Nagoya ]. med. Sci. 34: 143-161, 1971

EXPERIMENTAL STUDY ON SODIUM AND POTASSIUM CONCENTRATION GRADIENT AND Na-K DEPENDENT

ADENOSINE TRIPHOSPHATASE ACTIVITY IN THE RABBIT KIDNEY

PART I. ON SODIUM AND POTASSIUM CONCENTRATION GRADIENT IN THE KIDNEY UNDERGOING DIU­RESIS AND ANTIDIURESIS

SHINY A NAKAMURA

Department of Internal Medicine, Nagoya University, Branch Hospital (Director: .Associate Prof. Kaizo Kobayashi)

ABSTRACT

Several drugs were infused intravenously into rabbits to produce diuresis or antidiuresis, and the concentration of sodium and potassium was determined in the cortex, medulla and papilla of the kidney.

The papillary sodium concentration and the cortical potassium concentration were decreased in the furosemide-infused group, while the sodium content was decreased only in the papilla in the ethacrynic acid-infused group. These agents have no effect on the sodium content in the cortex in spite of producing marked diuresis.

The medullary and papillary sodium concentration and the medullary potas­sium concentration were decreased in the 20% mannitol-infused group. In the 50% glucose-infused group, the sodium content was reduced in the cortex, medulla, papilla, cortical mitochondrial fraction and microsomal fraction, whereas the potassium content was depressed in the cortex, medulla and cortical microsomal fractions. Changes in the sodium and potassium concentration were observed over wider range as compared with the furosemide and ethacrynic acid-infused groups. The above change could be due to the difference in the concentration of infusates.

The medullary potassium concentration was raised, but no change was observed in the sodium concentration in the group given low doses of vasopressin (0.1 U/kg ). The infusion of a high dose of vasopressin ( 10 U/kg ) caused an increase in the sodium concentration in the cortex, cortical mitochondrial and microsomal fractions, while even lower dose of vasopressin produced an increase in the medullary potassium. The above r esult would suggest that vasopressin might act on the reabsorption of potassium stronger than that of sodium. Judging from the fact that the r enal blood flow would be decreased by vasopressin, the energy r equired to reabsorb sodium and potassium may be partly supplied from other sources than A TP, as reported in Part II of this study.

* ti{$ -ill. Received for publication

143

144 S. NAKAMURA

INTRODUCTION

Recently the physiology of the kidney has made great progress, as is seen in the proposal of countercurrent multiplier system by Hargitay and Kuhn I).

The mechanism of concentration and dilution of urine by the renal tubules has also been made clear by the method of micropuncture z) and stop flow 3),

as well as clearance study. With the advancement of humoral physiology, renal tubular transport of salt and water has become a subject of interest. It was found that Na-K dependent Adenosine Triphosphatase (Na-K ATPase for short), which Skow•l reported in 1957, participates in the transportation of sodium. Na-K ATPase is widely present everywhere in the biological system such as in the brain, peripheral nerve, red blood cell, liver, and kidney. It is believed that this enzyme is involved in electrolyte transport and in the reabsorption of amino acid by the renal tubules and in the absorption of glucose by the intestine. This paper is undertaken to evaluate the correlation of Na-K ATPase activity and the concentration gradient of sodium and potas­sium in the cortex, medulla and papilla of rabbit kidney during diuresis and antidi uresis.

MATERIALS AND METHODS

Drug administration. Rabbits weighing 2.5 to 3.0 kg were used in this ex­periment. These rabbits had free access to water and were fed corn. All rabbits were deprived of water and food 12 hours before an experiment in which diuresis and antidiuresis were induced. For this purpose, furosemide and ethacrynic acid as saluretic agents, a 5 per cent and a 20 per cent solution of mannitol, and a 5 per cent and a 50 per cent solution of glucose as osmotic diuretic agents were used respectively, and then saluretic diuresis and osmotic diuresis were made. A 5 per cent solution of glucose was infused intravenonsly via the auricular vein into a rabbit at the rate of 1.8 ml per minute, per kg body weight for .30 minutes. Before this injection, furosemide and ethacrynic acid were dissolved in the 5% glucose solution in order to obtain doses of 0.8 mg and 1.2 mg, respectively per minute per kg body weight. A 5 per cent solution of mannitol was adjusted by dissolving a 20 per cent solution of mannitol (Manitou S, Kyorin Pharm. Co., Ltd.) in a 5 per cent solution of glucose. To produce osmotic diuresis, a 5 per cent and a 20 per cent solution of mannitol, a 5 per cent and a 20 per cent solution of glucose were administered intravenously at the rate of 1.8 ml per minutes per kg body weight over 30 minutes.

Urine volume was measured with Nelaton's catheter every 10 minutes and the urine was used to assay the sodium and potassium in it. Antidiuresis was induced by intravenous administration of vasopressin (Pitressin, Park, Davis & Co.) at the rate of 0.1 U or 10 U per kg body weight. Three rabbits

CHANGES OF SODIUM AND POTASSIUM IN THE KIDNEY 145

were used to collect and to measure the urine volume during antidiuresis. They were intramuscularly anesthetized by urethan (1.5/kg) and then the left ureter was exposed by a flank incision, into which was inserted a polyethylene catheter. The urine sample collected for 15 minutes before intravenous admi· nistration of vasopressin served as cont~ol. In consequence, vasopressin was infused and the urine sample was measured twice every 15 minutes. The rabbits which were subjected to the several conditions as mentioned above, were struck on the head and the kidneys were immediately removed.

