Ion Secretion Research Articles

Subclinical colitis associated with moderately hemolytic Brachyspira strains

Objective: Microbiological and virulence characterization of 2 moderately hemolytic Brachyspira strains. Materials and methods: Clinical isolates were obtained from diarrheic (3603-F2) and healthy (G79) pigs. Phenotypic characterization included assessment of hemolytic activity on blood agar and biochemical profiling. Genotyping was performed by sequencing the nicotinamide adenine dinucleotide oxidase (nox) gene, whole genome sequencing, and comparison to relevant Brachyspira. Pig inoculation included 4 treatment groups in 2 challenge experiments: negative control (sterile broth media; n = 12), positive control (Brachyspira hampsonii genomovar 2 strain 30446; n = 18), and 3603-F2 (n = 12) or G79 (n = 12). Fecal scoring and rectal swabbing for culture were performed daily. Animals were euthanized following onset of mucohemorrhagic diarrhea or between 21 and 28 days post inoculation (dpi). Gross and microscopic pathology were assessed. Terminal colon samples were used to characterize post-infection mucosal ion secretion. Results: Both strains were moderately hemolytic. Whole genome and nox sequencing identified 3603-F2 as Brachyspira murdochii and G79 as a novel strain. Both challenge trials revealed intestinal colonization, but no mucohemorrhagic diarrhea. Sporadic watery diarrhea was induced by 3603-F2 associated with a pattern of microscopic lesions similar to pigs with swine dysentery (positive controls). No diarrhea was observed in G79 inoculated pigs, but microscopic lesions were more severe than in controls. Both strains induced greater colonic anion secretory potential than negative controls 21 dpi. Implications: Allegedly avirulent Brachyspira species most closely related to B murdochii can be associated with subclinical colitis and may be a concern for grow-finish pigs.

Allicin Induces Electrogenic Secretion of Chloride and Bicarbonate Ions in Rat Colon via the TRPA1 Receptor.

Allicin, an antioxidant from garlic, is known to regulate intestinal contractions, but its effect on intestinal ion transport is unclear. The aim of this study was to examine the role of allicin in the regulation of electrogenic ion transport in rat intestine by measuring the transmural potential difference (ΔPD). Allicin induced significant positive ΔPD, when administered to the serosal side of the colonic mucosal-submucosal preparation. Allicin-induced colonic ΔPD was largely diminished by incubation in the chloride-free solution, although the transient peak of ΔPD after application of allicin remained. This transient peak of ΔPD was significantly diminished in both the chloride- and the bicarbonate-free incubation solution. Induction of ΔPD by allicin was greatly diminished by AP-18, an inhibitor of the transient receptor potential (TRP) cation channel subfamily A member 1, TRPA1. Both alliin and S-allylcysteine, the analogues of allicin, had no effect on ΔPD and did not affect allicin-induced ΔPD in the colon. These results suggest that allicin mainly evokes the electrogenic chloride secretion and only partially increases the electrogenic bicarbonate secretion via TRPA1.

Pancreatic ductal organoid cultures are a suitable model to study pancreatic ductal ion secretion

Pancreatic ductal organoid cultures are a suitable model to study pancreatic ductal ion secretion

Escherichia coli enterotoxigénica y enteroagregativa: prevalencia, patogénesis y modelos múridos.

Enterotoxigenic (ETEC) and enteroaggregative Escherichia coli (EAEC) pathotypes are important etiological agents causative of diarrhea in children younger than 5 years of age in Mexico and in developing countries, where they cause numerous deaths. Both have been associated with delayed growth in children and are the main causative agents of traveler's diarrhea. The pathogenesis of both bacteria starts by adhering to the intestinal epithelium by means of fimbriae, called colonization factors in human ETEC isolates and aggregative adherence fimbriae in EAEC isolates. Once ETEC adheres to the enterocyte, it produces one or both of its toxins and induces the secretion of chloride and sodium ions and water into the intestinal lumen, producing its characteristic watery diarrhea. EAEC binds to the intestinal epithelium forming a biofilm, induces the production of mucus, releases its toxins and promotes inflammation. EAEC and ETEC infection models with wild-type C57BL/6 and CD40 ligand-deficient mice (with intact microbiota), respectively, revealed that undernutrition and low-zinc diet increases EAEC infection, causing growth retardation, and that ETEC colonizes, persists and induces local and systemic humoral immune response.

