Urinary Kallikrein in Rats Bred for Their Susceptibility and Resistance to the Hypertensive Effect of Salt

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1 727 Urinary in Rats Bred fr Their Susceptibility and Resistance t the Hypertensive Effect f Salt A New Radiimmunassay fr Its Direct Determinatin OSCAR A. CARRETERO, VASUDEV M. AMIN, TERESA OCHOLIK, ALFONSO G. SCICLI, AND JOHN KOCH SUMMARY Urinary kallikrein was measured in rats bred t be susceptible (S) r resistant (R) t the hypertensive effect f salt. T determine kallikrein, three different methds were used: (1) a new direct radiimmunassay (RIA) fr the enzymic prtein; (2) a methd based n the capability f kallikrein, when incubated with kiningen, t generate kinins which were then measured by RIA (kiningenase activity); and (3) a methd based n the capability f kallikrein t break the ester bnd f /?-tsyl-l-arginine methyl ester HCI (TAMe). A significant crrelatin (r =.9) was fund between the direct RIA and the kiningenase methd. It was fund that urinary kallikrein was significantly decreased in the S as cmpared t the R rats by the use f these three methds. Urinary kallikrein in the S rats was much lwer when measured by the kiningenase methd than by direct RIA r esterlytic assay. This difference culd be due t excretin f pre-kallikrein and/r kallikrein bund t an inhibitr (inactive kallikrein). It is suggested that the decrease f urinary kallikrein excretin (active and inactive) in the S rats culd be a cnsequence f a genetic defect that may affect the develpment f hypertensin perhaps thrugh the alteratin f sdium and water excretin by the kidney. DAHL et al. 1 develped tw strains f rats, ne which is highly susceptible (S) and the ther which is highly resistant (R) t the hypertensive effect f salt. Althugh the inherited susceptibility r resistance t the effect f salt is plygenetic,' 2 the kidney appears t play a primary rle in the determinatin f bld pressure levels/ 1 " 5 Because the S rats develp hypertensin nly when fed a high-sdium diet, it is reasnable t assume that an abnrmal renal factr r factrs in the kidney may be invlved in regulating sdium and water excretin. It was f interest t investigate urinary kallikrein excretin in these tw strains f rats, since we had previusly pstulated that renal kallikrein culd be invlved in the regulatin f sdium and water excretin by the kidney.""" Therefre, urinary kallikrein excretin was measured in S and R rats by three different methds based n three different principles: 1. Direct Radiimmunassay (RIA) fr the Enzymic Prtein A RIA fr the direct measurement f urinary kallikrein was develped using antibdies against purified enzyme Frm the Department f Medicine, Henry Frd Hspital, Detrit, and the Department f Physilgy (J.K.), University f Michigan, Ann Arbr, Michigan. This research was partially supprted by a Frd Fundatin grant t Henry Frd Hspital, The Michigan Heart Assciatin, The American Heart Assciatin, and Natinal Institutes f Health Grant HL Dr. Carreter is an Established Investigatr f the American Heart Assciatin. Address fr reprints: Oscar A. Carreter, M.D., Hypertensin Research Labratry, Henry Frd Hspital, 2799 W. Grand Bulevard, Detrit, Michigan Received June 22, 1977; accepted fr publicatin January 9, and 125 I-kallikrein. This methd prvides a direct and highly sensitive measurement f rat urinary kallikrein, and thus culd be used in further studies f the kallikrein-kinin system in physilgical and pathlgical situatins. A descriptin f this methd is included here. 2. Kiningenase Activity In this methd, is measured by its capacity t generate kinins when incubated with kiningen (kallikrein substrate), and the kinins generated are measured by RIA Esterlytic Activity In this prcedure, the enzyme is determined by its capability t break an ester bnd f a synthetic substrate. 1 - " Methds Six S and six R male rats, 16 weeks f age, were btained frm the Brkhaven Natinal Labratry. They were placed in individual metablic cages and urine was cllected fr 6 days. Urinary vlume was recrded and samples were stred at -2 C fr kallikrein, prtein, sdium, and ptassium determinatins. At the end f the cllectin perid, bdy weight was recrded and bld pressure was measured by the tail-cuff methd. All f the rats were given tap water ad libitum and fed rat chw cntaining.45% sdium (nrmal sdium diet) and.89% ptassium. Urinary kallikrein was measured by the fllwing methds: 1. Direct RIA fr the enzymic prtein: Purified urinary

