Calcium transport across the basolateral membrane of isolated Malpighian tubules: a survey of several insect orders

The Malpighian tubules play a major role in haemolymph calcium homeostasis in insects by sequestering excess Ca²⁺ within the biomineralized granules that often accumulate in the tubule cells and/or lumen. Using the scanning ion‐selective microelectrode technique, measurements of basolateral Ca²⁺ transport are determined at several sites along the length of the Malpighian tubules isolated from the eight insects representing seven orders: Drosophila melanogaster (Diptera), Aedes aegypti (Diptera), Tenebrio molitor (Coleoptera), Acheta domesticus (Orthoptera), Trichoplusia ni (Lepidoptera), Periplaneta americana (Blattodea), Halyomorpha halys (Hemiptera) and Pogonomyrmex occidentalis (Hymenoptera). Ca²⁺ transport is specific to tubule segments containing Ca‐rich granules in D. melanogaster and A. aegypti, whereas Ca²⁺ transport is relatively uniform along the length of whole tubules in the remaining species. Generally, manipulation of second messenger pathways using cAMP and thapsigargin has little effect on rates of basolateral Ca²⁺ transport, suggesting that previous effects observed across midtubules of A. domesticus are unique to this species. In addition, the present study is the first to provide measurements of basolateral Ca²⁺ across single principal and secondary tubule cells, where Ca²⁺ uptake occurs only across principal cells. Estimated times for all tubules to eliminate the entire haemolymph Ca²⁺ content in each insect range from 6 min (D. melanogaster) to 19 h (H. halys) or more, indicating that rates of Ca²⁺ uptake by the Malpighian tubules are not always rapid. The results of the present study suggest that the principal cells of the Malpighian tubules contribute to haemolymph calcium homeostasis by sequestering excess Ca²⁺, often within specific tubule segments.     

Mechanisms of calcium sequestration by isolated Malpighian tubules of the house cricket Acheta domesticus

Hemolymph calcium homeostasis in insects is achieved by the Malpighian tubules, primarily by sequestering excess Ca²⁺ within internal calcium stores (Ca‐rich granules) most often located within type I (principal) tubule cells. Using both the scanning ion‐selective electrode technique and the Ramsay secretion assay this study provides the first measurements of basolateral and transepithelial Ca²⁺ fluxes across the Malpighian tubules of an Orthopteran insect, the house cricket Acheta domesticus. Ca²⁺ transport was specific to midtubule segments, where 97% of the Ca²⁺ entering the tubule is sequestered within intracellular calcium stores and the remaining 3% is secreted into the lumen. Antagonists of voltage‐gated (L‐type) calcium channels decreased Ca²⁺ influx ≥fivefold in adenosine 3′,5′‐cyclic monophosphate (cAMP)‐stimulated tubules, suggesting basolateral Ca²⁺ influx is facilitated by voltage‐gated Ca²⁺ channels. Increasing fluid secretion through manipulation of intracellular levels of cAMP or Ca²⁺ had opposite effects on tubule Ca²⁺ transport. The adenylyl cyclase‐cAMP‐PKA pathway promotes Ca²⁺ sequestration whereas both 5‐hydroxytryptamine and thapsigargin inhibited sequestration. Our results suggest that the midtubules of Acheta domesticus are dynamic calcium stores, which maintain hemolymph calcium concentration by manipulating rates of Ca²⁺ sequestration through stimulatory (cAMP) and inhibitory (Ca²⁺) regulatory pathways.     

Characterization of tetraethylammonium uptake across the basolateral membrane of the Drosophila Malpighian (renal) tubule

Basolateral transport of the prototypical type I organic cation tetraethylammonium (TEA) by the Malpighian tubules of Drosophila melanogaster was studied using measurements of basolateral membrane potential (V(bl)) and uptake of [(14)C]-labeled TEA. TEA uptake was metabolically dependent and saturable (maximal rate of mediated TEA uptake by all potential transport processes, reflecting the total transport capacity of the membrane, 0.87 pmol.tubule(-1).min(-1); concentration of TEA at 0.5 of the maximal rate of TEA uptake value, 24 muM). TEA uptake in Malpighian tubules was inhibited by a number of type I (e.g., cimetidine, quinine, and TEA) and type II (e.g., verapamil) organic cations and was dependent on V(bl). TEA uptake was reduced in response to conditions that depolarized V(bl) (high-K(+) saline, Na(+)-free saline, NaCN) and increased in conditions that hyperpolarized V(bl) (low-K(+) saline). Addition of TEA to the saline bathing Malpighian tubules rapidly depolarized the V(bl), indicating that TEA uptake was electrogenic. Blockade of K(+) channels with Ba(2+) did not block effects of TEA on V(bl) or TEA uptake indicating that TEA uptake does not occur through K(+) channels. This is the first study to provide physiological evidence for an electrogenic carrier-mediated basolateral organic cation transport mechanism in insect Malpighian tubules. Our results also suggest that the mechanism of basolateral TEA uptake by Malpighian tubules is distinct from that found in vertebrate renal tubules.     

Diuretic activity of Mas-DP II,an identified neuropeptide from Manduca sexta: An in vivo and in vitro examination in the adult moth

Mas-DP II, a recently identified 30 amino acid diuretic peptide isolated from the tobacco hornworm moth, Manduca sexta, was tested for its ability to increase fluid excretion in adult M. sexta, and for the ability to elevate the rate of fluid secretion from isolated Malpighian tubules cultured in vitro. Mas-DP II was found to increase fluid weight loss from decapitated adult moths in a dose-dependent manner; weight loss increased significantly at doses as low as 5 ng for female moths and 25 ng for male moths. Male moths injected with large doses of Mas-DP II continued to exhibit increased rates of fluid loss up to 4 h post-injection. In vitro, Mas-DP II stimulated fluid secretion from isolated Malpighian tubules at concentrations as low as 4 nM for tubules from both male and female moths. © 1994 Wiley-Liss, Inc.     

Proteomic changes in response to crystal formation in Drosophila Malpighian tubules

Kidney stone disease is a major health burden with a complex and poorly understood pathophysiology. Drosophila Malpighian tubules have been shown to resemble human renal tubules in their physiological function. Herein, we have used Drosophila as a model to study the proteomic response to crystal formation induced by dietary manipulation in Malpighian tubules. Wild-type male flies were reared in parallel groups on standard medium supplemented with lithogenic agents: control, Sodium Oxalate (NaOx) and Ethylene Glycol (EG). Malpighian tubules were dissected after 2 weeks to visualize crystals with polarized light microscopy. The parallel group was dissected for protein extraction. A new method of Gel Assisted Sample Preparation (GASP) was used for protein extraction. Differentially abundant proteins (p<0.05) were identified by label-free quantitative proteomic analysis in flies fed with NaOx and EG diet compared with control. Their molecular functions were further screened for transmembrane ion transporter, calcium or zinc ion binder. Among these, 11 candidate proteins were shortlisted in NaOx diet and 16 proteins in EG diet. We concluded that GASP is a proteomic sample preparation method that can be applied to individual Drosophila Malpighian tubules. Our results may further increase the understanding of the pathophysiology of human kidney stone disease.     

Immunocytochemical localization of a vacuolar-type ATPase in Malpighian tubules of the ant Formica polyctena

The presence of a vacuolar-type ATPase in Malpighian tubules of the ant Formica polyctena was investigated immunocytochemically, using antibodies to vacuolar ATPases of Manduca sexta midgut and bovine kidney. Specific labelling was observed at the brush border of the epithelium extending along the entire length of the tubules. These findings agree with the current view that a vacuolar ATPase is situated at the apical membrane of Malpighian tubule cells and other insect epithelial cells, being the energizing element of an electrogenic potassium pump. When antibodies were tested on tubules in different secretion conditions prior to fixation, no differences were observed in the distribution of the vacuolar ATPase.This work was supported by grants from the European Community (SCI-CT90-0480), from the Ministerio de Educación y Ciencia DGICYT, Spain (CE 91-0002), and from the Deutsche Forschungsgemeinschaft (Wi 698-3).     

