The Interactive Fly

Genes involved in tissue and organ development

Malpighian Tubules

Genes expressed in Malpighian tubule development

Transcription factors achaete caudal cut Dorsocross Ecdysone-induced protein 74EF Ecdysone-induced protein 75B Ecdysone receptor extra machrochaete forkhead GATAe Hepatocyte nuclear factor 4 homothorax Hormone receptor-like/Hr46 (common alternative name: DHR3) Hormone-receptor-like in 78 Krüppel numb odd-skipped PAR-domain protein 1 pebbled period pointed ribbon seven up tailless timeless X-box-binding protein 1 zeste Surface proteins, ligands or receptors argos (also known as giant lens) Dopamine 1-like receptor 2 Epidermal growth factor receptor (synonyms: DER or torpedo) 18 wheeler Fasciclin2 Frizzled-3 hodor (preferred name: pH-sensitive chloride channel 2) inebriated Inwardly rectifying potassium channel 1, 2, and 3 (Kir1, Kir2 and Kir3) Leucokinin receptor mesh Notch pH-sensitive chloride channel 2 (common alternative name: Hodor) shotgun slit spitz Star subdued Tachykinin Tachykinin-like receptor at 99D Toll Transient receptor potential cation channel γ veinlet (also known as rhomboid) white wingless

Subunits of vacuolar H+-ATPase Vacuolar H+-ATPase 68kd A subunit Vacuolar H+-ATPase 55kD B subunit: FlyBase ID FBgn0005671 Vacuolar H+-ATPase 26kD E subunit: FlyBase ID FBgn0015324 Vacuolar H+ ATPase 16kD subunit: FlyBase ID FBgn0004145 Vacuolar H+ ATPase 14kD subunit: FlyBase ID FBgn0010426 Other arc big brain calmodulin Clathrin heavy chain crossveinless c Cyclin E disc proliferation abnormal Exchange protein directly activated by cAMP futsch geminin Inositol-requiring enzyme-1 inscuteable (preferred name: not enough muscle) microRNA encoding gene let-7 mastermind Myoblast city Na pump α subunit Nitric oxide synthase Niemann-Pick Type C-2 not enough muscle Ornithine decarboxylase antizyme par-6 pitchoune atypical protein kinase C Rac1 raw rolling pebbles Synaptobrevin stardust Src homology 2, ankyrin repeat, tyrosine kinase Src oncogene at 42A string Tyrosine decarboxylase 1

Embryonic origin, functional domains and physiology of Malpighian tubules

The tubules arise during embryogenesis as four protuberances extending from the proctodeum. As this tissue is considered ectodermal, Malpighian tubules are classified as an ectodermally derived secretory tissue. The protuberances grow, first by cell proliferation and then by extensive rearrangement of the cells, to produce elongated blind end tubes composed of a single-cell-layered epithelium (Hoch, 1994).

The development of Malpighian tubules reveals an essential role for the gene Krüppel. In each Malpighian tubule, one cell is singled out, the tip cell, whose function during embryogenesis is to promote cell division in its neighbours. The tip cell arises by division of a tip mother cell, which is selected from a cluster of equivalent cells, each expressing Krüppel in each tubule primordium. Each cluster is delineated by the expression of proneural genes; the selection of a single cell from each group involves lateral inhibition, mediated by the neurogenic genes. achaete is responsible for tip cell allocation, but Kr acts as the selector gene, responsible for tip cell fate. The tip cell directs the growth of the Malpighian tubules and organizes the mitotic response and migration of the other cells forming each tubule (Hoch, 1994). Therefore Krüppel is responsible for cell fate in the Malpighian tubules, a function quite distinct from Krüppel's role as a gap gene.

Drosophila possesses two pair of Malpighian tubules. The right pair of tubules project forward from their point of insertion within the hindgut and lie at the anterior end of the abdomen, and the left pair extend backwards so that their tips become attached to the posterior part of the hindgut. Each tubule pair unites to form a common ureter, which enters the intestine between the midgut and hindgut. The two anterior Malpighian tubules are classically described as comprising a distal initial segment and a proximal main segment, joined by a narrow transitional segment; the two posterior tubules, in contrast, were thought to consist solely of a main segment. Contemporary studies, using enhancer trap lines, which place reporter genes under the control of tissue specific enhancers, confirm this viewpoint and thus the nomenclature "initial," "transitional," and "main" segments has been adopted to described these genetically deduced domains (Sözen, 1997).

Enhancer trap studies reveal an unexpected complexity in Malpighian tubules in terms of both regions and cell types. Enhancer trap lines that delineate the initial and transitional segments of anterior tubules, reveal previously undescribed analogous domains in posterior tubules. It is also possible to subdivide the main segments. While the transitional-main segment boundary has been established in accordance with classical studies, an additional domain is found marking the lower third of the tubule and the ureter. This latter region, in turn, can be resolved into three subregions: a lower tubule and an upper and lower ureter (Sözen, 1997).

Previous studies have described just two tubule cell types: principal (type I) and secondary or stellate (type II). Both can be further subdivided. Principal cells, for example, comprise at least two distinct subpopulations. Thus there appear to be differences in otherwise indistinguishable cells with respect to enhancer trap expression patterns and presumably with respect to function as well. Type II cells are distributed evenly throughout the initial, transitional and main segments of posterior tubules and within the main segment of anterior tubules. None of the enhancer trap markers mark cells in the lower tubule or ureter, suggesting that the mechanism by which type II cells are specified respects the newly defined lower tubule boundary. Several lines mark a "tiny" cell type found in lower tubules and posterior midgut but do not mark the same genetic domain as stellate cells. Possibly these previously undescribed cells are counterparts of the myoendocrine cells recently described in Formica. These cells may monitor the fluid collected in the ureter and secrete neurohormones basally into the hemolymph to regulate muscle contractility or ion transport (Sözen, 1997).

Do discrete physiological properties map to the genetic domains that have been identified? With respect to a number of different transport processes, this is indeed the case. The obvious functional property of the tubule is to secrete urine. It has been reported that the initial segment of Drosophila anterior tubule does not secrete detectable fluid, that the lower third of the tubule is reabsorptive, and that only the main segment is responsible for fluid production. High levels of proton-pumping V-ATPases energize apical plasm membranes of several epithelia, including Malpighian tubules. The B-subunit of Drosophila V-ATPase is expressed in the initial and transitional segments and is much weaker in the reabsorptive main segment. The main segment consists entirely of the large, principal cell. This provides the first evidence that cation transport into the lumen of Malpighian tubules may be a unique property of principal, rather than type II, cells. A putative aquaporin has been cloned in Drosophila: this channel is found in stellate cell basolateral membranes. Given that stellate cells are found in secretory but not reabsorptive tubule regions, they may well play an essential role in fluid secretion. Another function ascribed to Malpighian tubules is the secretion of organic metabolites. This function is confined to the main segment. It is clear that the staining pattern for alkaline phosphatase precisely matches the lower tubule boundary, and is associated with the reabsorptive, rather than the secretory, domain of the tubule (Sözen, 1997).

Secretion by Malpighian tubules is under hormonal control. CAP2b, a cardioacceleratory peptide, is present in Drosophila and stimulates Malpighian tubule fluid secretion via cGMP, which in turn stimulates the nitric oxide signaling pathway. Liquid chromatography analysis of adult Drosophila reveals the presence of a CAP2b-like peptide, that coelutes with Manduca sexta CAP2b and synthetic CAP2b and that has CAP2b-like effects on the M. sexta heart. CAP2b stimulation elevates tubule cGMP levels but not those of cAMP. Both CAP2b and cGMP increase the transepithelial potential difference, suggesting that stimulation of vacuolar ATP action underlies the corresponding increases in fluid secretion (Davies, 1995).

Calcium mobilization in identified cell types within an intact renal epithelium (the Drosophila Malpighian tubule) was studied by GAL4-directed expression of an aequorin transgene. Aequorin is a Ca2+ sensitive liminescent protein isolated from the coelenterate Aequorea victoria. It is a complex of apoaequorin, a 21 kDA polypeptide, and coelenterazine, a hydrophobic luminophore. Aequorin is used for monitoring Ca2+ changes. CAP2b, causes a rapid, dose-dependent rise in cytosolic calcium in only a single, genetically-defined, set of 77 principal cells in the main (secretory) segment of the tubule. In the absence of external calcium, the CAP2b-induced calcium response is abolished. In Ca2+-free medium, the endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin, elevates [Ca2+]i only in the smaller stellate cells, suggesting that principal cells do not contain a thapsigargin-sensitive intracellular pool. Assays for epithelial function confirm that calcium entry is essential for CAP2b to induce a physiological response in the whole organ. The data suggest a role for calcium signaling in the modulation of the nitric oxide signaling pathway in this epithelium. CAP2b must act to increase fluid secretion rates solely by an initial rise of [CA2+]i in principal cells. CAP2b stimulates tubule Nitric oxide synthase activity. It is probable that the CAP2b induced rise in [CA2+]i is sufficient to trigger the activation of Drosophila calcium sensitive Nitric oxide synthase. The maximal CAP2b concentrations employed elevate principal cell calcium levels from 87 to 255 nM, a value close to the EC50 of Drosophila NOS. This implies that Drosophila Nos is responsive over the range of the CAP2b concentrations employed. This may account for the observation that thapsigargin treatment results in increased basal cGMP levels that are not further increased on CAP2b stimulation. Thus the data provide strong evidence for a calcium-mediated link between CAP2b and NOS/cGMP activation of fluid secretion. The GAL4-targeting system allows general application to studies of cell-signaling and pharmacology that does not rely on invasive or cytotoxic techniques (Rosay, 1997).

Other hormones likely to be involved in Malpighian tubule function are the leucokinins. Leucokinins are a group of widespread insect hormones. In tubules, their major action is to raise chloride permeability through stellate cells by binding to receptors on the basolateral membrane, and so ultimately to enhance fluid secretion (Julian Dow, personal communication).

Ecdysone regulates morphogenesis and function of malpighian tubules in Drosophila melanogaster through EcR-B2 isoform

Malpighian tubules are the osmoregulatory and detoxifying organs of Drosophila and its proper development is critical for the survival of the organism. They are made up of two major cell types, the ectodermal principal cells and mesodermal stellate cells. The principal and stellate cells are structurally and physiologically distinct from each other, but coordinate together for production of isotonic fluid. Proper integration of these cells during the course of development is an important pre-requisite for the proper functioning of the tubules. This study conclusively determined an essential role of ecdysone hormone in the development and function of Malpighian tubules. Disruption of ecdysone signaling interferes with the organization of principal and stellate cells resulting in malformed tubules and early larval lethality. Abnormalities include reduction in the number of cells and the clustering of cells rather than their arrangement in characteristic wild type pattern. Organization of F-actin and beta-tubulin also show aberrant distribution pattern. Malformed tubules show reduced uric acid deposition and altered expression of Na+/K+-ATPase pump. B2 isoform of Ecdysone receptor is critical for the development of Malpighian tubules and is expressed from early stages of its development (Gautam, 2014).

Tip cells act as dynamic cellular anchors in the morphogenesis of looped renal tubules in Drosophila

Tissue morphogenesis involves both the sculpting of tissue shape and the positioning of tissues relative to one another in the body. Using the renal tubules of Drosophila, this study shows that a specific distal tubule cell regulates both tissue architecture and position in the body cavity. Focusing on the anterior tubules, it was demonstrated that tip cells make transient contacts with alary muscles at abdominal segment boundaries, moving progressively forward as convergent extension movements lengthen the tubule (see Tip-Cell-Dependent Anchorage of Anterior Tubules to Alary Muscles). Tip cell anchorage antagonizes forward-directed, TGF-beta-guided tubule elongation, thereby ensuring the looped morphology characteristic of renal tubules from worms to humans. Distinctive tip cell exploratory behavior, adhesion, and basement membrane clearing underlie target recognition and dynamic interactions. Defects in these features obliterate tip cell anchorage, producing misshapen and misplaced tubules with impaired physiological function (Weavers, 2013; see Graphical Abstract).

As the embryonic renal tubules assume their mature shape they interact with other tissues, responding to Dpp guidance cues as they take up their characteristic positions in the body cavity (Bunt, 2010). This study shows that, in addition, a single cell at the distal end of each renal tubule makes specific transitory, and finally long-term, contacts with target tissues. These cells express a distinctive pattern of genes and show characteristic exploratory activity, which is crucial for the stereotypical looped shape and position of the tubules in the body cavity. In turn, these features have profound consequences for the efficacy of fluid homeostasis in the whole animal (Weavers, 2013).

It is suggested that the elongation and forward extension of the tubules result from the combined effects of cell rearrangements that lengthen the tubule and the response of kink region cells to regional Dpp guidance cues. The evidence indicates that the tip cells act as anchors, through their interactions with alary muscles, so that tubules are tethered at both ends, the proximal end being attached through ureters to the hindgut. These attachments perform two functions: they stabilize the looped architecture, maintaining the kink close to the tubule midpoint, and they limit forward and ventral movement to ensure the stereotypical tubule arrangement in the body cavity (Weavers, 2013).

If tip cell contact with the alary muscles is lost, the kink 'unravels,' shifting distalward, and the tubule as a whole extends too far into the anterior, with the distal region lying more ventrally close to the Dpp-expressing gastric caeca. Confirming the existence of a forward tractive force responsible for tip cell detachment are the distortion of transient alary muscle targets before the tip cell detaches and the characteristic 'recoil' seen when the tip cell is ablated. Evidence that this results from the response to guidance cues is the failure of tip cells to detach from their first alary muscle contact (A5/A6) in the absence of the midgut Dpp guidance cue and the more anterior location of the kink region (close to the gastric caeca) in tubules where the tip cell stalk is greatly extended, for example when the activity of RhoA is repressed. The critical nature of the balance between these forward and restraining influences is also revealed when adhesion between the tip cells and alary muscles is increased by manipulating tip cell number or adhesive strength. In each case, the tip cells remain attached to alary muscles posterior to their normal final contacts, and this results in more posterior positioning of the whole tubule. Together, these results strongly suggest that tip cells detach because the forward movement of tubules overcomes the adhesive strength of their early transient contacts (Weavers, 2013).

The final tip cell/alary muscle target is highly reproducible, suggesting recognition through segmental identity, the A3/A4 target being the first encountered by the tip cell that expresses Ubx. However, altering Ubx expression in alary muscles has no effect on the final tip cell contact. Instead, it appears that tip cells adhere to each alary muscle they contact, and the final target depends on the balance between forward tubule movement and the strength of tip cell/target adhesion. Consistent with this view, when all the normal muscle targets are ablated, tip cells can make stable contacts with the A2/A3 alary muscle (Weavers, 2013).

From the time that they are specified, tip cells show distinctive patterns of gene expression, morphology, and behavior critical to their ability to make alary muscle contacts; they form dynamic filopodia, which explore the alary muscle surface, remain denuded of the BM that envelops the rest of the tubule, and express cell-adhesion proteins, including Neuromusculin (Nrm) and integrins. Protrusive activity depends on the rapid turnover of actin, mediated by regulators such as Rac GTPase and the actin-capping protein Enabled, which are active in tip cells (Weavers, 2013).

BM deposition basally around the tip cells severely inhibits protrusive behavior, and tip cells therefore employ multiple mechanisms to ensure that they remain denuded. These include the absence of expression of factors that promote BM deposition and stabilization (hemocyte attractants, BM components, or receptors), the removal by transcytosis of any BM that is deposited, and expression of MMP1, which is able to cleave matrix components. MMP1 is expressed late during tubule elongation and the protein is localized apically in tip cells, suggesting that its function might be to degrade transcytosed BM proteins (Weavers, 2013).

Protrusive exploratory behavior results in adhesive contacts made possible through tip cell expression of Nrm and integrins. Nrm is a homophilic cell-adhesion molecule of the Ig-domain superfamily. The binding partner for tip cell Nrm is unclear, as alary muscles do not express it. However, driving nrm expression in alary muscles induces strong adhesion, resulting in tip cells remaining bound to their first target in A5/A6. It is possible that Nrm in tip cells normally makes heterophilic associations with Ig domain-containing proteins such as Dumbfounded (Kirre), which is expressed in alary muscles and is sufficient, when overexpressed, to induce more posterior target adhesio (Weavers, 2013).

Tip cells express integrins, and complexes accumulate as each target contact is made, but initially they do not lead to long-term adhesion. It is suggested that the strength of adhesion increases with successive contacts, either through increased expression of integrins and their associated factors or by regulated adhesive complex turnover, as shown in other tissues. Once the final tip cell contact is made, BM accumulates around the tip cellalary muscle surface, increasing the concentration of integrin ligands at the junction. The accompanying decline in the protrusive activity of the tip cell could also result from integrin-mediated adhesion, which is known to reduce levels of the actin-capping protein Enabled. This sequence of events parallels the mechanism by which elongated myotubes and tendon cells establish their myotendinous junctions (Weavers, 2013).

Once the anterior tubule tip cells make their final alary muscle contact, they remain attached throughout development into adult life. Such interaction of excretory tubule tips with muscles is a common feature of renal systems in insects, either with alary muscles or with fine striated muscles that spiral along the tubule. Muscle contacts increase tubule movement, maximizing the effectiveness of excretion, by increasing hemolymph sampling and enhancing tubule flow. Similar contacts are found outside the arthropods; the flame cells that cap planarian protonephridial tubes develop prominent filopodia and interact with nearby muscle fibers, providing anchorage, thought to be important during branching morphogenesis in this system (Weavers, 2013).

Tip cells or groups of cells at the distal tips of outgrowing epithelial tubes act as organizers in tubular systems, from the migrating Dictyostelium slug to the branching epithelial scaffolds of human organs. As in fly renal tubules, these distinctive cells regulate cell division and guided tubule extension, and in mammalian systems they control branching morphogenesis (Weavers, 2013).

However, in distinct contrast to the role of tip cells in the morphogenesis of these systems, the tip cells of the anterior renal tubules play no role in leading outgrowth. Instead, they act to counteract outgrowth, and importantly this leads to the development of a looped tubular structure both by tethering the distal tips of tubules close to their proximal junction with the ureter and by maintaining the tightness of the tubule kink region. Looped tubular structures are relatively uncommon; a tubule tree as in the lung, pancreas, or liver or an anastomosing network as in the vascular system is more frequently seen. However, a striking example of looped tubules is found in the mammalian kidney, where the distal and proximal convoluted tubules together with the loop of Henle connect the tubule tip (at the glomerulus) to the collecting duct (close to the site of urine outflow). Looping of both the nephron and its vascular supply creates a countercurrent system that maximizes the efficiency of ion and fluid homeostasis. Such exchange systems also occur in insects with specialized diets or those living in dry conditions. Countercurrent exchange has not been demonstrated in Drosophila melanogaster tubules, where it is more likely that the looped tubule structure is important for effective hemolymph sampling (Weavers, 2013).

In the development of the mammalian nephron, as in fly renal tubules, both the site of connection to the ureter and the tubule tip, the renal corpuscle, are established early in organ development so that tubule extension, by both cell proliferation and rearrangements, occurs between these fixed points. It will be interesting to discover whether similar tissue interactions stabilize the position of the developing glomerulus, and so play a prominent role in maintaining the looped structure as kidney tubules extend, resulting in the final intricate and regular array of nephrons apparent in the mature mammalian kidney (Weavers, 2013).

The adult Drosophila malpighian tubules are maintained by multipotent stem cells

All animals must excrete the waste products of metabolism. Excretion is performed by the kidney in vertebrates and by the Malpighian tubules in Drosophila. The mammalian kidney has an inherent ability for recovery and regeneration after ischemic injury. Stem cells and progenitor cells have been proposed to be responsible for repair and regeneration of injured renal tissue. In Drosophila, the Malpighian tubules are thought to be very stable and no stem cells have been identified. This study has identified multipotent stem cells in the region of lower tubules and ureters of the Malpighian tubules. Using lineage tracing and molecular marker labeling, it was demonstrated that several differentiated cells in the Malpighian tubules arise from the stem cells and an autocrine JAK-STAT signaling regulates the stem cells' self-renewal. Identifying adult kidney stem cells in Drosophila may provide important clues for understanding mammalian kidney repair and regeneration during injury (Singh, 2008).

The regenerating renal cells may come from one of the three possible sources, based on previous studies. First, the circulating blood contains bone marrow-derived stem cells able to differentiate into non-haematopoietic cells, such as cells of the kidney. Second, the differentiated glomerular and tubular cells may also be able to dedifferentiate into stem-like cells to repair the damaged tissues. Third, large numbers of slowly cycling cells have recently been identified in the mouse renal papilla region; these cells may be adult kidney stem cells and may participate in renal regeneration after ischemic injury. Further, the ureter and the renal collecting ducts were formed from the epithelium originating from the ureteric bud, and the nephrons and glomeruli were formed from the metanephric mesoderm-derived portion during kidney development. Two distinguished stem cell types have been proposed as responsible for repairing the renal collecting tubules and the nephrons. This study identified a type of pluripotent stem cells (RNSCs) in the Drosophila renal organ. The stem cells are able to generate all cell types of the adult fly MTs. In the region of lower tubules and ureters, autocrine JAK-STAT signaling regulates the stem cell self-renewal. Weak JAK-STAT signaling may convert an RNSC into a renalblast (RB), which will differentiate into an RC in the region of lower tubules and ureters, and a type I or type II cell in the upper tubules. These data indicate that only one type of stem cell may be responsible for repair and regeneration of the whole damaged tissues in mammalian kidney (Singh, 2008).

The Drosophila RNSCs represent a unique model to study the molecular mechanisms that regulate stem cell or cancer stem cell behavior. In most of the stem cell systems that has been well characterized to date, stem cells always reside in a specialized microenvironment, called a niche. A niche is a subset of neighboring stromal cells and has a fixed anatomical location. The stromal cells often secrete growth factors to regulate stem cell behavior. The stem cell niche plays an essential role in maintaining stem cells, and stem cells will lose stem cell status once they are detached from the niche. The niche often provides the balanced (proliferation-inhibiting and proliferation-stimulating) signals that keep the stem cells dividing slowly. The inhibitory signals keep the stem cell quiescent most of the time while the stimulating signals promote stem cell division, to replenish lost differentiated cells. Maintaining the balance between proliferation-inhibiting and proliferation-stimulating signals is the key to maintaining tissue homeostasis (Singh, 2008).

Drosophila RNSCs are controlled differently. This study has demonstrated that the JAK-STAT signaling regulates the stem cell self-renewal. Both the ligand Upd and the receptor Dome are expressed in the RNSCs and the autocrine JAK-STAT signaling regulates the stem cell self-renewal; thus, the self-sufficient stem cells control their self-renewal or differentiation and do not need to constrained to a fixed niche. However, the RNSCs are still confined to the region of lower tubules and ureters even in the Upd overexpressed flies, suggesting that some other factors besides the JAK-STAT signaling may restrict the RNSCs to the region of the lower tubules and ureters (Singh, 2008).

Recent studies also suggest that tumors may arise from small populations of so-called cancer stem cells (CSCs). The CSCs probably have arisen from mutations that dysregulate normal stem cell self-renewal. For example, mutations that block the proliferation-inhibiting signals or promote the proliferation-stimulating signals can convert the normal stem cells into CSCs. This study demonstrates that amplifying the JAK-STAT signaling by overexpressing its ligand Upd stimulates the RNSCs to proliferate and also to differentiate into RC, which results in tumorous overgrowth in the MT. Therefore, the Drosophila RNSC system may also be a valuable in vivo system in which to study CSC regulation (Singh, 2008).

