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- The Company of Biologists
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- © 2022 The Company of Biologists. All rights reserved.
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- 0022-0949
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- 1477-9145
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- 10.1242/jeb.243293
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Abstract
© 2022. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2022) 225, jeb243293. doi:10.1242/jeb.243293 REVIEW Blending physiology and RNAseq to provide new insights into regulation of epithelial transport: switching between ion secretion and reabsorption 1 2, Dennis Kolosov and Michael J. O’Donnell * ABSTRACT compartment to another. Transepithelial transport of solutes is often an active process as shown in Fig. 1. Secretion of ions across animal This Review addresses the means by which epithelia change the epithelia is exemplified by NaCl secretion by the shark rectal gland, direction of vectorial ion transport. Recent studies have revealed that + + in which primary active transport by the basolateral Na /K -ATPase insect Malpighian (renal) tubules can switch from secreting to + couples the hydrolysis of ATP to the entry of K into the cell and reabsorbing K . When the gut of larval lepidopterans is empty + + + the exit of Na (Fig. 1A). The resultant lowering of Na activity (during the moult cycle) or when the larvae are reared on K -deficient + inside the cell energizes secondary active transport of Cl into the diet, the distal ileac plexus segment of the tubule secretes K from the + + − + cell through the Na /K /2Cl co-transporter. K is then recycled haemolymph into the tubule lumen. By contrast, in larvae reared + by leakage through basolateral K channels which contribute to the on K -rich diet, ions and fluid are reabsorbed from the rectal lumen inside-negative basolateral membrane potential, whereas Cl exits into the perinephric space surrounding the cryptonephridial tubules the cell through apical channels into the lumen of the gland, thus of the rectal complex. Ions and fluid are then transported from contributing to a favourable lumen-negative transepithelial the perinephric space into the lumen of the cryptonephridial tubules, electrical potential which drives movement of Na through the thus supplying the free segments of the tubule downstream. Under + paracellular pathway from blood to lumen. Reabsorption of NaCl by these conditions, some of the K and water in the tubule lumen is epithelia can be accomplished by rearrangement of the same suite of reabsorbed across the cells of the distal ileac plexus, allowing for transporters as for secretion, as in secondary active transport of Cl expansion of haemolymph volume in the rapidly growing larvae, as + in the thick ascending limb of the loop of Henle in the mammalian well as recycling of K and base equivalents. RNA sequencing data + + kidney (Fig. 1B). The Na /K -ATPase is again sited in the reveal large-scale changes in gene transcription that are associated + + − basolateral membrane, but in this case the Na /K /2Cl co- with the switch between ion secretion and ion reabsorption by the transporter and K channels are located in the apical membrane distal ileac plexus. An unexpected finding is the presence of voltage- and the Cl channels are in the basolateral membrane. gated, ligand-gated and mechanosensitive ion channels, normally Switching between ion secretion and ion reabsorption by seen in excitable cells, in Malpighian tubules. Transcriptomic surveys an epithelium is thus typically associated with a rearrangement indicate that these types of channels are also present in multiple other of ion transporter distribution such as that diagrammed in Fig. 1. types of vertebrate and invertebrate epithelia, suggesting that they Such a switch might therefore be expected to be relatively slow, may play novel roles in epithelial cell signalling and regulation of consistent with the time required for synthesis and/or insertion epithelial ion transport. of transporter proteins into the apical and basolateral cell KEY WORDS: Transporting epithelia, Electrophysiology, membranes. For instance, even though the gill epithelium of Next-generation sequencing, Voltage-gated ion channels, salinity-acclimated euryhaline teleosts can rapidly sense changes Transcriptomic survey and mount a rapid physiological response (Kültz, 2015), transcriptomic studies suggest that up to 21 days is required for a Introduction: secretion and reabsorption by epithelia full restructuring of the gill epithelium (Bonzi et al., 2021). Epithelia are sheets of cells that form a barrier between the Estuarine fishes such as Fundulus heteroclitus can quickly adjust to environment and the extracellular fluids. The cells which form the extremes in environmental salinity (Whitehead et al., 2012). And epithelium are joined by junctional complexes that provide a although Fundulus can take 6–48 h to fully adjust gill epithelium selectively permeable barrier between the solutions bathing each morphology (and presumably function) (Marshall et al., 1999; surface of the epithelium and thus mark the boundary between Whitehead et al., 2012), studies using their opercular epithelia the apical and basolateral surfaces of each cell. The hallmark of demonstrated that this estuarine species can rapidly (∼45–60 min) epithelial cell morphology is the asymmetric distribution of adjust their opercular epithelium conductivity (Wood and Marshall, transport proteins in the apical and basolateral membranes. This 1994; Marshall et al., 2000; Daborn et al., 2001). Thus, although an asymmetry polarizes the cell so that it is capable of vectorial immediate adjustment of ion transport in the fish gill can take transport of specific solutes and osmotically obliged water from one place rapidly, a complete ‘180 degree’ turnaround in its function takes days, owing primarily to the notion that different subsets of epithelial subtypes are responsible for giving rise to ion-secreting Department of Biological Sciences, California State University San Marcos, 333 S and ion-reabsorbing cells (Sakamoto et al., 2001; Evans et al., Twin Oaks Valley