Transepithelial transport of salicylate by the Malpighian tubules of insects from different orders

The organic anion salicylate is a plant secondary metabolite that protects plants against phytophagous insects. In this study, a combination of salicylate-selective microelectrodes and a radioisotope tracer technique was used to study the transepithelial transport of salicylate by the Malpighian tubules of 10 species of insects from five orders. Our results show that salicylate is transported into the lumen of the Malpighian tubules in all the species evaluated, except Rhodnius prolixus. The transepithelial transport of salicylate by the Malpighian tubules of Drosophila simulans, Drosophila erecta, Drosophila sechellia, and Acheta domesticus was saturable, Na⁺-dependent and inhibited by α-cyano-4-hydroxycinnamic acid. This transport system resembles that previously found in tubules of Drosophila melanogaster. In contrast, transepithelial transport of salicylate by Malpighian tubules of Tenebrio molitor, Plagiodera versicolora, Aedes aegypti, and Trichoplusia ni was unaffected by Na⁺-free bathing saline. The presence of both salicylate and salicylate metabolites in the secreted fluid samples from the Malpighian tubules of A. domesticus, R. prolixus, T. molitor, and T. ni indicates that insect Malpighian tubules may both transport and metabolize salicylate. The highest capacities to rid the hemolymph of salicylate were found in T. molitor, P. versicolora and Drosphila spp. Our results suggest that transport of salicylate by the Malpighian tubules might contribute to elimination of this organic anion from the hemolymph, particularly in some species that encounter high levels of organic anion in the diet.     

Fluid-phase endocytosis does not contribute to rapid fluid secretion in the malpighian tubules of the house cricket, Acheta domesticus.

When the Malpighian tubules (Mt) of the house cricket (Acheta domesticus) are treated with dibutyryl adenosine 3', 5'-cyclic monophosphate (db-cAMP; 1 mM), which causes a doubling in secretion rate, more than 50% of the cell volume is occupied by vesicles within 420 sec of exposure. In view of the fact that the increase in vesiculation occurs concomitantly with stimulated fluid transport, we set out to determine whether the vesicles are formed as a result of fluid-phase endocytosis (pinocytosis) and subsequently used to transport fluid to the lumen as one means of increasing transport rate. We used fluorescent fluid-phase markers (Lucifer Yellow Carbohydrazide [LYCH] and Alexa 488 hydrazide) and an electron dense marker (cationized ferritin) to elucidate the degree of endocytosis that occurred with db-cAMP stimulation. We found that, although some fluid is taken into the cells of the mid-tubule via endocytosis, it does not coincide with the level of vacuolation present in stimulated tubules. The amount of LYCH transported into the primary urine by the db-cAMP-stimulated Mt decreased by 40% as compared to the unstimulated transport, and the rate of transport of LYCH was only 30% of the unstimulated tubules. In summary, our findings do not support the theory that the majority of the vesicles or vacuoles comprise intracellular, endocytotic compartments formed via a basolateral endocytotic pathway. We also found no evidence to support the functioning of vesicles or vacuoles as transcellular "shuttling" mechanisms to move fluid from the basal region to the apical membrane and into the lumen.     

Characterization of salicylate uptake across the basolateral membrane of the malpighian tubules of Drosophila melanogaster

The organic anion salicylate is a plant secondary metabolite that can protect plants against herbivores. Transport of salicylate across the basolateral membrane of the Malpighian tubules of Drosophila melanogaster was studied using a radioisotope tracer technique. The uptake of [(14)C]salicylate by the Malpighian tubules was active, saturable and Na(+)-dependent; the maximum uptake rate (J(max)) and the half saturation concentration (K(t)) were 12.6 pmoltubule(-1)min(-1) and 30.7micromoll(-1), respectively. In contrast to organic anion transport by vertebrate renal tissues, salicylate uptake was not trans-stimulated by glutarate (0.01-1.0 mmoll(-1)) or cis-inhibited by high concentrations (5 mmoll(-1)) of various alpha-keto acids (glutaric acid, alpha-ketoglutaric acid, succinic acid, and citric acid). Changes in basolateral membrane potential or physiologically relevant changes in bathing saline pH did not affect the rate of [(14)C]salicylate uptake. Ring-structure monocarboxylic acids (benzoic acid, nicotinic acid, gentisic acid, unlabelled salicylic acid, alpha-cyano-4-hydroxycinnamic acid, probenecid, fluorescein, and P-aminohippuric acid) strongly inhibited [(14)C]salicylate uptake rate. In contrast, short-chain monocarboxylic acids had little (butyric acid) or no effect (lactic acid, pyruvic acid, and propionic acid). Our results suggest that salicylate uptake across the basolateral membrane of D. melanogaster Malpighian tubules is mediated by a non-electrogenic, alpha-cyano-4-hydroxycinnamic acid-sensitive, Na(+):salicylate cotransport system.     

