The effects of mechanical strain on adhesion in Myh9-ablated podocytes
Keller, Keith Hollis
MetadataShow full item record
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 glomerular sclerosis (FSGS) in patients with the mutation. Furthermore, a strong association has been discovered between genetic variation in the Myh9 region on chromosome 22, and the increased risk of chronic kidney disease in African Americans. Later studies by Johnstone et al. using podocyte specific, Myh9 KO in mice showed that knocking out this gene alone was not enough to cause proteinuria or glomerular sclerosis. 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. Based on these findings, we hypothesized that the mechanical strain in the form of stretch, occurring during hypertension, may act as a second hit to Myh9 ablation in podocytes, causing changes in podocytes ability to adhere to the glomerular basement membrane (GBM), ultimately leading to the pathology seen in patients. To test this, we knocked down Myh9 in immortalized mouse podocytes cultured on flexible silicon membranes and exposed them to mechanical stretch for 24hrs. Cell adhesion was evaluated via cell attachment assays. Cell morphology and focal adhesions (FA) were examined using immunofluorescence and quantified using imageJ. Our results showed a significant decrease in transduced cells attached to the membrane after stretch, as well as an increase in FA size and number in cells that underwent stretch, except in the Myh9 knockdown. Cells with Myh9 knockdown also showed marked increase in area, with a decrease in FA size and number after exposure to stretch. These results support our hypothesis that Myh9 mutations may be a contributing factor to podocyte loss in patients with hypertension and chronic kidney disease.