1. Volume 80, Issue 10, Pages 1035-1044 (November 2011)
Fibrinogen, acting as a mitogen for tubulointerstitial fibroblasts, promotes renal fibrosis 
Inga Sörensen, Nathan Susnik, Therese Inhester, Jay L. Degen, Anette Melk, Herrmann Haller, Roland Schmitt 
Kidney International 
Volume 80, Issue 10, Pages (November 2011)
DOI: /ki
Copyright © 2011 International Society of Nephrology Terms and Conditions

2. Figure 1
Fibrinogen accumulates in unilateral ureteral obstruction (UUO) kidneys and fibrinogen deficiency is associated with attenuated tubulointerstitial damage. (a) Immunoblot at 7 days of UUO in wild-type (WT) mice shows a massive increase in renal fibrinogen (Fib) in the obstructed vs contralateral kidney. (b) Fibrinogen immunosignal is confined to the vascular space in contralateral WT kidney, (c) but present throughout the tubulointerstitium after UUO. (d) Representative immunostaining for fibrinogen at 14 days of UUO in Fib+/− showing outer medulla and cortex. (e) Detail from the outer medulla of the same section. (f) As expected, no fibrinogen was found in kidneys from Fib−/− mice. Representative hematoxylin/eosin stains of cortex and outer medulla from Fib+/− and Fib−/− kidneys at (g, h) 7 and (j–m) 14 days of UUO. (i) At day 7, both groups showed dilated tubules with flattened epithelium but no significant difference in tubulointerstitial damage quantification. (i–m) At 14 days, loss of tubular structures and expansion of interstitial mass was significantly more severe in Fib+/− when compared with Fib−/− mice. Original magnifications: × 100 (j, k); × 200 (d); × 400 (b, c, f–h, l, m); × 630 (e). Scale bars represent mean values±s.e.m. *P<0.05; **P<0.01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

3. Figure 2
Fibrinogen deficiency is associated with reduced collagen accumulation. (a, a’) Representative microphotographs of collagen deposition as visualized by Picrosirius Red staining using (a) light microscopy and (a’) polarized light at 14 days of unilateral ureteral obstruction (UUO). (b) Quantification of Picrosirius Red polarized images in cortex and outer medulla (OM). (c) Immunoblot analysis for Collagen I using Fib+/− and Fib−/− kidneys at 14 days of UUO. (d) Representative microphotographs of immunostaining for CD45. (e) Quantification of CD45-positive leukocytes, CD4-positive lymphocytes, F4/80-positive macrophages, and Ly-6B.2-positive polymorphonuclear cells in UUO kidneys at 7 and/or 14 days of UUO. Original magnification × 400 (a, a’, d). Scale bars represent mean values±s.e.m. *P<0.05; **P<0.01.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

4. Figure 3
Fibrinogen deficiency is associated with a reduced upregulation of α-smooth muscle actin (α-SMA) and attenuated fibroblast proliferation. (a) Representative immunostaining for α-SMA, a marker for myofibroblasts, comparing Fib+/− and Fib−/− kidneys at 14 days of unilateral ureteral obstruction (UUO). (b) Immunoblot for α-SMA at 14 days of UUO. (c, d) Representative immuno-double staining for S100A4 and CD45. (c) Higher magnification of boxed area in d showing S100A4-positive/CD45-negative cells (arrows), CD45-positive/S100A4-negative cells (∼), and double-positive cells (asterisks). (e) S100A4-positive/CD45-negative cells were counted as fibroblasts comparing Fib+/− and Fib−/− kidneys at 14 days of UUO. (f, g) Immunostaining for fibroblast marker ER-TR7 and quantification of ER-TR7-positive cells in UUO kidneys at 14 days. Original magnifications: × 400 (a, f); × 630 (c, d). Scale bars represent mean values±s.e.m. *P<0.05.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

5. Figure 4
Fibrinogen has a direct effect on the proliferation of interstitial fibroblasts in vivo as well as in vitro. (a) Quantification of immunostaining for Ki67, a cell cycling marker, shows significantly lower rates of interstitial cell proliferation after 7 and 14 days of unilateral ureteral obstruction (UUO) in Fib−/− when compared with Fib+/− kidneys. (b) In vitro 5-bromodeoxyuridine (BrdU) proliferation analysis of rat renal fibroblasts (NRK49-F) after exposure to fibrinogen in different concentrations. (c) In vitro BrdU proliferation analysis of rat renal fibroblasts after exposure to plasma from Fib−/− and Fib+/− mice. (d) Representative laser scanning microphotograph of rat renal fibroblasts immunostained for α-smooth muscle actin (α-SMA; red) and fibronectin (green) after exposure to fibrinogen (1mg/ml) or to transforming growth factor-β (TGF-β; 2ng/ml; positive control). Fibrinogen did not significantly induce α-SMA or fibronectin expression. Original magnification (d) × 630. Scale bars represent mean values±s.e.m. *P<0.05; **P<0.01; ***P<
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

6. Figure 5
Mitogenic effect of fibrinogen on renal fibroblasts is mediated via Toll-like receptor (TLR)2, TLR4, and intercellular adhesion molecule 1 (ICAM-1). (a) 5-Bromodeoxyuridine (BrdU) proliferation assay in rat renal fibroblasts induced by fibrinogen after TLR2, TLR4, or MyD88 small interfering RNA (siRNA) knockdown (b). (c) BrdU proliferation of primary renal fibroblasts from wild-type (WT) and MyD88 knockout mice after exposure to fibrinogen. (d) BrdU proliferation in the presence of blocking antibodies to ICAM-1 (ICAM-1 Ab) after fibrinogen induction. (e) BrdU proliferation induced by fibrinogen after siRNA knockdown of ICAM-1. Effects of TLR2, TLR4, and ICAM-1 are additive (e). Fibrinogen concentration in all assays 1mg/ml. Scale bars represent mean values±s.e.m. *P<0.05; **P<0.01.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions