Comparative kidney tubule sources, isolation, perfusion, and function.
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Amphibians↗
Biomedical subjects
K W Beyenbach
Publications and source records attributed to K W Beyenbach.
Transport of magnesium across biological membranes.
The movement of Mg across biological membranes is reviewed from the perspectives of (1) passive transport, (2) primary active transport and (3) secondary active transport. Since all cells maintain intracellular Mg2+ at a lower electrochemical potential than extracellular Mg2+, active transport pumps bringing Mg2+ into the cell have neither been postulated nor been confirmed. Most evidence points to influx leaks, presumably via membrane channels and carriers which do not perfectly exclude Mg2+. However, Mg2+ currents have been measured in prokaryotic and eukaryotic cells suggesting the presence of Mg2+ channels. Mg2+ influx through channels is largely driven by the membrane voltage because transmembrane Mg2+ concentration differences are not very large. Efflux mechanisms have attracted most of the investigative focus. Secondary active transport by way of Na/Mg exchange appears to be widely distributed in eukaryotic cells. The early investigations of Na/Mg exchange had Mg2+ efflux driven solely by the Na+ influx (secondary active transport). However, recent studies have revealed an ATP dependence of Na/Mg exchange which may reflect the operation of an Mg2+ pump (primary active transport). Similar mechanisms of Mg2+ influx and efflux appear to operate in epithelial tissues which may net absorb or secrete Mg2+. In recent years, the intact red blood cell has emerged as the model of choice for studies of Mg2+ membrane transport. However, further probing of the details of individual transport mechanisms may be complicated by the presence of multiple parallel Mg2+ influx and efflux systems in intact cells. Accordingly, it would appear that the next round of advances in membrane transport of Mg2+ will come from studies at subcellular levels, which aim at the isolation of transporters and their reconstitution. These studies should now be possible at least for Na/Mg exchange given our fair understanding of this system in intact red blood cells.
Animals↗
Leucokinins, a new family of ion transport stimulators and inhibitors in insect Malpighian tubules.
Leucokinins are octapeptides isolated from heads of the cockroach Leucophaea maderae. In the cockroach they increase motility of the isolated hindgut. Surprisingly, synthetic leucokinins have biological activity in a different insect and in a different tissue. In isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti, leucokinins depolarize the transepithelial voltage. This effect on voltage is dependent on extracellular Cl. One leucokinin, LK-8, the effects of which were studied further in isolated Malpighian tubules, was found to inhibit transepithelial fluid secretion at low concentrations (10(-11) M threshold), and to stimulate fluid secretion at high concentrations (3.5 x 10(-9) M threshold). Together, the depolarizing effects on voltage and the stimulation of fluid secretion suggest that leucokinins increase the Cl permeability of the tubule wall thereby increasing the availability of Cl for secretion with Na, K and water. Structure-function comparisons of the seven leucokinins studied suggest that the active region of the octapeptide is segregated to the C-terminal pentapeptide. In view of the known effects of leucokinins on hindgut motility in the cockroach, our finding of effects in mosquito Malpighian tubules suggests that leucokinins may be widely distributed in insects where they may have diverse functions in a variety of organs.
Aedes↗
Fluid secretion in glomerular renal proximal tubules of freshwater-adapted fish.
Tubular secretion by renal proximal tubules, as a mechanism for delivering fluid and electrolytes to the urine, has received little attention in modern conceptions of renal function in vertebrates even though it is the mechanism for urine production in aglomerular fish. This report demonstrates that some proximal tubules of glomerular kidneys of freshwater-adapted euryhaline fish spontaneously secrete fluid. The fluid consists primarily of Na (138 mM) and Cl (160 mM). NaCl and fluid secretion can be stimulated by adenosine 3',5-cyclic monophosphate, suggesting that tubular fluid secretion is under hormonal control. Fluid secretion driven by NaCl secretion in glomerular proximal tubules of fish that already filter NaCl and water suggests that secretion of fluid and NaCl may play a fundamental role in vertebrate renal function beyond a preadaptation for aglomerular urine formation.
