Actions and metabolism of histamine in glomeruli and tubules of the human kidney

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1 Kidney Interntionl, Vol. 26 (1984), PP Actions nd metbolism of histmine in glomeruli nd tubules of the humn kidney JOHN R. SDOR nd HANNA. ABBOUD Division of Nephrology, Deprtment of Medicine, Veterns Administrtion Medicl enter, se Western Reserve University nd University Hospitls, levelnd, Ohio Actions nd metbolism of histmine in glomeruli nd tubules of the humn kidney. The effects of histmine on camp nd cgmp ccumultion nd the intrrenl metbolism of histmine were studied in glomeruli nd corticl tubules of nine humn kidneys. Histmine stimulted camp but not cgmp ccumultion in glomeruli ( + 1% to + 265%) in dose- (1 to 1 M rnge) nd time-dependent mnner. This effect of histmine ws inhibited by the histmine H2 ntgonist cimetidine but not the H1 ntgonist diphenhydrmine. Moreover, the H2 gonist dimprit but not the H1 gonist 2-pyridylethylmine stimulted camp ccumultion. Histmine hd no effect on camp or cgmp ccumultion in tubules. Becuse the content of histmine (=2 x 16 M) in glomeruli ws fr bove the circulting levels of plsm histmine in humns (<1_8 M), we explored whether histmine is formed in humn renl tissue. Incubtion of glomeruli with I mi of the histmine precursor L-histidine resulted in n increse in histmine levels ( pmoles/mg protein, N = 7 kidneys) while mrked drop in histmine levels ws observed in tubules ( M pmoles/mg protein. N = 7 kidneys). The increse in histmine levels in glomeruli ws bolished by the histidine decrboxylse inhibitor bromocresine. These results indicte tht humn glomeruli hve histmine H2 receptors, which medite enhnced camp ccumultion, nd tht glomeruli re mjor sites of histmine production in the humn kidney. Histmine cting vi camp my influence glomerulr function of the humn kidney. ffets et métbolisme de l'histmine dns les glomerules et les tubules de rein humin. Les effets de l'histmine sur l'ccumultion de camp et de cgmp, et Ic mdtbolisme intr-rénl de l'histmine ont été étudiés dns les glomérules et les tubules corticux de neuf reins humins. L'histmine stimulit l'ccumultion de camp mis non celle de cgmp dns les glomrules ( + 1% 265%) de fcon dose- (de l6 io M) et temps-dpendnte. et effet de l'histmine étit inhibé pr I cimdtidine, ntgoniste H2 de l'histmine mis non pr I diphenhydrmine, un ntgoniste H1. n outre, le dimprit, un goniste H2, mis non I 2-pyridylethylmine, un goniste H1, stimulé l'ccumultion de camp. L'histmine étit sns effet sur l'ccumultion tissulire de camp ou de cgmp. Prce que Ic contenu d'histmine dns les glomrules (2 x l6 M) étit bien supérieur ux niveux circulnts d'histmine plsmtique chez l'homme (<l M). nous vons explore si l'histmine se forme dns Ic tissu humin norml. L'incubtion de glomérules vec 1 mri d'histidine, le prdcurseur de l'histmine, entrin une ugmenttion des niveux d'histmine (+6,8,5 pmoles/mg protéines, N = 7 reins) lors quune chute mrquee des niveux d'histmine dtit ohserve dns les tubules ( l3,8 2,4 pmoles/mg protéines, N = 7 reins). L'ugmenttion des niveux d'histmine dns les glomrules té bolie pr I bromocrésine, un inhibiteur de l'histidinedécrhoxylse. es résultts indiquent que les glomrules humins possdent des récepteurs H2 Received for publiction August 17, 1983 nd in revised form Februry 7, by the Interntionl Society of Nephrology pour l'histmine qui médient I stimultion de l'ccumultion de camp, et que les glomerules sont les sites principux de production d'histmine dns Ic rein humin. L'histmine gissrt pr l'intermédiire du camp pourrit influencer I fonction glomerulire dns le rein huniin. Severl recent observtions suggest tht the biogenic mine histmine my ply role in renl pthophysiology [1 9]. In the rt kidney, histmine hs been shown to lter renl hemodynmics nd glomerulr microcircultion [2] nd to stimulte renm relese [3]. In rt glomeruli, histmine elicits mrked increse in 3',5'-denosine cyclic monophosphte (camp) ccumultion [4, 5], nd histmine receptors linked to denylte cyclse re present on glomerulr cells [6]. We nd others hve recently shown tht glomeruli re the mjor sites of histmine production in this species [7]. In ddition, studies in some experimentl models of glomerulr disese strongly suggest role for histmine in modulting the renl injury [7, 8]. Very little informtion is vilble on the metbolism nd cellulr ctions of histmine in the humn kidney (HK) [1, 9]. Lindberg, Lindell, nd Westling [1] found tht minced tissue of humn fetl kidney converted lbeled histidine to lbeled histmine. Zeller et l [11] described dimine oxidse ctivity in the humn renl cortex nd medull. Lindell [12] found tht whole renl tissue produces rdiolbeled methylimidzole cetic cid from "-histmine suggesting tht the methyltion pthwy is lso present in the humn kidney. More recent studies provide indirect evidence tht histmine my be involved in renl pthophysiology in humns. In norml subjects, loding studies with the histmine substrte L histidine show n increse in urine but not blood histmine [13] nd provide indirect evidence tht the humn kidney is cpble of histmine production. Moore, Thompson, nd Glssock [14] found tht the urinry nd blood levels of histmine re elevted following renl trnsplnttion in humns. The histmine H2 ntgonist cimetidine hs been reported to cuse ctul reduction in glomerulr filtrtion rte (GFR) in ptients with impired renl function [15], implying role for histmine in modulting renl function in humns. Becuse certin metbolic properties of isolted nephron segments hve been reported to differ in humns compred to other species [16, 17], the present studies were undertken to exmine the cellulr ctions of histmine in isolted glomeruli 144

