Kallikrein-Kinin and Renin-Angiotensin Systems in Rat Renal Lymph

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Kidney International, Vol. 25 (1984). PP. 880—885

Kallikrein-kinin and renin-angiotensin systems in rat renal lymph

DAVID PROUD, SAKIE NAKAMURA, FRANK A. CARONE, PATRICIA L. HERRING, MINORU KAWAMURA, TADASHI INAGAMI, and JOHN J. PISANO

Section on Physiological Chemistry, National Heart, Lung, and Blood Institute, Bethesda, Maryland; Department of Pathology, Northwestern University, Chicago, Illinois; and Department of Biochemistry, Vanderbilt University Medical School, Nash yule, Tennessee

Kallikrein-kinin and renin-angiotensin systems in rat renal lymph. Rat kallikrein into the bloodstream, particularly since Roblero et al renal lymph contains 254 17 nglml (x SEM, N =20)of immunoreac- [31 detected kallikrein in the perfusate as well as in the urine of tive glandular kallikrein. Like the immunoreactive glandular kallikrein in plasma, it is biologically inactive. Gel filtration of renal lymph reveals the isolated perfused rat kidney. profiles for immunoreactive glandular kallikrein, protein, and inhibition Immunoreactive glandular kallikrein does indeed exist in rat of trypsin and kallikrein which resemble those seen for plasma except plasma, as first reported by Nustad, Orstavik, and Gautvik [4]. that high molecular weight plasma components are reduced or missing Studies in our laboratory suggest a role for the kidney in the in renal lymph. In contrast, gel filtration of thoracic lymph reveals immunoreactive glandular kallikrein and protein profiles which are clearance and/or metabolism of immunoreactive glandular kalli- indistinguishable from those seen with plasma. Renin levels are 170-fold krein from plasma [5], but this does not preclude the possibility higher in renal lymph than in thoracic lymph while angiotensin-that the kidney may also secrete kallikrein into the circulation. converting enzyme levels are only 16% those of thoracic lymph. In Since kallikrein is localized in the tubular cells of the distal keeping with the high renin and low converting enzyme activities, renal nephron [6], the major route by which the enzyme could enter lymph contains high levels of angiotensin I. Immunoreactive glandular kallikrein levels in renal lymph, thoracic lymph and plasma do not show the bloodstream would appear to be via the interstitium and the the striking differences observed for renin. lymph. To study this possibility, we looked at the components of the kallikrein-kinin system in rat renal lymph, using compo- Les systemes kallikréine-kinine et renine-angiotensine dans Ia lymphe nents of the renin-angiotensin system for comparison. As a rénale de rat. La lymphe rénale de rat contient 254 17 ng/ml (x SEM, N control, we also examined lymph obtained from the thoracic =20)de kallikréine glandulaire immunoréactive. Comme Ia kallikréine glandulaire immunoréactive plasmatique, elle est biologiquement inac- duct