Claudins of sea lamprey (Petromyzon marinus) – organ‐specific expression and transcriptional responses to water of varying ion content
The role of lamprey epithelium tight junctions (TJs) in the regulation of salt and water balance is poorly understood. This study reports on claudin (Cldn) TJ protein transcripts of pre‐metamorphic larval and post‐metamorphic juvenile sea lamprey (Petromyzon marinus) and the transcriptional response of genes encoding Cldns to changed environmental ion levels. Transcripts encoding Cldn‐3b, −4, −5, −10, −14, −18 and − 19 were identified and mRNA expression profiles revealed organ‐specific presence of cldn‐5and ‐14, broad expression of cldn‐3b,‐4, −10, −18 and − 19, as well as spatial differences in the mRNA abundance of cldn‐4, −3b and − 14 along the ammocoete intestine. Expression profiles were qualitatively similar in ammocoetes and juvenile fishes. Transcript abundance of genes encoding Cldns in osmoregulatory organs (gill, kidney, intestine and skin) were subsequently investigated following exposure of ammocoetes to ion‐poor water (IPW) and juveniles to hyperosmotic conditions (60% seawater, SW). IPW‐acclimated ammocoetes increased mRNA abundance of nearly all cldns in the gill. Simultaneously, cldn‐10 abundance increased in the skin, while cldn‐4, −14 and − 18decreased in the kidney. Ammocoete cldn mRNA abundance in the intestine was altered in a region‐specific manner. In contrast, cldn transcript abundance was mostly downregulated in osmoregulatory organs of juvenile fish acclimated to SW – cldn‐3b, −10and − 19 in the gill; cldn‐3b, −4, −10, and − 19 in the skin; cldn‐3b in the kidney; and cldn‐3band − 14 in the intestine. Data support the idea that Cldn TJ proteins play an important role in the osmoregulatory physiology of pre‐ and post‐metamorphic sea lamprey and that Cldn participation can occur across organs, in an organ‐specific manner as well as differ spatially within organs that contribute to the regulation of salt and water balance in these fishes.
C-type natriuretic peptide regulates the molecular components of the rainbow trout gill epithelium tight junction complex
• C-type natriuretic peptide (CNP) directly targets gill epithelium of fishes.
• CNP induces a reduction in paracellular permeability of the gill.
• treatment with CNP alters mRNA abundance of claudins.
• CNP reduces epithelial permeability of freshwater fishes.
Mechanisms and regulation of chloride transport in the Malpighian tubules of the larval cabbage looper Trichoplusia ni
Malpighian tubules (MTs) and the hindgut together constitute the excretory system of insects. Larvae of lepidopterans (butterflies and moths) demonstrate the so-called cryptonephric arrangement, where the distal blind end of each MT is embedded into the rectal complex. The rest of the free tubule is modified into several distinct regions that differ greatly in the transport of cations and water. However, relatively little is known about the transport of counter-anions (e.g., Cl- and HCO3-) by the MTs of lepidopteran larvae. In the current study we used ion-selective microelectrodes to characterize Cl- transport in the distinct regions of the free MT of the larval Trichoplusia ni. Firstly, we note that Cl- transport in the MTs is sensitive to the Cl- concentration of the bathing saline, and several regions of the MTs are capable of either secreting or reabsorbing Cl-. In the distal ileac plexus (DIP), a region previously characterized by cellular heterogeneity and its ability to switch between cation secretion and reabsorption, principal cells (PCs) toggled between Cl- reabsorption (in high-Cl- saline) and Cl- secretion (in low-Cl- saline). In contrast, secondary cells (SCs) in the DIP secreted Cl- regardless of saline Cl-concentration. Mechanistically, we have detected a number of ‘leak’ and ligand-gated Cl- channels (ClC) and demonstrated that Cl- channels are involved in Cl- secretion. Additionally, we demonstrated that the lumen-positive transepithelial potential increased in response to glycine. Using the scanning ion-selective electrode technique we demonstrated that glycine stimulated Cl- secretion by SCs, but not by PCs. In contrast, when MTs were deprived of glycine, a decrease in Cl- secretion, coupled with a decrease in the TEP, was observed. In contrast to the effects of glycine, an active dose of helicokinin reduced Cl- secretion by PCs, but not by SCs. Lastly, we detected expression of chloride-bicarbonate exchangers (CBE) in all regions of the free tubule. Scans of H+ transport across the tubule indicated that base equivalents are likely reabsorbed across the ileac plexus. Blocking ClC or CBE led to secretion of a more basic fluid, indicating lack of base reabsorption. We suggest that the transport of Cl-in the MTs of larval lepidopterans (i) may be correlated with the reabsorption of base, (ii) may be sensitive to Cl- concentration in the haemolymph, and (iii) could be regulated by helicokinin and glycine.
