The housekeeping ribosomal transcripts vanish from the enrichment list (Table 2), which is now dominated by transporters. Intriguingly, these are not for the V-ATPase that is considered to dominate active transport by the tubule, but for organic and inorganic solutes. There is a range of broad-specificity transporters - for organic cations, anions, monocarboxylic acids, amino acids and multivitamins. There are also multiple inorganic anion co-transporters for phosphate and iodide. Most are not only very highly enriched, but also highly abundant. In more detail, the results are remarkable (Table 4). Nearly every class of transporter is represented, and almost all of these have at least one representative that is both abundant and enriched, implying a very specific renal role; indeed, this table contains the genes with the highest average enrichments of any class, frequently more than 30-fold. Some transporters have been documented implicitly as having a tubule role; many of the classical Drosophila eye-color mutants also have an effect on tubule color, and have since been shown to encode genes for transport of eye-pigment precursors 1236. These genes now turn out to be both abundant and enriched; among the ABC transporters are scarlet and white, and among the monocarboxylic acid transporters is CG12286, which we have recently argued to correspond to karmoisin, a probable kynurenine tranporter 37. Glucose and other sugar transporters are consistently abundant and enriched, implying that sugar transport is a major (and previously unsuspected) role of the tubule. Inorganic transporters are also included in the table; there are also copper and zinc transporters, which is consistent with electron-probe X-ray microanalysis data that heavy metals accumulate in tubule concretions 3839, and with the extreme abundance of metallothionein A (Table 1).

As well as specific transporters, the tubule is enriched for several families of broad-specificity transporters (organic anion and cation transporters, multivitamin transporters, ABC multidrug transporters and an oligopeptide transporter). When combined these would be capable of excreting a huge majority of organic solutes. These results invite a substantial revision of our interpretation of the role of the tubule. Classically, it is considered to be the tissue that excretes waste material, both metabolites and xenobiotics, and provides the first stage of osmoregulation. However, nearly all work on insect tubules in the last half-century has focused on the ionic basis of fluid secretion and its control, as these are easily measured experimentally. Although there have been sporadic reports on the active transport of organic solutes such as dyes 404142, the historical view was of a relatively leaky epithelium, with a paracellular default pathway for those solutes not recognized by specific transporters. While consistent with the more classical view of the tubule, our results also suggest that the insect is emulating a leaky epithelium to produce the primary urine by incorporating a vast array of broad-specificity active transporters in the plasma membranes of what is electrically rather a tight epithelium. Indeed, this interpretation is consistent with other independent data: the intercellular junctions in tubule are known to be of the pleated stellate variety, the invertebrate equivalent of tight junctions 43; and, like salivary glands, tubule cells are known to be highly polytene 44454647 or even binucleate 48, adaptations that maximize the size of cells and thus maximize their area/circumference ratios.

Physiological analysis of the tubule has concentrated on the secretion of primary urine, and the energizing transporter is a plasma membrane proton pump, the V-ATPase 13495051. This is a large holoenzyme of at least 13 subunits, encoded by 31 Drosophila genes 5253. V-ATPases have two distinct roles, one carried out at low levels in endomembrane compartments of all eukaryotic cells and the other in the plasma membranes of specialized epithelial cells of both insects and vertebrates 54. In such cells, the V-ATPases can pack the plasma membrane to such an extent that they resemble semi-crystalline arrays when observed by electron microscopy 55. It is clearly of interest to find out which genes contribute to the plasma-membrane role of the V-ATPase, though this would normally involve difficult and tedious generation of selective antibodies capable of distinguishing between very similar proteins. However, the mRNAs for those V-ATPase subunits enriched in epithelia should also be particularly abundant; one could thus predict that at least one gene encoding each V-ATPase subunit should show enrichment in tubule compared with the rest of the fly. This is indeed the case (Table 5): invariably, one gene for each subunit is both significantly enriched, and far more abundant, than any other gene encoding that subunit. The reason that the enrichment is not higher is probably because the whole-fly samples contain other epithelia, each with enriched V-ATPase, as minor parts of the overall sample.

The array data thus allow a rapid and authoritative prediction to be made on the subunit composition of the plasma membrane V-ATPase. It will be interesting to extend these data to other epithelia in which V-ATPase is known to be functionally significant.

