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Evolutionarily conserved developmental pathways
The nephron is the basic structural and functional unit of the vertebrate kidney. It is composed of a glomerulus, the site of ultrafiltration, and a renal tubule, along which the filtrate is modified. Although widely regarded as a vertebrate adaptation1 'nephron-like' features can be found in the excretory systems of many invertebrates, raising the possibility that components of the vertebrate excretory system were inherited from their invertebrate ancestors. This study shows that the insect nephrocyte has remarkable anatomical, molecular and functional similarity with the glomerular podocyte, a cell in the vertebrate kidney that forms the main size-selective barrier as blood is ultrafiltered to make urine. In particular, both cell types possess a specialised filtration diaphragm, known as the slit diaphragm in podocytes or the nephrocyte diaphragm in nephrocytes. Fly orthologues of the major constituents of the slit diaphragm, including nephrin, neph1, CD2AP, ZO-1 and podocin are expressed in the nephrocyte and form a complex of interacting proteins that closely mirrors the vertebrate slit diaphragm complex. Furthermore, the nephrocyte diaphragm is completely lost in flies mutant for nephrin or neph1 orthologues, a phenotype resembling loss of the slit diaphragm in the absence of either nephrin (as in the human kidney disease NPHS1) or neph1. These changes drastically impair filtration function in the nephrocyte. The similarities described between invertebrate nephrocytes and vertebrate podocytes provide evidence suggesting the two cell types are evolutionarily related and establish the nephrocyte as a simple model in which to study podocyte biology and podocyte-associated disease (Weavers, 2009).
Filtration of blood in the vertebrate kidney occurs within the glomerulus of the nephron (see The glomerular and nephrocyte filtration barriers are anatomically similar). The filtration barrier is formed by podocytes, specialised epithelial cells, which send out interdigitating foot processes to enwrap the glomerular capillaries. These processes are separated by 30-50nm wide slit pores spanned by the slit diaphragm, which together with the glomerular basement membrane (GBM), form a size- and charge-selective filtration barrier. Disruption to this barrier in disease leads to leakage of blood proteins into the urinary space and to kidney failure (Weavers, 2009).
Although invertebrate excretory systems are considered to lack nephrons, 'nephron-like' components, such as filtration cells and ducts in which the filtrate is modified, are widespread. Insect nephrocytes regulate haemolymph composition by filtration, followed by endocytosis and processing to sequester and/or secondarily metabolise toxic materials. Drosophila has two types - garland and pericardial nephrocytes. They are tethered to the oesophagus, and are bathed in haemolymph. Extensive infolding of the plasma membrane generates a network of labyrinthine channels or lacunae flanked by nephrocyte foot processes. The channel entrances are narrow slits 30nm in width, spanned by a single or double filament forming a specialised filtration junction; the nephrocyte diaphragm. Each nephrocyte is enveloped by basement membrane. The nephrocyte diaphragm and basement membrane behave as a size and charge-selective barrier and filtrate is endocytosed from the sides of the lacunae. Thus the anatomy of the nephrocyte and podocyte filtration barriers are remarkably similar (Weavers, 2009).
In view of this similarity, whether the nephrocyte diaphragm is molecularly related to the slit diaphragm was investigated. The major slit diaphragm components, the transmembrane Ig-domain superfamily proteins nephrin and neph1 are co-expressed in the podocyte and interact across the slit pore by homo- and hetero-typic binding to form the diaphragm. Mutations in nephrin, as in human congenital nephrotic syndrome of the Finnish type (NPHS1), cause slit diaphragm loss and foot process effacement, resulting in breakdown of the filtration barrier and proteinuria (Weavers, 2009).
