tissue k concentration in relation to the role of the kidney in hibernation and the cause of...

&np. Biochem. Physiol., 1971, Vol. 39A, pp. 437 to 445. Pergamon Press. Printed in Great Britain

TISSUE K CONCENTRATION IN RELATION TO THE ROLE OF THE KIDNEY IN HIBERNATION AND THE

CAUSE OF PERIODIC AROUSAL

J. S. WILLIS, S. S. GOLDMAN* and RACHEL F. FOSTER

Department of Physiology and Biophysics, University of Illinois, Urbana, Illinois

(Received 5 October 1970)

Abstract-In deep hibernation: 1. Cerebral cortex and diaphragm of hamsters and ground squirrels, liver and erythrocytes of hamsters and ventricle of ground squirrels retain constant K content.

2. Leg muscle of both species and erythrocytes of ground squirrels lose K. 3. Kidney cortex of both species and liver of ground squirrels accumulate K. Upon arousal: 4. The K content of kidney and of ground squirrel liver

returns to normal. 5. K uptake by kidney (and liver) may serve to compensate for loss due to

cold in other tissues. Prolonged loss of K from excitable tissues may be a trigger for natural periodic arousal.

INTRODUCTION

A CRUCIAL and perhaps the most important adaptation possessed by the cells of mammalian hibernators is their ability to retain a high K concentration and to exclude Na at the low temperatures characteristic of “deep” hibernation (5°C).

Surprisingly, there have been few attempts to establish what changes may occur

in cellular concentrations of Na and K during hibernation, despite numerous studies of blood plasma electrolytes (see Table 2). Two studies have shown that

no particular rise in Na content or fall in K content of muscular tissues is associated with the hypothermia of hibernation, although seasonal changes may occur (Eliassen & Leivestad, 1961a, b; Willis, 1964). Willis also found a remarkable rise in tissue K concentration in the kidney cortex of hibernating hamsters and ground

squirrels. At the time no particular significance was assigned to this observation. Another study in this laboratory has revealed that while red blood cells of

hamsters and ground squirrels possess very specific adaptations that allow the retention of K at low temperature, there is nevertheless a gradual fall in K concentration during longer periods of hibernation in ground squirrels (i.e. more than 5 days) and in the cells of both species stored at 5°C for prolonged periods (Kimzey

& Willis, 1971a, b). This finding seemed to require a re-evaluation of the results on kidney and muscle. In that earlier study only a small number of types of cells had been investigated and the longest period of hibernation even in ground

* Present address : Laboratory of Neurochemistry, National Institute of Neurological Diseases and Stroke, Bethesda, Maryland.

437

438 J. S. WILLIS, S. S. GOLDMAN AND RACHEL F. FOSTER

squirrels had only been 4 or 5 days. Conceivably, then, other tissues could have

lost significant quantities of K, particularly if the individuals had been allowed to hibernate for a longer interval before collection of the tissues. Viewed in this light,

it seemed that the increased K content of the kidney cortex might have been significant as a compensation for the loss of K from other tissues.

A further examination, encompassing other types of tissue, longer periods of

hibernation and changes associated with arousal was undertaken to clarify these

points.

MATERIALS AND METHODS

Animals

Two species were studied, Syrian (Golden) hamsters (Mesocricetus auratus) and thirteen- lined ground squirrels (Citellus tridecemlineatus). The hamsters were bred in our own animal rooms and the strain exhibits a high incidence of hibernation (about 90 per cent). The ground squirrels were caught on the Urbana campus of the University of Illinois. Individ- uals of both species were allowed to hibernate in a room with an ambient temperature of 57°C which was kept dark. Hamsters were given pellets of lab chow so that they could make a cache in the cedar flake nests. They were also provided with water ad lib. Ground squirrels in the cold room had no food or water. Animals in the warm room were fed ad lib. The light cycle was 14 : 10 and the temperature about 22°C.