Malpighian tubules of caterpillars: blending RNAseq and physiology to reveal regional functional diversity and novel epithelial ion transport control mechanisms
Malpighian tubules (MTs) and hindgut constitute the functional kidney of insects. MTs are outpouches of the gut and in most insects demonstrate proximodistal heterogeneity in function. In most insects, such heterogeneity is confined to ion/fluid secretion in the distal portion and ion/fluid reabsorption in the proximal portion. In contrast, MTs of larval Lepidoptera (caterpillars of butterflies and moths), are comprised of five regions that differ in their association with the gut, their structure, and ion/fluid transport function. Recent studies have shown that several regions can rapidly and reversibly switch between ion secretion and reabsorption. The current study employed RNAseq, pharmacology and electrophysiology to characterize four distinct regions of the MT in larval Trichoplusia ni. Luminal microelectrode measurements indicate changes in [K+], [Na+] and pH as fluid passes through different regions of the tubule. In addition, the regions examined differ in gene ontology enrichment, and demonstrate robust gradients in expression of ion transporters and endocrine ligand receptors. Lastly, the study provides evidence for direct involvement of voltage-gated and ligand-gated ion channels in epithelial ion transport of insect MTs.
Section-specific H+ fluxes in renal tubules of fasted and fed goldfish
A recent study demonstrated that in response to a feeding-induced metabolic acidosis, goldfish Carassius auratus adjust epithelial protein and/or mRNA expression in their kidney tubules for multiple transporters known to be relevant for acid-base regulation. These include Na+/H+-exchanger, V-H+-ATPase, cytoplasmic carbonic anhydrase, HCO3−-transporters, and Rhesus proteins. Consequently, renal acid output in the form of protons and NH4+ increases. Little, however, is known about mechanistic details of renal acid-base regulation in C. auratus and teleost fishes in general. The present study applied the Scanning Ion-selective Electrode Technique (SIET) to measure proton flux in proximal, distal and connecting tubules of goldfish. We detected increased H+ efflux into the extracellular fluid from the tubule in fed animals, resulting from paracellular back-flux of H+ through the tight junction. By applying inhibitors for selected acid-base regulatory epithelial transporters, we found that cytosolic carbonic anhydrase and HCO3− transporters were important in mediating H+ flux in all three tubule segments of fed goldfish. Contrastingly, V-H+-ATPase seemed to play a role for H+ flux only in proximal and distal tubule, and Na+/H+-exchanger in proximal and connecting tubule. We develop working models for transport of acid-base relevant equivalents (H+, HCO3−, NH3/NH4+) for each tubule segment in C. auratus kidney. While the proximal tubule appears to play a major role in both H+ secretion and HCO3− reabsorption, the distal and connecting tubules seem to mainly serve for HCO3− reabsorption and NH3/NH4+ secretion.
