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Abstract: TH-PO064

The Effects of Mechanical Strain on Adhesion in MYH9- Ablated Podocytes

Session Information

Category: Glomerular

  • 1002 Glomerular: Basic/Experimental Pathology

Authors

  • Keller, Keith H., Boston University School of Medicine , Boston, Massachusetts, United States
  • Belghasem, Mostafa, Boston University School of Medicine , Boston, Massachusetts, United States
  • Chen, Hui, BMC, Boston, Massachusetts, United States
  • Henderson, Joel M., Boston University Medical Center, Boston, Massachusetts, United States
Background

Myh9 is a gene that encodes for non-muscle myosin IIA (NM-IIA), an actin cytoskeleton component and protein involved in cell movement and adhesion in most cells, including podocytes. Autosomal dominant mutations in NM-IIA have been associated with focal segmental glomerulosclerosis (FSGS). Podocyte specific Myh9 knockout in mice showed that this gene alone was not enough to cause proteinuria or glomerulosclerosis. However, in our own laboratory we have found that when these same mice are exposed to models of glomerular hypertension, glomerular damage is promoted. This damage was preceded by evidence of podocyte loss in urine and tissue. Podocyte loss is a hallmark of kidney disease, and while it is known to occur in vivo, the mechanisms behind this phenomenon are unknown. It is believed that increase in glomerular capillary blood pressure is likely to be a strong contributing factor. Here we investigated the effect of mechanical strain on adhesion and cell morphology in Myh9 ablated podocytes.

Methods

Myh9 was knocked down, using RNAi, in immortalized mouse podocytes cultured on flexible silicone 6-well plates at a density of 6000 cells per well. Cells were mechanically stretched in a step-change fashion for 24hrs then cells were fixed or lysed for protein. Each experiment included three groups with two conditions (stretch/no stretch): WT; lentiviral control; KD. Changes in adhesion were assessed using cell counts, and morphologic changes were evaluated using immunofluorescence and quantified using imageJ.

Results

Transfected cells showed marked decrease in cellular attachment (V.CTL: -53%; V.CTL-ST: -53%; KD: -50%; KD-ST: -60%), compared to control cells (CTL: +77%; CTL-ST: +120%). Mean focal adhesion area increased with stretch in each experimental treatment group except KD (CTL: 1.41+ 1.62 μm2; CTL-ST: 2.12+ 3.13μm2; V.CTL: 0.54+ 0.47μm2; V.CTL-ST: 2.77+ 3.59μm2; KD: 3.04+ 2.57μm2; KD-ST: 2.09+ 1.13μm2). KD-ST cells showed large decrease in focal adhesion number compared to all other groups. Mean cell size was drastically larger in the KD groups (CTL: 773.6+489.μm2; CTL-ST: 505.5+377.7μm2; V.CTL: 817.6+1008,5μm2; V.CTL-ST: 1306.9+787μm2; KD: 3576.5+2694.7μm2; KD-ST: 5461.7+13,456.3μm2)

Conclusion

Myh9/NM-IIA may necessary for cells to bolster cell adhesion structures during events of mechanical stress, and its loss may compromise this protective adaptation.