Tissue collection

Hibernation periods were determined crudely by the ‘sawdust method’ (Lyman, 1948 ; Pengelley & Fisher, 1961) and daily records were kept for each individual. Although the procedure sometimes yields false results in golden-manteled ground squirrels (Twente & Twente, 196.5), we found that with hamsters and thirteen-lined ground squirrels individuals it showed fairly consistent patterns of entry and arousal. Before the animal was killed cheek pouch temperature was taken and was always found to be within about 1°C of the ambient for deeply hibernating individuals. For determinations on arousing animals, arousal was initiated by tapping animals on the nose and pulling whiskers. The animals were left in the cold room for varying periods (but longer than 3 hr) after initiation. Animals which had not been aroused by the roustering procedure were not used. All tissues (control and hibernating) were collected between December and March in 1967 through 1970.

Animals were stunned by a blow on the head, the neck was broken and the carotid arteries severed. Kidney, liver, diaphragm and ventricle of the heart were removed immediately and placed on Parafilm in a Petri dish lined top and bottom with moistened filter paper and placed in ice. In some cases the animal was killed by decapitation and slices from the brain were taken directly and placed in tissue beakers for analysis of cations. A leg muscle from the superficial part of the thigh was also dissected out; this procedure generally required about 5 min.

Liver and kidney cortex were sliced by hand with a dry razor blade. In many instances up to six slices were taken in order to provide a measure of variation within a single individual. Diaphragm and ventricle were handled as described previously (Willis, 1964). Initially, slices were rinsed briefly in an ice-cold phosphate buffered Krebs medium to remove excess blood, but comparison of slices with and without rinsing revealed that there was no difference in K content and the procedure was abandoned.

Tissues were dried in tissue beakers, digested with concentrated nitric acid and analyzed for K by flame photometry. Only tissue K concentrations are discussed in this report because tissue Na content depends largely on extracellular concentration and size of the extracellular compartment and also because determination of Na is more subject to

KIN HIBERNATION 439

extraneous experimental error. Such problems do not attach to K since more than 95 per cent of tissue K is intracellular and the possibility of spurious contamination of tissue by K is slight.

RESULTS

Changes during hibernation and arousal

The K concentrations of tissues of hamsters and ground squirrels, awake, deeply hibernating and aroused from hibernation, are summarized in Table 1. The earlier finding that the low body temperature of hibernation leads to no decline in K ~oncen~ation of tissues was generally confirmed for cerebral cortex, diaphragm skeletal muscle, ventricular heart muscle, kidney, liver and erythrocytes of hamsters (Table 1).

TABLE~-KCONTENTOFTISSUESOFWAKING,HIBBRNATINGANDAROUSINGGROUNDSQUIRRELS

AND HAMSTERS

Species Tissue

Tissue K concentration (CL-equivlg dry wt.)

-

N Awake Hibernating Arousing

Hamster Kidney (6,8,6) Liver (6,8,6) Thigh muscle (6,8,6) Cerebral cortex (11,6) Erythrocytes * (6,6)

Ground squirrel Kidney @,22,5) Liver (5,22,5> Thigh muscle (5,20,4) Diaphragm t6,16) Ventricle (6,161 Cerebral cortex (6,16) Erythrocytes * (6,9)

336i5 311+ 10 449+12 509fl2 106 + 3

334+3 291+9 416+8 335*11 337 + 10 529+9

89?3

435 + 13 378211 298 Z!I 10 314+9 348+15 372 rt 14 525+8 101+3

409rt7 347 * 10 347+5 28928 324~~17 339 (317-365) 314+8 3SOf7 550 ir 14

77rt2

* Kimzey & Willis (197la). Units for erythrocytes are EL-equiv/l. cells. Means + S.E. are shown (range is given for the four cases of Ieg muscle from

arousing ground squirrels). Number of cases (i.e. number of animals from which tissues were taken) are shown respectively for awake, hibernating and arousing groups.

