Testing the Podocyte Foot Process Proteome During Development and in Disease

Abstract Podocytes are kidney cells with cellular extensions, termed foot processes, that provide vital functions. Multiple podocytes interdigitate their foot processes to form a unique cell-to-cell contact termed a slit diaphragm (SD). The podocyte foot process / SD in conjunction with other cells of the kidney glomerulus and basement membrane wrap around the endothelium, functioning as a molecular sieve to filter blood. Podocytes and the complex architecture created by their interdigitating processes is vital for proper kidney function. Compromise to podocyte integrity is one of the most frequent clinical observations in kidney disease and cannot be repaired. Central to maintaining podocytes integrity is the slit diaphragm (SD). The SD transverses adjacent foot processes via a protein bridge and helps form the molecular sieve. As foot process integrity is essential to maintaining kidney physiology, it is clinically imperative to understand what at the molecular level enables them to withstand filtration forces and perform their indispensable role. Yet, the composition of the podocyte foot process is poorly defined. To this end we have generated a novel in vivo murine model able to identify the podocyte foot proteome through proximity based labeling technique. Podocin is one of the most highly expressed proteins within the foot process and localizes to the SD. Using podocin as a handle, we have modified the Nphs2 (podocin) locus to link the mutated, promiscuous BirA biotin ligase to podocin (podocin-BioID). A flexible linker allows for a generous proximity around podocin, thereby capturing the broad proteome of the podocyte foot process. Preliminary analysis has confirmed the proper function of the podocin-BioID in vitro. Furthermore, we have identified proper localization of the podocin-BioID to the glomerulus in our murine model. We hypothesize that proteomics analysis of the podocin-BioID will reveal novel molecular components interacting with podocin to maintain podocyte integrity. Candidate factors will be identified through correlation with in silico analyses and their localized expression verified in normal tissue sections. We will utilize zebrafish to test the requirement of our selected proteins for maintaining podocyte foot process architecture and kidney function. Zebrafish podocytes are structurally and functionally equivalent to their mammalian homologues by 48 hours post fertilization. Genes of interest identified by our studies will be subjected to CRISPR/Cas9 gene editing to abolish their activity. Embryos will be phenotypically assessed for compromise in filtration by initially screening for pericardial edema. Additionally, we will obtain mammalian diabetic nephropathy samples to determine whether candidate expression is altered during kidney disease. The results of our studies will provide novel insights into the spatially restricted foot process proteome and maintenance of podocyte architecture. These findings will pave the way to study comparative proteomics analysis occurring within podocytes upon disease initiation and subsequent progression. We anticipate our findings will provide new avenues of therapeutic intervention.