Cellular aspects of renal sodium transport and cell volume regulation

Kidney International, Vol. 9 (1976) p. 103—120

Cellular aspects of renal sodium transport and cell volume regulation GUILLERMO WHITTEMBURY and JARED J. GRANTFIAM Centro de Biofisica y Bioquiinica, Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela, and Nephrology Division, Department of Internal Medicine, University of Kansas Medical Center, Kansas City, Kansas

It is generally accepted that in the transtubular

or shrinkage. In this setting we will examine our

movement of matter in kidney tubules, a significant portion of the solute and water passes through the

knowledge of the factors which control the regulation

cytoplasm of the transporting cells. Since sodium and, to a lesser extent, potassium are the principal

across the peritubular side of the cell.

of renal cell solute and water transfer primarily

cations reabsorbed by nephrons, their extrusion from the cells may be importantly tied to transtubule so-

Theoretical considerations

lute absorption as well as maintenance of intracellular volume and solute concentrations of kidney tubule cells. Consequently, considerable effort

has been invested in studies of the mechanism of

Although the renal tubule cells are bipolar, i.e., the cells are anatomically and functionally asymmetric in contrast to blood cells (erythrocytes, leucocytes, etc.),

solute and water transport across the peritubular surface of kidney tubules. In the renal cortex in vivo, the

for the purpose of this discussion we will consider transport events across the peritubular aspect of the

cells ordinarily are bathed in isotonic medium on both the lumen and blood side. Accordingly, it is important to examine those factors which may perturb cell volume regulation to cause an increase or

cell only. In this way we may treat the renal tubule as a quasi-symmetrical system for the evaluation of factors influencing cell volume and electrolyte content.

In the first approximation, we consider all renal tubule cells to possess the following general properties in the steady state, as deduced from experimental

decrease in size, or a change in cellular ionic composition in an isotonic environment. The most common of these stresses encountered in the clinic are hypoxia (anoxia), acid-base disturbances and hypothermia (renal preservation for transplantation). On the other hand, renal, as well as other body cells, are subjected to anisotonic stresses which have an important impact on cell volume and ionic content. These include the hypoosmotic and hyperosmotic

findings: 1) Cell volume and intracellular concentration of solutes are constant. 2) The freezing point of fresh kidney tissue is similar to that of the extracellular material when no autolytic changes are

states encountered in heart and liver disease and diabetes mellitus. The delicate control of renal cell vol-

allowed to occur [1, 2]. However, there is an excess of intracellular macromolecules in relation to interstitial fluid. 3) The ionic concentration of cytoplasm differs from that of extracellular fluid, i.e., electrochemical gradients exist between cytoplasm and interstitium.

ume is important for at least two reasons. First,

4) The plasma membranes are permeable to water,

changes in cell volume may alter the normal func-

ions and organic solutes to a varying degree. 5) Some

tion of the tissue, potentially to an irreversible

mechanism(s) located in or near the plasma mem-

extent. Second, owing to the compact structure of the kidney, changes in size of renal cells may drastically

brane is (are) responsible for maintaining the volume and composition of the cells in a steady state away from the point of electrochemical equilibrium. Furthermore, we have learned by experimentation that

alter the flow of blood through the organ, and thereby intensify the injurious effects of cell swelling

the interior of the cells of most cortical tubules is electrically negative with respect to the interstitium and that the concentration of potassium is higher and

© 1976, by the International Society of Nephrology. Published by Springer-Verlag New York.

103

104

Whittembury/Grantham

that of sodium and chloride lower in cells than in blood serum.

For the general case, the steady state concentrations of cellular ions and the electrochemical potential difference have been formulated in a modification of the Goldman equation which is applicable

to renal tubule cells in the absence of tubular perfusion and net transtubular solute and fluid transport

between intracellular and extracellular fluids and ions (Na, K, Cl) are maintained by a "pump" mechanism sufficient to counterbalance the effect of impermeant

charged intracellular molecules. The condition of electroneutrality (i.e., the sum of positive and negative charges within the cell equals zero) is as follows: (K), + (Na)1 — (Cl)1 + (zA)/v = 0

(4)

where A is the amount of relatively impermeant molecules "trapped" in the cytoplasm (proteins,

[3—6]:

Vm = (RT/F) In (K)0 + a(Na)0 + b(Cl)1 (K)1 + a(Na)1 + b(Cl)0

(1)

HCO3-, S04, H2P04, etc.), z is the average valence and v is the cell volume, then, (K), + (Na)1 + (Cl)1 + (A/v) — C0 =

P/RT

where Vm is the computed transmembrane electrical potential in millivolts, and a and b are relative permeabilities (a = PNO/PK and b = Pcl/PK). K, Na and Cl are the concentrations of ions inside (I) or outside

where C0 is the total concentration of solutes in the external solution. In essence, equation 5 describes an

(o) of the cells assuming an activity coefficient of unity in both fluid phases. R, T, and F have their

chemical potential difference) between cytoplasm

usual meaning. If it is assumed further that chloride ion is passively

distributed across the peritubular surface of most renal cortical cells, then

effective osmotic difference (which is a function of the

and interstitium, i.e., C1 —

(2)

and upon further substitution and rearrangement of equation 1, Vm = (RT/F) In

= (RT/F) In

0

(3)

3 is a useful point of departure in consideration of the forces responsible for Equation

maintenance of the intracellular concentrations of the major ions in renal tubule cells. As we shall learn, an equation of this type does not necessarily accurately

C0,

that must be

counterbalanced by solute extrusion or the development of membrane tension in order to maintain the cell volume constant. (C1 — C0)

Vm =(RT/F)ln'

(5)

RT =

(6)

As shown in equation 6, "active transport" may be viewed in the context of a pressure (P) sufficient to maintain the number of intracellular osmotically active particles constant. The "pressure" may be real, as a derivative of high membrane tension, or the "pressure" may be obviated by the active process of solute extrusion. In the latter case, there would not be a measurable hydrostatic pressure difference. Methods of study

To analyze the forces which maintain renal cells in

reflect life processes; nevertheless, it provides a

a steady state with respect to their volume and intracellular ionic composition, it is necessary to have direct access to the tissue under study. In situ clear-

framework for hypothesis testing.

ance techniques of the classical variety do not permit

In another regard, it is important to portray the factors responsible for maintenance of cellular volume in the steady state. From considerations of

specific analysis of cellular events. Consequently,

Gibbs-Donnan equilibrium it can be shown that if, as mentioned above, the concentration of nondiffusible negatively charged macromolecules is higher inside the cell than outside it, the total solute concentration inside the cell will be larger than outside the cells. In

methods have been developed over the years to study the kidney tissue in vitro. Tissue slices. Thin slices of kidney cortex can be maintained in a steady state for reasonably long periods of time. The composition of the ambient medium can be controlled strictly. It is necessary, however, to perform the studies at or near room temperature, for

across the cell membrane is expected to exist. How-

at higher temperatures the tissue consumes oxygen and substrates at rates faster than these substances

ever, as mentioned above, the intracellular solute concentration is similar to that of the extracellular

can diffuse to the central region of the slice. As will be discussed subsequently, the absolute level of temper-

material [1, 2]. Therefore, in addition to Gibbs-Donnan forces it is necessary to assume that isosmolality

ature may have an important bearing on certain

consequence, an osmotic pressure difference, P,

transport processes. It is also important to note that

105

Cell sodium transport and volume regulation

nearly all studies utilizing slices of kidney have excluded colloid (serum proteins, dextran) from the external medium. The potential impact of colloid has not been studied seriously in slices. Slices of cortex

I'

r ..