In addition, Table 1 contains three other new results: 1. Thigh muscle of both hamsters and ground squirrels lost K in hibernation,

Kimzey & Willis (1971a) found that the loss of K from red blood cells of ground squirrels is progressive with length of time of the bout of hibernation, but in thigh muscle of ground squirrels there was no evidence of greater loss in the individuals that had hibernated longer periods. Red blood cells can be sampled successively in the same individual, a fact which permitted the observation that during arousal


the K which is lost in hibernation is immediately reaccumulated (Kimzey & Willis, 1971a). In the case of the thigh muscles successive sampling was not so feasible. The mean K content of the muscle was higher in the arousing hamsters and ground

squirrels than in the hibernating individuals, but the difference was not significant in either species.

2. The K content of liver was higher in hibernating ground squirrels than in awake squirrels.

3. As observed before, kidney cortex gained a large amount of K in hibernation in both species, but in addition the concentration of K in both kidney cortex of

hamsters and ground squirrels and liver of ground squirrels returned toward normal awake values during rewarming.

Time course of rise in kidney (K)

Since erythrocytes of ground squirrels lose K progressively over several days of hibernation, it was of interest to determine whether the K concentration of kidney cortex, the prime tissue accumulating K, exhibited a time-dependent rise.

Figure 1 shows the K concentration of kidneys from ground squirrels which were

k

i I I I ( I I I I I I J

0

0 2 4 6 8 10

DAYS IN HIBERNATION

FIG. 1. K content of kidney slices from ground squirrels in hibernation. Closed circles, mean values of 2-6 slices of kidney cortex from individual ground squirrels; open circle, mean value from 8 awake ground squirrels (Table 2); triangle, mean value from 5 ground squirrels that had been aroused from hibernation (Table 2).

killed at varying intervals within a period of hibernation. The regression line, which is based only on the data of actually hibernating animals (i.e. it omits the points at the origin, the average values of awake and of aroused animals) had a slope of 5.1 CL-equivlg dry wt. per day which was significantly different from a slope of zero (P~0.01). The extrapolated origin was 387. Thus, it appeared that after an initial increase at the beginning of hibernation there was a further, slower

K IN HIBERNATlON 441

progressive increase in K content of the kidney cortex of ground squirrels which is reversed upon arousal and resumes after the animal re-enters hibernation,

DISCUSSION

Simple as they are the results of this study lead to several interesting con- clusions :

1. Even though several of the tissues tested (erythrocytes, kidney, diaphragm, brain) are known to possess adaptations allowing for retention of cellular K at low temperature even in r&ro, some tissues are not so perfectly adapted as to allow retention of normal steady-state concentrations for indefinite periods of low body temperature.

2. The “over-adaptation” of the kidney cortex (and perhaps of other tissues) compensates somewhat for the loss of K from less well adapted tissues. This may indeed be the chief function of the kidney in hibernation.

3. The progressive loss of K from excitable cells would be expected to lead to hyperexcitability and eventually to arousal. Resoration of K balance may be one function of periodic spontaneous arousal in hibernation.

Each of these points will be discussed in order.

Adaptation to low temperature of retention of cellular K may consist of either increased ion transport at low temperature in hibernator tissues or diminished leakage of K and Na. In kidney and red blood cells, which have been studied in detail, both factors seem to be involved (Kimzey & Willis, 1971a, b; Willis, 1966). “Perfect” adaptation would involve maintenance of a balance between uptake and loss of K (or extrusion and inward leakage of Na) such that the steady-state concentration would remain unchanged. In diaphragm muscle this balance seems to be achieved in vivo, but it is lost in vitro and the muscle cannot retain K at low temperature as well as at high (Willis, 1967). In kidney, uptake of K is relatively greater than loss at low temperature and a new high steady state is achieved in vivo at about 5°C body temperature. fn vitro, the steady state at 5°C is the same as at 38°C and the temperature for maximal K retention is 15°C. Thus, in these two tissues removal from the organism and preparation for incubation leads to some loss of cold resistance, and the loss is even more severe in slices of cerebral cortex (Goldman, 1969). Erythrocytes, on the other hand, suffer relatively little trauma when being removed from the body and subjected to artificial media. Correspond- ingly, their progressive loss of K in vitro at low temperature is the same as that in Z&JO in hibernation at the same temperature (Kimzey & Willis, 1971a). This loss in vitro is much less than that which occurs in red cells of non-hibernators such as those of guinea pig or human. Consequently, we must think of various tissue as having “degrees of adaptation” which will vary from one tissue to another and which are determined by the relative balance of changes in permeability and active ion transport.