The mineralocorticoid receptor contributes to barrier function of a model fish gill epithelium
Cortisol-induced epithelial tightening of a primary cultured rainbow trout gill epithelium model occurs in association with reduced paracellular permeability and increased abundance of select barrier-forming TJ proteins. Corticosteroid receptor (CR) pharmacological blocker studies have suggested that to produce this tightening effect, cortisol acts on the mineralocorticoid receptor (MR) as well as glucocorticoid receptors (GRs). This study considered how cortisol influenced model gill epithelium permeability and TJ properties by transcriptional knockdown of the gene encoding MR (mr-KD) using double-stranded RNA. Following mr-KD a significant reduction in MR protein abundance was observed in the epithelium. The mr-KD epithelium demonstrated reduced transepithelial resistance (TER) and increased the paracellular flux of [3H] polyethylene glycol (MW 400 kDa, PEG-400). Concurrently, mRNA abundance of gr2 and 11βhsd increased, indicating a possible compensatory response to mr-KD. Transcript abundance of claudin (cldn) -6, -8d, 23a and -28b decreased while cldn-20a increased in mr-KD preparations. Cortisol-induced epithelial tightening was enhanced in mr-KD preparations, suggesting that alterations in CRs and TJ composition augmented model epithelium barrier function in response to lowered MR abundance. Cortisol treatment significantly increased the transcript and protein abundance of TJ proteins such as cldn/Cldn -8d, and -28b. However, in mr-KD preparations, Cldn-28b protein abundance did not significantly alter in response to cortisol treatment, while Cldn-8d significantly elevated. Data suggest that mr-KD compromises normal barrier function of a primary cultured rainbow trout gill epithelium both in the presence and absence of cortisol and that Cldn-28b protein abundance may be modulated by cortisol via the MR only.
Septate junction in the distal ileac plexus of larval lepidopteran Trichoplusia ni: alterations in paracellular permeability during ion transport reversal
The Malpighian tubules (MTs) and hindgut together act as the functional kidney in insects. MTs of caterpillars are notably complex and consist of several regions that display prominent differences in ion transport. The distal ileac plexus (DIP) is a region of Malpighian tubule that is of particular interest because it switches from ion secretion to ion reabsorption in larvae fed on ion-rich diets. The pathways of solute transport in the DIP are not well understood, but one potential route is the paracellular pathway between epithelial cells. This pathway is regulated by the septate junctions (SJs) in invertebrates, and in this study, we found regional and cellular heterogeneity in expression of several integral SJ proteins. DIP of larvae fed ion-rich diets demonstrated a reduction in paracellular permeability, coupled with alterations in both SJ morphology and the abundance of its molecular components. Similarly, treatment in vitro with helicokinin (HK), an antidiuretic hormone identified by previous studies, altered mRNA abundance of many SJ proteins and reduced paracellular permeability. HK was also shown to target a secondary cell-specific SJ protein Tsp2A. Taken together, our data suggest that dietary ion loading, known to cause ion transport reversal in the DIP of larval T. ni, leads to alterations in the paracellular permeability, SJ morphology and its molecular component abundance. The results suggest that HK is an important endocrine factor that co-regulates ion transport, water transport and paracellular permeability in MTs of larval lepidopterans. We propose that co-regulation of all three components of the MT function in larval lepidopterans allows for safe toggling between ion secretion and reabsorption in the DIP in response to variations in dietary ion availability.
Book chapter: The Malpighian tubules and cryptonephric complex in lepidopteran larvae
Lepidopterans (butterflies and moths) are an ecologically and agriculturally important group of holometabolous insects. Their larvae and adults exhibit trophic partitioning, which is reflected by the various modifications of their digestive and excretory systems. Adults are capable of flight and feed mostly on the nectar of plants, acting as pollinators. Larvae are voracious leaf eaters whose extremely alkaline midgut(pH ≈ 11) is an adaptation to high tannin levels in the host plants. Morphologically and functionally regionalized Malpighian tubules of the larva modify fluid as it flows through them. The larvae also exhibit the so-called cryptonephric condition, where the distal end of the tubule is juxtaposed to the rectum and enveloped by the perinephric membrane. The distal part of the free tubule adjacent to the ileum, termed the distal ileac plexus, is characterized by a high density of secondary cells. Recent studies have identified several unusual aspects of ion transportphysiology of the distal ileac plexus: (i) gap junctional coupling of principal and secondary cells that allows them to transport ions in opposite directions, (ii) the ability to switch between K+ secretion and K+reabsorption depending on the input from the cryptonephric tubule, (iii) the presence of voltage-gated, ligand-gated and mechanosensitive ion channels and (iv) coordinated regulation of water and septate junctionpermeability during the reversal from secretion to reabsorption (presumably aimed at retention of water and solute content in the distal ileac plexus lumen, while Na+ and K+ are being reabsorbed). We describe recent advances in understanding ion-transporting and regulatory mechanisms in the Malpighian tubules of larval Lepidoptera with a special emphasis on the distal ileac plexus segment.