Microhair on the adaxial leaf surface of salt secreting halophytic Oryza coarctata Roxb. show distinct morphotypes: Isolation for molecular and functional analysis

Halophytic Oryza coarctata is a good model system to examine mechanisms of salinity tolerance in rice. O. coarctata leaves show the presence of microhairs in adaxial leaf surface furrows that secrete salt under salinity. However, detailed molecular and physiological studies of O. coarctata microhairs are limited due to their relative inaccessibility. This work presents a detailed characterization of O. coarctata leaf features. O. coarctata has two types of microhairs on the adaxial leaf surface: longer microhairs (three morphotypes) lining epidermal furrow walls and shorter microhairs (reported first time) arising from bulliform cells. Microhair morphotypes include (i) finger-like, tubular structures, (ii) tubular hairs with bilobed and flattened heads and (iii) bi-or trifurcated hairs. The unicellular nature of microhairs was confirmed by propidium iodide (PI) staining. An efficient method for the isolation and enrichment of O. coarctata microhairs is presented (yield averaging ˜2 × 105/g leaf tissue). The robustness of the microhair isolation procedure was confirmed by subsequent viability staining (PI), total RNA isolation and RT-PCR amplification of O. coarctata trichome-specific WUSCHEL-related homeobox 3B (OcWox3B) and transporter gene-specific cDNA sequences. The present microhair isolation work from O. coarctata paves the way for examining genes involved in ion secretion in this halophytic wild rice model.

Comparison of Colonic Baseline Ion Secretion and Responses to Stress‐related Peptides between Male and Female Mice

Background and AimsSex plays an important role in the symptoms of irritable bowel syndrome (IBS) and responses to treatment. Constipation‐predominant IBS (IBS‐C) is more prevalent in women, whereas diarrhea‐predominant IBS (IBS‐D) is more common in men. Stress has been shown to increase intestinal ion secretion and may exacerbate symptoms in IBS‐D. Most of the previous preclinical studies investigating the effects of stress on gut function were performed in male animals. The aim of the present study was to determine if there was a sex difference in baseline ion secretion in the mouse colon and/or its responses to stress‐related peptides, including corticotrophin releasing hormone (CRF), urocortin 1 (Ucn1), Ucn2, and Ucn3.MethodsAdult C57BL/6J mice (47 males and 20 females) were used in the present study. Mucosa/submucosa preparations from the proximal colon were mounted in Ussing flux chambers for measurement of short‐circuit current (Isc) as an indicator of mucosal ion secretion.ResultsMale mice were found to exhibit a significantly (p < 0.05) higher baseline Isc (70.90 ± 4.84 μA/cm2, n = 47) in the proximal colon than females (52.92 ± 4.40 μA/cm2, n = 20). In addition, male mice exhibited a greater increase in colonic Isc (ΔIsc) than did female mice in response to 1 μM of CRF (10.26 ± 1.31 μA/cm2 vs. 4.59 ± 1.21 μA/cm2, n = 5/group, p < 0.05), 1 μM of Ucn1 (16.65 ± 2.23 μA/cm2 vs. 6.80 ± 1.16 μA/cm2, n = 5/group, p < 0.01), 1 μM of Ucn2 (21.76 ± 4.58 μA/cm2 vs. 6.72 ± 0.57 μA/cm2, n = 5/group, p < 0.05), or 1 μM of Ucn3 (41.41 ± 8.91 μA/cm2 vs. 7.86 ± 1.47 μA/cm2, n = 5, p < 0.01). Colonic Isc response to the selective CRF1 receptor agonist, stressin I (1 μM) was similar in male and female mice (10.19 ± 2.19 μA/cm2 vs. 7.08 ± 5.16 μA/cm2, n = 4–5/group, p > 0.05). In male mice, Ucn3 caused a significantly higher ΔIsc than stressin I, CRF, Ucn1, or Ucn2. Contrastingly, female mice displayed a similar and weaker ΔIsc response to these peptides.ConclusionsMarked sex differences were observed in colonic baseline ion secretion and responses to stress‐related peptides. Male mice demonstrated a significantly higher baseline ion secretion in the proximal colon when compared with their female counter parts. In addition, CRF, Ucn1, Unc2, and Ucn3 evoked a greater increase in colonic ion secretion in male mice than female mice. Ucn3 is more efficacious than the other stress‐related peptides to evoke ion secretion in the male mouse colon, suggesting a predominant role of the CRF2 receptors in stimulation colonic ion secretion in males. Female mice, however, exhibited low level responses to these stress‐related peptides, and no significant differences were seen between these peptides‐evoked responses. The results suggest that the greater colonic baseline ion secretion and responses to stress‐related peptides in male mice may contribute to the high prevalence of IBS‐D in males.Support or Funding InformationNIH R15 DK097460‐01A1 (SL) and UWL graduate research service and educational leadership grant (AK). Howard Hua is a summer research fellow from Wheeler High School, Marietta, GA.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synthesis and Pharmacological Screening of Pyridopyrimidines as Effective Anti-Diarrheal Agents through the Suppression of Cyclic Nucleotide Accumulation.

