Fig 1.
Histological assessment of glomerular lesions in experimental glomerulonephritis (GN).
(a) Periodic acid-Schiff staining in Habu venom-induced Hic-5+/+ and Hic-5-/- GN mice. On day 7, Hic-5+/+ GN Habu mice exhibited glomerular cell proliferation. Hic-5-/- Habu GN mice showed severe mesangial cell proliferation and intensive glomerular matrix expansion compared to Hic-5+/+ Habu GN mice. Original magnification x200, scale bar = 50 μm. (b) The number of glomerular cells was counted in 30 glomeruli per section and calculated. The data are shown as the means ± SD. *, P<0.01. There was no significant difference between Hic-5+/+ day 0 and Hic-5-/- day 0. (c) A matrix score was assessed in 30 glomeruli per section and calculated. The data are shown as the means ± SD. *, P<0.01. There was no significant difference between Hic-5+/+ day 0 and Hic-5-/- day 0.
Fig 2.
Immunohistochemistry for Ki-67 in Hic-5+/+ and Hic-5-/- glomerulonephritis (GN) mice.
(a) On day 7, Hic-5+/+ Habu GN mice showed a greater number of Ki-67-positive glomerular cells. In addition, more Ki-67-positive cells were observed in the glomeruli of Hic-5-/- Habu GN mice. There were few Ki-67-positive cells in the glomeruli of Hic-5+/+ and Hic-5-/- mice on day 0. Original magnification x200, scale bar = 50 μm. (b) The number of Ki-67-positive cells was counted in 30 glomeruli per section and calculated. The data are shown as the means ± SD. *, P<0.01. There was no significant difference between Hic-5+/+ day 0 and Hic-5-/- day 0.
Fig 3.
Glomerular expression of Hic-5, α-smooth muscle actin (SMA), and fibronectin (FN) in glomerulonephritis (GN) mice.
Representative immunofluorescence micrographs show the glomerular expression of Hic-5, α-SMA, and FN in Hic-5+/+ and Hic-5-/- GN mice on day 0 and day 7. Original magnification x200, scale bar = 50 μm.
Fig 4.
Semi-quantitative assessment of the glomerular expression of Hic-5, α-smooth muscle actin (SMA), and fibronectin (FN).
The glomerular expression of Hic-5 (a), α-SMA (b), and FN (c) in Hic-5+/+ and Hic-5-/- glomerulonephritis mice was determined as the positively immunoreactive fraction in the glomerular area based on the examination of 30 equatorially sectioned glomeruli for each section, and statistically analyzed. The data are shown as the means ± SD. *, P<0.01. There was no significant difference in the glomerular expression of α-SMA and FN between Hic-5+/+ day 0 and Hic-5-/- day 0, respectively. (d) The expression of Hic-5, α-SMA, and FN in kidney cortical tissues in GN was also investigated by western blotting. Expression of β-actin is shown as a loading control. Each experiment was performed at least three times.
Fig 5.
Glomerular expression of PDGF-B chain, PDGF receptor, TGF-β1, and TGF-β receptor in glomerulonephritis (GN) mice.
Representative immunofluorescence micrographs show the glomerular expression of PDGF-B chain, PDGF-receptor β subunit (PDGF-R-β), TGF-β1, and TGF-β receptor type II (TGF-β-R) in Hic-5+/+ and Hic-5-/- GN mice on day 0 or day 7. Original magnification x200, scale bar = 50 μm.
Fig 6.
Semi-quantitative assessment of the glomerular expression of PDGF-B chain, PDGF receptor, TGF-β1, and TGF-β receptor.
The glomerular expression of PDGF-B chain (a), PDGF receptor β subunit (PDGF-R-β) (b), TGF-β1 (c), and TGF-β receptor type II (TGF-β-R) (d) in Hic-5+/+ and Hic-5-/- glomerulonephritis mice was determined as the positively immunoreactive fraction in the glomerular area based on the examination of 30 equatorially sectioned glomeruli for each section, and statistically analyzed. The data are shown as the means ± SD. *, P<0.01. N.S., not significant. There was no significant difference between Hic-5+/+ day 0 and Hic-5-/- day 0.
