Luminal Solute Research Articles

A computational model of epithelial solute and water transport along a human nephron.

We have developed the first computational model of solute and water transport from Bowman space to the papillary tip of the nephron of a human kidney. The nephron is represented as a tubule lined by a layer of epithelial cells, with apical and basolateral transporters that vary according to cell type. The model is formulated for steady state, and consists of a large system of coupled ordinary differential equations and algebraic equations. Model solution describes luminal fluid flow, hydrostatic pressure, luminal fluid solute concentrations, cytosolic solute concentrations, epithelial membrane potential, and transcellular and paracellular fluxes. We found that if we assume that the transporter density and permeabilities are taken to be the same between the human and rat nephrons (with the exception of a glucose transporter along the proximal tubule and the H+-pump along the collecting duct), the model yields segmental deliveries and urinary excretion of volume and key solutes that are consistent with human data. The model predicted that the human nephron exhibits glomerulotubular balance, such that proximal tubular Na+ reabsorption varies proportionally to the single-nephron glomerular filtration rate. To simulate the action of a novel diabetic treatment, we inhibited the Na+-glucose cotransporter 2 (SGLT2) along the proximal convoluted tubule. Simulation results predicted that the segment’s Na+ reabsorption decreased significantly, resulting in natriuresis and osmotic diuresis.

Oligonucleotide Therapies for the Lung: Ready to Return to the Clinic?

The most promising targets for treating lung diseases are often intracellular molecules recalcitrant to conventional pharmacotherapy. In this respect, antisense oligonucleotides (ASO) would be valuable tools to treat airways disease, as the plurality of their potential mechanisms of action is well-established, their efficiency has been extensively validated in-vitro, and their potential clinical value has been demonstrated with numerous regulatory approvals. Yet in spite of high confidence in the treatment rationale and mechanism of action, it appears that oligonucleotides delivered topically to the lung either rapidly access circulation via epithelial transcytosis or are removed by alveolar macrophages, exerting minimal if any action in the cytosol of cells relevant to lung disease. Moreover, use of cell penetrating peptides, liposomes or nanoparticle delivery systems has not so far been shown to eliminate circulatory clearance, may activate immune responses, or drive macrophage phenotypic changes that, together or in isolation, may present risks to patients.

Isolation of Vacuoles and the Tonoplast.

Isolation of various subcellular compartments followed by a high-coverage proteomic analysis provides an unparalleled foundation for the functional analyses of proteins. Analyses of tonoplast preparations free of major contaminants provide insights into vesicular fusion machinery, solute transport, and the vacuole association with the cytoskeleton, whereas analyses of the vacuolar lumen have yielded numerous soluble glycosidases, proteases, and proteins involved in stress responses. In addition, vacuolar lumen preparations have also allowed characterization of a luminal solute content in response to various abiotic stresses. Here, I revisit and update one of the most successful methodologies for vacuole and tonoplast isolation.

Effects of NKCC2 isoform regulation on NaCl transport in thick ascending limb and macula densa: a modeling study.