The RNSCs are located in the region of the lower tubules and ureter of the MTs, while ISCs are located at the posterior midgut. The MTs' ureters connect to the posterior midgut. The two types of stem cells are at close anatomical locations in the adult fly digestion system and also share some properties. For example, both of them are small nuclear cells, Arm-positive, and express esg. However, RNSCs and ISCs produce distinctly different progenies. ISCs produce progenies that include either Su(H)GBE-lacZ- or Pros-positive cells, which are not among the progenies of RNSCs because Su(H)GBE-lacZ and Pros are not expressed in the MTs. RNSCs produce progenies that include Cut- or TSH-positive cells, which are not among the progenies of ISCs because Cut and TSH are not expressed in the posterior midgut. One possibility for this difference is that, although RNSCs and ISCs originate from the same stem cell pool, their particular environments restrict their differentiation patterns. Future experiments, such as transferring RNSCs to the posterior midgut and vice versa, should be able to test this model (Singh, 2008).

The JAK-STAT signaling regulates self-renewal of the male germline, the male somatic, female escort stem cells in fly. The signaling also regulates self-renewal and maintenance of mammalian embryonic stem cells. This study reports that the JAK-STAT signaling regulates self-renewal of RNSCs. The JAK-STAT signaling may be a general stem cell signaling and also regulate stem cell self-renewal in other, un-characterized stem cell systems (Singh, 2008).

esg has been used as a marker of both male germline stem cells. This study has demonstrated that the esg-Gal4. UAS-GFP transgene is specifically expressed in RNSCs. The function of the esg gene is to maintain cells as diploid in Drosophila imaginal cells. Stem cells may have to be diploid, and esg may be a general stem cell factor. Identifying a stem cell signaling pathway (such as the JAK-STAT signal transduction pathway) and a stem cell factor (such as esg) will provide useful tools for identifying stem cells in other systems and for understanding stem cell regulation in general (Singh, 2008).

Drosophila renal stem cells enhance fitness by delayed remodeling of adult Malpighian tubules

Drosophila renal stem cells (RSCs) contradict the common expectation that stem cells maintain tissue homeostasis. RSCs are abundant, quiescent, and confined to the peri-ureter region of the kidney-like Malpighian tubules (MTs). Although derived during pupation - as are intestinal stem cells - RSCs initially remodel the larval MTs only near the intestinal junction. However, following adult injury to the ureter by xanthine stones, RSCs remodel the damaged region in a similar manner. Thus, RSCs represent stem cells encoding a developmental redesign. The remodeled tubules have a larger luminal diameter and shorter brush border, changes linked to enhanced stone resistance. However, RSC-mediated modifications also raise salt sensitivity and reduce fecundity. These results suggest that RSCs arose by arresting developmental progenitors to preserve larval physiology until a time in adulthood when it becomes advantageous to complete the development by RSC activation (Wang, 2022).

Drosophila serotonin receptors stimulate fluid secretion in the Malpighian tubules

Every living cell must detect, and respond appropriately to, external signals. Thus, the functions of intracellular second messengers, such as guanosine 3'5'-cyclic monophosphate (cGMP), adenosine 3'5'-cyclic monophosphate (cAMP), and intracellular calcium, are intensively studied. However, artifact-free manipulation of these messengers is problematic, and simple pharmacology may not allow selective intervention in distinct cell types in a real, complex tissue. A method has been devised by which second messenger levels can be manipulated in cells of choice using the GAL4/UAS system. By placing different receptors (rat atrial natriuretic peptide [ANP] receptor and Drosophila serotonin receptors [5HT_Dro7 and 5HT_Dro1A]) under UAS control, they can be targeted to arbitrary defined populations of cells in any tissue of the fly, and second messenger levels can be manipulated simply by adding the natural ligand. The potential of the system is illustrated in the Drosophila renal (Malpighian) tubule, where each receptor has been shown to stimulate fluid secretion, to act through its cognate second messenger, and to be blocked by appropriate pharmacological antagonists. The results have uncovered a new role for cGMP signaling in tubules and also demonstrate the utility of the tubule as a possible in vivo test bed for novel receptors, ligands, or agonists/antagonists (Kerr, 2004).

Ectopic expression of the rat ANP receptor (GC-A) in tubules was achieved under control of both principal cell and stellate cell GAL4 drivers and a heat-shock (hs) promoter. Expression of the GC-A transgene was confirmed by RT-PCR. Expression of GC-A in tubules confers sensitivity to ANP, with resultant production of cGMP. Measurement of cAMP levels in tubules that express GC-A in principal cells (using the c42 GAL4 driver), stellate cells (using the c742 driver), or ubiquitously, show that cGMP levels are stimulated neither by GC-A receptor, nor by ANP, alone. ANP raises cGMP in a dose-dependent manner in c42-GC-A and c724-GC-A tubules, with a nearly 4-fold maximal increase in cGMP levels in c42-GC-A tubules and a 2-fold cGMP increase in c724-GC-A tubules. Since stellate cells make up only a small fraction of the tubule volume, the apparently lower fold increase in stellate cell cGMP probably reflects a larger absolute rise in cGMP in these cells. The EC50 for ANP in both principal and stellate cells is similar, 10^-8 M (Kerr, 2004).

Similar increases in fluid transport are observed upon stimulation with ANP, when GC-A is expressed either in only principal cells or in stellate cells. Although cGMP signaling has been shown in principal cells, a diuretic role for cGMP in stellate cells had not been demonstrated before. The effects of cGMP in principal and stellate cells are additive; when GC-A is expressed ubiquitously under hs control, maximal secretion rates are higher than when it is targeted to either cell alone (Kerr, 2004).

cGMP signals in principal cells may activate CNG-type calcium channels, resulting in calcium increase and fluid transport. Targeted expression of the calcium reporter, aequorin, was used to measure changes in intracellular calcium ([Ca²⁺]_i) in GC-A tubules. In tubule principal cells, a biphasic elevation of [Ca²⁺]_i is observed upon ANP stimulation, followed by a sustained secondary rise in [Ca²⁺]_i. By contrast, no change in stellate cell [Ca²⁺]_i was observed upon ANP challenge. Thus, major cellular targets for cGMP in principal cells are CNG channels. Stellate cells, however, must contain uncharacterized cGMP-activated targets that modulate fluid transport (Kerr, 2004).

ANP-mediated cGMP signaling and increased fluid transport are a result of specific ligand-receptor interactions, since the ANP antagonist, anantin, abolishes ANP stimulation of both cGMP and fluid transport in hs-GC-A tubules in a dose-dependent manner (Kerr, 2004).

Pilot experiments have shown that Drosophila tubules are insensitive to 5HT; there is thus scope to modulate second messengers by ectopic expression of the cognate GPCRs. The Drosophila 5HT_7Dro receptor raises cAMP levels in cultured cells by activating adenylate cyclase. When 5HT_7Dro is expressed in either principal or stellate cells using the appropriate GAL4 drivers, 5HT induces dose-dependent production of cAMP. EC₅₀ for 5HT in both lines was 10^-7 M. Similarly, the heat shock construct elicits elevation of [cAMP] upon 5HT treatment. Controls (non-heat-shocked hs-5HT_7Dro and heat-shocked wild-type or parental lines) show no response to 5HT. Furthermore, there is no detectable impact on cGMP levels when 5HT_7Dro is driven either in principal cells, stellate cells, or ubiquitously (Kerr, 2004).

As would be expected from the literature, increased [cAMP] in principal cells stimulates fluid transport. However, as for cGMP, a previously undocumented diuretic role of cAMP in stellate cells was uncovered, and the maximal rates of 5HT-induced fluid secretion observed with c42-5HT_7Dro and c724-5HT_7Dro tubules appear to be additive: the sum of maximal rates is approximately equal to the maximal 5HT-induced rate observed in heat-shocked hs-5HT_7Dro tubules. It was thus hypothesized that, if stellate cells have the machinery to respond functionally to cAMP and cGMP, then they are also likely to have the machinery to produce the signals (Kerr, 2004).

As with GC-A, the possibility that [Ca²⁺]_i may be altered upon activation of 5HT_7Dro was investigated. In principal cells of 5HT_7Dro/c42-aeq tubules, 5HT stimulation results in a biphasic elevation of [Ca²⁺]_i, similar to that seen in GC-A/c42-aeq tubules. However, no [Ca²⁺]_i rise was observed in 5HT-stimulated 5HT_7Dro/c710-aeq tubules. These results are consistent with both cAMP and cGMP acting on a CNG channel that is expressed only in principal, and not in stellate, cells (Kerr, 2004).

It proved possible to reproduce the known pharmacology of this 5HT receptor. The antagonist (+)-butaclamol almost completely attenuated 5HT-stimulated production of cAMP in a dose-dependent manner, with an IC₅₀ of 2.5 × 10^-8 M. This agrees precisely with values obtained for the receptor in cell lines. A maximal dose of (+)-butaclamol (10^-5 M) also reduced 5HT-stimulated fluid transport in hs-5HT_7Dro tubules (Kerr, 2004).

Another Drosophila 5HT receptor, 5HT_1ADro, is known to mobilize intracellular calcium. Expression of 5HT_1ADro in either principal or stellate cells results in 5HT-induced calcium responses. As would be expected from the actions of capa and leucokinin (ligands known to act through [Ca²⁺]_i), 5HT also stimulated fluid transport, when 5HT_1ADro was expressed in either principal cells or stellate cells, and no effect was seen without a GAL4 driver. Although a comprehensive dose-response curve was not performed, 5HT-stimulated fluid transport was inhibited by yohimbine. The effective concentration for yohimbine in tubules (10^-5 M) is comparable with the K_i (18 μM) obtained for this receptor in cell lines (Kerr, 2004).

The 5HT_1ADro receptor has been previously shown to inhibit adenylate cyclase in vitro. Accordingly, cAMP and cGMP were measured in 5HT-stimulated c42-5HT_1ADro and c724-5HT_1ADro tubules. Activation of the 5HT_1ADro receptor in tubules does not affect cyclic nucleotide levels, and thus the stimulatory effects of 5HT on fluid transport are solely due to stimulation of increased [Ca²⁺]_i (Kerr, 2004).

Although receptor guanylate cyclases (like GC-A) are self-sufficient, the strategy of modulating second messengers by ectopic expression of GPCRs depends on the expression of cognate G proteins in the target cell type. A priori, it was expected that most G proteins would be widely expressed, but this was confirmed in tubules by RT-PCR with primers against all known G protein α, β, and γ subunits. As predicted, all the Gα subunit genes found in Drosophila are expressed in tubules. Tubules also express the full complement of genes encoding the Gγ subunit; the only G protein subunit that does not appear to be expressed in tubules is Gβ76C (Kerr, 2004).

Although a particular target cell type might not express cognate G proteins, an RT-PCR strategy compared with measurement of second messengers should be informative. As well as the apoaequorin calcium reporter, others are now available, and successful cAMP and cGMP measurements in the tiny (160-cell) tubule suggest that radioimmunoassay is sufficiently sensitive for most Drosophila tissues of interest (Kerr, 2004).

Obviously, the target cell must not normally express receptors for, or respond to, the ligand of choice. In the case of Drosophila, this condition is satisfied; there is no atrial natriuretic peptide-like sequence encoded within the Drosophila genome. For 5HT receptors or other GPCRs, more caution must be exercised; nonetheless, standard experimental controls would quickly identify any problems. In the case of 5HT_Dro1A, expression has only been documented in some cells of the embryonic nervous system; 5HT_Dro7 expression has been found in cells of the embryonic ventral midline, and in adult head, but not body. It is thus likely that these constructs will be useful in most Drosophila nonnervous tissues (Kerr, 2004).

This study demonstrates successful ectopic expression of vertebrate and Drosophila receptors in Malpighian tubules and quantifies the effects of such expression on signal transduction pathways and physiological output. There are clear results from this approach: (1) the interactions between 3 second messenger pathways were studied in unprecedented detail, in two cell types, in an organotypic context; (2) the results validated all that was known about signaling through known neuropeptides in the tubule; (3) a diuretic role for cAMP and cGMP in stellate cells was demonstrated, inviting the intriguing question as to which extracellular ligands normally activate these pathways; (4) this conserved pharmacology of a vertebrate receptor (rat GC-A) expressed in a model organism illustrates another possibility for functional genomics, i.e., that novel genes could be characterized relatively cheaply and easily by ectopic expression in a model organism where detailed organotypic-phenotypic analysis is possible. The Drosophila Malpighian tubule is an ideal such 'test bed' for genes where organotypic analysis may be important for normal function (Kerr, 2004).

The utility of this experimental system is more general, allowing sensitive and specific intervention in second messenger signaling in any Drosophila tissue for which there exists a GAL4 driver. Combined with the ready availability of real-time calcium reporters in Drosophila and the possibility of measuring cAMP and cGMP similarly, using transgenic FRET reporters, this simple model organism now has an impressive genetic toolbox for cell signaling studies (Kerr, 2004).

For more information about Malpighian tubule function, see The Drosophila melanogaster Malpighian tubule WWW page, maintained by Julian Dow at the University of Glasgow. In addition, the Dow laboratory maintains a sensitive map illustrating the physiology of Malpighian tubule secretory cells. Functioning of the V-ATPase is described at Julian Dow's V-ATPase site.

White participates in vesicular transepithelial transport of cGMP

Guanosine 3'-5' cyclic monophosphate (cGMP) and adenosine 3'-5' cyclic monophosphate (cAMP) are important regulators of cell and tissue function. However, cGMP and cAMP transport have received relatively limited attention, especially in model organisms where such studies can be conducted in vivo. The Drosophila Malpighian (renal) tubule transports cGMP and cAMP and utilises these as signalling molecules. This study shows via substrate competition and drug inhibition studies that cAMP transport - but not cGMP transport - requires the presence of di- or tri-carboxylates; and that transport of both cyclic nucleotides occurs via ATP binding cassette sub-family G2 (ABCG2), but not via ABC sub-family C (ABCC), transporters. In Drosophila, the white (w) gene is known for the classic eye colour mutation. However, gene expression data show that of all adult tissues, w is most highly expressed in Malpighian tubules. Furthermore, as White is a member of the ABCG2 transporter class, it is a potential candidate for a tubule cGMP transporter. Assay of cGMP transport in w^- (mutant) tubules shows that w is required for cGMP transport but not cAMP transport. Targeted over-expression of w in w^- tubule principal cells significantly increases cGMP transport compared with that in w^- controls. Conversely, treatment of wild-type tubules with cGMP increases w mRNA expression levels, implying that cGMP is a physiologically relevant substrate for White. Immunocytochemical localisation reveals that White is expressed in intracellular vesicles in tubule principal cells, suggesting that White participates in vesicular transepithelial transport of cGMP (Evans, 2008).

The first Drosophila mutation to be identified was white (w), and it was instrumental to Morgan's description of genes and chromosomes. Despite over 3000 publications on w since then, few have investigated the biological function of White protein, tending instead to concentrate on the genetics of w. The most prominent phenotype of w mutants is the pronounced lack of eye colour. However, eye colour phenotypes can also reflect defects in Malpighian tubule structure and function, because several eye pigment precursors [notably the transport of compounds in the xanthommatin biosynthetic pathway are stored and processed in the larval tubule before being released into the pupal haemocoel for uptake by the developing adult eye. The Malpighian tubules of w^- Drosophila are clear or whitish in appearance due to the absence of tryptophan metabolites and pteridines, unlike wild-type tubules, which are yellow in appearance (Evans, 2008 and references therein).

Insect Malpighian tubules are critical for survival and play essential roles in osmoregulation, homeostasis and immune function (Dow, 2005). Excess fluid and solutes are transported and excreted largely across these blind-ended tubules, with selective re-absorption occurring in the rectum. Drosophila tubules provide a unique model for studying transport in a live polarised epithelial tissue, with added benefits of the availability of genetic tools. cGMP and cAMP signalling has been studied in the Drosophila Malpighian tubule for over 10 years, but the transport mechanisms of these cyclic nucleotides have received rather less attention. Nitric oxide (NO)-cGMP signalling was first identified to stimulate fluid transport, with more recent work implicating NO signalling in the immune response (McGettigan, 2005). Furthermore, NO-cGMP signalling can be induced by activation of the capa receptor in Dipteran tubules from several species (Pollock, 2004); and so is an important feature of tubule function in insect vectors of disease. Exogenous cAMP also stimulates fluid transport, with fluid transport also being stimulated by corticotropin-releasing factor (CRF)-like peptide (Cabrero, 2002) and calcitonin-like peptide (Evans, 2008 and references therein).

cGMP and cAMP are transported into the Malpighian tubules; cGMP transport across the tubule (efflux) is modulated by cGMP-dependent phosphodiesterases (cG-PDEs). In mammalian systems, cyclic nucleotide transport has been attributed to a number of ATP binding cassette (ABC) transporters and solute carriers (members of the SLC22 family). These transporters have been widely studied in mammals but equivalent transporters have not previously been identified in dipteran insects (Evans, 2008).

White is a member of the ABC transporter subfamily G, with greatest sequence similarity to human ABCG2. Investigations of the role of White in Malpighian tubules were carried out ~30 years ago, and tentatively identified location of and transport substrates for White. The location of White in the tubules was thought to be either the basolateral membrane or the pigment storage vesicle membranes. Potential substrates for White included tryptophan, kynurenine, 3-hydroxykynurenine, guanine and riboflavin (Evans, 2008 and references therein).

This study demonstrates that cGMP transported by the tubule occurs via ABCG2 transporters, and that White is required for cGMP but not cAMP, transport across the Malpighian tubule. Localisation of White to intracellular vesicles in the principal cells in the main, fluid-transporting segment of the tubule, indicates a possible storage-excretion mechanism of cGMP transport via vesicles (Evans, 2008).

Uptake and excretion of cGMP by Drosophila Malpighian tubules has been documented (Riegel, 1998). This study shows that the transport of cGMP is sensitive to the electrical gradient, suggesting that a secondary active transport mechanism is involved. Secreted fluid is capable of stimulating cGMP-dependent protein kinase (cGK) activity, suggesting that as in mammals, cGMP can be transported across the tubule membrane in unaltered form in Drosophila. Although primary active transport could not be shown unambiguously, the ABCG transport ATPase, White, is nonetheless necessary for significant cGMP flux; it must thus, at least, facilitate the diffusion of cGMP. In tubules, White is expressed throughout the cytoplasm in large vesicles of the main segment. These vesicles are probably the pigment vesicles which were suspected to be the location of White in a previous study (Sullivan, 1980). Other sub-families of ABC transporters also show intracellular localisation: ABCC4 transporters localise to the dense granules of platelets, and not at the plasma membrane. Thus, it seems that sub-families of the ABC transporter family, in both vertebrates and invertebrates, can be localised to vesicular structures in the cytoplasm of the cell. The first identified ABCG transporter in Leishmania, LiABCG4, has been shown to localise to the plasma membrane and to post-Golgi secretory vesicles when overexpressed in yeast (Castanys-Munoz, 2007). However, in the parasite, LiABCG4 is mainly localised to the plasma membrane, with some localisation in flagellar pockets; suggesting that the localisation in secretory vesicles in yeast may be due to over-expression of the ABCG4 transporter in the yeast system. Although the vesicular localisation of White in tubule cells may be due to targeted over-expression of w⁺, localisation of White in non-transgenic tubules (Sullivan, 1980) suggests that vesicular localisation for White is not associated with expression artifacts in vivo (Evans, 2008).

The primarily vesicular localisation of White may also indicate trafficking of cGMP across the Malpighian tubules in vesicles, a novel mechanism of transepithelial cGMP transport that would not compromise the integrity of intracellular cGMP signalling pathways. This would explain why fluid transport assays on tubules from w loss-of-function mutants show similar rates of cGMP-induced fluid transport (Davies, 1995; Dow, 1994b) to wild-type tubules. The current model is thus that cGMP is transported into the cell by a basolateral plasma membrane cGMP transporter that still remains to be discovered; once in the cell, it can act to stimulate fluid transport. However, White sequesters cGMP into vesicles, contributing [perhaps together with the action of DmPDE6 (Day, 2006) to its clearance from the cell; and these vesicles are excreted from the apical surface of the cell, presumably as part of a general purpose organic solute clearance mechanism. In the absence of White, cGMP is still transported into cells but due to reduced uptake into intracellular vesicles, is transported into the lumen at a much reduced rate. It will be interesting in due course to try to identify the plasma membrane transporter for cGMP. Importantly, this multi-stage transport model explains why it was not possible to demonstrate accumulation of cGMP beyond Nernst-predicted ratios, despite the involvement of a transport ATPase in the process (Evans, 2008).

Although the w gene has almost exclusively been researched in association with its role as an eye colour marker, recent microarray data of adult fly tissues (Chintapalli, 2007) has shown that w is most highly expressed in Malpighian tubule, a tissue enriched for organic anion transporters. Previous studies have shown that White can act as a heteromeric transporter: with Scarlet, it is a tryptophan transporter, responsible for brown eye colour; with Brown, it transports guanine, the precursor of the red pigment in eye. Mutation of key residues such as glycine 589 in the fifth transmembrane helix of White, significantly reduces guanine transport by White-Brown heterodimers, suggesting the importance of G589 in heterodimerisation and in guanine transport. Interestingly, mutation of amino acid 553 in TM5 of ABCG2 (a well-conserved residue corresponding to G589 in White) disrupts function and trafficking of ABCG2, implying conservation of dimerisation function of these residues across evolution (Evans, 2008 and references therein).

Even if White does play a key role in cGMP transport, organic solute transporters are heavily represented in the tubule transcriptome, and so there are other potential candidate transporters. In mammals, transporters of the ABCC class, notably MRP4 (ABCC4) and MRP5 (ABCC5) have been shown to transport cyclic nucleotides out of the cell. Treatment of Drosophila tubules with glibenclamide, a broad-spectrum inhibitor of ABC transporters, results in inhibition of both cAMP and cGMP transport. However, utilising either known inhibitors or competing substrates for ABBC transporters shows that such transporters are not involved in cAMP/cGMP transport by Malpighian tubules. Interestingly, tubules express five of the seven Drosophila homologues of mammalian ABCC transporters (Day, 2006); with one gene, CG9270, being expressed only in tubules of the adult fly. It would be interesting to screen the product of this gene for ABCC transporter function, and to determine its substrate specificity. In any case, the lack of effect of ABCC-specific drugs on the tubule could indicate evolutionary divergence between mammalian and Drosophila ABCC transporters, or may simply reflect inaccessibility of these transporters to specific drugs if the transporters are localised to the apical membrane (Evans, 2008).

This study has shown that cGMP transport by the tubule is specific and possesses distinct properties from cAMP transport. Previous investigations have suggested that there may be some overlapping function of cyclic nucleotide transporters. The different conclusions reached by these different studies may be a reflection of the concentration of each cyclic nucleotide used - the competing cyclic nucleotide was greatly in excess in the Riegel study. This study shows that cAMP transport requires the presence of di- or tri-carboxylates; but that cGMP transport is unaffected by these compounds. cAMP transport probably requires an OAT-like transporter at the basolateral membrane, whereas cGMP is transported via a different mechanism. Thus in the tubules, the mechanisms of transport of cGMP and cAMP are largely independent and specific. This could reflect the importance of these signalling molecules in the tubules. Alternatively, it could reflect the transport mechanisms necessary in a tissue for which there is a requirement for an established potential gradient to enable solute uptake and excretion (Evans, 2008).

