Portacaval shunt in the rat: An animal model for evaluation of uricase activity

Pharmacological Research Communications, VoL 10, No. 3, 1978

PORTACAVAL

195

SHUNT IN THE RAT: AN ANIMAL MODEL

FOR EVALUATION

OF URICASE ACTIVITY

Vincenzo

R. Olgiati*,

Umberto

Fox**,

Carmela Netti* and

Antonio Pecile* *

Department Medicine,

**

o£ Pharmacology University

Dep@rtment Medicine,

(3rd Chair),

o£ Milan,

Milan,

o£ Surgical Pathology University

o£ Milan,

School o9

Italy.

(3rd Chair),

Milan,

School o£

Italy.

Receivedin final form 21 February 1978

SUMMARY End-to-side portacaval

shunts were prepared

rats and their uricemia was measured the surgery.

7,14,21

between

and 30 days a£ter

Due to hepatic uricase exclusion,

crease in serum uric acid over preoperative 7 and 21 days a£ter operation.

~levels slowly declined,

in mature male

a sustained

levels was £ound

A£ter that, uric acid

probably as a consequence

tion o£ adhesions

between

The hyperuricemic

portacaval-shunted

in-

o£ the £orma-

the portal circulation and the liver.

model £or assaying exogenously help also in research studies

rat is a suitable animal

administere~

uricase,

and may

in the area o£ hyperuricemic

syn-

dromes.

INTRODUCTION In man,

higher apes,

main metabolic

birds and reptiles,

end-product

o£ xanthine metabolism.

mammalian species uric acid is converted case,

uric acid is the In the other

to al!antoin by uri-

an enzyme found mainly in the liver and lacking in those

0031-6989/78/0010-0195/$02.00/0

Pharmacological Research Communications, VoL 10, No. 3, 1978

196

species unable

to break doll uric acid to allantoin.

This ina-

bility and the occurence o£ gout only in man lead to the conclusion that the absence o£ uricase is a necessary primary condi[ion For the disease, abnormality.

in which hyperuricemia

Urica~e administration

riod may be o£ potential

For at least a limited pe-

therapeutic usefulness

ation o£ the activity o£ uricase preparations datory For drug controlling An attempt

is the main

and the evalu-

may become man-

pharmacological laboratories.

to produce an animal model For research

into

hyperuricemia and hyperuricosuria was made by Johnson et al. (1969),

who blocked the activity o£ hepatic uricase

in the la-

boratory rat by means o£ a selective inhibitor oF the enzyme. Oxonic acid,

or orally, was Found

when given intraperitoneally

to increase plasma and urine concentrations to decrease

o£ uric acid and

the excretion oF urinary allantoin.

quent injections

(or high oral doses,

for sustained uricase inhibition.

I g/day)

However Freare required

Moreover the experimental

condition proposed by Johnson et al. obviously

is not suitable

For evaluation oF the pharmacological

in vivo o£ exo-

genously administered uricase-like

activity

compounds

which might also

be inhibited by the oxonic acid as is the endogenous

enzyme.

In order to produce an animal model with sustained hyperuricemia,

suitable For pharmacological assays o£ various uricase

preparations,

we thought

might serve as a valuable (1972a) reported

that a rat with portacaval research tool.

that portacaval

dence o9 urate nephrolithiasis, els o£ aric acid,

shunt

Since Herz et al.

shunt produces a high incisuggesting

we decided to verify,

o9 the serum levels o9 uric acid,

sustained high lev-

by direct measurements

the eFFect o£ portacaval-

shunt in the rat, For a reasonable period o£ time after surgery (one month).

Once a sustained rise in uric acid had been ob-

tained in the portacaval-shunted

rat, the next step o£ the re-

Pharmacological Research Communications, VoL 10, No. 3, 1978

197

search program was to ascertain

if such a hyperuricemic animal

was siJitable for a q u a n t i t a t i v e

assay

MATERIALS

Sprague-Dawley" rats

~ere used.

der standard

(22+2°C

light 06:00-20:00)

et and water ad libitum.