This study aims to understand the extent to which modulation of the Na(+)-K(+)-2Cl(-) cotransporter NKCC2 differential splicing affects NaCl delivery to the macula densa. NaCl absorption by the thick ascending limb and macula densa cells is mediated by apical NKCC2. A recent study has indicated that differential splicing of NKCC2 is modulated by dietary salt (Schieβl IM, Rosenauer A, Kattler V, Minuth WW, Oppermann M, Castrop H. Am J Physiol Renal Physiol 305: F1139-F1148, 2013). Given the markedly different ion affinities of its splice variants, modulation of NKCC2 differential splicing is believed to impact NaCl reabsorption. To assess the validity of that hypothesis, we have developed a mathematical model of macula densa cell transport and incorporated that cell model into a previously applied model of the thick ascending limb (Weinstein AM, Krahn TA. Am J Physiol Renal Physiol 298: F525-F542, 2010). The macula densa model predicts a 27.4- and 13.1-mV depolarization of the basolateral membrane [as a surrogate for activation of tubuloglomerular feedback (TGF)] when luminal NaCl concentration is increased from 25 to 145 mM or luminal K(+) concentration is increased from 1.5 to 3.5 mM, respectively, consistent with experimental measurements. Simulations indicate that with luminal solute concentrations consistent with in vivo conditions near the macula densa, NKCC2 operates near its equilibrium state. Results also suggest that modulation of NKCC2 differential splicing by low salt, which induces a shift from NKCC2-A to NKCC2-B primarily in the cortical thick ascending limb and macula densa cells, significantly enhances salt reabsorption in the thick limb and reduces Na(+) and Cl(-) delivery to the macula densa by 3.7 and 12.5%, respectively. Simulation results also predict that the NKCC2 isoform shift hyperpolarizes the macula densa basolateral cell membrane, which, taken in isolation, may inhibit the release of the TGF signal. However, excessive early distal salt delivery and renal salt loss during a low-salt diet may be prevented by an asymmetric TGF response, which may be more sensitive to flow increases.

Chronic Diarrhea in Children

Chronic Diarrhea in Children

HIGHLIGHTS FROM THE LITERATURE

HIGHLIGHTS FROM THE LITERATURE

IRBIT governs epithelial secretion in mice by antagonizing the WNK/SPAK kinase pathway

Fluid and HCO(3)(-) secretion are fundamental functions of epithelia and determine bodily fluid volume and ionic composition, among other things. Secretion of ductal fluid and HCO(3)(-) in secretory glands is fueled by Na(+)/HCO(3)(-) cotransport mediated by basolateral solute carrier family 4 member 4 (NBCe1-B) and by Cl(-)/HCO(3)(-) exchange mediated by luminal solute carrier family 26, member 6 (Slc26a6) and CFTR. However, the mechanisms governing ductal secretion are not known. Here, we have shown that pancreatic ductal secretion in mice is suppressed by silencing of the NBCe1-B/CFTR activator inositol-1,4,5-trisphosphate (IP(3)) receptor-binding protein released with IP(3) (IRBIT) and by inhibition of protein phosphatase 1 (PP1). In contrast, silencing the with-no-lysine (WNK) kinases and Ste20-related proline/alanine-rich kinase (SPAK) increased secretion. Molecular analysis revealed that the WNK kinases acted as scaffolds to recruit SPAK, which phosphorylated CFTR and NBCe1-B, reducing their cell surface expression. IRBIT opposed the effects of WNKs and SPAK by recruiting PP1 to the complex to dephosphorylate CFTR and NBCe1-B, restoring their cell surface expression, in addition to stimulating their activities. Silencing of SPAK and IRBIT in the same ducts rescued ductal secretion due to silencing of IRBIT alone. These findings stress the pivotal role of IRBIT in epithelial fluid and HCO(3)(-) secretion and provide a molecular mechanism by which IRBIT coordinates these processes. They also have implications for WNK/SPAK kinase-regulated processes involved in systemic fluid homeostasis, hypertension, and cystic fibrosis.

A Generalized System for Photo-Responsive Membrane Rupture in Polymersomes.

Polymersomes are vesicles whose membranes are comprised of self-assembled block co-polymers. We recently showed that co-encapsulating conjugated multi-porphyrin dyes in a polymersome membrane with ferritin protein in the aqueous lumen confers photo-lability to the polymersome. In the present study, we illustrate that the photo-lability can be extended to vesicles containing dextran, an inert and inexpensive polysaccharide, as the luminal solute. Here we explore how structural features of the polymersome/porphyrin/dextran composite affect its photo-response. Increasing dextran molecular weight, decreasing block copolymer molecular weight, and altering fluorophore-membrane interactions results in increasing the photo-responsiveness of the polymersomes. Amphiphilic interactions of the luminal encapsulant with the membrane coupled with localized heat production in the hydrophobic bilayer likely cause differential thermal expansion in the membrane and the subsequent membrane rupture. This study suggests a general approach to impart photo-responsiveness to any biomimetic vesicle system without chemical modification, as well as a simple, bio-inert method for constructing photo-sensitive carriers for controlled release of encapsulants.