of rats in which contribution from the kidneys was tive. La filtration sur gel de lymphe rénale révèle des profils deeliminated by tying off the renal lymphatics. kallikréine glandulaire immunoréactive, de protéines, d'inhibiteurs de Ia trypsine et de kallikréine qui ressemblent a ceux vus pour Ic plasma, a l'exception que des constituants plasmatiques de haut poids molécu- Methods laire sont diminués ou absents dans Ia lymphe rénale. A l'opposé, Ia The following materials were obtained commercially: bovine filtration sur gel de lymphe thoracique révéle des profils de kallikréine glandulaire immunoréactive et de protéines qui ne sont pas distingua- trypsin and soybean trypsin inhibitor (Worthington Corp., bles de ceux du plasma. Les niveaux de refine sont 170 fois plus élevés Freehold, New Jersey); Sephadex G-150 and molecular weight dans Ia lymphe rénale que dans Ia lymphe thoracique, tandis que les marker proteins (Pharmacia, Piscataway, New Jersey); 3H- niveaux d'enzyme de conversion de l'angiotensine sont seulement 16% tosyl-L-arginine methyl ester (Amersham, Arlington Heights, de ceux de Ia lymphe thoracique. En mCme temps qu'une rCnine ClevCe Illinois); 3H-BZ-Phe-Ala-Pro (Ventrex, Portland, Maine); S- et que de faibles activités d'enzyme de conversion, Ia lymphe rénale contient des niveaux élevés d'angiotensine I. Les niveaux de kallikréine 2266 (D-Val-Leu-Arg-pNA) was obtained from Kabi Diagnos- glandulaire immunoréactive dans Ia lymphe rénale, Ia lymphe thoraci- tica, Stockholm, Sweden; ['4C]-hemoglobin (New England Nu- que et Ic plasma ne montrent pas les differences frappantes observées clear Corporation, Boston, Massachusetts); cathepsin D and pour Ia rénine. bovine pancreatic trypsin inhibitor (Sigma Chemical Company, St. Louis, Missouri); angiotensin I and lysylbradykinin (Penin- It has been suggested that the renin-angiotensin and kalli-sula, Belmont, California); captopril (SQ 14225) was obtained krein-kinin systems act as opposing forces in the regulation offrom Squibb, Princeton, New Jersey; mactin (Abbott, Chicago, blood pressure (BP) [1]. Renin is released into the circulation byIllinois); Trasylol was a gift from Bayer AG, Wueppertal, West the kidney and exists in very high levels in renal lymph [2].Germany; Affi-Gel 10 (BioRad, Richmond, California). All Because the kidney is a rich source of kallikrein, the questionother chemicals were of reagent or ultrapure grade. arises as to whether or not the kidney could also secrete Collectionofrat renal lymph. Male Sprague-Dawley rats (250 to 330 g) were anesthetized with mactin (150mg/kgbody weight) and placed on a heated, thermostatically controlled Received for publication May 16, 1983 table. Rectal temperature was monitored by a thermistor and and in revised form November 8, 1983 maintained at 37 to 38°C. A tracheotomy was performed and a © 1984 by the International Society of Nephrology catheter was placed in the right jugular vein. A solution of 5%

