Identification of Novel Therapeutic Targets for Alport Syndrome Using Near Super-Resolution Imaging of Podocytes

Background: Alport Syndrome is a rare disease currently without a cure. It is characterized by hematuria and proteinuria and eventual ESKD. Obtaining a deeper understanding of the alterations to the cytoskeletal architecture of podocytes from their interactions with the abnormal glomerular basement membrane (GBM) in Alport syndrome is one promising approach to identify novel therapeutic targets. Sarcomere-like structures (SLSs), are intertwining actin and myosin that form in areas of podocytes where foot processes become effaced. We hypothesize that SLSs are different in frequency in Alport mouse podocytes compared to COL4A3 rescued mouse podocytes. Methods: We performed magnified analysis of proteome (MAP) expansion microscopy of kidneys from wild type, Alport and rescued mice. Col4a3-/- Alport mice are rescued using a doxycycline-inducible Col4a3 transgene in podocytes, which normalizes the GBM and extends the time to ESKD. Doxycycline was started at 2 or 6 weeks of age and mice sacrificed 6 weeks later. Incubating paraffin embedded tissue in a high concentration of acrylamide, we expanded mouse kidney tissue near 4 times its original size. Antibody staining with synaptopodin and myosin was performed on expanded kidney sections. Airyscan confocal microscopy was used to image SLSs and images were analyzed using the ImageJ software. Results: Airyscan was used to demonstrate sarcomere-like structures (SLSs) in our mouse models. The percentage of the capillary wall covered with SLS was determined using the ImageJ software to identify alternating synaptopodin and myosin. Sarcomere-like structures were significantly less in mice rescued at 2 weeks compared to Alport mice. SLS were also less in mice induced at 2 weeks compared to those at 6 weeks of life. SLS were more organized throughout glomerular capillary loops in rescued mouse podocytes compared to Alport mouse podocytes. Conclusion: SLS are significantly reduced in podocyte foot processes in Alport mice successfully rescued at 2 weeks of life compared to those induced at 6 weeks of life. Utilizing restoration of the GBM in the inducible rescue mice to study its impact on podocyte cytoskeletal architecture, could provide the key to understanding mechanisms at play in maintenance of foot processes and the glomerular filtration barrier in Alport syndrome. Funding: Other NIH Support - NICHD