Life History and Electron Microscopy of a Haplosporidian,Nephridiophaga blattellae (Crawley) n. comb., in the Malphigian Tubules of the German Cockroach,Blattella germanica (L.)*

SYNOPSIS. Rearing of cockroaches without sporozoan parasites of the Malpighian tubules permitted the 1st experimental infections reported for these organisms as well as study of the intracellular early stages of infection. Infectivity tests show that the route of invasion is peroral rather than transovarian; that fresh spores obtained from either the Malpighian tubules or the hindgut are infective; that fresh spores held in aqueous suspension for 36 hr are not infective; that all ages and both sexes of cockroaches are susceptible, although older cockroaches are more readily infected experimentally; that a single peroral dose of a few hundred spores is sufficient to produce infection; that the earliest signs of progressive infection in the Malpighian tubules appear 15 days after ingestion of spores; and that the infection, spread mainly by schizogony, is well established by 23 days. Spores are acid-fast. The PAS-positive spore membrane is not dissolved by 20-min treatment with hot KOH, but tests for chitin were inconclusive. The life history of the parasite is described. “Encapsulation” of the parasite has been observed. Electron microscope studies, the first reported for the class Haplosporea, show similarities of the cytoplasmic structure of this haplosporidian with that of some members of the sporozoan class Telosporea, though no specialized structures were seen. Finding of intracellular stages together with failure of interspecific infection attempts suggest that the microorganism should be placed in the genus Nephridiophaga Ivanic, 1937 with the specific name blattellae Crawley, 1905.     

Dissolution of granules in the Malpighian tubules of Musca autumnalis DeGeer,during mineralization of the puparium

The dissolution of mineralized granules stored in the larval Malpighian tubules of the face fly, Musca autumnalis DeGeer, was studied both in situ and with isolated granules in vitro. The release of calcium, phosphorus, and magnesium from granules increased exponentially as the pH of the bathing medium was decreased. The pH measured in the distal region of the Malpighian tubules was 8.08 while that of the proximal region was 7.35. Thus, the decrease in pH of lumen contents from distal to proximal regions of the tubules appears to be a major effector of granule dissolution. Loss of structural integrity of the granules accompanied mineral release and also increased as pH of the bathing medium was lowered in vitro. This structural disintegration was similar to that observed in naturally dissolving granules isolated from the proximal region of the Malpighian tubules. The larval tubules, therefore, appear to have regional specialization in that granules are formed and stored in the distal lumen and dissolution takes place as granules move into the more acidic proximal region. No granules were found in the larval hindgut contents also indicating that dissolution and transport take place in the proximal region of the tubules. However, granules of similar composition were found in the meconium and in the most distal regions of adult Malpighian tubules.     

Genetic Control of Development of Malpighian Tubules in Drosophila melanogaster

Malpighian tubules of insects are a functional analog of mammalian kidneys and serve as a classical model for studying the structure and functions of transport epithelium. The review contains the data on structural organization, functioning, and formation of the Malpighian tubules during embryogenesis in Drosophila melanogaster. Various systems of genes are described that control the program of development of the renal (Malpighian) tubules in D. melanogaster. A special attention is paid to the ways of signal transduction and factors involved in cell differentiation, proliferation, and morphological transformation during development of the Malpighian tubules. Evolutionarily conservative genetic systems are considered that are involved in the control of development of both the renal epithelium ofDrosophila and mammalian kidneys. A relationship was noted between the disturbed balance of genetic material and congenital defects of the human excretory system.     

Evidence and bioassay for diuretic factors in the nervous system of larvalHeliothis virescens

