THE JOURNAL OP BIOLWIC~L CHEMISTRY Vol. 245, No. 7, Issue of April 10, pp. 1604-1612, 1970

Printed in U.S.A.

Gluconeogenesis in the Isolated Perfused Rat Kidney*

(Received for publication, October 22, 1969)

R. H. BOWMAN

WITH THE TECHNICAL ASSISTANCE OF FERN BARNEY

From the Veterans Administration Hospital, and Department of Pharmacology, Upstate Medical Center, Syracuse, New York 13210

SUMMARY

The perfused rat kidney was used for a study of gluco- neogenesis. The kidney was completely isolated and perfused with Krebs-Henseleit bicarbonate buffer containing 5% albumin. Perfusion flow rate and pressure remained stable for 2 to 3 hours, although the glomerular filtration rate diminished with time. The fractional reabsorption of both water and sodium was 0.92 during the 1st hour of perfusion.

The rate of glucose formation from pyruvate was linear and in excess of 1 pmole per min per g of tissue. Glucose production from L-lactate was less than that from pyruvate, and the uptake of lactate was also less. Fructose was converted to glucose more readily than was dihydroxyacetone, and at approximately the same rate as pyruvate. Glucose production from L-glutamate proceeded at about the same rate as that from L-lactate. L-Alanine produced no glucose, and no alanine uptake could be detected. A mixture of amino acids, pyruvate, L-lactate, and glycerol at normal plasma concentrations supported gluconeogenesis at an initial rate of about 0.6 Fmole per min per g of kidney.

The rate of gluconeogenesis was sensitive to small changes in the pH of the perfusate. With either lactate, pyruvate, glutamine, or a substrate mixture of predominantly amino acids, glucose production was greater at pH 7.23 than at pH 7.64. Conversely, gluconeogenesis from glycerol and fruc- tose was inhibited by a pH of 7.23 and increased by pH 7.64.

Addition of adenosine 3’,5’-monophosphate to the per- fusate stimulated gluconeogenesis from lactate, pyruvate, and amino acids, but not from glycerol. Neither glucagon, epinephrine, parathyroid extract, nor vasopressin altered the rate of gluconeogenesis.

The capacity of the kidney to synthesize glucose has been recognized for a number of years (2-9). Krebs (10) pointed out that on a weight basis the kidney cortex may, in fact, produce glucose more rapidly than the liver, and when consider- ation is given to the high rate of renal blood %ow the kidneys

* This work was supported in part by a grant-in-aid from the American Heart Association. A preliminary report has been published (1).

may make a substantial contribution to total body gluconeogen- esis. In the present work, renal gluconeogenesis has been studied in the isolated perfused rat kidney. The perfused kidney produced glucose from a number of substances; a phys- iologically significant rate ensued when the perfusate contained the normal complement of plasma substrates.

In the liver, the rate of gluconeogenesis is stimulated by glucagon (11-15) and epinephrine (11, 14-16), apparently via formation of adenosine 3’) 5’-monophosphate (14, 16-18). Nishiitsutsuji-Uwo, Ross, and Krebs (19) have observed that glucagon does not stimulate glucose formation by the perfused kidney. Our results confirm this, but show that cyclic AMP,I itself, is effective in increasing renal gluconeogenesis. The pII of the perfusate was also found to be a regulatory factor in glucose production, lending support to similar findings with kidney cortex slices from acidotic and alkalotic rats (20-23).

The use of isolated perfused organs, particularly the rat heart and rat liver, has produced an abundance of information con- cerning intermediary metabolism and its control. For the most part, metabolic functions of the kidney have been studied with slices and homogenates, and, except for one recent report (19), the perfused kidney has not been utilized for metabolic purposes. In the present communication a practical method for perfusion of the rat kidney is described. The preparation retains considerable physiological integrity and would appear to be useful for investigations of renal energy metabolism, especially as related to certain tubular functions.

EXPERIMENTAL PROCEDURES

ApparatusThe apparatus was the same as that described for rat heart perfusions by Morgan et al. (24) and later modified by Neely et al. (25). This device permits perfusion by gravity %ow or by recirculation using a peristaltic pump. For kidneys, an Allihn condenser (2.5 x 60 cm) was used for the perfusion chamber. A mixture of moistened 02:COz (95:5) was delivered by small diameter tubing to the lower portion of the chamber, and reoxygenation took place as the perfusate passed downward over the inner surface of the chamber An E-gauge hypodermic

needle, approximately 2.5 cm long, was used as the arterial cannula. The end was slightly beveled, with the edges filed to produce a smooth tip. The cannula was attached to a three- way stopcock to which recirculating perfusate was delivered

1 The abbreviations used are: cyclic AMP, adenosine 3’,5’-mono- phosphate; FDP, fructose 1, G-diphosphate.

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from the bubble trap. Also attached t’o the stopcock was tubing leading from an elevated reservoir which contained perfusate to be used for preliminary washout of the kidney. This chamber was at a height of approximately 76 cm above the kidney.

Perfusate Composition-Unless otherwise indicated, perfusate was composed of Krebs-Henseleit bicarbonate buffer (26) con- taining 5% Fraction V bovine serum albumin (Armour). The perfusate was dialyzed overnight against a large volume of buffer containing penicillin (4.8 mg per liter) and streptomycin (4 mg per liter) but without albumin. At the beginning and end of the dialysis period the dialysate was equilibrated with 02: COz (95 :5). The pH of the perfusate was measured prior to perfusion and adjusted to 7.45, if necessary.

Operative Procedure-Male rats weighing 350 to 450 g were anesthetized with sodium pentobarbital (approximately 5 mg/lOO g of body weight). A midline incision was made, and the major abdominal blood vessels were exposed. Two ligatures were passed around the left renal vein, into which heparin (200 units per rat) was injected. This blood vessel was then tied off. A ligature was passed around the right renal artery, and distal and proximal ligatures were placed around the mesenteric artery. When required, the right ureter was catheterized with PE-10 polyethylene tubing.

Cannulation-The right renal artery was cannulated via the mesenteric artery according to the method described by Nishiit- sutsuji-Uwo et al. (19). As the cannula was pushed into the renal artery, a hemostat holding back washout perfusate was released and perfusion was begun; there was immediate clearing of blood from the kidney. The inferior vena cava was rapidly severed and the kidney was completely freed from the animal. The kidney was trimmed of adhering tissue, and, after turning the stopcock to permit recirculation of the perfusate, the kidney was suspended in the perfusion chamber. The perfusion pres- sure was adjusted to 80 mm of mercury.

Perfusate and Tissue Sampling-A three-way stopcock located between the perfusion chamber and the perfusion pump was employed for drawing perfusate samples. For tissue analysis, the kidney was lifted from the chamber by means of the can- nula, and, while still being perfused, the kidney was frozen between blocks of aluminum (27) at Dry Ice temperature.

Analysis of Perjusate and Urine-Samples of perfusate col- lected during the perfusion were placed in tubes kept at 0”. For analysis of L-lactate, pyruvate, L-glutamate, and L-alanine aliquots were deproteinized with 5% HCIOI, neutralized to pH 3 to 5 with KzC03, and assayed enzymatically (28-31), using a Gilford recording spectrophotometer. Glucose in the perfusate was determined by the ferricyanide method with a Technicon Auto-Analyzer; simultaneous assays utilizing the glucose oxidase method (32) gave results similar to those obtained with the Auto-Analyzer. When either fructose or dihydroxyacetone was present in the medium, glucose was measured by glucose oxidase. Sodium and potassium were analyzed with an Instrumentation Laboratory (Boston, Massachusetts) flame photometer. Inulin- aH was measured by liquid scintillation counting. For this pur- pose, perfusate and urine were solubilized in 1 M Hyamine (Packard) before adding the scintillation fluid.

Tissue Analysis of iiTP, AMP, and Hexose Phosphates-The frozen kidneys were pulverized in a stainless steel mortar (33) at Dry Ice temperature, and a weighed aliquot of the frozen powder was extracted in 10% HC104. An aliquot of the

perchlorate extract was neutralized to pH 4 to 6 with KOH and assayed enzymatically for ATP (34), AMP (35), glucose 6-phosphate, and fructose g-phosphate (28), and fructose 1,6- diphosphate (36). Another portion of the frozen powder was desiccated for estimation of dry weight.

Chemicals-Substances used as substrates were: DL-lactate (Mallinckrodt Chemical Works); sodium L-lactate, fructose, and dihydroxyacetone (Nutritional Biochemicals); sodium pyruvate and amino acids (Sigma); glycerol (J. T. Baker Chemical Company) ; sodium n-lactate (Mann). Additional chemicals employed were: adenosine 3’) 5’-monophosphate and adenosine 5’-monophosphate (Sigma) and N6-2’-0-dibutyryl adenosine 3’,5’-monophosphate (Calbiochem and Boehringer Mannheim). Glucagon (lot 258-234B-167-1) and parathyroid extract were gifts from Drs. 0. K. Behrens and E. L. Grinnan of Lilly. Vaso- pressin and l-epinephrine were obtained from Parke-Davis and inulin-aH from Amersham/Searle.

Presentation of Results-At the end of 70 min of perfusion the kidneys weighed about 5% more than their paired, nonpeifused kidneys, both sets having been bisected and lightly blotted before weighing. On the other hand, the dry weight of the kidneys was about 10% less after perfusion. The wet weight to dry weight ratios of nonperfused and perfused kidneys from 24 animals were, respectively, 4.38 f 0.04 and 4.85 f 0.22 (mean and S.E.M.). There was no significant difference between fresh weights of right and left kidneys, and, therefore, the wet weight of the left, nonperfused kidney was used for expressing metabolic results when these were reduced to a gram basis. For the time course presentations, the results are expressed on the basis of the whole kidney.

Data are presented as means and standard errors of the mean. Probability values were derived from the Student t test.

RESULTS

Functional Integrity of Perfused Kidney-In the metabolic experiments reported in this paper, urine was allowed to mix with the recirculating perfusion medium. Compared with experiments in which urine did not mix with the perfusate, this procedure did not affect the rates of gluconeogenesis. In order to determine the functional performance of the perfused kidney, experiments were performed in which the ureter was catheterized and urine was collected quantitatively for multiple periods of 5 to 10 min for up to 2 hours (Table I). Inulin-3H was included in the perfusate and its clearance was used as a measure of glomeru- lar filtration rate.

The perfusion flow rate was high compared with renal plasma flow in z&o, which has been reported to be approximately 4 to 5 ml per min for the rat kidney (37). With the perfusion pressure maintained at 80 mm of mercury, the flow increased during the first 5 to 10 min of perfusion and then stabilized at about 30 ml per min per kidney. During the first 20 to 30 min glomerular filtration rate was approximately equal to that of the kidney in situ, i.e. about 0.75 ml per min per g (37), but it gradually de- creased with time so that the mean value for the entire 1st hour was about 60% of normal. Water and electrolyte reabsorption were depressed, giving values characteristic of decreased function of the distal and collecting tubules. However, the fractional reabsorption of water and sodium was compatible with normal functioning of the proximal tubule (38). The concentrations of ATP and AMP were in the normal range of kidney tissue frozen in situ (19).

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TABLE I

Functional behavior and ATP and AMP concentrations of isolated perfused rat kidney

During 1 hour of perfusion urine was collected for multiple periods of 5 to 10 min. The values are from a number of collection periods of 17 perfused kidneys. The quantities of sodium and potassium filtered were estimated from their concentrations in the perfusate and the rate of glomerular filtration (inulin-SH clear- ante).

Parameter No. of observations VdUCZ

Perfusion flow rate, ml/min/g.. 42 20.5 f 0.34 Glomerular filtration rate, pl/

min/g........................ 72 450 f. 18 Urine flow rate, pl/min/g.. 72 36.3 f 2.1 Fraction of filtered sodium ex-

creted........................ 56 0.078 f 0.007 Fraction of filtered potassium

excreted...................... 56 0.513 f 0.056 Inulin urine to perfusate ratio. 7s 19.2 f 1.9 ATP,” rmoles/g, dry tissue. 12 6.9 f 0.21 AMP,a pmoles/g, dry tissue. 9 0.63 f 0.05

D Kidneys frozen after 70 min of perfusion with 20 mM n-lactate.

TABLE II

Effect of substrate on rate of gluconeogenesis by isolated perfused rat kidney

Kidneys from 400- to 450-g rats, fasted for 18 to 24 hours, were perfused in a recirculating system with 35 ml of medium contain- ing one of the substrates listed below. The rates of glucose pro- duction were measured over a lo- to 20-min interval after an initial equilibration period of 10 min.

Substrate

nn-Lactate.. n-Lactate. n-Lactate. . n-lactate................. Pyruvate Pyruvate Fructose.................. Fructose. Dihydroxyacetone Glycerol.................. Glycerol.................. L-Glutamate. n-Alanine.

- I

--

-

oitial con- No. of Rate of glucose centration kidneys formation

mdl

10”

10 25 15 8

38 20 40 40 10 20 10 10

.- n

11 3 5 4 6 2 8 4 4 9 4 2 5

z~moles/g, wet wt/min

436 f 18 524 zk 26 582 f 14 235 f 8

1155 f 85 1424 f 115 1077 f 91 948 f 61 707 f 51 299 f 14 279 f 10 368 f 39

22 f 6

0 Concentration of the L isomer of lactate.

The rapid rate of perfusion flow eliminated the need for eryth-

rocytes in the perfusate. Oxygen tension in the venous effluent, measured by means of a polarographic needle electrode, ranged between 150 and 350 mm of Hg, depending upon the experimental conditions. Perfusate entered the kidney with an oxygen tension of approximately 450 mm of Hg.

Glucose Synthesis from Various PrecursorsThe glycogen concentration in the kidney has been reported to be low (7). In six kidneys in the present study it was found to be 1.05 f 0.09 pmoles (glucose equivalents, mean and S.E.M.) per whole organ

before perfusion. With pyruvate as the substrate there was no significant change in the glycogen level during 1 hour of perfusion. In the absence of substrate the glycogen decreased by over 50% during 1 hour, but quantitatively this was little more than 0.5 pmole. The glucose which appeared in the perfusate in the various experiments was assumed, therefore, to have arisen by gluconeogenesis. Without substrate, a very small amount of glucose appeared in the medium, and this ceased, for the most part, after 5 to 10 min (see, for example, Figs. 6 and 7). The glucose production rates shown in the accompanying tables and figures are net values and do not take into account the utilization of glucose. Our preliminary observations have shown the utilization rate to be about 12 pmoles of glucose per g per hour (see also Reference 19). It, therefore, is probable that true gluconeogenic rates would be higher than those given below.

Table II shows initial rates of glucose formation from several substrates. The rates were derived from a lo- to 20-min interval of perfusion after an equilibration period of 10 min. The maxi- mum rate of glucose formation from L-lactate was only 40 to 50% of that obtained from pyruvate. This result differs from observations in liver perfusions in which these two substrates have been found to be equally gluconeogenic2 (39, 40). Similar to findings with the liver, n-lactate produced only about one-half as much glucose as did the L isomer. Glycerol was less effective than lactate in producing glucose, whereas dihydroxyacetone was converted to glucose at a substantial rate. Fructose pro- duced glucose at about the same rate as pyruvate. Two amino acids were tested individually (see below for the effect of an amino acid mixture) for their gluconeogenic effectiveness. L- Alanine produced essentially no glucose, and no alanine uptake could be detected. On the other hand, glutamate was taken up; its disappearance and yield of glucose on an hourly basis were, respectively, 70.8 f 8 and 19.6 f 2.5 pmoles per g of kidney (mean and S.E.M. of two observations). This is slightly more than a 50% conversion, based on 1 mole of glucose arising from 2 of glutamate.

Table II shows that fructose was converted to glucose more readily than was dihydroxyacetone. This confirms earlier results of Krebs and Lund with kidney slices (41), but differs from the liver in which dihydroxyacetone phosphate is believed to be an intermediate in the synthesis of glucose from fructose (42). The result in the kidney suggested that fructose may be phosphorylated to fructose-6-P via hexokinase and, therefore, bypass several steps. That this may be the case is seen from the following experiment. Kidneys were perfused with either fructose or pyruvate and then frozen and analyzed for hexose phosphates (Table III). In kidneys perfused with fructose the levels of glucose-6-P and fructose-6-P were 4-fold greater than in those perfused with pyruvate, while there was little difference between the FDP values. Since the rates of gluconeogenesis

were similar, these results are indicative of direct phosphorylation of fructose to fructose-6-P.3

2 In the perfused liver, Exton and Park (42) have found that, at less than saturating levels of substrate, gluconeogenesis from pyruvate is greater than that from lactate. They suggest that limitation at the lactate dehydrogenase step is perhaps to be expected because of the high K, of the enzyme for lactate.

3 The rates of gluconeogenesis from pyruvate and fructose were linear and, therefore, the flux over the gluconeogenic pathway is assumed to have been in a relatively steady state condition. With this assumption, the rate of the fructose diphosphatase reac- tion in kidneys perfused with pyruvate will be equal to the rate

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TABLE III

Levels of hexose phosphates and rates of gluconeogenesis in kidneys perfused with pyruvate and fructose

Kidneys were perfused for 70 min and then clamp frozen for analysis.

Substratea

Pyruvate (4), 20 rnM

Fructose (4)) 20 rnM . . .

Rate of glucose

formation

62.0 f !

69.6 f !

1

42 -

FDP

Ratio of

gzT Fructose-

6-P to FDP

542 f 62 158 f 17 61 f 9 11.5

1350 f 272715 f 70105 f 10 29.2

a Number of kidneys in parentheses.

Time Course of Substrate Disappearance and Glucose Production

-In order to assess more accurately the rates of glucose forma- t,ion and substrate utilization, experiments were carried out in which the perfusate was assayed at intervals throughout the perfusion period. L-Lactate, pyruvate, and fructose were used separately as substrates and, unless otherwise indicated, the initial perfusion volume was 35 ml. The rate of formation of glucose from lactate was highest during the first 30 min and subsequently diminished (Fig. 1). The rate with 25 mM lac- tate (Fig. IB) was only slightly greater than with 9.5 mM (Fig. IA). In both cases, the disappearance of lactate proceeded slowly. Cessation of glucose production from lactate could not be explained by substrate exhaustion, since, in kidneys perfused with 25 mM lactate, the concentration in the perfusate was still 17 mM at the time glucose formation had nearly ceased. The possiblity existed that glucose accumulating in the medium decreased the gluconeogenic rate. Therefore, an experiment was performed in which glucose as well as lactate were present in the perfusate from the beginning. The initial lactate concentra- tion was 18 mM, and the glucose concentration was approximately 2.5 mM. This amounted to about 90 pmoles of glucose, which was twice as much as was terminally present in experiments performed with lactate alone. Nevertheless, the initial rate of glucose formation was essentially the same as in the absence of starting glucose (data not presented). Sustained production of glucose from lactate was obtained by using a larger perfusion vol- ume. With 70 ml rather than 35 ml and with 20 mM L-lactate, glucose was produced linearly for 85 min (Fig. 2). With this larger volume but at a lower level of lactate (8 mM), glucose production still declined after 40 min in spite of a terminal lactate concentration of nearly 5 mM.

Experiments to assess the rate of glucose formation from pyruvate are shown in Fig. 3; the initial perfusion volume was 35 ml. When 6 mM pyruvate was employed (Fig. 3A), uptake was rapid and cessation of glucose production paralleled sub- strate exhaustion. At a starting concentration of 38 mM (Fig.

of gluconeogenesis. With fructose, the rate of the diphosphatase reaction will be equal to or less than the rate of gluconeogenesis, since there are two pathways by which fructose can go to fructose- 6-P, only one of which involves the FDP to fructose-6-P step. Since the gluconeogenic rates from pyruvate and fructose were the same, an increase in the glucose-6-P + fructose-6-P to FDP ratio must indicate direct phosphorylation to fructose-6-P rather than greater fructose diphosphatase activity.

I

8. 25mM L-LACTATE

-40

-3O- I

1

,'? 400- d,Z

I

/o -200

1' P c

.z - 0

2 200- ,I lo= "7 2 I

P T;lS.EM. :

F .- 0 : 0: .5 ; 400-

A. 9.5mM L-LACTATE

H

-4o.g

Glucose s D ,P-----3(), a, 6

-20;

-10

P Oo5 20 35 50 65 80 95 I 0

Minutes of Perfusion

FIG. 1. Time course of glucose production from L-lactate. Kidneys from rats (400 to 500 g) fasted for 18 to 24 hours were perfused for 95 min. L-Lactate was present at zero time at a concentration of either 9.5 mM (A, one kidney) or 25 mM (B, mean of data from two kidneys). The initial volume of perfusate was 35 ml. Samples (1 ml) were withdrawn at the times indicated for determination of glucose and lactate. Results are given as micromoles of glucose produced and micromoles of lactate remain- ing. Kidney weights were approximately 1.7 g.

/ Pyruvate f 24mM -+

/

A

(0.7mMl

/' Pyruvate, err

2:tp: 0 IO 25 40 55 70 85

J

Minutes of Perfusion

FIG. 2. Comparison of gluconeogenic rates from pyruvate and L-lactate. Kidneys were from 350- to 400-g male rats fasted for 18 to 24 hours. The initial perfusion volume was 70 ml, and the initial substrate concentrations were as indicated by the arrows. The figures in parentheses were the terminal substrate concentra- tions (means) for each experiment. Each curve represents the mean of three to nine perfusions.

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1608 Renal Glucmeogenesis

B. 36mM PYRIJVATE _ I-r

1400

~-Id 3

- ,402 .

-120s

-100;

)-Bog- -

- 60 Y

I- 40:

; -A. 6mM PY RUVATE 70 2 0

7=I S.E.M. 60 2

Pyruvate 1 I" OFI

, -d--* I 0 5 20 35 50 65 80 95 ’

Minutes of Perfusion

FIG. 3. Time course of glucose and lactate production from pyruvate. Kidneys from 400- to 450-g rats, fasted for 18 to 24 hours, were perfused with an initial pyruvate concentration of either 6 rnM (A) or 38 mM (B). The starting perfusate volume was 35 ml, and samples were withdrawn for analysis at the times indicated. A, the means from four kidneys; B, a single perfusion.

FIG. 4. Time course of glucose production from fructose. Kid- neys from rats (400 to 450 g) fasted for 18 to 24 hours were perfused for 70 min. Fructose was present at an initial concentration of 19 mM in a perfusion volume of 35 ml. Perfusate was sampled at the times indicated and each point is the mean of four perfusions.

3B), the amount of pyruvate in the system was sufficient to allow glucose formation to proceed linearly for the entire 90 min perfusion. Pyruvate disappearance was also linear. In both experiments shown in Fig. 3 lactate was formed, and about 50%

Minutes of PerfusIon

FIG. 5. Time course of glucose production from a mixture of substrates at normal plasma concentrations. Kidneys from 350- to 400-g rats fasted for 18 to 24 hours were employed. The kidneys were initially perfused with 35 ml of medium containing no sub- strate. After 10 min the perfusate was sampled for glucose, and 1 ml of substrate-containing medium was added to bring the total concentrations to: amino acids, 3.8 mM; n-lactate, 1.0 m&l; pyru- vate, 0.1 mM; glycerol, 0.3 mM. Medium containing no substrate was added to other kidney perfusions at 10 min. Total perfusion time was 60 min. Each cww is the mean of four perfusions.

of the pyruvate which disappeared could be accounted for by glucose and lactate production. In kidneys perfused with 6 mM

pyruvate, the terminal lactate to pyruvate ratio was about 10; with 20 mM pyruvate the final ratio was 2 (data not shown), and when the starting pyruvate concentration was 38 InM the lactate to pyruvate ratio was between 0.3 and 1.3 after 90 min of per- fusion.

For comparison with the lactate experiments in which a larger perfusion volume was used, several kidneys were perfused with either 8 or 24 mM pyruvate in a volume of 70 ml (Fig. 2). The rate of glucose production from 8 mM pyruvate was initially the same as with 24 mM pyruvate and slightly greater than with 20 mivr lactate. At a starting pyruvate concentration of 8 MM,

glucose continued to be produced when the level of pyruvate had fallen to less than 1 mM. This is to be compared with lactate perfusions (Fig. 2) in which glucose production had ceased when the lactate concentration had fallen from 8 to 4.9 mM.

The time course of fructose conversion to glucose is shown in Fig. 4. The rates of fructose disappearance and glucose produc- tion were very rapid. During the first 10 min of perfusion the ratio of fructose uptake to glucose formation was lO:l, but for the last 20 min it was 1 :l.

Gluconeogenesis from Substrates at Normal Plasma Concentra- tions-The concentrations of substrates used in the above experi- ments were considerably above those found in normal rat plasma. It was of interest to determine if a mixture of substrates similar to that found in plasma could support renal gluconeogenesis. Kidneys were perfused for 10 min with no substrate, and then amino acids,4 lactate, pyruvate, and glycerol were added to give

4 The final concentrations (micromolar) of amino acids were: aspartate, 35; arginine, 200; proline, 180; glutamate, 200; glycine, 400; alanine, 450; valine, 200; isoleucine, 100; leucine, 170; phenyl- alanine, 80; lysine, 400; histidine, 80; tryptophan, 70; serine, 280;

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TABLE IV

Effect of perfusate pH on glucose production by isolated perfused rat kidney

Kidneys from 400. to 450-g rats, fasted for 18 to 24 hours, were perfused with Krebs-Henseleit NaCl solution which contained variable amounts of NaHC03 in order to give the indicated pH. Glucose production and substrate removal were measured over a 60.min interval of perfusion. Substrate concentrations are those which were present initially in the perfusion medium.

Substrate pH No. of GlUCW3 Substrate

kidneys production disappearance

pmoles/g, wet wt/lzr

DL-Lactate (10 mM L) 7.23 7 24.2 f 1.4 61.1 f 8.8 7.45 11 13.3 f 1.0 N.M.a 7.64 8 11.1 zt 1.1 56.7 f 4.2

Pyruvat,e (20 mM) 7.23 4 61.5 f 1.99 N.M. 7.64 4 49.4 f 2.15 N.M.

Glutamine (10 mM) 7.23 4 5.2 f 0.30 N.M. 7.64 4 1.3 f 0.44 N.M.

Substrate mixture* 7.23 4 35.8 f 2.2 N.M. 7.64 4 24.4 f 0.8 N.M.

Fructose (20 rnM)O 7.23 4 42.0 f. 6.2 116.0 f 1.7 7.64 4 69.6 f 4.0 115.1 f 10.9

Glycerol (10 mM) 7.23 5 4.6 f 0.89 N.M. 7.45 5 9.5 f 0.86 N.M. 7.64 5 19.7 f 1.6 N.M.

a N.M., not measured. b Amino acids (15.2 mM), L-lactate (4 mM), pyruvate (0.4 mM),

and glycerol (1.2 mM); see Footnote 4 for concentrations of con- stituent amino acids.

c Fed rats.

final concentrations of 3.8, 1.0, 0.1, and 0.3 mM, respectively. Perfusion was continued for an additional 50 min, and control kidneys were perfused for the entire 60 min without substrate.

After substrate addition, glucose production was approximately

1 pmole per min for the next 20 min (Fig. 5). Gluconeogenesis then became limited by the low level of precursors present in the medium.

Effect of Per&sate pH on Gluconeogenesis-Several recent reports indicate that kidney tissue from acidotic animals exhibits

an increased rate of gluconeogenesis (20-23). To investigate

this aspect in the perfused kidney, the pH of the perfusate was

decreased or increased slightly from the normal pH of 7.45. A

pH of 7.23 was achieved by changing the usual (25 mM) concen- tration of perfusate bicarbonate to 15 mM; 39 mM bicarbonate

was used to give a pH of 7.64. The gas phase was kept at 95% 02-5s CO% The data in Table IV show that, with lactate, pyruvate, glutamine, or a mixture of predominantly amino acids, glucose formation was more rapid at the lower pH. When fructose or glycerol was used, gluconeogenesis was greater at

pH 7.64. Renal uptake of lactate and fructose was not affected

threonine, 300; asparagine, 70; glutamine, 350; cysteine, 70; methionine, 70; tyrosine, 70; ornithine, 90. These are the levels of amino acids reported to be found in rat plasma (39). Taurine, a-aminobutyrate, and citrulline which are also found in rat plasma (39) were not present in the perfusate.

.g20- z

2 15-

&--m---J

Lactate+ Cyclic AMP

30 35 55 Minutes of Perfusvan

I 75 95

FIG. 6. Effect of adenosine 3’,5’-monophosphate on the time course of glucose production from lactate. Kidneys from rats (400 to 450 g) fasted for 18 to 24 hours were perfused for 30 min with no substrate. At 30 min the perfusate was sampled for glucose, and DL-lactate with or without cyclic AMP was added to bring the L-lactate concentration to approximately 20 mM and the cyclic AMP level to about 2 X low4 M. Five minutes later and at subsequent 20-min intervals a sample of perfusate was withdrawn for glucose analysis. Each cuTz)e is the mean of five perfusions.

by pH. Increased gluconeogenesis thus involved more efficient conversion of substrate to glucose rather than stimulation of substrate uptake.

Influence of Glucagon, Epinephrine, Parathyroid Extract, Vasopressin, Cyclic AMP, AMP, and N6-d’-0-Dibutyryl Cyclic AMP on Gluconeogenesis-The rate of gluconeogenesis from lac- tate or pyruvate was not increased by addition of glucagon (0.5 to 2.0 PM) or epinephrine (1 MM) to the perfusate (data not shown; see also Reference 19). Likewise, there was no effect from para- thyroid extract or vasopressin (data not shown). A response was obtained by adding cyclic AMP to the perfusate. Fig. 6 shows the effects of perfusing kidneys for 30 min with no sub- strate and then adding lactate with or without cyclic AMP. During the hour that the kidneys were exposed to cyclic AMP, gluconeogenesis proceeded at a rate much greater than with lactate alone. Table V shows the effect of cyclic AMP on gluconeogenesis from several substrates. The rate was acceler- ated with either lactate or pyruvate as substrates but not with glycerol. Adenosine 5’-monophosphate did not stimulate glucose production, and N6-2’.0-dibutyryl cyclic AMP was also ineffec- tive. In addition to the concentrations shown in Table V, dibutyryl cyclic AMP was tested at 0.025 mM and 0.10 m&l, and no response was observed.

DISCUSSION

Metabolic Viability of Perfused Kidney-The production of 1 pmole of glucose from lactate, pyruvate, or amino acids requires 6 pmoles of ATP. Table I shows that the concentration of ATP in the kidney was about 6 to 7 pmoles per g, dry weight, which reduces to 1 to 1.5 pmoles per g, wet tissue. Since the kidney can produce (from pyruvate) over 1 pmole of glucose per min per g, this means that ATP must turn over at least six times per min in order to satisfy the requirement for gluconeogenesis. The energy mechanism with which renal sodium reabsorption is coupled is not very clear (38,43), but if the energy transduction is

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1610 Renal Gluconeogenesis

TABLE V

Vol. 245, No. 7

Effect of adenine nucleotides on renal glueoneogenesis from several substrates

Substrates and nucleotides were added either at the beginning of the perfusion or after a 30-min perfusion without substrate; total perfusion times were 70 and 95 min, respectively. Except as noted, kidneys were from rats fasted for 18 to 24 hours.

Substrate

Substrate and nucleotide added at beginning of perfusion:

DL-Lactate (10 mM n)

Substrate and nucleotides added after prior perfusion for 30 min:

nn-Lactate (20 mM L)

L-Lactate (7 mM)

n-Lactate (20 mM)

Pyruvate (20 mM)

Glycerol (5 mrvr)

a Kidneys from fed rats.

No. of kidneys Nucleotide

70 None 55 Cyclic AMP 4 None 5 Cyclic AMP

4 None 4 Cyclic AMP 3 Dibutyryl cyclic AMP” 2 Dibutyryl cyclic AMPd 4 AMP

4 None 4 Cyclic AMP 4 Dibutyryl cyclic AMPd

4 None 4 Dibutyryl cyclic AMPd

6 None 6 Cyclic AMP

5 None 5 Cyclic AMP

Final concen- tration

1.0

0.25

0.2 0.2 0.25 0.2

0.2 0.05

0.01

0.2

0.2

Glucose production

pnotas/g, wet wt/kr

5.7 f 0.91 21.2 f 3.05” 11.9 f 1.80 21.7 f. 1.31”

11.0 f 0.72 20.7 f 1.36* 11.5 f 1.72 5.1 f 0.78 8.0 f 0.93

9.5 f 1.0 20.7 f 1.39b 13.0 f 1.39”

13.5 f 1.1 13.7 f 1.3

42.2 zk 2.85 55.3 f 4.771

5.8 f 0.26 6.1 zk 0.69

b p < 0.01 versus respective control perfused without nucleotide addition. c Procured from Calbiochem. d Procured from Boehringer Mannheim. * p > 0.05 versus respective control perfused without nucleotide addition. f p < 0.05 versus respective control perfused without nucleotide addition.

via ATP (44, 45) the turnover of ATP per min would be many fold greater than the 6 required for glucose formation. The maintenance of a normal steady state level of ATP appears, there- fore, to be good evidence of metabolic competence. It may be worthwhile to note that glucose formation was exquisitely dependent upon adequate oxygenation. Any interruption of oxygen supply to the system or interference with perfusate flow was clearly marked by diminished gluconeogenesis.

Substrates of Gluconeogenesis-Using kidney cortex slices (41, 46-50) and the perfused rat kidney (19), Krebs et al. have meas- ured the formation of glucose from various substrates. Both in slices and in the perfused preparation they found that pyruvate was taken up more avidly than was lactate. In turn, gluconeo- genesis from 5 mM L-lactate was only 25yo of that from 5 mM

pyruvate. In the present experiments also, pyruvate, at any concentration, was a considerably more effective substrate than lactate. With a perfusion volume of 70 ml, which was similar to that employed by Nishiitsutsuji-Uwo et al. (19), a concentra-

tion of 8 InM L-lactate supported a somewhat constant rate of glucose synthesis for little more than 30 min. Under the same conditions, 20 mu lactate led to linear production of glucose, but at a rate slightly less than that given by 8 mM pyruvate. That is, a concentration of lactate two or more times greater than that of pyruvate was required to maintain a similar rate of glucose production. Since pyruvate is an intermediate in the conversion of lactate to glucose, this appears to indicate some limitation on the oxidation of lactate to pyruvate. A high K, of lactate dehydrogenase for lactate,2 or limited availability of NAD+, or both might explain this result.

Glucose production from pyruvate and fructose proceeded at about equal rates, making it appear that no major limitation is imposed between pyruvate and the point at which fructose enters the gluconeogenic pathway. In regard to glucose forma- tion from fructose, the considerable rise in glucose-6-P conse- quent to perfusion with fructose (Table III) suggests that synthe- sis was limited by the activity of glucose 6-phosphatase.

