Effect ofAminonucleoside Nephrosis on Immune Complex … · 2014. 1. 30. · the GBIM. INTRODUCTION MIost immunologically mediated huinan renal dis-eases arebelievedto resultfrom

Effect of Aminonucleoside Nephrosison Immune Complex Localization in AutologousImmune Complex Nephropathy in Rats

WILLIAM G. COUSER, NANCYB. JERNIANOVICH, STEELE BELOK, andMAGDAMI. STILMANT, Evatns Memorial Department of Cliniical Research antd thleDepartments of AMedicinie and Pathology, Bostoni University Medical Center, andMallory Inistitute of Pathology, Boston City Hospital, Bostotn, Alassachusetts 02118

JOHN R. HOYER, Department of Pediatrics, Harvard Mledical School andChildrens Hospital Medical Cetnter, Bostoni, Mlassachusetts 02115

A B S T RA C T The effect of increased capillary per-ilmealility on1 glomerular immultine comiiplex localizationiwas studied in rats immiluniized with proximial tul)ularanitigeni (Fx1A) to induce autologouis imnmunie complexnephropathy (AICN). AICN rats were made proteinuric1)V- injection or uniilateral renal perfusion withamiiinoinucleoside of puromycin (PA) before developingsul)epithelial conmplex deposits. Control AICN kidneysdeveloped diffuse granular deposits of IgG and Fx1A oInthe subepithelial surf'ace of the glomerular b)asementmembrane (GBM) at 3 wk by immuiinofluiorescence andelectron microscopy, and dep(isits increased in sul)-se(quienit weekly biopsies. In conitrast, PA-nephroticAICN kidneys developed few or no GBMdeposits and asignificant increase in mesangial localization of IgG andFxlA during the period of PA-induced proteinuria.These alterationis in complex localizationi weredocumenited 1)oth in rats with PA nephrosis andl inunilaterally PA-inephrotic kidneys compared with coni-tralateral controls in the sanme animiials, thus excluidinigany effect of PA on the imnililiiopatlhogeneticmlechaniisnm in AICN as ani explaniation for thesefinidings. The absence of GBMi deposits closely corre-lated with reducedl stainiing for polyanionic glomierilarsialoproteini in proteinutiric kidneys, siniee PA-perfuisedkidneys studied 2 wk after resdoltutioin of proteinutriademiion strated retuirni of niormlial staininig for sialoprotein

Portionis of this work were presented at the 8th AinutalMeeting of the Amiericani Society of Nephrologv, Washington,D. C., 23 November 1976, and were published in abstractformn: 1975. Kidniey Juit. 8: 447; 1976. Kidtiey Int. 10: 541.

Dr. Couser is the recipient of Research Career DevelopmentAward 1-K04-AM-00102. Dr. Hoyer is an Established In-vestigator of the American Heart Associationi.

Recei';edfor publication 3 M11i 1977 and(c in revisedform 24October 1977.

and development of subepithelial comiiplex depositssimilar to those in contralateral conitrol kidneys. Thesestuidies demiionistrate that properties of the glomiierulusitself play ani inmportant role in determiniing the site ofcomplex depositioni in experimiienital AICN and suggestthat electrophysical characteristics of the glomerularcapillary wall may influence complex localization onthe GBIM.

INTRODUCTION

MIost immunologically mediated huinan renal dis-eases are believed to result from glornerular depositioniof circulatinig immune complexes (1). The type andseverity of the glomerular lesions produced are deter-mined largely by the quanititx and site of localizatioll ofimmuine reactanits within the glonmertultus (2). Studies inexperimental acute and clhronic seruml sickniess modelsin rabbits hiave indicatedl that several factors inayinlflueInce iiiimtmne complex localizationi, includinigcomplex size as determined -)y antigen:antibody ratio(1, 2), vasoactive amine release (2-4), anid hy-drodynamic (1, 3-5) and pharmnacologic (6) fiactors. Inmembranous nephropathy, the imiost comiiii on1 cause ofidiopatliic n ephrotic svildrome in aduilts (7), grainulardeposits conitaininlg IgG an(l halving the ultrastructturalcharacteristics of inintiiie complexes are localizedexcltusively along the sutbepithelial sturface of theglomeriular basement imembrane (GBNI).' In selected

' Abbreviatiotus tused in this paper: AICN, aultologouisimmune coomplex nephropathy, Hevmann niephritis; C3,p 1c--BLa, third comiiponent of conmplemiielnt; FxlA, proximaltubular brush border antigeni; GBM, glomerular I)asementmeml)rane; IF, immuniiiiiiofluioreseenice microscopy; PA,anuniiionucieleoside of puronix ci.

Tle Jouirnial of Clinical Itivestigation Volume 61 Marchi 1978.561-5572 561

patients, additional antigens have also been identifiedat this site and are believed to represent the antigeniccomponent of immune deposits (8-12). Complexes inthe subepithelial space are presumed to be derivedfrom the circulation, although the mechanisms whichregulate the localization of immune reactants at this siteare not known.

Autologous immune complex nephropathy in rats(AICN, Heymann nephritis) is an established experi-mental model of membranous nephropathy in whichthe antlgenic component of glomerular complex de-posits is derived from proximal tubular brush border(13, 14). Recent studies in this model have shown thatthe earliest detectable epimembranous deposits arelocalized on the subepithelial aspect of the GBMadjacent to the basilar portion of epithelial cells and inthe region of filtration slits beneath slit pore diaphragms(15, 16). This distribution of early epimembranouscomplex deposits corresponds to the localization ofpolyanionic glomerular sialoprotein on epithelial cellsand in filtration slits (17-19). Recent physiologic andultrastructural tracer studies have suggested a role forthis negatively charged material in regulating thepermeability of the glomerular capillary wall tocirculating macromolecules in both normal and diseasestates (20-24). However, the effect of alterations inintrinsic properties of the glomerulus on the localiza-tion of immune complexes has not been previouslyinvestigated.

Wehave presented preliminary data indicating thatAICN rats made proteinuric with aminonucleoside ofpuromycin (PA) before the earliest detectable complexdeposition in the subepithelial space demonstrate amarked alteration in subsequent localization of im-mune deposits (25, 26). In the present report, ourstudies of complex localization in AICN rats withPA-induced proteinuria are described in detail. Theseobservations indicate that the properties of theglomerulus itself which are altered by PA may beimportant determinants of glomerular complex localiza-tion.