2 Histmine nd the humn kidney 145 nd nonglomerulr corticl tissue (tubules) of the humn renl cortex nd to loclize the site(s) of histmine production in the humn kidney. Methods Studies were performed on tissue obtined from nine dult humn kidneys (HK): six cdver donor kidneys judged unsuitble for trnsplnttion, nd norml renl tissue obtined from three surgicl nephrectomy smples. All donor kidneys were initilly flushed with ice-cold ollin's solution nd then perfused vi the renl rtery with purified protein frction nd immersed in the sme solution (in the perfusion pprtus) up until the time of trnsfer to the lbortory. The surgicl smples were obtined immeditely following nephrectomy. The dt vilble on the nine ptients includes: HK I ws from 51-yer-old ptient who underwent nephrectomy for lrge hemtom involving the middle portion nd the upper pole of the kidney; the uninvolved lower pole of the kidney ws used. HK 2 ws cdver donor solitry kidney from 56-yer-old ptient who died in n uto ccident. HK 3 ws cdver donor kidney from 44-yer-old ptient; the cuse of deth ws not listed. HK 4 ws donor kidney from 41-yer-old ptient who died from stroke. HK 5 ws from 58-yer-old ptient who underwent nephrectomy for nonobstructing tumor of the renl pelvis. The renl prenchym ws not involved. HK 6 ws cdver donor kidney from 35-yer-old ptient who lso died in n uto ccident. HK 7 ws cdver donor kidney from 31-yer-old ptient who died from hed injuries in n uto ccident. HK 8 ws donor kidney from 5-yer-old ptient who died from trumtic subdurl hemtom. HK 9 ws from ptient who underwent nephrectomy for lrge hypernephrom. An uninvolved piece (s judged by light microscopy on frozen section) of the renl cortex most distnt from the tumor ws used. As soon s the tissue smples becme vilble, they were immersed in ice-cold modified Kreb's buffer (KRB) of the following composition: 14 mrvt Nl, 5 ms KI, 1.2 mm MgSO4, 2. mrvi I2, 1 m glucose, 1 m sodium cette, 2 mm sodium phosphte, nd 2 mm Tris (ph = 7.4), nd trnsferred to the lbortory. The renl cpsule ws stripped off the prenchym, the kidney ws bisected, nd the corticl tissue ws dissected with pir of scissors. All subsequent steps were performed t 4. Glomeruli nd nonglomerulr corticl tissue smples referred to here s "tubules" were prepred by sieving technique s described [5, 18] with minor modifictions. The corticl tissue smples were minced nd then pressed through stinless steel sieve (212 m) with metl spoon. The resulting suspension ws pssed in succession through 39-, 25-, nd l5-m nylon sieves; glomeruli were collected from the top of the l5-m sieve. The glomerulr suspension ws then llowed to settle by grvity for 5 mm nd the superntnt contining smll tubulr frgments or portions of glomeruli ws spirted with Psteur pipette. Glomeruli were then resuspended in fresh KRB. The glomerulr suspension ws greter thn 95% pure s judged by light microscopy. Unlike rt corticl tissue [5, 7], the humn nonglomerulr frction of the renl cortex frgmented into smll tubulr segments tht pssed through ll the sieves, with reltively little mteril collecting on top of the sieves. Therefore, ll the wshings subsequent to the first sieving (212 Im) were collected nd combined with the mteril tht settled on top of the sieves. The suspension ws pelieted by centrifugtion t x 15g for 2 mm nd resuspended in ice-cold KRB. This nonglomerulr suspension is referred to s "tubules." he resulting glomerulr nd tubulr preprtions were used for these studies. The bulk of the corticl nd glomerulr tissue from HK 1, 2, nd 3 ws used for enzyme (histidine decrboxylse) studies (to be reported elsewhere). yclic nucleotide studies. These studies were performed on glomerulr preprtions from six seprte kidneys. Incubtions were crried out in mnner nlogous to our studies in the rt [5, 18]. Freshly prepred glomeruli nd corticl tubules (5 to 1 mg wet wt) were resuspended in 1 d of ice-cold KRB nd distributed into 1-mi glss homogeniztion tubes (size A, Thoms Scientific Products, Phildelphi, Pennsylvni) contining pproprite volumes of KRB nd kept on ice. Tubes contining the vrious suspensions were then preincuhted t 37 for 2 mm in shking wter bth (12 cycles/mm, Dubnoff). At the end of the preincubtion, test gents dissolved in KRB were dded in 5-zl volumes. The ddition of test gents ws mde within time intervl of less thn 1 sec. The finl volume of ech incubtion ws 5 l, nd the finl concentrtion of test gents ws s indicted in Results. Tubes were then incubted for n dditionl 5 mm or s specified in Results t 37 in the metbolic shking wter bth. Drugs nd buffer were freshly prepred prior to ech experiment. At the end of the incubtion, the rection ws terminted by the ddition of 5 pj of ice-cold 1% TA; the tissue smples were immeditely homogenized. A smll liquot of the whole homogente ws tken for protein determintion, nd the rest of the homogente ws centrifuged to precipitte the proteins. TA ws removed from the supernte by repeted extrction (x 5) with wter-sturted ether. The remining ether ws then evported by heting nd smples were djusted to neutrl ph with.5 N NOH. Aliquots of these extrcts were used for determintion of camp nd cgmp. In one experiment, the homogente fter the incubtion ws divided into two seprte liquots for camp nd cgmp determintion. Trcer mount of 3[H]-cAMP ws dded to the respective uquot for monitoring recovery. The quntity of 3[H]-cAMP trcer dded for the determintion of recovery nd tht contributed to the finl concentrtion of camp ws clculted nd subtrcted from ech smple. Added 3H-cAMP (95% 4.) ws recovered in the finl extrct immeditely prior to RIA. Therefore, this step of monitoring the recovery ws omitted from the rest of the experiments. yclic nucleotide determintion by RIA ws crried out s originlly described by Steiner, Prker, nd Kipnis [19] nd modified to increse the sensitivity of camp [21 nd cgmp [21] by cetyltion nd succinyltion, respectively. Drugs used were tested for possible interference with RIA. At the highest concentrtion used in this study, none of the drugs interfered with either camp or cgmp ssy. In ech experiment, glomeruli nd tubules from the sme kidney were incubted on the sme dy nd under identicl conditions. Smples from the sme kidney were lso processed for extrction, RIA, nd protein determintion t the sme time with the sme btch of regents. Protein ws determined by the method of Lowry et l [22] nd cyclic nucleotide dt were expressed s picomoles per milligrm of protein.

