trapping by the kidney

Transcription

1 Kidney Interntionl, Vol. 14 (1978), pp ome hemodynmic determinnts of immune complex trpping by the kidney LEE A. HEBERT, ARIN L. ALLHIER, nd UAN M. KOETHE Deprtments of Medicine nd Pthology nd the Allen Brdley Medicl ciences Lbortory, Medicl ollege of Wisconsin nd the Milwukee ounty Generl Hospitl, Milwukee, Wisconsin ome hemodynmic determinnts of immune complex trpping by the kidney. This study ws undertken to help clrify the reltionship between cpillry hemodynmic events nd the tissue uptke of circulting immune complexes (I). In ech of 23 dogs, bovine serum lbumin (BA) nd rbbit ntiba soluble I lbeled with 125J were given by constnt iv. infusion, nd I uptke by normlly perfused kidney ws compred to tht of the contrlterl kidney in which renl blood flow (RBF) ws chnged by renl rtery constriction or rised ureterl pressure. In these sme nimls, I uptke in 15 other mjor orgn systems ws lso mesured simultneously. During I infusion microspheres of 85r were injected to mesure crdic output nd tissue blood flow, nd red cells lbeled with 51r were infused to mrk tissue vsculr volume. At completion of the I infusion, tissue smples were tken from the kidneys nd the 15 other mjor orgns systems. From the isotope content of ech tissue, we determined I content, blood flow rte, vsculr trnsit time, nd frctionl uptke of I (F1). In ddition, glomeruli were isolted from renl cortex to ssess I uptke in glomerulr versus renl nonglomerulr tissue. We found tht 1) for kidney, I delivery rte, cpillry hydrosttic pressure, nd cpillry ultrfiltrtion rte re less importnt thn the plsm I concentrtion in determining I uptke; 2) for ech orgn studied, the principl determinnt of I uptke per grm of tissue, t ny given P1, is vsculr volume per grm of tissue; 3) tissue vsculr volume per grm of tissue my determine I uptke per grm of tissue becuse tissue vsculr volume determines the cpillry surfce re in contct with circulting I or becuse tissue vsculr volume determines tissue vsculr trnsit time, t ny given tissue blood flow rte. Quelques determinnts hemodynmiques de I sequestrtion d'immuns-complexes pr le rein. e trvil été entrepris pour ider clrifier I reltion entre les phénomènes hémodynmiques cpillires et I cpttion tissulire des immuns complexes circulnts (I). hez 23 chiens des I solubles, mrquós pr 1251 de serum lbumine bovine (BA) et nti-ba de lpin, ont éte dministrés pr perfusion intrveineuse debit constnt et I cpttion d'l pr un rein normlement perfuse été cornprée celle du rein controltérl dont le debit snguin (RBF) té chnge pr I constriction de l'rtère ou l'ugmenttion de I pression urétérle. hez ces mêmes nimux I cpttion d'i pr d'utres orgnes été mesurée. Au cours de l'dministrtion Received for publiction December 29, 1977; nd in revised form April 6, / $ by the Interntionl ociety of Nephrology. d'i des microsphéres de 85r ont eté injectees pour mesurer les debits crdique et tissulires et des hémties mrquees pr 51r ont été dministrées pour évluer le volume vsculire. A I fin de l'dministrtion d'i des échntillons de tissu renl et de 15 utres orgnes importnts ont été prélevés, A prtir des rdioctivités de chque tissu, nous vons déterminé Ic contenu en I, le debit snguin, le temps de trnsit vsculire, et I cpttion frctionnelle d'i (F1). De plus, des glomerules ont ete isolés du cortex renl fin de comprer I cpttion pr les tissus glomerulire et non glomerulires. Nous vons constté que 1) pour le rein Ic debit d'i délivré, I pression hydrosttique cpilire et le debit d'ultrfiltrtion cpillire sont moms importnts que I concentrtion plsmtique d'i dns le déterminisme de I cpttion d'i; 2) pour chque orgne étudié Ic principl déterminnt de I cpttion d'i pr grmme de tissu, pour une P1c donnée, est le volume vsculire pr grmme de tissu; 3) le volume vsculire pr grmme de tissu peut determiner I cpttion d'i pr grmme de tissu puisque le volume vsculire determine I surfce cpillire en contct vec l'i circulnt ou bien prce que Ic volume vsculire du tissu determine le temps de trnsit vsculire pour chque vleur du debit snguin. The physicl nd biochemicl events which led to the trpping of circulting immune complexes (I) re, t present, only poorly understood. It hs been suggested tht high cpillry ultrfiltrtion rte, s occurs nturlly in the glomerulus [1] or s might be induced by relese of vsoctive mines from pltelets or mst cells, is n importnt determinnt of I-trpping [2 4]. Enhnced cpillry ultrfiltrtion rte cuses the plsm concentrtion of nonfilterble solutes to increse t the site of ultrfiltrtion nd cuses the cpillry flow velocity to diminish. These effects of ultrfiltrtion increse the concentrtion of I in contct with cpillry endothelium nd prolong contct time between circulting I nd endothelium. These events, singly or in combintion, might increse the rte t which circulting I re trpped by cpillry endothelium. If this hypothesis is correct, it follows tht plsm I concentrtion nd cpillry hemodynmic events, such s hydrosttic pressure, flow rte, nd tissue vsculr trnsit time, ech of which cn f- 452