880 Kallikrein-kinin and renin-angiotensin systems 881

mannitol in isotonic saline was infused at a constant rate of 5 p.1 of plasma mi/hrand maintained during the collection of renal lymph. The 3 6 12 24 48 left kidney was exposed by a flank incision and renal hilar 8 16 lymphatics were occluded by a fine silk ligature placed on and 95 3.0 around the outer aspect of a lymph node situated above the left p.1Of renal lymph renal artery near its origin from the aorta. The left kidney was 90 placed in a stabilized cup (Lucite®) and covered with mineral 2.0 oil. A major renal hilar lymphatic was cannulated with polyethylene tubing of approximately 0.25 mm outer diameter. Renal 80 lymph was collected in a small polyethylene tube packed in ice 1.0 for a 2- to 4-hr period and then frozen immediately. 70 Collectionof rat thoracic duct lymph. Following 60 0 an overnight Co fast, rats were anesthetized with ether and the thoracic and 50 0.0 renal areas were approached via left flank incision. To eliminate 40 any contribution from renal lymph to the thoracic duct lymph, 30 the right and left kidneys were each tied off with a single ligature —1.0 occluding the renal artery, vein, and ureter. Lymph from the 20 thoracic duct was then obtained via a PE-50 tubing cannula RUK: Slope = 0.99 according to the method of Bollman, Cain, and Gridlay [71. —2.0 10 Lymph: Slope = 0.94 (Parallel to standardl Followingthe surgical preparation, the animals were placed in a Plasma: Slope = 0.74 (Not parallel to standard) restraining cage [8] with free access to water. Lymph was collected in a plastic tube packed in ice for a 2- to 3-hr period 5 —3.0 (volume -4ml) and immediately frozen. Collectionof rat plasma. Pooledrat plasma was collected in 0.4 0.8 1.6 3.2 6.4 12.8 heparin as described previously [5]. RUK standard, ng/tube Kallikrein radioimmunoassay. Assayswere carried out as Fig. 1. Comparisonof logit-log plots for rat urinary kallikrein (RUK) described by Lawton et al [5]usinga new, highly purified rat standard (0—0),ratrenal lymph (•—•), and rat plasma (•—•). urinary kallikrein standard prepared by DE-52 ion-exchange chromatography, Sephadex G-100 gel filtration, and affinity chromatography using Trasylol immobilized on Affi-Gel 10.releasefrom highly purified human low molecular weight kinin- With this new standard, assay sensitivity was improved result- ogen [121. Samples were incubated with excess substrate for 1 ing in a minimum detectable dose of 100 pg/tube. hrat 37°C then quick-frozen on dry ice and stored at —20°C Assaysfor angiotensins I and II. Immunoreactiveangioten- until assayed. Generated kinin was measured by bioassay on sin I and angiotensin II were determined by specific radioimmu- the isolated rat uterus using synthetic lysylbradykinin as the noassays [9, 101 using 50 pi of tenfold diluted lymph samples.standard [13]. The presence of angiotensin-converting enzyme Goat antiserum to angiotensin I (a gift of Dr. Robert J.(ACE) was demonstrated by generation of angiotensin II from Workman, Gerontology Research Center, NICH, NIH, Balti-angiotensin I as previously described [141 and by the ability to more, Maryland) showed less than 0.5%cross-reactionwithdestroy bradykinin. angiotensin II while the goat antiserum to angiotensin II (also Angiotensin-convertingenzyme. ACEactivity was measured from Dr. Workman) showed less than 1% cross-reaction within lymph (diluted 1/10 to 1/100) and plasma (diluted 1/100 to angiotensin I. The identities of the immunoreactive angioten- 1/500) using [3H]-Bz-Phe-Ala-Pro (Ventrex) as described by the sins were confirmed by thin layer chromatography on silica gelmanufacturer. In brief, the assay relies on the hydrolysis of plates[11]. substrate to yield [3H]-Bz-Phe which is partitioned into the Renin assays. Renin activity was determined by the rate of organic phase for counting. The specificity of measured activity formationof angiotensin I from rat angiotensinogen duringfor ACE was demonstrated by inhibition with captopril or incubation with unfractionated plasma from nephrectomized SQ2O,88 1. rats. The incubation mixture consisted of 50 d of tenfold Otherenzyme assays. 3H-TAMEesterase activity was mea- diluted lymph, 20 1.d of plasma from nephrectomized rats, 5d sured by the method of Imanari et al [15] and cathepsin-like of 125 mri diisopropyl fluorophosphate in propanol, 5pJof 125 proteases were assayed by hydrolysis of ['4C]-hemoglobin as mM phenylmethanesulfonyl fluoride in propanol, and 420 d ofdescribed by Williams and Lin [161 (no catheptic activity was 0.1 M phosphate buffer, pH 7.0, containing EDTA (10 mM) anddetected in lymph samples). S-2266 assays of trypsin and 8-hydroxyquinoline sulfate (1.6 mM). After incubation for 3 hrkallikrein were performed at 37°C and pH 7.4 using 0.5 mM at 37°C, the reaction was stopped by chilling in ice; generatedsubstrate. angiotensin I was determined by radioimmunoassay [9]. Renin Inhibitorassays. Sampleswere preincubated with appropri- activity was calculated as the difference between angiotensin Iate enzymes for 30 mm at 25°C and residual enzymatic activity generated at 37°C; the control value obtained after an identicalwas determined as described above. mixture was incubated for 3 hr on ice. Renin values are Kininogenin lymph. A50-j.d sample of renal lymph contain- expressed in nanograms of angiotensin I per hour per millilitering 18 mi EDTA was incubated with 100 ng of rat urinary of sample. kallikrein at 37°C for 1 hr. Generated kinin was assayed by Bioassay.Kininogenaseactivity was determined by kininradioimmunoassay [17]. 882 Proud et a!

