Blunted natriuresis and abnormal systemic hemodynamic responses to C-type and brain natriuretic peptides in rats with cirrhosis

Journal of Hepatology 1995; 22: 319-325 Printed in Denmark . AN rights reserved

Copyright B Journal

of Heparology 1995

Journal of Hepatology

Munksgaard . Copenhagen

ISSN 0168-8278

Blunted natriuresis and abnormal systemic hemodynamic responses to C-type and brain natriuretic peptides in rats with cirrhosis Hirokazu

Komeichi,

Richard

Moreau,

Stephane

Cailmail, Christophe

Gaudin

Laboratoire d’H&modynamique Splanchnique, Unit6 de Recherches de Physiopathologie Hdpatique (INSERM Clichy, France

and Didier Lebrec U-24), H6pital Beaujon,

Background/Aims: The effects of C-type and brain natriuretic peptides (CNP and BNP, respectively) on renal excretion of sodium and hemodynamics have not yet been studied in cirrhosis. Methods: This study aimed to examine the effects of saline, CNP (300 ng - kg-’ - n&r-* i.v. for 30 min) and BNP (600 ng - kg-’ * min-’ i.v. for 30 min) on natriuresis and diuresis in normal and rats with cirrhosis. Moreover, regional and systemic hemodynamics were measured prior to and following CNP and BNP administration in normal rats and rats with cirrhosis with ascites. Results: In rats with cirrhosis, the effects of CNP or BNP on natriuresis and diuresis did not significantly differ from the effects of saline. CNP significantly decreased portal pressure and systemic vascular resistance and significantly increased the cardiac index. BNP significantly decreased portal tributary blood

flow, portal pressure and cardiac index. In normal rats, natriuresis and diuresis were significantly higher with CNP and BNP than with saline. Systemic hemodynamics were not changed by CNI! A decrease in arterial pressure was the sole BNP-induced hemodynamic change. Conclusions: In conclusion, this study shows that the natriuretic response to pharmacological doses of CNP and BNP is blunted in rats with cirrhosis. This blunting may be related to an activation of the endogenous anthratriuretic systems secondary to systemic vasodilation (by CNP) or to a decreased cardiac index (by BNP). Finally, this study shows that CNP and BNP have a portal hypotensive action.

I

CNP and ANP stimulate different receptors, the natriuretic response to CNP may be preserved in cirrhosis. In addition, since BNP may stimulate another receptor than NPR-A (see above), the natriuretic response to BNP may also be preserved in chronic liver disease. This hypothesis is supported by the finding that BNP increased natriuresis in patients with congestive heart failure who have a blunted natriuretic response to ANP (21). As a result, CNP or BNP may be new therapeutic tools for sodium retention and extracellular fluid accumulation in cirrhosis. The aim of the present study was to examine the effects of pharmacological doses of CNP and BNP on natriuresis and diuresis in rats with cirrhosis and in normal rats. Moreover, since both CNP and BNP are vasorelaxant substances in vitro (17-19), these peptides may accentuate the cirrhosis-induced vasodilation in vivo. Thus, the systemic and regional hemodynamic re-

patients and animals with cirrhosis, the administration of atria1 natriuretic peptide (ANP) does not increase sodium excretion (1-16). Thus, the utilization of this substance for the treatment of sodium retention and extracellular fluid accumulation is limited (3). On the other hand, the effects on sodium excretion of the two other members of the family of endogenous natriuretic peptides, i.e., C-type and brain natriuretic peptides (CNP and BNP respectively) (17,18) are unknown in cirrhosis. It should be kept in mind that ANP, CNP and BNP increase natriuresis by interacting with natriuretic peptide receptors (NPR), i.e., NPR-A for ANP, NPR-B for CNP and NPR-A or an as yet unidentified NPR for BNP (17-20). Since N CERTAIN

Received 5 April 1994 Correspondence: Dr. R. Moreau, INSERM U-24, Hopital Beaujon, 92118, Clichy, France.

Key words: Portal hypertension; Sodium excretion; Splanchnic circulation; Systemic circulation. 0 Journal of Hepatology.

319

H. Komeichi et al.

sponses to CNP and BNP were measured in normal rats and rats with cirrhosis.

