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J. Physiol. (1977), 271, pp. 505- 514 With 1 text-figure Printed in Great Britain
CHANGES IN RENAL CYCLIC NUCLEOTIDE CONTENT AS A POSSIBLE TRIGGER TO THE INITIATION OF COMPENSATORY RENAL HYPERTROPHY IN RATS
BY S. E. DICKER AND A. L. GREENBAUM From the Departments of Chemistry and of Biochemistry, University College London, London WC1E 6BT
(Received 25 January 1977) SUMMARY
1. Cyclic adenosine 3', 5'-monophosphate (cyclic AMP) and cyclic guanosine 3', 5'-monophosphate (cyclic GMP) have been estimated in the kidneys of rats. 2. Ten minutes after unilateral nephrectomy there was a threefold increase of cyclic GMP in the remaining kidney, which was accompanied by a moderate fall of cyclic AMP. 3. The changes in cyclic nucleotides in the remaining kidney after unilateral nephrectomy were of short duration. 4. When an anephric rat was cross-circulated with a normal litter-mate, there was an increase of cyclic GMP concentration in the kidneys of the latter, which reached its maximum 10 min after the establishment of the cross circulation. 5. In experiments where one kidney of a litter-mate was transplanted to the neck of another rat, unilateral nephrectomy was not followed by changes of the level of cyclic nucleotides in either the transplanted or the remaining kidney. Bilateral nephrectomy, however, resulted in a marked increase of cyclic GMP in the transplanted kidney. 6. The clamping of the blood vessels to one kidney for periods up to 10 min had the same effect as unilateral nephrectomy on the concentration of cyclic GMP in the remaining kidney. When the clamp was removed and the circulation restored, the concentrations of cyclic nucleotides returned to preoperative levels in both kidneys. INTRODUCTION
It has been suggested that the cyclic nucleotides, adenosine-3', 5'-monophosphate (cyclic AMP) and guanosine-3', 5'-monophosphate (cyclic GMP), may be involved in the regulation of cell growth. An increase of cyclic AMP
506 S. E. DICKER AND A. L. GREENBA UM has been shown to be associated with inhibition of cell growth, whereas increased cellular cyclic GMP concentrations appear to be associated with the initiation ofcellgrowth (Hardman, Robinscn & Sutherland, 1971; Posternak, 1974; Goldberg, Haddox & Nicol, 1975). Since it is well established that compensatory renal hypertrophy is an adaptive process which starts almost immediately after removal of one kidney it was of interest to see whether changes in the concentration of cyclic nucleotides could be detected in the early stages after unilateral nephrectomy. A preliminary account of some of the present findings has been published (Dicker, 1976). METHOD
Techniqum Adult male Wistar rats (220-250 g body weight) were used. For cross-over circulation experiments involving two or more animals litter-mates were used. All animals were anaesthetized with an i.P. injection of Inactin (Na-ethyl-methylpropyl-malonyl-thiourea), 10 mg/100 g body wt. Unilateral or bilateral nephrectomy was performed through a lumbar incision, care being taken to leave the adrenal gland intact. For cross-over circulation experiments, the technique described by Moolten & Bucher (1967) and used by van Vroonhoven, Soler-Montesinos & Malt (1972) was followed. As for the transplantation of kidneys to the carotid vessel of rat, the technique described initially by Govaerts for the dog (1936) was adopted. It consists essentially in an anastomosis between the lower aorta and the lower vena cava below the renal, vessels to the carotid and the jugular vessels of another animal. It allows for a perfusion of the 'transplanted' kidney without interruption of its blood supply, the kidney remaining in situ. Unilateral nephrectomy was performed in the minimum time and with the minimum handling of the kidney. The removed kidney was immediately frozen by clamping in Wollenberg tongs precooled in liquid nitrogen. The frozen tissues were plunged into liquid nitrogen and subsequently weighed. They were homogenized in 0-6 N perchloric acid solution (1: 5) with an ultra-turrax blender (Janke & Kunkel, Breisgau), spun in a refrigerated MSE centrifuge at 6000 revlmin for 5 min, and the precipitate discarded. The supernatant was neutralized to pH 6 6-6 8 with KOH and stood on ice for 30 min. After a second centrifugation, the supernatant was collected and stored at -20 °C, until used. All operations were carried out at 0 °C, and the cyclic nucleotides were estimated with kits prepared by the Radiochemical Centre (Amersham, England) for cyclic AMP and cyclic GMP respectively. Adenosine-5'-triphosphate (ATP) was estimated according to the method of Adam (1965). All assays were made in duplicate. Data are given as means and their standard errors.