Drosophila has two nephrin orthologues - sticks-and-stones (sns) and hibris (hbs) - and two neph1 orthologues - dumbfounded (duf) and roughest (rst). Since hbs and rst are expressed in only a subset of nephrocytes, focus was placed on sns and duf. Sns and Duf are expressed throughout life in both nephrocyte types, from midembryogenesis for garland cells and from the first larval instar for pericardial cells. Interestingly, the onset of Sns and Duf expression correlates in time with the appearance of the nephrocyte diaphragm at the ultrastructural level and double labelling reveals precise co-localisation. This finding is initially surprising because in most contexts Sns and Duf are expressed in complementary patterns and mediate interaction between cells of different type. The only other situation where the two types of Ig-domain proteins are co-expressed in the same cell is the vertebrate podocyte. Sns and Duf are dependent on one another for stabilization at the plasma membrane. Loss or knockdown of either protein in embryonic or larval nephrocytes leads to a loss, severe reduction or mislocalisation of the other. These data demonstrate an essential interaction between the two proteins in the same cell, similar to those between nephrin and neph1 in the podocyte. The precise subcellular location of the proteins was revealed by immuno-electron microscopy. Both Sns and Duf specifically localise to the nephrocyte diaphragm and double labelling reveals close colocalisation between the two proteins (Weavers, 2009).
Garland and pericardial nephrocytes are correctly specified in sns and duf mutants. However, given the importance of the Ig-domain proteins in slit diaphragm formation, the ultrastructure of the diaphragm was examined in sns and duf mutants. In wild-type garland cells, nephrocyte diaphragms and associated lacunae appear during mid-embryogenesis, progressively increasing in number. Diaphragms densely populate the cell periphery in third instar larvae. Strikingly, sns or duf mutant garland cells completely lack nephrocyte diaphragms at every stage and lacunae are rarely detected. Occasional infoldings do form, but are never bridged by diaphragms. Instead, the nephrocyte surface contains frequent, small patches of electron-dense subcortical material, possible remnants of undercoat normally associated with the wild-type diaphragm. These observations suggest that in the absence of the diaphragm, foot processes are unstable and undergo effacement. Scanning electron microscopy reveals the surface smoothening in mutant garland cells. These phenotypes are remarkably similar to those of podocytes lacking nephrin or neph1. Thus, by analogy with nephrin and neph1 in the slit diaphragm, it is suggested that Sns and Duf interact through their extracellular domains to form the nephrocyte diaphragm itself (Weavers, 2009).
It is noted that the basement membrane in sns knockdown and duf larval nephrocytes was irregular and dramatically expanded. The basement membrane in duf nephrocytes has an average depth of 202nm compared with 57nm for wild-type. This results from an increase in deposition of the Drosophila collagen IV (Viking). However this is unlikely to account for the four-fold thickening observed, and it is suggested that a further contributing factor is accumulation of haemolymph proteins that clog the basement membrane due to inefficient filtration (Weavers, 2009).
Given the similarities between the morphology and molecular requirements for podocyte and nephrocyte diaphragms, the ability of human nephrin to rescue the sns mutant phenotype was tested. However nephrocytes are sensitive to absolute levels of sns, so that even moderate overexpression produced abnormal phenotypes. Therefore the effects were compared of overexpressing Drosophila sns with human nephrin. Resulting phenotypes are strikingly similar, including abnormal nephrocyte foot process morphology and marked thickening of diaphragm filaments. These data indicate that precise levels of Sns are critical for diaphragm formation and more importantly that human nephrin and Drosophila Sns function in equivalent ways (Weavers, 2009).
The Immunoglobulin superfamily (IgSF) proteins Neph1 and Nephrin are co-expressed within podocytes in the kidney glomerulus, where they localize to the slit diaphragm (SD) and contribute to filtration between blood and urine. Their Drosophila orthologs Kirre (Duf) and Sns are co-expressed within binucleate garland cell nephrocytes (GCNs) that contribute to detoxification of the insect hemolymph by uptake of molecules through an SD-like nephrocyte diaphragm (ND) into labyrinthine channels that are active sites of endocytosis. The functions of Kirre and Sns in the embryonic musculature, to mediate adhesion and fusion between myoblasts to form multinucleate muscle fibers, have been conserved in the GCNs, where they contribute to adhesion of GCNs in the 'garland' and to their fusion into binucleate cells. Sns and Kirre proteins localize to the ND at the entry point into the labyrinthine channels and, like their vertebrate counterparts, are essential for its formation. Knockdown of Kirre or Sns drastically reduces the number of NDs at the cell surface. These defects are associated with a decrease in uptake of large proteins, suggesting that the ND distinguishes molecules of different sizes and controls access to the channels. Moreover, mutations in the Sns fibronectin-binding or immunoglobulin domains lead to morphologically abnormal NDs and to reduced passage of proteins into the labyrinthine channels for uptake by endocytosis, suggesting a crucial and direct role for Sns in ND formation and function. These data reveal significant similarities between the insect ND and the SD in mammalian podocytes at the level of structure and function (Zhuang, 2009).