Materials and Methods Animals

One hundred and ten male Sprague-Dawley rats (Charles River Laboratoires, Saint-Aubin-Lbs-Elbeuf, France) were divided into two groups. One group had secondary biliary cirrhosis with portal hypertension as a result of bile duct ligation, as previously described (22). Under ether anesthesia, the common bile duct was exposed by median laparotomy and occluded by double ligature with a nonresorbable suture (7-O silk). The first tie was made below the junction of the hepatic ducts, and the second was made above the entrance to the pancreatic ducts. The common bile duct was then resected between the two ligatures and the abdominal incision closed. Hemodynamic studies were performed 4-5 weeks after bile duct ligation, this delay being necessary for the development of so-called secondary biliary cirrhosis (23). The diagnosis of cirrhosis was confirmed by macroscopic examination of the liver and the existence of portal hypertension. In a second group, composed of normal rats, hemodynamic studies were performed 4-5 weeks after surgery. All rats were allowed free access to food and water until 14-16 h before the study, when food was withdrawn. Protocols performed in this laboratory were approved by the French Agricultural Office in conformity with European legislation for research involving animals. Protocols

Two sets of experiments were performed. First, the effects of saline (33 @min for 30 min), CNP (300 ng * kg-’ - min-‘i.v. for 30 min) and BNP (300 and 600 ng . kg-’ - min- ’ i.v. for 30 min) on natriuresis and diuresis were measured in normal rats and rats with cirrhosis. All rats with cirrhosis were killed and their abdominal cavity was then explored. Second, regional and systemic hemodynamics were measured prior to and following the administration of saline (33 ,ul/min for 30 min), CNP (300 ng * kg-’ * min-’ i.v. for 30 min) and BNP (300 and 600 ng . kg-’ - min-’ i.v. for 30 min) in normal rats and rats with cirrhosis. A 30-min infusion of 300 ng * kg-’ * min-’ (i.e., 9 &kg) of CNP and BNP was chosen because blunted natriuretic and hemodynamic responses to a similar dose of ANP have already been shown in rats with cirrhosis in our laboratory (ref. 24 and unpublished results). Moreover, a 9 &kg dose of CNP was chosen because preliminary results have shown that this dose did not decrease arterial pressure in rats with cirrhosis. Indeed, arterial pressure was 10123 mmHg, under baseline

320

conditions, and 99+3, 9822, 100~1, 98~2 and 9525 mmHg, following 3, 6, 9, 18 and 50 &kg of CNP (n= 7). In addition, a 600 ng * kg-’ . min-’ dose of BNP was chosen since a 300 ng * kg-’ . min-’ dose did not increase natriuresis in normal rats (see below). Urinary measurements

Animals were placed in metabolic cages where spontaneously voided urine was collected for 2 h. After this delay, and following an abdominal massage, a final urine sample was obtained (25). Urinary concentrations of sodium and potassium were measured using flame spectrophotometry. Hemodynamic measurements

Four hours before hemodynamic measurements, catheters were inserted under light diethyl ether anesthesia. Catheters were inserted into: a femoral artery to measure arterial pressure and heart rate; a femoral vein to give natriuretic peptides; and the portal vein to measure portal pressure. Briefly, the abdomen was opened and a polypropylene catheter (0.7 mm diameter) was inserted into a small ileal vein and gently advanced to the bifurcation of the superior mesenteric and the splenic veins. The abdominal incision was closed with catgut. The left ventricle was cannulated via the right carotid artery. All catheters were fixed to the external vascular walls and then tunneled subcutaneously to the back of the neck. Hemodynamic studies were performed in conscious unrestrained rats (26). Cardiac and regional blood flows were measured by the radioactive microsphere method and the reference sample method as previously described (22). For the first set of hemodynamic measurements, a precounted aliquot of approximately 60 000, 165 1 pm diameter, 141Ce-labeled microspheres (spec. act. 10 mCi/ g; New England Nuclear, Boston, MA, USA), suspended in Ficoll 70 (10% Pharmacia Fine Chemicals AB, Uppsala, Sweden) and Tween 80 (0.01%) and ultrasonically agitated, was injected into the ventricular catheter and flushed with 1 ml of isotonic saline for 45 s. During microsphere injection, a reference blood sample was drawn from the catheter in the femoral artery into a motor-driven syringe at 0.8 ml/min for 1 min. For the set of second hemodynamic measurements, an injection of 113Sn-labeled microspheres was given and the same technique was used. The animal was then killed with an overdose of pentobarbital sodium. Individual organs were dissected and placed in individual tubes for counting with a gamma-counter (Computer Gamma G 4000; Kontron, Montigny-LeBretonneux, France) at energy settings of 70-210 and 280-1000 keV for ‘13Sn and ‘41Ce, respectively. Errors