Road, San Marcos, CA 92096, USA. Department of Biology, 2005). In contrast, studies of the Malpighian (renal) tubule McMaster University, 1280 Main Street West, Hamilton, ON, Canada, L8S 4K1. epithelium in the cabbage looper caterpillar, Trichoplusia ni,have *Author for correspondence (odonnell@mcmaster.ca) shown that a switch between ion reabsorption and secretion can be accomplished in less than 10 min (Kolosov et al., 2018a). D.K., 0000-0003-3581-9991; M.J.O., 0000-0003-3988-6059 Journal of Experimental Biology REVIEW Journal of Experimental Biology (2022) 225, jeb243293. doi:10.1242/jeb.243293 Fig. 1. Model for secretion and reabsorption of NaCl in animal A B Secretory Reabsorptive + + epithelia involving active ion transport by Na /K -ATPase and + + − co-transport via Na /K /2Cl co-transporter. (A) Arrangement of transporters for NaCl secretion exemplified by shark rectal gland. Blood Lumen Blood Lumen (B) Arrangement of transporters for NaCl reabsorption by the thick ascending limb of the loop of Henle of the mammalian kidney. Adapted Na Na from Epstein et al. (1983). K + Na − K Cl + − Na Cl + − K Cl Cl Cl Cl + + Na Na As discussed below, recent work has indicated a role for voltage- of exceptionally high rates of KCl reabsorption from the tubule dependent ion channels in regulating the switch (Kolosov et al., lumen when stimulated (Maddrell, 1978). 2021; Kolosov and O’Donnell, 2019c). Two types of epithelial cell are common to the Malpighian In insects, the Malpighian tubules and hindgut provide the tubules of endopterygote insects such as flies, ants, beetles and functional analogue of the vertebrate kidney. As for other tubular butterflies/moths. Principal and secondary cells have different organs such as the kidney, pancreas and liver in vertebrates, the developmental origins: in Drosophila, principal cells develop tubular morphology of the insect Malpighian tubule provides an from the organ primordium, whereas secondary cells originate increased surface area that enhances interaction with the fluids from central visceral mesoderm recruited to the tubule during bathing the exterior and luminal surfaces and facilitates homeostatic organogenesis (Denholm, 2013). The maturation of principal and transport of solutes and water (Jung et al., 2005). The development secondary cells in the Malpighian tubules of insects which have of the tubule has been well described in the Drosophila larva, in them is governed by two distinct transcription factors: cut and which the Malpighian tubules consist of two pairs of epithelial tiptop/teashirt, respectively (Denholm, 2013). The larger and more tubes that bud from the hindgut during embryogenesis (Jung et al., numerous principal cells are involved in electrogenic cation + + 2005). In each bud, a specialized cell called the tip cell is singled out (Na ,K ) secretion that is energized by an apical vacuolar-type by activation of the mechanosensitive Notch signalling pathway H -ATPase. Cation transport through principal cells is stimulated early in development (Jung et al., 2005). The tip cell is required by diuretic hormones (DHs) which include functional homologs of for the normal pattern of cell division in the development of the the vertebrate corticotropin releasing factor (CRF) and calcitonin tubules (Skaer, 1989). It is worth noting that the Notch pathway is gene-related peptide (CGRP) families, as well as the capa peptides also implicated in regulating the interconversion of intercalated cells which share homology with the vertebrate neuromedin U peptides and principal cells via a transitional cell type in the collecting ducts (Cabrero et al., 2002; Coast et al., 2001; Terhzaz et al., 2012). The of the mammalian kidney nephron (Humphreys, 2018; Park et al., smaller, less numerous secondary cells are intercalated among the 2018). Another similarity with the vertebrate nephron is the division tubule principal cells. In tubules of fruit flies, Cl and water flow of the Malpighian tubules of many species into multiple segments through secondary cells that are called stellate cells for their that are adapted for specific functions. In Drosophila and Rhodnius, distinctive shape (Cabrero et al., 2020, 2014). Cl transport through downstream segments are implicated in reabsorption of K stellate cells is increased by kinin neuropeptides and the biogenic 2+ (Maddrell, 1978; O’Donnell and Maddrell, 1995). In other amine tyramine acting through increases in intracellular Ca insects, such as locusts, reabsorption of solutes and water is (Blumenthal, 2003; Cabrero et al., 2014). A striking difference in accomplished primarily by the hindgut (Phillips et al., 1994). the control of tubule function is seen in beetles (Order Coleoptera). Tubules of the water boatman have four segments differentiated on Beetle tubules lack the kinin pathway and the secondary cells are the the basis of rates of ion transport per unit length of tubule and site of action of CRF-related hormones DH37 and DH47 which capacity for secretion of alkaline equivalents and acidic dyes stimulate K flux via a cAMP-dependent mechanism (Koyama (Cooper et al., 1989), whereas larvae of many lepidopteran species et al., 2021). In conjunction with the presence of potent antidiuretic have at least five morphologically distinct segments that differ factors that act upon beetle tubules, these differences may provide + + − functionally with regards to transport of K ,Na ,Cl , acid–base tighter control of excretory water loss in beetles, a group which equivalents, toxins and uric acid (Irvine, 1969; Kolosov and thrives in osmotically stressful biomes (Koyama et al., 2021). O’Donnell, 2019c; Kolosov and O’Donnell, 2020; O’Donnell and Ruiz-Sanchez, 2015; Ruiz-Sanchez et al., 2015). Regional Changes in dietary ion availability mediate the switch specializations of Malpighian tubules of insects can thus rival from ion secretion to reabsorption in caterpillar those of the vertebrate nephron. Even within tubule regions of Malpighian tubules apparently uniform structure, there may be dramatic functional Lepidopteran larvae exhibit the so-called cryptonephric condition, differences. The upper region of the lower tubule of Rhodnius where the distal ends of the Malpighian (renal) tubules are applied