Canine Glomerulonephritis: Prevalence inDogs Submitted at Random for Euthanasia

B. T. Rouse and R. J. Lewis*

ABSTRACT

The kidneys from 71 stray dogs submittedfor euthanasia were examined by fluorescencemicroscopy (IF) for evidence of immunecom-plex glomerulonephritis (GN) and by histo-logy for evidence of renal pathology. Dogswere divided into three groups according toestimated age: less than one year old (A),adult (B) and aged (C). IgG deposits werefound in 0/31 dogs in group A, 6/20 in groupB and 10/20 in group C. Diffuse proliferativeGN was evident in 0/31, 5/19 and 7/15 dogsin group A, B and C respectively. One dog ingroup B and two in group C had histologicalsigns of membranous nephropathy.No definite correlation between results of

IF and clinical findings were noted and in theaged group the correlation between results ofIF and histopathology was poor. These re-sults serve to show that GN may be morecommon in dogs than previously recognizedand that pathological changes may not be al-ways accompanied by clinical nephritis.

RESUME

Cette etude portait sur les reins de 71 chienserrants soumis pour euthanasie. On les exa-mina par la technique d'immunofluorescence,pour verifier la presence du complexe de laglomerulo-nephrite immunitaire, et par la mi-croscopie ordinaire, pour verifier la presencede lesions renales. On divisa ces chiens entrois groupes, d'apres leur age approximatif:

*Department of Veterinary Microbiology (Rouse) andDepartment of Veterinary Pathology (Lewis), WesternCollege of Veterinary Medicine, University of Saskat-chewan, Saskatoon, Saskatchewan S7N OWO.

Submitted April 9, 1975.

A) ages de moins d'un an, B) adultes, C)vieux. Aucun des 31 chiens du groupe A, maissix des 20 du groupe B et dix des 20 du groupeC recelaient des depots d'IgG. Aucun des 31chiens du groupe A, mais cinq sur 19 de ceuxdu groupe B et sept sur 15 de ceux du groupeC presentaient des lesions diffuses de glome-rulo-nephrite proliferative. Un des chiens dugroupe B et deux du groupe C presentaientdes lesions histologiques de nephropathiemembraneuse.On ne releva aucune relation precise entre

les resultats de l'immunofluorescence et lesobservations cliniques. Chez les vieux chiens,la relation entre les resultats de l'immunofluo-rescence et ceux de l'histopathologie s'averamediocre. Les r'sultats de cette etude laissentpresumer que le complexe de la glomerulo-nephrite immunitaire canine pourrait s'avererplus frequent qu'on ne l'a deja cru et que leslesions renales ne correspondent pas toujoursa une nephrite clinique.

INTRODUCTION

In addition to playing a protective role,the immune response to an antigen may re-sult in tissue damage. At least fourmechanisms have been proposed to explainthe pathogenesis of immunological tissuedestruction (4). In one of these mechan-isms (Type III), complexes of antigen andantibody along with complement cause aninflammatory response. Although this re-sponse can occur in vessel walls in anyorgan it is frequently seen in the capil-laries of the glomerulus. In man, immunecomplex glomerulonephritis is one of thecommonest causes of renal damage (8) but

Vol. 39- October, 1975 365

in dogs until recently glomerulonephritishas not been recognized as an importantcause of renal impairment (6, 9, 11). Re-cently, however, several reports of canineglomerulonephritis have appeared (5, 6,12, 15) including one report (10) that de-scribes 42 cases amongst animals with pro-gressive renal disease or some other clin-ical syndrome. That glomerulonephritis,as detected by immunopathological tech-niques, is not a rare disease is also shownin the present communication. We report asurvey of 71 dogs randomly submitted foreuthanasia and demonstrate that immunecomplex deposits frequently occur in theglomerulus although most of the dogs hadno clinical evidence of nephritis. The etio-logy of these lesions and their clinicalsignificance remains to be assessed.

MATERIALS AND METHODS

DOGS

Most of the dogs in the study werestrays collected from numerous Saskatche-wan- communities. They were held at theAnimal Resources Center, University ofSaskatchewan, for a few days after whichthey were euthanized. Animals were agedafter physical examination and dividedinto three groups: less than one year, adultand aged (probably greater than six yearsold). Prior to euthanasia blood was col-lected for measurement of blood urea nitro-gen (BUN) and while under anaesthetica bladder urine sample was collected foranalysis. Blocks of kidney were snap-frozen in isopentane in liquid nitrogen andother blocks were fixed in 10%c neutralbuffered formalin for histological exam-ination.