METHODS

Induction of AICN. ACIN was induced in 50-100-g maleLewis rats (Charles River Breeding Laboratories, Wilmington,Mass.) by a single injection in the rear footpads of 0.2 ml of anemulsion of equal parts of incomplete Freunds adjuvantcontaining 4 mg/ml of pulverized mycobacterium tuberculosisH37RA (Difco Laboratories, Detroit, Mich.) and 0.02 Mphosphate-buffered saline, pH 7.3, containing 40 mg/ml ofproximal tubular brush border antigen (FxlA). FxlA wasisolated as described by Edgington et al. (27) from ahomogenate of freshly prepared Sprague-Dawley rat renalcortices and lyophilized before use. Our previous studies ofAICN induced by this protocol have demonstrated subepithe-lial glomerular deposits of IgG to be first detectable by directimmunofluorescence (IF) on day 21 and by electron micros-copy on day 28 (16).

Tissue processing. Open renal biopsies were performedunder ether anesthesia. Each cortical biopsy specimen wasdivided into three portions. Tissue for light microscopy wasfixed in 10%neutral buffered formalin, embedded in paraffin,and sectioned and stained with hematoxylin and eosin andperiodic-acid Schiff stains. Histochemical staining forglomerular sialoprotein was carried out with colloidal iron atpH 1.8 (28) and Alcian blue at pH 1.6 (19), and was recorded asnormal, reduced, or absent compared to controls. Tissue for IFwas snap-frozen in isopentane in a dry ice-acetone bath, and4-mm cryostat sections were prepared, stained, and examinedas described elsewhere (29). Tissue for electron microscopywas fixed in glutaraldehyde, then postfixed in osmium for 60min, followed by en bloc fixation in uranyl acetate for 30 minbefore embedding in Epon 812. Thin sections were studied ona Philips 300 electron microscope (N. V. Philips Gloeslampem-fabrieken, Einhoven, Netherlands). Results are based onanalysis of over 700 sections and 225 electron micrographstaken of different portions of several glomeruli from repre-sentative animals in each group.

IF procedures. Direct IF was performed using proceduresand controls previously described (29). Purified rat IgG (MilesResearch Division, Miles Laboratories, Inc., Elkhart, Ind.), ratalbumin (Schwartz/Mann Div., Becton, Dickinson & Co.,Orangeburg, N. Y.) further purified by agarose columnchromatography, and rat FxlA prepared as described abovewere used in the preparation of rabbit antisera to theseproteins. Antiserum to rat 81c-,31a (C3) was prepared by azymosan method (30). Rabbits immunized with FxlA receivedthree injections in the footpads and multiple sub- andintracutaneous sites of 10 mgof antigen in complete Freund'sadjuvant (Difco Laboratories) given at weekly intervals andwere bled 10 days after the last injection. Anti-FxlA was heatinactivated. (560C, 30 min) and absorbed extensively withlyophilized whole rat plasma (2 mg/ml, 37°C for 1 h, 40Covemight) until no precipitate developed and then with anequal volutne of fresh pooled rat blood cells including erythro-cytes, leukocytes, and platelets. Additional absorptions withpurified rat IgG did not alter the IF staining characteristics ofthis antiserum.

The IgG fractions of all antisera were isolated from a 50%saturated ammonium sulfate precipate by chromatography onDEAE-cellulose with a 0.02 M phosphate buffer, pH 7.5,concentrated by vacuum dialysis to 10 mg/ml of rabbit IgG asmeasured by radial immunodiffusion (31), conjugated withfluorescein isothiocyanate (BBL, Div. of Becton, Dickinson &Co., Cockeysville, Md.) by the dialysis method of Clark andShepard (32), rechromatographed, and concentrated to 10 mg/ml of IgG in 10%pooled normal rabbit serum. Conjugated anti-bodies to rat IgG, C3, and albumin were monospecific byimmunoelectrophoresis and micro-Ouchterlony double diffu-sion in 1% agarose; had fluorescein:antibody ratios of 0.140,0.168 and 0.152, respectively (29); and were adjusted toprecipitin titers of 1:4 before use. Anti-rat FxlA was notreactive with rat plasma by double diffusion in gel, but at adilution of 1:4 made two lines in 1%agarose against a 10 mg/mlsuspension of FxlA in saline (27,33). By direct IF, this reagentreacted specifically with the luminal brush borders of proximaltubular cells in normal rat kidney and with glomerular depositsin partially eluted (see below) sections of AICN kidneys with4+ glomerular IgG deposits. The titer of anti-FxlA by direct IFon proximal tubular epithelium (1:64) was equivalent to the IFtiter of antiserum to IgG determined on sections of AICNkidney with 4+ IgG deposits. Although glomerular staining forFxlA was often detectable in uneluted sections of AICNkidneys biopsied 3 or more wk after immunization, stainingwas enhanced by partial elution of washed, unfixed, cryostatsections in 2.5 M potassium thiocyanate at 37°C for 2 h (13).

562 Couser, Hoyer, Stilmant, Jermanovich, and Belok

This procedure was employed routinely before staining forFx1A. Specific staining with each antisera was blocked byprior incubation of sections with unconjugated antisera and byabsorption with specific antigen.

Specific glomerular IF for IgG and FxlA was recorded as0-4+ for granular GBMdeposits, with 4+ representing themaximal intensity of GBMdeposits seen in proteinuric AICNrats at 10-14 wk (16). The intensity of mesangial staining wasrelatively uniform compared with the variation in GBMdeposits. Mesangial staining was therefore graded by estimat-ing the amount of mesangial areas occupied by granulardeposits as follows: 0, no significant mesangial depositscompared to controls; 1+, 25%; 2+, 50%; and 3+, >50% ofmesangial regions of most glomeruli containing deposits. Allresults were recorded by photomicroscopy using 60-s expo-sure times on high-speed Ektachrome (Eastman Kodak Co.,Rochester, N. Y.) developed at ASA 400. Differences in IFfindings between groups were analyzed by Student's t test (34).

IgG antibody titers to Fx1A in groups A, B, and C weredetermined weekly by indirect IF. Serial dilutions of serumwere incubated for 60 min on normal rat kidney sections whichwere then washed and stained for rat IgG. Results wererecorded as the highest tube dilution producing detectabletubular brush border staining. Differences between groupswere analyzed by the Mann-Whitney test (34).

Production of PA nephrosis and experimental design. Thecharacteristics of the experimental and control groups in thisstudy are outlined for reference in Table I. The effects ofincreased glomerular permeability induced by systemicadministration of PA on the localization of early subepithelialcomplex deposits in AICN were studied in 12 AICN rats givenPA (ICN Pharmaceuticals Inc., Life Sciences Group, Cleve-land, Ohio) in normal saline, 100 mg/kg intravenously, ondays 14 and 28 after FxlA antigen injection (group A). Themaximal individual dose of PA injected was 25 mgregardlessof size. These rats had sustained proteinuria from days 21through 35. Eight control AICN rats were injected with anequal volume of normal saline on days 14 and 28 (group B). Asecond group (group C) of eight age- and weight-matchedLewis rats immunized with complete Freund's adjuvant onday 0 received PA, 100 mg/kg intravenously, on the same daysas rats in group A. Renal biopsies were performed on all rats ingroups A, B, and C on days 21, 28, and 35 after immunization.

