0021-972X/79/4905-0765$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1979 by The Endocrine Society
Vol. 49, No. 5 Printed in U.S.A.
Activation of Inactive Plasma Renin by Tissue Kallikreins* FRANS H. M. DERKX, H. LIES TAN-TJIONG, ARIE J. MAN IN'T VELD, MARGREETH P. A. SCHALEKAMP, AND MAARTEN A. D. H. SCHALEKAMP Department of Internal Medicine I, University Hospital Dijkzigt, Erasmus University, Rotterdam, The Netherlands
ABSTRACT. It has been reported that inactive (acid-activable) human renin could be converted into the active form by adding urinary kallikrein to acid-pretreated plasma. Without prior acidification, however, only a small portion of the total amount of inactive renin present in plasma was converted (activated) by kallikrein, probably because native plasma contains protease (kallikrein) inhibitors that are destroyed by acid. We have separated inactive and active renin by DEAE-Sepharose column chromatography of normal human plasma at pH 7.5 and a
linearly increasing sodium gradient. Inactive renin isolated in this way could be activated at pH 7.5 by highly purified pancreas and urinary kallikreins. With the semipurified preparation of inactive renin, prior acidification was not required for obtaining virtually complete activation by kallikrein. The kallikreins were effective at concentrations as low as 1 X 10~8 mol/liter. It is therefore possible that one or more tissue kallikreins act as physiological activators of inactive renin. J Clin Endocrinol Metab 49: 765, 1979)
R
ENIN ACTIVITY of normal human plasma increases by a factor of 3-7 after treatment at pH 3.0-4.0; this is caused by the conversion of enzymatically inactive renin or prorenin into the active form (1-7). Acid activation of inactive renin is a two-stage process; renin activity increases slightly during the acidification step, but most of the rise in activity occurs after pH has been restored to neutral, probably through the action of one or more serine proteases (6, 8-10). It seems unlikely that this pathway is operative in circulating plasma because of the inhibitory effect of various plasma proteins. But in vivo activation of inactive renin at the tissue level through a serine protease is a possibility, and there is now some evidence to support this. Preliminary experiments by Morris and Day (11) showed increased renin activity of amniotic fluid after the addition of large quantities of a crude preparation of pancreas kallikrein. Sealey et al. (12) reported that the rate of renin activation in acid-treated plasma was increased after the addition of urinary kallikrein, which is known to originate from the kidney (13,14); the renin activity ultimately attained was not higher than that attained with acid treatment alone. The possibility that kallikrein acts as an activator of
inactive renin is interesting, since renal kallikrein is produced close to the site where renin is synthesized (15-18) so that an effect of kallikrein on renin biosynthesis could influence renal circulation and sodium handling. Here we describe additional data on the activation of inactive renin by tissue kallikreins. We have separated inactive plasma renin from active renin by ion exchange chromatography and found that the inactive fraction could be activated at physiological pH by highly purified pancreas and urinary kallikreins without prior treatment with acid. Materials and Methods Ion exchange chromatography Blood from healthy male subjects was collected in plastic tubes containing disodium-EDTA (0.005 mol/liter blood). Within 5 min, the blood was centrifuged at 8000 X g for 10 min. The plasma was immediately frozen at —20 C. For ion exchange chromatography, EDTA-plasma was thawed and dialyzed for 24 h against Tris-acetate (0.024 mol/liter) buffer, pH 7.5, which contained NaCl (0.020 mol/liter). The dialyzed plasma (30 ml) was applied to 40 X 2.6-cm columns of DEAE-Sepharose CL6B (Pharmacia Fine Chemicals, Uppsala, Sweden), which had been equilibrated with the same buffer. Elution was performed with a linear gradient of NaCl up to 0.200 mol/liter. Flow rate was 20-22 ml/h, and the eluate was collected in 5.0- to 5.5-ml fractions. All procedures were carried out at 4 C.
Received January 23,1979. Address requests for reprints to: Dr. M. A. D. H. Schalekamp, Department of Internal Medicine I, Room P 434, University Hospital Dijkzigt, Erasmus University, Rotterdam, The Netherlands. * This work was supported by a grant from the Organization for Health Research (TNO), the Netherlands.
Activation of inactive renin by acid Samples (2 ml) of EDTA-plasma or fractions isolated from 765
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DERKX ET AL.
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plasma by ion exchange chromatography were dialyzed for 24 h at 4 C against a glycine-HCl buffer, pH 3.3, which contained EDTA (0.005 mol/liter) (19). After pH had rapidly been restored to 7.5 with NaOH (1.0 mol/liter), the samples were dialyzed for various periods, as indicated, at 4 C against a phosphate buffer, pH 7.5, containing EDTA (0.001 mol/liter) (19). Binding of kallikreins to Sepharose Highly purified hog pancreas kallikrein (Kallikrein, Bayer KZ 45/32, Leverkusen, West Germany) was a gift from Prof. G. L. Haberland and Dr. E. Wischhofer. This preparation had a specific activity of 1180 biological kallikrein units (Frey Units)/ mg protein, which corresponds with 9.4 enzymatic units. One enzymatic unit is defined here as the amount of enzyme hydrolyzing 1 /xmol of the synthetic chromogenic substrate H-D-valylL-leucyl-L-arginine-p-nitroanilide (S-2266, Kabi, Stockholm, Sweden)/min under the conditions of the spectrophotometric assay, as described below. Urinary kallikrein was isolated from human urine and purified according to Geiger et al. (20) using affinity chromatography on aprotinin-Sepharose; its specific activity was 6.3 enzymatic units/mg protein. Both kallikrein preparations were homogeneous at electrophoresis on 7.5% polyacrylamide gel at pH 8.6 (21). The enzymes were covalently bound to CNBr-activated Sepharose-4B (Pharmacia) at a ratio of 10 mg protein/g dry Sepharose (22). The advantage of binding the enzymes to an insoluble carrier is that they can be removed so that they cannot interfere with the assay of renin. The activities of the Sepharose-bound enzymes were measured through their amidolytic actions on the synthetic chromogenic substrate S-2266 (Kabi) (23) and compared with standard solutions of unbound pure hog pancreas kallikrein (Kallikrein, Bayer KZ 45/32). The enzyme suspensions (50 /xl) in Tris buffer (0.20 mol/liter), pH 8.2, were added to substrate (1.5 X 10"4 mol/liter), and the volume was adjusted to 1.1 ml with the same buffer. The mixtures were slowly shaken in a water bath for 30 min at 37 C. The reaction was then stopped with 50% acetic acid, and absorbance at 405 nm was measured in a 1-cm semimicrocuvette. Identical mixtures with aprotinin (Trasylol, Bayer) added (100 kallikrein-inhibiting units/ml incubate) served as blanks. Activation of inactive renin by Sepharose-bound kallikreins The immobilized kallikreins were suspended in phosphate (0.10 mol/liter) buffer, pH 7.5, containing NaCl (0.075 mol/ liter), and 0.1 ml of the suspension was added to EDTA-plasma (1 ml) or to fractions (1 or 2 ml) isolated from plasma by ion exchange chromatography. Pepsin (3000 U/mg protein; Sigma Chemical Co., St. Louis, MO), which was bound to Sepharose in the same way as the kallikreins but which is known to be inactive at neutral pH, was used as a control. The suspensions were slowly shaken at 4 C for various periods up to 48 h, as indicated. The enzymes were then removed by centrifugation at 8000 X g for 10 min. Experiments in which the immobilized enzymes were added to NaCl (0.15 mol/liter) did not result in detectable renin activity.
