Molecular and Cellular Endocrinology, 0 Elsevier/North-Holland

16 (1919)

29-37

29

Scientific Publishers, Ltd.

PROSTAGLANDIN SYNTHESIS BY ISOLATED RAT RENAL GLOMERULI J. SRAER r, J.D. SRAER ‘, D. CHANSEL ‘, F. RUSSO-MARIE B. KOUZNETZOVA 3 and R. ARDAILLOU r INSERM, Unite 64, Hbpital Tenon, 2 Unit& 90, HGpitai Necker et 3 Dipartement

de Radioimmunologie,

‘,

Institut Pasteur, Paris (France)

Received 22 May 1979; accepted 25 June 1979

The PGEa, PGF20 and 6-keto-PGFro contents of the incubation medium of glomeruli isolated from rat kidney were measured at different times with or without addition of arachidonic acid. These prostaglandins accumulated progressively with time and reached equilibrium after 60-120 min incubation. Synthesis of the 3 prostaglandins was inhibited when indomethacin was added whereas it was markedly enhanced, mainly for PGEa, at increasing doses of arachidonic acid. Plateaus were reached above 5 rg/ml and concentrations corresponding to 50% of the maximum values were 2 pg/ml for PGEa and PGFa,, and 0.8 pg/ml for 6-keto-PGFr,.. There were strictly linear relationships between PGEa or PGF ao productions and the concentration of glomerular protein. PGEa and PGF ao synthesis with or without arachidonic acid were maximum at 30-37°C. PGE2 glomerular content was almost undetectable initially and increased with time. These data demonstrate that PGEa, PGFa, and PGIa, in order of decreasing abundance, are synthesized by the glomerular cells and suggest that PGEa and PG12-sensitive glomerular adenylate cyclase activities and PGEa-sensitive renin synthesis may be stimulated by prostaglandins formed in the glomeruli themselves. Keywords:

renal glomeruh; prostaglandins.

Synthesis of prostaglandins (PG) in the kidneys has been demonstrated by histochemical techniques in collecting ducts (Janszen and Nugteren, 1971) and by tissue culture experiments in renal medullary interstitial cells (Muirhead et al., 1972; Dunn et al., 1976; Zusman and Keiser, 1977). PGs are also produced to a smaller extent in the renal cortex (Spector et al., 1974; Whorton et al., 1978) and several sites of production have been demonstrated: the arterial blood vessels (Terragno et al., 1978; Smith and Bell, 1978), the cortical collecting tubules (Smith and Bell, 1978) and also the glomeruli. Smith and Bell (1978) using immunohistochemical microscopy have found cyclooxygenase in ovine and bovine glomeruli. Folkert and Schlondorff (1979) and Hassid et al. (1979) have shown that [3H]arachidonic acid in the presence of isolated glomeruli was converted into several PCs identified by thin-layer chromatography. We have extended these studies and shown using specific radioimmunoassay for the 3 main renal PGs, PGE2, PGF2, and 6-keto-PGFr, that PGE2 was the major product synthesized by rat-isolated glomeruli.