To exclude possible effects of PA administration onextrarenal factors potentially influencing complex localization,studies were also performed after exposure of only one kidney

to PA. Unilateral PA nephrosis was produced by selectiveperfusion of the left kidney with PA, 15 mg in 1.5 ml of normalsaline, using techniques described in detail elsewhere (35,36). Previous studies by one of us (Dr. Hoyer) have shown thatproteinuria induced by this protocol develops 5-7 days afterperfusion, lasts for 14-21 days, and originates exclusively fromthe perfused left kidney (35, 36). Left kidneys of 10 AICN ratswere perfused with PA on day 14 after immunization withFx1A, and bilateral biopsies were obtained on days 21, 28, and49 (group D). The last biopsy was performed about 2 wk afterPA-induced proteinuria had resolved. Biopsies from theperfused left kidneys are designated group D-L. Controlbiopsies from the nonproteinuric right kidneys of theseanimals are designated group D-R. Two additional controlgroups of rats were studied in a similar fashion after unilateralrenal perfusion; left kidneys of four AICN rats were perfusedon day 14 with saline alone (group E), and left kidneys of fourage- and weight-matched Lewis rats immunized with adjuvantalone were perfused with PA on day 14 (group F).

Other procedures. 24-h urine collections were obtained inmetabolic cages immediately before each biopsy in allanimals, and urine protein excretion was measured by thesulfosalicylic acid method (37) using a whole serum standard.Urea and creatinine concentrations were determined bystandard autoanalyzer techniques on serum samples obtainedat the time of biopsy.The effect of PA on vasoactive amineactivity was determined by measuring the bluing reactioninduced in 30 min by intradermal injections of 1.0 ,ug ofhistamine base or 0.5 ,ug of serotonin in 0.1 ml of normal salinegiven after an intravenous injection of 1.0 ml of 0.5% Evansblue dye. Measurements were made 4 h and 1 and 5 days afteradministration of PA, 100 mg/kg intravenously, and 1 day aftera second injection of PA given 7 days after the first. Controlanimals were injected with saline alone. Differences betweengroups were analyzed by Student's t test.

RESULTS

Urine protein and renal function. All groupstreated with PAhad mean protein excretions exceeding60 mg/day when biopsied on days 21, 28, and 35, and allanimals in these groups were proteinuric. Mean urineprotein excretions on days before each biopsy areshown in Figs. 1 and 2. Control AICN rats injected or

TABLE IExperimental Protocol for PA-Treated and Control Groups

Group n Day 0 Day 14 Day 21 Day 28 Day 35 Day 49

Rats given PA,* 100 mg/kg intravenously, or NS 10 ml/kg intravenously

A 12 FxlA-CFA PA Biopsy 1 Biopsy 2, PA Biopsy 3B 8 FxlA-CFA NS Biopsy 1 Biopsy 2, NS Biopsy 3C 8 CFA PA Biopsy 1 Biopsy 2, PA Biopsy 3

Rats with left kidney perfused with PA, 15 mg, or NS, 1.5 ml

D-L 10 FxlA-CFA PA Biopsy 1 Biopsy 2 - Biopsy 3D-R 10 FxlA-CFA - Biopsy 1 Biopsy 2 Biopsy 3E 4 FxlA-CFA NS Biopsy 1 Biopsy 2 - Biopsy 3F 4 CFA PA Biopsy 1 Biopsy 2 Biopsy 3

* PA = aminonucleoside of puromycin; NS = normal saline.

Effect of Aminonucleoside on Immune Complex Localization 563

PMs iA B C A B C

±A B C

* /gG FxlA

I1L4LA CDAY21

A B C

DAY28

A C

DAY35

FIGURE 1 Urine protein excretion and mesangial and GBMdeposits of IgG and FxlA in grolps A,B, and C on days 21, 28, and 35. Groups A and C received PA, 100 mg/kg intravenously, onl days 14and 28. All values are mean+SEM.

perfused with saline alone (groups B and E) had nosignificant increase in protein excretion during thisperiod (Figs. 1, 2), a finding consistent with ourprevious results during the induction phase of AICN(16). Our previous studies have also indicated thatincreased proteinuria in unilaterally perfused rats isderived entirely from the perfused kidney (35), and thatthere is no significant increase in protein excretion dueto AICN alone at 21 and 28 days (16). Therefore,proteinuria in rats in group D is depicted as coming onlyfrom PA kidneys (group D-L) at 21 and 28 days in Fig. 2(recognizing that a very small percentage of the totalprotein excreted is derived from the right kidney). Themean urine protein excretions of perfused rats returnedto nearly normal values by day 35 (group D: 12+5,group F: 9+6 mg/day) and were not different fromcontrol values by day 49 (Fig. 2).

Serum creatinine and urea concentrations were notsignificantly different in rats receiving intravenous PAandcontrols at the time of biopsies on days 21 and 28 (TableII). By day 35, animals in groups A and C that hadreceived PA had significantly reduced renal function

manifested by douibling of the serum creatinineconcentrations and three-to-fourfold rise in ureanitrogen compared to nonproteinuric, sal.ine-injectedAICN controls in grouip B (Table II). Rats in groups D,E, and F perfuse(d tunilaterally with PA or saline had nosignificant increase in serum creatiniine or uirea(Table II).

IF microscopy. The patterns of complex locatliza-tion in AICN rats given systemic PAand control groupswere qualitatively similar to results in unilaterallyPA-perfuised animilals and controls. These results of IFstaining for IgG and FxlA are shown in Figs. 1-4.Deposits of IgG and FxlA in control AICN rats injectedwith saline (group B) and in nonperfused right kidneysof AICN rats (group D-R) were similar and corre-sponded closely with IF findings previously describedduring the early phase of AICN (16). Faint, finelygranular deposits of IgG and Fx1A were presentdiffusely along the GBMof all glonmeruli on day 21 aindincreased in biopsies on days 28 and 35 (Figs. 1-4). C3was; not demonstrable in GBMdeposits on day 21 andwas present in only trace amounts at days 28 and 35.

564 Couser, Hoyer, Stilmant, Jermanovich, and Belok

-125-

N 100-

E75-

- 50-

1 25-0eIL n

a

0ILwaUm

PiiSW

a

N

I54

0U

a.

0ILwaUSa

-I

_4 a(S(42Sinai

DAY21 DAY28 DAY49

-/

X O m /- - -ilD-L DAR E F D-L OR E F DL DOR E FDAY21 DAY28 DAY49

FIGuRE 2 Urine protein excretion and immunofluorescence deposits of IgG and Fx1A on GB.Mand in mesangium in groups D, E, and F on days 21,28, and 49. (Left) Kidneys in groups D-L and Fwere perfused unilaterally with PAon day 14, and group E was perfused with saline. All values aremean+SEM.

