scholarly journals Role of an apical K,Cl cotransporter in urine formation by renal tubules of the yellow fever mosquito (Aedes aegypti)

2011 ◽  
Vol 301 (5) ◽  
pp. R1318-R1337 ◽  
Author(s):  
Peter M. Piermarini ◽  
Rebecca M. Hine ◽  
Matthew Schepel ◽  
Jeremy Miyauchi ◽  
Klaus W. Beyenbach
Keyword(s):  
Aedes Aegypti ◽  
Epithelial Transport ◽  
Input Resistance ◽  
Fluid Secretion ◽  
Malpighian Tubules ◽  
Cell Swelling ◽  
Renal Tubules ◽  
Transport Pathway ◽  
Principal Cells

The K,Cl cotransporters (KCCs) of the SLC12 superfamily play critical roles in the regulation of cell volume, concentrations of intracellular Cl−, and epithelial transport in vertebrate tissues. To date, the role(s) of KCCs in the renal functions of mosquitoes and other insects is less clear. In the present study, we sought molecular and functional evidence for the presence of a KCC in renal (Malpighian) tubules of the mosquito Aedes aegypti . Using RT-PCR on Aedes Malpighian tubules, we identified five alternatively spliced partial cDNAs that encode putative SLC12-like KCCs. The majority transcript is AeKCC1-A1; its full-length cDNA was cloned. After expression of the AeKCC1-A protein in Xenopus oocytes, the Cl−-dependent uptake of 86Rb+ is 1) activated by 1 mM N-ethylmaleimide and cell swelling, 2) blocked by 100 μM dihydroindenyloxyalkanoic acid (DIOA), and 3) dependent upon N-glycosylation of AeKCC1-A. In Aedes Malpighian tubules, AeKCC1 immunoreactivity localizes to the apical brush border of principal cells, which are the predominant cell type in the epithelium. In vitro physiological assays of Malpighian tubules show that peritubular DIOA (10 μM): 1) significantly reduces both the control and diuretic rates of transepithelial fluid secretion and 2) has negligible effects on the membrane voltage and input resistance of principal cells. Taken together, the above observations indicate the presence of a KCC in the apical membrane of principal cells where it participates in a major electroneutral transport pathway for the transepithelial secretion of fluid in this highly electrogenic epithelium.

scholarly journals A SLC4-like anion exchanger from renal tubules of the mosquito (Aedes aegypti): evidence for a novel role of stellate cells in diuretic fluid secretion

2010 ◽  
Vol 298 (3) ◽  
pp. R642-R660 ◽  
Author(s):  
Peter M. Piermarini ◽  
Laura F. Grogan ◽  
Kenneth Lau ◽  
Li Wang ◽  
Klaus W. Beyenbach
Keyword(s):  
Aedes Aegypti ◽  
Anion Exchanger ◽  
Xenopus Oocytes ◽  
Malpighian Tubule ◽  
Stellate Cells ◽  
Fluid Secretion ◽  
Malpighian Tubules ◽  
Renal Tubules ◽  
Basal Region ◽  
Principal Cells

Transepithelial fluid secretion across the renal (Malpighian) tubule epithelium of the mosquito ( Aedes aegypti ) is energized by the vacuolar-type (V-type) H+-ATPase and not the Na+-K+-ATPase. Located at the apical membrane of principal cells, the V-type H+-ATPase translocates protons from the cytoplasm to the tubule lumen. Secreted protons are likely to derive from metabolic H2CO3, which raises questions about the handling of HCO3−by principal cells. Accordingly, we tested the hypothesis that a Cl/HCO3anion exchanger (AE) related to the solute-linked carrier 4 (SLC4) superfamily mediates the extrusion of HCO3−across the basal membrane of principal cells. We began by cloning from Aedes Malpighian tubules a full-length cDNA encoding an SLC4-like AE, termed AeAE. When expressed heterologously in Xenopus oocytes, AeAE is both N- and O-glycosylated and mediates Na+-independent intracellular pH changes that are sensitive to extracellular Cl−concentration and to DIDS. In Aedes Malpighian tubules, AeAE is expressed as two distinct forms: one is O-glycosylated, and the other is N-glycosylated. Significantly, AeAE immunoreactivity localizes to the basal regions of stellate cells but not principal cells. Concentrations of DIDS that inhibit AeAE activity in Xenopus oocytes have no effects on the unstimulated rates of fluid secretion mediated by Malpighian tubules as measured by the Ramsay assay. However, in Malpighian tubules stimulated with kinin or calcitonin-like diuretic peptides, DIDS reduces the diuretic rates of fluid secretion to basal levels. In conclusion, Aedes Malpighian tubules express AeAE in the basal region of stellate cells, where this transporter may participate in producing diuretic rates of transepithelial fluid secretion.

