kidney epithelium in 
 collecting duct contacts with hypotonic fluid which osmolality varies 
 significantly and the cells continually expose to osmotic stress . alterations 
 in luminal osmolality would influence cell volume . to avoid damage and perform 
 their   functions ,
these processes , termed regulatory volume decrease ( rvd ) , 
 occur through activation of specific transporters in the plasma membrane that 
 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 swelling in hypotonic medium [ 1 , 2 ] .  
cell swelling has the effect of increasing k efflux , this occurs through channels distinct from those responsible for basal k conductance .
obviously , both k and cl concentrations play key roles in governing these volume - regulatory responses . 

the predominant pathway for rvd is the opening of k channels which 
 are usually large conductance [ 3 , 4 ] .
these transporters can be quite nonselective and can 
 include more than one type of channels . in cells containing na channels 
 like collecting duct principal cells ,
na fluxes can influence the 
 volume - regulatory response but the role of intracellular sodium in rvd is 
 almost unstudied . from another hand , the mechanism by which cells sense changes 
 in cell volume and activates 

it is important to study the 
 changes of cell volume and [ na]i during the process of 
 rvd for better understanding of the mechanisms involved in this reaction .
the 
 current study was undertaken to investigate the time course of changes in cell 
 volume and sodium concentration during the volume - regulatory response after 
 acute swelling in hypotonic medium .
wistar rats weighting 150200 g ( breeding 
 laboratory of experimental animals , institute of cytology and genetics , 
 novosibirsk , russia ) 
 were kept in individual cages and received standard diet . before the 
 experiments
, the rats were anaesthetized with pentobarbital ( 50 mg / kg i.p . ) , 
 the kidneys were extracted and placed in ice - cold pbs , and a suspension of 
 collecting duct fragments was prepared .
a superfusion chamber was constructed as an acrylic block mounted on the 
 objective of the upright microscope ( water immersion 65 magnification , numerical 
 aperture 1.1 ) .
the perfusate flow rate was generally 20 ml / min , which gave 
 complete solution exchange in under 200 milliseconds .
fluorescence measurements 
 of cell volume were performed as it was described [ 6 , 7 ] .
fragments of outer medullary collecting duct ( omcd ) on glass plate were 
 loaded with calcein by incubation with calcein - am ( molecular probes inc . , ore , usa ) ( 5.0 m ) for 25 minutes at 23c .
the glass plate bearing the fragments of the omcd was positioned on the stage 
 of a microscope ( lomo - r8 , st .
calcein fluorescence was 
 measured continuously with halogen light source , calcein filter set ( 480 nm 
 excitation , 490 nm dichroic mirror , 535 nm emission ) , photomultiplier detector 
 has pinhole diaphragm to select the cells of interest at the end of fragment 
 where cells expose apical surface to bath solution , and 14-bit 
 analog - to - digital converter .
loading conditions and filter set for fluorescence 
 measurements were the same as for calcein .
tissue from the outer medulla zone was cut from the isolated kidneys and 
 squeezed through a needle ( 0.45 mm i.d . ) in the ice - cold calcium - free pbs .
the 
 resulting suspension was filtered through nylon mesh , diluted 10 times with 
 eagle mem culture medium and centrifuged ( 100 g , 10 minutes , 4c ) . 