Water and ion transport across the eversible vesicles in the collophore of a springtail Orchesella cincta (Collaborative study with Dr. Barbora Konopova, University of Göttingen, Germany)
Springtails (Collembola) are ancient close relatives of the insects. The eversible vesicles are their unique paired transporting organs, which consist of an epithelium located inside a tube-like structure on the first abdominal segment called the collophore. The vesicles can be protruded out of the collophore and several lines of evidence indicate that they have a vital function in water uptake and ion balance. However, the amount of water absorbed by the vesicles and which other ions apart from sodium are transported remain unknown. Using Orchesella cincta as a model, we developed protocols for two assays that enabled us to study water and ion movement across the eversible vesicles in whole living springtails. Using an inverse Ramsay assay we demonstrate that the eversible vesicles absorb water from a droplet applied onto their surface. Using the scanning ion-selective electrode technique (SIET) we show that the vesicles absorb Na+ and Cl- from the bathing medium, secrete NH4+, and both absorb and secrete K+, H+ is secreted at a low level in the anterior part and absorbed at the posterior. We did not detect transport of Ca2+ at significant levels. The highest flux was the absorption of Cl-, and the magnitude of ion fluxes were significantly lower in fully hydrated springtails. Our data demonstrate that the eversible vesicles are a transporting epithelium functioning in osmo- and ionoregulation, nitrogenous waste excretion and likely acid-base balance.
Transcriptomic analysis of the Malpighian tubules of Trichoplusia ni: clues to mechanisms for switching from ion secretion to ion reabsorption in the distal ileac plexus
Excretion of metabolic wastes and toxins in insect Malpighian tubules (MTs) is coupled to secretion of ions and fluid. Larval lepidopterans demonstrate a complex and regionalized MT morphology, and recent studies of larvae of the cabbage looper, Trichoplusia ni, have revealed several unusual aspects of ion transport in the MTs Firstly, cations are reabsorbed via secondary cells (SCs) in T. ni, whereas in most insects SCs secrete ions. Secondly, SCs are coupled to neighbouring principal cells (PCs) via gap junctions to enable such ion reabsorption. Thirdly, PCs in the SC-containing distal ileac plexus (DIP) region of the tubule reverse from cation secretion to reabsorption in response to dietary ion loading. Lastly, antidiuresis is observed in response to a kinin neuropeptide, which targets both PCs and SCs, whereas in most insects kinins are diuretics that act exclusively via SCs. Recent studies have generated a basic model of ion transport in the DIP of the larval T. ni. RNAseq was used to elucidate previously uncharacterised aspects of ion transport and endocrine regulation in the DIP, with the aim of painting a composite picture of ion transport and identifying putative regulatory mechanisms of ion transport reversal in this tissue. Results indicated an overall expression of 9103 transcripts in the DIP, 993 and 382 of which were differentially expressed in the DIP of larvae fed high-K+ and high-Na+ diets respectively. Differentially expressed transcripts include ion-motive ATPases, ion channels and co-transporters, aquaporins, nutrient and xenobiotic transporters, cell adhesion and junction components, and endocrine receptors. Notably, several transcripts for voltage-gated channels and cell volume regulation-associated products were detected in the DIP and differentially expressed in larvae fed ion-rich diet. The study provides insights into the transport of solutes (sugars, amino acids, xenobiotics, phosphate and inorganic ions) by the DIP of lepidopterans. Our data suggest that this region of the MT in lepidopterans (as previously reported) transports cations, fluid, and xenobiotics/toxic metals. Besides this, the DIP expresses genes coding for the machinery involved in Na+- and H+-dependent reabsorption of solutes, chloride transport, and base recovery. Additionally, many of the transcripts expressed by the DIP a capacity of this region to respond to, process, and sometimes produce, neuropeptides, steroid hormones and neurotransmitters. Lastly, the DIP appears to possess an arsenal of septate junction components, differential expression of which may indicate junctional restructuring in the DIP of ion-loaded larvae.
