1. Volume 84, Issue 1, Pages 78-89 (July 2013)
Fibrocytes develop outside the kidney but contribute to renal fibrosis in a mouse model 
Barbara Reich, Kathrin Schmidbauer, Manuel Rodriguez Gomez, Fabian Johannes Hermann, Nicole Göbel, Hilke Brühl, Isabel Ketelsen, Yvonne Talke, Matthias Mack 
Kidney International 
Volume 84, Issue 1, Pages (July 2013)
DOI: /ki
Copyright © 2013 International Society of Nephrology Terms and Conditions

2. Figure 1
Expression of surface markers on splenic and renal fibrocytes. (a) Gating strategy to identify fibrocytes by flow cytometry as shown for splenic fibrocytes: elimination of cell duplets (Gate A), identification of peripheral blood mononuclear cells (PBMCs) by light scatter properties (Gate B), gating for CD45-positive cells (Gate C), and identification of fibrocytes by intracellular staining of collagen I (Gate D) compared with control immunoglobulin G (IgG). (b) Numbers of fibrocytes in the obstructed kidney and spleen at various time points after unilateral ureteral obstruction (UUO) as quantified by flow cytometry with counting beads (mean±s.e.m., n=3 per group). (c) Fluorescence-activated cell sorting (FACS) plots showing expression of collagen-I in combination with CD115, CCR2, Gr-1, Ly-6G, and Ly-6C on splenic CD45+CD11b+ cells at day 7 after UUO (upper panel). (d) Percentage of collagen I+ fibrocytes expressing CD115, CCR2, Gr-1, Ly-6G, and Ly-6C at various time points after UUO (mean±s.e.m., n=3).
Kidney International  , 78-89DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

3. Figure 2
Depletion of monocytes with the monoclonal antibody MC-21 from day -1 to day 6. MC-21 (black bars) or isotype control antibodies (white bars) were injected from day -1 to day 6 (n=6 per group) in relation to unilateral ureteral obstruction (UUO) on day 0. Gr-1+ monocytes, Gr-1− monocytes, and fibrocytes were quantified by flow cytometry and counting beads (mean±s.e.m.). (a) Peripheral blood monocytes were analyzed at various time points after UUO. Representative fluorescence-activated cell sorting (FACS) plots of CD45+ peripheral blood mononuclear cells (PBMCs) are shown. (b) Number of monocytes in the spleen and UUO kidney at day 7 after UUO. (c) Number of fibrocytes in the obstructed (UUO), contralateral (Contra) kidney, or (d) spleen of mice at day 7 after UUO. Representative FACS dot plots are shown (gated on CD45+ cells). IgG, immunoglobulin G. (e) Expression of collagen type I mRNA in the obstructed (UUO) or Contra kidney. #P<0.005.
Kidney International  , 78-89DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

4. Figure 3
Depletion of monocytes with the monoclonal antibody MC-21 from day -1 to day 2. Unilateral ureteral obstruction (UUO) was performed on day 0. MC-21 (black bars) or isotype control antibodies (white bars) were injected from day -1 to day 2 (n=6 per group). Gr-1+ monocytes, Gr-1− monocytes, and fibrocytes were quantified by flow cytometry and counting beads (mean±s.e.m.). (a) Number of Gr-1+ and Gr-1− monocytes in the peripheral blood at day 0 and day 3 after UUO and in the spleen and UUO kidney at day 3 after UUO. (b) Number of fibrocytes in the spleen and in the obstructed (UUO) and contralateral (Contra) kidney at day 3 after UUO. Representative fluorescence-activated cell sorting (FACS) dot plots are shown for renal fibrocytes (gated on CD45+ cells). IgG, immunoglobulin G. #P<0.005; *P<0.05.
Kidney International  , 78-89DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