JCE & M • 1979 Vol49 • No 5
Assays of naturally occurring active renin and in vitro activated renin The method for measuring naturally occurring active renin, which has previously been described (3, 24), was slightly modified. Briefly, aliquots (0.1 or 0.2 ml) of EDTA-plasma or fractions isolated from plasma by ion exchange chromatography were mixed with purified sheep renin substrate (final concentration, 6.7 X 10~7 mol angiotensin I equivalents/liter), and the total volume was adjusted to 1.0 ml with phosphate buffer (0.10 mol/liter), pH 7.5, which contained NaCl (0.075 mol/liter). EDTA (0.001 mol/liter) was present in both substrate and buffer solutions. After the addition of 10 jul 8-hydroxyquinoline (0.34 mol/liter), 5 fil phenylmethylsulfonylfluoride (0.287 mol/ liter) in ethanol, and 10 /xl aprotinin (Trasylol, Bayer; 10,000 kallikrein-inhibiting units/ml), the mixtures were incubated for 3-12 h at 37 C. At the end of the incubation period, no more than 10% of the renin substrate had been consumed, and generation of angiotensin I was linear for the whole period. For measuring in vitro activated renin, the acid- or kallikrein-pretreated samples were similarly incubated with sheep renin substrate. The quantity of angiotensin I that was generated during incubation with renin substrate, was measured by RIA and compared with the quantity generated by standard human kidney renin (MRC standard 68/356, WHO International Laboratory for Biological Standards, Holly Hill, Hampstead, London, United Kingdom). With the protease (angiotensinase) inhibitors we have used, the recovery of angiotensin I, which was added to plasma after treatment with acid or kallikreins, was 98.8 ± 4.9% (mean ± SEM; n = 15). The recovery of standard renin, which was added to the samples before treatment with acid or kallikreins, was 97.0 ± 4% (n = 15). Renin concentration is expressed as microunits of the renin standard (MRC standard 68/356) per ml. Results In contrast with acid treatment, pancreas and urinary kallikreins had no effect on inactive renin in whole plasma (Fig. 1). The elution profile of DEAE-Sepharose chromatography showed two peaks of renin activity: peak A at a sodium concentration of 0.060 mol/liter, and peak B at 0.110 mol/liter (Fig. 2). The renin content of peak B was about 80% of the total quantity of naturally occurring active renin present in the plasma. Acid treatment of this peak did not alter its renin activity. Acid treatment doubled the renin activity of peak A, indicating that this peak contained inactive renin. However, acidification of this fraction did not activate more than 5% of the total quantity of inactive (acid-activable) renin present in the plasma. In contrast, the addition of Sepharosebound pancreas kallikrein to peak A (final concentration, 16 X 10~9 mol/liter), led to the activation of more than 80% of the inactive (acid-activable) renin present in the plasma. Similar results were obtained with urinary kallikrein (Fig. 1). Both pancreas and urinary kallikreins were effective at concentrations as low as 1 X 10~8 mol/
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767
KALLIKREINS AND RENIN PLAS MA
n=6 200 -
SODIUM
RENIN
n=4 -
f 1
150 -
100 -
50
-
-
-
o
^, -* -* —
0
i
1
i
1
1 A
n=6
1
n =2
PEA < A
25
200
y / V //
20 -
• O
300 400 500 ELUTION VOLUME ml • dialysis at pH 7.5 after dialysis at pH 3.3 o dialysis at pH 7.5
600
700
/
15 -
10
5 , •O
t
•
0
O
-
FIG. 2. Separation of inactive renin (peak A) and active renin (peak B) by DEAE-Sepharose column chromatography of plasma. Elution was carried out with a linear sodium gradient ( ). Eluates were treated as follows: 1) dialysis at pH 7.5 (48 h); 2) dialysis at pH 3.3 (24 h), followed by dialysis at pH 7.5 (24 h); or 3) incubation at pH 7.5 with Sepharose-bound pancreas kallikrein (24 h). After these procedures, which were carried out at 4 C, the samples were incubated (3-12 h) with an excess of sheep renin substrate at pH 7.5 and 37 C for measuring renin.
