(Pro)renin receptor is involved in mesangial fibrosis and matrix expansion

Figures

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  Figure 1. (A) (P)RR protein expression in SV40 MES13 cells assessed by western blot analysis. (B and C) SV40 MES13 cells were incubated with the indicated concentration of IS for 24 h (B) or 48 h (C). Mean ± SE (n = 6). ctl, control. *P < 0.05, by the Tukey–Kramer test. (C) SV40 MES13 cells were incubated with other uremic toxins for 48 h. Mean ± SE (n = 4). ctl, control. (E) Cell number after 250 µM IS stimulation (solid line) or 50 mM Tris-HCl incubation [control (ctl); dotted line] for the indicated periods. Mean ± SE (n = 6). (F) Cell viability of SV40 MES13 cells incubated with 250 µM IS assessed by the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (n = 6). Values are presented as the median (central line), interquartile range (box) and range (bars). NS, not significant, vs. control (ctl) by the Student’s t-test.

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  Figure 2. Immunofluorescence of (P)RR in SV40 MES13 cells. SV40 MES13 cells were stained with a (P)RR-specific antibody (red) and the nuclear stain, 4,5-diamidino-2-phenylindole (DAPI, blue). Left: low-power field, Middle: high-power field, Right: bright field. (A) Cells after 50 mM Tris-HCl stimulation for 48 h as a control. (B) Cells after 250 µM IS stimulation for 48 h. (C) Negative control. Scale bars, 20 µm (low-power field) and 5 µm (high-power field and bright field).

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  Figure 3. (A–C) Protein expression of type IV collagen (A), TGF-β (B) and fibronectin (C) in SV40 MES13 cells transfected with (P)RR small interfering RNA (siRNA) or nontargeting siRNA (scRNA) (n = 4 in each group). Cells were stimulated with 250 µM IS or 50 mM Tris-HCl (ctl) for 48 h. Mean ± SE (n = 4). *P < 0.05, **P < 0.01, by the Tukey–Kramer test. (D) (P)RR expression in SV40 MES13 cells transfected with (P)RR siRNA or scRNA. Mean ± SE (n = 4). *P < 0.05 compared with scRNA by the Student’s t-test. (E) Cell viability of SV40 MES13 cells treated with (P)RR siRNA or scRNA by the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (n = 6). Values are presented as the median (central line), interquartile range (box), and range (bars). NS, not significant, by the Student’s t-test.

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  Figure 4. (A and B) mRNA expression of Mmp9 (A) and Timp1 (B) in SV40 MES13 cells. (C and D) Immunoblotting of MMP9 (C) and TIMP1 (D). Cells were stimulated with 250 µM IS or 50 mM Tris-HCl (ctl) for 48 h. Mean ± SE (n = 4). *P < 0.05, **P < 0.01, A and B were examined by the Student’s t-test. C and D were examined by the Tukey–Kramer test.

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  Figure 5. (A) Representative immunoblotting of phospho-ERK1/2 (pERK1/2) and total ERK1/2. (B) ERK1/2 phosphorylation by 250 µM IS stimulation was decreased in SV40 MES13 with U0126 (ERK1/2 specific inhibitor). Mean ± SE (n = 4). **P < 0.01, compared with control by the Student t-test. Cells were stimulated with 250 µM IS. (C and D) Immunoblotting of MMP9 (C) and TIMP1 (D). Cells were stimulated with 250 µM IS or 50 mM Tris-HCl (ctl) for 48 h after treatment with U0126. Mean ± SE (n = 4). *P < 0.05, **P < 0.01, by the Tukey–Kramer test.

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  Figure 6. (A) Relationship between IGL levels and serum s(P)RR. Subjects were divided into four groups based on quartiles of IGL levels. The quartile ranges were: Q1, <1.2 (n = 14); Q2, 1.2–1.5 (n = 14); Q3, 1.5–1.8 (n = 13); Q4, ≥1.8 (n = 14). Mean ± SD (n = 55). The Kruscal-Wallis test was used (P = 0.001). *P < 0.05, **P < 0.01, by the the Steel–Dwass test. (B) Relationship between IS levels and serum s(P)RR. Subjects were divided into four groups based on quartiles of IS levels. The quartile ranges were: Q1, <0.74 (n = 14); Q2, 0.74–1.08 (n = 14); Q3, 1.08–1.62 (n = 14); Q4, ≥1.62 μg/mL (n = 13). Mean ± SD (n = 55). The Kruscal-Wallis test was used (P = 0.03). *P < 0.05, by the Steel–Dwass test.