The Effect of Diet on Midgut and Resulting Changes in Infectiousness of AcMNPV Baculovirus in the Cabbage Looper, Trichoplusia ni
Insecticide resistance has been reported in many important agricultural pests, and alternative management methods are required. Baculoviruses qualify as an effective, yet environmentally benign, biocontrol agent but their efficacy against generalist herbivores may be influenced by diet. However, few studies have investigated the tritrophic interactions of plant, pest, and pathogen from both a gene expression and physiological perspective. Here we use microscopy and transcriptomics to examine how diet affects the structure of peritrophic matrix (PM) in Trichoplusia ni larvae and consequently their susceptibility to the baculovirus, AcMNPV. Larvae raised on potato leaves had lower transcript levels for chitinase and chitin deacetylase genes, and possessed a thicker and more multi-layered PM than those raised on cabbage or artificial diet, which could contribute to their significantly lower susceptibility to the baculovirus. The consequences of these changes underline the importance of considering dietary influences on pathogen susceptibility in pest management strategies.
Helicokinin alters ion transport in the secondary cell-containing region of the Malpighian tubule of the larval cabbage looper Trichoplusia ni
Excretion in insects is accomplished by the combined actions of the Malpighian tubules (MTs) and hindgut, which together form the functional kidney. MTs of many insect groups consist of principal cells (PC) and secondary cells (SC). In most insect groups SCs are reported to secrete ions from haemolymph into the tubule lumen. Paradoxically, SCs in the MTs of the lepidopteran cabbage looper T. ni are used to reabsorb Na+ and K+ back into haemolymph. The current study was designed to investigate the effects and mode of action of the lepidopteran kinin, Helicokinin (HK), on ion transport in the SC-containing region of MT of T. ni. We identified a HK receptor (HK-R) homologue in T. ni and detected its expression in the SC-containing region of the MTs. The mRNA abundance of hk-r altered in response to changes in dietary K+ and Na+ content. HK-R immunolocalized to both PCs and SCs. Ramsay assays of preparations of the isolated distal ileac plexus (DIP) indicated that [HK] = 10−8 M: (i) decreased fluid secretion rate in unstimulated and serotonin-stimulated preparations, and (ii) increased [Na+]/[K+] ratio in the secreted fluid. Scanning ion-selective electrode technique measurements revealed that HK reduced: (i) K+ secretion by the PCs, and (ii) Na+ reabsorption by the SCs in intact tubules. In vitro incubation of the DIP with HK resulted in reduced mRNA abundance of hk-r as well as Na+/K+-ATPase subunit α (NKAα), Na+/K+/Cl− co-transporter (nkcc), Na+/H+ exchangers (nhe) 7 and 8, and aquaporin (aqp) 1. Taken together, results of the current study suggest that HK is capable of altering fluid secretion rate and [Na+]/[K+] ratio of the fluid, and that HK targets both PCs and SCs in the DIP of T. ni.
Molecular mechanisms of bi-directional ion transport in the Malpighian tubules of a lepidopteran crop pest, Trichoplusia ni
Classical studies have described in detail the complex and regionalized morphology of the Malpighian tubule (MT) in larval Lepidoptera. Recent studies revealed unusual aspects of ion transport in the Malpighian tubules of the larva of the cabbage looper, Trichoplusia ni. These included: cation reabsorption via secondary cells (SC); coupling of SCs to neighbouring PCs via gap junctions to enable reabsorption; and a reversal from cation secretion to reabsorption by the principal cells in the distal ileac plexus region of the in situ tubule in response to dietary ion loading. The current paper aimed to identify molecular components of ion transport in the MTs of T. ni and to describe their role in the recently reported reversal of ion transport in ion-loaded animals. Using a combination of molecular, immunohistochemical and electrophysiological techniques, we assigned roles to Na+/K+-ATPase (NKA), V-type H+-ATPase (VA), Na+/K+/Cl- co-transporter (NKCC), K+/Cl- co-transporter (KCC), inward-rectifying K+ channel (Kir), and Na+/H+ exchangers (NHE)-7 and -8 in the transport of Na+ and K+ by the distal ileac plexus of T. ni. The yellow region of the tubule lacked all of the above except VA, and the white region lacked all of the above transporters but expressed an amiloride-sensitive Na+ channel (NaC). Overall, the ion transport machinery in the distal ileac plexus of the T. ni tubule shows remarkable similarity to that in tubules of other groups of insects, yet this region transports ions very differently. Shutdown of secretory ATPases and utilisation of the same molecular machinery in the face of changing ion gradients may enable ion transport reversal in lepidopteran MTs. We propose that gap junction-based coupling of the two cell types likely aids in toggling between ion secretion and ion reabsorption in this segment.
