Quantification of osmotic water transport in vivo using fluorescent albumin

2014 ◽  
Vol 307 (8) ◽  
pp. F981-F989 ◽  
Author(s):  
Johann Morelle ◽  
Amadou Sow ◽  
Didier Vertommen ◽  
François Jamar ◽  
Bengt Rippe ◽  
...  
Keyword(s):  
Peritoneal Dialysis ◽  
Water Transport ◽  
Molecular Mechanisms ◽  
Water Channel ◽  
Transgenic Animals ◽  
Peritoneal Membrane ◽  
Intraperitoneal Pressure ◽  
Alexa Fluor ◽  
Osmotic Water

Osmotic water transport across the peritoneal membrane is applied during peritoneal dialysis to remove the excess water accumulated in patients with end-stage renal disease. The discovery of aquaporin water channels and the generation of transgenic animals have stressed the need for novel and accurate methods to unravel molecular mechanisms of water permeability in vivo. Here, we describe the use of fluorescently labeled albumin as a reliable indicator of osmotic water transport across the peritoneal membrane in a well-established mouse model of peritoneal dialysis. After detailed evaluation of intraperitoneal tracer mass kinetics, the technique was validated against direct volumetry, considered as the gold standard. The pH-insensitive dye Alexa Fluor 555-albumin was applied to quantify osmotic water transport across the mouse peritoneal membrane resulting from modulating dialysate osmolality and genetic silencing of the water channel aquaporin-1 (AQP1). Quantification of osmotic water transport using Alexa Fluor 555-albumin closely correlated with direct volumetry and with estimations based on radioiodinated (125I) serum albumin (RISA). The low intraperitoneal pressure probably accounts for the negligible disappearance of the tracer from the peritoneal cavity in this model. Taken together, these data demonstrate the appropriateness of pH-insensitive Alexa Fluor 555-albumin as a practical and reliable intraperitoneal volume tracer to quantify osmotic water transport in vivo.

scholarly journals Mechanisms of Crystalloid versus Colloid Osmosis across the Peritoneal Membrane

2018 ◽  
Vol 29 (7) ◽  
pp. 1875-1886 ◽  
Author(s):  
Johann Morelle ◽  
Amadou Sow ◽  
Charles-André Fustin ◽  
Catherine Fillée ◽  
Elvia Garcia-Lopez ◽  
...  
Keyword(s):  
Peritoneal Dialysis ◽  
Water Flow ◽  
Molecular Mechanisms ◽  
Experimental Models ◽  
Peritoneal Membrane ◽  
Aquaporin 1 ◽  
Dialysis Solution ◽  
Osmotic Water ◽  
Osmotic Agents

Background Osmosis drives transcapillary ultrafiltration and water removal in patients treated with peritoneal dialysis. Crystalloid osmosis, typically induced by glucose, relies on dialysate tonicity and occurs through endothelial aquaporin-1 water channels and interendothelial clefts. In contrast, the mechanisms mediating water flow driven by colloidal agents, such as icodextrin, and combinations of osmotic agents have not been evaluated.Methods We used experimental models of peritoneal dialysis in mouse and biophysical studies combined with mathematical modeling to evaluate the mechanisms of colloid versus crystalloid osmosis across the peritoneal membrane and to investigate the pathways mediating water flow generated by the glucose polymer icodextrin.ResultsIn silico modeling and in vivo studies showed that deletion of aquaporin-1 did not influence osmotic water transport induced by icodextrin but did affect that induced by crystalloid agents. Water flow induced by icodextrin was dependent upon the presence of large, colloidal fractions, with a reflection coefficient close to unity, a low diffusion capacity, and a minimal effect on dialysate osmolality. Combining crystalloid and colloid osmotic agents in the same dialysis solution strikingly enhanced water and sodium transport across the peritoneal membrane, improving ultrafiltration efficiency over that obtained with either type of agent alone.Conclusions These data cast light on the molecular mechanisms involved in colloid versus crystalloid osmosis and characterize novel osmotic agents. Dialysis solutions combining crystalloid and colloid particles may help restore fluid balance in patients treated with peritoneal dialysis.

scholarly journals Aquaporin-1 Facilitates Transmesothelial Water Permeability: In Vitro and Ex Vivo Evidence and Possible Implications in Peritoneal Dialysis