-, ,,

U

0

temperature and left there until the electrolyte and water content is stable. After the initial stabiliza-

—50

of the cells, Mudge [7] and many workers subsequently have increased the sodium and water content of the tissue upon removal from the animal by incubating the cortex in "isotonic" medium near 0°C, containing no external potassium (Fig. I). In this manner, it is possible to evaluate factors which modify the extrusion of NaCI and water, and the uptake of potassium on rewarming the tissue in "normal" potassium medium [8]. The electrolyte and water composition of cortical renal slices from a number of animal species is listed in Table 1. Fresh tissue, i.e., that immediately immersed in warm "physiologic" medium is compared to slices that were "leached" in low potassium cold

25°C

25°C

150

I

2

, 'I -y

principal ways: (1) Immediately after the normal kidney is removed from the animal, a slice of cortex is immersed in isotonic "physiologic" medium at room

experimental manipulation may be examined. (2). In order to study net movement of solute and water out

150

0—3°C

500

slice is generally inferred by including an extracellular

tion period, the response of the cells to further

101

I

(K)0 0

from proximal tubules. The contribution of cellula electrolytes to the total ionic content of an individual

volume marker, such as inulin, in the external medium. The slice studies have been performed in two

temp.

(Na)0 150

contain an admixture of tubules although in most species more than 80% of the cortical cells derive

Rise K

Rise

Fresh tissue

— —

/—

g of cell watei

12

gofsolid

C)

0 0. C)

U

PD —100

0

1

2

3

Time, hr Fig. 1. Experimental procedure to study net sodium efflux and its relationship to net K, net CI and water movements. Cell Na, K, Cl and water content and cell electrical potentials of the outermost slices from the guinea pig kidney cortex (cortex corticis) incubated in vitro are shown as a function of time of incubation. Some slices were separated for fresh analysis, others were immersed in a chilled medium (0 to 3°C) containing 150 ms Na and no K in order to

load the cells with Na and make them lose K and gain Cl and water. After two-hour immersion the slices were reimmersed in a similar medium at 25°C. In the experiment shown here, the reimmersion medium was subsequently replaced by another containing 2 mi K. The continuous lines refer to steady states (from [35J).

medium initially. Table 1 also includes some intracellular ion concentrations calculated from total tissue analysis and extiacellular space determina-

composition of cortical suspensions as reported from several laboratories is shown in Table 1. Isolated tubules. It is possible to obviate the prob-

tions.

lems of tubular heterogeneity by studying specific segments of dissected nephrons in vitro [20]. One disadvantage of the method is that the very small quantities of tissue limit the breadth and precision of chemical analysis. Consequently, only a few studies

Suspensions of tubules. Burg and Orloff, disenchanted with the complexity of isotopic kinetic studies in slices, developed a different approach to the study of renal tubule cells [18]. In this method, pieces of kidney cortex are incubated in collagenase solution until individual fragments of tubule are freed-up. The suspension of tubule fragments obviates the problem

of impaired diffusion of oxygen and substrates to the cells. Consequently, kinetic studies of bidirectional isotope flux are susceptible to more rigid interpretation. The suspension method eliminates

of cell electrolyte analysis have been reported. A summary of these findings is included in Table 1. Methodological limitations, a) Intracellular concen-

trations: Since the composition of the cell interior cannot be assessed directly, the composition of the extracellular space or trapped extracellular volume

of transport across the peritubular membrane pri-

has to be subtracted from the analysis of whole slices or suspensions of isolated tubules, respectively. Errors in extracellular space or trapped volume determinations are therefore critical in estimation of intracellular concentrations of ions like Na or Cl with a high extracellular to intracellular concentration ratio

marily, since the lumens are not filled with fluid. The

[23].

neither the problem of cellular heterogeneity, nor the

necessity to evaluate the contribution of the extracellular space (trapped within the suspension). Also, as in the slice method, one observes the consequence

106 Table

Whitteinbury/Grantham

I. Ion and water composition of kidney cortical slices, guinea pig kidney cortical slices and separated rabbit renal Na

Water

Condition

%

Incubation 0°C

Rabbit Incubation 0°C

Rat

Dog

/2moles/g of wet tissue

Kidney cortical slices° 70 77.6 61 24 79.0 118

Rabbit Fresh

Reincubation 25°C Reincubation 25°C +

Cl

K

ouabain

Incubation 25°C Same + inhibitor Same + ouabain + inhibitor Effect of inhibitor Effect of ouabain

Fresh Incubated 0°C Change due to incubation Incubation 25°C Incubation 25°C + ouabain Change due to ouabain

Change due to temøerature rise Change due to (K). rise

311

7

562

116

7 524

9

252

9

436

9

291

213

10

223

281

10

90

306

+68 +25

10 10 10

188

II

651

2.97

279

(149)

( 48)

(110)

3.73

531

148 344 135

2.63

251

3.12

399

3.19

536

+0.49 +148 —68 +0.07 +137 —133

77 78

44

12

310 273 68

34

194

73 65

3.25

248

3.55

255

386

4.55

634

127

12 12 12

13 13

53

89

386

299

14, 15

76

SI

3.33 3.48

231

63

284

343

222

14, 15

86 46

64

3.22

272

6

4.16

350 579

365

81

240

417

6

(a)

76

83

(b) - a)

81

112

+0.94 +229 —125 +145

(a)

(b)

3.10

270

3.80

460

340 130

(a)

(b)

(2.68) (2.03) (1.97)

(489) (367) (168)

(152) (126) (323)

(c)

a) (c - b)

—0.65 —0.06

—122

Separated tubules

3.00

Slices

3.35

260 297

(b

16 16

(119) (113)

17 17 17

—6

17

(246)

—26 —127

—199 +197

6 16

+0.70 +190 —210

a)

Incubation 0°C, (K). = 0mM Reincubation 25°C, (K)0 = 0mM Reincubation 25°C, (K),, 16mM

271

4.57

Guinea pig kidney cortex slices° 85 78 56 82 114 23

(b

3.47

59)

58

Reference

3.76

(

76.5

Cl

/2moles/g of solids

(lOS)

(c — b)

(b

K

(172)

(b - a)

Outer slice Fresh Inner slice Fresh

solids

Na

( 41)

(b) (c)

Fresh Incubation at 0°C

g/goj

(139) 77)

(a)

Fresh Fresh Incubated 0°C Incubated 35°C

Water

tubules

17

Separated rabbit renal tubulesab

Separated tubules 0°C (K) = 4mM Separated tubules 25°C (K)0 4mM

308 313

18

18

13 137

80 52

3.17 2.24

733

54

333

19

239

496

110

56

2.37

165

261

189 132

20 20

Separatd tubules

3.53

221

321

237

21

Slices

2.83

189

339

220

21

2.57

285 207

150

290 204

22 22

1.69

96

250

—0.63

—78

+I +98

142 —86

22 22 22

176

66 70

Isolated tubules

Separated tubules 0.5°C (K)0 = 0mM Separated tubules 28°C (K)0 = 0mM Separated tubules 28°C (K)0 = 5 mM Effect of rewarming Effect of (K)0 rise

(Ill)

(

(107)

( 78)

(c)

(

—

a)

(c —

b)

(b

58)

(a)

(b)

56)

(112) (105)

(148) (

83)

1.94

—0.25 —112

151

—63

° Figures for ions have been rounded to integers. In parenthesis are given cell ion concentrations and not tissue ion concentrations.