scholarly journals NHE8 is an intracellular cation/H+ exchanger in renal tubules of the yellow fever mosquito Aedes aegypti

2009 ◽  
Vol 296 (4) ◽  
pp. F730-F750 ◽  
Author(s):  
Peter M. Piermarini ◽  
Dirk Weihrauch ◽  
Heiko Meyer ◽  
Markus Huss ◽  
Klaus W. Beyenbach
Keyword(s):  
Aedes Aegypti ◽  
Yellow Fever ◽  
Xenopus Oocytes ◽  
Intracellular Localization ◽  
Malpighian Tubules ◽  
Xenopus Laevis Oocytes ◽  
Renal Tubules ◽  
Yellow Fever Mosquito ◽  
Western Blots ◽  
Principal Cells

The goal of this study was to identify and characterize the hypothesized apical cation/H+ exchanger responsible for K+ and/or Na+ secretion in the renal (Malpighian) tubules of the yellow fever mosquito Aedes aegypti. From Aedes Malpighian tubules, we cloned “ AeNHE8,” a full-length cDNA encoding an ortholog of mammalian Na+/H+ exchanger 8 (NHE8). The expression of AeNHE8 transcripts is ubiquitous among mosquito tissues and is not enriched in Malpighian tubules. Western blots of Malpighian tubules suggest that AeNHE8 is expressed primarily as an intracellular protein, which was confirmed by immunohistochemical localizations in Malpighian tubules. AeNHE8 immunoreactivity is expressed in principal cells of the secretory, distal segments, where it localizes to a subapical compartment (e.g., vesicles or endosomes), but not in the apical brush border. Furthermore, feeding mosquitoes a blood meal or treating isolated tubules with dibutyryl-cAMP, both of which stimulate a natriuresis by Malpighian tubules, do not influence the intracellular localization of AeNHE8 in principal cells. When expressed heterologously in Xenopus laevis oocytes, AeNHE8 mediates EIPA-sensitive Na/H exchange, in which Li+ partially and K+ poorly replace Na+. The expression of AeNHE8 in Xenopus oocytes is associated with the development of a conductive pathway that closely resembles the known endogenous nonselective cation conductances of Xenopus oocytes. In conclusion, AeNHE8 does not mediate cation/H+ exchange in the apical membrane of Aedes Malpighian tubules; it is more likely involved with an intracellular function.

Analysis of epithelial transport by measurement of K+, Cl- and pH gradients in extracellular unstirred layers: ion secretion and reabsorption by Malpighian tubules of Rhodnius prolixus

1997 ◽  
Vol 200 (11) ◽  
pp. 1627-1638 ◽  
Author(s):  
KA Collier ◽  
MJ O'Donnell
Keyword(s):  
Epithelial Transport ◽  
Malpighian Tubule ◽  
Fluid Secretion ◽  
Rhodnius Prolixus ◽  
Malpighian Tubules ◽  
Unstirred Layer ◽  
Ph Gradients ◽  
In Situ Preparation

