The Drosophila Nephrocyte
Authors: Jianbo Na, Ross Cagan
Journal: Journal of the American Society of Nephrology (2013)

Drosophila nephrocytes have been an object of study for more than a century. They were named by Bruntz, based on observations of cells around the heart, the central nervous system, and in the sternal area in scorpions.1 Kowalevsky suggested that they function as a storage kidney, because they can absorb ammonia carmine from the hemolymph.2 Despite decades of study, however, the precise function of the nephrocyte has remained elusive. Two papers in the current issue of JASN now extend their function, making these remarkable cells still more surprising. The adult Drosophila nephrocytes are categorized as thoracic nephrocytes or abdominal nephrocytes.3 They derive from larval nephrocytes, among the largest cells in the larval body, which are classified as garland cells or pericardial cells based on their location. Garland cells lie close to the esophagus or between the salivary glands.4,5 Pericardial nephrocytes develop from the cardiogenic mesoderm by the late embryo/early larval stage6,7; they arrange into two rows of 20–25 flanking each side of the heart from the first to the sixth segment. Using transmission electron microscopy of adult Drosophila nephrocytes, Mills and King found that the plasma membrane of the pericardial nephrocyte is invaginated to form elaborate sheets and tubules along with organelles that resemble lysosomes.8 Functional lysosomes were confirmed by histochemical methods9,10 and were presumed to act as a garbage disposal system. By studying the third-instar larva of Calliphora, Crossley documented the ultrastructure of pericardial cells and demonstrated a desmosome-like structure, very similar to the podocyte slit diaphragm that is required for proper filtration within the vertebrate kidney.4 Although Schwinck observed that pericardial cells of Panorpa synthesize proteins and export them to hemolymph,11,12 more attention has been paid to the role of nephrocytes in taking up materials from the hemolymph. Pericardial nephrocytes were demonstrated to absorb foreign materials from the hemolymph, providing selection by size and charge.13 Based on dye injections, Hollande proposed that the Lepidopteran pericardial cell absorbs and stores toxic compounds from the hemolymph, hydrolyzing them to soluble, nontoxic molecules that are released into the hemolymph, perhaps to be excreted by the Malpighian tubules.14 This hypothesis was later supported by Wigglesworth's observation in bloodsucking insects that hemoglobin protein constituents consumed in a blood meal are broken down into biliverdin and stored in the nephrocytes, while the iron is released to the hemolymph.15 Recently, Drosophila nephrocytes have returned to center stage. Weavers et al. and Zhuang et al.16,17 independently showed that the molecular structure of the nephrocyte diaphragm is similar to that of the podocyte slit diaphragm. Reducing key components of the nephrocyte diaphragm, the Nephrin or Neph1 orthologs SNS and Kirre/Duf, respectively, led to structural defects. This strongly suggests that nephrocytes act in a manner analogous to our podocytes, the fly nephrocyte diaphragm presumably functioning similar to the mammalian slit diaphragm to regulate filtration. Weavers et al. therefore proposed that the nephrocyte diaphragm functions as a filter to exclude large hemolymph constituents from the labyrinthine channels, and that this filtration depends on the proper functioning of SNS/Nephrin and Kirre/Neph1. This led to the attractive hypothesis that the nephrocyte acts primarily as a fly podocyte (filtration), perhaps without the need for an associated renal proximal tubule (re-absorption). This work broadened interest in nephrocytes as a useful model for understanding kidney filtration. The charge and size-selectivity of nephrocytes is reminiscent of podocytes, a crucial component of the mammalian glomerular filtration barrier. Genetic mutations that affect constituent proteins within the slit diaphragm lead to severe proteinuria and kidney failure in humans.18,19 Proteinuria can also be caused by defects in the re-absorption machinery in the proximal tubule, involving the major proteins in the endocytic receptor complex (e.g., megalin, cubilin, and amnionless).20,21 In a pair of articles in the current issue of JASN, Zhang et al. use genetic approaches to further explore the functions of the Drosophila nephrocytes.22,23 Establishing a stable transgenic fly line producing a secreted fluorescently tagged protein (ANF-RFP) that accumulates in nephrocytes—reminiscent of the secreted GFP system described by Ferrandon et al.,24—they assessed the ability of loci to regulate protein uptake. For example, they demonstrated that mutating Sns/Nephrin, Kirre/Neph1, or Drosophila podocin abolished secreted protein accumulation in nephrocytes. Using an unbiased screen, they then identified >70 genes required for nephrocyte function. These provide an important resource for exploring nephrocyte function. Their screen identified the Drosophila orthologs of mammalian cubilin and amnionless as required for nephrocyte function. Reducing cubilin or amnionless function in the nephrocyte abolished both toxin and ANF-RFP uptake, whereas overexpression of amnionless increased protein uptake. Cubilin and amnionless are both specifically expressed in nephrocytes. Mammalian amnionless does not bind ligands directly, and the authors postulate that amnionless acts to enrich cubilin at the nephrocyte membrane, leading to increased protein uptake. Evidence from transmission electron microscopy further suggests that a cubilin/amnionless receptor complex is required for maintaining endocytic trafficking machinery within the nephrocyte. Of note, overexpressing human amnionless rescued ANF-RFP uptake in amnionless-knockdown flies, demonstrating that a cubilin/amnionless receptor complex is conserved between flies and humans. This work led the authors to propose the intriguing model that the Drosophila nephrocyte encompasses two functions in one cell: filtration and protein re-absorption. These would make nephrocytes analogous to podocyte plus proximal tubule. In mammals, the filtration system is composed of three layers: fenestrated endothelium, glomerular basement membrane, and podocyte foot processes. In Drosophila, the filtration system is composed of two: the nephrocyte basement membrane and nephrocyte diaphragm. Proteins, particles, or toxins enter the lacuna network by the filtration membrane, where they are degraded or reused. If correct, this model indicates that nephrocytes are a remarkably conserved yet parsimonious solution to the problem of blood filtration. Emerging structural, molecular, and functional similarities between the Drosophila nephrocyte and the mammalian nephron emphasize its promise for dissecting the mechanisms that underlie proper and compromised kidney function. Given its powerful genetic and molecular tools, several fundamental questions may prove usefully addressed by the fly system. For example, cubilin and megalin lie within the endocytosis receptor complex and have many ligands including vitamin-binding proteins, enzymes, drugs, hormones, toxins, calcium, albumin, lipoproteins, and hemoglobin.21 Identifying new ligands may help elucidate the mechanism of nutrient recycling in the proximal renal tubule, a process central to proteinuria. Flies can help with this task. Megalin is an enormous protein that includes an intracellular region composed of a menagerie of domains including endocytic motifs (NPXY), a NPXY-like motif (NQNY), SH2 and SH3 recognition motifs, and consensus phosphorylation sites.21 Flies provide a tool for understanding structure/function in vivo. Third, the amn locus yields five protein products, but the one that interacts with cubilin is unknown. Human amnionless is able to rescue loss of Drosophila amnionless, providing an interesting tool for exploring the details of cubilin/amnionless interactions as well as their downstream targets. Kidney disease is the eighth leading cause of death in the United States.25 Although they are useful, cell culture systems are limited in their ability to recapitulate the in vivo characteristics and functions of the podocyte.26,27 As Han and colleagues demonstrate, insect nephrocytes continue to amaze, taking the stage yet again as a promising in vivo tool for studying podocyte function. We anticipate more surprises will be in store for our understanding of this remarkable cell. Disclosures R.C. declares financial interest in Medros Inc. We thank Jay Pendse for critically reviewing the manuscript. This work was supported in part by a grant from the Nephcure foundation.