trypsin and kallikrein by lymph and plasma showed that kallikrein inhibition was weaker than trypsin inhibition in each case (Fig. 3). With plasma, inhibition of both enzymes reached a plateau which was not observed with lymph. Initial experiments in which a low level of rat urinary kallikrein was mixed with lymph and subsequently incubated with kininogen resulted in measurable released kinin. This was unexpected since the levels of kallikrein used should have been inhibited by the lymph. It was subsequently shown that the :1, 44 kinin was actually released from endogenous kininogen before Volume added (tl): 25 25 25 25 25 inhibition occurred. In fact, examination of the lymph sample pooled from five rats (containing 285 ng kallikrein antigen/ml) Incubation mixture revealed the presence of considerable amounts of kininogen (1 hr at 37°C) (Table 1). Kininogen was determined on the basis of kinin Al 2ag/m/ 200 200 200 200 200 released by the addition of excess kallikrein; kinin was measured by radioimmunoassay. Assay of lymph before the addi- Buffera 25 25 tion of kallikrein revealed the apparent presence of 20 ng/ml free kinin. This finding was not verified by bioassay and can Lymph 25 25 probably be explained by the cross-reaction of kininogen (< Lymph/18 mM EDTA 25 2%) in the radioimmunoassay. When excess kallikrein was incubated with the lymph, a kinin concentration of 130 nglml °0.1 M Tris/0.025% NaN3/0.1% BSA, pH 7.4 was detected. When human kininogen was added to lymph prior Fig. 2. Conversion of angiotensin I to angiotensin II by rat renal lymph to the incubation with kallikrein, recovery of kinin from the and inhibition of the conversion by EDTAasdemonstrated on the rat added kininogen was 94%. uterus. Upon Sephadex G-150 gel filtration of renal lymph, immunoreactive glandular kallikrein eluted as two peaks with apparent molecular weights of 85,000 and 35,000 daltons (Fig. 4, [18]). Results The absorbance profile suggested that relatively little high Rat renal lymph contained immunoreactive glandular kalli-molecular weight protein was present in renal lymph. The major krein which cross-reacted in a manner parallel to the standardprotein peak corresponded to the elution volume of alubmin curve in a radioimmunoassay (RIA) for rat urinary kallikrein (67,000 daltons). Although some weak inhibitory activities were (Fig. 1). As previously reported [5] rat plasma did not cross-observed at the void volume, trypsin and kallikrein inhibitory react in a parallel fashion. Lymph samples contaminated withactivities each eluted as one major peak with apparent molecu- blood also deviated from parallelism. Measurement of 20 indi- lar weights of 65,000 and 80,000 daltons, respectively. The vidual lymph samples revealed 254 17ng antigen/mi (x major peak of [3H]-TAME esterase activity also occurred at an SEM) andrecovery of standard kallikrein added to lymph was 92 apparent molecular weight of 80,000 daltons. No esterase 10%(N =4). activity was observed associated with the 35,000-dalton peak of Initial attempts to see if the immunoreactive kallikrein hadimmunoreactive kallikrein. biological activity were complicated by the presence of kininase In comparison, gel filtration of thoracic lymph (Fig. 5) activity. When 25 d of pooled lymph from seven rats (contain-revealed profiles for protein and immunoreactive glandular ing 400 ng kallikrein antigen/mi) were incubated with 500 ng ofkallikrein which were virtually identical to those seen for kinin for 15 mm at 37°C and pH 7.4, 100 ng of kinin wereplasma. Thoracic lymph, like plasma, showed nonparallel destroyed. All of this activity was due to angiotensin-converting cross-reaction in the radioimmunoassay. enzyme (kininase II) since it could be completely inhibited by An extension of the comparison between renal and thoracic 18 mM EDTA or by l0- Mcaptopril(ID50 -5x l0 M).In lymph is given in Table 2. Both sets of lymph values are related addition, it could be shown that renal lymph converted angio-to plasma values when appropriate. In all parameters studied, tensin I to angiotensin II, as measured on the isolated ratthoracic lymph was very similar to plasma. Immunoreactive uterus, and this conversion could be inhibited by EDTA (Fig. glandular kallikrein levels are similar in renal lymph, thoracic 2). Therefore, to abolish kininase effects, all lymph sampleslymph, and plasma. In contrast, renal lymph contains levels of were made 18 m in EDTA prior to incubation with kininogen. renin which are 170-fold those of thoracic lymph. In addition, After kininase inhibition, however, neither kininogenase activi-angiotensin I is present in high levels in EDTA-treated renal ty nor direct oxytocic activity was detected in any of threelymph but could not be detected in thoracic lymph while separate lymph pools studied, although all three pools pos-angiotensin-converting enzyme activity in renal lymph is only sessed [3H]-TAME esterase activity. This esterase activity was16% of those of thoracic lymph and plasma. not inhibited by SBTI and was only weakly inhibited by a level of Trasylol sufficient to totally inhibit an amount of rat urinary Discussion kallikrein with equivalent esterase activity. The presence of kininogen as well as kallikrein in human Attempts to use trypsin to see if the antigen represented aurine [19], together with the localization of kallikrein [6] and proenzyme were thwarted by the high levels of trypsin inhibi-kininogen [20] in tubular cells of the distal nephron of rat and tory activity in lymph. A comparison of the inhibition of bothhuman kidneys, respectively, suggests that generation of kinin Kallikrein-kinin and renin-angiotensin systems 883

