Fig 2: Immunostaining and Western blotting of the GBM in Col4a5 deficient kidneys. (a–c) Immunofluorescence analyses of rat kidney sections with antibodies against (a): Laminin ß2 (red), Nestin (green; glomeruli); (b): CTGF (red), Nephrin (green); (c): MMP3/10 (red), Nephrin (green); and DAPI (blue; nuclei) in wildtype (WT) and Col4a5 mutant (Hemi) male rats at 8 weeks (left) and 20 weeks (right) of age. Scale bars, 50 µm. (d) Detection of phospho- and total Smad3, and MMP12 by Western blot analyses of the kidneys in wildtype (W) and Col4a5 mutant (H) male rats from 8, 12, 16, to 20 weeks of age. Tubulin was evaluated as an internal control.

Fig 3: The suppression effect of ligustrazine is achieved by the activated PPAR? on the transrepression of SMRT-mediated HIF-1a. (a) Western blot analysis of protein expression of profibrotic cytokines (VEGF and CTGF) in hypoxia-induced PMCs treated with KC7F2 for 24 h. (b, c) FDA staining assay of cell viability in hypoxia-induced PMCs treated with KC7F2 for 24 h (200x). Compared with the control, *P < 0.05 and **P < 0.01. (d) Western blot analysis of protein expression of migration and adhesion-associated molecules (VCAM-1 and ICAM-1) in hypoxia-induced PMCs treated with KC7F2 for 24 h. (e) Western blot analysis of cytoskeleton protein (vinculin) expression in hypoxia-induced PMCs treated with KC7F2 for 24 h. (f) Western blot analysis of protein expression of FSP1 in hypoxia-induced PMCs treated with KC7F2 for 24 h. (g) Western blot analysis of protein expression of ECM-associated molecules (MMP2 and TIMP-1), mesothelial-related phenotypic biomarkers (E-cadherin and cytokeratin 18), and mesenchymal-related phenotypic biomarkers (Snail and a-SMA) in hypoxia-induced PMCs treated with KC7F2 for 24 h. (h, i) qRT-PCR and Western blot analysis of HIF-1a levels in hypoxia-induced PMCs treated with 20 µM ligustrazine and/or 1 µM GW9662 for 24 h. Compared with the control, *P < 0.05 and **P < 0.01. Compared with the ligustrazine group, #P < 0.05 and ##P < 0.01. (j, k) qRT-PCR analysis of mRNA levels of SMRT and NCoR in hypoxia-induced HPMCs after different transfection for 24 h. Compared with the control, *P < 0.05 and **P < 0.01. (l) qRT-PCR analysis of mRNA levels of HIF-1a in hypoxia-induced HPMCs treated with 20 µM ligustrazine and/or with different transfection for 24 h. Compared with the control, *P < 0.05 and **P < 0.01. Compared with the ligustrazine group, #P < 0.05 and ##P < 0.01. (m) Western blot analysis of protein expression of HIF-1a in hypoxia-induced HPMCs treated with 20 µM ligustrazine and/or with different transfection for 24 h.

Fig 4: Knockdown of Ddah1 or Ddah2 gene inhibited renal fibrosis at day 7 after UUO operation. Fifty microliters of NC (n = 7), Ddah1 (n = 7) or Ddah2 (n = 7) siRNA was injected intrarenally to the left kidney, which was subjected to the UUO operation thereafter. Renal tissues were collected at day 7 after UUO operation. The expression of FN, Col-I, CTGF, a-SMA and pSmad3 at day 7 in the UUO kidney was measured by western blotting, and quantification of more than three blots is shown (A, B). Renal fibrosis was further evaluated by Masson’s trichrome staining at day 7 in the UUO kidney (C). One representative of three independent experiments is shown. Data represent mean ± SD. *P < 0.05 versus NC; **P < 0.01 versus NC; ***P < 0.001 versus NC. Scale bars: 100 µm. Statistical differences were analyzed by one-way ANOVA or unpaired Student’s t-test. A P value <0.05 was considered statistically significant.

Fig 5: Effect of sauchinone on AngII-induced mesangial cell fibrosis. (A) Western blotting and (B) real-time PCR showed protein and mRNA levels of fibronectin, Collagen IV, and CTGF in sauchinone-treated and AngII-stimulated cells at 48 h. (C,D) Effect of sauchinone on the relative levels of TGF-ß1/Smads. The results were detected by Western blotting and real-time PCR. (E) Immunofluorescent images of p-Smad-2 nuclear translocation under the laser scanning confocal microscopy are show (magnification. 400×). Nuclei were stained with DAPI (blue) and p-smad-2 was stained with Alexa Fluor 488 (green). Green fluorescence indicates localization of p-Smad-2. Respective blot data were obtained from five independent experiments. Each value represents the means ± S.E. of five independent experiments. ** p < 0.01 vs. control and ## p < 0.01, # p < 0.05 vs. AngII alone.