The increased levels of cyclic nucleotides (cGMP and cAMP) in enterocytes trigger intracellular mechanisms of ion and fluid secretion into the lumen, causing secretory diarrhea. Twelve novel pyridopyrimidines derived from 5‐(3,5‐bistrifluoromethylphenyl)‐1,3‐dimethyl‐5,11‐dihydro‐1H‐indeno[2,1 : 5,6]pyrido[2,3‐d]pyrimidine‐2,4,6‐trione (FPIPP) were synthesized and evaluated on intracellular cyclic nucleotide accumulation. All compounds had no effect on either cyclic nucleotide basal levels or on pre‐contracted aortic rings. The metabolic activity and viability in T84 cells, assessed by MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide) and the LDH (lactate dehydrogenase) assays, respectively, were not affected by incubation with the compounds (50 μM). Compound VI almost abolished cGMP accumulation (94 % inhibition) induced by STa toxin in T834 cells and significantly reduced (69 %) forskolin‐induced cAMP accumulation in Jurkat cells. Compound VI was active in an in vivo model for diarrhea in rabbits. These results prompted us to perform a microscopic histopathological analysis of intestinal tissues, showing that only compound VI preserves the intestine without significant pathological changes and with a decreased inflammatory pattern in comparison to FPIPP. In vitro stability test revealed that compound VI is resistant to oxidation promoted by atmospheric oxygen.

Intestinal Epithelial AMPK Regulates Paracellular Electrolyte Flux and Electrogenic Ion Transport Responses in Mouse Colon

Ion transport within the colonic epithelium is crucial to maintain colonic homeostasis. During pathological states such as intestinal inflammation, alteration of ion transport processes contributes to the development of diarrhea and constipation. These pathways are tightly regulated by expression and localization to ensure fluid balance. AMP‐Activated Protein Kinase (AMPK) is a highly conserved energy‐sensing kinase that has previously been shown to regulate ion channels and transporters such as CFTR and NKCC1, in both intestinal epithelium and other epithelia. The aim of this study was to specifically test the impact of AMPK activity on ion flux across intestinal epithelia by utilizing a tissue‐specific knockout mouse model lacking both AMPK‐α catalytic isoforms.MethodsIntestinal epithelial cell‐specific knockout of AMPK activity (AMPKαΔIEC KO) was achieved by crossing Prkaa1 and Prkaa2 floxed mice (AMPKαfl/fl) with Villin‐Cre mice. For Ussing chamber experiments, large intestinal segments were stripped of the serosal layer and the mucosal layer was mounted in Ussing chambers containing Kreb's Ringer's buffer. Chloride secretion was stimulated using Forskolin (300μM) applied bilaterally, followed by Carbachol (10μM) applied basolaterally. For immunostaining, whole intestinal tissue was isolated and fixed in 4% paraformaldehyde overnight, washed with 1X PBS, and stored in 30% sucrose in 1X until OCT embedding. 5μm frozen sections of intestinal tissues were sliced and mounted onto glass slides.ResultsWe previously demonstrated AMPKαΔIEC KO mice do not have a barrier defect indicated by macromolecular intestinal permeability, histological analysis, and response to dextran sulfate sodium (DSS) compared to AMPKαfl/fl mice. We next determined if AMPK alters electrolye permeability in different regions of the large intestine. Indeed, loss of AMPK decreased transepithelial electrical resistance (TEER) in mouse proximal colon (35.21 ± 3.3 Ω·cm2, n = 6–7, p = 0.0007). Conversely, AMPKαΔIEC KO mice exhibited an increase in basal TEER in the distal colon (62.5 ± 4.5 Ω·cm2, n = 2–3, p = 0.2683), although this was not statistically significant. We next tested if AMPKαΔIEC KO mice exhibited changes in electrogenic ion transport by intestinal region. Preliminary evidence in the cecum indicated increased electrogenic ion transport responses to carbachol and Forskolin in AMPKαΔIEC KO mice (n = 1–3) while only forskolin‐dependent ion transport was increased in the distal colon (n = 2–3). In the proximal colon, Ca2+‐dependent ion transport was increased two‐fold (n = 6–7, p = 0.1131) while there was no alteration in cAMP‐dependent ion transport (n = 6–7). To determine if these region‐specific effects were associated with changes in transporter expression, colonic sections were stained for the Na+‐K+‐Cl− Cotransporter 1 (NKCC1) as a key contributor to electrogenic ion secretion. NKCC1 expression in AMPKαΔIEC KO mice was similar to that of AMPKαfl/fl mice (n = 2–3).ConclusionsAMP‐activated protein kinase mediates region‐specific maintenance of the intestinal barrier and regulation of electrogenic ion transport in mice.Support or Funding InformationNIH 2R01DK091281 (DFM)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of Sensitization and Inflammation on the Interaction of Mast Cells With the Intestinal Epithelium in Rats.

