Fig 1.
Glomerular structure and summary of findings.
The diagram represents a section though the center of a glomerulus showing capillary walls made up of podocyte foot processes, endothelial cells, and the GBM. Podocytes are shown in blue (cell body, processes, and foot process as round structures on the surface of capillary walls attached to the GBM), endothelial cells in green (broken circle inside capillaries signifying fenestrated endothelium attached to the GBM), mesangial cells in grey, and the GBM in black. In reality, but not shown in the diagram, podocyte processes that contain vinculin wrap around the capillaries and appear to provide structural support. Below the label for each structure are listed the cell components affected by the various experimental interventions described in the results section. ATP production is not shown because it is essential to the function of all cellular components. The scale bars below the glomerulus show the approximate size range of glomerululi before (normal diameter, 80–90 μm) and after compression (compressed diameter, 65–75 μm), and indicate that compression (15 μm) by micro-indentation involves capillaries and not the mesangial region of the glomerulus.
Fig 2.
Representative glomeruli isolated by sieving.
A. Mouse glomeruli were isolated by sieving, embedded in agarose, sectioned, and stained with PAS. B. Mouse glomeruli were isolated by sieving, treated as indicated, and stained with propidium iodide (red) and calcein AM (green). Glomeruli shown are representative of 10 glomeruli in each group.
Fig 3.
Effect of cytoskeleton-active agents and metabolic inhibitors on glomerular elastic modulus and F/G actin ratios.
The top panel (3A) shows the E of isolated mouse glomeruli treated with DMSO, jasplakinolide (1 μM), latrunculin (1 μM), blebbistatin (50 μM). The second panel (3B) shows the E of isolated mouse glomeruli treated with DMSO, 2-deoxyglucose (11. 1 mM), rotenone, (1 μM), and Na azide (10 mM). Each bar in 2A and 2B represents the E mean +/- SD of three separate experiments, in each of which the E of 6–8 glomeruli was measured. The bottom panels 3A and B shows representative phase contrast images of glomeruli from each condition (40 X magnification). The third panel (3C) shows F/G actin ratios in glomeruli treated with DMSO, blebbistatin (50 μM), latrunculin (1 μM), jasplakinolide (1 μM),), 2-deoxyglucose (11.1 mM), rotenone, (1 μM), or Na azide (10 mM). Each bar represents the mean of three to four separate experiments +/- SD and is expressed as the percent of control for each experiment. * p < 0.01 (Paired t test vs control). The top of 3C is a representative western blot showing F and G actin fractions from each condition. + p < 0.02 (Paired t test) vs control.
Fig 4.
Elastic modulus of glomeruli treated with Wfa and latrunculin, EDTA and Mg, and decellularized glomeruli (GBMs).
4A Isolated glomeruli were treated with Wfa 6 μm, latrunculin 1 μm, or Wfa and latrunculin for 2 hrs in RPMI, and their E values determined by microindentation. Each bar represents the mean of six separate experiments +/- SD, in which four to six glomeruli were measured. In Fig 4B, glomeruli were treated with 10 mM EDTA or 5.0 mM MgCl2 for 15 min, and their E was measured using microindentation. Each bar represents the mean value +/- SD from three separate experiments in which 4–5 individual glomeruli were measured. * p < 0.001 (paired t test vs control). 4C shows the mean +/- SD of eight separate measurements of decellularized glomeruli using microindentation. Glomeruli were isolated with magnetic beads, and beads can be seen in the glomerular structures.
Fig 5.
Measurement of GBM E using magnetic bead displacement.
Glomeruli were isolated by perfusion with 4.5 μm magnetic beads, and decellularized with detergents and DNase. The GBMs were stored in PBS at 4°C where they formed aggregates. Fragments of the aggregates on the order of 30 x 150 x 200 μm were attached to APTES-coated glass coverslips and imaged using a confocal video microscope (Leica) where the beads seen as dark spots (arrows) were localized in X, Y, and Z dimensions (Panel 5A, XY plane). Panel 5B shows a bead in the XZ plane before activation of the magnet. Panel 5C shows the same bead in the XZ plane 5 min after activation of the magnet. The movement of the bead is evident from its relationship to the bottom of the image. The magnetic force and bead displacement were calculated in the Z axis for four beads, and the Young’s modulus, E, was calculated with the method of Kamgoue et al [18].
Fig 6.
Effects of matrices with different E values on structure and gene expression in cultured mouse podocytes.
6A and 6B show podocytes stained with rhodamine-phalloidin (red) and vinculin (green) (5A) or WT-1 (green) (6B) antibodies, rhodamine-phalloidin (red), and DAPI (nuclear stain) at 40X on glass or PA gels coated with collagen (5 kPa, 3.0 kPa, 1.8 kPa, and 0.5 kPa).
Fig 7.
Panels 7A, B, and C show mRNA levels for filamin, non-muscle myosin IIa, and WT-1, respectively, grown on matrices with E values of 10, 5, 1.8, 1.1, and 0.5 kPa, or glass treated with 100 μm blebbistatin, measured by qRT-PCR.
Differentiated podocytes were plated on the matrix described and studied 18–24 hrs later. Each bar represents the mean +/- SEM of 4–6 separate experiments. * p < 0.02, + p < 0.05 (paired T test vs control (glass)). 7D shows a representative western blot for WT-1 in podocytes grown on plastic, 10, 3, and 1.1 kPa collagen-coated PA gels. 7E shows a representative western blot for WT-1 from podocytes grown in collagen-coated plastic tissue culture plates, in suspension (bacterial culture plates to which the cells do not adhere), or in collagen-coated plastic tissue culture plates with 100 μm blebbistatin for 18–24 hrs.
Fig 8.
Effects of matrices with different E values on structure and gene expression in cultured mouse mesangial cells.
8A shows mesangial cells stained with rhodamine-phalloidin (red), vinculin antibodies (green), and DAPI (nuclear stain, blue) at 40X on glass or PA gels coated with collagen (5 kPa, 3.0 kPa, 1.8 kPa, and 0.5 kPa). Fig 8B, and 8C show mRNA levels for filamin and non-muscle myosin IIa, respectively, grown on matrices with E values of 10, 5, 1.8, 1.1, and 0.5 kPa, measured by qRT-PCR. Each bar represents the mean +/- SEM of 4–6 separate experiments. *p < 0.05 (paired T test vs control (glass)).
Fig 9.
Elastic modulus and structure of glomeruli with ischemia and ischemia-reperfusion injuries.
Panels 9A and B show representative H and E stained sections from mouse kidneys featuring glomeruli from control mice (A) and mice with 20 min of ischemia (B) (40 X), and representative transmission electron micrographs of mouse kidney glomeruli from control mice (C) and mice with 20 min of ischemia (D) (1,400 X). 9E shows the E of control mouse glomeruli and glomeruli following 20 min of ischemia. Panels 9F and G show representative H and E stained sections of kidneys following 20 min of ischemia with 2 hr reperfusion (40 X), and 9H and I show representative transmission electron micrographs (1,400 X) from the same kidneys. Panel J shows E or Glomeruli from control kidneys and kidneys following 20 min of ischemia with 2 hrs of reperfusion. Each bar in E and J represents the E +/- SEM derived from separate measurement of 6–8 glomeruli. * P < 0.05 (Paired T test vs control).