RESULTS
Values of concentrations of cyclic 3',5' monophosphate (cyclic AMP) and of cyclic guanosine 3',5' monophosphate (cyclic GMP) in control kidneys were 681-2 + 14-36 (12) and 59-1 + 3-98 (23) x 10-12 mole/g wet tissue, respectively, with an average cyclic AMP/cyclic GMP ratio of the
CYCLIC NUCLEOTIDES IN KIDNEYS
507
TABLE 1. Content of cyclic nucleotides in the remaining kidney after unilateral nephrectomy
Time after unilateral nephrectomy Omin 40 min 10 min 5m 20 min 82-2 45-9 59.1 64-1 160-3 Cyclic GMP ± 22-95 (10) ± 11-60 (4) ± 2-32 (4) ± 8-72 (5) ± 3-98 (23) 511-2 546-5 691-0 681-2 478-0 Cyclic AMP ± 31-76 (4) ± 19-08 (4) + 14-36 (12) ± 12-71 (5) ± 41-08 (7) Cyclic GMP=cyclic-3',5'-guanosine monophosphate, cyclic AMP=cyclic-3',5'adenosine monophosphate. Results are means+s.E. and are expressed as 10-12 mole/g kidney wet wt. In parentheses, number of experiments. There was no significant difference between values found in control kidneys (time 0) and in those of sham-operated animals. For the values of cyclic GMP at 5 min, see test.
order of 11. These values agree well with those reported by Goldberg, Dietz & O'Toole (1969), Steiner, Pagliara, Chase & Kipnis (1972), and Kobayashi & Fang (1975).
Effect of unilateral nephrectomy on cyclic AMP and cyclic OMP Rats were killed 5, 10, 20, 40, 80, 120 and 240 min after unilateral nephrectomy and the contents of cyclic AMP and cyclic GMP in the remaining kidneys were compared with those of the removed kidneys (taken as controls) or those found in kidneys of sham-operated animals, killed after 5, 10, 80 and 120 min. Levels of both cyclic nucleotides were similar in control kidneys and in kidneys from sham-operated animals. The measurements of the cyclic nucleotides were most variable in rats killed 5 min after unilateral nephrectomy. In two of the five rats killed at this time, the cyclic GMP content had fallen by 20 and 30 %, with no significant changes in the level of cyclic AMP, while in the remaining three animals the concentration of cyclic GMP had increased by between 30 and 40 %, and the level of cyclic AMP had fallen by about 50 %. Taking the group of five rats as a whole, however, 5 min after unilateral nephrectomy there was an increase of 8 % in cyclic GMP level, and a fall of 30 % in the concentration of cyclic AMP in the remaining kidney (Table 1). In contrast, in all rats killed 10 min after unilateral nephrectomy there was a threefold increase of the cyclic GMP level, accompanied by a moderate (25 %) decrease of cyclic AMP, in the remaining kidney. The rise of cyclic GMP was of short duration, and was only 36 % higher than the control values, 20 min after the operation (Table 1). Changes of cyclic AMP/cyclic GMP ratios in the remaining kidneys over the whole experimental period of 4 hr are represented in Fig. 1, from which it will be seen that 80 min after
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unilateral nephrectomy the renal levels of both cyclic nucleotides were nearly back to preoperative levels. Effects of cross-circulations between anephric and normal rats Two series of experiments were performed. In the first series, bilaterally nephrectomized rats were cross-circulated with normal animals. Ten to fifteen min were allowed between the bilateral nephrectomy and the onset of the cross-circulation which usually lasted 10 min. The removed kidneys of the anephric animal served as controls, and their levels of cyclic AMP and cyclic GMP were compared with those of the intact animal at the end of the cross-circulation. The results showed that at the end of the crosscirculation, the level of cyclic GMP in the kidneys of the normal animals had increased from 65-7 + 5-75 (8) in the control kidneys to 169-2 + 8-49 (8) x 10-12 mole/g. This rise was accompanied by a moderate fall of cyclic AMP from 718 + 12-22 (8) to 671-0 + 7.59 (8) x 10-12 mole/g wet wt. In another series of experiments, one anephric rat was cross-circulated first with one normal animal for 10 min. As soon as the kidneys of the latter had been removed and frozen in liquid nitrogen, the cross-circulation
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S. E. DICKER AND A. L. GREENBAUM was switched to another normal rat. After 10 mi, its kidneys having been removed and frozen, cross-circulation was switched to a third normal animal and the circulation was maintained for another period of 10 min. Allowing about 2 min for removing the kidneys of each test animal, freezing and weighing them, the total duration of the cross-circulation was of the order of 35 min. The level of cyclic GMP in the kidneys of the three normal rats cross-circulated with one anephric animal increased in keeping with the above results although for the 3rd cross-circulation the level of cyclic GMP had declined almost to the control value and was not statistically different from it (Table 2). 510
Effect of 'transplanting' a kidney to a unilaterally nephrectomized rat The kidney of a litter-mate was 'transplanted' to the carotid of another rat. As soon as the circulation to the 'transplanted' organ was established, the left kidney of the recipient animal was removed. The circulation to the 'transplanted' kidney was maintained for 10 or 20 min, when both the recipient's own and the 'transplanted' kidneys were removed, and immediately frozen in liquid nitrogen. In five out of six experiments, the levels of cyclic AMP and cyclic GMP were normal: 702.2 ± 12*25 and 56'0 ± 1.04 x 10-12 mole/g in the 'transplanted' organ and 698-4 ± 947 and 62*7 ± 2*09 x 10-12 mole/g, in the recipient's own kidney. In the sixth experiment, blood clotting in the carotid prevented normal circulation to the 'transplanted' kidney. In this case, cyclic GMP concentration in the recipient's kidney was raised to 186-6 x 10-12 mole/g, while that of cyclic AMP was 607 x 10-12 mole/g kidney wet wt. Finally, in two experiments, a kidney was 'transplanted' to the neck of an anephric rat, and its circulation maintained for 10 and 12 min. The levels of cyclic GMP in the transplanted kidneys were similar to those found in the remaining kidney 10 min after unilateral nephrectomy: 151*7 and 145*8 x 10-12 mole/g, respectively. No estimation of cyclic AMP was performed in these two experiments.