Values for slices are included for comparison.

b) The possibility that intracellular ions may be affected by cellular compartmentalization remains unresolved in the kidney. The supposition that concentration in the cellular compartment is uniform is implicit in the estimation of electrochemical potential

gradients across the cell membrane. Kinetic studies indicate that cell ions are compartmentalized [24] and

that oxygen supply, temperature of incubation and use of inhibitors change markedly the kinetics of Na, K and Cl isotope distribution [15, 25—28]. There are

107

Cell sodium transport and volume regulation

no convincing studies of intracellular Na activities

0

although valuable attempts have been made to meas-

ure K activities (c.f., [29] and references therein).

—-10 -

Other limitations of the kidney slice technique when extrapolated to in vivo studies have been pointed out

—20

[30].

c) Tissue solid weight, DNA and tissue protein content may be required as references to calculate ionic movements when cells gain or lose isotonic solutions or when the amount of solids within the cell varies during the experimental procedure [6, 31]

—30

Distribution of potassium ions. The measured cell membrane potential has been studied as a function of

[K'] =—59 log—

c0

1)

0

[K']0

—40 -

(33)

0. c-I

Distribution of ions between cells and extracellular space

(24)\•(44)

50 —60 -

•(67)

—70 -

\P) •(19) (11)

the intracellular to extracellular distribution of K ions in the amphibian kidney [32, 33]. Similar studies

have been performed in mammalian kidney slices allowed to recover at 25°C after a period of leaching in the cold). As illustrated in Fig. 2, it has been found

that over a wide range (10 to 100 mEq/liter) of changes in the extracellular K concentration, the log-

arithm of the K ratio across the cell membrane is linearly related to the cell membrane potential (cell negative). This indicated that at these (K)0 values, K distributes passively across the cell membrane since the electrochemical potential for K ions inside the cell equals that outside the cell. All experimental data (even those at lower K concentrations) may be fitted by equation 3 with a value for a = 0.015. The departure from the straight line at the lower extra-

cellular K concentrations (see legend, Fig. 2) indicates that at these low (K)0 values the values of(K)j

are too high to be accounted for by the membrane potential. This may be taken to be evidence that, upon rewarming at these reduced K concentrations, K ions must be taken actively into the cell [13, 34]. This aspect of the K distribution is illustrated in Fig. 3 in a different manner. It may be observed that the

intracellular K activity at (K)0 lower than 10 mEq/liter is higher than that expected for a passive distribution. Besides the possibility that K ions are taken up actively by kidney cells, the possibility has

also been considered that some K is compart-

mentalized and does not contribute to the intracellular K ion activity [36]. Distribution of chloride ions. It has long been real-

ized that Cl ions do not distribute across the cell membrane as expected from equation 2 [13, 15, 17, 32, 34]. (Cl)0/(Cl)1 ratios of about 3 have been computed; however, from the values of the membrane potential Vm (which range from —60 to —70/mv

2

357w 2030 5070

K activity (cell/bath), [K']1/[K']0 Fig. 2. Cell membrane potential Vm as a function of the cell to rei,nmersion mediu,n K activity ratio. Number of measurements are given in parenthesis. The continuous line has been drawn according to the Nernst equation and represents the K equilibrium potential, EK = —59 log (K)1/(K)0 where the prime symbols denote calculated K activities. The three experimental points to the right cor-

respond to (K). of 5.3, 2.0 and 1.0 mM. At these (K)0 Vm is 7, 17 and 25 my less negative than EK, respectively. These differences between Vm and E would be larger if concentrations instead of activity ratios are used (see insert). The point labelled (P) corresponds to Vm measured with slices bathed in a solution containing 2% albumin (reprinted from [13] with permission from the Rockefeller University Press).

inside negative), ratios between 10 and 15 are to be expected. No clear explanation exists for this discrepancy. The possibility of intracellular Cl sequestration within some sübcellular compartment (as re-

cently proposed in other tissue [37]) should be investigated before it can be definitely concluded

that Cl distribution is not passive. Kleinzeller, Nedundkova and Knotkova have found evidence indicating that Cl distributes passively across the cell membrane if a large separate intracellular Cl com-

partment is considered to exist [38]. Furthermore (see section on electrogenic Na extrusion), net Cl efflux observed upon rewarming guinea pig kidney cortex slices follows the membrane potential as expected from the passive movement of Cl ions [34]. However, the existence of active chloride transport in some parts of the nephron must be considered since a mechanism that actively reabsorbs Cl from the lumen of the thick ascending limb of Henle's loop has been described [39, 40]. Distribution of sodium ions. As illustrated in Fig. 3, the Na activity within the cell is much lower than that expected from a passive distribution of Na ions. This

108

Whittembury/Grantham

2000

I /

/

1000

600

I

[Na']1 = [Na'].

—FVrn

exp RT

/

/ / //

/ ,/ 0a

that, in the steady state, the experimental data can be fitted by equation 3 (with finite permeabilities for K and Na) means that in the channels used for active ion movement, Na ions moving outwards are being exchanged for inward movement of K ions [5]. The observation that at (K)0 > 10 mEq/liter, the simple Nernst equation EK = Vm = —59 log (K)1/(K)0 is followed (see Fig. 2) means that at these (K)0 values

the existing membrane potential results in forces which are sufficient to account for the experimentally observed distribution of K in the steady state: Active sodium extrusion

0 a 0

The sodium for potassium exchange pump. Kidney slices and isolated tubules have been useful to study

I) U

the relationship between Na and K movements across the peritubular cell border. In these preparations, several lines of evidence show the existence of Na for K exchange by the Na pump. (1) Sodium and potassium move in opposite directions 40 80 60 100 120 Reimmersion fluid cation activity, mmoles/kg

Fig. 3. Cell K and cell Na activities plotted as afunction of the K and Na ion activities in the (extracellular) medium used to reimmerse guinea pig kidney cortex slices. Slices were immersed first in the cold in a medium containing no K. Then they were reimmersed at 25°C with variable Na and K concentrations. These Na and K concentrations are plotted on the abscissa. The dashed line shows the corresponding intracellular ionic activities calculated using the extracellular K and Na activities and the membrane potential with the equations given in the figure. Activities were estimated using the activity coefficients for KCI and NaCI in free solution. To the left are the K data. Notice that cell K distributes passively except at low external K activities in which cell K activities are higher than expected for a passive distribution. Consequently, during reimmersion at this low external K activities the cells either have gained more K than expected from the membrane potential or some K is sequestered within the cell. To the right are Na data. Notice that cell Na activities are at least an order of magnitude lower than the calculated values. This is generally accepted to be due to active Na extrusion (reprinted from [I 3] with permission from the Rockefeller University Press).

indicates that the electrochemical potential for Na ions is much lower inside than outside the cell. In the case of K or Cl ions, compartmentalization of the cell or sequestration within the cell would give a fit of the distribution of K and Cl ions across cell membrane closer to that expected from a passive distribution. On the contrary, in the case of Na ions, the fit would depart even more from a passive one. This indicates that to maintain this low intracellular Na activity Na ions must be extruded. Active Na ion movement can

be envisaged as fluxes in parallel with passive Na movement. By definition, in the steady state net fluxes across the cell membrane should equal zero and active ion movement must be equal and opposite to passive ion leaks. In consequence, the observation

across tubular cell membranes. Thus, in slices which

have been loaded with Na and made to lose K by

leaching, the following can be shown upon rewarming: (a) Cell sodium decreases when cell potassium increases; i.e., a clear relationship can be established between net movement of Na from cell to extracellular space and net entry of K and vice versa (Fig. 1 and Fig. 4) [7, 8, 13, 14, 16]. (b) Net Na extru-

sion depends on the K concentration in the

extracellular medium [13, 16, 22, 35]. c) Net K entry depends on intracellular Na concentration [16, 41]. (2) As illustrated in Fig. 4, ouabain inhibits extrusion of Na and K uptake in a magnitude to suggest a one to one relation [16, 17, 22, 35]. (3) Finally, as also illustrated in Fig. 4, the ouabain dose-response curve

is closely paralleled by inhibitory action of the (sodium plus potassium-stimulated, magnesiumdependent hydrolysis of adenosine triphosphate) Nat, K-ATPase activity of a cell membrane preparation from the same tissue [16, 42, 43].