0.4 B Table 1.Demonstrationof kininogen in rat renal lymph based on Lymph + 1 p.g Lymph + 1.7 g RUK increased kinin content following incubation with kallikrein trypsin Kinin contenta 0.3 0.3 Sample ng/ml Lymph alone 20 Lymph + RUK 130 0.2 0.2 Kininogenb + RUK 197 Lymph + Kininogen + RUK 315c Buffer +RUK 0 Abbreviation: RUK, rat urinary kallikrein. 0.1 0.1 The content was determined by radioimmunoassay. Highly purified human low molecular weight kininogen was used. The value represents 94% recovery.

0 2 3 4 5 0 4 8 12 16 20 E differential inhibition by Trasylol, and the absence of any Volume of lymph, /2/ esterase activity in the 35,000-dalton peak of antigen seen upon •1 0.4 gel filtration, clearly indicate that the TAME esterase activity Plasma + 0.9 g Plasma+ 1.7 RUK doesnot represent kallikrein. trypsin The high levels of trypsin inhibitory activity in lymph pre- vented attempts to see if the antigen represented a proenzyme. 0.3 0.3 Kallikrein inhibition by lymph, however, was considerably weaker than that by plasma and the absence of any plateau phase in the lymph inhibition profiles (Fig. 3) suggested that t2- 0.2 0.2 macroglobulin was not present in the lymph. These low levels of kallikrein inhibition may well explain the parallel cross- reaction of lymph in the RIA, since it is the kallikrein inhibitors