2 728 CIRCULATION RESEARCH VOL. 42, N. 5, MAY 1978 kallikrein and its antiserum were prepared as previusly described. 12 was idinated by a mdificatin f the methd f Hunter and Greenwd. 13 Fr this, 87 /xg f chlramine T (25 /xl), 35 /xg f the purified kallikrein (1 /xl), and 1 mci f 125 NaI (5 /xl) were incubated at rm temperature fr 1 secnds. The reactin was stpped by the additin f 4 /xg f sdium metabisulfite (5 /xl) in.5 M phsphate buffer, ph 7.5. The mixture was then diluted with 2 /xl f carrier ptassium idide (1%) and 2 /xl f.5 M phsphate buffer, ph 7.5, cntaining 1 mg f dextran blue 2 and 1% bvine serum albumin (BSA). The separatin f labeled kallikrein frm the reactin mixture was carried ut by gel filtratin using Sephadex G15 (1-ml bed vlume). Equilibratin and elutin frm the gel were carried ut in.5 M phsphate buffer, ph 7.5, and the vid fractin with the dextran blue was cllected. Tw hundred micrliters f this fractin were purified further n a clumn f Sephadex G1 (5 x 1 cm). Equilibratin and elutin frm the gel were carried in.5 M Tris-HCl buffer, ph 7.5, cntaining.1% BSA (Tris buffer). All subsequent dilutins fr the RIA prcedure were made with this buffer except the kallikrein antiserum, which was diluted with Tris buffer cntaining 1:4 nrmal rabbit serum instead f BSA. Fr the radiimmunassay prcedure, 1-4 ng f purified urinary kallikrein (standard curve) r 1-1 /xl f rat urine (unknwn) were transferred by pipette int dispsable plyethylene tubes. T each tube, 1 /xl f the I25 I- kallikrein having apprximately 3 cunts/min and 1 /xl f 1:4, dilutin f kallikrein antiserum were added, and the final vlume was adjusted t 5 /xl with Tris buffer. After 4 hurs f incubatin at rm temperature, 1 /xl f a 3:1 dilutin f a previusly titered sheep antirabbit gamma glbulin antiserum were added t each tube. The tubes were subsequently incubated fr 18 hurs at 4 C. After the incubatin, they were centrifuged at 3 g fr 4 minutes and the precipitate cunted in an autmatic, well-type gamma cunter. Bth the supernatant extract and precipitate f the first tw tubes, in which antibdy against kallikrein was mitted, were cunted t determine ttal cunt and nnspecific binding. Each pint n the standard curve, as well as fr the unknwn samples, was assayed in duplicate. In additin, an internal standard f crude rat urinary kallikrein was run in every assay. Recvery studies were dne by adding 2 ng f purified kallikrein t 2, 3, 4, and 5 /xl f rat urine. The percent recvery was calculated by using the fllwing equatin: (U 2 - U,/PUK) x 1 = percent recvery. \J t is the enzyme quantity in the urine sample, U 2 is the enzyme quantity in the urine sample cntaining the purified kallikrein, and PUK is the enzyme quantity f the purified kallikrein. Results were calculated by cmputer using "lgit-lg" linerizatin f the standard plt as described by Rdbard et al." Results were expressed as micrgrams f enzymic prtein per 24-hur urinary vlume. 2. Kiningenase activity: The kiningenase activity f the urine was determined as previusly described. 9 Results were expressed as micrgrams f kinins generated per minute f incubatin per 24-hur urinary vlume. 3. Esterlytic activity: In this prcedure, kallikrein was determined by the capacity f the enzyme t break an ester bnd f p-tsyl-l-arginine methyl ester HC1 (TAMe). The unhydrlyzed ester was measured by a mdificatin f Rbert's clrimetric methd. 1 - " Results were expressed in micrmles f substrate cnsumed per minute per 24-hur urinary vlume. Sdium and ptassium were measured by flame phtmetry using lithium as the internal standard. Urinary prtein cncentratin was determined by the Lwry prcedure. 