Abstract: PO1680
Novel Ex Vivo Culture System Reveals Mechanosensitive "Sarcomere-Like Structures" During Early Podocyte Spreading
Session Information
- Podocyte Pathobiology: Basic Science Studies and Animal Models
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1204 Podocyte Biology
Authors
- Jiang, Shumeng, Washington University in St Louis, St Louis, Missouri, United States
- Miner, Jeffrey H., Washington University in St Louis School of Medicine, St Louis, Missouri, United States
- Genin, Guy M., Washington University in St Louis, St Louis, Missouri, United States
- Suleiman, Hani, Washington University in St Louis School of Medicine, St Louis, Missouri, United States
Background
Chronic kidney disease and ESKD are widespread health problems with no cure, in part because the biophysics underlying them are still not clear. A recently discovered feature of injured podocytes includes the de novo assembly of sarcomere-like stress fibers, identified by their alternating myosin and synaptopodin/a-actinin-4. It is not known whether these indicate a transient healing phenotype or are a feature of the cascade leading to foot process effacement.
Methods
To model the early events of podocyte injury, we developed a new in vitro system that enables the study of podocytes outside of their native microenvironment, but with in vivo-like mechanobiological and extracellular matrix (ECM) features. This system includes controllable stiffness, micropatterned substrates for spreading, and the use of primary podocytes as they migrate from freshly isolated glomeruli.
Results
When cultured on micropatterns of physiologically relevant extracellular matrix proteins and appropriate stiffness, myosin- and synaptopodin-positive stress fibers developed over two days of culture, then disappeared after six days, and the appearance of these stress fibers was sensitive to substrate stiffness and could be disrupted by inhibiting actomyosin contraction (blebbistatin), culturing cells with stiffness outside of the physiologic range, or presenting cells with substrates associated with pathology.
Conclusion
These results reveal the role of mechanobiological factors in podocytes represented by the mechanoresponsive sarcomere-like structure and establish a novel system for characterizing this mechanobiology in vitro.
(a) Protocol for preparing the micropatterns of ECM proteins. (b, c) Primary podocytes prefer surfaces with laminin-521, the normal GBM laminin. (d, e) Sarcomere-like structures were observed at the early stages of podocyte spreading on micropatterns. (f, g) The sarcomere-like structure is mechanosensitive: a soft substrate (0.9 kPa) or myosin inhibition (Blebbistatin) increases the periodicity in the pattern.
Funding
- Private Foundation Support