Byung-Ju Jin

Aquaporin-4–dependent K+ and water transport modeled in brain extracellular space following neuroexcitation

Potassium (K+) ions released into brain extracellular space (ECS) during neuroexcitation are efficiently taken up by astrocytes. Deletion of astrocyte water channel aquaporin-4 (AQP4) in mice alters neuroexcitation by reducing ECS [K+] accumulation and slowing K+ reuptake. These effects could involve AQP4-dependent: (a) K+ permeability, (b) resting ECS volume, (c) ECS contraction during K+ reuptake, and (d) diffusion-limited water/K+ transport coupling. To investigate the role of these mechanisms, we compared experimental data to predictions of a model of K+ and water uptake into astrocytes after neuronal release of K+ into the ECS. The model computed the kinetics of ECS [K+] and volume, with input parameters including initial ECS volume, astrocyte K+ conductance and water permeability, and diffusion in astrocyte cytoplasm. Numerical methods were developed to compute transport and diffusion for a nonstationary astrocyte–ECS interface. The modeling showed that mechanisms b–d, together, can predict experimentally observed impairment in K+ reuptake from the ECS in AQP4 deficiency, as well as altered K+ accumulation in the ECS after neuroexcitation, provided that astrocyte water permeability is sufficiently reduced in AQP4 deficiency and that solute diffusion in astrocyte cytoplasm is sufficiently low. The modeling thus provides a potential explanation for AQP4-dependent K+/water coupling in the ECS without requiring AQP4-dependent astrocyte K+ permeability. Our model links the physical and ion/water transport properties of brain cells with the dynamics of neuroexcitation, and supports the conclusion that reduced AQP4-dependent water transport is responsible for defective neuroexcitation in AQP4 deficiency.

Spatial model of convective solute transport in brain extracellular space does not support a "glymphatic" mechanism.

A “glymphatic mechanism” has been proposed to mediate convective fluid transport from para-arterial to paravenous extracellular space in the brain. Jin et al. model such a system and find that diffusion, rather than convection, can account for the transport of solutes.

Hyperviscous airway periciliary and mucous liquid layers in cystic fibrosis measured by confocal fluorescence photobleaching

Airway surface liquid (ASL) volume depletion and mucus accumulation occur in cystic fibrosis (CF). The ASL comprises a superficial mucus layer (ML) overlying a periciliary fluid layer (PCL) that contacts surface epithelial cells. We measured viscosity of the ML and PCL from the diffusion of FITC‐dextran dissolved in the ASL of unperturbed, well‐differentiated primary cultures of human bronchial epithelia grown at an air‐liquid interface. Diffusion was measured by fluorescence recovery after photobleaching, using a perfluorocarbon immersion lens and confocal fluorescence detection. Bleaching of an in‐plane 6‐μm‐wide region was done in which diffusion coefficients were computed using solution standards of specified viscosity and finite‐element computations of 2‐layer dye diffusion in 3 dimensions. We found remarkably elevated viscosity in both ML and PCL of CF vs. non‐CF bronchial epithelial cell cultures. Relative viscosities (with saline = 1) were in the range 7–10 in the non‐CF ML and PCL, and 25–30 in both ML and PCL in CF, and greatly reduced by amiloride treatment or mucin washout. These data indicate that the CF airway surface epithelium, even without hyperviscous secretions from submucosal glands, produces an intrinsically hyperviscous PCL and ML, which likely contributes to CF lung disease by impairment of mucociliary clearance. Our results challenge the view that the PCL is a relatively watery, nonviscous fluid layer in contact with a more viscous ML, and offer an explanation for CF lung disease in the gland‐free lower airways.—Derichs, N., Jin, B. ‐J., Song, Y., Finkbeiner, W. E., Verkman, A. S. Hyperviscous airway periciliary and mucous liquid layers in cystic fibrosis measured by confocal fluorescence photobleaching. FASEB J. 25, 2325–2332 (2011). www.fasebj.org

Test of the 'glymphatic' hypothesis demonstrates diffusive and aquaporin-4-independent solute transport in rodent brain parenchyma

Transport of solutes through brain involves diffusion and convection. The importance of convective flow in the subarachnoid and paravascular spaces has long been recognized; a recently proposed ‘glymphatic’ clearance mechanism additionally suggests that aquaporin-4 (AQP4) water channels facilitate convective transport through brain parenchyma. Here, the major experimental underpinnings of the glymphatic mechanism were re-examined by measurements of solute movement in mouse brain following intracisternal or intraparenchymal solute injection. We found that: (i) transport of fluorescent dextrans in brain parenchyma depended on dextran size in a manner consistent with diffusive rather than convective transport; (ii) transport of dextrans in the parenchymal extracellular space, measured by 2-photon fluorescence recovery after photobleaching, was not affected just after cardiorespiratory arrest; and (iii) Aqp4 gene deletion did not impair transport of fluorescent solutes from sub-arachnoid space to brain in mice or rats. Our results do not support the proposed glymphatic mechanism of convective solute transport in brain parenchyma.

Chloride channel inhibition by a red wine extract and a synthetic small molecule prevents rotaviral secretory diarrhoea in neonatal mice

Background Rotavirus is the most common cause of severe secretory diarrhoea in infants and young children globally. The rotaviral enterotoxin, NSP4, has been proposed to stimulate calcium-activated chloride channels (CaCC) on the apical plasma membrane of intestinal epithelial cells. We previously identified red wine and small molecule CaCC inhibitors. Objective To investigate the efficacy of a red wine extract and a synthetic small molecule, CaCCinh-A01, in inhibiting intestinal CaCCs and rotaviral diarrhoea. Design Inhibition of CaCC-dependent current was measured in T84 cells and mouse ileum. The effectiveness of an orally administered wine extract and CaCCinh-A01 in inhibiting diarrhoea in vivo was determined in a neonatal mouse model of rotaviral infection. Results Screening of ∼150 red wines revealed a Cabernet Sauvignon that inhibited CaCC current in T84 cells with IC50 at a ∼1:200 dilution, and higher concentrations producing 100% inhibition. A >1 kdalton wine extract prepared by dialysis, which retained full inhibition activity, blocked CaCC current in T84 cells and mouse intestine. In rotavirus-inoculated mice, oral administration of the wine extract prevented diarrhoea by inhibition of intestinal fluid secretion without affecting rotaviral infection. The wine extract did not inhibit the cystic fibrosis chloride channel (CFTR) in cell cultures, nor did it prevent watery stools in neonatal mice administered cholera toxin, which activates CFTR-dependent fluid secretion. CaCCinh-A01 also inhibited rotaviral diarrhoea. Conclusions Our results support a pathogenic role for enterocyte CaCCs in rotaviral diarrhoea and demonstrate the antidiarrhoeal action of CaCC inhibition by an alcohol-free, red wine extract and by a synthetic small molecule.

Model of Aquaporin-4 Supramolecular Assembly in Orthogonal Arrays Based on Heterotetrameric Association of M1-M23 Isoforms

Tetramers of aquaporin-4 (AQP4) water channels form supramolecular assemblies in cell membranes called orthogonal arrays of particles (OAPs). We previously reported evidence that a short (M23) AQP4 isoform produced by alternative splicing forms OAPs by an intermolecular N-terminus interaction, whereas the full-length (M1) AQP4 isoform does not by itself form OAPs but can coassemble with M23 in OAPs as heterotetramers. Here, we developed a model to predict number distributions of OAP size, shape, and composition as a function M23:M1 molar ratio. Model specifications included: random tetrameric assembly of M1 with M23; intertetramer associations between M23 and M23, but not between M1 and M23 or M1; and a free energy constraint limiting OAP size. Model predictions were tested by total internal reflection fluorescence microscopy of AQP4-green-fluorescent protein chimeras and native gel electrophoresis of cells expressing different M23:M1 ratios. Experimentally validated model predictions included: 1), greatly increased OAP size with increasing M23:M1 ratio; 2), marked heterogeneity in OAP size at fixed M23:M1, with increased M23 fraction in larger OAPs; and 3), preferential M1 localization at the periphery of OAPs. The model was also applied to test predictions about binding to AQP4 OAPs of a pathogenic AQP4 autoantibody found in the neuroinflammatory demyelinating disease neuromyelitis optica. Our model of AQP4 OAPs links a molecular-level interaction of AQP4 with its supramolecular assembly in cell membranes.