0 J
12
24
1
36
L^J_
48 72 0 TIME (hr)
12
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36
48
pH 3.3 pH7.5 dialysis at pH 7.5 after dialysis at pH 3.3 dialysis at pH 7.5 incubation with pancreas kallikrein 16x10 mol/l incubation with pancreas kallikrein 1.6x.lO"'mol/l incubation with urinary kallikrein 7xlO~°mol/l incubation with urinary kallikrein 0.7x 10~'mol/l incubation with pepsin
FIG. 1. Activation of inactive renin as a function of time in whole plasma and in a fraction isolated by DEAE-Sepharose column chromatography with a linear sodium gradient. Six tubes (5 ml each) with the highest renin content from the renin peak which was eluted at a sodium concentration of about 0.060 mol/liter (peak A; see Fig. 2) were pooled. Samples were treated as follows: 1) dialysis at pH 7.5; 2) dialysis at pH 3.3, followed by dialysis at pH 7.5; or 3) incubation at pH 7.5 with Sepharose-bound pancreas kallikrein, urinary kallikrein, and pepsin. After these procedures, which were carried out at 4 C, the samples were incubated (3-12 h) with an excess of sheep renin substrate at pH 7.5 and 37 C for measuring renin.
liter. The kallikreins had no effect on the renin activity of peak B, probably because this peak did not contain any inactive renin. The inability of added kallikreins to activate inactive renin in whole plasma can be explained by the presence of protease inhibitors (25). Thus, ion exchange chromatography has resulted in the separation of inactive renin from active renin as well as from inhibitors that interfere with the proteolytic activation of inactive renin.
Discussion The results demonstrate that pancreas and urinary kallikreins in concentrations as low as 1 X 10~8 mol/liter are capable of activating inactive plasma renin at neutral pH without prior acidification. The findings with urinary kallikrein confirm and extend the results of Sealey et al. (12). The precise chemical relationship between active and inactive plasma renin is still unknown. While there is agreement that the molecular weights of both naturally occurring active renin and in vitro activated renin are about 44,000, there is no consensus on whether inactive (acid-activable) renin in plasma represents a high molecular weight form (4, 7, 26-29). Recent findings seem to indicate that the difference in molecular weight with active renin is small or even absent (4, 7). With column chromatography on Sepharose G-100 and G-200, we could not detect any difference in molecular weight between naturally occurring active renin and acid- or trypsin-activable renin in plasma (Derkx, F. H. M., H. L. Tan-Tjiong, A. J. Man in't Veld, M. P. A. Schalekamp, and M. A. D. H. Schalekamp, unpublished observations). Our findings confirm the results of Shulkes et al. (7), who have shown that the active and inactive forms of renin can be readily separated on the basis of their difference in net electrical charge. The present study was restricted to tissue kallikreins. They differ in substrate specificity from plasma kallikrein
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(30). Nevertheless, we have recently found that prekallikrein (Fletcher factor)-deficient plasma had much lower renin activity after acid treatment than after trypsin treatment (31), whereas in normal plasma, identical results were obtained with the two procedures (32). This is strong evidence that plasma kallikrein is involved in the acid activation of the inactive renin. Under these artificial conditions, plasma kallikrein can unfold its action on renin, probably because the protease (kallikrein) inhibitors have been destroyed by acid. Under physiological conditions, however, any newly formed kallikrein will be rapidly inactivated in the circulating plasma. The ability of tissue kallikreins to act as activators of inactive renin may have physiological implications. Evidence is accumulating from studies in animals that both hormones are produced in close anatomical association with a strategically important part of the nephron, the juxtaglomerular apparatus (17, 18). Intrarenal activation of inactive renin by kallikrein might therefore affect some aspects of renal function, particularly sodium handling. Recent observations in patients with renovascular hypertension have provided some evidence that inactive plasma renin can be activated as it passes through the kidney (33). Furthermore, several studies have revealed some striking quantitative correlations among plasma renin activity, plasma bradykinin, and urinary kallikrein. These hormones increased in parallel after both standing and sodium depletion (34-37). Plasma renin and bradykinin were suppressed by excessive mineralocorticoid activity, whereas urinary kallikrein was increased (37). Another link is formed by the enzyme that converts angiotensin I into angiotensin II. This enzyme also inactivates bradykinin (30). Thus, there is growing evidence from both in vitro and in vivo work favoring the existence of intricate relationships between two important vasoactive systems, i.e. the renin-angiotensin system and the kallikrein-kinin system. References 1. Lumbers, E. R., Activation of renin in human amniotic fluid by low pH, Enzymologia 40: 329, 1971. 2. Skinner, S. L., E. J. Cran, R. Gibson, R. Taylor, W. A. W. Walters, and K. J. Catt, Angiotensin I and II, active and inactive renin, renin substrate, renin activity and angiotensinase in human liquor amnii and plasma, Am J Obstet Gynecol 121: 626, 1975. 3. Derkx, F. H. M., G. J. Wenting, A. J. Man in't Veld, J. M. G. van Gool, R. P. Verhoeven, and M. A. D. H. Schalekamp, Inactive renin in human plasma, Lancet 2: 496, 1976. 4. Boyd, G. W., An inactive higher-molecular-weight renin in normal subjects and hypertensive patients, Lancet 1: 215, 1977. 5. Leckie, B. J., A. McConnel, J. Grant, J. J. Morton, M. Tree, and J. J. Brown, An inactive renin in human plasma, Circ Res [Suppl 1] 40: 46, 1977. 6. Atlas, S. A., J. E. Sealey, and J. H. Laragh, "Acid"- and "cryo"activated inactive plasma renin, Circ Res [Suppl 1] 43: 128, 1978. 7. Shulkes, A. A., R. R. Gibson, and S. L. Skinner, The nature of inactive renin in human plasma and amniotic fluid, Clin Sci Mol Med 55: 41, 1978.