Natriuretic peptides in rats with cirrhosis

due to the spillover of the 113Sn and 141Cechannel were corrected using ‘13Sn and i41Ce standards. Adequate microsphere mixing was assumed with a difference ~10% between the left and right kidneys. Cardiac index (CI) was calculated from the following formula: CI (ml * min-’ . 100 g-‘)=[radioactivity (cpm)/reference blood sample radioactivity [loo/body wt (g)]XO.8 (ml/min). Systemic vascular resistance from the following formula:

.

.

injected (cpm)]~ &Mm

!

qNP

@oon#.kf#-!d’ loraoomh)

BNP (ma w #lo-?mh-’ loraomh,

Fig. 1. Values for natriuresis following the administration of saline (n=5), C-type natriuretic peptide (CNP, 300 ng * kg-’ * min-’ t.v. . for 30 min. n=8) or brain natriuretic peptide (BNP, 300 and 600 ng * kg-’ * min-’ i.v. for 30 min, n=8 and 6, respectively) in rats with cirrhosis. Mean values for natriuresis (horizontal bars) were 1.3+-0.8 ,umol/ min following saline, 1.2r0.5 pmol/min following CNP, 1.4kO.2 pmobmin and 1.81fI1.2 ,umol/min following a 300 and 600 ng * kg-’ . min- ’ BNP infusion, respectively.

SVR [(dyn * s . cmW5 * 100 g)X 103]=mean arterial pressure (mmHg)X80/CI (ml . min-’ - 100 g-i). Regional blood flows were calculated from the following formula: Organ blood flow (ml - min- 1 * 100 g- ‘)=[organ radioactivity (cpm)/radioactivity injected (cpm)]XCI (ml * min-’ * 100 g-l). Portal tributary blood flow was calculated as the sum of stomach, intestine, colon, spleen, and mesentericpancreas flows. Portal territory vascular resistance (PTVR) was calculated from the following formula:

(mmHg)X80/portal min-’ - 100 g-l).

tributary

blood

flow

(ml -

Renal vascular resistance (RVR) was calculated from the following formula:

PTVR [(dyn . s . cmp5 - 100 g)X 103]= [mean arterial pressure (mmHg)-portal pressure (mmHg)] X 80/partal tributary blood flow (ml - min-’ * 100 g-l). Hepatic artery vascular resistance (HAVR) was calculated from the following formula:

RVR [(dyn * s - cms5 * 100 g-‘)X 103]=(mean arterial pressure (mmHg) X 80/renal blood flow (ml * min-’ * 100 g-1). Substances CNP-22 (27) and rat BNP-32 were purchased

HAVR [(dyn * s . cmp5 * 100 gg’)X lO]=mean arterial pressure (mmHg)X80/renal blood flow (ml * min-’ * 100 g-1).

from

Sigma Chemicals (St. Louis, MO, USA). Statistical

Hepatocollateral vascular resistance (HVR) was calculated from the following formula:

TABLE

CNP (anollg rg-! lnlc loralmh)

(SVR) was calculated

HVR [(dyn * s * cmp5 * 100 gg’)X lO’]=portal

-G-

*.

analysis

Values are mean+S.E. The Mann-Whitney U-test and Student’s paired t-test were performed where appropriate.

pressure

1

Diuresis and natriuresis following saline or a 30-min infusion of C-type natriuretic peptide (BNP; 300 and 600 ng . kg-’ . mint) in normal rats and rats with cirrhosis

peptide

Normal

Urinary concentrations Sodium Potassium Diuresis @urnin) Natriuresis @moVmin) Kaliuresis @rnoYmin) a Means?S.E.