Animals↗
The everted renal tubule: a methodology for direct assessment of apical membrane function.
A technique is described whereby it is possible to evert a 0.5- to 1.0-mm segment of an amphibian renal tubule and perfuse it in vitro. Consequently, the apical membranes of an intact nephron fragment are directly accessible for electrophysiological study. Viability of the cells of everted diluting segments taken from Ambystoma kidney was indicated by 1) failure of the cells to take up trypan blue and 2) the existence of an apical membrane voltage (average 66 mV, cell negative), which decreased predictably in the presence of either 5 mM barium or elevated potassium in the luminal bathing solution. The utility of the everted tubule to patch clamp studies was tested. A large conductance channel that appeared to be selective for potassium could be demonstrated in a cell-attached patch of the apical membrane of an everted initial collecting tubule. The everted tubule preparation not only provides large quantities of apical membrane for patch clamp studies but, more importantly, allows the investigator to control the solutions bathing each membrane surface independently. The application of patch clamp techniques to perfused, everted tubules may then serve to more completely describe the role of the apical membrane in transcellular ion transport.
Ambystoma↗
High-conductance K+ channel in apical membranes of principal cells cultured from rabbit renal cortical collecting duct anlagen.
Using the patch clamp technique, one type of K+ channel was identified in the apical cell membrane of cultured principal cells of rabbit renal collecting ducts in the cell-attached or excised-patch configuration. The channel was highly selective for K+ over Na+ (typically 30-70-fold) and had a conductance of 180, SD +/- 39 pS (n = 6), referred to a situation of 140 mmolar K+-Ringer solution present on either surface of the patch membrane. Channel activity was completely blocked by Ba2+ (5 mmol/l) and partially inhibited by Na+. The latter was evidenced by a deviation from Goldman rectification at high cytoplasm-positive membrane potentials, which was observed when Na+ competed with K+ for channel entrance from the cytoplasmic surface. Channel open probability depended strongly on membrane voltage and cytoplasmic Ca2+ concentration. Open-close kinetics exhibited double exponential behaviour, with a strong voltage dependence of the slow open time constant. Infrequently also a substate conductance level was identified. The voltage and calcium dependence suggest that the channel plays a role in adjusting K+ secretion to Na+ absorption in the fine regulation of cation excretion in renal collecting ducts.
Animals↗
Hormone-controlled cAMP-mediated fluid secretion in yellow-fever mosquito.
Evidence is presented for hormone-controlled adenosine 3',5'-cyclic monophosphate (cAMP)-mediated NaCl diuresis in Malpighian tubules of the blood-feeding yellow-fever mosquito Aedes aegypti. Studies in isolated Malpighian tubules reveal that cAMP added to the peritubular bath selectively stimulates NaCl secretion and not KCl secretion by increasing the Na conductance of the basolateral membrane of primary cells. These effects are duplicated by forskolin and theophylline in parallel with increased intracellular concentrations of endogenous cAMP. Two natriuretic peptides that we have isolated by high-pressure liquid chromatography (HPLC) methods from mosquito heads also increase NaCl and fluid secretion in isolated Malpighian tubules together with increased intracellular levels of cAMP. These results are consistent with a mechanism of NaCl diuresis in which the natriuretic peptides and cAMP are respectively the primary and secondary messengers that couple the ingestion of a blood meal to the excretion of the unwanted salt and water fraction of the meal. This hypothesis is supported by in vivo studies that reveal elevated intracellular cAMP levels in Malpighian tubules at the time of maximum NaCl diuresis.
Aedes↗
Peptide nature of two mosquito natriuretic factors.