J C E & M . 1979 Vol 49 ( No 5
8. Atlas, S. A., J. H. Laragh, and J. E. Sealey, Activation of inactive plasma renin: evidence that both cryoactivation and acid-activation work by liberating a neutral serine protease from endogenous inhibitors, Clin Sci Mol Med 55: 1353, 1978. 9. Derkx, F. H. M., H. L. Tan-Tjiong, and M. A. D. H. Schalekamp, Endogenous activator of plasma-inactive-renin, Lancet 2: 218,1978. 10. Leckie, B. J., An endogenous protease activating plasma inactive renin, Clin Sci Mol Med 55: 1335, 1978. 11. Morris, B. J., and R. P. Day, Activation of inactive renin by kallikrein, Int Res Commun Series Med Sci 6: 348, 1978. 12. Sealey, J. E., S. A. Atlas, J. H. Laragh, N. B. Oza, and J. W. Ryan, Human urinary kallikrein converts inactive to active renin and is a possible physiological activator of renin, Nature 275: 144, 1978. 13. Nustad, K., The relationship between kidney and urinary kininogenase, Br J Pharmacol 39: 73, 1970. 14. Roblero, J., H. Croxatto, R. Garcia, J. Corthorn, and E. De Vito, Kallikrein like activity in perfusates and urine of isolated rat kidneys, Am J Physio I 231: 1383, 1976. 15. Carvalho, I. F., and C. R. Diniz, Cellular localization of renin and kininogenin, Ciencia Cult (Sao Paulo) 16: 263, 1964. ' 16. Webster, M. E., Kallikreins in glandular tissues, In Erdos, E. G. (ed.), Bradykinin, Kallidin and Kallikrein, Handbook of Experimental Pharmacology, vol. 25, New York, Springer-Verlag, 1970, p. 131. 17. 0rstavik, T. B., K. Nustadt, P. Brandtzaeg, and J. V. Pierce, Cellular origin of urinary kallikreins, J Histochem Cytochem 24: 1037, 1976. 18. Tyler, D. W., Localization of renal kallikrein in the dog, Experienta 34: 621, 1978. 19. Skinner, S. L., Improved assay methods for renin 'concentration' and 'activity' in human plasma. Methods using selective denaturation of renin substrate, Circ Res 20: 392, 1967. 20. Geiger, R., K. Mann, and T. Bettels, Isolation of human urinary kallikrein by affinity chromatography, J Clin Chem Clin Biochem 15:479,1977. 21. Heber, H., R. Geiger, and N. Heimburger, Human plasma kallikrein: purification, enzyme characterization and quantitative determination in plasma, Hoppe Seylers Z Physiol Chem 359: 659, 1978. 22. Affinity Chromatography. Principles and Methods, Laboratory Manual, Pharmacia Fine Chemicals, Uppsala, Sweden. 23. Claeson, G., P. Friberger, M. Knbs, and E. Eriksson, Methods for determination of prekallikrein in plasma, glandular kallikrein and urokinase, Haemostasis 7: 76, 1978. 24. Derkx, F. H. M., G. J. Wenting, A. J. Man in't Veld, R. P. Verhoeven, and M. A. D. H. Schalekamp, Control of enzymatically inactive renin in man under various pathological conditions: implications for the interpretation of renin measurements in peripheral and renal venous plasma, Clin Sci Mol. Med 54: 529, 1978. 25. McConnell, D. J., Inhibitors of kallikrein in human plasma, J Clin Invest 51: 1611, 1972. 26. Day, R. P., J. A. Luetcher, and C. M. Gonsalvez, Occurrence of big renin in human plasma, amniotic fluid and kidney extracts, J Clin Endocrinol Metab 40: 1078, 1975. 27. Day, R. P., and J. A. Luetcher, Biochemical properties of big renin extracted from human plasma, J Clin Endocrinol Metab 40: 1085, 1975. 28. Hsueh, W. A., E. J. Carlson, J. A. Luetcher, and G. Grislis, Big renin in plasma of healthy subjects on high sodium intake, Lancet 2: 1281, 1978. 29. Mailing, C, and K. Poulsen, Direct measurement of high molecular weight forms of renin in plasma, Biochim. Biophys Acta 49: 542, 1977. 30. Erdos, E. G., The kinins. A status report, Biochem Pharmacol 25: 1563, 1976. 31. Derkx, F. H. M.( A. J. Man in't Veld, and M. A. D. H. Schalekamp, Prekallikrein (Fletcher factor)-dependent pathway for activating inactive renin, Int Res Commun Series Med Sci 7: 135, 1979. 32. Leckie, B. J., A. McConnel, and J. Jordan, Inactive renin—a renin proenzyme? Adv Exp Med Biol 95: 249, 1977. 33. Derkx, F. H. M., G. J. Wenting, A. J. Man in't Veld, R. P. Verhoeven, and M. A. D. H. Schalekamp, Evidence for activation
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KALLIKREINS AND RENIN of circulating inactive renin by the human kidney, Clin Sci Mol Med 56: 115, 1979. 34. Wong, P. Y., R. C. Talamo, G. H. Williams, and R. W. Colman, Response of the kallikrein-kinin and renin-angiotensin systems to saline infusion and upright posture, J Clin Invest 55: 691, 1975. 35. Margolius, H. S., D. Horwitz, R. G. Geller, R. W. Alexander, J. R. Gill, J. J. Pisano, and H. R. Reiser, Urinary kallikrein excretion in normal man. Relationships to sodium intake and sodium-retaining
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steroids, Circ Res 35: 812, 1974. 36. Levy, S. B., R. P. Frigon, and R. A. Stone, The relationship of urinary kallikrein activity to renal salt and water excretion, Clin Sci Mol Med 54: 39, 1978. 37. Vinci, J. M., R. M. Zusman, J. L. Izzo, R. E. Bowden, D. Horwitz, J. J. Pisano, and H. R. Keiser, Human urinary and plasma kinins. Relationship to sodium-retaining steroids and plasma renin activity, Circ Res 44: 228, 1979.
Combined First Annual Scientific Session of the Society for Clinical Trials and Seventh Annual Symposium for Coordinating Clinical Trials May 6-8, 1980; Philadelphia, Pennsylvania The Sessions will focus on the design, organization, management, and analyses of clinical trials. Abstracts must be received by January 21, 1980. For information write to: Christian R. Klimt, M.D., Secretary, Society for Clinical Trials, Inc., 600 Wyndhurst Avenue, Baltimore, Maryland 21210.