Preparation of slices and fractions. The kidneys which were immediately re­moved were cut on the Toyo filter paper into slices the breadth of 1 to 1.5 mm, which were classified into slices of cortex, medulla and papilla. These slices were blotted lightly on the filter paper and weighed. The slices weighing 100 to 300 mg were placed into a flask. To obtain both a cortical mitochondrial fraction and a cortical microsomal fraction, the cortex slices were added to ice cold 0.25 mol sucrose with 10 per cent (W /V) of homogenate, and then were homogenized for about twenty seconds, sitting in ice cold water. This homogenate was centrifuged at zoe by the method of Charnock et al.5> and a mitochondrial fraction [10000 x g, for 30 minutes] and a microsomal fraction [35000 x g, for 30 minutes] were obtained. The wet weight of these fractions was 500 to 800 mg. These samples were also put into a flask.

Assay of sodium and potassium contents. The fractions were added 1 ml of concentrated sulfuric acid and heated quietly on the sand. To them were subsequently added concentrated nitrogen acid to made them transparent. The samples were distilled with exactly 10 ml of water, and the contents of sodium and potassium were determined by Coleman's flame photometer. The dry weights of tissues and fractions were examined after drying at 120°C for twelve hours. Concentrations of sodium and potassium in the kidney tissues were expressed in mEq per dry kg weight of tissue. Contents of sodium and potassium in the urine sampling as well as in the kidney tissue were determined by Coleman's flame photometer and expressed in mEq per liter of urine volume.

RESULTS

The urinary volume, excretion of sodium and potassium and Na/ Kin the urine. Thirty minutes after the intravenous infusion of furosemide, the mean urine volume and the mean Na/ K in urine were 27.3 ml and 6.4, respectively. With respect to ethacrynic acid, the mean urine volume and the mean Na/K in the urine were 31.8 ml and 6.3, respectively. With respect to 5% mannitol, the

. mean urine volume and the mean Na/K in the urine were 20.9 ml and 3.9, respectively. With respect to 20% mannitol, the mean urine volume and the mean Na/K in the urine were 40.1 ml and 6.4, respectively. With respect to

146 S. NAKAMURA

(mRl Urine Volume Na/ K in Urine

60 ,. ........-.. Furosemide

' ~-X Ethacrynic acid ' <>--0 5'/oMannitol I

' 50'/oGlucose : - 5'/o Glucose

40 ' .<> e ....... s 20'/oMannitol 8 ' ...

' I c:/

,' !d 20 /Jj 4 . .

;

~' ... e

' ' 0 0

Time 0 10 20 30 (min.) 0 10 20 30

FIG. 1. Changes of urine volume and Na/K in urine by

intravenous infusion of drugs.

TABLE 1. Changes of Urine Volume and Na/K in Urine

Administration of drugs

Urine volume (ml) I Na/K in urine

0-10' I 10-20' I 20-30' Before I 0-10' I 10-20' I 20-30'

Furosemide 10.4 20.9 27.3 0.03 2.3 4.5 6.4

Ethacrynic acid 13.9 22.8 31.8 0.03 2.4 3.9 6.3

5% mannitol 1.4 13.9 20.9 0.05 1.5 2.3 3.9

20% mannitol 11..5 28.3 40.1 0.03 2.2 4.3 6.4

50% glucose 15.5 45.3 56.5 0.03 1.7 5.8 7.7

5% glucose 2.3 6.5 10.7 0.03 0.6 1.1 3.1

50% glucose, the mean urine volume and the mean Na/K in the urine were

56.5 ml and 7.7, respectively. With respect to 5% glucose, the mean urine

volume and the mean Na/K were 10.7 ml and 3.1, respectively (Fig. 1, Table

1). Before injection of vasopressin (10 U/kg) the 15 minutes urine volumes

obtained with a Nelaton's catheter were 0.8 to 2.8 ml (Fig. 2, Table 2). The

mean sodium and potassium contents in these samples were 0.013 mg and 0.932

mg, respectively. Urine volume was decreased markedly thirty minutes after

the injection of vasopressin, and the sodium and potassium excretion was also

decreased in the urine, as illustrated in Fig. 2. To summarize these above data, urine volume and Na/K in urine was

elevated slightly thirty minutes after the injection of a 5 per cent solution of

glucose and a 5 per cent solution of mannitol. It was also apparently enhanced

by furosemide, ethacrynic acid, a 20 per cent solution of mannitol and a 50

CHANGES OF SODIUM AND POTASSIUM IN THE KIDNEY 147

(m/)

3.0

2.0

1.0

Urine Volume

I

I L ___ _ I I

Excretion of Na & Kin Urine

Na (x70-3mg) K (xiO"'mg)

20 20

15

10

5

q ' I

I I I I

h. I I

I

I• I I I I I I

·-A

I I I I I I

I I I I

I I

I I

o Na

D.K

0 \

\

' -.I \\ \ ... \

....... \ .... _ \ \1 . ·. \

t;... -.• 0

15

10

5

1~·.::·.::·:.:·.::-~, o '-------.----'r===; o L---,-::=.==;===L:.J o -15 0 15 30 -15 0 15 30

FIG. 2. Effect of vasopressin (10 unit/kg) on urinary volume and excretion of sodium and potassium in urine.