111 Esterlytic activity, prtein, sdium, and ptassium in the urine were determined nly n days 1,3, and 5 f the urine cllectin. Cefficient f crrelatin r percent calculatin was calculated with the data cllected frm these 3 days. Results in the text and figures are expressed as mean ± SE. Differences between mean values were evaluated by Student's Mest. Results Direct RIA fr the Enzymic Prtein A typical standard curve shwing significant displacement f 125 l-kallikrein with nly 2 ng f purified kallikrein is shwn in Figure 1. Figure 2 shws displacement f 125 I- kallikrein by rat, muse, dg, and human urine. Figure 3 shws results btained when the kallikrein in the urine f five different rats was measured by six assays n different days (interassay variability). The intraassay cefficient f variatin was 2.4%. In additin, an internal standard f crude urinary kallikrein was run n each assay fr quality cntrl. If the standard was less than 1.9 /xg r mre than 2.4 /xg/mg f rat urinary prtein, the assay was repeated. The recvery f purified kallikrein added t untreated urine was 16 ± 4%., measured by the kiningenase activity (kinin RIA) and the direct RIA fr the enzymic prtein in 36 urine specimens f R rats and 36 urine specimens f S rats, is shwn in Figure 4. Table 1 shws the cefficients S TO O z CO 6 i t 5 Z \ ng KALLIKREIN FIGURE 1 Standard curve fr the direct RIA f kallikrein. Upper right panel shws linearizatin f this plt by using "lgit" scale in the rdinate fb/b and lg scale f standards in the abscissa.

3 URINARY KALLIKREIN/Carr«er et al H.O RAT KALLIKREIN RAT "I ng KALLIKREIN r//i URINE (Lg Scale) FIGURE 2 Displacement f ' 2i l-kallikrein by rat kallikrein (standard curve) and rat, muse, dg, and human urine. Ordinate: lgit scale fb/b; abscissa: lg scale f standards. f crrelatin between the three different methds used t measure the urinary kallikrein and between kallikrein and urinary prtein. The r was calculated fr the S and R grup cmbined and separately. In additin, 11 different dilutins f pure kallikrein ranging frm 1 ng t 1 /ig/ml were measured by the three different methds. The crrelatin amng the results btained by these three methds were: enzymic prtein (direct RIA) vs. kiningenase, r =.98; enzymic prtein vs. esterlytic activity, r =.99; and kiningenase vs. esterlytic activity, / =.99. Urinary, Prtein, Sdium, and Ptassium Excretin by the S and R Rats Figure 5 shws: urinary kallikrein excretin as measured by the three methds; 24-hur urinary prtein S 12 1 ' 'e 6 4 I «> ASSAY NUMBER FIGURE 3 Between-assay variance fr five different rat urines. Each urine was tested by duplicate in six different assays n different days. >.- T3 ^ < S c?m imn r =.9 p<.l y = I.I9X+ (-23.58) 7 6 ' 5 / < 3 4 / CO 3 2 / 1 / - * ' 1 ZO (147-93) [fjq /U.V) Direct RIA fr measurement f enzymic prtein FIGURE 4 Cmparisn f 24-hur kallikrein excretin measured by the direct RIA fr the enzymic prtein and by the kiningenase activity. Filled circles indicate urine frm S rats; pen circles indicate urine frm R rats. Brken line indicates identity line. excretin; and bld pressure measured at the end f the experiment. Figure 6 shws kallikrein excretin by the S rats expressed as a percent f the kallikrein excretin in the R rats. Urinary sdium excretin per day was 3.28 ±.1 meq in the S rats and 3.9 ±.2 meq in the R rats. Urinary ptassium was 3.75 ±.1 meq and 3.41 ±.2 meq, and urinary vlume was 22 ±.6 ml and 36 ± 8 ml in the S and R rats, respectively. Bdy weight at the end f the experiment was 383 ± 5 g in the S rats and 353 ± 9 g in the R rats. Discussin There has been n reprt, up t the present, f a direct RIA fr urinary kallikrein. The purificatin f rat urinary kallikrein and the prductin f antibdies against this enzyme were instrumental in develping the direct RIA. The antiserum, having a very high titer, was used in a final dilutin f 1:2,. This RIA is able t detect 1 ng f TABLE 1 Crrelatin between Values Obtained by Three Different Methds and between These and Ttal Urinary Prtein S&R* S R Enzymic prtein vs. kiningenase Enzymic prtein vs. esterlytic activity Kiningenase vs. esterlytic activity Enzymic prtein vs. urinary prtein Kiningenase vs. urinary prtein Esterlytic activity vs. urinary prtein S&R indicates that r was calculated fr the S and R grups cmbined.

