Metabolism of parathyroid hormone in isolated perfused rat kidney and liver combined

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1 Kidney Interntionl, Vol. 38 (199), pp Metbolism of prthyroid hormone in isolted perfused rt kidney nd liver combined HENRIK DAUGAARD, MARTIN EGFJORD, nd KLAUS OLGAARD Medicl Deprtment P. Division of Nephrology, nd Deprtment of Experimentl Pthology, Rigshospitlet, Copenhgen, Denmrk Metbolism of prthyroid hormone in isolted perfused rt kidney nd liver combined. Metbolism of synthetic, intct, humn prthyroid hormone (PTH) l 54 ws studied in new experimentl model using the isolted perfused rt kidney nd liver combined. The combined orgns clered intct PTH significntly fster thn the single kidneys (P <.2) or livers (P <.2), but not fster thn the sum of the clernces in the single orgns. The kidneys clered intct PTH without ccumultion of NH2-terminl, mid-molecule or COOH-terminl ipth, nd high-performnce liquid chromtogrphy (HPLC) studies did not revel ny PTH frgments. The livers clered mid-molecule nd COOH-terminl ipth significntly slower (P <.2) thn intct PTH, nd HPLC demonstrted genertion of mid-molecule nd COOHterminl PTH frgments. The combined orgns ccumulted significntly less mid-molecule (P <.1) nd COOH-terminl (P <.3) ipth thn the single livers, nd HPLC demonstrted mid-molecule peks tht were smller but not qulittively different. In conclusion, the predominnce of COOH-terminl PTH frgments in plsm my be mintined by differentil clernce minly in the liver, excessive ccumultion being prevented by filtrtion in the kidneys. Metbolism of intct prthyroid hormone (PTH) hs ttrcted much interest since frgments with high biologicl ctivity my be generted in this process [1, 2] nd frgments of PTH my ct s uremic toxins [2, 3]. PTH frgments re generted nd secreted by the prthyroid glnds [4 6], but under norml conditions the peripherl metbolism of secreted intct PTH my be the min source of PTH frgments in plsm [5, 7]. A widely ccepted model of the peripherl metbolism of PTH ws proposed by Mrtin et! [8] bsed on severl niml studies in vivo nd vitro (Mrtin et l [9, 1], Hrusk et l [11, 12]). According to this theory, intct PTH is cleved by the liver into equl mounts of NH2-terminl nd COOH-terminl frgments tht re not further metbolized by the liver. The much lower concentrtions of NH2-terminl thn of COOH-terminl frgments normlly found in the circultion were explined by further peritubulr extrction nd filtrtion of NH2-terminl frgments in the kidneys nd by extrction of NH2-terminl frgments by bone, in contrst to COOH-terminl frgments only being clered by filtrtion in the kidneys. Thus, in this model the end-product of the metbolism of intct PTH in the Received for publiction June 5, 1989 nd in revised form Jnury 5, 199 Accepted for publiction Jnury 29, by the Interntionl Society of Nephrology liver would be the substrte for further degrdtion in the kidney, nd the two orgns might exert synergistic effect on ech other in the metbolism of intct PTH. Studies exmining only the influence of one orgn, especilly the reltively few investigtions to dte of the metbolism of intct PTH in the isolted perfused liver [13, 141 or kidney [1, 12, 14, 15], were not designed to elucidte the effects of possible interction between the two orgns. We therefore developed new experimentl model [161, combining the isolted perfused rt kidney nd rt liver (Fig. 1). This model permitted comprison of the metbolism of dded synthetic intct PTH in the combined orgns with the metbolism in the single orgns under identicl conditions. Clernces were mesured by n ssy specific for intct PTH. Genertion of PTH frgments in the perfusion models ws investigted by high-performnce liquid chromtogrphy (HPLC) nd ssys highly specific for NH2-terminl, mid-molecule, or COOHterminl ipth. The combined kidney nd liver compred with the single perfused liver proved to be n interesting model of fundmentl spect of uremi, tht is, the bsence of functioning kidney. Thus, this model my be used to explin the pttern of NH2- nd COOH-terminl ipth plsm levels seen in renl filure. Methods Sources of PTH nd PTH frgments Synthetic humn prthyroid hormone were: synhpth-(1-84)[asp76] (synthesized t the Peptide Institute, the Protein Reserch Foundtion, Minoh, Osk, Jpn); synhpth-(39-68), synhpth-(39-84), synhpth-(53-84), nd synhpth-(69-84) were obtined from Peninsul Lbortories (Belmont, Cliforni, USA). SynhPTH-(13-34), synhpth-(1-34), synhptll-(1-44), synhpth-(28-48), synhpth-(44-68), nd synhpth-(64-84) were obtined from Bchem (Bubendorf, Switzerlnd). Highly purified intct bovine prthyroid hormone ws gift from Jerry Morrissey, Ph.D., Renl Division, Wshington University (St. Louis, Missouri, USA). Rdioimmunossys (RIAs) for intct PTH nd PTH frgments 1-84 RIA, (N-Tct PTH, Immuno Nucler, Stiliwter, Minnesot, USA). Smples were extrcted nd concentrted on columns contining chicken nti-hpth-(1-34) ntibody coupled to Sephrose beds. After wshing, column elutes were me- 55

2 56 Dugrd et!: PTH metbolism in kidney nd liver combined I I Fig. 1. Schemtic outline of the system for 2 2 combined kidney nd liver perfusion. (Figure reprinted with permission from Eur J Physiol 49:22 222, 1987). A, Perfuste reservoir with stirrer; B, Roller pumps; C, 8 m filters; D, Silstic lungs; E, Bubble trps; F, Pressure trnsducer; G, Vil for urine collection; H, Kidney chmber; I, Mnometer connected to portl vein ctheter; J, Cvl ctheter with smpling port; K, Bile ctheter nd syringe for bile collection. Smples for disppernce curves were drwn from the centrl reservoir. sured in RIA using chicken ntibody binding only hpth- (43-68) RIA, (INS-PTH, Nichols Institute, Sn Jun Cpistrno, Cliforni, USA). This RIA used chicken ntibody rised ginst synhpth-(1-34) nd specific for the NH2-terminl prt of the PTH molecule RIA, (PTH OMEGA, Cmbridge Medicl Dignostics, Cmbridge, Msschusetts, USA). This RIA used got ntiserum specific for the hpth-(53-68) sequence RIA, (MILAB, Mlmö Immunlbortorium AB, Mlmö, Sweden). This ssy used chicken ntibody highly specific for the COOH-terminl prt of the PTH molecule. The performnce of these ssys hs been vlidted in our lbortory before [14], including dilution series to test prllelism ginst synhpth-(1-84) in perfusion medium. The 1-84 RIA ws found to be specific for intct PTH, the 1-34 RIA ws specific for the NH2-terminl region, the RIA ws specific for the mid-molecule region, nd the RIA ws specific for the COOH-terminl region of PTH (Fig. 2). Medium for orgn perfusions All experiments were performed with modified Krebs-Henseleit bicrbonte buffer contining 5 m glucose, 67 glliter bovine serum lbumin (frction V, Miles Lbortories, Elkhrt, Indin, USA) nd ll 2 physiologicl L-mino cids, Electrolyte composition ws (in mm) 141 N, 5 K, 3 C,.7 Mg,.7 SO4, 25 HCO3, 122 Cl, 3 P4, nd 4 lctte. Kidney perfusion technique Experiments were performed on mle Wistr rts mintined on stndrd pellet diet nd llowed wter d libitum. The nimls were nesthetized with 2.5% Thiopentl sodium 1.6 to 2.2 ml intrperitonelly. The right kidney ws cnnulted with 19-guge blunted needle vi the superior mesenteric rtery without interruption of flow s described before [16]. Liver perfusion technique Femle Wistr rts were fsted 24 hours before the experiments but llowed free ccess to wter. Pentobrbitl sodium nesthesi, 6 mg per 1 g rt body weight, ws given intrperitonelly. The bile duct ws cnnulted with PE-5 tubing for bile collection. The portl vein nd the inferior cvl vein were cnnulted with 14 G ctheters nd perfusion ws conducted s described before [161. Combined perfusions These experiments were performed by two opertors, permitting the two orgns (one from ech rt) to be cnnulted t the sme time. After n equilibrtion period of 3 minutes, the isolted perfused kidney nd the isolted perfused liver contined within the sme thermostticlly-controlled plexiglss chmber were juxtposed to shre the sme beker with recirculting perfuste (Fig. 1). A detiled technicl description of the perfusion circuit for combined perfusions hs been published before [16]. Priming volume in the combined circuit ws only 8 ml. HPLC Reverse-phse HPLC ws performed using wter/cetonitrile/trifluorocetic cid (TFA) solvent system, nd Wters NOVA-PAK C18 RdilPk 4 i columns.8 x 1 cm compressed in Z-MODULE (Wters Assocites, Rochester, Minnesot, USA), nd operted t