Efficiency of sodium transport in the thick ascending limb

Thick ascending limb (TAL) cells are thought to transport Na+ efficiently owing to passive paracellular Na+ absorption. Transport efficiency is of particular importance in the outer medulla where O2 availability is limited by low blood flow. We used a mathematical model of TAL cells to estimate the efficiency of Na+ transport along the TAL, and to examine what determines transport efficiency. The TAL cell model includes all major solutes and transporters and channels, and is based on mass conservation and electroneutrality constraints. We analyzed TAL transport in cells with conditions relevant to the inner stripe of the outer medulla, the corticomedullary junction, and in the distal cortical TAL. At each location, we varied luminal [Na+] and [Cl−] and simulated a stop flow condition, where the luminal solute concentrations decrease to limiting, static‐head values. Na+ transport efficiency was calculated as the ratio of total net Na+ transport to transcellular Na+ transport. The results show that transport efficiency decreases as the transepithelial Na+ gradient increases. Based on predictions from countercurrent models, transport efficiency is predicted to be highest at the corticomedullary boundary where the transepithelial Na+ gradient is the smallest. As transepithelial Na+ gradient increases towards the deep inner stripe, transport efficiency decreases. Transport efficiency also decreases in the cortex as the luminal ion concentrations approach static head. Funded by NIH Grants SC1GM084744 and DK42091 and NSF grant DMS‐0715021

Modeling Proximal Tubule Cell Homeostasis: Tracking Changes in Luminal Flow

During normal kidney function, there are routinely wide swings in proximal tubule fluid flow and proportional changes in Na(+) reabsorption across tubule epithelial cells. This "glomerulotubular balance" occurs in the absence of any substantial change in cell volume, and is thus a challenge to coordinate luminal membrane solute entry with peritubular membrane solute exit. In this work, linear optimal control theory is applied to generate a configuration of regulated transporters that could achieve this result. A previously developed model of rat proximal tubule epithelium is linearized about a physiologic reference condition; the approximate linear system is recast as a dynamical system; and a Riccati equation is solved to yield the optimal linear feedback that stabilizes Na(+) flux, cell volume, and cell pH. The first observation is that optimal feedback control is largely consigned to three physiologic variables, cell volume, cell electrical potential, and lateral intercellular hydrostatic pressure. Parameter modulation by cell volume stabilizes cell volume; parameter modulation by electrical potential or interspace pressure act to stabilize Na(+) flux and cell pH. This feedback control is utilized in a tracking problem, in which reabsorptive Na(+) flux varies over a factor of two, in order to represent a substantial excursion of glomerulotubular balance. The resulting control parameters consist of two terms, an autonomous term and a feedback term, and both terms include transporters on both luminal and peritubular cell membranes. Overall, the increase in Na(+) flux is achieved with upregulation of luminal Na(+)/H(+) exchange and Na(+)-glucose cotransport, with increased peritubular Na(+)-3HCO(3)(-) and K(+)-Cl(-) cotransport, and with increased Na(+), K(+)-ATPase activity. The configuration of activated transporters emerges as a testable hypothesis of the molecular basis for glomerulotubular balance. It is suggested that the autonomous control component at each cell membrane could represent the cytoskeletal effects of luminal flow.