prolixus is osmotically very permeable and plays no part in to the outer surface of the rectum and enveloped by a perinephric reabsorbing KCl from the primary excretory fluid, whereas membrane, forming the rectal complex (Fig. 2). Emerging from morphologically identical cells in the lowermost 30% of the the rectal complex are the free segments of the tubule; whereas the length of the lower tubule are osmotically impermeable and capable cryptonephric tubules are bathed in the fluid contained with the Journal of Experimental Biology REVIEW Journal of Experimental Biology (2022) 225, jeb243293. doi:10.1242/jeb.243293 Fig. 2. Anatomical arrangements of caterpillar Malpighian tubules, their associations with regions of the gut and the functional switch between two sources of ions – haemolymph and gut. (A) In the larva, the distal end of each tubule, termed the cryptonephridial Malpighian tubule (cMT), lies within the rectal complex (in longitudinal Rectum Ileum section, dashed blue line, top panel). The short segment termed the rectal lead (RL) connects each cMT to the ‘free’ tubule, of which there are four distinct regions. The PNS downstream distal ileac plexus (DIP, purple) is arranged close to the ileum and contains most of the secondary cells DIP PIP YR cMT RL (purple circles). The proximal ileac plexus (PIP) then terminates in the downstream yellow and white regions (YR and WR, respectively) that are closely applied to the posterior midgut. The Malpighian tubule (MT) then terminates in the urinary bladder, which empties into the midgut–hindgut junction. The DIP, PIP, YR and WR show Ileum Bladder heterogeneity in ultrastructure and gene expression. PNS, perinephric space. (B) Larvae have two options as a Midgut source of ions and water for secreting fluid into the MT. The reabsorptive DIP epithelium (in purple) sources ions and water from the gut by way of the cMT when the caterpillars Rectal complex Ileac plexus WR are well fed with ion-rich diets. Some ions reabsorbed from the gut are transferred into the haemolymph across the free regions of the MT. The secretory DIP epithelium (in red) sources ions and water from the larva’s haemolymph, when Reabsorptive DIP Secretory DIP dietary ions and water are unavailable (e.g. during moulting) (ions sourced from gut) (ions sourced from haemolymph) or are in short supply. Haemolymph Haemolymph Lumen Lumen To PIP DIP Rectum Ileum Midgut perinephric space, the free tubules are bathed in haemolymph. allows the haemolymph to be continuously cleared of toxins When the larvae are not feeding (e.g. during the moult cycle) or and metabolic waste irrespective of dietary ion and water intake. when the diet is deficient in K , the distal ileac plexus region of the Recent studies have examined the switch between the two + + − free tubule functions in secretory mode: secretion of K ,Na ,Cl ion transport modes using tissue and cell-specific functional and osmotically obliged water by the principal cells drives fluid assays, immunohistochemical identification of ion transporters, secretion, flushing the tubule lumen so that toxins and waste can be electron microscopy, quantitative PCR and transcriptomic RNAseq cleared from the haemolymph. When the larvae are feeding on K - approaches. The following sections describe the suite of cellular and + − rich foods, transport of K and HCO into the midgut lumen molecular changes that occur during the switch from ion secretion to elevates the pH of the gut contents, breaking tannin–protein bonds reabsorption. to enhance digestion of the plant proteins in the diet (Berenbaum, There is a pronounced downregulation of transcript abundance of 1980; Moffett, 1994). Water and ions are subsequently reabsorbed secretory ATPases and K transporters in principal cells of the distal downstream from the rectal lumen and secreted into the lumen of the ileac plexus during the switch to reabsorption elicited by increased cryptonephridial segment of the Malpighian tubule. Some of the levels of K in the diet (Fig. 3). Shutting down secretory ATPases water and ions are then reabsorbed into the haemolymph across the and changes in abundance of passive transporters allows ion downstream free segments of the tubule, allowing both expansion of transport to reverse from ion secretion to reabsorption within 24 h in haemolymph volume in rapidly growing larvae and recycling of K response to increased ion availability in the diet (Kolosov et al., and base (HCO ). Switching between secretion and reabsorption 2019a). Some transporters such as the potassium/chloride co- by the distal ileac plexus means that either the haemolymph or the transporter (KCC) and sodium/proton exchangers (NHEs) may hindgut can provide a source of ions and water for fluid secretion by reverse the direction in which they transport ions when the tubule the tubules (Fig. 2). One advantage of this arrangement is that it switches from ion secretion to reabsorption. Journal of Experimental Biology REVIEW Journal of Experimental Biology (2022) 225, jeb243293. doi:10.1242/jeb.243293 Secretory Toxins Toxins Xenobiotics Xenobiotics Haemolymph Cl OC , Cl + + – – + + K K Cl Cl Na K – NH H O 2+ 2+ Cl 3 2 Mg ,Zn ? Gly CBE OCT/ NKCC K ATOX/ NKA ir CBE Rh b AQP MRP Na CIC – HCO CBE 3 + + + H Na /K – – Cl Cl HCO OCT/ + + 3 CIC CPA Na /K VA ATOX/ CBE Sym Rh b AQP MRP Na Sugar HCO AA XCOO Lumen Reabsorptive Haemolymph Cl OC , Cl NH H O 2+ 2+ – 3 2 Mg ,Zn Cl HCO ? ? Cl HCO + + Na /K + + – Na /K HCO Na Sugar H AA XCOO Toxins Toxins Lumen Xenobiotics Xenobiotics Haemolymph Tubule lumen AQP SC SC Ion flow H O flow H O flow Ion flow SC + + + + Haemolymph Na K Na K INX INX PC PC PC PC Tubule lumen Tubule lumen Fig. 3. Cellular and molecular changes between the distal ileac plexus in secretory mode versus reabsorptive mode. (A) In secretory mode, K is secreted + + + + + − by the principal cells and reabsorbed by the secondary cells. K secretion is enabled by a combined action of basolateral Na /K -ATPase (NKA), Na /K /2Cl + + co-transporter (NKCC) and inward-rectifier K channels (K ) of the principal cells and apical V-type H -ATPase (VA) and cation–proton antiporters (CPA). ir Some potassium ions are diverted (blue arrows) from surrounding principal cells to the secondary cell