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Issue of April 10, 1970 R. H. Bowman 1611

Comparison of Rates of Renal and Hepatic Gluconeogenesis-In the perfused rat liver, the rate of gluconeogenesis from pyruvate or L-lactate is about 1 pmole per min per g of tissue (39, 40). The present results show a rate of glucose formation from pyru- vate slightly in excess of 1 pmole per min per g for the perfused kidney. With a mixture of substrates at normal plasma levels, initial glucose production approximated this same rate, which was at least as rapid as that obtained with a similar mixture of sub- strates in the perfused liver (39). These results lend support to the contention of Krebs (lo), referred to above, that on a weight basis the gluconeogenic capacity of the kidney cortex probably equals or exceeds that of the liver.

InJEuence of Perfusate pH on Rate of GEuconeogene.&--An increase in renal gluconeogenesis induced by metabolic acidosis was first demonstrated by Goodman, Fuisz, and Cahill (20), using rat kidney cortex slices. There were subsequent confirmatory reports (21, 22), and recently Kamm and Cahill (23) have shown that an enhanced rate of glucose formation by cortical slices from diabetic and starved rats was returned to normal by alkali feed- ing. The present studies show that formation of glucose by the perfused kidney is sensitive to the pH of the perfusate. The direction of the pH effect depended upon the substrate employed. Goodman et al. (20) had earlier observed this substrate specificity, and, based upon their findings, those of Goorno, Rector, and Seldin (21), and the present observations, it may be postulated that the selective substrate response to pH change is due to at least two pH sensitive steps. One, accelerated by a low pH, appears to be located in the pathway prior to the point at which glycerol enters. Another affects precursors closer to glucose and has greater activity at a more alkaline pH. The latter step must be only weakly inhibited by a low pH, since, with substrates such as lactate, the more acid perfusate resulted in greater flux through the entire gluconeogenic pathway. If pH influences gluconeogenesis from glycerol and fructose by altering the rate of their initial phosphorylation, glucose produc- tion from lactate would, of course, not be affected at all by this latter pH sensitive step.

Hormonal and Nucleotide Effects on Renal Gluconeogenesis Glucagon and epinephrine are believed to potentiate hepatic gluconeogenesis by causing increased formation of cyclic AMP (14, 16-18). In the perfused kidney, exogenous cyclic AMP was found to stimulate glucose production, although glucagon and epinephrine by themselves were without effect (see also Reference 19). It would appear from this that the appropriate receptors (presumably glucagon- and epinephrine-sensitive adenyl cyclases) are absent from renal tissue, but this has not been tested directly. It is possible that endogenous and exoge- nous cyclic AMP may not be available to the same reactive sites. As evidence of this, neither parathyroid extract nor vasopressin altered the rate of gluconeogenesis in spite of the fact that both of these hormones activate adenyl cyclases in the kidney (51,52), and parathyroid extract increases the level of cyclic AMP in the perfused kidney.6 The problem remains as to the physiological significance of the stimulation of renal gluconeogenesis by exoge- nous cyclic AMP6 and as to what, if any, may be the natural

6 R. W. Butcher! R. H. Bowman, and E. W. Sutherland, un- published observations.

6 While this paper was in preparation, Pagliara and Goodman (53) reported that cyclic AMP stimulates gluconeogenesis in rat kidney cortex slices. They also found that, in slices from para-

activator of cylcic AMP formation in this regard. A possibility, not yet examined, is that a renal adenyl cyclase may respond to changes in perfusate pH.

The gluconeogenic response to cyclic AMP was much greater from lactate than from pyruvate. This may reflect the fact that the basal rate of glucose formation from pyruvate was already fairly rapid. The small additional effect of cyclic AMP would appear to indicate that the step (between pyruvate and glucose) accelerated by this nucleotide does not impose a major limitation on gluconeogenesis. A stimulation of reactions leading to in- creased availability of NADf might explain the action of cyclic AMP on lactate gluconeogenesis. The failure of cyclic AMP to augment glucose production from glycerol points to control by this nucleotide at an earlier step in gluconeogenesis. It is similar to results obtained in the perfused liver in which glucagon stimulated glucose formation from lactate and pyruvate but not from dihydroxyacetone or fructose (14).

Failure of the dibutyryl derivative of cyclic AMP to enhance glucose production was somewhat unexpected in view of the positive action of the parent compound. In other tissues, di- butyryl cyclic AMP has been found generally to be much more potent than cyclic AMP, presumably owing to better penetration (54, 55) and greater resistance to hydrolysis by the diesterase. Since it is known that deacylation must occur in order for the dibutyryl derivative to be effective (56, 57), the present findings raise the question as to whether the deacylation reaction may be limiting in some tissues. Precedence for this possibility has been observed (58) in fibroblasts whose growth is inhibited by cyclic AMP. Dibutyryl cyclic AMP was found to be only weakly inhibitory to cell growth, and the authors suggested that the enzyme required for conversion of dibutyryl cyclic AMP to cyclic AMP may be absent from these cells.

Acknowledgments-The excellent and conscientious assist,ance of Fern Barney, Jean Bowman, and Ellen Matcha in all phases of this work is gratefully acknowledged.

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R. H. Bowman and With the technical assistance of Fern BarneyGluconeogenesis in the Isolated Perfused Rat Kidney

1970, 245:1604-1612.J. Biol. Chem. 

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