Isolation of viable rat kidney tubules

Pergamon Press

Life Sciences Vol . 10, Part II, pp. 223-232, 1971 . Printed in Great Britain

ISOLATION OF VIABLE RAT RIDNEY TUBULES Ho Sam Ahn and Helen R . Strausser Department of Zoology and Physiology, Rutgera University, Newark, N . J .

(Received 28 November 1970; in final form 31 December 1970) In addition to its extensively studied role in the maintenance of fluid and electrolyte homeostasis, the kidney has long been recognized as a locus of many important metabolic processes . Rat kidney cortex slices have been extensively used for in vitro studies of kidney intermediary metabolism, not only because of the high rate of aerobic metabolism (1), but also because the cortex is an active site of glucose formation (2), deamination (3) and fatty acid utilization (4) . Except for some studies on the electrolyte transport of isolated rabbit and flounder renal tubules (5,6,7,8), there are no reports of studies which utilize isolated rat kidney tubules for the metabolic and functional studies of the kidney . advantages of the use of these tubules are :

The

1) there is no

cellular barrier between the bathing medium and the inner cells, such as would be present in rat renal slices ; and 2) parts of the nephron with particular function can be isolated . The purpose of this paper is to report a successful isolation technique of renal tubules from rat kidney cortex and to examine its viability by studying the effects of substrates, cofactors and insulin on the respiration of the isolated tubule . Procedure Sprague-Dawley or Charles River adult male rats, weighing between 400 and 500 g, were used .

223

The rats were killed by

Isolation a~f Kidney Tubules

224

carotid exsanguination .

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Four ml of enzyme solution (consisting

of 0 .058 collagenase, Type I, Sigma Co ., and 0 .18 hyaluronidase, Type I, Sigma Co ., dissolved in calcium- and glucose-free Hanks solution at pH 7 .4 (9) with 0 .1 q/R magnesium chloride added to the original formula) was injected into each kidney through the renal artery for approximately 2 min while the renal vein was clamped.

In experiments involving the use of alloxan and insu-

lin, Robinson's medium (10) was used in place of the Hanks . Robinson's medium appeared to have a better buffering capacity in these experiments and prevented a rise in pH with time .

A gas

mixture of 58 carbon dioxide and 958 oxygen was bubbled through the medium for 5 min prior to incubation . The renal cortex was then dissected out and sliced into pieces about 1 mm 3 in size .

These pieces were then incubated at

37°C for 65 min in the same enzyme solution as used for perfusion in a Dubnoff Metabolic Shaking Incubator .

The digested tissue

was then filtered through two layers of nylon stocking .

The

residue was discarded and the filtrate centrifuged for 1 min at The supernatant solution was discarded and the pellet

50 x g .

washed three times with the Hanks or Robinaon solution .

The

final pellet was diluted to 58 by packed volume in the same Approximately 100 mg dry wt of tubular segments and

buffer .

glomeruli were obtained from two kidneys .

Kidneys from three

rats were used for each experiment . Oxygen consumption was measured with a Warburq apparatus at 30 °C .

The oxygen consumed was measured at 10-min intervals

for periods up to 120 min .

Protein determfnationa were performed

by the biuret procedure (11) .

Results are expressed as uß of

oxygen consumed per mg of dry wt per min .

The following sub-

stances were used in one or more of the respiration experiments :

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Isolation ad Kidney Zwbules

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5 .56 mM dextrose, reagent grade (Schering Research Division) ; 20 mM succinate, reagent grade (Nutritional Biochemical Co .) ; 0 .05 uq/ml glucaqon-free insulin obtained through the courtesy of Dr . W . R . Kirtley of the Lilly Laboratories, Indianapolis, Indiana ; 10 mg alloxan, reagent grade (Nutritional Biochemical Co .) ; calcium chloride, reagent grade, to a final concentration of 16 .8 mM (Fischer) .

In experiments in which alloxan was used,

the suspension was incubated for 10 min at 37°C in the presence of alloxan .

The tubules were then washed three times before use

in the Warburg .

In some experiments the tubules were disrupted

by vibrating the suspension on a Vortex mixer at full speed . Results The suspension was found by microscopic examination to contain more than 908 tubules which had a smooth tubular boundary and less than 108 glomeruli .

Based on the length and width of

the tubules in the final preparation (proximal tubules are about three times longer and much wider than other tubular parts of the nephron (12)), approximately 708 were proximal .

In the rabbit,

proximal tubules comprise about 808 of the mass of the kidney cortex (7) .

Phenol red test for viability showed that almost all

the tubules were viable and took up the stain . The rate of oxygen uptake is considerably stimulated with the addition of 5 .6 mM glucose (Fig . 1) .

This enhanced respira-

tory activity was maintained in a linear pattern throughout the incubation period, but there was also significant endogenous respiration in the absence of added substrate .

An average

Q02 (uR 02 consumed per mg dry wt of tissue per hr at 30°C in the

presence of glucose) of 10 .6 was calculated .

In experiments in

which the tissue concentration was varied between 1 .7 to 4 .2 mg dry wt, in the presence of glucose, oxygen uptake increased

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Isolation auf Kidney 1~bules

with increasing amounts of tissue added .

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The oxygen uptake of

disrupted tubules was lower than that of intact tubules and plateaued in 30 min (Table I) .

The addition of glucose increased

the oxygen uptake of both intact and disrupted tubules .

Fig . 1 .

Effect of glucose on the respiratory rate of isolated renal tubules . TABLE I

Effect of tubular disruption on respiration of isolated renal tubules Tubular condition and medium*

Tbtai 02 consumption in yß/mg dry wt 30 min 60 min 90 min

Undisrupted tubules with 4 .2 mM glucose

5 .7 t 0 .1

10 .7 t 0 .1

Disrupted tubules with 4 .2 mM glucose

4 .7 t 1 .8

14 .8 t 0 .1

7 .7 t 0 .1

10 .1 t 0 .7

Undisrupted tubules without glucose

4 .6 t 0 .1

7 .3 t 0 .5

8 .9 ± 1 .1

4 .9 t 0 .4

6 .3 t 0 .3

6 .8 t 0 .3

Disrupted tubules without glucose

*Basic medium was calcium- and glucose-free Banks solution .

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227

The addition of calcium to the medium increased the rate of respiration of intact tubules over and above that of glucose alone (Fig . 2) .

The addition of 48 bovine albumin also stimu

lated tubular respiration in calcium-free Hanks solution .

i

i

i i

ii ii

i

ii

!~

Ca t+ -free Hanks

J;

Fig . 2 .

i:

Complete Hanks solution

,.

Disrupted tubules ~"~' in Cat+-free Hanks Cat+- and glucosefree Hanks

Effect of calcium on tubular respiration .

The use of succinate as a substrate in the glucose- and calcium-free Hanks solution markedly enhanced the oxygen uptake of the tubular suspension .

At 90 min the total oxygen uptake

in the presence of succinate was 83 u~R/mq dry wt, whereas without the substrate the uptake was only 7 uR .

Larger amounts

of succinate, e .g ., 26 .7 mM and 53 .2 mM, were inhibitory (Table II) .

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TABLE II Effect of substrates on respiration of isolated renal tubules Substrate added*

~

None 5 .6 mM glucose

Total 02 consumption in uR/mg dzy wt 30 min 60 min 90 min 3 .5 t p,7

5 .1 t 1 .9

5 .9 t 1 .4

5 .4 t 0 .3

10 .6 t 0 .2

15 .6 t 0 .6

57 .3 ± 0 .2

87 .6 ± 0 .3

13 .3 mM succinate

30 .2 t 0 .4

26 .7 mM succinate

24 .5 t 0 .3

53 .0 t 0 .2

82 .1 t 0 .2

53 .2 mM succinate

20 .6 t 0 .1

40 .5 t 0 .1

64 .4 t 0 .2

*Basic medium was calcium- and glucose-free Hanks solution . When insulin was added to the medium containing glucose, the respiratory activity of the isolated tubules was greatly enhanced (Fig . 3) .

Respiration was depressed in tubules that

3

Fig . 3 .

The effect of alloxan and/or insulin on tubular respiration .

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Isolation of Kidney fibules

were pretreated with alloxan, but the addition of insulin to the alloxan-treated tubules approximately doubled the oxygen uptake . Discussion The technique used in this study is a modification of the method used to isolate intact rat liver cells (13) and that used to study fragments of rabbit nephrons

(5) .

Hyaluronidase was

used in conjunction with collagenase for separating the kidney tubules on the basis that hyaluronic acid was found to be part of the intercellular matrix of kidney tubules (14) .

Also, since

kidney contains more mucopolysaccharide per gram of tissue than most other organs of the body (15), the use of hyaluronidase in addition to collagenase for the separation of tubules appeared to be desirable . The effect of calcium in stimulating respiration of the tubular fragments was similar to that previously demonstrated (10) in which it was found that both calcium and glucose were necessary for steady endogenous respiration of adult rat kidney slices .

If either one of these was not present in the suspension

medium, the respiratory rate fell to about three quarters of the initial rate in 4 hr and, if both were absent, the rate fell to half its initial value . value obtained is comparable to the of 11 .6 Q02 Q02 for rat kidney cortical slices using Rrebs-Ringer phosphate bufThe

fer (16) and 22 .2 for slices in Ringer phosphate buffer (17) . This suggests that relatively little damage was done to the tubules during the isolation procedures .

Also, the fact that

disrupted tubules did not respire to the same degree as intact tubules indicates that the tubular preparation retained good physiologic characteristics . The experiments with kidney tubules and those with

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230

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kidney slices (10) indicate that glucose stimulates the respiration of tubules .

This would suggest that kidney tubules retain

the necessary enzymes and energy requirements in an intact intracellular milieu to undergo glycolysis .

In addition, succinate

markedly improved the oxygen uptake of the isolated kidney tubules .

While the main energy source for the kidney cortex is

lipid, the kidney cortex can also oxidize the intermediates of the tricarboxylic acid cycle and glycolysis

(18,19,20) .