FLUORESCENCE MICROSCOPY

Sections 3-6,u thick were cut with a cry-ostat, fixed for two minutes at 20°C inacetone and washed for ten minutes in twochanges of phosphate buffered saline(PBS-0.01 M phosphate, 0.15 M sodiumchloride pH 7.2). All sections were stainedwith fluorescein-conjugated IgG fraction

Fig. 1. Positive fluorescence for dog IgG in renal glome-rulus of male aged dog. Note irregular deposits alongcapillary glomerular basement membrane. X205.

of rabbit anti-dog IgG' and some sectionsalso with rabbit anti-dog #XC' or rabbitanti-dog albumen' for 30 minutes at roomtemperature. A counterstain of rhodamine-conjugated bovine serum albumen2 wasadded to the diluted fluoresceinated re-agents. Following staining, sections werewashed for five minutes in each of threechanges of PBS, then mounted with gly-cerol (1 part) and PBS (9 parts). Fluor-escent microscopy was performed with aCarl Zeiss Ultraphot III using incidentlight from an Osram HBO 200 lamp withBG 3 exciter filter in combination with apermanent BG 38 filter and 50/44 barrierfilter. These filters gave a peak excitationintensity of 320-400 nm. Both anti-IgG andanti-,81C reagents were absorbed withwashed dog erythrocytes and washed dogbuffy coat cells. Certain control sectionswere reacted first with unlabelled rablbitanti-dog IgG or anti-albumen'.

LIGHT MICROSCOPY

Kidney sections were fixed in 10% neu-tral buffered formalin, sectioned at 5pand stained with haematoxylin and eosin(H&E). All slides were evaluated forpathological changes with emphasis placedon the glomerulus. Selected tissues weresectioned at 2,u and stained with H&E,periodic acid Schiff and methenamine sil-ver. Classification of glomerular lesions

'Cappel Laboratories, Downingtown, Pennsylvania 19335.

2BBL, Cockelsville, Maryland 21039.

:Cappel Laboratories, Downingtown, Pennsylvania 19335.

Can. J. comp. Med.366

followed that of Murray and Wright whichdivided glomerular lesions into two mor-phological types, proliferative glomerulo-nephritis (GN) and membranous nephro-pathy (10). Proliferative GN was charac-terized by glomerular hypercellularity re-sulting predominantly from mesangial cellproliferation. Polymorphonuclear leucocyte(PMN) infiltration and epithelial cell pro-liferation constituted a minor componentof the hypercellularity. The glomerularbasement membrane (GMB) of some capil-lary loops was thickened and the glomeru-lar space almost obliterated. Adhesionsbetween GBM and Bowman's capsule occa-sionally occurred. On the basis of lesiondistribution, proliferative GN was furthersubdivided into diffuse and segmentalforms. Hypercellularity affecting the wholeglomerular tuft characterized diffuse pro-liferative GN whereas mesangial cell pro-liferation affecting only one or two lobulesof the glomerulus indicated segmentalproliferation GN.Membranous nephropathy was charac-

terized by a diffuse irregular thickening ofthe GBM of all capillary loops, a decreasein the cellularity of the glomerulus andpossibly occasional capsular adhesions.

ELUTION OF IMMUNOGLOBULINS FROMKIDNEYThe method of Fong and Drummond (3)

was used. Renal cortical tissue was ex-pressed through an 80 mesh stainless sieveand suspended in PBS. The suspension waswashed six times in cold PBS and the finalsediment which was composed mainly ofglomeruli was ground in a Tenbroeketissue grinder. Glomeruli were suspendedin approximately ten volumes of 0.02 Mcitrate buffer (pH 3.2) and held at roomtemperature for two hours with occasionalshaking. The glomeruli were removed bycentrifugation at 5,000 g for ten minutesand the pH of the glomerular eluate ad-justed to pH 7.4 with NaOH. After dialysisat 4°C against many changes of PBS, theeluate was concentrated to approximately5 mg of protein per ml. The indirect fluor-escent antibody technique was used to de-tect anti-GMB activity. Sections of youngdog kidneys, negative by direct fluores-cence for IgG, were reacted with the eluateeither from dogs with positive fluorescenceor with dogs with negative fluorescence for30 minutes at room temperature. Aftertwo washes in PBS, fluorescein labelledrabbit anti-dog IgG was added.

Fig. 2. Diffuse proliferative G!N in an adult male dog.Note glomerular hypercellularity and thickening of theGBM of many capillary loops. 2L H&E. X186.

