MOESM1 of Pentoxifylline Attenuates Proteinuria in Anti-Thy1 Glomerulonephritis via Downregulation of Nuclear Factor-ÎşB and Smad2/3 Signaling

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Localization of Cyr61 expression in the UUO kidneys.

<p>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.</p

Cysteine-Rich Protein 61 Plays a Proinflammatory Role in Obstructive Kidney Fibrosis

<div><p>Cysteine-rich protein 61 (Cyr61) is a secreted matrix-associated protein that regulates a broad spectrum of biological and cellular activities. This study aimed to investigate the role of Cyr61 in progressive kidney fibrosis induced by unilateral ureteral obstruction (UUO) surgery in mice. The expression of Cyr61 transcripts and proteins in the obstructed kidneys were increased from day 1 and remained high until day 10 after surgery. Immunohistochemistry indicated that Cyr61 was expressed mainly in renal tubular epithelial cells. The upregulated Cyr61 in UUO kidneys was reduced in mice treated with pan-transforming growth factor-β (TGF-β) antibody. The role of TGF-β in tubular Cyr61 upregulation after obstructive kidney injury was further supported by experiments showing that TGF-β1 stimulated Cyr61 expression in cultured tubular epithelial cells. Notably, the upregulation of Cyr61 in UUO kidneys was followed by a marked increase in monocyte chemoattractant protein 1 (<em>MCP-1</em>) transcripts and macrophage infiltration, which were attenuated in mice treated with anti-Cyr61 antibodies. This proinflammatory property of Cyr61 in inducing <em>MCP-1</em> expression was further confirmed in tubular epithelial cells cultured with Cyr61 protein. The anti-Cyr61 antibody in UUO mice also reduced the levels of collagen type 1-α1 transcripts, collagen fibril accumulation evaluated by picrosirius red staining, and the levels of α-smooth muscle actin (<em>α-SMA</em>) transcripts and proteins on day 4 after surgery; however, the antifibrotic effect was not sustained. In conclusion, the TGF-β-mediated increase in tubular Cyr61 expression involved renal inflammatory cell infiltration through MCP-1 induction during obstructive kidney injury. The Cyr61 blockade attenuated kidney fibrosis in the early phase, but the antifibrotic effect could not be sustained.</p> </div

Experimental design and time course of the study.

<p>Experimental design and time course of the study.</p

Primer sequences used for quantitative-PCR.

<p>Primer sequences used for quantitative-PCR.</p

Effect of Cyr61 blockade on renal inflammation in the UUO kidneys.

<p>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. (<b>A–C</b>) Graphs showing a summary of <i>MCP-1</i> (A), chemokine (C-C motif) receptor-2 (<i>CCR-2</i>) (B), and <i>F4/80</i> (C) transcripts normalized to <i>18S</i>. The data are expressed as the fold differences compared to the sham operation kidneys (gray bar). N = 8/group. (<b>D</b>) 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. (<b>E and F</b>) Quantifying the expression of the profibrogenic macrophage-associated chemokine (C-C motif) ligand 17 (<i>CCL17</i>) and <i>CCL22</i> transcripts by Q-PCR. Graph showing the ratios of <i>CCL17</i> to <i>F4/80</i> (D) and <i>CCL22</i> to <i>F4/80</i> (E). All of the values are the mean+SD; *P<0.05 vs. sham operation; #P<0.05 vs. control IgG; N = 8/group.</p

Structure specificity and neutralizing activity of anti-Cyr61 antibody.

<p>(<b>A</b>) 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>B</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 <i>MCP-1</i> expression after anti-Cyr61 antibody pretreatment. N = 4/group. (<b>C</b>) 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 <i>MCP-1</i> upregulation. N = 4/group. The values are the mean+SD. *P<0.05.</p

Effect of SheaFlex75 treatment on the knee joint width in rats that underwent ACLT plus MMx.

<p> The widths of the bilateral joints were measured before and weekly after surgery. The data are the difference between the widths of the contralateral and ipsilateral knees, expressed as the mean ± S.E.M. Two-way ANOVA and Tukey’s multiple comparisons test were used to analyze the data. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, when compared to the OA group.</p

Cyr61 expression in cultured renal tubular epithelial cells.

<p>(<b>A</b>) Cultured rat proximal tubular epithelial cells (NRK-52E) were treated with various concentrations of recombinant TGF-β1 for 2 hours. Q-PCR showed <i>Cyr61</i> upregulation by TGF-β1 at doses of 0.25 ng/mL or greater. N = 4/group. (<b>B</b>) Recombinant Cyr61 was added to NRK-52E cells for 2 or 6 hours. Q-PCR showed increased expression of <i>MCP-1</i> transcripts by Cyr61 in a dose-dependent manner. N = 4/group. The values are the mean+SD. *P<0.05 vs. control.</p

Effect of Cyr61 blockade on renal fibrosis in UUO kidneys.

<p>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. (<b>A–B</b>) Q-PCR showing renal <i>Col 1-α1</i> (A) and α-smooth muscle actin (<i>α-SMA</i>) (B) transcripts after UUO. N = 8/group. (<b>C</b>) 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. (<b>D</b>) Representative images of Western blots of renal α-SMA protein expression in the UUO kidneys. (<b>E–F</b>) Q-PCR showing no difference in renal <i>CTGF</i> (E) and <i>TGF-β1</i> (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.</p