Vasopressin was infused intravenously in amounts of 10 unit per kg.

TABLE 2. Effect of Vasopressin (10 U/kg) on Urine Volume and Excretion of Na and K in Urine

Case I

1

2

3

I

Time (min.)

-15- 0 0-15

15-30

-15- 0 0-15

15-30

-15- 0 0-15

15-30

I Urine volume I Na excretion (ml) x1o-s mg

' 0.8 870.0 0.2 396.5 0.1 178.0

i 2.8

I

1904.4 I 0.8 796.9 i 0.2 202.4 !

0.8

I 1231.0

0.2 623.8 0.1

I 286.3

I

I

K excretion X lQ-4 mg

6824.0 471.0 372.5

16182.0 611.2 512.0

5952.0 378.0 227:l

per cent solution of glucose. A 50 per cent solution of glucose produced the strongest diuresis among these agents. In contrast, urine volume and urinary excretion of sodium and potassium were lowered distinctly by vasopressin.

The concentration of sodium and potassium of kidney tissue Normal group. The content of sodium and potassium in kidney tissue was

148 S. NAKAMURA

expressed in mEq per kg dry weight of tissue. The sodium and potassium in the cortex was 259.3±23.6 and 290.4±15.2, respectively. The salt content in the medulla was 567.0±36.9 for sodium and 328.2±16.2 for potassium. The papillary sodium and potassium was 851.5 :r: 105.6 and 345.0 ± 103.0, respectively.

The concentration of sodium in the cortical mitochondrial fraction was 92.7 ± 5.3 and that of potassium was 99.5 ± 11.3. The content of sodium and potassium in the cortical microsomal fraction was 123.7 ± 11.4 and 102.4± 14.3, respectively (Fig. 3, Table 3). The sodium concentration was higher in the microsomal fraction than the mitochondrial fraction. (P < 0.05) .

1 2 3 4 5 6 7 8

E Ow m q' k9

200 400 600 800 1000

• • ----+-----. •

Na

K

FIG. 3. Sodium and potassium concentration in nomal rabbit k idney.

TABLE 3. Sodium and Potassium Concentration in Normal Rabbit Kidney

Na (mEq/Dw•kg)

c M p

I 214.5 633.5 732.1 254.6 521.2 764.5 271.6 540.9 820.9 229.2 607.1 969.5 305.5 616.3 951.0 277.7 532.4 655.3 263.5 542.6 999.4 258.1 541.6 919.2

I

Mt j Me I 69.7 146.1 75.0 123.3 83.5 102.5 79.1 124.5 98.9 134.1

131.8 182.0 92.0 120.4

101.4 110.2

K (mEqjDw•kg)

c M p Mt

303.6 315.2 390.5 61.0 270.1 314.6 381.7 86.4 298.8 308.4 301.9 86.5 268.1 325.6 326.5 87.8 278.4 309.8 293.4 101.6 320.3 351.9 319.4 119.5 299.9 353.3 372.0 115.9 284.3 346.4 374.5 107.9

\ Me

i 89.2

I

95.1 77.1

107.8 91.4

126.4 121.3 110.7

-mean I 259.3 I 567.0 1 8::>1.5 I 92.7 1123.7 1290.4 1 328.3 I 345.0 I 99.5 1 102.4 --~'--'±23.6 ± 36.9 ±105.6 ±5.3 ±11.4 ± 15.2 ±16.2 ±103.0 ± 11.3 ±14.3

:Notes; C: cortica l slice, M: medullary slice, P: papillary slice, Mt; <;orti<:l!l :mitocbo:ndria1

fraction, Me: c()rti<:al mi<;rosc,>mal fra<;tio:n,

CHANGES OF SODIUM AND POTASSIUM IN THE KIDNEY 149

Furosemide infused group. Thirty minutes after administration of furosemide,

the sodium and potassium in the cortex was 294.3±34.2 and 230.6 ± 29.7 (which

was lower than normal, P < 0.05), respectively. The salt content in the medulla

was 439.1 ± 150.0 for sodium and 312.5 ± 127.9 for potassium. The papillary

sodium and potassium was 570.7 ± 71.0 (which was decreased than normal, P<

0.05) and 345.8 ± 113.1, respectively. The concentration of sodium in the cortical

mitochondrial fraction was 113.7 ± 31.7 and that of potassium was 108.0 ± 28.9.

The content of sodium and potassium in the cortical microsomal fraction was

126.9± 27.6 and 111.6 ± 9.5, respectively (Fig. 4, Table 4).

1 2 3 4

Cortex I 0 3 0

10 Medulla ll) :::J 1!.1 .... ll)

Papilla

M ito -

::rr chondri a

"' ::!. c;· Mi CT 0 -" some

0 200 400 E Ow

m q 1 kg

600 800

•

•

0 Na

h':?/J K

1000

FIG. 4: Effect of furosemide on sodium and potassium concentration in

rabbit kidney. Furosemide was infused intravenously at the rate of 0.8 mg per kg per

minute.

