Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Sci Rep
2017 Jun 13;71:3395. doi: 10.1038/s41598-017-03595-y.
Show Gene links
Show Anatomy links
Raman micro-spectroscopy as a viable tool to monitor and estimate the ionic transport in epithelial cells.
Puppulin L
,
Pezzotti G
,
Sun H
,
Hosogi S
,
Nakahari T
,
Inui T
,
Kumamoto Y
,
Tanaka H
,
Marunaka Y
.
???displayArticle.abstract???
The typical response to the lowering of plasma Na+ concentration and blood pressure in our body involves the release of aldosterone from the adrenal glands, which triggers the reabsorption of sodium in the kidney. Although the effects of aldosterone on this physiological mechanism were extensively studied in the past decades, there are still some aspects to be fully elucidated. In the present study, we propose for the first time a new approach based on Raman spectroscopy to monitor the ionic activity in aldosterone-treated A6 renal epithelial cells. This spectroscopic technique is capable of probing the cells through their thickness in a non-destructive and nimble way. The spectroscopic variations of the Raman bands associated to the O-H stretching of water were correlated to the variations of ionic concentration in the intracellular and extracellular fluids. The increase of Na+ concentration gradients was clearly visualized in the cytosol of aldosterone-treated cells. The enhancement of the Na+ current density induced by aldosterone was estimated from the variation of the ionic chemical potential across the intracellular space. In addition, the variation of the O-H Raman bands of water was used to quantify the cell thickness, which was not affected by aldosterone.
Figure 1. (a) Explanation of the z-axis scanning protocol adopted in the Raman spectroscopy experiments. The incident laser and the back-scattered Raman signal collected by the same lens are separated using a beam splitter. The confocal cross-slit enables to increase the spatial resolution of the measurements by cutting off the signal coming from regions that are more than a few hundred of nm out of the focal plane. A diffraction grating is used to split the light into its components, whose intensities are measured by the detector. (b) Schematic of the A6 renal epithelial cell under investigation, in which are described the ionic transport mechanism of sodium reabsorption.
Figure 2. Examples of bright-field images of aldosterone treated cells: (a) focus on the permeable membrane (z = 0); (b) above the membrane at z = 5 and (c) at z = 15 μm.
Figure 3. (a) Typical Raman spectrum collected from water solutions in the region 2400–4200 cm−1 and the 5 sub-bands used to interpret the structure of liquid water. (b) Example of spectral variation induced by ionic concentration in the millimolar range (total integrated intensity of each spectrum was normalized to 100).
Figure 4. Liner dependence between the ionic concentration and the intensity ratio of the bands DA and DDAA, r = I
DA
/I
DDAA, for solutions of NaCl (a), KCl (b), MgCl2 (c), CaCl2 (d), NaHCO3 (e), Hepes (f), glucose (g) and proteins (bovine serum albumin) (h).
Figure 5. Typical Raman spectrum obtained from A6 cells, focusing the laser probe close to the polycarbonate substrate. Assignments of the labeled bands are reported in Table 2.
Figure 6. Profiles of the intensity ratio r, as retrieved from control (a) and aldosterone treated (b) A6 cells, respectively.
Figure 7. (a) Difference of the ratio r between treated and untreated cells as calculated in the cytosol of A6 epithelial cells. (b) The data reported in (a) can be converted to variation of [Na+] or [Cl−] in the cytosol using the linear fitting equation reported in the inset of Fig. 3(a). At each z-axis coordinate, the statistically meaningful variations of r and [Na] (or [Cl−]) are marked by *(unpaired t-test, 95% confidence, p < 0.05).
Alvarez de la Rosa,
Effects of aldosterone on biosynthesis, traffic, and functional expression of epithelial sodium channels in A6 cells.
2002, Pubmed,
Xenbase
Alvarez de la Rosa,
Effects of aldosterone on biosynthesis, traffic, and functional expression of epithelial sodium channels in A6 cells.
2002,
Pubmed
,
Xenbase
Amuzescu,
Zinc is a voltage-dependent blocker of native and heterologously expressed epithelial Na+ channels.
2003,
Pubmed
,
Xenbase
Bindels,
Stimulation of sodium transport by aldosterone and arginine vasotocin in A6 cells.