The muscular tissues examined in the earlier study (Willis, 1969) were all essential for survival (cardiac muscle, diaphragm). It is of interest that leg skeletal muscle which is presumably not so active during hibernation does not show the

same retention of K as ventricular cardiac and diaphragm skeletal muscle. One possible complication in interpretation of ion changes which might arise

in muscle and liver are changes in dry weight due to gain or loss of glycogen. Thus,

glycogen content of muscle and liver may be greater in hibernation (Hannon & Vaughan, 1961) and is broken down during arousal (Lyman & Leduc, 1953), which could give spuriously low and high values for K during hibernation and arousal respectively. While this problem might alter the interpretation of the

results for the hamster liver and leg skeletal muscle of both species, it would not detract from the increase in K observed in ground squirrel liver during hibernation and loss during arousal.

2. Kidney K vs. plasma K

The previous section suggested that cold adaptation or ion regulation consists

of a maintenance of balance between loss and gain of ions; the point of this section is that at the level of the whole organism, adaptation consists of a balance between

some tissues losing K and others (notably kidney cortex) gaining it. Two questions are important here; is plasma K kept from rising and is the amount of uptake by

the kidney cortex and other tissues of a magnitude to be significant ? With regard to the first question, there is a fairly large literature on ion contents

of plasma or serum. A perversely persistent tendency has manifested itself for the results to be interpreted either as equivocal or as suggesting a rise in serum K during hibernation. Fisher & Manery (1967) reviewed this literature very thor-

oughly, but curiously came to this same conclusion. We have examined the same material again in detail (Table 2) and we conclude that where there have been careful observations repeated sufficient times to make valid comparison there is no

compelling evidence for a steady state rise in serum K associated with the periodic hypothermia of hibernation (that is, comparing cold room awake with actually

hibernating individuals). The painstaking determinations of Pengelley & Kelley (1961) indeed suggested a fall in serum K.

The ability and capacity of kidney cortex to accumulate K at low temperature is not only greater in hibernating species, than in non-hibernators, but it also

probably increases in an animal of a hibernating species when it begins to hibernate. Thus, the K uptake in slices of kidney cortex of hibernating hamsters is twice as rapid at 5°C as that in those of kidneys from awake hamsters and K accumulates to a higher steady state level even in vitro (Willis, 1966). Recent studies have shown that the activity of NaK ATPase also increases in hibernating hamsters (Fang & Willis, 1970; Goldman & Willis, 1970).

The absolute amount of K which can be taken up by kidney cortex can be computed by multiplying the weight of both cortices (about l-2 g in hamsters) by the net increase in K content (So-100 p-equiv/g dry wt.) and by the fraction of solid in the cortex (O-25) to give between 12 and 50 p-equiv. of K. With a plasma

K IN HIBERNATION 443

volume of 10 ml the net uptake would be equal to the entire steady-state content

of the plasma. Of course the volume of concern is the entire extracellular space

which would be about three times larger. On the other hand if the liver, in ground

squirrel, or heart, in hamsters (Willis, 1964), also participates there is ample

TABLE 2-K IN PLASMA OF HIBERNATORS

References Species Condition (K) Remarks

Biorck et al. (1962)

Erinaceous europaeus (hedgehog)

Riedesel & Folk Myotis lucifugus (1958) (little brown bat)

