An ex vivo culture model of kidney podocyte injury reveals mechanosensitive, synaptopodin-templating, sarcomere-like structures

Chronic kidney diseases are widespread and incurable. The biophysical mechanisms underlying them are unclear, in part because material systems for reconstituting the microenvironment of relevant kidney cells are limited. A critical question is how kidney podocytes (glomerular epithelial cells) regenerate foot processes of the filtration apparatus following injury. Recently identified sarcomere-like structures (SLSs) with periodically spaced myosin IIA and synaptopodin appear in injured podocytes in vivo. We hypothesized that SLSs template synaptopodin in the initial stages of recovery in response to microenvironmental stimuli and tested this hypothesis by developing an ex vivo culture system that allows control of the podocyte microenvironment. Results supported our hypothesis. SLSs in podocytes that migrated from isolated kidney glomeruli presented periodic synaptopodin-positive clusters that nucleated peripheral, foot process–like extensions. SLSs were mechanoresponsive to actomyosin inhibitors and substrate stiffness. Results suggest SLSs as mechanobiological mediators of podocyte recovery and as potential targets for therapeutic intervention.


Fig. S1.
Evidence that podocytes were contractile. (a) Upon disruption of the actin cytoskeleton and inhibition of further actin polymerization using cytochalasin D (CytoD), most cells elongated along their axes, indicated that they were in a state of contraction. Shown here is a cell on a 0.9 kPa substrate just before (a) and 15 min after (a') application of CytoD. (b) Strain mapping based on fluorescent microbeads embedded within the 0.9 kPa hydrogel revealed that podocytes were contractile. The contour plot shows the peak eigenvalue of the Green-Lagrange strain tensor field associated with movement of beads as cells released tension over 15 minutes following treatment with CytoD on the 0.9 kPa hydrogels shown. Strains shown take the post-treatment state (a') as the reference configuration. (c) To verify that the contraction was predominantly associated with contractility in the actin cytoskeleton, an additional strain mapping was performed following elimination of the cells using the detergent sodium dodecyl sulfate (SDS). The strains associated with this subsequent SDS treatment were very small compared to those associated with CytoD. (d) Strains associated with contraction transmitted through actin cytoskeleton, calculated as in (b) and (c), were smaller on stiffer (6 kPa) substrates. Again, strains associated with CytoD treatment were much larger than those associated with subsequent SDS treatment. (e) Paired ttests revealed that the increases in cell length associated with 15 min of CytoD treatment were statistically significant. (f) Less cell relaxation is recorded on a hydrogel with a stiffness of 6 kPa (f). Scale bar, 20 µm.  The preferential adhesion/migration on laminin-521 is specific for podocytes, as there are many cells without synaptopodin attached to the fibronectin between the laminin-521 micropatterns. Scale bars, 10 µm.

Fig. S4.
When cultured on Laminin 521-micropatterned hydrogels with Fibronectin outside the micropatterns, the podocytes send out synaptopodin-positive protrusions (Marked by red arrows); these seem to enable migration to a nearby micropattern. Scale bars, 20 µm.

Fig. S9.
In differentiated immortalized podocytes, the SLSs showed dramatically larger spacings between synaptopodin positive bands compared to primary podocytes, whereas myosin showed multiple bands in between synaptopodin bands.   Modeling the shear stress at the adhesion surface shows dramatically reduced shear stress when the contractility inside of the cells is inhibited.

Fig. S13.
Mild myosin inhibition is not enough to eliminate all the SLSs inside the spreading podocytes, but SLSs in the center of the cells were disturbed due to shear lag effects. Scale bars, 10 µm.

Fig. S14.
Blebbistatin wash-out led to the regeneration of SLSs in podocytes within 2 hours. Longer (1 day) recovery allowed restoration of the SLSs even at the center of the cells. Scale bars, 5 µm.