via gap junctions (in red) and reabsorbed back into the + + haemolymph via the basolateral membrane of the secondary cells. Apical entry of K and Na from the tubule lumen through unspecified channels (labelled ‘?’)is − − − also possible (dashed blue arrow). Cl is secreted by both principal cells and secondary cells by the combined action of NKCC, Cl channels (ClC) and Cl / − − HCO exchangers (CBE). Base reabsorption is connected with Cl secretion via CBEs. Reabsorption of sugars, amino acids and carboxylates is enabled by symporters (Sym). Water is transported by aquaporins (AQP) and ammonia is transported by Rhesus protein b (Rh b). Organic cations and heavy metals are secreted by organic cation transporters (OCT), multidrug resistance proteins (MRP) and metal trafficking proteins (ATOX). Toxins and xenobiotics with no specific transporter can move into the lumen via the paracellular pathway through the septate junctions between epithelial cells. (B) In reabsorptive mode, both principal and secondary cells reabsorb K , although exact molecular mechanisms of this process remain largely unstudied. Dashed black lines denote reduced rates of transport through the indicated pathway or transporter. All K -secretory transporters are transcriptionally downregulated. Paracellular permeability and water permeability are downregulated as well to prevent the back-flux of water and toxins from the tubule lumen. Secondary processes connected to ion secretion in this segment (e.g. excretion of nitrogenous waste and nutrient reabsorption) are downregulated as well. Transcriptomic evidence suggests that while Cl -coupled base reabsorption via principal cells is minimized during the switch-over from ion secretion to ion reabsorption, the secondary cells provide a separate pathway for base reabsorption. Gap junctional coupling between principal and secondary cells also decreases. (C) In secretory mode, increased gap junctional coupling provides a way for the secondary cells (SC) to return ions and water (secreted by principal cells, PC) back into the larva’s haemolymph. (D) The MTs clear the haemolymph of waste and toxins more efficiently because of the presence of recycling loops of ions and water (in blue) as the fluid flows through the tubule. Journal of Experimental Biology REVIEW Journal of Experimental Biology (2022) 225, jeb243293. doi:10.1242/jeb.243293 The mRNA abundance of xenobiotic/metal transporters in the Several regions of the free Malpighian tubules can switch − − distal ileac plexus also decreases substantially when larvae are between Cl secretion and Cl reabsorption within minutes in a + + reared (for ∼2 weeks) on K -rich or Na -rich diets and are thus in manner similar to that seen for cation transport reversal in the distal reabsorptive mode (Kolosov et al., 2019a). Similarly, transcript ileac plexus (Kolosov and O’Donnell, 2020). Within the distal ileac abundance of the ammonium transporter Rhesus protein b (Rh b), plexus, both principal cells and secondary cells secrete Cl , + + − and components of transporters for H - and Na -dependent although principal cells can switch to Cl reabsorption when the reabsorption of trehalose and amino acids decreases. Together, the bathing saline Cl concentration is increased. The mechanism results suggest that less toxin excretion and nutrient reabsorption underlying the reversal of Cl transport from secretion to + + occurs when the distal ileac plexus is reabsorbing K and/or Na and reabsorption is unclear. However, an important role of the water. secondary cells in the distal ileac plexus is to provide a pathway Gap junctional coupling between principal cells and secondary for HCO reabsorption that is independent of the principal cells, + + + − cells is also downregulated when larvae are reared on K -rich diet which may switch between ion secretion (Na ,K ,Cl ) and (for ∼2 weeks) and the principal cells are reabsorbing (Kolosov reabsorption depending upon dietary ion availability. HCO et al., 2018a). By contrast, when the tubules are in secretory mode, reabsorption likely involves chloride/bicarbonate exchangers principal cells of the distal ileac plexus secrete K whereas adjacent (CBEs) in the distal ileac plexus (Kolosov and O’Donnell, 2020). + − secondary cells reabsorb K , part of which is routed from the Application of drugs which block Cl channels or CBEs leads to an neighbouring principal cells through gap junctions (Fig. 3C). The increase in lumen pH, consistent with coupling between Cl juxtaposition of secondary cells and principal cells in the loops of secretion into the lumen and HCO reabsorption into the tubule within the ileac plexus allows for local recycling of ions and haemolymph (Kolosov and O’Donnell, 2020). water; K reabsorbed by secondary cells may be secreted back into the lumen by principal cells of a juxtaposed loop of tubule (Fig. 3D). Neuropeptides may orchestrate a well-coordinated switch + + This recirculation system for Na ,K and osmotically obliged water between ion secretion and reabsorption and provide may enhance clearance of waste and toxins from the haemolymph. potential links for autocrine and paracrine control of The switch from ion secretion to reabsorption is also associated Malpighian tubule function with a reduction in paracellular permeability and water The widespread changes in cell structure and function in the distal permeability. Measurements using a fluorescent extracellular ileac plexus during the switch from ion secretion to reabsorption permeability marker (FITC-dextran) revealed that paracellular may be orchestrated, in part, by helicokinins, which are members of permeability in the distal ileac plexus is reduced in larvae reared the kinin family of myotropic and diuretic peptides. Helicokinin I + + + − (for ∼2 weeks) on Na -rich or K -rich diet (Kolosov et al., 2019b). acts as a K - and Cl -sparing natriuretic on the distal ileac plexus Electron micrographs show that the distal ileac plexus epithelium of (Kolosov and O’Donnell, 2019a; Kolosov and O’Donnell, 2020) larvae fed ion-rich diets (for ∼24 h) has longer and more convoluted and produces many of the effects seen during the switch from ion septate junctions between adjacent principal cells, consistent with secretion to reabsorption