Fig 7.
Detection of apoptotic glomerular cells in Hic-5+/+ and Hic-5-/- glomerulonephritis (GN) mice.
(a) Representative TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining (upper) and cleaved caspase-3 positive cells (lower). Apoptotic glomerular cells in GN were identified by fluorescence-labeled nuclei (yellow), indicated by arrows. The nuclei of glomerular cells were counterstained with propidium iodide (red). In addition, cleaved caspase-3 positive cells were detected by immunohistochemistry and indicated by arrows. Original magnification x200, scale bar = 50 μm. (b) The number of apoptotic glomerular cells was counted and calculated in 30 full-size glomeruli. (c) The number of cleaved caspase-3 positive cells was counted and calculated in 30 full-size glomeruli. The data are shown as the means ± SD. *, P<0.01. N.S., not significant. There was no significant difference between Hic-5+/+ day 0 and Hic-5-/- day 0 on both apoptotic glomerular cells and cleaved caspase-3 positive cells.
Fig 8.
Morphology and characterization of cultured mesangial cells (MCs) isolated from Hic-5+/+ and Hic-5-/- mice.
(a) MCs isolated from both Hic-5+/+ and Hic-5-/- mice were cultured after the isolation of glomeruli using magnetic beads. Cell morphology is shown in sparse (upper panel) and confluent (middle panel) conditions. Representative images were captured by an inverted microscope (Olympus CKX41). Original magnification x100 (upper) and x40 (middle), respectively. Lower panels show rhodamine α-smooth muscle actin (SMA) staining by fluorescence microscopy (Nikon Eclipse E600). Original magnification x400. (b) MC adhesion to collagen type I (COL:10 ng/mL) and fibronectin (FN:10 ng/mL) were shown as absorbance (630 nm). The data are shown as the means ± SD. N.S., not significant.
Fig 9.
Assessment of cell proliferation in cultured mesangial cells (MCs) isolated from Hic-5+/+ and Hic-5-/- mice.
(a) Quiescent Hic-5+/+ and Hic-5-/- MCs (5x104/plate) were stimulated with appropriate concentrations of PDGF-BB (50 ng/ml) and TGF-β1 (10 ng/ml) for 48 h. MCs were counted after harvesting and assessed as the mean cell number. The data are shown as the means ± SD. *, P<0.01. N.S., not significant. Control Hic-5-/- MCs showed a significantly increased cell number compared to control Hic-5+/+ MCs (P<0.01). (b) Quiescent MCs (6x103/100 μl) in 96-well culture plates were stimulated with PDGF-BB (50 ng/mL) or TGF-β1 (10 ng/mL) for 24 hours. The stimulated MCs were pulsed with 10 μl CCK-8 solution for 3 hours, and absorbance was measured at 450 nm. The data are shown as the means ± SD. *, P<0.01. Control Hic-5-/- MCs showed significantly increased absorbance compared to control Hic-5+/+ MCs (P<0.01). Each experiment was performed independently a minimum of three times.
Fig 10.
Expression of cyclins A and D1, and p21 in cultured mesangial cells (MCs).
(a) Representative western blotting images show levels of cyclins A and D1, and p21 in Hic-5+/+ and Hic-5-/- MCs with or without stimulation by PDGF-BB (50 ng/ml) or TGF-β1 (10 ng/ml). Expression of β-actin is shown as a loading control. Quantitative data obtained by ImageJ 1.47v (National Institutes of Health) are shown as the means ± SD. The bar graphs b, c, and d indicate the expression of cyclin A, cyclin D1, and p21, respectively. *, P<0.01. #, P<0.05. N.S., not significant. The results shown are representative of three independent experiments. There were significant differences in the expression of cyclin D1 and p21 between control Hic-5+/+ and Hic-5-/- MCs without stimulation (P<0.01). There was no significant difference in the expression of cyclin A between control Hic-5+/+ and Hic-5-/- MCs without stimulation.