J. Phy'siol. (1977), 271, pp. 625-639 With 6 text-flgurew Printed in Great Britain
625
THE EFFECT OF INDOMETHACIN ON AUTOREGULATION OF RENAL BLOOD FLOW IN THE ANAESTHETIZED DOG
BY L. J. BEILIN* AND J. BHATTACHARYA From the Department of the Regius Professor of Medicine, Radcliffe Infirmary, Oxford
(Received 19 October 1976) SUMMARY
1. Renal blood flow autoregulation was studied in anaesthetized greyhounds, using an electromagnetic flowmeter, before and after the administration of the prostaglandin-synthesis inhibitor, indomethacin, or phosphate buffer. 2. Indomethacin caused a reduction in renal blood flow at all levels of perfusion pressure, but did not affect the ability of the kidney to autoregulate. 3. The abrupt reinstatement of renal perfusion pressure from previously reduced levels caused a triphasic transient response in flow. Peak hyperaemia at the beginning of the transient was not affected by indomethacin. After indomethacin, the second phase of this flow transient showed an oscillatory pattern during which flow fell initially to levels significantly lower than control. 4. It is concluded that although indomethacin did not abolish steadystate autoregulation, renal prostaglandins may damp rapid oscillations in renal blood flow and thus contribute to the efficiency of autoregulation. INTRODUCTION
Considerable interest has been evinced as regards the possible role of prostaglandins in the control of renal blood flow. For instance, there is evidence that renal prostaglandins are concerned with the intrarenal distribution of blood flow, and help to maintain flow both to the deep cortex (Itskovitz, Terragno & McGiff, 1974; Larsson & Angaard, 1974) as well as the superficial regions (Beilin & Bhattacharya, 1976, 1977). Claims have been made that renal prostaglandins are crucial in renal blood flow autoregulation (Herbaczynska-Cedro & Vane, 1973) and that they may subserve the process of reactive hyperaemia in the kidney (Herbaczynska* Present address University Department of Medicine, Royal Perth Hospital, Perth, Western Australia 6000.
L. J. BEILIN AND J. BHATTACHARYA 626 Cedro & Vane, 1974). However, there is difficulty in interpreting the data from studies where the preparations used have involved extensive surgical trauma, for instance with use of isolated perfused kidneys, or have been subject to excessive ischaemia from progressive stepwise reductions in perfusion pressure, without intermediate relief (Herbaczynska-Cedro & Vane, 1973; Anderson, Taher, Cronin, McDonald & Schrier, 1975). Such manoeuvres can alter the profile of the renal autoregulation curve (Kircheim, Gross & Keintzel, 1972). Further, experiments to investigate the mechanism of reactive hyperaemia have usually involved total ischaemia of the kidney for appreciable lengths of time prior to reinstatement of flow (Herbaczynska-Cedro & Vane, 1974; Swain, Heyndrickx, Boettcher & Vatner, 1975). Autoregulatory correction of flow begins within 3 sec of the pressure disturbance and is well established in 30 see (Thurau, 1966). A role for prostaglandins in this early phase of autoregulation has not been studied adequately. The present study was designed to examine the effects of the prostaglandin synethesis inhibitor indomethacin on both the profile of the steadystate autoregulation of renal blood flow, and the dynamic response of the renal circulation, subsequent to a sudden change in perfusion pressure. Particular features of the present study were the recording of flow during brief periods of pressure reduction, and the adoption of a relatively atraumatic retroperitoneal approach to the kidney, while maintaining its natural perfusion. A preliminary account of this work has been published elsewhere (Bhattacharya & Beilin, 1975). METHODS
Experiments were performed on male greyhounds with a mean weight of 29 kg + 1-2 S.D. of observations (n= 11). Following premeditation with chlorpromazine (1 mg/kg, I.M.), dogs were anaesthetized with i.v. pentobarbitone (20 mg/kg), intubated, and ventilated artificially by a Starling Pump ajdusted to give an arterial of approx. 100 mmHg and an arterial Pco, of ca. 40 mmHg. Moderately light Po. anaesthesia (corneal reflex present) was maintained by bolus injections of 60 mg pentobarbitone. In order to compensate for fluid losses, I.v. normal saline was given at a rate of 1.0 ml./min. Rectal temperature was maintained at 37 00 by electric blankets. The left renal artery was approached by a muscle-splitting incision over the left loin. No attempt was made to dissect the adventitia of the artery more than necessary in order to place a 3 or 5 mm flow probe around it. The abdominal aorta was loosely snared by silk thread, the free ends of which were pulled through a 10 cm length of Polythene tubing. Aortic pressure was recorded at the level of the left renal artery with Polythene tubing (i.d. 0 9 mm) introduced via the right femoral artery. The bladder was catheterized and drained occasionally.
RENAL AUTOREGULATION
627
Pressure was recorded by means of a strain gauge pressure transducer (Consolidated Electro Dynamics), and flow with a square wave electromagnetic flowmeter (S.E.M. 275, S.E. LABS), the signals from which were displayed on a multi-channel recorder (Devices, M4, Welwyn Garden City, Herts).