(CNP;

300 ng

. kg-’ . min-‘)

or brain

natriuretic

Cirrhosis

Saline

CNP

BNP

(n=6)

(n=8)

300 (n=8)

37213” 57219 16+4 0.520.1 0.9*0.3

4659 31?8 39*7b 2.120.7s 1.220.3

12+-4 28t7 33-c7 0.520.2 0.7?0.1

Saline

CNP

BNP

;nt 6)

(n=S)

(n=8)

300 (n=8)

EZ6)

3524 51?15 64+13b 2.0?0.3b 2.420.4

47%14 62511 3O-clO 1.3kO.8 1.9-r-0.8

29?10 56210 39?4 1.2kO.5 2.220.5

2828 36t5 42%5 1.420.2 1.4eO.2

41218 42210 3529 1.821.2 1.4kO.3

@mol/ml)

b Significantly

different

from saline (p
321

H. Komeichi et al.

Results In rats with cirrhosis, mild ascites was found in all but four animals which had large ascites (one rat per group, saline, CNP and each dose of BNP). In rats with cirrhosis, the effects of CNP or BNP on natriuresis and diuresis did not differ significantly from the effects of saline (Table 1 and Fig. 1). In normal rats, natriuresis and diuresis were significantly higher with CNP and the highest dose of BNP than with saline (Table 1 and Fig. 2). Natriuresis measured following saline was not significantly different between normal rats and rats with cirrhosis. In rats with cirrhosis, CNP significantly decreased portal pressure (923%) and systemic vascular resistance (112 5%) and significantly increased cardiac index (1424%) (Table 2). The lowest dose of BNP induced no significant changes in regional and systemic hemodynamics. The highest dose of BNP significantly decreased portal tributary blood flow (19+5%), portal pressure (12+ 1%) and cardiac index (1123%). Saline did not significantly modify hemodynamics (Table 2). In normal rats, saline did not significantly modify systemic hemodynamics, while it significantly decreased portal tributary blood flow and increased portal territory vascular resistance (Table 3). The lowest dose of BNP significantly decreased portal tributary blood flow and increased portal territory vascular resistance. A decrease in arterial pressure was the sole

6-

.

Natriuresio (pmolhnln) . .

3.

f

I

J.

P.zfl.05 I

Fig. 2. Values for natriuresis following the administration of saline (n=6), C-type natriuretic peptide (CNP, 300 ngekg-‘emin -’ iv. for 30 min. n=8) or brain natriuretic peptide (BNP, 300 and 600 ng * kg-’ . mine1 i.v. for 30 min. n=8 and 6, respectively) in normal rats. Mean values for natriuresis (horizontal bars) were 0.520.1 ,umollmin following saline, 2.120.7 ,umol/min following CNP (pcO.O.5 vs. saline), 0.5kO.2 pmol/min following a 300 ng * kg-’ * min- t BNP infusion, and 2.OkO.3 pmobmin following a 600 ng . kg-’ . mint BNP infusion (p
322

hemodynamic change following the highest dose of BNI? Regional and systemic hemodynamics were not significantly changed by CNP (Table 3). Under baseline conditions, portal pressure, cardiac index, portal tributary and hepatic artery blood flows were significantly higher in rats with cirrhosis than in normal rats. Vascular resistance in systemic, portal, hepatic arterial and renal territories was significantly lower in rats with cirrhosis than in normal rats.