Summary The pH and concentrations of K+ and Cl- in the unstirred layer (USL) associated with the basolateral surfaces of the upper and lower Malpighian tubules of Rhodnius prolixus were measured using extracellular ion-selective microelectrodes. When stimulated with 5-hydroxytryptamine (5-HT) in vitro, the upper Malpighian tubule secretes Na+, K+, Cl- and water at high rates; the lower Malpighian tubule reabsorbs K+ and Cl- but not water. Concentrations of K+ and Cl- in the unstirred layer of the lower Malpighian tubule ([K+]USL, [Cl-]USL) were greater than those in the bathing saline, consistent with the accumulation of K+ and Cl- in the USL during 5-HT-stimulated KCl reabsorption. [K+]USL exceeded [K+]Bath by as much as 5.3-fold. Calculations of K+ flux based on measurements of [K+]USL at various distances from the tubule surface agreed well with flux calculated from the rate of fluid secretion and the change in K+ concentration of the secreted fluid during passage through the lower tubule. Concentrations of K+ in the unstirred layer of the upper Malpighian tubule were reduced relative to those in the bathing saline, consistent with depletion of K+ from the USL during 5-HT-stimulated secretion of K+ from bath to lumen. Changes in [K+]USL during 5-HT-stimulated K+ secretion from single upper Malpighian tubule cells could be resolved. Although differences between [K+]USL and [K+]Bath were apparent for upper and lower tubules in an in situ preparation, they were reduced relative to the differences measured using isolated tubules. We suggest that convective mixing of the fluids around the tubules by contractions of the midgut and hindgut reduces, but does not eliminate, differences between [K+]USL and [K+]Bath in situ. The USL was slightly acidic relative to the bath in 5-HT-stimulated upper and lower tubules; contributions to USL acidification are discussed. The results also show that the techniques described in this paper can resolve rapid and localized changes in ion transport across different regions of Malpighian tubules in response to stimulants or inhibitors of specific membrane transporters.

The anterior and posterior ‘stomach’ regions of larval Aedes aegypti midgut: regional specialization of ion transport and stimulation by 5-hydroxytryptamine

1999 ◽  
Vol 202 (3) ◽  
pp. 247-252 ◽  
Author(s):  
T.M. Clark ◽  
A. Koch ◽  
D.F. Moffett
Keyword(s):  
Steady State ◽  
Aedes Aegypti ◽  
Ion Transport ◽  
Malpighian Tubules ◽  
Transport Mechanisms ◽  
Regional Specialization ◽  
Negative Deflection ◽  
Electrical Polarity

The ‘stomach’ region of the larval mosquito midgut is divided into histologically distinct anterior and posterior regions. Anterior stomach perfused symmetrically with saline in vitro had an initial transepithelial potential (TEP) of −66 mV (lumen negative) that decayed within 10–15 min to a steady-state TEP near −10 mV that was maintained for at least 1 h. Lumen-positive TEPs were never observed in the anterior stomach. The initial TEP of the perfused posterior stomach was opposite in polarity, but similar in magnitude, to that of the anterior stomach, measuring +75 mV (lumen positive). This initial TEP of the posterior stomach decayed rapidly at first, then more slowly, eventually reversing the electrical polarity of the epithelium as lumen-negative TEPs were recorded in all preparations within 70 min. Nanomolar concentrations of the biogenic amine 5-hydroxytryptamine (5-HT, serotonin) stimulated both regions, causing a negative deflection of the TEP of the anterior stomach and a positive deflection of the TEP of the posterior stomach. Phorbol 12,13-diacetate also caused a negative deflection of the TEP of the anterior stomach, but had no effect on the TEP of the posterior stomach. These data demonstrate that 5-HT stimulates region-specific ion-transport mechanisms in the stomach of Aedes aegypti and suggest that 5-HT coordinates the actions of the Malpighian tubules and midgut in the maintenance of an appropriate hemolymph composition in vivo.