2 Determinnts of immune complex trpping 453 fect either cpillry ultrfiltrtion rte or contct time of I with endothelil cells, might be importnt determinnts of tissue uptke of circulting I. The present study exmines severl spects of this problem by ssessing the reltionship between rteril plsm I concentrtion, cpillry hemodynmic events, nd I-trpping by the kidney nd other orgns. Methods Preprtion of i/n mnune coin plexes rystllized bovine serum lbumin (BA; Miles Lbortory) ws lbeled with iodine 125 (1251) s previously described [5]. Lbeling efficiency ws bout 45%, nd specific ctivity rnged from 2 to 3 Mi/mg. AntiBA ntibody ws produced by immunizing New Zelnd rbbits, s previously described [5]. The ser were tested for ntibody concentrtions by the quntittive precipitin method. I were formed by combining the rbbit ntiserum with '251-BA t equivlence nd incubting for 1 hr t 37 nd then t 48 hr t 4. The resulting precipitte ws centrifuged, wshed, nd then incubted with.1 M phosphte buffered (ph, 7.).9% sline (PB) to which five times the ntigen excess ws dded. The solution ws stirred for 3 mm t room temperture, then centrifuged t x 1,g for 1 mm nd the precipitte discrded. About one hlf of I precipitte ws solubilized by this method. Gel filtrtion on ephdex G-2 showed tht 7 to 9% (men, 82%; N = 5) of 125I. BA in this solution ws incorported into single dominnt pek composed of complexes 19. The reminder of the 1251-BA ws uncomplexed. Merthiolte (1/1,) ws dded to ll btches to retrd bcteril growth. The I were stored t 4 nd used within 2 weeks. BA-rbbit ntiba I produced in this fshion re biologiclly ctive, s shown by their bility to fix complement nd increse cpillry permebility nd by their pttern of systemic uptke [51. urgiclpreprtion Twenty-three mongrel dogs weighing 5 to 15 kg were deprived of food nd given sodium iodide in drinking wter pproximtely 2 hr prior to study. They were nesthesized with i.v. pentobrbitl (3 mg/kg) nd given dditionl smll doses s required to mintin constnt level of nesthesi. An endotrchel tube ws inserted nd connected to constnt-volume positive-pressure ventiltor. theteriztion of the following blood vessels ws performed: right brchil vein (for constnt infusion of I solution); right femorl vein to superior ven cv (to mesure concentrtion of I delivered to the lungs); left femorl vein (for constnt infusion of norml sline t 4 ml/min nd cretinine to mesure cretinine clernce; this infusion ws begun t the strt of the surgicl preprtion); left femorl rtery (to mesure blood pressure); right femorl rtery (to obtin rteril blood smples); nd left crotid rtery (to ctheterize left ventricle for injection of microspheres [strontium 85 (85r), 15 tm, 3-M ompny, t. Pul, Minn.]). Both kidneys were exposed by midline bdominl incision nd freed of their perirenl ttchments, nd both ureters were cnnulted. In 16 experiments, Bllock clmp ws loosely plced round either the right or left renl rtery in preprtion for ppliction lter in the experiment. In four experiments, renl interstitil fluid ws collected from the subcpsulr spce from both kidneys to ssess for entry of iodine 125 (1251) into the interstitil spce [5]. Experimentl protocol About 45 mm before strting the experiment proper, bout 2 ml of blood ws removed, nd the red blood cells (RB) were lbeled with 3 i of chromium 51 (51r). ontrol period. The orifices of both ureterl ctheters were fixed t the level of the superior surfces of the kidney. This ws defined s ureterl pressure (UP) = mm Hg. Two 15-mirL periods of urine collection from both kidneys were begun 3 mm before the I infusion to mesure cretinine clernce. In the 16 experiments in which RBF ws chnged by mnipulting renl perfusion pressure, the Bllock clmp ws then djusted until urine flow rte from the experimentl kidney ws pproximtely one hlf tht observed in the contrlterl control kidney. The men urine flow rte from the control kidney during the control period ws mllmin. After estblishing stedy-stte urine flow rtes (15 to 3 mm of djustment), the experimentl period ws begun. In those experiments in which RBF ws chnged by rising UP, the orifice of the ureterl ctheter of either the right or left kidney ws rised to 42 cm bove the superior surfce of tht kidney so tht when the ctheter ws completely filled with urine, UP = 3 mm Hg. After stedy-stte urine flow rtes were estblished (15 to 3 mm), the experimentl period ws begun. Experimentl period. The I infusion ws given s follows: Ech niml received 1 to 2 /Li of I (6 to 8 mg of protein) in 3 ml of PB. Approximte-

3 454 Hehert! cii ly one third of the I solution ws infused over 5 mm; the reminder ws given over the next 55 mm by constnt infusion pump. Twenty minutes before completing the I infusion, the 5tr-RB were infused. The microspheres (1 to 3 i) were suspended by ultrsonic mixing in 5 ml of sline to which 1 drop of Tween-8 ws dded, nd they were infused s follows: one third of the microspheres were given by bolus infusion t 1, 3, nd 5 mm, during the I infusion. Arteril smples were obtined during the 1- nd 5-mm smples to mesure crdic output. At the conclusion of the I infusion, the nimls were killed, nd the following tissue smples were tken for nlysis: renl cortex (Three midcoronl sections bout.5-cm thick were tken from both kidneys, From ech coronl section, two cubes of full-thickness cortex bout 1-cm wide were cut. From ech section, one of the cubes ws divided into outer nd inner cortex nd tken for isotopecounting, nd the other cube ws tken for isoltion of glomeruli by sieving techniques [5, 6].), brin (frontl cortex), choroidplexus, thymus, lrge bowel, smll bowel, stomch, ft (peritonel), diphrgm, skin (bdominl), gonds (ovries or testes), drenl glnd, hert (ventricle), spleen, liver, nd lung. mples weighed 2 to 6 mg, except for choroid plexus where the entire orgn ws tken for nlysis. The entire weight of lung, spleen, stomch, lrge nd smll bowel were lso mesured so tht frctionl uptke of I (F1) in lung nd liver could be clculted, s discussed below. pecilprotocol To ssess whether the tissue distribution of 1251 observed fter the infusion of the '251-BA-rbbit ntiba I might be importntly ffected by n bnorml sequestrtion of free '251-BA in tissues, in eight seprte experiments we exmined the stedy-stte distribution of 1251-BA in liver, spleen, nd kidney. '251-BA (3 to 5 Mi) ws infused i.v. nd llowed to circulte for 2 hr. At tht time, smples of liver nd spleen were obtined by clmping the edge of the orgn, incising the tissue, nd llowing it to fll directly into liquid nitrogen to freeze the tissue quickly nd thereby prevent fluid loss. Kidney ws removed without fluid loss by clmping the hilr structures prior to removl. The kidney ws then frozen. The 1251 concentrtion of tissue nd simultneously obtined plsm smples were then