Kidney Week

Abstract: SA-PO581

Visualization of Force Dynamics and Actin Remodeling in ACTN4 Mutant Podocytes Subjected to Stretch

Session Information

• Noncystic Mendelian Diseases
  November 04, 2017 | Location: Hall H, Morial Convention Center
  Abstract Time: 10:00 AM - 10:00 AM

Category: Genetic Diseases of the Kidney

• 802 Non-Cystic Mendelian Diseases

Authors

• Feng, Di, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, United States

Di Feng,

• Benjamin, Ava, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, United States

Ava Benjamin,

• Notbohm, Jacob, University of Wisconsin-Madison, Madison, Wisconsin, United States

Jacob Notbohm,

• Wang, Minxian, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, United States

Minxian Wang,

• Krishnan, Ramaswamy, BIDMC/ Harvard Medical School, Boston, Massachusetts, United States

Ramaswamy Krishnan,

• Pollak, Martin R., Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, United States

Martin R. Pollak,

Background

Alpha-actinin-4 gene (ACTN4) mutations cause a rare form of familial focal segmental glomerulosclerosis (FSGS) and podocyte injury in humans. Our study aimed to better understand the mechanism by which mutant ACTN4 contributes to podocyte dysfunction by assessing the effect of the mutation on a podocyte’s response to transient stretch.

Methods

We used primary podocytes isolated from Actn4 K256E Knock in mutant mice (n=3) and WT littermates (n=3) as a cellular model. Measurements were performed in isolated single cells and data were pooled across multiple mice. We used traction force microscopy to quantify contractile forces exerted by podocytes on their underlying substrate in response to transient stretch. We used live cell imaging to examine the distribution of actin.

Results

Compared to WT, mutant ACTN4 podocytes bear more actin stress fibers bundles and exert greater contractility before stretch. After the first transient stretch, WT and mutant podocytes demonstrated similar reductions in their contractile forces. During the recovery period, WT podocytes demonstrated recovery of their contractile forces close to pre-stretch baseline values, whereas the majority of mutant podocytes showed impaired recovery. After a total of 3 transient stretches, WT podocytes on average recovered 79% of their baseline contractile forces, whereas the majority of mutant podocytes (10/15) recovered less than 50% of their baseline, with 7 failing entirely (completely losing their ability to generate contraction forces). Additionally, representative WT podocytes demonstrated cracks in their cytoskeletons after the first stretch that were subsequently repaired during the recovery period, whereas representative mutant podocytes whose contractile forces failed demonstrated cracks that persisted during the recovery period.

Conclusion

Our findings provide the first direct evidence of a mutant ACTN4 podocyte’s inability to maintain its structure and function in response to mechanical stress, suggesting a clearer path through which mutant ACTN4 leads to podocyte detachment and kidney injury.

Funding

• NIDDK Support