Hindawi Publishing CorporationJournal of BiophysicsVolume 2008, Article ID 420963, 5 pagesdoi:10.1155/2008/420963

Research Article

Cell Volume and Sodium Content in Rat Kidney Collecting DuctPrincipal Cells During Hypotonic Shock

Evgeny I. Solenov

Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, Novosibirsk State University,Novosibirsk 630090, Russia

Correspondence should be addressed to Evgeny I. Solenov, eugsol@bionet.nsc.ru

Received 3 March 2008; Accepted 3 July 2008

Recommended by Walter Stuhmer

The purpose of this study was to investigate the time course of the volume-regulatory response and intracellular sodiumconcentration ([Na+]i) in the principal cells of rat kidney outer medulla collecting duct (OMCD) epithelia during acute swellingin hypotonic medium. Hypotonic shock was created by PBS diluted with 50% of water. Changes in cell volume were measuredwith calcein quenching method. Intracellular sodium concentration was studied with fluorescence dye Sodium Green. Principalcells of microdissected OMCD fragments swelled very fast. The characteristic time of swelling (τ1) was 0.65 ± 0.05 seconds, andthe volume increased more than 60% (92.9± 5.6 and 151.3± 9.8μm3 control and peak volumes correspondently, P < .01). Aftercell volume reached the peak of swelling, the RVD began without lag period. The characteristic time of volume decreasing tonew steady-state level (τ2) was 8.9 ± 1.1 seconds. In hypoosmotic medium, cell volume stabilized on higher level in comparisonwith control (110.3 ± 8.3μm3, P < .01). After restoration of the medium osmolality to normotonic, cell volume stabilized onsignificantly low level in comparison with control level (71.4±6.1μm3, P < .01). During the hypoosmotic shock, [Na+]i decreasedfrom control level in isotonic PBS to the low level in hypoosmotic solution (27.7± 1.4 and 5.8± 0.23 mM, P < .01). Calculation ofsodium content per cell has shown the significant sodium entry into the cells, which caused a temporary increase correlated withthe peak of cell volume caused by swelling. The conclusion is made that in our model of hypoosmotic shock, swelling activatestransporters with high permeability for Na+ that provides sodium flux into the cells.

Copyright © 2008 Evgeny I. Solenov. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Kidney epithelium in collecting duct contacts with hypotonicfluid which osmolality varies significantly and the cellscontinually expose to osmotic stress. Alterations in luminalosmolality would influence cell volume. To avoid damageand perform their functions, principal cells clearly require aneffective cell volume regulation mechanism. These processes,termed regulatory volume decrease (RVD), occur throughactivation of specific transporters in the plasma membranethat mediate net fluxes of osmotically active molecules.Despite its importance, very little is known about cell-volume regulation inkidney outer medulla collecting duct(OMCD) cells.

Cells recruit special mechanisms to correct acute swellingin hypotonic medium [1, 2]. Cell swelling has the effect ofincreasing K+ efflux, this occurs through channels distinct

from those responsible for basal K+ conductance. Obviously,both K+ and Cl− concentrations play key roles in govern-ing these volume-regulatory responses. The predominantpathway for RVD is the opening of K+ channels which areusually large conductance [3, 4]. Little is known about themolecular basis of the anion conductance. These transporterscan be quite nonselective and can include more than onetype of channels [5]. In cells containing Na+ channels likecollecting duct principal cells, Na+ fluxes can influence thevolume-regulatory response but the role of intracellularsodium in RVD is almost unstudied. From another hand,the mechanism by which cells sense changes in cell volumeand activates the appropriate transporters remains unknown.It is important to study the changes of cell volume and[Na+]i during the process of RVD for better understandingof the mechanisms involved in this reaction. The currentstudy was undertaken to investigate the time course of

2 Journal of Biophysics

changes in cell volume and sodium concentration during thevolume-regulatory response after acute swelling in hypotonicmedium.

2. Methods

2.1. Animals

Wistar rats weighting 150–200 g (Breeding Laboratory ofExperimental Animals, Institute of Cytology and Genetics,Novosibirsk, Russia) were kept in individual cages andreceived standard diet. Before the experiments, the rats wereanaesthetized with pentobarbital (50 mg/kg i.p.), the kidneyswere extracted and placed in ice-cold PBS, and a suspensionof collecting duct fragments was prepared.