30

J. Sraer et al.

METHODS Isolation of rat renal glomeruli Renal glomeruli were isolated from Sprague-Dawley rats weighing 150-220 g according to the technique of Fong and Drummond (1968) with minor modifications. After pentobarbital anesthesia (5 mg/lOO g body weight i.p.) the kidneys of 2 rats were removed. The cortex from the 4 kidneys was dissected and minced to a paste-like consistency. The homogenate, suspended in 20 mM Tris-HCl buffer, pH 7.4, containing 5 mM glucose, 135 mM NaCl, 10 mM KC1 and 10 mM NaCHeCOO, was pushed successively through a 106+rn sieve which excluded the tubules and a SO-pm sieve which retained the glomeruli. The suspension was then passed through a 25-gauge needle and centrifuged at 120 Xg for 90 sec. The supernatant was discarded, the pellet resuspended in the same buffer solution and passed again through the needle and centrifuged. This operation was repeated 5 times. Each individual preparation was checked for purity under light microscopy. Virtually no afferent and efferent arterioles could be detected. Tubular fragments were always below 2% of the total number of glomeruli. Chemicals and radioimmunoassay reagents PGE2, PGFzo, and 6-keto-PGFr, were gifts from Dr. J. Pike (Upjohn, Kalamazoo, MI) and indomethacin from Merck, Sharp and Dohme (Paris). Arachidonic acid (sodium salt) was obtained from Sigma (St. Louis, MO), kept under nitrogen at -20°C and extemporaneously dissolved in ethanol-water (7 : 10, v/v). [3H]PGEz and [3H]PGFzQl (1 lo-170 Ci/mmole) were purchased from the Radiochemical was coupled to histamine using carbodiCentre (Amersham, UK). 6-Keto-PGF,, imide (Maclouf et al., 1976). The iodination with 1251 of this conjugate using the chloramine-T procedure and the subsequent purification of the tracer were performed as previously described (Maclouf et al., 1976). AntiPGE,, anti-PGFr,, and anti&keto-PGFr, antiserums were obtained from Institut Pasteur (Paris). These antibodies cross-react only slightly with other prostaglandins and could be considered as specific (Dray et al., 1975; Dray et al., 1978). All other chemicals were analytical grade. Incubation of isolated glomeruli and measurement of PGE2 content of the incubation medium Isolated glomeruli (500-1000 kg/ml of glomerular protein) were incubated at 37°C in room atmosphere under continuous agitation (100 cycles/min) in 200 /..4.l of buffer similar to that employed in the purification procedure except for the addition of 1 mM CaC12. After usually 60 min incubation, the preparation was centrifuged at 3000 Xg for 10 min and the supernatant collected. PGEs and PGF2, synthesis by isolated glomeruli and its stimulation in the presence of arachidonic acid was demonstrated in early experiments using radioimmunoassay after extraction and chromatography according to Dray et al. (1975). In these conditions [3H]-

Prostaglandin synthesis by glomeruli

31

PGEz and [3H]PGF2a, were added to the sample for subsequent recovery prior to extraction. Since the incubation medium is essentially protein-free, these results were compared with parallel data directly obtained without prior extraction and chromatography. For this comparison, various experimental conditions (incubation time and arachidonic acid concentration) were used to obtain a wide range of prostaglandin concentrations. The results from the two techniques were closely related as shown by the parameters of the regression lines (results obtained from the indirect technique as a function of those obtained from the direct technique and corresponding to 13 coupled values). The correlation coefficients were to.992 and t0.993, the slopes were to.975 and t1.056, and the ordinate intercepts were t121 and -27 for PGE? and PGFzo, respectively. Both slopes were not statistically different from 1 and both ordinate intercepts were not statistically different from zero. Therefore all subsequent experiments were performed using a direct assay. 6-KetoPGF,, was also determined by radioimmunoassay directly without purification. The technique was different from that used for PGEz and PGF?,. [“‘1]6-KetoPGFr, and the corresponding specific antibody at a final dilution of l/30 000 were incubated for 18 h at 4°C in 300 ~1 of 0.05 M phosphate buffer, pH 7.4, containing 160 mM NaCl and 2 mg/ml of bovine gammaglobulins (Calbiochem, San Diego, CA). Free and bound radioactivity were separated after addition of 300 /J of 30% polyethylene glycol in distilled water. In these conditions, 50% inhibition of binding was obtained in the presence of 22 ng, 350 ng and 2000 ng/tube of 6-ketoPGFr,, PGEz and PGF?, respectively. PGEz, PGFzo, and 6-keto-PGFr, syntheses were studied as functions of time, arachidonic acid concentration and presence of indomethacin respectively. Supplementary experiments (effects of temperature and of glomerular protein concentration) were performed including only determinations of PGE2 and PGFZU. Each value given in the figures and in the tables is the mean of 6 determinations performed at increasing dilutions of the same sample of the incubation medium. A blank value corresponding to the incubation medium not exposed to glomeruli was prepared at each of the same dilutions. This blank value was nil in the absence of arachidonic acid or at low concentrations of this precursor in the incubation medium (smaller than 5 pg/ml). A non-specific displacement of the tracer was observed at the greatest concentrations (10 pg/ml) in the 3 radioimmunoassays. This corresponded to a blank value of approximately 7% of the total PG concentration. Glomerular protein was determined according to Lowry et al. (1951). Measurement of PGE, content of the isolated glomentli The intraglomerular content of PGEz was estimated as the difference between total PGEz of a glomerular suspension homogenized in a Sorvall omnimixer apparatus and PGEz present in the medium. Parallel studies were done in the presence of 1 .l 1 mM acetylsalicylic acid. In all these experiments, PGE? was assessed by radioimmunoassay after extraction and chromatography.