The density of intestinal mast cells has been reported to increase during inflammatory bowel disease (IBD). As mast cell mediators are known to increase the permeability of epithelial tight junctions, we hypothesized that antigen responses in sensitized animals might be enhanced under inflammatory conditions. This would contribute to a vicious circle by further enhancing the entry of luminal antigens into the colonic wall and thereby continuing the inadequate immune response during IBD. Therefore, one group of rats was sensitized against ovalbumin. In a second group of animals additionally a colitis was induced by rectal administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) dissolved in ethanol. Specimens from distal colon and jejunum (as intestinal segment located distantly from the inflamed area) were mounted in Ussing chambers to measure tissue conductance, short-circuit current (Isc) induced by antigen exposure and paracellular permeability (fluorescein flux). This was paralleled by determination of mast cell markers and tight junction proteins with immunofluorescence and qPCR. In contrast to the initial hypothesis, antigen-induced Isc was not upregulated, but tended to be downregulated in the tissues from the colitis animals, both in colon and in jejunum. Only in the jejunum mast cell degranulation evoked an increase in fluorescein flux. Mast cell density was not altered significantly in the colon of the colitis animals. In the jejunum, sensitization induced a strong increase in mast cell density, which was unaffected by additional induction of colitis. Expression of sealing tight junction components claudin-3 and -4 were increased on the protein level in the sensitized animals in comparison to non-sensitized animals. Additional induction of colitis evoked a downregulation of claudin-3 in both intestinal segments and an upregulation of claudin-4 in the jejunum. Consequently, these data indicate segment differences in mast cell – epithelium interaction, but no enhancement of ion secretion in the TNBS/ethanol model of acute colitis after prior sensitization.

Differential effects of Clostridium difficile toxins on ion secretion and cell integrity in human intestinal cells.

Toxin A (TcdA), toxin B (TcdB), and binary toxin (CDT) produced by Clostridium difficile (CD) are thought to play a key role in inducing diarrhea. The aim of this study was to investigate the individual and combined roles of CD toxins in inducing enterotoxic and cytotoxic effect. Ion secretion and epithelial damage were evaluated in the Ussing chambers as measure of enterotoxic or cytotoxic effect, respectively, in human-derived intestinal cells. When added to the mucosal side of Caco-2 cells, TcdB, but not TcdA, induced ion secretion and its effects increased in the presence of TcdA. CDT also induced ion secretion when added to either the mucosal or serosal compartment. Serosal addition of TcdB induced epithelial damage consistent with its cytotoxic effect. However, mucosal addition of TcdB had similar effects, but only in the presence of TcdA. CDT induced epithelial damage when added to the serosal side of cell monolayers, and this was associated with a late onset but prolonged effect. All data were replicated using human colon biopsies. These data indicate that CD, through the combined and direct activity of its three toxins, induces integrated and synergic enterotoxic and cytotoxic effects on the intestinal epithelium.