4 73 CIRCULATION RESEARCH VOL. 42, N. 5, MAY 1978 Enzymic Prtein (//g/u.v Kiningenase Activity (//gkinin/min/uv) Esterlytic Activity (//M/min./U.V) Urinary Prtein (mg/uv) h i 4 n BP (mmhg SL R = 93t7 S=113*9 n R S R S R S I 4 5 FIGURE 5 excretin measured by three different methds and urinary prtein excretin in the R (hatched bars) and in the S (dtted bars) rats. Number under RS in the lwer prtin f the figure indicates date f urine cllectin. Bx in the upper right crner shws bld pressure fr the S and R rats at the end f the experiment. purified rat urinary kallikrein, and nly 1-5 /u.1 f nrmal rat urine are necessary fr the determinatin f the kallikrein. Althugh displacement f 125 I-kallikrein with urine f ther species was bserved (Fig. 2), there is insufficient evidence t cnclude that the crss-reactin f urinary kallikrein f different species is ne-t-ne when cmpared t rat urinary kallikrein. Therefre, at the present time, the use f this RIA is limited nly t the determinatin f rat urinary kallikrein. The validity f this RIA methd was verified as fllws. 5 4 Ul c "a; Kiningenase Activity Enzymic Esterlytic Prtein Activity FIGURE 6 Percent f excretin in the S rats when cmpared with the R rats. The kallikrein was measured by three different methds (abscissa) and the values btained fr the R rats with each f the methds were arbitrarily assigned 1%. (1) the displacement prduced by rat urine was similar t that prduced by purified rat urinary kallikrein (Fig. 2); (2) when the same urine samples were tested repeatedly, results were cnsistent (Fig. 3); (3) recvery f purified kallikrein added t untreated urine was 1%; and (4) a highly significant crrelatin was fund between urinary kallikrein activity measured by direct RIA and by kiningenase activity (kinin RIA) (Fig. 4). When crrelatins were made between the results btained by the direct RIA and by the esterlytic assay, the crrelatin was strng nly when the results f the S and R rats were cmbined (Table 1). The crrelatin between the direct RIA r the kiningenase assay and the esterlytic assay fr the S rats was nt significant (P >.1). It may be that when the levels f urinary kallikrein are lw, as in the case f the S rats, mst f the esterlytic activity f the urine may nt be due t urinary kallikrein but t ther enzymes. Urinary kallikrein excretin, measured by the three different methds, was significantly lwer in the S rats when cmpared t the R rats (P <.1) (Fig. 5). Urinary vlume in the S rats was als lwer than in the R rats, but this difference was nt statistically significant (P >.5). The fact that urinary kallikrein excretin was lwer in the S rats culd nt be explained by lwer urinary vlume, since the cncentratin f urinary kallikrein per milliliter f urine in the S rat als was lwer. This was mst clear when the kallikrein was measured by the kiningenase methd. The bld pressure in the S and R rats was similar. Hypertensin did nt develp in the S rats because they were fed a diet cntaining nly.45% sdium and they had been selectively bred t develp hypertensin nly when fed a high-sdium diet (8% sdium cntent). 