mbient temperture. The two limit solvents,.1% TFA (TFA, CF3 COOR) in wter nd 5% cetonitrile in.1% TFA, were mixed by two pumps in proportions determined by Wters 721 System Controller. The grdient used in ll seprtions is shown in Figure 3. Flowrte ws 1 mi/mm. The elution positions of vrious PTH frgments ws estblished by injecting 5 to 2 g quntities of the different frgments dissolved in 2 1td.1% TFA into the system nd mesuring ultrviolet bsorption of column elutes t 215 nm (Fig. 3). Perfuste smples (6 ml ech) were extrcted on three C18 Sep-Pks before HPLC. Priming nd rinsing procedures for the Sep-Pks were s described by Bennett, Solomon nd Goltzmn [171. Lyophilized Sep-Pk elutes were redissolved in 3 d.1% TFA, 2 pj of which were injected on the HPLC column. Column elutes were collected in 1 ml frctions, lyophilized, redissoived in 1 ml of pentobrbitl sodium buffer with EDTA nd humn serum proteins, nd nlyzed in the

3 Dugrd et t: PTH metbolism in kidney nd liver combined 57 ( > F A Kidney B Liver C Kidney nd liver combined Time, minutes Fig. 2. Disppernce of immunorective prthyroid hormone (ipth) from the perfustes of (A) 5 isolted kidneys, (II) 6 isolted livers, nd (C) 6 kidneys nd livers combined. Synthetic humn PTH- (1-84) ws dded t 1, pmol/liter t time. ipth ws mesured in four ssys: (D) 1-84 RIA; () 1-34 RIA; () RIA; ( ) RIA. Mesured vlues corrected for smpling re expressed s percent of estimted initil vlues. Results re mens SE. Liver: Significntly different from ipth (% of initil rectivity) mesured in 1-84 rdioimmunossy (RIA) t sme time: *P <.5; ** <.1; *** <.1. Kidney nd liver combined: Significntly different from ipth (% of initil rectivity) mesured in 1-84 RIA t sme time: *D <.5. PTH ssys. Recovery of 1 to 1, pmol/liter of different NH2- nd COOH-terminl synhpth frgments in perfuste ws lso tested in this system. S.. 9 Experimentl design Orgn perfusions. The circulting volume ws 2 ml in ll experiments to llow smpling for ll ssys nd HPLC seprtions. The initil stbilizing nd control period ws fifty minutes for both kidneys, livers, nd combined perfusions. Perfuste ws smpled 5, 15, 3, 6, nd 9 minutes fter the ddition of PTH, nd nlyzed in four PTH ssys. The results were corrected for smpling nd plotted versus time on semilogrithmic scle. The clernces were clculted from the regression lines for ech ssy. To be ble to mesure the production of frgments with HPLC nd the different ssys, synhpth-(1-84) ws dded to the perfustes t n initil concentrtion of 1 pmolll (5 kidneys, 6 livers, nd 6 combined perfusions). Furthermore, 25 control perfusions were conducted under vrious other experimentl conditions. These re summrized in Tble 4, nd described in the Results section. The five single kidneys nd the six single livers used s controls for the six combined perfusiops in this study (Tble 3) were selected mong twenty single kidneys nd eighteen single livers perfused the sme wy in our lbortory. The results obtined with the remining fifteen kidneys nd twelve livers were not significntly different from the results shown in Tble 3. However, only the perfusions presented were conducted during the sme period of time s the combined perfusions nd lternting with these, using the sme stocks of buffers nd BSA s the combined perfusions. This selection implied, tht results obtined with four of the single kidneys nd four of the single livers presented here hve been used s prt of mteril presented before [14]. HPLC studies Perfuste smples from five kidney perfusions, six liver perfusions, nd six combined perfusions were nlyzed by HPLC. Smples drwn 6 or 9 minutes fter the ddition of synhpth-(1-84) t 1 pmol/liter were frctionted by HPLC nd nlyzed in the PTH ssys specific for NH2-terminl, mid-molecule nd COOH-terminl IPTH, respectively. Dt nd sttisticl nlysis The dt re given s mens sr where nothing else is stted. Prmetric liner regression, nd Student's I-test for pired or unpired smples, s pproprite, were used. The vlue of Student's t in the comprisons between the sum of the clernces in the single kidneys (K) plus livers (L) versus the clernces in the combined orgns (C) ws clculted by the formul: t = (MenK + MenL MenC)/\/(sEMK2 + SEML2 semc2). All significnce limits re two-sided. Results Function of the perfused kidneys Results from the kidney perfusions with synhpth-(1-84) dded t n initil concentrtion of 1. i9 M re given in Tble 1. No significnt differences were found between the single kidneys nd the kidneys together with livers, when the functionl prmeters were determined s verges of the whole experimentl period of 9 minutes for ech kidney. When ech urine smpling period of 3 minutes ws nlyzed seprtely, C excretion ws found to be significntly (P <.5) lower in the single kidneys, 42 3 nmol. min'. g', thn in the kidneys combined with livers, nmol. min. g, during the lst 3 minutes of the experimentl period. Ultrfil-

4 58 Dugrdet!: PTH metbolism in kidney nd liver combined.1 9co 5 C,, 5 C U, (C (C C (C C) C (C. C, Time, minutes 4 3 Fig. 3. High-performnce liquid 2 chromtogrphy (HPLC) seprtion of synthetic humn prthyroid hormone (PTH) < frgments. Between 1 nd 2 sg of ech 1 peptide ws chromtogrphed. Elution positions were determined by seprte chromtogrms of ech frgment preceding the chromtogrm shown. Amino cid sequences of the frgments re indicted; Ul nd U2 were unspecific impurities of the frgments. Ultrviolet bsorption ws mesured t 215 nm. Acetonitrile/. 1% trifluorocetic cid grdient used in ll seprtions is shown. Tble 1. Function of perfused rt kidney Kidney lone Kidney combined with liver Number of perfusions 5 6 Kidney weight, g Perfusion pressure, mm Hg Perfuste flow, ml min'. g' Urine production, d min'. g' p-aminohippuric cid clernce, ml. min'.g' Inulin clernce, id. min'.g' Filtrtion frction, % N excretion, mo!. min'.g' 3.2 1, Frctionl rebsorption of N, % K excretion, i.&mol. min'. g' Frctionl excretion of K, % C excretion, nmol. min'.g' Frctionl excretion of C, % Pi excretion, nmol. min'. g' Frctionl excretion of Pi, % Vlues re mens SD bsed on verges for ech kidney of results obtined during the whole experimentl period with synhpth-(1-84) l t. This ws 5 to 14 minutes fter cnnultion. None of the differences between the two groups re significnt. terble clcium ws ssumed to be 56% of totl clcium in the clcultions [181. None of the other functionl prmeters exhibited significnt differences between single kidneys nd combined kidneys nd livers t ny time. Function of the perfused livers No significnt differences were found t ny time during the experimentl period between the functionl prmeters of the single livers nd of the livers combined with kidneys (Tble 2). Clernce of PTH-(1-84) in the perfusion models Results from the perfusions with synhpth-(l-84) dded t n initil concentrtion of 1. l M to five kidneys, six livers, nd six kidneys nd livers combined re shown in Figure 2. In the kidneys, the disppernce of intct ipth, nd of NH2-terminl, mid-molecule nd COOH-terminl ipth ws Tble 2. Function of perfused rt liver Liver lone Liver combined with kidney Number of perfusions 6 6 Rt weight, g Liver wet weight, g Portl vein pressure, cm Ff2 Bile flow, j.d. hour' Oxygen consumption, smol min' 2 pmol. min' g' Perfuste flow (fixed), ml min' Vlues re mens SD bsed on verges for ech liver of results obtined during the whole experimentl period with synhpth-(l-84) io 54. This ws 5 to 14 minutes fter cnnultion. None of the differences between the two groups re significnt. prllel. In the livers, the disppernce of mid-molecule nd COOH-terminl ipth ws significntly (Fig. 2) slower thn the disppernce of NH2-terminl nd intct ipth, the two ltter disppering in prllel fshion. In the kidney nd the liver combined, the disppernce of intct ipth nd NH2-terminl ipth ws not significntly different. Accumultion of midmolecule ipth ws not significnt (P.8 t 6 mm, P.13 t 9 mm) either, wheres COOH-terminl ipth disppered significntly (Fig. 2) slower thn intct ipth. From these dt it ws lso found tht t ninety minutes, significntly less mid-molecule ipth (P <.1) nd COOH-terminl ipth (P <.3) ws present in the perfustes of the combined perfusions thn in the perfustes of the single livers. The metbolic clernces clculted from the disppernce curves re given in Tble 3. No significnt differences were found between the clernce of intct PTH nd the clernces of NH2-terminl, mid-molecule, or COOH-terminl ipth in the kidneys. Also, the clernce of intct PTH ws not significntly different from the clernce of inulin (P.48). The delyed disppernce of mid-molecule nd of COOHterminl ipth in the liver, nd of COOH-terminl ipth in the