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Vol. 49, No. 5 Printed in U.S.A.
Activation of Inactive Plasma Renin by Tissue Kallikreins* FRANS H. M. DERKX, H. LIES TAN-TJIONG, ARIE J. MAN IN'T VELD, MARGREETH P. A. SCHALEKAMP, AND MAARTEN A. D. H. SCHALEKAMP Department of Internal Medicine I, University Hospital Dijkzigt, Erasmus University, Rotterdam, The Netherlands
ABSTRACT. It has been reported that inactive (acid-activable) human renin could be converted into the active form by adding urinary kallikrein to acid-pretreated plasma. Without prior acidification, however, only a small portion of the total amount of inactive renin present in plasma was converted (activated) by kallikrein, probably because native plasma contains protease (kallikrein) inhibitors that are destroyed by acid. We have separated inactive and active renin by DEAE-Sepharose column chromatography of normal human plasma at pH 7.5 and a
linearly increasing sodium gradient. Inactive renin isolated in this way could be activated at pH 7.5 by highly purified pancreas and urinary kallikreins. With the semipurified preparation of inactive renin, prior acidification was not required for obtaining virtually complete activation by kallikrein. The kallikreins were effective at concentrations as low as 1 X 10~8 mol/liter. It is therefore possible that one or more tissue kallikreins act as physiological activators of inactive renin. J Clin Endocrinol Metab 49: 765, 1979)
R
ENIN ACTIVITY of normal human plasma increases by a factor of 3-7 after treatment at pH 3.0-4.0; this is caused by the conversion of enzymatically inactive renin or prorenin into the active form (1-7). Acid activation of inactive renin is a two-stage process; renin activity increases slightly during the acidification step, but most of the rise in activity occurs after pH has been restored to neutral, probably through the action of one or more serine proteases (6, 8-10). It seems unlikely that this pathway is operative in circulating plasma because of the inhibitory effect of various plasma proteins. But in vivo activation of inactive renin at the tissue level through a serine protease is a possibility, and there is now some evidence to support this. Preliminary experiments by Morris and Day (11) showed increased renin activity of amniotic fluid after the addition of large quantities of a crude preparation of pancreas kallikrein. Sealey et al. (12) reported that the rate of renin activation in acid-treated plasma was increased after the addition of urinary kallikrein, which is known to originate from the kidney (13,14); the renin activity ultimately attained was not higher than that attained with acid treatment alone. The possibility that kallikrein acts as an activator of
inactive renin is interesting, since renal kallikrein is produced close to the site where renin is synthesized (15-18) so that an effect of kallikrein on renin biosynthesis could influence renal circulation and sodium handling. Here we describe additional data on the activation of inactive renin by tissue kallikreins. We have separated inactive plasma renin from active renin by ion exchange chromatography and found that the inactive fraction could be activated at physiological pH by highly purified pancreas and urinary kallikreins without prior treatment with acid. Materials and Methods Ion exchange chromatography Blood from healthy male subjects was collected in plastic tubes containing disodium-EDTA (0.005 mol/liter blood). Within 5 min, the blood was centrifuged at 8000 X g for 10 min. The plasma was immediately frozen at —20 C. For ion exchange chromatography, EDTA-plasma was thawed and dialyzed for 24 h against Tris-acetate (0.024 mol/liter) buffer, pH 7.5, which contained NaCl (0.020 mol/liter). The dialyzed plasma (30 ml) was applied to 40 X 2.6-cm columns of DEAE-Sepharose CL6B (Pharmacia Fine Chemicals, Uppsala, Sweden), which had been equilibrated with the same buffer. Elution was performed with a linear gradient of NaCl up to 0.200 mol/liter. Flow rate was 20-22 ml/h, and the eluate was collected in 5.0- to 5.5-ml fractions. All procedures were carried out at 4 C.
Received January 23,1979. Address requests for reprints to: Dr. M. A. D. H. Schalekamp, Department of Internal Medicine I, Room P 434, University Hospital Dijkzigt, Erasmus University, Rotterdam, The Netherlands. * This work was supported by a grant from the Organization for Health Research (TNO), the Netherlands.
Activation of inactive renin by acid Samples (2 ml) of EDTA-plasma or fractions isolated from 765
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DERKX ET AL.