1 Thus, the cnspicuusly lwer urinary kallikrein excretin in the S rats was nt secndary t an increase in bld pressure. It is ntewrthy that, when kallikrein excretin in the S rats was measured by the kiningenase methd, it was much lwer than when measured with the direct RIA r the esterlytic methd (Fig. 5). This difference remained even after the nrmalizatin f the data (Fig. 6). The presence f an inhibitr in the urine f the S rats culd explain this phenmenn. This hypthetical inhibitr may either be attached t a site in the kallikrein necessary fr the binding f the kiningen (natural substrate) r it may induce an allsteric change in the enzyme that impairs the binding f the kiningen. Hwever, this inhibitr culd leave the antigenic site f the enzyme unaffected, allwing the direct RIA t detect this inactive kallikrein. In the case f the esterlytic methd, it culd be, as mentined previusly, that mst f the esterlytic activity in the urine f the S rat is nt due t kallikrein but t ther enzymes. It is als pssible that the inhibitr-kallikrein cmplex dissciates in the presence f a high cncentratin f TAMe, r that the inhibitr des nt ccupy a site clse t the active regin f the enzyme. This wuld allw the binding f a lw mlecular weight substrate such as TAMe (ml wt 379) but wuld still impair the binding f the high mlecular weight substrate such as the kiningen (ml wt 4,-6,). This assertin is, in part, supprted by a recent study which shwed that a specific kallikrein inhib-

5 URINARY KALLIKREINICarreter et al. 731 itr was islated frm the rat kidney tubules. 15 This inhibitr was able t inhibit the kiningenase activity f the urinary kallikrein but nt the esterlytic activity. Hwever, it was nt reprted whether this inhibitr is secreted in the urine. In the present study, we fund an increase in the urinary prtein in the S rats (Fig. 6) and a significant (P <.1) negative crrelatin between the prteinuria and the kiningenase activity. It culd be that these urinary prteins are respnsible fr the inhibitin f the kiningenase activity, since they are prbably plasmatic prteins, and plasma cntains a cnsiderable amunt f kallikrein inhibitrs. 1 " Anther pssible explanatin f the disprprtinately lwer kiningenase activity in the S rats is that part f the kallikrein excreted in the urine is inactive pre-kallikrein that crss-reacts with the antibdy against kallikrein. Recently, Crthrn et al.' 7 reprted that a significant amunt f pre-kallikrein is excreted in the urine. It appears that, in certain situatins, the direct RIA measures ttal kallikrein (active and inactive) and that the kiningenase methd measures nly active kallikrein. Frm the physilgical pint f view, ne culd pstulate that measurement f kallikrein by the kinngenase methd is preferable, since the pssible effects f this enzyme in the kidney are nt direct but mediated thrugh the release f kinins. Hwever, it has nt yet been determined whether the amunt f kallikrein is the limiting factr in the intrarenal frmatin f kinins. It culd be that in the S rats with higher urinary prtein, mre kiningen is present in the distal nephrn where kallikrein is incrprated int the urine' 8 and kinins are frmed. 19 Then the S rats with lw kallikrein excretin culd be frming the same amunt f kinins as the R rats if the reactin is f first rder in relatin t the substrate. It seems unlikely, hwever, that the decrease f 1 t 2 times in kiningenase activity fund in the S rat, when cmpared t R rats, culd be explained by an increase in the availability f kiningen. The answers t many f these questins will have t wait until accurate prcedures t measure intrarenal kinin frmatin are develped. Dahl et al., 2 " 5 using interstrain renal transplants, cncluded that genetically cntrlled factrs perating thrugh the kidney are respnsible in part fr the hypertensin in the S rats. Since the S rats develp hypertensin nly when fed a high-sdium diet, it is reasnable t assume that at least ne f these abnrmal renal factrs in the kidney is invlved in regulating sdium and water excretin. It may be that ne f these lci cntrls the prductin f urinary kallikrein. The alteratin f this lcus culd be at the level f the distal tubule, where it was fund that urinary kallikrein is prduced. 