In Drosophila, the Immunoglobulin superfamily (IgSF) proteinsencoded by kin of irre [kirre; also known asdumbfounded (duf)], roughest (rst),sticks and stones (sns) and hibris (hbs)function as ligand-receptor pairs on the surface of founder cells and fusioncompetent myoblasts. These proteins mediate recognition, adhesion and fusion toform multinucleate syncitia through direct interaction at sites of myoblastcontact. However, neither their action nor their expression is exclusive tothe musculature, and previous studies noted their role in cell recognition andadhesion in the Drosophila eye. Moreover, multiple studies have confirmed the presence of the kirre transcript and sns transcript in the binucleate garland cell nephrocytes (GCNs). These nephrocytes possess a structure visible by transmission electron microscopy (TEM) reminiscent of the slit diaphragm (SD) in the vertebrate kidney, and process waste products from the hemolymph. It is therefore compelling that the fly detoxificationmachinery may have similarities to that in mammals, and that Sns and Kirreplay roles similar to those of their vertebrate counterparts (Zhuang, 2009).
Removal of waste products from the closed circulatory system of vertebratestakes place in the kidney glomerulus. Podocytes, kidney epithelial cells thatsurround the capillary blood vessels, extend foot processes that contact thesurface of these vessels. Filtration then occurs as molecules flow out of thebloodstream through slits between adjacent foot processes into the urine. Neph1, vertebrate orthologs of the above Drosophila IgSF proteins, localize to this filter and appear to be an important determinant of glomerular permeability (Hamano, 2002; Liu, 2003). Mutations in nephrin and neph1 are associated with congenital nephrotic syndrome as a consequence of defects in this filtration diaphragm. Lack of either nephrin or neph1 leads to podocyte foot process effacement and detachment of podocytes from the glomerular basement membrane, loss of SDs, and proteinuria (Donoviel, 2001; Putaala, 2001; Zhuang, 2009 and references therein).
In addition to their high degree of homology, Nephrin and Neph1 have otherfeatures in common with Sns and Kirre. Heterophilic interactions occur intrans between the extracellular domains of Nephrin and Neph1, and Sns andKirre. Studies have suggested that, in addition to serving as a scaffold onto which other proteins in the SD assemble, Nephrin and Neph1 function as signaling molecules to direct downstream cytoplasmic events(Benzing, 2004). They cooperate to transduce a signal that directs actin polymerization (Garg, 2007), and activation of this pathway occurs through interaction of phosphorylated tyrosines in the cytoplasmic domains of Nephrin and Neph1 to adaptor proteins (Jones, 2006; Verma, 2006). Theseadaptor proteins recruit components of the actin polymerization machinery thatinclude N-WASp and Arp2/3. Similar phosphotyrosine modifications are important for Sns function and studies have shown that the WASp and Arp2/3 actinpolymerization machinery functions in Drosophila myoblast fusion,probably downstream of Sns and Kirre (Zhuang, 2009).
The pericardial cells and garland cells comprise two subpopulations ofDrosophila nephrocytes that, along with Malpighian tubules, form theexcretory system. Approximately 25-30 tightly associated binucleate GCNsencircle the anterior end of the proventriculus in a 'garland' at its junctionwith the esophagus. The cortical region of the cytoplasm includes elaboratechannels that are generated by invagination of the plasma membrane duringembryogenesis and early larval instar stages. The initial invagination isassociated with formation of a junction between two sites on the plasmamembrane that are visible by TEM. Through a mechanism that is not entirely clear, this initial invagination expands into an extensive array of labyrinthine channels by the third-instar larval stage. The GCNs are very active in endocytosis via coated vesicles at sites deep within these labyrinthine channels. Thus, molecules to be eliminated must gain access to the endocytic machinery deep in these channels. These studies also identified a thin bridge spanning the channel opening that is visually similar to the vertebrate SD. The presence of Sns and Kirre and a slit diaphragm-like structure in these binucleate cells raised the possibility that these IgSF proteins might function in GCN fusion and/or in formation of this structure (Zhuang, 2009).