3 146 Sedor nd Abboud Histmine content. Tissue smples from two seprte kidneys (one surgicl smple nd nother cdver donor kidney) were used for the determintion of histmine content. As soon s the tissue becme vilble, smll piece of the renl cortex, freshly prepred glomeruli, nd tubules were homogenized in ice-cold.5 M sodium phosphte buffer ph 7.9. A smll liquot of the homogente ws tken from protein determintion; the rest of the homogente ws heted in boiling wter for 5 mm nd centrifuged t x 12,8g for 3 mm in microcentrifuge (ppendorf). The superntnt ws frozen nd stored t 2 for histmine determintion by rdioenzymtic microssy [23] (see below). Histmine production studies. These studies were performed on glomerulr nd tubulr preprtions from seven seprte kidneys. The incubtion of freshly prepred glomeruli nd corticl tubules ws crried out in mnner similr in principle to our previous studies in the rt [7]. Freshly prepred glomeruli (SO-p.l liquots) nd corticl tubules (15 to 25 mg wet wt) suspended in KRB were distributed into l.5-ml microcentrifuge tubes (ppendor kept on crushed ice. The incubtion mixture lso contined.1 mm pyridoxl phosphte, coenzyme of histidine decrboxylse, the histminse inhibitor minogunidine (.1 mm), nd 1. m L-histidine, the histmine substrte. The finl volume of ech incubtion ws brought up to 2 pa with KRB buffer ph 7.4. In ech experiment, control incubtions were mde using glomerulr nd tubulr suspensions heted in boiling wter for 5 mm before regents were dded. In some experiments, dditionl control incubtions were crried out in the presence of D-histidine (not histmine substrte). Three experiments included incubtions crried out in the presence or bsence of the histidine decrboxylse inhibitor bromocresine. Tubes were shken mnully nd incubted for 18 mm t 37 in wter bth. The tubes were dditionlly shken every 3 mm during the incubtion. At the end of the incubtion, the rection ws stopped by heting the tubes in boiling wter for 5 mm. The tubes were then frozen nd stored t 2. Prior to the determintion of histmine, suspensions of glomeruli nd tubules were frozen nd thwed in n ethnol/dry ice bth (four times) to insure relese of histmine. Tubes were then centrifuged t x 12,8g for 3 mm in microcentrifuge (ppendorf). The supernte ws trnsferred to clen tubes. ph ws djusted to 7.9 to 8. by the ddition of 5 pa of.5 N NOH (optimum ph for the histmine ssy). Protein ws determined in the pellets which were solubilized in 1. N NOH. Histmine ws determined on 2 p1 liquots of the supernte by specific nd sensitive rdioenzymtic microssy [23]. Briefly, this ssy uses histmine methyltrnsferse (HMT), which methyltes histmine to form Nr-methylhistrnine. The smple or histmine stndrd (2 jd) is incubted with 1 pi of guine pig HMT (2 mg/mi), contining.22 pmoles of 3H-histmine, nd.28 nmoles of '4 SAMe. The finl concentrtions of the rectnts were s follows: 3H-histmine 7.35 n nd '4-SAMe 9.35 LM. The totl rection volume ws 3 pa. The mixture ws incubted for 1 hr t 37. The rection ws terminted by dding 1 1d 2.4 N perchloric cid. The rection product Nr-methylhistmine ws extrcted s described [231 with minor modifictions [24]. NL (2 pa) sturted 3.3 N NOH, nd 4 pa of chloroform were dded to the tubes. Tubes were then tightly cpped nd shken on mechnicl shker (ppendorf, Brinkmnn) for 2 mm. Then they were centrifuged t >< l2,8g for 2 mm in microcentrifuge (ppendorf 5412). The upper queous phse ws removed by spirtion, nd to the orgnic phse 1 pa of N1 sturted 1 N NOH were dded. Tubes were shken for n dditionl 5 mm nd centrifuged. The NOH lyer ws removed crefully by spirtion. A 3-pA liquot of the chloroform phse ws trnsferred to counting vil; chloroform ws evported to dryness in strem of ir. Then 5 ml of Aqusol (New nglnd Nucler orp., Boston, Msschusetts) were dded, nd the rdioctivity of '4 nd 3H ws counted in scintilltion spectrometer (Serle Mrk III Liquid Scintilltion System, Model 688, Serle Anlyticl, hicgo, Illinois) until the S of counting ws 5% or for 2 mm, whichever ws shorter. A stndrd curve for uthentc histmine bse ws determined for ech ssy. The rtio of '4/3H is directly proportionl to the mount of unlbeled histmine present in the incubtion mixture. The ssy used ws liner between 2.5 nd 5 pmoles histmine per smple or stndrd. Histmine dded to boiled tissue extrct ws recovered to the extent of 85 to 95%. Vlues for histmine were expressed s picomoles per milligrms of protein. Mterils Histmine dihydrochloride, L-histidine, D-histidine, 1-methyl-3-isobutylxnthine (MIX), nd diphenhydrmine HI were purchsed from Sigm hemicl o., St. Louis, Missouri. Aminogunidine sulphte ws purchsed from stmn Orgnic hemicls, stmn Kodk o., Rochester, New York, Bromocresine (4-bromo-3-hydroxybenzylozymmne diphydrogen phosphte) ws kindly supplied by Lederle Lbortories, Division of Americn ynmid o., Perl River, New York. imetidine, dimprit, nd 2-pyridylethylmine were kindly supplied by Mr.. Porter of Smith, Kline nd French, Phildelphi, Pennsylvni. [3H]-cAMP for the mesurement of recoveries ws purchsed from New nglnd Nucler orp. Antibodies nd l251 lbeled ntigens for camp were from RIA kits purchsed from Schwrtz/Mnn, Division of Becton, Dickinson nd o., Orngeburg, New York. Antibodies nd 3H-lbeled ntigens for cgmp nd S-Adenosyl-L-methionine-['4-methyl], 55 to 6 mi/mmole, were purchsed from Amershm orp., Arlington Heights, Illinois [(/3-side chin lbel)-3h] histmine ws prepred from [(/3-side chin lbel)-3h1 L-histidine 1 i/mmole (New nglnd Nucler orp.) s described [7, 25]. Sttisticl significnce ws determined by nlysis of vrince. Results The effect of histmine on camp production ws exmined in glomeruli isolted from six seprte humn kidneys (Tble 1). In the presence of.5 mii MIX, phosphodiesterse inhibitor, io M histmine incresed camp ccumultion in ll six glomerulr preprtions tested. After 5-mm incubtion, the stimultory effect of histmine on camp ccumultion rnged between 1 to 263% nd did not pper to depend on the durtion of the perfusion or the source of the tissue (Tble 1). The bsl concentrtion of camp nd cgmp in glomeruli incubted without MIX ws much lower thn in the presence of MIX (.5 mm). However, even when glomeruli were incubted with histmine lone (in the bsence of MIX), n increse in camp ws lso observed but ws of lower mgnitude thn in