sediment containing the tubules was diluted with culture medium to an 
 appropriate concentration of about 10 fragments per microliter .
the solutions used were based on pbs 
 ( 137 mm nacl , 4.7 mm na2hpo4 , 2.7 mm kcl , 1.5 mm kh2hpo4 , 0.5 mm mgcl2 , 0.05 mm cacl2 , 
 280 mosm / l , ph = 7 , 27 , 1 ) and contained 1.0 mg / ml glucose .
this solution was chosen so as to be able to 
 degas it without affecting its ph . to create osmotic challenges ,
bath solutions were changed 
 from normal pbs to pbs diluted with distilled water ( 1:1 ) .
isotonic solutions 
 with decreased [ na]o were prepared by substitution part of 
 sodium in pbs by nmdg ( n - methyl - d - glucamine , 
 sigma - aldrich , moscow , russia ) . for 
 measurements of sodium efflux in isotonic medium
fluorescent measurements were calibrated 
 plotting the relative values of fluorescence as an x value , against synchronized data of 
 microscopic image analysis , obtained by the method published before .
a 
 perfusion chamber , described above , was mounted on the objective of the 
 microscope ( water immersion 65 magnification , numerical aperture 1.1 , 
 1.6 magnification photo adapter ) .
the glass plate bearing the fragments of the 
 omcd was positioned on the stage of a microscope for acquisition of transmitted 
 light images by a ccd camera .
twelve - bit monochrome images were captured at a 
 rate of 15 frames per second , stored on a personal computer .
cell volume was calculated from 
 measurements of calcein fluorescence according this calibration . in vivocalibration of sodium green
was accomplished by 
 exposing the fragments to various extracellular [ na ] ( [ na]o ) in the presence of 0.1 u/l nystatin .
the solutions with various [ na]o were prepared by substitution part of sodium in pbs by nmdg . for calibration were used solutions with 
 138 , 14 , and 5 mm [ na]o . 

a calibration was performed at the end of each experiment ( see figure 2 ) .
ion fluxes that 
 appeared during cell swelling seem to be critically involved in the regulation 
 of cell volume . however , the nature of the channels and the mechanism of their 
 activation are poorly understood .
it is also not clear to what extent this 
 activation can explain the regulatory volume decrease .
the goal of this study 
 is to examine the time course of cell volume changes and concomitant variations 
 in [ na]i after hypotonic shock . in 
 hypotonic medium , principal cells in omcd fragments
swell very fast , and the 
 volume increases more than 60% ( see figures 2 and 3 ) .
the evaluation 
 of characteristic time of swelling ( 1 ) gives the value 0.65      0.05 seconds ( n = 12 ) , which is in a good 
 agreement with our previous results . in our experiments , basolateral and apical 
 surfaces of the cells on the end of microdissected fragment are in contact with 
 medium .
fast swelling of the principal cells is the consequence of the high 
 water permeability determined by the water channels that are expressed in 
 basolateral and apical membranes [ 8 , 9 ] . 

it was shown that abundance of water channels correlates with faster rvd 
 [ 10 , 11 ] .
probably the water channels are part of the mechanism , which is 
 activated in cell volume decrease response .
after cell volume reaches the peak 
 of swelling , the rvd begins without lag period .
the kinetics of this process is slower than swelling , 
 and the characteristic time of volume decreasing to steady - state level ( 2 ) is 8.9      1.1 seconds ( n = 12 ) ( see figure 4 ) . 

the published data show that cells in cell lines derived from kidney epithelia , 
 rccd , cos-7 , a6 have a significantly slower rate of rvd , and 2 is 
 at least one order more [ 1113 ] .
there is a discrepancy of results obtained on microdissected fragments of 
 omcd and published data obtained on cell lines .
most probably the difference is 
 due to the loss of some ion transporters , which could not be activated by hypoosmotic 
 shock in cultured cells .
after restoration of the medium osmolality to normotonic , 
 the cells shrink and the volume stabilizes on significantly lower level in 
 comparison with control level ( see figure 4 ) .
this decrease probably is due to 
 the loss of cellular osmolites during rvd . 