End-to-side

clamp was then removed,

In all

the animals

preparation

the abdominal

extracted

14 h/ rat diwere

(1963).

A

the procedure

contents

The assays

replaced

The in the

was used.

into shunted rats,

and 320-mg/Kg

and 7,14,21

o£ serum uric acid

fla~!s

KH 2 PO 4 7.5 mg;

uricase

were drawn before

intravenously

treatment

10 mg and

or intramuscuIn intrave-

of 40,

volume of solvent

80,

160

(2.5 ml/Kg).

and 15, 30,

60 and

120 min.

In intramuscular experiments

after enzyme administration.

Lot

in a buffering

glucose

was given at doses

Au u r i c a s e

(CB 8129,

21 days postoperatively.

in a constant

uri-

at doses

o£ 80, 160,

320 and 640 mg/Kg in a

o9 solvent

(,2.5 ml/Kg)

and blood was d-awn be-

case was injected volume

before

The enzyme was dissolved

0.9% NaCI to 20 ml) and injected

nous experiments,

for uric acid de-

by Henry et ai.(1957).

from Aspergillus

(K2HPO 4 11.25 mg;

fore and 60,

et al.

in which

blood was collected

out as described

Midy)

constant

and

portacaval-shunts

from the cut tip o£ the tail,

were carried

Bloods

cages un-

the portal vein For 15 min.

and 30 days postoperatively.

larly

- 65% humidity

g body

cavity and the wound closed with clips.

termination

solvent

oE 250-300

in individual

by Bismuth

was done on 8 animals

was taken as far as clamping

peritoneal

River)

and given a standard

in 60 rats as described

sham operation

004-C,

(Charles

They were maintained

room conditions

day artificial

prepared

preparations.

AND METHODS

Male weight

of u r i c a s e

120,

240,

360 min.

[er surgery all the surviving

after treatment.

rats were weighed

One month aEand their tails

were cut £or blood collection and then they were killed by de-

Pharmacological Research Communications, VoL 10, No. 3, 1978

198

capitation. Patency and adequacy o£ the portacaval-shunt were verified in each instance by introduction of a probe From the vena cava to the portal vein. The livers From shunted and shamoperated rats were removed and weighed. All data were expressed as means + S.E. and the results were analyzed For probability of significance according to the Student t test.

RESULTS All animals remained in good health after the immediate postoperative recovery period. A marked loss o£ body hair, which became apparent two weeks after surgery, was noticed in ten shunted rats. Hair did not regrow in the area clipped For the midline incision and hair loss was observed in large ventral and dorsal areas. The body weight gain observed in shunted rats

(5+4 g) was

significantly smaller (p
(85+10 g).

In rats bearing portacaval-shunts, mean l i v e ~ weight decreased to 55% o£ that of sham-operated animals

(8.1 g vs

14.5 g, p < O.001). This tendency to hepatic atrophy may be viewed as a result o£ portal blood diversion,

causing the total

blood Flow through the liver to be reduced (Flynn and Kennan, 1968; Herz et al., 1972b). In agreement w i t h previous reports (Assal et al., 1971; Herz et al., 1972b), macroscopic evaluation of the livers of shunted rats did not reveal gross pathologic alterations. Serum uric acid levels, determined weekly £o~ one month (Fig. I), increased progressively,

reaching at 21 days post-

operatively a peak c6rmesponding to twice the basal (preoperative) values

(1.69!0.I vs 3.11~0.13, p < 0 . 0 0 1 ) .

From the 21st

day after portacaval-shunt onwards, serum uric acid levels

Pharmacological Research Communications, VoL 10, No. 3, I978 ..J

E

199

4 Shunted

rats

Sham-operated

5~

rats

@

4 S

S~ S

-

uJ

1

_

I[

0

. . . . . . . . . . . . . . . . . . . . . . . . . . .

i

-,,,.

-

_

.

I

7 Days

~ , _ _

~

_

14 after

I

.

.

.

.

.

.

.

.

.

.

.