J. Sraer et al.

32

RESULTS Initial experiments indicated that PGEz and PGF?, syntheses were linearly related to the concentrations of glomerular protein from 0 to 2 mg/ml (r to.99 and to.97 for each of these prostaglandins respectively with 12 coupled values in both cases). PGEl and PGFza progressively accumulated as a function of time as shown in Fig. 1. When 2.5 pg/ml of arachidonic acid was added, the production of both prostaglandins was clearly enhanced, but more markedly for PGEz (6 times the control value at 60 min) than for PGFzo, (1.5 times the control value at 60 min). A plateau was rapidly reached for both prostaglandins and in each of the experimental conditions. Addition of 1 mM indomethacin to the incubation medium strongly inhibited PGEz and PGF?, productions whether or not arachidonic acid was present. PGEz and PGFzo, accumulations after 60 min incubation were studied at increasing doses of arachidonic acid. S-shaped curves (Fig. 2) were obtained. Maximum stimulations occurred above 5 pg/ml in both cases. They represented approx. 4 and 2.5 times the basal value for PGEz and PGFza respectively. Concentrations corresponding to 50% of the maximum stimulations were close to 2 pg/ml for both prostaglandins. PGEz and PGFzO productions were temperature-dependent both in

PGE2 hrm9-fl

30

60

90

120

min

Fig. 1. Time-course of PGEs (left) and PGF ao (right) accumulation with (closed triangles) and without (closed circles) 2.5 fig/ml arachidonic acid. The effect of 1 mM indomethacin is also shown at 60 mm incubation with (open triangles) and without (open circles) arachidonic acid. Each point is the mean of 6 values and each vertical bar represents twice the standard error of the mean.

33

Prostaglandin synthesis by glomeruli

f’GF2a (ngdrl.mg-l)

PGE2 (ng.h-‘.mg-‘)



I

I /f N

O.dl

0.1 ’

12

10

o.oi

100

0.1

1 2

arachidonic

Fig. 2. PGEa (left) and PGF2, (right) productions after 60 min increasing doses of arachidonic acid. Each point is the mean of 6 represents twice the standard error of the mean. The dotted lines corresponding to 50% of the maximum stimulations for each of tively.

10

acid

(pg.ml.l)

incubation as a function of values and each vertical bar indicate the concentrations these prostaglandins respec-

the presence and in the absence of arachidonic acid and were maximum in the range 30-37’C (Table 1). 6-KetoPGF,, progessively accumulated with time as shown in Fig. 3. A plateau Table 1 Effects of temperature on glomerular production without 2 pg/ml arachidonic acid Temperature (“C)

0 22 30 31

PGE2 (pg . h-t . mg-r )

of PGEz and PGFao (mean f S.E.M.) with or

PGFza (pg. h-r . mg-r )

Without arachidonic acid

With arachidonic acid

239 f 21 1191t81 1503 f 57 1188k41

614 3600 4611 3095

f 6 + 116 f 159 f 187

Without arachidonic acid

With arachidonic acid

321*44 838 f 50 759 f 37 888 + 52

520 5 65 1865 f 53 2130*48 2103 + 96

J. Sraer et al.

34 !Keto.PGFta h-1 mg-7

6Keto.PGF1, (“9

t?g.

mg-1)

T

3.

3-

I, 7

15

30

45

60

go

120

min

II

II..

. 0.01

0.1

1 2

. 10 arachldomc (vg.ml-l)

acid

Fig. 3. Time-course of accumulation (left) and production after 60 min incubation as a function of increasing doses of arachidonic acid (right) for 6-keto-PGFro. Time-course accumulation is shown with (closed triangles) and without (closed circles) 2.5 pg/ml arachidonic acid. The effect of 1 mM indomethacin is given at 60 min incubation with (open triangles) and without (open circles) arachidonic acid. Each point is the mean of 6 values and each vertical bar represents twice the standard error of the mean.