-

I

TABLE 4. Effect of Furosemide on Sodium and Potassium Concentration

in the Rabbit Kidney

Na (mEq/Dw•kg) K (mEq/Dw·kg)

c I

M I p I Mt I Me c I M I p

I Mt

I I

I

I 313.8 366.7 524.7 102.3 104.0 239.2 290.9 451.8 102.3 264.1 576.2 564.5

I

143.4 123.1 211.8 430.5 305.8 I 121.5 295.0 390.4 631.8 107.3 138.9 252.4 277.8 321.0

I

85.4 303.9 423.2 561.8 101.9 I 141.7 218.8 250.8 304.4 122.8

I

I Me

119.7 105.4 111.1 110.1

1294.3 1439.1 1570.7 1113.7 1126.9 1230.6 1312.5 1345.8 1108.0 1111.6 mean + 34.2 + 150.0 + 71.0 + 31.7 + 27.6 + 29.7 + 127.9 + 113.1 + 28.9 ± 9.5

150 S. NAKAMURA

Ethacrynic acid infused group. Thirty minutes after intravenous administra­tion of ethacrynic acid, the sodium and potassium in the cortex was 271.5 ± 23.0 and 245.6 ± 23.6, respectively. The salt content in the medulla was 371.6 ± 23.6 for sodium and 304.6±30.7 for potassium. The papillary sodium and potassium was 591.3±36.6 (which was lowered than normal, P<0.05) and 387.0±87.1, respectively. The concentration of sodium in the cortical mitochondrial fraction was 98.7 ± 19.0 and that of potassium was 89.5 ± 22.1. The content of sodium and potassium in the microsomal fraction was 137.6±32.7 and 90.4±8.9, respec­tively (Fig. 5, Table 5).

1 2 3 4

I 0 3 0 IJl (j) :::J Ill .... Ill

-n iil A. c;· ::>

Cortex

Medulla

Papilla

M ito -

chondria

Micro -

so me

0 200 400 mEqt~g"

600 800

Na

K

FIG. 5. Effect of ethacrynic acid on sodium and potassium concentration in rabbit kidney. Ethacrynic acid was infused intravenously at the rate of 1.2 mg per kg per minute.

TABLE 5. Effect of Ethacrynic Acid on Sodium and Potassium Concentration in the Rabbit Kidney

Na (mEqJDw•kg)

c M p

276.9 376.3 615.9 289.4 371.7 605.9 261.6 396.0 568.0 258.2 360.4 576.4

Mt

115.8 95.8 94.9 88.0

Me

1 1 1 1

60.2 21.9 49.8 18.5

K (mEqJDw•kg)

c M p Mt

237.0 328.1 401.7 71.7 238.4 288.2 456.5 92.6 267.4 312.7 359.4 105.4 239.4 289.3 330.5 88.3

Me

70.4 88.2

113.8 89.3

mean 1271.5 1376.1 1591.3 198.7 [137.6 1245.6 1304.6 1387.0 189.5 19(!:"4-±23.0 I ±23.6 ±36.6 +19.0 I +32.7 +23.6 +30.7 +87.1, +22.1 +8.9

CHANGES OF SODIUM AND POTASSIUM IN THE KIDNEY 151

50 3b glucose infused group. Thirty minutes after intravenous injection of a 50 per cent solution of glucose, the sodium and potassium in the cortex was 227.7 :±:37.0 and 235.8±11.8, respectively. The salt content in the medulla was 369.4±37.4 for sodium and 295.2:±:37.3 for potassium. The papillary sodium and potassium was 499.1±286.3 and 392.4±60.0, respectively. The concentration of sodium in the cortical mitochondrial fraction was 81.4±17.9 and that of potassium was 88.0±11.2. The content of sodium and potassium in the micro­somal fraction was 95.1 ± 6.0 and 82.0 ::t: 1 1.7, respectively (Fig. 6, Table 6). The sodium was depressed in all parts and potassium was decreased in the cortex,

1 2 3 4 5

E Ow m q 1 l<.g

0 200 400 600 800

Cortex :c 0 3 0

Medulla lO .., :::J Ill -11>

Papilla

M ito -

"T1 chondri a

;;; !l 0 Micro -:::0

D Na

some UJ!Yi/{j K

FIG. 6. Effect of 503(; glucose on sodium and potassium concentration in rabbit kidney.

503(; glucose was infused intravenously at the rate of 0.7 ml per kg per minute.

TABLE 6. Effect of 503(; Glucose on Sodium and Potassium Concentration in the Rabbit Kidney

Na (mEqJDw·kg) K (mEqjDw•kg)

I c I M I p

I Mt I Me c I M I p

I Mt

268.2 375.2 575.5 71.3 98.3 240.5

I

282.2 423.2 76.1 232.1 325.5 524.0 83.8 98.1 224.7 252.3 376.6 85.7 207.7 386.3 471.5 69.8 86.8 230.4 304.2 449.4 85.6 188.8 355.7 425.4 76.8 97.2 232.6

I 304.0 320.4 92.6

241.7 404.1 510.6 105.2 I

95.2 249.2 33.!.2 394.3 I 100.2

'

I Me

74.6 91.4 74.4 93.2 76.6

1227.7 1369.4 1499.1 I 81.4 I 95.1 1235.8 1295.2 1392.4 188.0 182.0 mean +37.0 +37.4 +286.31 +17.9 +6.0 +11.8 +37.3 +60.0 ±11.2 ±11.7

152 S. NAKAMURA

medulla and cortical microsomal fraction than normal (P<0.05).