0.1 0.1 in plasma which probably account for deviation from parallelism. To try and gain some additional information about the immunoreactive glandular kallikrein in lymph, gel filtration 0 0.1 0.2 0.30.4 0.5 0 2 4 6 8 10 studies were performed. Nustad, Orstavik, and Gautvik [4] first Volume of plasma. /2/ reported that, upon gel filtration of rat plasma, three peaks of immunoreactive glandular kallikrein were seen. Studies in our Fig. 3.Inhibition of trypsin and kallikrein by rat renal lymph and rat laboratory [5] subsequently showed that the first two peaks, plasma.Residual enzymic activity was measured against D-Val-Leu- Arg-pNA. A Lymph + I pg IPypsin. B Lymph + 1.7 p.g RUK. C Plasma representing enzyme-inhibitor complexes, showed nonparallel + 0.9 p.g trypsin.DPlasma + 1.7 pg RUK. Note the differing scales on cross-reaction in the radioimmunoassay. The third peak, which the abscissas. had the same elution volume as rat urinary kallikrein, cross- reacted in a parallel fashion but was devoid of biological activity. This peak was found to increase dramatically following could occur in the lumen of the distal nephron as well asbilateral nephrectomy but to decrease after sialoadenectomy, intracellularly. The recent report that rat renal kallikrein existssuggesting that the kidney plays an important role in the in fractions enriched in basolateral membranes [21], togetherclearance and/or metabolism of glandular kallikrein from rat with the finding that kallikrein occurs in the perfusate as well asplasma, while the salivary glands are a major source of antigen. in the urine of the isolated perfused rat kidney [3] suggests that This view is supported by the finding of Rabito et al [23] who kallikrein may be secreted into the bloodstream by way of the detected a negative arteriovenous difference across the kidney lymph and raises the possibility of a third site of kinin forma-but a positive difference across the salivary glands. Upon gel tion, namely the interstitium. To examine this possibility more filtration of renal lymph, immunoreactive kallikrein eluted as closely, we looked at components of the kallikrein-kinin systemtwo peaks corresponding to the two smaller molecular weight in rat renal lymph. peaks present in plasma. Negligible high molecular weight Immunoreactive glandular kallikrein was detected in renalprotein is present in renal lymph, as judged by the absorbance lymph and showed parallel cross-reaction in the radioim-profile, and very little inhibitory activity was seen at the void munoassay. The presence of kininogen in renal lymph wouldvolume. The absence of a2-macroglobulin had already been also tend to support the possible interstitial formation of kinins. suggested by the lack of plateau phase in the inhibition curves However, even after kininase inhibition, no kininogenase activ-for lymph (Fig. 3). The major peak of kallikrein inhibition in ity could be detected in any of three renal lymph pools studied,renal lymph eluted earlier than that for trypsin inhibition which despite the presence of TAME esterase activity in all threeagrees with the data obtained by Hojima, Isobe, and Moriya pools. DeBono and Mills [22] reported on the presence of[24] for rat plasma and suggests that the kallikrein inhibitor is TAME esterase activity in dog renal lymph and concluded thatnot cs1-antitrypsin. The overall pattern seen for renal lymph is it represented kallikrein. In our present study in the rat, one in which the profiles for immunoreactive glandular kalli- however, the lack of correlation with biological activity, thekrein, protein, and trypsin and kallikrein inhibition all resemble 884 Proudet al

0.20 100 Fig. 4. SephadexG-150 ge/filtration of rat re- na/lymph. Column,1.6 x 91 cm; eluant, 0.16 80 0.01 MTris-HCI/O.15MNaCI/literm EDTA, pH 7.4; flow rate, 8 ml/hr; fraction volume, c 2.65 ml.The column was standardized using >. 0.12 3.0 60 a bluedextran, ferritin, catalase, bovine serum > 2.5 albumin, ovalbumin, chymotrypsinogen, and g ribonuclease. Sample was a pool from five 2000 0.08 40 rats. Applied 0.4 ml lymph (4.1 A280 U) in a E final volume of 2 ml. Symbols are: (0—0) II.;. A20; (A—A) kallikrein antigen, ng/ml; (I—I) 1000 - 0.04 20 [3HJ-TAME esterase activity (cpm); (U—LI) % trypsin inhibition; (U—U)%kallikrein inhibition. (Published with permission from 0- 0 0 [18].) 20 30 Fraction number