21

portacaval

30

shunt

F i ~ . 1 - Serum uric acid concentration in 55 shunted rats (---) and in 8 sham-operated controls ( - - ) on di££erent days a£ter portacaval-shunt or sham-operation. The vertical bars represent standard errors. *Signi£icantly di££erent £rom sham-operated:p( 0.001. sho~ed a slight continuous decrease.

In sham-operated animals,

serum uric acid levels did not rise signi£icantly £rom the basal ones during the entire experimental period

(£ig.

I).

Uricase activity on lowering serum uricemia was tested on shunted rats 21 days pos~toperatively

(Table I). 0nly a slight

reduction in serum uric acid was seen as a result o£ intravenous solvent injection.

A dramatic decrease in uricemia imme-

diately £o!lowed the intravenous

I

injection o£ uricase enzyme

80

160

320

Uricase

Uricase

Uricase

**

40

Uricase

-

Di£Perence

- Dzfference

-

Solvent

Prom

from

(mg/Kg)

Dose

-2.02+0.28"

6

.

.

.

.

.

~

p z. O. 001'

p z-.0.01

.

-2.43+0.29**

~reate'J only ~ith sol'vent

control

inco rats

-2.11 +_0.24**

animals

6

-2.52+O. 3 1 ~

-2.48_+0.2 ,~**

-I ,35+0.25*

-0.91+0.27

-0.28÷0.14_

treated only with solvent

-2.59+O.25-*

-2.52+0.24**

-I ,95+0,48*

-I, 24+0.21-

-O,45+O,10

control animals

6

6

-2,38+0,32**

-O.73+O.14

6

injected

,

. _

. . . . . . . . . . .

~ -1.98+O. 38-

-I .24+0.21

-0.92+0.24

-0.98+0.24

-0,54+0,23

,,,,

Changes in serum uric acid (mg/IOO ml) versus pretreatment levels at various time Prom drug administration (Mean + S .E.) 15' 30' 60' ~ 120' --

6

rats

of'

Number

I - Effect on serum uric acid o£ uricase intravenously subjected to portacaval shunt 21 days earlier.

Treatment

TABLE

:m

G O

Pharmacological Research Comn~umcations, Vol. 10, No. 3, 197S

at all concentrations,

201

with a dose-related effect clearly ap-

parent at 30, 60 and 120 min. after drug adminis~nationo

At the

Final time (120 mi%.), only the larger dose produced a significant decrease~(p< O.O1). Urolytic ~ctivity o£ intramuscularly given enzyme (Table 2) was less impressive and was significant a

only at.the higher doses

(320 and 640 mg/Kg). At these doses

given by the ~ntramuscular route~ being sTil~ significant X injection of the enzyme.

~sting the

the effect was, however, more

(p< 0.05) even 360 rain. after

DISCUSSION Thep~esent por~acaval-shunt

investigation has shown that in the rat with a an increase in serum uric acid becomes appar-

@nt From'the FirSt postoperative values

days. Uric acid levels reach

twice the basal ones and the increase i~ sustained For

abo~t 20 days an i £ollo~ed by a slow decline thereafter. Although in shunted rats urate nephrolithiasi~=

and hydroneph-

rotic.kidneys containing urate crystals have been observed (Herz et al.,

1972a; Kyu and Cavanagh,

changes in uric acid metabolism, uric acid in portacaval-shunted

1970), suggesting

no measurements

o£ serum

rats have previously been re-

ported. As is known, in the rat uric acid is not'the main metab01fc endproduct of xanthine metabolism, to allantoin by the hepatic enzyme uricaseo

but is broken down The reduction o£

the hepatic circul~tiQn and of the Functioning consequent to portacaval-shunt conversion_-o~u~ic-acid~

liver cell mass

is associated with diminished

to a ~ l a n t o i ~

probably through hepatic

urfcase exclu~i0n, so that sustained hyperuricemia and increased uric acid excretion ensue. The tendency.of s@~um uric acid levels to-decline From the third Week after surgery sugI

gest the £ormaZion Qf_adhesi0ns

between the portal circulation

and the liver, This lowering of hyperumicemia might therefore represent

in Shunted r a t s

good indirect~evidence

For the degree

160

320

640

Uricase

Uricase

Uricase

5

5

5

5

5

Number of rats

injected into rats

subjected

-1.30+0.35"