determined nd the tissue-to-plsm 1251 rtios clculted. Anlyticl methods Rdioisotope counting ws done in refrigerted utomtic Pckrd dul chnnel gmm counter. ounts for ech isotope were corrected for the presence of the other isotopes by using pproprite correction fctors nd simultneous equtions, which were solved by progrm dpted to Monroe 186 desk top computer. Protein concentrtions of the I preprtions were done by the Lowry method. Other nlyticl techniques re s previously reported [71. /c,, I tioil Frction of glomeruli isolted from smple of cortex. The frction of glomeruli isolted from smple of cortex [5, 6] = (85r cpm in glomerulr isolte/g of cortex from which glomeruli isolted)! (85r epinig of comprble smple of cortex), where cpm = counts per mm. I content of tissue. The I content of tissue (Qic) = totl 1251 cpmlg of whole tissue vsculr spce correction. Vsculr spce correction. The vsculr spce correctio& = [51rcpm!g of tissue]. [(1251 cpmlg of blood)/(51r cpmlg of blood)]. rdic output. The crdic output (O) = r cpin injected/i(85r cp,nlg of blood) x t] = g!min, where 85r cpm!g of blood is determined from n rteril blood smple withdrwn by constnt withdrwl pump t the rte of 1 mi/mm for time t (in these experiments, t = 6 sec) beginning 1 sec before the microsphere infusion, which ws given over pproximtely 1 sec. Frctionl uptke of im,nune complexes. The frctionl uptke of immune complexes (F1) = Q1I (Pw x O x R), where Pw = men rteril plsm I concentrtion. For ech experiment, this ws clculted by determining the re under the curve of plot of plsm 1251 cpm!g vs. time nd dividing this vlue by the time intervl. R = the rtio [(85r cp,n/g of tissue smple)/85r cpm injected]. To clculte F1 of lung, c ws mesured from superior ven cv blood, R ws ssigned vlue of 1., nd O ws divided by totl lung weight. These modifictions of the eqution re necessry becuse the lung receives immune complexes t slightly higher concentrtion thn the rest of the body does nd becuse microspheres injected rterilly cnnot be used to mesure lung blood The vsculr correction ssumes tht the rteril hemtocrit is the sme s the introrgri hemtocrit. In fct, the intrrenl hemtocrit (renl cortex red cell volume/renl cortex blood volume) nd the whole body hemtocrit (whole body red cell volume/whole body blood volume) re both bout 9 / of the rteril hemtocrit [8, 9]. This smll difference between lrge vessel nd smll vessel hemtocrit hs no importnt effect on the vsculr correction nd, therefore, ws ignored.

4 Determinnts of immune co,np/ex trpping 455 flow becuse the microspheres re removed en route to the lungs. Lung, however, receives the entire crdic output. Knowing these vribles permits clcultion of F1 by lung [6]. To clculte the F1 of liver, the totl microsphere (in counts per mm) in spleen, stomch, lrge nd smll intestine re dded to the totl microsphere (counts per mm) of liver. This modifiction of the eqution is necessry becuse the microspheres mesure only heptic rtery flow. The reminder of blood flow to liver is vi the portl vein which receives venous return from gut nd spleen. Thus, microspheres in these splnchnic orgns lso represent blood flow to liver. F1 clculted s bove is n pprent frctionl uptke of I, since the frction of rteril plsm 1251 counts which represent I tht re susceptible to trpping is less thn the ctul 125j plsm concentrtion nd my be less thn the ctul rteril plsm concentrtion of I s ssessed by gel filtrtion, since not ll I my be susceptible to trpping [11. By the end of the I infusion, only one third to one hlf of the 1251 counts in rteril plsm represent I (rnge of three observtions), wheres initilly s much s 9% of the 1251 counts represent I. Thus, the true F1 is greter thn F1 clculted s bove. The F1 rtio (F1 of given tissue/f1 control kidney outer cortex, however, should be resonble estimte of the true F1 rtio since the term P1 ppers in both numertor nd denomintor nd therefore cncels from the rtio. The F1 rtio for liver is n underestimte since the liver receives I t lower concentrtion thn other orgns do, becuse bout three fourths of heptic blood flow is vi the portl vein [11] nd some of the I hve been removed from the blood during pssge through gut nd spleen. Nevertheless, portl blood flow is lrgely from gut [11], which, s will be shown, hs reltively low F1. Thus, the liver F1 rtio, lthough n underestimte, is resonble index of the true liver Fi( rtio. Tissue vsculr trnsit time. Tissue vsculr trnsit time (TT) = V/F, where V = frction of tissue volume which is vsculr volume, vsculr volume is clculted from the 51r-RB spce in tissue, s described bove, nd F = blood flow per grm of tissue = O x R. TT clculted s bove is not true tissue vsculr trnsit time since TT is clculted from tissues in which blood ws llowed to drin spontneously. Nevertheless, the vsculr volume of these excised tissues is proportionl to the vsculr volume of these sme tissues in vivo, s shown in Fig. 1. Furthermore, the mesurement of tissue blood flow with the microsphere technique is vlid under the hemodynmic conditions imposed by immune complex infusion, since we found no evidence of excessive shunting of microspheres through the systemic vsculr circuits. This ws shown by the fct tht only 2.8.5% of the microspheres injected into the left ventricle ws found in lung, nd over one third of this microsphere rdioctivity cn be ccounted for by bronchil rtery U >< 3 Liver Lung E > E > 5 Y = O.62x P < Previous studies: men tissue vsculr volume (in vivo estimtes), % Fig. 1. Reltionship between tissue vsculr volume of excised tissues nd the vsculr volume oft/me sme tissues, in vivo. The men vlues of vsculr volume for excised tissues re from the present study. The in vivo vlues re men vlues previously reported in the literture: brin [12], hert [131, kidney [14], liver [151, nd lung [16]. The vsculr volume for brin is bsed on studies in mn. All of the other vsculr volumes re bsed on studies in the doe.