Discussion In this study, the natriuretic and diuretic responses to pharmacological doses of CNP or BNP occurred in normal animals but not in animals with cirrhosis. A blunted natriuretic response to ANP has also been shown in patients and animals with cirrhosis (1-16). Taken together, the findings with ANP (1-16) and the results of the present study with CNP and BNP indicate that cirrhosis is associated with a decreased natriuretic response to all members of the natriuretic peptide family. Natriuresis measured following saline tended to be higher in rats with cirrhosis than in normal rats. In other words, there was no evidence that rats with cirrhosis had decreased sodium excretion after saline. Since the natriuretic responses to CNP and BNP were compared to natriuresis following saline, no information on the natriuretic responses to CNP or BNP in rats with cirrhosis and “low sodium excretion following saline” was provided. Because of this limitation, the results obtained with CNP and BNP in this study cannot be extrapolated to patients or animals with low sodium excretion (under baseline conditions or following saline administration). However, it would be surprising if CNP or BNP significantly increased natriuresis in animals with cirrhosis and low “saline-induced natriuresis”. Indeed, in this case, a blunted natriuresis to natriuretic peptides should be expected. In patients with cirrhosis, the lower the baseline natriuresis, the lower is the natriuretic response to ANP (2). The finding that natriuresis measured after saline tended to be higher in rats with cirrhosis than in normal rats may indicate differences in baseline natriuresis between these two groups of animals. Such a difference, in turn, could partly explain why natriuretic responses to CNP and BNP were blunted in rats with cirrhosis. In this study, CNP and BNP altered the hyperdynamic circulation observed under baseline conditions in rats with cirrhosis. In these animals, CNP enhanced the systemic vasodilation while BNP de-

Natriuretic peptides in rats with cirrhosis TABLE

2

Effects of saline or a 30-min infusion of C-type ng . kg-’ . mint) in rats with cirrhosis

natriuretic

peptide

Saline

Mean arterial pressure (mmHg) Heart rate (beats/min) Cardiac index (ml . min-’ . 100 g) Systemic vascular resistance (dyn . s . CIII-~. 100 gX 10’) Portal pressure (mmHg) Portal tributary blood flow (ml . min-’ f 100 g) Portal territory vascular resistance (dyn . s. cme5. 100 gX103) Hepatocollateral vascular resistance (dyn . s cme5 . 100 gX 103) Hepatic artery blood flow (ml. min-’ . 100 g) Hepatic artery vascular resistance (dyn . s . cmm5 . 100 gX 103) Renal blood flow (ml. min-’ . 100 g) Renal vascular resistance (dyn . s . cmes 100 gX 103) a Means+S.E.

TABLE

b Significantly

. kg-’ ’ min-‘)

CNP

(n=5) Baseline

(CNP, 300 ng

peptide

(BNP

300 and 600

BNP

(n=8) After

on brain natriuretic

Baseline

300 (n=5) After

Baseline

600 (n=7) After

Baseline

After

9123” 413213 48.2L3.6

92+4 420? 13 46.523.1

9823 386? 15 46.923.0

9822 432*17b 53.0-c2.2b

94k-3 390+13 58.023.2

8925 4142 19 58.722.4

156217

162+18

171*11

150*7b

13157

123210

1942 14

14.620.8 6.420.6

14.4kO.9 5.920.5

14.1kO.7 6.520.6

12.7+0.6b 6.220.4

16.621.3 7.5eo.5

14.821.5 7.5-cO.8

18.01r1.4 5.9kO.4

15.921.4b 4.8?0.5b

1003+307

1084&112

10202 173

1022% 147

829%30

815573

1178299

1443*193

190?25

200515

189223

170216

18028

162220

24328

277?17

2.7~0.5

3.220.2

2.820.7

3.220.6

3.320.6

4.2kO.6

2.0+0.2

2.1kO.l

42592960

3338,868

2589%511

1928?417

4519?634

36312124

3.650.2

3.820.3

4.650.6

4.520.5

3.520.3

3.220.3

22232110

2155?164

17192194

16332123

2463e-201

3182?627 3.620.1 20535115

different

2292? 173 3.6%0.1 20592 102

from baseline

10222 369215 43.2k2.3

9423 410? 12s 38.4?2.6b 19929

2483?246

($0.05).

3

Effects of saline or a 30-min infusion ng. kg-‘. mint) in normal rats

of C-type

a Means2S.E.

b Significantly

peptide

(CNP

300 ng

. kg-’ . mint)

on brain natriuretic

Saline

CNP

BNP

(n=6)

(n=8)

300 (n=8)