Effect of clamping renal blood ves8els of one kidney A small clamp was put on the blood vessels of the left kidney for limited periods of time of 10 or 20 min after which both kidneys were removed for estimation of the cyclic nucleotides. In some cases, the clamp was removed after 10 min and the blood was allowed to circulate through the kidney again for 10 min. In others, resumption of blood flow, after a period of 10 min clamping, was accompanied by right unilateral nephrectomy. From these experiments (Table 3) four effects were observed. First, the clamping of the renal vessels of one kidney had the same effect on the
CYCLIC NUCLEOTIDES IN KIDNEYS 51J level of cyclic nucleotides as unilateral nephrectomy. Secondly, after releasing the clamp and allowing the blood to circulate again, levels of cyclic nucleotides were similar to those found in control kidneys. Thirdly, when clamps were released from renal vessels simultaneously with unilateral nephrectomy, 10 min later, there was a rise of cyclic GMP in the remaining kidney, though somewhat smaller than usual. Fourthly, the changes in cyclic nucleotides concentration were independent of those of ATP concentrations. DISCUSSION
From the results of the present investigation it would appear that the level of both cyclic nucleotides remains unchanged in the kidneys of a rat only if it has two functional kidneys, even if one of these is a transplanted organ from a litter-mate. If, however, one kidney is removed, or if the circulation to one kidney has been stopped, there is a characteristically sharp increase in the level of cyclic GMP in the remaining kidney, usually accompanied by a small decrease of cyclic AMP. Schlondorff & Weber (1976) also found that after unilateral nephrectomy there was a considerable rise of cyclic GMP content accompanied by a moderate decrease in the level of cyclic AMP in the remaining kidney. According to Schlondorff & Weber (1976) the increase of cyclic GMP lasted several days: however, from the evidence presented here the increase is short-lived, and has disappeared 40 min after unilateral nephrectomy (Table 1). This agrees with Solomon, Wise, Ratner & Sanburn (1976), who did not find any change in the concentration of cyclic nucleotides in the remaining kidney 1 h after nephrectomy. Compensatory renal hypertrophy is characterized essentially by cell hypertrophy, hyperplasia being minimal (Malt, 1969): it is of interest that whereas the onset of compensatory renal hypertrophy appears to be associated with a sharp increase of cyclic GMP level in the remaining kidney, renal hyperplasia as observed after administration of folic acid, or liver hyperplasia after partial hepatectomy, are both associated with a marked increase of cyclic AMP and a decrease of cyclic GMP (Berridge, 1975; Solomon et al. 1976). The earliest known changes occurring after unilateral nephrectomy are a small but significant increase of the systemic blood pressure and an enhanced renal blood flow. Though these may have an effect on subsequent renal hypertrophy. it is unlikely that they are the cause of an elevated level of cyclic GMP in the remaining kidney, since increases of cyclic GMP content were observed during cross-circulations between one anephric and a normal rat where it is unlikely that the renal blood flow would be increased. Nor can an increase in cyclic GMP be attributed to a nervous I7-2
S. E. DICKER AND A. L. GREENBAUM 512 stimulation, since it was observed in kidneys 'transplanted' to the carotid of an anephric animal. What then produces the changes in cyclic nucleotides in the remaining kidney? The evidence presented here suggests that the effect may be due to the existence of a humoral factor, the identity of which is at present unknown. This is indicated by the observations that there was a marked increase of cyclic GMP in both kidneys of a normal animal cross-circulated with an anephric rat, and that the switching of the cross-circulation from one anephric animal to a second or even third normal rat still evoked an increase of the particular cyclic nucleotide in their kidneys. It will also be remembered that van Vroonhoven et al. (1972) showed that after 48 hr of cross-circulation between an anephric animal and normal rat, there was an increase of RNA concentration in the kidneys of the latter, which disappeared when the cross-circulation was interrupted (Dykhuis, van Urk, Malamud & Malt, 1975). The existence of a humoral factor has also been postulated by Lyons, Evan, McLaren & Solomon (1974) who observed that the plasma from a unilaterally nephrectomized hamster stimulated the incorporation of [3H]uridine into RNA in cultures of hamster renal cortical cells in vitro, through the plasma of a partially hepatectomized animal did not have that effect. If a humoral renotrophic factor exists, why should it appear to be effective when one kidney only is functional, e.g. after unilateral nephrectomy or after the circulation to one kidney has been stopped? Since according to micro-puncture studies the first signs of renal adaptation after the exclusion of one kidney do not appear before 5-10 min (Dirks, 1976), it is conceivable that immediately after the removal of one kidney the normal 'clearance' activity of the remaining kidney is impaired, and that the temporarily decreased 'clearance' of the humoral renotrophic factor is responsible for the changes in cyclic nucleotides. This view is supported by the observation that complete adaptation of the remaining kidney appears to be effective about 60-80 min after functional exclusion of one kidney (Diezi, 1976), a time when the concentration of renal cyclic nucleotides, especially that of cyclic GMP, has returned to normal levels. This concept of a short-lived decreased renal clearance of a renotrophic factor would explain first, the rapid increase in the content of cyclic GMP in the remaining kidney after unilateral nephrectomy, as well as the short duration of the changes in the levels of cyclic nucleotides; secondly, the increase of cyclic GMP in one kidney after the interruption of the blood flow to the other and the rapid return to normal levels of cyclic GMP, as soon as its blood supply is restored; thirdly, the increased renal level of cyclic GMP observed during a cross-circulation between normal and
513 CYCLIC NUCLEOTIDES IN KIDNEYS anephric rats; fourthly, the absence of changes in the concentration of cyclic nucleotides in the remaining kidney when unilateral nephrectomy was performed in an animal which had a 'transplanted' kidney.
The support of an Emeritus Leverhulme Fellowship and of a grant from the Medical Research Council are gratefully acknowledged by one of us (S.E.D.). S. E. D. would like to thank Dr B. Banks for allowing him to operate in her laboratory and Professor M. McGlashan for his hospitality. We would like to express our appreciation to Miss C. A. Morris for her skill and technical help.
REFERENCES ADAM, H. (1965). Adenosine 5'-triphosphate. Determination with phosphoglycerate kinase. In Method8 of Enzymatic Analysis, ed. BERGMEYER, H. U., p. 539. New York, London: Academic Press. BERRIDGE, M. J. (1975). The interaction of cyclic nuoleotides and calcium in the control of cellular activity. Adv. cyclic nucleotide Res. 6, 1-98. DICKER, S. E. (1976). Changes in renal cyclic nucleotides as a trigger to the onset of compensatory renal hypertrophy. Int. Symp. on Renal Adaptation to Nephron Loe8. Montreux: to be published. DIEzI, J. (1976). Acute functional compensatory adaptation in rats: micropuncture and clearance studies. Int. Symp. on Renal Adaptation to Nephron Lo88, Montreux: to be published. Dmxs, J. H. (1976). Acute functional adaptation to nephron loss: micropuncture studies. Int. Symp. on Renal Adaptation toNephron Lom8, Montreux: to be published
UI4, H., MALAMUD, D. & MALT, R. A. (1975). Rapid reversal of compensatory renal hypertrophy after withdrawal of the stimulus. Surgery 78, 476-480. GOLDBERa, N. D., DiETZ, S. B. & O'TooLE, A. G. (1969). Cyclic guanosine 3',5'monophosphate in mammalian tissues and urine. J. biol. Chem. 244, 4458-4468. GOLDBERG, N. D., HADDOX, M. K. & NICOL, S. E. (1975). Biologic regulation through opposing influences of cyclio GMP and cyclic AMP: the Yin-Yang hypothesis. Adv. cyclic nucleotide8 Re8. 5, 307-330. GOVARTS, P. (1936). Le fonotionnement du rein malade. Recherche8 Experimentale et Clinique8. Paris: Masson. HARDMN, J. G., ROBINSON, G. A. & SUTHERLAND, E. W. (1971). Cyclic nucleotides. Rev. Physiol. Biochem. Pharmacol. 33, 311-316. KOBAYASHI, R. & FANG, V. S. (1975). A simple and sensitive competitive proteinbinding assay for cyclic GMP. Biochem. biophys. re8. Common. 67, 473-500. LyoNs, H. J., EVAN, A. R., McLAREN, L. C. & SOLOMON, S. (1974). In vitro evidence for a renotrophic factor in renal compensatory hypertrophy. Nephron 13, 198-21 1. MALT, R. A. (1969). RNA metabolism in compensatory renal growth. In Compen. satory Renal Hypertrophy, ed. NownNsiu, W. W. & Goss, R. J. London: Academic
DYKuuiS, C. M., VA
Press.