2021 ◽  
Vol 22 (22) ◽  
pp. 12535
Author(s):  
Francesca Piccapane ◽  
Andrea Gerbino ◽  
Monica Carmosino ◽  
Serena Milano ◽  
Arduino Arduini ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Peritoneal Dialysis ◽  
Tight Junctions ◽  
Water Transport ◽  
Water Flux ◽  
Water Channel ◽  
Bathing Solution ◽  
Ussing Chambers ◽  
Aquaporin 1 ◽  
Osmotic Water

We previously showed that mesothelial cells in human peritoneum express the water channel aquaporin 1 (AQP1) at the plasma membrane, suggesting that, although in a non-physiological context, it may facilitate osmotic water exchange during peritoneal dialysis (PD). According to the three-pore model that predicts the transport of water during PD, the endothelium of peritoneal capillaries is the major limiting barrier to water transport across peritoneum, assuming the functional role of the mesothelium, as a semipermeable barrier, to be negligible. We hypothesized that an intact mesothelial layer is poorly permeable to water unless AQP1 is expressed at the plasma membrane. To demonstrate that, we characterized an immortalized cell line of human mesothelium (HMC) and measured the osmotically-driven transmesothelial water flux in the absence or in the presence of AQP1. The presence of tight junctions between HMC was investigated by immunofluorescence. Bioelectrical parameters of HMC monolayers were studied by Ussing Chambers and transepithelial water transport was investigated by an electrophysiological approach based on measurements of TEA+ dilution in the apical bathing solution, through TEA+-sensitive microelectrodes. HMCs express Zo-1 and occludin at the tight junctions and a transepithelial vectorial Na+ transport. Real-time transmesothelial water flux, in response to an increase of osmolarity in the apical solution, indicated that, in the presence of AQP1, the rate of TEA+ dilution was up to four-fold higher than in its absence. Of note, we confirmed our data in isolated mouse mesentery patches, where we measured an AQP1-dependent transmesothelial osmotic water transport. These results suggest that the mesothelium may represent an additional selective barrier regulating water transport in PD through functional expression of the water channel AQP1.

Transgenic Mouse Models

2007 ◽  
Vol 27 (6) ◽  
pp. 625-633 ◽  
Author(s):  
Tomoya Nishino ◽  
Jie Ni ◽  
Olivier Devuyst
Keyword(s):  
Peritoneal Dialysis ◽  
Mouse Models ◽  
Molecular Mechanisms ◽  
Experimental Models ◽  
The Body ◽  
In Vivo Studies ◽  
Limiting Factor ◽  
Peritoneal Membrane ◽  
Pharmacological Agents

The development of peritoneal dialysis has been paralleled by a growing interest in establishing suitable experimental models to better understand the functional and structural processes operating in the peritoneal membrane. Thus far, most investigations have been performed in rat and rabbit models, with mechanistic insights essentially based on intervention studies using pharmacological agents, blocking antibodies, or transient expression systems. Since the body size of a species is no longer a limiting factor in the performance of in vivo studies related to peritoneal dialysis, it has been considered that mice, particularly once they have been genetically modified, could provide an attractive tool to investigate the molecular mechanisms operating in the peritoneal membrane. The purpose of this review is to illustrate how investigators in peritoneal dialysis research, catching up with other fields of biomedical research, are increasingly taking advantage of mouse models to provide direct evidence of basic mechanisms involved in the major complications of peritoneal dialysis.

Serial analysis of gene expression (SAGE) during porcine embryo development

2004 ◽  
Vol 16 (2) ◽  
pp. 87 ◽  
Author(s):  
Le Ann Blomberg ◽  
Kurt A. Zuelke
Keyword(s):  
Gene Expression ◽  
Functional Genomics ◽  
Embryo Development ◽  
Molecular Mechanisms ◽  
Physiological Role ◽  
Transgenic Animals ◽  
Specific Gene ◽  
Research Strategies

Functional genomics provides a powerful means for delving into the molecular mechanisms involved in pre-implantation development of porcine embryos. High rates of embryonic mortality (30%), following either natural mating or artificial insemination, emphasise the need to improve the efficiency of reproduction in the pig. The poor success rate of live offspring from in vitro-manipulated pig embryos also hampers efforts to generate transgenic animals for biotechnology applications. Previous analysis of differential gene expression has demonstrated stage-specific gene expression for in vivo-derived embryos and altered gene expression for in vitro-derived embryos. However, the methods used to date examine relatively few genes simultaneously and, thus, provide an incomplete glimpse of the physiological role of these genes during embryogenesis. The present review will focus on two aspects of applying functional genomics research strategies for analysing the expression of genes during elongation of pig embryos between gestational day (D) 11 and D12. First, we compare and contrast current methodologies that are being used for gene discovery and expression analysis during pig embryo development. Second, we establish a paradigm for applying serial analysis of gene expression as a functional genomics tool to obtain preliminary information essential for discovering the physiological mechanisms by which distinct embryonic phenotypes are derived.