2.2. Perfusion Chamber and Microscopy

A superfusion chamber was constructed as an acrylic blockmounted on the objective of the upright microscope (waterimmersion 65 magnification, numerical aperture 1.1) [6].The perfusate flow rate was generally 20 mL/min, whichgave complete solution exchange in under 200 milliseconds.Fluorescence measurements of cell volume were performedas it was described [6, 7] . Fragments of outer medullarycollecting duct (OMCD) on glass plate were loaded withcalcein by incubation with calcein-AM (Molecular ProbesInc., Ore, USA) (5.0 μM) for 25 minutes at 23◦C . The glassplate bearing the fragments of the OMCD was positioned onthe stage of a microscope (LOMO-R8, St. Petersburg, Rus-sia). Calcein fluorescence was measured continuously withhalogen light source, calcein filter set (480 nm excitation,490 nm dichroic mirror, 535 nm emission), photomultiplierdetector has pinhole diaphragm to select the cells of interestat the end of fragment where cells expose apical surfaceto bath solution, and 14-bit analog-to-digital converter.The rate of data acquisition was 200 milliseconds. In theseexperiments, [Na+]i was measured using the fluorescentindicator Sodium Green. Loading conditions and filter set forfluorescence measurements were the same as for calcein.

2.3. Collecting Duct Fragments

Tissue from the outer medulla zone was cut from the isolatedkidneys and squeezed through a needle (0.45 mm i.d.) inthe ice-cold calcium-free PBS. The resulting suspension wasfiltered through nylon mesh, diluted 10 times with EagleMEM culture medium and centrifuged (100 g, 10 minutes,4◦C). Sediment containing the tubules was diluted withculture medium to an appropriate concentration of about10 fragments per microliter. This suspension was used inexperiments as a preparation of OMCD fragments [6].

2.4. Solutions

The solutions used were based on PBS (137 mM NaCl,4.7 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2HPO4, 0.5 mMMgCl2, 0.05 mM CaCl2, 280 mOsm/l, pH = 7, 2–7, 1) and

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(μm

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0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7

F/F0

Figure 1: Calibration curve for calcein fluorescence quenchingmethod. Absciss: relative fluorescence (F/F0). F0: fluorescence ofintact cells in isotonic PBS. F: current fluorescence. Ordinate:absolute cell volume calculated from cells images.

contained 1.0 mg/mL glucose. This solution was chosenso as to be able to degas it without affecting its pH.To create osmotic challenges, bath solutions were changedfrom normal PBS to PBS diluted with distilled water (1:1).Isotonic solutions with decreased [Na+]o were preparedby substitution part of sodium in PBS by NMDG (n-methyl-D-glucamine, Sigma-Aldrich, Moscow, Russia). Formeasurements of sodium efflux in isotonic medium was usedan extracellular isotonic solution with 69 mM [Na+]o.

2.5. Calibration of Cell Volume Measurementswith Calcein Fluorescence

Fluorescent measurements were calibrated plotting the rel-ative values of fluorescence as an X value, against syn-chronized data of microscopic image analysis, obtained bythe method published before [7]. A perfusion chamber,described above, was mounted on the objective of themicroscope (water immersion 65 magnification, numericalaperture 1.1, 1.6 magnification photo adapter). The glassplate bearing the fragments of the OMCD was positioned onthe stage of a microscope for acquisition of transmitted lightimages by a CCD camera. Twelve-bit monochrome imageswere captured at a rate of 15 frames per second, stored on apersonal computer. The result was linear calibration plot (seeFigure 1). Cell volume was calculated from measurements ofcalcein fluorescence according this calibration.

2.6. Calibration of [Na+]i

In vivocalibration of Sodium Green was accomplishedby exposing the fragments to various extracellular [Na+]([Na+]o) in the presence of 0.1 U/μl Nystatin. The solutionswith various [Na+]o were prepared by substitution partof sodium in PBS by NMDG. For calibration were used

Journal of Biophysics 3

0.8

0.9

1

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F(A

U)

0 50 100 150 200 250

(Seconds)

138 mM

14 mM

5 mM

138 mM

Figure 2: Calibration curve for Sodium Green fluorescence (rep-resentative curve). Absciss: time (seconds). Ordinate: intensity offluorescence (F) in arbitrary units.

solutions with 138, 14, and 5 mM [Na+]o. A calibration wasperformed at the end of each experiment (see Figure 2).

