European Journal of Pharmacology, 60 (1979) 181--187

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© Elsevier/North-Holland Biomedical Press

CHARACTERIZATION OF FUROSEMIDE-INDUCED ACTIVATION OF THE RENAL PROSTAGLANDIN SYSTEM * GIOVANNI CIABATTONI **, FRANCESCO PUGLIESE, GIULIO A. CINOTTI, GIOVANNI STIRATI, ROBERTO RONCI, GIOACCHINO CASTRUCCI, ALESSANDRO PIERUCCI and CARLO PATRONO

Department of Pharmacology, Catholic University and Department of Medicine H, University of Rome, Italy Received 19 July 1979, accepted 6 September 1979

G. C I A B A T T O N I , F. PUGLIESE, G.A. CINOTTI, G. STIRATI, R. RONCI, G. C A S T R U C C I , A. PIERUCCI and C. P A T R O N O , Characterization of furosemide-induced activation of the renal prostaglandin system. European J. Pharmacol. 60 (1979) 181--187. A detailed time course of changes in plasma renin activity (PRA), urinary prostaglandin (PG) E2, PGF2a, thromboxane (TX) B2 and sodium excretion rates following furosemide was obtained in 7 women. P R A increased within the first 15 min and remained elevated all through the experiment. PGE2, PGF2a, T X B 2 and sodium increased simultaneously, reached a peak between 15 and 45 rain after furosemide and declined thereafter. It is concluded that furosemide induces a generalized activation of the renal P G system temporally related to the increase of renin release and natriuresis.

Renal prostaglandins

Thromboxane B2

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1. Introduction Several lines of evidence from data obtained in humans indicate that the increase of renin release, renal blood flow (RBF) and natriuresis following high ceiling diuretics may be mediated, at least in part, by increased PG biosynthesis in the kidney (Patak et al., 1975; Weber et al., 1977). In particular it has been claimed that (a) furosemide stimulates arachidonic acid release and, by these means, the formation of PG endoperoxides and PGE2 which, in turn, increase renin release and RBF in the kidney; (b) a delayed increase in PGF2a synthesis induced by raised PGE2-9-keto-reductase activity may * This study was presented in part at the 7th International Congress of Pharmacology, Paris, July 16-21, 1978 (Abstract No. 2182). ** Address for correspondence: Dr. G. Ciabattoni, Dept. of Pharmacology, Catholic University, Via Pineta Sacchetti 644, 00168 Rome, Italy.

Renin radioimmunoassay

be involved in the mechanism of termination of furosemide-induced renin release (Weber et al., 1977). The aim of the present study was to obtain detailed information on the occurrence and time course of activation of the renal PG system in relation to the increase of renin release and natriuresis induced by furosemide. Since urinary PGs appear to r e f l e c t - within l i m i t s - their renal synthesis (FrSlich et al., 1975), measurement of PGE2 and PGF:a excretion rates by radioimmunoassay represents a convenient tool to investigate the effect of drugs on the renal PG system (Ciabattoni et al., 1979b). Consistency of data obtained with various antisera and with gas chromatography/mass spectrometry was used to validate the specificity of such measurements (Ciabattoni et al., 1979a). The applicability of urinary PG measurements to human studies appears, however, to be restricted to female subjects since seminal fluid may contribute a highly variable fraction

182 of the measured urinary PGs in men (Patrono et al., 1979c). Accordingly, the present study was performed in healthy women.

2. Materials and methods

2.1. Experimental Informed written consent was obtained from 7 healthy female volunteers (aged 20-40 years). They were placed on a controlled sodium and potassium intake (100 and 80 mEq/day, respectively) for 5 days prior to the study. They were catheterized after an overnight fast and remained recumbent for the whole duration of the experiment. A 4 h control sample before and 16 consecutive 15 min urine samples after an i.v. injection of furosemide (50 mg) were collected and immediately frozen. Sodium balance and plasma volume were kept as constant as possible, by continuous replacement with an isotonic saline solution i.v. The infusion rate of this solution was adjusted according to sodium and urinary volume determinations performed every 15 min. Samples of peripheral venous blood (10 ml) were collected in chilled tubes containing EDTA (0.02 ml of a 5% solution per ml of blood), before and 15, 30, 60, 120, 180 and 240 min after furosemide. The separated plasma was frozen immediately. Both urine and plasma samples were kept a t - - 2 0 ° C until the time of the assay.