Secretory NaCl and volume flow in renal tubules

1986 ◽  
Vol 250 (5) ◽  
pp. R753-R763 ◽  
Author(s):  
K. W. Beyenbach
Keyword(s):  
Fluid Secretion ◽  
Renal Tissue ◽  
Tubular Secretion ◽  
Proximal Tubules ◽  
Epithelial Cell Line ◽  
Renal Tubules ◽  
Genetic Potential ◽  
Dog Kidney ◽  
Nacl Secretion

This review attempts to give a retrospective survey of the available evidence concerning the secretion of NaCl and fluid in renal tubules of the vertebrate kidney. In the absence of glomerular filtration, epithelial secretory mechanisms, which to this date have not been elucidated, are responsible for the renal excretion of NaCl and water in aglomerular fish. However, proximal tubules isolated from glomerular fish kidneys of the flounder, killifish, and the shark also have the capacity to secrete NaCl and fluid. In shark proximal tubules, fluid secretion appears to be driven via secondary active transport of Cl. In another marine vertebrate, the sea snake, secretion of Na (presumably NaCl) and fluid is observed in freshwater-adapted and water-loaded animals. Proximal tubules of mammals can be made to secrete NaCl in vitro together with secretion of aryl acids. An epithelial cell line derived from dog kidney exhibits secondary active secretion of Cl when stimulated with catecholamines. Tubular secretion of NaCl and fluid may serve a variety of renal functions, all of which are considered here. The occurrence of NaCl and fluid secretion in glomerular proximal tubules of teleosts, elasmobranchs, and reptiles and in mammalian renal tissue cultures suggests that the genetic potential for NaCl secretion is present in every vertebrate kidney.

Voltage clamping single cells in intact Malpighian tubules of mosquitoes

2000 ◽  
Vol 279 (4) ◽  
pp. F747-F754 ◽  
Author(s):  
R. Masia ◽  
D. Aneshansley ◽  
W. Nagel ◽  
R. J. Nachman ◽  
K. W. Beyenbach
Keyword(s):  
Apical Membrane ◽  
Single Cells ◽  
Basolateral Membrane ◽  
Malpighian Tubule ◽  
Input Resistance ◽  
Membrane Resistance ◽  
Malpighian Tubules ◽  
Shunt Resistance ◽  
Transport Pathways ◽  
Principal Cells

Principal cells of the Malpighian tubule of the yellow fever mosquito were studied with the methods of two-electrode voltage clamp (TEVC). Intracellular voltage ( V pc) was −86.7 mV, and input resistance ( R pc) was 388.5 kΩ ( n = 49 cells). In six cells, Ba2+ (15 mM) had negligible effects on V pc, but it increased R pc from 325.3 to 684.5 kΩ ( P< 0.001). In the presence of Ba2+, leucokinin-VIII (1 μM) increased V pc to −101.8 mV ( P < 0.001) and reduced R pc to 340.2 kΩ ( P < 0.002). Circuit analysis yields the following: basolateral membrane resistance, 652.0 kΩ; apical membrane resistance, 340.2 kΩ; shunt resistance ( R sh), 344.3 kΩ; transcellular resistance, 992.2 kΩ. The fractional resistance of the apical membrane (0.35) and the ratio of transcellular resistance and R sh (3.53) agree closely with values obtained by cable analysis in isolated perfused tubules and confirm the usefulness of TEVC methods in single principal cells of the intact Malpighian tubule. Dinitrophenol (0.1 mM) reversibly depolarized V pc from −94.3 to −10.7 mV ( P< 0.001) and reversibly increased R pc from 412 to 2,879 kΩ ( P < 0.001), effects that were duplicated by cyanide (0.3 mM). Significant effects of metabolic inhibition on voltage and resistance suggest a role of ATP in electrogenesis and the maintenance of conductive transport pathways.

Net fluid secretion in proximal straight renal tubules in vitro: role of PAH

1974 ◽  
Vol 226 (1) ◽  
pp. 191-197 ◽  
Author(s):  
JJ Grantham ◽  
PB Qualizza ◽  
RL Irwin
Keyword(s):  
Fluid Secretion ◽  
Renal Tubules

scholarly journals Excretion in the House Cricket (Acheta Domesticus): Stimulation of Diuresis by Tissue Homogenates