Monitoring of Long-Term Peritoneal Membrane Function

2001 ◽  
Vol 21 (2) ◽  
pp. 225-232 ◽  
Author(s):  
Simon J. Davies
Keyword(s):  
Peritoneal Dialysis ◽  
Solute Transport ◽  
Water Transport ◽  
Drop Out ◽  
Published Data ◽  
Peritoneal Membrane ◽  
Membrane Function ◽  
Technique Survival ◽  
Ultrafiltration Failure

Objective Peritoneal membrane function influences dialysis prescription and clinical outcome and may change with time on treatment. Increasingly sophisticated tools, ranging from the peritoneal equilibration test (PET) to the standard permeability analysis (SPA) and personal dialysis capacity (PDC) test, are available to the clinician and clinical researcher. These tests allow assessment of a number of aspects of membrane function, including solute transport rates, ultrafiltration capacity, effective reabsorption, transcellular water transport, and permeability to macromolecules. In considering which tests are of greatest value in monitoring long-term membrane function, two criteria were set: those that result in clinically relevant interpatient differences in achieved ultrafiltration or solute clearances, and those that change with time in treatment. Study Selection Clinical validation studies of the PET, SPA, and PDC tests. Studies reporting membrane function using these methods in either long-term (5 years) peritoneal dialysis patients or longitudinal observations (> 2 years). Data Extraction Directly from published data. Additional, previously unpublished analysis of data from the Stoke PD Study. Results Solute transport is the most important parameter. In addition to predicting patient and technique survival at baseline, there is strong evidence that it can increase with time on treatment. Whereas patients with initially high solute transport drop out early from treatment, those with low transport remain longer on treatment, although, over 5 years, a proportion develop increasing transport rates. Ultrafiltration capacity, while being a composite measure of membrane function, is a useful guide for the clinician. Using the PET (2.27% glucose), a net ultrafiltration capacity of < 200 mL is associated with a 50% chance of achieving less than 1 L daily ultrafiltration at the expense of 1.8 hypertonic (3.86%) exchanges in anuric patients. Using a SPA (3.86% glucose), a net ultrafiltration capacity of < 400 mL indicates ultrafiltration failure. While there is circumstantial evidence that, with time on peritoneal dialysis, loss of transcellular water transport might contribute to ultrafiltration failure, none of the current tests is able to demonstrate this unequivocally. Of the other membrane parameters, evidence that interpatient differences are clinically relevant (permeability to macro-molecules), or that they change significantly with time on treatment (effective reabsorption), is lacking. Conclusion A strong case can be made for the regular assessment by clinicians of solute transport and ultrafiltration capacity, a task made simple to achieve using any of the three tools available.

scholarly journals Intraperitoneal pressure: Stability over time and validation of Durand’s measurement method

2020 ◽  
pp. 089686082097312
Author(s):  
Alicia Sobrino-Pérez ◽  
Alfonso Pérez-Escudero ◽  
Lucila Fernández-Arroyo ◽  
Ana Dorado-García ◽  
Berta Martín-Alcón ◽  
...  
Keyword(s):  
Peritoneal Dialysis ◽  
Venous Pressure ◽  
Relevant Parameter ◽  
Pressure System ◽  
Intraperitoneal Pressure ◽  
In Vitro Measurements ◽  
Long Timescales ◽  
Over Time