These observations agree with the view that energy for the function of the Na pump comes from hydrolysis of AlP at the level of the cell membrane, stimulated by Na from inside and by K from the outside and sensitive to the cardiac glycoside ouabain. These

views are illustrated in Fig. 5. Figure 5 also incorporates other Na and Ca extruding mechanisms related to ATP hydrolysis. They are dealt with later on. Electrogenic nature of the sodium pump: Studies out

of the steady state. The knowledge of whether Na extrusion is neutral or electrogenic is relevant to understanding the way in which K and Cl passive move-

109

Cell sodium transport and volume regulation

into the cell (and the small leak of K out of the cell). Because of this small pump activity, there is a strong

— —.o

Mg

-o

0)

'0

6.3 X 1O6 MI

0 ISO

K

lOO 50

.j10O• —

150k .6

(K)0 =8mM

I

7

6

5

—log

4

possibility that the potential due to K diffusion will mask any electrogenic Na extrusion which, if existent, will remain unnoticed. Consequently, to study this phenomenon experimental conditions have been chosen in which the pump activity is enhanced. After kidney cells have been made Na-rich by chilling, rewarming is performed in a medium rich in K. Thus, the electrochemical potential opposing Na extrusion is diminished and Na extrusion is enhanced. Net ion mociement of Na and Cl outwards and of K inwards can be easily assessed from changes in cell ion concentrations. Figure 6 illustrates an experiment of this sort. As may be seen in Fig. 6, under these conditions the negativity of Vm increases up to 30 my beyond EK. It was found that Vm values measured during each minute of this transitory hyperpolarization are

directly proportional to the net Na effiux values measured concomitantly at one-minute intervals (r =

(ouabain) Mitochondria membrane

Fig. 4. Dose-response curves of the inhibition by ouabain of the

residual A TPase (circles) and of the Na + K —stimulated A TPase (dots) are shown at the top. The dose for half inhibition of the latter is 6.3 X lO-6M ouabain. These curves are to be compared with those depicting the effect of different ouabain concentrations

Ca — Stim. Ouab—Insens.

on the changes in cell ionic content of slices rewarmed in the presence of 8 mvi K shown at the bottom. Net influx (gain) of ions subsequent to rewarming is represented by values above, and net efflux (loss) of ions by values below the zero line. The stars show movement of Na accompanied by Cl and water. These correspond to loss of volume (water) and of Na and Cl by cells upon rewarm-

I

ADP+Pi

H2

Na + K — Stim. Ouab—Sens.

ing which was affected little by ouabain. The empty diamonds represent movements of K and full diamonds represent calculated movements of Na in exchange for K. These ionic movements are clearly affected by ouabain. Dose for half inhibition is 1.4 X lO-5M

ouabain (reprinted from 142] with permission from Elsevier Publishing Company).

L_ATP

Na') Na

Na — Stim. Ouab—Insens.

-ADP + P,

ments evolve. (a) If Na extrusion is neutral, K could be taken up into the cell by the Na pump mechanism and the ensuing increase in (K)1 would increase the negativity of Vm by diffusion of the ion through the passive pathways. The increased negativity of the cell interior could drive Cl passively out of the cell. (b) On the other hand, if Na extrusion is electrogenic, i.e., if the Na pump separates charges across the cell membrane, Na extrusion itself would increase the negativity of Vm. This could drive Cl passively out of the

cell and/or K into the cell to maintain the

electroneutrality. These ion movements would use pathways different from the Na pump. In kidney slices and suspensions examined in the steady state it seems difficult to decide between these alternatives, since under these circumstances the Na pump needs to transport ions only to a very limited extent, in order to balance the small leak of Na ions

Fig. 5. Scheme for coupling between active transport and respiration

in slices of kidney cortex. Oxidative phosphorylation in mitochondria provides ATP required to support active movements of Na, K, Ca and other energy-requiring processes in the cell 116,441.

The ADP resulting from this ATP hydrolysis stimulates oxygen consumption which is coupled to active transport. I) ATP hydrolysis by membrane Na + K-ATPase provides energy for the widely accepted ouabain-sensitive active Na extrusion and K uptake. 11) Since the membrane potential is negative, intracellular Ca concentrations higher than those obtaining in the extracellular fluid are expected from a passive behavior. Measured intracellular calcium concentrations are lower than in the extracellular fluid. Therefore, a mechanism for calcium extrusion is required. A calcium ATPase has been described in microsomal fractions. Thus, energy arising from ATP hydrolysis may be used for Ca extrusion, which regulates (Ca)1. Increased (Ca)j inhibits Na-K-ATPase dnd this may

influence active Na-K transport. (Ca)0 controls passive permeability of kidney cell membranes 145]. The hypothetical mechanochemical system of Kleinzeller is proposed to be related with a Ca ATPase [46]. 111) A Na-stimulated ouabain-insensitive ATPase has been described recently. It may be related to the second mode of Na extrusion described in the text. Systems not widely accepted or hypothetical are depicted with dotted lines.

110

Whittembury/Grant ham

(K)0 lower than 10 mEq/liter, K ions may be actively

16mM

[K]0 16

taken up into cells. Probably the two types of K

Temp 0.6°

25°C

movements use parallel channels, active uptake being in closer relationship with the Na pump while passive

K movement is related to Na extrusion only by

!

means of the change in the electrochemical potential that Na extrusion originates [34]. 50

—30 -

—40-

40

-

: 11 0

—50 -

0

0

2

4

6

8

11—1530—50

Rewarining time, mm

Fig. 6. Time course of membrane potential (Vm) (bottom) and net

Na efflux (top) in guinea pig kidney cortex slices. Cells of kidney slices had been loaded with Na and allowed to lose K at 0.6°C by immersion in a medium containing 150 mEq of Na/liter and no K. Measurements of Vm were undertaken in a medium containing 16 mEq of K/liter still at 0.6°C. The temperature was suddenly in-

creased to 25°C in medium containing 16 mEq of K/liter thus enhancing Na extrusion. Circles represent values calculated for E, the K equilibrium potential. At 0° and at 25°C after the steady State had been reached Vm = E. However, during the first ten minutes of rewarming, the rate of Na extrusion (shown with the

Energy supply for sodium transport. The Na-K pump requires renewed synthesis of ATP since it is inhibited by 2,4-dinitrophenol and by anoxia [17, 35, 47]. Studies of the energy supply for the Na pump have stressed the relationship between ion movement, oxygen consumption and ATPase activity [48] which have been studied in kidney slices and subcellular fractions in an effort to pinpoint the strict location of these relationships [16, 44]. Oxygen consumption consists of two fractions, a constant basal rate and a variable rate [48] which is related to the energy requirements for Na extrusion and uptake of K. Thus diminution of cell Na extrusion (attained by replacing Na by choline) resulted in reduced oxygen consumption. Increase in extrusion of Na and K uptake obtained by increasing the Na concentrations of the

bathing medium resulted in a parallel increase in oxygen consumption. Enhancement and inhibition of exchange of Na for K by the Na pump (obtained by

changing the bathing medium K and ouabain concentrations) were associated with parallel changes in

scale downwards) paralleled Vm rather than EK (from [34]).

