Modeling Monogenic Human Nephrotic Syndrome in the Drosophila Garland Cell Nephrocyte

Abstract

Steroid-resistant nephrotic syndrome is characterized by podocyte dysfunction. garland cell nephrocytes are podocyte-like cells and thus provide a potential model in which to study the pathogenesis of nephrotic syndrome. However, relevant pathomechanisms of nephrotic syndrome have not been studied in nephrocytes. Here, we discovered that two slit diaphragm proteins, orthologs of the human genes encoding nephrin and nephrin-like protein 1, colocalize within a fingerprint-like staining pattern that correlates with ultrastructural morphology. Using RNAi and conditional CRISPR/Cas9 in nephrocytes, we found this pattern depends on the expression of both orthologs. Tracer endocytosis by nephrocytes required Cubilin and reflected size selectivity analogous to that of glomerular function. Using RNAi and tracer endocytosis as a functional read-out, we screened orthologs of human monogenic causes of nephrotic syndrome and observed conservation of the central pathogenetic alterations. We focused on the coenzyme Q (CoQ) biosynthesis gene , the silencing of which disrupted slit diaphragm morphology. Restoration of CoQ synthesis by vanillic acid partially rescued the phenotypic and functional alterations induced by -RNAi. Notably, colocalized with mitochondria, and silencing increased the formation of reactive oxygen species (ROS). Silencing of, a subunit of the mitochondrial respiratory chain that controls ROS formation independently of CoQ, phenocopied the effect of -RNAi. Moreover, the ROS scavenger glutathione partially rescued the effects of-RNAi. In conclusion, garland cell nephrocytes provide a model with which to study the pathogenesis of nephrotic syndrome, and ROS formation may be a pathomechanism of-nephropathy.

GAPVD1 and ANKFY1 Mutations Implicate RAB5 Regulation in Nephrotic Syndrome

“…Fluorescent tracer uptake in nephrocytes was performed as previously described. 55 Briefly, nephrocytes were dissected in PBS and incubated with FITC-albumin (Sigma) for 30 seconds. After a fixation of 5 minutes in 8% paraformaldehyde, cells were rinsed in PBS and exposed to Hoechst 33342 (1:1000) for 20 seconds and mounted in Antifade Diamond.…”

“…Garland cell nephrocytes are podocyte-like cells that are an established model for glomerular disease, 54,78 including monogenic SRNS. 55 In nephrocytes, the ortholog of nephrin forms autocellular slit diaphragms across membrane invaginations called labyrinthine channels. 54,55,78 By sequence analysis, we identified the uncharacterized Drosophila gene CG1657 as the ortholog of human GAPVD1 ( Figure 6A).…”

New Insights into Podocyte Biology in Glomerular Health and Disease

“…Drosophila provides a model that is readily amenable to genetic manipulation, and therefore, it is used for disease gene validation by various groups. 47 Han and colleagues 48 investigated uptake of Red Fluorescent Protein in the pericardial nephrocytes by screening 3800 genes with 268 hits, such as RabGTPases Rab5, -7, and -11, leading to changes in nephrocyte morphology and endocytosis. They furthermore showed that knockdown of dKANK, the Drosophila homolog of kidney Ankyrin repeat-containing proteins KANK1, -2, and -4 (newly identified as disease-causing genes in patients with steroid-resistant nephrotic syndrome [SRNS]), has evolutionary conserved functions in the nephrocyte.…”

A deregulated stress response underlies distinct INF2 associated disease profiles

“…Importantly, the degree of intracellular actin accumulation is quantitatively correlated with a perturbed, discontinuous distribution of nephrin in the plasma membrane of fly nephrocytes. This phenotype is reminiscent of other nephropathy models in fly nephrocytes, such as nephrotic syndrome 28 . The defect in nephrin distribution could either be directly linked to a function of cortical actin in the organization of the slit diaphragm 29 or be due to defects in nephrin trafficking.…”