Intraperitoneal pressure (IPP) is gaining consideration as a relevant parameter of peritoneal dialysis (PD) in adults, although many of its aspects are still pending clarification. We address here its stability over time and the validity of the usual method of clinical measurement, as proposed by Durand in 1992 but never specifically validated. We performed this validation by comparing Durand’s method and direct measurements with a central venous pressure system. We performed a total of 250 measurement pairs in 50 patients with different intraperitoneal volumes plus in-vitro measurements with a simulated peritoneum. Absolute differences between the two systems in vivo were 0.87 ± 0.91 cmH2O (range 0–5 cmH2O); only 6.4% of them were ≥3 cmH2O. In vitro results for both methods were identical. We also compared IPP measurements in the same patient separated by 1–4 h (514 measurement pairs in 136 patients), 1 week (92 pairs in 92 patients), and 2 years (34 pairs in 17 patients). Net differences of measurements separated by hours or 1 week were close to 0 cmH2O, with oscillations of 1.5 cmH2O in hours and 2.3 cmH2O in 1 week. IPP measured 2 years apart presented a net decrease of 2.5 ± 4.9 cmH2O, without correlation with body mass index changes or any other usual parameter of PD. In hours, 7% of IPP differences were >3 cmH2O, 22% in 1 week, and 50% in 2 years. In conclusion, Durand’s method is precise enough to measure IPP in peritoneal dialysis. This parameter is not stable over long timescales, so it is necessary to use recent measurements.

scholarly journals P1238EVALUATION OF AN IN VITRO CO-CULTURE MODEL FOR TESTING EFFECTS OF CYTOPROTECTIVE ADDITIVES IN PERITONEAL DIALYSIS FLUIDS ON CARDIOVASCULAR OUTCOME

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Juan Manuel Sacnun ◽  
Rebecca Herzog ◽  
Maria Bartosova ◽  
Claus Schmitt ◽  
Klaus Kratochwill
Keyword(s):  
Endothelial Cells ◽  
Cell Death ◽  
Peritoneal Dialysis ◽  
Cardiovascular Risk ◽  
Cell Damage ◽  
Peritoneal Membrane ◽  
Cytoskeleton Reorganization ◽  
Harmful Effects

Abstract Background and Aims The composition of all currently available peritoneal dialysis (PD) fluids triggers morphological and functional changes in the peritoneal membrane. Periodic exposure leads to vasculopathy, hypervascularization, and diabetes-like damage of vessels, eventually leading to failure of the technique. Patients undergoing dialysis generally, have a high risk of cardiovascular events. It is currently unclear if there is a mechanistic link between peritoneal membrane failure and cardiovascular risk. In vitro and in vivo studies have shown that cytoprotective additives (e.g. dipeptide alanyl-glutamine (AlaGln) or kinase inhibitor lithium chloride (LiCl)) to PDF reduce peritoneal damage. Here, we developed an experimental model for investigating effects of these cytoprotective additives in PDF in the cardiovascular context. Method For modelling the peritoneal membrane in vitro, mesothelial and endothelial cells were co-cultured in transwell plates. Mesothelial cells were grown in the upper compartment and primary human umbilical vein endothelial cells (HUVEc) or primary microvascular cells were grown in the lower compartment. PDF with or without cytoprotective compounds, was added to the upper compartment to only expose mesothelial cells directly to different dilutions of the fluid. Effects on cell damage was assessed by quantification of lactate-dehydrogenase (LDH) release and live-dead staining of cells. Proteome profiles were analysed for both cell-types separately and in combination using two-dimensional difference gel electrophoresis (2D-DiGE) and liquid chromatography coupled to mass spectrometry (LC-MS). In vitro findings were related to PD-induced arteriolar changes based on abundance profiles of micro-dissected omental arterioles of children treated with conventional PD-fluids and age-matched controls with normal renal function. Results Marked cellular injury of HUVEc after PD-fluid exposure was associated with a molecular landscape of the enriched biological process clusters ‘glucose catabolic process’, ‘cell redox homeostasis’, ‘RNA metabolic process’, ‘protein folding’, ‘regulation of cell death’, and ‘actin cytoskeleton reorganization’ that characterize PD-fluid cytotoxicity and counteracting cellular repair process respectively. PDF-induced cell damage was reduced by AlaGln and LiCl both in mesothelial and endothelial cells. Proteome analysis revealed perturbation of major cellular processes including regulation of cell death and cytoskeleton reorganization. Selected markers of angiogenesis, oxidative stress, cell junctions and transdifferentiation were counter-regulated by the additives. Co-cultured cells yielded differently regulated pathways following PDF exposure compared to separate culture. Comparison to human arterioles confirmed overlapping protein regulation between endothelial cells in vitro and in vivo, proving harmful effects of PD-fluids on endothelial cells leading to drastic changes of the cellular process landscape. Conclusion In summary, this study shows harmful effects of PD-fluids also effecting endothelial cells and elucidates potential mechanisms by which cytoprotective additives may counteract the signalling axis between local peritoneal damage and systemic vasculopathy. An in vitro co-culture system may be an attractive approach to simulate the peritoneal membrane for testing direct and indirect effects of cytoprotective additives in PDF. When cultured and stressed in close proximity cells may respond differently. Characterisation of PD-induced perturbations will allow identifying molecular mechanisms linking the peritoneal and cardiovascular context, offering therapeutic targets to reduce current limitations of PD and ultimately decreasing cardiovascular risk of dialysis patients.