Flow probe calibration The main difficulty in calibrating a flow probe on the renal artery lay in ensuring that the cuff maintained its position throughout. During the experiment, zero flow was calibrated by occluding the renal artery with the constriction distal to the cuff by means of a previously placed ligature. The flow signal was aligned with the baseline output of the flowmeter, and a match of the two made for every pair of experimental readings. In order to obtain a calibration plot of known volume against that measured by the flow probe in vivo at the end of each experiment, a wide bore Polythene tube i.d. 4*9 mm) was passed via the femoral artery into the aorta to about the level of the left renal artery. The other end of the tube was fitted to one limb of a Y piece which further connected it to the strain-gauge manometer on the one hand, and via a two-way tap, to a calibrated syringe on the other. It was possible therefore to monitor the pressure of an injection up the wide-bore tube. The aorta was tied off about a finger's breadth on either side of the renal artery, which was kept distended by constricting the vessel distal to the cuff and applying pressure through the wide-bore tube, to prevent collapse and hence dislocation of the cuff. This also proved a test for the absence of collateral channels connecting with the tube artery conduit. Varying measured volumes of heparinized blood from the animal were injected at near systolic pressure. A plot of volume, as recorded off the cuff signal, consistently over-estimated the actual injected volume in all experiments, but the calibration was always linear and passed through zero. The error estimated from this calibration served to correct flow measurements for the experiment. After the surgical procedure, which took ca. 2 hr, a time (ca. 1 hr) was allowed to elapse before the first readings were taken, in order to permit transient effects due to surgery to subside. An experiment consisted of two runs. The first, or 'control' run, was separated in time from the second, or 'test' run, by 30 min. At the end of the control run in six dogs, an i.v. injection of indomethacin 10 mglkg (Merck, Sharp and Dohme) was given in 0-2 M-potassium phosphate buffer (pH 7-4). Five dogs did not receive indomethacin between control and test runs but were given an equivalent volume of buffer alone (30 ml.). Dogs given indomethacin alternated for study with those given only buffer. In a separate group of seven greyhounds, indomethacin was given at a dose of 2 mglkg to four animals, while the remaining dogs received buffer (10 ml.) between runs.
Steady-state records A 'run' consisted of steady-state blood flow readings at different levels of aortic pressure and some transient flow responses to a step pressure change. Typically, subsequent to records for zero flow and zero pressure, a run was begun with a reading of flow at resting pressure. Following this, the renal artery pressure was reduced by pulling on the aortic snare, and constricting the aorta. After holding the pressure reduction at a steady level for 30 sec, flow and pressure tracings were obtained at a paper speed of 2-5-5 cm/sec, and then the snare was released. Resting pressure and zero readings were taken after every two such manoeuvres. Following aortic or renal artery constriction, a time gap of 3 min was allowed before taking any further readings. The general aim in a run was to obtain flow records at ca. six to seven evenly spaced pressure intervals, both above and below 100 mmHg.
628
L. J. BEILIN AND J. BHATTACHARYA
'Transient' records On completion of the steady-state records, pressure was reduced with the snare by ca. 60 mmHg from control systolic levels and held steadily for 30 sec. The recording chart was run at 2-5 or 5 cm/sec, and a few seconds later the aortic constriction abruptly relieved. The subsequent pressure and flow profiles were recorded for ca. 45 sec. A second transient was recorded in exactly the same way, but at a slower paper speed of 10 cm/min. Again, resting pressure and zero levels for pressure and flow were taken before and after each transient. Finally, the animals were killed with i.v. pentobarbitone, and the left kidney was removed and weighed. Mean flow was estimated from the charts by planimetry. Mean pressure was obtained by adding a third of the pulse pressure to the diastolic pressure. Pressure and flow transients were analysed from records run at 2-5-5 cm/sec. Paired comparisons were done using Student's t test. Mean renal blood flow was plotted against mean aortic pressure for each series of pressure changes. As the graphs showed that autoregulation of blood flow was not seen at pressures below ca. 80 mmHg, whereas flow was relatively constant for pressures above ca. 100 mmHg (Fig. 1), regression of flow on pressure was calculated separately for points above B.P. = 100 mmHg, and for those below B.P. = 80 mmHg. The adequacy of autoregulation was established by: (i) Lack of a statistically significant correlation between flow and pressure above 100 mmHg, and (ii) the presence of such a correlation below 80 mmHg. RESULTS
The main effect of indomethacin at doses of 2 and 10mg/kg was to reduce renal blood flow. At a dose of 10 mg/kg the group mean renal blood flow at resting pressure decreased by ca. 22 % (P < 0-01), and at 2 mg/kg by ca. 10 % (P < 0-03) (Table 1). In contrast, in dogs given buffer, renal blood flow (if anything) increased, while group averages for pressure and heart rate were lower after the injection of buffer (Table 1). A 10 % increase in mean B.P. (P < 0-02) and 25 % reduction in mean heart rate (P < 0-02) was seen after indomethacin (2 mg/kg) but not after buffer (Table 1). In the dogs receiving indomethacin at 10 mg/kg, the fall in heart rate was more pronounced, although the rise in B.P. was less pronounced.
Steady-state responses By the criteria for autoregulation described above, every plot of flow against pressure showed good autoregulation, whether taken before or after buffer or indomethacin (2 and 10 mg/kg) (Fig. 2). The calculated slopes of the flow pressure diagram for points taken above pressures of 100 mmHg were not significantly different from zero (group average slopes - 0-003, s.E. of mean + 0-001 before buffer and - 0-002, s.E. of mean + 0-002 after buffer; - 0-006, s.E. of mean + 0-002 before indomethacin and - 0-001, s.E. of mean + 0-002 after indomethacin 10 mg/kg),
629 RENAL AUTOREGULATION whereas the relationship was highly significant in every case for the low pressure points, with a significant reduction in the regression slopes after indomethacin 10 mg/kg (P < 0.02) compared with before (group average slopes 005, s.E. of mean + 0 007 before buffer, - 004, s.E. of mean + 0-007 after buffer; 005, s.E. of mean + 0008 before indomethacin and 0-036, s.E. of mean + 0-009 after indomethacin 10 mg/kg). A -
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RENAL AUTOREGULATION 631 and for those given buffer alone were calculated at intervals of 10 mmHg along the X-axis. These were then plotted as shown in Fig. 3. The resetting of the flow pressure curves at a lower level of flow after indomethacin, and a tendency towards an upward shift after buffer, is depicted in this diagram. 5
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The transient response The response of flow to a sudden increase in pressure after 30 see of aortic constriction of moderate severity was usually characterized by a triphasic response (Figs. 4, 5). In dogs given buffer, a sudden increase in pressure following abrupt release of aortic constriction caused the following: (1) A short period of transient hyperaemia in which flow increased by ca. 40 % of preconstriction control values in the first two flow pulses. (2) Subsequently, a progressive decrease in flow took place over approx. 5 see to a steady plateau at about 20 % above control level. 23-2
L. J. BEILIN AND J. BHATTACHARYA 632 The duration of this elevated plateau was variable in the runs analysed. On average, it lasted up to the 19th see from the step, although on occasions being more prolonged. (3) The end of the response, as a whole, was signalled by a further lowering of flow to within 10 % of control levels. Averages of flow and pressure over intervals of approx. 2 see from the relief of aortic constriction, expressed as percentage difference from the 4.0
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resting value, were plotted for each run. After indomethacin the most noticeable difference was a swifter fall in flow, following the initial hyperaemia and the attainment of a flow minimum in the first 10 see (Fig. 6). Paired tests on flow from control and test runs showed a significant reduction after indomethacin (P < 0-001) on the 6th, 8th and 10th see after relief of aortic constriction. Pressure usually returned to resting levels over 20 see following the step increase. Control flows were similar before and after the transient changes.