5 Dugrd et!: PTH metbolism in kidney nd liver combined 59 Tble 3. Clernce of synhpth-(l-84) i M in perfused rt orgns mesured in four rdioimmunossys N5 N6 N=6 Kidney nd Kidney Liver liver together; Assy id min' P 1-84 RIA <.5 l-34r1a < RIA " < RIA l l88c <.5 Inulin <.1 Vlues re mens SD. Significntly different from 1-84 clernce in the liver: p <.2, "P < o.ooi. Significntly different from 1-84 clernce in the combined orgns: P <.2. P-vlues in right column relte to the sum of the clernces in the single kidneys nd the single livers, compred with the clernces in the combined orgns. combined orgns, ws reflected in clernces tht were significntly lower thn the clernces of intct ipth in these perfusion models. The kidney nd the liver combined clered intct IPTH significntly fster thn the single kidneys (P <.2) or the single livers (P <.2), but the clernces of PTH in the combined model ws significntly (Tble 3, right column) lower thn the tenttive sum of the clernces obtined with the single orgns. Results from twenty-five control perfusions re summrized in Tble 4. SynhPTH-(l-84) dded t 5. i M to one kidney, one liver, nd one combined perfusion resulted in clernces within the sme rnges s in the experiments with synhptfl-(l-84) 1.l M, s did synhpth-(l-84) dded t 2. 1 M to one kidney, one liver, nd one combined perfusion. These perfusions were performed to further substntite tht the metbolic clernce rtes could be clculted ssuming first order kinetics. Highly-purified intct bovine PTH dded t 1. io M to one kidney, one liver, nd one combined perfusion gve similr results. These perfusions were performed to fcilitte comprison with erlier studies, nd to test the influence of the PTH preprtion used. To test tht the dditive chrcteristic of the clernce concept held true in the perfusion model, one perfusion ws conducted with two kidneys combined. Frctionl excretion C,H/C,flUIjfl in this model ws.9, sme s in the single kidneys. Thus the system ccurtely reflected the presence of two filtering kidneys. One perfusion ws conducted with two livers combined, The oxygen consumption in this model ws 14 mol. min' + 15 mol. min'. One kidney ws perfused for 6 minutes, the perfuste ws then trnsferred to liver tht ws perfused for 9 minutes. A similr perfusion ws conducted with trnsfer of perfuste from liver to kidney. The clernces in these models were of the sme order s in the corresponding single orgns. One kidney perfusion, two liver perfusions, nd one combined perfusion were performed with removl of the orgns fter 3 to 45 minutes nd continued smpling for 6 minutes. After removl of the orgns, the clernce of ipth ws indistinguishble from zero. The clernces were lso zero in two control experiments without ny orgns in the perfusion circuit. HPLC studies The cpbility of the HPLC system used to seprte vrious NH2-terminl nd COOH-terminl synhpth frgments is demonstrted in Figure 3. Even 2 pg synhpth-(l-84) could not be visulized t 215 nm, but by frctiontion nd ssying of column elutes s described it ws shown to elute four minutes lter thn synhpth-(l-34). Using this system, 1 pmol/liter of synhpth-(l-34), synhpth-(l3-34) nd synhpth-(l-44) in perfuste could be distinguished from synhpth-(l-84) 1 pmoll liter. Perfuste smples from three kidneys, four livers nd four combined perfusions drwn t 6 minutes were nlyzed by HPLC s described in the legend to Figure 4. In the kidney perfuste only one pek ws found, recting in the NH2-terminl, the mid-molecule, nd the COOH-terminl RIA. This pek, representing the remining synhpth-(l-84), served s internl stndrd in ll seprtions. Thus no NH2- terminl or mid-molecule/cooh-terminl PTH frgments were demonstrted. The chromtogrphic pttern ws the sme in ll three kidney perfusions nlyzed. In the liver perfuste, only the intct PTH pek ws found in the NH2-terminl RIA. In the mid-molecule RIA one more brod pek ws detected eluting ner the estblished elution positions of vrious