4 Hist,nine nd the humn kidney 147 Tble 1. ffect of histmine on camp nd cgmp ccumultion in isolted humn glomeruli camp, pmoleslmg protein cgmp, pmoles/mg protein Kidney source/no. Bsl Histmine % Bsl Histmine % 1. Surgicl smple NT NT 2. Perfused donor kidney (12 hr)b % Perfused donor kidney (18 hr)b % Perfused donor kidney (46 hr)b NT NT 5. Surgicl smple % Perfused donor kidney (16 hr)b % Perfused donor kidney (36 hr)' % Perfused donor kidney (12 hr)b % Surgicl smple NT NT +48% 2% 3% +7% 1% +3% " 181 1% % Abbrevition: NT, not tested. Isolted glomeruli were incubted for 5 mm in the presence of.5 mm MIX. b The number of hours in prentheses denotes perfusion time. Vlues represent smple mens SM of duplicte incubtion smples ech ssyed in duplicte. "The vlue is significntly different from bsl vlues (without histmine), P <.5 or higher degree of significnce (pired t test). Tble 2. ffect of histmine on camp nd cgmp ccumultion in isolted glomeruli nd tubules of HK 3 Glomeruli Histmine Tubules Histmine Bsl (1 n M) % Bsl (l M) camp,pmoles/mg protein 2.34b ± % cgmp,pmoles/mg protein % Isolted glomeruli nd tubules were incubted for 5 mm t 37 in the presence of.5 msi MIX. b The vlue represents the men rnge of duplicte incubtion smples tht were ech ssyed in duplicte. %_- +21% 23.8% identicl incubtions crried out in the presence of.5 mti MIX (% + 34%, + 54% nd + 74% in HK, 2, 3 nd 7, respectively). Histmine did not influence cgmp ccumultion in glomeruli (Tble 1), nd camp nor cgmp in one tubulr preprtion tested (Tble 2). Histmine stimulted camp ccumultion in glomeruli in time- nd dose-dependent mnner. Stimultion ws seen s erly s 3 sec nd reched mximum t 5 mm (Fig. 1A). In three seprte glomerulr preprtions, totl camp ws determined t 5 mm s function of histmine concentrtion. There ws dose-dependent increse in camp ccumultion between 1 6 to l M histmine (Fig. 1B), with hlf-norml stimultionts x 15M. The effect of histmine specific gonists nd ntgonists on camp ccumultion ws tested in three seprte glomerulr preprtions (Fig. 2A). The histmine (l M) stimulted camp ccumultion ws bolished by cimetidine (l- M), n H2 ntgonist, but not by equimolr concentrtions of the H1 ntgonist diphenhydrmine. imetidine (1 M) nd diphenhydrmine (l M) lone, hd no sigificnt effect on camp ccumultion. In one glomerulr preprtion tested (ilk 8), the /3-drenergic blocker proprnolol (l M) lso hd no effect on bsl camp levels or on the stimultory effect of histmine (1 M) on camp ccumultion (dt not shown). The effects of the specific H1 gonist 2-pyridylethylmine nd the H2 gonist dimprit were lso compred in the sme three seprte glomerulr preprtions (Fig. 2A). Dimprit (1O M) cused more thn twofold increse in camp ccumultion, while 2- pyridylethylmine (l M) hd no effect. Neither histmine nor histmine specific gonists or ntgonists influenced cgmp ccumultion in glomeruli (Fig. 2B). The stimultory effect of dimprit on camp ccumultion, similr to the effect of histmine, ws dose-dependent (Fig. 3A) with hlf-mximl stimultion chieved t l M. Figure 3B shows tht cimetidine inhibits camp ccumultion in dose-dependent mnner, with 5% inhibition chieved t l M, drug level chieved in the blood fter cliniclly employed orl doses [26]. quimolr concentrtions of diphenhydrmine, on the other hnd, hd no effect on camp ccumultion. The concentrtion of circulting histmine in humn plsm (<1 M) is more thn two orders of mgnitude lower thn the concentrtion (1-6 M) tht elicited enhnced camp ccumultion in glomeruli. Therefore, histmine content ws determined in the renl cortex, glomeruli, nd tubules of the sme kidney. Histmine content verged in the cortex, in glomeruli, nd in tubules (pmoles/mg protein; N = 4 smples from ilk 1) nd in the cortex, in glomeruli, nd in tubules (pmoles/mg protein; N = 4 smples from HK 2). These tissue levels of histmine mrkedly exceed plsm levels. To determine the source of histmine in the humn renl cortex, glomeruli nd tubules isolted from the sme kidneys were incubted with the histmine substrte L-histidine nd histmine levels were determined. Incubtions with 1 mtvt L-histidine (Fig. 4A) resulted in significnt increse in histmine levels in seven seprte glomerulr preprtions tested (+ M.8.5 pmoles/mg protein). Unlike incubtions with L-histidine, incubtions of glomeruli with D-histidine (not precursor of