5 456 Hebert et! blood flow [11]. The vlidity of the microsphere techniques in the present experimentl setting is lso supported by comprison of previously reported microsphere dt on tissue blood flow in the dog [11] with the men vlues of the present study. This comprison showed excellent greement (r =.97, P <.1). Thus TT clculted s bove is resonble index of the true tissue vsculr trnsit time. Results All men vlues re shown 1 EM. vste,nic uptke of immune complexes. I were infused by giving one third over 5-mm period s loding dose nd two thirds over the next 55 mm by constnt infusion. On the verge, this chieved nerly stedy-stte conditions in the vsculr comprtment, since for ll experiments the men rtio of the 125J counts per grm of blood t 5 mm, to the 125J counts per grm of blood t the completion of the loding dose (5 mm) ws Virtully ll (> 98%) of the 125J in plsm obtined t the end of the experiment ws trichlorcetic cid precipitble, nd < 2% of the 125j infused ws excreted in the urine during the experiments (observtions from three rndomly selected experiments). Thus, there ws no evidence of importnt loss of the 125! from the lbeled proteins. We cn conclude, therefore, tht during the period of constnt infusion, lbeled proteins were leving the vsculr spce t rte lmost equl to the rte of infusion. ince 7 to 9% of the 1251 infused represented lbeled I, it follows tht lmost ll of the lbeled proteins leving the vsculr spce during the I infusion represented the systemic uptke of I. Finlly, since two thirds of the I solution ws given during the period of constnt infusion, it follows tht bout two thirds of the I infused were tken up systemiclly. Tble 1 shows the pttern of systemic uptke of the infused I. The dt in Tble 1 were derived s follows: The rtio of the 125j counts per grm of given tissue (corrected for the vsculr spce 125! counts) to the 1251 counts per grm of control kidney outer cortex (corrected for vsculr spce 1251 counts) of the sme niml ws determined for ech tissue in ech niml. The vlues shown in Tble 1 re the men of the individul rtios for ech orgn. The dt re expressed s the rtio of 125Jcontent in given orgn to tht of renl cortex of the sme niml, rther thn s the bsolute 1251 counts per grm of ech tissue, becuse the dose of complex infused vried from niml to niml. This ws unvoidble becuse, lthough the specific ctivity of the I mixture ws known, the percent of the 1251 BA incorported into complexes declined with time of storge (bout 5 to 15% per week, rnge of three observtions), nd it ws not possible to perform gel filtrtion nlysis prior to ech experiment. The tissue distribution of 125j shown in Tble I, is not importntly ffected by ctbolism of the trpped immune complexes with relese of the 125! lbel into the extrcellulr fluid. If this process hd occurred to ny significnt extent, the 125! would hve to be either in blood or urine (the nimls were given sodium iodide prior to the experiments; thus, free lj ws not tken up by the thyroid glnd). As discussed bove, however, only trivil mounts of free 1251 were found in blood nd urine. It is lso unlikely tht the tissue distribution of 125j shown in Tble 1 is importntly ffected by bnorml tissue retention of '251-BA. This is shown by the specil protocol" studies exmining the stedy stte tissue! plsm distribution of 1251-BA in the orgns principlly responsible for the clernce of I from plsm. From these experiments, the following men rtios for 125J were obtined: renl cortex! plsm =.27.2, liver/plsm.32.3, nd spleen/plsm These tissue-to-plsm rtios re those to be expected from the pproprite distribution of lbumin in the tissue vsculr nd interstitil spce. These rtios contrst shrply with the corresponding tissue-to-plsm rtios for 1251 mesured t 1 hr following the infusion of the 125j BA-rbbit nti-ba I. These rtios were: renl outer cortex/plsm =.9.14, liver/plsm = , nd spleen/plsm = , These rtios re mny fold higher thn those observed for the tissue-to-plsm distribution of 125j. BA ( protein tht does not deposit in tissue) nd, thus, indicte the presence of 1251-lbeled I which hve been trpped nd re tissue-bound (fixed I [5]). The dt in Tble 1 show tht the kidney is mjor site of I uptke since only liver, spleen, nd lung hd higher concentrtions. The findings in Tble 1 re consistent with previous observtions on the pttern of systemic uptke of biologiclly ctive I [5, 1, Tble 2 shows the men of the individul F1( rtios (F1 of given tissue/f1 of control kidney outer cortex of the sme niml) for ll tissues studied. As cn be seen, most tissues hve F1 rtios greter thn 1.. Tht is, per unit of blood flow, these tissues trp more I thn does control kidney outer cortex. The men vlue for F1c for control kidney outer cortex from ll experiments ws For lung, it ws possible to directly ssess the F1 since the 1251 content of both superior ven cv nd