High-pressure liquid chromatography (HPLC) of saline extracts of Aedes aegypti heads yields three fractions (from a total of 108) that affect transepithelial voltage and/or fluid secretion in isolated Aedes Malpighian tubules. In this study we investigated the physical and chemical nature of the active materials in these fractions. Gel-filtration chromatography revealed that the molecular weights of the three fractions were between 1,900 and 2,700. To test their thermostability the fractions were repeatedly frozen and thawed over a period of 110 days without loss of biological activity. Boiling at 100 degrees C for 5 min failed to significantly reduce their biological effects in isolated Malpighian tubules. In contrast, treatment with the proteolytic enzyme mixture, pronase, destroyed activity in all three. Fraction I no longer depolarized the transepithelial voltage of in vitro perfused Malpighian tubules, and fractions II and III completely lost their ability to stimulate fluid secretion and to affect transepithelial voltage. We conclude that our HPLC isolation yields a heterogeneous group of three polar low-molecular weight peptides. Expression of their biological activities in Malpighian tubules depends on intact peptide bonds.
Aedes↗
Renal proximal tubule of flounder. I. Physiological properties.
The proximal segment of the winter flounder, Pseudopleuronectes americanus, was investigated. Isolated tubules net secrete fluid, although at low rates, 37 pl X min-1 X mm-1. The dominant ions in secreted fluid are Na and Cl, with [Cl] significantly higher than in the bath. Mg and SO4 concentrations in secreted fluid are more than 10-fold greater than in the bath. The transepithelial voltage (-1.9 mV) and resistance (26 omega X cm2) indicate an electrically leaky epithelium, and transepithelial diffusion potentials suggest the Na selectivity of the paracellular pathway. Transepithelial electrochemical potentials point to active transport of Mg, SO4, and probably also Cl and to transepithelial equilibrium of Na. Failure to observe any secretory transport in tubules perfused in vitro at the commonly used perfusion rates illustrates the necessity of low, preferably subnanoliter, perfusion rates in detecting and studying low-capacity epithelial transport systems by the method of Burg.
Animals↗
Renal proximal tubule of flounder II. Transepithelial Mg secretion.
The kinetics of transepithelial Mg secretion were studied in isolated perfused proximal tubule II of the flounder, Pseudopleuronectes americanus. To detect measurable changes of luminal [Mg] as a function of bath [Mg], the lumen had to be perfused at subnanoliter rates. Transepithelial Mg secretion did not obey first-order reaction kinetics; hence values for half saturation of transport (0.22 mM) and transport maximum (1.5 pmol X min-1 X mm-1) are apparent and suggest a high-affinity low-capacity transport system. Because all experiments were done in the absence of bath SO4, the independence of Mg transport from SO4 transport is established. In the absence of perfusion, when tubules secrete fluid spontaneously, secreted fluid contained Cl (156 +/- 3 mM), Na (130 +/- 6 mM), and Mg (27 +/- 5 mM), all significantly different from the bath. Rates of Cl, Na, and Mg secretion were all positively correlated with fluid secretion, but Na and Mg concentrations in secreted fluid were inversely proportional. The results indicate that NaCl secretion provides basal rates of fluid secretion, and when MgCl2 is secreted in addition, fluid secretion increases with the effect of generating inverse relationships between luminal Na and Mg concentrations.
Animals↗
Secretory NaCl and volume flow in renal tubules.
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.
Animals↗
Unresolved questions of renal magnesium homeostasis.
While the basic tenets of Mg handling by the mammalian kidney have been clearly outlined, Mg homeostasis in the body, the mechanism of extracellular fluid Mg regulation by the kidneys, and the regulation of intracellular Mg concentrations are largely unknown. Only a small fraction of total body Mg is present in the extracellular fluid and handled by the kidney, raising the question whether other organs, intestine, muscle and bone participate in Mg balance as well. In view of the tight regulation of Mg concentration in the plasma and the life-threatening derangements when plasma Mg concentration falls outside the physiological range it is surprising that a hormone responsible for general Mg homeostasis has not yet been identified. A hormone or factor which regulates tubular Mg reabsorption and hence renal Mg excretion is suspected, but it has not been found in any animal. Renal regulation of extracellular Mg involves filtration at the glomerulus and reabsorption in the proximal tubule and the thick ascending limb of the loop of Henle, where respectively, 30 and 60% of the filtered Mg load are reabsorbed. In the nongrowing individual the quantities of Mg excreted match the quantities absorbed from the gut. Mechanisms of tubular Mg transport are largely unknown for lack of understanding of Mg transport across epithelial cell membranes. The unexplored nature of Mg physiology can be attributed to analytical limitations. However, with the advent of a new Mg exchanger suitable for microelectrode work and with the use of Mg-sensitive dyes, Mg research is about to enter the renaissance period.