5 148 Sedor nd Abboud A 12.5 with L-histidine but hd no effect on histmine levels in tubules incubted under identicl conditions. 1. ) 7.5 ) ) 9) ) Istmine (1 mm) /TTiTIiIi 1- MIX (.5mM) B Time, minutes (%+113±33) (% + 57±13) camp (% + 159± _J(% + 162±41) (% BycGMP io 1 6 i io i- Histmine, M Fig. 1. A Time course of the eff'ect of histmine on camp ccumultion. isolted glomeruli from HK 7 nd 8 were incubted for vrious time periods t 37. Totl camp ws determined by RIA. For detils see Methods. Points indicte the men SM of dt from four incubtion smples (two from ech kidney). ch smple ws ssyed in duplicte. B Dose-response curve for the ejfrct qf histmine on camp nd cgmp ccu,nultion. isolted glomeruli from HK 5, 6, nd 7 were incubted for 5 mm t 37. Totl camp nd comp were determined by RIA. For detils see Methods. Points indicte the men SM of dt from six seprte incubtion smples (two from ech kidney), with the exception of the io- M (four seprte incubtion smples). ch smple ws ssyed in duplicte. histmine) did not result in chnge in histmine levels (Fig. 4B). In contrst to glomeruli, suspension of corticl tubules incubted with L-histidine (Fig. 4A) under identicl conditions s glomeruli showed no increse but rther shrp decline in histmine levels ( M pmoles/mg protein). A similr drop in histmine levels were observed in tubules incubted with D-histidine (Fig. 4B). To test whether the differences in the bility of glomeruli or tubules to ccumulte histmine ws due to different rte of histmine synthesis rther thn to differentil ctbolism or other fctors tht my influence histmine levels, the effect of bromocresine, n inhibitor of histidine decrboxylse [27], on histmine ccumultion ws exmined. In three seprte preprtions tested (Fig. 5), the ddition of bromocresine bolished the ccumultion of histmine in glomeruli incubted Discussion These studies demonstrte tht the camp system in isolted humn glomeruli is sensitive to stimultion by histmine nd tht glomeruli re sites of ctive histmine synthesis within the humn renl cortex. The mgnitude of the stimultory effect of histmine on camp ccumultion does not pper to be relted to the source of tissue (surgicl smple vs. perfused cdver donor kidney) nor to the durtion of the perfusion. Of interest is tht glomeruli isolted from kidneys perfused for s long s 18 hr showed similr or higher responsiveness in terms of camp ccumultion s glomeruli isolted from freshly obtined surgicl smples. Whether this difference in responsiveness to histmine represents inherent individul vrition, ltertion in the levels of the camp substrte ATP or chnges in the ctivities of denylte cyclse or camp phosphodiesterse 28] during the perfusion procedure or clmping of the renl rtery prior to the nephrectomy cnnot be determined from our studies. The effect of histmine on camp ccumultion is dose-dependent; the concentrtion of histmine (= 5 X 1 M) eliciting hlf-mximl elevtion in camp levels (reflecting indirectly the ffinity of the receptor for histmine) pproximtes the concentrtion required for hlf-mximl stimultion of camp ccumultion in humn prthyroid tissue [29] nd the pprent Km of histmine-sensitive denylte cyclse in humn gstric mucos [3]. Severl criteri suggest tht the effect of histmine is medited by distinct histmine receptors. Inhibition of the stimultory effect of histmine on camp ccumultion by cimetidine but not by diphenhydrmine or proprnolol indictes tht the effect of histmine is medited by n H2 receptor linked to denylte cyclse tht is lso distinct from the B-drenergic receptor. This conclusion is supported further by the finding tht the H2 receptor gonist dimprit lso stimultes camp production in glomeruli in dose-dependent mnner. In contrst, the histmine H1 gonist 2-pyridylethylmine t equimolr concentrtions hd no effect on camp ccumultion. While the present studies focused on glomeruli, the limited observtions showing no effect of histmine on camp or cgmp ccumultion in corticl tubules suggest tht the presence of histmine receptors in the humn renl cortex is distinct feture of glomeruli. The cell type or types within the renl glomerulus bering histmine receptors remins to be identified. These studies in humn glomeruli differ from previous observtions [4, 5] on the effect of histmine on cyclic nucleotides in rt glomeruli in two respects. First, the increse in camp ccumultion in response to histmine is lower in humn glomeruli s compred to rt glomeruli. Second, histmine hd no effect on cgmp ccumultion in humn glomeruli even fter 2-mm incubtions (dt not shown), unlike the smll but consistent increse in cgmp levels in response to histmine observed in the rt. Whether these differences could be ccounted for by species or ge differences or the mnner in which the kidneys were hndled prior to the glomerulr isoltion cnnot be determined from our experiments. If the effect of histmine on cgmp in rt glomeruli is secondry to the inhibitory effect of generted camp on cgmp phosphodiester-

6 Histmine nd the humn kidney 149 A 2 ) U, * * Fig. 2. A nd B ffects of histmine nd histmine-specific gonists nd ntgonists on camp (A) nd cgmp (B) ccumultion. Isolted glomeruli from HK 5, 6, nd 7 were incubted for 5 mm t 37 (in the presence of.5 mrvi MIX). Totl camp nd cgmp were mesured by RIA. For detils see Methods. ch br represents men SM of dt from six seprte incubtion smples (two from ech kidney). ch smple ws ssyed in duplicte. Asterisks denote vlues significntly different from bsl vlue (MIX lone) t P <.5 or higher degree of significnce (nlysis of vrince). Abbrevitions re: H, histmine; Dimp, dimprit (H2 gonist); PA, 2- pyridylethylmine (H1 gonist); Diph, diphenhydrmine (H1 ntgonist); im, cimetidine (H2 ntgonist). )., ) B 15 1: Bsl Diph H im H (14M) (1 4M) (1 4M( (14M) + + Diph im (14M) (14M( Bsl H Dimp PA Diph H im H (14M) (14M) (1O4M) (14M) (14M) (1O4M) (14M( + + Diph im (1 4M( (14M( se, s suggested by Torres et! [4], the bsence of such n effect in humn glomeruli my be ccounted for by the lesser increse in camp