1988,
Pubmed
,
Xenbase
Chen,
Epithelial sodium channel regulated by aldosterone-induced protein sgk.
1999,
Pubmed
,
Xenbase
Collins,
The Hofmeister effect and the behaviour of water at interfaces.
1985,
Pubmed
Devuyst,
Aldosterone interaction on sodium transport and chloride permeability: influence of epithelial structure.
1995,
Pubmed
,
Xenbase
Eaton,
Regulation of epithelial sodium channel trafficking by ubiquitination.
2010,
Pubmed
GAUNT,
Aldosterone: a review.
1955,
Pubmed
Guyton,
Blood pressure control--special role of the kidneys and body fluids.
1991,
Pubmed
Handler,
The effect of adrenal steroid hormones on epithelia formed in culture by A6 cells.
1981,
Pubmed
,
Xenbase
Handler,
Hormone effects on transport in cultured epithelia with high electrical resistance.
1981,
Pubmed
,
Xenbase
Howell,
Raman spectral analysis in the C-H stretching region of proteins and amino acids for investigation of hydrophobic interactions.
1999,
Pubmed
Izzo,
Angiotensin-converting enzyme inhibitors.
2011,
Pubmed
Kitamura,
Proteolytic activation of the epithelial sodium channel and therapeutic application of a serine protease inhibitor for the treatment of salt-sensitive hypertension.
2012,
Pubmed
Mancinelli,
Hydration of sodium, potassium, and chloride ions in solution and the concept of structure maker/breaker.
2007,
Pubmed
Marunaka,
Characteristics and pharmacological regulation of epithelial Na+ channel (ENaC) and epithelial Na+ transport.
2014,
Pubmed
Marunaka,
Effects of vasopressin and cAMP on single amiloride-blockable Na channels.
1991,
Pubmed
Mähler,
A study of the hydration of the alkali metal ions in aqueous solution.
2012,
Pubmed
Nickolov,
Water structure in aqueous solutions of alkali halide salts: FTIR spectroscopy of the OD stretching band.
2005,
Pubmed
Niisato,
Activation of the Na+-K+ pump by hyposmolality through tyrosine kinase-dependent Cl- conductance in Xenopus renal epithelial A6 cells.
1999,
Pubmed
,
Xenbase
Perkins,
Transport properties of toad kidney epithelia in culture.
1981,
Pubmed
,
Xenbase
Rotin,
Regulation of the epithelial sodium channel (ENaC) by accessory proteins.
2000,
Pubmed
Sasamoto,
Analysis of Aprotinin, a Protease Inhibitor, Action on the Trafficking of Epithelial Na+ Channels (ENaC) in Renal Epithelial Cells Using a Mathematical Model.
2017,
Pubmed
Schneider,
Rapid aldosterone-induced cell volume increase of endothelial cells measured by the atomic force microscope.
1997,
Pubmed
Sevá Pessôa,
Key developments in renin-angiotensin-aldosterone system inhibition.
2013,
Pubmed
Soundararajan,
Role of epithelial sodium channels and their regulators in hypertension.
2010,
Pubmed
Staub,
Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination.
1997,
Pubmed
,
Xenbase
Staub,
Regulation of the epithelial Na+ channel by Nedd4 and ubiquitination.
2000,
Pubmed
Tait,
The discovery, isolation and identification of aldosterone: reflections on emerging regulation and function.
2004,
Pubmed
Taruno,
Analysis of blocker-labeled channels reveals the dependence of recycling rates of ENaC on the total amount of recycled channels.
2010,
Pubmed
,
Xenbase
USSING,
Active transport of sodium as the source of electric current in the short-circuited isolated frog skin.
1951,
Pubmed
Verdonk,
Angiotensin II type 2 receptor agonists: where should they be applied?
2012,
Pubmed
Xie,
A Simple Theory for the Hofmeister Series.
2013,
Pubmed
Zhao,
Changes of water hydrogen bond network with different externalities.
2015,
Pubmed
van Kats,
Angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade prevent cardiac remodeling in pigs after myocardial infarction: role of tissue angiotensin II.
2000,
Pubmed