18 Furthermre, it is pssible that this is the primary alteratin in the kidney and, perhaps, is als the cause f the hypertensin. On the basis f ur results, hwever, we cannt exclude the pssibility that the decrease in urinary kallikrein excretin in the S rats was secndary t an unidentified alteratin in the kidney. In cnclusin, urinary kallikrein excretin was measured by three different methds and fund t be significantly decreased in the S rats when cmpared t the R rats. A significant crrelatin was bserved between kallikrein measurement by a new direct RIA fr the enzymic prtein and by the kiningenase methd. Hwever, when urinary kallikrein in the S rats was measured by its kiningenase activity, the decrease in urinary kallikrein excretin was mre prnunced than when measured by the direct RIA. It is pstulated that this phenmenn was due t the excretin f inactive kallikrein by the S rat. This inactive kallikrein culd be detected by the direct RIA but nt by the kiningenase methd. The decrease f urinary kallikrein excretin in the S rats culd be a cnsequence f a genetic defect that may play a rle in the develpment f hypertensin, perhaps thrugh the alteratin f sdium and water excretin by the kidney. References 1. Dahl LK, Heine M, Tassinari L: Effects f chrnic excess salt ingestin: Evidence that genetic factrs play an imprtant rle in susceptibility t experimental hypertensin. J Exp Med 115: , Knudsen KD, Dahl LK, Thmpsn K, Iwai J, Heine M, Leitl G: Effects f chrnic excess salt ingestin: Inheritance f hypertensin in the rat. J Exp Med 132: 976-1, Dahl LK, Heine M, Thmpsn K: Genetic influence f renal hmgrafts n bld pressure f rats frm different strains. Prc Sc Exp Bil Med 14: , Dahl LK, Heine M, Thmpsn K: Genetic influence f the kidneys n bld pressure: Evidence frm chrnic renal hmgrafts in rats with ppsite predispsitins t hypertensin. Ore Res 34: 94-11, Dahl LK, Heine M: Primary rle f renal hmgrafts in setting chrnic bld pressure levels in rats. Circ Res 36: , Marin-Grez M, Cttne P, Carreter OA: Evidence fr an invlvement f kinins in regulatin f sdium excretin. Am J Physil 233: , Carreter OA, Oza NB: Urinary kallikrein, sdium metablism and hypertensin. In Mechanisms f Hypertensin. Amsterdam, Excerpta Medica, 1974, pp Carreter OA, Scicli AG: Renal kallikrein: Its lcalizatin and pssible rle in renal functin. Fed Prc 35: , Carreter OA, Oza NB, Piwnska A, Ochlik T, Scicli AG: Measurement f urinary kallikrein activity by kinin radiimmunassay. Bichem Pharmacl 25: , Rberts PS: Measurement f the rate f plasmin actin n synthetic substrates. J Bil Chem 232: , Nustad K, Pierce JV: Purificatin f rat urinary kallikreins and their specific antibdy. Bichemistry 13: , Oza NB, Amin VM, McGregr RK, Scicli AG, Carreter OA: Islatin f rat urinary kallikrein and prperties f its antibdies. Bichem Pharmacl 25: , Hunter WM, Greenwd FC: Preparatin f idine-131 labelled human grwth hrmne f high specific activity. Nature 194: , Lwry OH, Rsebrugh NJ, Farr AL, Randall RJ: Prtein measurement with the flin phenl reagent. J Bil Chem 193: , Geiger R, Mann K: A kallikrein-specific inhibitr in rat kidney tubules. Z Physil Chem 357: , Vgel R, Werle E: Inhibitrs. In Handbk f Experimental Pharmaclgy XXV. Berlin, Springer-Verlag, 197, pp Crthrn J, Imanari T, Yshida H, Kaizu T, Pierce J, Pisan J: Inactive kallikrein in human urine (abstr). Prc 36: 893, Scicli AG, Carreter OA, Hamptn A, Crtes P, Oza NB: Site f kiningenase secretin in the dg nephrn. Am J Physil 23: , Scicli AG, Gandlfi R, Carreter OA: Site f frmatin f kinins in the dg nephrn. Am J Physil 234: F36-F4, 1978