Mesangial deposits were not present in nonproteinuricAICN kidneys with IgG deposits on the GBM. Thefindings in saline-perfused kidneys in group E were notdifferent from those in groups B and D-R, indicatingthat the procedure of unilateral perfusion had no effecton subsequent complex localization (Fig. 2).

The patterns of staining for IgG and Fx1A inproteinuric AICN kidneys in groups A and D-L werestrikingly different from those of the control kidneysdescribed above. The kidneys with proteinuria aftersystemic PA injection (group A) showed similarfindings to kidneys selectively perfused with PA (groupD-L). Glomeruli in AICN kidneys made proteinuricwith PA before day 21 (groups A and D-L) hadessentially no demonstrable deposits of IgG, FxlA, orC3 on the GBM during the period of increasedproteinuria (Figs. 1-4). In addition to the markedreduction in GBMdeposits, proteinuiric AICN kidneysin groups A and D-L also manifested a significantincrease in deposition of both IgG and FxlA in agranular pattern in the mesangium compared withnonproteinuric AICN controls in groups B, D-R, and E(Figs. 1-4). Mesangial staining for IgG was alsosignificantly greater and more finely granular than that

seen in non-AICN control groups C and F that receivedPA (P < 0.05 on days 21, 28, and 35) (Figs. 1-4).Mesangial staining seen in groups C and F with PAnephrosis alone was more confluenit and nodular thanthat in AICN rats, as described previouslI by others (35,38, 39). Mesangial staining for FxlA paralleled that forIgG in proteinuric AICN kidneys and was in a siimilarpattern, although less intense (Figs. 1-4). No Fx1Astaining was seeIn in glomeruli of control ainimals ingroups C and F with PAnephrosis alone. No groups hadsignificant staining for C3 in the mesanigiumiii. Pro-teinuric AICN kidneys in groups A and D-L frequentlyhad staining for IgG on the lumiinial border of proximaltubular cells, presumably reflecting glomerular filtra-tion of circulating anti-FxlA antibody. IgG aiid albuuminwere present in rare tubular casts, aind somile granularand nodular staining for these proteins was present inmesangial and epithelial areas in groups A, C, D-L, and(lF as described by others in PA nephrosis (35, 38, 39).

These mlarked differences in comiiplex localizationibetweeni proteinuric and nonproteiniuric kidneys dle-scribed above were clearly apparenit in each animalstudied in group D wheni the proteinutric left kidneywas comppared with the noniproteiniuric right kidney ill

Effect of Aminonucleoside o01 Inmuniie Complex LocaliZation _,_65

TABLE IISerumi1 Creatinine, Urea, and Anti-FxlA Levels at the Time of Each Biopsy

Day 21 Day 28 Day 35 Day 49

Serum creatinine,mg/llOO ml

Group A 1.14+(0.18 (12)* 0.96+(0.15 (8) 2.38±0.42 (8)4 NNDGroup B 0.78+(0.16 (8) 1.00+(0.17 (8) 0.91+(0.11 (7) NDGroup C 1.07+0.21 (8) 1.04+0.19 (6) 2.41±0.34 (6)t NDGroup D 1.01±0.14 (10) 1.14±0.14 (10) ND§ 1.06±0.18 (10)Group E 0.94±0.17 (4) 0.98±0.21 (4) ND 1.01±0.17 (4)Group F 1.12±0.19 (4) 1.17+0.19 (4) ND 1.18±0.21 (4)

Serum urea nitrogenmg/1i00 ml

Group A 33.9±1.2 (12) 26.8±2.4 (8) 82.6±12.9 (8)4 NDGroup B 30.8±2.4 (18) 26.2+0.97 (8) 25.2±4.7 (7) NDGroup C 31.9±1.8 (8) 29.1±1.9 (6) 76.1±+10.8 (6)4 NDGroup D 34.1±1.4 (10) 28.9±2.6 (10) ND 27.4±2.1 (10)Group E 30.8±1.6 (4) 29.1±2.3 (4) ND 28.4±2.6 (4)Group F 36.2±2.9 (4) 32.8±3.1 (4) ND 33.1±2.9 (4)

Anti-FxJA antibodylevels

Group A 2.50±0.26 (12) 2.17±0.79 (8) 1.17±0.48 (8)4 NDGroup B 3.50±0.49 (8) 4.25±0.88 (8) 5.57±0.65 (7) NDGroup C 0 (8) 0 (6) 0 (6)4 ND

Mean (±SEM) serum creatinine and urea concentrations in all groups, and anti-FxlA antibodylevels in groups A, B, and C, measured just before each biopsy. Groups A and C receivedPA systemically on days 14 and 28 and groups D and F were perfused unilaterally withPA on Day 14.* = N.4 P value compared to grouip B

FIGURE3 Imniunofluoreseent photomicrographs from biopsies on day 28. (A) NonproteinuricAICN rat in group B with 2+ GBMdeposits of IgG. (B) Sameanimal as A showing GBMdeposits ofFxlA. (C) AICN rat in group A made proteinuric with PA and stained for IgG showing increasedmeesangial deposits. No GBMdeposits are detectable. (D) Same animal as C showing staining forFxlA in mesangium and absence of FxlA on GBM. (x450)

gium were seen in AICN rats with mesangial depositsby IF compared with controls. PA nephrotic kidneyshad only minor morphologic changes by light micros-copy, including focal tubular dilatation, focal tubularcasts, and periodic-acid Schiff-positive droplets inoccasional glomerular and proximal tubular epithelialcells.

Anti-FxJA antibodyl levels. Titers of circulatingantibody to proximal tubular bnrish border determined1v indirect IF in groups A, B, and C are expressed astube dilutions in Table II. PA-treated AICN aniimals ingroup A had lower titers of antibody than noni-proteinuric AICN controls in group B, but thesedifferences dlid not reach statistical significance untilday 3.5 (Table II). Despite the lower mean values ingrouip A, there Nwas considerable overlap, and several

animals without detectable GBMdeposits in group Ahad higher antibody levels at days 21 and 28 than someanimals in group B with 1-2+ GBMdeposits. Animalsin group C had no detectable circulating antibody bythis technique (Table II).

Vasoactive amine activity. Measurements of themean diameter of bluing induced by histamine andserotonin in PA-treated and normal rats at 4 h, 1 and 5days, and 1 day after a second injection of PA 1 wk afterthe first demonstrated no effect of PA on increasedcapillary permeability induced by vasoactive amineseither acutely or after PA-induced proteinuria.