oxygen consumption [16, 35, 50]. Several of these experimental maneuvers did not alter respiration of

0.96). In other words, the largest amount of Na was extruded when the magnitude of the electrical force

isolated mitochondria, indicating that the linkage between oxygen consumption and ion transport (and, for example, the action of ouabain and K) occurs at the cell membrane level. Therefore, the rate of active

opposing net Na extrusion was the largest. Furthermore, inhibitors of Na extrusion curtailed the hyperpolarization significantly. Although, neither

ouabain nor ethacrynic acid added separately completely abolished this hyperpolarization, both agents added together blocked it completely. Therefore, the conclusion may be reached that Na extrusion is electrogenic and contributes noticeably to the generation of Vm so that Vm is more negative than EK [34]. A good correlation between net K influx and net

Cl- efflux (ionic movements taking place concomitantly during the first ten minutes of rewarming) and the measured values of Vm was also observed. However, the correlation coefficients are negative (r = —0.98 for K and r = —0.93 per Cl, respectively) indicating that these ions move passively, secondarily to the observed hyperpolarization

[34]. This observation indicates that at external K concentrations of 16 mEq/liter, K ions movepassively. However, it was also pointed out above that at

cation transport is a factor in the control of tissue respiration, a finding that is compatible with the hypothesis of obligatory coupling between ATP production and utilization. Hydrolysis of ATP has to be

mediated by an extramitochondrial ATPase. This ATPase, located at the cell membrane level and sensi-

tive to Na, K, Ca and ouabain, is one of the pacemakers of respiration in kidney cortex. Role of Na,K-A TPase in cell sodium transport.

Some of the common ion transport properties of kidney slices and microsomal preparations [51] from the same tissue already have been described in pre-

vious sections. There are further reasons to link Na,K-ATPase with Na-K transport in the kidney. Thus, Na,K-ATPase is found in very high concentrations in the kidney. This has led to the use of kidney ATPase preparations in attempts to purify this enzyme preparation [52]. Its activity increases

when animals have been adapted to conditions in which the Na pump activity is expected to be en-

Cell sodium transport and volume regulation

hanced because Na reabsorption is increased (i.e., administration of adrenal steroids, unilateral nephrectomy, high protein diet) or more K is excreted (chronic K loading; c.f. [53]). As mentioned before (see Fig. 4), its activity decreases upon addition of cardiac glycosides in a manner which closely follows

inhibition by cardiac glycosides of Na-K transport [16, 17]. Its activity also decreases when the Na-K pump is expected to diminish, i.e., adrenalectomy [53, 54], and with extracellular volume expansion [55] which diminishes Na reabsorption in the kidney [53] and in postobstructive diuresis in the rat [56]. Finally,

microdissection and membrane fractionation methods have shown that all Na, K-ATPase activity is strategically located at the basal border of the cell (where one expects the Na-K pump to be located) and not at the luminal side [57—61].

Other requirements of the transport ATPase sys-

tem have not yet been described in the kidney, namely its vectorial aspects [62] and the possibility that reversal of the process can synthesize ATP [63]. It is pertinent to point out other limitations in the concept of the Na,K-ATPase system. Na,K-ATPase activity is defined as the difference between the ATP hydrolyzing activity observed in the presence of Mg + Na + K minus that observed in the presence of Mg alone or minus that observed in the presence of Mg +

Na + K + ouabain. Since the method of prepara-

tion and the inhibition of its activity by ouabain are part of the mode of defining the Na,KATPase system, observation of AlP hydrolyzing activities which might be related to other modes of ion

transport may be precluded. It is still unclear how much of the ATPase activity observed under given experimental conditions depends on changes in the fraction of sites exposed by the experimental procedure or in changes in the absolute number of active sites present in the preparation. It is accepted that intracellular pH is different from extracellular pH. Yet, ATPase preparation and the reactions to assess hydrolysis of ATP are carried out with both sides of the system at same pH. Also, the concentrations of Mg ions, Na ions, K ions and of ATP used do not necessarily correspond to those present in vivo [53]. There are differences in the Na,K-ATPase activity in different regions of the kidney [53, 57—59, 64—66].

Procedures to obtain preparations richer in per-

itubular membranes [60, 67] should be incorporated into the ATPase technique. Unresolved problems. Although a combination of a single Na-K pump (with a variable coupling ratio) and ion leak system can explain several experimental observations relative to ion and water movements in kidney cells, there is a puzzling set of unexpected

111

observations if one is to consider a single pump and leak system.

Some observations arise from kidney studies in vivo. (1) If there were only one system to effect Na reabsorption, complete inhibition of the energy supply to this system (i.e., inhibition of the Na,K-AT-

Pase activity by ouabain) should block Na reabsorption completely. However, about 50% of the filtered Na (and Cl) continues to be reabsorbed when the Na,K-ATPase activity is 100% inhibited in vivo

and in the perfused kidney. The remaining reab-

sorption can be blocked with cyanide and iodoacetate. Thus, the ouabain-resistant sodium reabsorption is dependent on metabolic energy [68—70]. The obvious conclusion is that other reabsorptive mechanisms may be in operation. (2) Although under control conditions net sodium reabsorption in the perfused amphibian kidney correlates well with peritubular K uptake, this relationship can be markedly upset by different means so as to have near normal Na reabsorption with very low K uptake, and under other conditions negligible Na and Cl reabsorption with normal K uptake [41]. Na reabsorption can also be dissociated from maintenance of cell ionic and volume composition [71]. Thus, either the Na movement is loosely linked to K flux or the two ions move independently. Other observations arise from work with kidney slices. (1) In kidney slices single tubules may be perfused and their reabsorptive transport properties under various conditions may be studied in conjunction with observed changes in cell ion concentration. This

method has the weakness that only a few tubule segments are indeed perfused while cell composition is investigated in cells from mostly nonperfused tubules which constitute the large majority of the slice.

However, the observation that widely different Na reabsorptive properties have been observed in cases in which cells have similar intracellular Na concentrations has lead to the conclusion that transcellular and homocellular transport processes may be different [72]. (2) Working with mammalian kidney

slices Kleinzeller and Knotkova [9] observed that kidney slices can extrude Na with Cl even in the presence of concentrations of ouabain known to in-

hibit exchange of Na for K. These observations have been confirmed and extended [17, 21, 22, 35, 73—75]. The body of experimental evidence is summarized in the following section. Two modes of sodium extrusion in kidney slices. As may be seen in Fig. 1, once cells of guinea pig kidney cortex slices have been loaded with Na and allowed

to lose K by immersion in the cold, rewarming to 25°C in a medium without K results in expulsion of

112

Whittembury/Granthani

Na with Cl and water from the cells. Once a steady state has been reached, K may be added to the bathing medium. Under these conditions, further Na extrusion is observed accompanied by entry of K without volume change (i.e., without further efflux of Cl and water). Thus, two modes of Na extrusion may be described. One in which Na is expelled with Cl and water isosmotically (and therefore with an important reduction in cell volume), and another in which Na is expelled in exchange for K. If ouabain is added to the bathing medium prior to rewarming, extrusion of Na with Cl and water continues (even in the presence of 10 mrvi ouabain) although the glycoside com-

pletely inhibits extrusion of Na in exchange for K [42]. If ethacrynic acid is added prior to rewarming, cells are able to take up K; however, extrusion of Na

(with Cl and water) stops. Both inhibitors added together have additive effects to block both modes of Na extrusion. Dinitrophenol and anoxia also inhibit both modes of Na extrusion [17, 35]. It may be concluded that hydrolysis of ATP is the source of energy for both modes of Na extrusion.