Animals↗
Patch clamp studies of apical membranes of renal cortical collecting ducts.
Patch clamp techniques were used in study of the apical membrane of isolated renal cortical collecting ducts. Whereas on-the-cell patches (reported previously) gave channel activity due primarily to K+, isolated inside-out patches of the same membranes bathed with 150 mM Na+, 5 mM K+, and 1 mM Ca2+ gave channel activity due primarily to Na+. Na+ channels could remain either open or closed for periods of milliseconds to minutes. Although unitary currents of tenths of a pA were observed routinely, patch activity could change spontaneously between quiescent and violent with 'apparent' unitary currents of several pA. It was not possible to rule out the idea that the complex activity of the patches was due to synchronous or near synchronous openings/closings of several channels of identical unitary current. The unpredictable spontaneous changes of patch activity make difficult the design of experiments to test for the influence of agents expected or suspected to alter channel behavior.
Animals↗
Dibutyryl-cAMP increases basolateral sodium conductance of mosquito Malpighian tubules.
Dibutyryladenosine 3',5'-cyclic monophosphate (cAMP) stimulates fluid secretion in isolated Malpighian tubules of the mosquito Aedes aegypti. In the present study the effects of cAMP on the basolateral membrane were studied with conventional microelectrodes. Membrane conductances were evaluated from the changes of the basolateral membrane potential (Vbl) consequent to ion changes in the bath. Under control conditions, Vbl measured -65.2 +/- 1.5 mV [83 impalements, 67 tubules]. A fivefold decrease in the bath Na concentration hyperpolarized Vbl by 10.2 +/- 0.6 mV [7], whereas a 4.4-fold increase in the bath K concentration depolarized Vbl by 7.9 +/- 1.0 mV [9]. In the presence of cAMP (10(-4) M) Vbl depolarized to -24.8 +/- 2.7 mV [9]. Vbl now hyperpolarized by 22.7 +/- 1.5 mV [7] for the bath Na change and depolarized by only 3.8 +/- 1.1 mV [6] for the bath K change. Thus the dominant effect of cAMP is the increase of the basolateral membrane Na conductance. This increase is consistent with 1) the depolarization of Vbl and 2) the hyperpolarization of the transepithelial voltage, the decrease of the transepithelial resistance, and the increase of Na and fluid secretion observed previously. Spontaneous oscillations of Vbl were observed and could not be attributed to cyclical changes of the basolateral membrane Na conductance.
Aedes↗
Preliminary isolation of mosquito natriuretic factor.
A natriuretic factor that triggers diuresis in isolated Malpighian tubules of the mosquito was isolated from the head of the yellow-fever mosquito Aedes aegypti by passing a saline extract of mosquito heads through low-pressure and then high-pressure liquid chromatography (HPLC) columns. Three fractions with biologic activity eluted during a reverse-phase HPLC linear acetonitrile gradient run. Fraction I depolarized the transepithelial voltage (Vt) of isolated perfused Malpighian tubules but did not not stimulate fluid secretion in the Ramsay assay (J. A. Ramsay, J. Exp. Biol. 31: 104-113, 1954). Fraction II depolarized and fraction III hyperpolarized Vt, and both stimulated fluid secretion three- to fourfold. Even though the effects of fractions II and III on Vt differed, both stimulated fluid secretion by increasing the rate of NaCl secretion without affecting K secretion. The selective stimulation of active secretory Na transport by fraction III is mimicked by cyclic AMP (cAMP), suggesting the second messenger role of cAMP in the effects of fraction III. Because fraction III stimulates a NaCl-rich, as opposed to KCl-rich, fluid, the term mosquito natriuretic factor is proposed for this active fraction.