salt withdrawal procedure could result in responses that depend on the 
 type of cation removed . in this study
, we reduced the osmolality by diluting 
 pbs with water or removing nacl from the bath .
the procedure causes drastic 
 changes in ionic strength that could interfere with the mechanisms involved in 
 volume recovery .
hypotonic medium causes decrease of [ na]i and stabilization on the low level ( see figures 2 and 5 ) . the established level of [ na]i during the hypotonic shock is the result of interaction of three main processes : dilution of osmolites with water that enters the cell , 
 activity of na , k - atpase , and ion flux into the cell through channels activated 
 by swelling , which could be permeable for sodium . comparing the rates of normalized cell volume and [ na]i shows that 
 cell volume grows about two times faster than [ na]i decreases ( 0.72      0.05 and   0.30      0.02 s correspondingly , p < .01 ) .
this could be 
 explained if during swelling water enters the cell accompanied by sodium .
to 
 block cell swelling and water entry , we created the transmembrane sodium 
 gradient with normotonic pbs where 69 mm of na was substituted by 
 nmdg . in the absence of cell swelling with virtually zero water entry , the  of [ na]i decrease is significantly faster in comparison with the same parameter in hypoosmotic 
 shock ( 2.6      0.26 and 8.9      0.98 seconds ,    n = 8 
 and n = 12 , correspondently , p < .01 ) . these results could be accepted as 
 evaluation of sodium efflux through na , k - atpase .  
the evaluation of sodium content per cell , made 
 by multiplication of synchronized measurements of calcein and sodium green 
 fluorescence , shows that peak of cell volume correlates with the temporary 
 increase of this calculated parameter ( see figures 2 and 5 ) .
these results indicate 
 that the water comes into cell during the swelling together with sodium .
this could happen if swelling activated several 
 types of channels which could pass ions with high conductivity and probably 
 there are some channels which could pass water .
probably , this sodium flux aggravates the cell 
 swelling in the hypotonic medium if [ na]o is higher than [ na]i but during the phase of cell 
 volume decreasing these channels being active could promote the outcome of 
 potassium from the cell .
the outcome of the ions , mainly potassium , should be 
 facilitated by partial depolarization which could happened in these cells during 
 swelling .
one of the most probable transporters active in 
 normal conditions and which probably takes a part in volume regulating response 
 is epithelial sodium channel ( enac ) .
this channel is expressed in apical 
 membranes of principal cells , and there are data that indicate that enac could 
 be involved in rvd [ 15 , 16 ] .  
to evaluate 
 the putative influence of enac on [ na]i during hypoosmotic challenge , we blocked it with 
 amiloride .
the addition of 
 amiloride to the concentration of 10 m in bathing solutions caused 
 decrease of control steady - state level of [ na]i ( see figure 6 ) , 
 but did not influence this parameter in hypotonic medium and after returning 
 the normotonic pbs .
amiloride did 
 not influence significantly the relative swelling during hypoosmotic challenge 
 ( 0.65      0.01 control , 0.77      0.05 amiloride , ns ) . 

the ineffectiveness of amiloride means that contribution of enac in 
 sodium entry in our model of hypotonic shock is negligible and can not be 
 detected in our experiments .
the results of these 
 experiments indicate that in principal cells hypoosmotic shock causes 
 activation of the transporters , which have high permeability for water and 
 probably provide sodium and potassium transport across cell membrane in 
 dependence of the ion electrochemical gradient which is forming during swelling .
 the purpose of this study was to investigate the time course of the volume - regulatory response and intracellular sodium concentration ( [ na+]i ) in the principal cells of rat kidney outer medulla collecting duct ( omcd ) epithelia during acute swelling in hypotonic medium . 
 hypotonic shock was created by pbs diluted with 50% of water . 
 changes in cell volume were measured with calcein quenching method . 
 intracellular sodium concentration was studied with fluorescence dye sodium green . 
 principal cells of microdissected omcd fragments swelled very fast . 
 the characteristic time of swelling ( 1 ) was 0.65  0.05 seconds , and the volume increased more than 60% ( 92.9  5.6 and 151.3  9.8 m3 control and peak volumes correspondently , p < .01 ) . 
 after cell volume reached the peak of swelling , the rvd began without lag period . 
 the characteristic time of volume decreasing to new steady - state level ( 2 ) was   8.9  1.1 seconds . 
 in hypoosmotic medium , cell volume stabilized on higher level in comparison with control ( 110.3  8.3 m3 , p < .01 ) . 
 after restoration of the medium osmolality to normotonic , cell volume stabilized on significantly low level in comparison with control level ( 71.4  6.1 m3 , p < .01 ) . during the hypoosmotic shock ,   
 [ na+]i decreased from 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 of sodium content per cell has shown the significant sodium entry into the cells , which caused a temporary increase correlated with the peak of cell volume caused by swelling . 
 the conclusion is made that in our model of hypoosmotic shock , swelling activates transporters with high permeability for na+ that provides sodium flux into the cells . 
1. Introduction
2. Methods
3. Results and Discussion