Summary Diuretic factors were studied in the central nervous system of larvae of the tobacco budworm,Heliothis virescens, using [¹⁴C]urea as a sensitive indicator for water movement through isolated Malpighian tubules. The assay required Na⁺ and a pH of 6.0–6.2 for maximum activity. Malpighian tubules had high secretory activity in feeding larvae of the fifth instar, but the activity declined during the burrowing-digging stage that preceded pupation. Malpighian tubules from starved larvae showed a greater response to extracts of nervous tissues than did tubules from feeding larvae, and extracts showed a dose-response relationship with fluid secretion. Diuretic activity was distributed throughout all parts of the central nervous system with the brain having the most activity. Brain extracts increased fluid secretion by in vitro Malpighian tubules by more than 3-fold and doubled the rate of dye clearance from the hemolymph in vivo. Diuretic activity in nervous tissue extracts was unaffected by boiling but sensitive to proteases. Fluid secretion by in vitro tubules was increased by cAMP, dbcAMP, theophylline, octopamine and dopa. These studies provide evidence for the presence of diuretic factors in the central nervous system ofH. virescens larvae and describe a sensitive bioassay for these factors.Abbreviations AR activation ratio - cAMP cyclic AMP - dbcAMP dibutyryl cyclic AMP - dbcGMP dibutyryl cyclic GMP - Dopa dihydroxyphenylalanine - 5-HT 5-hydroxytryptamine - L1 larval instar - VCNS ventral central nervous system     

Evidence for hormonal control of diuresis after a blood meal in the mosquito Aedes aegypti

In previous studies we have presented evidence for the role of peptides, isolated from heads of the mosquito Aedes aegypti, in stimulating fluid secretion by isolated Malpighian tubules. In the present study we conducted experiments to investigate whether these peptides are involved in hormone-mediated diuresis after a blood meal. In vivo experiments showed that the head was required to maintain diuresis after the blood meal. Whereas feeding on blood triggered a prompt diuresis in the intact mosquito, subsequent decapitation caused a gradual, not an abrupt, decline in urine excretion rate. Hemolymph collected from mosquitoes fed blood significantly stimulated fluid secretion in vitro by isolated Malpighian tubules, whereas hemolymph from unfed or blood-fed decapitated mosquitoes did not. These results indicate that a diuretic factor was released into the hemolymph after a blood meal. This factor was not present in the hemolymph of decapitated females. We identified the head as a source of diuretic factors. Peptides isolated from a head extract by high-performance liquid chromatography, when injected into the hemocoel of blood-fed decapitated mosquitoes, triggered diuresis in vivo and also stimulated fluid secretion in isolated Malpighian tubules. These studies support the hypothesis that the head is a storage site for diuretic peptides that may be released after a blood meal to control diuresis.     

Excretory role of the midgut in larvae of the tobacco hornworm, Manduca sexta (L.).

Caterpillars of Manduca sexta use two distinct transport mechanisms for the excretion of dyes. One pump (Type A) has a high affinity for acid (anionic) dyes and occurs in the midgut and medial Malpighian tubules. Acid dyes accumulate rapidly in the lumen of the midgut while the Malpighian tubules appear to play only a minor role in the excretion of these dyes. The other pump (Type B) excretes basic (cationic) dyes and is located primarily in the proximal Malpighian tubules. Evidence is presented that hippuric acid competes with acid dyes for excretion by both midgut and Malpighian tubules. After the final-instar larva purges its gut the ability of the midgut and Malpighian tubules to excrete dyes gradually decreases. Sixty hours after the purge only the Malpighian tubules retain some dye excreting activity.     

Analysis of malpighian tubule membrane proteins from Drosophila melanogaster

A method had been developed for radioactively labelling and analyzing the membrane proteins of Malpighian tubules and other tissues obtained by dissections from Drosophila melanogaster larvae. A fraction was identified on sucrose gradients which binds concanavalin A, and is labelled by galactose oxidase reduction. This fraction was examined in the electron microscope and found to contain membraneous structures.The membrane proteins were analyzed following fractionation of dissected tissues using two dimensional gel electrophoresis and fluorography. Animals were made radioactive by feeding larvae on yeast which was grown in a medium containing [³²S]sulphate. The membrane fraction of Malpighian tubules contains approx. 125 major spots. Of these, about 50% seem to be common to the membranes of several cell types. The remainder of the membrane proteins appear to be tissue type specific.     

The ultrastructure of the anal vesicle of the parasitoid,Microplitis croceipes (Hymenoptera:Braconidae)

The fine structural characteristics of epithelial cells of the anal vesicle in the hymenopteran parasitoid, Microplitis croceipes (Cresson), are similar to those of transport cells. Apical and basal infoldings, an abundance of mitochondria, ribosomes, rough endoplasmic reticulum, Golgi complexes and pinocytotic vesicles all indicate a transport function for these epithelial cells. The medial portions of both Malpighian tubules located within the anal vesicle also were examined and on the basis of morphology appear to be active. These observations support earlier physiological data which indicate that the anal vesicle functions in absorption of nutrients and excretion.     