Overall, though, the results suggest that the extraordinary abundance of White in the adult Malpighian tubule may reflect a novel role that continues beyond the need to handle visual pigment precursors in the larva and pupa. White is thus a more versatile transport protein that previously suspected (Evans, 2008).

SLC22 Transporters in the Fly Renal System Regulate Response to Oxidative Stress In Vivo

Several SLC22 transporters in the human kidney and other tissues are thought to regulate endogenous small antioxidant molecules such as uric acid, ergothioneine, carnitine, and carnitine derivatives. These transporters include those from the organic anion transporter (OAT), OCTN/OCTN-related, and organic cation transporter (OCT) subgroups. In mammals, it has been difficult to show a clear in vivo role for these transporters during oxidative stress. Ubiquitous knockdowns of related Drosophila SLC22s-including transporters homologous to those previously identified in mammals such as the "Fly-Like Putative Transporters" FLIPT1 (SLC22A15) and FLIPT2 (SLC22A16)-have shown modest protection against oxidative stress. However, these fly transporters tend to be broadly expressed, and it is unclear if there is an organ in which their expression is critical. Using two tissue-selective knockdown strategies, this study was able to demonstrate much greater and longer protection from oxidative stress compared to previous whole fly knockdowns as well as both parent and WT strains. Expression in the Malpighian tubule and likely other tissues as well (e.g., gut, fat body, nervous system) appear critical for managing oxidative stress. These four Drosophila SLC22 genes are similar to human SLC22 transporters (CG6126: SLC22A16, CG16727: SLC22A7, CG4630: SLC22A3, and CG6006: SLC22A1, etc.)-many of which are highly expressed in the kidney. Consistent with the Remote Sensing and Signaling Theory, this indicates an important in vivo role in the oxidative stress response for multiple SLC22 transporters within the fly renal system, perhaps through interaction with SLC22 counterparts in non-renal tissues. Many of the human relatives are well-known drug transporters. This work not only indicates the importance of SLC22 transporters in the fly renal system but also sets the stage for in vivo studies by examining their role in mammalian oxidative stress and organ crosstalk (Zhang, 2021).

Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis

Epithelia frequently segregate transport processes to specific cell types, presumably for improved efficiency and control. The molecular players underlying this functional specialization are of particular interest. In Drosophila, the renal (Malpighian) tubule displays the highest per-cell transport rates known and has two main secretory cell types, principal and stellate. Electrogenic cation transport is known to reside in the principal cells, whereas stellate cells control the anion conductance, but by an as-yet-undefined route. This study resolved this issue by showing that a plasma membrane chloride channel, encoded by ClC-a, is exclusively expressed in the stellate cell and is required for Drosophila kinin-mediated induction of diuresis and chloride shunt conductance, evidenced by chloride ion movement through the stellate cells, leading to depolarization of the transepithelial potential. By contrast, ClC-a knockdown had no impact on resting secretion levels. Knockdown of a second CLC gene showing highly abundant expression in adult Malpighian tubules, ClC-c, did not impact depolarization of transepithelial potential after kinin (see Leucokinin) stimulation. Therefore, the diuretic action of kinin in Drosophila can be explained by an increase in ClC-a–mediated chloride conductance, over and above a resting fluid transport level that relies on other (ClC-a–independent) mechanisms or routes. This key segregation of cation and anion transport could explain the extraordinary fluid transport rates displayed by some epithelia (Cabrero, 2014).

Epithelia provide essential barrier and vectorial transport capabilities intrinsic to the success of higher organisms. Depending on their roles and specializations, epithelia may be termed tight or leaky, based on their electrical conductance, which in turn depends on the patency of their paracellular spaces. Tight epithelia are thought to have a highly restricted paracellular route because of prominent tight junctions (septate junctions in insects), so constraining transepithelial fluxes to a transcellular route. Leaky epithelia, traditionally associated with high flux rates, lack such prominent tight junctions (Cabrero, 2014).

Insect renal (Malpighian) tubules move fluid at the highest rates observed in biology, and thus would seem to be ideal candidates for leaky epithelia. However, insect tubule cells are typically large and multinucleate or polyploid (large cell diameters minimize the junctional circumference per unit transporting membrane), and the cells are also surrounded by prominent septate junctions. Nonetheless, it has been argued that, in tubules of the dengue fever mosquito Aedes aegypti, the rapid neurohormonally controlled chloride shunt conductance is paracellular, caused by remodeling of the septate junctions with switch-like speed under the influence of diuretic peptides of the kinin family (Cabrero, 2014).

Drosophila melanogaster is a member of one of the largest insect orders, the Diptera, and these tubules are distinguished by a prominent secondary cell type, the stellate cell. The powerful transgenic toolbox available for Drosophila allows cell-specific contributions to tissue-level function to be probed; by expressing the luminescent calcium reporter apoaequorin transgenically in specific cell types, it was possible to show that kinin signals specifically through intracellular calcium in only the stellate cells-and not the principal cells-consistent with the observed expression of the kinin receptor in just stellate cells. The biogenic amine tyramine also acts similarly; that is, it stimulates fluid secretion by activating a chloride shunt conductance by raising intracellular calcium levels in the stellate cells. Thus-at least in Drosophila-the stellate cell plays a crucial role in transducing the diuretic kinin signal into a rapid increase in chloride conductance (Cabrero, 2014).

In practice, both transcellular and paracellular routes of chloride conductance are likely to contribute to both resting and kinin-stimulated fluid secretion, but the relative importance of the two routes is not known. This study used Drosophila transgenics-combined with physiology, electrophysiology, and imaging-to show that a CLC chloride channel encoded by ClCa/ CG31116 is a basolateral and apical plasma membrane chloride channel, uniquely localized to stellate cells, that is essential for neuropeptide-stimulated, but not resting, levels of secretion. Furthermore, by generating Drosophila that were transgenic for a membrane-targeted fluorescent chloride reporter, this study demonstrated that tubule stellate cells displayed a characteristic intracellular chloride signature upon kinin stimulation. Therefore, this issue has bee resolved in Drosophila; kinin stimulates chloride flux by a transcellular route that is confined to the stellate cells (Cabrero, 2014).

Only two of the three CLC genes encoded by the D. melanogaster genome show highly abundant expression in adult Malpighian tubule. By contrast, ClC-b shows low-level expression consistent with a predicted role as a housekeeping endomembrane channel. Drosophila ClC-b (CG8594) is a homolog of human CLCN7 (CLC-7), an endosomal and lysosomal Cl- channel that is thought to act as a H+/2Cl- exchanger that provides the major chloride conductance of lysosomes. Based on these distributions- and the functions of their respective homologs in humans-this study focused attention on the functional roles of ClC-a and -c (Cabrero, 2014).

Drosophila ClC-a (CG31116) is a homolog of human CLCN2 (CLC-2), a plasma membrane inwardly rectifying chloride channel functionally significant in airway epithelia and the central nervous system. In epithelia, CLCN2 is apical and has been suggested to play a role in Cl- efflux (Cabrero, 2014).

The GAL4/UAS binary expression system allows the knockdown of specific transcripts in specific cells within an otherwise normal organism, under control of the appropriate GAL4 driver lines. The Vienna UAS-dsRNA line ClC-akk101247 produced a significant knockdown (>70%) in overall tubule expression of ClC-a when driven in stellate cells, but no knockdown when driven in principal cells, suggesting that ClC-a is expressed uniquely in the stellate cells (Cabrero, 2014).

To determine whether ClC-a knockdown in stellate cells affected tubule physiology, tubule secretion was measured in control and ClC-a knockdown tubules. ClC-a RNAi knockdown in stellate cells had no effect on basal fluid secretion levels, but abolished the stimulation of fluid secretion normally induced by Drosophila kinin, a neuropeptide that activates the tubule chloride shunt pathway in Drosophila and other insects. By contrast, knockdown of ClC-a in principal cells had no effect on either resting or stimulated secretion. Therefore, ClC-a in stellate cells is essential for kinin-stimulated, but not resting, fluid secretion (Cabrero, 2014).

ClC-a knockdown might have an impact indirectly on fluid secretion, rather than directly on chloride conductance. The electrophysiological signature of kinin action is a rapid abolition of by a completely different route-for example, a paracellular conductance (Cabrero, 2014).

Stellate-cell-specific ClC-a knockdown also produced an inflated abdomen phenotype in adult flies, implying defective osmoregulation. To confirm that the abdominal bloating was due to increased haemolymph water content, wet-dry weight measurements were performed. Wet weight measurements revealed that ClC-a knockdown male and female adults are significantly heavier compared with control flies, whereas dry weight measurements are equivalent. Interestingly, the same distended abdomen phenotype was observed upon knockdown of teashirt, a transcription factor that regulates stellate cell differentiation and renal physiology in Drosophila (Cabrero, 2014).

If kinin is indeed activating a stellate-cell-specific chloride shunt conductance, it may be possible to detect transient changes in intracellular chloride on kinin stimulation. To monitor stellate cell intracellular concentration of Cl- ([Cl-]i), flies were made transgenic for a genetically encoded fluorescent combined Cl-/pH - biosensor (ClopHensor), under UAS control, allowing its cell-specific expression. The fluorescence of the PalmPalm-ClopHensor confirms that this transgenic sensor is typically localized to plasma and intracellular membranes, as seen in other systems (Cabrero, 2014).

First, the ratiometric changes were measured in RCl (F458/F545) of the PalmPalm-ClopHensor, in response to application of 75 mM KCl to either single stellate cells or individual Malpighian tubules, using confocal microscopy or spectrophotometer recording, respectively, to confirm the chloride sensitivity of the system. Kinin stimulation in the tubule caused a strong increase of [Cl-]i, likely due to chloride ion passage through the basolateral plasma membrane. After peaking, [Cl-]i rapidly stabilized to values similar to normal, possibly due to partial desensitization of the kinin response and redistribution of chloride ions through the ClC-a chloride channel in the stellate cells or a transient mismatch between activation of basal and apical conductances. These changes were dramatically reduced in reporter lines in which ClC-a was specifically knocked down in only in tubule stellate cells, confirming that the changes seen were due to ClC-a chloride channel activation by kinin (Cabrero, 2014).

Real-time PCR data show that ClC-a expression is primarily in the stellate cells, because only knockdown in stellate cells significantly impinges on overall tubule expression level. Although ClC-a is a homolog of a plasmamembrane chloride channel, and its knockdown leads to specific effects on transepithelial transport, it is conceivable that it could be an intracellular channel that in some way impinges indirectly on transepithelial chloride flux. Accordingly, it is important to establish where in the stellate cell it resides (Cabrero, 2014).

A specific antibody was raised against ClC-a, and it was validated by Western blotting. Immunocytochemistry showed specific labeling of only stellate cells within the tubule, and confocal microscopy revealed that the location was on the plasma membrane. By overexpressing in the stellate cell a known apical membrane aquaporin channel, Drip, labeled with enhanced YFP (eYFP), it was possible to show that ClC-a both colocalizes apically with DRIP, and prominently labels the basal membrane. So although additional chloride transport routes cannot be excluded, ClC-a is present in both apical and basal plasma membranes and is both necessary and sufficient for kinin response. Therefore, a plasma membrane ClC-a chloride channel, uniquely in the stellate cells, is essential for the action of kinin to stimulate fluid production by increasing the chloride conductance, so collapsing the transepithelial membrane potential (Cabrero, 2014).

It was also possible to confirm by immunocytochemistry the efficiency of the knockdown of ClC-a expression at the protein level. Only down-regulation of ClC-a in the stellate cell was able to replicate the effect of blocking the antibody with the antigenic peptide (Cabrero, 2014).

ClC-a is not the only chloride channel enriched in the tubule; ClC-c (CG5284), a homolog of human CLCN3 (CLC-3) known to play both plasma membrane and epithelial roles, is also tubule-enriched. In the plasma membrane of epithelia, it is implicated in cell swelling. It is also strongly expressed in the nervous system, where it is found in endosomal compartments and synaptic vesicles; mice deficient for CLCN3 show gross neurological defects, such as blindness and loss of the hippocampus. Accordingly, the equivalent experiments were performed on a knockdown of ClC-c. By contrast with ClC-a, knockdown of ClC-c in principal cells knocked down overall tubule Clc-c expression levels, whereas knockdown in stellate cells produced compensatory up-regulation of whole tubule expression, suggesting that ClC-c is expressed in both cell types and does not play a stellate-specific role. Similarly, fluid secretion was only partially reduced in stellate-specific ClC-c knockdown. Significantly, electrophysiological analysis showed that knockdown of ClC-c in either principal or stellate cells did not have an impact on kinin-induced depolarization of the transepithelial potential, implying that ClC-c is not involved in the chloride shunt pathway, and so any impact on fluid secretion is through some other mechanism. Consistent with the quantitative PCR (q-PCR) data, a specific antibody against ClC-c, stained both principal and stellate cells uniformly. Therefore, it is concluded that, although ClC-c is important for tubule function, it is not a direct participant in transepithelial chloride ion transport (Cabrero, 2014).

The relative contributions of transcellular and paracellular flux and of specialized cell types in the integrated performance of an epithelium is a fundamental and important issue, in which experimental techniques have been limiting. By selecting the Drosophila as an experimental subject, powerful cell-type–specific transgenics provide valuable methodologies to address such questions. Previously studies used cell-type–specific calcium reporters to show that the kinin-induced calcium signal is exclusively expressed in stellate cells, implying that they alone mediate the tissue response to kinin; this study directly show that kinin actions on chloride flux- evidenced independently by increase in fluid secretion, increase in intracellular chloride, and decrease in transepithelial potential- all depend obligatorily on the presence of the plasma membrane chloride channel ClC-a. These results confirm the proposed spatial segregation of cation and anion transport in Drosophila into two distinct cell types and suggest that the paracellular pathway is more likely to account for basal, rather than kinin-stimulated, fluid secretion (Cabrero, 2014).

Although these transgenic technologies are not readily applied to other species, the model developed in Drosophila is likely to have more general application. In both the dengue vector Aedes aegypti, and the malaria vector Anopheles gambiae, kinin receptors have been localized to the stellate cells. Indeed, stellate cells are considered general to the Diptera. Excitingly, differentiated characters of the stellate role are under control of teashirt in Drosophila, and orthologs of teashirt have been found to be localized to previously undocumented secondary cells in highly diverged insect groups such as Orthoptera and Coleoptera. It may well be that the uniquely high secretory rates of many insect renal tubules can be explained by the model shown in this study (Cabrero, 2014).

Specialized stellate cells offer a privileged route for rapid water flux in Drosophila renal tubule.

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, four genes of the major intrinsic protein family are expressed at a very high level in the fly renal tissue: the aquaporins (AQPs) 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 result in impaired hormone-induced fluid secretion. The Drosophila tubule has 2 main secretory cell types: active cation-transporting principal cells, wherein the aquaglyceroporins localize to opposite plasma membranes, and small stellate cells, the site of the chloride shunt conductance, with these AQPs localizing to opposite plasma membranes. This suggests a model in which osmotically obliged water flows through the stellate cells. Consistent with this model, fluorescently labeled 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 in regulating their internal environments (Cabrero, 2020).

There are more species of insects than all other forms of life combined. In part, this is because of the exceptional ability of the simple body plan to operate in a wide range of environments, and osmoregulation is a key component of this success. Remarkably, the insect Malpighian (renal) tubule is capable of secreting fluid faster (on a per cell volume basis) than any other epithelium known, and shows an extremely high osmotic water permeability (Posm). In Drosophila, the renal tubule has 2 major cell types; the mitochondria-rich principal cell actively transports protons via an apical, plasma membrane vacuolar H+-ATPase (V-ATPase), setting up a gradient which is exchanged primarily for potassium, which enters the cell basolaterally through the combined activity of Na+, K+-ATPase, inward rectifier potassium channels and potassium cotransports. The smaller stellate cell provides a route for hormone-stimulated chloride conductance through a basolateral ClC-a chloride channel, partnered with secCl, an apical cys-loop chloride channel, to balance the lumen-positive charge, and so effect a net movement of salt. Aquaporins (AQPs; the water transporting major intrinsic proteins [MIPs]) are known to be highly expressed in insect tubules, and global knockdown of an AQP in the Aedes mosquito , or in the beetle Tribolium, impacts water loss. Although in situ hybridization of Drip showed expression in stellate cells, the route or mechanism of the very high osmotically obliged water fluxes that produce such remarkable fluid output has not been characterized. Using the powerful cell-specific transgenic technologies unique to Drosophila melanogaster, this study shows that this flux is transcellular, and, selectively through the stellate cells, mediated by 2 AQPs, in response to diuretic hormone stimulation. Knockdown of AQPs in stellate cells impacts survival under stress, and comparative studies suggest that water flux is confined to specific cell types in tubules from a broad phylogenetic range of insects (Cabrero, 2020).

MIPs are a multigene family of 6-transmembrane domain proteins that assemble as tetramers to form pores. Most members of the family are true water channels (AQPs); others can facilitate movement of water or small organic molecules (aquaglyceroporins); but the substrates of some are still obscure. In Drosophila, 8 genes make up the MIP family, but the FlyAtlas and FlyAtlas2 gene expression online resources independently report that only 4 are expressed at high levels in epithelia such as the salivary gland, midgut, hindgut, and Malpighian tubules. Two of these highly expressed genes (Drip and Prip) are similar to classical AQPs in structure, whereas the other 2 (Eglp2 and Eglp4) align with the aquaglyceroporins. Comparison of the protein sequence of D. melanogaster AQPs (Drip and Prip) and aquaglyceroprins (Eglp2 and Eglp4) in a Clustal Omega alignment shows that key active-site residues, including those required for water selectivity and those involved for their regulation, have been conserved. There are thus at least 2 candidates that could mediate high water flux rates in polarized epithelia (Cabrero, 2020).

Water and solutes transport is achieved by an apicobasally polarized distribution of membrane proteins, and, accordingly, it is important to establish where in the tubule principal and stellate cells MIPs reside. Specific antibodies were raised against the 4 tubule-expressed MIPs, and they were validated by Western blotting. Immunocytochemistry showed clear segregation of MIP expression, with the 2 AQPs expressed on opposite sides of the specialized stellate cell (Drip and Prip are localized to the apical and basolateral membranes, respectively), and the 2 aquaglyceroporins expressed on opposite sides of the main principal cell (Eglp2 and Eglp4 are localized to the apical and basolateral membranes, respectively). Accordingly, overexpression of all 4 MIPs labeled with Venus (eYFP) recapitulate the pattern of expression observed by immunocytochemistry. These data are consistent with other reports that Drip and Prip show spatial separation in other insects, such as silkworm. It would thus be tempting to surmise that the stellate cell provides a major route for water flux through the tissue, but only the transport properties of one of these MIPs (Drip) has been established; how many of them are, in fact, functional AQPs (Cabrero, 2020)?

Each of the 4 candidate genes was expressed in Xenopus oocytes, and tested both for classical swelling under hypoosmotic stress and for facilitated flux of organic solutes. The 2 channels expressed in tubules (Drip and Prip) both acted as classical AQPs, showing rapid water fluxes but only barely detectable fluxes of organic solutes. By contrast, the Eglp2 channel showed fluxes of water comparable to Prip, but also very rapid fluxes of small organic solutes, such as glycerol and urea; Eglp4 did not permit water flux, but showed extremely rapid flux of organic solutes. These fluxes are consistent with the predicted classification of Eglp2 and Eglp4 as aquaglyceroporins. These data are thus in agreement with Drip and Prip providing a transcellular route for water through the stellate cells, and, as the tubule provides a range of physiological readouts, this prediction can be tested experimentally (Cabrero, 2020).

Although epithelial polarization of some AQPs has been shown in other insects, Drosophila genetic technology allows their physiological roles to be dissected with great precision. Using the GAL4/UAS system, which uses the yeast GAL4 transcription factor, a regulator of gene expression of galactose-induced genes, and its recognition site, UAS (Upstream Activating Sequence), and renal cell-type-specific drivers, it is possible to generate transgenic flies in which a single candidate gene is knocked down in only the tubule cell type in which it is expressed, leaving expression throughout the rest of the fly untouched. Accordingly, each of the 4 genes was knocked down in the cell type in which its proteins had been shown to be expressed, and it was possible to confirm by qPCR and immunocytochemistry the efficiency of the knockdown of MIPs expression at the gene and protein levels. The resulting fluid output was then measured under baseline conditions, and when maximally stimulated with diuretic peptides of the capa and kinin families. Knockdown of either Drip or Prip in just the stellate cell significantly impeded fluid secretion, confirming functional roles in rapid fluid movement across the tissue. However, knockdown of Eglp2 or Eglp4 in the principal cells also elicited reduced fluid secretion rates. This suggested 2 possibilities: either that all 4 MIPs could produce water conductance, through both cell types (at variance with the biophysical characterization), or that one pair of channels provided the main route for water, while the other pair allowed flux of a necessary organic osmolyte, or metabolic substrate, such that blockade could reduce overall function of the tissue. In a simple saline containing glucose and glutamine, which has been shown to increase secretion rates in amino acid-free saline, similar results were obtained. The very high rates of generation of primary urine by the tubule could become a liability under dry conditions, and so knockdown of AQPs would be predicted to impact survival under desiccation. This had been shown by global knockdown of the Drip ortholog in Anopheles gambiae, the malaria vector; however, Drip is broadly expressed, and so the effect could not be attributed solely to the tubules. Using GAL4/UAS technology, it was possible to knock down Drip or Prip expression in just the tubule stellate cells of an otherwise normal insect and show that knockdown of either AQP was sufficient to produce enhanced survival under desiccation stress in female flies. Interestingly, knockdown of either Eglp2 or Eglp4 in principal cells did not impact survival to desiccation. Water flux across the tubule is thus limiting for terrestrial insects under desiccation stress, as previously suggested, and the reduction in water loss by Malpighian tubules through the stellate cells appears to be an important mechanism for desiccation resistance (Cabrero, 2020).

To distinguish the roles of the aquaglyceroporins from the AQPs, it would be necessary to determine the route of water flux through the tubule. The complex polyglucan, dextran, can be readily fluorescently labeled, and can be size-selected to ranges that can be swept along by water flux, but then trapped in a pathway of restricted permeability. Both the principal and stellate cells have apical microvilli, which, in principal cells, are stabilized by the cell adhesion molecule Fasciclin2 and contain mitochondria to support intense activity of the V-ATPase; both cell types also possess basal infoldings that increase the available surface area for transport. Thus tubules were stimulated in the presence of fluorescently labeled 40- to 70-kDa dextran, which pilot experiments had shown was too large to move across the epithelium. Dextran would thus accumulate in a compartment diagnostic of the route of water movement, be it the principal or stellate cells, or the paracellular route between the tight ('septate') junctions. The results showed that only the basal labyrinths of the stellate cells became labeled with 40-kDa dextran, and that the percentage of stellate cell population displaying accumulation of dextran was significantly higher after kinin stimulation of fluid secretion. Although the possibility cannot be excluded, for example, that apical Eglp2 in the principal cells can allow a water flux, perhaps via gap junctions from basolateral Prip in the stellate cells, only the stellate cell has a basolateral functional AQP, and so the pathway provided by Prip and Drip in the stellate cells is likely to be the major route for transepithelial water movement through the tubule (Cabrero, 2020).