scholarly journals Effect ofbalanceSolution on the Peritoneal Membrane in Automated Peritoneal Dialysis

2016 ◽  
Vol 36 (5) ◽  
pp. 569-572 ◽  
Author(s):  
Tatiana De los Ríos ◽  
Juan Pérez-Martínez ◽  
Jose Portoles ◽  
Monika Lichodziejewska-Niemierko ◽  
Maite Rivera ◽  
...  
Keyword(s):  
Peritoneal Dialysis ◽  
Degradation Products ◽  
Peritoneal Membrane ◽  
Open Label ◽  
Cell Functions ◽  
Neutral Ph ◽  
Appearance Rate ◽  
Glycation End Products ◽  
Membrane Cell

Interference of conventional peritoneal dialysis fluids (cPDFs) with peritoneal membrane cell functions may be attributed to the dialysis fluid's low pH, high glucose concentration, and/or the presence of glucose degradation products (GDPs), the last of which leads to higher levels of advanced glycation end-products (AGEs). It has been suggested that the peritoneal membrane might be better preserved by using biocompatible solutions, including cancer antigetn 125 (CA125). This prospective, open-label, multicentre, randomized, controlled, cross-over phase IV study compared the in vivo biocompatibility of a neutral-pH, low-GDP peritoneal dialysis (PD) solution ( balance) with a cPDF in automated PD (APD) patients. Our study revealed a significantly increased appearance rate and concentration of CA125 in the peritoneal effluent of APD patients treated with the neutral-pH, low-GDP solution balance versus a conventional PD solution.

Permeability properties of rat renal lysosomes

1994 ◽  
Vol 266 (1) ◽  
pp. C121-C133 ◽  
Author(s):  
A. I. Piqueras ◽  
M. Somers ◽  
T. G. Hammond ◽  
K. Strange ◽  
H. W. Harris ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Lipid Bilayer ◽  
Gradient Centrifugation ◽  
Water Flux ◽  
Water Channel ◽  
Water Channels ◽  
Water Proton ◽  
Ph Gradients ◽  
Osmotic Water

Although lysosomes maintain large pH gradients and may be subjected to significant osmotic gradients in vivo, little is known about their passive permeability properties. In recent studies, vacuolar H(+)-adenosine-triphosphatases (ATPases), such as those found in lysosomes, have been suggested to act as water channels. In addition, the erythrocyte and proximal tubule water channel CHIP28 is present on the plasma membrane of proximal tubule cells and may undergo endocytosis so that it is incorporated in lysosomes. We therefore examined water, proton, and small nonelectrolyte permeabilities in freshly purified lysosomes from rat renal proximal tubule. Lysosomes were purified by differential and Percoll gradient centrifugation. The preparation contained only lysosomes when examined by electron microscopy. Moreover, analysis by flow cytometry showed virtually all particles to be positive for acid phosphatase and cathepsin B activities. Permeabilities were measured on a stopped-flow fluorimeter by monitoring the self-quenching or pH-sensitive quenching of entrapped fluorescein derivatives. Osmotic water permeability (Pf) averaged 0.011 +/- 0.003 cm/s (n = 6), a value similar to that of biological membranes containing water channels. However, Pf was insensitive to the organic mercurial reagent p-chloromercuribenzene-sulfonate and to HgCl2 and exhibited an activation energy of 10.8 +/- 0.8 kcal/mol. These results indicate that water flux in lysosomes occurred via the lipid bilayer, and not via water channels. Addition of ATP led to lysosomal acidification (proton flux = 4.6 +/- 0.8 x 10(-11) mmol H+.s-1.cm-2), which was completely inhibited by 0.1 microM bafilomycin. Pf was insensitive to this agent as was the passive proton permeability (0.36 +/- 0.18 cm/s, n = 4). Permeabilities to small nonelectrolytes varied in proportion to the oil-water partition coefficient, confirming the applicability of Overton's rule to lysosomes. We conclude that proximal tubular lysosomes exhibit high Pf, which occurs via the lipid bilayer and not via vacuolar H(+)-ATPase.

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