Podocytes Respond to Mechanical Stress In Vitro

Mechanical stress—induced reorganization of the podocyte actin cytoskeleton (A and B; F-actin). Unstressed control cells (A) often exhibited transversal stress fibers (arrowheads). Stress fibers in mechanically stressed podocytes were radially organized (arrow-heads) and connected to an actin-rich center (ARC, arrow). Mechanical stress did not induce major changes of the microtubule and vimentin cytoskeleton (C through F; C and D, α-tubulin; E and F, vimentin). Magnification, ×160.

The three-dimensional organization of F-actin in mechanically stressed podocytes was reconstructed with the aid of confocal microscopy (A, xy plane; B, xz plane). The ARC resembled an ellipsoid, and stress fibers were connected to the ARC at the equatorial plane. Three-dimensional reconstruction was done from 36 optical sections of 0.2 μm thickness. Magnification, ×1000. Quantitative analysis confirmed that the number of podocytes with transversal stress fibers was significantly diminished by mechanical stress, whereas the percentage of podocytes that possessed an ARC increased to 100% (C). Mechanical stress increased not only the number of ARC-positive cells but also the size of the ARC. The larger diameter of the slightly elliptical ARC is shown. For the frequency of transversal stress fibers and the ARC, 135 control cells and 106 stressed cells were classified. ARC diameter was measured in 30 control cells and 106 stressed cells. Data of one experiment are presented. Similar results were obtained in two additional experiments. ^*, P < 0.05.

Transmission electron microscopy of mechanically stressed podocytes revealed the ellipsoidal and dense filamentous structure of the ARC. A fiber bundle (arrow) extends from the ARC to a focal contact (arrowhead) at the cell membrane (A). The single actin filaments, which are densely wound up in the ARC, can be resolved at higher magnification (B). The electron micrographs show a horizontal section through a podocyte. Magnifications: ×9500 in A; ×34,000 in B.

Mechanical stress did not induce apoptosis in stressed podocytes. DNA staining with Hoechst 33342 showed intact nuclei in podocytes, which were mechanically stressed fo r 1 d (B), and in unstressed controls (A). Mechanical stress did not induce an ARC or radial stress fibers in epithelial LLC-PK1 cells. F-actin was visualized in mechanically stressed LLC-PK1 cells (D) and unstressed controls (C). The F-actin cytoskeleton of unstressed primary cultured podocytes (E), which stained positively for nuclear Wilms' tumor-1 (WT-1; F), occasionally showed an ARC (arrow). Magnifications: ×180 in A and B; ×340 in C and D; ×230 in E and F.

Reorganization of the F-actin cytoskeleton in response to mechanical stress depended on Ca²⁺ influx and Rho kinase but not on Gd³⁺-sensitive cation channels. Representative images of mechanically stressed podocytes are shown for the different conditions: no treatment (A), 1 mM Ni²⁺ (B), 50 μM Gd³⁺ (C), and Rho kinase inhibition with 10 μM Y-27632 (D). The inhibitory effect of Ni²⁺ and Y-27632 on the stress-induced actin reorganization was quantified by counting of ARC-positive cells after application of mechanical stress for 1 d (E). Data are means ± SEM of three experiments examining 100 to 113 cells per experiment. ^*, P < 0.05. Magnification, ×230.

Typical actin-associated proteins of stress fibers were not enriched in the ARC. Confocal sections through the ARC in the plane of stress fiber attachment revealed that the fluorescence intensity of myosin II (A), α-actinin (B), and synaptopodin (C) was considerably lower in the ARC (arrow) than in radial stress fibers. Co-localization of synaptopodin (D) and F-actin (E) in a mechanically stressed podocyte is shown in F. Whereas synaptopodin was co-localized with F-actin in stress fibers, it was practically absent from the center of the ARC. Co-localization was done using conventional fluorescence microscopy. Magnifications: ×720 in A through C; ×600 in D through F.