Focal segmental glomerulosclerosis (FSGS) is a kidney disease defined by morphological changes to, and eventual loss of, podocytes: specialized cells that form the renal filtration barrier. Podocytes have a unique morphology, with cytoskeleton-rich "foot processes" that interdigitate to form the renal slit diaphragm. Podocyte loss due to FSGS causes nephrotic syndrome (NS), which, if untreated, progresses to end-stage kidney disease. Genetic studies have identified mutations in >20 genes associated with FSGS. Current therapies to treat NS are ineffective when the underlying FSGS is due to a single genetic cause (i.e., monogenic). Therefore, for monogenic FSGS it is critical to understand 1) how do the proteins encoded by FSGS-causing genes function? 2) how do disease-causing mutations affect them? and 3) do they function in a network that could be manipulated to provide new therapeutic options? One of the most commonly mutated genes in monogenic FSGS is INF2, a member of the formin family of actin-polymerizing factors. Previous models for the role of INF2 in FSGS centered on its canonical role as an F-actin regulator. However, our work showed that INF2 function is more complex. We characterized the two C. elegans INF2 orthologs,
exc-6 and
inft-2, by studying their function in the excretory canal (ExCa): a unicellular tube required for osmoregulation. We showed that
exc-6 not only regulates F-actin accumulation, but also microtubule (MT) dynamics, raising the possibility that MTs may be affected in FSGS. We also showed that disease-causing forms of human INF2 rescue
exc-6, demonstrating conserved function, and, for the first time, the gain-of-function nature of these mutations (Shaye and Greenwald, 2015). As for
inft-2, we showed that it promotes F-actin accumulation and that this activity is inhibited by
cyk-1, the sole ortholog of the mammalian formin Diaphanous (Shaye and Greenwald, 2016). To date, how gain-of-function mutations in INF2 lead to the changes in podocyte architecture that underlie FSGS remains unknown. To study the consequences of FSGS-causing mutations, we are mutating the conserved, disease-associated, residues in
inft-2, to ask how they affect INFT-2 localization and the cellular functions regulated by
inft-2. FSGS-causing mutations have also been identified in seven other genes whose products are cytoskeletal regulators, or cytoskeleton-associated proteins (TRPC6, CD2AP, ANLN, ACTN4, ARHGAP24, ARHGDIA and MYOE1). However, whether these genes, and INF2, function in a common process, or in different pathways, to regulate the podocyte cytoskeleton has not been explored. C. elegans encodes orthologs for all these genes. We are assessing their expression and function in the ExCa, as we did for INF2 homologs, and are testing genetic interactions between these genes, and
inft-2, to define a functional network of FSGS-associated genes.