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plasma by ion exchange chromatography were dialyzed for 24 h at 4 C against a glycine-HCl buffer, pH 3.3, which contained EDTA (0.005 mol/liter) (19). After pH had rapidly been restored to 7.5 with NaOH (1.0 mol/liter), the samples were dialyzed for various periods, as indicated, at 4 C against a phosphate buffer, pH 7.5, containing EDTA (0.001 mol/liter) (19). Binding of kallikreins to Sepharose Highly purified hog pancreas kallikrein (Kallikrein, Bayer KZ 45/32, Leverkusen, West Germany) was a gift from Prof. G. L. Haberland and Dr. E. Wischhofer. This preparation had a specific activity of 1180 biological kallikrein units (Frey Units)/ mg protein, which corresponds with 9.4 enzymatic units. One enzymatic unit is defined here as the amount of enzyme hydrolyzing 1 /xmol of the synthetic chromogenic substrate H-D-valylL-leucyl-L-arginine-p-nitroanilide (S-2266, Kabi, Stockholm, Sweden)/min under the conditions of the spectrophotometric assay, as described below. Urinary kallikrein was isolated from human urine and purified according to Geiger et al. (20) using affinity chromatography on aprotinin-Sepharose; its specific activity was 6.3 enzymatic units/mg protein. Both kallikrein preparations were homogeneous at electrophoresis on 7.5% polyacrylamide gel at pH 8.6 (21). The enzymes were covalently bound to CNBr-activated Sepharose-4B (Pharmacia) at a ratio of 10 mg protein/g dry Sepharose (22). The advantage of binding the enzymes to an insoluble carrier is that they can be removed so that they cannot interfere with the assay of renin. The activities of the Sepharose-bound enzymes were measured through their amidolytic actions on the synthetic chromogenic substrate S-2266 (Kabi) (23) and compared with standard solutions of unbound pure hog pancreas kallikrein (Kallikrein, Bayer KZ 45/32). The enzyme suspensions (50 /xl) in Tris buffer (0.20 mol/liter), pH 8.2, were added to substrate (1.5 X 10"4 mol/liter), and the volume was adjusted to 1.1 ml with the same buffer. The mixtures were slowly shaken in a water bath for 30 min at 37 C. The reaction was then stopped with 50% acetic acid, and absorbance at 405 nm was measured in a 1-cm semimicrocuvette. Identical mixtures with aprotinin (Trasylol, Bayer) added (100 kallikrein-inhibiting units/ml incubate) served as blanks. Activation of inactive renin by Sepharose-bound kallikreins The immobilized kallikreins were suspended in phosphate (0.10 mol/liter) buffer, pH 7.5, containing NaCl (0.075 mol/ liter), and 0.1 ml of the suspension was added to EDTA-plasma (1 ml) or to fractions (1 or 2 ml) isolated from plasma by ion exchange chromatography. Pepsin (3000 U/mg protein; Sigma Chemical Co., St. Louis, MO), which was bound to Sepharose in the same way as the kallikreins but which is known to be inactive at neutral pH, was used as a control. The suspensions were slowly shaken at 4 C for various periods up to 48 h, as indicated. The enzymes were then removed by centrifugation at 8000 X g for 10 min. Experiments in which the immobilized enzymes were added to NaCl (0.15 mol/liter) did not result in detectable renin activity.
JCE & M • 1979 Vol49 • No 5
Assays of naturally occurring active renin and in vitro activated renin The method for measuring naturally occurring active renin, which has previously been described (3, 24), was slightly modified. Briefly, aliquots (0.1 or 0.2 ml) of EDTA-plasma or fractions isolated from plasma by ion exchange chromatography were mixed with purified sheep renin substrate (final concentration, 6.7 X 10~7 mol angiotensin I equivalents/liter), and the total volume was adjusted to 1.0 ml with phosphate buffer (0.10 mol/liter), pH 7.5, which contained NaCl (0.075 mol/liter). EDTA (0.001 mol/liter) was present in both substrate and buffer solutions. After the addition of 10 jul 8-hydroxyquinoline (0.34 mol/liter), 5 fil phenylmethylsulfonylfluoride (0.287 mol/ liter) in ethanol, and 10 /xl aprotinin (Trasylol, Bayer; 10,000 kallikrein-inhibiting units/ml), the mixtures were incubated for 3-12 h at 37 C. At the end of the incubation period, no more than 10% of the renin substrate had been consumed, and generation of angiotensin I was linear for the whole period. For measuring in vitro activated renin, the acid- or kallikrein-pretreated samples were similarly incubated with sheep renin substrate. The quantity of angiotensin I that was generated during incubation with renin substrate, was measured by RIA and compared with the quantity generated by standard human kidney renin (MRC standard 68/356, WHO International Laboratory for Biological Standards, Holly Hill, Hampstead, London, United Kingdom). With the protease (angiotensinase) inhibitors we have used, the recovery of angiotensin I, which was added to plasma after treatment with acid or kallikreins, was 98.8 ± 4.9% (mean ± SEM; n = 15). The recovery of standard renin, which was added to the samples before treatment with acid or kallikreins, was 97.0 ± 4% (n = 15). Renin concentration is expressed as microunits of the renin standard (MRC standard 68/356) per ml. Results In contrast with acid treatment, pancreas and urinary kallikreins had no effect on inactive renin in whole plasma (Fig. 1). The elution profile of DEAE-Sepharose chromatography showed two peaks of renin activity: peak A at a sodium concentration of 0.060 mol/liter, and peak B at 0.110 mol/liter (Fig. 2). The renin content of peak B was about 80% of the total quantity of naturally occurring active renin present in the plasma. Acid treatment of this peak did not alter its renin activity. Acid treatment doubled the renin activity of peak A, indicating that this peak contained inactive renin. However, acidification of this fraction did not activate more than 5% of the total quantity of inactive (acid-activable) renin present in the plasma. In contrast, the addition of Sepharosebound pancreas kallikrein to peak A (final concentration, 16 X 10~9 mol/liter), led to the activation of more than 80% of the inactive (acid-activable) renin present in the plasma. Similar results were obtained with urinary kallikrein (Fig. 1). Both pancreas and urinary kallikreins were effective at concentrations as low as 1 X 10~8 mol/
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767
KALLIKREINS AND RENIN PLAS MA
n=6 200 -
SODIUM
RENIN
n=4 -
f 1
150 -
100 -
50
-
-
-
o
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0
i
1
i
1
1 A
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1
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20 -
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600
700
/
15 -
10
5 , •O
t
•
0
O
-
FIG. 2. Separation of inactive renin (peak A) and active renin (peak B) by DEAE-Sepharose column chromatography of plasma. Elution was carried out with a linear sodium gradient ( ). Eluates were treated as follows: 1) dialysis at pH 7.5 (48 h); 2) dialysis at pH 3.3 (24 h), followed by dialysis at pH 7.5 (24 h); or 3) incubation at pH 7.5 with Sepharose-bound pancreas kallikrein (24 h). After these procedures, which were carried out at 4 C, the samples were incubated (3-12 h) with an excess of sheep renin substrate at pH 7.5 and 37 C for measuring renin.