6 Frontl brin Determinnts of immune complex trpping 457 Tble 1. Rtio of 1251 in given tissue to tht of renl outer cortex, Tissues with men 125J countslg rtio significntly less thn 1. (lt P < horoid plexus Hert Adrenl.1) Thymus.18.2 Lrge bowel N=18 N=17 N=17 N=15 N=l4 N=22 mll bowel tomch Ft Diphrgm kin Gonds.2.1 N= N=22 N=15 N=18 Tissues with 125J counisig rtio significntly greter thn 1. Lung Liver pleen N=22 N=23 N=23 P <.1 P <.1 P <.1 The 1251 counts of ech tissue is corrected for the 125J counts in the vsculr spce. i N = number of experiments in which mesurements were mde N= N=17 rteril blood ws mesured during the I infusion. We found smll, but insignificnt grdient for 125J cross the lungs [( ] x lo vs. ( x los, cpm, P >.5]. This outcome could hve been expected for the following resons: 1) Lung F, is reltively smll (Tble 2), nd it is difficult to detect ccurtely smll differences between two lrge numbers (the 1251 counts in superior ven cv nd rteril blood), 2) I probbly re not dequtely mixed in superior ven cv blood since the I re infused only reltively short distnce below the superior ven cv. The bove exmple lso serves to emphsize the difficulty in mesuring ccurtely the F1 by me- suring the grdient of 125J cross given orgn since, like lung, most orgns hve low F1. At the sme time, the bove exmple underscores the vlue of the present method for estimting F1. Tht is, lthough the 125! grdient cross n orgn my be difficult to mesure, the 125J trpped in tissue [corrected for nonfixed 125J [5] ] divided by the tissue delivery rte of 125j, is reltively esy to mesure. Effect of reduced renl blood flow on immune complex uptke by the kidney. Figs. 2 nd 3 show for outer nd inner cortex, respectively, the reltionship between the rtio of RBF (RBF experimentl kidney/rbf control kidney) nd the rtio of I uptke (I uptke of experimentl kidney cortex/i Tble 2. Rtio of the frctionl uptke of immune complexes (F,) in given tissue to the frctionl uptke of immune complexes in renl outer cortex Liver P <.1 N = 2 tomch 13.6± 1.98 P <.1 N = 19 pleen P <.1 N = 21 Ft P <.1 N = 13 Tissues with men F1 rtios significntly greter thn 1. Thymus P <.1 N = 12 Diphrgm P <.1 N= II Tissues wit!, men F, rtios significntly less thn 1. Frontl horoid brin plexus P <.1 P <.1 N=17 N=14 Lrge bowel P <.2 N = 19 kin P <.1 N = 13 Tissues with men F, rtios not significntly different from 1. Hert Lung Adrenl <P<.1 P>.9.1<P<.2 N=14 N=23 N=14 N = number of experiments in which mesurements were mde. mll bowel P <.1 N = 22 Gonds P <.1 N 16

7 458 1-Jebert et l 5- -o c 5. ) -.2) ( ). x ) Outer cortex Experimentl kidney renl rtery constriction o Experimentl kidney elevted ureterl pressure :777:72 I I I I I I I I I mr counts/q rtio )experiment) kidney/control kidney) Fig. 2. Effect of chnges in renl outer corticl blood flow on renl outer corticl immune complex uptke. Ech experiment is represented by single point or by pir of points which re joined by n rrow (s discussed in Text). The ttr rtio represents the rtio of the renl blood flow in the experimentl versus the control kidney. The 1251 rtio (corrected for vsculr 125J counts) represents the rtio of immune complex uptke in the experimentl versus control kidney. uptke of control kidney cortex). Ech experiment is represented by single point.2 As cn be seen, in virtully ll experiments with 85r rtios < 1. the 2 This ws true except for four experiments in which renl interstitil fluid ws collected simultneously from the experimentl nd control kidney to correct corticl 129 counts for entry of diffusible t25j (s lbeled ntigen, I or free 1251) intothe corticl interstitil spce. The entry of these lbeled molecules into the interstitil spce results in n overestimte of the mount of I which hs been trpped nd fixed to renl cortex nd, therefore, cpble of inducing tissue dmge. The four experiments in which renl interstitil fluid ws collected re identified by the points joined by n rrow. The rrow indicted the mgnitude nd direction of the chnge in the 125J rtio s result of the correction for entry of diffusible 125J into the interstitil spce. As cn be seen, in the present experimentl setting, the 1251 rtios re not gretly ffected by this correction. Furthermore, s in our previous report, the interstitil spce correction ws reltively smll (men c 15% of totl 1251 content of tissue). ince we previously found tht the vsculr nd interstitil spce corrections for 1251 re pproximtely equl [5], we cn conclude tht, on the verge, in the present study bout 85% of the 125J counts of renl cortex (corrected for vsculr spce 125J counts) represents I which re trpped by renl cortex nd tissue bound. Y =.6: r =.789 <.1 ) 5- t E - 2< ) Inner cortex: Experimentl kidney renl rtery constriction o Experimentl kidney elevted ureterl pressure y =.53 x =.548 p<.2.2. I I I I I I I I I r counts/g rtio )experimentl kidney/control kidney) Fig. 3. Effect of chnges in renl inner corticl blood flow on renl inner corticl immune complex uptke. The conventions used re the sme s in Fig. 2. I uptke of the experimentl kidney cortex is less thn the simultneous I uptke of the contrlterl control kidney cortex. The regression lines drwn through these points show slopes significntly different from. for both outer nd inner cortex. For experiments with 55r rtios greter thn 1., the dt re indequte in mount for nlysis. In both the control nd experimentl kidney, outer cortex ppered to trp I in higher concentrtions thn did inner cortex. The men of the individul rtios: (t25i cpm/g of outer cortex)/(t551 cpm/g of inner cortex) for control nd experimentl kidneys ws the sme (1.16.4). Both rtios re significntly greter thn 1. (P <.1 for both). This interprettion, however, ssumes tht the interstitil correction for t25j (see footnote 2) for inner nd outer cortex is similr. Although I uptke diminished s RBF ws reduced, uptke of I fell proportiontely less thn did RBF (nd delivery rte of I). Thus, s shown in Fig. 4, Ftc in renl cortex incresed s RBF ws decresed. Fig. 4 shows the reltionship between the rtio of RBF (RBF of experimentl kidney/rb F of control kidney) nd the rtio of Fic (Ftc of experimentl kidney cortex/f1 of control kidney cortex).

