Elastic modulus and structure of glomeruli with ischemia and ischemia-reperfusion injuries.

<p>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).</p

Effects of matrices with different E values on structure and gene expression in cultured mouse mesangial cells.

<p>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)).</p

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.

<p>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.</p

Effects of matrices with different E values on structure and gene expression in cultured mouse podocytes.

<p>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).</p

Elastic modulus of glomeruli treated with Wfa and latrunculin, EDTA and Mg, and decellularized glomeruli (GBMs).

<p>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.</p

Glomerular structure and summary of findings.

<p>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.</p

Measurement of GBM E using magnetic bead displacement.

<p>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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167924#pone.0167924.ref018" target="_blank">18</a>].</p

Effect of cytoskeleton-active agents and metabolic inhibitors on glomerular elastic modulus and F/G actin ratios.

<p>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.</p

Identification of critical residues of human (h)AQP4_281-300 for presentation in the context of <i>HLA-DRB1*03</i>:<i>01</i> and recognition by the B.10 T cell receptor (TCR).

<p>(A) First, the ability of hAQP4_281-300-reactive lymph node cells to recognize the alanine screening peptides was determined by ELISpot. 5.0x10⁵ cells/well lymph node cells taken ten days post immunization of <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice with hAQP4_281-300 were restimulated with hAQP4 alanine scanning peptides (2 5 μg/mL) for 48 hours in IFNγ and IL-17 ELISpot plates (* = P-value < 0.05 and ** = P-value < 0.01). (B) Alanine screening peptides that not result in an increased frequency of IFNγ and IL-17 secreting lymph node cells were identified as the key residue peptides, and were subsequently tested in a MHC binding assay. Splenocytes taken from <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice were incubated for 12 hours in the presence of biotinylated hAQP4 alanine scanning peptides. Post incubation, cells were stained utilizing FITC-Avidin, and antigen positive cells were quantified by flow cytometry (* = P-value < 0.05 and ** = P-value < 0.01). (C) There was no Ig isotype class switch in mice immunized with mAQP4_284-299 with regard to antibody responses against whole-length AQP4 protein. (D) Critical <i>HLA-DRB1*03</i>:<i>01</i> anchor residues, and B.10 TCR contact amino acids are specified. E₂₈₈ and L₂₉₄ are required as <i>HLA-DRB1*03</i>:<i>01</i> anchor residues, while T₂₈₉, D₂₉₀, D₂₉₁, and I₂₉₃ are critical B.10 TCR interacting residues.</p

Immunization with human (h)AQP4_281-300 leads to an expansion of antigen-specific CD4⁺ T cells <i>in vivo</i>, and an Ig isotype switch in <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice.

<p>(A) Following immunization with human (h)AQP4_281-300, an expansion of antigen-specific CD4⁺ T helper cells was detected by tetramer staining of lymph node cells. The fluorescent signal of <i>HLA-DRB1*03</i>:<i>01</i>-loaded tetramers minus the fluorescent signal of empty <i>HLA-DRB1*03</i>:<i>01</i> tetramers is shown. CD4⁺ T helper cells provide soluble mediators that drive B cell differentiation immunoglobulin (Ig) class switching. To determine whether hAQP4_281-300-reactive CD4⁺ T cells are capable of causing IgM to IgG isotype switching in <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice, the concentration of Ig against hAQP4_281-300, mAQP4284-299, or with whole-length hAQP4 protein in serum of immunized mice was quantified longitudinally. Since the NMO-IgG is a human IgG1 isotype, both, the murine IgG2a and IgG2b isotype were examined as they have similar properties with regard to complement binding and the Fcγ receptor. A switch from IgM to IgG2b was detected in mice immunized with hAQP4_281-300 peptide with regard to (B) antibody responses against hAQP4_281-300 and (C) whole-length AQP4 protein. An Ig isotype switch from IgM to IgG2b was also detectable in mice immunized with whole-length AQP4 protein with regard to (D) antibody responses against hAQP4_281-300 and (E) whole-length AQP4 protein.</p