Three main types of explanations which have in common the proposition of mechanisms working in parallel have been offered to account for such observations:

1) The cryptic pump hypothesis considers that there is difference in the accessibility of the external

bathing solutions to the Na pump in the surface of the kidney cell facing the basement membrane and in those pumps located deep in the crypts of the clefts

present at the basal aspects of kidney cells. In this conception, Na extrusion in a medium without K can be accounted for because the cell K that is being lost into the crypts is readily taken up by the Na pump so that the K concentration is not really zero within the crypts [75, 76]. This allows Na to be expelled from the cells even when the K concentration in the exter-

nal medium is ostensibly zero. Ouabain would be unable to block part of Na extrusion because it does not reach the pumps located deep in the clefts (which would continue to work uninhibited) but only those present at the surface. This possibility was put aside [76] since it is difficult to think of cryptic sites which are inaccessible to ouabain but which can be reached by ethacrynic acid. Important to ruling out the crypt pump idea is the observation that ouabain (at concentrations of 1 and even 10 mM) does not inhibit extrusion of Na with Cl despite the fact that extrusion

of Na in exchange for K is already maximally blocked at ouabain concentrations several orders of magnitude lower (see Fig. 4) [17, 42]. The cryptic pump hypothesis has received recent support since it has been found that isolated tubules cannot expel Na

with Cl and water if they are dialyzed so as to produce greater K loss or if they are reincubated in the presence of ouabain [22]. Further experiments are required to settle this matter since extrusion of Na

with Cl and water has been observed to be maintained in isolated tubules at low extracellular pH, while it was apparently lost at high pH values [211.

2) Kleinzeller has provided evidence for the existence of a mechanochemical (contractile) mechanism with physical properties determined by Ca and ATP metabolism which may counterbalance a major hydrostatic pressure gradient in kidney cells. This mechanism can qualitatively explain most of the present observations, namely extrusion of Na or Li with Cl

and water; the relationship between Ca, ATP and volume regulation; the finding of Ca ATPase (vide infra) and the presence of filamentous contractile structures at the basal aspects of the cells [46]. However, it is necessary to ascertain whether this mechanism can account quantitatively for the magnitude of the cell volume changes observed and whether it is not located in the basement membrane (instead of at

the plasma membrane). Of further concern in this regard is the possible role of protein oncotic forces in

the determination of cell volume. Serum proteins clearly affect the rate of transtubular fluid absorption in proximal tubules from the peritubular side [125,

77]. If, as Kleinzeller suggests, small gradients of hydrostatic pressure are important in cell volume regulation, then it is entirely possible that colloid osmotic pressure could have a role as well. 3) The existence of a second Na pump which would extrude Na has also been considered. In support of

this view are the observations that ethacrynic acid inhibits mostly the expulsion of Na with Cl and water

out of kidney cells which can still take up K. This idea has been criticized in view of the observation that ethacrynic acid does not act exclusively at the level of the cell membrane, but that it inhibits cell function n onspecifIcally at progressively higher levels

[46, 78, 79]. On the other hand, the addition of the inhibitory effect of ouabain and ethacrynic acid (at different concentrations) upon respiration and K uptake do support the dual pump hypothesis [50]. Also, the ability of kidney cells to expel Na with Cl and water can be stimulated by angiotensin even in the presence of ouabain under conditions in which Na for K exchange is inhibited [80]. Finally, a ouabainresistant, Na- and/or Li-stimulated ATPase has been described in kidney membrane preparation. This ATPase might be related to the mechanism that would link ouabain-insensitive Na extrusion with ATP hydrolysis [65].

Cell sodium transport and volume regulation

113

4) In addition, experiments are required to explore the possibility that water and/or ionic compartments

Regulation of cell volume in anisotonic media

within the cell may vary in size under some

Osmometric behavior, i.e., an alteration in volume proportionate to the change in osmolality of the external medium, appears to be a fundamental property of animal cells. The rapid change of cell volume in anisotonic solutions is dependent on the relatively more rapid penetration of water than solutes through the plasma membranes. In mammals, the cells of the distal renal tubule and collecting duct are probably subjected to the widest fluctuations in ambient os-

experimental conditins in such a way that a given amount of Na, Cl and water becomes available or unavailable for Na extrusion. Other A TPases and sodium transports. In view of the general agreement about linkage of energy coming from ATP hydrolysis and sodium transport, attempts have been made to find modes of ATP hydrolysis in kidney cell membrane preparations different from the Na,K-ATPase. Two lines of research have been fruitful.

(a) The first is the demonstration of an ATPase activated by Ca in the brush border [67] and basal lateral plasma membranes [61] and one activated by Ca and Mg (neither Mg nor Na or K are necessary for Ca activation; ouabain and ethacrynic acid do not inhibit activation by Ca) [81]. This ATPase activity differs from that found in red blood cells [82]. Since Na,K-ATPase is inhibited at high Ca concentrations,

it is tempting to suggest that Na pumping activity may be modulated by intracellular calcium concen-

trations so that at high intracellular Ca concentrations the Na-K pump activity would slow down.

(b) Recently another mode of activity of Mg-dependent ATPase activity has been described in kidney membrane fractions aged by storage at 4°C for several days [65]. This ATPase activity is different from

molality in consequence of the diluting and concentrating functions of the kidney. For example, the sudden conversion from water diuresis to anti-

diuresis in response to elaboration of antidiuretic hormone causes the cells of the distal tubule [83, 84] and collecting system [85, 86] to swell owing to the entry of water from the relatively hypotonic urine. Unbridled cellular swelling could have profound effects on hydrodynamic flow of urine along the full extent of the nephron owing to a diminution in lumen diameter. In contrast to this acute setting, the gradual development of dilutional hyponatremia in association with heart and liver disease conceivably could

cause the cells of all nephron segments (as well as other body cells) to swell and thereby compromise renal hydrodynamics. Thus, it is of some importance to understand the extent to which renal tubule cells respond osmometrically, and also if adjustments are made to regulate cell volume in anisotonic environm ents.

On the basis of studies of kidney cortex in vitro, it

other Na-stimulated ATPase activities described in

had been suggested that kidney cells were "poor"

the literature [661 and has the following characteristics: (1) It is stimulated by Na and Li but

osmometers at 25°C, but were "good" osmometers at lower temperature [38]. However, recent studies suggest that kidney cells are osmometrically active even at 37°C. Isolated rabbit renal tubules were found to change volume rapidly in response to sudden altera-

not by K, Rb, Cs, NH4 or chloride. (2) Its activity is independent of the anion accompanying Na or Li. (3) It is not inhibited by 1 and 10 mrvi ouabain. (4) It is

100% inhibited by 2 mvi ethacrynic acid, a concentration which inhibits only 30% of the Mgdependent ATPase and 50% of the Na,K-ATPase. (5) It is inhibited by furosemide (I0-5M) and by triflocin under conditions in which neither Na,K-ATPase nor Mg-ATPase activites are inhibited. (6) Its appearance is accelerated if kidney tissue is first incubated with angiotensin II before the membrane fraction is prepared so that one to three days of aging are required

tions in the osmolality of the external bathing medium [87]. Perhaps even more important was the finding that swollen cells rapidly adjusted the cell volume towards the initial resting volume. The central features of the experiments are shown in Fig. 7 from the work of Dellasega and Grantham. Three nephron portions were studied in vitro: the convoluted and straight portions of proximal tubule and the cortical collecting tubule. The tubules were

as compared to two to three weeks in the controls. Although much work is required to further improve the yield of ATPase activity of this preparation and

not perfused; therefore, changes in tubule cell volume were due to movements of fluid across the peritubular

to further define its characteristics, the characteristics

segments, tubule cell volume increased rapidly when

described coincide with the requirements expected from the ouabain-resistant mode of Na extrusion described above.