8 Determinnts of immune complex trpping 459 > ) x 5) 5) ) ) '5 E E E U Experimentl kidney renl rtery constriction: outer cortex inner ortex Experimentl kidney rised ureterl pressure: outer ortex inner ortex A 'A Ȧ Log Y.46 x +.46 r.77 p <.1 o r counts/g rtio (cortex, experimentl kidney/cortex, control kidney) Fig. 4. Reltionship between the rtio of renl blood flow in the experimentl versus control kidney nd the rtio of frctionl uptke of immune complexes in the experimentl versus control kidney. Vlues for both outer nd inner cortex re shown from 21 experiments. The dshed line indictes the chnge in F1 which would hve to occur in the experimentl kidney to mintin immune complex uptke in the experimentl kidney t the sme level s the normlly perfused contrlterl control kidney. The vlues for both outer nd inner cortex re shown. The increse in renl cortex F1 with decresing RBF could hve been the result of prolongtion of TT. It ws not possible to test this hypothesis for renl cortex becuse of the reltively smll rnge of F1 observed in the kidney nd the imprecision in the method used to mesure TT. When ll tissues were considered together, however, wide rnge of vlues for F1 nd TT were observed. These re compred in Fig. 5, which shows the men vlues for TT nd F1 for ech orgn exmined. As cn be seen, high degree of correltion ws found. ince the term, tissue blood flow, ppers, however, in the denomintor of both the expression for TT nd F1, the fct tht the two vribles re correlted indictes only tht cuse-nd-effect reltionship hs not been excluded by this nlysis. A second possible explntion for the rise in renl cortex F1 s RBF ws reduced (i.e., the reltive A A A stbility of renl I uptke in the fce of decresing I delivery rte) is tht the rte of I-trpping my be determined lrgely by the concentrtion of I in contct with cpillry endothelium. To ssess this possibility, in ech experiment we exmined the reltionship between the men rteril plsm 1251 concentrtion during the I infusion (n index of the men plsm I concentrtion [P1] during I infusion) nd 1251 cpm per grm of control kidney cortex (corrected for 1251 counts in vsculr spce). This reltionship is shown in Fig. 6. As cn be seen, in generl, the higher the P, the greter the mount of I trpped by cortex. The sme pproximte liner reltionship ws observed for ll orgns (r>.8 for ll orgns); of course, the slope of the line describing this reltionship, however, vried from orgn to orgn ccording to I content per grm of tissue. Those tissues with high concentrtions of 1251 exhibited steeper slopes thn did those with low concentrtions of 125J The reson for the differences from orgn to orgn in the mount of I trpped per grm of tissue t ny given rteril P1 ppers to be relted to differences in the cpillry mss per grm of tissue. This is suggested by the reltionship shown in Fig. 7 in which, for ech tissue, the men rtio of 1251 counts per grm of the given tissue to tht of renl outer cortex (dt of Tble 1) is shown in reltionship to the men tissue vsculr volume (n index of cpillry mss) for ech orgn. As cn be seen, in generl, the greter the cpillry mss per grm of tissue, the greter is the mount of I trpped per grm of tissue. This nlysis suggests tht under condition of norml tissue perfusion the mount of I trpped per grm of tissue, t ny given P1, is determined by the cpillry surfce re in contct with the circulting I. The reltionship, however, between tissue vsculr volume nd I trpped per grm of tissue, shown in Fig. 7, could lso be explined by the reltionship demonstrted between TT nd F1 (Fig. 5). This cn be shown s follows: From Fig. 7: I uptke/g of tissue = k V (1) where k is the proportionlity constnt nd V is vsculr volume per grm of tissue. ince I uptke per grm tissue = F >< F1, where F =blood flow per grm of tissue, it follows tht F X F1 = k V 2) Rerrnging Eqution 2, F1 = k V/F but (3) V/F=TT (4)

9 46 1-lebert et! ). 6 ) E E.1-5).5 RO-EK LB RI-EK B RI-EK R K Lu.1 H F t 'K p eg Di 'Li y =.62 x +.37 r.9 p < Tissue vsculr trnsit time, loin Fig. 5. Reltionship between tissue vsculr trnsit time nd the frctionl uptke of immune complexes. Note tht the bciss scle is broken to ccommodte the vlues for thymus. The vlue of thymus ws not included in the clcultion of the regression line. The tissues re identified s follows: drenl glnd (A), brin-frontl cortex (B), choroid plexus (P), diphrgm (D), ft (F), gonds (U), hert (H), lrge bowel (LB), liver (L), lung (Lu), renl outer cortex of experimentl kidney (RO-EK), renl outer cortex of control kidney (RO-K), renl inner cortex of experimentl kidney (RI-EK), renl inner cortex of control kidney (RI-K), skin (k), smll bowel (B), spleen (p), stomch (t), thymus (T). see Methods. ombining Eqution 3 nd 4, we obtm, F1 = k TT This is the reltionship demonstrted in Fig. 5. Thus, the reltionship between F1 nd TT demonstrted in Fig. 5 follows from the reltionship between I uptke nd V demonstrted in Fig. 7. This nlysis, however, does not estblish which reltionship is primry. Tht is, t ny given P1 nd tissue blood flow rte, does V determine I uptke becuse V is proportionl to cpillry surfce re or becuse V determines TT? Glomerulr versus renl non glomerulr uptke of immune complexes. To ssess the site of I uptke within the kidney, in 19 experiments glomeruli were isolted from both the control nd experimentl kidney. The men recovery of glomeruli from given corticl smple ws 24..8% in control kidneys nd % in experimentl kidneys. The percent of corticl 1251 found in the glomerulr isoltes in the control versus the experimentl kidneys ws.54.2% vs %. Thus, s we hve previously observed, during cute infusions of I or I-like mcromolecules, the vst mjority of I trpped by the kidney re trpped in nonglomerulr sites [5, 6]. Hemodynmic events during immune complex infusion. Blood pressure fell significntly during the I infusion from men vlue of 13 5 mm Hg during the first one third of the I infusion to men vlue 76 4 mm Hg during the lst one third of the I infusion (P <.1). During I infusion, crdic output incresed modestly, but significntly,from g/min t 1 mm to 1,32 l4ogi mm t 5 mm. Thus, the decrese in systemic blood pressure ws the result of fll in peripherl resistnce. Other investigtors [2, 21] nd we ourselves [5, 6] hve previously noted fll in systemic blood pressure during the infusion of biologiclly ctive complexes. Presumbly, the fll in systemic blood pressure is result of I-induced relese of vsoctive substnces. Glomerulr filtrtion ws not significntly different in the control versus experimentl kidney priorto I infusion ( vs. 2.2 l.9mllmin). During the I infusion (fter rising ureterl pressure or lowering renl perfusion pressure, in the experimentl kidney), GFR ws significntly lower in the experimentl kidney versus the control kidney ( vs , P <.1). Discussion In the present study, biologiclly ctive I were infused i.v. under conditions in which RBF ws chnged in one kidney (the experimentl kidney) by constricting the renl rtery or rising ureterl pressure. I uptke by the experimentl kidney ws compred to the simultneously determined I uptke by the contrlterl (control) kidney, nd the reltionship between the observed chnges in RBF nd renl I uptke ws exmined. The present study ppers to be vlid ssessment of the effect of RBF reductions on renl I uptke for the following resons: 1) The dose of I used ws fr below the concentrtions which re thought to cuse sturtion of the reticuloendothehl system [19] nd pprently below the concentrtions which cuse sturtion in other tissues, since we found tht the tissue I content for ll tissues studied, including kidney cortex (Fig. 6), incresed progressively with incresing P'. Thus, we ssume tht in the present study the effect of RBF (delivery rte of I) on I uptke ws not obscured becuse

10 Determinnts of immune complex trpping 461 '1 x ontrol kidney: outer cortex inner cortex ) ' Y = 166 x 26,4 r =.93 P <.1 U 1 4 Men plsm 1!,counts/2Omin/g X 1U Fig. 6. Reltionship between the plsm 125J concentrtion, n index of plsm immune complex concentrtion, nd renl cortex 125J counts/g of tissue (corrected for vsculr spce 125J counts), n index of immune complex uptke by renl cortex. tissue binding sites for I were sturted. 2) The I were infused for only 1 hr. The renl ctbolism of trpped I is probbly miniml over this time period [5, 221; thus, t the end of 1-hr I infusion, the tissue I content is determined principlly by I uptke. 3) The present pproch uses vlid mens for estimting tissue I content, since we hve previously shown tht the I content of renl cortex, clculted s totl 125J cprnlg of tissue minus the vsculr 125J cpm/g of tissue, is close estimte of the mount of I which hs been trpped nd tissue-bound [51. 4) The microsphere method vlidly mesures tissue blood flow under conditions of I infusion, s discussed in the Methods section. ).9 4. ' 3. p Li ) 2. Lu > 1. LB Y = 11.1 x.16 r =.84 P <.1 B.1 I.2 Tissue vsculr volume, g/g of tissue.3 Fig. 7. Reltionship between tissue vsculr volume nd immune complex uptke per grm of tissue for ech of the mjor orgn systems studied. Note tht the ordinte scle is broken to ccommodte the vlues for liver nd spleen.