in response to histmine. In ddition to the presence of histmine receptors, humn renl corticl tissue contins reltively high levels of histmine.' While these concentrtions of histmine [1] probbly underestimte its in situ concentrtion, they re still within the rnge cpble of eliciting n increse in camp in glomeruli. Norml circulting levels of plsm histmine (< 18 M) [32] on the other hnd re t lest 1 times lower thn the miniml concentrtion of histmine cpble of stimulting camp ccumultion in this tissue in vitro, nd plsm levels sufficient to hve stimultory effect on glomeriilr camp system re unlikely to be Assuming tht tissue content of wter is bout 8% of wet weight 131], the estimted histmine concentrtion in humn glomerulr tissue fluid would be bout 2 nmoles/ml of tissue wter or 2 x l M. chieved in plsm since smll elevtions in plsm histmine evoke numerous nd severe systemic rections in humns [13, 33]. ndogenous histmine is more likely therefore to influence glomerulr function thn circulting histmine, However, the extent to which endogenous histmine is quntittively ccessible to histmine receptors remins to be determined. There re two potentil sources of histmine in renl tissue [27]. Histmine my be synthesized loclly from L-histidine or it my conceivbly be extrcted from the rteril blood nd concentrted in the tissue. Our finding tht incubtion with L- histidine but not D-histidine increses histmine levels in glomeruli but not in corticl tubules incubted under identicl conditions suggests tht locl synthesis in glomeruli is the mjor source of histmine in this nephron structure. This conclusion is supported by the observtion tht the histidine decrboxylse inhibitor bromocresine bolished the increse in histmine levels observed in glomeruli. The reltively high levels of histmine in corticl tubules vries with our recent findings in the rt [71 where the histmine levels in corticl tubules

7 15 Sedor nd Abboud A 17.5 A 4 8 ) ' io io Agonist, M camp I 2 1 Boiled L-hist. 4 2 Boiled L-hist B B 4 Glomeruli 8 Tubules 3 5 ) io io io Antgonist, M Fig. 3. A Dose-response curve (f the effects of histmine nd histmine gonists on camp nd cgmp ccumultion. Isolted glomeruli from HK 7 were incubted for 5 mm t 37, with incresing concentrtion of gonists (in the presence of.5 mt MIX). Totl camp nd cgmp were determined by RIA. Fr detils see Methods. Points represent the men of four determintions from two incubtion smples. The rnge for the incubtion smples ws 29% or less. Symbols re:,histmine; I, pyridylethylmine; A A, dimprit. B Dose-response curve of the effects of histmine ntgonists on histmine-stimulted camp ccumultion. Isolted glomeruli from HK 8 were incubted for 2 mm t 37 with 1 M histmine nd incresing concentrtion of ntgonists (in the presence of.5 msi MIX). Points represent the men rnge of four determintions from two incubtion smples. Symbols re:, cimetidine;, diphenhydrmine. were much lower thn tht in glomeruli, The reson for this discrepncy is not cler. Severl fctors my influence histmine levels during the perfusion or the nephrectomy, such s chnges in the ctivity of the synthesizing or degrding enzymes or even influx or effiux of histmine from the tissue. Furthermore, the nonglomerulr frction of the renl cortex referred to here s "tubules" represents not only mteril tht sedimented on top of the sieves, but lso ll nonglomerulr corticl tissue which might substntilly differ in its cellulr content (interstitil cells or mst cells) from the "tubulr" frction. ) ) I 3I ' (5) ) o 6 Boiled D-hlst Boiled D-hist Fig. 4. A Histmine content in glomeruli nd tubules in control (boiled) incuhtions nd fter incubtion with 1. mm L-histidine for 18 mm t 37. All incubtions contined.1 ms'i pyridoxl phosphte nd.1 mm minogunidine. ch point indictes men rnge of t lest two incubtion smples from ech kidney. ch smple ws ssyed in duplicte. Numbers between prentheses indicte kidney smple (for key see Tble 1). The men histmine level (pmoles/mg protein) in boiled glomeruli ws nd in incubted glomeruli (men SM of seven experiments; P <,5 nlysis of vrince). orresponding levels in tubules (pmoles/mg protein) were nd (men SM of seven experiments; P <.5 nlysis of vrince). B Histmine content in gloineruli nd tubules in control (boiled) incubtions nd fter incubtion with 1. mm D-histidine (not histmine substrte) for 18 mm t 37. All incubtions contined.1 msi pyridoxl phosphte nd.1 m minogunidine. ch point indictes men rnge of dt from two incubtion smples from ech kidney. ch smple ws ssyed in duplicte. Numbers between prentheses indicte kidney smples (for key see 'fble 1). Histmine levels (pmoles/mg protein) in boiled glomeruli ws nd in incubted glomeruli (men SM of four experiments). orresponding levels in tubules (pmoles/mg protein) were nd (men SM of four experiments) (9). 2 in the rt studies. Therefore, whether or not similr pttern of distribution of histmine exists in situ in the humn kidney tubules or glomeruli cnnot be determined from these studies. The shrp decline in histmine levels observed when tubules