Tricellular tight junction-associated angulins in the gill epithelium of rainbow trout
The molecular physiology of tricellular tight junction (tTJ)-associated proteins; lipolysis-stimulated lipoprotein receptor (lsr, = angulin-1) and an immunoglobulin-like domain-containing receptor (ildr2, ≈ angulin-3), were examined in model trout gill epithelia. Transcripts encoding lsr and ildr2 are broadly expressed in trout organs. A reduction in lsr and ildr2 mRNA abundance was observed during and following the formation of confluence in flask cultured gill cells. In contrast, as high resistance and low permeability characteristics developed in a model gill epithelium cultured on permeable polyethylene terephthalate membrane inserts, lsr and ildr2 transcript abundance exhibited an initial increase. However, as epithelia entered the developmental plateau phase, lsr abundance fell back to initial values while ildr2 transcript remained elevated. When mitochondrion-rich cells were introduced to model preparations, lsr mRNA abundance was unaltered and ildr2 abundance significantly increased. Transcript abundance of ildr2 was not altered in association with corticosteroid-induced tightening of the gill epithelium while lsr mRNA abundance decreased. Transcriptional knockdown of the tTJ protein tricelluin (tric-KD) reduced Tric abundance, increased gill epithelium permeability and caused an increase in lsr without significantly altering ildr2 transcript abundance. Data suggest that angulins contribute to fish gill epithelium barrier properties but that Lsr and Ildr2 seem likely to play different roles. This is because ildr2 typically exhibited increased abundance in association with decreased model permeability while lsr abundance altered in a manner that suggested a role in Tric recruitment to the tTJ.
Malpighian tubules of Trichoplusia ni: recycling ions via gap junctions and switching between secretion and reabsorption of Na+ and K+ in the distal ileac plexus.
The functional kidney in insects consists of the Malpighian tubules and hindgut. Malpighian tubules secrete ions and fluid aiding in hydromineral homeostasis, acid-base balance, and metabolic waste excretion. In many insects, including lepidopterans, the Malpighian tubule epithelium consists of principal cells (PCs) and secondary cells (SCs). The SCs in the Malpighian tubules of larvae of the lepidopteran Trichoplusia ni have been shown to reabsorb K+, transporting it in a direction opposite to that in the neighbouring PCs that secrete K+. One of the mechanisms that could enable such an arrangement is a gap junction (GJ)-based coupling of the two cell types. In the current study, we have immunolocalised GJ protein Innexin-2 to the PC-PC and SC-PC cell-cell borders. We have demonstrated that GJs in the SC-containing region of the Malpighian tubules enable Na+ and K+ reabsorption by the SCs. We also demonstrated that in ion-loaded animals PCs switch from Na+/K+ secretion to reabsorption, resulting in an ion- transporting phenotype similar to that of tubules with pharmacologically blocked GJs. Concomitantly, mRNA abundance encoding GJ proteins was downregulated. Finally, we observed that such PC-based reabsorption was only present in the distal ileac plexus connected to the rectal complex. We propose that this plasticity in the PC function in the distal ileac plexus is likely to be aimed at providing ion supply for the SC function in this segment of the tubule.