11 462 Hebert er! The present study shows tht reductions in RBF result in decresed renl I uptke. The effect ws similr whether RBF ws reduced by renl rtery constriction or by ureterl pressure elevtion (Figs. 2 nd 3). ince these mneuvers hve opposite effects on cpillry hydrosttic pressure3, it seems likely tht cpillry hydrosttic pressure, itself, is not criticl determinnt of I-trpping. Although renl I uptke decresed with decresing RBF, the fll in I uptke ws proportiontely less thn the fll in I delivery rte to the kidney. Thus, the mount of I trpped per unit of blood flow rose, nd, s consequence, renl F1 incresed. Indeed, for those experiments in which only moderte reductions in RBF occurred, the increse in F1 in the experimentl kidney ws sufficient to ccount for n I uptke in the experimentl kidney which ws only slightly less thn tht of the contrlterl control kidney. The increse in F1 with decresing RBF could hve been due to chnge in cpillry hemodynmics in which, s renl blood flow decresed, TT ws prolonged resulting in incresed contct time between circulting I nd cpillry endothehum. In ddition, it hs been shown tht decresing glomerulr cpillry flow rte increses the frction of mcromolecules trnsferred cross the glomerulus, pprently by incresing time for mcromolecule diffusion 26II. These possible effects of decresed RBF might hve led to the observed increse in renl F1. It ws not, however, possible to evlute this question ccurtely for kidney, since, s discussed in Methods, the method used to clculte TT does not relibly detect smll chnges in TT. It ws possible, however, to evlute the reltionship between F1 nd TT over wide rnge of vlues by exmining this reltionship for ll tissues studied. As shown in Fig. 5, there ws strong correltion between TT nd F1. This correltion, however, cn only be interpreted s evidence tht TT cnnot be discrded s possible determinnt of F1. The fct tht F1 nd TT re correlted could be cusl reltionship or could reflect entirely the fct tht the term "tissue blood flow" is in the denomintor of We hve shown previously tht renl rtery constriction nd ureterl pressure elevtion of the mgnitude used in this study chnge renl subcpsulr pressure (RP), n index of pentubulr cpillry pressure [23], significntly. During renl rtery constriction the men decrese in RP ws 5 mm Hg During ureterl pressure elevtion, the men increse in RP ws +2 mm Hg 125]. both the expression for TT nd the expression for F1. Thus, even if there were no cusl reltionship between F1 nd TT, there is nturl tendency for the two vlues to chnge together s the term "tissue blood flow" chnges. A second possible explntion for the rise in F1 (nd the tendency of renl I uptke to remin nerly constnt) s RBF is diminished, is tht renl I uptke might be proportionl to the concentrtion of circulting I in contct with cpillry endothelium. If this were true, the rise in F1 with decresing RBF would not be evidence tht the vidity with which I re being trpped incresed with decresing RBF. Rther, the increse in F1 would simply be the mthemticl consequence of the fct tht, s RBF is reduced, I uptke is held nerly constnt (by nerly constnt introrgn P1) s I delivery rte is decresed. The hypothesis tht renl I uptke is principlly determined by rteril P1 could explin the observtion tht smll RBF reductions re ssocited with only smll reductions in renl I uptke, while lrger RBF reductions re ssocited with substntil reductions in renl I uptke. The rtionle is s follows: Under conditions of norml or ner norml renl perfusion, P1 is reltively constnt within the kidney, since only smll frction (probbly less thn 5%) of the 1 delivered to the kidney is removed in single pssge through the kidney. With modest decreses in RBF, only smll further reduction in P occurs within the kidney; thus, only smll decreses in I uptke re observed. With lrge reductions in RBF, however, the mount of I removed from renl plsm becomes higher frction of the I delivered; thus, greter intrrenl reduction in P1 occurs. Becuse of the greter intrrenl decline in Plc with lrge reductions in RBF, renl I uptke is decresed substntilly. Evidence supporting the hypothesis tht rteril P'c is n importnt determinnt of renl I uptke is provided by the demonstrtion tht I uptke per grm of renl cortex ws proportionl to the men rteril plsm 1251 concentrtion (n index of Pic, during the I infusion). A similr liner nd significnt reltionship ws observed for ll other orgns, however; of course, the slope of the reltionship between P1c nd tissue I concentrtion vried gretly from tissue to tissue ccording to the I content of the given tissue, s discussed in the Results section. The reson for the differences in I uptke per grm of tissue, t ny given P1, my be relted to differences in the cpillry mss per grm of tissue, since I uptke per grm of tissue nd tissue vscu-

