Elucidating the role of cyclin G-associated kinase as a regulator of podocyte autophagy

Project Summary: An estimated 37 million people in the US and over 800 million people worldwide are affected by chronic kidney disease (CKD), which is defined as persistent abnormalities in the structure and/or function of the kidneys. Integral to the filtering function of the kidney are podocytes, which are highly specialized, post-mitotic epithelial cells that line the glomerular filtration barrier. Although damage to podocytes has been implicated in the pathophysiology of CKD, significant gaps in our understanding of podocyte repair and maintenance hinder the development of novel therapies that specifically mediate podocyte injury. To address this problem, I will use a genetically engineered mouse model with a podocyte-specific knockout of cyclin G-associated kinase (GAK) to study cellular mechanisms involved in podocyte maintenance. Our group has previously demonstrated that podocyte-specific loss of Gak results in podocyte damage and kidney failure. Interestingly, we found that re- expression of the truncated 62-kDa C-terminal end of GAK (C62) in mice completely rescues this severe phenotype. I hypothesize that the 62-kDa C-terminus of GAK regulates podocyte autophagy and is indispensable for proper turnover of cellular debris and maintenance of homeostasis. In Aim 1, I will test my hypothesis by measuring autophagic flux in primary podocytes derived from wildtype, Gak knockout, and GAK C62 rescue mice via western blot and immunofluorescence. Furthermore, I will assess the ability of GAK C62 to bind and activate key autophagy regulators via western blot. In Aim 2, I will perform translating ribosome affinity purification sequencing (TRAP-seq) on wildtype, Gak knockout, and C62 podocytes in vivo. This data-driven approach will provide an unbiased view of pathways that are regulated by the C-terminal end of GAK in podocytes. I will validate the results of the TRAP-seq experiment using western blot and qRT-PCR, as well as in vitro knockdown assays in primary podocytes, targeting pathways that exhibit the highest upregulation and downregulation between the three groups. Given that GAK is implicated in other cellular processes, this approach would enable us to both solidify its role in autophagy and determine additional pathways specifically mediated by GAK C62. This proposal to investigate the role of GAK in regulating autophagy in podocytes will advance our collective understanding of mechanisms that contribute to podocyte repair and maintenance and potentially uncover promising therapeutic targets for treating podocytopathies. Furthermore, this project is an integral component of a comprehensive training plan that will provide me with plenty of new learning opportunities and necessary mentorship towards my long-term goal of becoming a physician-scientist in nephrology.