A role for tight junction-associated MARVEL proteins in larval sea lamprey (Petromyzon marinus) osmoregulation
This study reports on tight junction-associated MARVEL proteins of larval sea lamprey (Petromyzon marinus) and their potential role in ammocoete osmoregulation. Two occludin isoforms (designated Ocln and Ocln-a) and a tricellulin (Tric) were identified. Transcripts encoding ocln, ocln-a and tric were broadly expressed in larval lamprey, with the greatest abundance of ocln in the gut, liver and kidney, ocln-a in the gill and skin, and tric in the kidney. Ocln and Ocln-a resolved as ∼63 kDa and ∼35 kDa MW proteins, respectively, while Tric resolved as a ∼50 kDa protein. Ocln immunolocalized to the gill vasculature and in gill mucous cells while Ocln-a localized to the gill pouch and gill epithelium. Both Ocln and Ocln-a localized in the nephron, the epidermis and the luminal side of the gut. In branchial tissue, Tric exhibited punctate localization, consistent with its presence at regions of tricellular contact. Following ion-poor water (IPW) acclimation of ammocoetes, serum [Na+] and [Cl−] decreased, but not [Ca2+], and carcass moisture content increased. In association, Ocln abundance increased in the skin and kidney, but reduced in the gill of IPW-acclimated ammocoetes while Ocln-a abundance reduced in the kidney only. Tric abundance increased in the gill. Region-specific alterations in ocln, ocln-a and tric mRNA abundance were also observed in the gut. Data support a role for Ocln, Ocln-a and Tric in the osmoregulatory strategies of a basal vertebrate.
Claudin-8d is a cortisol-responsive barrier protein in the gill epithelium of trout
The influence of claudin (Cldn) 8 tight junction (TJ) proteins on cortisol-mediated alterations in gill epithelium permeability were examined using a primary cultured trout gill epithelium model. Genes encoding three Cldn-8 proteins (cldn-8b, -8c and -8d) have been identified in trout and all are expressed in the model gill epithelium. Cortisol treatment 'tightened' the gill epithelium, as indicated by increased transepithelial resistance (TER) and reduced paracellular [3H]polyethylene glycol (MW 400 kDa; PEG-400) flux. This occurred in association with elevated cldn-8d mRNA abundance, but no alterations in cldn-8b and -8c mRNA abundance were observed. Transcriptional knockdown (KD) of cldn-8d inhibited a cortisol-induced increase in Cldn-8d abundance and reduced the 'epithelium tightening' effect of cortisol in association with increased paracellular PEG-400 flux. Under simulated in vivo conditions (i.e. apical freshwater), cldn-8d KD hindered a cortisol-mediated reduction in basolateral to apical Na+ and Cl- flux (i.e reduced the ability of cortisol to mitigate ion loss). But cldn-8d KD did not abolish the tightening effect of cortisol on the gill epithelium. This is likely due, in part, to the effect of cortisol on genes encoding other TJ proteins, which in some cases appeared to exhibit a compensatory response. Data support the idea that Cldn-8d is a barrier protein of the gill epithelium TJ that contributes significantly to corticosteroid-mediated alterations in gill epithelium permeability.
Claudin-31 contributes to corticosteroid-induced alterations in the barrier properties of the gill epithelium
The contribution of Claudin-31 (Cldn-31) to corticosteroid-induced tightening of the trout gill epithelium was examined using a primary cultured model preparation. Cldn-31 is a ∼23 kDa protein that localizes to the periphery of gill epithelial cells and diffusely in select gill cells that are Na+-K+-ATPase-immunoreactive. Transcriptional knockdown (KD) of cldn-31 reduced Cldn-31 abundance and increased epithelium permeability. Under simulated in vivo conditions (apical freshwater), cldn-31 KD increased net ion flux rates (≡ efflux). Cortisol treatment increased Cldn-31 abundance and decreased epithelium permeability. This tightening effect was diminished, but not eliminated, by cldn-31 KD, most likely due to other cortisol-sensitive TJ proteins that were transcriptionally unperturbed or enhanced in cortisol-treated cldn-31 KD preparations. However, cldn-31 KD abolished a cortisol-induced increase in Cldn-8d abundance, which may contribute to compromised cldn-31 KD epithelium permeability. Data suggest an important barrier function for Cldn-31 and an integral role for Cldn-31 in corticosteroid-induced gill epithelium tightening.