The insect nephrocyte is a podocyte-like cell with a filtration slit diaphragm

Abstract

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 adaptation, '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. Here we show that the insect nephrocyte has remarkable anatomical, molecular and functional similarity to 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 specialized filtration diaphragm, known as the slit diaphragm in podocytes or the nephrocyte diaphragm in nephrocytes. We find that fly (Drosophila melanogaster) orthologues of the major constituents of the slit diaphragm, including nephrin, NEPH1 (also known as KIRREL), CD2AP, ZO-1 (TJP1) and podocin, are expressed in the nephrocyte and form a complex of interacting proteins that closely mirrors the vertebrate slit diaphragm complex. Furthermore, we find that the nephrocyte diaphragm is completely lost in flies lacking the orthologues of nephrin or NEPH1-a phenotype resembling loss of the slit diaphragm in the absence of either nephrin (as in human congenital nephrotic syndrome of the Finnish type, NPHS1) or NEPH1. These changes markedly impair filtration function in the nephrocyte. The similarities we describe between invertebrate nephrocytes and vertebrate podocytes provide evidence suggesting that the two cell types are evolutionarily related, and establish the nephrocyte as a simple model in which to study podocyte biology and podocyte-associated diseases.

Figure 1. The glomerular and nephrocyte filtration barriers are anatomically similar

a-d, Schematic drawings of the vertebrate nephron (a), glomerular filtration barrier (b), insect excretory system (c) and nephrocyte filtration barrier (d). Ultrafiltration (red arrow), filtrate flow (black arrow) and urinary space (b) or extracellular lacunae (d) (asterisk) are shown. e,f, Drosophila garland (anti-HRP, e) and pericardial (anti-Pericardin, f) nephrocytes. Higher magnification images are shown in eⁱ and fⁱ. g-i, TEMs of stage 16 embryonic garland nephrocytes. g, Four garland nephrocytes surrounding the proventiculus (pv), connective fibres (arrowhead). h and i, High magnification of garland nephrocyte cell surface (h) and nephrocyte diaphragm (i) showing nephrocyte diaphragm (arrowhead), extracellular lacunae (asterisk). Scale bars 2μm (g), 100nm (h,i). fp, foot process; sd, slit diaphragm; nd, nephrocyte diaphragm; bm, basement membrane.

Figure 2. Sns and Duf are expressed in Drosophila nephrocytes

a-h, Sns (a,d,f,h) and Duf (b,c,e,g,h) expression in garland (a,b,d,e) and pericardial (c,f,g,h) nephrocytes. Embryonic (a,b, arrowheads), first instar larva (c, green, arrowheads), and third instar larvae (d-h, green). The actin cytoskeleton has been counterstained in c,d,f,g (red). h, Sns (h, green) and Duf (hⁱ, red) co-localise (hⁱⁱ, yellow). i-l, Clusters of ~6-8 wild-type (i,k), sns (j) or duf,rst (l) embryonic garland cells stained with anti-Duf (i,j) or anti-Sns (k,l). m-p, wild-type (m,o), sns-RNAi (n) and duf (p) third instar garland cells stained for anti-Duf (red) and anti-Sns (green) (merge appears yellow) and DNA (blue). Single optical section (m,n) or z-projection of cell surface (o,p) are shown. q-u, TEMs of wild-type third instar garland cells immunogold-stained for anti-Sns (q), anti-Duf (r,s) or double labelled (t,u). For double labelling 5nm (arrowhead) and 10nm (arrow) gold particles are used for Sns and Duf respectively. Scale bars 100nm (q-t) and 10nm (u).