when larvae are fed ion-rich diets: + − the observed reduction in paracellular permeability (Kolosov et al., helicokinin I reduces K and Cl secretion by principal cells and 2019b). Reduction in paracellular permeability may be aimed at Na reabsorption by secondary cells and it also reduces water minimizing the back-flux of organic ions, waste metabolites and permeability and paracellular junction permeability. These effects secreted toxins into the haemolymph. Concomitant reductions in may be explained in part by reduced transcript abundance for a Na / + + − water permeability will limit water flux accompanying K K /2Cl co-transporter, two NHEs (nhe-7 and -8) as well as the + + reabsorption (Kolosov and O’Donnell, 2019b), thus retaining alpha subunit of the Na /K -ATPase and aquaporin 1 (Kolosov and water in the lumen so that the concentrations of toxins and waste O’Donnell, 2019a). The effects of helicokinin I on both principal remain low, further limiting diffusive back-flux of these solutes into and secondary cells of the distal ileac plexus differ dramatically the haemolymph, as does the reduction in expression of xenobiotic from the diuretic effects of kinins on dipteran Malpighian tubules. In and metal transporters. tubules of fruit flies, for example, kinins act specifically on the From the experimental evidence described above, it is evident stellate cells to increase fluid secretion through effects on Cl that the switch from ion secretion to ion reabsorption can take place channels and aquaporins (Cabrero et al., 2020, 2014). in as little as 24 h and as long as 2 weeks, where transcriptional changes (presumably aimed at restructuring the Malpighian tubule Controlling the direction of vectorial ion transport: voltage- epithelium) take place, coinciding with the changes in ion transport, gated, mechanosensitive and ligand-gated ion channels are paracellular permeability and water permeability. Despite all of implicated in the switch between ion secretion and ion these long-term changes, the switch from ion reabsorption to ion reabsorption secretion can take as little as ∼10 min, indicating that although the The presence of voltage-gated ion channels in non-excitable cells phenotype of the epithelium is adjusted, it is not restructured challenges the paradigm that such channels are involved primarily permanently. Not much is known about the time course of this in generating signals in nervous tissues (Pitt et al., 2020). RNAseq process as animal dissection, isolation and mounting of the has identified multiple voltage-gated, mechanosensitive and ligand- preparation takes at least 10 min. Time course measurements will gated ion channels in the Malpighian tubules of T. ni (Kolosov et al., 2+ require an intricate setup with intracellular electrodes and/or luminal 2019a,c) (Fig. 4). A recent study has shown that Ca entry through perfusions. We know that the first thing a tubule experiences upon voltage-gated calcium channels enables robust ion secretion by the excision from the animals is a drop in hydrostatic pressure in the distal ileac plexus (Kolosov et al., 2021) (Fig. 4A). lumen and a subsequent drop in fluid flow. We believe these may be Immunohistochemistry has revealed voltage-gated calcium the triggers behind the short-term changes in the direction of ion channels in both apical and basolateral membranes of the transport, which act through voltage-gated ion channels, as when principal cells. In addition, transcript abundance of the voltage- 2+ the latter are blocked, robust ion secretion reverts back to ion sensing subunit of a voltage-gated Ca channel is downregulated in reabsorption. the distal ileac plexus of larvae reared on a K -rich diet, and this Journal of Experimental Biology REVIEW Journal of Experimental Biology (2022) 225, jeb243293. doi:10.1242/jeb.243293 Fig. 4. Voltage-gated, mechanosensitive and Haemolymph ligand-gated ion channels regulate ion transport in the Malpighian tubules of larval lepidopterans. Extracellular cues? + (A) Activity of voltage-gated Ca 1 channel and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is necessary for robust secretion in NKA NKCC HCN the Malpighian tubules (MT) of larval lepidopterans. Ca Both are thought to stimulate active ion transport machinery in the distal ileac plexus. Ca 1 relays its 2+ action via an increase in intracellular [Ca ]. The [Ca ] contribution of most voltage-gated ion channels to the regulation of ion transport remains unstudied (see text). (B) Mechanosensitive degenerin/epithelial Na VA CPA channels (Deg/ENaC), transient receptor potential Ca (TRP) and piezo channels are expressed in the MT and may translate changes in hydrostatic pressure and/or fluid flow in the lumen into changes in membrane potential (flash sign in a black circle), which Extracellular cues? can be detected by the voltage-gated ion channels. Lumen Transcriptomic evidence also suggests that several neuropeptides (indicated by black dots in a white circle) are encoded by the epithelia and that their release may be used for autocrine/paracrine regulation Haemolymph of ion transport by as yet unknown pathways (indicated by ‘?’). (C) Several ligand-gated ion channels and metabotropic receptors for the same ligands have been detected in larval MTs. Stimulation of MTs with glycine (purple square) leads to increased Cl secretion by the secondary cells without affecting principal cells. In contrast, application of γ- aminobutyric acid (GABA, purple triangle) reduces K + − secretion and Na reabsorption, and increases Cl secretion by the principal cells. The effects of Deg/ Piezo TRP ENaC glutamate (blue hexagon) on MT ion transport remain unstudied, despite the fact that several glutamate receptors (GluR) are expressed in the MTs. Lumen Glycine Cl Glutamate Glutamate GABA Haemolymph + + Na K – Cl Gly Glu GABA Glu R R CIC Lumen change, together with other transcriptional changes, enables the receive the depolarizing stimulus necessary to open and support 2+ switch to ion reabsorption. In vitro, pharmacological inhibition of Ca influx in the absence of an action potential? 