Eptesicus (big brown bat)

Ground squirrel

Mesocricetus auratus (Syrian hamster)

Denyes & Hassett Syrian hamster (1960)

Pengelley & Kelley Citellus lateralis (1967) (golden-mantled

ground squirrel) Raths (1964) Cricetus cricetus

A 6.6 (5.9-7.7) June H 7.0 (68-7.1) January

7.1 (64-80) March 6.0 (5~7-7.1)

WA 6.4 + 0.9 (S.D.) CA 7.2 f 0.6 H 6.5 + 0.6 Through season

7.2 + 0.6 Authors call this “increase in hibernation”

WA 6.2 & 0.2 (SE.) H 5.9 f 0.2 (SE.) WA 5.8 + 0.3 (S.E.) CA 8.2 f 0.1 H 6.5 f 0.5 WA 8.6 f 0.05 (S.E.) CA 6.5 + 0.3 H 9.5 2 determinations Fasted WA 5.1 -t 0.3 (S.E.) Fasted CA 5.2 + 0.2 Nonfasted CA 7.2 + 0.7 H 6.9 + 0.2 A 9.6 + 0.7 (SE.) Very careful H 6.0 f 0.6 determination

A; H 5.5-6.0 No change in (European hamster) deep hiberna-

tion ; transitory increase while entering

A = active, W = warm room, C = cold room, H = hibernating.

reserve for considerable “buffering” of K. With regard to K balance, it is important that there should be little excretion since most hibernators are essentially closed systems, and have no opportunity to restore progressive urinary loss. If there is a tendency for tissues to lose K in hibernation, a kidney whose cells capture the lost K and return it to the plasma upon rewarming, instead of to the urine, has much to recommend it.


3. K and arousal

The loss of K from excitable cells, particularly if it resulted in an increase in

concentration immediately outside the cell membrane, would have the effect of depolarizing cells. Ultimately, depolarization would lead to inexcitability of nerves

and muscles and therefore to death of the organism. Before that would occur, however, depolarization would first result in an approach of the membrane toward threshold and therefore to an increase in excitability. An increase in irritability

of ground squirrels in later phases of each bout of hibernation has been noted by Twente & Twente (1968a, b). Lyman & O’Brien (1969) have reported a hyper-

responsiveness of skeletal muscles and reflexes in hibernation of thirteen-lined

ground squirrels and have suggested this as a key to spontaneous periodic arousal. We endorse that hypothesis with the further suggestion that tendency for loss of ion-balance underlies the hyperresponsiveness.

Other hypotheses which have been advanced to account for periodic arousal have centered on depletion of a metabolic intermediate or accumulation of a toxic

substance. Not only have such key substances not been identified (with the possible exception of urea) (E. W. Pfeiffer, personal communication) but it has also

not been demonstrated that receptors exist for these substances which could trigger arousal. The present hypothesis has the advantage that it not only indicates

a need (teleologically) for arousal but also accounts for the mode of stimulation which triggers that arousal.

REFERENCES

BIORCK G., JOHANSSON B. W. & NILSON I. M. (1962) Blood coagulation studies in hedgehogs, in a hibernating and a non-hibernating state, and in dogs hypothermic and normo- thermic. Acta physiol. scund. 56, 334-348.

DENYES A. & HASSETT J. (1960) A study of the metabolism of liver, diaphragm and kidney in cold-exposed and hibernating hamsters. Bull. Mus. camp. Zool. Harv. 124, 437-456.

ELIASSEN E. & LEIVESTAD H. (196Ia) The effect of hibernating hypothermia on the potassium and sodium content of the muscles in the hedgehog, Erinaceus europaeus L. Arb. Univ. Bergen I962 Mat. Naturv. Serie No. 5.

ELIASSEN E. & LEIVESTAD H. (1961 b) Sodium and potassium content in the muscles of hibernating animals. Nature, Lond. 192,459-460.