Figure 3. Sns and Duf are required for nephrocyte diaphragm formation and normal morphology

a,b, sns (a, aⁱ) and duf,rst (b) embryonic garland cells lack diaphragms and lacunae. aⁱ, higher magnification of a, showing electron-dense subcortical material (arrowheads). Small lacunae (asterisk) lacking diaphragms are occasionally found (b, arrowhead). c,d, Wild-type (c) and duf (d) third instar garland cells. c, diaphragms (arrowheads) and lacunae (asterisk) densely populate the nephrocyte surface. d, duf nephrocytes have small lacunae (arrowheads) lacking diaphragms and a substantially thickened basement membrane (bm). e,f, SEMs of wild-type (e) and duf (f) third instar garland nephrocytes stripped of basement membrane by collagenase treatment. duf nephrocytes lack the furrows corresponding to diaphragm rows. g,h, Wild-type (g) and duf (h) Viking-GFP (collagen IV) third instar garland cells, stained with anti-GFP (green) showing greater Viking deposition around duf nephrocytes (arrowheads and inset). Garland cell number is also reduced in duf larvae, suggesting that mutant cells ultimately die. i,j, Diaphragm and foot process morphology are abnormal (arrowheads) in sns (i) and human nephrin (j) embryonic overexpression. Scale bars 200nm (a,c,d), 100nm (aⁱ,b), 50nm (i-j), 5μm (e,f). os, oesophagus, pv, proventriculus, tr, trachea.

Figure 4. Analysis of slit diaphragm-associated protein orthologues in the fly nephrocyte

a-f, Third instar garland (a,c,e) and pericardial (b,d,f) nephrocytes hybridised with probes directed against Mec2 (a,b), pyd (c,d) and CG31012 (e,f). g, Pyd (g, green) and Duf (gⁱ, red) co-localise (gⁱⁱ, yellow) in third instar garland nephrocytes. h, Schematic drawing of the major components of the podocyte slit diaphragm (black arrows) and nephrocyte diaphragm (described here and elsewhere, red arrows). i, Schematic drawing of Drosophila orthologues of slit diaphragm-associated proteins: PDZ-binding domain (THV), PDZ domain (PDZ), stomatin domain (STO) and SH3 domain (SH3). Region of the protein used in the yeast two-hybrid analysis is outlined in red. j, Yeast two-hybrid analysis of Sns or Duf with Pyd, CG31012, Mec2 and negative control (empty vector). Direct protein interaction is indicated by growth of yeast on selective media (H- 5mM 3′AT). k, Duf and Pyd-V5 coimmunoprecipitate with one another from Drosophila cells (unlabelled arrowhead on left corresponds to Pyd, asterisk indicates cleaved form of Duf that is also coimmunoprecipitated with Pyd). l, Schematic drawing of molecular interactions at the nephrocyte diaphragm, Sns (green), Duf (black), Mec2 (yellow), Pyd (blue), CG31012 (red), direction of filtration (red arrow), extracellular lacuna (asterisk). Putative links to signalling or the actin cytoskeleton based on analogy with the equivalent complex at the slit diaphragm are shown. bm, basement membrane.

Figure 5. Sns and Duf are required for nephrocyte filtration

a,c,d, Third instar garland nephrocytes from wild-type (a), duf (c) and sns RNAi knockdown (d) animals co-incubated with 10,000mw (magenta) and 500,000mw (green) fluorescently-labelled dextran. Inset in c,d shows merged image of transmitted light and 500,000mw channels. b,e, Schematic drawing of filtration and endocytosis in wildtype (b) and sns or duf mutant (e) nephrocytes. f, Quantification of small (magenta) and large (green) dextran uptake in wild-type, duf and sns RNAi knockdown garland cells. Pixel number exceeding threshold is shown on Y-axis (error bars, S.E.M). nb, the molar ratio of dye:dextran is 1:1 (for 10,000mw) and 64:1 (for 500,000mw). g, control nephrocyte incubated with fluorescent dextran at 4°C showing no uptake (gⁱ). h, 10μm section of garland nephrocytes from a wild-type larva fed with AgNO₃ (brown granular staining). i, Percentage of eclosing sibling control or duf adults fed yeast paste (grey) or yeast paste with AgNO₃ (blue) (n = 65, 68, 55 and 57).