20% mannitol infused group. Thirty minutes after intravenous infusion of a 20 per cent solution of mannitol, the sodium and potassium in the cortex was 277.3±31.7 and 255.0±33.4, respectively. The salt content in the medulla was 390.7±99.9 for sodium and 282.5±17.2 for potassium. These were decreased than normal (P<0.05). The papillary sodium and potassium was 511.9±104.8 (which was depressed than normal, P<0.05) and 345.6±35.7, respectively. The concentration of sodium in the cortical mitochondrial fraction was 93.6 ± 18.3 and that of potassium was 93.4 ± 11.9. The content of sodium and potassium

1 2 3 4

mEq I ~9' 0 200 400 600 800

Cortex I 0 3 0

1.0 Medulla fl) :J e. fl)

Papilla

M ito -

chondr ;a ::jl n .. g a ;;; 0

X

::J Micro - D Na some li::C::F;~fH K

FIG. 7. Effect of 20% mannitol on sodium and potassium concentra­tion in rabbit kidney.

20% mannitol was infused intravenously at the rate of 0.7 ml per kg per minute.

TABLE 7. Effect of 20% Mannitol on Sodium and Potassium Concentration in the Rabbit Kidney

Na (mEq/Dw·kg) K (mEq/Dw•kg)

c M p Mt Me C I M p Mt Me ~~-----,~-·--·--------------··-- ~~~'--~--'

278.9 447.9 553.1 94.2 98.3 270.31 290.4 338.7 102.8 84.3 269.6 322.6 449.0 77.6 127.8 274.1 291.9 362.6 94.3 112.0 258.5 440.2 582.3 98.1 115.4 245.6 278.9 306.4 91.5 119.9 3o2.1 352.2 463.o 104.5 98.8 229.8 1 268.9 374.5 84.8 94.6

_m_e_a_n-----c-1 =27=7.3--~390. 7 1511.9 193.6 1110.1 1255.0 1282.5 '1345.6 1 93.4 1· fo2:7--+31.7 +99.9 +104.8 +18.3 +22.7 +33.4 +17.2 +35.7] ±11.9 ±25.6

CHANGES OF SODIUM AND POTASSIUM IN THE KIDNEY 153

in the microsomal fraction was 110.1 ±22.7 and 102.7 ±25.6, respectively (Fig. 7, Table 7).

The low dose of vasopressin infused group. Thirty minutes after intravenous administration of vasopressin (0.1 Ujkg), the sodium and potassium in the cortex was 235.0 ± 42.6 and 276.0 ± 14.8, respectively. The salt content in the medulla was 514.2±63.8 for sodium and 390.0±53.8 for potassium (which was enhanced than normal, P<0.05). The papillary sodium and potassium was 726.4 ± 227.7 and 408.0 ± 89.5, respectively. The concentration of sodium in the cortical mitochondrial fraction was 76.3 ± 10.8 and that of potassium was 98.6 ±

1 2 3 4

:::c 0 3 0

tO tD :::J et. tD

"T1 iil ~ c;·

"

0

Cortex

Medulla

Papilla

Mito -

chondria

Micro-

some

200 400

Dw mEq I kg

600 800

• •

D Na

t,::;;;cl K

1000

FIG. 8. Effect of vasopressin (0.1 unit/kg) on sodium and potassium con· centration in rabbit kidney.

Vasopressin ( 0.1 unit/kg) was infused intravenously.

TABLE 8. Effect of Vasopressin (0.1 unit/kg) on Sodium and Potassium Concentration in the Rabbit Kidney

Na (mEqjDw•kg) K (mEq/Dw·kg) --.----

c M P Mt c M p I Mt 1 Me -·

I i I 241.4 550.0 657.2 84.5 114.4 272.6 393.2 400.6 1044 103.4

I 267.9 555.6 928.9 69.2 109.4

I

270.4 387.8 440.4 82.2 \ 91.8

I 226.8 470.9 601.5 78.9 91.9 289.8 437.6 459.6 102.4 87.0 203.9 480.3 717.9 72.6 99.6 271.0 341.0 332.0 105.2 97.8

I ··--~----- ~-···-·~---

1235.0 1514.2 1726.4 176.3 1103.8 1276.0 1390.0 1408.0 198.6 193.8 me~±42.6. :±:Ji~:8 ±227.7 ±10.8 ±16.0 ±14.8 ±53.8 ±89.5 ±17.4 ±11.0

-·

154 S. NAKAMURA

17.4. The content of sodium and potassium in the microsomal fraction was 103.8± 16.0 and 93.8± 11.0, respectively (Fig. 8, Table 8) .

