-regulation of cdk 2 (33 kD) after 48 and 72 hours. Tk 173 cells had a much higher basal level of cdk 2 protein expression compared with primary Tk 455 fibroblasts. Conversely, levels of the cyclin-dependent kinase inhibitor p27kip1 (27 kD) were similar at base line in both cell lines and were decreased after 24 and 48 hours before returning to basal levels after 72 hours.
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions

10. Figure 9
Proposed mechanism of FGF-2 in renal fibrogenesis. Release of TGF-β1 by glomerular, tubular epithelial, and infiltrating cells results in an increased synthesis of FGF-2 in fibroblasts possibly mediating autocrine proliferation in these cells.
Kidney International  , DOI: ( /j x)
Copyright © 2001 International Society of Nephrology Terms and Conditions