-0.54!0.57

-0.54+0.41

-0.43+0.25

-0.20+0.04

-2.32+0.22**

-I .31+O.55

-I. 37+0.40

-0.88+0.56

-O. 79+0.2 7

** Difference From control animals

treated only with solvent

p
p,~ 0 . 0 %

-I .97+0.4!**

-I. 72+0.45*

-1.26+0.45

-0.38+0.28

-O. 06+0.32

-I. 52+0.342

-1.50+0.42*

-1.09+0.45

-0.61+0.22

-O. 08+0.21

Changes in serum acid (mg/100 ml) versus pretreatment levels at various time from drug administration (Mean + S.E.) 60' 120' 240' 360'

earlier.

Difference from control animals treated only ~ith solvent

80

Uricase

*

-

(mg/Kg)-

Dose

Solvent

Treatment

to portacaval shunt 21 days

TABLE 2 - Effect on serum uric acid of uricase intramuscularly

~O

<)"

~o

o

Pharmacological Research Communications, Vol. 10, No. 3, 1978

203

to which portal blood is Finding its way back to the liver. As For the use o£ the hyperuricemic portacaval-shunted

rat

For testing the pharmacological effects on uricemia o£ uricase preparations,

we have chosen an enzyme of Fungal origin that

has already been Found to be effective in clinical experiments (Royer et al.,

1967; Royer et al.,

1968; Kissel et al.,

1968)

and that was also tested in anesthetized dogs surviving a Few hours after a portoFemoral anastomosis

(Brunaud et al.,

1969).

Our reported Findings show that the proposed animal model is suitable For the assay of the activity o£ uricase preparations to be used as therapeutic agents in hyperuricemic syndromes.

Considering that the sustained hyperuricemia

in portacaval-

shunted rats simulates a condition associated with a numer o£ human diseases and systemic disorders psoriasis,

glycogen ~torage disease,

Nyhan syndrome and leukemia),

(gout, diabetesmellitus,

sarcoidosis, the Lesch -

we hope that the animal model

proposed by us in which hyperuricemia

is not obtained by ad-

ministration o£ any inhibitor o£ uric acid metabolism may be useful not only For assays of uricase preparations, out also as a valuable research tool in a large a r e ~ o £ medical research.

ACKNOWLEDGEME~[TS We are very grateful to Midy S.p.A.

(Milan) For providing

the uricase enzyme. The skillFui-assistance

of Dr. Ottorino

Pozzi and Miss Marina Pucci is greatly appreciated.

REFERENCES Assal, J., tevrat, R., Cahn, T. and Renold, A E., (1971), Z.ges.exp.Med., 154, 87 B~smuth, H.~ Benhamon, J.P. and Lataste, J., (1963), Pr@sse m@d., 39~ 1859 Brunaud, M., Gros, P., Navarro, I. and Raveux, R., (1969), Th~rapie, 24, 785

Pharmacological'Research. Communications, VoL 10, No. 3, 1978

204

Flynn, P.J. and Kennan, A.L., (1968), Archs.Path., 85, 138 Henry, R.J., Sobel, C. and Kim, J., (1957), Am.J.clin. Path., 28, 152 Herz. R., Sautter, V. and Bircher, J., (1972a), Experientia, 28, 2"/ Herz, R., Sautter, V., Robert, F. and Bircher, J., (1972b), Eur.J.clin. Invest., 2, 390 Johnson, W.J., Stavric, B. and Chartrand, A., (1969), Proc. Soc. exp. Biol.Med., 131, 8 Kissel, P., Lamarche, M. and Royer, R., (1968), Nature, 217, 72 Kyu, M.H. and Cavanagh, J.B., (19'70), Br.J.exp. Path., 51, 217 Royer, R., Lamarche, M and Kissel, P , (1967), Th_rapze, 22, 1113

Royer, R., Vindel, J., Lamarche, Pr@sse m~d., 76, 2325

M. and Kissel, P., (1968),