5. Figure 4
Analysis of fibrocytes in CCR2−/− mice. Unilateral ureteral obstruction (UUO) was performed on day 0 in wild-type (WT, white bars) or CCR2-deficient mice (CCR2−/−, black bars) (n=5 per group). Gr-1+ monocytes, Gr-1− monocytes, and fibrocytes were quantified by flow cytometry and counting beads (mean±s.e.m.). (a) Number of Gr-1+ and Gr-1− monocytes in the peripheral blood at day 0 and day 7 with representative fluorescence-activated cell sorting (FACS) plots of CD45+ peripheral blood mononuclear cells (PBMCs). (b, c) Number of Gr-1+ and Gr-1− monocytes in the spleen and UUO kidney at day 7 after UUO. (d, e) Number of fibrocytes in the spleen, obstructed (UUO), and contralateral (Contra) kidney at day 7 after UUO. Representative FACS dot plots are shown for splenic and renal fibrocytes (gated on CD45+ cells). IgG, immunoglobulin G. (f) Expression of collagen type I mRNA in the obstructed (UUO) or Contra kidney. (g) Quantification of collagen type I staining (red) in the obstructed kidney measured by immunofluorescence as percentage of collagen-I (Col-1)-positive area (original magnification × 200, scale bar=100μm; mean±s.e.m.). *P<0.05; #P<0.005.
Kidney International  , 78-89DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

6. Figure 5
Depletion of fibrocytes in CD11b-DTR bone marrow chimeric mice. Mice were treated with phosphate-buffered saline (PBS; Control) or diphtheria toxin (DT) on day -1 and days 1 to 6 (n=5 per group). Unilateral ureteral obstruction (UUO) was performed on day 0. Gr-1+ monocytes, Gr-1− monocytes, and fibrocytes were quantified by flow cytometry and counting beads (mean±s.e.m.). (a) Number of Gr-1+ and Gr-1− monocytes in the peripheral blood at days 0, 3, and 7 after UUO together with a representative fluorescence-activated cell sorting (FACS) plot of CD45+ peripheral blood mononuclear cells (PBMCs) at day 7. (b) Number of Gr-1+ and Gr-1− monocytes in the UUO kidney at day 7 after UUO. (c, d) Number of fibrocytes in the spleen, obstructed (UUO), and contralateral (Contra) kidney at day 7 after UUO. Representative FACS dot plots are shown for splenic and renal fibrocytes (gated on CD45+ cells). IgG, immunoglobulin G. (f) Expression of collagen type I mRNA in the obstructed (UUO) or Contra kidney. (g) Quantification of collagen type I staining (red) in the obstructed kidney measured by immunofluorescence as percentage of collagen-I (Col-1)-positive area (original magnification × 200, scale bar=100μm; mean±s.e.m.). *P<0.05; #P<0.005.
Kidney International  , 78-89DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

7. Figure 6
Depletion of monocytes and fibrocytes with the antibody Gr-1. Anti-Gr-1 (black bars) or isotype control antibodies (white bars) were injected from day -1 to day 6 (n=5 per group) and unilateral ureteral obstruction (UUO) was performed on day 0. Gr-1+ monocytes, Gr-1− monocytes, and fibrocytes were quantified by flow cytometry and counting beads (mean±s.e.m.). (a) Number of Gr-1+ and Gr-1− monocytes in the peripheral blood at days 0, 3, and 7 after UUO together with representative fluorescence-activated cell sorting (FACS) plots of CD45+ peripheral blood mononuclear cells (PBMCs). (b) Number of neutrophils in the peripheral blood at days 0, 3, and 7 after UUO. (c) Number of Gr-1+ and Gr-1− monocytes in the UUO kidney at day 7 after UUO. (d, e) Number of fibrocytes in the spleen, obstructed (UUO), and contralateral (Contra) kidney at day 7 after UUO. Representative FACS dot plots are shown for splenic and renal fibrocytes (gated on CD45+ cells). (f) Quantification of collagen type I staining (red) in the obstructed kidney measured by immunofluorescence as percentage of collagen-I (Col-1)-positive area (original magnification × 200, scale bar=100μm; mean±s.e.m.). *P<0.05; #P<0.005.
Kidney International  , 78-89DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions