Increased hepatic capacity of urea synthesis in acute and chronic uraemia in rats

CIinicd Nutrition (1991) 10: 206-212 0 Longman Group UK Ltd 1991

Increased hepatic capacity of urea synthesis in acute and chronic uraemia in rats T. ALMDAL*,

M. EGFJORDt,

6. A. HANSEN*

and H. VILSTRUP*

“Divison of Hepatology, Department of Medicine A-2151, tDivision of Nephrology, Department of Medicine P-2131 and *tDepartment of Experimental Pathology, Rigshospitalet, Copenhagen, Denmark (Correspondence to T.A., Department of Medicine A2151, Rigshospitalet, 9 Blegdamsvej, 2100 Copenhagen, Denmark)

Presented at The 10th Congress Leipzig, August 1988.

of the European

Society of Parenteral and Enteral Nutrition,

ABSTRACT-We studied whole body nitrogen balance in female rats for 28 days after induction of experimental uraemia by 516 nephrectomy and at the same time the kinetics of hepatic urea synthesis by means of the in vivo capacity of urea synthesis. The N-balance of 5 uraemic rats kept in metabolic cages was negative on day 2, and positive but only half of control on day 28. In between, it was normal. The uraemic rats lost weight during the first week, and later only slowly regained their initial weight. The capacity for urea synthesis was determined during alanine loading in 5 unoperated controls, in uraemic rats in groups of 5 on days 2,7,14,21, and 28, and correspondingly in sham operated rats. In the control rats the capacity was 8.9 + 0.7t.~mol/(min 1009 BW) and the same in sham operated rats. In uraemic rats the capacity increased to 17.1 + 2.0Fmol/(min 1009 BW) (p c 0.01) 2 days after partial nephrectomy. On day 7, the capacity fell to 5.5 + 1.O (not different from initial values), and thereafter again gradually increased to 16.5 * 1.5tJ,mol/(min 1009 BW) on day 28. The early increase in the capacity may be related to glucagon, that nearly doubled on day 2, but not on day 28. The hepatic capacity for urea synthesis doubles biphasically: acutely and after 4 weeks of experimental uraemia. This may play a role in the reduction in N-balance, since an increase in the capacity implies larger hepatic amino-N conversion at any blood amino-acid concentration.

Introduction

dent, and reflects the enzyme activity. Determination of the in vivo maximum rate of urea synthesis allows studies of substrate independent changes in urea synthesis. In intact rats the capacity for urea synthesis can be measured during loading with exogenous amino-acids (5). The purpose of the present investigation was to study the capacity of urea synthesis sequentially for 28 days after induction of uraemia, and to relate this to the nitrogen balance during the same period.

Clinical and experimental uraemia is associated with catabolism (1). Amino-acids released from organ proteins are taken up by the liver. Nitrogen from the metabolised amino-acids is incorporated into urea, accumulated in the body water, and finally excreted in the urine. Urea synthesis in uraemic rat has been studied in vitro in liver slices (2), isolated hepatocytes (3), and isolated perfused livers (4). Some studies report an increased rate of urea synthesis and others a normal rate. However, the studies are not comparable since they were undertaken at varying time after induction of uraemia, and using different amino-acid compositions and concentrations. Moreover, none of these estimated the Vmax of the process, which is substrate indepen-

Material and methods Animals

Female Wistar rats with an initial average body weight of 225 g were kept in a thermostated room 206

CLINICALNUTRITION

with an artificial 12 h light/dark cycle and with free access to stock rat pellets (Altromin, Altromi Werke, Lage, FRG). Uraemia

Rats were made uraemic by means of 5/6 partial nephrectomy as previously described (6). They were anaesthetised with pentobarbital i.p., 5 mgl lOOg, and the left and 2/3 of the right kidney were removed. Control rats were sham operated. Protocols The Capacity

for Urea Nitrogen Synthesis (CUNS) was determined in the following groups of rats: 1. Control rats (n = 5) 2. Sham operated rats (groups of 5), examined on post-operative days 2,7, and 14 3. Uraemic rats (groups of 5), examined on post-operative days 2,5, 14 and 28. Food intake and N-balance were determined in the following two groups of rats kept in metabolic cages: 4. Sham operated rats followed for 28 days after the operation (n = 5) 5. Uraemic rats followed for 28 days after the operation (n = 5). Rats of protocol 2 were followed for only 2 weeks since it was assumed that no metabolic changes would occur later in those groups. The capacity for urea nitrogen synthesis (CUNS) (protocols 1, 2 and 3). Before determination of

CUNS the rats were fasted overnight, anaesthetised by thiopental 50mg/kg body weight i.p., tracheotomised, and nephrectomised (bilaterally or removal of the kidney remnant in uraemic rats). Catheters were placed in a jugular vein for infusions and in a common carotid artery for blood sampling. Alanine was administered as a priming dose of 0.4-0.7ml of a 1007mmol/l solution in sterile water followed by a constant infusion for 90min of 2.0-3.5 ml/h of a 224 mmoY1 solution by means of a roller pump (Perfusor Secura, Braun, Melsungen, FRG) adjusted so as to obtain steady state within the amino-acid concentration interval 7.6-11.3mmol/l, where the urea synthesis is at maximum (5), i.e. saturated and substrate independent. After 20min, blood was sampled (100 ~1) every 10min for determination of blood urea concentration, and CUNS was calculated as:

207

CUNS = dcu/dt x 0.63 BW x 1/(1-L) where dcu/dt is the slope of the linear regression analysis of blood urea concentration on time, 0.63 BW is the volume of distribution of urea (7), and L is the fractional loss of urea in gut, with values of L as given below (pilot experiment). Food nitrogen intake and nitrogen balance (protocols 4 and 5). Animals were kept in metabolic

cages for a total of 5 weeks. On the last day of the first week the food intake and the amounts of excreta were determined. On the following days the rats were sham operated (protocol 4) or partially nephrectomised (protocol 5). On days 2, 7, 14, 21, and 28 after operations the food intake and the amounts of excreta were determined. Samples of urine and faeces were kept for analysis of total N-content. The N-balance was calculated as: N-balance = Food amount (g) x food-N concentration - (urine-N + faecal-N) Analyses

Blood and urine urea concentration were measured by the urease-Berthelot method (8), total blood alpha-amino-nitrogen concentration by the dinitrofluoro-benzene method (9), and blood glucose by the glucose oxidase technique, using a rapid glucose analyser (Yellow Springs Instruments Co., Yellow Springs, Ohio, USA). Plasma insulin and glucagon concentrations were determined by radioimmunoassays (10, 11), glucagon after extraction by ethanol. The inter-individual coefficient of variation of these assays is approximately 18%. Nitrogen contents of food and excreta were determined by the micro Kjeldahl technique. Pilot experiments Intestinal hydrolysis of urea. Metabolism

of urea only takes place in gut bacteria, and this metabolism may be influenced by uraemia. Therefore, the fraction of newly synthesised urea lost in gut (7) was determined in control rats (n = 5) and in uraemic rats on post-operative days 2 (n = 5), 7 (n = 5), and 28 (n = 5). The rats were kept in metabolic cages and were injected i.m. with 0.25 l&u of i4C urea. Urine was collected quantitatively for 4 days, every day into a separate container acidified with 5 ml of 3 mol/l of sulphuric acid. The radioactivity in the urine of each container was counted in a liquid scintillation counter

208

UREA SYNTHESIS IN URAEMIA

20.0 =: z g m 4 c

15.0

5 -

10.0

!

Controls

2 days

7 days

14 days

21 days

28 days

Fig. The capacity of urea N synthesis (CUNS) in control rats, in sham operated rats 2,7, and 14 days after sham operation (Cl), and in uraemicrats (U) 2,7, 14,21, and 28 days after partial nephrectomy. Columns are means with 95% confidence limits.

(Tricarb Packard, USA), and the total amount of 14C urea in the urine calculated. In all animals the radioactivity was excreted within the first 3 days. The fraction of injected radioactivity not recovered in urine and thus lost in gut, L, in control rats was: 0.20 f 0.02 (mean f SEM), and similar with values previously found in normal rats (5). After 2 days in the uraemic rats L was 0.42 + 0.04 (significantly higher than in the conrols p < 0.001). After both 7 and 28 days in the uraemic rats L was 0.32 If: 0.02 (significantly higher than controls, p < 0.01, and lower than after 2 days, p < 0.02). Effect of total nephrectomy. Nephrectomy undertaken immediately before the determination of CUNS may influence the results. In 5 control rats therefore the total nephrectomy was omitted and catheters were placed in the urinary bladder for quantitative collection of urine. In these rats CUNS was calculated as:

CUNS = (dcu/dtw x 0.63 + Eu/dt) x (l/l-L) Eu is the urinary excretion of urea during the infusion. The notation is otherwise as above. CUNS was 9.15 + 1.23 Fmol/(min x 1OOgBW) in the rats with bladder catheters vs. 8.89 + 0.67 kmol/(min 1OOg SW) in the nephrectomised animals, i.e. identical. Therefore, the common mean is used as control value in the results section.

The established method involving total nephrectomy was maintained, since it is less technically demanding and results in smaller experimental variation. Statistical methods

Results are given as mean f SEM. Difference between two groups was evaluated by two-tailed t-test. Differences among more than two groups were evaluated by one way analysis of variance. p < 0.05 was considered statistically significant. Results

Uraemia for 2 days doubled the capacity for urea-N synthesis (Fig. p < 0.01). The capacity fell to low normal values on day 7, and then again increased with time to two times control value after 28 days (p < 0.05). Sham operation did not change the capacity of urea-N synthesis (Fig). Fasting blood glucose concentration did not change in any group (Table 1). Fasting blood total alpha-amino-N concentration did not change in the sham operated groups (Table 1). In the uraemic rats the concentration increased by about 50% from day 14 and on (p < 0.05). Fasting blood urea concentration did not change in sham operated rats. In uraemic rats it initially increased four-fold, and later only two-fold (p < 0.05) (Table 1).

CLINICAL NUTRITION Table 1 Fasting blood concentrations

in control rats and in sham operated and partially nephrectomised

209

rats at intervals after

surgery. Results are mean + SEM. Control Sham operated Glucose (mmoV1) Nephrectomised

21 day

28 day

4.7 f 0.1

4.5 f 0.2

6.3 + 0.5” 7.1 f 0.3

6.5 f 0.1” _

7.4 * 0.5” _

14.3 f 2.3”

18.8 + 2.3”

14.9 f 0.9”

2 day

7 day

14 day

5.2 + 0.3

4.4 + 0.1

4.3 + 0.1

5.1 + 0.5

4.6 f 0.5

4.3 f 0.3

5.9 * 0.4

5.0 f 0.2

4.7 f 0.2 6.5 f 0.4

5.4 * 0.5 6.7 f 0.4

27.7 + 4.0”

19.3 * 3.0”

4.1 + 0.3

Sham operated A-A-N (mmol/l) Nephrectomised

4.8 + 0.3

Sham operated Urea-N (mmol/l) Nephrectomised

7.0 + 0.7

4.1 f 0.2

a higher than control rats (p < 0.05) A-A-N = Blood alpha amino-nitogen concentration

Table 2 Fasting glucagon and insulin concentrations in control rats, in sham operated and partially nephrectomised after surgery. Results are mean f SEM. Control Sham operated Glucagon (q/l) Nephrectomised Sham operated Insulin (mu/l) Nephrectomised

rats at intervals

2 day

7 day

14 day

21 day

28 day

122 f 25

75 ? 20

107 f 23

_

152 + 22” 12.8 f 5.5

48 + 16” 14.7 f 2.9

112 f 20 lO.Of1.4

-

_

29.0 + 5.1”

11.5 f 4.8

20.5 z!z3.8”

13.3 * 2.7”

22.9 + 3.8”

90 + 5 842 11

92+ 11

9.0 f 1.1

a higher than control rats (p < 0.05) b lower than control rts (p < 0.05)

Table 3 Body weight, food intake and N-balance in control rats and in sham operated and partially nephrectomised rats at intervals after surgery. Results are mean f SEM. Pre-operative

2 day

7 day

14 day

21 day

28 day

Sham operated Body weight (g) Nephrectomised

225 f 3

222 f 4

235 + 7

235 f 5

245 f 5

257 + 7

232 + 4

209 f 3”

208 + 8”

226 + 6”

226 f 6”

228 * 7”

Sham operated Food-N intake (mmol/24 h) Nephrectomised

35.6 + 1.1

28.9 f 2.7

34.5 + 2.2

32.1 + 1.6

32.1 + 1.6

38.0 Y!Y 0.8

Sham operated N-balance (mmoU24 h) Nephrectomised

31.2 + 1.6

8.7 + 2.3”