Probing Structure, Topology and Dynamics of KCNE3 in Proteoliposomes using Site-Direct Spin Labeling and EPR Spectroscopy

KCNE3 (E3) is a β-subunit of voltage gated potassium ion channels (Kv) that modulates the function of Kv by interacting with its a subunits including KCNQ1 and KCNQ4 (known as Q1 and Q4). Compared to other Kv channels, E3-Q1 interactions have been well studied in intestinal and tracheal epithelial cells where the complex maintains ion homeostasis. E3 interaction with Q1 allows Ky to act as a voltage independent and constitutively active channel that facilitates transepithelial Cl- ion secretion. Mutations in KCNE3 are responsible for diseases related to salt and fluid disorders including cystic fibrosis. Hence, it is important to elucidate KCNE3 structural and dynamic information with functional studies. Previously, the structure of E3 has been studied in a bicelle environment. However, in physiological conditions, lipids are organized into bilayers instead of single sheets of molecules. This research has been focused on protein structure, dynamic and topology of KCNE3 membrane protein in POPC/POPG vesicle usingSite-Directed Spin Labeling (SDSL) and Electron Paramagnetic Resonance Spectroscopy (EPR)techniques. EPR coupled with SDSL provides structural and dynamic information about proteins. In this study we have successfully introduced S-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate spin label (MTSL) on several site-specific position of E3 by site-directed mutagenesis to obtain EPR spectra. CW-EPR (Continuous Wave Electron Paramagnetic Resonance)line shape analysis has been performed to investigate the dynamic of the protein in a lipid bilayer, while EPR power saturation has been used to study the topology of KCNE3 in a vesicle. Our data are consistent with the model of KCNE3 in a bicelle and revealed more promising structure and dynamic information for the protein in a native membrane environment.

Malpighian tubules of caterpillars: blending RNAseq and physiology to reveal regional functional diversity and novel epithelial ion transport control mechanisms.

The Malpighian tubules (MTs) and hindgut constitute the functional kidney of insects. MTs are outpouchings 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 composed 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 present 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- and ligand-gated ion channels in epithelial ion transport of insect MTs.

Septate junction in the distal ileac plexus of larval lepidopteranTrichoplusia 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 MT 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 the 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 Trichoplusia ni, leads to alterations in paracellular permeability, SJ morphology and the abundance of its molecular components. 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.

Cystic Fibrosis of the Pancreas: The Role of CFTR Channel in the Regulation of Intracellular Ca2+ Signaling and Mitochondrial Function in the Exocrine Pancreas.

Cystic fibrosis (CF) is the most common genetic disorder that causes a significant damage in secretory epithelial cells due to the defective ion flux across the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Pancreas is one of the organs most frequently damaged by the disease leading to pancreatic insufficiency, abdominal pain and an increased risk of acute pancreatitis in CF patients causing a significant decrease in the quality of life. CFTR plays a central role in the pancreatic ductal secretory functions by carrying Cl- and HCO3- ions across the apical membrane. Therefore pathophysiological studies in CF mostly focused on the effects of impaired ion secretion by pancreatic ductal epithelial cells leading to exocrine pancreatic damage. However, several studies indicated that CFTR has a central role in the regulation of intracellular signaling processes and is now more widely considered as a signaling hub in epithelial cells. In contrast, elevated intracellular Ca2+ level was observed in the lack of functional CFTR in different cell types including airway epithelial cells. In addition, impaired CFTR expression has been correlated with damaged mitochondrial function in epithelial cells. These alterations of intracellular signaling in CF are not well characterized in the exocrine pancreas yet. Therefore in this review we would like to summarize the complex role of CFTR in the exocrine pancreas with a special focus on the intracellular signaling and mitochondrial function.

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 ion 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.

AMP-Activated Protein Kinase (AMPK)-Dependent Regulation of Renal Transport.

AMP-activated kinase (AMPK) is a serine/threonine kinase that is expressed in most cells and activated by a high cellular AMP/ATP ratio (indicating energy deficiency) or by Ca2+. In general, AMPK turns on energy-generating pathways (e.g., glucose uptake, glycolysis, fatty acid oxidation) and stops energy-consuming processes (e.g., lipogenesis, glycogenesis), thereby helping cells survive low energy states. The functional element of the kidney, the nephron, consists of the glomerulus, where the primary urine is filtered, and the proximal tubule, Henle’s loop, the distal tubule, and the collecting duct. In the tubular system of the kidney, the composition of primary urine is modified by the reabsorption and secretion of ions and molecules to yield final excreted urine. The underlying membrane transport processes are mainly energy-consuming (active transport) and in some cases passive. Since active transport accounts for a large part of the cell’s ATP demands, it is an important target for AMPK. Here, we review the AMPK-dependent regulation of membrane transport along nephron segments and discuss physiological and pathophysiological implications.