Baseline Mean arterial pressure (mmHg) Heart rate (beats/min) Cardiac index (ml . min 'X 100 g) Systemic vascular resistance (dyn . s. cmm5 . 100 gX 103) Portal pressure (mmHg) Portal tributary blood flow (ml . min-’ 100 g) Portal territory vascular resistance (dyn . s . cmm5 . 100 gX 103) Hepatocollateral vascular resistance (dyn . s . cmm5 . 100 gX 103) Hepatic artery blood flow (ml min-’ . 100 g) Hepatic artery vascular resistance (dyn . s . cmd5 100 gX 103) Renal blood flow (ml. min-’ . 100 g) Renal vascular resistance (dyn s cmm5 . 100 gX 103)

natriuretic

After

Baseline

11052a 385215 35.222.0 254215

10922 380214 32.7~ 1.5 2702 14

11123 388?12 33.62 1.2 266212

8.720.2 5.220.4

8.7’0.2 4.6-c0.3b

7.lkO.5 5.520.3

16255 144

17732 127b

15622125

139+13

152?11

0.56?0.14

0.74?0.13b

2283326482

1392422689

different

2.720.1 3254% 152

After 11323 39926 34.121.0 268213 7.OeO.6 4.8kO.2 1813297

Baseline

peptide

(BNP

300 and 600

600 (n=8) After

Baseline

After

114*5 35928 37.521.6 251+14

11524 403?19 36.42 1.6 255?9

11323 357? 14 35.022.4 267?19

109?3b 3942 14 33.22 1.6 2662 12

7.7kO.3 6.820.5

7.1 kO.2 5.0%0.5b

7.6kO.2 5.820.4

7.020.4 4.6kO.4

1270272

1852t187b

1503tllO

1875+158

10828

129?13

107?11

119211

94?8

122-t 12

0.60?0.10

0.70?0.21

0.81?0.16

0.76+0.16

0.89+0.10

1.11+0.17

1955923576

1383122330

1985527455

27451212131

11100~1215

955321718

2.6kO.l

2.720.2

2.7kO.l

3.8kO.3

3.1eo.4

3.020.2

2.820.1

34672244

3319?246

3471~242

25242264

3028?208

3173?323

3161+231

from baseline

(~K0.05).

323

H. Komeichi et al.

creased the high cardiac output state. Since these effects are known to evoke a reflex activation in the endogenous antinatriuretic systems (28,29), this may be the mechanism for the loss of the natriuretic properties of CNP and BNP in rats with cirrhosis. On the other hand, renal hypoperfusion was not the cause of the cirrhosis-induced reduction in the natriuretic response to CNP or BNP since these peptides did not significantly change renal hemodynamics. Finally, since the present study did not examine the characteristics of NPRs, a downregulation of these receptors cannot be ruled out in the mechanism of the cirrhosis-induced blunting of natriuretic responses to CNP and BNP Since CNP and BNP are cleared, in part, from the circulation by receptors termed NPR-C (17,18), an upregulation of these receptors may increase plasma clearance of natriuretic peptides and decrease their availability at the level of their biologically active renal receptors. This decreased availability may induce blunted renal responses to natriuretic peptides. Some evidence for a cirrhosis-induced upregulation of NPR-C has been shown in isolated glomeruli from rats with cirrhosis (30). This study shows that CNP and BNP induced a portal hypotensive effect. The BNP-induced decrease in portal pressure was secondary to the reduction in portal tributary blood flow. The mechanism for the CNP-induced portal hypotensive effect is unclear, since it was not associated with significant changes in portal tributary blood flow or hepatocollateral vascular resistance. In certain studies, CNP did not increase natriuresis and induced a systemic vasoconstriction ‘in normal animals (31,32). Thus, the pattern of the hemodynamic and renal responses to CNP in normal animals differed between these findings (31,32) and the present study. These discrepant results may be due to differences in species (dogs vs. rats), doses (10, 50 or 100 ng * kg-’ . mini vs. 300 ng * kg-’ - mini), conscious state (anesthetized vs. conscious animals). On the other hand, the results of the present study confirm previous findings which showed that CNP is a natriuretic and vasorelaxant substance (27). In conclusion, this study has shown that the natriuretic response to pharmacological doses of CNP and BNP is blunted in rats with cirrhosis. Blunting of the natriuretic response to these peptides may be related to an activation of the endogenous antinatriuretie systems secondary to systemic vasodilation (by CNP) or a decreased cardiac index (by BNP). Finally, this study has shown that CNP and BNP have a portal hypotensive action. 324

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