Modeling Proximal Tubule Cell Homeostasis: Tracking Changes in Luminal Flow

Normally, there are wide swings in proximal tubule fluid flow and proportional changes in Na reabsorption across tubule epithelial cells. This “glomerulotubular balance” occurs in the absence of substantial change in cell volume, and is a challenge to coordinate luminal solute entry with peritubular solute exit. In this work, linear optimal control theory is applied to generate a configuration of regulated transporters that could achieve this result. A model of rat proximal tubule epithelium is linearized about a reference condition; the approximate linear system is recast as a dynamical system; and a Riccati equation is solved to yield the optimal linear feedback that stabilizes Na flux, cell volume, and cell pH. The first observation is that optimal feedback control is largely consigned to three variables: cell volume, cell electrical potential, and lateral intercellular hydrostatic pressure. Parameter modulation by cell volume stabilizes cell volume; parameter modulation by electrical potential or interspace pressure stabilizes Na flux and cell pH. This feedback control is utilized in a tracking problem, in which reabsorptive Na flux varies over a factor of two. The resulting control parameters consist of two terms, an autonomous term and a feedback term, and both terms include transporters on both luminal and peritubular cell membranes. The increase in Na flux is achieved with upregulation of luminal Na/H exchange and Na‐glucose cotransport, with increased peritubular Na‐3HCO3 and K‐Cl cotransport, and with increased Na,K‐ATPase activity. The configuration of activated transporters emerges as testable hypothesis of the molecular basis for glomerulotubular balance. It is suggested that the autonomous control component at each cell membrane could represent cytoskeletal effects of luminal flow. Supported by NIH RO1‐DK‐29857.

Making sense of the sensor: Mysteries of the macula densa

Increases in luminal NaCl concentration at the macula densa (MD), the sensing element, activate tubuloglomerular feedback (TGF). MD cell volume increases when increments are isosmotic and shrinks if osmolality increases. This interesting finding introduces additional complexity to the role of the MD in TGF.

Unraveling the relationship between macula densa cell volume and luminal solute concentration/osmolality

At the macula densa, flow-dependent changes in luminal composition lead to tubuloglomerular feedback and renin release. Apical entry of sodium chloride in both macula densa and cortical thick ascending limb (cTAL) cells occurs via furosemide-sensitive sodium-chloride-potassium cotransport. In macula densa, apical entry of sodium chloride leads to changes in cell volume, although there are conflicting data regarding the directional change in macula densa cell volume with increases in luminal sodium chloride concentration. To further assess volume changes in macula densa cells, cTAL-glomerular preparations were isolated and perfused from rabbits, and macula densa cells were loaded with fluorescent dyes calcein and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate. Cell volume was determined with wide-field and multiphoton fluorescence microscopy. Increases in luminal sodium chloride concentration from 0 to 80 mmol/l at constant osmolality led to cell swelling in macula densa and cTAL cells, an effect that was blocked by luminal application of furosemide. However, increases in luminal sodium chloride concentration from 0 to 80 mmol/l with concomitant increases in osmolality caused sustained decreases in macula densa cell volume but transient increases in cTAL cell volume. Increases in luminal osmolality with urea also resulted in macula densa cell shrinkage. These studies suggest that, under physiologically relevant conditions of concurrent increases in luminal sodium chloride concentration and osmolality, there is macula densa cell shrinkage, which may play a role in the macula densa cell signaling process.

A mathematical model of rat collecting duct. I. Flow effects on transport and urinary acidification.

A mathematical model of the rat collecting duct (CD) has been developed by concatenating previously published models of cortical (Weinstein AM. Am J Physiol Renal Physiol 280: F1072-F1092, 2001); outer medullary (Weinstein AM. Am J Physiol Renal Physiol 279: F24-F45, 2000); and inner medullary segments (Weinstein AM. Am J Physiol Renal Physiol 274: F841-F855, 1998). Starting with end-distal tubular flow rate and composition, plus interstitial solute profiles, the model predicts urinary solute flows, including the buffer concentrations required to assess net acid excretion. In the model CD, the interstitial corticomedullary osmotic gradient provides the basis for the flow effect on the transport of several solutes. For substances that have an interstitial accumulation and that can have diffusive secretion (e.g., urea and NH(4)(+)), enhanced luminal flow increases excretion by decreasing luminal accumulation. For substances that are reabsorbed (e.g., K+ and HCO(3)(-)), and for which luminal accumulation can enhance reabsorption, increasing luminal flow again increases excretion by decreasing luminal solute concentration. In model calculations, flow-dependent increases in HCO(3)(-) and NH(4)(+) approximately balance, so net acid excretion is little changed by flow, albeit at a higher urinary pH. The model identifies delivery flow rate to the CD as a potent determinant of urinary pH, with high flows blunting maximal acidification. At even modestly high flows (9 nl x min-1. tubule-1, with 6% of filtered Na+ entering the CD), the model cannot achieve a urinary pH <5.5 unless the delivered HCO(3)(-) concentration is extremely low (<2 mM). Nevertheless, simulation of Na2SO4 diuresis does yield both an increase in net acid excretion and a decrease in urinary HCO(3)(-) (i.e., a decrease in pH) despite the increase in urinary flow. This model should provide a tool for examining hypotheses regarding transport defects underlying distal renal tubular acidosis.