was obtained

60 min incubation. When 2.5 pg/ml of arachidonic acid was of 6-ketoPGF1, at equilibrium was slightly greater than two times the control value. Addition of 1 mM indomethacin to the incubation medium markedly inhibited 6-ketoPGFr, production whether or not arachidonic acid was present. 6-KetoPGFr, accumulation at 60 min was stimulated in the presence of increasing concentrations of arachidonic acid. The maximum value occurred above 2 pg/ml and represented slightly more than twice the basal value. The concentration corresponding to 50% of this maximum stimulation was 0.8 E.cg/ml. PGEz content of the isolated glomeruli was negligible. Indeed, at the beginning of the incubation when 200.6 * 17.1 pg/ml of PGEz could be detected in the total glomerular homogenate, as much as 192.3 + 23.7 pg/ml could be accounted for by PGEz content of the supernatant. After 60 min incubation, the corresponding values were 1182.0 f 58.3 and 941.2 + 32.0 pg/mg showing that, although increased, the glomerular content represented only 20% of the total PGEz present in the glomerular homogenate. In the presence of acetylsalicylic acid, there was a clear added,

after

the production

Prostaglandinsynthesis by glomeruli

decrease of the PGEz content of the glomerular homogenate, undetectable beginning of the incubation and reaching 331 .O f. 79.4 pg/mg at 60 min.

35

at the

DISCUSSION Our data clearly indicate that glomeruli isolated from the rat-kidney cortex synthesize PGEz, PGFzO and PGIz. The two first prostaglandins were directly measured in the incubation medium. The presence of the latter was demonstrated by the assay of its stable metabolic product, 6-keto-PGFr,. The extracellular accumulation of these 3 prostaglandins reflects new synthesis rather than release of stored material since it was strongly enhanced by arachidonic acid and clearly inhibited by indomethacin. Moreover, PGEz glomerular content, almost undetectable at the end of the purification process when the incubation was started, increased as well as PGEz of the incubation medium after 60 mm. The amounts of PGEz and PGF2, synthesized measured in the same conditions varied according to the day of the experiment. This may depend on the glomerular ability itself to produce PGs and also reflect variability in the incorporation into tissue phospholipids of arachidonic acid added. The maximum percentage conversions of arachidonic acid to PGEz and to PGF,, calculated from Fig. 2 were 0.20-0.25% per hour; the same parameter for 6-keto-PGF,, calculated from Fig. 3 was smaller, approximately 0.12%. Zusman and Keiser (1977) using rabbit medullary interstitial cells in tissue culture, reported a similar value for PGE,, but a clearly smaller one for PGF,,. In the presence of 2.5 pg/ml of arachidonic acid, stimulation of the basal production of PGEz was clearly greater than for PGFzol (Fig. 1) and 6-ketoPGF,, (Fig. 3). The irregular shapes of the time-course curves, mainly for PGEz and PGFzo, could be due to the multi-focal origin of the prostaglandins compatible with the heterogenous structure of the glomerulus composed of 3 cell types. Two or all of them may synthesize prostaglandins at different rates. The fmal plateaus after 60 min incubation could reflect the progressive degradation with time of the enzymes intervening in the syntheses, the decreased availability of arachidonic acid and/or equilibrium between prostaglandin formation and degradation. The absolute amounts of PGEz and PGF2, synthesized per hour and per mg of tissue protein were clearly smaller than those observed in the studies with medullary interstitial cells (Zusman and Keiser, 1977). However, as opposed to isolated glomeruli, cultured cells represent a single cellular type, and thus PGEz and PGFza productions are not strictly comparable on this basis. 6-Keto-PGFr, production by the renal cortex has recently been demonstrated (Wharton et al., 1978; Terragno et al., 1978). In these studies, the distribution of 3H radioactivity after incubation of renal tissue with [3H]arachidonic acid was only shown. This mode of expression allows comparison of the syntheses of the different prostaglandins but does not provide the absolute values of prostaglandins synthesized. 6-Keto-PGF,, was the principal arachidonic acid metabolite recovered from the cortical arteries (Terragno