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to pressure during a value certain of difficult. is pressure is denoted Usually, autoregulation as a limiting index above which autoregulation is seen (Nashat, 1974), although it is unlikely that autoregulatory processes are switched on abruptly at a fixed point in pressure. In the present study a graphical
Quantification of the steady-state flow response
L. J. BEILIN AND J. BHATTACHARYA 634 approach was used which involved the demonstration that above a pressure of 100 mmHg, where visual evidence of autoregulation was apparent on the flow-pressure plots, flow consistently failed to correlate Dog 40 before indomethacin I
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with pressure. On the other hand, below 80 mmHg a correlation was always obtained. Further, comparison of flow at control pressure with that measured at ca. 105 mmHg yielded no significant differences. The plots of renal blood flow against pressure agree well with those obtained previously (Selkurt, 1946; Guyton, Langston & Navar, 1964).
RENAL AUTOREGULATION 635 Both the steady-state and transient data negate a central role for prostaglandins as being mediators of autoregulation in the kidney. Thus, after indomethacin, there was constancy of blood flow with pressure change above 100 mmHg in the steady state, and the dynamic-flow response to a sudden increase in pressure subsided with a delay not exceeding that seen in the normal. If anything, the transient increase in flow after a steep 50 T
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L. J. BEILIN AND J. BHATTACHARYA 636 rise of pressure is relatively short-lived after the drug, and there are attempts at restoring the control level very early on in the sequence. This is not seen in the pre-indomethacin transient. In order to exclude the effects of anaesthesia, haemorrhage and other time-dependent variables on these measurements, some dogs were given only phosphate buffer. There was good autoregulation of flow before and after buffer and no remarkable alterations occurred in heart rate or blood pressure. This suggests that the changes seen in the flow-pressure relationships after indomethacin were probably not due to influences other than those due to the drug. Indomethacin inhibits a number of enzymes, in addition to those concerned with prostaglandin synthesis, although with considerably less potency (Flower & Vane, 1974), so that an effect of this drug unrelated to that on prostaglandins must be considered a possibility. Although we had no direct measurement of renal prostaglandin synthesis there is evidence that the doses of indomethacin used here were sufficient to reduce prostaglandin synthesis (Vane, 1971; Ferreira, Moncada & Vane, 1971). Indomethacin, in doses less than those used in the present experiments, has been shown to reduce significantly the efflux of prostaglandins into dog renal venous blood (Aiken & Vane, 1973; Herbaczynska-Cedro & Vane, 1973; Lonigro, Itskovitz, Crowshaw & McGiff, 1973) and to enhance the vasoconstriction caused by angiotensin, which normally promotes prostaglandin release in the absence of indomethacin. Indomethacin abolishes the conversion of labelled arachidonic acid to prostaglandins in homogenates of rabbit kidney (Larsson & Angaard, 1973), and reduces urinary efflux of prostaglandins by 40 %. White, Kirschenbaum, Stein & Ferris (1974), using specific radio-immunoassay, have shown that a single 2 mg/kg dose of indomethacin in anaesthetized dogs reduces the renal venous efflux of prostaglandins of the E series for over 2 hr. Both indomethacin and meclofenamate, two inhibitors of prostaglandin synthesis which differ in chemical structure, increase renal vascular resistance in conscious rabbits (Beilin & Bhattacharya, 1976, 1977). The maintenance of renal blood flow autoregulation, in the presence of indomethacin, is in contradiction to the findings of Herbaczynska-Cedro & Vane (1973) but is in agreement with the recent publication of Swain et al. (1975). The former study was carried out using pump perfused kidneys and it is very likely that the effects of anaesthesia, surgical trauma and severe intermittent hypoxia combined to exaggerate the contribution that prostaglandins appeared to make the renal blood flow in those experiments. It is not possible to conclude whether the reduction in renal blood flow with indomethacin was secondary to loss of a vasodilator action of prosta-