MOOLTEN, F. L. & BUCHER, N. L. R. (1967). Regeneration of rat liver: transfer of humoral agent by cross-circulation. Science, N.Y. 158, 272-273. PosTERNAR, T. (1974). Cyclic AMP and cyclic GMP. A. Rev. Pharmac. 14, 23-33. ScHLONDORrF, D. & WEBER, H. (1976). Cyclic nucleotide metabolism in compen. satory renal hypertrophy and neonatal kidney growth. Proc. natn. Acad. Sci. U.S.A. 73, 524-528.
614
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SOLOMON, S., WIsE, P. M., RATNER, A. & SANBORN, C. (1976). Cyclic nucleotide concentrations in relation to renal growth and hypertrophy. Int. Symp. on Renal Adaptation to Nephron Lose, Montreux: to be published. STEINER, A.. L., PAGLIARA, A. S., CHASE, L. R. & KIPNIs, D. M. (1972). Radioimmunoassay for cyclic nucleotides. Adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate in mammalian tissues and body fluids. J. biol. Chem. 247, 1114-1120. VAN VROONHOVEN, T. J., SOLER-MONTESIOS, L. & MALT, R. A. (1972). Humoral regulation of renal mass. Surgery 72, 300-305.
J. Physiol. (1977), 271, pp. 505- 514 With 1 text-figure Printed in Great Britain
CHANGES IN RENAL CYCLIC NUCLEOTIDE CONTENT AS A POSSIBLE TRIGGER TO THE INITIATION OF COMPENSATORY RENAL HYPERTROPHY IN RATS
BY S. E. DICKER AND A. L. GREENBAUM From the Departments of Chemistry and of Biochemistry, University College London, London WC1E 6BT
(Received 25 January 1977) SUMMARY
1. Cyclic adenosine 3', 5'-monophosphate (cyclic AMP) and cyclic guanosine 3', 5'-monophosphate (cyclic GMP) have been estimated in the kidneys of rats. 2. Ten minutes after unilateral nephrectomy there was a threefold increase of cyclic GMP in the remaining kidney, which was accompanied by a moderate fall of cyclic AMP. 3. The changes in cyclic nucleotides in the remaining kidney after unilateral nephrectomy were of short duration. 4. When an anephric rat was cross-circulated with a normal litter-mate, there was an increase of cyclic GMP concentration in the kidneys of the latter, which reached its maximum 10 min after the establishment of the cross circulation. 5. In experiments where one kidney of a litter-mate was transplanted to the neck of another rat, unilateral nephrectomy was not followed by changes of the level of cyclic nucleotides in either the transplanted or the remaining kidney. Bilateral nephrectomy, however, resulted in a marked increase of cyclic GMP in the transplanted kidney. 6. The clamping of the blood vessels to one kidney for periods up to 10 min had the same effect as unilateral nephrectomy on the concentration of cyclic GMP in the remaining kidney. When the clamp was removed and the circulation restored, the concentrations of cyclic nucleotides returned to preoperative levels in both kidneys. INTRODUCTION
It has been suggested that the cyclic nucleotides, adenosine-3', 5'-monophosphate (cyclic AMP) and guanosine-3', 5'-monophosphate (cyclic GMP), may be involved in the regulation of cell growth. An increase of cyclic AMP
506 S. E. DICKER AND A. L. GREENBA UM has been shown to be associated with inhibition of cell growth, whereas increased cellular cyclic GMP concentrations appear to be associated with the initiation ofcellgrowth (Hardman, Robinscn & Sutherland, 1971; Posternak, 1974; Goldberg, Haddox & Nicol, 1975). Since it is well established that compensatory renal hypertrophy is an adaptive process which starts almost immediately after removal of one kidney it was of interest to see whether changes in the concentration of cyclic nucleotides could be detected in the early stages after unilateral nephrectomy. A preliminary account of some of the present findings has been published (Dicker, 1976). METHOD
Techniqum Adult male Wistar rats (220-250 g body weight) were used. For cross-over circulation experiments involving two or more animals litter-mates were used. All animals were anaesthetized with an i.P. injection of Inactin (Na-ethyl-methylpropyl-malonyl-thiourea), 10 mg/100 g body wt. Unilateral or bilateral nephrectomy was performed through a lumbar incision, care being taken to leave the adrenal gland intact. For cross-over circulation experiments, the technique described by Moolten & Bucher (1967) and used by van Vroonhoven, Soler-Montesinos & Malt (1972) was followed. As for the transplantation of kidneys to the carotid vessel of rat, the technique described initially by Govaerts for the dog (1936) was adopted. It consists essentially in an anastomosis between the lower aorta and the lower vena cava below the renal, vessels to the carotid and the jugular vessels of another animal. It allows for a perfusion of the 'transplanted' kidney without interruption of its blood supply, the kidney remaining in situ. Unilateral nephrectomy was performed in the minimum time and with the minimum handling of the kidney. The removed kidney was immediately frozen by clamping in Wollenberg tongs precooled in liquid nitrogen. The frozen tissues were plunged into liquid nitrogen and subsequently weighed. They were homogenized in 0-6 N perchloric acid solution (1: 5) with an ultra-turrax blender (Janke & Kunkel, Breisgau), spun in a refrigerated MSE centrifuge at 6000 revlmin for 5 min, and the precipitate discarded. The supernatant was neutralized to pH 6 6-6 8 with KOH and stood on ice for 30 min. After a second centrifugation, the supernatant was collected and stored at -20 °C, until used. All operations were carried out at 0 °C, and the cyclic nucleotides were estimated with kits prepared by the Radiochemical Centre (Amersham, England) for cyclic AMP and cyclic GMP respectively. Adenosine-5'-triphosphate (ATP) was estimated according to the method of Adam (1965). All assays were made in duplicate. Data are given as means and their standard errors.
RESULTS
Values of concentrations of cyclic 3',5' monophosphate (cyclic AMP) and of cyclic guanosine 3',5' monophosphate (cyclic GMP) in control kidneys were 681-2 + 14-36 (12) and 59-1 + 3-98 (23) x 10-12 mole/g wet tissue, respectively, with an average cyclic AMP/cyclic GMP ratio of the
CYCLIC NUCLEOTIDES IN KIDNEYS
507
TABLE 1. Content of cyclic nucleotides in the remaining kidney after unilateral nephrectomy
Time after unilateral nephrectomy Omin 40 min 10 min 5m 20 min 82-2 45-9 59.1 64-1 160-3 Cyclic GMP ± 22-95 (10) ± 11-60 (4) ± 2-32 (4) ± 8-72 (5) ± 3-98 (23) 511-2 546-5 691-0 681-2 478-0 Cyclic AMP ± 31-76 (4) ± 19-08 (4) + 14-36 (12) ± 12-71 (5) ± 41-08 (7) Cyclic GMP=cyclic-3',5'-guanosine monophosphate, cyclic AMP=cyclic-3',5'adenosine monophosphate. Results are means+s.E. and are expressed as 10-12 mole/g kidney wet wt. In parentheses, number of experiments. There was no significant difference between values found in control kidneys (time 0) and in those of sham-operated animals. For the values of cyclic GMP at 5 min, see test.
order of 11. These values agree well with those reported by Goldberg, Dietz & O'Toole (1969), Steiner, Pagliara, Chase & Kipnis (1972), and Kobayashi & Fang (1975).
Effect of unilateral nephrectomy on cyclic AMP and cyclic OMP Rats were killed 5, 10, 20, 40, 80, 120 and 240 min after unilateral nephrectomy and the contents of cyclic AMP and cyclic GMP in the remaining kidneys were compared with those of the removed kidneys (taken as controls) or those found in kidneys of sham-operated animals, killed after 5, 10, 80 and 120 min. Levels of both cyclic nucleotides were similar in control kidneys and in kidneys from sham-operated animals. The measurements of the cyclic nucleotides were most variable in rats killed 5 min after unilateral nephrectomy. In two of the five rats killed at this time, the cyclic GMP content had fallen by 20 and 30 %, with no significant changes in the level of cyclic AMP, while in the remaining three animals the concentration of cyclic GMP had increased by between 30 and 40 %, and the level of cyclic AMP had fallen by about 50 %. Taking the group of five rats as a whole, however, 5 min after unilateral nephrectomy there was an increase of 8 % in cyclic GMP level, and a fall of 30 % in the concentration of cyclic AMP in the remaining kidney (Table 1). In contrast, in all rats killed 10 min after unilateral nephrectomy there was a threefold increase of the cyclic GMP level, accompanied by a moderate (25 %) decrease of cyclic AMP, in the remaining kidney. The rise of cyclic GMP was of short duration, and was only 36 % higher than the control values, 20 min after the operation (Table 1). Changes of cyclic AMP/cyclic GMP ratios in the remaining kidneys over the whole experimental period of 4 hr are represented in Fig. 1, from which it will be seen that 80 min after
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unilateral nephrectomy the renal levels of both cyclic nucleotides were nearly back to preoperative levels. Effects of cross-circulations between anephric and normal rats Two series of experiments were performed. In the first series, bilaterally nephrectomized rats were cross-circulated with normal animals. Ten to fifteen min were allowed between the bilateral nephrectomy and the onset of the cross-circulation which usually lasted 10 min. The removed kidneys of the anephric animal served as controls, and their levels of cyclic AMP and cyclic GMP were compared with those of the intact animal at the end of the cross-circulation. The results showed that at the end of the crosscirculation, the level of cyclic GMP in the kidneys of the normal animals had increased from 65-7 + 5-75 (8) in the control kidneys to 169-2 + 8-49 (8) x 10-12 mole/g. This rise was accompanied by a moderate fall of cyclic AMP from 718 + 12-22 (8) to 671-0 + 7.59 (8) x 10-12 mole/g wet wt. In another series of experiments, one anephric rat was cross-circulated first with one normal animal for 10 min. As soon as the kidneys of the latter had been removed and frozen in liquid nitrogen, the cross-circulation
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PHY 27I
S. E. DICKER AND A. L. GREENBAUM was switched to another normal rat. After 10 mi, its kidneys having been removed and frozen, cross-circulation was switched to a third normal animal and the circulation was maintained for another period of 10 min. Allowing about 2 min for removing the kidneys of each test animal, freezing and weighing them, the total duration of the cross-circulation was of the order of 35 min. The level of cyclic GMP in the kidneys of the three normal rats cross-circulated with one anephric animal increased in keeping with the above results although for the 3rd cross-circulation the level of cyclic GMP had declined almost to the control value and was not statistically different from it (Table 2). 510
Effect of 'transplanting' a kidney to a unilaterally nephrectomized rat The kidney of a litter-mate was 'transplanted' to the carotid of another rat. As soon as the circulation to the 'transplanted' organ was established, the left kidney of the recipient animal was removed. The circulation to the 'transplanted' kidney was maintained for 10 or 20 min, when both the recipient's own and the 'transplanted' kidneys were removed, and immediately frozen in liquid nitrogen. In five out of six experiments, the levels of cyclic AMP and cyclic GMP were normal: 702.2 ± 12*25 and 56'0 ± 1.04 x 10-12 mole/g in the 'transplanted' organ and 698-4 ± 947 and 62*7 ± 2*09 x 10-12 mole/g, in the recipient's own kidney. In the sixth experiment, blood clotting in the carotid prevented normal circulation to the 'transplanted' kidney. In this case, cyclic GMP concentration in the recipient's kidney was raised to 186-6 x 10-12 mole/g, while that of cyclic AMP was 607 x 10-12 mole/g kidney wet wt. Finally, in two experiments, a kidney was 'transplanted' to the neck of an anephric rat, and its circulation maintained for 10 and 12 min. The levels of cyclic GMP in the transplanted kidneys were similar to those found in the remaining kidney 10 min after unilateral nephrectomy: 151*7 and 145*8 x 10-12 mole/g, respectively. No estimation of cyclic AMP was performed in these two experiments.
Effect of clamping renal blood ves8els of one kidney A small clamp was put on the blood vessels of the left kidney for limited periods of time of 10 or 20 min after which both kidneys were removed for estimation of the cyclic nucleotides. In some cases, the clamp was removed after 10 min and the blood was allowed to circulate through the kidney again for 10 min. In others, resumption of blood flow, after a period of 10 min clamping, was accompanied by right unilateral nephrectomy. From these experiments (Table 3) four effects were observed. First, the clamping of the renal vessels of one kidney had the same effect on the
CYCLIC NUCLEOTIDES IN KIDNEYS 51J level of cyclic nucleotides as unilateral nephrectomy. Secondly, after releasing the clamp and allowing the blood to circulate again, levels of cyclic nucleotides were similar to those found in control kidneys. Thirdly, when clamps were released from renal vessels simultaneously with unilateral nephrectomy, 10 min later, there was a rise of cyclic GMP in the remaining kidney, though somewhat smaller than usual. Fourthly, the changes in cyclic nucleotides concentration were independent of those of ATP concentrations. DISCUSSION
From the results of the present investigation it would appear that the level of both cyclic nucleotides remains unchanged in the kidneys of a rat only if it has two functional kidneys, even if one of these is a transplanted organ from a litter-mate. If, however, one kidney is removed, or if the circulation to one kidney has been stopped, there is a characteristically sharp increase in the level of cyclic GMP in the remaining kidney, usually accompanied by a small decrease of cyclic AMP. Schlondorff & Weber (1976) also found that after unilateral nephrectomy there was a considerable rise of cyclic GMP content accompanied by a moderate decrease in the level of cyclic AMP in the remaining kidney. According to Schlondorff & Weber (1976) the increase of cyclic GMP lasted several days: however, from the evidence presented here the increase is short-lived, and has disappeared 40 min after unilateral nephrectomy (Table 1). This agrees with Solomon, Wise, Ratner & Sanburn (1976), who did not find any change in the concentration of cyclic nucleotides in the remaining kidney 1 h after nephrectomy. Compensatory renal hypertrophy is characterized essentially by cell hypertrophy, hyperplasia being minimal (Malt, 1969): it is of interest that whereas the onset of compensatory renal hypertrophy appears to be associated with a sharp increase of cyclic GMP level in the remaining kidney, renal hyperplasia as observed after administration of folic acid, or liver hyperplasia after partial hepatectomy, are both associated with a marked increase of cyclic AMP and a decrease of cyclic GMP (Berridge, 1975; Solomon et al. 1976). The earliest known changes occurring after unilateral nephrectomy are a small but significant increase of the systemic blood pressure and an enhanced renal blood flow. Though these may have an effect on subsequent renal hypertrophy. it is unlikely that they are the cause of an elevated level of cyclic GMP in the remaining kidney, since increases of cyclic GMP content were observed during cross-circulations between one anephric and a normal rat where it is unlikely that the renal blood flow would be increased. Nor can an increase in cyclic GMP be attributed to a nervous I7-2
S. E. DICKER AND A. L. GREENBAUM 512 stimulation, since it was observed in kidneys 'transplanted' to the carotid of an anephric animal. What then produces the changes in cyclic nucleotides in the remaining kidney? The evidence presented here suggests that the effect may be due to the existence of a humoral factor, the identity of which is at present unknown. This is indicated by the observations that there was a marked increase of cyclic GMP in both kidneys of a normal animal cross-circulated with an anephric rat, and that the switching of the cross-circulation from one anephric animal to a second or even third normal rat still evoked an increase of the particular cyclic nucleotide in their kidneys. It will also be remembered that van Vroonhoven et al. (1972) showed that after 48 hr of cross-circulation between an anephric animal and normal rat, there was an increase of RNA concentration in the kidneys of the latter, which disappeared when the cross-circulation was interrupted (Dykhuis, van Urk, Malamud & Malt, 1975). The existence of a humoral factor has also been postulated by Lyons, Evan, McLaren & Solomon (1974) who observed that the plasma from a unilaterally nephrectomized hamster stimulated the incorporation of [3H]uridine into RNA in cultures of hamster renal cortical cells in vitro, through the plasma of a partially hepatectomized animal did not have that effect. If a humoral renotrophic factor exists, why should it appear to be effective when one kidney only is functional, e.g. after unilateral nephrectomy or after the circulation to one kidney has been stopped? Since according to micro-puncture studies the first signs of renal adaptation after the exclusion of one kidney do not appear before 5-10 min (Dirks, 1976), it is conceivable that immediately after the removal of one kidney the normal 'clearance' activity of the remaining kidney is impaired, and that the temporarily decreased 'clearance' of the humoral renotrophic factor is responsible for the changes in cyclic nucleotides. This view is supported by the observation that complete adaptation of the remaining kidney appears to be effective about 60-80 min after functional exclusion of one kidney (Diezi, 1976), a time when the concentration of renal cyclic nucleotides, especially that of cyclic GMP, has returned to normal levels. This concept of a short-lived decreased renal clearance of a renotrophic factor would explain first, the rapid increase in the content of cyclic GMP in the remaining kidney after unilateral nephrectomy, as well as the short duration of the changes in the levels of cyclic nucleotides; secondly, the increase of cyclic GMP in one kidney after the interruption of the blood flow to the other and the rapid return to normal levels of cyclic GMP, as soon as its blood supply is restored; thirdly, the increased renal level of cyclic GMP observed during a cross-circulation between normal and
513 CYCLIC NUCLEOTIDES IN KIDNEYS anephric rats; fourthly, the absence of changes in the concentration of cyclic nucleotides in the remaining kidney when unilateral nephrectomy was performed in an animal which had a 'transplanted' kidney.
The support of an Emeritus Leverhulme Fellowship and of a grant from the Medical Research Council are gratefully acknowledged by one of us (S.E.D.). S. E. D. would like to thank Dr B. Banks for allowing him to operate in her laboratory and Professor M. McGlashan for his hospitality. We would like to express our appreciation to Miss C. A. Morris for her skill and technical help.
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