mid-molecule/cooh-terminl synhpth frgments. Some frctions within this pek lso rected in the COOH-terminl RIA. The chromtogrphic pttern ws the sme in ll four liver perfusions nlyzed. In the perfuste from kidney nd liver combined, no NH2- terminl frgments were found either. Besides the slim intct PTH pek brod pek ws found in the mid-molecule ssy, eluting in the sme frctions s the mid-molecule pek found in the livers, but somewht smller. This pek ws not found in the COOH-terminl RIA, but s seen from Figure 4 compring with the liver it might hve disppered just below the detection limit. The chromtogrphic pttern ws the sme in ll four combined perfusions nlyzed except tht in one cse the mid-molecule pek ws not found. Smples drwn t 9 minutes from two liver perfusions nd two combined perfusions were lso seprted on HPLC. Frctions were only nlyzed in the mid-molecule RIA, permitting lower dilution. These studies demonstrted tht both in the livers nd in the combined perfusions the brod mid-molecule!cooh-terminl pek consisted of severl, poorly defined peks without ny consistent pttern. Discussion Previous studies from our lbortory hve delt with the metbolism of synthetic humn, intct prthyroid hormone [synhpth-( 1-84)] in single isolted perfused rt kidneys nd livers [14]. The dvntges of the use of isolted perfused orgns nd the limittions imposed by these models nd the use of higher thn physiologic levels of PTH hve been discussed in detil before [14]. It should lso be noted, tht the conditions in vivo develop during stedy stte sitution mesured over dys nd weeks, wheres the results of the present investigtion were obtined over short nd limited period of perfusion.

6 6 Dugrd et l: PTH metbolism in kidney nd liver combined Perfusion model Tble 4. Summry of 25 control perfusions Number of perfusions PTH dded Clernce, d. min' Intct PTH (1-84) Inulin Single kidney I synhpth-(l-84) 5 pmol/liter Single liver I synhpth-(l-84) 5 pmol/liter 7 Kidney nd liver combined I synhpth-(l-84) 5 pmol/liter Single kidney I synhpth-(1-84) 2 pmol/liter Single liver I synhpth-(l-84) 2 pmollliter 629 Kidney nd liver combined I synhpth-(1-84) 2 pmol/liter Single kidney I bpth-(1-84) 1 pmol/liter Single liver I bpth-(i-84) 1 pmollliter 684 Kidney nd liver combined I bpth-(i-84) 1 pmollliter Two kidneys combined I synhpth-(1-84) 1 pmollliter Two livers combined I synhpth-(l-84) 1 pmollliter 1284 Sequentil perfusion: Single kidney 1 synhpth-(1-84) 1 pmollliter Sequentil perfusion: Single liver 1 Perfuste trnsferred from kidney 564 Sequentil perfusion: Single liver I synhpth-(1-84) 1 pmol/liter 58 Sequentil perfusion: Single kidney 1 Perfuste trnsferred from liver Single kidney I synhpth-(l-84) 1 pmol/liter After removl of the kidney I Sme perfuste s bove Single liver 2 synhpth-(l-84) 1 pmol/liter 129 After removl of the liver 2 Sme perfuste s bove Kidney nd liver combined I synhpth-(i-84) 1 pmollliter After removl of the kidney nd liver 1 Sme perfuste s bove Perfusion circuit without orgns 2 synhpth-(l-84) 1 pmol/liter No significnt correltion between time nd the disppernce of PTH or inulin s indicted, tht is, no clernce, even if clculted vlues were slightly positive or negtive. Where two perfusions were performed, the results given re verges of the two perfusions. I- C) E. : A Kidney B Liver C Kidney nd liver combined 5 4 E 3 2 : E : C') IC) L Time, minutes Fig. 4. High-performnce liquid chromtogrphy seprtion (HPLC) of one perfuste smple from one isolted perfused kidney (A), one smple from one liver (B), nd one smple from one combined perfusion (C). The smples were tken 6 minutes fter ddition of synthetic humn prthyroid hormone [synhpth-(1-84)j t 1, pmol/liter to perfuste. After lyophiliztion, the 7 collected frctions were redissolved in ssy buffer. Different further dilutions were needed for ech of the three PTH ssys. Curves re ctul ssy results not multiplied by dilution fctors. Dshed lines indicte sensitivity limits of the different rdioimmunossys (RIAs)