RESU LTS

CLINICAL FINDINGS

None of the 71 animals in the studywere considered clinically ill from renaldisease. However, of the 71 animals exam-ined, three of those in the aged group hadsigns of possible renal dysfunction indi-cated by proteinuria (30, 60 and 80 mg/100 ml). The levels, however, were relative-ly low and the specific gravity of eachsample was within the normal range (1.048,1.027 and 1.047 respectively). One otheranimal in the aged group had a trace ofproteinuria. In the adult group, one ani-mal had a proteinuria of 750 mg/100 mland a trace of proteinuria was present intwo other animals. In the dogs less thanone year of age, one animal had a trace ofprotein in its urine. Three animals hadmild nasal and ocular discharges and feversuggestive of distemper.

FLU)ORESCENCE MICROSCOPY

The degree of glomerular staining was

judged subjectively into four categories:completely negative (0), glomeruli lightlystained throughout the glomerulus or inpatches (1+), definite fluorescence affect-ing the whole glomerulus (2+) and heavyfluorescence (3+) (Fig. 1). One plus re-

actions were considered of doubtful signi-ficance.

In the 2+ and 3+ categories, the pat-tern of staining was considered granular

Vol. 39 - October, 1975 367

TABLE I. Results of Fluorescence and Light Microscopy Examination of 71 Dogs

Degree of Fluorescencea Glomerular HistopathologybAge Neg + ±+ +++ Normal DPGN MN NE Total

1 year ............ 30 1 0 0 31 0 0 0 31Adult ............ 6 8 5(1)c ld 13 5 1 1 20Aged ............. 6 4(3) 5 5(1) 6 7 2 5 20

aCriteria described in resultsbCriteria described in methodscNumbers in brackets refer to animalswith proteinuria or azotemadAlso positive for complement deposition.This animal appeared to have distemper

with the deposit usually situated on theendothelial aspect of the GBM. In someanimals the pattern appeared linear butthis was probably a fine granular patternand not indicative of true antiglomerularbasement membrane antibody deposition.This conception was supported by negativeattempts to eluate antiGBM antibody fromsuch kidneys. In order to determine if the2+ and 3+ staining reactions with anti-IgG were specific, a number of controlswere included on a few selected specimens.Firstly, treatment with unlabelled anti-dogIgG prior to reaction with fluorescein la-belled anti-IgG yielded negative or veryweak staining. However, treatment withnormal rabbit serum or rabbit anti-dogalbumen failed to prevent staining by thefluorescein labelled anti-dog IgG. Second-ly, the labelling activity of anti-dog IgGcould be removed by prior absorption ofthe fluoresceinated reagent with dog IgG.Thirdly, treatment of sections positive withanti-IgG were not positive when stainedwith fluoresceinated anti-rabbit IgG orwith anti-dog albumen. Finally, treatmentof positive sections with unlabelled rabbitanti-dog IgG followed by fluoresceinatedsheep anti-rabbit4 resulted in fluorescence.The results of the examination of the

71 dogs are shown in Table I. All of thekidneys from dogs younger than one yearold were judged negative. In the adultgroup, six of 20 showed 2+ and 3+ stain-ing with anti-IgG and eight had slight glo-merular (1+) staining. The one dog inthe adult group with 3+ staining withanti-IgG appeared clinically to have dis-temper. The kidney also showed a 2+ re-action with anti-,/3C but was negative withanti-albumen. A further dog in this agegroup with possible distemper had 2+positive fluorescence. One dog in this

4Wellcome, Beckenham, England.

DPGN - diffuse proliferative GNMN membranous nephropathyNE - not examined by histopathology

group had proteinuria (750 mg/%) and,in addition, had a 2+ positive reactionwith anti-IgG.

In the aged group, half of the kidneysshowed IgG deposits in the glomerulus. Infive of these, the reaction was consideredheavy (3+) but only one of these dogshad proteinuria (80 mg/100 ml). Two dogsin the group had elevated BUN (50 and116 mg/100 ml) but neither dog had signsof IgG deposition in their glomeruli. Onedog with positive immunofluorescenc'-(2+) was suspected to have distemper.

LIGHT MICROSCOPY

The results are summarized in Table I.In animals less than one year of age, theglomeruli of 16/31 appeared more cellularthan the glomeruli of adult animals. How-ever, this change was unassociated withany alteration in GBM thickness, increasein mesangial matrix, capsular adhesions orPMN infiltration. The degree of cellulari-ty was not considered abnormal for younganimals.