0 J
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1
36
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48 72 0 TIME (hr)
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48
pH 3.3 pH7.5 dialysis at pH 7.5 after dialysis at pH 3.3 dialysis at pH 7.5 incubation with pancreas kallikrein 16x10 mol/l incubation with pancreas kallikrein 1.6x.lO"'mol/l incubation with urinary kallikrein 7xlO~°mol/l incubation with urinary kallikrein 0.7x 10~'mol/l incubation with pepsin
FIG. 1. Activation of inactive renin as a function of time in whole plasma and in a fraction isolated by DEAE-Sepharose column chromatography with a linear sodium gradient. Six tubes (5 ml each) with the highest renin content from the renin peak which was eluted at a sodium concentration of about 0.060 mol/liter (peak A; see Fig. 2) were pooled. Samples were treated as follows: 1) dialysis at pH 7.5; 2) dialysis at pH 3.3, followed by dialysis at pH 7.5; or 3) incubation at pH 7.5 with Sepharose-bound pancreas kallikrein, urinary kallikrein, and pepsin. After these procedures, which were carried out at 4 C, the samples were incubated (3-12 h) with an excess of sheep renin substrate at pH 7.5 and 37 C for measuring renin.
liter. The kallikreins had no effect on the renin activity of peak B, probably because this peak did not contain any inactive renin. The inability of added kallikreins to activate inactive renin in whole plasma can be explained by the presence of protease inhibitors (25). Thus, ion exchange chromatography has resulted in the separation of inactive renin from active renin as well as from inhibitors that interfere with the proteolytic activation of inactive renin.
Discussion The results demonstrate that pancreas and urinary kallikreins in concentrations as low as 1 X 10~8 mol/liter are capable of activating inactive plasma renin at neutral pH without prior acidification. The findings with urinary kallikrein confirm and extend the results of Sealey et al. (12). The precise chemical relationship between active and inactive plasma renin is still unknown. While there is agreement that the molecular weights of both naturally occurring active renin and in vitro activated renin are about 44,000, there is no consensus on whether inactive (acid-activable) renin in plasma represents a high molecular weight form (4, 7, 26-29). Recent findings seem to indicate that the difference in molecular weight with active renin is small or even absent (4, 7). With column chromatography on Sepharose G-100 and G-200, we could not detect any difference in molecular weight between naturally occurring active renin and acid- or trypsin-activable renin in plasma (Derkx, F. H. M., H. L. Tan-Tjiong, A. J. Man in't Veld, M. P. A. Schalekamp, and M. A. D. H. Schalekamp, unpublished observations). Our findings confirm the results of Shulkes et al. (7), who have shown that the active and inactive forms of renin can be readily separated on the basis of their difference in net electrical charge. The present study was restricted to tissue kallikreins. They differ in substrate specificity from plasma kallikrein
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(30). Nevertheless, we have recently found that prekallikrein (Fletcher factor)-deficient plasma had much lower renin activity after acid treatment than after trypsin treatment (31), whereas in normal plasma, identical results were obtained with the two procedures (32). This is strong evidence that plasma kallikrein is involved in the acid activation of the inactive renin. Under these artificial conditions, plasma kallikrein can unfold its action on renin, probably because the protease (kallikrein) inhibitors have been destroyed by acid. Under physiological conditions, however, any newly formed kallikrein will be rapidly inactivated in the circulating plasma. The ability of tissue kallikreins to act as activators of inactive renin may have physiological implications. Evidence is accumulating from studies in animals that both hormones are produced in close anatomical association with a strategically important part of the nephron, the juxtaglomerular apparatus (17, 18). Intrarenal activation of inactive renin by kallikrein might therefore affect some aspects of renal function, particularly sodium handling. Recent observations in patients with renovascular hypertension have provided some evidence that inactive plasma renin can be activated as it passes through the kidney (33). Furthermore, several studies have revealed some striking quantitative correlations among plasma renin activity, plasma bradykinin, and urinary kallikrein. These hormones increased in parallel after both standing and sodium depletion (34-37). Plasma renin and bradykinin were suppressed by excessive mineralocorticoid activity, whereas urinary kallikrein was increased (37). Another link is formed by the enzyme that converts angiotensin I into angiotensin II. This enzyme also inactivates bradykinin (30). Thus, there is growing evidence from both in vitro and in vivo work favoring the existence of intricate relationships between two important vasoactive systems, i.e. the renin-angiotensin system and the kallikrein-kinin system. References 1. Lumbers, E. R., Activation of renin in human amniotic fluid by low pH, Enzymologia 40: 329, 1971. 2. Skinner, S. L., E. J. Cran, R. Gibson, R. Taylor, W. A. W. Walters, and K. J. Catt, Angiotensin I and II, active and inactive renin, renin substrate, renin activity and angiotensinase in human liquor amnii and plasma, Am J Obstet Gynecol 121: 626, 1975. 3. Derkx, F. H. M., G. J. Wenting, A. J. Man in't Veld, J. M. G. van Gool, R. P. Verhoeven, and M. A. D. H. Schalekamp, Inactive renin in human plasma, Lancet 2: 496, 1976. 4. Boyd, G. W., An inactive higher-molecular-weight renin in normal subjects and hypertensive patients, Lancet 1: 215, 1977. 5. Leckie, B. J., A. McConnel, J. Grant, J. J. Morton, M. Tree, and J. J. Brown, An inactive renin in human plasma, Circ Res [Suppl 1] 40: 46, 1977. 6. Atlas, S. A., J. E. Sealey, and J. H. Laragh, "Acid"- and "cryo"activated inactive plasma renin, Circ Res [Suppl 1] 43: 128, 1978. 7. Shulkes, A. A., R. R. Gibson, and S. L. Skinner, The nature of inactive renin in human plasma and amniotic fluid, Clin Sci Mol Med 55: 41, 1978.