Aggregation state determines the localization and function of M1- and M23-aquaporin-4 in astrocytes

An aggregation state–dependent mechanism for segregation of plasma membrane protein complexes confers specific functional roles to the M1 and M23 isoforms of the water channel AQP4.

Aquaporin-1 gene deletion reduces breast tumor growth and lung metastasis in tumor-producing MMTV-PyVT mice

Aquaporin 1 (AQP1) is a plasma membrane water‐transporting protein expressed strongly in tumor microvascular endothelia. We previously reported impaired angiogenesis in implanted tumors in AQP1‐deficient mice and reduced migration of AQP1‐deficient endothelial cells in vitro. Here, we investigated the consequences of AQP1 deficiency in mice that spontaneously develop well‐differentiated, luminal‐type breast adenomas with lung metastases [mouse mammary tumor virus‐driven polyoma virus middle T oncogene (MMTV‐PyVT)]. AQP1+/+ MMTV‐PyVT mice developed large breast tumors with total tumor mass 3.5 ± 0.5 g and volume 265 ± 36 mm3 (se, 11 mice) at age 98 d. Tumor mass (1.6±0.2 g) and volume (131±15 mm3, 12 mice) were greatly reduced in AQP1–/– MMTV‐PyVT mice (P<0.005). CD31 immunofluorescence showed abnormal microvascular anatomy in tumors of AQP1–/– MMTV‐PyVT mice, with reduced vessel density. HIF‐1α expression was increased in tumors in AQP1–/– MMTV‐PyVT mice. The number of lung metastases (5± 1/mouse) was much lower than in AQP1+/+ MMTV‐PyVT mice (31±8/mouse, P<0.005). These results implicate AQP1 as an important determinant of tumor angiogenesis and, hence, as a potential drug target for adjuvant therapy of solid tumors.—Esteva‐Font, C., Jin, B.‐J., Verkman, A. S. Aquaporin‐1 gene deletion reduces breast tumor growth and lung metastasis in tumor‐producing MMTV‐PyVT mice. FASEB J. 28, 1446–1453 (2014). www.fasebj.org