RENAL A UTOREGULATION 637 glandins alone, or also to an associated increase in over-all sympathetic tone. Hedqvist (1974) has demonstrated decreased noradrenaline reuptake in sympathetic nerves in various organs, including the kidney, in the presence of indomethacin; and increased sympathetic activity reduces total renal blood flow (Pomeranz, Birtch & Barger, 1968). The increase in arterial pressure seen after indomethacin may reflect such a generalized change in sympathetic tone to arterioles. The situation during the periods of aortic constriction used to assess autoregulation is further complicated by nervous and humoral changes. For example, the increase in arterial pressure, proximal to the constriction, will have activated carotid and aortic baroreflexes leading to withdrawal of sympathetic tone and increased vagal tone, while below the constriction the more severe reductions in renal perfusion pressure would tend to stimulate release of renin. The demonstration of efficient autoregulation of renal blood flow in the presence of these changes, and despite the administration of large doses of a prostaglandin synthesis inhibitor, leaves the myogenic hypothesis (Folkow, 1949) unassailed. The transient The normal transient flow profile obtained was a triphasic one in most cases and consisted of a period of hyperaemia, lasting ca. 5 sec, in which flow rose to a peak of ca. 40 % above control, and then progressively declined; a plateau at ca. 20 % above control, and further lowering of flow after 20 sec to within 10 % of control flow. Held, Niedermayer, Schaffer, Schwartzkopf & Weiss (1971) observed a similar triphasic flow response, with comparable time course, to pump induced increases in pressure. Such responses also have been recorded from the isolated kidney and heart (Basar, Ruedas, Schartzkopf & Weiss, 1968; Basar & Weiss, 1969; Basar, Tischner & Weiss, 1968). After indomethacin, in what is presumed to be a state of relative depletion of prostaglandins, the second phase of the transient response was biphasic, contrasting with the normal hyperaemic plateau. This swift descent to near control levels was soon followed by a rebound to ca. 20 % above control. Flow instability with indomethacin has been commented on earlier, but not quantified (Herbaczynska-Cedro & Vane, 1973; Swain et al. 1975). The subsequent delay (third phase) in the return to control levels was comparable with the pre-indomethacin flow transient. Thus, the early phase of autoregulation was (if anything) augmented after indomethacin, the reverse of what Herbaczynska-Cedro & Vane (1973) suggested. Herbaczynska-Cedro & Vane (1974) and Swain et al.(1975) concluded that the prostaglandins are important in establishing reactive hyperaemia. The present findings make it difficult to assign a role for prostaglandins as mediators of reactive
L. J. BEILIN AND J. BHATTACHARYA hyperaemia in previously non-ischaemic kidneys. Prior to eliciting reactive hyperaemia, previous workers caused total cessation of blood flow in the kidney for 45 sec or more. It is possible that severe anoxia, which can relax vasculature (Selkurt, 1946), may also cause greater prostaglandindependant vasodilation (Wennmalm, Pham-hull-Chanh & Junstad, 1974) and hence more pronounced hyperaemia on relief of stress. In the present experiments, flow restriction was considerably less severe. A role of prostaglandins as determinants of resting renal vascular tone is compatible with the present data. In the anaesthetized dog, the downward resetting of the steady-state flow-pressure relationship following administration of a prostaglandin synthesis inhibitor, may be attributable to a modulating action of prostaglandins on vascular tone, leaving unaffected the ability of the kidney to maintain stable flow rates over a wide range of perfusion pressures. 638
This work formed part of an Oxford D.Phil. thesis by Dr J. Bhattacharya, who was in receipt of a Schorstein Scholarship. We are grateful for financial support from the National Kidney Research Fund and I.C.I. Ltd and the Wellcome Trust.
REFERENCES AixEN, J. W. & VANE, J. R. (1973). Intrarenal prostaglandin release attenuates the renal vasoconstrictor activity of angiotensin. J. Pharmac. exp. Ther. 184, 678-687. ANDERSON, R. J.,TAPR, M. S., CRONIN, R. B., MCDONALD, K. M. & SCHRIER, R. W. (1975). Effect of / adrenergic blockade and inhibitors of angiotensin II and prostaglandin on renal autoregulation. Am. J. Phyiol. 229, 731-736. BAsAR, E., RUEDAS, 0., SCHWARZKOPF, H. J. & WEISS, CH. (1968). Untersuchungen des zeiltlichen Verhaltens druckabhangiger stromungswider standes im coronargefabsystem des Rattemjerzens. Pflugers Arch. gem. Physiol. 304, 189-202. BASAR, E., TISORNER, H. & WEISS, CH. (1968). Untersuchungen zur Dynamik druckinduzierter Anderungen des Stromungswiderstandes der autoregulierenden, isolierten Rattenhiere. Pfliger8 Arch. gea. Phy8iol. 299, 191-213. BASAR, E. & WEISS, CH. (1969). Rate sensitivity of the mechanisms of pressure induced changes of vascular resistance. Kybernetik, 5, 241-247. BEILIN, L. J. & BHATrACHARYA, J. (1976). The effects of prostaglandin synthesis inhibitors on renal blood flow distribution within the kidney. J. Phy8iol. 256, 9-10. BEILIN, L. J. & BHATTACHARYA, J. (1977). The effect of prostaglandin synthesis inhibitors on renal blood flow distribution in conscious rabbits. J. Phyeiol. (in the press). BHATrACHARYA, J. & BEILIN, L. J. (1975). Inhibition of prostaglandin synthesis and renal blood flow autoregulation in the dog. Blood Ve88el8, 12, 354-355. FERREIRA, S. H., MONCADA, S. & VANE, J. R. (1971). Indomethacin and aspirin abolish prostaglandin release from the spleen. Nature, New Biol. 231, 237-239. FLOWER, R. J. & VANE, J. R. (1974). Some pharmacologic and biochemical aspects of prostaglandin biosynthesis and its inhibition. In Prodkaglandin Synthetase Inhibitors, ed. ROBINSON, H. J. & VANE, J. R. New York: Raven.
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FoLKow, B. (1949). Intravascular pressure as a factor regulating the tone of the small vessels. Acta physiol. scand. 17, 289-310. FRYuu, K. H. & HEDQVIST, P. (1975). Evidence for prostaglandin mediated prejunctional control of renal sympathetic transmitter release and vascular tone. Br. J. Pharmacol. 54, 189-196. GUYTON, A. C., LANGSTON, J. B. & NAVAR, G. (1964). Theory of renal autoregulation by feedback at the juxtaglomerular apparatus. Circulation Res. 15 (suppl.), 187-197. HEDQVIST, P. (1974). Effect of prostaglandins and prostaglandin synthesis inhibitors on norepinephrine release from vascular tissue. In Prostaglandin Synthesis Inhibitors, ed. ROBINSON, H. & VANE, J. R., p. 303. New York: Raven. HELD, K., NIEDERMAYER, W., SCHAEFER, J., SCHWARTZKOPF, H. J. & WEISS, CH. (1971). Pressure induced changes of renal blood flow resistance in the dog kidney in situ. Pflugers Arch. ges. Physiol. 325, 95-102. HERBACZYNSKA-CEDRO, K. & VANE, J. R. (1974). Prostaglandins as mediators of reactive hyperaemia in the kidney. Nature, Lond. 247, 492. HERBACZYNSKA-CEDRO, K. & VANE, J. R. (1973). Contribution of intrarenal generation of prostaglandin to autoregulation of renal blood flow in the dog. Circulation Res. 33, 428-436. ITSKOvITZ, H. D., TERRAGNO, N. A. & MCGIFF, J. C. (1974). Effect of a renal prostaglandin on distribution of blood flow in the isolated canine kidney. Circulation Res. 34, 770-776. KIRCHEIM, H., GROSS, R. & KEINTZEL, B. (1972). Dynamic pressure-flow curves in the autoregulating kidney vasculature of conscious dog. Pflugers Arch. ges. Physiol. 335, 29-45. LARSSON, C. & ANGAARD, E. (1973). Regional differences in the formation and metabolism of prostaglandins in the rabbit kidney. Eur. Jl. Pharmac. 21, 30-36. LARSSON, C. & ANGAARD, E. (1974). Increased juxtamedullary blood flow on stimulation of prostaglandin biosynthesis. Eur. JX. Pharmac. 25, 326-334. LONIGRO, A. J., ITSKOVITZ, H. D., CROWSHAW, K. & McGIFF, J. C. (1973). Dependancy of renal blood flow on prostaglandin synthesis in the dog. Circulation Res. 32, 721-727. NASHAT, F. S. (1974). Topics in renal physiology. In Recent Advances in Physiology, ed. LINDEN, R. J., p. 191. London: Churchill. POMERANZ, H. H., BRITCH A. G. & BARGER, A. C. (1968). Neural control of intrarenal blood flow. Am. J. Physiol. 215, 1067-1081. SELKURT, E. E. (1946). Relationship of renal blood to effective arterial pressure in the intact kidney of the dog. Am. J. Physiol. 147, 537-549. SWAIN, J. A., HEYNDRICKX, G. R., BOETTCHER, D. H. & VATNER, S. F. (1975). Prostaglandin control of renal circulation in the unanaesthetised dog and baboon. Am. J. Physiol. 229, 826-830. THURAU, K. (1967). Nature of autoregulation of renal blood flow. In Proc. 3rd Int. Congr. Nephrol. Washington, vol. 1, pp. 162-173. Basel, New York: Kerger. VANE, J. R. (1971). Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature, New Biol. 231, 232-235. WENNMALM, A., PHAM-HULL-CHANH & JUSTAD, M. (1974). Hypoxia causes prostaglandin release from perfused rabbit hearts. Acta physiol. scand. 91, 133-135. WHITE, N., KIRSCHENBAUM, M. A., STEIN, J. H. & FERRIS, T. F. (1974). Role of prostaglandin relate on the haemodynamic and natriuretic effect of renal vasodilation. (Abstr.) Clin. Res. 22, 550.
625
THE EFFECT OF INDOMETHACIN ON AUTOREGULATION OF RENAL BLOOD FLOW IN THE ANAESTHETIZED DOG
BY L. J. BEILIN* AND J. BHATTACHARYA From the Department of the Regius Professor of Medicine, Radcliffe Infirmary, Oxford
(Received 19 October 1976) SUMMARY
1. Renal blood flow autoregulation was studied in anaesthetized greyhounds, using an electromagnetic flowmeter, before and after the administration of the prostaglandin-synthesis inhibitor, indomethacin, or phosphate buffer. 2. Indomethacin caused a reduction in renal blood flow at all levels of perfusion pressure, but did not affect the ability of the kidney to autoregulate. 3. The abrupt reinstatement of renal perfusion pressure from previously reduced levels caused a triphasic transient response in flow. Peak hyperaemia at the beginning of the transient was not affected by indomethacin. After indomethacin, the second phase of this flow transient showed an oscillatory pattern during which flow fell initially to levels significantly lower than control. 4. It is concluded that although indomethacin did not abolish steadystate autoregulation, renal prostaglandins may damp rapid oscillations in renal blood flow and thus contribute to the efficiency of autoregulation. INTRODUCTION
Considerable interest has been evinced as regards the possible role of prostaglandins in the control of renal blood flow. For instance, there is evidence that renal prostaglandins are concerned with the intrarenal distribution of blood flow, and help to maintain flow both to the deep cortex (Itskovitz, Terragno & McGiff, 1974; Larsson & Angaard, 1974) as well as the superficial regions (Beilin & Bhattacharya, 1976, 1977). Claims have been made that renal prostaglandins are crucial in renal blood flow autoregulation (Herbaczynska-Cedro & Vane, 1973) and that they may subserve the process of reactive hyperaemia in the kidney (Herbaczynska* Present address University Department of Medicine, Royal Perth Hospital, Perth, Western Australia 6000.
L. J. BEILIN AND J. BHATTACHARYA 626 Cedro & Vane, 1974). However, there is difficulty in interpreting the data from studies where the preparations used have involved extensive surgical trauma, for instance with use of isolated perfused kidneys, or have been subject to excessive ischaemia from progressive stepwise reductions in perfusion pressure, without intermediate relief (Herbaczynska-Cedro & Vane, 1973; Anderson, Taher, Cronin, McDonald & Schrier, 1975). Such manoeuvres can alter the profile of the renal autoregulation curve (Kircheim, Gross & Keintzel, 1972). Further, experiments to investigate the mechanism of reactive hyperaemia have usually involved total ischaemia of the kidney for appreciable lengths of time prior to reinstatement of flow (Herbaczynska-Cedro & Vane, 1974; Swain, Heyndrickx, Boettcher & Vatner, 1975). Autoregulatory correction of flow begins within 3 sec of the pressure disturbance and is well established in 30 see (Thurau, 1966). A role for prostaglandins in this early phase of autoregulation has not been studied adequately. The present study was designed to examine the effects of the prostaglandin synethesis inhibitor indomethacin on both the profile of the steadystate autoregulation of renal blood flow, and the dynamic response of the renal circulation, subsequent to a sudden change in perfusion pressure. Particular features of the present study were the recording of flow during brief periods of pressure reduction, and the adoption of a relatively atraumatic retroperitoneal approach to the kidney, while maintaining its natural perfusion. A preliminary account of this work has been published elsewhere (Bhattacharya & Beilin, 1975). METHODS
Experiments were performed on male greyhounds with a mean weight of 29 kg + 1-2 S.D. of observations (n= 11). Following premeditation with chlorpromazine (1 mg/kg, I.M.), dogs were anaesthetized with i.v. pentobarbitone (20 mg/kg), intubated, and ventilated artificially by a Starling Pump ajdusted to give an arterial of approx. 100 mmHg and an arterial Pco, of ca. 40 mmHg. Moderately light Po. anaesthesia (corneal reflex present) was maintained by bolus injections of 60 mg pentobarbitone. In order to compensate for fluid losses, I.v. normal saline was given at a rate of 1.0 ml./min. Rectal temperature was maintained at 37 00 by electric blankets. The left renal artery was approached by a muscle-splitting incision over the left loin. No attempt was made to dissect the adventitia of the artery more than necessary in order to place a 3 or 5 mm flow probe around it. The abdominal aorta was loosely snared by silk thread, the free ends of which were pulled through a 10 cm length of Polythene tubing. Aortic pressure was recorded at the level of the left renal artery with Polythene tubing (i.d. 0 9 mm) introduced via the right femoral artery. The bladder was catheterized and drained occasionally.
RENAL AUTOREGULATION
627
Pressure was recorded by means of a strain gauge pressure transducer (Consolidated Electro Dynamics), and flow with a square wave electromagnetic flowmeter (S.E.M. 275, S.E. LABS), the signals from which were displayed on a multi-channel recorder (Devices, M4, Welwyn Garden City, Herts).
Flow probe calibration The main difficulty in calibrating a flow probe on the renal artery lay in ensuring that the cuff maintained its position throughout. During the experiment, zero flow was calibrated by occluding the renal artery with the constriction distal to the cuff by means of a previously placed ligature. The flow signal was aligned with the baseline output of the flowmeter, and a match of the two made for every pair of experimental readings. In order to obtain a calibration plot of known volume against that measured by the flow probe in vivo at the end of each experiment, a wide bore Polythene tube i.d. 4*9 mm) was passed via the femoral artery into the aorta to about the level of the left renal artery. The other end of the tube was fitted to one limb of a Y piece which further connected it to the strain-gauge manometer on the one hand, and via a two-way tap, to a calibrated syringe on the other. It was possible therefore to monitor the pressure of an injection up the wide-bore tube. The aorta was tied off about a finger's breadth on either side of the renal artery, which was kept distended by constricting the vessel distal to the cuff and applying pressure through the wide-bore tube, to prevent collapse and hence dislocation of the cuff. This also proved a test for the absence of collateral channels connecting with the tube artery conduit. Varying measured volumes of heparinized blood from the animal were injected at near systolic pressure. A plot of volume, as recorded off the cuff signal, consistently over-estimated the actual injected volume in all experiments, but the calibration was always linear and passed through zero. The error estimated from this calibration served to correct flow measurements for the experiment. After the surgical procedure, which took ca. 2 hr, a time (ca. 1 hr) was allowed to elapse before the first readings were taken, in order to permit transient effects due to surgery to subside. An experiment consisted of two runs. The first, or 'control' run, was separated in time from the second, or 'test' run, by 30 min. At the end of the control run in six dogs, an i.v. injection of indomethacin 10 mglkg (Merck, Sharp and Dohme) was given in 0-2 M-potassium phosphate buffer (pH 7-4). Five dogs did not receive indomethacin between control and test runs but were given an equivalent volume of buffer alone (30 ml.). Dogs given indomethacin alternated for study with those given only buffer. In a separate group of seven greyhounds, indomethacin was given at a dose of 2 mglkg to four animals, while the remaining dogs received buffer (10 ml.) between runs.
Steady-state records A 'run' consisted of steady-state blood flow readings at different levels of aortic pressure and some transient flow responses to a step pressure change. Typically, subsequent to records for zero flow and zero pressure, a run was begun with a reading of flow at resting pressure. Following this, the renal artery pressure was reduced by pulling on the aortic snare, and constricting the aorta. After holding the pressure reduction at a steady level for 30 sec, flow and pressure tracings were obtained at a paper speed of 2-5-5 cm/sec, and then the snare was released. Resting pressure and zero readings were taken after every two such manoeuvres. Following aortic or renal artery constriction, a time gap of 3 min was allowed before taking any further readings. The general aim in a run was to obtain flow records at ca. six to seven evenly spaced pressure intervals, both above and below 100 mmHg.
628
L. J. BEILIN AND J. BHATTACHARYA
'Transient' records On completion of the steady-state records, pressure was reduced with the snare by ca. 60 mmHg from control systolic levels and held steadily for 30 sec. The recording chart was run at 2-5 or 5 cm/sec, and a few seconds later the aortic constriction abruptly relieved. The subsequent pressure and flow profiles were recorded for ca. 45 sec. A second transient was recorded in exactly the same way, but at a slower paper speed of 10 cm/min. Again, resting pressure and zero levels for pressure and flow were taken before and after each transient. Finally, the animals were killed with i.v. pentobarbitone, and the left kidney was removed and weighed. Mean flow was estimated from the charts by planimetry. Mean pressure was obtained by adding a third of the pulse pressure to the diastolic pressure. Pressure and flow transients were analysed from records run at 2-5-5 cm/sec. Paired comparisons were done using Student's t test. Mean renal blood flow was plotted against mean aortic pressure for each series of pressure changes. As the graphs showed that autoregulation of blood flow was not seen at pressures below ca. 80 mmHg, whereas flow was relatively constant for pressures above ca. 100 mmHg (Fig. 1), regression of flow on pressure was calculated separately for points above B.P. = 100 mmHg, and for those below B.P. = 80 mmHg. The adequacy of autoregulation was established by: (i) Lack of a statistically significant correlation between flow and pressure above 100 mmHg, and (ii) the presence of such a correlation below 80 mmHg. RESULTS
The main effect of indomethacin at doses of 2 and 10mg/kg was to reduce renal blood flow. At a dose of 10 mg/kg the group mean renal blood flow at resting pressure decreased by ca. 22 % (P < 0-01), and at 2 mg/kg by ca. 10 % (P < 0-03) (Table 1). In contrast, in dogs given buffer, renal blood flow (if anything) increased, while group averages for pressure and heart rate were lower after the injection of buffer (Table 1). A 10 % increase in mean B.P. (P < 0-02) and 25 % reduction in mean heart rate (P < 0-02) was seen after indomethacin (2 mg/kg) but not after buffer (Table 1). In the dogs receiving indomethacin at 10 mg/kg, the fall in heart rate was more pronounced, although the rise in B.P. was less pronounced.
Steady-state responses By the criteria for autoregulation described above, every plot of flow against pressure showed good autoregulation, whether taken before or after buffer or indomethacin (2 and 10 mg/kg) (Fig. 2). The calculated slopes of the flow pressure diagram for points taken above pressures of 100 mmHg were not significantly different from zero (group average slopes - 0-003, s.E. of mean + 0-001 before buffer and - 0-002, s.E. of mean + 0-002 after buffer; - 0-006, s.E. of mean + 0-002 before indomethacin and - 0-001, s.E. of mean + 0-002 after indomethacin 10 mg/kg),
629 RENAL AUTOREGULATION whereas the relationship was highly significant in every case for the low pressure points, with a significant reduction in the regression slopes after indomethacin 10 mg/kg (P < 0.02) compared with before (group average slopes 005, s.E. of mean + 0 007 before buffer, - 004, s.E. of mean + 0-007 after buffer; 005, s.E. of mean + 0008 before indomethacin and 0-036, s.E. of mean + 0-009 after indomethacin 10 mg/kg). A -
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parabolic regression seemed to suit the plots of flow against pressure (P < 0-01 in each case)*, so parabolas were drawn from the data from each experiment on an X-Y plotter (Hewlett-Packard). Group means of flow during control and test runs for dogs given indomethacin (10 mg/kg) *
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The transient response The response of flow to a sudden increase in pressure after 30 see of aortic constriction of moderate severity was usually characterized by a triphasic response (Figs. 4, 5). In dogs given buffer, a sudden increase in pressure following abrupt release of aortic constriction caused the following: (1) A short period of transient hyperaemia in which flow increased by ca. 40 % of preconstriction control values in the first two flow pulses. (2) Subsequently, a progressive decrease in flow took place over approx. 5 see to a steady plateau at about 20 % above control level. 23-2
L. J. BEILIN AND J. BHATTACHARYA 632 The duration of this elevated plateau was variable in the runs analysed. On average, it lasted up to the 19th see from the step, although on occasions being more prolonged. (3) The end of the response, as a whole, was signalled by a further lowering of flow to within 10 % of control levels. Averages of flow and pressure over intervals of approx. 2 see from the relief of aortic constriction, expressed as percentage difference from the 4.0
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resting value, were plotted for each run. After indomethacin the most noticeable difference was a swifter fall in flow, following the initial hyperaemia and the attainment of a flow minimum in the first 10 see (Fig. 6). Paired tests on flow from control and test runs showed a significant reduction after indomethacin (P < 0-001) on the 6th, 8th and 10th see after relief of aortic constriction. Pressure usually returned to resting levels over 20 see following the step increase. Control flows were similar before and after the transient changes.
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to pressure during a value certain of difficult. is pressure is denoted Usually, autoregulation as a limiting index above which autoregulation is seen (Nashat, 1974), although it is unlikely that autoregulatory processes are switched on abruptly at a fixed point in pressure. In the present study a graphical
Quantification of the steady-state flow response
L. J. BEILIN AND J. BHATTACHARYA 634 approach was used which involved the demonstration that above a pressure of 100 mmHg, where visual evidence of autoregulation was apparent on the flow-pressure plots, flow consistently failed to correlate Dog 40 before indomethacin I
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with pressure. On the other hand, below 80 mmHg a correlation was always obtained. Further, comparison of flow at control pressure with that measured at ca. 105 mmHg yielded no significant differences. The plots of renal blood flow against pressure agree well with those obtained previously (Selkurt, 1946; Guyton, Langston & Navar, 1964).
RENAL AUTOREGULATION 635 Both the steady-state and transient data negate a central role for prostaglandins as being mediators of autoregulation in the kidney. Thus, after indomethacin, there was constancy of blood flow with pressure change above 100 mmHg in the steady state, and the dynamic-flow response to a sudden increase in pressure subsided with a delay not exceeding that seen in the normal. If anything, the transient increase in flow after a steep 50 T
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L. J. BEILIN AND J. BHATTACHARYA 636 rise of pressure is relatively short-lived after the drug, and there are attempts at restoring the control level very early on in the sequence. This is not seen in the pre-indomethacin transient. In order to exclude the effects of anaesthesia, haemorrhage and other time-dependent variables on these measurements, some dogs were given only phosphate buffer. There was good autoregulation of flow before and after buffer and no remarkable alterations occurred in heart rate or blood pressure. This suggests that the changes seen in the flow-pressure relationships after indomethacin were probably not due to influences other than those due to the drug. Indomethacin inhibits a number of enzymes, in addition to those concerned with prostaglandin synthesis, although with considerably less potency (Flower & Vane, 1974), so that an effect of this drug unrelated to that on prostaglandins must be considered a possibility. Although we had no direct measurement of renal prostaglandin synthesis there is evidence that the doses of indomethacin used here were sufficient to reduce prostaglandin synthesis (Vane, 1971; Ferreira, Moncada & Vane, 1971). Indomethacin, in doses less than those used in the present experiments, has been shown to reduce significantly the efflux of prostaglandins into dog renal venous blood (Aiken & Vane, 1973; Herbaczynska-Cedro & Vane, 1973; Lonigro, Itskovitz, Crowshaw & McGiff, 1973) and to enhance the vasoconstriction caused by angiotensin, which normally promotes prostaglandin release in the absence of indomethacin. Indomethacin abolishes the conversion of labelled arachidonic acid to prostaglandins in homogenates of rabbit kidney (Larsson & Angaard, 1973), and reduces urinary efflux of prostaglandins by 40 %. White, Kirschenbaum, Stein & Ferris (1974), using specific radio-immunoassay, have shown that a single 2 mg/kg dose of indomethacin in anaesthetized dogs reduces the renal venous efflux of prostaglandins of the E series for over 2 hr. Both indomethacin and meclofenamate, two inhibitors of prostaglandin synthesis which differ in chemical structure, increase renal vascular resistance in conscious rabbits (Beilin & Bhattacharya, 1976, 1977). The maintenance of renal blood flow autoregulation, in the presence of indomethacin, is in contradiction to the findings of Herbaczynska-Cedro & Vane (1973) but is in agreement with the recent publication of Swain et al. (1975). The former study was carried out using pump perfused kidneys and it is very likely that the effects of anaesthesia, surgical trauma and severe intermittent hypoxia combined to exaggerate the contribution that prostaglandins appeared to make the renal blood flow in those experiments. It is not possible to conclude whether the reduction in renal blood flow with indomethacin was secondary to loss of a vasodilator action of prosta-
RENAL A UTOREGULATION 637 glandins alone, or also to an associated increase in over-all sympathetic tone. Hedqvist (1974) has demonstrated decreased noradrenaline reuptake in sympathetic nerves in various organs, including the kidney, in the presence of indomethacin; and increased sympathetic activity reduces total renal blood flow (Pomeranz, Birtch & Barger, 1968). The increase in arterial pressure seen after indomethacin may reflect such a generalized change in sympathetic tone to arterioles. The situation during the periods of aortic constriction used to assess autoregulation is further complicated by nervous and humoral changes. For example, the increase in arterial pressure, proximal to the constriction, will have activated carotid and aortic baroreflexes leading to withdrawal of sympathetic tone and increased vagal tone, while below the constriction the more severe reductions in renal perfusion pressure would tend to stimulate release of renin. The demonstration of efficient autoregulation of renal blood flow in the presence of these changes, and despite the administration of large doses of a prostaglandin synthesis inhibitor, leaves the myogenic hypothesis (Folkow, 1949) unassailed. The transient The normal transient flow profile obtained was a triphasic one in most cases and consisted of a period of hyperaemia, lasting ca. 5 sec, in which flow rose to a peak of ca. 40 % above control, and then progressively declined; a plateau at ca. 20 % above control, and further lowering of flow after 20 sec to within 10 % of control flow. Held, Niedermayer, Schaffer, Schwartzkopf & Weiss (1971) observed a similar triphasic flow response, with comparable time course, to pump induced increases in pressure. Such responses also have been recorded from the isolated kidney and heart (Basar, Ruedas, Schartzkopf & Weiss, 1968; Basar & Weiss, 1969; Basar, Tischner & Weiss, 1968). After indomethacin, in what is presumed to be a state of relative depletion of prostaglandins, the second phase of the transient response was biphasic, contrasting with the normal hyperaemic plateau. This swift descent to near control levels was soon followed by a rebound to ca. 20 % above control. Flow instability with indomethacin has been commented on earlier, but not quantified (Herbaczynska-Cedro & Vane, 1973; Swain et al. 1975). The subsequent delay (third phase) in the return to control levels was comparable with the pre-indomethacin flow transient. Thus, the early phase of autoregulation was (if anything) augmented after indomethacin, the reverse of what Herbaczynska-Cedro & Vane (1973) suggested. Herbaczynska-Cedro & Vane (1974) and Swain et al.(1975) concluded that the prostaglandins are important in establishing reactive hyperaemia. The present findings make it difficult to assign a role for prostaglandins as mediators of reactive
L. J. BEILIN AND J. BHATTACHARYA hyperaemia in previously non-ischaemic kidneys. Prior to eliciting reactive hyperaemia, previous workers caused total cessation of blood flow in the kidney for 45 sec or more. It is possible that severe anoxia, which can relax vasculature (Selkurt, 1946), may also cause greater prostaglandindependant vasodilation (Wennmalm, Pham-hull-Chanh & Junstad, 1974) and hence more pronounced hyperaemia on relief of stress. In the present experiments, flow restriction was considerably less severe. A role of prostaglandins as determinants of resting renal vascular tone is compatible with the present data. In the anaesthetized dog, the downward resetting of the steady-state flow-pressure relationship following administration of a prostaglandin synthesis inhibitor, may be attributable to a modulating action of prostaglandins on vascular tone, leaving unaffected the ability of the kidney to maintain stable flow rates over a wide range of perfusion pressures. 638
This work formed part of an Oxford D.Phil. thesis by Dr J. Bhattacharya, who was in receipt of a Schorstein Scholarship. We are grateful for financial support from the National Kidney Research Fund and I.C.I. Ltd and the Wellcome Trust.
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