2.2. Analyses Urinary excretion of unmetabolized PGE2, PGF2a and TXB2 was determined b y radioimmunoassay (RIA), after prior extraction with an organic solvent system and group separation of the urinary PGs b y silicic acid column chromatography, as described elsewhere (Ciabattoni et al., 1979b). Anti-PGE2 (GP 356) and anti-PGF2a (GP 705) sera were obtained from guinea-pigs immunized with human serum albumin-PG conjugates. The immunizing procedure and cross-reactivity of

G. CIABATTONI ET AL. each antiserum have been described elsewhere (Ciabattoni et al., 1979b). 3H-PGE2 and 3HPGF2a (120-170 Ci/mM) were purchased from Amersham Radiochemical Centre. 3H-TXB2 was kindly provided by Dr. B.A. Peskar (Anhut et al., 1977), and anti-TXB2 serum was a gift of Dr. L. Levine. The cross-reactivity of primary PGs and PG metabolites with this antiserum is less than 1% (Patrono et al., 1979b). The concentration of TXB2 required to displace 50% of b o u n d 3H-TXB2 is 50 pg/ ml. TXB2 RIA conditions were the same as for PGE2 and PGF2~. The nature of PG- and TXB2-1ike immunoreactivity present in purified urinary extracts after furosemide injection was studied by means of thin layer chromatography (TLC), as previously reported (Patrono et al., 1978; Ciabattoni et al., 1979b). Plasma renin activity (PRA) was measured by RIA of angiotensin I, as described by Haber et al. (1969), using a commercially available kit (Sorin Biomedica, Saluggia, Italy). Urinary sodium was determined by flame photometry. Statistical analysis of the data was performed using the paired Student's t-test.

3. Results The time course of PRA and of urinary PGE2, PGF2a, TXB2 and sodium after furosemide injection is depicted in fig. 1. Furosemide caused a statistically significant increase of urinary PGF2~, PGE2, TXB2 and sodium excretion rates and of PRA levels, within the first 15 min. The basal urinary excretion rate of PGF2~, PGE2 and TXB2 averaged 560 + 79, 286 +- 81 and 94 + 20 pg/min (mean + S.E.M.) respectively. During the first 15 min after furosemide, both PGs and TXB2 urinary excretion rate increased to 1719 + 354 pg/ min for PGF2~, 1016 + 287 for PGE2 and 505 + 208 for TXB2. The excretion rate of both PGs and TXB2 remained elevated over a period of 45 min, with no statistically significant differences among the first three collections and declined thereafter. The sodium

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Fig. 2. Chromatographic behaviour of furosemidestimulated urinary PGF2a-, P G E 2 - a n d TXB2-1ike immunoreactivity. A 100 ml aliquot of urine obtained during the 45 rain following furosemide injection was extracted and purified by silicic acid column chromatography. The dried residue of the column eluate was recovered with 0.25 ml of methanol and subjected to TLC, in the solvent system described by Cottee et al. (1977). After development, each lane of the plate was divided into 0.5 c m segments, the silica gel scraped off and eluted with 1 ml of methanol. The mixtures obtained after removing silica gel by centrifugation were dried and recovered in 0.02 M P O 4 buffer p H 7.4 and tested in the R I A systems. The total amount of immunoreactivity detected in each 0.5 c m segment is reported. The dots indicate the location of co-chromatographed authentic PGs: o, origin; 6kF, 6-keto-PGFla; F, PGF2a; TXB, TXB2; E, PGE2; D, PGD2; sf, solvent front. Ordinate: Immunoreactivity in eluates of silica gel segments (ng).

184

excretion rate showed a parallel increase as compared to PGs and TXB2, though it declined more slowly. PRA rose significantly after 15 min and remained elevated for the whole period of observation. Fig. 2 shows the pattern of TLC distribution of PG- and TXB2-1ike immunoreactivity present in the urine after furosemide injection, as determined by anti-PGF2a, anti-PGE2 and anti-TXB2 sera. The immunoreactivity detected by each antiserum in the methanol eluates of silica gel segments of the TLC plate showed an identical chromatographic behaviour with authentic PGF2~, PGE2 and TXB2, respectively. The presence of PGE 2-like immunoreactivity in an overlapping area of the TLC plate did not contribute to the measured TXB2-1ike immunoreactivity to any significant extent, in view of the very low cross-reactivity of PGE2 with anti-TXB2 serum (0.19%). Failure of the three antisera to detect any appreciable amount of immunoreactivity in areas other than those corresponding to the homologous compounds strongly supports the identification of the urinary immunoreactivity as PGF2~, PGE2 and TXB2, respectively.

4. Discussion Previous studies from different laboratories have clearly demonstrated that the increase of PRA induced by loop diuretics is blocked by pretreatment with indomethacin (a potent inhibitor of PG synthesis) in dogs (Olsen and Ahnfelt-R~ne, 1976), rabbits (Oliw et al., 1976) and in humans (Patak et al., 1975; Rumpf et al., 1975; FrSlich et ai., 1976; Weber et al., 1977). Moreover, direct evidence has been provided that these drugs can affect renal venous or urinary PG release (Williamson et al., 1976; Abe et al., 1976, 1977; Weber et al., 1977; Scherer et al., 1978; Abe et al., 1978). However, the reported changes were not characterized in a detailed fashion in terms of their temporal relation to other concomitant effects. In order to investigate the behaviour of various members of the

G. CIABATTONI ET AL.

renal PG system after furosemide, urinary PGE2, PGF2~ and TXB2 were measured at short intervals during a 4 h period following the injection. The main finding of the present study was the demonstration that furosemide induced a prompt and generalized activation of the renal PG system concomitant with increased renin release and enhanced natriuresis. The parallel behaviour of both PGs and their unchanged ratio throughout the study suggests that these compounds originate independently from common PG endoperoxide intermediates rather than by their interconversion via the 9-keto-reductase pathway. An additional novel finding of this investigation was the demonstration of relatively low though readily measurable levels of TXB2 in human female urine. The TLC data showing a single peak of immunoreactivity with the same chromatographic behaviour as authentic TXB2 strongly supports the identification of the urinary immunoreactive material as TXB2. The observation of an excretory pattern following furosemide substantially similar to that of PGE2 and PGF2~ strongly suggests that urinary TXB2 may reflect the renal synthesis of TXA2, breaking down non-enzymatically to TXB2 in urine. Morrison et al. (1977)have described the unmasking of thromboxane synthetase activity by ureteral obstruction in the rabbit kidney. Moreover, TXA2 production in glycerol-induced acute renal failure has been reported recently (Benabe et al., 1979). Using a more sensitive assay, Larsson (1978) has measured TXB2 in outer and inner cortex and medulla of the rabbit kidney. Very low levels could be detected in the outer and inner medulla, amounting to 1-2% of total PG measured. In addition, the synthesis of substantial amounts of TXB2 has been described in isolated rat kidney glomeruli (Hassid et al., 1979). Based on our urinary measurements in healthy women, TXB2 would amount to approximately 10% of total PG measured. Although it is conceivable that TXB2 may be formed by platelets circulating in the kidney, we have obtained evidence which argues against a platelet origin of urinary

FUROSEMIDE AND RENAL PROSTAGLANDINSYSTEM TXB2 (Patrono et al., in preparation). As for the mechanism(s) underlying the prompt and concomitant increase of the urinary excretion rate of both PGs and TXB: following furosemide injection, at least two separate effects of the drug might contribute to the observed changes: (a) an increased renal synthesis of these compounds, due to increased arachidonic acid release {Weber et al., 1977); (b) a diminished renal metabolism of these compounds as a result of inhibition of the 15-hydroxy-PG-dehydrogenase (PGDH) {Stone and Hart, 1976). Although the latter effect has only been demonstrated in vitro at high drug concentrations it might perhaps explain the simultaneous and comparable increase of three different substances, all of which are substrates for PGDH. A generalized inhibition of this enzyme would be compatible with the finding by Abe et al. {1976) of a decreased excretion of the main urinary metabolite of PGF2, after furosemide in man. In addition, since very limited information is available on the parameters possibly affecting PG excretion, one should perhaps not disregard the possibility that changes in urinary pH and flow rate might contribute to the observed excretory pattern following furosemide. That the increased diuresis per se is responsible for the increased PG and TXB2 excretion seems, however, unlikely in view of their substantially unmodified excretion following chlorothiazide administration {unpublished observation). An additional link between furosemide action and increased urinary excretion of members of the renal PG-system might be represented by activation of the renal kailikrein-kinin system (Abe et al., 1978), in view of the close interrelationship between the two systems (McGiff et al., 1976). We have recently characterized the time course of urinary kallikrein excretion following furosemide, under the same experimental conditions as in the present study (Cinotti et ai., 1979), and found that furosemide caused a prompt and short-lived increase of urinary kallikrein excretion.

185

As for the close temporal relationship between PG and sodium excretion found in the present study, it seems unlikely that the two events are linked by a causal connexion, since it has been previously shown that indomethacin has no influence on furosemideinduced natriuresis in man {Weber et al., 1977). Conflicting results have been reported by other investigators, with no conclusive evidence that renal PG synthesis and sodium excretion are closely interrelated {Dunn and Hood, 1977). On the contrary, the concomitant increase of PRA and urinary excretion of PGs and TXB2 is consistent with previous observations indicating that arachidonic acid metabolism via the cyclooxygenase pathway plays an essential role in furosemide-induced renin release. The reason for a sustained elevation of PRA despite restitution of fluid and electrolytes is not readily apparent. Although such replacement may have been incomplete, previous studies have shown that furosemideand ethacrynic acid-induced renin secretion was not modified when volume depletion was prevented by returning urinary flow to the femoral vein {Meyer et al., 1968; Cooke et al., 1970). The persistence of elevated PRA levels long after the return of both PGs and TXB2 to basal excretory rates might reflect at least two distinct mechanisms: (a) the existence of a late phase of furosemide-induced renin release which is not PG-mediated; (b) the occurrence of major changes in PG synthesis, involving particularly prostacyclin (PGI2) and its metabolites, which would not be evident from examining urine, because of the functional and anatomical compartmentalization of the renal vasculature and the nephron. PGI2 has been shown to stimulate renin release from rabbit renal cortical slices in vitro (Whorton et al., 1977) and from the dog denervated kidney in vivo {Gerber et al., 1978), and to increase PRA in healthy men (Patrono et al., 1979a). PGI2 infused into healthy men at a rate of 2.5-20 ng/kg/min does not increase the urinary excretion rate of 6-keto-PGFl~, thus

186

suggesting that this compound may originate from hydrolysis of a fraction of renal PGI2 escaping intrarenal metabolism, similarly to PGE2, PGF2~ and TXA2 (Patrono et al., 1979a). A five-fold increase of the excretion rate of 6-keto-PGFl~ was measured during the first 15 min following furosemide injection (Patrono et al., 1979a), thus providing additional evidence for the existence of a generalized activation of the renal PG system by this drug. The behaviour of urinary 6-ketoPGFla closely mimicked that of other members of the renal PG system, thus suggesting a common site of synthesis. It is, however, entirely possible that stimulation of PGI2 synthesis (or inhibition of its degradation) sufficient to sustain renin release may have continued in the vascular compartment despite the observed reduction in urinary excretion of 6-keto-PGFla. A clarification of this point might also contribute to the understanding of extra-renal effects of furosemide. Studies of this nature are now in progress in our laboratory.

Acknowledgement We are indebted to Dr. Carin Larsson for helpful discussion and criticism, and to Dr. John E. Pike (Upjohn Company, Kalamazoo) for several generous gifts of PGs and TXB2. This study was supported by Grants from Consiglio Nazionale delle Ricerche to G.A.C. (74.00171.04 and 75.00523.04) and to C.P. (Progetto finalizzato Tecnologie Biomediche, Subprogetto CHIM-2).

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G. CIABATTONI ET AL. in man: effect of furosemide on the excretion of the main metabolite of PGF2a, Prostaglandins 12, 843. Abe, K., M. Yasujima, S. Chiba, N. Irokawa, T. Ito, and K. Yoshinaga, 1977, Effect of furosemide on urinary excretion of Prostaglandin E in normal volunteers and patients with essential hypertension, Prostaglandins 14,513. Anhut, H., W. Bernauer and B.A. Peskar, 1977, Radioimmunological determination of Thromboxane release in cardiac anaphylaxis, European J. Pharmacol. 44, 85. Benabe, J.E., S. Klahr and A.R. Morrison, 1979, Thromboxane A2 production in glycerol-induced acute renal failure, Clin. Res. 2 7 , 4 0 9 A . Ciabattoni, G., F. Pugliese, E. Pinca, G.A. Cinotti, A. De Salvo, M.A. Satta and C. Patrono, 1979a, Biologic and methodologic variables affecting urinary prostaglandin measurement, Advan. Prost. Thromb. Res. (in press). Ciabattoni, G., F. Pugliese, M. Spaldi, G.A. Cinotti and C. Patrono. 1979b, Radioimmunoassay measurement of Prostaglandins E2 and F2c~ in h u m a n urine, J. Endocrinol. Invest. (in press). Cinotti, G.A., G. Stirati, F. Taggi, R. Ronci, B.M. Simonetti and A. Pierucci, 1979, Relationship between kallikrein and P R A after intravenous furosemide, J. Endocrinol. Invest. (in press). Cooke, C.R., T.C. Brown, B.J. Zacherle and W.G. Walker, 1970, The effect of altered sodium concentration in the distal nephron segment on renin release,J. Clin. Invest. 49, 1630. Cottee, F., R.J. Flower, S. Moncada, J.A. Salmon and J.R. Vane, 1977, Synthesis of 6-keto-PGF1a by ram seminal vesicle microsomes, Prostaglandins 14, 413. Dunn, M.J. and V.L. Hood, 1977, Prostaglandins and the kidney, Amer. J. Physiol. 233, F. 169. FrSlich, J.C., J.W. Hollifield, J.C. Dormois, B.L. FrSlich, H. Seyberth, A.M. Michelakis and J.A. Oates, 1976, Suppression of plasma renin activity by indomethacin in man, Circulation Res. 39,447. FrSlich, J.C., T.W. Wilson, B.J. Sweetman, M. Smigel, A.S. Nies, K. Carr, J.T. Watson and J.A. Oates, 1975, Urinary prostaglandins. Identification and origin, J. Clin. Invest. 55,763. Gerber, J.G., R.A. Branch, A.S. Nies, J.G. Gerkens, D.G. Shand, J. Hollifield and J.A. Oates, 1978, Prostaglandins and renin release: II. Assessment of renin secretion following infusion of PGI2, E2 and D2 into the renal artery of anesthetized dogs, Prostaglandins 15, 81. Haber, E., T. Koerner, L.B. Page, B. Kliman and A. Purnode, 1969, Application of a radioimmunoassay for angiotensin I to the physiologic measurement of plasma renin activity in normal h u m a n subjects, J. Clin. Endocrinol. 29, 1349.

FUROSEMIDE AND RENAL PROSTAGLANDIN SYSTEM Hassid, A., M. Konieczkowski and M.J. Dunn, 1979, Prostaglandin synthesis in isolated rat kidney glomeruli, Proc. Natl. Acad. Sci. USA, 76, 1155. Larsson, C., 1978, The renal Prostaglandin system; localization and some biological effects, Contrib. Nephrol. 12, 82. McGiff, J.C., H.D. Itskovitz, A. Terragno and P.Y.K. Wong, 1976, Modulation and mediation of the action of the renal kallikrein-kinin system by prostaglandins, Federation Proc. 3 5 , 1 7 5 . Meyer, P., J. Menard, N. Papanicolaou, J.M. Alexandre, C. Devaux and P. Milliez, 1968, Mechanism of renin release following furosemide diuresis in rabbit, Amer. J. Physiol. 2 1 5 , 9 0 8 . Morrison, A.R., K. Nishikawa and P. Needleman, 1977, Unmasking of Thromboxane A2 synthesis by ureteral obstruction in the rabbit kidney, Nature 2 6 7 , 2 5 9 . Oliw, E., G. K6ver, C. Larsson and E..~ngg~rd, 1976, Reduction by indomethacin of furosemide effects in the rabbit, European J. Pharmacol. 38, 95. Olsen, U.B. and J. Ahnfelt-R~nne, 1976, Bumetamide-induced increase of renal blood flow in conscious dogs and its relation to local renal hormones (PGE, Kallikrein and Renin), Acta Pharmacol. Toxicol. 3 8 , 2 1 9 . Patak, R.V., B.K. Mookerjee, C.J. Bentzel, P.E. Hysert, M. Babej and J.B. Lee, 1975, Antagonism of the effects of furosemide by indomethacin in normal and hypertensive man, Prostaglandins 10, 649. Patrono, C., G. Ciabattoni, G.A. Cinotti, F. Pugliese, A. De Salvo and G. Castrucci, 1978, Characterization of human urinary Prostaglandin-like immunoreactivity, Prostaglandins 1 5 , 7 0 0 (Abstract). Patrono, C., G. Ciabattoni, G.A. Cinotti, F. Pugliese, A. Maseri and S. Chierchia, 1979a, Prostacyclin and renin release in man, Clin. Res. 2 7 , 4 2 6 A .

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Patrono, C., G. Ciabattoni, F. Pugliese, E. Pinca, G. Castrucci, A. De Salvo, M.A. Satta and M. Parachini, 1979b, Radioimmunoassay of serum thromboxane B2 : a simple method to assess pharmacologic effects on platelet function, Advan. Prost. Thromb. Res. (in press). Patrono, C., A. Wennmalm, G. Ciabattoni, J. Nowak, F. Pugliese and G.A. Cinotti, 1979c, Evidence for an extra-renal origin of urinary Prostaglandin E 2 in healthy men, Prostaglandins (in press). Rumpf, K.W., S. Frenzel, H.D. Lowitz and F. Scheler, 1975, The effect of indomethacin on plasma renin activity in man under normal conditions and after stimulation of the renin angiotensin system, Prostaglandins 1 0 , 6 4 1 . Scherer, B., J. Schnermann, M. Sofroniew and P.C. Weber, 1978, Prostaglandin (PG) analysis in urine of humans and rats by different radioimmunoassays: effect on PG-excretion by PG-synthetase inhibitors, laparotomy and furosemide, Prostaglandins 1 5 , 2 5 5 . Stone, K.J. and M. Hart, 1976, Inhibition of renal PGE2-9-keto-reductase by diuretics, Prostaglandins 12,197. Weber, P.C., B. Scherer and C. Larsson, 1977, Increase of free arachidonic acid by furosemide in man as the cause of prostaglandin and renin release, European J. Pharmacol. 4 1 , 3 2 9 . Whorton, A.R., K. Misono, J. Hollifield, J.C. FrSlich, T. Inagami and J.A. Oates, 1977, Prostaglandins and renin release: I. Stimulation of renin release from rabbit renal cortical slices by PGI2, Prostaglandins 14, 1095. Williamson, H.E., G.R. Marchand, W.A. Bourland, D.B. Farley and D.E. Van Orden, 1976, Ethacrynic acid induced release of prostaglandin E to increase renal blood flow, Prostaglandins 11,519.