The high dose of vasopressin infused group. Thirty minutes after intravenous infusion of vasopressin (10 Uj kg), the sodium and potassium in the cortex was 307.0 ± 13.2 (which was elevated than normal, P < 0.05) and 293.1 ±27.1, respectively. The salt content in the medulla was 601.9 ± 74.7 for sodium and 361.0±18.3 for potassium (which was higher than normal, P < 0.05). The papillary sodium and potassium was 936.8 ± 36.7 and 414.6 ± 31.5, respectively. The concentration of sodium in the cortical mitochondrial fraction was 114.7 ±

Cortex :I: 0 3 0

10 Medulla I)) ::J Ill -I))

Papilla

Mito _

~ chondri a.

ll c;· Micro-" some

u 200 400

Ow mE9 I kg

600 800

Na

K

1000

• •

FIG. 9. Effect of vasopressin ( 10 unit/ kg) on sodium and potassium content in rabbit kidney.

Vasopressin ( 10 unit/kg) was infused intravenously.

TABLE 9. Effect of Vasopressin ( 10 unit/ kg) on Sodium and Potassium Concentration in the Rabbit Kidney

Na (mEq/Dw•kg) I K (mEq/ Dw·kg) - --,--- - -,---,----- -,--··-- -i-~-·--- - ----c---c-- -

M P Mt ] Me j C \ M P Mt \ Me c

I

I

I I I 1 320.9 586.1 897.1

I

105.9 161.7 290.2 386.3 438.3 128.1 117.5 2 284.8 568.3 919.2 108.3 148.0 278.3 344.0 361.1 125.0 101.6 3 302.2 642.7 936.4 110.1 167.0 317.1

I

347.0 419.4 113.7 129.7 4 314.6 646.9 956.4 118.9

I

154.6 323.7 363.9 401.3 I

112.0 114.1 5

I 307.8 596.7 997.3

I 115.9 150.3 253.2 376.1 442.1 105.1 115.1

6 311.8 570.8 914.4 129.0 144.7 296.0 348.7 425.4 I 90.5 I 110.0 I -

mea. n 1307.0 1601.9 I 936.8 11l4.7 1154.5 1293.1 1361.0 1414.6 ·!112.5 1114.7 + 13.2 . + 74.7 1 + 36.71 + 8.9 +9.1 + 27.1 + 18.3 + 31.5 + 14.4 + 9.7

CHANGES OF SODIUM AND POTASSIUM IN THE KIDNEY 155

8.9 (which was enhanced than normal, P < 0.05) and that of potassium was 112.4 ± 14.7. The content of sodium and potassium in the microsomal fraction was 154.5±9.1 (which was increased than normal, P < 0.05) and 114.7 ±9.7, respectively (Fig. 9, Table 9).

DISCUSSION

The sodium and potassium concentration gradient increases gradually from the cortex and the medulla toward the papilla in the normal rabbit kidney. This phenomenon is supported by the countercurrent theory and has also been reported by Nishida 6> and Nocenti et af.?>. In this experiment, in comparing

the mitochondrial fraction with the microsomal fraction, the sodium concentra­tion was higher in the latter than in the former, but there was no difference

in the potassium concentration between them (?<0.05). Today it is believed that the microsomal fraction arises from the endoplasmic reticulm, which is connected to the cell membrane taking part in the transport of sodium and potassium by the renal tubules. In order to obtain the preparations of the mitochondrial and microsomal fractions, we have to cut tissues into small pieces on ice and to prepare a homogenate by centrifugation. Accordingly, it is to be expected that degeneration and destruction of the cell membrane would break out, and as a result, the alternation of electrolyte and enzymatic

activity of the cell membrane would issue. Therefore it might be difficult to say that these preparations obtained by the method cited above retained the net sodium and potassium content and enzymatic activity in them. The fact that the sodium concentration was higher in the cortical microsomal fraction than the mitochondrial fraction would suggest that microsome plays a more important role in sodium transport than mitochondria.

In a 5 per cent glucose solution injected group, which served as control, the mean urine volume and the mean NajK in the urine were 10.7 ml and 3.1, respectively. In this case the diuresis and NajK in the urine were raised slightly. In the present experiment, furosemide, ethacrynic acid and mannitol

were infused and they increased the diuresis and Na/K in the urine, when they were dissolved in a 5 per cent solution of glucose. Judging from the

result, there was no influence by the 5 per cent solution of glucose.

According to the stop-flow theory by Suzuki et al.8>, furosemide inhibits the reabsorption of sodium in the proximal and distal tubules, and facilitates the excretion of potassium in the proximal tubules. Therefore, the finding that the sodium in the papilla and the potassium in the cortex were decreased

by furosemide would partly support the report of Suzuki et al ..

Thirty minutes after the infusion of ethacrynic acid, the sodium concentra­tion was decreased in the papilla, but no decrease of the potassium was observed (P<0.05). At this time the urine volume and NajK in the urine

156 S. NAKAMURA

were raised to 31.8 ml and 6.3, respectively, when a marked diuresis was observed as with furosemide. Ethacrynic acid inhibits the reabsorption of sodium in the proximal and distal renal tubules according to Cannon et al. 9l. No depression of the potassium content would show that it has little action on the potassium excretion which is different from furosemide.