2.7. Statistical Analysis

Data are expressed as means ± SEM. Statistical discrimina-tions were performed with Student’s unpaired t-test. Valuesof P < .05 were considered as significant.

3. Results and Discussion

Ion fluxes that appeared during cell swelling seem to becritically involved in the regulation of cell volume. However,the nature of the channels and the mechanism of theiractivation are poorly understood. It is also not clear towhat extent this activation can explain the regulatory volumedecrease. The goal of this study is to examine the timecourse of cell volume changes and concomitant variationsin [Na+]i after hypotonic shock. In hypotonic medium,principal cells in OMCD fragments swell very fast, and thevolume increases more than 60% (see Figures 2 and 3). Theevaluation of characteristic time of swelling (τ1) gives thevalue 0.65 ± 0.05 seconds (n = 12), which is in a goodagreement with our previous results. In our experiments,basolateral and apical surfaces of the cells on the end ofmicrodissected fragment are in contact with medium. Fastswelling of the principal cells is the consequence of the highwater permeability determined by the water channels thatare expressed in basolateral and apical membranes [8, 9].It was shown that abundance of water channels correlateswith faster RVD [10, 11]. Probably the water channels arepart of the mechanism, which is activated in cell volumedecrease response. After cell volume reaches the peak ofswelling, the RVD begins without lag period. The kineticsof this process is slower than swelling, and the characteristictime of volume decreasing to steady-state level (τ2) is 8.9±1.1seconds (n = 12) (see Figure 4). The published data show

0.40.50.60.70.80.9

11.11.21.31.41.51.61.71.81.9

Nor

mal

ized

para

met

ers

0 1 2 3 4 5

(Seconds)

Figure 3: Synchronized time courses of cell volume and [Na+]i

in rat kidney OMCD principal cells. �: gradient signal from thevalve: switch from normal PBS [Na+]i 280 mM to hypotonic 0.5 PBS[Na+]i 140 mM. Absciss: time (seconds). Ordinate: (•) cell volume(μm3). (�) intracellular sodium concentration [Na+]i (mM). (�)calculated content of intracellular sodium (×10 fmol/cell).

that cells in cell lines derived from kidney epithelia, RCCD,COS-7, A6 have a significantly slower rate of RVD, and τ2

is at least one order more [11–13]. There is a discrepancyof results obtained on microdissected fragments of OMCDand published data obtained on cell lines. Most probablythe difference is due to the loss of some ion transporters,which could not be activated by hypoosmotic shock incultured cells. After restoration of the medium osmolalityto normotonic, the cells shrink and the volume stabilizeson significantly lower level in comparison with control level(see Figure 4). This decrease probably is due to the loss ofcellular osmolites during RVD. Salt withdrawal procedurecould result in responses that depend on the type of cationremoved. In this study, we reduced the osmolality by dilutingPBS with water or removing NaCl from the bath. Theprocedure causes drastic changes in ionic strength that couldinterfere with the mechanisms involved in volume recovery.

Hypotonic medium causes decrease of [Na+]i and stabi-lization on the low level (see Figures 2 and 5). The establishedlevel of [Na+]i during the hypotonic shock is the result ofinteraction of three main processes: dilution of osmoliteswith water that enters the cell, activity of Na, K-ATPase,and ion flux into the cell through channels activated byswelling, which could be permeable for sodium. Comparingthe rates of normalized cell volume and [Na+]i shows that cellvolume grows about two times faster than [Na+]i decreases(0.72± 0.05 and −0.30± 0.02 s−1 correspondingly, P < .01).This could be explained if during swelling water enters thecell accompanied by sodium. To block cell swelling and waterentry, we created the transmembrane sodium gradient withnormotonic PBS where 69 mM of Na+ was substituted byNMDG. In the absence of cell swelling with virtually zerowater entry, the τ of [Na+]i decrease is significantly fasterin comparison with the same parameter in hypoosmotic

4 Journal of Biophysics

60

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(μm

3)

0 10 20 30 40

(Seconds)