1.0 5.0 Table 2. Comparison of components of kallikrein-kinin and renin- 0.9 4.5 angiotensin systems in rat renal lymph, thoracic lymph, and plasma 0.8 4.0 Renal lymph Thoracic lymph Plasma 0.7 3.5 c 16.5 2 (10) 44.2 50.0 3 (6) C A280 3 (7) 0.6 3.0 , Kallikrein, J 0.5 2.5 ng/ml 254 17(20) 230 12 (7)b 180 5 (6) Renin, 0.4 2.0 c ngAi/ 0.3 1.5 . mi/hr 172 52(10) 1.0 0.3 (5) ND 1.0 Al, ng/ml 191 78 (10) < 0.4 (5) ND ACE, U 690 100 (13) 4150 230(7) 4500 160 (3) 0.1 0.5 0 Abbreviations: ACE, angiotensin-converting enzyme; Al, angioten- 10 20 30 40 50 60 70 sin 1; ND, not determined. Fractionnumber Values aregivenas x sEM;figuresin parenthesis indicate N values. Fig. 5. SephadexG-150 gel fl//ration of rat ihoracic lymph. Conditions bValueswere not paralleled in RIA; data are only semi-quantitive. were as described for Figure 4. Applied 0.5 ml lymph (20 A280 U) was in a final volume of 2 ml. Symbols are: (0—0) A280; (A—A) kallikrein antigen (ng/ml). The levels of immunoreactive kallikrein in renal lymph, thoracic lymph, and plasma did not show the striking differences observed for renin. While this does not preclude the those seen for plasma except that high molecular weight plasmapossibility that there is some passage of kallikrein from the components are reduced or missing. This is consistent with thetubular cells into the interstitium and the lymph, we feel that idea of lymph representing plasma which has passed through athis data together with the low levels of angiotensin-converting molecular weight filter. enzyme (another tubular enzyme) in renal lymph and the When thoracic lymph was gel-filtered, the immunoreactiveobserved gel filtration profiles can all be explained if renal glandular kallikrein and protein profiles were indistinguishablelymph is assumed to represent primarily plasma which has from those seen with plasma. Indeed, in all the parameterspassed through a molecular weight filter (peritubular studied in Table 2, thoracic lymph was very similar to plasma,vasculature?). suggesting that there may be free exchange between lymph and plasma at the level of the thoracic duct. The 170-fold difference Acknowledgments in renin values and thoracic lymph is striking. While we cannot Part of this work was presented in preliminary form at the Interna- eliminate the possibility that these differences in values aretional Conference on Kallikrein, Kinins, Kininogens, and Kininases in reflective of different levels of renin inhibitor (no such inhibitorMunich, November 2—5, 1981. As part of the symposium presentation, the preliminary form of this work was published in AdvExp Med Biol has ever been reported in either plasma or lymph), we consider 156B:889—896,1983. This work was supported by National Institutes of the possibility extremely unlikely. Health grant HL-14192. Dr. D. Proud's present address is the John Angiotensin I was also present in renal but not thoracic Hopkins University School of Medicine, Good Samaritan Hospital, lymph. The angiotensin II content of renal lymph, however, Baltimore, Maryland. was low (3 1 1 2 ng/ml; N =10).These data are consistent Reprintrequests to Dr. J. J. Pisano, Physiological Chemistry, Labo- with the high levels of renin and low levels of converting ratoy of Chemistry, NHLBJ, Department of Health &HumanSen- enzyme in renal lymph, although effects of angiotensinases ices, National Institutes of Health, Bethesda, Maryland 20205, USA cannot be discounted. It is particularly interesting that angiotensin-converting enzyme is so low compared to thoracic lymph References and plasma, since the proximal tubules of the kidney are a rich I. VINCI JM, ZUSMAN RM, Izzo JL, BOWDEN RE, HoRwITz D, source of this enzyme [25]. PI5AN0 JJ, KEI5ERHR:Human urinary and plasma kinins. Rela- Kal!ikrein-kinin andrenin-angiotensin systems 885

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