With

kidney cortex slices from the well-fed rat, 5 mNl glucose has been shown to supply 25-308 of the respiratory fuel, but the corresponding value of slices of the starved rat was only 108 (4) . In our experiments with tubules obtained from starved rats, glucose did not stimulate respirationf this is probably due to In kidney cortex slices from starved

gluconeogenetic activity .

rats, there is increased glucose production by increased activation of major gluconeogenetic pathway enzymes, such as phosphoenolpyruvate carboxykinase and pyruvatecarboxylase (21) . Alloxan treatment significantly reduced the respiratory In

activity of the tubules, even in the presence of glucose .

similar experiments with kidney slices from alloxan-diabetic rats, the metabolism of glucose was markedly reduced (22) .

It

has been theorized that alloxan lowers the concentration of glutathione in kidney and liver, and that this reduction inhibited the activity of an insulin-degradation system (23) .

This

allowed for a parallel enhancement of insulin action on the uptake of glucose (24) .

Slices from non-alloxanized animals did

not demonstrate this effect unless the kidney slices were incubated at lower temperatures

(e .g ., 20°C) .

At this temperature

the activity of the insulin-degradation system was slowed . The results presented here indicate that the respiration

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Isolation ad Kidney Tubules

291

of the tubules in the presence of glucose was enhanced by the addition of insulin to the medium .

The stimulatory effect of

insulin on tubular respiration, without alloxan treatment, may be due to the fact that collagenase treatment facilitates the tubular response to insulin by altering the membrane surface, or that collagenase affects the insulin-degradation system .

It should be

noted also that our experiments with kidney tubules were performed at 30°C . Summary A method has been devised for the isolation of viable tubules from the rat renal cortex .

More than 70B of the prepara-

tion consisted of fragments of proximal tubules which respired in calcium- and glucose-free buffer solution .

The addition of glu-

cose or succinate to the medium enhanced the oxygen uptake and allowed for a linear respiration for periods up to 120 min .

The

addition of calcium ion in the presence of substrate significantly enhanced the respiration .

Disruption of the tubules with

consequent fragmentation of the kidney cells markedly decreased the oxygen uptake .

In the presence of glucose the respiration of

the tubules was enhanced by the addition of insulin to the medium : This increase was approximately the same for alloxan-treated as for control preparations . References 1.

0 . Warburg, Biochem. Z . lf~, 484 (1927) .

2.

H. A. Krebs, D . A. H . Bennett, P . deGasquet, T . Gascoyne and T . Yoshida, Bîochem. J . ~6, 22 (1963) .

3.

H. A. Krebs, Biochem . J . 2~, 1620 . (1935) .

4.

M . J . Weidemann and H . A . Krebs, Biochem . J . 112, 149 (1969) .

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M. B . Burg and J . Orloff, Am . J . Physiol . 2Q3, 327 (1962) .

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M. B . Burg, J . Grantham, M. Abramow and J . Orloff, Am . J . Physiol . 210, 1293 (1966) .

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Isolation of Kidney ~trules

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7.

M. B . Burg and J . Orloff, Am . J . Physio l . 211, 1005

8.

R. P . Fors ter and J . V . Taqgart, J . Cell . and Comp . Physiol . 36, 251 (1950) .

9.

J . H . Hanks and R. E . Wallace, Pro . Soc . Exp . Biol . Med . 19 6 (1949) .

(1966) .

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J . R. Robinson, Biochem. J . 4~5 , 68 (1949) .

11 .

A . G. Gornall, C . J . Bardawill and M . M . David, J . Biol . Chem . ~, 751 (1949) .

12 .

H . W . Smith, in The Kidney . Press (1935) .

13 .

R. B . Howard and L . A . Peach, J . Biol . Chem . 243, 3105 (1968) .

14 .

J . C . Morard, Compt . Rend . Acad . Sci . D2 4, 2166

15 .

W . Bartley, L . M. Hint and P . Banks, in The Biochemistry of the Tissues . P . 24 . New York, John Wiley & Sons, Ltd . (1968) .

16 .

J . H . Quastel and 2 . J. Bickis, Nature ~, 281 (1959) .

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R. L . Russell and B . A. Westfall, Am . J . Physiol . ~7 , 468 (1954) .

18 .

H . A . Krebs, Biochem. J . ~, 225 (1961) .

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J . B . Lee, V. K . Vance and G. F . Cahill, Am . J . Physiol . 2~3, 27 (1962) .

20 .

C . T . Teng, Arch . Biochem. Biophys . 48, 415 (1954) .

21 .

H . M . Tepperman, P . Fabxy and J . Tepperman, J. Nutrition ,~,Q,Q, 837 (1970) .

22 .

R. B . Flinn, B . Leboeuf and G . F. Cahill, Jr ., J . Hiol . Chem . 2 p, 508 (1961) .

23 .

R. J . Mahlen and O . Szabo, Endocrinoloc~r 83, 1166

24 .

R . J. Mahlen and O . Szabo, Pro . Soc . Biol . Med . 125, 879 (1967) .

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(1968) .