The segregation of active cation transport to principal cells, and chloride and water flux to stellate cells, may confer selective advantages and could potentially extend to other insects. Stellate cells are more widely distributed than initially thought, and previous work has shown that fluorescently labeled kinin [the neuropeptide that stimulates the chloride conductance] marks stellate-like cells in most advanced endopterygote insects, suggesting an ancient and conserved role. To probe the route of water flux in insects without the benefits of Drosophila transgenics, the dextran flux labeling technique was applied to a panel of insects selected to represent the major exopterygote and endopterygote orders, so providing an initial view on the 2-cell model. Among the endopterygote insects, dextran selectively labeled stellate-like cells of all insect orders except the beetles, where an extensive network was observed. Significantly, kinin genes are almost never found in Coleoptera, consistent with a lack of stellate cell specialization. In the more primitive exopterygotes, the story is more varied; although kinin had labeled the epithelium rather generally, this general pattern was seen in the locust, but not a cockroach. As a first approximation, therefore, the 2-cell model, that links chloride flux, kinin stimulation, and water flux, seems to have broad applicability across the higher insects (Cabrero, 2020).

The tubule shows a remarkable ability to secrete primary urine at very high rates, and, together with other recent results, it is becoming clear that this success relies on the functional segregation of transport between different cell types. The main, principal cell has long apical microvilli, each containing a mitochondrion and loaded with proton-pumping V-ATPase, and is thought to drive an exchanger from the NHA family to produce a net K+ flux. Basolaterally, the infoldings contain high levels of Na+, K+-ATPase, inward rectifier K+ channels, and Na+/K+/Cl- cotransporters. This metabolically active cell is likely the route for excretion of a wide range of solutes via ABC transporters and other organic solute transporters, many of which are abundantly expressed in the tubule. The rarer stellate cells, by contrast, have shorter microvilli and fewer mitochondria, but are the gatekeepers for the hormone-stimulated chloride shunt conductance (through basolateral ClC-a and apical secCl), and now also for the passage of osmotically obliged water through basolateral Prip and apical Drip AQPs. The metabolically active principal cell is thus sheltered from these very high, and potentially disruptive, fluxes of water (Cabrero, 2020).

A model is presented for tubule function. The mitochondria-rich principal cell is specialized for metabolically intensive cation and solute transport. The apical V-ATPase sets up a proton electrochemical gradient which drives net K+ secretion via NHA or NHE exchangers. Basolateral K+ entry is afforded by inward rectifier K+ channels, Na+,K+ ATPase and an Na+,K+,2Cl- cotransport. The resulting lumen-positive potential drives a chloride shunt conductance, mainly via basolateral ClC-a and apical secCl channels in the stellate cell. The net transport of KCl drives osmotically obliged water, which is primarily via basolateral Prip and apical Drip in the stellate cells. In this way, the metabolically active principal cell is sheltered from the required high flux rates of water (Cabrero, 2020).

Given the severe consequences of unregulated fluid loss to a small terrestrial insect, it is not surprising that the tubule is under sophisticated neurohormonal control. Whereas cation pumping by the principal cells is under control of DH31, DH44, and Capa neuropeptides, the stellate cells are independently controlled by the neuropeptide kinin and by the biogenic amine tyramine; both act indistinguishably through intracellular calcium. The chloride shunt conductance is a known target of kinin and tyramine, as both rapidly collapse the lumen-positive potential; however, it will be interesting to investigate whether one or both of these messengers have an independent action to regulate stellate cell AQPs, perhaps through phosphorylation or recruitment of AQPs to the plasma membrane (Cabrero, 2020).

This 2-cell model is likely to be widely applicably through the higher insects, the endopterygotes, which include flies, butterflies, and bees, in which a secondary cell type has been observed either directly, or by mapping an AQP, or by visualization with fluorescently labeled kinin (the hormone which regulates chloride flux), or by otherwise mapping the kinin receptor. However, a universal model is unlikely, as most members of one higher insect order (the Coleoptera) do not use kinin signaling, and, in the lower exopterygote insects, such as crickets, there is no evidence for specialized secondary cells. The next challenge will be to map out the generality of this 2-cell model, and its alternatives, across the tens of millions of species that make up the insects (Cabrero, 2020).

Sulphonylurea sensitivity and enriched expression implicate inward rectifier K+ channels in Drosophila melanogaster renal function

Insect Malpighian (renal) tubules are capable of transporting fluid at remarkable rates. Secondary active transport of potassium at the apical surface of the principal cell must be matched by a high-capacity basolateral potassium entry route. A recent microarray analysis of Drosophila tubule identified three extremely abundant and enriched K(+) channel genes encoding the three inward rectifier channels of Drosophila: irk1, irk2 and irk3. Enriched expression of inward rectifier channels in tubule was verified by quantitative RT-PCR, and all three IRKs localised to principal cells of the main segment (and ir and irk3 to the lower tubule) by in situ hybridisation, suggesting roles both in primary secretion and reabsorption. A new splice form of irk2 was also identified. The role of inward rectifiers in fluid secretion was assessed with a panel of selective inhibitors of inward rectifier channels, the antidiabetic sulphonylureas. All completely inhibited fluid secretion, with IC(50)s of 0.78 mmol l(-1) for glibenclamide and approximately 5 mmol l(-1) for tolbutamide, 0.01 mmol l(-1) for minoxidil and 0.1 mmol l(-1) for diazoxide. This pharmacology is consistent with a lower-affinity class of inward rectifier channel that does not form an obligate multimer with the sulphonylurea receptor (SUR), although effects on non-IRK targets cannot be excluded. Glibenclamide inhibited fluid secretion similarly to basolateral K(+)-free saline. Radiolabelled glibenclamide is both potently transported and metabolised by tubule. Furthermore, glibenclamide is capable of blocking transport of the organic dye amaranth (azorubin S), at concentrations of glibenclamide much lower than required to impact on fluid secretion. Glibenclamide thus interacts with tubule in three separate ways; as a potent inhibitor of fluid secretion, as an inhibitor (possibly competitive) of an organic solute transporter and as a substrate for excretion and metabolism (Evans, 2005).

Two inwardly rectifying potassium channels, Irk1 and Irk2, play redundant roles in Drosophila renal tubule function

Inwardly rectifying potassium channels play essential roles in renal physiology across phyla. Barium-sensitive K+ conductances are found on the basolateral membrane of a variety of insect Malpighian (renal) tubules, including Drosophila melanogaster. This study found that barium decreases the lumen-positive transepithelial potential difference in isolated perfused Drosophila tubules, and decreases fluid secretion and transepithelial K+ flux. In those insect species in which it has been studied, transcripts from multiple genes encoding inwardly rectifying K+ channels are expressed in the renal (Malpighian) tubule. In Drosophila melanogaster, this includes transcripts of the Kir1, Kir2, and Kir3. The role of each of these gene products in renal tubule function is unknown. Simultaneous knockdown of Irk1 and Irk2 in the principal cell of the fly tubule decreases transepithelial K+ flux, with no additive effect of Irk3 knockdown, and decreases barium sensitivity of transepithelial K+ flux by approximately 50%. Knockdown of any of the three inwardly rectifying K+ channels individually has no effect, nor does knocking down Irk3 simultaneously with Irk1 or Irk2. Irk1/Irk2 principal cell double knockdown tubules remain sensitive to the kaliuretic effect of cAMP. Inhibition of the Na+/K+-ATPase with ouabain and Irk1/Irk2 double knockdown have additive effects on K+ flux, and 75% of transepithelial K+ transport is due to Irk1/Irk2 or ouabain-sensitive pathways. In conclusion, Irk1 and Irk2 play redundant roles in transepithelial ion transport in the Drosophila melanogaster renal tubule, and are additive to Na+/K+-ATPase-dependent pathways (Wu, 2015).

Inwardly rectifying K+ channels play important roles in invertebrate and vertebrate renal physiology. In insects, basolateral membrane barium-sensitive K+ conductances have been demonstrated in a variety of insect renal tubules. Examples include the yellow fever mosquito Aedes aegypti, the forest ant Formica polyctena, the Chagas vector Rhodnius prolixus, the agricultural pest Locusta migratoria, and the mealworm Tenebrio molitor. In addition, transcripts for inwardly rectifying K+ channels are expressed in the Malpighian tubules of Aedes aegypti, the vector for yellow fever, dengue, and chikungunya; Anopheles gambiae, the malaria vector; and the bed bug Cimex lectularius. Drugs targeting renal tubule inwardly rectifying K+ channels are currently being developed as mosquitocidal insecticides in Aedes aegypti. These channels may represent targets for the control of other insect pests as well, although killing of benign or beneficial insects will need to be avoided. This study extends the understanding of the role of these channels in insect renal tubule function (Wu, 2015).

Barium has been extensively used to probe the function of inwardly rectifying K+ channels in insects. This study replicated previous findings that barium decreases fluid secretion in the fly renal tubule and showed that it decreases transepithelial K+ flux. A decrease (lumen less positive) was observed in the transepithelial potential difference of 21 mV. This is consistent with prior reports of hyperpolarization of the basolateral membrane potential of similar magnitude, likely explaining the effect on the transepithelial potential difference. Both Irk1 and Irk2 are sensitive to barium when expressed in heterologous cells, and barium blocks the basolateral K+ conductance of the Drosophila tubule. Barium may also have additional effects on other, undefined ion channels on the basolateral membrane of the fly tubule. Indeed, Irk1 and Irk2 appear to account for only half of the barium sensitivity observed for transepithelial K+ flux. Genetic studies, in which specific channels can be manipulated, therefore provide complementary information to pharmacological studies. This is particularly important given the number of genes encoding inwardly rectifying K+ channels. The genomes of the mosquito species Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus encode Irk1 and Irk3 homologs, whereas the Irk2/Kir2 gene has undergone a duplication event, resulting in Kir2A and Kir2B genes. Additional duplication events have resulted in Kir2A and Kir2A' genes in Anopheles and Culex, and Kir2B and Kir2B' genes in Aedes. In Culex and Anopheles, the Kir3 gene has also been duplicated to result in Kir3A and Kir3B genes (Wu, 2015).

This study observed that Irk1 and Irk2, but not Irk3, are important for transepithelial fluid secretion and K+ flux. Interestingly, no functional channel activity was observed with attempts at expression of Irk3 in Xenopus oocytes or Drosophila S2 cultured cells, whereas both Irk1 and Irk2 possessed inwardly rectifying K+ channel activity. Similarly, no channel activity was observed in Xenopus oocytes expressing the Aedes aegypti Irk3 homolog, AeKir3, although it is highly expressed in the mosquito tubule. In addition, recent immunolocalization data indicates that AeKir3 is expressed in intracellular punctae in the mosquito tubule. In bed bugs, the Irk3 homolog ClKir3 transcript is expressed at very high levels in the Malpighian tubules, yet whole-organism silencing using RNA interference had no effect on bed bug viability. The functional role of Irk3 and its homologs in other insects in the renal tubule is therefore unclear (Wu, 2015).

In contrast, compounds that inhibit AeKir1 and AeKir2B have inhibitory effects on whole-mosquito urine excretion and on transepithelial fluid secretion and K+ flux in the Ramsay assay. This is broadly consistent with the current results that both Irk1 and Irk2 play roles in the fly tubule although there are interesting differences; in Aedes, AeKir1 is located in the stellate cell, whereas AeKir2B is in the principal cell, and inhibition of both AeKir1 and AeKir2 has additive effects on K+ flux. However, this may also reflect the effects of acutely inhibiting the channels pharmacologically, as opposed to the longer-term genetic knockdown used in this study. Additional open questions remain. For example, why is expression of AeKir2B enriched in the mosquito tubule, rather than the fly Irk2 homolog, AeKir2A? Does AeKir2B play a specific role in fluid secretion and ion flux after a blood meal, a situation not faced by Drosophila? Do specific splice isoforms of Irk2/Kir2A, demonstrated in Drosophila, Aedes aegypti, and Anopheles gambiae, have specific functional roles in the tubule? Given the ease of genetic manipulation and transgenesis in Drosophila, the fly renal tubule could serve as a platform to explore, not only the role of the Drosophila channels, but potentially also the physiological roles and/or the drug sensitivities of various inwardly rectifying K+ channels of other insects, aiding in the development of pharmacological agents to control insect disease vectors or insect pests (Wu, 2015).

Irk1 and Irk2 both have K+ channel activity as homomeric channels when heterologously expressed. It is possible that they could also function as heteromeric channels, as is the case for some other inwardly rectifying K+ channels. However, the fact that Irk1 and Irk2 must simultaneously be knocked down to see a change in transepithelial flux suggests that, even if the two channels do have heteromeric interactions, these are not absolutely required for their function in the tubule (Wu, 2015).

What roles are Irk1 and Irk2 playing in transepithelial ion flux? One possibility is that Irk1 and Irk2 constitute all or part of the basolateral K+ conductance and are important for maintaining the basolateral membrane potential. Could they also serve as a conductive pathway for K+ entry from the hemolymph into the principal cell? EK (-52 mV) is close to the basolateral membrane potential (-43 mV), with a net outward driving force for K+ movement from cell to bath. Because EK and the basolateral membrane potential are close to one another, relatively modest changes in conditions could affect the direction of the driving force. Indeed, the bathing solution for Ramsay assay studies of genetically modified tubules differed from those used in another study. In the Formica tubule, EK was also close to the basolateral membrane potential, and, depending on the measurement technique as well as the bath K+ concentration, driving forces were observed that were either inward, outward, or zero. Another subsequent study proposed that, at high hemolymph K+ concentrations, K+ uptake occurs through basolateral K+ channels. Similarly, studies in the deoxycorticosterone-treated rabbit cortical collecting duct demonstrated an inward (bath to cell) driving force for potassium across the basolateral membrane. In this preparation, transepithelial K+ flux from bath to lumen increased when bath K+ concentration increased, and this increase was attenuated by the basolateral application of barium, indicating that basolateral K+ channels allow K+ uptake into the cortical collecting duct principal cell. Similarly, application of an AeKir1/AeKir2B inhibitor to the basolateral membrane of the Aedes aegypti tubule depolarized the basolateral membrane potential and decreased input conductance under high bath K+ (34 mM) conditions (Wu, 2015).

It was found in a previous study that the Na+/K+-ATPase generates the driving force for NKCC activity in the fly renal tubule. Another potential role for K+ channels could be to recycle the K+ entering through the Na+/K+-ATPase. However, this study observed additive effects of the Na+/K+-ATPase inhibitor ouabain and knockdown of Irk1 and Irk2, indicating that Irk1 and Irk2 have functions beyond recycling the K+ entering through the Na+/K+-ATPase. Indeed, about 75% of transepithelial K+ flux was mediated by Irk1, Irk2, and ouabain-sensitive pathways, which could include the Na+/K+-ATPase itself as well as secondary active K+ uptake by the NKCC (Wu, 2015).

These genetic and pharmacological results are most consistent with a role for the inwardly rectifying K+ channels, Irk1 and Irk2, on the basolateral membrane of the Drosophila melanogaster main segment principal cell. Possible roles of Irk1 and Irk2 are the maintenance of the basolateral membrane potential or to allow the movement of K+ from the hemolymph into the principal cell during transepithelial K+ flux. Because flies eat a K+-rich diet, the existence of multiple mechanisms to allow principal cell K+ uptake, Irk1 and Irk2 as well as the Na+/K+-ATPase and NKCC, builds redundancy into the system for renal K+ excretion (Wu, 2015).

Early gene Broad complex plays a key role in regulating the immune response triggered by ecdysone in the Malpighian tubules of Drosophila melanogaster

In insects, humoral response to injury is accomplished by the production of antimicrobial peptides (AMPs) which are secreted in the hemolymph to eliminate the pathogen. Drosophila Malpighian tubules (MTs), however, are unique immune organs that show constitutive expression of AMPs even in unchallenged conditions and the onset of immune response is developmental stage dependent. Earlier reports have shown ecdysone positively regulates immune response after pathogenic challenge however, a robust response requires prior potentiation by the hormone. This study provides evidence to show that MTs do not require prior potentiation with ecdysone hormone for expression of AMPs and they respond to ecdysone very fast even without immune challenge, although the different AMPs Diptericin, Cecropin, Attacin, Drosocin show differential expression in response to ecdysone. Early gene Broad complex (BR-C) could be regulating the IMD pathway by activating Relish and physically interacting with it to activate AMPs expression. BR-C depletion from Malpighian tubules renders the flies susceptible to infection. It was also shown that in MTs ecdysone signaling is transduced by EcR-B1 and B2. In the absence of ecdysone signaling the IMD pathway associated genes are down-regulated and activation and translocation of transcription factor Relish is also affected (Verma, 2015).

Expression of polyQ aggregates in Malpighian tubules leads to degeneration in Drosophila melanogaster

Polyglutamine (polyQ) disorders are caused by expanded CAG (Glutamine) repeats in neurons in the brain. The expanded repeats are also expressed in the non-neuronal cells, however, their contribution to disease pathogenesis is not very well studied. This study expressed a stretch of 127 Glutamine repeats in Malpighian tubules (MTs) of Drosophila melanogaster as these tissues do not undergo ecdysone induced histolysis during larval to pupal transition at metamorphosis. Progressive degeneration, which is the hallmark of neurodegeneration was also observed in MTs. The mutant protein forms inclusion bodies in the nucleus resulting in expansion of the nucleus and affect chromatin organization which appear loose and open, eventually resulting in DNA fragmentation and blebbing. A virtual absence of tubule lumen was observed followed by functional abnormalities. As development progressed, severe abnormalities affecting pupal epithelial morphogenesis processes were observed resulting in complete lethality. Distribution of heterogeneous RNA binding protein (hnRNP), HRB87F, Wnt/wingless and JNK signaling and expression of Relish was also found to be affected. Expression of multi-drug resistance genes following polyQ expression was up regulated. The study gives an insight into the effects of polyQ aggregates in non-neuronal tissues (Yadav, 2015).

Segment-specific Ca transport by isolated Malpighian tubules of Drosophila melanogaster: A comparison of larval and adult stages

Haemolymph calcium homeostasis in insects is achieved through the regulation of calcium excretion by Malpighian tubules in two ways: (1) sequestration of calcium within biomineralized granules and (2) secretion of calcium in soluble form within the primary urine. Using the scanning ion-selective electrode technique (SIET), basolateral Ca2+ transport was measured at the distal, transitional, main and proximal tubular segments of anterior tubules isolated from both 3rd instar larvae and adults of Drosophila. Basolateral Ca2+ transport exceeded transepithelial secretion by 800-fold and 11-fold in anterior tubules of larvae and adults, respectively. The magnitude of Ca2+ fluxes across the distal tubule of larvae and adults were larger than fluxes across the downstream segments by 10 and 40 times, respectively, indicating a dominant role for the distal segment in whole animal Ca2+ regulation. Basolateral Ca2+ transport across distal tubules of Drosophila varied throughout the life cycle; Ca2+ was released by distal tubules of larvae, taken up by distal tubules of young adults and was released once again by tubules of adults 168h post-eclosion. In adults and larvae, SIET measurements revealed sites of both Ca2+ uptake and Ca2+ release across the basolateral surface of the distal segment of the same tubule, indicating that Ca2+ transport is bidirectional. Ca2+ uptake across the distal segment of tubules of young adults and Ca2+ release across the distal segment of tubules of older adults was also suggestive of reversible Ca2+ storage. These results suggest that the distal tubules of Drosophila are dynamic calcium stores which allow efficient haemolymph calcium regulation through active Ca2+ sequestration during periods of high dietary calcium intake and passive Ca2+ release during periods of calcium deficiency (Browne, 2016).

The corticotropin-releasing factor-like diuretic hormone 44 (DH) and kinin neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster

Malpighian tubules are critical organs for epithelial fluid transport and stress tolerance in insects, and are under neuroendocrine control by multiple neuropeptides secreted by identified neurons. This study demonstrates roles for CRF-like diuretic hormone 44 (DH44) and Leukokinin (Lk) in desiccation and starvation tolerance. Gene expression and labelled DH44 ligand binding data, as well as highly selective knockdowns and/or neuronal ablations of DH44 in neurons of the pars intercerebralis and DH44 receptor (DH44-R2) in Malpighian tubule principal cells, indicate that suppression of DH44 signalling improves desiccation tolerance of the intact fly. Leucokinin receptor (Lkr), is expressed in DH44 neurons as well as in stellate cells of the Malpighian tubules. Lkr knockdown in DH44-expressing neurons reduces Malpighian tubule-specific Lkr, suggesting interactions between DH44 and LK signalling pathways. Finally, although a role for DK in desiccation tolerance was not defined, a novel role was demonstrated for Malpighian tubule cell-specific Lkr in starvation tolerance. Starvation increases gene expression of epithelial LKR. Also, Malpighian tubule stellate cell-specific knockdown of LKR significantly reduced starvation tolerance, demonstrating a role for neuropeptide signalling during starvation stress (Cannell, 2016).

Characterization of a set of abdominal neuroendocrine cells that regulate stress physiology using colocalized diuretic peptides in Drosophila

Multiple neuropeptides are known to regulate water and ion balance in Drosophila melanogaster. Several of these peptides also have other functions in physiology and behavior. Examples are corticotropin-releasing factor-like diuretic hormone (diuretic hormone 44; DH44) and leucokinin (LK), both of which induce fluid secretion by Malpighian tubules (MTs), but also regulate stress responses, feeding, circadian activity and other behaviors. This study investigated the functional relations between the LK and DH44 signaling systems. DH44 and LK peptides are only colocalized in a set of abdominal neurosecretory cells (ABLKs). Targeted knockdown of each of these peptides in ABLKs leads to increased resistance to desiccation, starvation and ionic stress. Food ingestion is diminished by knockdown of DH44, but not LK, and water retention is increased by LK knockdown only. Thus, the two colocalized peptides display similar systemic actions, but differ with respect to regulation of feeding and body water retention. It was also demonstrated that DH44 and LK have additive effects on fluid secretion by MTs. It is likely that the colocalized peptides are coreleased from ABLKs into the circulation and act on the tubules where they target different cell types and signaling systems to regulate diuresis and stress tolerance. Additional targets seem to be specific for each of the two peptides and subserve regulation of feeding and water retention. These data suggest that the ABLKs and hormonal actions are sufficient for many of the known DH44 and LK functions, and that the remaining neurons in the CNS play other functional roles (Zandawala, 2017).

This study reveals that a portion of the LK-expressing neurosecretory cells (ABLKs) in abdominal ganglia co-express DH44, similar to earlier findings in the moth Manduca sexta, the locust Locusta migratoria, and blood-sucking bug Rhodnius prolixus. Colocalization of these peptides in multiple insect orders, including basal orders, suggests that this colocalization and the subsequent functional interaction between these signaling systems evolved early on during insect evolution. Since ABLKs are the sole neurons producing both peptides in Drosophila, it was possible to use GAL4 lines to knock down each of the two peptides in these cells only and thereby isolate the contribution of the ABLKs to the physiology. This enabled establishing that these neuroendocrine cells are sufficient for many of the functions assigned to DH44 and LK and therefore these functions are hormonally mediated. In contrast, earlier studies were based upon altering peptide levels or activity in entire populations of DH44 and LK neurons. Also, this study showed that the LK-GAL4 driver includes a set of ectopic brain cells (ipc-1) that do not express LK, but another peptide ITP. The ipc-1 neurons produce sNPF and tachykinin in addition to ITP and have been found to regulate stress responses. This means that in earlier studies, where the LK-GAL4 line was used to inactivate or activate neurons, additional phenotypes are likely to have arisen. Using the current approach, only ABLK neurons were targetted, it was found that both DH44-RNAi and Lk-RNAi in these cells increase resistance to desiccation, starvation, and ionic stress. This suggests that diminishing the release of these two peptides from ABLKs is sufficient for this phenotype to occur. However, food intake is not affected by LK knockdown in ABLKs, whereas DH44 knockdown diminishes feeding, and conversely the knockdown of LK in ABLKs results in increased body water content that is not seen after DH44-RNAi. Thus, the two colocalized peptides appear to display similar systemic actions, but differ with respect to feeding and water retention. When knocking down LK in all LK neurons, a very similar set of effects was obtained to those when only the ABLKs were targeted, indicating that in the assays that were performed in this study the other two sets of LK neurons (LHLK and SELK) played a minimal role (Zandawala, 2017).