8 I 2 I 4.. s. e r. (I.6 I Boiled L-hist + Bromocresine Boiled L-hist Fig. 5. hnges in histmine content in glomeruli nd tubules in control (boiled) incubtions nd fter incubtions with 1. mw L-histidine for 18 mm t 37 in the bsence ( ) nd in the presence ( ) of.1 mm bromocresine. All incubtions contined.1 msi pyridoxl phosphte nd.1 msi minogunidine. Incubtions were crried out for 18 mm t 37. ch point indictes the men rnge of two incubtion smples from ech kidney. ch smple ws ssyed in duplicte. Numbers between prentheses indicte kidney smple (for key see Tble 1). I Histmine nd the humn kidney 151 Tubules (5) (6) be medited by camp. xperimentl evidence in rt s well s humn glomeruli suggest tht certin hormones cting vi camp my influence the contrctile properties of the glomerulr mesngium thereby regulting renl blood flow nd GFR [42]. A similr role of histmine nd camp in modulting glomerulr microcircultion my be opertive in humns. it should lso be emphsized tht histmine cting vi camp my hve numerous other effects, besides its hemodynmic effects, on both intrinsic glomerulr cells or cells infiltrting the glomerulus in the course of immune-medited inflmmtory injury [43]. For exmple, histmine my modulte lysosoml enzyme relese, phgocytosis, relese of other meditors, nd metbolism of extrcellulr glomerulr mtrix by intrinsic glomerulr cells, s hs been demonstrted in extrrenl tissues [43 46]. Furthermore, since histmine receptors hve been demonstrted on number of inflmmtory cells [47] tht hve been shown to infiltrte glomeruli in vrious forms of gbmerulonephritis in experimentl nimls [48, 49] nd humns [5], histmine my profoundly modulte vrious phses of glomerulr immune inflmmtory injury. Bromocresine. Recent studies [511 showing tht inflmmtory meditors such s lymphocyte products my enhnce histmine production in trget orgns suggest tht locl synthesis of histmine my be n importnt mechnism of histmine vilbility in glomeruli, sites of histmine receptors. Phrmcologic mnipultion of endogenous histmine nd cyclic nucleotides my potentilly prove to be effective in influencing the course of immune-medited glomerulr injury. re incubted with L-histidine (similr to those with D-histidine) excludes tubulr synthesis of histmine s mjor source of histmine in the humn kidney. Active uptke of circulting histmine by tubules hs been demonstrted in the rt [341 or in ddition histmine synthesized in glomeruli tht my prtilly lek to the peritubulr circultion my contribute to tubulr histmine content. However, regrdless of the presence nd source of histmine in tubules, our studies show tht glomeruli re not only trgets for histmine's ction on camp but lso sites of ctive synthesis. The cell type(s) within the renl glomerulus cpble of histmine synthesis is not known. Of mjor interest is tht humn glomeruli re devoid of mst cells [351, the mjor site of tissue histmine synthesis. It is conceivble tht histmine synthesized in humn glomeruli represents non-mst cell or nscent histmine. The presence of this pool of histmine ws proposed severl yers go [36, 37]. Recent observtions in mouse model deficient in mst cells [381 support the existence of this pool of non-mst cell histmine in mny tissues including the kidney. The presence of histidine decrboxylse in isolted cultured endothelil nd smooth muscles from rbbit ort [39, 4] nd the bility of humn endothelil cells cultured from umbilicl veins to synthesize histmine [41] suggest tht glomerulr endothelil nd perhps mesngil (smooth, muscle-like) cells re the source of glomerulr histmine synthesis. Precise identifiction of the cell type responsible for histmine synthesis wits studies utilizing glomerulr cells in culture. The presence of histmine H2 receptors tht medite the enhnced camp ccumultion in humn glomeruli suggest tht t lest some ctions of histmine in this nephron structure my Acknowledgments Preliminry results of this work were published in bstrct form in lin Res 31 :44lA, The work ws supported by grnt-in-id from the Americn Hert Assocition, Northest Ohio Affilite, nd through Veterns Administrtion Reserch Funds to Dr. H.. Abboud. Dr. J. Sedor is fellow of the Kidney Foundtion of Ohio. The uthors thnk Mr.. Willims nd the personnel t the Orgn Preservtion Lbortory t the levelnd linic Foundtion nd the Urology Stff t the Veterns Administrtion for providing the kidneys, J. Murry nd A. Hung for technicl ssistnce, nd Mrs. L. Polcek nd Mrs. J. Smrt for typing ssistnce. Reprint requests to Dr. H.. Abboud, Division of Nephrology, Deprtment of Medicine, Veterns Administrtion Medicl enter, 17! st Boulevrd, levelnd, Ohio 4416, USA References 1. ABBOUD H: Histmine nd serotonin, chp 12, in Renl ndocrinology, edited by DUNN MJ, Bltimore, Willims nd Wilkins, 1983, pp IHIKAWA I, BRNNR BM: Mechnisms of ction of histmine nd histmine ntgonists on the glomerulr microcircultion in the rt. irc Res 45: , SHWRTSHLAG U, HAKNTHAL : Histmine stimultes renin relese from the isolted perfused rt kidney. Nunyn Schmiedebergs Arch Phrmcol 319: , TORRS V, NORTHRUP T, DWARDS RM, SHAH SV, DousA TP: Modultion of cyclic nucleotides in isolted rt glomeruli: Role of histmine, crbmyicholine, prthyroid hormone nd ngiotensin II. J lin Invest 62: , ABBOUD H, SHAH SV, DOUSA TP: ffects of dexmethsone on cyclic nucleotide ccumultion in glomeruli. J Lb lin Med 94:78 717, HANSL D, OUDINT J-P, NIVX M-P, ARDAILLOU R: Histmine H2 receptors in glomeruli isolted from rt renl cortex. Biochern Phrmcol 3 1: , 1982