30.0 f 2.4

32.5 + 2.0

30.9 + 2.8

36.0 f 2.8

11.2 f 1.6

8.8 f 2.9

10.0 + 2.2

11.5 + 2.9

11.0 f 1.4

11.0 f 1.4

9.2 f 2.1

-9.7 + 1.4”

a higher than control rats (p < 0.05) A-A-N = Blood alpha amino-nitrogen

concentration

8.9 + 1.6”

8.0 + 2.1”

7.6 + 1.5”

5.5 + 0.8”

210

UREA SYNTHESIS IN URAEMIA

Fasting plasma glucagon and plasma insulin (Table 2) were not changed by sham operation. Uraemia nearly doubled glucagon on day 2 (p < 0.05). Later, plasma glucagon levels did not differ systematically from initial values. Uraemia increased plasma insulin (p < 0.05), except on day 7. The body weight of the sham operated rats increased by about 8 g per week (Table 3)) whereas the uraemic rats lost 24g within the first week, and only gradually approached their initial body weight during the rest of the observation period. Food N-intake did not change in any of the sham operated animals (Table 3). Uraemic rats reduced their food intake to 20% on day 2 (p < O.Ol), and later ate as normal. Nitrogen balance remained constant and positive in the sham operated rats (Table 3). In the uraemic rats it shifted to negative on day 2. Thereafter, the N-balance was positive but increasingly lower, and on day 28 significantly so, than in sham operated rats (p < 0.05).

Experimental uraemia changed nitrogen balance and the hepatic capacity for urea synthesis biphasitally. In the early phase the capacity for urea synthesis doubled and the nitrogen balance became negative. In the late phase the capacity after a transient normalisation again doubled, and the nitrogen balance, although positive, was only half of normal. The model of uraemia was a surgical 5/6 partial nephrectomy (6). This was compatible with survival and induced a moderate degree of renal failure as evidenced by the increased blood urea concenimmediately tration. The total nephrectomy before the amino-acid load did not influence the results. The calculation of urea synthesis includes a measure of microbiotic urea metabolism. In the uraemic rats this fraction increased from 0.20 to 0.42 two days after partial nephrectomy, and later it stabilised at 0.32. This is in accordance with most other reports (12, 13, 14) but not all (15). The measurements of urea synthesis were standardised as to amino-acid concentration drive on the process by means of the exogenous amino-acid load. Since urea synthesis only takes place in hepatocytes, the capacity of urea synthesis then reflects the enzyme activities in the liver. The advantage of measuring the capacity for urea synthesis, which is the Vmax of the process, is that changes in this theoretically also apply to changes

at any other substrate concentrations. If CUNS is increased, this means that urea synthesis is also increased at any other amino-acid concentration. This technique has not been previously applied to uraemia, and the lack of standardisation of urea synthesis adds to the variation among reported results. In the acutely uraemic rats, i.e. 2 days after induction of uraemia, the capacity for urea synthesis doubled. This is in agreement with Persike et al an increase in urea (16) 7 who demonstrated excretion by acutely uraemic rats. Moreover, a number of studies report increased urea formation during acute uraemia, in liver slices (2) and in perfused rat livers (4, 17, 18). That the trauma of the nephrectomy is itself partly responsible for the increased CUNS cannot be excluded as it has been demonstrated that surgical trauma leads to an increased CUNS after 24h (19). After the early increase, the capacity for urea synthesis decreased transiently to low-normal values, and then again gradually increased to a value similar to the initial increase. This bi-phasic time course of the in vivo kinetics of urea synthesis may further explain the varying results reported from other studies. Brown et al (20) found increased activity of the urea cycle enzyme arginino-succinate synthetase 14 days after nephrectomy. Likewise, Klim et al (4) in hepatocytes isolated from 14 days uraemic rats found increased rate of amino-acid stimulated urea synthesis, and Perez et al found a slight increase in urea synthesis in isolated livers obtained from rats which had been uraemic for 14 days (18). Abitbol et al (12) found identical rates of urea synthesis in uraemic and control rats in vivo 2 - 6 weeks after partial nephrectomy. These rates were not standardised with regard to amino-acid concentration. Glucagon is an important regulator of the in vivo kinetics of urea synthesis (21), and sustained hyperglucagonaemia leads to increased synthesis and excretion of urea (22). Increased concentrations of glucagon have been demonstrated previously in acutely uraemic rats (23), due to an increase in release as well as a decrease in renal elimination. Major surgical trauma increased glucagon (19), but glucagon did not increase in the present sham operated rats. Uraemia changed glucagon in a time-dependent manner. The early increase was followed by a decrease and later normalisation. Thus only the early change in the capacity for urea synthesis can be explained by an increased glucagon concentration. Seven days after induction of uraemia reduced glucagon con-

CLINICAL NUTRITION

were seen. The explanation for this may be that the rats, during the first week of uraemia, took less food. It has been demonstrated that reduced protein intake results in decreased glucagon levels. After 2 weeks and onwards the blood aminoacid concentration was increased. It has been demonstrated that feeding with a high protein diet induces the capacity for urea synthesis (24), and it is therefore possible that the high capacity after 4 weeks is a result of the increased amino-acid concentration. Blood glucose concentrations were the same in all groups, but in the uraemic rats were associated with higher insulin concentrations, confirming relative insulin insensitivity in experimental uraemia (25). Insulin in itself reduces the capacity for urea synthesis linearly (26). This effect, however, seemed to be overridden by opposite effects in uraemia. The acutely uraemic rats had negative nitrogen balance, and lost weight during the first week. This catabolism is partly ascribable to the decrease in food intake (Table 3), but the increase in the capacity for urea synthesis also contributed, by leading to an increased elimination of endogenous amino-acids. On days 7-21 the N balance, urea synthesis capacity and body weight gain of uraemic animals did not differ from those in controls. Later, the N-balance, although still positive, fell in spite of normal food intake. This may be explained by the increase in the capacity for urea synthesis, in itself implying increased hepatic amino-acid elimination. However, the total blood amino-acid concentration also increased with time in uraemic rats. This is due to an increase in net organ amino-acid release exceeding hepatic elimination. The increase in amino-acid concentration may further increase the urea synthesis rate, by a substrate effect, independent of changes in the kinetics. The present study demonstrates that the hepatic capacity of urea synthesis increases in both acutely and chronically uraemic rats. The early increase is attributable to an effect of glucagon and the late increase may be due to induction of urea synthesis by high amino-acid concentrations. Nitrogen balance decreased when the capacity for urea synthesis increased. This suggests that increased urea synthesis may play a primary role in the nitrogen wasting of uraemia. Comparable data on human uraemia is not as yet available. centrations

Acknowledgements The skillful assistance by technicians B Krog and K Prissholm

D

211

is gratefully acknowledged. The antisera and iodinated hormones were generously supplied by NOVO, Denmark.

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19. Heindorff H, Almdal T, Vilstrup H 1990 Contradictory effects of uncomplicated versus complicated abdominal surgery on the hepatic efficacy for urea synthesis in rats. Jou%l of Surgical Research 49: 239-243 20. Brown C L. Houehton B J. Souhami R L. Richards P 1972 The effects of low-protein diet and uraemia upon urea cycle enzymes and transaminases in rats. Clinical Science 43: 371-376 21. Petersen K F, Hansen B A, Vilstrup H 1987 Time dependent stimulatory effect of glucagon on the capacity of urea-N synthesis in rats. Hormone and Metabolic Research 19: 53-56 22. Almdal T P, Vilstrup H 1988 Loss of nitrogen from organs in rats induced by exogenous glucagon. Endocrinology 123: 2182-2186

Submission

date: 5 September

1990; Accepted

after revision:

23. Emmanouel D S, Jaspan J B, Kuku S F, Rubenstein A H, Katz A I 1976 Pathogenesis and characterization of hyperglucagonaemia in the uraemic rat. Journal of Clinical Investigation 58: 12661272 24. Petersen K F, Vilstrup H, Tygstrup N 1990 Effects of dietary protein on the capacity of ,urea synthesis in rats. Hormone and Metabolic Research 22,612-615 25. Mondon A E, Dolkas C B, Reaven G 1978 The site of insulin resistance in acute uraemia. Diabetes 27: 571-576 26. Hansen B A, Krog B, Vilstrup H 1986 Insulin and glucose decreases the capacity of urea-N synthesis in the rat. Scandinavian Journal of Clinical and Laboratory Investigation 46: 599-603

15 March 1991