When two cells are better than one: specialized stellate cells provide a privileged route for uniquely rapid water flux inDrosophilarenal tubule

Abstract

Insects are highly successful, in part through an excellent ability to osmoregulate. The renal (Malpighian) tubules can secrete fluid faster on a per-cell basis than any other epithelium, but the route for these remarkable water fluxes has not been established. In Drosophila melanogaster, we show that 4 members of the Major Intrinsic Protein family are expressed at very high level in the fly renal tissue; the aquaporins Drip and Prip, and the aquaglyceroporins Eglp2 and Eglp4. As predicted from their structure and by their transport function by expressing these proteins in Xenopus oocytes, Drip, Prip and Eglp2 show significant and specific water permeability, whereas Eglp2 and Eglp4 show very high permeability to glycerol and urea. Knockdowns of any of these genes impacts tubule performance resulting in impaired hormone-induced fluid secretion. The Drosophila tubule has two main secretory cell types: active cation-transporting principal cells with the aquaglyceroporins localize to opposite plasma membranes and small stellate cells, the site of the chloride shunt conductance, with these aquaporins localising to opposite plasma membranes. This suggests a model in which cations are pumped by the principal cells, causing chloride to follow through the stellate cells in order to balance the charge. As a consequence, osmotically obliged water follows through the stellate cells. Consistent with this model, fluorescently labelled dextran, an in vivo marker of membrane water permeability, is trapped in the basal infoldings of the stellate cells after kinin diuretic peptide stimulation, confirming that these cells provide the major route for transepithelial water flux. The spatial segregation of these components of epithelial water transport may help to explain the unique success of the higher insects.Significance statementThe tiny insect renal (Malpighian) tubule can transport fluid at unparalleled speed, suggesting unique specialisations. Here we show that strategic allocation of Major Intrinsic Proteins (MIPs) to specific cells within the polarized tubule allow the separation of metabolically intense active cation transport from chloride and water conductance. This body plan is general to at least many higher insects, providing a clue to the unique success of the class Insecta.

Compromised junctional integrity phenocopies age-dependent renal dysfunction in Drosophila Snakeskin mutants

“…Notably, these results were also observed using a second SC-specific GAL4 driver, ClC-aGAL4 (Cabrero et al, 2020), which reiterated the phenotypic profile associated with SC-specific knockdown of Ssk, albeit to a Journal of Cell Science • Accepted manuscript lesser extent likely due to ClC-aGAL4 acting as a weaker driver compared with c724GAL4. That is ClC-aGAL4>GAL80 ts >Ssk RNAi experimental adults developed a bloated abdomen due to an accumulation of haemolymph in the abdomen, indicative of loss of tubule epithelial integrity, distended MTs with disorganised junctional complexes, and cuboidal SCs with associated loss of apico-basal polarity and the ClC-a channel (Figure S3).…”

“…Dysregulation of Dlg, and associated loss of cytoarchitecure, also results in failure of apicobasal polarity (Laprise and Tepass, 2011). To test for loss of cell polarity we employed two antibodies directed against the aquaporin channels Prip and Drip, which are specific, respectively, to the SCs baso-lateral and apical membranes (Figure 4A and D) (Cabrero et al, 2020).…”

“…In SCs, kinin-modulated Clshunt conductance occurs specifically through the chloride channel ClC-a localizing to the basolateral membrane (Figure S6A) (Cabrero et al, 2020;. We attempted to employ anti-ClC-a to assay polarity in SC populations in experimental adult MTs and found ClCa expression completely absent in the cuboidal SC population in SC SskRNAi (Furuse) MTs (Figure S6B) but apparently unaffected in SCs in PC SskRNAi (Furuse) MTs (Figure S6C).…”

“…In the fruit fly Drosophila melanogaster, the MTs are comprised mainly of two physiologically distinct secretory cell-types; the principal (PC) cell and intercalated or 'stellate' (SC) cell (Beyenbach et al, 2010;Cohen et al, 2020;Dow, 2012;Sözen et al, 1997). PCs are the sites of active cation transport, energised by V-ATPases localized apically to a prominent brush border (Cohen et al, 2020;Halberg et al, 2016;Naseem et al, 2022), whereas the smaller SCs control channel-mediated Cland water fluxes (Beyenbach et al, 2010;Cabrero et al, 2020;Cohen et al, 2020;Dow, 2012). Primary urine production in Drosophila is achieved through the integrated actions of PCs and SCs, driving transepithelial transport of ions and water.…”

Adipose Triglyceride Lipase protects the endocytosis of renal cells on a high fat diet inDrosophila

“…In Drosophila, the renal system comprises two anatomically distinct components -Malpighian tubules and nephrocytes [26][27][28] (Fig 1A). Malpighian tubules are excretory cells that also function in salt and water balance, similar to mammalian renal tubules [29,30]. Nephrocytes are podocyte-like cells that regulate the composition of the hemolymph (blood) via a filtration barrier consisting of Sns and Kirre, orthologs of the mammalian slit diaphragm proteins Nephrin and Neph1 [31,32].…”