Interestingly, knockdown of DH44 in ABLKs increases resistance to desiccation and decreases feeding, but these effects were not seen when DH44 was diminished in all DH44 neurons. This is consistent with previous work where inactivation or activation of DH44 neurons had no effect on food intake. Perhaps, the effects seen following ABLK manipulations could be compensated by action of the six DH44-expressing MNCs in the brain. Similarly, reduction of LK in ABLKs causes a slight increase in time of recovery from chill coma, but this is not noted after global knockdown of LK. This minor difference could possibly be attributed to the strength of the two GAL4 driver lines used and, thus, the efficiency of LK knockdown in ABLKs (Zandawala, 2017).

This study also demonstrated that DH44 and LK have additive effects on fluid secretion in MTs. It is likely that these two colocalized peptides are released together and act on the MTs where they target different cell types, receptors, signaling systems, and effectors to regulate fluid secretion. The action of these peptides on the MTs may also in part be responsible for the regulation of stress responses seen in the assays, as shown earlier for CAPA peptide and DH44. It is, however, not clear whether the altered food intake and water retention after DH44 and LK knockdown, respectively, are direct actions on target tissues or indirect effects caused by altered water and ion regulation in the fly (Zandawala, 2017).

Not only do the ABLKs produce two diuretic hormones, but they also seem to be under tight neuronal and hormonal control. Receptors for several neurotransmitters and peptides have been identified on these cells in adults: the serotonin receptor 5-HT1B, LK receptor (LkR), and the insulin receptor dInR. Knockdown of the 5-HT1B receptor in ABLK neurons diminished LK expression, increased desiccation resistance, and diminished food intake, but manipulations of dInR expression in these cells generated no changes in physiology in the tests performed. In larvae, all ABLKs colocalize LK and DH44, and several receptors have been detected in addition to 5-HT1B and dInR [29], namely RYamide receptor, SIFamide receptor, and the ecdysis-triggering hormone (ETH) receptor ETHR-A. However, the expression of these receptors on adult ABLKs has so far not been investigated. Interestingly, the functions of ABLKs in larvae, studied so far, seem to be primarily related to regulating muscle activity and ecdysis motor patterns. The 5-HT-1B receptor on ABLKs was shown to modulate locomotor turning behavior, whereas ETH-mediated activation of ETHR-A on ABLKs initiates the pre-ecdysis motor activity. In this context, it is worth noting that during metamorphosis six to eight novel ABLKs differentiate anteriorly in the abdominal ganglia, and these are the ones that display the strongest expression of DH44. In adult flies, the ABLKs are neurosecretory cells with restricted arborizations in the CNS, but widespread axon terminations along the abdominal body wall and in the lateral heart nerves, whereas in larvae the same cells send axons that terminate on segmental abdominal muscles, muscle 8. It is not yet known whether larval ABLKs are involved in the regulation of diuresis and other related physiological functions in vivo, but certainly larval functions in locomotion and ecdysis behavior are specific to that developmental stage. Thus, it seems that there is a developmental switch of function in this set of peptidergic neuroendocrine cells (Zandawala, 2017).

In summary, this study shows that a set of abdominal neuroendocrine cells, ABLKs, co-expressing DH44 and LK, are sufficient for regulation of resistance to desiccation, starvation and ionic stress, as well as modulating feeding and water content in the body. These ABLKs represent a subset of neurons that express DH44 and LK, and the functions of the remaining neurons are yet to be determined (Zandawala, 2017).

The orphan pentameric ligand-gated ion channel pHCl-2 is gated by pH and regulates fluid secretion in Drosophila Malpighian tubules

Pentameric ligand-gated ion channels (pLGICs) constitute a large protein superfamily in metazoa whose role as neurotransmitter receptors mediating rapid, ionotropic synaptic transmission has been extensively studied. Although the vast majority of pLGICs appear to be neurotransmitter receptors, the identification of pLGICs in non-neuronal tissues and homologous pLGIC-like proteins in prokaryotes points to biological functions, possibly ancestral, that are independent of neuronal signaling. This study reports the molecular and physiological characterization of a highly divergent, orphan pLGIC subunit, pHCl-2 (CG11340), in Drosophila melanogaster. pHCl-2 forms a channel that is insensitive to a wide array of neurotransmitters, but is instead gated by changes in extracellular pH. pHCl-2 is expressed in the Malpighian tubules, which are non-innervated renal-type secretory tissues. pHCl-2 is localized to the apical membrane of the epithelial principal cells of the tubules, and loss of pHCl-2 reduces urine production during diuresis. These data implicate pHCl-2 as an important source of chloride conductance required for proper urine production, highlighting a novel role for pLGICs in epithelial tissues regulating fluid secretion and osmotic homeostasis (Feingold, 2016).

The orphan pentameric ligand-gated ion channel pHCl-2 is gated by pH and regulates fluid secretion in Drosophila Malpighian tubules

Pentameric ligand-gated ion channels (pLGICs) constitute a large protein superfamily in metazoa whose role as neurotransmitter receptors mediating rapid, ionotropic synaptic transmission has been extensively studied. Although the vast majority of pLGICs appear to be neurotransmitter receptors, the identification of pLGICs in non-neuronal tissues and homologous pLGIC-like proteins in prokaryotes points to biological functions, possibly ancestral, that are independent of neuronal signalling. This study reports the molecular and physiological characterization of a highly divergent, orphan pLGIC subunit encoded by the pHCl-2 (CG11340/Hodor) gene, in Drosophila melanogaster. pHCl-2 forms a channel that is insensitive to a wide array of neurotransmitters, but is instead gated by changes in extracellular pH. pHCl-2 is expressed in the Malpighian tubules, which are non-innervated renal-type secretory tissues. This study demonstrates that pHCl-2 is localized to the apical membrane of the epithelial principal cells of the tubules and that loss of pHCl-2 reduces urine production during diuresis. The data implicate pHCl-2 as an important source of chloride conductance required for proper urine production, highlighting a novel role for pLGICs in epithelial tissues regulating fluid secretion and osmotic homeostasis (Feingold, 2016).

Sequencing of invertebrate genomes has led to the recognition that the pLGIC subunit superfamily is much larger and more diverse than was previously realized based on work in vertebrate nervous systems. Vertebrate genomes encode five main classes of pLGICs that have been defined based on ligand specificity: the cation-selective nicotinic acetylcholine receptors, serotonin 5HT3 receptors and zinc-activated receptors and the anion-selective GABA and glycine receptors. Invertebrate genomes, in contrast, encode a greater assortment of channel types with a wider range of ligand specificities and ligand-ion combinations than those found in vertebrates (Dent, 2006). In addition to the nicotinic acetylcholine and GABA receptors found in vertebrates, invertebrate genomes encode anion-selective acetylcholine, glutamate, serotonin, dopamine, tyramine and pH channels, as well as cation-selective GABA and proton channels. Moreover, multiple putative invertebrate pLGICs have been identified that cannot be assigned to any neurotransmitter family based on sequence homology (Feingold, 2016).

The biological functions of pLGICs are also likely to be much more diverse than has generally been appreciated. For instance, the cation-selective, proton-activated PBO-5,-6 channel in Caenorhabditis elegans mediates an intercellular pH signal that stimulates muscle contraction. The proton signal is generated by a proton pump in the intestine rather than by synaptic release from neurons. The function of the Drosophila melanogaster pHCl channel, which is open under alkaline conditions, is not known but its expression in the nervous system and the hindgut suggests non-canonical roles in signalling and/or ion regulation. Finally, the discovery of the proton-gated channel from the cyanobacterium Gloeobacter violaceus suggests that pLGICs originally evolved to regulate ion homeostasis in response to environmental changes (Feingold, 2016).

This study shows that CG11340, a putative pLGIC subunit in D. melanogaster which this study has named pHCl-2, forms a pH-gated chloride channel that is expressed in the Malpighian tubules, which are non-innervated secretory tissues. pHCl-2 channels are localized to the apical (lumen-facing) membrane of Malpighian tubule principal cells, precluding a role in responding to humoral signals originating in the haemolymph. Evidence is presented that, instead, pHCl-2 regulates fluid secretion by the Malpighian tubules in response to the pH of urine by controlling chloride counter-ion availability. Based on these data, a new role is proposed for pLGICs in ion homeostasis and implicate pHCl-2 in a previously unrecognized mechanism regulating urine secretion, a mechanism that will enrich current models of insect secretion (Feingold, 2016).

Previous phylogenetic analysis identified pHCl-2 as a member of an arthropod-specific clade of divergent orphan Cys-loop pLGICs. In Drosophila, pHCl-2 groups with two other orphan pLGIC subunits, Secretory chloride channel and CG6927, which together most closely resemble the Drosophila pH-sensitive chloride channel (pHCl) and the pH-sensitive chloride channel in S. scabiei (SsCl). Clades of channel subunits orthologous to the clade of subunits defined by pHCl-2, CG7589 and CG6927 have been reported in other insects such as Apis mellifera, A. aegypti, Nasonia vitripennis and Tribolium castaneums and in non-insect arthropods such as the deer tick Ixodes scapularis, but not in nematodes, molluscs, annelids or chordates (Dent, 2006). The pH response of pHCl-2 closely resembles that of the two other characterized pH-sensitive arthropod pLGICs, Drosophila pHCl and Sarcoptes SsCl; both pHCl and SsCl are inhibited by protons and are increasingly activated by a rise in alkalinity, exhibiting half-maximal activity at pH 7.33±0.16 and 7.55±0.06, respectively. In contrast, the other well-characterized pH-gated pLGICs identified to date, the PBO-5,-6 heteromeric cation channel in C. elegans and GLIC from cyanobacterium G. violaceus, are inhibited by alkaline conditions, and instead, are increasingly activated by a rise in proton concentration, displaying half-maximal responses at pH 6.83±0.01 and 5.1±0.1, respectively. While this study has shown that standard neurotransmitters do not gate pHCl-2 channels expressed in oocytes, it cannot be ruled out that pHCl-2 channels are sensitive to other ligands that might be found in gastric fluid or urine (Feingold, 2016).

Fluid secretion in the Malpighian tubules is mediated by active transepithelial ion transport, which establishes the osmotic gradient necessary for the formation of the primary urine. This active transport is powered by an apically localized, electrogenic H+-ATPase that pumps protons into the tubule lumen and generates a net positive apical membrane potential. The proton gradient is used to drive alkali metal cation/H+ antiporters, which replace luminal protons with sodium and potassium. Chloride enters the lumen passively, following its electrochemical gradient, and is a critical regulator of secretion because, in the absence of this negative counter-ion, cation transport into the lumen would result in an increasingly positive apical membrane potential, which would oppose transport by the ATPase before a significant osmotic gradient has formed (Feingold, 2016).

A role for pHCl-2 as an important source of chloride conductance in the Malpighian tubules is supported by the observation that urine production is affected in pHCl-2 mutants. Loss of pHCl-2 did not obviously impair fluid secretion in unstimulated Malpighian tubules, consistent with alternative routes of chloride flow into the lumen, either via known channels in the stellate cells or through a putative paracellular route via septate junctions. However, upon stimulation of the Malpighian tubules with cAMP, a second messenger that enhances the output of the H+ ATPase, pHCl-2 mutant Malpighian tubules showed a significantly reduced diuretic response compared with wild-type. These data suggest that under conditions of enhanced cation transport into the lumen, pHCl-2 provides a necessary source of chloride conductance in the principal cells, without which maximal secretion rates are not achieved (Feingold, 2016).

The expression of pHCl-2 in the apical membrane of principal cells, together with electrophysiology data demonstrating that pHCl-2 forms a pH-sensitive channel, suggests that the pHCl-2-mediated chloride conductance may be regulated by the pH of the luminal environment. Luminal pH is strongly influenced by the relative activities of the proton ATPase and the cation/H+ antiporter, which transport protons into and out of the tubule lumen, respectively, and it is proposed that the gating of pHCl-2 by pH may reveal a homeostatic mechanism that maintains an appropriate balance between these two cation transporters. Under conditions where the activity of the ATPase is high relative to the antiporter, the pH of the lumen would drop because the rate of proton transport into the lumen would exceed proton removal by the antiporter. As secondary active transport of sodium and potassium into the tubule lumen by the antiporter is coupled to active proton transport, an accumulation of excess protons in the tubule lumen would reflect a decrease in the energy efficiency of sodium and potassium secretion. It is proposed that the presence of a pH-sensitive chloride channel like pHCl-2 would counteract such an imbalance. If the pH of the lumen were too acidic, pHCl-2 would be antagonized, chloride permeability would become rate limiting for secretion and the increasingly positive apical membrane potential generated by the electrogenic ATPase would oppose further transport of protons into the lumen. The proton gradient, however, would continue to drive the antiporter, increasing luminal pH, which would, in turn, increase pHCl-2 conductance, and decrease the electrical barrier opposing the ATPase, thus re-establishing homeostasis. pHCl-2 would therefore serve as a 'brake' on the H+-ATPase, providing an upper limit to which the ATPase can operate relative to the antiporter, thus minimizing the expenditure of ATP under conditions where protons are not being put to metabolically efficient use (Feingold, 2016).

If pHCl-2 is in fact inhibited by acidic luminal pH, then one might predict that the stimulatory effects of cAMP on the H+-ATPase would lead to inhibition of pHCl-2-mediated chloride conductance. cAMP signalling in the Malpighian tubules leads to the secretion of a more acidic urine, with pH decreasing from 7.8 to 7.4, consistent with an increase in proton transport by the ATPase. Based on its pH sensitivity in oocytes, the chloride conductance of pHCl-2 channels should decrease ∼80% in response to cAMP, and the normalized chloride conductance-limited secretion rate increase should be significantly smaller in the presence of a pH-sensitive channel (i.e. wild-type) than in its absence (i.e. pHCl-2 knockout). Yet, the opposite was observed: the normalized response of secretion to cAMP in the pHCl-2 knockout was smaller than in wild-type, indicating that a simple physiological model does not account for the pHCl-2 phenotype. Instead, the pHCl-2 knockout may have indirect feedback effects on lumen pH or membrane potential, for example by directly affecting the ability of the H+-ATPase to respond to cAMP (Feingold, 2016).

Another effect of down-regulating pHCl-2 may be to increase the relative chloride current through stellate cell chloride channels. Decreased luminal pH that inhibits pHCl-2, resulting in a rise in apical membrane potential, should increase the driving force for chloride through apically localized chloride channels in stellate cells, thus increasing the relative contribution of stellate cells to apical chloride current. The increased activity of stellate cell chloride channels could in turn drive anion exchange via the basal membrane-localized Cl-/HCO3- transporter in stellate cells, thereby alkalinizing the haemolymph. Interestingly, pharmacological block of the Cl-/HCO3- transporter in A. aegypti has little effect on resting secretion rate but inhibits stimulated secretion, similar to the pHCl-2 mutant phenotype (Piermarini, 2010). As bicarbonate (HCO3-) is thought to be produced by carbonic anhydrase in the principal cells and enter the stellate cells through intracellular junctions, the effect of the pHCl-2 mutant on stimulated transport may be an indirect effect on the Cl-/HCO3- balance in principal cells feeding back and affecting chloride transport through stellate cells (Feingold, 2016).

Recent work demonstrated that pHCl-2 is expressed in the copper cells of the midgut and influences sensitivity to dietary copper: flies deficient for pHCl-2 display increased copper tolerance, whereas the opposite is observed when pHCl-2 is over-expressed. Copper cells are the principal site of acid secretion in the midgut, and, like the Malpighian tubules, are thought to transport protons via apically localized V-type H+-ATPases. While a clear relationship between H+-ATPase output and copper uptake has yet to be elucidated, it has been shown that copper uptake is impaired in flies whose copper cells are deficient in acid secretion, and that the copper cell midgut region is less acidic following copper feeding. Similar to the proposed model in the Malpighian tubules, pHCl-2 may influence ATPase output in copper cells by regulating chloride counter-ion availability, which, in turn, could affect the rate of copper uptake. Curiously, the role of pHCl-2 both in the Malpighian tubules and in the copper cells points to a somewhat counter-intuitive physiological model in which the activity of an alkaline-gated chloride channel provides the necessary counter-current for an acid-secreting transporter (Feingold, 2016).

Demonstration that pHCl-2 regulates secretion in the Malpighian tubules, a polarized epithelial tissue that is not directly associated with the nervous system, underscores the ability of pLGICs to function in a wide array of biological contexts beyond their canonical function in the nervous system. Previous work, for instance the discovery of pLGIC-like proteins in bacteria, has also hinted at possible roles for this ion channel superfamily that are entirely independent of neuronal signalling. There are also examples of pLGICs with well-characterized roles in the nervous system that appear to function in non-neuronal tissues. For example, the mammalian immune system is rich in pLGICs, including nicotinic acetylcholine (nACh), GABAA and glycine receptors. While such non-neuronal roles of the GABA and glycine receptors are poorly understood, the α7 nicotinic acetylcholine receptor (nAChR) is expressed in macrophages where it regulates tumour necrosis factor-α in response to acetylcholine released from spleen lymphocytes. nAChR function has also been reported in bronchial epithelia, where nAChRs expressed on the apical membrane respond to non-neuronal autocrine/paracrine ACh release to regulate chloride permeability through the cystic fibrosis transmembrane conductance regulator (CFTR) channel. Non-neural roles for nAChRs have also been identified in vascular endothelia and in keratinocytes (Grando, 1995) (Feingold, 2016).

pHCl-2 represents an extreme in the evolution of pLGIC functions. Like epithelial nAChRs, it is expressed in non-innervated tissues, but pHCl-2 is unique in that it is not obviously responding to an autocrine/paracrine signal. Whether pHCl-2 has an additional function in the nervous system remains unclear; microarray data suggest that it is not expressed in the head, brain and eyes of adult flies, or in the larval central nervous system, but this broad survey would not necessarily detect expression in a small subset of neurons. Additionally, althoug pHCl-2 localizes to the apical membrane of principal cells, the possibility cannot be ruled out that it functions in apically enriched endosomal vesicles to regulate secretion, similar to CUP-4 in C. elegans, which is localized to endosomes and is necessary for endosomal trafficking, although its activating ligand, if any, is unknown. Nevertheless, whether it acts in endosomes or the apical membrane, characterization of pHCl-2 illustrates the remarkable ability of the pLGICs to evolve diverse physiological functions (Feingold, 2016).

Drosophila imaginal disc growth factor 2 is a trophic factor involved in energy balance, detoxification, and innate immunity

Drosophila imaginal disc growth factor 2 (IDGF2) is a member of chitinase-like protein family (CLPs) able to induce the proliferation of imaginal disc cells in vitro. This study characterized physiological concentrations and expression of IDGF2 in vivo as well as its impact on the viability and transcriptional profile of Drosophila cells in vitro. IDGF2 was shown to be independent of insulin and protects cells from death caused by serum deprivation, toxicity of xenobiotics or high concentrations of extracellular adenosine (Ado) and deoxyadenosine (dAdo). Transcriptional profiling suggested that such cytoprotection is connected with the induction of genes involved in energy metabolism, detoxification and innate immunity. IDGF2 was hown to be an abundant haemolymph component, which is further induced by injury in larval stages. The highest IDGF2 accumulation was found at garland and pericardial nephrocytes supporting its role in organismal defence and detoxification. These findings provide evidence that IDGF2 is an important trophic factor promoting cellular and organismal survival (Broz, 2017).

Hsp27 over expression protect against cadmium induced nephrotoxicity in Drosophila melanogaster

Cadmium (Cd) exposure to the animals including humans is reported as nephrotoxic compounds i.e., disturbing redox status (increase oxidative stress), mitochondrial dysfunction, renal cell death and altered transporters in the renal system. Hsp27 (a small heat shock protein) has been shown as one of the modulators in the renal dysfunction and increased against the Cd induced toxicity. However, no studies are reported on the genetic modulation of stress protein against the Cd-induced nephrotoxicity. The current study aimed to examine the protective role of hsp27 overexpression against the Cd-induced nephrotoxicity using Drosophila melanogaster as an animal model. D. melanogaster renal system includes nephrocytes and Malpighian tubules (MTs) that show the functional similarity with mammalian kidney nephron. Overexpression of the hsp27 was found to reduce the Cd induced oxidative stress, rescue cell death in MTs of Cd exposed D. melanogaster larvae. The rescued GSH (Glutathione) level, NADPH level and glucose 6 phosphate dehydrogenase (G6PD) activity were also observed in the MTs of the Cd exposed organism. Function (efflux activity and fluid secretion rate) of the MTs was restored in Cd exposed hsp27 overexpressed larvae. Further, results were confirmed by restored brush border microvilli density and reduced uric acid level. Tissue specific knockdown of hsp27 developed Cd like phenotypes in MTs and the phenotypes enhanced in Cd exposed condition. The present study clearly shows the role of hsp27 overexpression in restoration of the MTs function and protection against the Cd induced renal toxicity (Saini, 2023).

Identification of multiple functional receptors for tyramine on an insect secretory epithelium

The biogenic amine tyramine (TA) regulates many aspects of invertebrate physiology and development. Although three TA receptor subtypes have been identified (TAR1-3), specific receptors have not been linked to physiological responses in native tissue. In the Malpighian (renal) tubule of Drosophila melanogaster, TA activates a transepithelial chloride conductance, resulting in diuresis and depolarization of the transepithelial potential. In the current work, mutation or RNAi-mediated knockdown in the stellate cells of the tubule of TAR2 (tyrR, CG7431) resulted in a dramatic reduction, but not elimination, of the TA-mediated depolarization. Mutation or knockdown of TAR3 (tyrRII, CG16766) had no effect. However, deletion of both genes, or knockdown of TAR3 on a TAR2 mutant background, eliminated the TA responses. Thus while TAR2 is responsible for the majority of the TA sensitivity of the tubule, TAR3 also contributes to the response. Knockdown or mutation of TAR2 also eliminated the response of tubules to the related amine octopamine (OA), indicating that OA can activate TAR2. This finding contrasts to reports that heterologously expressed TAR2 is highly selective for TA over OA. This is the first report of TA receptor function in a native tissue and indicates unexpected complexity in the physiology of the Malpighian tubule (Zhang, 2017).

Cold tolerance of Drosophila species is tightly linked to epithelial K+ transport capacity of the Malpighian tubules and rectal pads

Insect chill tolerance is strongly associated with the ability to maintain ion and water homeostasis during cold exposure. Maintenance of K+ balance is particularly important due to its role in setting the cell membrane potential that is involved in many aspects of cellular function and viability. In most insects, K+ balance is maintained through secretion at the Malpighian tubules balancing reabsorption from the hindgut and passive leak arising from the gut lumen. This study examined K+ flux across the Malpighian tubules and the rectal pads in the hindgut in five Drosophila species that differ in cold tolerance. Chill tolerant species were found to be better at maintaining K+ secretion and suppressing reabsorption during cold exposure. In contrast, chill susceptible species exhibited large reductions in secretion with no change, or a paradoxical increase, in K+ reabsorption. Using an assay to measure paracellular leak, chill susceptible species were found to experience a large increase in leak during cold exposure, which could explain the increased K+ reabsorption found in these species. The data therefore strongly support the hypothesis that cold tolerant Drosophila species are better at maintaining K+ homeostasis through an increased ability to maintain K+ secretion rates and through reduced leakage of K+ towards the hemolymph. These adaptations are manifested both at the Malpighian tubule and at the rectal pads in the hindgut and ensure that cold tolerant species experience less perturbation of K+ homeostasis during cold stress (Andersen, 2017).