FANG L. S. T. & WILLIS J. S. (1970) Further analysis of cold adaptation of Na-K ATPase of hibernating mammals. The Physiologist 13, 193.

FISHER K. C. & MANERY J. F. (1967) Water and electrolyte metabolism in heterotherms. In Mammalian Hibernation (Edited by FISHER K. C. et al.), Vol. III, pp. 235-279. American Elsevier, New York.

GOLDMAN S. (1970) Cold resistance of cation regulation in the brain during hibernation. Ph.D. thesis, University of Illinois.

GOLDMAN S. S. & WILLIS J. S. (1970) Acclimation of active cation transport in the central nervous system during hibernation. Fedn Proc. Fedn Am. Sots exp. Biol. 29, 718 (abstract).

HANNON J. P. & VAUGHAN D. A. (1961) Initial stages of intermediary glucose catabolism in the hibernator and non-hibernator. Am. J. Physiol. 201, 217-223.

KIMZEY S. L. & WILLIS J. S. (197Ia) Resistance of erythrocytes of hibernating mammals to loss of K during hibernation and during cold storage. r. gen. Physiol. (In press.)

K IN HIBERNATION 445

KIMZEY S. L. & WILLIS J. S. (1971b) Temperature adaptation of active Na-K transport and of passive permeability in erythrocytes of ground squirrels. J. gen. Physiol. (In press.)

LYMAN C. P. (1948) The oxygen consumption and temperature regulation of hibernating hamsters. J. exp. Zool. 109,55-78.

LYMAN C. P. & LEDUC E. H. (1953) Changes in blood sugar and tissue glycogen in the hamster during arousal from hibernation. J. cell. camp. Physiol. 41,471-492.

LYMAN C. P. & O’BRIEN R. C. (1969) Hyperresponsiveness in hibernation. Symp. S.E.B. 23,489-509.

PENGELLEY E. T. & FISHER K. C. (1961) Rhythm’ ica arousal from hibernation in the golden- 1 manteled ground squirrel. Citellus lateralis tescorum. Can. J. Zool. 39, 98-120.

PENCELLEY E. T. & KELLEY K. H. (1967) Plasma potassium and sodium concentration in active and hibernating golden-manteled ground squirrels, Citellus lateralis. Comp. Biochem. Physiol. 20, 299-305.

RATHS P. (1964) Mineralhaushalt und hormonale Aktivitst in Winterschlaf. Experentia 20, 178-190.

RIEDESEL M. L. & FOLK G. E. (1958) Serum electrolyte levels in hibernating mammals. Am. Nat. 92, 307-312.

TWENTE J. W. & TWENTE J. A. (1965) Effects of core temperature upon duration of hiberna- ation of Citellus lateralis. J. appl. Physiol. 20,411-416.

TWENTE J. W. & T~ENTE J. A. (1968a) Effects of epinephrine upon progressive irritability of hibernating Citellus lateralis. Comp. Biochem. Physiol. 25,475-483.

T~ENTE J. W. & TWENTE J. A. (1968b) Progressive irritability of hibernating Citellus lateralis. Comp. Biochem. Physiol. 25, 467-474.

WILLIS J. S. (1964) Potassium and sodium content of tissues of hamsters and ground squirrels during hibernation. Science 146, 546-547.

WILLIS J. S. (1966) Characteristics of ion transport in kidney cortex of mammalian hibernators. J. gen. Physiol. 49, 1221-1239.

WILLIS J. S. (1967) Cold adaptation of activities of tissues of hibernating mammals. In Mammalian Hibernation (Edited by FISHER K. C. et al.), Vol. III, pp. 356-381. American Elsevier, New York.

Key Word Index-Hibernation; liver; diaphragm; cerebral cortex; RBC; kidney; potassium ions ; hamster; Mesocricetus auratus-hibernation; Citellus tridecemlineatus- hibernation.

tissue k concentration in relation to the role of the kidney in hibernation and the cause of...

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