Mechanism by which glucose stimulates the passive absorption of small solutes by the human jejunum in vivo

Background/Aims: Active absorption of glucose stimulates passive absorption of small solutes. Part of this effect may be caused by glucose-induced water absorption. Increased water absorption can enhance passive solute absorption by solvent drag and by passive diffusion if the luminal solute concentration increases as water is removed from the lumen. The purpose of this research was to quantitate the contribution of these two forces when glucose enhances the absorption of l-xylose. Methods: The effect of solvent drag on l-xylose absorption was determined by measuring the effect of water absorption stimulated by hypotonicity on l-xylose absorption when the l-xylose concentration was constant. The effect of diffusion on l-xylose absorption was determined by measuring the effect of l-xylose concentration on l-xylose absorption when water absorption was nil. Results: Glucose increased l-xylose absorption by 1.8 mmol·h−1·30 cm−1 (from 1.4 to 3.2 mmol·h−1·30 cm−1). The increase attributable to solvent drag was 1.03 mmol·h−1·30 cm−1; the increase attributable to passive diffusion was 0.75 mmol·h−1·30 cm−1. Conclusions: When glucose stimulates the passive absorption of l-xylose, 57% of the increase can be attributed to solvent drag and 42% to passive diffusion. Because the combined effect of these two forces can account for 99% of the observed effect, virtually all of the glucose effect on l-xylose absorption can be explained by glucose-induced water absorption.

Cell volume regulation in rabbit proximal straight tubule perfused in vitro.

Volume regulation in the perfused proximal nephron of the rabbit was examined quantitatively with a computer-based method for estimating cell volume from differential interference-contrast microscopic images of isolated nephron segments. Following a hyperosmotic challenge (290-390 mosmol), the cells shrank as simple osmometers without a subsequent regulatory volume increase. Conversely, cell swelling induced by a hyposmotic challenge (290-190 mosmol) was completely reversed with a triphasic time course in which a rapid (less than 2 min) initial volume decline was followed by secondary swelling and shrinking phases. A similar regulatory volume decrease was observed following isosmotic cell swelling that was induced by exposure to 290 mosmol, urea-containing solutions. In addition, the cells partially reversed isosmotic swelling that was induced by the luminal replacement of a relatively impermeant cation (i.e., choline) with Na+ and a concomitant increase in luminal solute entry. Our results support two conclusions. First, there exist quantitative differences between the volume regulatory behaviors of perfused and nonperfused proximal tubules, the latter of which exhibit an incomplete and monotonic reversal of hyposmotic cell swelling (M. Dellasega and J. Grantham, Am. J. Physiol. 224: 1288-1294, 1973). Second, the primary physiological role of cell volume regulation in the proximal nephron may be to minimize isosmotic cell swelling associated with acute imbalances in the rates of cell solute entry and exit.

Effects of pH on potassium transport by renal distal tubule.