It would be expected that the sodium content decreases not only in the papilla but also in the cortex and medulla by the infusion of furosemide and ethacrynic acid in consideration of the mechanism confirmed by the stop-flow theory. Using the micropuncture technique, that is, inserting several times a micropipette into the same tubule segments of the dog kidney, Dirks et al.10l

collected samplings of the tubule fluid and examined their inulin concentration. And it was found that the ratio of the tubular fluid to plasma inulin did not fluctuate. Thereafter, experiments were successively carried out involving administration of hydropenia, loaded with the isotonic or hypertonic saline, vasopressin and desoxycorticosterone, and the inulin concentration was also assayed in each obtained sampling. The result showed that the ratio of the tubular fluid to plasma inulin still did not fluctuate. It is considered HJ that this finding shows that diuretics act stronger on the distal tubule or the loop of Henle than on the proximal tubule, although it has been conventionally believed that diuretics strongly affects the proximal tubule where the transport of water is most prominent. In the present experiment in which furosemide and ethacrynic acid were intravenously administered, the sodium content was not decreased in the cortex and medulla. These findings partly support the data that the ratio of the tubule fluid to plasma inulin did not change by micropuncture. It is, therefore, unreasonable to compare the result by micro­puncture with that by free hand cutting, which we adopted to prepare renal slices, namely, of the cortex, medulla and papilla. Both the above data and the present results imply that diuretics such as furosemide and ethacrynic acid have little effect on the proximal tubule.

Mannitol is considered to have a mode of action of osmotic diuresis 12l as well as urea and glucose. There was a slight increase in the urine volume and NajK in the urine when a 5 per cent solution of mannitol was intravenously infused. Moreover, the urine volume and Na/K in the urine reached 40.1 ml and 6.4, respectively, with a marked increase thirty minutes after the injection of a 20 per cent solution of mannitol. At this time it was found that mannitol abolished the medullary and papillary sodium, and the medullary potassium concentration gradients (P<0.05).

Next, with respect to the 50% glucose infusion, the urine volume and Na/K in the urine rose to 56.5 ml and 7.7, respectively. The sodium content was decreased in the cortex, medulla, papilla, cortical mitochondrial and microsomal fractions, and the potassium content was also depressed in the cortex, medulla and cortical microsome (P<0.05). This is believed to indicate that both sodium

CHANGES OF SODIUM AND POTASSIUM IN THE KIDNEY 157

and potassium are rapidly washed out by osmotic diuresis as with mannitol. It would be induced by a stronger osmotic diuresis than with mannitol that the decrease of sodium and potassium in the kidney tissue was found over wider range by infusion of 50% glucose.

There are several theories on the mode of action of ADH: i) ADH causes an apocrine secretion of hyaluronidase by the renal tubule which makes the structure separating the tubule lumen from the interstitial tissue permeable to water (Ginetinsky 13>) . ii) Linkage involving the disulfide (S-S) bridge of vasopressin and the free sulfhydryl (SH) groups on the membrane makes aqueous channels or pores in the luminal membrane enlarged to increase permeability to water (Rassmussen et a[.14>) . iii) Vasopressin induces its effect on permeability by stimulating the production and accumulation of cyclic AMP in the receptor tissue. This cyclic AMP makes the membrane structure · permit the accelerated flow of water along an osmotic gradient (Orloff et al.15>). Besides these actions, Gottschalk 16> assumed (i) increased transport of sodium by the thin, ascending limb (2) increased permeability to water of the thin, ascending limb or (3) decreased medullary blood flow. Tomomatsu et al.17>

found that the administration of pitressin lowered the urine volume, and free water clearance in humans as well as in dogs, but on the contrary the total amount of sodium excretion was increased in four hours.

In the present paper, as illustrated in Fig. 2, immediately after the large administration of vasopressin (10 Ujkg) the excretion of sodium and potassium were decreased, accompanied by a decrease in urine volume. This phenomenon would involve the decrease of GER by the contraction of vessels and the increase of permeability to water by the distal tubule and collecting duct. Moreover, examining the sodium and potassium in the tissues, besides measure­ment of the urine volume, the sodium concentration was observed to increase apparently in the cortex, cortical mitochondrial fraction and microsomal fraction (P< 0.05). This shows that the decrease of sodium is involved in reabsorption of water and sodium by the cortex.

Darmedy et al.18> reported that radioautograms of the isolated nephron from rats injected with P 31 pitressin show radioactivity to be located over the distal convoluted tubule and the upper two thirds of the collecting tubule. The reports of Darmedy and Gottschalk, and the present data led the author to the conclusion that ADH acts in the reabsorbtion of sodium in the ascending limb and distal tubule. It is supposed that the sodium increment occurs in the parenchymatous cell, juding from the mitochondrial and microsomal increments. This r esult suggest that the reason why high Na in the subcellular fractions occurs may be the comparative increase of passive permeability to sodium ion in the luminal membrane of tubules with or without the acceleration of active Na pump located in the peritubular membrane. But it is doubtful whether only permeability of the luminal wall to sodium ion would be accelerated ir-

158 S. NAKAMURA

respective with sodium pump. Solomon 19> has reported that the tubular fluid

is free from sodium by the replacement of sodium chloride in the tubular

fluid with choline chloride, by the micropuncture oil method. Under this

condition, sodium flows from the cell back into the tubular lumen. As the

sodium concentration of cells decreases, the sodium pump slowes down. In

consequence, the potassium concentration gradually decreases because of not

being exchanged with sodium ion. In this experiment, the sodium and potas­

sium concentration of the sliced tissue and fractions were determined, but no

distinctive decrease of potassium ion was observed. Therefore, it would not

be necessary to take into account that the permeability of the tubular wall to

sodium ion increases, which the sodium pump is depressed.

