Changes in plasma renin activity and renal immunohistochemically demonstrated renin in carbadox treated pigs

Research in Veterinary Science /989. 46. 40/-405

Changes in plasma renin activity and renal immunohistochemically demonstrated renin in car bad ox treated pigs E. J. VAN DER MOLEN*, J. H. L. M. VAN LlESHOUT, M. J. A. NABUURS, Central Veterinary Institute, PO Box 54, 8200 AB Lelystad, The Netherlands, F. H. M. DERKX, Erasmus University, Rotterdam, The Netherlands, A. P. M. LAMERS, University of Nijmegen, Nijmegen, The Netherlands, A. M. MICHELAKIS, Michigan State University, USA

Carbadox is known to induce toxic effects on the adrenal cortex, resulting in hypoaldosteronism. To study the involvement of carbadox on the reninangiotensin system, weaned piglets of five weeks old received feed supplemented with 0 (control group), 50, 100, 150 or 200 ppm carbadox. After four weeks the 100 and 150 ppm groups had significantly higher plasma renin activity levels than the control group and after nine weeks plasma renin activity levels of all treated groups were significantly higher than the control group. Five and 10 weeks after carbadox administration, three and two pigs, respectively, of all groups were necropsied and the kidneys were screened for immunohistochemically demonstrated renin. All dosed pigs demonstrated an increase of immunoreactive renin, which was dose- and time-related. From these results it is concluded that carbadox induces activation of the renin-angiotensin system, secondary to the suppressing effect on mineralocorticoid secretion and that these changes rna}' be responsible for part of the clinical picture. CARBADOX, a quinoxaline di-N-oxide compound, is widely used in the pig industry as a growth promotant and as an antibacterial drug to prevent gastrointestinal infections especially in weaned pigs (Gropp et al 1971, Aumaitre and Raynaud 1978, Blobel and Schliesser 1979). A few years after its introduction in the Netherlands, carbadox became suspected of inducing toxic effects in weaned pigs. The most important clinical signs were: growth retardation, wasting, urine drinking and a strong interest in salt products. In an earlier study (Van der Molen et al 1986) a suppressing effect of carbadox on aldesterone blood levels in weaned pigs was found, combined with changes of plasma sodium and potassium. The biochemical changes were correlated with pathomorphological changes in the adrenal cortex. Carbadox could induce specific changes in the zona glomerulosa, which suggested a hyper- rather

'Dr Van der Molen died on September 19. 1987

than a hypofunction of the glomerular cells (Van der Molen 1987). Therefore it was supposed that the blocking effect of carbadox on aldosterone biosynthesis or release was followed by stimulation of the zona glomerulosa via negative feedback mechanisms. One of the mechanisms involved in this stimulation might be the renin-angiotensin system (RAS), which is one of the main aldosterone regulating mechanisms (Muller 1971, Stockigt 1975, Nelson 1980). This study was designed to investigate the involvement of the RAS in carbadox intoxication. Plasma renin activity levels were measured in weaned pigs which received different doses of carbadox. In addition, in kidneys of necropsied pigs renin activity was estimated, using an immunohistochemical method. Materials and methods For this study pigs were used from an earlier experiment, in which the effect of car bad ox on aldosterone biosynthesis was studied (Van der Molen et al 1986). The pigs came from a commercial farm and were weaned at four weeks. After one week of acclimatisation they were randomly divided into five groups of 13 animals each. One group served as the control and received a commercially available baby piglet feed unsupplemented. The remaining four groups received the same diet supplemented with 50, 100, 150 or 200 ppm (mg kg- J) carbadox (Mecadox; Pfizer). The feed was allowed ad libitum for 10 weeks. Once a week heparinised blood samples were taken from the same 10 animals in each group. In the 200 ppm group, however, nine and eight pigs were sampled, respectively, because mortality occurred in week I (n = 12) and week 9 (n = 8). The samples taken at weeks 3, 4, 6 and 9 were analysed for plasma renin activity (PRA) according to the method described by Bailie et al (1980). The data were statistically analysed by the Wilcoxon test. At five and 10 weeks, three and two pigs, respectively, of the control, 50, 100 and 200 ppm groups were necropsied. The kidneys were removed under