CRISPR-induced null alleles show that Frost protects Drosophila melanogaster reproduction after cold exposure

The ability to survive and reproduce after cold exposure is important in all kingdoms of life. However, even in a sophisticated genetic model system like Drosophila melanogaster, few genes have been identified as functioning in cold tolerance. The accumulation of the Frost (Fst) gene transcript increases after cold exposure, making it a good candidate for a gene that has a role in cold tolerance. Despite extensive RNAi knockdown analysis, no role in cold tolerance has been assigned to Fst. CRISPR is an effective technique for completely knocking down genes, and is less likely to produce off-target effects than GAL4-UAS RNAi systems. CRISPR-mediated homologous recombination was used to generate Fst-null alleles, and these Fst alleles uncovered a requirement for FST protein in maintaining female fecundity following cold exposure. However, FST does not have a direct role in survival following cold exposure. FST mRNA accumulates in the Malpighian tubules, and the FST protein is a highly disordered protein with a putative signal peptide for export from the cell. Future work is needed to determine whether FST is exported from the Malpighian tubules and directly interacts with female reproductive tissues post-cold exposure, or whether it is required for other repair/recovery functions that indirectly alter energy allocation to reproduction (Newman, 2017).

Functional plasticity of the gut and the Malpighian tubules underlies cold acclimation and mitigates cold-induced hyperkalemia in Drosophila melanogaster

At low temperatures, Drosophila, like most insects, lose the ability to regulate ion and water balance across the gut epithelia, which can lead to a lethal increase of [K(+)] in the hemolymph (hyperkalemia). Cold-acclimation, the physiological response to a prior low temperature exposure, can mitigate or entirely prevent these ion imbalances, but the physiological mechanisms that facilitate this process are not well understood. This study tested whether plasticity in the ionoregulatory physiology of the gut and Malpighian tubules of Drosophila may aid in preserving ion homeostasis in the cold. Upon adult emergence, D. melanogaster females were subjected to seven days at warm (25 ° C) or cold (10 ° C) acclimation conditions. The cold acclimated flies had a lower critical thermal minimum (CTmin), recovered from chill coma more quickly, and better maintained hemolymph K(+) balance in the cold. The improvements in chill tolerance coincided with increased Malpighian tubule fluid secretion and better maintenance of K(+) secretion rates in the cold, as well as reduced rectal K(+) reabsorption in cold-acclimated flies. To test whether modulation of ion-motive ATPases, the main drivers of epithelial transport in the alimentary canal, mediate these changes, the activities were measured of Na(+)-K(+)-ATPase and V-type H(+)-ATPase at the Malpighian tubules, midgut, and hindgut. Na(+)/K(+)-ATPase and V-type H(+)-ATPase activities were lower in the midgut and the Malpighian tubules of cold-acclimated flies, but unchanged in the hindgut of cold acclimated flies, and were not predictive of the observed alterations in K(+) transport. These results suggest that modification of Malpighian tubule and gut ion and water transport likely prevents cold-induced hyperkalemia in cold-acclimated flies and that this process is not directly related to the activities of the main drivers of ion transport in these organs, Na(+)/K(+)- and V-type H(+)-ATPases (Yerushalmi, 2018).

Separate roles of PKA and EPAC in renal function unraveled by the optogenetic control of cAMP levels in vivo

Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates a variety of essential processes in diverse cell types, functioning via cAMP-dependent effectors such as protein kinase A (PKA) and/or exchange proteins directly activated by cAMP (EPAC). In an intact tissue it is difficult to separate the contribution of each cAMP effector in a particular cell type using genetic or pharmacological approaches alone. This study, therefore, utilized optogenetics to overcome the difficulties associated with examining a multicellular tissue. The transgenic photoactive adenylyl cyclase bPAC can be activated to rapidly and reversibly generate cAMP pulses in a cell-type-specific manner. This optogenetic approach to cAMP manipulation was validated in vivo using GAL4-driven UAS-bPAC in a simple epithelium, the Drosophila renal (Malpighian) tubules. As bPAC was expressed under the control of cell-type-specific promoters, each cAMP signal could be directed to either the stellate or principal cells, the two major cell types of the Drosophila renal tubule. By combining the bPAC transgene with genetic and pharmacological manipulation of either PKA or EPAC it was possible to investigate the functional impact of PKA and EPAC independently of each other. The results of this investigation suggest that both PKA and EPAC are involved in cAMP sensing, but are engaged in very different downstream physiological functions in each cell type: PKA is necessary for basal secretion in principal cells only, and for stimulated fluid secretion in stellate cells only. By contrast, EPAC is important in stimulated fluid secretion in both cell types. It is proposed that such optogenetic control of cellular cAMP levels can be applied to other systems, for example the heart or the central nervous system, to investigate the physiological impact of cAMP-dependent signaling pathways with unprecedented precision (Efetova, 2013).

This study has pioneered the use of bPAC, a photoactive adenylyl cyclase, as an optogenetic tool to distinguish between the functions of alternative cAMP effectors in the regulation of a physiological process in vivo. To validate bPAC as an in vivo tool Drosophila renal tubules were used to confirm the bPAC transgene could be stimulated with blue light to generate cAMP signals in a cell-type-specific manner. This optogenetic approach was combined with standard techniques that targeted PKA or EPAC to resolve the complex regulatory network of discrete cAMP pathways involved in the control of fluid secretion (Efetova, 2013).

Primary urine is generated within the main segment of the Malpighian tubules, where the principal cells establish an electrochemical gradient that provides the driving force for fluid secretion, by actively transporting potassium from the basolateral to the apical surface via a defined array of ion transporters. In parallel, the stellate cells control the anion shunt conductance and water flux of the tubules, via the action of tightly regulated aquaporins and chloride channels (Efetova, 2013).

As revealed by the current analysis, two distinct cAMP pathways are deployed within the principal cells to sustain fluid secretion: firstly, the basal principal cell PKA pathway, which regulates the rate of basal fluid secretion; and secondly the stimulatory principal cell EPAC pathway, which stimulates fluid secretion above basal levels in a cAMP-dependent manner. Manipulation of EPAC activity altered stimulated secretion but not basal secretion, and manipulation of PKA altered basal secretion but not stimulated secretion. In this respect, the two principal cell secretory control pathways appear to be independent of one another (Efetova, 2013).

Could these downstream pathways be controlled independently in vivo, through a single second messenger? While imposed cAMP signals feeding into each pathway could be generated by activation of the bPAC transgene with a defined light intensity, in vivo the neuropeptides DH44, related to corticotropin releasing factor (CRF), and DH31, related to calcitonin/calcitonin gene-related peptide (CGRP), both increase fluid secretion by raising cAMP in the principal cells. However, there is evidence in other insects that these two neuropeptides might have distinct downstream effects; in the related malarial mosquito Anopheles gambiae, DH31, but not DH44, acts as a natriuretic peptide by increasing basolateral Na+ conductance. Moreover, DH31 and DH44 have an additive stimulatory effect on fluid secretion, suggesting that they target different transport processes. Cellular association of specific GPCRs with either PKA or EPAC might well account for the different outputs observed from each GPCR. Another tempting possibility involves a class of soluble adenylyl cyclases (sACs) that are localized near the apical membrane and activated by cellular ionic concentrations rather than GPCRs, as seen in the mammalian kidney (Efetova, 2013).

The apical plasma membrane H+ V-ATPase is the driving force for ion transport in the principal cells, and is therefore an obvious downstream target for stimulatory or inhibitory cAMP signals. Formation of a functional V-ATPase complex requires PKA-dependent phosphorylation, which prevents the complex from disassembly. In blowfly salivary gland (another insect epithelium energized by a V-ATPase), cAMP has been shown to promote assembly of the V-ATPase complex. However, V-ATPase assembly - and thus activation - has also been reported via EPAC signaling within the rat renal collecting duct. By contrast, intracellular calcium has been shown to activate tubule H+ V-ATPase by directly activating mitochondria, and so increasing the ATP supply. In this complex field, optogenetic control of cellular cAMP levels in the principal cells will provide a valuable analytical tool to investigate such issues (Efetova, 2013).

A surprising feature of cAMP-dependent fluid secretion is the complete inhibition (below basal) observed with millimolar levels of cell-permeable 8-Br-cAMP, or at very high illumination levels in bPAC-transgenic tubules. Through targeted use of bPAC this effect was localized to the principal cells, and formally established an inhibitory principal cell cAMP signal. It is likely that these manipulations bring intracellular cAMP levels to abnormally high levels that are unlikely to be reached in vivo, where the resting intracellular cAMP concentration is typically in the range 0.1-1.5 μM; nonetheless, there is a real effect to be explained. At present, it is only possible to speculate on the underlying mechanisms, but it is likely that saturation or desensitization of some component of the signaling pathway is occurring; or that there is cross-talk to, for example calcium signaling via cyclic nucleotide gated calcium channels, which are known to play a role in tubule (Efetova, 2013).

In the stellate cells this study identified a stimulatory stellate cAMP signal that stimulates fluid secretion via PKA, with moderate illuminations of bPAC. In contrast, high illuminations return fluid secretion to the baseline level, suggesting that dual modulation, i.e., augmentation with low levels and inhibition with high levels of cAMP, is a common theme within the stellate and principal cells. However, further experiments will be required to substantiate this speculation (Efetova, 2013).

Interestingly, the stellate cells are known to be controlled by leucokinin, which acts though calcium, rather than cAMP, so no extracellular ligand for the stellate cAMP pathway is presently known. Tyramine has also been shown to act on stellate cells, but its second messenger is yet to be established (Efetova, 2013).

Selective elevation of cAMP in stellate cells shows that both PKA and EPAC can stimulate fluid secretion. However, these pathways do not act in parallel in the stellate cells; PKA must be upstream of EPAC, because RNAi knockdown of DC0 in stellate cells abolishes the ability of bPAC to stimulate fluid secretion. In contrast, EPAC is sufficient for secretion when activated in a cAMP-independent manner via the EPAC-specific agonist 8-pCPT-2'-O-Me-cAMP. Therefore, cAMP is likely to signal through PKA to EPAC. In turn, EPAC levels are likely to be rate limiting, as stellate-specific overexpression of epac enormously enhanced secretion (Efetova, 2013).

This study has established the use of photoactive adenylyl cyclases (PACs) as a potent tool for investigating organotypic physiological processes in vivo. A unique advantage of this optogenetic transgene is that it acts as a 'Trojan horse', allowing cell-type-specific control of cellular cAMP levels with temporal and spatial precision, through simple blue light illumination. It is this feature that has allowed deconstruction of the complex regulatory network of cAMP pathways involved in fluid secretion control, and to assign function within the Drosophila renal (Malpighian) tubule. This experimental approach can easily be adapted to other physiological preparations, for example the central nervous system or the cardiac system, to address similar physiological questions (Efetova, 2013).

Further improvements to bPAC could be achieved; for example it would be beneficial to further reduce the residual dark activity, which must be considered during experimental analysis. Although functional imaging of cAMP has been achieved, further development of this complementary technology would be advantageous for studying complex cellular signaling networks. Another feature of light-induced cAMP signals is that, as bPAC is cytoplasmic, the elevation of cAMP is uniform across the cell. In contrast, naturally occurring cAMP is often unevenly distributed on a sub-cellular level, and concentrated in local microdomains. In addition to the compartmentalization of cAMP, the cAMP sensors PKA and EPAC are also spatially regulated by binding to scaffolding proteins, such as A-kinase anchoring proteins (AKAPs). In future, it should be possible to localize genetically encoded PACs to specific subcellular domains, and embark on a new era of precision optogenetics (Efetova, 2013).

Epithelial function in the Drosophila Malpighian tubule: An in vivo renal model

The insect renal (Malpighian) tubule has long been a model system for the study of fluid secretion and its neurohormonal control, as well as studies on ion transport mechanisms. To extend these studies beyond the boundaries of classical physiology, a molecular genetic approach together with the 'omics technologies is required. To achieve this in any vertebrate transporting epithelium remains a daunting task, as the genetic tools available are still relatively unsophisticated. Drosophila melanogaster, however, is an outstanding model organism for molecular genetics. This study describes a technique for fluid secretion assays in the D. melanogaster equivalent of the kidney nephron. The development of this first physiological assay for a Drosophila epithelium, allowing combined approaches of integrative physiology and functional genomics, has now provided biologists with an entirely new model system, the Drosophila Malpighian tubule, which is utilized in multiple fields as diverse as kidney disease research and development of new modes of pest insect control (Davies, 2019).

The Septate Junction Protein Tetraspanin 2A is critical to the Structure and Function of Malpighian tubules in Drosophila melanogaster

Tetraspanin-2A (Tsp2A) is an integral membrane protein of smooth septate junctions in Drosophila melanogaster. To elucidate its structural and functional roles in Malpighian tubules, this study used the GAL4/UAS system to selectively knockdown Tsp2A in principal cells of the tubule. Tsp2A localizes to smooth septate junctions (sSJ) in Malpighian tubules in a complex shared with partner proteins Snakeskin (Ssk), Mesh and Discs Large (Dlg). Knockdown of Tsp2A led to the intracellular retention of Tsp2A, Ssk, Mesh and Dlg, gaps and widening spaces in remaining sSJ, and tumorous and cystic tubules. Elevated protein levels in Malpighian tubules together with diminished V-type H(+)-ATPase activity is consistent with cell proliferation and reduced transport activity. Indeed, Malpighian tubules isolated from Tsp2A knockdown flies failed to secrete fluid in vitro. The absence of significant transepithelial voltages and resistances manifest an extremely leaky epithelium that allows secreted solutes and water to leak back to the peritubular side. The tubular failure to excrete fluid leads to extracellular volume expansion in the fly and to death within the first week of adult life. Expression of the c42-GAL4 driver begins in Malpighian tubules in the late embryo and progresses upstream to distal tubules in third instar larvae, which can explain why larvae survive Tsp2A knockdown and adults do not. Uncontrolled cell proliferation upon Tsp2A knockdown confirms the role of Tsp2A as tumor suppressor in addition to its role in sSJ structure and transepithelial transport (Beyenbach, 2020).

The tetraspanins form a subset of the transmembrane 4 superfamily (TM4SF) of proteins that are highly conserved in eukaryotic organisms from plants to mammals. They are present in virtually every cell, and some cells have up to 100,000 copies of protein. Integral membrane proteins of small size (20–50 kDa), the tetraspanins first drew attention as 'clusters of differentiation' proteins (CD9, CD81, CD151, etc.) before they were found to share the sequence of tetraspanins. Protruding only for a short distance (3–5 nm) from membranes, tetraspanins are suited better for lateral than linear associations with each other and with other proteins. However, binding laterally to membrane proteins that extend into the extracellular space, tetraspanins may have enabled contact with other cells as the first step toward multicellular organisms. Today, tetraspanins are known to associate with integrins, cadherins, Rac, matrix-metalloproteases, EGF receptors and immunoglobulins, thereby serving scaffolding, cell adhesion, signal transduction, intracellular signaling, cell motility and migration, cell proliferation and differentiation, cell-cell fusion, endocytic trafficking, host-parasite interactions, metastasis, and viral infection (Beyenbach, 2020).

Tetraspanins are also found at paracellular junctions of epithelia. The tetraspanins Tspan3, CO-029, CD9, CD15, CD81, and CD181 associate with claudins in tight junctions of vertebrates. In invertebrates, tetraspanins are found in septate junctions. In particular, Tsp2A was found to localize to smooth septate junctions in the midgut and Malpighian tubules of Drosophila. The present study confirmed the location of Tsp2A in Malpighian tubules by localizing this protein in a well-defined band surrounding epithelial cells near the tubule lumen of Malpighian tubules of Drosophila. Sharing this band with Ssk and Mesh identifies Tsp2A as a protein of the smooth septate junction, as in Drosophila midgut epithelial cells. With length along the Malpighian tubule, the band of Tsp2A, Ssk, or Mesh forms a trapezoid ribbon dominated by principal cells that comprise 80% of tubule cells. The present study shows further that Dlg, the membrane-associated guanylate kinase (MAGUK) required for the formation of both septate and tricellular junctions and for epithelial polarization in Drosophila, is part of the trapezoid ribbon in Malpighian tubules. It suggests that Ssk, Mesh, Tsp2A, and Dlg form a complex at sSJs in Malpighian tubules and perhaps also in the midgut of Drosophila. Although Malpighian tubules and midgut utilize a single-layered epithelium for mediating transepithelial transport, they differ in the relative location of SJs and adherens junctions (AJs). In the midgut, sSJs are apical to AJs as in vertebrate tight junctions, while enigmatically the reverse is true for Malpighian tubules, even though Malpighian tubules and midgut share the same canonical polarization factors (Beyenbach, 2020).

The trapezoid ribbon of Tsp2A, Ssk, Mesh, and Dlg no longer forms upon the developmental knockdown of just one of these proteins, Tsp2A. The four septate junctional proteins remain largely in the cytoplasm of principal cells in close association, consistent with the need for the full complement of sSJ proteins before they can move to their usual paracellular location near the apical membrane. The few sSJs that can still be observed in Tsp2A knockdown tubules now spot the paracellular pathway for long distances with frequent gaps, wide gaps between the cell membranes of adjacent cells, and the loss of rungs in the ladder-like sSJ architecture. Junctions appearing at several places along the apical/basal axis indicate the diffuse distribution of sSJs along the paracellular pathway. Principal cells are also compromised. At basal surfaces, the basal lamina is unraveling consistent with the loss of structural support indicated by the increase in tubule diameter and hyperplasia. At the apical membrane, microvilli can be entirely absent; those present are shorter, less dense, and often devoid of mitochondria compared with control tubules. Thus the effects of knocking down Tsp2A extend beyond sSJs to the breakdown of the structural features of basolateral and apical plasma membranes in principal cells (Beyenbach, 2020).

The physiological consequences of the structural defects are severe. Malpighian tubules isolated from Tsp2A knockdown flies reared at 27°C failed to secrete fluid during their short life span less than a week. For one reason, apical microvilli, the sites that generate the driving force for transepithelial electrolyte secretion, are far less developed in Tsp2A knockdown tubules than in control tubules consistent with the diminished V-type H+-ATPase activity and the absence of significant lumen-positive transepithelial voltages in Tsp2A knockdown tubules. For another reason, the paracellular pathway has completely lost its barrier property in view of transepithelial input resistances that are not significantly different from the input resistances measured between the two measuring electrodes in Ringer solution. In the absence of paracellular barriers, the little solute and water still secreted into the tubule lumen via transcellular transport likely leak back to the peritubular bath (or hemolymph) via the paracellular pathway instead of flowing downstream for excretion. The tubular failure to excrete fluid is expected to disrupt extracellular fluid homeostasis as solute and water accumulate in the extracellular fluid compartment. The volume expansion leads to severe generalized edema and to death in the first week of adult life (Beyenbach, 2020).

In a recent study we found that Tsp2A, Ssk, and Dlg also remain largely in principal cells upon the developmental knockdown of mesh. Furthermore, mesh knockdown tubules fail to secrete fluid, and the flies gain extracellular fluid volume, become edematous, and die in early adulthood, as in the knockdown of Tsp2A. Similar effects of the knockdown of Tsp2A and mesh suggest that the full complement of septate junctional proteins consisting of Mesh, Tsp2A, Ssk, Dlg (and possibly other proteins) must be expressed before the sSJ can form in apicolateral regions in Malpighian tubules. The full complement of septate junctional proteins is indeed required for the formation sSJs in the midgut. There, the three proteins, Tsp2A, Mesh, and Ssk, are mutually dependent for their proper localization at the sSJ (Beyenbach, 2020).

The molecular mechanisms that bring about the structural and functional defects caused by the knockdown of Tsp2A were beyond the aims of the present study. Nevertheless, studies by others are relevant. Malpighian tubules are formed in the embryonic stage and become functional in the second half of the first day of development, responding in part to the 100-fold increase in uric acid by secreting this xanthine into the tubule lumen. From then on, the tubules do not undergo metamorphosis. Instead, they grow during the larval stages by increasing cell size associated with the endoreplication of DNA, and they acquire renal stem cells as late as the first day of pupation. Accordingly, the development of tumors and cysts upon the knockdown of Tsp2A must take place late in the development of the fly, which may explain why larvae survive the knockdown of Tsp2A or mesh (or other sSJ proteins) but not adults (Beyenbach, 2020).

Renal stem cells (RSCs) were first identified in Malpighian tubules as 'tiny cells' in the ureter and lower tubule of adult flies but not larvae. RSCs are thought to be multipotent, capable of generating all types of Malpighian tubule cells under autocrine regulation of JAK-STAT signaling, which stimulates RSCs to form renal blasts (RBs) that migrate and differentiate to stellate cells and principal cells in the upper tubule. Thus RSC activity can be expected to replace Tsp2A-deficient principal cells from the pupal stage on. Alas, the knockdown of Tsp2A may cause RSCs to produce Tsp2A-deficient principal cells, and failing to replace defective principal cells with normal cells, RSCs may remain active. Unstopped cell proliferation may then produce the hypertrophy, tumor- and cyst-like structures observed in the present study. Support for this hypothetical scenario in Malpighian tubules comes from the recent study of the role of sSJs in cell proliferation in the midgut of Drosophila. Here, enterocytes deficient of Tsp2A, Ssk, or Mesh exhibit defective sSJs, epithelial barrier dysfunction, abnormal enterocytes, and increased cell proliferation and intestinal hypertrophy, which the flies do not survive for more than 10 days. Thus the phenotype produced by knocking down Tsp2A in the intestine is similar to that of knocking down Tsp2A in Malpighian tubules. Moreover, the proliferation of intestinal stem cells (ISCs) was activated by Ras-MAP kinase and by the cytokines Unpaired2 (Upd2) and Unpaired3 (Upd3); the latter are known to activate JAK-STAT pathway involved in regulating RSC activity in Malpighian tubules. Similar molecular mechanisms in the intestine and Malpighian tubules suggest that septate junctional proteins and sSJs are critically involved in the regulation of stem cell proliferation in both tissues in as much as midgut intestinal stem cells (ISCs) arise from the same pool of midgut progenitor as RSCs in Malpighian tubules (Beyenbach, 2020).