In the adult group, the glomeruli of allsix dogs with positive fluorescence hadhistological evidence of glomerular patho-logy. Five of these had diffuse prolifera-tive GN (Fig. 2) and one demonstratedmembranous nephropathy (Fig. 3). Thislatter animal had heavy granular depositsof both IgG and complement along theGBM.

In the aged group, diffuse proliferativeGN was present in seven of 15 animalsexamined histologically (five animalswere examined by immunofluorescenceonly). Four of the seven histologically posi-tive dogs were judged definitely positive(3+) by immunofluorescence and threewere considered negative. A further fouranimals had definite or heavy glomerular

Can. J. comp. Med.368

Fig. 3. Membranous nephropathy in an aged male. Thereis a decrease in the cellularity of the glomerulus andthe GBM of the capillary loops is thickened. 2 t H&E.X290.

fluorescence (2 or 3+) but were judgedhistologically normal. Two animals in thisgroup had membranous nephropathy andboth had heavy deposits of IgG. Theseanimals were not examined for comple-ment deposition.

Segmental proliferative GN was notpresent in any of the animals examinedhistologically.

DISCUSSION

Evidence is accumulating to refute thepreviously held opinion (13) that glomeru-lonephritis (GN) is a rare condition inthe dog. In the present survey of 71 kid-neys examined by immunofluorescence(IF), 16 were judged to have GN sincedeposits of IgG were detected along theGMB. Several control studies indicate thatthe positive fluorescence was indeed attri-butable to IgG deposition. We assume fronthe irregular "lumpy bumpy" (2) patternof IgG deposition that the dogs had im-mune complex glomerulonephritis. Ourfailure to further confirm this notion byregularly demonstrating similar depositsof complement may be explained by theweak reactivity of our anticomplement re-agent.The dogs used in the study were all

strays collected for euthanasia and con-sequently we had no history. During thethree days holding prior to euthanasia all

but three dogs appeared physically nor-mal. However, five other animals possiblyhad mild renal dysfunction as indicated byproteinuria or azotemia. Two of these dogshad GN detectable by IF. The other 14zIF-positive dogs in the study showed no in-dication of renal dysfunction. Although pre-vious reports of canine GN, detectable byIF, have been largely from dogs with clin-ical nephritis, the lack of correlation be-tween IF and clinical pathology findingshas been reported in other animal species(1, 7). Presumably, even if the immunecomplex build up along the glomerularbasement membrane (GBM) is sufficient tocause damage to the glomerulus the otherstructures of the kidney can compensatefor the damage.

Glomerulonephritis was also detectableby histopathology. Two forms were dis-tinguished, diffuse proliferative GN andmembranous nephropathy. Of these twoforms which have previously been de-scribed in detail by Murray and Wright(10) proliferative GN was more common,occurring in 12 of the 15 animals with glo-merular histopathology. Both types ofglomerular lesions are assumed to be theresuLlt of immunological damage to theglomerulus (2, 10). In our study, discre-pancies were found between the results ofIF and histopathology. Such discrepancieshave also been noted in other species exam-ined (1, 7). Thus, in the mouse, immunecomplex GN may be common in older ani-mals but no glomerular damage is detect-able by histopathology (7). From ourexperimental design, it is highly possiblethat many of the deposits found could haveresulted from the glomerular trapping ofcomplexes present in the circulation foronly a brief period, perhaps of insufficientduration to cause severe glomerular dam-age. If this were the case, we assume thatwith time the turnover and removal ofGBM would lead to the disappearance ofthe immune complex deposition from thekidney (14). In those dogs with glomeru-lar histopathology and negative fluores-cence we could either assume that thepathogenesis did not involve immune com-plexes or that the previously pathologicalimmune complexes had disappeared. Theseconceptions could be evaluated by the se-quential sampling of dogs over severalmonths. Such studies are under way inour laboratory.

In the present study, although we haveassumed that the glomerular IgG deposits

Vol. 39 - October, 1975 369

represent immune complexes, we do notknow the nature of the antigen. Antigensof diverse nature including infectiousagents, self components and dietary sub-stances have been incriminated in isolatedinstances in the dog as well as in otherspecies (5, 9, 10). However, in the greatmajority of cases of immune complex GNthe antigen is not identified. Further stu-dies are needed to decide if the immunecomplex GN described results from com-plexes of immunoglobulin with a commonantigen or whether several different anti-gens are involved. Studies are in progressusing antisera against several infectiousagents to decide if these agents can act inthe initiation and development of canineglomerulonephritis.

ACKNOWLEDGMENTS

We appreciate the technical assistanceof Brian Manns, Lynn Franson and EdBueckert and the encouragement and cri-ticism of Dean N. 0. Nielsen.

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