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8. Atlas, S. A., J. H. Laragh, and J. E. Sealey, Activation of inactive plasma renin: evidence that both cryoactivation and acid-activation work by liberating a neutral serine protease from endogenous inhibitors, Clin Sci Mol Med 55: 1353, 1978. 9. Derkx, F. H. M., H. L. Tan-Tjiong, and M. A. D. H. Schalekamp, Endogenous activator of plasma-inactive-renin, Lancet 2: 218,1978. 10. Leckie, B. J., An endogenous protease activating plasma inactive renin, Clin Sci Mol Med 55: 1335, 1978. 11. Morris, B. J., and R. P. Day, Activation of inactive renin by kallikrein, Int Res Commun Series Med Sci 6: 348, 1978. 12. Sealey, J. E., S. A. Atlas, J. H. Laragh, N. B. Oza, and J. W. Ryan, Human urinary kallikrein converts inactive to active renin and is a possible physiological activator of renin, Nature 275: 144, 1978. 13. Nustad, K., The relationship between kidney and urinary kininogenase, Br J Pharmacol 39: 73, 1970. 14. Roblero, J., H. Croxatto, R. Garcia, J. Corthorn, and E. De Vito, Kallikrein like activity in perfusates and urine of isolated rat kidneys, Am J Physio I 231: 1383, 1976. 15. Carvalho, I. F., and C. R. Diniz, Cellular localization of renin and kininogenin, Ciencia Cult (Sao Paulo) 16: 263, 1964. ' 16. Webster, M. E., Kallikreins in glandular tissues, In Erdos, E. G. (ed.), Bradykinin, Kallidin and Kallikrein, Handbook of Experimental Pharmacology, vol. 25, New York, Springer-Verlag, 1970, p. 131. 17. 0rstavik, T. B., K. Nustadt, P. Brandtzaeg, and J. V. Pierce, Cellular origin of urinary kallikreins, J Histochem Cytochem 24: 1037, 1976. 18. Tyler, D. W., Localization of renal kallikrein in the dog, Experienta 34: 621, 1978. 19. Skinner, S. L., Improved assay methods for renin 'concentration' and 'activity' in human plasma. Methods using selective denaturation of renin substrate, Circ Res 20: 392, 1967. 20. Geiger, R., K. Mann, and T. Bettels, Isolation of human urinary kallikrein by affinity chromatography, J Clin Chem Clin Biochem 15:479,1977. 21. Heber, H., R. Geiger, and N. Heimburger, Human plasma kallikrein: purification, enzyme characterization and quantitative determination in plasma, Hoppe Seylers Z Physiol Chem 359: 659, 1978. 22. Affinity Chromatography. Principles and Methods, Laboratory Manual, Pharmacia Fine Chemicals, Uppsala, Sweden. 23. Claeson, G., P. Friberger, M. Knbs, and E. Eriksson, Methods for determination of prekallikrein in plasma, glandular kallikrein and urokinase, Haemostasis 7: 76, 1978. 24. Derkx, F. H. M., G. J. Wenting, A. J. Man in't Veld, R. P. Verhoeven, and M. A. D. H. Schalekamp, Control of enzymatically inactive renin in man under various pathological conditions: implications for the interpretation of renin measurements in peripheral and renal venous plasma, Clin Sci Mol. Med 54: 529, 1978. 25. McConnell, D. J., Inhibitors of kallikrein in human plasma, J Clin Invest 51: 1611, 1972. 26. Day, R. P., J. A. Luetcher, and C. M. Gonsalvez, Occurrence of big renin in human plasma, amniotic fluid and kidney extracts, J Clin Endocrinol Metab 40: 1078, 1975. 27. Day, R. P., and J. A. Luetcher, Biochemical properties of big renin extracted from human plasma, J Clin Endocrinol Metab 40: 1085, 1975. 28. Hsueh, W. A., E. J. Carlson, J. A. Luetcher, and G. Grislis, Big renin in plasma of healthy subjects on high sodium intake, Lancet 2: 1281, 1978. 29. Mailing, C, and K. Poulsen, Direct measurement of high molecular weight forms of renin in plasma, Biochim. Biophys Acta 49: 542, 1977. 30. Erdos, E. G., The kinins. A status report, Biochem Pharmacol 25: 1563, 1976. 31. Derkx, F. H. M.( A. J. Man in't Veld, and M. A. D. H. Schalekamp, Prekallikrein (Fletcher factor)-dependent pathway for activating inactive renin, Int Res Commun Series Med Sci 7: 135, 1979. 32. Leckie, B. J., A. McConnel, and J. Jordan, Inactive renin—a renin proenzyme? Adv Exp Med Biol 95: 249, 1977. 33. Derkx, F. H. M., G. J. Wenting, A. J. Man in't Veld, R. P. Verhoeven, and M. A. D. H. Schalekamp, Evidence for activation
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KALLIKREINS AND RENIN of circulating inactive renin by the human kidney, Clin Sci Mol Med 56: 115, 1979. 34. Wong, P. Y., R. C. Talamo, G. H. Williams, and R. W. Colman, Response of the kallikrein-kinin and renin-angiotensin systems to saline infusion and upright posture, J Clin Invest 55: 691, 1975. 35. Margolius, H. S., D. Horwitz, R. G. Geller, R. W. Alexander, J. R. Gill, J. J. Pisano, and H. R. Reiser, Urinary kallikrein excretion in normal man. Relationships to sodium intake and sodium-retaining
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steroids, Circ Res 35: 812, 1974. 36. Levy, S. B., R. P. Frigon, and R. A. Stone, The relationship of urinary kallikrein activity to renal salt and water excretion, Clin Sci Mol Med 54: 39, 1978. 37. Vinci, J. M., R. M. Zusman, J. L. Izzo, R. E. Bowden, D. Horwitz, J. J. Pisano, and H. R. Keiser, Human urinary and plasma kinins. Relationship to sodium-retaining steroids and plasma renin activity, Circ Res 44: 228, 1979.
Combined First Annual Scientific Session of the Society for Clinical Trials and Seventh Annual Symposium for Coordinating Clinical Trials May 6-8, 1980; Philadelphia, Pennsylvania The Sessions will focus on the design, organization, management, and analyses of clinical trials. Abstracts must be received by January 21, 1980. For information write to: Christian R. Klimt, M.D., Secretary, Society for Clinical Trials, Inc., 600 Wyndhurst Avenue, Baltimore, Maryland 21210.
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