An In Vivo Functional Analysis System for Renal Gene Discovery in Drosophila Pericardial Nephrocytes

Abstract

The difficulty in accessing mammalian nephrons in vivo hinders the study of podocyte biology. The Drosophila nephrocyte shares remarkable similarities to the glomerular podocyte, but the lack of a functional readout for nephrocytes makes it challenging to study this model of the podocyte, which could potentially harness the power of Drosophila genetics. Here, we present a functional analysis of nephrocytes and establish an in vivo system to screen for renal genes. We found that nephrocytes efficiently take up secreted fluorescent protein, and therefore, we generated a transgenic line carrying secreted fluorescent protein and combined it with a nephrocyte-specific driver for targeted gene knockdown, allowing the identification of genes required for nephrocyte function. To validate this system, we examined the effects of knocking down sns and duf, the Drosophila homologs of nephrin and Neph1, respectively, in pericardial nephrocytes. Knockdown of sns or duf completely abolished the accumulation of the fluorescent protein in pericardial nephrocytes. Examining the ultrastructure revealed that the formation of the nephrocyte diaphragm and lacunar structure, which is essential for protein uptake, requires sns. Our preliminary genetic screen also identified Mec2, which encodes the homolog of mammalian Podocin. Taken together, these data suggest that the Drosophila pericardial nephrocyte is a useful in vivo model to help identify genes involved in podocyte biology and facilitate the discovery of renal disease genes.

ADCK4 mutations promote steroid-resistant nephrotic syndrome through CoQ10 biosynthesis disruption

“…The mitochondrial fractionation of podocytes was done using Mitochondrial Isolation Kit for Cultured Cells and UAS-RNAi transgenes (14,41). Nephrocyte function was measured by the amount of secreted red fluorescent proteins accumulated in nephrocytes at the second instar larvae stage (14,41).…”

“…In addition, we performed knockdown of Drosophila coq8 (dcoq8) in Drosophila pericardial nephrocytes, which share remarkable similarities to the glomerular podocytes (14). The protein uptake assay showed that knockdown of dcoq8 in nephrocytes reduced the uptake ability of a secreted fusion protein of rat atrial natriuretic factor (ANF) and GFP, suggesting that dcoq8 is required for nephrocyte function in Drosophila (Supplemental Figure 2, A and B).…”

ARHGDIA mutations cause nephrotic syndrome via defective RHO GTPase signaling

“…The proteinuric effect of the arhgdia MO was confirmed using an established zebrafish proteinuria ELISA assay (38) against a fusion protein of vitamin D-binding protein and GFP in /-fabp::VDBP-GFP transgenic zebrafish ( Figure 5E). Similarly, the protein uptake assay using Drosophila pericardial nephrocytes, which share remarkable similarities to the glomerular podocytes (39), showed that knockdown of Drosophila RhoGDI (CG7823) in nephrocytes reduced the uptake ability of the secreted fusion protein of rat atrial natriuretic factor (ANF) and GFP, suggesting that CG7823 is required for nephrocyte function in Drosophila (Supplemental Figure 9).…”

Src64B phosphorylates Dumbfounded and regulates slit diaphragm dynamics: Drosophila as a model to study nephropathies

“…In Drosophila, mutations in duf and sns also result in loss of filtration diaphragms and in radical changes in nephrocyte architecture and function (Prieto-Sánchez, 2009;Weavers et al, 2009;Zhuang et al, 2009). Taking into account all the similarities previously described, we proposed that the nephrocyte filtration diaphragm could be a suitable model with which to study the formation, maintenance and repair of the vertebrate SD (Weavers et al, 2009) (a view supported by Dow and Romero, 2010;Simons and Huber, 2009;Zhang et al, 2013).…”