2+ 2+ the voltage-gated Ca channel Ca 1 reduces intracellular Ca There are several mechanisms by which changes in hydrostatic concentration and reverses the direction of K transport from pressure, ion concentration or pH inside the tubule lumen in 2+ secretion to reabsorption (Kolosov et al., 2021). Although Ca response to dietary ion loading could lead to the switch between entry through voltage-gated channels is clearly required for ion secretory and reabsorptive modes of the distal ileac plexus. One of secretion by the tubule, an important question remains: as the these is a potential link of voltage-gated ion channels and tubules are non-excitable, how do voltage-gated calcium channels mechanosensation in the tubule (Fig. 4B). There are three Journal of Experimental Biology REVIEW Journal of Experimental Biology (2022) 225, jeb243293. doi:10.1242/jeb.243293 mechanosensitive channels expressed in the distal ileac plexus: (Fig. 5). Back-flux of K from the tubule lumen to the haemolymph piezo, transient receptor potential channel painless (TRP painless), through the paracellular pathway provides a mechanism by which and a member of the degenerin/epithelial Na channels (Deg/ENaC) the basolateral membrane voltage can generate a current that flows family (Kolosov et al., 2019a). In studies of other epithelia, stretch through the septate junctions and charges up the apical membrane, activation of piezo in alveolar type I cells of the mammalian lung and vice versa (Fig. 5). For example, depolarization of the 2+ + leads to increases in intracellular Ca concentration and release of basolateral membrane by K inflow through K channels in the ir ATP that in turn acts as a paracrine stimulation of surfactant principal cells could lead through crosstalk to a depolarization of the 2+ secretion by alveolar type II cells (Diem et al., 2020). Piezo1 is also apical membrane that may, in turn, open voltage-gated Ca 2+ implicated in urinary dilution and the decrease in urea concentration channels, allowing an influx of Ca and consequent intracellular in the kidney following rehydration in mammals, possibly through signalling. The contributions of other K channels to ion transport ir 2+ Piezo1-dependent changes in intracellular [Ca ], leading to regulation and their cellular/membrane localization remain decreased cAMP levels and enhanced retrieval of aquaporin 2 unstudied in the distal ileac plexus of T. ni. from the plasma membrane (Martins et al., 2016). An important The paracellular pathway provides another means by which aspect of future work will be to determine how piezo channels in the voltage-gated ion channels may regulate vectorial ion transport in distal ileac plexus of caterpillar Malpighian tubules are activated so the tubules. MTs of Aedes aegypti transport Cl (in part) via septate 2+ as to lead to Ca entry. Increased fluid secretion rates in response to junctions (Beyenbach, 2003), which can in principle offset the 2+ higher levels of intracellular Ca will require both higher levels of apical membrane potential enough for voltage-gated ion channels to + + active ion transport by the basolateral Na /K -ATPase and the react to this change. apical V-ATPase in the principal cells, as well as increased K and There are receptors for multiple neurotransmitters in the distal Cl entry through channels, co-transporters or exchangers. In this ileac plexus, including glycine, γ-amino butyric acid (GABA), 2+ + − context, it is worth noting that there are Ca -gated K and Cl serotonin, glutamate (metabotropic and ionotropic), acetylcholine channels expressed in the distal ileac plexus (Kolosov and and catecholamines (α1A adrenergic) (Kolosov et al., 2019a). O’Donnell, 2020), and these can be activated by fluctuations in Application of serotonin to the yellow region isolated together with intracellular calcium concentration. In addition to piezo, TRP the ileac plexus increases the secretion of fluid and Na and painless channels are non-selective cation channels which could decreases secretion of K (Ruiz-Sanchez et al., 2015). Both glycine 2+ − also mediate Ca entry in response to membrane stretch (Goodman, and GABA alter Cl secretion by the distal ileac plexus. Glycine 2003). Similar to piezo, studies connect TRP V family ion channels stimulates Cl secretion by the secondary cells (Kolosov and 2+ + to Ca -mediated activation of BK channels in K -secreting renal O’Donnell, 2019c), whereas GABA acts upon the principal cells, + + − collecting duct cells in mice (Li et al., 2018). Although Deg/ENaC reducing K secretion and Na reabsorption and increasing Cl channels are not voltage gated and are sodium selective (Eastwood secretion (Kolosov and O’Donnell, 2019c). The advantage of such a and Goodman, 2012), enhanced Na current through the channel multiplicity of messengers is unclear, but it is worth noting that 2+ could lead to a depolarization that would open voltage-gated Ca multiple peptides and amines also stimulate fluid secretion in 2+ channels in the apical membrane and thus lead indirectly to a Ca tubules of the adult tobacco hawkmoth, Manduca sexta (Skaer et al., influx. 2002). In view of the large number of compounds that stimulate RNAseq has also revealed transcripts for hyperpolarization- fluid secretion by the tubules, Skaer et al. (2002) suggest that rather activated cyclic nucleotide gated (HCN) channels in the distal ileac than single compounds eliciting stereotypical responses from a plexus. HCN channels are unusual in that they are activated by single tissue, there is continuous broadcast of information in the hyperpolarization, leading typically to an influx of Na , primarily, form of a ‘chemical language’ within the extracellular fluids; this + 2+ with smaller amounts of K and Ca (Wahl-Schott and Biel, 2009) language coordinates the functions of multiple tissues, including the (Fig. 4A). HCN channels are also regulated by intracellular and Malpighian tubules. extracellular pH, raising the possibility that pH changes in the tubule The discussions above indicate that there are multiple lumen during feeding could alter their opening. HCN channels have mechanisms which play a role in the switch between ion secretion been directly implicated in the regulation of ion transport by the and ion reabsorption. The switch takes place under physiologically distal ileac plexus; application of the HCN blocker ZD7288 to relevant conditions (e.g. ingesting ion-rich diet, moulting), and + + isolated tubules results in a switch from K secretion to K some of the above-described mechanisms are able on their own to reabsorption by the principal cells, while secondary cells remained induce a change in the direction of ion transport. For instance, unaffected (Kolosov and O’Donnell, 2019c). Current flow through pharmacological closing of gap junctions (Kolosov et al., 2018a) voltage-dependent HCN channels could also be altered as a and inhibition of HCN channels (Kolosov and O’Donnell, 2019c) secondary response to the operation of electrogenic chloride/ induce switching from ion secretion to ion reabsorption in the distal − − bicarbonate exchangers (Cl /2HCO ). For example, increased ileac plexus. In contrast, pharmacological inhibition of Ca 1 3 V bicarbonate concentration in fluid reabsorbed from the rectal lumen (Kolosov et al., 2021), application of HK-1 (Kolosov and and then into the cryptonephridial tubules in feeding larvae could O’Donnell, 2019a) and stimulation of ligand-gated ion channels increase lumen pH in the distal ileac plexus, thus modulating pH- (Kolosov and O’Donnell, 2019c, 2020) regulate transport of sensitive HCN channels. specific ions without switching the distal ileac plexus between ion Several inward-rectifier K channels are also expressed in the secretion and ion reabsorption. There are several potential reasons region of the tubule that switches between ion secretion and ion for this complexity. Firstly, the magnitude of the cellular response reabsorption. Basolateral K 1 channels that are blocked by the small may matter – blocking gap junctions and hyperpolarization- ir molecule inhibitor VU591 play an important role in enabling activated channels is likely to change the membrane potential of secretion (Kolosov et al., 2018b). As in vertebrate epithelia such as the whole cell and cause a downstream response, resulting in the ion the kidney nephron, leakage of current through paracellular shunt transport switch from ion secretion to ion reabsorption. In contrast, pathways allows for crosstalk between the apical and basolateral cell pharmacologically blocking or activating ion channels that conduct membranes of Malpighian tubules (Pannabecker et al., 1992) a single ion species may change the membrane potential and fine- Journal of Experimental Biology REVIEW Journal of Experimental Biology (2022) 225, jeb243293. doi:10.1242/jeb.243293 V +8 +70 mV te V –25 +25 mV bl V –95 +17 mV? ap –25 mV –65 mV +40 mV –1 ∼120 mmol l NKCC + + K K NKA –1 80 mmol l CPA VA K ir –1 25 mmol l Fig. 5. Cross-talk between apical and basolateral membrane and typical membrane potential values and luminal, haemolymph and intracellular K + + −1 concentrations. K is secreted across principal cells in the distal ileac plexus of larval lepidopterans. Typical haemolymph [K ] is 25 mmol l . Basolateral entry + + + + − + can be active via combined action of Na /K -ATPase (NKA) and Na /K /2Cl co-transporter (NKCC), and/or passive via inward-rectifier K (K ) channels. Active ir + + + + transport across the apical membrane occurs by combined action of V-type H -ATPase and K /H antiporters (CPA). As K accumulates in the lumen −1 (∼80 mmol l ), paracellular back-flux via septate junctions can allow for cross-talk between apical and basolateral membrane potential (V and V ), so changes ap bl in membrane potential on either membrane can affect each other. Transepithelial potential (V ) across the distal ileac plexus can fluctuate between +8 mV and te +70 mV lumen-positive and V can fluctuate between −25 mV inside-negative to +25 mV inside-positive. Therefore, V may fluctuate between −95 mV inside- bl ap negative and +17 mV inside-positive. These wide voltage ranges across basolateral and apical membranes span the activation ranges for many voltage- dependent ion channels. Intracellular [K ] has not been measured directly in tubules of T. ni, but most reports in insect tubules indicate values of 60 to −1 120 mmol l . Resistance across septate junctions and both membranes is illustrated with resistor symbols. The ion pumps, exchangers, co-transporters and channels in the apical and basolateral membranes behave as an electromotive force, indicated by the battery symbols, that makes the cell interior negative. tune ion transport without bringing about the more widespread to ion reabsorption, may be important – depending on how far cellular changes required for switching the direction of ion downstream the molecular machinery is, its experimental transport. Secondly, the downstream/upstream position in the manipulation may result in a complete or partial switch from signalling cascade that controls the switch-over from ion secretion transepithelial ion secretion to reabsorption. Journal of Experimental Biology REVIEW Journal of Experimental Biology (2022) 225, jeb243293. doi:10.1242/jeb.243293 ‘Big Data’ provide new insights into regulation of epithelial identified, as well as expression of voltage-gated channels that are ion transport: the impact of RNAseq on studies of epithelial non-selective and cation permeable. Many of these channels are 2+ transport in less-studied species involved in the regulation of intracellular Ca signalling, osmotic For epithelia in model species such as Drosophila, earlier stress response, extracellular ion sensing and modulation of microarray analyses of gene expression have been greatly vectorial ion transport (Abbott, 2014; Bleich and Warth, 2000; extended by the more recent application of RNAseq (Chintapalli Demolombe et al., 2001; Morera et al., 2015; Nilius and et al., 2007; Leader et al., 2018; Robinson et al., 2013). Mammalian Droogmans, 2001; Schönherr et al., 2000; Shi et al., 1997; Siroky epithelia such as the kidney were early candidates for RNAseq et al., 2017; Yang and Cui, 2015; Zhu et al., 2010). (Mimura et al., 2014), as were model organisms and tissues used in A likely advantage of the presence of voltage-gated, ligand- studies of vertebrate transporting epithelia, such as the pufferfish gill gated and mechanosensitive ion channels in animal epithelia is the (Cui et al., 2014). The decreasing cost of next-generation ability of these channels to respond quickly to changing sequencing