DISCUSSION

These studies demonstrate that kidneys of ratsimmnllliiized with FxlA fail to develop glomerular

Effect of Aminonucleoside on Immune Complex Localization 567

FIGuRE 4 IF photomicrograph from AICN rat unilaterally perfused with PA. (A) IgG on GBNI Itday 28 in nonperftused R kidney in group D-R. (B) PA-perftused left kidney at day 28 from the saimeanimal as A showing absence of GBMdeposits and increased localization of IgG, predominanitly inthe mesangium. (C) Same biopsy as B, stained for FxlA aind showing localization of FxlA inmesaingium aind no GBNI staining. (D) Samekidney as in B at day 49, stained for IgG aind showingdevelopment of typical rneml)ranous deposits after cessationi of proteinuria. Mesangial depositsare no longer appatrent. Some residual tubular brush border staininig is present. (x450)

subepithelial complex deposits while proteinuric as aresult of treatment with PA before the oniset ofglomerular complex depositioni. After PA-inducedproteinuria subsides, subepithelial deposits similar tothose observed in control kidnieys of AICN rats may bedetected in these same kidneys. The studies in theunilateral model fu-rther demonstrate that this lack ofepi men)branious complex deposition in PA proteinurickidneys occurs despite persistenice of' an ongoingimniunopathogenetic process that results in epimem-l)ranious GBMdeposits in nonlproteinuiric contralateralkidneys of these samne aiiinials. In addition, granulardepositioni of IgG and FxlA were observed within theglomieruilar mesangium inl AICN rats during the periodof PA-indtuced proteinuria. These studies clearly

exclude anl effect of PA on systemic factors thalt nmightinfluence the formlation or composition of immnunecomplexes. Hence, the changes in complex localizatioinobserve(l in PA-treated AICN rats demonstrate thatproperties of the glomerulus itself significantly affectthe site and (quantity of comiiplex deposition. Such anleffect hals not been previously de inon strattedl.

The identification of antigenis (9-12) anid specific anti-body to thenm (9, 40), in subepithelial GBMI deposits inmemlbranous nephropathy in man is consistenit with theview that these deposits represen-t glomertular trappingof circulatin-ig, soluble immune complexes as apparenitlyoccurs in the acute and chronic serum sickniess mlodelsin rabbits (1, 2, 41). Immune complexes have beendirectly visuialized in the circulationi, crossing the

568 Couser, Hoyer, Stilniatnt, Jermlatnovich, and Belok

1.---

*.

'.1

4. -~C

t-!~~~- .-. CL-4-

FIGURE 5 Electron micrographs from representative AICN rat in group D at day 28 comparingnoniperfused right kidney (A) Nwith PA-perfused proteinuric left kidney (B). Early complex depositsare present in the subepithelial space aind in filtrationi slits in the right kidney (A, arrows). Depositsatre absenit in the proteintiric left kidney (B), which has extensive epithelial cell foot process fusion.BM, basemlenit membrane; CL, capillary lutmen; EP, epithelial cell; EN, endothelial cell; uiranvland lead. x 19,700.

GBMI, and localized in the subepithelial space in miceimmunized with ferritin by Stilmant et al. (42). Theview that sutbepithelial comiiplex deposits in AICN alsorepresent glomerular trapping of circutlating iimiiiineconmplexes is supported by the presence of subepithe-lial granular deposits of tubular antigeni and antibodyafter active (13, 34) or passive (33, 44) immunizationagainst tubular antigens and by demonstration oftubular antigen (45) and antil)ody to it (14, 43, 45) in thecirculation of AICN rats. However, recent stuidies byVan Dammeet al. (46) have provided evidence thatsubepithelial immunle deposits simiiilar to those inAICN can be produced by direct perfusioll of ratkidneys with antibody to FxlA. These latter findinigssuggest strongly that the deposits seeni in AICN mayresult from the reaction- of circulating antibody withantigens within the glomerulus rather than depositionof circulating immunie comiplexes.

Regardless of the precise mechainism by whichsubepithelial GBMdeposits are formed in AICN, thebasis for the marked reductioni in epimembranousdeposits in PA-treated rats in our studies has not been

established. Thus, several systemic variables whichhave been shown to influence the site and quantity ofglomlerular conmplex localization must be considered.These include changes in complex size and latticeformation due to alterations in the molecular weight ofantigen or antibody or the antigen:antibody ratio (1, 2).In addition, alterations in clearance kinetics of circulat-ing complexes due to changes in reticuloendothelialsystem functioni (47, 48), reduction and alkylation of theantibody component of circulating complexes (49), oradministration of pharmacologic agents such as cor-tisonie (6, 50) and drugs which affect vasoactive amineactivity (1, 3, 4, 51) may alter complex localization.However, in the uniilateral PA model, both kidneyswere exposed to the same systemic milieu, and changesin complex localization were observed only in thePA-treated kidneys. These findings thus effectivelyexcludle possible effects of PA on such systemicvariables as the basis for the altered complex localiza-tion observed.

Several effects of PA on the structural and functionalcharacteristics of the glomerultus warrant consideration

Effect of Aminonucleoside on1 Imnmune Complex Localization 6569

in interpreting our findings. Ultrastructural studies ofglomeruli of rats made proteinuric with PAdemonstrateextensive morphologic alterations in the epithelialsurface of the capillary wall including separation of theepithelial cell layer from GBM(52, 53). However, suchzones of epithelial detachment are focal and are presentonly in a minority of glomeruli in PA nephrosis (53).Hence they could not be responsible for the uniformabsence of GBMdeposits in PA-treated kidneys in ourstudies.

Hemodynamic changes have been shown to alterglomerular complex localization in other models (1, 5).A marked reduction in antibody or complex delivery toglomeruli consequent to PA-induced alterations inglomerular hemodynamics could decrease deposits inPA-treated kidneys. Bohrer et al. have characterized thehemodynamic changes induced by administration ofPA in a dose comparable to that received by animals inour group A at the time of biopsy at 21 days (24). Theirstudies document a 40% reduction in glomerularfiltration rate primarily due to a 60% reduction in theglomerular ultrafiltration coefficient (Kf) and, to a muchlesser extent, to a 20% reduction in glomerular plasmaflow rate (24). It appears unlikely that a 20% reductionin glomerular plasma flow, and therefore delivery ofantibody or complexes, could account for the verymarked reduction in epimembranous deposits toessentially zero in PA-treated kidneys observed here.This conclusion also appears justified in light of otherstudies of the role of renal blood flow in glomerularcomplex deposition (54). Moreover, the presence ofextensive antibody deposits on proximal tubular brushborders in kidneys of AICN rats with PA-inducedproteinuria demonstrates that substantial glomerulardelivery and filtration of antibody occurred withoutformation of GBMdeposits. A possible contribution ofPA-induced changes in Kf to our findings cannot beexcluded with certainty, since the role of this propertyof the glomerular capillary wall in regulating localiza-tion of macromolecules is not known.