401

Van der Molen, Van Lieshout, Nabuurs, Derkx, Lamers, Michelakis

402 40

* *

.,

30

s: I

E

c: cQ) 20

'iii

o

Oppm

[ill]

100 ppm

~

150 ppm

. 2 0 0 ppm

0

'OJ c: III

0

E

0-

10

*

o FIG 1: Plasma renin activity (PRAI in control. 100, 150 and 200 ppm dosed groups after three, four. six and nine weeks carbadox administration (mean ± SEM). 'Significantly different from the control group (P<0·05)

anaesthesia induced with azaperone (Stresnil; Janssen Pharmaceutica) 2 mg kg" I intramuscularly followed by metomidate (Hypnodil; Janssen Pharmaceutica) 10 mg kg" I intravenously. Tissue samples were immediately fixed in a 10 per cent formalin solution. The tissues were dehydrated by standard techniques and embedded in paraplast , Sections (5 J.lm) were prepared for the immunohistochemical procedure as described by Lamers et al (1985). The antiserum, used in a I :400 dilution, had been raised in rabbits with highly purified renin isolated from the submandibular gland of the mouse (Michelakis et al 1974). In two stained sections from each pig counts were performed of the total number of glomeruli and of the number with immunoreactive juxtaglomerular cells (JGc). This was achieved using an eye piece graticule (integration plate II 100125; Zeiss) at magnification x 100. In each section 10 fields were counted, five in the outer and five in the inner zone of the renal cortex. The percentage of glomeruli with immunoreactive JGC was calculated from the counts in the two sections. Moreover, the degree of granulation in the JGC in each section was graded (+ slight, + + moderate, + + + strong and + + + + very strong immunoreactivity). All histology was done 'blind'. Results

Plasma renin activity (Fig 1) No significant differences in PRA between carbadox-treated groups and controls were observed

after three weeks of carbadox administration. The 100 ppm dosed group showed a somewhat lower mean PRA, but the value was not significantly different from the control group. There was a moderate spread of the values in all groups, with the largest spread in the 200 ppm group (0' I to 45' 0) and the smallest in the control group (5'4 to 40'0) . After four weeks significantly higher renin concentrations were seen in the 100 and ISOppm group, but not in the 200 ppm group. The latter, like the control group, demonstrated a decrease of renin concentrations compared with the three weeks value, but the decrease was less, and the mean value in this group remained about twice the control value. The spread in the 100 and ISOppm groups was about five times that of the control group (4'9 to 50,7, 2· 3 to 51·6 and I· 5 to 11'1, respectively). The 200 ppm group showed an intermediate position (0' I to 26· 2) with four values lower than the lowest control value. After six and nine weeks all carbadox-treated groups had significantly higher renin concentrations than the controls. The mean values were not, however, dose-related. After six weeks the 200 ppm group showed a lower value than the 100 and ISOppm groups. The spread in the treated groups was nearly of the same magnitude (2'1 to 27· 3,0'8 to 28· 3 and O' 8 to 28' 5 in 100, ISOand 200 ppm groups, respectively), and about seven times that of the control group (I ·2 to 4'9). The 200 ppm group had five animals, the values of which fell within the spread of the control group, against two in the ISO ppm and one in the 100 ppm group. After nine weeks the 100 and 200 ppm groups demonstrated a nearly similar mean level of PRA while the ISO ppm group showed an approximately 40 per cent lower value than the 100 ppm group. The spread in all treated groups was

TABLE 1: Renin immunoreactivity after five weeks carbadox administration

Animal number

1685 1686

1688

1700 1702 1710 1641 1648 1649 1669 1671 1675

+

Dose (ppml

Percentage of glomeruli with positive JGC

o o o

50 50 50 100 100 100 200 200 200

Slight immunoreactivity

Moderate immunoreactivity ++ Strong immunoreactivity +++ + + + + Very strong immunoreactivity

24 32 23 56 44 38 66 68

54 66

64 61

Grade of granulation in JGC

+

+ + ++ ++

++

++++ +++ +++ +++ +++ +++

Renin activity in carbadox intoxication in pigs

403

'1/1:

"

..