7 The nticlciuric response to the dded synhpth-(1-84) found in the single kidneys ws bolished during the perfusions where the liver ws lso present. Thus in the combined perfusions the clcium excretion becme of the sme order s in single kidneys without PTH dded [14]. The disppernce of intct PTH ws significntly fster in the combined perfusions thn in the single kidneys (P <.2), but s much s pmollliter synhpth-(l-84) ws mesured in the intct PTH RIA t 9 minutes in the combined perfusions, nd this concentrtion might still hve been expected to exert biologicl ctivity. However, Keutmnn et l [19] found tht humn PTH-(1-34) ws bout ten times less potent thn rt PTH-(l-34) in the rt denylte cyclse ssy, so in terms of bioctivity the present investigtion my hve been performed t ner-norml physiologicl PTH concentrtions. The 1-84 RIA used in the present investigtion hs previously been shown by our lbortory to be specific for intct PTH [141. Thus, this ssy ccurtely reflected the disppernce of the dded synhpth-(l-84) unffected by ny PTH frgments tht might hve been generted. Glomerulr filtrtion rte (GFR), determined s the disppernce of inulin from the perfustes, ws significntly (P <.1) lower in the kidneys combined with livers thn in the single kidneys (Tble 3). We hve found the sme significnt difference in perfusion series without PTH dded [16]. A possible explntion is genertion of vsoctive substnces by the combined kidney nd liver. Thus, we hve been ble to mesure production of ngiotensin I by the combined orgns, wheres the single orgns did not produce mesurble mounts of this precursor of ngiotensin II. The combined orgns clered intct PTH significntly fster thn the single kidneys (P <.2) or livers (P <.2). but the clernce in the combined model ws significntly (Tble 3, right column) lower thn the tenttive sum of the clernces obtined with the single orgns. However, we hve previously shown tht synhpth-(l-84) is minly removed by filtrtion in the isolted perfused rt kidney with glomerulr sieving coefficient of t lest.8 [14]. Therefore, it would be pproprite to correct for the higher inulin clernces found in the single kidneys in this study. Inulin clernces in combined nd single kidneys respectively, clculted from the disppernce of inulin from the perfustes the sme wy s intct PTH clernces were clculted, differed more thn the inulin clernces clculted from urine excretions. While the ltter method my be best suited for the clcultions of frctionl electrolyte hndling by the kidney, it my not be precise mesure of GFR over longer periods of time, s hs been discussed in detil by Mck [2]. The sum of the intct PTH clernces in the single livers nd kidneys, corrected for GFR, ws 132 1d min, versus 124 d. min' in the combined orgns, not significntly Dugrd et!: PTH metbolism in kidney nd liver combined 61 different (P.78). Thus, no synergistic effect ws ttined by combining the orgns. As discussed in the introduction such n effect might hve been expected if the liver selectively took up intct PTH, s proposed by Mrtin et t [8 1]. Although the NH2-terminl RIA, the mid-molecule RIA, nd the COOH-terminl RIA used were specific for the respective min regions of the PTH molecule, they rected with ll frgments contining the necessry ntigenic sequence, nd immunorectivity of smll PTH frgments ws unpredictble [14]. So, in contrst to the intct PTH RIA, no simple stoichiometric reltionship could be ssumed between the mounts of frgments present in the perfuste nd immunorectivity mesured in these ssys. However, they were ll clibrted ginst synhpth-( 1-84), so if the dded synhpth-( 1-84) disppered without relese of ny frgments bck to the perfustes, then the disppernce curves mesured with these ssys should be prllel with the intct PTH disppernce curve. This ws the cse in the kidney perfusions (Fig. 2), nd the HPLC studies (Fig. 4) confirmed tht no detectble frgments were produced by the kidneys. Conversely, the livers relesed mid-molecule nd COOH-terminl PTH frgments to the perfuste s demonstrted by the HPLC studies, nd this ccumultion of immunorective frgments of the dded intct PTH ws reflected in the significnt distnce between the intct PTH disppernce curve nd the mid-molecule nd COOH-terminl disppernce curves. The combined orgns ccumulted significntly less mid-molecule (P <.1) nd COOH-terminl (P <.3) ipth thn the single