This study, along with that of Weavers (2009) demonstrates thatSns and Kirre are present in, and crucial for, the nephrocyte diaphragm (ND).Knockdown of Kirre or Sns results in a severely diminished number of NDs andsmoothening of ND-associated furrows on the GCN surface, implicating Sns andKirre in their formation. Mutations in the extracellular domain of Sns causemajor perturbations in the ND, establishing that Sns also dictates fundamentalaspects of its structure. Similar smoothening of the GCN surface occurs uponknockdown of Polychaetoid (Pyd), the Drosophila ortholog of the zonula occludens (ZO-1) tight junction protein that interacts with Neph1, providing strong support for functional conservation of these molecules. The ND controls access of molecules to the labyrinthine channels for uptake by endocytosis, and can discriminate between molecules of different sizes in a rate-dependent manner. Finally, in contrast to that reported by Weavers (2009) and reminiscent of their action in the embryonic musculature, Sns and Kirre contribute to the adhesion of the GCNs into an organized garland and their fusion into binucleate cells (Zhuang, 2009).
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Donoviel, D. B., Freed, D. D., Vogel, H., Potter, D. G., Hawkins, E., Barrish, J. P., Mathur, B. N., Turner, C. A., Geske, R., Montgomery, C. A. et al. (2001). Proteinuria and perinatal lethality in mice lacking NEPH1, a novel protein with homology to NEPHRIN. Mol. Cell. Biol. 21: 4829-4836. PubMed Citation: 11416156
Garg, P., Verma, R., Nihalani, D., Johnstone, D. B. and Holzman, L. B. (2007). Neph1 cooperates with nephrin to transduce a signal that induces actin polymerization. Mol. Cell. Biol. 27: 8698-8712. PubMed Citation: 17923684
Hamano, Y., Grunkemeyer, J. A., Sudhakar, A., Zeisberg, M., Cosgrove, D., Morello, R., Lee, B., Sugimoto, H. and Kalluri, R. (2002). Determinants of vascular permeability in the kidney glomerulus. J. Biol. Chem. 277: 31154-31162. PubMed Citation: 12039968
Jones, N., Blasutig, I. M., Eremina, V., Ruston, J. M., Bladt, F., Li, H., Huang, H., Larose, L., Li, S. S., Takano, T., et al. (2006). Nck adaptor proteins link nephrin to the actin cytoskeleton of kidney podocytes. Nature 440: 818-823. PubMed Citation: 16525419
Liu, G., Kaw, B., Kurfis, J., Rahmanuddin, S., Kanwar, Y. S. and Chugh, S. S. (2003). Neph1 and nephrin interaction in the slit diaphragm is an important determinant of glomerular permeability. J. Clin. Invest. 112: 209-221. PubMed Citation: 12865409
Putaala, H., Soininen, R., Kilpelainen, P., Wartiovaara, J. and Tryggvason, K. (2001). The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death. Hum. Mol. Genet. 10: 1-8. PubMed Citation: 11136707
Verma, R., Kovari, I., Soofi, A., Nihalani, D., Patrie, K. and Holzman, L. B. (2006). Nephrin ectodomain engagement results in Src kinase activation, nephrin phosphorylation, Nck recruitment, and actin polymerization. J. Clin. Invest. 116: 1346-1359. PubMed Citation: 16543952
Weavers, H., Prieto-Sanchez, S., Grawe, F., Garcia-Lopez, A., Artero, R., Wilsch-Brauninger, M., Ruiz-Gomez, M., Skaer, H. and Denholm, B. (2009). The insect nephrocyte is a podocyte-like cell with a filtration slit diaphragm. Nature 457: 322-326. PubMed Citation: 18971929
Zhuang, S., Shao, H., Guo, F., Trimble, R., Pearce, E. and Abmayr, S. M. (2009). Sns and Kirre, the Drosophila orthologs of Nephrin and Neph1, direct adhesion, fusion and formation of a slit diaphragm-like structure in insect nephrocytes. Development 136(14):2335-44. PubMed Citation: 19515699
date revised: 20 February 2010
Developmental Pathways conserved in Evolution
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