Recent work has uncovered the critical role of Tsp2A in regulating stem cell proliferation in the Drosophila midgut via Hippo signaling. From fly to mammals, the Hippo pathway modulates cell proliferation, differentiation, and migration in developing tissues and limits growth in adults. First identified in Drosophila in 2003, the Hippo pathway has now grown to a signaling network of more than 40 players. One critical player is the transcription cofactor and oncogene Yorkie (Yki), which has been associated with Yki tumors and excessive body fluid retention described as 'bloating syndrome' in Drosophila. The Hippo pathway is regulated by components at or near cell junctions such as tight, septate, and adherens junctions. Transitions of components of the pathway [atypical protein kinase C (aPKC); Warts (Wts); and Hippo kinase (Hpo)] between the membrane and cytoplasm are thought to activate/inactivate the Hippo pathway while the shuttling of Yki between the cytoplasm and the nucleus is thought to regulate transcription and cell proliferation and apoptosis. Damage to the Drosophila midgut has been proposed to increase Yki activity and stimulate ISC proliferation. As the wound heals, Tsp2A undergoes internalization to facilitate the endocytic degradation of aPKC. Reduced aPKC activity allows Hpo to dimerize at the membrane, setting off a series of phosphorylations that ends with phosphorylated Yki and its cytoplasmic restriction, which stops ISC proliferation. Indeed, defects in Tsp2A expression or defective Tsp2A-sSJ assembly cause excessive aPKC-Yki-JAK-STAT activity and make the midgut epithelium highly proliferative, like a wound that cannot heal. Whether Hippo signaling is similarly activated in Tsp2A knockdown tubules will be of interest for future studies (Beyenbach, 2020).

An abundant quiescent stem cell population in Drosophila Malpighian tubules protects principal cells from kidney stones

Adult Drosophila Malpighian tubules have low rates of cell turnover but are vulnerable to damage caused by stones, like their mammalian counterparts, kidneys. This study shows that Drosophila renal stem cells (RSCs) in the ureter and lower tubules comprise a unique, unipotent regenerative compartment. RSCs respond only to loss of nearby principal cells (PCs), cells critical for maintaining ionic balance. Large polyploid PCs are outnumbered by RSCs, which replace each lost cell with multiple PCs of lower ploidy. Notably, RSCs do not replenish principal cells or stellate cells in the upper tubules. RSCs generate daughters by asymmetric Notch signaling, yet RSCs remain quiescent (cell cycle-arrested) without damage. Nevertheless, the capacity for RSC-mediated repair extends the lifespan of flies carrying kidney stones. It is proposed that abundant, RSC-like stem cells exist in other tissues with low rates of turnover where they may have been mistaken for differentiated tissue cells (Wang, 2020).

Nonapoptotic role of caspase-3 in regulating Rho1GTPase-mediated morphogenesis of epithelial tubes of Drosophila renal system

Cells trigger caspase-mediated apoptosis to eliminate themselves from the system when tissue needs to be sculptured, or they detect any abnormality within them, thus preventing irreparable damage to the host. However, nonapoptotic activities of caspases are also involved in many cellular functions. Interestingly, Drosophila Malpighian tubules (MTs) express apoptotic proteins, without succumbing to cell death. This study showed apoptosis-independent role of executioner caspase-3, Drice, in MT morphogenesis. Drice is required for precise cytoskeleton organization and convergent extension, failing which morphology, size, cell number, and arrangement get affected. Furthermore, characteristic stellate cell shape transformation in MTs is also governed by Drice. Genetic interaction study shows that Drice mediates its action by regulating Rho1GTPase functionally, and localization of polarity protein Disc large. Subsequently, downregulation of Rho1GTPase in Drice mutants significantly rescues the cystic MTs phenotype. The study shows a mechanism by which Drice governs tubulogenesis via Rho1GTPase-mediated coordinated organization of actin cytoskeleton and membrane stabilization. Collectively these findings suggest a nonapoptotic function of caspase-3 in fine-tuning of cellular rearrangement during tubule development, and these results will add to the growing understanding of diverse roles of caspases during its evolution in metazoans (Ojha, 2022).

The transcription factor ATF4 mediates endoplasmic reticulum stress-related podocyte injury and slit diaphragm defects

Mutations in OSGEP and four other genes that encode subunits of the KEOPS complex cause Galloway-Mowat syndrome, a severe, inherited kidney-neurological disease. The complex catalyzes an essential posttranscriptional modification of tRNA and its loss of function induces endoplasmic reticulum (ER) stress. Using Drosophila garland nephrocytes and cultured human podocytes, this study aimed to elucidate the molecular pathogenic mechanisms of KEOPS-related glomerular disease and to test pharmacological inhibition of ER stress-related signaling as a therapeutic principle. ATF4, an ER stress-mediating transcription factor, or its fly orthologue Crc, were upregulated in both fly nephrocytes and human podocytes. Knockdown of Tcs3, a fly orthologue of OSGEP, caused slit diaphragm defects, recapitulating the human kidney phenotype. OSGEP cDNA with mutations found in patients lacked the capacity for rescue. Genetic interaction studies in Tcs3-deficient nephrocytes revealed that Crc mediates not only cell injury, but surprisingly also slit diaphragm defects, and that genetic or pharmacological inhibition of Crc activation attenuates both phenotypes. These findings are conserved in human podocytes where ATF4 inhibition improved the viability of podocytes with OSGEP knockdown, with chemically induced ER stress, and where ATF4 target genes and pro-apoptotic gene clusters are upregulated upon OSGEP knockdown. Thus, these data identify ATF4-mediated signaling as a molecular link among ER stress, slit diaphragm defects, and podocyte injury, and the data suggest that modulation of ATF4 signaling may be a potential therapeutic target for certain podocyte diseases (Krausel, 2023).

Voltages and resistances of the anterior Malpighian tubule of Drosophila melanogaster

The small size of Malpighian tubules in the fruit fly Drosophila melanogaster has discouraged measurements of the transepithelial electrical resistance. The present study introduces two methods for measuring the transepithelial resistance in isolated D . melanogaster Malpighian tubules using conventional microelectrodes and PClamp hardware and software. The first method uses three microelectrodes to measure the specific transepithelial resistance normalized to tubule length or luminal surface area for comparison with resistances of other epithelia. The second method uses only two microelectrodes to measure the relative resistance for comparing before and after effects in a single Malpighian tubule. Knowledge of the specific transepithelial resistance allows the first electrical model of electrolyte secretion by the main segment of the anterior Malpighian tubule of D . melanogaster. The electrical model is remarkably similar to that of the distal Malpighian tubule of Aedes aegypti when tubules of Drosophila and Aedes are studied in vitro under the same experimental conditions. Thus, despite 189 millions of years of evolution separating these two genera, the electrophysiological properties of their Malpighian tubules remains remarkably conserved (Beyenbach, 2019).

Active transport of brilliant blue FCF across the Drosophila midgut and Malpighian tubule epithelia

Under conditions of stress, many animals suffer from epithelial barrier disruption that can cause molecules to leak down their concentration gradients, potentially causing a loss of organismal homeostasis, further injury or death. Drosophila is a common insect model, used to study barrier disruption related to aging, traumatic injury, or environmental stress. Net leak of a non-toxic dye (Brilliant blue FCF) from the gut lumen to the hemolymph is often used to identify barrier failure under these conditions, but Drosophila are capable of actively transporting structurally-similar compounds. This study examined whether cold stress (like other stresses) causes Brilliant blue FCF (BB-FCF) to appear in the hemolymph of flies fed the dye, and if so whether Drosophila are capable of clearing this dye from their body following chilling. Using in situ midgut leak and transport assays as well as Ramsay assays of Malpighian tubule transport, this study tested whether these ionoregulatory epithelia can actively transport BB-FCF. In doing so, it was found that the Drosophila midgut and Malpighian tubules can mobilize BB-FCF via an active transcellular pathway, suggesting that elevated concentrations of the dye in the hemolymph may occur from increased paracellular permeability, reduced transcellular clearance, or both. It is concluded that Drosophila are able to actively secrete Brilliant blue FCF, a commonly used marker of barrier dysfunction (Livingston, 2019).

LIM and SH3 protein 1 (LASP-1): A novel link between the slit membrane and actin cytoskeleton dynamics in podocytes

The foot processes of podocytes exhibit a dynamic actin cytoskeleton, which maintains their complex cell structure and antagonizes the elastic forces of the glomerular capillary. Interdigitating secondary foot processes form a highly selective filter for proteins in the kidney, the slit membrane. Knockdown of slit membrane components such as Nephrin or Neph1 and cytoskeletal adaptor proteins such as CD2AP in mice leads to breakdown of the filtration barrier with foot process effacement, proteinuria, and early death of the mice. Less is known about the crosstalk between the slit membrane-associated proteins and cytoskeletal components inside the podocyte foot processes. This study shows that LASP-1, an actin-binding protein, is highly expressed in podocytes. Electron microscopy studies demonstrate that LASP-1 is found at the slit membrane suggesting a role in anchoring slit membrane components to the actin cytoskeleton. Live cell imaging experiments with transfected podocytes reveal that LASP-1 is either part of a highly dynamic granular complex or a static, actin cytoskeleton-bound protein. This study identified CD2AP as a novel LASP-1 binding partner that regulates its association with the actin cytoskeleton. Activation of the renin-angiotensin-aldosterone system, which is crucial for podocyte function, leads to phosphorylation and altered localization of LASP-1. In vivo studies using the Drosophila nephrocyte model indicate that Lasp is necessary for the slit membrane integrity and functional filtration (Lepa, 2020).

Exocyst Genes Are Essential for Recycling Membrane Proteins and Maintaining Slit Diaphragm in Drosophila Nephrocytes

Studies have linked mutations in genes encoding the eight-protein exocyst protein complex to kidney disease, but the underlying mechanism is unclear. Because Drosophila nephrocytes share molecular and structural features with mammalian podocytes, they provide an efficient model for studying this issue. Genes encoding exocyst complex proteins were silenced specifically in Drosophila nephrocytes, and the effects on protein reabsorption by lacuna channels and filtration by the slit diaphragm were studied. Nephrocyte functional assays were performed, super-resolution confocal microscopy of slit diaphragm proteins was carried out, and transmission electron microscopy was used to analyze ultrastructural changes. The colocalization of slit diaphragm proteins with exocyst protein Sec15 and with endocytosis and recycling regulators Rab5, Rab7, and Rab11 was also studied. Silencing exocyst genes in nephrocytes led to profound changes in structure and function. Abolition of cellular accumulation of hemolymph proteins with dramatically reduced lacuna channel membrane invaginations offered a strong indication of reabsorption defects. Moreover, the slit diaphragm's highly organized surface structure-essential for filtration-was disrupted, and key proteins were mislocalized. Ultrastructural analysis revealed that exocyst gene silencing led to the striking appearance of novel electron-dense structures that were named "exocyst rods," which likely represent accumulated membrane proteins following defective exocytosis or recycling. The slit diaphragm proteins partially colocalized with Sec15, Rab5, and Rab11. These findings suggest that the slit diaphragm of Drosophila nephrocytes requires balanced endocytosis and recycling to maintain its structural integrity and that impairment of the exocyst complex leads to disruption of the slit diaphragm and nephrocyte malfunction. This model may help identify therapeutic targets for treating kidney diseases featuring molecular defects in vesicle endocytosis, exocytosis, and recycling (Wen, 2020).

Renal Purge of Hemolymphatic Lipids Prevents the Accumulation of ROS-Induced Inflammatory Oxidized Lipids and Protects Drosophila from Tissue Damage

Animals require complex metabolic and physiological adaptations to maintain the function of vital organs in response to environmental stresses and infection. This study found that infection or injury in Drosophila induced the excretion of hemolymphatic lipids by Malpighian tubules, the insect kidney. This lipid purge was mediated by a stress-induced lipid-binding protein, Materazzi, which was enriched in Malpighian tubules. Flies lacking materazzi had higher hemolymph concentrations of reactive oxygen species (ROS) and increased lipid peroxidation. These flies also displayed Malpighian tubule dysfunction and were susceptible to infections and environmental stress. Feeding flies with antioxidants rescued the materazzi phenotype, indicating that the main role of Materazzi is to protect the organism from damage caused by stress-induced ROS. These findings suggest that purging hemolymphatic lipids presents a physiological adaptation to protect host tissues from excessive ROS during immune and stress responses, a process that is likely to apply to other organisms (Li, 2020).

CG4928 Is Vital for Renal Function in Fruit Flies and Membrane Potential in Cells: A First In-Depth Characterization of the Putative Solute Carrier UNC93A

The number of transporter proteins that are not fully characterized is immense. This study used Drosophila melanogaster and human cell lines to perform a first in-depth characterization of CG4928, an ortholog to the human UNC93A, of which little is known. Solute carriers regulate and maintain biochemical pathways important for the body, and malfunctioning transport is associated with multiple diseases. Based on phylogenetic analysis, CG4928 is closely related to human UNC93A and has a secondary and a tertiary protein structure and folding similar to major facilitator superfamily transporters. Ubiquitous knockdown of CG4928 causes flies to have a reduced secretion rate from the Malpighian tubules; altering potassium content in the body and in the Malpighian tubules, homologous to the renal system; and results in the development of edema. The edema could be rescued by using amiloride, a common diuretic, and by maintaining the flies on ion-free diets. CG4928-overexpressing cells did not facilitate the transport of sugars and amino acids; however, proximity ligation assay revealed that CG4928 co-localized with TASK(1) channels. Overexpression of CG4928 resulted in induced apoptosis and cytotoxicity, which could be restored when cells were kept in high-sodium media. Furthermore, the basal membrane potential was observed to be disrupted. Taken together, the results indicate that CG4928 is of importance for generating the cellular membrane potential by an unknown manner. However, it is speculated that it most likely acts as a regulator or transporter of potassium flows over the membrane (Ceder, 2020).

Multidrug Resistance Like Protein 1 Activity in Malpighian Tubules Regulates Lipid Homeostasis in Drosophila

Multidrug resistance proteins (MRPs), members of the ATP-binding cassette transporter (ABC transporter) family, are pivotal for transporting endo- and xenobiotics, which confer resistance to anticancer agents and contribute to the clearance of oxidative products. However, their function in many biological processes is still unclear. This study investigated the role of an evolutionarily conserved MRP in metabolic homeostasis by knocking down the expression of Drosophila multidrug-resistance like protein 1 (MRP) in several tissues involved in regulating metabolism, including the gut, fat body, and Malpighian tubules. Interestingly, only suppression of MRP in the Malpighian tubules, the functional equivalent to the human kidney, was sufficient to cause abnormal lipid accumulation and disrupt feeding behavior. Furthermore, reduced Malpighian tubule MRP expression resulted in increased Hr96 (homolog of human pregnane X receptor) expression. Hr96 is known to play a role in detoxification and lipid metabolism processes. Reduced expression of MRP in the Malpighian tubules also conveyed resistance to oxidative stress, as well as reduced normal levels of reactive oxygen species in adult flies. This study reveals that an evolutionarily conserved MRP is required in Drosophila Malpighian tubules for proper metabolic homeostasis (Liu, 2021).

Tryptophan regulates Drosophila zinc stores

Zinc deficiency is commonly attributed to inadequate absorption of the metal. Instead, this study shows that body zinc stores in Drosophila melanogaster depend on tryptophan consumption. Hence, a dietary amino acid regulates zinc status of the whole insect—a finding consistent with the widespread requirement of zinc as a protein cofactor. Specifically, the tryptophan metabolite kynurenine is released from insect fat bodies and induces the formation of zinc storage granules in Malpighian tubules, where 3-hydroxykynurenine and xanthurenic acid act as endogenous zinc chelators. Kynurenine functions as a peripheral zinc-regulating hormone and is converted into a 3-hydroxykynurenine–zinc–chloride complex, precipitating within the storage granules. Thus, zinc and the kynurenine pathway—well-known modulators of immunity, blood pressure, aging, and neurodegeneration—are physiologically connected (Garay, 2022).

Chloroquine disrupts zinc storage granules in primary Malpighian tubule cells of Drosophila melanogaster

Contrasting reports exist in the literature regarding the effect of chloroquine treatment on cellular zinc uptake or secretion. This study tested the effect of chloroquine administration in the Drosophila model organism. Larvae grown on a diet supplemented with 2.5 mg/ml chloroquine were shown to lose up to 50% of their stored zinc and around 10% of their total potassium content. This defect in chloroquine-treated animals correlates with the appearance of abnormal autophagolysosomes in the principal cells of the Malpighian tubules, where zinc storage granules reside. It was further shown that the reported increase of Fluozin-3 fluorescence following treatment of cells with 300 μM chloroquine for 1 h may not reflect increased zinc accumulation, since a similar treatment in Madin-Darby canine kidney cells results in a 36% decrease in their total zinc content. Thus, chloroquine should not be considered a zinc ionophore. Zinc supplementation plus chloroquine treatment restored zinc content both in vivo and in vitro, without correcting autophagic or other ionic alterations, notably in potassium, associated with the chloroquine treatment. It is suggest that chloroquine or hydroxychloroquine administration to patients could reduce intracellular zinc storage pools and be part of the drug's mechanism of action (Campos-Blazquez, 2022).

Sequence analysis and function of mosquito aeCCC2 and Drosophila Ncc83 orthologs

Dipteran insects have genes that code for two different Na(+)-dependent cation-chloride cotransporter (CCC) paralogs. Aedes aegypti aeNKCC1 is an ortholog of Drosophila melanogaster Ncc69, a bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC). Aedes aegypti aeCCC2 and aeCCC3 are orthologs of Drosophila Ncc83. Prior work suggests that the transport properties of aeCCC2 differ from canonical NKCCs. In particular, Xenopus oocytes expressing aeCCC2 have increased Na(+)-dependent membrane currents compared to controls, whereas NKCCs are electroneutral. This study further evaluated the function and localization of aeCCC2 and Ncc83. In oocytes expressing aeCCC2 or Ncc83, membrane potential (V(m)) hyperpolarized upon Na(+) removal; following hypotonic exposure the change in V(m) was greater than it was in controls. In voltage-clamp experiments, membrane currents were concentration dependent on Na(+) with an apparent affinity (K(m)) of approximately 4.6 mM. In Malpighian tubules of larval and adult mosquitoes, aeCCC2 was localized along the basolateral aspect of principal cells. Sequence comparisons among transporters from Drosophila, Aedes, Anopheles, and Culex revealed 33 residues within the transmembrane domains (TMDs) that are fully conserved within paralogs but that differ between orthologs of NKCC1 and orthologs of aeCCC2/Ncc83. These residues are distributed across all 12 TMDs. These results provide a foundation for further exploration of the structural basis for functional differences between insect Na(+)-dependent CCCs (Duong, 2022).

KANK deficiency leads to podocyte dysfunction and nephrotic syndrome

Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of progressive renal function decline and affects millions of people. This study identified recessive mutations in kidney ankyrin repeat-containing protein 1 (KANK1), KANK2, and KANK4 in individuals with nephrotic syndrome. In an independent functional genetic screen of Drosophila cardiac nephrocytes, which are equivalents of mammalian podocytes, it was determined that the Drosophila KANK homolog (dKank) is essential for nephrocyte function. RNAi-mediated knockdown of dKank in nephrocytes disrupted slit diaphragm filtration structures and lacuna channel structures. In rats, KANK1, KANK2, and KANK4 all localized to podocytes in glomeruli, and KANK1 partially colocalized with synaptopodin. Knockdown of kank2 in zebrafish recapitulated a nephrotic syndrome phenotype, resulting in proteinuria and podocyte foot process effacement. In rat glomeruli and cultured human podocytes, KANK2 interacted with ARHGDIA, a known regulator of RHO GTPases in podocytes that is dysfunctional in some types of nephrotic syndrome. Knockdown of KANK2 in cultured podocytes increased active GTP-bound RHOA and decreased migration. Together, these data suggest that KANK family genes play evolutionarily conserved roles in podocyte function, likely through regulating RHO GTPase signaling (Gee, 2002).

Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes

The Drosophila nephrocyte is a critical component of the fly renal system and bears structural and functional homology to podocytes and proximal tubule cells of the mammalian kidney. Nephrocytes are highly active in endocytosis and vesicle trafficking. Rab GTPases regulate endocytosis and trafficking but specific functions of nephrocyte Rabs remain undefined. This study analyzed Rab GTPase expression and function in Drosophila nephrocytes and found that 11 out of 27 Drosophila Rabs were required for normal activity. Rabs 1, 5, 7, 11 and 35 were most important. Gene silencing of the nephrocyte-specific Rab5 eliminated all intracellular vesicles and the specialized plasma membrane structures essential for nephrocyte function. Rab7 silencing dramatically increased clear vacuoles and reduced lysosomes. Rab11 silencing increased lysosomes and reduced clear vacuoles. These results suggest that Rab5 mediates endocytosis that is essential for the maintenance of functionally critical nephrocyte plasma membrane structures and that Rabs 7 and 11 mediate alternative downstream vesicle trafficking pathways leading to protein degradation and membrane recycling, respectively. Elucidating molecular pathways underlying nephrocyte function has the potential to yield important insights into human kidney cell physiology and mechanisms of cell injury that lead to disease (Fu, 2017).

Targeted renal knockdown of Na(+)/H(+) exchanger regulatory factor Sip1 produces uric acid nephrolithiasis in Drosophila

Nephrolithiasis is one of the most common kidney diseases with poorly understood pathophysiology, but experimental study has been hindered by lack of experimentally tractable models. Drosophila melanogaster is a useful model organism for renal diseases because of genetic and functional similarities of Malpighian (renal) tubules with the human kidney. This study demonstrates the function of Sip1 (SRY-interacting protein 1) gene, an orthologue of human NHERF1 in Drosophila MTs, and its impact on nephrolithiasis. Abundant birefringent calculi were observed in Sip1 mutant flies, and the phenotype was also observed in renal stellate cell-specific RNAi Sip1 knockdowns in otherwise normal flies, confirming a renal aetiology. This phenotype was abolished in rosy flies (which model human xanthinuria) and by the xanthine oxidase inhibitor allopurinol, suggesting that the calculi were of uric acid. This was confirmed by direct assay for urate. Stones rapidly dissolved when the tubule was bathed in alkaline media, suggesting that Sip1 knockdown was acidifying the tubule. SIP1 was shown to co-locate with Na(+)/H(+) exchanger NHE2, and with moesin, in stellate cells; and so a model was developed in which Sip1 normally regulates NHE2 activity and thus luminal pH. Drosophila renal tubule thus offers a useful model for urate nephrolithiasis (Ghimirie, 2019).

Nephrocytes remove microbiota-derived peptidoglycan from systemic circulation to maintain immune homeostasis

Preventing aberrant immune responses against the microbiota is essential for the health of the host. Microbiota-shed pathogen-associated molecular patterns translocate from the gut lumen into systemic circulation. This study examined the role of hemolymph (insect blood) filtration in regulating systemic responses to microbiota-derived peptidoglycan. Drosophila deficient for the transcription factor Klf15 (Klf15(NN)) are viable but lack nephrocytes-cells structurally and functionally homologous to the glomerular podocytes of the kidney. Klf15(NN) flies were more resistant to infection than wild-type (WT) counterparts but exhibited a shortened lifespan. This was associated with constitutive Toll pathway activation triggered by excess peptidoglycan circulating in Klf15(NN) flies. In WT flies, peptidoglycan was removed from systemic circulation by nephrocytes through endocytosis and subsequent lysosomal degradation. Thus, renal filtration of microbiota-derived peptidoglycan maintains immune homeostasis in Drosophila, a function likely conserved in mammals and potentially relevant to the chronic immune activation seen in settings of impaired blood filtration (Troha, 2019).