To determine the relative importance of plasma and luminal pH changes as factors regulating potassium secretion by rat distal tubule, superficial tubules were continuously microperfused in vivo. The effects of changes in plasma pH were examined by producing acute systemic metabolic acidosis or alkalosis and holding luminal flow rate, solute composition, and pH constant by microperfusion. Alternatively, the effect of luminal solution pH was evaluated by microperfusing tubules with solutions buffered to either pH 6.5 or 8.0 at constant systemic acid-base balance. Net transport of Na and K and the pH of the luminal fluid were measured. Results showed that metabolic acidosis inhibited and metabolic alkalosis stimulated potassium secretion. Increased luminal fluid pH, in contrast, did not stimulate potassium transport. In experiments in which metabolic acidosis produced a diuresis, urinary potassium excretion was enhanced compared with hydropenic controls. Free-flow micropuncture studies revealed that the rate of fluid delivery to the distal tubule was 45% greater during acidosis compared with control and that potassium secretion increased in both the distal and collecting tubule. Since the rate of fluid delivery is a potent stimulus of potassium secretion in the distal tubule, it is concluded that the stimulus of increased delivery of fluid, observed in free-flow conditions, masked the inhibitory effect of acidosis on potassium transport. Potassium transport by the distal tubule, during acid-base disorders, is regulated by plasma pH and the rate of delivery of fluid but is not stimulated by alkalinization of the luminal fluid.

Flow dependence of nonelectrolyte absorption in the nephron

The axial flow dependence of nonelectrolyte absorption was examined in terms of a model incorporating interactions between net volume absorption and both saturable and nonsaturable solute absorption. The model solutions demonstrated that changes in transepithelial solute transport are produced by changes in the average luminal solute concentration. Even passive non-saturable solute absorption was shown to exhibit dependence on the perfusion rate, and, therefore, on the solute delivery rate, which could be incorrectly interpreted as demonstrating the presence of a saturable absorptive mechanism. For a unidirectional lumen-to-bath solute flux mediated in part by a saturable mechanism, the observed flux is dependent on the permeability of any parallel nonsaturable permeation pathway. This permeability also sets a lower bound on the luminal solute concentration which may be achieved during active net solute absorption by determining the rate of passive solute backleak. Extension of the model to incorporate dependence of net volume absorption on the delivery of nonelectrolytes predicted a relationship between perfusion rate and net volume absorption equivalent to approximately one-third of complete glomerulotubular balance.

Sodium chloride, urea, and water transport in the thin ascending limb of Henle. Generation of osmotic gradients by passive diffusion of solutes.

Studies were designed to examine whether the thin ascending limb of Henle (tALH) decreases its luminal solute concentration by an active or a passive transport process. In all experiments isolated segments of rabbit tALH were perfused in vitro. When tubules were perfused with solutions identical to the bath, active transport of NaCl was excluded by the following: (a) osmolality of the collected fluid remained unchanged and the same as the bath. (b) net water reabsorption could not be demonstrated, and (c) transtubular potential difference was zero. Isotopic permeability coefficients (x 10(-5) cm s-1) were calculated from the disappearance rate of the respective isotope added to the perfusate. These values indicate that tALH is moderately permeable to [14C]urea (6.97 +/- 1.95) while having a higher permeability to 22Na (25.5 +/- 1.8) and [not readable: see text]Cl (117 +/- 9.1) than any other segment similarly studied. The influx (bath-to-lumen) isotopic permeabilities were not statistically different from the above efflux permeabilities. Osmotic water permeability was immeasurably small. When tALH were perfused with a 600 mosmol/liter solution predominantly of NaCl against a 600 mosmol/liter bath in which 50% of osmolality was NaCl and 50% urea (to simulate in vivo papillary interstitium), the collected fluid osmolality was decreased significantly below that of the bath (300 mosmol/liter/mm of tubule). The decrease in osmolality was due to greater efflux of NaCl as compared to influx of urea. We conclude that active transport of salt by the tALH was not detected by the experimental protocol of the current studies, and that the unique membrane characteristics of tALH allows for generation of osmotic gradients (lumen less concentrated than adjacent surroundings) on purely passive mechanisms when perfused with isosmolal salt solutions in a bath with appropriate salt and urea concentrations. These findings are consistent with the passive counter-current model previously proposed from this laboratory.