Recent studies have suggested a role for complementreceptors demonstrated on the epithelial aspect of theGBMin man in contributing to complex localization atthis site (55-57). However, glomerular complementreceptors have not been demonstrable in the rat, andthe presence of definite subepithelial complex depositsin AICN before localization of C3 (14, 16) furthermitigates against a role for such receptors in this model.

An additional possibility is that the decrease inepimembranous complex localization observed wasconsequent to PA-induced alterations in electrophysi-cal properties of the capillary wall. Histochemical andbiochemical studies have shown a marked reduction inglomerular polyanion associated with the onset ofproteinuria in PA nephrosis (19). The increased

fractional clearances of anionic dextran sulfates in PAnephrosis without increased clearances of neutraldextrans of the same sizes is also consistent with a lossof capillary wall charge in this model (24). Histochemi-cal and ultrastructural studies demonstrate the majorsite of negative charge to be or! the subepithelial aspectof the capillary wall in the polyanionic sialoproteincoating of epithelial cells and filtration slit diaphragms(17-19). Our present and earlier studies (16) and thestudies of Schneeberger et al. (15, 58) demonstrate thatthe earliest detectable localization of complex depositsin AICN occurs at this site beneath epithelial cells andin filtration slits. Rennke et al. have shown that neutraland anionic ferritin molecules do not penetrate normalGBM, but that cationic ferritin molecules of the samesize reach the subepithelial surface and accumulate asaggregates in filtration slits similar to the distribution ofearly deposits in AICN (21). Heparin-protaminepolyelectrolyte complexes also localize in the sub-epithelial space and slit pores (59, 60). These studiessuggest a role for capillary wall charge in the subepithe-lial localization of macromolecules in the glomerulus.Complex deposits in our studies were reduced orabsent during the period of PA-induced reduction inpolyanion staining and proteinuria and developednormally when proteinuria resolved and polyanionstaining returned to normal. These observationssuggest that glomerular capillary wall charge is also animportant determinant of subepithelial complex locali-zation in AICN.

The finding of granular deposits of IgG and FxlA inthe mesangium of PAkidneys in AICN rats is of interest.Schneeberger et al. have recently reported diminishedmesangial uptake of colloidal carbon in AICN (61), afinding which may be related to the relative lack ofmesangial immune deposits in rats with proteinuria dueto AICN alone. Previous studies have demonstrated amarked increase in mesangial uptake of exogenousmacromolecules from the circulation in glomerulitreated with PA or nephrotoxic serum (35, 36, 62).Although mesangial deposition of immunoglobulin hasbeen noted in PAnephrosis by us and others (35,38,39),the mesangial deposits in PA-treated kidneys of AICNrats in this study exceeded those in PA-treated controls,were clearly granular as well as than nodular, and con-tained FxlA, which was not found in the mesangium ofcontrols. Since uptake of nonaggregated IgG is notincreased in PA nephrosis (62), the mesangial IgG andFxlA seen in AICN rats was presumably in amacromolecular, probably immune complex form. Thisfinding suggests that circulating immune complexescontaining tubular antigens are present in AICN. Itfurther demonstrates that increased mesangial uptakeof endogenous immune complexes in PA nephrosisappears similar to that previously shown with exoge-

570 Couser, Hoyer, Stilmant, Jermanovich, and Belok

nous macromolecules. As previously suggested (35, 36,63, 64), this latter finding may be relevant to thepathogenesis of focal sclerotic mesangial lesions thatdevelop in several chronic proteinuric disorders.

ACKNOWLEDGMENTS

Weare grateful to Christine Darby aind Mary NMorani for experttechnical assistance and to Lisa Easterdav for secretarialsupport in preparation of the manuscript.

Support for this work was provided by research grantsAM-17722 and AM-19097, General Research Support grantRR-05487 (University Hospital), National Research Traininggrant AM-07053 (Dr. Jermanovich and Dr. Belok) from theU. S. Public Health Service, and a research grant from theAmerican Heart Association.

REFERENCES

1. Cochrane, C. G., and D. Koffler. 1973. Immunle comiiplexdisease in experimental animals and man. Adv. Imununol.16: 185-264.

2. Germuth, F. G., Jr., and E. Rodriguez. 1973. Im-munopathology of the Renal Glomerulus. Little, Brownand Company, Boston. 227 pp.

3. Kniker, W. T., and C. G. Cochrane. 1968. The localizationof circulating immune complexes in experimental serumilsickness: the role of vasoactive amines aind hydrodyniamicforces. J. Exp. Med. 127: 119-135.

4. Kniker, W. T. 1972. Modulation of the inflammatoryresponse in vivo: prevention or anmelioration of immllunecomplex disease. In Inflamniatory Mechanisms andControl. I. H. Lepow and P. A. Ward, editors. AcademicPress, Inc., New York. 335-367.

5. Germuth, F. G., Jr., W. A. Kelemen, and A. D. Pollack.1967. Immune complex disease. II. The role of circulatorydynamics and glomerular filtration in the development ofexperimental glomerulonephritis. Johns Hopkinis Med. J.120: 252-257.

6. Germuth, F. G., A. J. Valdes, L. B. Senterfit, and A. D.Pollack. 1968. A unique influence of cortisone on thetransit of specific macromolecules across vascular walls inimmune complex disease. Johns Hopkins iMed. J. 122:137-153.

7. Coggins, C. H. 1975. An interhospital study of the adultidiopathic nephrotic syndrome and its response totreatment. Kidney Int. 8: 408. (Abstr.)

8. Ehrenreich, T., and J. Churg. 1968. Pathology of mem-branous nephropathy. Pathol. Annu. 3: 145-186.

9. Koffler, D., P. H. Schur, and H. G. Kunkel. 1967.Immunological studies concerning the nephritis of sys-temic lupus erythematosus. J. Exp. Med. 126: 607-623.

10. Combes, B., P. Stastny, J. Shorey, E. H. Eigenbrout, A.Barrera, A. R. Hull, and N. W. Carter. 1971. Glomeruloine-phritis with deposition of Australia antigen-antibodycomplexes in glomerular basement membrane. Lan cet. 2:234-237.

11. Couser. W. G., J. B. Wagonfeld, B. H. Spargo, anid E. J.Lewis. 1974. Glomerular deposition of tumiiior aintigeni inmembranous nephropathy associated with colonic car-cinoma. Am. J. Med. 57: 962-970.

12. Naruse, T., K. Kitamura, Y. Miyakawa, aind S. Shibata.1973. Deposition of renal tubular epithelial antigeni alongthe glomerular capillary walls of patients with membra-nous glomerulonephritis. J. Immunol. 110: 1163-1166.