~

..

FIG 2: Kidney; 0 ppm carbadox, five weeks. Normal renin immunoreactivity; small proportion of the glomeruli demonstrates immunostained afferent arterioles. x 55

FIG 3: Kidney; 200 ppm carbadox, five weeks. Increased renin immunoreactivity; a larger number of glomeruli have immunostained afferent arterioles. x 55

of the same order (100 ppm: 0'6 to 25'3; ISO ppm: O' 8 to 19'Oand 200 ppm: O' 8 to 21'6) and moderately higher than in the control group (0' 4 to 2· 2). When comparing the values within each group, the control group showed a marked decrease from three weeks treatment. This was also true for the 200 ppm group, though the decline occurred much slower and was very slight from four weeks treatment. The 100 and 150 ppm groups demonstrated an initial increase from week 3 to week 4, followed by a moderate decline between four and six weeks and a slighter decrease between six and nine weeks.

of renin immunoreactivity compared with the control values (Table 1, Figs 2,3,4 and 5). The percentage of glomeruli with immunoreactive JGC was moderately higher in both groups. All individual pigs showed a higher percentage than the controls. Moreover, the grade of granulation in the JGC had increased too in both groups (Figs 2 and 3). A dose-response effect could not be observed when comparing the data of the 100 and 200 ppm dosed pigs. The groups showed a similar response of both parameters. The 50 ppm group had a weaker response than the other groups, but for both parameters the response was clear. All pigs in this group had a higher percentage of glomeruli with immunoreactive JGC combined with a higher grade of granulation compared with the controls. After 10 weeks of carbadox administration all dosed groups showed an increase of renin immuno-

Immunohistochemical demonstration of renin in the kidney After five weeks of carbadox administration the 100 and 200 ppm groups showed an obvious increase

.~:

,.

FIG 4: Kidney; 0 ppm carbadox, five weeks. Normal renin immunoreactivity in juxtaglomerular cells of afferent arterioles. x 140

FIG 5: Kidney; 100 ppm carbadox, five weeks. Strongly increased renin immunoreactivity in juxtaglomerular cells of afferent arterioles. x 140

404

Van der Molen, Van Lieshout, Nabuurs, Derkx, Lamers, Michelakis

.-

'"

,

....

\~r

. ., 'I ~.,

.: -I'

.>

'~J":'.,

I

FIG 6: Kidney; 0 ppm carbadox, 10 weeks. Normal renin immunereactivity in small proportion of glomeruli's afferent arterioles. x 55

FIG 7: Kidney; 200 ppm carbadox, 10 weeks. Increased renin immunoreactivity. A large number of glomeruli demonstrate afferent arterioles with increased immunostaining. x 55

reactivity demonstrated by a higher percentage of glomeruli with immunoreactive JGC compared with the controls (Table 2, Figs 6 and 7). There was a tendency that at higher dosages a higher percentage of immunoreactive glomeruli was found. In the 50 and 100 ppm groups the average percentage was about twice and in the 200 ppm group about three times the average in the control group. The grade of granulation in JGC increased in all dosed groups, though in the 50 ppm group only one of two pigs showed a higher granulation than the controls. When comparing the dosed groups a doseresponse effect was visible, most clearly at the higher dosages.

week of administration of 100 ppm carbadox or more, while a clear response of PRA at these dose levels was not seen until four weeks of treatment in this study. There appears to be a delay in renin response in the 100 and 150 ppm treated pigs of about three weeks. The lower response in the 200 ppm group at four and, to a lesser degree, at six weeks, must be attributed to very low PRA levels seen in some members of this group (lower than or equal to control values). The reason for this remains unclear, but it might be that the renin release in the kidney in these pigs was suppressed, or angiotensinogen production in the liver might become partly exhausted at this dosage. In this case the peak of PRA might have been earlier in this group and therefore not measured. From the immunohistochemical data it appears that renin production at 200 ppm dosage was not suppressed as similar renin immunoreactivity was found as at 100 ppm dosage after five weeks. After 10 weeks the activity was even higher in the 200 ppm dosed pigs. Stimulation of the RAS causes plasma angiotensin II levels to increase, which enhances the production of

Discussion These combined biochemical and immunohistochemical studies suggest that the RAS is involved in carbadox intoxication in pigs. The results of this study confirm the hypothesis, based on earlier results, that the stimulated picture of the adrenal glomerulosa is induced by a stimulated RAS (Van der Molen 1987). Thus suppression of aldosterone production or release by carbadox must be the primary event. Decreased aldosterone secretion stimulates the reninangiotensin system due to sodium depletion and extracellular volume contraction (Vecsei et al 1978, Dzau and Pratt 1986). Because of the negative feedback mechanism between aldosterone and renin production (Eisenstein 1967) the results of this study can be considered as a secondary response to the inhibiting effect of carbadox on aldosterone production or release. This becomes clear when combining the data on the aldosterone decline of these pigs from our earlier study (Van der Molen et al 1986) with the I'RA levels in this study. The aldosterone decline occurred within one

TABLE 2: Renin immunoreactivity after 10 weeks carbadox administration

Animal number 1689 1691 1706 1715 1646 1653 1667 1668

Dose (ppml 0 0

50 50 100 100 200 200

Percentage of glomeruli with positive JGC 18 26 47 38

56

37 57 64

• Degree of immunoreactivity as for Table 1

Grade of granulation in JGC'

+ ++ + +++ +++

+ + + + + + + +

Renin activity in carbadox intoxication in pigs aldosterone from the adrenal glomerulosa (Nelson 1980). This may, therefore, be the main factor inducing the enlarged glomerular zone found histologically after five weeks treatment with 50 ppm and above (Van der Molen 1988). It may explain the physiological paradox seen in these pigs, namely reduced blood aldosterone combined with enlargement of the glomerular zone. Therefore the carbadoxinduced aldosterone decline can be considered as a hyperreninaemic form of hypoaldosteronism, with the adrenal as the primary target organ for the drug. RAS stimulation seen in this study may also be responsible for the recovery of aldosterone blood levels found in the present authors' earlier study after longer administration of carbadox (Van der Molen et al 1986). The clinical condition then changes from a hyperreninaemic form of hypoaldosteronism to normal aldosterone blood levels, but still with an elevated PRA. The damaged glomerular zone is apparently able to maintain a normal aldosterone blood level if PRA is elevated. So the aldosterone data in fact reflect an apparent clinical recovery (Nabuurs and Van der Molen 1989). The pigs still suffer from an activated RAS with high angiotensin II plasma levels with potential consequences for blood pressure, blood volume and renal function. The elevated PRA might also be responsible for a specific symptom seen in carbadox intoxication, ie, depraved appetite, wall licking, urine drinking and an appetite for salt (Nabuurs and Van der Molen 1989) This sign ~dlso persisted when aldosterone levels had returned' to normal values and carbadox treatment was stopped. It is known from studies in rats that intracerebral or intravenous administration of angiotensin II will arouse a salt appetite which can be blocked by captopril, an angiotensin-converting enzyme inhibitor (Bryant et al 1980, Findlay and Epstein 1980, Moe et al 1984, Weiss et al 1986, Sakai et al 1986). Elevated PRA with consequently increased angiotensin II levels might also be the pathophysiological base for this typical clinical feature, even after carbadox administration was stopped. Summarising the results in this study and those from earlier work it can be concluded that in addition to the disturbed mineralocorticoid secretion of the adrenals, carbadox has a secondary effect on the RAS. This may explain, at least partly, the recovery from the state of hypoaldosteronism. Moreover, it might be responsible for part of the clinical picture, especially those signs which persist after aldosterone has returned to normal levels.

405

Acknowledgements

The authors wish to express their gratitude to Mr F. J. Propsma for preparing the figures, and to Ms H. Pees for typing the manuscript. References AU MAITRE, A. & RAYNAUD, J. P. \1978) Zeitschrift fur Tierphysiologie, Tierernahrung und Futtermittelkunde 40. 67-74 BAILIE, M.P .• DERKX, F. H. M. & SCHALEKAMP. M. A. D. N. \1980) Developmental Pharmacology and Therapeutics I. 47-57 BLOBEL, H. & SCHLIESSER, T. \1979) Handbuch der bakteriellen Infektionen bei Tieren, Band I. Jena, VEB Gustav Fischer Verlag. PI' 506-509 BRYANT. R. W., EPSTEIN, A. N .• FITZSIMONIS, J. T. & FLUHARTY. S. J. \1980) Journal of Physiology, London 301. 365-382 DZAU. V. J. & PRATT. R. E. \1986) The Heart and Cardiovascular System. Vols I and 2. Ed H. A. Fozzard. New York. Raven Press. 1'1'1631-1662 EISENSTEIN, A. B. \1967) The Adrenal Cortex. London. J. and A. Churchill. PI' 217-232 FINDLEY. A. L. R. & EPSTEIN. A. N. \1980) Hormone Behaviour 14.86-92 GROPP. J., TlEUWS, J. & HEIDECKE. F. W. (1971) Zeitschrijt fur Tierphysiologie, Tierernahrung und Futtermittelkunde 28. 218-223 LAMERS, A. P. M., VERHOFSTAD, A. A. J., STADHOUDERS, A. M. & MICHELAKlS, A. M. (1985) Cell Tissue Research 239. 677-682 MICHELAKIS. A. M., YOSHIDA. N.. MENZIE. J .. MURAKAMI. K. & INAGAMI. T. \1974) Endocrinology 94. 1101-1105 MOE, K. E .• WEISS. M. L. & EPSTEIN, A. N. (1984) American Journal of Physiology 247. R356-365 MULLER. J. \1971) Regulation of Aldosterone Biosynthesis. Heidelberg. New York. Springer Verlag NABUURS, M. J. A. & VAN DER MOLEN, E. J. \1989) Journal of Veterinary Medicine (Series A) NELSON. D. H. (1980) The Adrenal Cortex: Physiological Function and Disease. Vol 18. Philadelphia. W. B. Saunders. 1'1' 89-101 SAKAI. R. R., NICOLAIDIS. S. & EPSTEIN, A. N. (1986) American Journal of Physiology 251. R762-768 STOCKIGT. J. R. (1975) Mineralocorticoid Hormones. Advances in Steroid Biochemistry and Pharmacology. Vol 5. Eds M. H. Briggs and G. A. Christie. London, Academic Press. PI' 161-230 VAN DER MOLEN. E. J .• DE GRAAF. G. J .• BAARS. A. J. & SCHOPMAN, W. \1986) Journal of Veterinary Medicine (Series A) 33, 617-623 VAN DER MOLEN, E. 1. \1988) Journal of Comparative Pathology 98, 55-67 VECSEI. 1'., HACKENTHAL. F. & GANTEN, D. \1978) Klinische Wochenschrift 56. 5-21 WEISS, 1\1. L., MOE, K. E. & EPSTEIN, A. N. (1986) American Journal of Physiology 250. R250-259

Received September 4. /987 Accepted November 30. /988