9 152 Sedor nd Abbud 7. Aouo H, Ou SL, VLOSA JA, SHAH S. DousA TP: Dynmics of histmine in the norml rt kidney nd mino nucleoside nephrosis. J /in Invest 69: , KNIKR W. OHRAN : The locliztion of circulting immune complexes in experimentl serum sickness: The role of vsoctive mines nd hydrodynmic forces. J rp Med 127: , ABBOUD H, DOUSA TP: Renl metbolism nd ctions of histmine nd serotonin. Miner lectrolyte Metb 9: LINDBRG S, LINDI,L S. WSTLING H: Formtion nd inctivtion of histmine by humn foetl tissues in vitro. Act Obstet Gynecol Scnd 42(suppl l):49 54, ZLLR A, BIRKHAUSR H. MIsLIN H. WNK M: Uber ds Vorkommen der Dimin-Oxydse bei Mcnsch, Sugetier nd Vogel. Mit einem Anhng Oher ds Vorkommen der holinesterse beim Vogel. He/v him Act 22: , LINDLL S: Observtions on the inctivtion of -histmine by humn renl tissue in vitro. Kong! Fvsiogrufisk Sä/iskpets Hndlingr 28:15, BAVN MA, JAOBSN S. HORAKOVA Z: Modifiction of the enzymtic isotopic ssy of histmine nd its ppliction to mesurements of histmine in tissues, serum nd urine. /in him Act 37:91 13, MooR T, THOMPSON PP, GLASSOK RJ: levtion in urinry nd blood histmine following clinicl renl trnsplnttion. Ann Surg 173: , DUH MK, MoonY P. NORTUFILD T: ffect of cimetidine on renl function in mn. Br J /in Phrmcol 12:47 5, FIABARDS D, GAGNAN-BRUNTT G. IMBRT-TBOUL M, GONT- HARVSKAIA, MONTGUT M. LIQU A, MORL F: Adenylte cyclse responsiveness to hormones in vrious portions of the humn nephron. J /in invest 65: , SRAR J, RIGAUD M, BNS M, RABINOVITH H, ARDAILLOU R: Metbolism of rchidonic cid vi the lipoxygense pthwy in humn nd murine glomeruli. J Biol hem 258: , ABBOUD H, DousA TP: Actions of denosine on cyclic nucleotides in rt glomeruli. Am J Phvsioi 13:F633 F STINR AL, PARKR W, KIPNIS DM: Rdioimniunossy for cyclic nucleotides: I. Preprtion of ntibodies nd iodinted cyclic nucleotides. J Biol hem 247:116 I 113, HARPR IF, BROOKR G: Fcmtomole sensitive rdioimmunossy for cyclic AMP nd cyclic GMP fter 2 cetyltion by cetic nhydride in queous solution. J yclic Nucleotide Res 1:27 218, FRANDSN K, KRISHNA G: A simple ultrsensitive method for ssy of cyclic AMP nd cyclic GMP in tissues. Life Sc! 18: , LOWRY OH, ROSNBROUGH NJ, FARR AL, RANDALL Ri: Protein mesurement with the folin phenol regent. J Biol hem 193: , TAYLOR KM, SNYDR SH: Isotopic microssy of histmine, histidine, histidine decrboxylse nd histmine methyltrnsferse in brin tissue, J Neurochem 19: , GUILLOU L. HARTMANN D, VILL G: nzymtic isotopic ssy for humn plsm histmine. /in him Act 116: , BAVN MA, HORAKOVA Z: The enzymtic isotopic ssy of histmine, chp 3, in Hndbook of xperimentl Phrmcology, edited by ROHA de SILVA M. Berlin, Springer-Verlg, pp GRIFFITHS R, L RM, TAYLOR D: Kinetics of cimetidine in mn nd experimentl nimls, in imetidine.' Proc 2nd mt Symp Histmine H2-receptor An/g. Amsterdm, xcerpt Medic, pp BAVN M: Fctors regulting vilbility of histmine t tissue receptors, chp 3, in Phrmcology ofhistmine Receptors, edited by GANLLIN R, PARSONS M. London. Wright PSG, 1982, pp KIM JK, FRORNRT PP, Hui YSF, BARNS LK, FARROW GM, DOUSA TP: nzymes of cyclic 3',5'-nucleotide metbolism in humn renl cortex nd renl denocrcinom. Kidney mt 12: , ABBOUD H, ZIMMRMAN D. DIS Al, HATH H, DousA TP: Histmine nd humn prthyroid denom: ffect on denosine 3',5'-monophosphte ccumultion in vitro. J /in ndocrinol Metb 53: SIMON B, KATHR H: Histmine-sensitive denylte cyclse of humn gstric mucos. Gstroentero/ogy 73: , GIFS A: ell physiology, in Biochemistry of the e/i. Phildelphi. W.B. Sunders, o., 1973, pp SFIAFF R, BAVN M: Incresed sensitivity of the enzymtic isotopic ssy of histmine in plsm nd serum. Anl Biochem 94:425 43, Moss J. Roscow, SAVAR5 ii, PHILBIN DM, KNIFFIN KJ: Role of histmine in the hypotensive ction of d-tubocurrine in humn nesthesiology. Anesthesiology 55: 19 25, SHWARTZ I: pttion et inctivtion de l'histmine pr le rein de rt. J Physiol (Pris) 59:68 95, SYL H: The Mst e/is. Wshington, D.., Buttersworths, SHAYR R: nzymtic formtion of histmine from histidine, in Hndbook of xperiment/ Phrmcology, vol 18, Histtnine nd Antihistmines, Prt 1, edited by ROHA de SILVA M, Berlin, Springer-Verlg, 1966, pp KAHLSON G, ROSNGRN : New pproches to the physiology of histmine. Physiol Rev 48: , YAMATODANI A, MAYAMA K, WATANAB T, WADA H, KIT- AMURA Y: Tissue distribution of histmine in mutnt mouse deficient in mst cells. Biochem Phrmcol 31:35 39, OWNS GK, HOLLIS TM: Locl ortic histmine metbolism nd lbumin ccumultion: Differences between blue nd white res. Arteriosclerosis 1: , 198! 4t). ORLIDG A, HOLLIS TM: Aortic endothelil nd smooth muscle histmine metbolism in experimentl dibetes. Arteriosclerosis 2:142 15, ABBOUD H, MURRAY J, TI-IORGIRSSON G: l-iistidine decrboxylse medited histmine synthesis in cultured humn endothelil cells (bstrct). /in Res 3:l66A, SHOR NI, IHIKAWA 1, BRNNR BM: Mechnism of ction of vrious hormones nd vsoctive substnces on glomerulr ultrfiltrtion in the rt. Kidney mt 2: , Dous, TP, SHAH SV, ABBOUD H: Potentil role of cyclic nucleotides in glomerulr pthophysiology. Adv yc/ic Nucleotide Res 12: , MLMON KL, RKLIN R, ROBRTO R: Autcoids s modultors of the inflmmtory nd immune response. Am J Med 71:1 16, LIHTNSTIN LM, PLANT M: Histmine nd Immune Responses, chp 1, in Phrmco/ogy of Histmine Receptors, edited by GANNLLIN R, PARSONS M, Boston, Wright, PSG, 1982, pp ROKLIN R, BR Di: Histmine nd immune modultion, in Advnces in Internl Medicine, edited by STOI.LRMAN GH, hicgo, Yer Book Medicl Publishers, 1983, vol 28, pp BOURN HR, LIHTNSTIN LM, MLMON KL, HNNY S, WINSTIN Y, SHARR GM: Receptors for vsoctive hormones nd meditors of inflmmtion regulte mny leukocyte functions. Science 184:19 28, SHRJNR GF, OTRAN RS. PARDO J, UNANU R: A mononucler cell component in experimentl immunologic glomerulonephritis. J xp Med 147: , OHRAN G. UNANU R, DIxoN Fi: A role of polymorphonucler leukocytes nd complement in nephrotoxic nephritis. J xp Med 122: MLUSKY RT, BHAN AK: ell-medited mechnisms in renl disese. Kidney mt 21(suppl I I):S-6 S-l2, DY M, LBL F, KAMAUU P, HAMBURGR J: Histmine production during the nti llogrft response. J xp Med 153:293 39, 1981