12 tr volume (n index of cpillry mss) were correlted (Fig. 7). This suggests tht I uptke per grm of tissue, t ny given P, nd tissue blood flow rte re lrgely determined by the cpillry surfce re per grm of tissue in contct with the circulting I. As more rigorously discussed in Results, it is lso possible, however, tht the effect of vsculr volume per grm of tissue to ffect I uptke per grm of tissue is relted to the fct tht vsculr volume per grm of tissue determines TI, t ny given tissue blood flow rte. Thus, if contct time or diffusion time of I ffects I cpillry uptke, vsculr volume per grm of tissue could lso exert its effect on I uptke by determining TT, t ny given tissue blood flow rte.4 As cn be seen in Fig. 7 spleen nd liver hve especilly high I concentrtions for the level of vsculr volume. For these tissues, however, there is evidence tht tissue-bound mcrophges possess receptors for trpping I [19, 27]. This my increse the efficiency with which these orgns trp circulting I nd my ccount for the especilly high tissue I content t ny given P. The hypothesis tht, t ny given tissue blood flow rte, I uptke per grm of tissue in given orgn is lrgely determined by P, nd the vsculr mss of tht orgn, cn lso explin why norml glomeruli re not the mjor intrrenl site of uptke of i or Ic-like mcromolecules during cute i.v. infusions of these mcromolecules [5, 6]. Tht is, glomerulr cpillry volume is only bout one tenth to one fortieth of the peritubulr cpillry volume It should be mde cler tht tissues which differ with respect to TT do not necessrily differ with respect to cpillry flow velocity. This cn be demonstrted s follows: TT = V/F onsidering flow thru single tube of cross-sectionl re = A nd length = L, However, TT = (A)< L)/F flow velocity () = F/A ubstituting Eqution 8 into 7 yields: TT = LI Thus, in tissue with given vsculr volume nd blood flow rte per grm of tissue, TT could be prolonged becuse of low cpillry flow velocity (5) or becuse the cpillries hve norml flow velocity but the cpillries re longer (nd, therefore, more nrrow) thn norml. In either cse, TT would be prolonged nd would result in incresed contct between plsm I nd cpillry endothelium nd result in incresed time for diffusion of I into cpillry endothelium with incresed permebility. Determinnts of immune complex trpping 463 [28]; thus, if cpillry surfce re in contct with circulting I is n importnt determinnt of I uptke, glomeruli would be expected to be minor intrrenl site of I uptke. Differences in TT, however, could lso explin why the glomeruli re not the mjor intrrenl site of I uptke. Tht is, glomeruli hve the sme blood flow rte s the renl peritubulr cpillry system, but becuse of the much smller glomerulr vsculr volume, TT through glomeruli is much less thn IT through the peritubulr cpillry system. Thus, if contct time of I with cpillry endothelium is determinnt of I uptke, glomeruli would be expected to be minor intrrenl site of I uptke. Two importnt conclusions follow from the observtion tht glomeruli re not the mjor intrrenl site of I uptke during cute i.v. I infusion: 1) If glomeruli re the mjor intrrenl site of I uptke in glomerulonephritis, the glomeruli must first be ltered in some wy so tht glomeruli cn trp I more vidly. 2) cpillry ultrfiltrtion rte is not criticl determinnt of I uptke since glomerulr cpillries, which exhibit very high rtes of cpillry ultrfiltrtion [1], do not vidly trp I; but the peritubulr cpillry system, which exhibits no net ultrfiltrtion (i.e., these cpillries pprently undergo net uptke of fluid cross their entire length [29]), pprently trp lrge mounts of I. We do not suggest, however, tht cpillry ultrfiltrtion rte plys no role in the tissue uptke of I, since, ll other things being equl, n increse in cpillry ultrfiltrtion rte would loclly increse plsm I concentrtion nd increse the contct time of I with cpillry endothelium. These events, s discussed bove, might increse I uptke. The effect of decresed RBF to diminish renl i (6) uptke might explin the protection fforded ginst glomerulonephritis by conditions such s renl rtery stenosis or ureterl obstruction [3, 31]. Although those observtions suggest tht severe reductions in RBF re necessry to protect the kidney from I-medited injury, the findings of the present (8) study, tht even modest reductions in RBF result in decresed renl i uptke, suggest tht less severe reductions in RBF might lso exert protective effect ginst glomerulonephritis. Nevertheless, s mens to decrese renl I uptke, the present dt indicte tht lowering P1c would be fr more efficient thn would decresing RBF. By contrst, chnging tissue blood flow rte to liver or spleen might gretly ffect uptke by these orgns. For exmple, lthough I uptke is only

13 464 Hehert et! modertely flow-dependent in kidney (which hs low F1c, i.e., smll introrgn decrese in P1), in liver nd spleen (which hve high F1, i.e., lrge introrgn decrese in P1), I uptke might be highly flow-dependent. Tht is, incresing flow to these orgns might tend to mintin introrgn P, t higher levels (i.e., incresing blood flow might tend to minimize the introrgn decrese in P1), nd this might substntilly increse the mount of I clered by these orgns. Thus, chnging blood flow to liver or spleen might be mens for regulting the systemic uptke of I. In summry, the present study indictes tht P1 is key determinnt of I uptke by the kidney. By contrst, I delivery rte, cpillry hydrosttic pressure, nd ultrfiltrtion rte re less importnt. Tissues differ gretly with respect to I uptke per grm of tissue t ny given P1g. These differences from orgn to orgn pper to be lrgely due to differences in the vsculr volume per grm of tissue. The effect of vsculr volume per grm of tissue might be medited through the effects of vsculr volume to determine cpillry surfce re per grm of tissue in contct with plsm I or by the effect of vsculr volume to determine tissue vsculr trnsit time, t ny given tissue blood flow rte. Acknowledgments This work ws supported by NIH Grnt HE We grtefully cknowledge the expert secretril ssistnce of Miss herry Ngel. Reprint requests to Dr. L, A. Hebert, Renl ection, Deprtment of Medicine, Medicl ollege of Wisconsin, nd Milwukee ounty Medicl omplex, 87 West Wisconsin Avenue, Milwukee, Wisconsin, 53226, U..A. References 1. BRENNER BM, TROY JL, DAUGI-IARTY TM: The dynmics of glomerulr ultrfiltrtion in the rt. 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