1987 ◽  
Vol 129 (1) ◽  
pp. 63-81 ◽  
Author(s):  
JEFFREY H. SPRING ◽  
SHELIA R. HAZELTON
Keyword(s):  
Lethal Dose ◽  
Fluid Secretion ◽  
Sodium Concentration ◽  
Inhibitory Effect ◽  
Acheta Domesticus ◽  
Malpighian Tubules ◽  
Secretion Rate ◽  
Tissue Homogenates ◽  
Extreme Sensitivity

1. A new method is described for maintaining cricket Malpighian tubules in vitro. Warmed, oxygenated saline is circulated rapidly past the tubules, while the secreted urine is collected under oil for analysis. This technique allows the cricket tubules to be observed and manipulated for extended periods (6 h), in contrast to their short life (&gt;1 h) using conventional methods. 2. Cricket tubules show extreme sensitivity to oxygen deprivation, such that 15 min of anoxia represents the median lethal dose (LD50) for in vitro preparations. 3. Homogenates of corpus cardiacum (CC) cause the rate of fluid secretion by the tubules to double. The maximum stimulation is dose-dependent over the range 0.01 to 1.0 CC. Homogenates of brain and other ganglia show much smaller stimulatory effects (0.01-0.02 CC-equivalents). Cyclic AMP mimics the increase in secretion rate, but has an inhibitory effect on the smooth muscle of the ureter. 4. Control preparations maintain a urine osmotic pressure (OP) that is hyperosmotic to the bath by 5–10 mosmol l−1. CC homogenate produces a decrease in urine OP to 10–12 mosmol l−1 hypo-osmotic to the bath. This suggests that active solute reabsorption is occurring in the lower tubule or ampulla. 5. Stimulation by CC homogenate increases the urine potassium concentration slightly less than two-fold, whereas the sodium concentration increases by a maximum of five-fold and remains at a higher concentration than potassium throughout the experiment. Tubule secretion rate is drastically inhibited in nominally sodium-free saline.

scholarly journals Excretion of NaCl and KCl loads in mosquitoes. 2. Effects of the small molecule Kir channel modulator VU573 and its inactive analog VU342

2014 ◽  
Vol 307 (7) ◽  
pp. R850-R861 ◽  
Author(s):  
Matthew F. Rouhier ◽  
Rebecca M. Hine ◽  
Seokhwan Terry Park ◽  
Rene Raphemot ◽  
Jerod Denton ◽  
...  
Keyword(s):  
Urinary Excretion ◽  
Small Molecules ◽  
Fluid Secretion ◽  
Ringer Solution ◽  
Malpighian Tubules ◽  
Kir Channel ◽  
Channel Modulator ◽  
Inactive Analog

The effect of two small molecules VU342 and VU573 on renal functions in the yellow fever mosquito Aedes aegypti was investigated in vitro and in vivo. In isolated Malpighian tubules, VU342 (10 μM) had no effect on the transepithelial secretion of Na+, K+, Cl−, and water. In contrast, 10 μM VU573 first stimulated and then inhibited the transepithelial secretion of fluid when the tubules were bathed in Na+-rich or K+-rich Ringer solution. The early stimulation was blocked by bumetanide, suggesting the transient stimulation of Na-K-2Cl cotransport, and the late inhibition of fluid secretion was consistent with the known block of AeKir1, an Aedes inward rectifier K+ channel, by VU573. VU342 and VU573 at a hemolymph concentration of about 11 μM had no effect on the diuresis triggered by hemolymph Na+ or K+ loads. VU342 at a hemolymph concentration of 420 μM had no effect on the diuresis elicited by hemolymph Na+ or K+ loads. In contrast, the same concentration of VU573 significantly diminished the Na+ diuresis by inhibiting the urinary excretion of Na+, Cl−, and water. In K+-loaded mosquitoes, 420 μM VU573 significantly diminished the K+ diuresis by inhibiting the urinary excretion of K+, Na+, Cl−, and water. We conclude that 1) the effects of VU573 observed in isolated Malpighian tubules are overwhelmed in vivo by the diuresis triggered with the coinjection of Na+ and K+ loads, and 2) at a hemolymph concentration of 420 μM VU573 affects Kir channels systemically, including those that might be involved in the release of diuretic hormones.

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