the bath was changed from isotonic to hypotonic

membrane, only. As shown in Fig. 7 in all three medium. The peak swelling response was inversely related to bath osmolality, indicating osmometric be-

114

Whiuembury/Grantham 294 mOsm

I 294

147 mOsm

18

mOsm

16 -

I)

that intracellular anion was decreased as well. Thus, it would appear than an important factor in the volume regulation of hypotonic medium is the net loss of intracellular solute, notably potassium. The mecha-

PCT

14 -

nism of net solute loss is unresolved presently. In 12 -

duck erythrocytes, the first cells from warm-blooded

animals observed to regulate volume in hypotonic medium, the net loss of KCI appeared to be passive

0 I) 10 -

0

[89]. In contrast to that in kidney tubules, intracellular sodium did not change appreciably. Yannet may have described a similar volume regu-

.0

F-

hypotonic medium there was a marked decrease in intracellular potassium; intracellular sodium decreased also, but to a lesser extent. It may be assumed

8-

latory phenomenon in brain tissue many years ago

6-

[90]. Brain cells of rats were protected from sustained osmotic swelling in hyponatremic states owing to the I 5

0

I 10

15

Al/n Fig. 7. Effect of hypotonic medium on renal tubule cell volume. Note the sudden increase in tubule cell volume followed by a period in which the cell volume returned toward the initial value. PCT. proximal convoluted tubule; PST, proximal straight tubule; CCT, cortical collecting tubule dissected from rabbit kidney (reprinted

from [87] with permission from the American Physiological Society).

havior of both proximal and distal elements of the nephron. The surprising finding in this study was the

observation that the tubule cells rapidly adjusted their volume towards normal when kept in the hypotonic bath. In the proximal segments the adjustment of cell volume was relatively rapid, requiring three to five minutes, whereas, in the collecting tubule reduc-

tion of tubule volume proceeded at a much slower rate. These cell volume adjustments occurred independently of net transtubular solute and water absorption since the tubules were not perfused and the

massive loss of intracellular potassium chloride. Thus, it would appear that volume regulation in hypotonic medium may be a fundamental property of many types of mammalian cells. In this regard it is important to consider some of the factors which may

impair the volume regulatory response. Dellasega and Grantham observed that cooling renal tubules to 10°C virtually abolished volume regulation of proximal tubules in hypotonic media (Fig. 8). The pattern of intracellular sodium and potassium (unpublished work) is interesting in this regard for, as shown

in Table 3, the net loss of K was detected, as in tubules incubated in hypotonic medium at 37°C. However, in contrast to the normothermic state, the intra-

cellular content of sodium increased, rather than decreased. Thus, the failure of volume regulation at 10°C in the hypotonic medium is probably due to the failure to extrude intracellular cation. Further, findings of the hypothermia experiment may be interpreted to in-

lumens were collapsed. Thus, the mechanism for volume regulation in hypotonic medium is located in or about the basolateral plasma membrane. Recently, preliminary information has been presented in regard to the mechanism of volume adjust-

ment in hypotonic media. Intracellular Na and K were measured in isolated segments of proximal straight tubule immersed acutely in hypotonic medium [88]. As shown in Table 2, after 15 mm in

5 — 7°C mOsm 5 — 7°i

14 C)

12

F

0 V

.0 0

Table 2. Effect of hypotonic medium on cellular content of

294 mOsm

16

F-

10

8

proximal straight tubulesa

Isotonic (294 mOsm kg')

Hypotonic

Change

(147 mOsm kg-1)

mEqmm 1O mEqmm X /O K

Na

100.9 32.8

Twenty-one experiments.

79.5 24.6

0

4

8

12

16

0

4

8 15—300

4

Mm

X

—21.3

— 8.2

Fig. 8. Effect of cooling on volume regulation of proximal straight tubules in hypotonic medium. The dashed/inc depicts the changes in volume observed at 37°C in the hypotonic medium (reprinted from [87] with permission from the American Physiological Society).

115

Cell sodium transport and volume regulation Table 3. Effect of hypothermia on electrolyte content of proximal

-

straight tubules in hypotonic media

K

Na

Control isotonic 37°C

Hypotonic

Hypotonic

37°C

10°C

100.0 28.9

—12.4 —3.9

—12.5

+20.2

mOsm + I 0 M Oubain'l46mOsm +

I

ü M Oubaij

18 16 C)

14

dicate that sodium transport out of the cell (presumably by an active mechanism) is importantly

291

-

C C)

tied to the net extrusion of solute in hypotonic medium. If active sodium transport is importantly related to

volume regulation in hypotonic medium, then ouabain, the classic inhibitor of the ATPase-dependent component of sodium transport, should have blocked volume regulation. However, ouabain failed to abolish volume regulation of renal tubules in hypotonic medium ([87], Fig. 9). Findings in the ouabain experiment cast doubt on the primacy of potassium loss in the hypotonic volume regulatory mechanism. It is well known that ouabain causes cells to lose potas-

sium and gain sodium owing to inhibition of the peritubular "exchange" pump. In recent studies it was shown that ouabain-treated cells exhibited inhibited volume regulation in hypotonic medium as was

observed previously, but that intracellular Na was lost primarily, rather than potassium [88]. Thus, at this juncture it is clear that cation loss, either potas-

sium or sodium or both, is involved in the contraction of cell volume in hypotonic medium; however, the mechanism of solute extrusion in normal and ouabain-treated tubules is very much in doubt. There are several implications of these obser-

I

I

0

4

8

I

12 16 20

I

0

4

8

10

Mm

Fig. 9. Effect of ouabain on regulation of cell volume (PST) in hypotonic medium. Tubules were incubated in ouabain initially in isotonic medium, after which the bath was changed to hypotonic medium (reprinted from [871 with permission from The American Physiological Society).

relative stiffness of the renal capsule, there would be compression of parenchyma, and compromise of vascular flow. More than likely, the hypotonic volume regulatory mechanism prevents marked fluctuations in renal cell volume. There is another clinical situation in which blockade of the volume regulatory mechanism could be

of critical importance. As will be discussed subsequently, in the course of renal transplantation, donor organs often are cleared of residual blood by perfusing the vessels with isotonic solution. It is reasonable to expect the organs removed from an antidiuretic patient excreting highly concentrated urine

appreciation of the volume regulatory feature of

would have hypertonic medullary and papillary zones. Infusion of cold isotonic saline into such a kidney would potentially cause massive osmotic

mammalian cells provides a rational explanation for why brain, blood and kidney cells do not swell to a large extent in patients with chronic severe dilutional hyponatremia. If the brain is osmometrically active,

volume into the normal range. More than likely, prolonged preservation in the swollen state would

vations to clinical nephrology. First of all, an

like the kidney, a reduction in the serum sodium concentration from 140 to 120 mEq/liter could cause

an increase in intracellular brain volume of approximately 17% were no volume regulation to occur. Such an increase in volume, considering that the brain is housed in a rigid framework, could have disastrous consequences. Since it is well known that

patients with serum sodium concentrations in the neighborhood of 120 mEq/liter may be entirely asymptomatic, it seems reasonable to conclude that important adjustments have taken place to prevent massive swelling.