livers. Mid-molecule PTH frgments could still be mesured in the HPLC studies, nd they exhibited the sme pttern of severl mid-molecule/coohterminl subfrctions s found in the single liver perfusions. The metbolism of intct PTH in the single kidney nd liver hs been discussed before [14]. When compring the results from the combined orgns (Fig. 2) with those obtined with the single kidney nd liver, respectively, it is cler tht in this model the heterogeneity of immunorective PTH resulting from the dded intct PTH, including the lower concentrtions of intct nd NH,-terminl PTH thn of mid-molecule nd COOHterminl PTH, ws due to differentil clernce in the liver rther thn in the kidney. Segre et! [21] nd Bringhurst et l [22] found clevge of intct bovine PTH by isolted rt Kupifer cells, fst degrdtion of the resulting NH2-terminl frgments, nd slow if ny degrdtion of COOH-terminl frgments. These findings were repeted by Pilli nd Zull [23] who chrcterized slightly different COOH-terminl frgments nd discussed the limittions of these models using centrifugl methods or colloidl iron to seprte the liver cells. The present investigtion using the intct liver in vitro supports the findings with isolted Kupffer cells. Studies in vivo by D'Amour et l [24, 25], nd Segre et l [26], nd n erlier study by Cnterbury et l [131 with the isolted perfused liver lso give evidence for differentil clernce by the liver. Mrtin et l [27], nd Hrusk et l [11] found significnt peritubulr uptke of intct nd NH2-terminl bovine PTH by kidneys of rts nd dogs in vivo nd vitro. However, Ku nd Mck [15] found only filtrtion of intct bovine PTH in the isolted perfused rt kidney, nd Mrtin et l [1] concluded tht under norml conditions the kidney minly removes PTH by filtrtion. In the present investigtion the kidneys removed synhpth-(l-84) t the sme rte s inulin, together with vrious mid-molecule/cooh-terminl PTH frgments generted by the isolted perfused rt liver. The kidneys in this study did not relese frgments of the filtered intct PTH bck to the perfuste. In the studies by Brling nd Christie [28], nd Hrusk et l [12] PTH frgments were generted, but t very vrible rte s some kidneys produced only smll mounts of PTH frgments. The combined perfused kidney nd liver compred with the perfused liver lone (Fig. 2) illustrte one fundmentl spect of uremi, tht is, the bsence of functioning kidney. It is

8 62 Dugrd et l: PTH metbolism in kidney nd liver combined suggestive tht the PTH profile in the perfustes of the combined orgns with intct PTH dded ended up looking very much like the norml sitution in vivo with low levels of intct PTH nd NH2-terminl ipth nd higher levels of mid-molecule nd COOH-terminl ipth. In comprison, the perfustes from the single livers demonstrted the sme ltertions s seen in uremic ser with higher levels of intct PTH nd NH2-terminl ipth nd very high levels of mid-molecule nd COOH-terminl ipth. If the different levels of NH2-terminl nd COOHterminl ipth were mintined by the kidney rther thn the liver s proposed by Mrtin et l [8], other systems such s bone or low-cpcity processes in serum would hve to tke over this function in uremic ptients. The results of the present investigtion indicte tht the predominnce of COOH-terminl ipth in plsm my be mintined by differentil clernce of PTH frgments minly in the liver, n excessive ccumultion being prevented by filtrtion in the kidney. In ccordnce with this, no synergistic effect ws observed by combining the isolted perfused rt kidney nd liver, nd the HPLC profile of mid-molecule/cooh-terminl ipth in the combined orgns ws not qulittively different from tht of the single perfused livers. This model offers simple explntion of some of the chnges in the peripherl metbolism of PTH found in uremi. Acknowledgments The present investigtion ws supported by grnts from the Dnish Medicl Reserch Council, Novo Foundtion, Foundtion of 187, Krl Mrie JØrgensen of Kerteminde Foundtion, Peter Ryholt Foundtion, Dnish Foundtion for the Advncement of Medicl Science, Johnn nd Hnne Weimnn born Seedorif's Foundtion, Jcob nd Olg Mdsen Foundtion, nd Ruth I.E. Konig-Petersen Foundtion. 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