The role of the classical Na⁺, K⁺-ATPase in tubule is enigmatic. In nearly all animal epithelia, transport is energized by a basolateral Na⁺, K⁺-ATPase, which establishes a sodium gradient that drives secondary transport processes. By contrast, insect epithelia are energized by a proton gradient from the apical V-ATPase 5657 and, consistent with this, many insect tissues are paradoxically refractory to ouabain, the specific Na⁺, K⁺-ATPase inhibitor 58. Accordingly, models of insect epithelial function tend not to include the Na⁺, K⁺-ATPase. It is thus interesting to note that both Atpalpha and Nervana 1 (encoding isoforms of the α and β subunits, respectively) are among the most abundant transcripts in tubule (Table 6). Both are about as enriched in tubule as the V-ATPase subunits, but are significantly more abundant (compare Table 5). By contrast, a novel alpha-like subunit (CG3701), and both Nrv2 (the neuronal β-subunit) and other novel β-like subunits are at near-zero levels. As Na⁺, K⁺-ATPase has previously been documented as being particularly abundant in Drosophila tubule 59, it may thus be prudent to re-include the Na⁺, K⁺-ATPase as an important part of models of tubule function.

Potassium is actively pumped across the tubule, and the main basolateral entry step is via barium-sensitive potassium channels, both in tubule 506061 and in other V-ATPase-driven insect epithelia 6263. Of the ion channels, the potassium channel family is by far the most diverse in all animals: in Drosophila, there are at least 28, and in human 255, K⁺-channel genes 64. Inspection of the potassium channels on the array (Table 7) clearly identifies just four that are expressed at appreciable levels. Irk3, Ir, Irk2 and NCKQ are all both very abundant and highly enriched in tubule. Irk3 in particular is 80-fold enriched over the rest of the fly, implying a unique role in tubule. Three of these genes are members of the inward rectifier family of potassium channels: supporting the hypothesis that they are critical for potassium entry, these channels are known to be highly barium-sensitive 65. An inward rectification of potassium current (meaning that potassium would pass much more easily into the cell than out) would be ideal for a basolateral entry step. Inward rectifier channels normally associate with the sulfonylurea receptor (SUR), an ABC transporter, in order to make functional channels 6667. In tubules, SUR mRNA is present at extremely low abundance (signal 6, enrichment 0.9 times). However, CG9270, a gene with very close similarity to SUR (1 × 10^-28 by BLASTP) is very abundant in tubule (see Table 4), (signal 422, enrichment 21 times). A second very similar gene, CG31793 (previously also known as CG10441 and CG17338), is very much less abundant (signal 24, enrichment 0.5). We therefore predict that novel inward rectifiers, formed between Irk3, Ir or Ir2 and CG9270, may provide the major basolateral K⁺ entry path in tubule. In contrast, the other classes of K⁺ channel, and the Na/K/Cl co-transporter that has been documented in tubule, are all relatively low in both abundance and enrichment.

In a fluid-secreting epithelium, a necessary correlate of the active transport of cations must be the provision of a shunt pathway for anions and a relatively high permeability to water. In Drosophila tubules, a hormonally regulated chloride conductance pathway has been shown to occur in the stellate cells, although the molecular correlate of the currents has not been determined. There are three ClC-type chloride channels in the Drosophila genome, and RT-PCR has shown that all three are expressed in tubule 12. The array data present a prime candidate (Table 8). Although all three genes are expressed, only one (CG6942) is both very abundant and enriched in tubule (signal 251, enrichment 4). It is thus an obvious candidate partner to provide a shunt pathway for the epithelial V-ATPase.

Water flux through the tubule is also phenomenally fast: each cell can clear its own volume of fluid every 10 seconds 12. Although traditionally it was thought that only a leaky epithelium could sustain such rates, the identification of aquaporins (AQP) (the predominant members of the major intrinsic protein (MIP) family) as major water channels in both animals and plants 68 provides an obvious counter-explanation. There is physiological and molecular data for the presence of aquaporins in Drosophila tubule 69, and AQP-like immunoreactivity has been demonstrated in stellate cells 12. Table 9 shows that only four of the seven AQP/MIP genes are abundant, and only three enriched. One can thus tentatively assign an organism-wide role to CG7777 (signal 243, enrichment 0.6), but tubule-specific roles to CG4019, CG17664 and DRIP. In particular, CG17664, is both highly abundant and very highly enriched (signal 705, enrichment 7.9).