Muddying the water in brain edema

In their recent review in TiNS, Thrane et al. [1] discussed the role of a proposed ‘glymphatic’ system of convective solute flow in the pathology of acute brain edema. Controversies relating to the relative importance of convective versus diffusive transport in clearing toxic metabolic byproducts from brain parenchyma have been comprehensively reviewed elsewhere [2, 3] and it is not our intention to repeat the arguments made in these papers but, instead, to clarify aspects of the physiological function of the glial water channel aquaporin-4 (AQP4) and its contribution to brain edema that we believe have been muddied by the glymphatic hypothesis. As originally stated [4], the glymphatic hypothesis pro-poses convective flow in brain parenchyma generated by hydrostatic pressure-driven trans-astrocytic water move-ment facilitated by AQP4 in perivascular endfeet. There are several problems with this hypothesis (Figure 1). First, the hydrostatic pressure is extremely small compared with the osmotic pressure generated by ion transport across cell membranes. Arterial wall pulsations that have been pro-posed to generate hydrostatic pressure in the paravascular space are on the order of 1 µm [5]; modeling studies with larger wall displacements of 5 µm suggest maximal hydro-static pressure transients of approximately 1 kPa in the paravascular space [6], equivalent to the osmotic pressure generated by a 0.4-mOsm difference across the endfoot membrane. Osmotic gradients of 20 mOsm or more are routinely generated across the astrocyte plasma mem-brane in response to metabolic activity and K+ redistribution, which provide the dominant driving force for water transport. A related issue is the ‘salt accumulation problem’ [3, 7], where pressure-driven water flow across a salt-impermeable membrane concentrates solutes on the high-pressure side and dilutes solutes on the low-pressure side, creating an opposing osmotic gradient that largely counter-acts the effectiveness of hydrostatic pressure in driving water transport within confined volumes. Furthermore, because AQP4 transports water only and is concentrated on the perivascular face of endfeet, where it facilitates uptake of water into the astrocyte and cellular swelling, it is hard to envision how convective flow across the endfoot itself could occur. Finally, the cell impermeant tracers used to track convective flow move through the small gaps between endfeet and not through the endfeet themselves. Any pulsation-driven flow into endfeet would reduce the driving force for convection through the gaps and, hence, tracer transport into the interstitial space. Therefore, pulsation-driven water flow through AQP4 in astrocyte endfeet is unlikely at physiologically relevant hydrostatic pressures and, if it did occur, would not be expected to generate convective flow in the interstitial space or in-crease tracer movement from paravascular cerebrospinal fluid (CSF) to interstitial fluid (ISF). Figure 1 The glymphatic hypothesis versus conventional understanding of the role of aquaporin-4 (AQP4) in brain water movement. (A) The glymphatic hypothesis proposes a major role for hydrostatic pressure-driven water flow through astrocyte endfeet; conventionally, ... Application of the glymphatic hypothesis in the context of brain edema led Thrane et al. to propose that convective pumping of CSF into ischemic areas, rather than osmotic mechanisms, is responsible for acute edema following ischemia. As the authors indicate, studies to measure astrocyte volume changes using two-photon optical micros-copy are limited by spatial resolution; however, electron microscopy consistently demonstrates endfoot swelling as an early consequence of ischemia [8, 9], supporting AQP4-mediated osmotic uptake of water into astrocytes during the early stages of edema. As the authors correctly point out, much remains to be understood about the functional significance of AQP4 enrichment in astrocyte endfeet, both in normal physiology and during edema. Experimental data suggesting that AQP4 facilitates fluid exchange between CSF and ISF are intriguing but difficult to interpret because of baseline differences in parenchymal extracellular volume fraction between wild type and AQP4-null mice [10, 11]. Quantitative studies have failed to find evidence for directional convective flow in cortical gray matter under normal conditions [12, 13], so perhaps the results of Iliff et al. [4] are a consequence of differences in extracellular space (ECS) structure between wild type and AQP4-null mice. Future studies of the role of AQP4 in diffusive and convective fluid transport in the brain will benefit from the application of quantitative imaging methods and biophysically realistic spatial modeling.

Convective washout reduces the antidiarrheal efficacy of enterocyte surface-targeted antisecretory drugs.

Secretory diarrheas such as cholera are a major cause of morbidity and mortality in developing countries. We previously introduced the concept of antisecretory therapy for diarrhea using chloride channel inhibitors targeting the cystic fibrosis transmembrane conductance regulator channel pore on the extracellular surface of enterocytes. However, a concern with this strategy is that rapid fluid secretion could cause convective drug washout that would limit the efficacy of extracellularly targeted inhibitors. Here, we developed a convection–diffusion model of washout in an anatomically accurate three-dimensional model of human intestine comprising cylindrical crypts and villi secreting fluid into a central lumen. Input parameters included initial lumen flow and inhibitor concentration, inhibitor dissociation constant (Kd), crypt/villus secretion, and inhibitor diffusion. We modeled both membrane-impermeant and permeable inhibitors. The model predicted greatly reduced inhibitor efficacy for high crypt fluid secretion as occurs in cholera. We conclude that the antisecretory efficacy of an orally administered membrane-impermeant, surface-targeted inhibitor requires both (a) high inhibitor affinity (low nanomolar Kd) to obtain sufficiently high luminal inhibitor concentration (>100-fold Kd), and (b) sustained high luminal inhibitor concentration or slow inhibitor dissociation compared with oral administration frequency. Efficacy of a surface-targeted permeable inhibitor delivered from the blood requires high inhibitor permeability and blood concentration (relative to Kd).