Table 1.
Primer sequences used for quantitative-PCR.
Figure 1.
Increased Cyr61 expression in the kidneys after unilateral ureteral obstruction (UUO) surgery.
(A–D) The Q-PCR time-course for transcripts of whole kidney Cyr61 (A), monocyte chemoattractant protein-1 (MCP-1) (B), connective tissue growth factor (CTGF) (C), and collagen type I -α1 (Co l 1-α1) (D) following UUO surgery. The expression levels were normalized by 18S ribosomal RNA. N = 8/time point. (E) Representative images of the time-course Western blot of whole kidney lysates for Cyr61 after surgery. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (F) Bar chart showing the Cyr61 protein expression time-course normalized by GAPDH. N = 8/time point. The values are the mean+SD. *P<0.05 vs. sham operation; #P<0.05 vs. contralateral (CLK) kidney.
Figure 2.
Localization of Cyr61 expression in the UUO kidneys.
Representative immunohistochemical staining for Cyr61 protein in the sham operation kidneys (A, D, G, J), and the UUO kidneys at day 1 (B, E, H, K) and day 10 (C, F, I, L) after surgery. Low-power field images (A–F) show the major distribution of Cyr61 in renal tubular epithelial cells. Images of corticomedullary junctions (A–C) disclose that Cyr61 staining was positive in all renal tubule segments within the renal cortex and medulla. High-power field images (G–I) reveal that Cyr61 expression in the tubular epithelial cells was prominent in the cytoplasm. The star marks (*) indicate the lack of significant positive stain in the fibrotic area. Negative control (NC) staining showed no signal (J–L). (M) Masson trichrome stain of the corresponding day 10 UUO kidney shows the blue staining of collagen. Scale bar = 100 µm.
Figure 3.
Transforming growth factor-β (TGF-β) activity is associated with Cyr61 gene expression after UUO.
(A) Expression of TGF-β1 transcripts normalized to 18S in the UUO kidneys, measured by Q-PCR. N = 8/time point. (B–C) Active (B) and total (C) TGF-β1 protein concentrations as determined by immunoassay in kidneys of mice receiving sham operation or UUO surgery. N = 4/time point. (D) Representative Western blot of Smad2/3 proteins in the UUO kidneys. (E) The graph summarizes the analysis for renal phosphorylated Smad2/3 normalized to total Smad2/3 after UUO. N = 4/time point. (F) The mice received an intraperitoneal injection of 10 µg/g BW pan-TGF-β monoclonal antibody (1D11) or control antibody (13C4) 2 hours before the UUO surgery and were euthanized the next day. Q-PCR showed that the increased Cyr61 transcripts in the UUO kidneys were attenuated by 1D11 treatment. N = 4/group. The values are the mean+SD. *P<0.05 vs. sham operation kidneys; #P<0.05 vs. 1D11 treated.
Figure 4.
Cyr61 expression in cultured renal tubular epithelial cells.
(A) Cultured rat proximal tubular epithelial cells (NRK-52E) were treated with various concentrations of recombinant TGF-β1 for 2 hours. Q-PCR showed Cyr61 upregulation by TGF-β1 at doses of 0.25 ng/mL or greater. N = 4/group. (B) Recombinant Cyr61 was added to NRK-52E cells for 2 or 6 hours. Q-PCR showed increased expression of MCP-1 transcripts by Cyr61 in a dose-dependent manner. N = 4/group. The values are the mean+SD. *P<0.05 vs. control.
Figure 5.
Structure specificity and neutralizing activity of anti-Cyr61 antibody.
(A) Western blot of 50 ng recombinant CTGF and Cyr61 protein detected by an anti-Cyr61 antibody. This anti-Cyr61 antibody could specifically recognize Cyr61 but not structurally relevant CTGF. (B) Culture medium containing 400 ng/mL of recombinant Cyr61, pretreated with 50 µg/mL anti-Cyr61 antibody or control rabbit IgG for 1 hour, were added to NRK-52E cells for 2 hours. Q-PCR showed a significant reduction in Cyr61-enhanced MCP-1 expression after anti-Cyr61 antibody pretreatment. N = 4/group. (C) The NRK-52E cells were treated with 1 ng/mL of TGF-β1 and 50 µg/mL of anti-Cyr61 antibody or control rabbit IgG for 24 hours. Q-PCR showed that anti-Cyr61 antibody attenuated TGF-β1-induced MCP-1 upregulation. N = 4/group. The values are the mean+SD. *P<0.05.
Figure 6.
Effect of Cyr61 blockade on renal inflammation in the UUO kidneys.
The mice received an intraperitoneal injection of 10 µg/g BW of the control rabbit IgG (filled bar) or anti-Cyr61 antibody (open bar) 2 hours before the UUO surgery and then one dose per day until euthanasia. (A–C) Graphs showing a summary of MCP-1 (A), chemokine (C-C motif) receptor-2 (CCR-2) (B), and F4/80 (C) transcripts normalized to 18S. The data are expressed as the fold differences compared to the sham operation kidneys (gray bar). N = 8/group. (D) Representative F4/80 immunofluorescence photomicrographs (magnification 400×, scale bar = 100 µm) of kidneys from mice at days 4 and 7 after UUO. The bar charts are a summary of the percentage of positive F4/80-stained areas. N = 4/group. (E and F) Quantifying the expression of the profibrogenic macrophage-associated chemokine (C-C motif) ligand 17 (CCL17) and CCL22 transcripts by Q-PCR. Graph showing the ratios of CCL17 to F4/80 (D) and CCL22 to F4/80 (E). All of the values are the mean+SD; *P<0.05 vs. sham operation; #P<0.05 vs. control IgG; N = 8/group.
Figure 7.
Effect of Cyr61 blockade on renal fibrosis in UUO kidneys.
The mice received an intraperitoneal injection of 10 µg/g BW of the control rabbit IgG (filled bar) or anti-Cyr61 antibody (open bar) 2 hours before the UUO surgery and then one dose per day until euthanasia. (A–B) Q-PCR showing renal Col 1-α1 (A) and α-smooth muscle actin (α-SMA) (B) transcripts after UUO. N = 8/group. (C) Representative images of picrosirius red staining for interstitial fibrillar collagen (red) in the UUO kidneys (magnification 200×, scale bar = 100 µm). The bar charts are a summary of the picrosirius red-stained percentage in the field. N = 8/group. (D) Representative images of Western blots of renal α-SMA protein expression in the UUO kidneys. (E–F) Q-PCR showing no difference in renal CTGF (E) and TGF-β1 (F) transcripts between the 2 groups. N = 8/group. All of the Q-PCR data are expressed as the fold differences compared to the sham operation kidneys (gray bar). The values are the mean+SD. *P<0.05 vs. sham operation; #P<0.05 vs. control IgG.