Figure 4: Effect of hypotonic medium on cell volume of OMCDprincipal cells (representative curve). Cell volumes were estimatedaccording calcein fluorescence intensity. �: gradient signal from thevalve: switch from normal PBS [Na+]i 280 mM to hypotonic 0.5 PBS[Na+]i 140 mM, and back to normal PBS. (1) control normal PBS[Na+]i 280 mM. (2) peak of cell volume during swelling. (3) steadystate in hypotonic 0.5 PBS [Na+]i 140 mM.

shock (2.6 ± 0.26 and 8.9 ± 0.98 seconds, n = 8 andn = 12, correspondently, P < .01). These results couldbe accepted as evaluation of sodium efflux through Na, K-ATPase. The evaluation of sodium content per cell, made bymultiplication of synchronized measurements of calcein andSodium Green fluorescence, shows that peak of cell volumecorrelates with the temporary increase of this calculatedparameter (see Figures 2 and 5). These results indicate thatthe water comes into cell during the swelling together withsodium. This could happen if swelling activated several typesof channels which could pass ions with high conductivity andprobably there are some channels which could pass water.Probably, this sodium flux aggravates the cell swelling inthe hypotonic medium if [Na+]o is higher than [Na+]i butduring the phase of cell volume decreasing these channelsbeing active could promote the outcome of potassium fromthe cell. The outcome of the ions, mainly potassium, shouldbe facilitated by partial depolarization which could happenedin these cells during swelling [14].

One of the most probable transporters active in normalconditions and which probably takes a part in volumeregulating response is epithelial sodium channel (ENaC).This channel is expressed in apical membranes of principalcells, and there are data that indicate that ENaC could beinvolved in RVD [15, 16]. To evaluate the putative influenceof ENaC on [Na+]i during hypoosmotic challenge, weblocked it with amiloride. The addition of amiloride to theconcentration of 10−5 M in bathing solutions caused decreaseof control steady-state level of [Na+]i (see Figure 6), butdid not influence this parameter in hypotonic medium andafter returning the normotonic PBS. Amiloride did not influ-ence significantly the relative swelling during hypoosmoticchallenge (0.65 ± 0.01 control, 0.77 ± 0.05 amiloride, NS).

4

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[Na+

] i(p

mol

)

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(Seconds)

Figure 5: Time course of intracellular sodium concentration inOMCD principal cells. Effect of hypotonic medium (representativecurve). �: gradient signal from the valve (same as in Figure 4). (1)control normal PBS [Na+]i 280 mM. (2) steady state in hypotonic0.5 PBS [Na+]i 140 mM.

0

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[Na+

] i(m

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1 2

∗ ∗

∗

Figure 6: Intracellular sodium concentration in OMCD principalcells. Effect of amiloride. Gray columns: control, light gray columns:10−5 M amiloride. (1) control normal PBS [Na+]i 280 mM. (2)steady state in hypotonic 0.5 PBS [Na+]i 140 mM. (∗): P < .05difference with intact control.

The ineffectiveness of amiloride means that contribution ofENaC in sodium entry in our model of hypotonic shock isnegligible and cannot be detected in our experiments.

The results of these experiments indicate that in prin-cipal cells hypoosmotic shock causes activation of thetransporters, which have high permeability for water andprobably provide sodium and potassium transport acrosscell membrane in dependence of the ion electrochemicalgradient which is forming during swelling.

Acknowledgment

The work was supported by Grant RFBR 05-04-48213.

Journal of Biophysics 5

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[13] B. Nilius, J. Sehrer, P. De Smet, W. Van Driessche, and G.Droogmans, “Volume regulation in a toad epithelial cell line:role of coactivation of K+ and Cl− channels,” Journal ofPhysiology, vol. 487, no. 2, pp. 367–378, 1995.

[14] M. Paulmichl, F. Friedrich, K. Maly, and F. Lang, “Theeffect of hypoosmolarity on the electrical properties of MadinDarby canine kidney cells,” Pflugers Archiv European Journal ofPhysiology, vol. 413, no. 5, pp. 456–462, 1989.

[15] H. Hager, T.-H. Kwon, A. K. Vinnikova, et al., “Immunocyto-chemical and immunoelectron microscopic localization of α-,β-, and γ-ENaC in rat kidney,” American Journal of Physiology,vol. 280, no. 6, pp. F1093–F1106, 2001.

[16] D. J. Benos, “Sensing tension: recognizing ENaC as a stretchsensor,” Hypertension, vol. 44, no. 5, pp. 616–617, 2004.

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