By the same token the kidney could swell to the same extent in hypoosmotic states, and owing to the

swelling of the medullary and papillary renal tubule cells and block the mechanism for regulating the cell

adversely affect the function of the organ upon implantation. These considerations may help to explain the clinical observation that organs from diuretic donors (water diuresis or mannitol) withstand hypo-

thermic preservation to a better extent than antidiuretic kidneys. Application of principles to kidney transplantation'

During the process of transplantation, the kidney removed from a living or cadaver donor and The David M. Hume Memorial Symposium on Transplantation (Transplant Proc 6, No. 4 (suppl I), Dec., 1974) contains a good collection of articles on the subject. We will not attempt to cover immunologic aspects (c.f. [1001).

116

Whiuembury/Grantham

mVQ Na

implanted in the recipient must recover normal function. An unavoidable period of warm ischemia affects several cortical functions [9 1—94]. Impaired active

transport (specifically Na extrusion) and cell autolysis increase the intracellular osmotic pressure [1, 2, 95—97]. This leads to increase in the volume of the epithelial and vascular cells of the kidney [96]. The lumina of kidney tubules and capillaries are reduced, thus hindering tubular and vascular fluid flow. Since oxygen consumption by the kidney diminishes at low

Normal function

temperature while several functions are unharmed [99, 101, 102], the kidney temperature is reduced. However, although hypothermia will limit ischemic damage, it will not insure that kidney damage may be reversed since it induces changes [95, 102—107] (Fig. 10) which also lead to cell swelling further occluding

lumina of tubules and capillaries. Therefore, other steps have to be undertaken. (a) Cell volume can be restored by increasing the osmotic pressure of the extracellular fluids, for example with mannitol [98]

Chilling

which seems more effective than glucose (c.f. [109—112]. Pretreatment of donors with mannitol [96,

117, 118] and with steroids [114, 119] is recommended. (b) Use of macromolecules, which will exert osmotic pressure from within the capillary beds thus increasing the capillary lumina, seems also desirable [108, 109, 115, 116]. (c) In addition, a suitable extracellular ionic composition is required to reduce the external electrochemical potential for Na in order

to allow the Na pump to work downhill [35]. This suitable composition may be achieved by replace-

Chilling +

t(K)0, (Na)0 (Cr)0, (SO) t Nonelect

ment of Na and Cl in the extracellular environment by K salts of sulfate [11, 109, 111] or phosphate [109, 112, 113].

Two methods are in vogue to preserve kidneys

removed for transplantation, namely continuous pulsatile perfusion, and single hypothermic washout of the kidney with hyperosmotic fluid [120, 123]. In the first method, cold perfusate of a composition similar

to that of the extracellular fluids and containing plasma proteins is used [115, 124]. In view of the reduced temperature, it may be advisable for the solutions to contain a (K)0 of 10 to 20 mEq of K/liter. This (K)0 is higher than that usually found in the extracellular fluid, but not as high as those recommended for the second procedure and shown in Table

4. Pulsatile perfusion takes advantage of the use of proteins to increase the oncotic pressure of the circulating fluid2 (see also [77, 109, 115, 125, 126]) result-

ing in the maintenance of a controlled kidney water

Fig. 10. Effect ojiowering the temparature on some mechanisms that regulate cell volume and ionic concentrations. Top. During normal

function sodium-extruding mechanisms expel sodium and take up potassium, thus maintaining high intracellular potassium and low intracellular sodium concentratuon. The cell membrane potential of about —7OmV (inside negative) tends to reject anions (chloride) from the cell interior. The volume-regulating mechanism is depicted as a second sodium pump. Middle. With hypothermia the sodium pump slows down. As a consequence, intracellular sodium rises, intracellular potassium decreases, the negativity of the cell membrane diminishes, chloride ions are not rejected and enter the cell. Thus, the cell gains effectively sodium and chloride. The cell water content increases because volume-regulating mechanisms also slow down. Bottom. Three modifications in the extracellular fluid help to restore cell ionic composition and volume. (a) The extracellular fluid is made rich in potassium and poor in sodium ions. Thus, the sodium pump works downhill expelling accumulated sodium. The high external potassium concentration lowers the cell membrane potential to values near zero and limits even more the tendency of cells to reject anions. Therefore, (b) the anion (sulfate, phosphate) making up the solution should penetrate the cell membrane at most only slowly as compared to chloride. (c) The solution is made very hyperosmotic by use of nonelectrolytes

that slowly permeate the cell membrane (glucose, mannitol or 2 Proteins are also known to stabilize the cell membrane of tubular cells as judged from membrane electrical potential measurements (see Fig. 2; also, 132]).

other nonelectrolyte) to compensate for the tendency of the cells to

swell (reprinted from [97] with permission from S. Karper AG, Basel).

Cell sodium transport and volume regulation Table 4. Fluids for kidney single perfusion (flush-out solutions), mmoles/liter

accumulation in the proximal conouluted tubules of the

I

II

60

K2S04

KH2PO

15

15

35

K2HPO4

42.5 IS

42.5

42.5

KCI KHCO3 NaHCO3 MgCl2

15

—

23 20

10

250

140

440

350

10

15 16

280 410

210 430

30

MgSO4

Nonelectrolyte Osmolality, mosm/kg

frog's kidney. J Gen Physiol 29:305—334, 1946 AL: Ionic movements and electrical activity in giant nerve fibres. Proc R Soc Lond [Bid] 148:1—37, 1957 6. LEAF A: On the mechanism of fluid exchange of tissues in vitro. Biochem J 62:24 1—248, 1956 7. MUDGE GH: Studies on potassium accumulation by rabbit 5. HODGKIN

Sackse

Acquatellaa Collinsb

117

kidney slices: Effect of metabolic activity. Am J Physiol 165:113—127, 1951

8. CORT JH, KI.EINZELLER A: Transport of alkali cations by kidney cortex slices. Biochim Biophys Ada 23: 321—326, 1957 9. KLEINZELLER A, KNOTROVA A: The effect of ouabain on the

electrolyte and water transport in kidney cortex and liver slices. J Physiol 175:172—192, 1964 10. MACKNIGHT ADC:

use of a small amount of colloid to improve vascular

Water and electrolyte contents of rat renal cortical slices incubated in medium containing p-

filling without increasing inordinately the viscosity of the perfusion

chloromercuribenzoic acid or p-chloromercuribenzoic acid

a.b.c The

fluid is desirable [109]. If used, Collins solution should be made more hyperosmotic by addition of more glucose (or mannitol) than the amount recommended by Collins [110—114]. a [11, 109, 1101; [113]; [110, 112].

and ouabain. Biochim Biophys Acta 163:500—505, 1968 II. ACQUATEI.I,A H, PEREZ-GONZALEZ M, MORALES JM, WHITTEMBLJRY G:

Ionic and histological changes in the kidney

after perfusion and storage for transplantation. Transplantation 14:480—489, 1972

content and of high perfusate flow rates [116]. The second method, much simpler than the first, involves single perfusion and storage with solutions rich in K and in impermeant anions and poor in Na and Cl (see Table 4). These solutions do not presently include serum proteins in the perfusion fluid. Although the use of macromolecules increases the viscosity of the perfusion fluids and renders the initial washout

procedure more difficult, inclusion of these substances in the initial wash to clear the kidney of blood cells may prove to be advantageous [109, 115]. Acknowledgments

Dr. Whittembury was supported in part by grants for project 0296 from CON CIT and from OEA. Dr. Grantham was supported in part by grants from the U.S. Public Health Service, AM-13476 and Career Development award AM-22760. Dr. Ernesto Gon-

zalez helped in the preparation of this review. Reprint requests to Dr. Guillermo Whittembury. Instituto Venezo. lano de Investigaciones Cienlificas, P. 0. Box 1827, Caracas 101, Venezuela.

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