Acid-Base Disorders

Under physiologic conditions, the acid-base balance of the body is maintained via changes in ventilation that eliminate carbon dioxide, buffering of acid loads, and renal excretion of hydrogen ions. Failure to maintain the pH of the blood between 7.35 and 7.45 can result in life-threatening conditions. This review details the pathophysiology, stabilization and assessment, diagnosis and treatment, and disposition and outcomes of acid-base disorders. Figures show the relationship between hydrogen ions and blood pH, proximal tubular bicarbonate reabsorption, the secretion of hydrogen ions, renal ammonia production, ammonium diffusion, metabolic alkalosis, electrocardiographic changes in hypokalemia and hyperkalemia, pseudoinfarction caused by hyperkalemia, and an algorithmic approach to suspected acid-base disorders. Tables list causes of high–anion gap metabolic acidosis, metabolic acidosis with a normal anion gap, type 1 renal tubular acidosis, type 4 renal tubular acidosis and aldosterone resistance, metabolic alkalosis, respiratory acidosis, and respiratory alkalosis; treatment of hyperkalemia; and a stepwise approach for the evaluation of suspected acid-base disorders. This review contains 9 highly rendered figures, 9 tables, 64 references, and a list of pertinent Web sites.

Adenylyl cyclase 6 is involved in the hyposecretory status of experimental colitis.

One of the cardinal symptoms of intestinal inflammation is diarrhea. Acute intestinal inflammation is associated with inhibition of ion absorption and increased secretion, along with fluid leakage due to epithelial injury and changes in permeability. However, in the chronic situation, a downregulation of both absorptive and secretory transport has been reported. We investigated how experimental colitis reduces cAMP levels in intestinal epithelial cells through modulation of adenylyl cyclases (AC). Primary colonic epithelial cells obtained from rats with trinitrobenzenesulfonic acid colitis and non-colitic controls were analyzed for AC expression by RT-qPCR and Western blot, following a preliminary microarray analysis. AC6 and AC5 were found to be expressed in colonocytes, and downregulated by inflammation, with the former exhibiting considerably higher mRNA levels in both cases. To test the hypothesis that inflammatory cytokines may account for this effect, Caco 2 cells were treated with IL-1β, TNF-α, or IFN-γ. All three cytokines inhibited forskolin evoked short-circuit currents in Ussing chambers and lowered intracellular cAMP, but failed to alter AC6 mRNA levels. AC5/AC6 expression was however inhibited in mouse jejunal organoids treated with IFN-γ and TNF-α, but not IL-1β. Gene knockdown of AC6 resulted in a significant decrease of ion secretion in T84 cells. We conclude that the disturbances in ion secretion observed in rat TNBS colitis are associated with low intracellular levels of cAMP in the epithelium, which may be explained in part by the downregulation of AC5/AC6 expression by proinflammatory cytokines.

Hyperkalemic Forms of Renal Tubular Acidosis: Clinical and Pathophysiological Aspects.

In contrast to distal type I or classic renal tubular acidosis (RTA) that is associated with hypokalemia, hyperkalemic forms of RTA also occur usually in the setting of mild-to-moderate CKD. Two pathogenic types of hyperkalemic metabolic acidosis are frequently encountered in adults with underlying CKD. One type, which corresponds to some extent to the animal model of selective aldosterone deficiency (SAD) created experimentally by adrenalectomy and glucocorticoid replacement, is manifested in humans by low plasma and urinary aldosterone levels, reduced ammonium excretion, and preserved ability to lower urine pH below 5.5. This type of hyperkalemic RTA is also referred to as type IV RTA. It should be noted that the mere deficiency of aldosterone when glomerular filtration rate is completely normal only causes a modest decline in plasma bicarbonate which emphasizes the importance of reduced glomerular filtration rate in the development of the hyperchloremic metabolic acidosis associated with SAD. Another type of hyperkalemic RTA distinctive from SAD in which plasma aldosterone is not reduced is referred to as hyperkalemic distal renal tubular acidosis because urine pH cannot be reduced despite acidemia or after provocative tests aimed at increasing sodium-dependent distal acidification such as the administration of sodium sulfate or loop diuretics with or without concurrent mineralocorticoid administration. This type of hyperkalemic RTA (also referred to as voltage-dependent distal renal tubular acidosis) has been best described in patients with obstructive uropathy and resembles the impairment in both hydrogen ion and potassium secretion that are induced experimentally by urinary tract obstruction and when sodium transport in the cortical collecting tubule is blocked by amiloride.

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.