1. Volume 84, Issue 2, Pages 317-326 (August 2013)
Mast cell chymase protects against renal fibrosis in murine unilateral ureteral obstruction 
Walid Beghdadi, Lydia C. Madjene, Julien Claver, Gunnar Pejler, Lucie Beaudoin, Agnès Lehuen, Eric Daugas, Ulrich Blank 
Kidney International 
Volume 84, Issue 2, Pages (August 2013)
DOI: /ki
Copyright © 2013 International Society of Nephrology Terms and Conditions

2. Figure 1
mMCP4 chymase-deficient mice show increased renal pathology after unilateral ureteral obstruction (UUO). (a) Upper panel shows representative photomicrographs of Masson’s Trichrome–stained kidney sections from wild-type (WT) and mMCP4−/− mice at day 9 after UUO or sham treatment. Scale bar=50μm. Lower panels represent the quantitative histological evaluation of interstitial fibrosis, cellular infiltrates, and tubular dilatation. Note that sham-treated animals do not show any signs of pathology. (b) Upper panels show representative immunofluorescence light microscopy photomicrographs of kidney cryosections from WT and mMCP4−/− mice at day 9 after UUO or sham treatment after staining with anti-type I collagen. Scale bar=50μm. The corresponding quantitative evaluation of type I collagen expression is shown below. Results are the mean±s.e.m. of four independent experiments involving a total of at least 14 mice. *P<0.05, **P<0.01, ***P<0.001.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

3. Figure 2
Reconstitution of MC-deficient mice with wild-type (WT) but not mouse mast cell protease 4 (mMCP4)-deficient bone marrow–derived MC (BMMC) restores protection from pathology after unilateral ureteral obstruction (UUO). MC-deficient mice were reconstituted with either WT or mMCP4-deficient BMMC as described in Materials and Methods section. Twelve weeks after engraftment, mice were subjected to UUO together with age-matched WT, KitWsh/Wsh, and mMCP4−/−. (a) Day 9 UUO kidney sections were stained with Masson’s Trichrome before quantitative histological evaluation of interstitial fibrosis, tubular dilatation, and cellular infiltrates. (b) Day 9 UUO kidney cryosections were immunofluorescence stained with anti-type I collagen before quantitative evaluation. Results are expressed as the mean±s.e.m. of four experiments involving sets of, respectively, three mice. *P<0.05, **P<0.01, ***P<0.001.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

4. Figure 3
Early stages of fibrosis are accelerated in mouse mast cell protease 4−/− (mMCP4−/−) mice. (a) Representative images of western blot analysis examining E-cadherin and α-SMA expression in kidney lysates of wild-type (WT) and mMCP4−/− mice prepared 9 days after unilateral ureteral obstruction (UUO) or sham treatment. (b) Quantitative densitometric analysis of the ratio of α-SMA/E-cadherin versus actin loading control. Results are expressed as the mean±s.e.m. obtained from eight mice. *P<0.05, **P<0.01, ***P<0.001.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

5. Figure 4
Analysis of the inflammatory cell infiltrate. (a, left panel) Photomicrographs of kidney capsules stained with anti-mouse mast cell protease 6 (mMCP6) showing the presence of a non-activated MC in sham- and an activated degranulated mast cell in unilateral ureteral obstruction (UUO)-treated kidneys. Scale bar=25μm. Below are shown toluidine blue positive MC counts in kidney capsules from wild-type (WT) and mMCP4−/− mice (n=6) in sham- and UUO-treated mice. Note the significant decrease in countable granulated MC. (a, right panel) Representative images of western blot analysis examining the expression of the FcεRIβ chain in kidney capsule lysates of WT and mMCP4−/− mice prepared 9 days after UUO or sham treatment. The panel below shows a quantitative analysis of FcεRIβ expression relative to the actin loading control. Results are expressed as the mean±s.e.m. from eight mice. *P<0.05, **P<0.01, ***P< (b) Representative images of western blot analysis showing the specificity of the anti-mMCP4 antibody and examining the expression of mMCP4 in kidney capsule lysates of WT mice prepared 9 days after UUO or sham treatment. The panel below shows a quantitative analysis of mMCP4 expression relative to the actin loading control obtained from six mice. (c) Representative immunofluorescence staining Mac-1+ macrophages and immunohistochemistry staining of CD3+ T cells in kidney sections from WT and mMCP4−/− mice at day 9 after UUO or sham treatment. Scale bar=50μm. The corresponding quantitative evaluation is shown to the right. Results are the mean±s.e.m. from at least nine mice. **P<0.01, ***P<0.001.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

6. Figure 5
CCL2 and transforming growth factor-β1 (TGF-β1) local levels in unilateral ureteral obstruction (UUO) kidneys are increased in mMCP4−/− mice. Nine days after UUO or sham treatment, CCL2 and immunoreactive TGF-β1 concentrations in kidney lysates of wild-type (WT) and mouse mast cell protease 4−/− (mMCP4−/−) mice were determined by enzyme-linked immunosorbent assay (ELISA). Results are normalized to the protein concentrations of lysates and are the mean±s.e.m. of five independent experiments involving a total of at least 11 mice. *P<0.05, **P<0.01.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

7. Figure 6
Increased expression of fibronectin in mouse mast cell protease 4−/− (mMCP4−/−) mice after unilateral ureteral obstruction (UUO). (a) Representative photomicrographs of fibronectin immunofluorescence staining of kidney cryosections from wild-type (WT) and mMCP4−/− mice at day 9 after UUO. Scale bar=50μm. The corresponding quantitative evaluation is shown below. Results are the mean±s.e.m. of three independent experiments involving a total of at least 11 mice. **P<0.01. (b) Left panel: western blot analysis of fibronectin or fibronectin treated with 40ng of recombinant human chymase. Right panel: western blot of the kidney homogenates obtained at day 9 after sham or UUO treatment. Note the increase of unprocessed fibronectin in mMCP4−/− mice kidney. Arrows indicate possible degradation products recognized by the anti-fibronectin antibody. Data are representative of three similar experiments.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions