PROSTACYCLIN ACTIVITY IN RAT KIDNEY STIMULATED BY ANGIOTENSIN II

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1 Br. J. exp. Path. (1979) 60, 38 PROSTACYCLIN ACTIVITY IN RAT KIDNEY STIMULATED BY ANGIOTENSIN II K. SILBERBAUER, H. SINZINGER AND M. WINTER From the 2nd Department of Internal Medicine, Department of Physiology, and 2nd Department of Gastroenterology. Univresity of Vienna, Medical School, Vienna, Austria Received for publication May 14, 1978 Summary.-Prostacyclin (PGx, PG12) activity can be found in renal tissue as indicated by platelet aggregation inhibition. The activity of the medullary tissue in terms of wet weight is significantly higher (P<001) than that of cortex. The activity decreases with time and has almost entirely gone within 1 h. Boiling for 30 s destroys the inhibitory effect of the prostacyclin on platelet aggregation. Angiotensin II is able to stimulate the prostacyclin availability of the tissue after incubation for 3 min. Addition of angiotensin II to platelet-rich plasma (PRP) has no significant effect on ADP-induced platelet aggregation. The spontaneous generation of prostacyclin as well as that stimulated by angiotensin II can be suppressed by previous incubation of the tissue with a prostaglandin synthetase inhibitor such as ketoprofen. Tissues which have only a small amount of basal release in buffer show an increased platelet aggregation inhibitory effect after addition to PRP. The difference between medulla and cortex is statistically significant. This different release of prostacyclin cannot be related to different endothelial surface area, because the endothelial surface in medul - la and cortex is quite similar. It is suggested that prostacyclin has an important influence on the renal function. The different capacity of renal medulla and cortex in generation of prostacyclin could be of great importance for a fuller understanding of the physiology of renal function. PROSTAGLANDINS play an important role in renal function, i.e. the regulation of renal blood flow (Itskovitz et al., 1973, 1974), water and salt excretion (Tannenbaum etal., 1975; Kauker, 1977), and renin release (Hinman, 1972). The kidney has the capacity to synthesize various prostaglandins (McGiff et al., 1970). The interrelation of the renin-angiotensin, kallikrein-kinin and prostaglandin systems is one of the main determinants of renal function under physiological (McGiff and Itskovitz, 1973) and pathological conditions (Gill et al., 1976; McGiff, 1977). A new unstable prostaglandin (PGx, PG12), generated in vascular tissue, was described by Moncada et al. (1976a, 1977a, b; Johnson et al., 1977). The enzyme responsible for prostacyclin biosynthesis is mainly localized in the intima of the vascular wall (Salmou et al., 1977). Prostacyclin has 2 major biological characteristics; it is the most potent endogenous inhibitor of platelet aggregation as far as is known and is a strong vasodilator more potent than PGE2 (Moncada and Vane, 1978). In recent publications the existence of prostacyclin activity was reported in other tissues (placenta-myatt and Elder, 1977; heart-dedeckere, Nugteren and ten Hoor, 1977). The synthesis, metabolism and degradation of prostaglandin is quite different in the cortical and medullary part of the kidney (Weber and Mann, 1977). The aim of the present study was to evaluate the basal prostacyclin activity in Address for reprints: H. Sinzinger, Department of Physiology, University of Vienna, Medical School, 1090 Vienna, Schwarzspanierstrasse 17, Austria.

2 PROSTACYCLIN ACTIVITY IN RAT KIDNEY 39 rat kidney (medulla and cortex) and the influence of angiotensin II on prostacyclin availability. MATERIAL AND METHODS Animals.-I0 Wistar male rats (weight g, aged 6 months) were anaesthetized with 50 mg/kg body wt Pentothal sodium (5- ethyl-5-(1-methylbutyl) thiobarbituric acid Na, Abbott Labs, USA) i.p. After laparatomy the renal arteries and veins were clamped and the kidneys removed. The kidneys were kept in tris-hcl buffer (0-05 mol/l, ph 7-5) at 20 (ph 7.1). The investigations were performed between 15 and 60 min after removal. To test the time stability of prostacyclin availability from the tissue some experiments were done up to 4 h after removal. Kidney preparation. Histologically controlled samples (85 from 10 kidneys) were cut with a scalpel from the medulla (mean wt 19 ± 4 mg) and cortex ( 17 ± 3 mg) of the kidney and transferred without mechanical injury into incubation solution. Samples were incubated (220) for 3 min in 0-2 ml tris-hcl buffer, angiotensin II (Hypertensin, CIBA) diluted with buffer (2 ug/0i 1 ml), and ketoprofen (Profenid, Heilmittelwerke 2 ug/0i 1 ml), a potent inhibitor of prostaglandin synthesis. Platelet preparation.-human blood was drawn from healthy volunteers who had not taken any medication for at least 14 days (1 volume of sodium citrate 3-8% to 9 volumes blood). PRP (platelet-rich plasma) and PPP (platelet-poor plasma) was obtained by centrifugation at 150 g (8 min) and 1600 g (15 min). PRP samples are adjusted with PPP to a definite platelet number (250, ,000/mm3). Platelet aggregation in 1 ml samples was induced by ADP (final concentration 2,umol/l). Rat PRP was prepared according to the method of Sinakos and Caen (1967) using sodium citrate 3% and also adjusted with PPP to the same platelet count. Methods of measurement. The estimation of the prostacyclin activity was performed according to the method of Moncada et al. (1976a). The very potent inhibition of platelet aggregation by prostacyclin was measured in a platelet aggregometer (Born-type) under standardized conditions. The aggregation process was recorded continuously with an Omniscribe TM (Houston Instruments) recorder. We determined the maximal extent of platelet aggregation (ATmax) and the extent 4 min after the addition of ADP (AT4 mi) by assuming that PPP represents 100% aggregation and PRP 0%. As a measure of the rate of platelet aggregation the tan cx (oc i.e. the angle between the tangent of the steepest portion of the initial increase of the response curve and the basal line) was evaluated. Experiments.-100,u of the supernatant of the incubated tissue samples were added to human PRP 1 min before the addition of ADP and stirred at 370 with 1000 u/min. The antiaggregatory activity was measured. The time course of the anti-aggregatory ability was tested over a period of 1 h; the heat stability was evaluated by boiling the supernatant for 30 s and its effect on aggregation was retested. Preincubation with ketoprofen, washing with buffer twice, followed by incubation in 200,ul buffer were also carried out. In other experiments medullary and cortical samples of the kidney were incubated in PRP itself and stirred for 3 min at 37. Some analogous experiments were also carried out with rat PRP (0-5,tmol/l ADP). Statistical analysis.-the differences between group means were analysed using Student's t test. RESULTS The supernatant of renal cortex shows inhibition of ADP-induced platelet aggregation in comparison to buffer control, indicative of prostacyclin activity (Fig. 1, Table I). The inhibitory activity extracted from renal medulla (in terms of wet wt) was significantly stronger (tan (x: P<0-02; ATmax: P<0-05; ATmin: P) than that extracted from the renal cortex (Fig. 1, Table I, II). It seems therefore that the basal prostacyclin release in the Fic. 1. Effect of cortical and medullary tissue supernatants on platelet aggregation. 1, buffer control; 2, 28 mg cortex in buffer; 3, 28 mg medulla in buffer. Aggregation induced by ADP (human PRP); final concentration 2,umol/l. Ui 2

3 40 K. SILBERBAUER, H. SINZINGER AND M. WINTER TABLE I.-Inhibition of ADP-induced Platelet Aggregation by Renal Cortex Extract Platelet aggregation tan o ETmax ET4min Wet weight (mg; X±s. d.) TABLE II. x±s.e. mean. Platelet aggregation tan cx T max T4min Wet weight (mg; ±s.d.) R±s.e. mean. A Control 6-31 ±0 * 67 C Angiotensin II Cortex 5 00±0*43 40±1 16±6 17±1 A:B p A:C P B:C P n.s n.s n.s n.s. <0 01 <0-05 <0-05 <0 01 <0-05 Inhibition of ADP-induced Platelet Aggregation by Renal Medulla Extract A Control 6-31±0-67 B Cortex * 54 51±4 44± 7 17±3 B Medulla ±4 13±5 19±4 C Angiotensin II Medulla 1-08±0-46 7±3 1±1 18±5 A:B p <001 A:C p n.s. B:C p <0-05 <0-05 n.s. I I %T I40 30 ao I min FIG. 2.-Availability of prostacyclin during long-term incubation. 1, buffer control; 2, 24 mg medullary tissue (2 min) in buffer; 3, 24 mg medullary tissue (20 min) in buffer; 4, 24 mg medullary tissue (30 min) in buffer; 5, 24 mg medullary tissue (60 min) in buffer. Aggregation induced by a final concentration of 2,umol/l ADP. min FIG. 3.-Effect of boiling on the antiaggregatory activity produced by medullary tissue. 1, buffer control; 2, 15 mg medulla in buffer; 3, 16 mg medulla in buffer, supernatant boiled for 30 s; 4, 19 mg medulla in angiotensin II, boiled for 30 s. Aggregation induced by 2 pmol/l ADP; human PRP.

4 PROSTACYCLIN ACTIVITY IN RAT KIDNEY 41 I min Vie. 4. Reversal of ADP-induced platelet aggregation ( J ) by supernatant of medulla and cortex. 1, 18 mg cortex in buffer, addition of stupernatant at the top of the curve; 2, 16 mg medulla in buffer, addition of supernatant at the top of the curve; 3, 16 mg medulla in buffer; 4, buffer control. Human PRP; aggregation induced by ADP in a finial concentration of 2,umol/l. 4 within 1 h. Boiling the supernatant for 30 s destroys the inhibitory potency (Fig. 3). Platelets aggregated by previous addition of ADP can be disaggregated by addition of 041 ml of "active" supernatant. The expected irreversible curve becomes reversible. This reversibility is more pronounced by the medullary supernatant than by the cortex (Fig. 4). Lowering of the ph of the incubation solution is followed by decreased availability of prostacyclin (Fig. 5). Different buffers (sodium barbiturate buffer, tris-hcl buffer, imidazole buffer, sodium phosphate buffer) show in the same ph range no influence on prostacyclin activity. Addition of angiotensin II to PRP had no significant influence on ADP-induced platelet aggregation; however, incubation of renal medulla in angiotensin II leads to a significantly stronger inhibitory activity than incubation in buffer alone (Fig. 6, Tables I, II). Boiling the "active" supernatant is also able to destroy the prostacyclin activity (Fig. 3). The time 1>~~~# _ Fie 5. Influence of ph on prostacyclin availability. 1, buffer control (ph 7 4); 2, 23 mg medulla in buffer (ph 7.4); 3, 21 mg medulla in buffer (ph 6.5); 4, 18 mg medulla (ph 5.5). cortex is only about 25 0 that of medullary activity. The availability of prostacyclin decreases during incubation at 22 (Fig. 2) and has almost completely disappeared Fia. 6.-Enhanced prostacyclin generation by angiotensin II. 1, buffer control; 2, 16 mg medulla incubated in buffer; 3, 19 mg medulla incubated in angiotensin II. Aggregation induced by a final concentration of 2 jimol/i ADP with hu-man PRP. 2

5 42 K. SILBERBAUER, H. SINZINGER AND M. WINTER &_2' _. curve of PG12 after angiotensin II stimulation is similar to that obtained for basal release of prostacyclin (by incubation with buffer only). Ketoprofen inhibits the second wave of platelet aggregation. Incubation of cortex and medulla in buffer containing ketoprofen for 2 min has no additional inhibitory activity. After preincubation of the tissue in ketoprofen for 2 min, twice washing, followed by incubation in buffer for 3 min, no PGI2 is available (Fig. 7). In contrast a control experiment with washing of tissue twice in buffer followed by incubation in buffer for 3 min does show prostacyclin activity. Incubation of cortical and medullary samples directly in PRP for 3 min in the aggregometer reduces platelet aggregation induced by ADP. Tissues which on incubation in buffer show little basal release of prostacyclin also have an increased inhibitory effect when added to PRP. The marked difference between medulla and cortex can be found too. Various experiments were performed with human PRP. Controls carried out with rat PRP showed similar results _04 I min FIG. 7.-Inhibition of prostacyclin formation by preincubation of tissue in ketoprofen. 1, buffer control; 2, preincubation (2 min) of 15 mg medullary tissue in ketoprofen, washing 2 x, incubation in buffer for 3 min; 3, 16 mg medulla incubated in ketoprofen (3 min); 4, addition of ketoprofen to human PRP 1 min before ADP; 5, 16 mg medulla in angiotensin II. Aggregation in(luce(l by ADP in a concentration of 2,umol/l. DISCUSSION The results presented in this study demonstrate the capacity of normal rat kidney tissue to generate an unstable substance, which has the ability to inhibit ADP-induced platelet aggregation. The properties exhibited by this compound indicate that it is prostacyclin (PGx, PG12). The highly significant difference of prostacyclin activity between the renal, cortical and medullary tissue correlates with the known distribution of the "stable" compounds of prostaglandin synthesis in the kidney (Bohmann and Larsson, 1975; Weber and Mann, 1977). Moncada et al. (197 6b) reported that the main part of prostacyclin activity found is generated by the endothelium. According to Siegenthaler (1977) the endothelial surface area in medulla and cortex per mg weight is not significantly different. Therefore it can be stated that the different prostacyclin activities in medullary and cortical kidney tissue cannot be related to differences in endothelial surface area. In accordance with recent work done with blood vessels (Moncada et al., 1976a, b, 1977a, b; Moncada and Vane, 1977; Moncada, 1977; DeGaetano and Villa, 1977; DeGaetano, 1977; DeDeckere et al., 1977; Myatt and Elder, 1977; Gryglewski et al., 1976; Szczeklik, 1977), the properties of prostacyclin found in vessels and in kidney are quite similar. The very potent platelet-aggregation inhibitory activity of this substance lasts not longer than 1 h (Moncada et al., 1977a) and is destroyed by short-term boiling (Moncada et al., 1977). As our results demonstrate, prostacyclin availability decreases at a ph below 6-5. A

6 PROSTACYCLIN ACTIVITY IN RAT KIDNEY 43 suppressive effect of a cyclo-oxygenase inhibitor (ketoprofen) on prostacyclin activity can be found. An additional effect of other PG compounds on platelet inhibition in this bioassay cannot be completely excluded (Bunting et al., 1976). Rat platelets are, however, insensitive to PGE2 and PGD2. There is strong evidence therefore against interference of "stable" prostaglandins in this test (DeDeckere et al., 1977). Our assumption is supported by the known reactivity of human platelets to PGE2. In low concentration PGE2 somewhat enhances ADPinduced platelet aggregation and only in extremely high concentrations is it a weak inhibitor of shape change and aggregation (Shio et al., 1970). The interaction between the products of PG synthesis seems to be of great importance for renal function. McGiff et al. (1970) and Danon et al. (1975) reported that angiotensin II is able to stimulate the release of prostaglandins. On the other hand an intermediate PG G2 was shown to release renin directly (Weber et al., 1976). One could suggest that prostacyclin may contribute to the regulation of intrarenal haemodynamics. Gimbrone and Alexander (1975) were able to show generation of an immunoreactive PG E-like substance by angiotensin II; however, they did not measure prostacyclin. The different capacity of renal medulla and cortex to produce prostacyclin could probably be of importance for an understanding of various physiological and pathophysiological conditions. The Bartter syndrome (Bartter et al., 1962) for example is of particular interest for the study of prostacyclin, because overproduction of PGs may represent the key mechanism for the pathogenesis of this disease. We wish to thank Professor Ian Rannie for his kind help with this paper. REFERENCES BARTTER, F. C., P1RONOVE, P., GILL, J. R. & MCCARDLE, R. C. (1962) Hyperplasia of the Juxtaglomerular Complex with Hyperaldostero- 4 nism and Hypokalemic Alkalosis. A New Syndrome. Am. J. Med., 33, 811. BOHMANN, S. 0. & LARSSON, C. (1975) Prostaglandin Synthesis in Membrane Fractions from the Rabbit Renal Medulla. Acta physiol. scand., 94, 244. BUNTING, S., GRYCGLEWSKI, S., MONCADA, S. & VANE, J. R ) Arterial Wall Generate from Prostaglandin Endoperoxides a Substance (Prostaglandin X) Which Relaxes Strips of Mesenteric and Coeliac Arteries and Inhibits Platelet Aggregation. Prostaglandins, 12, 897. DANON, A., CHANG, L. C. T., SWEETMAN, B. J., NIES, A. S. & OATES, J. A. (1975) Synthesis of Prostaglandins by the Rat Renal Papilla in vitro. Mechanism of Stimulation by Angiotensin II. Biochim. biophys. Acta, 388, 71. DEDECKERE, E. A. M., NUGTEREN, D. H. & TEN HooR, F. (1977) Prostacyclin is the Major Prostaglandlin Released from the Isolated Perfused Rabbit and Rat Heart. Nature, 268, 160. DEGAETANO, G. & VILLA, S. (1977) Prostacyclinlike Activity in Vascular Tissues from Normal and Thrombocytopenic Rats is Inhibited by Aspirin. Proc. IJt. Atheroscler. Conf., Milan, November (in press). DEGAETANO, G. (1977). Proc. International Symposium on Platelets: A multidisciplinary approach. Florence, September. Remark in discussion. GILL, J. R., FROLICH, J. C. BOWDEN, R. E., TAYLOR, A. A., KEISER, H. R., SEYBERTH, H. W. OATES, J. A. & BARTTER, F. C. (1976) Bartter's Syndrome: a Disordler Characterized by High Urinary Prostaglandins and a Dependence of Hyperreninemia oin Prostaglandin Synthesis. Am. J. Med., 61, 43. GIMBRONE, M. A. & ALEXANDER, R. W. (1975) Angiotensin II Stimulation of Prostaglandin Prodluction in Culturecl Human Vascular Endothelium. Scienice, 189, 219. GRYCGLEWSKI, R., BUNTING, S., MONCADA, S., FLOWER, R. J. & VANE, J. R. (1976) Arterial Walls are Protected against, Deposition of Platelet Thrombi by a substance (Prostaglandin X) Which They Make from Prostaglandin Endoperoxidles. Prostaglandits, 12, 685. HINMAN, J. W. (1972) Prostaglandins. Ann. Rev. Biochem., 41, 161. ITSKOVITZ, H. D., TERRAGNO, N. A. & MCGIFF, J. C. (1974) Effect of Renal Prostaglandins on Distribution of Blood Flow in the Isolated Canine Kidney. Circ. Res., 34, 770. ITSKOVITZ, H. D., STEMPER, J., PACHOLCYZYK, D. & McGIFF, J. C. (1973) Renal Prostaglandins: Determinants of Intrarenal Distribution of Blood Flow. Clin. Sci. mol. Med., 45, 321. JOHNSON, R. A., MORTON, D. R., KINNER, J. H., GORMIAN, R. R., MCGIFIRE, J. R., SUN, F. F., WHITTACKER, N., BU-NTING, S., SALTMON, J. A., MONCADA, S. & VANE, J. R. (1977) The Chemical Characterization of Prostaglandin X (Prostacyclin). Prostaglantdins, 12, 915. KAIKER, M. L. (1977) Prostaglandin E2 Effect from the Luminal Side oin Renal Tubular 22Na-efflux: Tracer Microinjection Studies (39653). Proc. Soc. exp. Biol. Med., 154, 274. McGIFF, J. C. (1977) Bartter's Syndrome Results from an Imbalance of Vasoactive Hormones. Ann. mnt. Med., 87, 369. MCGIFF, J. C., CROWSHAW, K., TERRAGNO, N. A. &

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