With respect to the potassium pump, Fujimoto 20l has reported that it is

not clear whether the potassium pump (H+ secrete) is present in the tubular

wall or not, and the secretion of hydrogen ion coupled with the intake of potassium ion. Solomon 19> implied with respect to the potassium pump: that

besides the sodium pump, another small pump which lies in the tubular wall,

transports the potassium ion into the cell. In the future, it is necessary to

evaluate whether this pump transports the potassium ion actively or passively.

With respect to the potassium content after the infusion of vasopressin

(0.1 U/kg), it was found that the potassium concentration was elevated in the

medulla. This increase is considered to have issued in the medullary mito­

chondrial fraction and the microsomal fraction as well as in the cortex with

the sodium concentration. According to Berliner 19>, potassium is filtered through

the glomerulus and is reabsorbed completely by the proximal tubule. Solomon19l

reported that it is doubtful whether potassium is completely reabsorbed by

the proximal tubule. In the present experiment the elevation of the potassium

content in the medulla implies that potassium is also reabsorbed in the loop

of Henle, as Solomon reported. The fact that the increase of the potassium

content is observed after the administration of vasopressin with small doses

or large doses indicates that vasopressin or its mediator would promote the

potassium pump in the medulla to reabsorb potassium. Vasopressin seems to

have a stronger effect on potassium reabsorption than on sodium reabsorption,

since potassium increased in the medulla without the increase of sodium in

the cortex after the intravenous infusion of small doses of vasopressin.

The action of ADH on vessel contracture has been reported by Oliver et

a/. 21) and by Thurau et a/. 22> It is believed that the blood flow in the kidney

probably diminished in this experiment after the administration of vasopressin.

The finding that sodium and potassium are accumulated in the kidney tissue,

although the ischemic condition is thought to be present, is consistent with

the concept that the energy essential to the reabsorption of sodium and potas­

sium would be supplied at least in part by a pathway other than of A TP.

CHANGES OF SODIUM AND POTASSIUM IN THE KIDNEY 159

CONCLUSION

By using several agents diuresis and antidiuresis were induced in order to evaluate the correlation of sodium and potassium transport and Na-K ATPase activity by the renal tubules, and to evaluate how these diuretic and antidiuretic agents effect the sodium and potassium content in the kidney tissue and urinary excretion of sodium and potassium. In Part I, the results obtained were as follows:

1) After the administration of furosemide, the sodium concentration in the papilla and the potassium content in the cortex were decreased. This finding partly supports the theory that furosemide inhibits reabsorption of sodium by the proximal and distal tubules and excretes potassium by the distal tubules.

2) After the administration of ethacrynic acid, the sodium concentration was decreased in the papilla but the potassium content did not change in contrast to the control. The result that the potassium concentration did not change indicates that the action of ethacrynic acid on the excretion of potas­sium is not stronger than that of furosemide.

3) After the administration of 20% mannitol, the sodium concentration was decreased in the medulla and papilla, and the potassium concentration was reduced in the medulla only.

4) After the administration of 50% glucose, the sodium concentration was depressed in the cortex, medulla, papilla, cortical mitochondrial fraction and microsomal fractions. On the other hand, the potassium content was decreased in the cortex, medulla and cortical microsomal fraction.

5) After the administration of a large dose of vasopressin, urine volume and urinary excretion of sodium and potassium was decreased. Nevertheless, it was observed that the sodium concentration was raised in the cortex, cortical mitochondrial fraction and microsomal fraction. It was also observed that the potassium content was increased in the medulla after intravenous administra­tion of vasopressin in small doses or large doses. (Table 10)

TABLE 10. Changes of Na and K Concentration in the Rabbit Kidney

Administration of drugs Me

Furosemide -> -> t -> -> t -> -> -> ->

Ethacrynic acid -> -> t -> -> -> -> -> -> ->

50% glucose t t t t t ,j, t -> -> ,j,

20% mannitol --> t t -> -> -> t -> -> ->

Vasopressin (0.1 U/kg) -> -> -> __,. -> -> t ->

Vasopressin ( 10 U/kg) t -> -> t t -> t ->

ln c:;ontr(lst to normal, t inc:;rease, -> no <;hange, t dec:;rea!)e ( P<0.05 ).

160 S. NAKAMURA

SUMMARY

The results mentioned above indicate that the effect of a saluretic agent on tissue electrolyte is observed to occur on the papillary electrolyte mostly. On the other hand action of osmotic diuresis is stronger than that of saluretic agents. The effect of osmotic diuresis acts not only on the papillary electro­lyte but also on the medullary and cortical electrolyte. Judging from the result that cortical sodium and medullary potassium were increased in the antidiuretic condition by vasopressin, the both sodium pump and potassium pump participate in reabsorption of these electrolytes in any way and vasopressin would facilitate the potassium pump more sensitively than the sodium pump.

(The outline of this paper was presented at the 69th Proceedings of the Tokai and Hokuriku Regional Meeting of the Japanese Society of Internal Medicine.)

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CHANGES OF SODIUM AND POTASSIUM IN THE KIDNEY 161

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