13. Edgington, T. S., R. J. Glassock, and F. J. Dixon. 1967.

Autologous immuine comiiplex pathogenesis of'experimiien-tal allergic glomileruloniephritis. Scieice (WVash. D.C.). 155:1432-1434.

14. Glassock, R. J., T. S. Edgingtoni, J. I. Wcatsoni, and F. J.Dixon. 1968. Autologous immllune complex nephritisiIl(lltced with renal tubular anitigeni. II. The palthogenieticmechainism.J. Exp. Med. 127: 573-588.

15. Schneeberger, E. E., P. D. Leber, MI. J. Karnovsky, and R.T. McCluskev. 1974. Altered funcetionial properties of therenal glomerulus in autologous immuntie complex ne-phritis: anl ultrastructural tracer study.J. Exp). Mled. 139:1283-1302.

16. Couser, W. G., M. M. Stilmiant, and C. Darby. 1976.Autologous immune comiiplex nephropathv. I. Sequentialstudy of immune complex deposition, ultrastrueturalchaniges, proteinuria and alterations in glomerular sialo-protein. Lab. Incvest. 34: 23-30.

17. Jones, D. B. 1969. Mucosubstances of the glomlerulus.Lab. Itnvest. 21: 119-125.

18. 'Mohos, S. C., and L. Skoza. 1969. Glomerular sialoproteini.Scienice (Wash. D. C.). 164: 1519-1521.

19. Michael, A. F., E. Blau, and R. L. V'ernier. 1970.Glomerular polvanion: alteration in amiinlonticleosidlenephrosis. Lab. Invest. 23: 649-657.

20. Chang, R. L. S., XV. NI. Deen, C. R. Robertson, andl B. NI.Brennier. 1975. Permselectivity of the glomiieruilar capillarywall. III. Restricted transport of polvanions. Kidntety Itlt. 8:212-218.

21. Rennke, H. G., R. S. Cotran, and MI. A. V'enkataclalamii.1975. Role of molecular charge in glomiierular permeabil-itv: tracer stuidies with cationized ferritinis.J. Cell Biol. 67:638-646.

22. Blau, E. B., and J. E. Haas. 1973. Glomerular sialic aci(dand proteinuria in human rencal disease. Lab. Invest. 28:477-481.

23. Bennlett, C. MI., R. J. Glassock, R. L. S. Chanig, WV. NI. Deeni,C. R. Robertson, and B. NI. Brennler. 1976. Permselectivitvof the glomerular capillary wall: studies of experimentalglomerulonephritis in the rat using dextran sulfate.J. Clin.Incvest. 57: 1287-1294.

24. Bohrer, NI. P., C. Baylis, C. R. Robertson, an(l B. NI.Brenner. 1977. Mlechanisms of puromycin-induced de-fects in the transglomerular passage of water andlmacromolecules.J. Clin. Incest. 60: 152-161.

25. Couser, W. G., and NI. NI. Stilmant. 1975. Effects of alteredglomerular permeability on immune coomplex (IC) locali-zationi. Kidnei hIt. 8: 447. (Abstr.)

26. Couser, W. G., J. R. Hoyer, NI. NI. Stilmlant, and N. B.Jermanovich. 1976. Altered localizationi of immunlliie coml-plexes (IC) in unilateral animonuceleoside (AN) nephrosis.Kidtney lot. 10: 541. (Abstr.)

27. Edgingtoni, T. S., R. J. Glassock, andl F. J. Dixon. 1968.Auitologois immunie complex niephritis indtuced withrencal tubular antigeni. I. Identificationi and isolationl of thepathogenietic anitigeni. J. Ex). Aled. 127: 555-572.

28. Rinehart, J. F., and S. K. Abul-Haj. 1951. An improvedmethod for histologic demiion strationi of acid m11ucopolyvsac-charides in tissues. Arch. Pat/iol. 52: 189-194.

29. Couiser, W. G., and E. J. Lewis. 1974. A mlethodl forpreservation of immnofluoreseeniee in renial tisstue. Amii. J.Clitn. Pathol. 61: 873-876.

30. NMardiney, NI. R., Jr., and H. J. MIiiller-Eberhard. 1965.NIouse Blc-globulin: prodtuction of anitiserumil and char-acterization in the coomplemenit reactioni. J. Imumuol.94: 877-882.

31. Nlancini, D., A. 0. Carbonara, and J. R. Heremlanis. 1965.

Effect of A ii ion ucleoside o011 Iin nUtine Complex Localization 571571

Immuniochemical (quantitationi of antigens b)y sinigle radialimmunodiffuision. Imniunocheminstrn. 2: 235-254.

32. Clark, H. F., and N. C. Shepard. 1963. A dialysis techniqluefor preparinig fluiorescenit antil)odlies. Virology. 20:642-644.

33. Van Es, L. A., A. P. R. Blok, L. Schoenfeld, andl R. J.Glassock. 1977. Chronic nephritis induiced ly an)til)odiesreacting with glomerular-bounld inuntune comp)lexes.Kidniey Itmt. 11: 106-115.

34. Snedecor, G. W., and W. C. Cochranie. 1967. StatisticalMethods. The Iowa State University Press, Ames. 6thedition. 120-134.

35. Hoyer, J. R., S. M. Mauer, and A. F. Michael. 1975. Uni-lateral renal disease in the rat. I. Clinical, morphologicand glomerular mesangial functional features of the ex-perimiiental model producedl hy renial perfulsion with theaminonucleoside of puromncin. J. Lab. Clini. Med. 85:756-768.

36. Hover, J. R., J. C. Elema, and( R. L. V'erniier. 1976.Unilateral renal disease in the rat. II. Glomerularmesangial uptake of colloidlal carbon in unilateral amino-nucleoside nephrosis and( nephrotoxic seruim nephritis.Lab. Invest. 34: 250-255.

37. Bradley, G. M., and E. S. Bensoni. 1974. Examinationiof the urine. In Todd-Sanford Clinical lDiagnosis byLaboratory Methods. I. Davidsorn and J. B. Henry,editors. W. B. Sacuniders Company, Philadelphia. 15thedition. 74-75.

38. Unanue, E., and( F. J. Dixon. 1964. Experiimienitalglomerulonephritis. IV. Participationi of complement innephrotoxic nephritis.J. Exp). Med. 119: 965-987.

39. Okuda, R., M. H. Kaplan, F. E. Cuppage, and W. Heymann.1965. Deposition of gammia globulin in kidneys of ratswith nephrotic renal disease of various etiologies.]J. Lab.Clin. Med. 66: 204-215.

40. Lewis, M. G., L. W. Loughridge, and T. NI. Phillips.1971. Immunological studies of nephrotic syndrome as-sociated with extra renal malignant disease. Lancet. 2:134-135.