Adipose triglyceride lipase protects renal cell endocytosis in a Drosophila dietary model of chronic kidney disease

Obesity-related renal lipotoxicity and chronic kidney disease (CKD) are prevalent pathologies with complex aetiologies. One hallmark of renal lipotoxicity is the ectopic accumulation of lipid droplets in kidney podocytes and in proximal tubule cells. Renal lipid droplets are observed in human CKD patients and in high-fat diet (HFD) rodent models, but their precise role remains unclear. This study establish a HFD model in Drosophila that recapitulates renal lipid droplets and several other aspects of mammalian CKD. Cell type-specific genetic manipulations show that lipid can overflow from adipose tissue and is taken up by renal cells called nephrocytes. A HFD drives nephrocyte lipid uptake via the multiligand receptor Cubilin (Cubn), leading to the ectopic accumulation of lipid droplets. These nephrocyte lipid droplets correlate with endoplasmic reticulum (ER) and mitochondrial deficits, as well as with impaired macromolecular endocytosis, a key conserved function of renal cells. Nephrocyte knockdown of diglyceride acyltransferase 1 (DGAT1), overexpression of adipose triglyceride lipase (ATGL; brummer), and epistasis tests together reveal that fatty acid flux through the lipid droplet triglyceride compartment protects the ER, mitochondria, and endocytosis of renal cells. Strikingly, boosting nephrocyte expression of the lipid droplet resident enzyme ATGL is sufficient to rescue HFD-induced defects in renal endocytosis. Moreover, endocytic rescue requires a conserved mitochondrial regulator, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC1α). This study demonstrates that lipid droplet lipolysis counteracts the harmful effects of a HFD via a mitochondrial pathway that protects renal endocytosis. It also provides a genetic strategy for determining whether lipid droplets in different biological contexts function primarily to release beneficial or to sequester toxic lipids (Lubojemska, 2021).

The Basolateral Polarity Module Promotes Slit Diaphragm Formation in Drosophila Nephrocytes, a Model of Vertebrate Podocytes

Podocyte slit diaphragms (SDs) are intercellular junctions that function as size-selective filters, excluding most proteins from urine. Abnormalities in SDs cause proteinuria and nephrotic syndrome. Podocytes exhibit apicobasal polarity, which can affect fundamental aspects of cell biology, including morphology, intercellular junction formation, and asymmetric protein distribution along the plasma membrane. Apical polarity protein mutations cause nephrotic syndrome, and data suggest apical polarity proteins regulate SD formation. However, there is no evidence that basolateral polarity proteins regulate SDs. Thus, the role of apicobasal polarity in podocytes remains unclear. Genetic manipulations and transgenic reporters determined the effects of disrupting apicobasal polarity proteins in Drosophila nephrocytes, which have SDs similar to those of mammalian podocytes. Confocal and electron microscopy were used to characterize SD integrity after loss of basolateral polarity proteins, and genetic-interaction studies illuminated relationships among apicobasal polarity proteins. The study identified four novel regulators of nephrocyte SDs: Dlg, Lgl, Scrib, and Par-1. These proteins comprise the basolateral polarity module and its effector kinase. The data suggest these proteins work together, with apical polarity proteins, to regulate SDs by promoting normal endocytosis and trafficking of SD proteins. Given the recognized importance of apical polarity proteins and SD protein trafficking in podocytopathies, the findings connecting basolateral polarity proteins to these processes significantly advance understanding of SD regulation (Mysh, 2021). ">

Activation of Hippo Pathway Damages Slit Diaphragm by Deprivation of Ajuba Proteins

The highly conserved Hippo pathway, which regulates organ growth and cell proliferation by inhibiting transcriptional cofactors YAP/TAZ, plays a special role in podocytes, where activation of the pathway leads to apoptosis. The Ajuba family proteins (Ajuba, LIM domain-containing protein 1 (LIMD1) and Wilms tumor protein 1-interacting protein [WTIP]) can bind and inactivate large tumor suppressor kinases 1 and 2, (LATS1/2) two of the Hippo pathway key kinases. WTIP, furthermore, connects the slit diaphragm (SD), the specialized cell-cell junction between podocytes, with the actin cytoskeleton. This study used garland cell nephrocytes of Drosophila to monitor the role of Ajuba proteins in Hippo pathway regulation and structural integrity of the SD. In nephrocytes, the Ajuba homolog Djub recruited Warts (LATS2 homolog) to the SD. Knockdown of Djub activated the Hippo pathway. Reciprocally, Hippo activation reduced the Djub level. Both Djub knockdown and Hippo activation led to morphological changes in the SD, rearrangement of the cortical actin cytoskeleton, and increased SD permeability. Knockdown of Warts or overexpression of constitutively active Yki prevented these effects. In podocytes, Hippo pathway activation or knockdown of YAP also decreased the level of Ajuba proteins. It is concluded that Ajuba proteins regulate the structure and function of the SD in nephrocytes, connecting the SD protein complex to the actin cytoskeleton and maintaining the Hippo pathway in an inactive state. Hippo pathway activation directly influencing Djub expression suggests a self-amplifying feedback mechanism (Gilhaus, 2023).

Disruption of the Rab7-Dependent Final Common Pathway of Endosomal and Autophagic Processing Results in a Severe Podocytopathy

Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates, such as podocytes. How disturbances within these trafficking pathways may act as factors in proteinuric glomerular diseases is poorly understood. To explore how disturbances in trafficking pathways may act as factors in proteinuric glomerular diseases, this study focused on Rab7, a highly conserved GTPase that controls the homeostasis of late endolysosomal and autophagic processes. Mouse and Drosophila in vivo models lacking Rab7 were generated exclusively in podocytes or nephrocytes, and histologic and ultrastructural analyses was performed. To further investigate Rab7 function on lysosomal and autophagic structures, immortalized human cell lines depleted for Rab were used. Depletion of Rab7 in mice, Drosophila, and immortalized human cell lines resulted in an accumulation of diverse vesicular structures resembling multivesicular bodies, autophagosomes, and autoendolysosomes. Mice lacking Rab7 developed a severe and lethal renal phenotype with early-onset proteinuria and global or focal segmental glomerulosclerosis, accompanied by an altered distribution of slit diaphragm proteins. Remarkably, structures resembling multivesicular bodies began forming within 2 weeks after birth, prior to the glomerular injuries. In Drosophila nephrocytes, rab7 knockdown resulted in the accumulation of vesicles and reduced slit diaphragms. In vitro, RAB7 knockout led to similar enlarged vesicles and altered lysosomal pH values, accompanied by an accumulation of lysosomal marker proteins. It is concluded that isruption within the final common pathway of endocytic and autophagic processes may be a novel and insufficiently understood mechanism regulating podocyte health and disease (Voing, 2023).

Slit diaphragm maintenance requires dynamic clathrin-mediated endocytosis facilitated by AP-2, Lap, Aux and Hsc70-4 in nephrocytes

The Slit diaphragm (SD) is the key filtration structure in human glomerular kidney that is affected in many types of renal diseases. SD proteins are known to undergo endocytosis and recycling to maintain the integrity of the filtration structure. However, the key components of this pathway remain unclear. Using the Drosophila nephrocyte as a genetic screen platform, most genes involved in endocytosis and cell trafficking were screened, and the key components were identified of the cell trafficking pathway required for SD protein endocytosis and recycling. The SD protein endocytosis and recycling pathway was found to contain clathrin, dynamin, AP-2 complex, like-AP180 (Lap), auxilin and Hsc70-4 (the endocytosis part) followed by Rab11 and the exocyst complex (the recycling part). Disrupting any component in this pathway led to disrupted SD on the cell surface and intracellular accumulation of mislocalized SD proteins. This study provides the first in vivo evidence of trapped SD proteins in clathrin-coated pits at the plasma membrane when this pathway is disrupted. All genes in this SD protein endocytosis and recycling pathway, as well as SD proteins themselves, are highly conserved from flies to humans. Thus, these results suggest that the SD proteins in human kidney undergo the same endocytosis and recycling pathway to maintain the filtration structure, and mutations in any genes in this pathway could lead to abnormal SD and renal diseases (Wang, 2021).

dCubilin/dAMN- mediated protein reabsorption in Drosophila nephrocytes modulates longevity

Aging is a multi-faceted process regulated by multiple cellular pathways, including the proteostasis network. Pharmacological or genetic enhancement of the intracellular proteostasis network extends lifespan and prevents age-related diseases. However, how proteostasis is regulated in different tissues throughout the aging process remains unclear. This study shows that Drosophila homologs for Cubilin/Amnionless (dCubilin/dAMN)-mediated protein reabsorption from hemolymph (fly equivalent of blood) by nephrocytes modulates longevity through regulating proteostasis in muscle and brain tissues in Drosophila. Overexpression of dAMN receptor in nephrocytes extends lifespan, whereas nephrocyte-specific dCubilin or dAMN RNAi knockdown results in a protein reabsorption defect and shortens lifespan in flies. dCubilin/dAMN-mediated protein reabsorption in nephrocytes regulates proteostasis in hemolymph and improves healthspan. In addition, it was shown that enhanced dCubilin/dAMN-mediated protein reabsorption in nephrocytes slows down the aging process in muscle and brain by maintaining the proteostasis network in these tissues. Furthermore, this study shows that dCubilin/dAMN -mediated protein reabsorption in nephrocytes affects proteasome activity in the whole body and muscle tissues. Altogether, this work has revealed an inter-organ communication network across nephrocytes and muscle/neuronal tissue which is essential to maintain proteostasis and to delay senescence in these organs. The findings have provided insights into the role of renal protein reabsorption in the aging process via this tele-proteostasis network (Feng, 2021).

Rabphilin silencing causes dilated cardiomyopathy in a Drosophila model of nephrocyte damage

Heart failure (HF) and the development of chronic kidney disease (CKD) have a direct association. Both can be cause and consequence of the other. Many factors are known, such as diabetes or hypertension, which can lead to the appearance and/or development of these two conditions. However, it is suspected that other factors, namely genetic ones, may explain the differences in the manifestation and progression of HF and CKD among patients. One candidate factor is Rph, a gene expressed in the nervous and excretory system in mammals and Drosophila, encoding a Rab small GTPase family effector protein implicated in vesicular trafficking. Rph is expressed in the Drosophila heart, and the silencing of Rph gene expression in this organ had a strong impact in the organization of fibers and functional cardiac parameters. Specifically, a significant increase was observed in diastolic and systolic diameters of the heart tube, which is a phenotype that resembles dilated cardiomyopathy in humans. Importantly, this study also showed that silencing of Rabphilin (Rph) expression exclusively in the pericardial nephrocytes, which are part of the flies' excretory system, brings about a non-cell-autonomous effect on the Drosophila cardiac system. In summary, this work demonstrates the importance of Rph in the fly cardiac system and how silencing Rph expression in nephrocytes affects the Drosophila cardiac system (Selma-Soriano, 2021).

Endocytosis mediated by an atypical CUBAM complex modulates slit diaphragm dynamics in nephrocytes

The vertebrate endocytic receptor CUBAM, consisting of three cubilin monomers complexed with a single amnionless molecule, plays a major role in protein reabsorption in the renal proximal tubule. This study shows that Drosophila CUBAM is a tripartite complex composed of dAmnionless and two cubilin paralogues Cubilin and Cubilin-2, and that it is required for nephrocyte slit diaphragm (SD) dynamics. Loss of CUBAM-mediated endocytosis induces dramatic morphological changes in nephrocytes and promotes enlarged ingressions of the external membrane and SD mislocalisation. These phenotypes result in part from an imbalance between endocytosis, strongly impaired in CUBAM mutants, and exocytosis in these highly active cells. Noteworthy, rescuing receptor-mediated endocytosis by Megalin/LRP2 or Rab5 expression only partially restores SD-positioning in CUBAM mutants, suggesting a specific requirement of CUBAM in SD degradation and/or recycling. This finding and the reported expression of CUBAM in podocytes argue for a possible unexpected conserved role of this endocytic receptor in vertebrate SD remodelling (Atienza-Manuel, 2021).

Exploring the relevance of NUP93 variants in steroid-resistant nephrotic syndrome using next generation sequencing and a fly kidney model

Variants in genes encoding nuclear pore complex (NPC) proteins are a newly identified cause of paediatric steroid-resistant nephrotic syndrome (SRNS). Recent reports describing NUP93 variants suggest these could be a significant cause of paediatric onset SRNS. This study report NUP93 cases in the UK and demonstrate in vivo functional effects of Nup93 depletion in a fly (Drosophila melanogaster) nephrocyte model. Three hundred thirty-seven paediatric SRNS patients from the National cohort of patients with Nephrotic Syndrome (NephroS) were whole exome and/or whole genome sequenced. Patients were screened for over 70 genes known to be associated with Nephrotic Syndrome (NS). D. melanogaster Nup93 knockdown was achieved by RNA interference using nephrocyte-restricted drivers. Six novel homozygous and compound heterozygous NUP93 variants were detected in 3 sporadic and 2 familial paediatric onset SRNS characterised histologically by focal segmental glomerulosclerosis (FSGS) and progressing to kidney failure by 12 months from clinical diagnosis. Silencing of the two orthologs of human NUP93 expressed in D. melanogaster, Nup93-1, and Nup93-2 resulted in significant signal reduction of up to 82% in adult pericardial nephrocytes with concomitant disruption of NPC protein expression. Additionally, nephrocyte morphology was highly abnormal in Nup93-1 and Nup93-2 silenced flies surviving to adulthood. This study expanded the spectrum of NUP93 variants detected in paediatric onset SRNS and demonstrate its incidence within a national cohort. Silencing of either D. melanogaster Nup93 ortholog caused a severe nephrocyte phenotype, signaling an important role for the nucleoporin complex in podocyte biology (Bierzynska, 2022).

Insect nephrocyte function is regulated by a store operated calcium entry mechanism controlling endocytosis and Amnionless turnover

Insect nephrocytes are ultrafiltration cells that remove circulating proteins and exogenous toxins from the haemolymph. Experimental disruption of nephrocyte development or function leads to systemic impairment of insect physiology as evidenced by cardiomyopathy, chronic activation of immune signalling and shortening of lifespan. The genetic and structural basis of the nephrocyte's ultrafiltration mechanism is conserved between arthropods and mammals, making them an attractive model for studying human renal function and systemic clearance mechanisms in general. Although dynamic changes to intracellular calcium are fundamental to the function of many cell types, there are currently no studies of intracellular calcium signalling in nephrocytes. This work aimed to characterise calcium signalling in the pericardial nephrocytes of Drosophila melanogaster. To achieve this, a genetically encoded calcium reporter (GCaMP6) was expressed in nephrocytes to monitor intracellular calcium both in vivo within larvae and in vitro within dissected adults. Larval nephrocytes exhibited stochastically timed calcium waves. A calcium signal could be initiated in preparations of adult nephrocytes and abolished by EGTA, or the store operated calcium entry (SOCE) blocker 2-APB, as well as RNAi mediated knockdown of the SOCE genes Stim and Orai. Neither the presence of calcium-free buffer nor EGTA affected the binding of the endocytic cargo albumin to nephrocytes but they did impair the subsequent accumulation of albumin within nephrocytes. Pre-treatment with EGTA, calcium-free buffer or 2-APB led to significantly reduced albumin binding. Knock-down of Stim and Orai was non-lethal, caused an increase to nephrocyte size and reduced albumin binding, reduced the abundance of the endocytic cargo receptor Amnionless and disrupted the localisation of Dumbfounded at the filtration slit diaphragm. These data indicate that pericardial nephrocytes exhibit stochastically timed calcium waves in vivo and that SOCE mediates the localisation of the endocytic co-receptor Amnionless. Identifying the signals both up and downstream of SOCE may highlight mechanisms relevant to the renal and excretory functions of a broad range of species, including humans (Sivakumar, 2022).

PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes

Drosophila nephrocytes are an emerging model system for mammalian podocytes and proximal tubules as well as for the investigation of kidney diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia, phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. This study demonstrates that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development (Gass, 2022).

A protective role for Drosophila Filamin in nephrocytes via Yorkie mediated hypertrophy

Podocytes are specialized epithelial cells of the kidney glomerulus and are an essential part of the filtration barrier. Because of their position, they are exposed to constant biomechanical forces such as shear stress and hydrostatic pressure. These forces increase during disease, resulting in podocyte injury. It is likely podocytes have adaptative responses to help buffer against deleterious mechanical force and thus reduce injury. However, these responses remain largely unknown. In this study, using the Drosophila model, this study show the mechanosensor Cheerio (dFilamin) provides a key protective role in nephrocytes. Expression of an activated mechanosensitive variant of Cheerio rescued filtration function and induced compensatory and hypertrophic growth in nephrocytes depleted of the nephrocyte diaphragm proteins Sns or Duf. Delineating the protective pathway downstream of Cheerio it was found that repression of the Hippo pathway induces nephrocyte hypertrophy, whereas Hippo activation reversed the Cheerio-mediated hypertrophy. Furthermore, it was found that Yorkie was activated upon expression of active Cheerio. Taken together, these data suggest that Cheerio acts via the Hippo pathway to induce hypertrophic growth, as a protective response in abnormal nephrocytes (Koehler, 2022).

Nephrotic Syndrome Gene TBC1D8B is Required for Endosomal Maturation and Nephrin Endocytosis in Drosophila

Variants in TBC1D8B cause nephrotic syndrome. TBC1D8B is a GTPase-activating protein for Rab11 (RAB11-GAP) that interacts with nephrin, but how it controls nephrin trafficking or other podocyte functions remains unclear. A stable deletion was generated in TBC1D8B using microhomology-mediated end joining genome editing. Ex vivo functional assays utilized slit diaphragms in podocyte-like Drosophila nephrocytes. Manipulated endocytic regulators in transgenic mice provided a comprehensive functional analysis of TBC1D8B. A null allele of Drosophila TBC1D8B exhibited nephrocyte-restricted nephrin mislocalization, similar to patients with isolated nephrotic syndrome who have variants in the gene. The protein was required for rapid nephrin turnover in nephrocytes and for endocytosis of nephrin induced by excessive Rab5 activity. The protein expressed from TBC1D8B bearing the edited deletion predominantly localized to mature early endosomes and late endosomes and was required for endocytic cargo processing and degradation. Silencing Hrs, a regulator of endosomal maturation, phenocopied loss of TBC1D8B Low-level expression of murine TBC1D8B rescued loss of the Drosophila gene, indicating evolutionary conservation. Excessive murine TBC1D8B selectively disturbed nephrin dynamics. Finally, four novel TBC1D8B variants were discovered within a cohort of 363 FSGS patients, and functional impact was validated of two variants in Drosophila, suggesting a personalized platform for TBC1D8B-associated FSGS. It is concluded that variants in TBC1D8B are not infrequent among FSGS patients. TBC1D8B, functioning in endosomal maturation and degradation, is essential for nephrin trafficking (Milosavljevic, 2022).

Interaction of the sorting nexin 25 homologue Snazarus with Rab11 balances endocytic and secretory transport and maintains the ultrafiltration diaphragm in nephrocytes

Proper balance of exocytosis and endocytosis is important for the maintenance of plasma membrane lipid and protein homeostasis. This is especially critical in human podocytes and the podocyte-like Drosophila nephrocytes that both use a delicate diaphragm system with evolutionarily conserved components for ultrafiltration. This study shows that the sorting nexin 25 homologue Snazarus (Snz) binds to Rab11 and localizes to Rab11-positive recycling endosomes in Drosophila nephrocytes, unlike in fat cells where it is present in plasma membrane/lipid droplet/endoplasmic reticulum contact sites. loss of Snz leads to redistribution of Rab11 vesicles from the cell periphery and increases endocytic activity in nephrocytes. These changes are accompanied by defects in diaphragm protein distribution that resemble those seen in Rab11 gain-of-function cells. Of note, co-overexpression of Snz rescues diaphragm defects in Rab11 overexpressing cells, whereas snz knockdown in Rab11 overexpressing nephrocytes or simultaneous knockdown of snz and tbc1d8b encoding a Rab11 GTPase-activating protein (GAP) leads to massive expansion of the lacunar system that contains mislocalized diaphragm components: Sns and Pyd/ZO-1. Loss of Snz enhances while its overexpression impairs secretion, which, together with genetic epistasis analyses, suggest that Snz counteracts Rab11 to maintain the diaphragm via setting the proper balance of exocytosis and endocytosis (Maruzs, 2023).

Hydrostatic Pressure Sensing by WNK kinases

Previous work has demonstrated that the WNK kinases 1 and 3 are direct osmosensors consistent with their established role in cell volume control. WNK kinases may also be regulated by hydrostatic pressure. Hydrostatic pressure applied to cells in culture with N(2) gas or to Drosophila Malpighian tubules by centrifugation induces phosphorylation of downstream effectors of endogenous WNKs. In vitro, the autophosphorylation and activity of the unphosphorylated kinase domain of WNK3 (uWNK3) is enhanced to a lesser extent than in cells by 190 kPa applied with N(2) gas. Hydrostatic pressure measurably alters the structure of uWNK3. Data from size exclusion chromatography in line with multi-angle light scattering (SEC-MALS), SEC alone at different back pressures, analytical ultracentrifugation (AUC), NMR, and chemical crosslinking indicate a change in oligomeric structure in the presence of hydrostatic pressure from a WNK3 dimer to a monomer. The effects on the structure are related to those seen with osmolytes. Potential mechanisms of hydrostatic pressure activation of uWNK3 and the relationships of pressure activation to WNK osmosensing are discussed (Humphreys, 2023).

Regulatory sites in the Mon1-Ccz1 complex control Rab5 to Rab7 transition and endosome maturation

Maturation from early to late endosomes depends on the exchange of their marker proteins Rab5 to Rab7. This requires Rab7 activation by its specific guanine nucleotide exchange factor (GEF) Mon1-Ccz1. Efficient GEF activity of this complex on membranes depends on Rab5, thus driving Rab-GTPase exchange on endosomes. However, molecular details on the role of Rab5 in Mon1-Ccz1 activation are unclear. This study identified key features in Mon1 involved in GEF regulation. The intrinsically disordered N-terminal domain of Mon1 was shown to autoinhibit Rab5-dependent GEF activity on membranes. Consequently, Mon1 truncations result in higher GEF activity in vitro and alterations in early endosomal structures in Drosophila nephrocytes. A shift from Rab5 to more Rab7-positive structures in yeast suggests faster endosomal maturation. Using modeling, a conserved Rab5-binding site was identified in Mon1. Mutations impairing Rab5 interaction result in poor GEF activity on membranes and growth defects in vivo. This analysis provides a framework to understand the mechanism of Ras-related in brain (Rab) conversion and organelle maturation along the endomembrane system (Borchers, 2023).

Modeling of ACTN4-Based Podocytopathy Using Drosophila Nephrocytes

Genetic disorders are among the most prevalent causes leading to progressive glomerular disease and, ultimately, end-stage renal disease (ESRD) in children and adolescents. Identification of underlying genetic causes is indispensable for targeted treatment strategies and counseling of affected patients and their families. This study reports on a boy who presented at 4 years of age with proteinuria and biopsy-proven focal segmental glomerulosclerosis (FSGS) that was temporarily responsive to treatment with ciclosporin A. Molecular genetic testing identified a novel mutation in alpha-actinin-4 (p.M240T). A feasible and efficient experimental approach is descibed to test its pathogenicity by combining in silico, in vitro, and in vivo analyses. The de novo p.M240T mutation led to decreased alpha-actinin-4 stability as well as protein mislocalization and actin cytoskeleton rearrangements. Transgenic expression of wild-type human alpha-actinin-4 in Drosophila melanogaster nephrocytes was able to ameliorate phenotypes associated with the knockdown of endogenous actinin. In contrast, p.M240T, as well as other established disease variants p.W59R and p.K255E, failed to rescue these phenotypes, underlining the pathogenicity of the novel alpha-actinin-4 variant. These data highlight that the newly identified alpha-actinin-4 mutation indeed encodes for a disease-causing variant of the protein and promote the Drosophila model as a simple and convenient tool to study monogenic kidney disease in vivo (Odenthal, 2023).

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