The Malpighian (renal) tubules of insects are functionally analogous to mammalian kidneys, as they are involved in the homeostatic maintenance of the insects’ fluid balance by controlling the volume and ion/solute composition of the urine they produce. The production of urine (diuresis) is under the control of different neuropeptide/receptor systems whose evolutionary origin and functional relation to the renal tubule's architecture is largely unexplored. In a study published recently in Nature Communications, a team of scientists led by Julian Dow from the University of Glasgow, UK, examined the origin of insect renal function using an exceptional approach based on fluorescent neuropeptide analogues to localize neuropeptide receptors in renal cells.
Renal tubules of insects have been extensively studied in flies and mosquitoes. They are formed by a single-layered epithelium which typically consists of two cell types, so-called principal and secondary (or stellate) cells. According to the classical two-cell-type model, diuresis is driven by a V-type ATPase that resides in the apical membranes of the principal cells. This pump powers the exchange of potassium ions against protons so that net potassium ions that are secreted into the lumen are passively followed by water. Several diuretic and antidiuretic hormones control fluid secretion, including the highly conserved diuretic neuropeptides kinin, capa and DH31. While the last two act on the principal cells stimulating V-type ATPase activity, kinin increases diuresis by stimulating the transcellular chloride transport in secondary cells, which facilitates potassium ion secretion. But does this model of fluid secretion account for insects other than flies and mosquitoes and when was this system established during the evolution of insects?
To answer these questions, Dow's team designed a set of nifty experiments that allowed them to test neuropeptide function along with their sites of action. For this purpose, they synthesized the kinin, capa and DH31 neuropeptides and coupled them to a red fluorophore, hoping that the artificial neuropeptides would work like the natural ones and stimulate diuresis in isolated renal tubules. They further reasoned that if the neuropeptides were functional, they should be able to use them to identify principal and secondary cells by fluorescence microscopy after they had bound to the corresponding receptors in the basal membranes of these cells.
Testing their concept in the well-characterized model fly Drosophila melanogaster, the team found that the neuropeptides were functional, binding to the corresponding receptor in the correct target cell. Next, they traced the evolutionary origin of the two-cell-type model by probing renal tubules from various strategically selected insect species representing different phylogenetic orders. By comparing these data with genomic (presence/absence of neuropeptide/receptor genes) and functional data (neuropeptide effects on diuresis), they constructed a clear picture on the structure–function relationship of the hormonal system regulating diuresis in the insect's renal tubules.
It turned out that secondary cells are much more widespread than previously believed, because they could detect kinin labelling in secondary cells of most of the more advanced holometabolous insects. In the more primitive hemimetabolous insects, however, kinin bound uniformly to the renal tubule, with no evidence for secondary cells, suggesting that the kinin signalling pathway evolved before the radiation of insects and that holometabolous insects have evolved kinin-responding secondary cells. A more surprising finding was that diuretic control is organized differently in the largest insect order, the beetles, as these holometabolous insects obviously have lost the kinin signalling pathway secondarily, and only a subset of their renal cells respond to capa and DH31 signals.
Dow and his team have convincingly demonstrated that the two-cell-type model describing renal function and control accounts for most advanced insects, except for beetles, and that it may have evolved from a single cell-type renal system already capable of responding to kinin signals.