in recent years and the availability of genomic data environmental and systemic variables. In contrast to motile cilia for non-model species has made it possible to design transcriptomic in specialized cell types, the immotile, primary cilia that protrude experiments to uncover novel and previously unaccounted for ion from the surface of most types of mammalian epithelial cell receive transport and regulatory mechanisms in epithelia other than those of stimuli from the environment and transduce the information into model insect species such as Drosophila. an intracellular response (Wachten and Mick, 2021). Primary For example, transcriptomic analysis of Malpighian tubules of cilia on renal cells are thought to act as mechanosensors that + − larval and adult mosquitoes (A. aegypti) identified Na and Cl detect fluid flow. Although molecular mechanisms underlying the channels whose expression is highly enriched in the tubules of the sensory function of primary cilia are not well understood, blood-feeding adult females (Li et al., 2017). These channels are G-protein-coupled receptors (GPCRs) that produce changes in the 2+ candidates for transporters hypothesized in a current model of fluid concentrations of intracellular second messengers (cAMP, Ca )are transport by the tubules, namely a basolateral sodium channel, generally thought to be involved. In intestinal epithelia, GPCRs which allows the entry of Na into the principal cells from the that are responsive to a range of nutrients have been identified haemolymph, and an apical chloride channel, which allows the (Moran et al., 2021). One of these GPCRs, gustducin, can stimulate movement of Cl from the stellate cells to the tubule lumen (Hine both phosphodiesterase, to cause cAMP degradation, and 2+ et al., 2014). RNAseq studies of the Asian tiger mosquito, Aedes phospholipase C, leading to inositol trisphosphate-mediated Ca 2+ albopictus, indicate that blood feeding alters the Malpighian tubule release. The subsequent increase in cytoplasmic Ca activates a 2+ epithelium from one specializing in active transepithelial fluid Ca -sensitive transient receptor potential (TRP) channel M5, secretion to one specialized for detoxification and metabolic waste thus triggering membrane depolarization and opening of voltage- 2+ 2+ excretion (Esquivel et al., 2014). Digestion of haemoglobin from gated Ca channels, amplifying the Ca signal. In kidney and the blood meal leads to the production of haeme, a toxic metabolite intestinal epithelia, the calcium-sensing receptor (CaSR) is a GPCR that causes cell and tissue damage through oxidative stress, so that senses several key nutrients. The primary ligand for CaSR is 2+ activation of multiple mechanisms for haeme detoxification is extracellular Ca , but in intestinal epithelia it can also be activated adaptive. allosterically by L-amino acids (Moran et al., 2021). In the proximal Previous studies of the control of fluid secretion by Malpighian tubule of the kidney, activation of CaSR by an increase in luminal 2+ tubules of the caterpillar T. ni have identified multiple molecules Ca concentration leads to an increase in sodium-dependent proton with diuretic effects, including amines and peptides. RNAseq of the extrusion and fluid reabsorption (Capasso et al., 2013). tubules led to the serendipitous discovery of transcriptomic evidence for a glycine receptor in the distal ileac plexus segment Future directions of the tubule. This discovery prompted subsequent functional tests In summary, although expression of voltage-gated, mechanosensitive which revealed glycine modulation of ion transport (Kolosov and and ligand-gated ion channels in epithelia of different animal clades is O’Donnell, 2020). The progression of understanding in epithelial well reported, there is very little understanding of: (i) whether the role physiology often fits a sequence: observation of a phenomenon, of a particular ion channel is conserved across epithelia of different followed by identification of the tissues and cells involved, animal clades, (ii) whether these novel ion transport regulators are pharmacological evidence for involvement of particular proteins connected to any other aspects of epithelial function (e.g. junctional (e.g. ion transporters), and identification of the genes which code for or water permeability), and (iii) what precisely activates voltage- those proteins. RNAseq offers the opportunity to turn this sequence gated ion channels in animal epithelia, and specifically whether of events around, so that identification of genes that are richly mechanosensation and voltage-gated ion channels are connected or expressed or unexpectedly present in a tissue (e.g. glycine receptors) are parts of two separate signalling networks aimed at adjustment of may lead to functional assays that reveal previously unobserved epithelial ion transport. phenomena (e.g. glycine stimulation of tubule Cl secretion). For example, switching between ion secretion and ion reabsorption in the Malpighian tubules of larval lepidopterans involves Functions of voltage-gated, ligand-gated and simultaneous fine-tuning of ion transport, water permeability, mechanosensitive ion channels in epithelia of other clades paracellular permeability and gap junctional coupling. There is a Voltage-gated, ligand-gated and mechanosensitive ion channels gap in our understanding of whether voltage-gated ion channels have been detected in epithelia of several vertebrates and regulate only ion transport, or whether they regulate all four aspects of invertebrates using publicly available RNAseq datasets (Kapoor the switch-over. Any links between the presence of voltage-gated, et al., 2021). Studies on vertebrate (and most often mammalian) ligand-gated and mechanosensitive ion channels and the regulation of epithelia have reported expression of voltage-gated ion channels in paracellular permeability, gap junctional coupling and water epithelia of the lung, intestine (Barshack et al., 2008), kidney permeability even in the Malpighian tubules of larval lepidopterans (Siroky et al., 2017) and skin (Pitt et al., 2021). 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Journal of Experimental Biology – The Company of Biologists
Published: Mar 8, 2022