41. Dixon, F. J., J. D. Feldmain, and J. J. Vaz(luez. 1961. Experi-mental glomerulonephritis. The pathogeniesis of a labora-tory model resembling the spectrumil of humicain glo-merulonephritis. J. Ex). Med. 113: 899-920.

42. Stilmant, M. M., W. G. Couser, and R. S. Cotranl. 1975.Experimental glomlerulonephritis in the mouse associate(lwith mesanigial depositioni of 'autologonis ferritin imimiiunllecomplexes. Lab. Itnvest. 32: 746-756.

43. Grupe, W. E., anid M. H. Kaplan. 1969. Demiionistrationi ofani antibody to proximial tubular anitigeni in thepathogenesis of experimiiental autoimmunie nephlrosis inthe rat. j. Lab. Clitn. Med. 74: 400-409.

44. Feenstra, K., R. V. D. Lee, H. A. Creber, A. Arcid(ls, and P.J. floedemiiaeker. 1975. Experimental glomerulonephritisin the rat ind(uiced I)b antibodies directed againist tubularanitigeni. I. The natural history: a hiistologic and im-inunlnohistologic study at the lighlt microscopic and theultrastructural level. Lab. Incvest. 32: 235-242.

45. Naruse, T., T. Ftukasawa, N. Hirokawa, S. Oike, anid Yl.Mivakawa. 1976. The pathogenesis of experimenitalmemrbranouis glomerulonephritis in duced with homolo-gous nephritogeinie tubular aintigeni. J. Exp. Mled. 144:1347-1362.

46. Vani Dammlle, B., G. J. Fleuireni, W. W. Bakker, P. J.Hoedemaeker, and R. L. Vernier. 1976. Fixecl glomiierularanitigens in the pathogeinesis of heterologouis immilunItecomplex niephritis. Kidniey IJut. 10: 551. (Abstr.)

47. Ford, P. N1. 1975. The effect of maniipuilationi of re-

tictuloenidothelial system activity onl glomeruilar deposi-tion of aggregated proteini anid immulne complexes in two(lifferent strainis of mlice. Br. J. Ex). Pat lol. 56: 523-529.

48. Swerdlin, A., P). Hoff'steni, C. Hill, A. Tavel, anid S. Klahr.1976. Retictuloenidothelial ftunctioni in the LCNI mousemiiodel of iiimmunle complex glomerulloniephritis (ICGN).Kidtnetl Itit. 10: 551. (Abstr.)

49. Haakenistad, A. O., G. E. Striker, anid MI. Mlannik. 1976.The glomerulatr deposition of soluble immuntie coomplexesprepared with re(duced and alkylate(l antibodies ani(d withintact antibodies in mice. Lab. Intiest. 35: 293-301.

50. Haakenstad, A. O., J. B. Case, anid NI. Man-inick. 1975.Effect of cortisone oni the disappearance kinetics anidtissuie localizationi of soluble immulllne con)iplexes. J.Ilmimuniol. 114: 1153-1160.

51. Boltoni, W. K., B. H. Spargo, and E. J. Lewis. 1974. Chroniietutologotis immuinuiie coiomplex glomerulopathv: effect of

cyproheptacline.j. Lab. Clitn. Mled. 83: 695-704.52. Ryan, G. B., and( MI. J. Karnovsky. 1975. An ultrastrtuettural

stuidyl of the miieclhansnisms of proteiniuria in amiinioiuni-cleoside nephrosis. Kidcney Juit. 8: 219-232.

53. Caulfield, J. P., J. J. Reid, and NI. G. Far(quihar. 1976.Alterations of glomeruilar epitheliumll in acuite aminionu-cleosidle nieplhrosis. Lab. Invest. 34: 43-59.

54. Hebert, L. A., C. L. Allhisenl, S. MI. Koethe, and G. E.Rodev. 1976. Uptake of immunie comlplexes by the kidney:effect of decreasinig delivery rate 1y decreasinig renialblood How. Clini. Res. 24: 589A. (Abstr.)

55. Gelfanld, NI. C., NI. NI. Franik, and I. Creen. 1975. Areceptor for the third conmponenit of comlplemiienit in thehuman glomerulus.j. Ex). Med. 142: 1029-10)34.

56. Burkholder, P. NI., T. D. Oberly, T. A. Barber, A. Beacomii,antcd C. Koehler. 1977. Immiiunie adherence in renalglomerutili. Comlplemiienit receptor sites onl glomerular-capillary epithelial cells. AmI J. Pathiol. 86: 635-654.

57. Gelfacld. NI. C., NI. L. Shini, R. B. Nagle, I. Creen, and(I M.NI. Fraink. 1976. The glonmerular comsplemiienit receptor inimmunologicallyi mediated renal glomeruilar injury. N.Etigl. J. Med. 295: 10-14.

58. Schneeberger, E. E., and(I XV. E. Grupe. 1976. I'heultrastructure of the glonmeruilar slit dliaphragmii in auitolo-gouis immunie complex nephlitis. Lab. Incest. 34: 298-305.

59. Seile-, NI. XV., NI. A. Venkatachalam, andl R. S. C'otrain.1975. Glomlerullar epitheliumll: structural alterationis in-clucetc by polvcations. Scienice (W1lash. D). C.). 189:390-393.

60. Seiler, MI. \V., H. G. Reinnke, NI. A. Venkatachalam, and R.S. Cotrani. 1975. Pathogenesis of polycation-inclucedalterations ("ftision'") of glomerular epitheliuim. Lab.Itncest. 36: 48-61.

61. Schhneeberger, E. E., A. B. Collins, H. Latta, ancd R. T.McCluskev. 1977. Diminished glomerular accunmulationiof colloidal carbon in autologotis immiinitie complexnel)hritis. Lab. Invest. 37: 9-19.

62. Mlaier, S. NI., A. J. Fish, E. B. Blatn, and A. F. MIichalel.1972. The glomerular nmesaniigiuml. I. Kinetic studclies ofmiiacromiiolecular uptake in normalcll and nephrotic rats. J.Clitn. Itncest. 51: 1092-1101.

63. Couser, 1W. C., and NI. NI. Stilmanit. 1976. NMesangiallesionis acnd( focal glomiierular sclerosis in the aginig rat. Lab.Itncest. 33: 491-501.

64. Velosa, J. A., R. J. Glasser, 1. E. Nev ins, and( A. F. Nlichael.1977. Exerpimiientatl model offiocal sclerosis. II. Correla-tion with immunopathologic chalnges, mlacromolecuilarkinietics and polvanion loss. Lab. Incest. 36: 527-534.

.572 Con.ser, Hloler, Stilnait, Jermnanocich, anid Belok