Number of Malpighian Tubules in Larvae and Adults of Stingless Bees (Hymenoptera: Apidae) from Amazonia

The number of Malpighian tubules in larvae and adults of bees is variable. Larvae of Apis mellifera L. have four Malpighian tubules, while adults have 100 tubules. In stingless bees, this number varies from four to eight. The objectives of this study were to provide characteristics of the Malpighian tubules as well as to quantify their number in larvae and adults of six species of Meliponinae, Melipona seminigra merrillae Cockerell, Melipona compressipes manaosensis Schwarz, Melipona rufiventris Lepeletier, Scaptotrigona Moure, Frieseomelitta Ihering, and Trigona williana Friese. Malpighian tubules were dissected from larvae and adults, measured, quantified, and maintained in microtubes with Dietrich??s solution. The numbers of Malpighian tubules were constant only for larvae of M. rufiventris (four and eight) and Scaptotrigona sp. (four). The most frequent number of tubules in the Melipona group was seven and eight in larvae, and 70 and 90 in adults. In the Trigona group were four and 20 to 40, for larvae and adults, respectively. The results showed differences in the number of Malpighian tubules among the species analyzed and also between the larvae and adults of the same species. Despite the variation observed, species of the group Melipona always have a larger number and longer Malpighian tubules in both larvae and adults as compared to the Trigona group, which may indicate an evolutionary trend of differentiation between these groups.     

Malpighian tubules of larval Aedes aegypti are hormonally stimulated by 5-hydroxytryptamine in response to increased salinity

Two environmental parameters, feeding status and salinity, are expected to affect water and ion balance of the aquatic larvae of Aedes aegypti. Evidence was obtained for regulation of Malpighian tubule fluid secretion rates in response to changes in each of these parameters. Exposure to increased salinity induces release into the hemolymph of material with diuretic effects on Malpighian tubules. Diuretic material is present in hemolymph of larvae raised in higher salinities, rapidly appears in the hemolymph of larvae following transfer from dilute water to higher salinity, and rapidly disappears from the hemolymph following transfer from higher salinity to dilute water. Feeding status affects diuretic properties of both hemolymph and Malpighian tubules. Feeding causes hemolymph to become diuretic relative to hemolymph from nonfeeding larvae. Malpighian tubules removed from feeding larvae have greater basal fluid secretion rates and also appear to have greater maximal fluid secretion capacity than do tubules removed from nonfeeding larvae. Larval hemolymph [5-HT] was found to increase fivefold in response to elevated salinity but was unaffected by feeding status. Methiothepin, a 5-HT receptor antagonist, inhibited stimulation of fluid secretion by 5-HT and blocked the diuretic effects of hemolymph from larvae exposed to higher salinity but was without effect on stimulation of fluid secretion by diuretic peptide. During the course of this investigation, a preliminary pharmacological characterization of the 5-HT receptor on Aedes Malpighian tubules, suggesting that this receptor may be pharmacologically distinct from other described insect 5-HT receptors, was obtained. Arch. Insect Biochem. Physiol. 34:123–141, 1997. © 1997 Wiley-Liss, Inc.     

The use of eryopreservation for convenient bioassay of insect organs in vitro

Abstract Pheromone glands of the female moth Helicoverpa (Heliothis) armigera and Malpighian tubules of the locust Locusta migratoria migratorioides (R&F) retained partial biological activity after eryopreservation. Pheromone glands were frozen to -70^oC in a tissue culture medium (TC-199) containing 10% of the cryoprotectant dimethylsulfoxide. Malpighian tubules, however, required initial preincubation for 24 h in a medium of high osmolarity before being frozen to -70^oC in the presence of 10% of the cryoprotectant glycerol. After rapid thawing of both organs, biological activity was compared with the respective fresh organs. Using a radiochemical assay, it was shown that previously frozen pheromone glands were stimulated by pheromone biosynthesis activating neuropeptide to the same degree as fresh pheromone glands. In addition, previously frozen Malpighian tubules were stimulated by an extract of corpora cardiaca to produce c-AMP to the same extent as fresh Malpighian tubules.