36

J. Sraer et al.

et al., 1978) and was approximately equivalent to PGEz in microsomes prepared from rabbit renal cortex (Wharton et al., 1978). This is different from our results with glomeruli in the presence of arachidonic acid which show a clear preponderance of PGEz and PGFza over 6-keto-PGF,, when syntheses are expressed on a weight basis. More recently, Folkert and Schlondorff (1979) and Hassid et al. (1979) demonstrated PG production by isolated rat renal glomeruli. Their results differ from ours in several aspects. These authors used in most of their experiments thin-layer chromatography to identify the PGs produced after incubation of the glomeruli with [3H]arachidonic acid. This technique does not allow estimation of the amounts of PG produced and. thus cannot provide precise studies of accumulation of PG with time, PG synthesis as a function of arachidonic acid concentration or of glomerular protein. Moreover the necessary presence of exogenous arachidonic acid in the medium prevents from studying the basal conditions. In contradistinction with these two studies we observed a greater production of PGEz and not of PGF2,. PGEa and PGIz have been shown to stimulate glomerular adenylate cyclase (Schlondorff et al., 1978). The effective concentration of PGEz was 20 PM and thus clearly greater than those observed in the incubation medium, even in the presence of arachidonic acid. On the contrary, PGIz stimulated glomerular adenylate cyclase at 1 nM which is in the range of the concentrations of 6-ketoPGFi, obtained in the present report. This observation suggests a physiological role for PGIz on glomerular vasoreactivity. Other effects of glomerular prostaglandins, either directly or through cyclic AMP production, could be the decrease in the glomerular ultrafiltration coefficient (Baylis et al., 1976; Ichikawa and Brenner, 1977) and the stimulation of renin production by the juxtaglomerular apparatus (Yun et al., 1978).

ACKNOWLEDGMENTS The authors wish to express their sincere gratitude to Dr. P. Verroust for critical review of the manuscript. Miss Bizien and Miss Knobloch provided excellent secretarial service.

REFERENCES Baylis, C., Deen, W.M., Myers, B.D., and Brenner, B.M. (1976) Am. J. Physiol. 230, 1148. Dray, F., Charbonnel, B., and Maclouf, J. (1975) Eur. J. Clin. Invest. 5, 311. Dray, F., Pradelles, P., Maclouf, J., Sors, H., and Bringuier, A. (1978) Prostaglandins 15, 715. Dunn, M.J., Staley, R.S., and Harrison, M. (1976) Prostaglandins 12,37. Folkert, V.W., and Schlondorff, D. (1979) Prostaglandins 17, 79. Fong, J.S.C., and Drummond, K.N. (1968) J. Lab. Clin. Med. 71, 1034. Hassid, A., Konieczkowski, M., and Dunn, M.J. (1979) Proc. Natl. Acad. Sci. (U.S.A.) 76, 155.

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Ichikawa, I., and Brenner, B.M. (1977) Am. J. Physiol. 233, F102. Janszen, F.H., and Nugteren, D.H. (1971) Histochemistry 27,159. Lowry, O.H., Rosebrough, N.J., Fax, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265. Maclouf, J., Prader, M., PradeIles, P., and Dray, F. (1976) Biochim. Biophys. Acta 431, 139. Muirhead, E.E., Germain, G., Leach, B.E., Pitcock, J.A., Stephenson, P., Brooks, B., Brosius, W.L., Daniels, E.G., and Kinman, J.W. (1972) Circ. Res. 30-31, Suppl. II, 161. Schlondorff, D., Yoo, P., and Alpert, B.E. (1978) Am. J. Physiol. 235, F458. Smith, W.L., and Bell, T.G. (1978) Am. J. Physiol. 235, F451. Spector, D., Zusman, R.M., Caldwell, B.V., and Speroff, L. (1974) Prostaglandins 6,263. Terragno, N.A., Terragno, A., Early, J.A., Roberts, M.A., and McGiff, J.C. (1978) Clin. Sci. Mol. Med. 55, 199s. Whorton, A.R., Smigel, M., Oates, J.A., and Frolich, J.C. (1978) Biochim. Biophys. Acta 529, 176. Yun, J.C.H., Kelly, G.D., Bartter, F.C., and Smith, G.W. (1978) Life Sci. 23, 945. Zusman, R.M., and Kejser, H.R. (1977) J. Clin. Invest. 60, 215.

Prostaglandin synthesis by isolated rat renal glomeruli.

Molecular and Cellular Endocrinology, 0 Elsevier/North-Holland 16 (1919) 29-37 29 Scientific Publishers, Ltd. PROSTAGLANDIN SYNTHESIS BY ISOLATED...
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