Aedes↗
Electrophysiological evidence for Cl secretion in shark renal proximal tubules.
The electrophysiology of shark proximal tubules (Squalus acanthias) was investigated using conventional microelectrodes and cable analysis. Under in vitro perfusion with symmetrical Ringer solutions, tubule transepithelial resistance was 36.3 +/- 2.3 omega X cm2 (means +/- SE, n = 44). Other electrophysiological variables varied widely under control conditions. In unstimulated tubules (n = 16) the transepithelial voltage (VT,o) was lumen positive (1.2 +/- 0.2 mV), the basolateral membrane potential (Vbl,x) was -61.3 +/- 1.6 mV, and the fractional resistance of the apical membrane (fRa) was 0.67 +/- 0.02. Spontaneously stimulated tubules (n = 28) had lumen-negative VT,o values (-1.5 +/- 0.4 mV), low Vbl,x values (-41.3 +/- 1.7 mV), and low fRa values (0.30 +/- 0.02). The stimulated state can be induced in unstimulated tubules via treatment with cAMP. Multiple microelectrode impalements in a single tubule revealed epithelial cells sharing similar electrophysiological properties. Selective ion substitutions in the tubule lumen and peritubular bath uncovered an increased Cl conductance in the apical membrane of spontaneously and cAMP-stimulated tubules. Anthracene-9-carboxylic acid tended to reverse the stimulated state, and furosemide hyperpolarized Vbl,x. These results constitute the first evidence for secretory Cl transport in a renal proximal tubule. The electrophysiological responses to ion substitutions, stimulators, and inhibitors are strikingly similar to those of known Cl-transporting epithelia.
Animals↗
Mechanism of fluid secretion in isolated shark renal proximal tubules.
Renal proximal tubules from the glomerular Squalus acanthias were studied for evidence of fluid secretion by closing one end of isolated tubules and leaving the other end open so that secreted fluid could be collected. When tubules were bathed in shark Ringer, fluid secretion rate was 27.6 +/- 3.9 (SE) pl X min-1 X mm-1 (21 tubules). Dibutyryl cAMP stimulated fluid secretion 50% (P less than 0.02, n = 14), furosemide inhibited fluid secretion 50% (P less than 0.01, n = 6), and metabolic inhibitors blocked fluid secretion nearly 100%. Secreted fluid was slightly hyperosmotic to peritubular bath (P less than 0.01, n = 7) but Na, Cl, S, K, and Ca concentrations were not significantly different from bath concentrations (wavelength-dispersive spectroscopy, electron probe analysis). cAMP had no effect on secreted fluid composition, and in some tubules cAMP did not stimulate fluid secretion. In conjunction with previous data we propose that spontaneous fluid secretion is driven by secretion of NaCl. However, finding the mechanism of NaCl and fluid secretion in glomerular renal tubules offers new perspectives of some previously inexplicable phenomena in the renal physiology of fish.
2,4-Dinitrophenol↗
Comparative physiology of the renal proximal tubule.
The comparative physiology of the renal proximal tubule (PT) is reviewed in the context of vertebrate evolution and vertebrate strategies of salt and water balance. Though extrarenal mechanisms of salt and water balance contribute importantly to extracellular fluid (ECF) homeostasis in the lower vertebrates, the kidney acquires these functions with evolutionary progress and becomes the dominant organ of ECF homeostasis in mammals. In acquiring the major responsibility over the ECF compartment the kidney favored filtration-reabsorption as the preferred mechanism for the rapid turnover of ECF with the advantage of providing quick renal regulatory responses. In spite of this specialization the structure and function of the PT do not appear to have undergone major evolutionary changes. In present-day vertebrates the PT remains as an immensely diverse transport epithelium with impressive capacities for both reabsorptive and secretory work, as exemplified by the mammalian PT with mostly reabsorptive functions and the PT of aglomerular kidneys with mostly secretory functions. The recent evidence for NaCl and fluid secretion in the PT of, unexpectedly, glomerular kidneys is consistent with the functional diversity and the conservative nature of evolution in the case of the PT.
Animals↗